研究者詳細 - 鳴海　覚志

論文 - 鳴海　覚志

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Familial Non-Autoimmune Hyperthyroidism Caused by an Extracellular Domain Variant (p.Leu267Phe) of the TSH Receptor.

Shimura K, Ichihashi Y, Abe K, Ishii T, Hasegawa T, Narumi S

Hormone research in paediatrics 1 - 9 2025年02月

ISSN 1663-2818

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Recovery from Atrophic Autoimmune Thyroiditis in a Child: Thyroid Stimulation-Blocking Antibody as a Prognostic Marker.

Kusano C, Hori N, Hasegawa T, Narumi S

Hormone research in paediatrics 1 - 5 2025年01月

ISSN 1663-2818

Introduction: Atrophic autoimmune thyroiditis (AAT) is a form of autoimmune hypothyroidism characterized by the absence of a goiter. Thyroid stimulation-blocking antibody (TSBAb) has been detected in a subset of pediatric AAT cases. Although the disappearance of TSBAb has been related with the recovery of thyroid function in adult AAT cases, similar outcomes have not been documented in pediatric cases. Case Presentation: A 2-year-old Japanese boy presented for evaluation of stunted growth from1 year 10 months of age. Tests for congenital hypothyroidism were negative on newborn screening, and he had no significant medical history. However, he showed symptoms of hypothyroidism (inactiveness, hair loss, dry skin), and primary hypothyroidism was confirmed by blood test (serum TSH level, 818 mU/L; serum free T4 level, <0.40 ng/ dL). The patient exhibited a unique antibody profile: positive for TSH receptor antibody (TRAb) and TSBAb and negative for anti-thyroglobulin antibody (TgAb) and antiperoxidase antibody (TPOAb). He was treated with levothyroxine, after which his growth was normalized. During the 8-year follow-up, the patient's TSBAb levels decreased, allowing for the discontinuation of levothyroxine therapy. Conclusion: We reported the case of a 2-year-old boy diagnosed with AAT who presented with a characteristic antibody profile, negative for TgAb and TPOAb, but positive for TRAb and TSBAb. During 8 years of follow-up, TSBAb seroconversion to negative was observed, leading to treatment discontinuation at age 10 years. This case suggests that monitoring of TSBAb after a diagnosis of AAT may be used to determine treatment discontinuation even in children.

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Sex education to an adolescent male with Down syndrome in a single-mother family in Japan

Saito A., Sato T., Shima H., Yamagishi H., Narumi S., Ishii T., Hasegawa T.

Pediatrics International 67 （ 1 ） e15896 2025年01月

ISSN 13288067

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Two Novel SLC5A5 Variants (Q263L and G350D) Causing Congenital Hypothyroidism

Abe K., Koizumi M., Kogai T., Ida S., Sugisawa C., Kawai M., Hasegawa T., Narumi S.

Thyroid 35 （ 3 ） 335 - 337 2025年

ISSN 10507256

SLC5A5 encodes sodium-iodide symporter (NIS), which transports inorganic iodide into thyroid cells. Biallelic loss-of-function variants in SLC5A5 cause thyroid dyshormonogenesis due to iodide transport defect (ITD). We report a Japanese sibling with ITD carrying novel compound heterozygous SLC5A5 variants (p. [Gln263Leu]; [Gly350Asp]). The elder brother was diagnosed with congenital hypothyroidism (CH) through newborn screening (NBS), while the younger brother, with a negative NBS result, developed CH-related symptoms at age 3 months. We characterized the two variant NIS proteins in vitro and negligible iodide transport capacity of both proteins. These findings provide unique evidence for the structure-function relationship of the NIS protein.

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Establishment of Star-edited Y1 cells as a novel in vitro functional assay for STAR

Sato T., Narumi S., Sakuma T., Shimura K., Ichihashi Y., Yamamoto T., Ishii T., Hasegawa T.

Journal of Molecular Endocrinology （Bioscientifica） 73 （ 4 ） 2024年11月

ISSN 09525041

Genetic variants involving steroidogenic acute regulatory protein cause lipoid congenital adrenal hyperplasia, which is characterized by impaired steroidogenesis in the adrenal glands and gonads. Functional assessment of variant STAR proteins is necessary for an accurate genetic diagnosis. Ideally, steroidogenic cells should be used to assess the functionality of STAR proteins, but the presence of endogenous STARs in steroidogenic cells precludes such a method. Here, we generated Star-edited cells from steroidogenic Y1 mouse adrenocortical tumor cells by genome editing. Star-edited Y1 cells exhibited very low but measurable cAMP-dependent pregnenolone production. Furthermore, stimulation of the cAMP pathway for 2 weeks resulted in the formation of lipid droplets in the cytoplasm of Star-edited Y1 cells, which resembled the histology of the adrenal glands of patients with lipoid congenital adrenal hyperplasia. The steroidogenic defect of Star-edited Y1 cells can be restored by transient overexpression of mouse Star. We found that human STAR can also restore defective steroidogenesis in Star-edited Y1 cells, and we were able to construct a novel in vitro system to evaluate human STAR variants. Collectively, we established Star-edited Y1 cells that retain the steroidogenic pathway downstream of the Star protein. Star-edited Y1 cells recapitulate the functional and morphological changes of lipoid congenital adrenal hyperplasia and can be used to evaluate the functionality of human STAR variants.

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Znrf3 exon 2 deletion mice do not recapitulate congenital adrenal hypoplasia

Uchida N., Ishii T., Amano N., Takada S., Kobayashi K., Murakami T., Narumi S., Hasegawa T.

Journal of Molecular Endocrinology 73 （ 4 ） 2024年11月

ISSN 09525041

Wnt/β-catenin signaling is essential for adrenocortical development. Zinc and ring finger 3 (ZNRF3), an E3 ubiquitin ligase that attenuates Wnt/β-catenin signaling, is negatively regulated by R-spondin via an extracellular domain that is partially encoded by exon 2 of ZNRF3. We recently identified ZNRF3 exon 2 deletions in three individuals with congenital adrenal hypoplasia. ZNRF3 exon 2 deletion impairs R-spondin binding, thereby attenuating β-catenin expression and eventually leading to the development of congenital adrenal hypoplasia. To elucidate the influence of ZNRF3/Znrf3 exon 2 deletion on adrenocortical development, we generated homozygous Znrf3 exon 2 deletion (Znrf3Δ2/Δ2) mice. Whereas the adrenal glands of Znrf3Δ2/Δ2 mice did not show gross morphological changes at birth, moderate hyperplasia of the zona fasciculata (ZF), dispersed medulla arrangement, and a radially spreading zone with macrophage infiltration between the ZF and medulla were observed at 6 weeks of age. 20α-hydroxysteroid dehydrogenase, a marker of the adrenal X-zone, was hardly detected by immunostaining, and gene expression was significantly downregulated. The number of activated β-catenin-positive cells decreased in the zona glomerulosa, consistent with the results of in situ hybridization for Axin2, a Wnt/β-catenin target gene. Plasma ACTH and serum corticosterone levels in Znrf3Δ2/Δ2 mice did not differ significantly from those in wild-type mice. These results show a species-specific difference in the effects of ZNRF3/Znrf3 exon 2 deletions in humans and mice; Znrf3Δ2/Δ2 mice do not develop congenital adrenal hypoplasia but instead exhibit moderate ZF hyperplasia, dispersed medulla arrangement, X-zone dysplasia, and macrophage infiltration occurred in the inner cortex.

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Effectiveness of inactivated influenza vaccine in children during the 2023/24 season: The first season after relaxation of intensive COVID-19 measures

Shinjoh M., Yaginuma M., Yamaguchi Y., Tamura K., Furuichi M., Tsumura Y., Itaki R., Iqbal A., Maeda N., Narabayashi A., Kamei A., Shibata A., Yamada G., Nishida M., Kenichiro T., Chiga M., Shimoyamada M., Yoshida M., Fukushima N., Nakata Y., Fukushima H., Kawakami C., Narumi S., Sugaya N.

Vaccine 42 （ 23 ） 126241 2024年10月

ISSN 0264410X

Background: The annual administration of the influenza vaccine is the most effective method for preventing influenza. We have evaluated the effectiveness of the inactivated influenza vaccine in children aged 6 months to 15 years across the seasons from 2013/2014 to 2022/2023. This study aims to investigate the effectiveness of the inactivated influenza vaccine in the 2023/2024 season, the first year following the easing of strict COVID-19 measures, and possibly the last season when only the inactivated vaccine is available on the market. Methods: Adjusted vaccine effectiveness for the 2023/2024 season was assessed using a test-negative case-control design, with results based on polymerase chain reaction and rapid influenza diagnostic tests. Vaccine effectiveness was calculated by influenza type and patient hospitalization/outpatient status. Results: A total of 1832 children were recruited. The inactivated influenza vaccine was effective in preventing both symptomatic influenza A and B in both inpatient and outpatient settings. Overall vaccine effectiveness for influenza A was 51% (95% confidence interval [CI], 23%–69%, n = 930) in inpatient settings and 54% (95%CI, 27%–71%, n = 559) in outpatient settings. For influenza B, effectiveness was 60% (95%CI, 22%–79%, n = 859) in inpatient settings and 56% (95%CI, 26%–74%, n = 558) in outpatient settings. Analysis suggested that administering two doses enhanced effectiveness specifically against influenza B. Conclusions: This is the first study to demonstrate influenza vaccine effectiveness in children after the relaxation of strict COVID-19 measures in Japan (2023/2024). We recommend the current inactivated vaccine for preventing both influenza A and B in children, with consideration for the potential use of two doses to enhance effectiveness against influenza B.

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Digital clubbing without hypoxia for lysinuric protein intolerance

Watanabe D., Tsujioka Y., Nakato D., Yamada M., Suzuki H., Ohnishi T., Tamai N., Kijima T., Takenouchi T., Miya F., Narumi S., Kosaki K.

European Journal of Medical Genetics 71 104967 2024年10月

ISSN 17697212

Digital clubbing is characterized by bulbous enlargement of the terminal segments of the fingers. Hypotheses including hypoxia have been proposed for the pathogenesis of digital clubbing, but the exact pathogenesis of digital clubbing is still uncertain. Lysinuric protein intolerance (LPI) is caused by pathogenic variants in SLC7A7 and is often associated with interstitial lung disease. Previously two patients of LPI with digital clubbing but without hypoxia have been reported. It is unclear whether digital clubbing in LPI is secondary to hypoxia or directly related to SLC7A7 deficiency. Here we report a 6-year-old Japanese boy presented with digital clubbing without hypoxia. He had episodic vomiting, each episode consisting of a single vomiting event occurring once a month, and his growth had been delayed. He had interstitial lung disease and hepatomegaly. He had compound heterozygous pathogenic variants in the SLC7A7, leading to the diagnosis of LPI. Together with the two previously reported patients mentioned above, we conclude that digital clubbing can occur in the absence of hypoxia. Digital clubbing in the absence of hypoxia has been observed in two genetic disorders related to prostaglandin (PG) E2, HPGD and SLCO2A1. PGE2 synthesis is primarily regulated by the cyclooxygenase 2, which plays a critical role in the control of inflammation. A high urine PGE level in the patient was compatible with the notion that PGE2 production may be increased in LPI. The occurrence of digital clubbing in the absence of hypoxia in LPI patients with SLC7A7 may be attributed to the mechanism of increased PGE2 production.

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Comprehensive study on central precocious puberty: molecular and clinical analyses in 90 patients.

Narusawa H, Ogawa T, Yagasaki H, Nagasaki K, Urakawa T, Saito T, Soneda S, Kinjo S, Sano S, Mamada M, Terashita S, Dateki S, Narumi S, Naiki Y, Horikawa R, Ogata T, Fukami M, Kagami M

The Journal of clinical endocrinology and metabolism 110 （ 4 ） 1023 - 1036 2024年09月

ISSN 0021-972X

Context: Defects in MKRN3, DLK1, KISS1, and KISS1R and some disorders, such as Temple syndrome (TS14), cause central precocious puberty (CPP). Recently, pathogenic variants (PVs) in MECP2 have been reported to be associated with CPP. Objective: We aimed to clarify the contribution of (epi)genetic abnormalities to CPP and clinical and hormonal features in each etiology. Methods: We conducted targeted sequencing for MKRN3, DLK1, MECP2, KISS1, and KISS1R and methylation analysis for screening of imprinting disorders such as TS14 associated with CPP in 90 patients with CPP (no history of brain injuries and negative brain magnetic resonance imaging) and collected their clinical and laboratory data. We measured serum DLK1 levels in 3 patients with TS14 and serum MKRN3 levels in 2 patients with MKRN3 genetic defects, together with some etiology-unknown patients with CPP and controls. Results: We detected 8 patients with TS14 (6, epimutation; 1, mosaic maternal uniparental disomy chromosome 14; 1, microdeletion) and 3 patients with MKRN3 genetic defects (1, PV; 1, 13-bp deletion in the 5′-untranslated region [5′-UTR]; 1, microdeletion) with family histories of paternal early puberty. There were no patients with PVs identified in MECP2, KISS1, or KISS1R. We confirmed low serum MKRN3 level in the patient with a deletion in 5′-UTR. The median height at initial evaluation of TS14 patients was lower than that of all patients. Six patients with TS14 were born small for gestational age (SGA). Conclusion: (Epi)genetic causes were identified in 12.2% of patients with CPP at our center. For patients with CPP born SGA or together with family histories of paternal early puberty, (epi)genetic testing for TS14 and MKRN3 genetic defects should be considered.

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Genotype–Phenotype Correlations in 30 Japanese Patients With Congenital Hypothyroidism Attributable to TG Defects

Tanase-Nakao K., Iwahashi-Odano M., Sugisawa C., Abe K., Muroya K., Yamamoto Y., Kawada Y., Mushimoto Y., Ohkubo K., Kinjo S., Shimura K., Aoyama K., Mizuno H., Hotsubo T., Takahashi C., Isojima T., Kina Y., Takakuwa S., Hamada J., Sawaki M., Shigehara K., Sugimoto S., Etani Y., Narumi-Wakayama H., Mine Y., Hasegawa T., Hishinuma A., Narumi S.

Journal of Clinical Endocrinology and Metabolism 109 （ 9 ） 2358 - 2365 2024年09月

ISSN 0021972X

Context: Thyroglobulin (Tg), encoded by TG, is essential for thyroid hormone synthesis. TG defects result in congenital hypothyroidism (CH). Most reported patients were born before the introduction of newborn screening (NBS). Objective: We aimed to clarify the phenotypic features of patients with TG defects diagnosed and treated since the neonatal period. Methods: We screened 1061 patients with CH for 13 CH-related genes and identified 30 patients with TG defects. One patient was diagnosed due to hypothyroidism-related symptoms and the rest were diagnosed via NBS. Patients were divided into 2 groups according to their genotypes, and clinical characteristics were compared. We evaluated the functionality of the 7 missense variants using HEK293 cells. Results: Twenty-seven rare TG variants were detected, including 15 nonsense, 3 frameshift, 2 splice-site, and 7 missense variants. Patients were divided into 2 groups: 13 patients with biallelic truncating variants and 17 patients with monoallelic/biallelic missense variants. Patients with missense variants were more likely to develop thyroid enlargement with thyrotropin stimulation than patients with biallelic truncating variants. Patients with biallelic truncating variants invariably required full hormone replacement, whereas patients with missense variants required variable doses of levothyroxine. Loss of function of the 7 missense variants was confirmed in vitro. Conclusion: To our knowledge, this is the largest investigation on the clinical presentation of TG defects diagnosed in the neonatal period. Patients with missense variants showed relatively mild hypothyroidism with compensative goiter. Patients with only truncating variants showed minimal or no compensative goiter and required full hormone replacement.

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Molecular and Clinical Features of Congenital Hypothyroidism Due to Multiple DUOX2 Variants

Uehara E., Abe K., Tanase-Nakao K., Muroya K., Hattori A., Matsubara K., Fukami M., Narumi S.

Thyroid 34 （ 7 ） 827 - 836 2024年07月

ISSN 10507256

Background: DUOX2 is one of the major causative genes of congenital hypothyroidism (CH). Still, the mutation spectrum and clinical outcomes of biallelic DUOX2 variants are not fully understood. This study aimed to elucidate the molecular features and long-term clinical manifestations of CH caused by multiple pathogenic DUOX2 variants. Methods: A total of 255 patients with CH were screened for rare variants of 11 known causative genes. DUOX2 variants were classified according to their protein structure and residual activity. In vitro assays were performed for several variants of unknown functions. Clinical analyses were conducted for patients with multiple pathogenic variants of DUOX2 but not of other genes. Results: We identified 24 pathogenic variants of DUOX2, together with two benign variants and seven variants of uncertain significance, in 63 patients. The pathogenic variants included three missense substitutions and one frameshift variant that have not yet been linked to CH. Twenty-one patients carried multiple pathogenic DUOX2 variants without any other pathogenic gene variants. Three of the 21 patients harbored homozygous variants. Family analysis, long-read amplicon sequencing, and haplotype phasing confirmed compound heterozygosity of the DUOX2 variants in 14 patients, whereas the allelic positions of the variants in the remaining four patients could not be determined. Of the 21 patients, 19 were treated with levothyroxine; their ages at drug withdrawal ranged from 9 months to 21.4 years. Three patients required retreatment after drug-free intervals of 6 months, 8 months, and 10 years. There were no differences in clinical severity among patients with DUOX2 amorphic/amorphic, amorphic/hypomorphic, and hypomorphic/hypomorphic variants. Conclusions: These results broaden the mutational spectrum of DUOX2. Furthermore, our data imply that patients with multiple pathogenic DUOX2 variants typically exhibit transient CH without significant genotype-phenotype correlations. Most importantly, this study demonstrated for the first time that these patients are at risk of developing recurrent hypothyroidism after a long drug-free interval.

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15q26.1マイクロサテライトリピート数減少による家族性甲状腺異常症の5家系の新生児期の特徴

丸尾良浩, 塚村篤史, 筒井英美, 長井静世, 長崎啓祐, 鳴海覚志

日本マス・スクリーニング学会誌（(一社)日本マススクリーニング学会） 34 （ 2 ） 234 - 234 2024年07月

ISSN 0917-3803

Disulfide Bonds of Thyroid Peroxidase Are Critical Elements for Subcellular Localization, Proteasome-Dependent Degradation, and Enzyme Activity

Iwasaki H., Suwanai H., Yakou F., Sakai H., Ishii K., Hara N., Buckle A.M., Kanekura K., Miyagi T., Narumi S., Suzuki R.

Thyroid （Mary Ann Liebert Inc） 34 （ 5 ） 659 - 667 2024年05月

ISSN 10507256

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) (p < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.

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4世代にわたり特異的甲状腺表現型を示した家系とその病因PAX8新規バリアントG56Sの多階層的機能解析

阿部清美, 西岡淳子, 小田野めぐみ, 岩野麗子, 藏本純子, 鳴海覚志, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 100 （ 1 ） 294 - 294 2024年05月

ISSN 0029-0661

Functional variants in a TTTG microsatellite on 15q26.1 cause familial nonautoimmune thyroid abnormalities

Narumi S., Nagasaki K., Kiriya M., Uehara E., Akiba K., Tanase-Nakao K., Shimura K., Abe K., Sugisawa C., Ishii T., Miyako K., Hasegawa Y., Maruo Y., Muroya K., Watanabe N., Nishihara E., Ito Y., Kogai T., Kameyama K., Nakabayashi K., Hata K., Fukami M., Shima H., Kikuchi A., Takayama J., Tamiya G., Hasegawa T.

Nature Genetics 56 （ 5 ） 869 - 876 2024年05月

ISSN 10614036

Insufficient thyroid hormone production in newborns is referred to as congenital hypothyroidism. Multinodular goiter (MNG), characterized by an enlarged thyroid gland with multiple nodules, is usually seen in adults and is recognized as a separate disorder from congenital hypothyroidism. Here we performed a linkage analysis of a family with both nongoitrous congenital hypothyroidism and MNG and identified a signal at 15q26.1. Follow-up analyses with whole-genome sequencing and genetic screening in congenital hypothyroidism and MNG cohorts showed that changes in a noncoding TTTG microsatellite on 15q26.1 were frequently observed in congenital hypothyroidism (137 in 989) and MNG (3 in 33) compared with controls (3 in 38,722). Characterization of the noncoding variants with epigenomic data and in vitro experiments suggested that the microsatellite is located in a thyroid-specific transcriptional repressor, and its activity is disrupted by the variants. Collectively, we presented genetic evidence linking nongoitrous congenital hypothyroidism and MNG, providing unique insights into thyroid abnormalities.

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Letter to the Editor regarding “Serum steroid metabolite profiling by LC-MS/MS in two phenotypic male patients with HSD17B3 deficiency: Implications for hormonal diagnosis”

Sato T., Nakano S., Honda M., Narumi S., Ishii T., Hasegawa T.

Journal of Steroid Biochemistry and Molecular Biology （Elsevier BV） 239 106484 - 106484 2024年05月

ISSN 09600760

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中枢性思春期早発症患者90症例の遺伝学的解析と臨床像の検討

成澤宏宗, 浦川立貴, 長崎啓祐, 矢ヶ崎英晃, 内木康博, 佐野伸一朗, 齋藤朋洋, 曽根田瞬, 鳴海覚志, 堀川玲子, 緒方勤, 深見真紀, 鏡雅代

日本内分泌学会雑誌（(一社)日本内分泌学会） 100 （ 1 ） 355 - 355 2024年05月

ISSN 0029-0661

日本人若年発症原発性アルドステロン症の遺伝学的・臨床的特徴

内田登, 石井智弘, 西本紘嗣郎, 小原收, 鳴海覚志, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 100 （ 1 ） 323 - 323 2024年05月

ISSN 0029-0661

先天性甲状腺機能低下症の次世代再発率は最大30%である

長崎啓祐, 鳴海覚志, 中尾佳奈子, 廣嶋省太, 澤野堅太郎, 小貫孝則, 柴田奈央, 入月浩美

日本内分泌学会雑誌（(一社)日本内分泌学会） 99 （ 5 ） 1301 - 1301 2024年04月

ISSN 0029-0661

甲状腺ペルオキシダーゼ活性におけるジスルフィド結合の重要性の検討

岩崎源, 諏訪内浩紹, 酒井裕幸, 谷古宇史芳, 鳴海覚志, 金蔵孝介, 鈴木亮

日本内分泌学会雑誌（(一社)日本内分泌学会） 99 （ 5 ） 1254 - 1254 2024年04月

ISSN 0029-0661

Single-Exon Deletions of ZNRF3 Exon 2 Cause Congenital Adrenal Hypoplasia

Amano N., Narumi S., Aizu K., Miyazawa M., Okamura K., Ohashi H., Katsumata N., Ishii T., Hasegawa T.

Journal of Clinical Endocrinology and Metabolism 109 （ 3 ） 641 - 648 2024年03月

ISSN 0021972X

Context: Primary adrenal insufficiency (PAI) is a life-threatening condition characterized by the inability of the adrenal cortex to produce sufficient steroid hormones. E3 ubiquitin protein ligase zinc and ring finger 3 (ZNRF3) is a negative regulator of Wnt/β-catenin signaling. R-spondin 1 (RSPO1) enhances Wnt/β-catenin signaling via binding and removal of ZNRF3 from the cell surface. Objective: This work aimed to explore a novel genetic form of PAI. Methods: We analyzed 9 patients with childhood-onset PAI of biochemically and genetically unknown etiology using array comparative genomic hybridization. To examine the functionality of the identified single-exon deletions of ZNRF3 exon 2, we performed three-dimensional (3D) structure modeling and in vitro functional studies. Results: We identified various-sized single-exon deletions encompassing ZNRF3 exon 2 in 3 patients who showed neonatal-onset adrenal hypoplasia with glucocorticoid and mineralocorticoid deficiencies. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the 3 distinct single-exon deletions were commonly transcribed into a 126-nucleotide deleted mRNA and translated into 42-amino acid deleted protein (ΔEx2-ZNRF3). Based on 3D structure modeling, we predicted that interaction between ZNRF3 and RSPO1 would be disturbed in ΔEx2-ZNRF3, suggesting loss of RSPO1-dependent activation of Wnt/β-catenin signaling. Cell-based functional assays with the TCF-LEF reporter showed that RSPO1-dependent activation of Wnt/β-catenin signaling was attenuated in cells expressing ΔEx2-ZNRF3 as compared with those expressing wild-type ZNRF3. Conclusion: We provided genetic evidence linking deletions encompassing ZNRF3 exon 2 and congenital adrenal hypoplasia, which might be related to constitutive inactivation of Wnt/β-catenin signaling by ΔEx2-ZNRF3.

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Prolongation of the Menstrual Cycle after Receipt of the Primary Series and Booster Doses of mRNA Coronavirus Disease 2019 (COVID-19) Vaccination

Hosoya S., Piedvache A., Nakamura A., Nasu R., Hine M., Itoi S., Yokomizo R., Umezawa A., Hiraike O., Koga K., Osuga Y., Narumi S., Morisaki N.

Obstetrics and Gynecology 143 （ 2 ） 284 - 293 2024年02月

ISSN 00297844

OBJECTIVE:To quantitatively evaluate the effect of a booster vaccination dose against coronavirus disease 2019 (COVID-19) on menstrual cycle in a large-scale retrospective cohort study using a menstrual cycle tracking smartphone application (app).METHODS:Prospectively or retrospectively recorded data, including the start and finish dates of menstrual cycles, were collected with the app. Detailed data on vaccinations, side effects, and participants' characteristics were retrospectively collected from a questionnaire on the app. For each COVID-19 vaccination shot (first, second, and third), within-individual changes in menstrual cycle length up to the fourth postvaccination cycle were evaluated.RESULTS:Among the 7,376 and 6,873 participants who had the first and second COVID-19 vaccine doses in different menstrual cycles, respectively, menstrual cycles immediately after the vaccination (first postvaccination cycles) were an average of 0.22 days (95% CI, 0.06-0.39) and 0.37 days (95% CI, 0.20-0.54) longer than the prevaccination cycle. In contrast, among the 1,672 participants who received the first and second doses in the same cycle, the first postvaccination cycle was an average of 4.21 days (95% CI, 3.69-4.72) longer. The second to fourth postvaccination cycles returned to the level of the prevaccination cycle. However, among the 4,768 participants who had the third COVID-19 vaccine dose, the menstrual cycle immediately after the vaccination was an average of 1.20 days (95% CI, 1.00-1.40) longer, with prolongation of cycles of 0.27 days (95% CI, 0.10-0.44) to 0.41 days (95% CI, 0.22-0.59) persisting from the second to the fourth postvaccination cycle.CONCLUSION:The booster shot against COVID-19 may have a greater and longer-lasting effect on menstrual cycles than the primary-series shots. Although the effect size was small, evidence on the side effects of immunization on menstruation should be accumulated.

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A thyroid adenoma in a pubertal male with thyroxine-binding globulin deficiency

Kim H., Naiki Y., Iwahashi-Odano M., Narumi S., Ito K., Ishiguro A.

Clinical Pediatric Endocrinology （Clinical Pediatric Endocrinology） 33 （ 1 ） 23 - 26 2024年01月

ISSN 09185739

Complete deficiency of thyroxin-binding globulin (TBG-CD) is not commonly associated with clinical symptoms, and little is known about thyroid tumors associated with TBG-CD. We present a case report of an asymptomatic follicular adenoma that spontaneously shrank in a patient with TBG-CD. A previously healthy 13-yr-old male presented with a diffusely swollen thyroid gland. Thyroid function tests revealed low total thyroxin and TBG concentrations, indicating a TBG deficiency. Ultrasonography revealed a mildly swollen thyroid gland with a nodule (14 × 12 × 19 mm) in the left lobe. Genetic analysis of peripheral blood revealed a previously reported SERPINA7 variant, which resulted in complete loss of TBG function. The nodule was identified as a follicular adenoma using fine-needle aspiration. Subsequently, the adenoma shrank without treatment. This pubertal case suggests that careful observation with ultrasonography is warranted for follicular adenoma in patients with TBG deficiency and that treatment may not be required.

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Usefulness of a mobile airwayscope with a monitor in examination of the external genitalia of a 483 g female neonate

Hiraizumi T., Sato T., Kobayashi H., Arimitsu T., Narumi S., Ishii T., Hasegawa T.

Pediatrics International （Wiley） 66 （ 1 ） e15759 2024年01月

ISSN 13288067

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Concurrent THRB and DUOX2 variants in a patient detected via newborn screening for congenital hypothyroidism: a case of resistance to thyroid hormone

Hatano M., Tanase-Nakao K., Uehara E., Iwano R., Muroya K., Narumi S.

Clinical Pediatric Endocrinology 33 （ 2 ） 94 - 100 2024年

ISSN 09185739

Most patients with resistance to thyroid hormone (RTH) test negative in newborn screening (NBS) for congenital hypothyroidism (CH). Here, we present a case of RTH diagnosed through NBS. The patient presented to us after her NBS for CH revealed high TSH (23.4 µIU/mL) and free T4 (FT4) (5.40 ng/dL) levels. Apart from tachycardia, she exhibited no other manifestations related to excess or deficiency of thyroid hormones. A confirmatory test replicated the findings, showing elevated serum TSH levels (35.7 µIU/mL) along with high FT4 levels (5.84 ng/ dL). Ultrasonography showed marked thyroid gland enlargement (> +4 SD). Targeted next-generation sequencing of genes associated with genetic thyroid disorders revealed a previously reported THRB variant, p.Gly345Cys. Unexpectedly, two biallelic DUOX2 variants (p.His678Arg and p.Arg1334Trp) were also detected. At her last visit, no significant issues were observed with neurological development, growth, bone maturation, or gastrointestinal symptoms related to thyroid function at the age of 1 year, without treatment for RTH and CH. During follow-up, the TSH and FT4 levels gradually decreased. In conclusion, we report a patient with simultaneous RTH and DUOX2 defects, demonstrating the value of conducting a comprehensive analysis of multiple genes associated with thyroid diseases to better comprehend the pathogenesis in patients with atypical thyroid-related phenotypes.

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Impact of COVID-19 on paediatric care in Japan: Analysis of national health insurance claims data

Ohnishi T., Kinoshita M., Narumi S.

Acta Paediatrica, International Journal of Paediatrics 2024年

ISSN 08035253

Aim: To quantify the impact of the COVID-19 pandemic on paediatric care use compared to adult care and identify affected subdomains. Methods: Data from the National Database of Health Insurance Claims and Specific Health Checkups of Japan (NDB) for six fiscal years (FY2016-2021) were analysed. Pre-pandemic (FY2016-FY2019) and pandemic years (FY2020, FY2021) were compared to delineate changes in medical services for paediatric and adult patients. Virtually all individuals who received medical care in Japan during this period were included. Results: In FY2019, healthcare utilisation by the paediatric population (under 15 years) accounted for 24% of 245 million first outpatient visits, 7% of 1.2 billion follow-up visits, and 2.6% of 297 million patient-days hospitalisations. In FY2020, outpatient visits of those aged 0–4 years decreased by 44% (first visit) and 24% (follow-up visit) compared to FY2019, and hospitalisations decreased by 47%, with the largest declines observed in community medical institutions across all age groups. Conclusion: The COVID-19 pandemic led to a substantial reduction in paediatric healthcare utilisation in Japan, varying by age and type of care. These findings may inform the rational allocation of healthcare resources during public health emergencies.

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DHX37 Variant Is One of the Common Genetic Causes in Japanese Patients with Testicular Regression Syndrome/Partial Gonadal Dysgenesis without Müllerian Derivatives

Shimura K., Ichihashi Y., Nakano S., Sato T., Hamajima T., Numasawa K., Narumi S., Hasegawa T., Ishii T.

Hormone Research in Paediatrics （S. Karger AG） 98 （ 2 ） 1 - 8 2024年

ISSN 16632818

Introduction: The testicular regression syndrome (TRS) is a form of differences of sex development (DSD) in which the testes differentiate and function during early embryonic development, but subsequently regress. The clinical phenotype of TRS often overlaps with that of partial gonadal dysgenesis (PGD). Previous studies have demonstrated a causal association between TRS/ PGD and heterozygous missense variants of DHX37. Methods: We enrolled 11 Japanese 46,XY individuals (from 10 families) with TRS/PGD who exhibited undetected or hypoplastic testes, Möllerian duct regression, and low serum testosterone or anti-Möllerian hormone levels. The subjects underwent targeted sequencing of 36 known causative genes for DSD, PCR-based Sanger sequencing of DHX37, or whole-exome sequencing. Results: Previously described pathogenic variants or novel nonsense variants (SRY, NR5A1, and DMRT1) were observed in four out of 10 families. Additionally, we identified two heterozygous rare variants of DHX37 in four families: A previously reported pathogenic variant (c.923G>A, p.Arg308Gln) in three and a novel likely pathogenic variant (c.1882A>C, p.Thr628Pro) in one. The external genitalia of patients with the DHX37 variants varied from female-Type to male-Type without micropenis. Eighty percent of Japanese patients with TRS/PGD had monogenic disorders including DHX37 variant being the most commonly identified (40%). The external or internal genital phenotype of TRS/PGD overlaps between DHX37 variant carriers and others. Conclusions: DHX37 variant is one of the common genetic causes in Japanese patients with TRS/PGD without Möllerian derivatives. Genetic test is helpful in detecting DHX37-related TRS/PGD because of the phenotypic diversity of the external genitalia in this disorder.

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Deciphering the mystery of CHNG3

Narumi S.

Annals of Pediatric Endocrinology and Metabolism 29 （ 5 ） 279 - 283 2024年

ISSN 22871012

Congenital hypothyroidism (CH), characterized by insufficient thyroid hormone production due to abnormalities in the hypothalamic-pituitary-thyroid axis, is the most common congenital endocrine disorder. We previously conducted comprehensive genetic screening of 102 patients with permanent CH born in Kanagawa Prefecture, Japan and identified mutations in several genes in 19 CH patients, including defects in genes encoding dual oxidase 2, thyroglobulin, thyrotropin receptor, thyroid peroxidase, and paired-box 8. Despite these findings, approximately 80% of cases remain unexplained. CH pedigrees unexplained by known genetic forms of CH have been reported in the literature and registered as congenital hypothyroidism, nongoitrous, 3 (CHNG3; %609893) in Online Mendelian Inheritance in Man. We also identified a Japanese pedigree of CH that was compatible with CHNG3. However, the exact genetic cause of CHNG3 was not revealed by standard analysis methods such as exome sequencing and array comparative genomic hybridization. We therefore took a combined approach and analyzed a total of 11 undiagnosed CH pedigrees by whole genome sequencing to analyze a 3-Mb linkage region, and found a disease-causing variant affecting a TTTG microsatellite in a noncoding region on chromosome 15. Further analysis revealed that 13.9% of 989 Japanese CH patients had abnormalities involving the TTTG microsatellite, with a substantial proportion (41.5%) of familial CH cases carrying these mutations. Identification of the genetic cause of CHNG3 provides new insights into the pathogenesis of CH, and highlights the need for continued exploration of noncoding genomic regions in Mendelian disorders of unknown etiology.

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Congenital Hypothyroidism with Thyroid in situ: A Case Report with NKX2-1 and DUOX2 Hypomorphic Variants

Uehara E., Hori N., Tanase-Nakao K., Akiba K., Sueoka H., Matsubara K., Narumi S.

Hormone Research in Paediatrics 1 - 7 2024年

ISSN 16632818

Introduction: NK2 homeobox 1 (NKX2-1) encodes a transcription factor, NKX2-1, that is expressed in the thyroid gland, lung, and brain. Dual oxidase 2 (DUOX2) encodes an enzyme which generates hydrogen peroxide and is involved in the thyroid hormone synthesis. Cases of congenital hypothyroidism (CH) with dyshormonogenesis showing two or more genetic variants are increasingly reported. We describe the first case of transient dyshormonogenesis who had experimentally verified a loss-of-function NKX2-1 variant and DUOX2 variants. Case Presentation: The proband was a 15-year-old female patient with CH who was diagnosed in the frame of newborn screening for CH. She had a mildly elevated serum TSH level (14.56 mU/L), a low free thyroxine level (0.87 ng/dL), and a high thyroglobulin (Tg) level (>800 ng/mL). Ultrasonography revealed goiter. She was followed clinically without levothyroxine treatment and showed normal growth and development. She had slightly high Tg levels throughout the clinical course. Next-generation sequencing-based genetic analysis revealed that the patient was heterozygous for an NKX2-1 variant (p.Ile228Ser), a nonsense DUOX2 variant (p.[Lys530∗His678Arg]), and a functional DUOX2 polymorphism (p.His678Arg). NKX2-1 p.Ile228Ser showed about 50% reduced residual activity on the Tg promoter. Conclusion: A partial loss-of-function NKX2-1 variant with a monoallelic nonsense DUOX2 variant and a DUOX2 functional polymorphism can cause transient CH with high serum Tg levels.

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Siblings of neonatal hyperbilirubinemia with UGT1A1 double missense variants

Kubota Y., Sato T., Matsuyama M., Maruo Y., Narumi S., Ishii T., Hasegawa T.

Clinical Pediatric Endocrinology （Japanese Society for Pediatric Endocrinology） 33 （ 3 ） 195 - 198 2024年

ISSN 09185739

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A Small-for-Gestational-Age Infant with MIRAGE Syndrome Who Developed Heat Stroke and Rhabdomyolysis due to Severe Temperature Instability

Saito K., Nakagawa R., Narumi S., Ohashi H., Ishiguro A., Kabe K.

Neonatology （Neonatology） 120 （ 3 ） 390 - 394 2023年07月

ISSN 16617800

MIRAGE syndrome is characterized by myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy. This report describes heat stroke and rhabdomyolysis caused by anhidrosis as a symptom of MIRAGE syndrome in a small-for-gestational-age (SGA) female neonate born at 32 weeks of gestation (birth weight, 911 g [-3.8 SD]). She developed severe temperature instability with anhidrosis, growth failure, mild developmental delay, hypothyroidism, and intractable enteropathy. On day 156, her temperature reached 42.0°C; her fever persisted for 2 h with prolonged irritability. Her serum creatine kinase level increased to a peak value of 12,716 (normal range, 43-321) IU/L. The clinical feature was diagnosed as rhabdomyolysis caused by heat stroke, which resulted from physical exertion with anhidrosis. Her SAMD9 variant was c.2945G>A, p. (Arg982His). Neonatologists should be aware of MIRAGE syndrome as a differential diagnosis of SGA with temperature instability.

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Differential toxicity and localization of arginine-rich C9ORF72 dipeptide repeat proteins depend on de-clustering of positive charges

Miyagi T., Ueda K., Sugimoto M., Yagi T., Ito D., Yamazaki R., Narumi S., Hayamizu Y., Uji-i H., Kuroda M., Kanekura K.

iScience （iScience） 26 （ 6 ） 106957 - 106957 2023年06月

ISSN 2589-0042

Arginine-rich dipeptide repeat proteins (R-DPRs), poly(PR) and poly(GR), translated from the hexanucleotide repeat expansion in the amyotrophic lateral sclerosis (ALS)-causative C9ORF72 gene, contribute significantly to pathogenesis of ALS. Although both R-DPRs share many similarities, there are critical differences in their subcellular localization, phase separation, and toxicity mechanisms. We analyzed localization, protein-protein interactions, and phase separation of R-DPR variants and found that sufficient segregation of arginine charges is necessary for nucleolar distribution. Proline not only efficiently separated the charges, but also allowed for weak, but highly multivalent binding. In contrast, because of its high flexibility, glycine cannot fully separate the charges, and poly(GR) behaves similarly to the contiguous arginines, being trapped in the cytoplasm. We conclude that the amino acid that spaces the arginine charges determines the strength and multivalency of the binding, leading to differences in localization and toxicity mechanisms.

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Adult Thyroid Outcomes of Congenital Hypothyroidism

Sugisawa C., Narumi S., Tanase-Nakao K., Hoshiyama A., Suzuki N., Ohye H., Fukushita M., Matsumoto M., Yoshihara A., Watanabe N., Sugino K., Hishinuma A., Noh J.Y., Katoh R., Taniyama M., Ito K.

Thyroid （Thyroid） 33 （ 5 ） 556 - 565 2023年05月

ISSN 10507256

Background: More than 40 years have passed since the introduction of newborn screening (NBS) for congenital hypothyroidism (CH), and many early diagnosed patients have reached adulthood. Their thyroid morphology and function have been little studied. This cross-sectional, observational study was conducted to characterize the thyroid morphology and function of adult CH patients diagnosed in the framework of NBS for CH. Methods: A total of 103 adult CH patients born after 1979 were enrolled at Ito Hospital, Tokyo, Japan, and were classified into Goiter, Normal gland, and Dysgenesis groups based on ultrasonographic findings. For 60 patients, genetic analysis was performed. Thyroid function test results and the proportion of patients with thyroid nodules were compared among the three groups and between 56 female CH patients and 168 non-CH women matched for thyrotropin levels. Results: A significantly low serum free triiodothyronine/free thyroxine ratio (0.22) was observed in the Dysgenesis group. Thyroid nodules were detected in 14.3% (8/56) of female CH patients, more frequently than in non-CH women. Thyroid nodules were detected most frequently in the Goiter group (71%, 10/14). Genetic defects were identified in 89% (8/9) of patients belonging to the Goiter group, including thyroglobulin defect (33%, 3/9), thyroid peroxidase defect (33%, 3/9), and dual oxidase 2 defect (22%, 2/9). Conclusions: Our results suggest that adults with thyroid dysgenesis on levothyroxine replacement therapy have relative triiodothyronine deficiency. Most adults with goitrous CH have genetic dyshormonogenesis. They are at high risk of developing thyroid nodules. Our findings support the current guideline recommendation that CH patients with dyshormonogenesis should undergo periodic thyroid ultrasonography.

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Cord Blood Transplantation in 2 Infants Presenting Monosomy 7 Clonal Hematopoiesis: SAMD9 / SAMD9L Germline Mutation

Hirai M., Yagasaki H., Kanezawa K., Ueno M., Shimozawa K., Imai K., Morio T., Kato M., Gocho Y., Narumi S., Ebihara Y., Morioka I.

Journal of Pediatric Hematology/Oncology （Journal of Pediatric Hematology/Oncology） 45 （ 2 ） E290 - E293 2023年03月

ISSN 10774114

Recently, germline mutations in SAMD9 and SAMD9L were increasingly found in children with monosomy 7. We report the outcomes in 2 infants with the SAMD9/SAMD9L variant, who presented with anemia and thrombocytopenia (patient 1), and neutropenia and nonsymptomatic white-matter-encephalopathy (patient 2). Both patients received cord blood transplantation and experienced critical post-cord blood transplantation adverse events; patients 1 and 2 developed fulminant engraftment syndrome and life-threatening graft-versus-host disease, respectively. Of note, selective loss of chromosome 7 in bone marrow-derived CD34+ cells was inferred.

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若年者バセドウ病のMMI単独治療、MMI+KI併用治療の有効性と安全性に関する多施設共同観察研究(後ろ向きコホート研究)の現状報告

大江秀美, 南谷幹史, 宇野希世子, 金子徹治, 荒田尚子, 長谷川奉延, 伊藤順庸, 猪俣弘明, 内野眞也, 鬼形和道, 佐藤浩一, 杉原茂孝, 長崎啓祐, 鳴海覚志, 長谷川行洋, 原田正平, 深田修司, 久門真子, 御前隆, 横谷進, 吉村弘, 金城さおり, 春名英典, 松下理恵, 宮田市郎, 三善陽子, 虫本雄一, 堀川玲子, 室谷浩二, 日本甲状腺学会小児甲状腺疾患診療委員会

日本内分泌学会雑誌（(一社)日本内分泌学会） 98 （ 5 ） 1277 - 1277 2023年03月

ISSN 0029-0661

POU1F1/Pou1f1 c.143-83A > G Variant Disrupts the Branch Site in Pre-mRNA and Leads to Dwarfism

Akiba K., Hasegawa Y., Katoh-Fukui Y., Terao M., Takada S., Hasegawa T., Fukami M., Narumi S.

Endocrinology (United States) （Endocrinology (United States)） 164 （ 2 ） 2023年02月

査読有り, ISSN 00137227

POU Class 1 Homeobox1 (POU1F1/Pou1f1) is a well-established pituitary-specific transcription factor, and causes, when mutated, combined pituitary hormone deficiency in humans and mice. POU1F1/Pou1f1 has 2 isoforms: the alpha and beta isoforms. Recently, pathogenic variants in the unique coding region of the beta isoform (beta domain) and the intron near the exon-intron boundary for the beta domain were reported, although their functional consequences remain obscure. In this study, we generated mice carrying the Pou1f1 c.143-83A>G substitution that recapitulates the human intronic variant near the exon-intron boundary for the beta domain. Homozygous mice showed postnatal growth failure, with an average body weight that was 35% of wild-type littermates at 12 weeks, which was accompanied by anterior pituitary hypoplasia and deficiency of circulating insulin-like growth factor 1 and thyroxine. The results of RNA-seq analysis of the pituitary gland were consistent with reduction of somatotrophs, and this was confirmed immunohistochemically. Reverse transcription polymerase chain reaction of pituitary Pou1f1 mRNA showed abnormal splicing in homozygous mice, with a decrease in the alpha isoform, an increase in the beta isoform, and the emergence of the exon-skipped transcript. We further characterized artificial variants in or near the beta domain, which were candidate positions of the branch site in pre-mRNA, using cultured cell-basis analysis and found that only c.143-83A>G produced transcripts similar to the mice model. Our report is the first to show that the c.143-83A>G variant leads to splicing disruption and causes morphological and functional abnormalities in the pituitary gland. Furthermore, our mice will contribute understanding the role of POU1F1/Pou1f1 transcripts in pituitary development.

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女児鼠径ヘルニアを契機に診断された46,XY DSD症例の臨床遺伝学的検討

木村妙, 小田野めぐみ, 齋藤真希, 鳴海覚志, 長谷川奉延, 宮田市郎, 大石公彦

日本小児科学会雑誌（(公社)日本小児科学会） 127 （ 2 ） 199 - 199 2023年02月

ISSN 0001-6543

Graves' disease as an emerging complication of MIRAGE syndrome

Wada T., Mitani-Konno M., Tanase-Nakao K., Etani Y., Narumi S., Hasegawa Y., Kawai M.

Pediatrics International （Pediatrics International） 65 （ 1 ） e15678 2023年01月

ISSN 13288067

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Congenital anaemia associated with loss-of-function variants in DNA polymerase epsilon 1

Takeuchi I., Tanase-Nakao K., Ogawa A., Sugawara T., Migita O., Kashima M., Yamazaki T., Iguchi A., Naiki Y., Uchiyama T., Tamaoki J., Maeda H., Shimizu H., Kawai T., Taniguchi K., Hirata H., Kobayashi M., Matsumoto K., Naruse K., Hata K., Akutsu H., Kato T., Narumi S., Arai K., Ishiguro A.

Journal of Medical Genetics （Journal of Medical Genetics） 61 （ 3 ） 239 - 243 2023年

ISSN 00222593

DNA polymerase epsilon (Pol ϵ), a component of the core replisome, is involved in DNA replication. Although genetic defects of Pol ϵ have been reported to cause immunodeficiency syndromes, its role in haematopoiesis remains unknown. Here, we identified compound heterozygous variants (p.[Asp1131fs];[Thr1891del]) in POLE, encoding Pol ϵ catalytic subunit A (POLE1), in siblings with a syndromic form of severe congenital transfusion-dependent anaemia. In contrast to Diamond-Blackfan anaemia, marked reticulocytopenia or marked erythroid hypoplasia was not found. Their bone marrow aspirates during infancy revealed erythroid dysplasia with strongly positive TP53 in immunostaining. Repetitive examinations demonstrated trilineage myelodysplasia within 2 years from birth. They had short stature and facial dysmorphism. HEK293 cell-based expression experiments and analyses of patient-derived induced pluripotent stem cells (iPSCs) disclosed a reduced mRNA level of Asp1131fs-POLE1 and defective nuclear translocation of Thr1891del-POLE1. Analysis of iPSCs showed compensatory mRNA upregulation of the other replisome components and increase of the TP53 protein, both suggesting dysfunction of the replisome. We created Pole-knockout medaka fish and found that heterozygous fishes were viable, but with decreased RBCs. Our observations expand the phenotypic spectrum of the Pol ϵ defect in humans, additionally providing unique evidence linking Pol ϵ to haematopoiesis.

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A case of syndromic congenital hypothyroidism with a 15.2 Mb interstitial deletion on 2q12.3q14.2 involving PAX8

Iwahashi-Odano M., Kitamura M., Narumi S.

Clinical Pediatric Endocrinology （Clinical Pediatric Endocrinology） 32 （ 1 ） 65 - 71 2023年

ISSN 09185739

Paired box 8 (PAX8) mutations are an established genetic cause of congenital hypothyroidism (CH). The majority of these mutations are found in the protein-coding exons of the gene. The proband, a 3-yr-old girl, had tetralogy of Fallot and polydactyly soon after birth. She was diagnosed with CH in the newborn screening for CH. She had a high serum TSH level (239 mU/L) and low free T4 level (0.7 ng/dL). Ultrasonography revealed thyroid hypoplasia. We performed array comparative genomic hybridization because the patient exhibited a variety of symptoms across multiple organ systems. The analysis revealed a novel heterozygous deletion that spanned a 15.2 Mb region in 2q12.3q14.3 (GRCh37; chr2:109,568,260–124,779,449). There were 71 protein-coding genes in this region, including two genes (PAX8 and GLI2) associated with congenital endocrine disorders. The common clinical features of the two previously reported patients with a total PAX8 deletion and our case were CH, short stature and intellectual disability, but the severity of hypothyroidism and other clinical features were variable. In conclusion, we describe a syndromic CH patient with a novel 2q12.3q14.3 deletion involving PAX8. Patients with CH, whose unifying diagnosis is not obvious, could have a genomic deletion involving PAX8.

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A case of 46,XY complete gonadal dysgenesis with a novel missense variant in SRY

Narita C., Takubo N., Sammori M., Matsumura Y., Shimura K., Ozaki R., Haruna H., Narumi S., Ishii T., Hasegawa T., Shimizu T.

Clinical Pediatric Endocrinology （Clinical Pediatric Endocrinology） 32 （ 4 ） 235 - 238 2023年

ISSN 09185739

Disorders of sex development (DSD) with mild external genital abnormalities may be diagnosed after puberty. Here, we report a case of 46,XY complete gonadal dysgenesis with a novel missense variant in sex-determining region Y (SRY), diagnosed after primary amenorrhea. A 15-yr-old patient presented to our gynecology department with a chief complaint of amenorrhea. The patient was diagnosed with a 46,XY karyotype, and SRY gene positivity. Gonadotropin levels were high, whereas testosterone levels were low. A pelvic magnetic resonance imaging (MRI) revealed a hypoplastic uterus; however, no gonads could be identified. Laparoscopy revealed bilateral streak gonads, fallopian tube-like structures, and the uterus. The gonads were removed based on the risk of gonadal malignancy. Comprehensive genetic analysis of DSD revealed a previously unreported SRY variant, c.271A>T, p.Ser91Cys, and in silico analysis predicted the variant to be pathogenic. The patient was diagnosed with 46,XY complete gonadal dysgenesis with a novel missense variant in SRY. The patient continued female hormone replacement therapy and experienced breast enlargement and cyclic menstruation. Determining the etiology of DSD can be difficult, causing anxiety in patients and their families. In addition to surgical scrutiny, genetic analysis is important to aid in diagnosis and reassure patients and their families.

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Genome-wide association studies for thyroid physiology and diseases

Narumi S.

Endocrine Journal （Endocrine Journal） 70 （ 1 ） 9 - 17 2023年

ISSN 09188959

Genetic factors are involved in the etiology of most diseases, but prior to 2000, the methods for identifying such factors were very limited. Genome-wide association study (GWAS), developed in the 2000s, is an analytical method that can be applied to most diseases, including endocrine disorders. GWAS has provided a wealth of information on disease risks and the molecular pathogenesis of many human diseases. This review summarizes key findings from GWAS for thyroid physiology and diseases, and illustrates how GWAS is a powerful research tool to elucidate the molecular mechanisms of the diseases.

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GWAS of thyroid dysgenesis identifies a risk locus at 2q33.3 linked to regulation of Wnt signaling

Narumi S., Opitz R., Nagasaki K., Muroya K., Asakura Y., Adachi M., Abe K., Sugisawa C., Kuhnen P., Ishii T., Nothen M.M., Krude H., Hasegawa T.

Human Molecular Genetics （Human Molecular Genetics） 31 （ 23 ） 3967 - 3974 2022年12月

ISSN 09646906

Congenital hypothyroidism due to thyroid dysgenesis (TD), presented as thyroid aplasia, hypoplasia or ectopia, is one of the most prevalent rare diseases with an isolated organ malformation. The pathogenesis of TD is largely unknown, although a genetic predisposition has been suggested. We performed a genome-wide association study (GWAS) with 142 Japanese TD cases and 8380 controls and found a significant locus at 2q33.3 (top single nucleotide polymorphism, rs9789446: P = 4.4 × 10-12), which was replicated in a German patient cohort (P = 0.0056). A subgroup analysis showed that rs9789446 confers a risk for thyroid aplasia (per allele odds ratio = 3.17) and ectopia (3.12) but not for hypoplasia. Comprehensive epigenomic characterization of the 72-kb disease-associated region revealed that it was enriched for active enhancer signatures in human thyroid. Analysis of chromosome conformation capture data showed long-range chromatin interactions of this region with promoters of two genes, FZD5 and CCNYL1, mediating Wnt signaling. Moreover, rs9789446 was found to be a thyroid-specific quantitative trait locus, adding further evidence for a cis-regulatory function of this region in thyroid tissue. Specifically, because the risk rs9789446 allele is associated with increased thyroidal expression of FDZ5 and CCNYL1 and given the recent demonstration of perturbed early thyroid development following overactivation of Wnt signaling in zebrafish embryos, an enhanced Wnt signaling in risk allele carriers provides a biologically plausible TD mechanism. In conclusion, our work found the first risk locus for TD, exemplifying that in rare diseases with relatively low biological complexity, GWAS may provide mechanistic insights even with a small sample size.

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Whole-exome analysis of 177 pediatric patients with undiagnosed diseases

Narita K., Muramatsu H., Narumi S., Nakamura Y., Okuno Y., Suzuki K., Hamada M., Yamaguchi N., Suzuki A., Nishio Y., Shiraki A., Yamamori A., Tsumura Y., Sawamura F., Kawaguchi M., Wakamatsu M., Kataoka S., Kato K., Asada H., Kubota T., Muramatsu Y., Kidokoro H., Natsume J., Mizuno S., Nakata T., Inagaki H., Ishihara N., Yonekawa T., Okumura A., Ogi T., Kojima S., Kaname T., Hasegawa T., Saitoh S., Takahashi Y.

Scientific Reports （Scientific Reports） 12 （ 1 ） 14589 2022年12月

Recently, whole-exome sequencing (WES) has been used for genetic diagnoses of patients who remain otherwise undiagnosed. WES was performed in 177 Japanese patients with undiagnosed conditions who were referred to the Tokai regional branch of the Initiative on Rare and Undiagnosed Diseases (IRUD) (TOKAI-IRUD). This study included only patients who had not previously received genome-wide testing. Review meetings with specialists in various medical fields were held to evaluate the genetic diagnosis in each case, which was based on the guidelines of the American College of Medical Genetics and Genomics. WES identified diagnostic single-nucleotide variants in 66 patients and copy number variants (CNVs) in 11 patients. Additionally, a patient was diagnosed with Angelman syndrome with a complex clinical phenotype upon detection of a paternally derived uniparental disomy (UPD) [upd(15)pat] wherein the patient carried a homozygous DUOX2 p.E520D variant in the UPD region. Functional analysis confirmed that this DUOX2 variant was a loss-of-function missense substitution and the primary cause of congenital hypothyroidism. A significantly higher proportion of genetic diagnoses was achieved compared to previous reports (44%, 78/177 vs. 24–35%, respectively), probably due to detailed discussions and the higher rate of CNV detection.

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小児Basedow病に対する放射性ヨウ素内用療法に関するアンケート調査結果

御前隆, 南谷幹史, 溝上哲也, 荒田尚子, 伊藤順庸, 猪股弘明, 内野眞也, 大江秀美, 鬼形和道, 佐藤浩一, 杉原茂孝, 長崎啓祐, 鳴海覚志, 長谷川奉延, 長谷川行洋, 原田正平, 深田修司, 久門真子, 横谷進, 吉村弘, 日本甲状腺学会小児甲状腺疾患診療委員会

日本甲状腺学会雑誌（(一社)日本甲状腺学会） 13 （ 2 ） 112 - 117 2022年12月

ISSN 2185-3126

小児Basedow病への放射性ヨウ素内用療法の実態を知るため,日本甲状腺学会の評議員を対象に電子メールでアンケートを行った。有効回答46通(回収率28%)。10年間で小児(18歳以下)に内用療法を施行した回答者は14人で,患児の年齢別では16～18歳に対する症例数が多かった。39/46の回答者が5歳未満を禁忌としていた。適応は他に方法がない場合に限定するが25/54,成人と同様は10/54であった(重複回答あり)。小児が対象であるための配慮としては,安全性を詳しく説明・必要性を説明し同意書を取得・長期休暇中に施行などが挙げられた。眼症があれば他法を優先するとの回答が26/46を占めた。患児に放射線治療であることを伝えないとする回答は3/46と少数であった。内用療法に起因すると思われる腫瘍発生を経験した回答者はなかった。小児の特性に配慮しつつ,概ね学会ガイドラインに沿って治療が行われていることが確認され,今後検討すべき課題として年少児への説明の工夫や同胞の被ばく防護についての配慮が浮かんできた。(著者抄録)

小児Basedow病に対する放射性ヨウ素内用療法に関するアンケート調査結果

日本甲状腺学会雑誌（(一社)日本甲状腺学会） 13 （ 2 ） 112 - 117 2022年12月

ISSN 2185-3126

A patient with congenital hypothyroidism due to a PAX8 frameshift variant accompanying a urogenital malformation

Tanase-Nakao K., Muroya K., Adachi M., Abe K., Hasegawa T., Narumi S.

Clinical Pediatric Endocrinology （Clinical Pediatric Endocrinology） 31 （ 4 ） 250 - 255 2022年10月

ISSN 09185739

PAX8 is a transcription factor that is expressed in the thyroid gland and kidneys. Monoallelic loss-of-function PAX8 variants cause congenital hypothyroidism (CH), and urogenital malformations are infrequent complications seen in less than 10% of PAX8 variant carriers. Herein, we report the case of a 3-yr-old female patient with CH who was diagnosed during newborn screening. She was treated with levothyroxine, and she showed normal growth and development at a minimal dose (0.7 µg/kg/d of levothyroxine at 3 yr of age). At 5 mo of age, she visited an emergency department for fever and was incidentally found to have differently sized kidneys by ultrasonography, which was subsequently diagnosed as unilateral multicystic dysplastic kidney. Her serum creatinine and cystatin C levels were normal. Next-generation sequencing-based genetic analysis revealed that the patient was heterozygous for a PAX8 frameshift variant (p.Thr320ProfsTer106) and a DUOX2 missense variant (p.Arg885Gln). Our patient is the first truncating PAX8 variant carrier to have a urogenital malformation with CH. Genetic analysis for PAX8 should be considered in patients with CH and urogenital malformations.

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Two ovarian candidate enhancers, identified by time series enhancer RNA analyses, harbor rare genetic variations identified in ovarian insufficiency

Nakagawa R., Takasawa K., Gau M., Tsuji-Hosokawa A., Kawaji H., Murakawa Y., Takada S., Mikami M., Narumi S., Fukami M., Sreenivasan R., Maruyama T., Tucker E.J., Zhao L., Bowles J., Sinclair A., Koopman P., Hayashizaki Y., Morio T., Kashimada K.

Human Molecular Genetics （Human Molecular Genetics） 31 （ 13 ） 2223 - 2235 2022年07月

ISSN 09646906

The genetic regulation of ovarian development remains largely unclear. Indeed, in most cases of impaired ovarian development-such as 46,XX disorders of sex development (DSD) without SRY, and premature ovarian insufficiency (POI)-the genetic causes have not been identified, and the vast majority of disease-Associated sequence variants could lie within non-coding regulatory sequences. In this study, we aimed to identify enhancers of five ovarian genes known to play key roles in early ovarian development, basing our analysis on the expression of enhancer derived transcripts (eRNAs), which are considered to characterize active enhancers. Temporal expression profile changes in mouse WT1-positive ovarian cells were obtained from cap analysis of gene expression at E13.5, E16.5 and P0. We compared the chronological expression profiles of ovarian-specific eRNA with expression profiles for each of the ovarian-specific genes, yielding two candidate sequences for enhancers of Wnt4 and Rspo1. Both sequences are conserved between mouse and human, and we confirmed their enhancer activities using transient expression assays in murine granulosa cells. Furthermore, by sequencing the region in patients with impaired ovarian development in 24 patients, such as POI, gonadal dysgenesis and 46,XX DSD, we identified rare single nucleotide variants in both sequences. Our results demonstrate that combined analysis of the temporal expression profiles of eRNA and mRNA of target genes presents a powerful tool for locating cis-element enhancers, and a means of identifying disease-Associated sequence variants that lie within non-coding regulatory sequences, thus advancing an important unmet need in forward human genetics.

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The Patterning and Proportion of Charged Residues in the Arginine-Rich Mixed-Charge Domain Determine the Membrane-Less Organelle Targeted by the Protein

Miyagi T., Yamazaki R., Ueda K., Narumi S., Hayamizu Y., Uji-i H., Kuroda M., Kanekura K.

International Journal of Molecular Sciences （International Journal of Molecular Sciences） 23 （ 14 ） 2022年07月

ISSN 16616596

Membrane-less organelles (MLOs) are formed by biomolecular liquid–liquid phase separation (LLPS). Proteins with charged low-complexity domains (LCDs) are prone to phase separation and localize to MLOs, but the mechanism underlying the distributions of such proteins to specific MLOs remains poorly understood. Recently, proteins with Arg-enriched mixed-charge domains (R-MCDs), primarily composed of R and Asp (D), were found to accumulate in nuclear speckles via LLPS. However, the process by which R-MCDs selectively incorporate into nuclear speckles is unknown. Here, we demonstrate that the patterning of charged amino acids and net charge determines the targeting of specific MLOs, including nuclear speckles and the nucleolus, by proteins. The redistribution of R and D residues from an alternately sequenced pattern to uneven blocky sequences caused a shift in R-MCD distribution from nuclear speckles to the nucleolus. In addition, the incorporation of basic residues in the R-MCDs promoted their localization to the MLOs and their apparent accumulation in the nucleolus. The R-MCD peptide with alternating amino acids did not undergo LLPS, whereas the blocky R-MCD peptide underwent LLPS with affinity to RNA, acidic poly-Glu, and the acidic nucleolar protein nucleophosmin, suggesting that the clustering of R residues helps avoid their neutralization by D residues and eventually induces R-MCD migration to the nucleolus. Therefore, the distribution of proteins to nuclear speckles requires the proximal positioning of D and R for the mutual neutralization of their charges.

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Achievement of Developmental Milestones Recorded in Real Time: A Mobile App-Based Study

Matsubara K., Hattori T., Narumi S.

Journal of Pediatrics （Journal of Pediatrics） 245 201 - 207.e9 2022年06月

ISSN 00223476

Objective: To clarify the characteristics of milestone achievement of Japanese infants using data assessed and recorded in real time via a Web-based app by caregivers. Study design: Between 2014 and 2019, the achievement of developmental milestones of 16 627 Japanese infants were recorded via the mobile app Papatto Ikuji along with pertinent information including sex, birth date, anthropometric measurements, and information on feeding. The milestones consisted of 20 items belonging to 4 domains: personal-social, fine motor-adaptative, language, and gross motor. The distribution of age at milestone achievement was compared with 4 conventional normative data. Subgroup analyses according to sex, season of birth, and feeding methods were performed with restricted mean survival time analysis. Results: Age distributions of milestone achievement were generally similar in the app-based and conventional data. The exception was “Smile responsively,” for which the age at achievement seemed higher in the app-based data. Subgroup analyses showed female-dominant sex differences in the achievement of person-social and language milestones. The results also showed seasonality in achievement of gross motor milestones, with earlier achievement in infants born in winter compared with those born in summer. Conclusions: We describe mobile app-based data on developmental milestone achievements that were recorded in real time. The app-based data were generally comparable with conventional normative data, suggesting that its potential to assess the development of children in real time. This could be used complementarily with the current well-child visits at scheduled time points.

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Novel biallelic mutations in the DUOX2 gene underlying very early-onset inflammatory bowel disease: A case report

Kyodo R., Takeuchi I., Narumi S., Shimizu H., Hata K., Yoshioka T., Tanase-Nakao K., Shimizu T., Arai K.

Clinical Immunology （Clinical Immunology） 238 109015 - 109015 2022年05月

ISSN 15216616

Genetic variants affecting the function of dual oxidase 2 (DUOX2), the catalytic subunit of membrane-bound enzymes that produce hydrogen peroxide, are associated with very early-onset inflammatory bowel disease (VEO-IBD). We report the case of a 1-year-old boy diagnosed with VEO-IBD after presenting with bloody diarrhea. He had pancolitis and an extensive small intestinal ulcerative lesion at age 4 years. Infliximab treatment was successful but was discontinued due to delayed reaction. At age 7 years, treatment with ustekinumab was started, and remission has been maintained for more than 2 years. Whole-exome sequencing identified compound heterozygous missense DUOX2 variants of unknown significance (p.[R1212H];[F1490Y]). Protein expression in the whole-cell lysate and plasma membrane was lower in F1490Y-DUOX2 than in wild-type (WT)-DUOX2. Hydrogen peroxide generation upon ionomycin stimulation was lower in cells expressing R1212H-DUOX2 and F1490Y-DUOX2 than in those expressing WT-DUOX2. The novel, inherited, biallelic DUOX2 mutations may be molecular risk factors of VEO-IBD.

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High-dose fludrocortisone therapy was transiently required in a female neonate with 21-hydroxylase deficiency

Kawasaki Y., Sato T., Nakano S., Usui T., Narumi S., Ishii T., Hasegawa T.

Clinical Pediatric Endocrinology （Clinical Pediatric Endocrinology） 31 （ 2 ） 93 - 97 2022年04月

ISSN 09185739

For salt-wasting 21-hydroxylase deficiency (21OHD), fludrocortisone (FC) is usually supplemented at 0.05–0.2 mg/d dose. To date, no report has described 21OHD neonates requiring > 0.4 mg/d of FC. Our female 21OHD patient was lethargic and experienced weight loss with hyponatremia (133 mEq/L), hyperkalemia (6.5 mEq/L), and elevated active renin concentration (ARC, 1942.2 pg/mL) at 6 days of life. Hydrocortisone and FC replacement were initiated. FC dose was gradually increased to 0.4 mg/d at 21 days of life, but her hyperkalemia (6.4 mEq/L) and high ARC (372.3 pg/mL) persisted. We increased FC to 0.6 mg/d and used a low-potassium and high-sodium formula. Hyperkalemia subsequently improved. At 33 days of life, the ARC decreased to 0.6 pg/mL and FC dosage was gradually decreased. At 3 months of age, the low-potassium and high-sodium formula was discontinued, but the serum potassium level was normal and ARC remained low at 0.1 mg/d of FC. We speculated that severe mineralocorticoid resistance was the reason why her hyperkalemia persisted even with 0.4 mg/d of FC; however, the pathophysiology of transiently severe resistance to FC in this patient is unknown. In conclusion, 21OHD neonates may show severe salt-wasting that transiently require > 0.4 mg/d of FC.

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若年者バセドウ病のチアマゾール単独治療、チアマゾール+無機ヨウ素併用治療の有効性と安全性に関する多施設共同観察研究(後ろ向きコホート研究)の現状報告

大江秀美, 南谷幹史, 荒田尚子, 長谷川奉延, 伊藤順庸, 猪股弘明, 内野眞也, 鬼形和道, 佐藤浩一, 杉原茂孝, 長崎啓祐, 鳴海覚志, 長谷川行洋, 原田正平, 深田修司, 久門真子, 御前隆, 横谷進, 吉村弘, 金城さおり, 春名英典, 松下理恵, 宮田市郎, 三善陽子, 虫本雄一, 堀川玲子, 室谷浩二, 日本甲状腺学会小児甲状腺疾患診療委員会

日本内分泌学会雑誌（(一社)日本内分泌学会） 97 （ 5 ） 1108 - 1108 2022年03月

ISSN 0029-0661

先天性甲状腺機能低下症原因遺伝子ヘテロ変異症例の臨床像

阿部清美, 鳴海覚志, 室谷浩二, 朝倉由美, 安達昌功, 小田野めぐみ, 中尾佳奈子, 杉澤千穂, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 97 （ 5 ） 1130 - 1130 2022年03月

ISSN 0029-0661

成人期DUOX2異常症の甲状腺組織像は正常である世界初の甲状腺組織所見の報告

杉澤千穂, 鳴海覚志, 中尾佳奈子, 都研一, 阿部清美, 星山綾子, 渡邊奈津子, 吉村弘, 加藤良平, 伊藤公一, 長坂昌一郎

日本内分泌学会雑誌（(一社)日本内分泌学会） 97 （ 5 ） 1149 - 1149 2022年03月

ISSN 0029-0661

小児バセドウ病に対する放射性ヨウ素内用療法に関するアンケート調査結果

御前隆, 南谷幹史, 溝上哲也, 荒田尚子, 長谷川奉延, 伊藤順庸, 猪股弘明, 内野眞也, 大江秀美, 鬼形和道, 佐藤浩一, 杉原茂孝, 長崎啓祐, 鳴海覚志, 日本甲状腺学会小児甲状腺疾患診療委員会

日本内分泌学会雑誌（(一社)日本内分泌学会） 97 （ 5 ） 1158 - 1158 2022年03月

ISSN 0029-0661

乳児期早期からの間質性肺炎と多臓器石灰化を認めI型インターフェロン異常症を疑った男児例

相原健志, 石川尊士, 岡井真史, 中尾寛, 玉井直敬, 肥沼悟郎, 木戸口千晶, 庄司健介, 西健太朗, 太田知子, 曽根田京子, 大北恵子, 山田全毅, 今留謙一, 鳴海覚志, 今井耕輔, 小野寺雅史, 窪田満, 石黒精, 河合利尚

日本小児科学会雑誌（(公社)日本小児科学会） 126 （ 2 ） 284 - 284 2022年02月

ISSN 0001-6543

Discovery of MIRAGE syndrome

Narumi S.

Pediatrics International （Pediatrics International） 64 （ 1 ） e15283 2022年01月

ISSN 13288067

Since the first report in 2009, whole exome sequencing has become the most effective and efficient research tool in human genetics. MIRAGE syndrome is a novel single-gene disorder discovered through whole-exome sequencing for pediatric patients with adrenal insufficiency of unknown etiology, and is caused by de novo heterozygous variants in SAMD9. MIRAGE syndrome was initially discovered as a systemic disease affecting multiple systems, including hematopoietic, immune, endocrine, and gastrointestinal systems but later studies revealed a subset of patients with myelodysplastic syndrome as the sole manifestation. In addition, pathogenic variants in SAMD9L, a paralog gene of SAMD9, were reported to cause an inherited disorder of the hematopoietic system and central nervous system, called ataxia-pancytopenia syndrome. This article reviews the history of MIRAGE syndrome from its discovery to the proposal of SAMD9/SAMD9L syndromes, and discusses directions for future research.

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Biallelic PROKR2 variants and congenital hypogonadotropic hypogonadism: a case report and a literature review

Sugisawa C., Taniyama M., Sato T., Takahashi Y., Hasegawa T., Narumi S.

Endocrine Journal （Endocrine Journal） 69 （ 7 ） 831 - 838 2022年

ISSN 09188959

Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder that causes gonadotropin-releasing hormone (GnRH) deficiency and sexual immaturity. CHH may accompany an abnormal sense of smell (Kallmann syndrome, KS) or no such manifestation (normosmic-CHH). This unusual combination of manifestations is explained by the fact that GnRH neurons originate in the olfactory placode and migrate to the forebrain during embryogenesis. We describe the case of a 31-year-old man with normosmic-CHH, who also had obesity, type 2 diabetes and intellectual disability. He was noticed to have sexual immaturity (small testes with no pubic hair) at age 20 years, when diabetic ketoacidosis developed. Basal and GnRH-stimulated levels of LH (1.0→12.0 IU/L) and FSH (1.9→6.1 IU/L) were detectable but low. The results of the T&T olfactometer and the Alinamin test were definitely normal, with an anatomically normal olfactory system on MRI. Sequencing of 22 CHH-related genes was performed, and compound heterozygous PROKR2 variants were identified: one was a previously known loss-of-function variant (p.Trp178Ser) and the other was a nonsense variant (p.Trp212*). Through a literature review, we found 22 patients (including our patient) with CHH due to biallelic PROKR2 variants, which led us to recognize that most of the patients (86%) were diagnosed with KS. Clinical observations in this study indicate that, even though they have CHH, biallelic PROKR2 variant carriers may have a normal olfactory system as well as presumably normal migration of GnRH neurons. This suggests that the PROK2-PROKR2 pathway affects the function of GnRH neurons after their migration.

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An improved macromolecular crowding sensor CRONOS for detection of crowding changes in membrane-less organelles under stressed conditions

Miyagi T., Yamanaka Y., Harada Y., Narumi S., Hayamizu Y., Kuroda M., Kanekura K.

Biochemical and Biophysical Research Communications （Biochemical and Biophysical Research Communications） 583 29 - 34 2021年12月

ISSN 0006291X

Membrane-less organelles (MLOs) formed by liquid-liquid phase separation (LLPS) play pivotal roles in biological processes. During LLPS, proteins and nucleotides are extremely condensed, resulting in changes in their conformation and biological functions. Disturbed LLPS homeostasis in MLOs is thought to associate with fatal diseases such as amyotrophic lateral sclerosis. Therefore, it is important to detect changes in the degree of crowding in MLOs. However, it has not been investigated well due to the lack of an appropriate method. To address this, we developed a genetically encoded macromolecular crowding sensor CRONOS (crowding sensor with mNeonGreen and mScarlet-I) that senses the degree of macromolecular crowding in MLOs using a fluorescence resonance energy transfer (FRET) system. CRONOS is a bright biosensor with a wide dynamic range and successfully detects changes in the macromolecular volume fraction in solution. By fusing to the scaffold protein of each MLO, we delivered CRONOS to MLO of interest and detected previously undescribed differences in the degree of crowding in each MLO. CRONOS also detected changes in the degree of macromolecular crowding in nucleolus induced by environmental stress or inhibition of transcription. These findings suggest that CRONOS can be a useful tool for the determination of molecular crowding and detection of pathological changes in MLOs in live cells.

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The case of a patient with MIRAGE syndrome with familial dysautonomia-like symptoms

Kawashima-Sonoyama Y., Okuno K., Dohmoto T., Tanase-Nakao K., Narumi S., Namba N.

Human Genome Variation （Human Genome Variation） 8 （ 1 ） 27 - 27 2021年12月

ISSN 2054-345X

We describe a case of posthumously diagnosed MIRAGE syndrome (Myelodysplasia, Infection, Restriction of growth, Adrenal hypoplasia, Genital problems, and Enteropathy) in a girl with a new pathogenic SAMD9 variant (p.F437S), who was initially considered to have familial dysautonomia (FD)-like disease due to increased levels of catecholamine metabolites. Functional analyses of F437S-SAMD9 were performed, showing characteristics of disease-causing variants. This new SAMD9 variant (p.F437S) also causes MIRAGE syndrome.

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ZNF445: a homozygous truncating variant in a patient with Temple syndrome and multilocus imprinting disturbance

Kagami M., Hara-Isono K., Matsubara K., Nakabayashi K., Narumi S., Fukami M., Ohkubo Y., Saitsu H., Takada S., Ogata T.

Clinical Epigenetics （Clinical Epigenetics） 13 （ 1 ） 119 - 119 2021年12月

ISSN 18687075

Background: ZNF445, as well as ZFP57, is involved in the postfertilization methylation maintenance of multiple imprinting-associated differentially methylated regions (iDMRs). Thus, ZNF445 pathogenic variants are predicted to cause multilocus imprinting disturbances (MLIDs), as do ZFP57 pathogenic variants. In particular, the MEG3/DLK1:IG-DMR would be affected, because the postzygotic methylation imprint of the MEG3/DLK1:IG-DMR is maintained primarily by ZNF445, whereas that of most iDMRs is preserved by both ZFP57 and ZNF445 or primarily by ZFP57. Results: We searched for a ZNF445 variant(s) in six patients with various imprinting disorders (IDs) caused by epimutations and MLIDs revealed by pyrosequencing for nine iDMRs, without a selection for the original IDs. Re-analysis of the previously obtained whole exome sequencing data identified a homozygous ZNF445 variant (NM_181489.6:c.2803C>T:p.(Gln935*)) producing a truncated protein missing two of 14 zinc finger domains in a patient with Temple syndrome and MLID. In this patient, array-based genomewide methylation analysis revealed severe hypomethylation of most CpGs at the MEG3:TSS-DMR, moderate hypomethylation of roughly two-thirds of CpGs at the H19/IGF2:IG-DMR, and mild-to-moderate hypomethylation of a few CpGs at the DIRAS3:TSS-DMR, MEST:alt-TSS-DMR, IGF2:Ex9-DMR, IGF2:alt-TSS, and GNAS-AS1:TSS-DMR. Furthermore, bisulfite sequencing analysis for the MEG3/DLK1:IG-DMR delineated a markedly hypomethylated segment (CG-A). The heterozygous parents were clinically normal and had virtually no aberrant methylation pattern. Conclusions: We identified a ZNF445 pathogenic variant for the first time. Since ZNF445 binds to the MEG3/DLK1:IG-DMR and other iDMRs affected in this patient, the development of Temple syndrome and MLID would primarily be explained by the ZNF445 variant. Furthermore, CG-A may be the target site for ZNF445 within the MEG3/DLK1:IG-DMR.

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Hereditary paraganglioma presenting with atypical symptoms: Case report

Eguchi S., Ono R., Sato T., Yada K., Umehara N., Narumi S., Ichihashi Y., Nozaki T., Kanomata N., Hasegawa T., Ozawa M., Hasegawa D.

Medicine (United States) （Medicine (United States)） 100 （ 46 ） e27888 2021年11月

ISSN 00257974

Rationale:Paraganglioma (PGL), an extra-adrenal pheochromocytoma, is a rare tumor, especially in children. While hypersecretion of catecholamines causes the classic triad of headaches, palpitations, and profuse sweating, prompt diagnosis is still challenging.Patient concerns:For 7 months, an 8-year-old boy complained of polyuria and weight loss, followed by proteinuria and headache for 1 month prior to admission. He was admitted to our hospital due to an afebrile seizure.Diagnosis:His blood pressure remained markedly elevated even after cessation of the convulsion. Magnetic resonance imaging of the brain revealed posterior reversible encephalopathy syndrome. Abdominal computed tomography showed a mass lesion encasing the left renal artery, measuring 41 mm in length along its major axis. The plasma and urine levels of normetanephrine were elevated. Additionally, iodine-123-metaiodobenzylguanidine scintigraphy showed an abnormal uptake in the abdominal mass with no evidence of metastasis. Based on these findings, we tentatively diagnosed him with PGL.Intervention:Substantial alpha-and beta-blocking procedures were performed, followed by a tumor resection and an extended left nephrectomy on day 31 of hospitalization. Pathological findings confirmed the diagnosis of PGL.Outcome:The postoperative course was uneventful, and his blood pressure normalized without the use of antihypertensive agents. Genetic testing revealed a known SDHB germline mutation. The same mutation was also detected on his father and paternal grandfather without any history of hypertension or malignant tumor.Lesson:It remains challenging to diagnose pheochromocytoma/paraganglioma (PPGL) promptly because PPGL can present with a variety of symptoms. Preceding symptoms of the presented case might be caused by PGL. Although PPGL is a rare disease, especially in children, it should be considered in differential diagnosis when various unexplained symptoms persist.

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Phase separation and toxicity of c9orf72 poly(Pr) depends on alternate distribution of arginine

Chen C., Yamanaka Y., Ueda K., Li P., Miyagi T., Harada Y., Tezuka S., Narumi S., Sugimoto M., Kuroda M., Hayamizu Y., Kanekura K.

Journal of Cell Biology （Journal of Cell Biology） 220 （ 11 ） e202103160 2021年11月

査読有り, ISSN 00219525

Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid–liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein–protein interactions.

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【小児遺伝子疾患事典】免疫疾患 SAMD9(関連疾患:MIRAGE症候群)

中尾佳奈子, 鳴海覚志

小児科診療（(株)診断と治療社） 84 （ 11 ） 1683 - 1684 2021年11月

ISSN 0386-9806

Role of Liquid-Liquid Separation in Endocrine and Living Cells

Akiba K., Katoh-Fukui Y., Yoshida K., Narumi S., Miyado M., Hasegawa Y., Fukami M.

Journal of the Endocrine Society （Journal of the Endocrine Society） 5 （ 10 ） bvab126 2021年10月

Context: Recent studies have revealed that every eukaryotic cell contains several membraneless organelles created via liquid-liquid phase separation (LLPS). LLPS is a physical phenomenon that transiently compartmentalizes the subcellular space and thereby facilitates various biological reactions. LLPS is indispensable for cellular functions; however, dysregulated LLPS has the potential to cause irreversible protein aggregation leading to degenerative disorders. To date, there is no systematic review on the role of LLPS in endocrinology. Evidence acquisition: We explored previous studies which addressed roles of LLPS in living cells, particularly from the viewpoint of endocrinology. To this end, we screened relevant literature in PubMed published between 2009 and 2021 using LLPS-associated keywords including "membraneless organelle,""phase transition,"and "intrinsically disordered,"and endocrinological keywords such as "hormone,""ovary,""androgen,"and "diabetes."We also referred to the articles in the reference lists of identified papers. Evidence synthesis: Based on 67 articles selected from 449 papers, we provided a concise overview of the current understanding of LLPS in living cells. Then, we summarized recent articles documenting the physiological or pathological roles of LLPS in endocrine cells. Conclusions: The discovery of LLPS in cells has resulted in a paradigm shift in molecular biology. Recent studies indicate that LLPS contributes to male sex development by providing a functional platform for SOX9 and CBX2 in testicular cells. In addition, dysregulated LLPS has been implicated in aberrant protein aggregation in pancreatic β-cells, leading to type 2 diabetes. Still, we are just beginning to understand the significance of LLPS in endocrine cells.

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A case report with functional characterization of a HNF1B mutation (P.Leu168Pro) causing MODY5

Yoshida K., Mushimoto Y., Tanase-Nakao K., Akiba K., Ishii K., Urakami T., Sugihara S., Kikuchi T., Fukami M., Narumi S.

Clinical Pediatric Endocrinology （Clinical Pediatric Endocrinology） 30 （ 4 ） 179 - 185 2021年10月

ISSN 09185739

We previously performed next-generation sequencing-based genetic screening in patients with autoantibodynegative type 1 diabetes, and identified the p.Leu168Pro mutation in HNF1B. Here, we report the clinical course of the patient and the results of functional characterization of this mutation. The proband had bilateral renal hypodysplasia and developed insulin-dependent diabetes during childhood. The pathogenicity of Leu168Pro-HNF1B was evaluated with three-dimensional structure modeling, Western blotting, immunofluorescence analysis and luciferase reporter assays using human embryonic kidney 293 cells. Three-dimensional structure modeling predicted that the Leu168 residue is buried in the DNA-binding Pit-Oct-Unc-specific (POUS) domain and forms a hydrophobic core. Western blotting showed that the protein expression level of Leu168Pro-HNF1B was lower than that of wild-type (WT) HNF1B. Immunofluorescence staining showed that both WT- and Leu168Pro-HNF1B were normally localized in the nucleus. The cells transfected with WT-HNF1B exhibited 5-fold higher luciferase reporter activity than cells transfected with an empty vector. The luciferase activities were comparable between WT-HNF1B/Leu168Pro-HNF1B and WT-HNF1B/empty vector co-transfection. In conclusion, Leu168Pro is a protein-destabilizing HNF1B mutation, and the destabilization is likely due to the structural changes involving the hydrophobic core of POUS. The disease-causing Leu168Pro HNF1B mutation is a loss-of-function mutation without a dominant-negative effect.

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Acquired uniparental disomy of chromosome 7 in a patient with mirage syndrome that veiled a pathogenic samd9 variant

Tanase-Nakao K., Kawai M., Wada K., Kagami M., Narumi S.

Clinical Pediatric Endocrinology （Clinical Pediatric Endocrinology） 30 （ 4 ） 163 - 169 2021年10月

ISSN 09185739

Gain-of-function variants in SAMD9, which resides on chromosome 7, cause MIRAGE syndrome that is associated with congenital adrenal insufficiency and gonadal dysgenesis. We previously reported a Japanese patient with MIRAGE syndrome carrying a de novo heterozygous SAMD9 variant (p.Ala1479Ser). In this study, we confirmed the pathogenicity of Ala1479Ser-SAMD9 in vitro. Genetic study results revealed an atypically low variant allele frequency (26%) and we suspected of genomic rearrangement(s) involving chromosome 7. Single nucleotide polymorphism (SNP) array and short tandem repeat analysis showed presence of mosaic maternal isodisomic uniparental disomy 7 (UPD7). Deep sequencing using DNA samples obtained at 0, 6, 10, and 25 mo of age revealed that the percentage of cells with UPD7 increased constantly from 6% to 82% over 25 mo, and this increase coincided with a decrease in the percentage of cells with p.Ala1479Ser from 94% to nearly undetectable levels. We further screened for low-allele-frequency and rare SAMD9 variants in eight patients with Silver-Russel syndrome and maternal UPD7; however, none of the patients harbored such a variant. In conclusion, our case demonstrates that genetic findings can vary considerably in patients with MIRAGE syndrome and that a comprehensive diagnostic approach, including SNP array and deep sequencing, is important in such cases.

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SOX10 Mutation Screening for 117 Patients with Kallmann Syndrome

Shima H., Tokuhiro E., Okamoto S., Nagamori M., Ogata T., Narumi S., Nakamura A., Izumi Y., Jinno T., Suzuki E., Fukami M.

Journal of the Endocrine Society （Journal of the Endocrine Society） 5 （ 7 ） 1 - 7 2021年07月

Introduction: Kallmann syndrome (KS) is a genetically heterogeneous condition characterized by hypogonadotropic hypogonadism (HH) and olfactory dysfunction. Although SOX10, a causative gene for Waardenburg syndrome (WS) and peripheral demyelinating neuropathy, central demyelination, WS, and Hirschsprung disease (PCWH) has previously been implicated in KS, the clinical significance of SOX10 variants as the cause of KS remains uncertain. Patients and Methods: A total of 117 patients with KS underwent mutation screening of SOX10 and 14 other causative genes for KS/HH. Rare SOX10 variants were subjected to in silico and in vitro analyses. We also examined clinical data of the patients and their parents with SOX10 variants. Results: Sequence analysis identified 2 heterozygous variants of SOX10 (c.1225G > T, p.Gly409∗ and c.475C > T, p.Arg159Trp) in patients 1-3, as well as in the parents of patients 1 and 3. The variants were assessed as pathogenic/likely pathogenic, according to the American College of Medical Genomics guidelines. Both variants lacked in vitro transactivating activity for the MITF promoter and exerted no dominant-negative effects. Patients 1-3 carried no pathogenic variants in other genes examined. The patients presented with typical KS, while such features were absent in the parents of patients 1 and 3. None of the 5 variant-positive individuals exhibited hypopigmentation, while 1 and 2 individuals exhibited complete and partial hearing loss, respectively. Conclusion: These results provide evidence that SOX10 haploinsufficiency accounts for a small percentage of KS cases. SOX10 haploinsufficiency is likely to be associated with a broad phenotypic spectrum, which includes KS without other clinical features of WS/PCWH.

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Identification of the first promoter-specific gain-of-function SOX9 missense variant (p.E50K) in a patient with 46,XX ovotesticular disorder of sex development

Ushijima K., Ogawa Y., Terao M., Asakura Y., Muroya K., Hayashi M., Ishii T., Hasegawa T., Sekido R., Fukami M., Takada S., Narumi S.

American Journal of Medical Genetics, Part A （American Journal of Medical Genetics, Part A） 185 （ 4 ） 1067 - 1075 2021年04月

ISSN 15524825

SOX9, a transcription factor, is expressed in the undifferentiated XX and XY gonads. SRY induces significant upregulation of SOX9 expression in XY gonads. Loss-of-function SOX9 variants cause testicular dysgenesis in 46,XY patients, while duplication of the total gene or the upstream regulatory region results in testicular development in 46,XX patients. However, gain-of-function (GoF) SOX9 variants have not been reported previously. We report the case of a 16-year-old female patient with a 46,XX karyotype who had masculinized external genitalia and unilateral ovotestis. Next-generation sequencing-based genetic screening for disorders of sex development led to the identification of a novel SOX9 variant (p.Glu50Lys), transmitted from the phenotypically normal father. Expression analysis showed that E50K-SOX9 enhanced transactivation of the luciferase reporter containing the testis enhancer sequence core element compared with that containing the wildtype-SOX9. This GoF activity was not observed in the luciferase reporter containing Amh, the gene for anti-Müllerian hormone. We genetically engineered female mice (Sox9E50K/E50K), and they showed no abnormalities in the external genitalia or ovaries. In conclusion, a novel SOX9 variant with a promoter-specific GoF activity was identified in vitro; however, the disease phenotype was not recapitulated by the mouse model. At present, the association between the GoF SOX9 variant and the ovotestis phenotype remains unclear. Future studies are needed to verify the possible association.

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若年者バセドウ病のチアマゾール単独治療、チアマゾール+無機ヨウ素併用治療の有効性と安全性に関する多施設共同観察研究後ろ向きコホート研究

大江秀美, 南谷幹史, 荒田尚子, 長谷川奉延, 伊藤順庸, 猪俣弘明, 内野眞也, 鬼形和道, 佐藤浩一, 杉原茂孝, 長崎啓祐, 鳴海覚志, 長谷川行洋, 原田正平, 深田修司, 久門真子, 御前隆, 横谷進, 吉村弘, 金城さおり, 春名英典, 松下理恵, 宮田市郎, 三善陽子, 虫本雄一, 堀川玲子, 室谷浩二, 日本甲状腺学会小児甲状腺疾患診療委員会

日本内分泌学会雑誌（(一社)日本内分泌学会） 96 （ 4 ） 927 - 927 2021年04月

ISSN 0029-0661

若年者バセドウ病のチアマゾール単独治療、チアマゾール+無機ヨウ素併用治療の有効性と安全性に関する多施設共同観察研究後ろ向きコホート研究

日本内分泌学会雑誌（(一社)日本内分泌学会） 96 （ 4 ） 927 - 927 2021年04月

ISSN 0029-0661

先天性甲状腺機能低下症をきたすジスルフィド結合の消失を伴う新規甲状腺ペルオキシダーゼ変異の機能解析

谷古宇史芳, 諏訪内浩紹, 石川卓也, 伊藤真理子, 志熊淳平, 三輪隆, 酒井裕幸, 金蔵孝介, 鳴海覚志, 鈴木亮, 小田原雅人

日本内分泌学会雑誌（(一社)日本内分泌学会） 96 （ 4 ） 955 - 955 2021年04月

ISSN 0029-0661

A Case of Luscan-Lumish Syndrome: Possible Involvement of Enhanced GH Signaling

Suda K., Fukuoka H., Iguchi G., Kanie K., Fujita Y., Odake Y., Matsumoto R., Bando H., Ito H., Takahashi M., Chihara K., Nagai H., Narumi S., Hasegawa T., Ogawa W., Takahashi Y.

Journal of Clinical Endocrinology and Metabolism （Journal of Clinical Endocrinology and Metabolism） 106 （ 3 ） 718 - 723 2021年03月

ISSN 0021972X

Context: Luscan-Lumish syndrome (LLS) is characterized by postnatal overgrowth, obesity, Chiari I malformation, seizures, and intellectual disability. SET domain-containing protein 2 (SETD2) is a histone methyltransferase, where mutations in the gene are associated with the development of LLS. However, mechanisms underlying LLS remain unclear. Case Description: A 20-year-old man was referred to our hospital because of tall stature. His body height was 188.2 cm (+3.18 SD) and he showed obesity with a body mass index of 28.4 kg/m2. He exhibited acral overgrowth, jaw malocclusion, and prognathism, but no history of seizures, intellectual disability, or speech delay. Serum growth hormone (GH), insulin-like growth factor 1 (IGF-1), and nadir GH levels after administration of 75 g oral glucose were within normal range. Pituitary magnetic resonance imaging showed no pituitary adenoma, but Chiari I malformation. Whole exome sequencing analysis of the proband revealed a de novo heterozygous germline mutation in SETD2 (c.236T>A, p.L79H). Skin fibroblasts derived from the patient grew faster than those from his father and the control subject. In addition, these cells showed enhanced tyrosine phosphorylation and transcriptional activity of signal transducer and activator of transcription 5b (STAT5b) and increased IGF-1 expression induced by GH. Conclusion: This is a mild case of LLS with a novel mutation in SETD2 without neurological symptoms. LLS should be differentiated in a patient with gigantism without pituitary tumors. Although further investigation is necessary, this is the first study to suggest the involvement of aberrant GH signaling in the development of LLS.

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Inactivation of a Frameshift TSH Receptor Variant Val711Phefs∗18 is Due to Acquisition of a Hydrophobic Degron

Sugisawa C., Ono M., Kashimada K., Hasegawa T., Narumi S.

Journal of Clinical Endocrinology and Metabolism （Journal of Clinical Endocrinology and Metabolism） 106 （ 1 ） E265 - E272 2021年01月

ISSN 0021972X

Context: Inactivating variants of thyrotropin (thyroid-stimulating hormone; TSH) receptor (TSHR) cause congenital hypothyroidism. More than 60 such variants have been reported so far, most of which were located in the extracellular or transmembrane domain. Objective: We report the identification and characterization of a frameshift TSHR variant in the intracytoplasmic C-tail region. Methods: Sequencing of TSHR was performed in a patient with congenital hypothyroidism. The functionality of the identified variants was assessed by expressing TSHR in HEK293 cells and measuring TSH-dependent activation of the cAMP-response element-luciferase reporter. A series of systematic mutagenesis experiments were performed to characterize the frameshifted amino acid sequence. Results: The proband was heterozygous for a known TSHR variant (p.Arg519His) and a novel frameshift TSHR variant (p.Val711Phefs∗18), which removed 54 C-terminal residues and added a 17-amino acid frameshifted sequence. The loss of function of Val711Phefs∗18-TSHR was confirmed in vitro, but the function of Val711∗-TSHR was found to be normal. Western blotting showed the low protein expression of Val711Phefs∗18-TSHR. Fusion of the frameshift sequence to green fluorescent protein or luciferase induced inactivation of them, indicating that the sequence acted as a degron. A systematic mutagenesis study revealed that the density of hydrophobic residues in the frameshift sequence determined the stability. Eight additional frameshift TSHR variants that covered all possible shifted frames in C-tail were created, and another frameshift variant (Thr748Profs∗27) with similar effect was found. Conclusions: We characterized a naturally occurring frameshift TSHR variant located in C-tail, and provided a unique evidence that hydrophobicity in the C-terminal region of the receptor affects protein stability.

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Complete androgen insensitivity syndrome with accelerated onset of puberty due to a sertoli cell tumor

Izawa M., Hisamatsu E., Yoshino K., Yoshida M., Sato T., Narumi S., Hasegawa T., Hamajima T.

Clinical Pediatric Endocrinology （Clinical Pediatric Endocrinology） 30 （ 2 ） 99 - 104 2021年

ISSN 09185739

Complete androgen insensitivity syndrome (CAIS) is caused by mutations in the androgen receptor gene. Patients with this syndrome have a 46,XY karyotype, male gonads, and normal female external genitalia. While the pre-pubertal risk of developing gonadal tumors is low in these patients, it increases with age. Most gonadal tumors arise from germ cells; stromal cell tumors are uncommon. Herein, we report a CAIS patient with a feminizing Sertoli cell tumor. The patient presented at 8 yr of age with breast enlargement and growth acceleration, concomitant with elevated serum estradiol levels and suppressed serum gonadotropin levels; these findings were inconsistent with CAIS. The patient underwent gonadectomy at 10 yr of age, and histology demonstrated presence of a non-malignant Sertoli cell tumor in the right gonad. We conclude that this is the first reported case of CAIS with accelerated onset of puberty resulting from a Sertoli cell tumor.

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Prevalence of germline GATA2 and SAMD9/9L variants in paediatric haematological disorders with monosomy 7.

Yoshida M, Tanase-Nakao K, Shima H, Shirai R, Yoshida K, Osumi T, Deguchi T, Mori M, Arakawa Y, Takagi M, Miyamura T, Sakaguchi K, Toyoda H, Ishida H, Sakata N, Imamura T, Kawahara Y, Morimoto A, Koike T, Yagasaki H, Ito S, Tomizawa D, Kiyokawa N, Narumi S, Kato M

British journal of haematology 191 （ 5 ） 835 - 843 2020年12月

査読有り, ISSN 0007-1048

Monosomy 7 (-7) occurs in various types of paediatric myeloid disorders and has a poor prognosis. Recent studies have demonstrated that patients with germline gain-of-function SAMD9/9L variants and loss-of-function GATA2 variants are prone to developing myelodysplastic syndrome (MDS) associated with -7. However, the prevalence of the genetic variants among paediatric haematologic disorders with -7 is unknown. The present study screened germline variants of GATA2 and SAMD9/9L in 25 patients with various types of paediatric haematological disorders associated with -7. The diagnoses of the 25 patients included MDS (n = 10), acute myeloid leukaemia (AML) and myeloid sarcomas (n = 9), juvenile myelomonocytic leukaemia (n = 3) and other disorders (n = 3). Seven patients with a germline pathogenic GATA2 variant were found. For SAMD9/9L screening, next-generation sequencing was used to detect low-abundance variants and found four novel germline variants. Functional analysis revealed that three out of the four variants showed growth-restricting capacity in vitro and thus, were judged to be pathogenic. Cases with GATA2 mutation tended to be older, compared to those with SAMD9/9L mutations. In conclusion, GATA2 and SAMD9/9L were sequenced in 25 patients with paediatric haematologic disorders associated with -7, and 40% of them were found to have some pathogenic germline variants in the three genes.

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Congenital Hypothyroidism Due to Truncating PAX8 Mutations: A Case Series and Molecular Function Studies.

Iwahashi-Odano M, Nagasaki K, Fukami M, Nishioka J, Yatsuga S, Asakura Y, Adachi M, Muroya K, Hasegawa T, Narumi S

The Journal of clinical endocrinology and metabolism 105 （ 11 ） 2020年11月

査読有り, ISSN 0021-972X

CONTEXT: PAX8 is a transcription factor required for thyroid development, and its mutation causes congenital hypothyroidism (CH). More than 20 experimentally verified loss-of-function PAX8 mutations have been described, and all but one were located in the DNA-binding paired domain. OBJECTIVE: We report the identification and functional characterization of 3 novel truncating PAX8 mutations located outside the paired domain. METHODS: Three CH probands, diagnosed in the frame of newborn screening, had thyroid hypoplasia and were treated with levothyroxine. Next-generation sequencing-based mutation screening was performed. Functionality of the identified mutations were verified with Western blotting, intracellular localization assays, and transactivation assays with use of HeLa cells. Luciferase complementation assays were used to evaluate the effect of mutations on the interaction between PAX8 and its partner, NKX2-1. RESULTS: Each proband had novel truncating PAX8 mutations that were I160Sfs*52, Q213Efs*27, and F342Rfs*85. Western blotting showed destabilization of the I160fs-PAX8 protein. Q213fs-PAX8 and F342fs-PAX8 showed normal protein expression levels and normal nuclear localization, but showed loss of transactivation of the luciferase reporter. By luciferase complementation assays, we showed that PAX8-NKX2-1 interaction was defective in Q213fs-PAX8. We also characterized the recombinant PAX8 proteins, and found that the protein sequence corresponding to exon 10 (363-400 aa residues) was essential for the PAX8-NKX2-1 interaction. CONCLUSIONS: Clinical and molecular findings of 3 novel truncating PAX8 mutations located outside the paired domain were reported. Experiments using cultured cells and recombinant proteins showed that the C-terminal portion (ie, 363-400 aa) of PAX8 is required for the PAX8-NKX2-1 interaction.

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SOX9 is colocalized with paraspeckle protein NONO in cultured murine sertoli cells and features structural characteristics of intrinsically disordered proteins.

Akiba K, Narumi S, Nishimura R, Kato-Fukui Y, Takada S, Hasegawa Y, Fukami M

Molecular reproduction and development 87 （ 11 ） 1124 - 1125 2020年11月

ISSN 1040-452X

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Age-Dependent and Seasonal Changes in Menstrual Cycle Length and Body Temperature Based on Big Data.

Tatsumi T, Sampei M, Saito K, Honda Y, Okazaki Y, Arata N, Narumi K, Morisaki N, Ishikawa T, Narumi S

Obstetrics and gynecology 136 （ 4 ） 666 - 674 2020年10月

査読有り, ISSN 0029-7844

OBJECTIVE: To evaluate the effects of age and season on menstrual cycle length and basal body temperature (BBT). We also examined the effects of climate on cycle length and BBT, taking into account Japanese geographic and social characteristics. METHODS: In this retrospective cohort study, we analyzed data from 6 million menstrual cycles entered into a smartphone application from 310,000 females from 2016 to 2017. Only those who entered more than 10 cycles in 2 years were included. Generalized estimation equations were used to adjust for confounding factors and for within-person correlations of multiple records. Multiple regression analysis was conducted, with age, external average temperature, precipitation amount, and sunshine hours as confounding factors. RESULTS: The mean menstrual cycle length increased from age 15-23 years, subsequently decreased up to age 45 years, and then increased again. Average follicular phase body temperature showed no significant age-dependent changes, but luteal phase body temperature gradually increased up to 29 years and then stabilized and started to decrease after age 42 years. A significant association between external temperature and body temperature (follicular and luteal phase) was observed, though menstrual cycle length did not show such an association. CONCLUSION: These results, derived from data self-entered into a smartphone application, revealed underrecognized age-dependent and seasonal changes in menstrual cycle length and BBT, which will contribute to a better understanding of female reproductive health in the modern world.

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A girl with MIRAGE syndrome who developed steroid-resistant nephrotic syndrome: a case report.

Ishiwa S, Kamei K, Tanase-Nakao K, Shibata S, Matsunami K, Takeuchi I, Sato M, Ishikura K, Narumi S

BMC nephrology 21 （ 1 ） 340 - 340 2020年08月

査読有り

BACKGROUND: MIRAGE syndrome is a recently discovered rare genetic disease characterized by myelodysplasia (M), infection (I), growth restriction (R), adrenal hypoplasia (A), genital phenotypes (G), and enteropathy (E), caused by a gain-of-function mutation in the SAMD9 gene. We encountered a girl with molecularly-confirmed MIRAGE syndrome who developed steroid-resistant nephrotic syndrome. CASE PRESENTATION: She was born at 33 weeks gestational age with a birth weight of 1064 g. She showed growth failure, mild developmental delays, intractable enteropathy and recurrent pneumonia. She was diagnosed as MIRAGE syndrome by whole exome sequencing and a novel SAMD9 variant (c.4615 T > A, p.Leu1539Ile) was identified at age four. Biopsied skin fibroblast cells showed changes in the endosome system that are characteristic of MIRAGE syndrome, supporting the genetic diagnosis. Proteinuria was noted at age one, following nephrotic syndrome at age five. A renal biopsy showed focal segmental glomerulosclerosis (FSGS) with immune deposits. Steroid treatment was ineffective. Because we speculated that her nephrosis was a result of genetic FSGS, we decided not to introduce immunosuppressive agents and instead started enalapril to reduce proteinuria. Although her proteinuria persisted, her renal function was normal at age eight. CONCLUSIONS: This is the first detailed report of a MIRAGE syndrome patient with nephrotic syndrome. Because patients with MIRAGE syndrome have structural abnormalities in the endosomal system, we speculate that dysfunction of endocytosis in podocytes might be a possible mechanism for proteinuria.

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Contribution of gene mutations to Silver-Russell syndrome phenotype: multigene sequencing analysis in 92 etiology-unknown patients.

Inoue T, Nakamura A, Iwahashi-Odano M, Tanase-Nakao K, Matsubara K, Nishioka J, Maruo Y, Hasegawa Y, Suzumura H, Sato S, Kobayashi Y, Murakami N, Nakabayashi K, Yamazawa K, Fuke T, Narumi S, Oka A, Ogata T, Fukami M, Kagami M

Clinical epigenetics 12 （ 1 ） 86 - 86 2020年06月

査読有り, ISSN 1868-7075

BACKGROUND: Silver-Russell syndrome (SRS) is characterized by growth failure and dysmorphic features. Major (epi)genetic causes of SRS are loss of methylation on chromosome 11p15 (11p15 LOM) and maternal uniparental disomy of chromosome 7 (upd(7)mat). However, IGF2, CDKN1C, HMGA2, and PLAG1 mutations infrequently cause SRS. In addition, other imprinting disturbances, pathogenic copy number variations (PCNVs), and monogenic disorders sometimes lead to SRS phenotype. This study aimed to clarify the frequency and clinical features of the patients with gene mutations among etiology-unknown patients with SRS phenotype. RESULTS: Multigene sequencing was performed in 92 out of 336 patients referred to us for genetic testing for SRS. The clinical features of the patients were evaluated based on the Netchine-Harbison clinical scoring system. None of the patients showed 11p15 LOM, upd(7)mat, abnormal methylation levels for six differentially methylated regions (DMRs), namely, PLAGL1:alt-TSS-DMR on chromosome 6, KCNQ1OT1:TSS-DMR on chromosome 11, MEG3/DLK1:IG-DMR on chromosome 14, MEG3:TSS-DMR on chromosome 14, SNURF:TSS-DMR on chromosome 15, and GNAS A/B:TSS-DMR on chromosome 20, PCNVs, or maternal uniparental disomy of chromosome 16. Using next-generation sequencing and Sanger sequencing, we screened four SRS-causative genes and 406 genes related to growth failure and/or skeletal dysplasia. We identified four pathogenic or likely pathogenic variants in responsible genes for SRS (4.3%: IGF2 in two patients, CDKN1C, and PLAG1), and five pathogenic variants in causative genes for known genetic syndromes presenting with growth failure (5.4%: IGF1R abnormality (IGF1R), SHORT syndrome (PIK3R1), Floating-Harbor syndrome (SRCAP), Pitt-Hopkins syndrome (TCF4), and Noonan syndrome (PTPN11)). Functional analysis indicated the pathogenicity of the CDKN1C variant. The variants we detected in CDKN1C and PLAG1 were the second and third variants leading to SRS, respectively. Our patients with CDKN1C and PLAG1 variants showed similar phenotypes to previously reported patients. Furthermore, our data confirmed IGF1R abnormality, SHORT syndrome, and Floating-Harbor syndrome are differential diagnoses of SRS because of the shared phenotypes among these syndromes and SRS. On the other hand, the patients with pathogenic variants in causative genes for Pitt-Hopkins syndrome and Noonan syndrome were atypical of these syndromes and showed partial clinical features of SRS. CONCLUSIONS: We identified nine patients (9.8%) with pathogenic or likely pathogenic variants out of 92 etiology-unknown patients with SRS phenotype. This study expands the molecular spectrum of SRS phenotype.

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Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells.

Matsumoto R, Suga H, Aoi T, Bando H, Fukuoka H, Iguchi G, Narumi S, Hasegawa T, Muguruma K, Ogawa W, Takahashi Y

The Journal of clinical investigation 130 （ 2 ） 641 - 654 2020年02月

査読有り, ISSN 0021-9738

Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone-producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.

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TSH単独欠損症の分子基盤小児患者13例の系統的遺伝子解析と文献レビュー

杉澤千穂, 阿部清美, 志賀健太郎, 菅原秀典, 大杉康司, 室谷浩二, 朝倉由美, 安達昌功, 大通尚, 沼倉周彦, 小池明美, 椿淳子, 橘田一輝, 松浦信夫, 谷山松雄, 長谷川奉延, 鳴海覚志

日本内分泌学会雑誌（(一社)日本内分泌学会） 95 （ 4 ） 1333 - 1333 2020年02月

ISSN 0029-0661

A Novel Homozygous Mutation of Thyroid Peroxidase Gene Abolishes a Disulfide Bond Leading to Congenital Hypothyroidism.

Yakou F, Suwanai H, Ishikawa T, Itou M, Shikuma J, Miwa T, Sakai H, Kanekura K, Narumi S, Suzuki R, Odawara M

International journal of endocrinology 2020 9132372 - 9132372 2020年

査読有り, ISSN 1687-8337

Congenital hypothyroidism (CH) is the most prevalent congenital endocrine disorder and causes mental retardation. A male Japanese patient with first cousin marriage parents was diagnosed as CH at 10 months. He was born before introduction of mass screening for CH. With continuous thyroid hormone replacement therapy, normal thyroid hormone status was maintained until adulthood. Genetic screening of next-generation sequencing was performed at the age of 52 years, and we identified a new homozygous thyroid peroxidase (TPO) gene mutation (GRCh38.p13, chromosome 2 at position 1493997, c.1964 G>T, p.Cys655Phe). TPO is an important enzyme to produce thyroid hormone. As demonstrated by a homology analysis of TPO proteins among different species, cysteine 655 residue is highly conserved, suggesting an important role in maintaining TPO function and structure. An in silico study with three-dimensional structure of the novel mutation was performed and suggested that the mutation abolished disulfide bond between cysteines at positions 598 and 655. An in vitro functional analysis using HEK293 cells revealed that TPO activity of the mutant was significantly impaired compared with that of the wild type. Furthermore, study of immunohistochemistry showed that localization of TPO in cells did not differ between the wild type and the mutant. In conclusion, this single disulfide bond loss mutation of a new TPO homozygous mutation, p.Cys655Phe, reduced TPO activity and caused congenital hypothyroidism without affecting subcellular localization of TPO proteins.

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Predicting the pathogenicity of NKX2-1 and IGSF1 variants with in silico bioinformatic tools.

Narumi S

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 29 （ 3 ） 123 - 126 2020年

査読有り, ISSN 0918-5739

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MIRAGE syndrome caused by a novel missense variant (p.Ala1479Ser) in the SAMD9 gene.

Onuma S, Wada T, Araki R, Wada K, Tanase-Nakao K, Narumi S, Fukui M, Shoji Y, Etani Y, Ida S, Kawai M

Human genome variation 7 4 - 4 2020年

査読有り, ISSN 2054-345X

MIRAGE syndrome is a recently identified disorder characterized by myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy. It is caused by a gain-of-function variant in the SAMD9 gene, but there is limited knowledge regarding the genotype-phenotype correlation. We herein report a Japanese patient with MIRAGE syndrome carrying a novel de novo heterozygous missense variant in the SAMD9 gene (c.4435 G > T; p.Ala1479Ser).

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Identification and functional characterization of a novel PAX8 mutation (p.His39Pro) causing familial thyroid hypoplasia.

Iwahashi-Odano M, Fujisawa Y, Ogata T, Nakashima S, Muramatsu M, Narumi S

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 29 （ 4 ） 173 - 178 2020年

ISSN 0918-5739

Mutations in PAX8, the gene for a thyroid-specific transcription factor, causes congenital hypothyroidism (CH) with autosomal dominant inheritance. All previously detected PAX8 mutations except one are located in the DNA-binding paired domain. The proband, a 1-yr-old boy, was diagnosed with CH in the frame of newborn screening. He had high serum TSH level (180 mU/L) and low serum free T4 level (0.4 ng/dL). Ultrasonography revealed that the proband had thyroid hypoplasia. Importantly, he had a family history of CH, i.e., his mother also had CH and hypoplasia. Next generation sequencing-based mutation screening revealed a novel heterozygous PAX8 mutation (c.116A>C, p.His39Pro) that was transmitted to the proband from the mother. Expression experiments with HeLa cells confirmed that His39Pro-PAX8 exhibited defective transactivation of the TG promoter-luciferase reporter. In conclusion, we identified and described a novel loss-of-function PAX8 mutation in a family with thyroid hypoplasia. Patients with dominantly inherited CH and no extrathyroidal abnormalities could have PAX8 mutations.

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Evaluating the seasonality of growth in infants using a mobile phone application.

Narumi S, Ohnuma T, Takehara K, Morisaki N, Urayama KY, Hattori T

NPJ digital medicine 3 138 - 138 2020年

It has been observed that growth velocity of toddlers and school children shows seasonal variation, while such seasonality is unknown in infants. The aim of this study was to examine whether growth velocity (length and weight) of infants differs by seasons. We assessed longitudinal measurement data obtained for 9,409 Japanese infants whose parents used the mobile phone application, "Papatto Ikuji", during the period from January 2014 to October 2017. On average, each infant had 4.8 entries for length and 5.4 entries for weight. The mean daily change in sex- and age-adjusted z-scores between two time points was estimated as the growth velocity during that period: ΔLAZ/day and ΔWAZ/day for length and weight, respectively. We analyzed 20,007 ΔLAZ/day (mean, -0.0022) and 33,236 ΔWAZ/day (mean, 0.0005) measurements, and found that ΔLAZ/day showed seasonal differences with increases during summer. We conducted a multilevel linear regression analysis, in which effects of age, sex, nutrition and season of birth were adjusted, showing significant difference in ΔLAZ/day between winter and summer with a mean ΔLAZ/day difference of 0.0026 (95%CI 0.0015 to 0.0036; P < 0.001). This seasonal difference corresponded to 13% of the average linear growth velocity in 6-month-old infants. A modest effect of nutrition on linear growth was observed with a mean ΔLAZ/day difference of 0.0015 (95%CI 0.0006 to 0.0025; P < 0.001) between predominantly formula-fed infants and breastfed infants. In conclusion, we observed that linear growth, but not weight gain, of Japanese infants showed significant seasonality effects represented by increases in summer and decreases in winter.

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A novel NPR2 mutation (p.Arg388Gln) in a patient with acromesomelic dysplasia, type Maroteaux.

Amano N, Kitoh H, Narumi S, Nishimura G, Hasegawa T

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 29 （ 3 ） 99 - 103 2020年

査読有り, ISSN 0918-5739

Acromesomelic dysplasia, type Maroteaux (AMDM) is a congenital bone dysplasia characterized by disproportionate, acromesomelic shortening of the limbs and mild spondylar dysplasia. AMDM is caused by biallelic loss-of-function mutations in NPR2 encoding natriuretic peptide receptor-B. We report on a 25-yr-old Japanese woman with AMDM. Her height was 119.0 cm (-7.4 SD) and weight 35 kg (-2.3 SD). She had acromesomelic shortening of limbs and severe brachydactyly. Radiological examination showed that her metacarpals and phalanges were short and wide, and her vertebral bodies were mildly flattened. Molecular analysis revealed a novel homozygous NPR2 mutation (c.1163G>A, p.Arg388Gln). We performed in vitro functional studies using HA-tagged wild-type (WT) and Arg388Gln vectors (HA-WT-NPRB and HA-R388Q-NPRB). Cells expressing HA-R388Q-NPRB showed negligible cGMP responses to C-type natriuretic peptide (CNP) stimulation, indicating that the mutation led to severe loss-of-function. By immunofluorescence experiments under permeabilized conditions, HA-WT-NPRB was expressed on plasma membrane, while HA-R388Q-NPRB co-localized with an Endoplasmic Reticulum marker. Cells co-expressing R388Q and the WT exhibited lower responses under CNP treatment than cells co-expressing the WT and empty vectors. Thus, it was thought that R388Q caused a dominant-negative effect with a defect in cellular trafficking to the plasma membrane.

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Genetics of Congenital Isolated TSH Deficiency: Mutation Screening of the Known Causative Genes and a Literature Review.

Sugisawa C, Takamizawa T, Abe K, Hasegawa T, Shiga K, Sugawara H, Ohsugi K, Muroya K, Asakura Y, Adachi M, Daitsu T, Numakura C, Koike A, Tsubaki J, Kitsuda K, Matsuura N, Taniyama M, Ishii S, Satoh T, Yamada M, Narumi S

The Journal of clinical endocrinology and metabolism 104 （ 12 ） 6229 - 6237 2019年12月

査読有り, ISSN 0021-972X

CONTEXT: Congenital isolated TSH deficiency (i-TSHD) is a rare form of congenital hypothyroidism. Five genes (IGSF1, IRS4, TBL1X, TRHR, and TSHB) responsible for the disease have been identified, although their relative frequencies and hypothalamic/pituitary unit phenotypes have remained to be clarified. OBJECTIVES: To define the relative frequencies and hypothalamic/pituitary unit phenotypes of congenital i-TSHD resulting from single gene mutations. PATIENTS AND METHODS: Thirteen Japanese patients (11 boys and 2 girls) with congenital i-TSHD were enrolled. IGSF1, IRS4, TBL1X, TRHR, and TSHB were sequenced. For a TBL1X mutation (p.Asn382del), its pathogenicity was verified in vitro. For a literature review, published clinical data derived from 74 patients with congenital i-TSHD resulting from single-gene mutations were retrieved and analyzed. RESULTS: Genetic screening of the 13 study subjects revealed six mutation-carrying patients (46%), including five hemizygous IGSF1 mutation carriers and one hemizygous TBL1X mutation carrier. Among the six mutation carriers, one had intellectual disability and the other one had obesity, but the remaining four did not show nonendocrine phenotypes. Loss of function of the TBL1X mutation (p.Asn382del) was confirmed in vitro. The literature review demonstrated etiology-specific relationship between serum prolactin (PRL) levels and TRH-stimulated TSH levels with some degree of overlap. CONCLUSIONS: The mutation screening study covering the five causative genes of congenital i-TSHD was performed, showing that the IGSF1 defect was the leading genetic cause of the disease. Assessing relationships between serum PRL levels and TRH-stimulated TSH levels would contribute to predict the etiologies of congenital i-TSHD.

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KLF11 variant in a family clinically diagnosed with early childhood-onset type 1B diabetes.

Ushijima K, Narumi S, Ogata T, Yokota I, Sugihara S, Kaname T, Horikawa Y, Matsubara Y, Fukami M, Kawamura T, Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes

Pediatric diabetes （Wiley） 20 （ 6 ） 712 - 719 2019年09月

査読有り, ISSN 1399-543X

KLF11 is the causative gene for maturity-onset diabetes of the young 7 (MODY7). KLF11 regulates insulin gene expression through binding to the GC box in the promoter. To date, only two KLF11 mutations have been identified in three families with early-onset type 2 diabetes. Here, we report a novel KLF11 variant associated with early childhood-onset type 1B diabetes. The proband and his younger sister exhibited hyperglycemia at age 1 year, and their mother developed diabetes at age 4 years. These three individuals required insulin injection from the initial phase of the disease. Being negative for islet cell autoantibodies, they were diagnosed with type 1B diabetes. Mutation screening for 30 diabetes-associated genes identified a heterozygous KLF11 variant (p.His418Gln) in the proband and his sister. The variant was also detected in the affected mother, as well as in the allegedly unaffected maternal grandmother. In silico analyses indicated that this variant involves a highly conserved histidine residue in the first C2 H2 zinc finger domain which ligates a zinc ion. In vitro analyses showed that expression levels and intracellular localization of His418Gln-KLF11 were comparable to those of wildtype (WT)-KLF11. Luciferase assays demonstrated that while WT-KLF11 suppressed the activity of a 6 × GC box-containing reporter, His418Gln-KLF11 lacked the suppressive effect. Notably, His418Gln-KLF11 canceled the suppressive effect of co-transfected WT-KLF11. Such a dominant-negative effect was absent in the previously reported Ala347Ser-KLF11 variant. These results indicate that specific variants of KLF11 (MODY7) with a dominant-negative effect underlie early childhood-onset type 1B diabetes with incomplete penetrance. This study documents a novel monogenic mutation associated with diabetes in children.

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Activity-Dependent Secretion of Synaptic Organizer Cbln1 from Lysosomes in Granule Cell Axons.

Ibata K, Kono M, Narumi S, Motohashi J, Kakegawa W, Kohda K, Yuzaki M

Neuron 102 （ 6 ） 1184 - 1198.e10 2019年06月

ISSN 0896-6273

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Molecular and clinical analyses of two patients with UPD(16)mat detected by screening 94 patients with Silver-Russell syndrome phenotype of unknown aetiology.

Inoue T, Yagasaki H, Nishioka J, Nakamura A, Matsubara K, Narumi S, Nakabayashi K, Yamazawa K, Fuke T, Oka A, Ogata T, Fukami M, Kagami M

Journal of medical genetics 56 （ 6 ） 413 - 418 2019年06月

査読有り, ISSN 0022-2593

BACKGROUND: Recently, a patient with maternal uniparental disomy of chromosome 16 (UPD(16)mat) presenting with Silver-Russell syndrome (SRS) phenotype was reported. SRS is characterised by growth failure and dysmorphic features. OBJECTIVE: To clarify the prevalence of UPD(16)mat in aetiology-unknown patients with SRS phenotype and phenotypic differences between UPD(16)mat and SRS. METHODS: We studied 94 patients with SRS phenotype of unknown aetiology. Sixty-three satisfied the Netchine-Harbison clinical scoring system (NH-CSS) criteria, and 25 out of 63 patients showed both protruding forehead and relative macrocephaly (clinical SRS). The remaining 31 patients met only three NH-CSS criteria, but were clinically suspected as having SRS. To detect UPD(16)mat, we performed methylation analysis for the ZNF597:TSS-differentially methylated region (DMR) on chromosome 16 and subsequently performed microsatellite, SNP array and exome analyses in the patients with hypomethylated ZNF597:TSS-DMR. RESULTS: We identified two patients (2.1%) with a mixture of maternal isodisomy and heterodisomy of chromosome 16 in 94 aetiology-unknown patients with SRS phenotype. Both patients exhibited preterm birth and prenatal and postnatal growth failure. The male patient had ventricular septal defect and hypospadias. Whole-exome sequencing detected no gene mutations related to their phenotypes. CONCLUSION: We suggest considering genetic testing for UPD(16)mat in SRS phenotypic patients without known aetiology.

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Systematic alanine scanning of PAX8 paired domain reveals functional importance of the N-subdomain.

Iwahashi M, Narumi S

Journal of molecular endocrinology 62 （ 3 ） 129 - 135 2019年04月

査読有り, ISSN 0952-5041

Thyroid-specific transcription factor PAX8 has an indispensable role in the thyroid gland development, which is evidenced by the facts that PAX8/Pax8 mutations cause congenital hypothyroidism in humans and mice. More than 90% of known PAX8 mutations were located in the paired domain, suggesting the central role of the domain in exerting the molecular function. Structure-function relationships of PAX8, as well as other PAX family transcription factors, have never been investigated in a systematic manner. Here, we conducted the first alanine scanning mutagenesis study, in which 132 alanine variants located in the paired domain of PAX8 were created and systematically evaluated in vitro. We found that 76 alanine variants (55%) were loss of function (LOF) variants (defined by <30% activity as compared with wild type PAX8). Importantly, the distribution of LOF variants were skewed, with more frequently observed in the N-subdomain (65% of the alanine variants in the N-subdomain) than in the C-subdomain (45%). Twelve out of 13 alanine variants in residues that have been affected in patients with congenital hypothyroidism were actually LOF, suggesting that the alanine scanning data can be used to evaluate the functional importance of mutated residues. Using our in vitro data, we tested the accuracy of seven computational algorithms for pathogenicity prediction, showing that they are sensitive but not specific to evaluate on the paired domain alanine variants. Collectively, our experiment-based data would help better understand the structure-function relationships of the paired domain, and would provide a unique resource for pathogenicity prediction of future PAX8 variants.

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DUOX2異常症におけるレボチロキシンNaの投与中止時期についての検討

柴田奈央, 入月浩美, 佐々木直, 佐藤英利, 小川洋平, 鳴海覚志, 長崎啓祐

日本内分泌学会雑誌（(一社)日本内分泌学会） 95 （ 1 ） 419 - 419 2019年04月

ISSN 0029-0661

Somatic mosaic monosomy 7 and UPD7q in a child with MIRAGE syndrome caused by a novel SAMD9 mutation.

Csillag B, Ilencikova D, Meissl M, Webersinke G, Laccone F, Narumi S, Haas O, Duba HC

Pediatric blood & cancer 66 （ 4 ） e27589 2019年04月

査読有り, ISSN 1545-5009

MIRAGE syndrome caused by mutations in SAMD9 is associated with potential loss of chromosome 7 (-7/7q-) and an increased risk to develop myelodysplastic syndrome (MDS). We report a case of MIRAGE syndrome, caused by a novel SAMD9 mutation p.Leu641Pro, leading to characteristic clinical features as well as to the coexistence of cells with monosomy 7 (20%) and with uniparental disomy of long arm of chromosome 7 (UPD7q). In contrast to previously reported MIRAGE patients with -7/7q- developing MDS, our patient achieved complete cytogenetic remission of monosomy 7. As UPD7q remained unchanged, it seems to be a protective factor against MDS.

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新たに発見された変異TBL1X遺伝子の機能解析

高見澤哲也, 鳴海覚志, 杉澤千穂, 谷山松雄, 錦戸彩加, 岡村孝志, 土岐明子, 石田恵美, 松本俊一, 堀口和彦, 中島康代, 登丸琢也, 石井角保, 小澤厚志, 渋沢信行, 石塚高広, 佐藤哲郎, 長谷川奉延, 山田正信

日本内分泌学会雑誌（(一社)日本内分泌学会） 95 （ 1 ） 397 - 397 2019年04月

ISSN 0029-0661

MIRAGE syndrome with recurrent pneumonia probably associated with gastroesophageal reflux and achalasia: A case report.

Yoshizaki K, Hachiya R, Tomobe Y, Kaku U, Akiba K, Shima H, Narumi S, Hasegawa Y

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 28 （ 4 ） 147 - 153 2019年

査読有り, ISSN 0918-5739

Aspiration pneumonia is a common complication of myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy (MIRAGE) syndrome. However, the detailed clinical course of aspiration pneumonia in neonates and infants diagnosed with this disorder remains unclear. We report a case of a 2-yr-old girl diagnosed with MIRAGE syndrome during the early neonatal period. The patient developed 3 episodes of aspiration pneumonia until 4 mo of age, and this complication was attributed to esophageal hypoperistalsis secondary to achalasia and gastroesophageal reflux. Enteral feeding via a duodenal tube effectively prevented further episodes of aspiration pneumonia in this patient.

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A novel truncating mutation in MYH3 causes spondylocarpotarsal synostosis syndrome with basilar invagination.

Takagi M, Shimomura S, Fukuzawa R, Narumi S, Nishimura G, Hasegawa T

Journal of human genetics 63 （ 12 ） 1277 - 1281 2018年12月

ISSN 1434-5161

Spondylocarpotarsal synostosis syndrome (SCT) is a rare group of skeletal dysplasias, characterized by disproportionate short stature with a short trunk, abnormal segmentation of the spine with vertebral fusion, scoliosis and lordosis, carpal and tarsal synostosis, and mild facial dysmorphisms. While the majority of the cases show autosomal recessive inheritance, only a few cases of vertical transmissions, with MYH3 mutations, have been reported. Here we report a case with typical SCT, carrying a novel heterozygous mutation in MYH3. This observation supports the hypothesis of a pathogenic link between autosomal dominant SCT and heterozygous mutations in MYH3. Of note, our case showed basilar invagination on brain magnetic resonance imaging at the age of 10 years. Basilar invagination could be a rare complication of both autosomal recessive and dominant SCT, indicating that prompt investigation are warranted for SCT patients.

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An association with hypopituitarism and 9q subtelomere deletion syndrome.

Higuchi S, Takagi M, Takeda R, Yoshihashi H, Narumi S, Hasegawa T

Clinical case reports 6 （ 12 ） 2371 - 2375 2018年12月

査読有り, ISSN 2050-0904

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中枢性先天性甲状腺機能低下症男児における新規TBL1X変異の同定と機能解析

杉澤千穂, 志賀健太郎, 菅原秀典, 大杉康司, 谷山松雄, 長谷川奉延, 高見澤哲也, 石井角保, 佐藤哲郎, 山田正信, 鳴海覚志

日本内分泌学会雑誌（(一社)日本内分泌学会） 94 （ 4 ） 1228 - 1228 2018年12月

ISSN 0029-0661

Germline loss-of-function SAMD9 and SAMD9L alterations in adult myelodysplastic syndromes.

Nagata Y, Narumi S, Guan Y, Przychodzen BP, Hirsch CM, Makishima H, Shima H, Aly M, Pastor V, Kuzmanovic T, Radivoyevitch T, Adema V, Awada H, Yoshida K, Li S, Sole F, Hanna R, Jha BK, LaFramboise T, Ogawa S, Sekeres MA, Wlodarski MW, Cammenga J, Maciejewski JP

Blood 132 （ 21 ） 2309 - 2313 2018年11月

査読有り, ISSN 0006-4971

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GATA4 mutations are uncommon in patients with 46,XY disorders of sex development without heart anomaly.

Igarashi M, Mizuno K, Kon M, Narumi S, Kojima Y, Hayashi Y, Ogata T, Fukami M

Asian journal of andrology 20 （ 6 ） 629 - 631 2018年11月

査読有り, ISSN 1008-682X

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Optogenetic Control of Synaptic AMPA Receptor Endocytosis Reveals Roles of LTD in Motor Learning.

Kakegawa W, Katoh A, Narumi S, Miura E, Motohashi J, Takahashi A, Kohda K, Fukazawa Y, Yuzaki M, Matsuda S

Neuron 99 （ 5 ） 985 - 998.e6 2018年09月

ISSN 0896-6273

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Rare monogenic causes of primary adrenal insufficiency.

Narumi S

Current opinion in endocrinology, diabetes, and obesity 25 （ 3 ） 172 - 177 2018年06月

査読有り, ISSN 1752-296X

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Nodular Lymphocyte-predominant Hodgkin Lymphoma in a 15-Year-Old Boy With Li-Fraumeni Syndrome Having a Germline TP53 D49H Mutation.

Yamazaki F, Shima H, Osumi T, Narumi S, Kuroda T, Shimada H

Journal of pediatric hematology/oncology 40 （ 3 ） e195 - e197 2018年04月

査読有り, ISSN 1077-4114

Germline mutations in TP53 are the primary cause of Li-Fraumeni syndrome (LFS). Most mutations are reported within the DNA-binding domain. We report a case of a 15-year-old boy with LFS who developed early-stage nodular lymphocyte-predominant Hodgkin lymphoma, a rare subtype of Hodgkin lymphomas. His sister was diagnosed with embryonal rhabdomyosarcoma at the age of 1.5 years. Sequence analysis revealed a germline mutation in the transactivation domain of TP53, c.145G>C (p.D49H), in the patient, his sister, and father. One family with LFS with a germline TP53 D49H mutation has previously been reported. This report supports the pathogenicity of this mutation.

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胎児甲状腺腫性甲状腺機能低下症を呈し胎内治療を行うも幼児期に軽快したDUOXA2異常症世界初の報告例

宮田市郎, 寺内綾子, 齋藤真希, 和田誠司, 岩橋めぐみ, 中尾佳奈子, 鳴海覚志

日本内分泌学会雑誌（(一社)日本内分泌学会） 94 （ 1 ） 333 - 333 2018年04月

ISSN 0029-0661

Comprehensive screening for monogenic diabetes in 89 Japanese children with insulin-requiring antibody-negative type 1 diabetes.

Ushijima K, Fukami M, Ayabe T, Narumi S, Okuno M, Nakamura A, Takahashi T, Ihara K, Ohkubo K, Tachikawa E, Nakayama S, Arai J, Kikuchi N, Kikuchi T, Kawamura T, Urakami T, Hata K, Nakabayashi K, Matsubara Y, Amemiya S, Ogata T, Yokota I, Sugihara S, Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes

Pediatric diabetes 19 （ 2 ） 243 - 250 2018年03月

査読有り, ISSN 1399-543X

BACKGROUND: Mutations in causative genes for neonatal diabetes or maturity-onset diabetes of the young have been identified in multiple patients with autoantibody-negative type 1 diabetes (T1D). OBJECTIVES: We aimed to clarify the prevalence and phenotypic characteristics of monogenic abnormalities among 89 children with autoantibody-negative insulin-requiring T1D. METHODS: Mutations in 30 genes were screened using next-generation sequencing, and copy-number alterations of 4 major causative genes were examined using multiplex-ligation-dependent probe amplification. We compared the clinical characteristics between mutation carriers and non-carriers. RESULTS: We identified 11 probable pathogenic substitutions (6 in INS , 2 in HNF1A , 2 in HNF4A , and 1 in HNF1B ) in 11 cases, but no copy-number abnormalities. Only 2 mutation carriers had affected parents. De novo occurrence was confirmed for 3 mutations. The non-carrier group, but not the carrier group, was enriched with susceptible HLA alleles. Mutation carriers exhibited comparable phenotypes to those of non-carriers, except for a relatively normal body mass index (BMI) at diagnosis. CONCLUSIONS: This study demonstrated significant genetic overlap between autoantibody-negative T1D and monogenic diabetes. Mutations in INS and HNF genes, but not those in GCK and other monogenic diabetes genes, likely play critical roles in children with insulin-requiring T1D. This study also suggests the relatively high de novo rates of INS and HNF mutations, and the etiological link between autoimmune abnormalities and T1D in the non-carrier group. Carriers of monogenic mutations show non-specific phenotypes among all T1D cases, although they are more likely to have a normal BMI at diagnosis than non-carriers.

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A novel SAMD9 mutation causing MIRAGE syndrome: An expansion and review of phenotype, dysmorphology, and natural history.

Jeffries L, Shima H, Ji W, Panisello-Manterola D, McGrath J, Bird LM, Konstantino M, Narumi S, Lakhani S

American journal of medical genetics. Part A （Wiley-Liss Inc.） 176 （ 2 ） 415 - 420 2018年02月

査読有り, ISSN 1552-4825

Germline gain-of-function variants in SAMD9 have been associated with a high risk of mortality and a newly recognized constellation of symptoms described by the acronym MIRAGE: Myelodysplasia, Infection, Restriction of growth, Adrenal insufficiency, Genital phenotypes, and Enteropathy. Here, we describe two additional patients currently living with the syndrome, including one patient with a novel de novo variant for which we provide functional data supporting its pathogenicity. We discuss features of dysmorphology, contrasting with previously described patients as well as drawing attention to additional clinical features, dysautonomia and hearing loss that have not previously been reported. We detail both patients’ courses following diagnosis, with attention to treatment plans and recommended specialist care. Our patients are the oldest known with arginine-substituting amino acid variants, and we conclude that early diagnosis and multidisciplinary management may positively impact outcomes for this vulnerable group of patients.

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Association between monoallelic TSHR mutations and congenital hypothyroidism: a statistical approach.

Abe K, Narumi S, Suwanai AS, Adachi M, Muroya K, Asakura Y, Nagasaki K, Abe T, Hasegawa T

European journal of endocrinology （BioScientifica Ltd.） 178 （ 2 ） 137 - 144 2018年02月

査読有り, ISSN 0804-4643

Objective: Biallelic TSHR mutations cause congenital hypothyroidism (CH). Serum TSH levels of monoallelic mutation carriers range from normal to mildly elevated, and thus the size of its effect remains unclear. The objectives were to examine the association between monoallelic TSHR mutations and positivity at newborn screening (NBS) for CH, and to test whether the association was modified by another genetic factor. Subjects and methods: We enrolled 395 patients that had a positive result in NBS and sequenced TSHR. Monoallelic TSHR mutation carriers were further sequenced for DUOX2. Molecular functions of the mutations were verified in vitro. The frequency of the mutations in the study subjects was compared with a theoretical value in the Japanese general population. Odds ratio (OR) for NBS positivity associated with the mutation was calculated. Using Bayes' theorem, we estimated a posterior probability of NBS positivity given the mutation. Results: Twenty-six monoallelic TSHR mutation carriers were found. Four out of the 26 also had a monoallelic DUOX2 mutation (double heterozygotes). The frequencies of monoallelic TSHR mutation carriers (6.6%) and double heterozygotes (1.0%) were significantly higher than those in the general population (0.58% and 0.0087%, respectively). OR for NBS positivity of having a monoallelic TSHR mutation or being a double heterozygote was 12.0 or 117.9, respectively. Posterior probability of NBS positivity was 0.38% in monoallelic TSHR mutation carriers and 3.8% in double heterozygotes. Conclusions: Monoallelic TSHR mutations are significantly associated with NBS positivity, and the association is further strengthened by the coexistence of monoallelic DUOX2 mutations.

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Two patients with MIRAGE syndrome lacking haematological features: role of somatic second-site reversion SAMD9 mutations.

Shima H, Koehler K, Nomura Y, Sugimoto K, Satoh A, Ogata T, Fukami M, Jühlen R, Schuelke M, Mohnike K, Huebner A, Narumi S

Journal of medical genetics 55 （ 2 ） 81 - 85 2018年02月

査読有り, ISSN 0022-2593

BACKGROUND: Myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (MIRAGE) syndrome is a recently described congenital disorder caused by heterozygous SAMD9 mutations. The phenotypic spectrum of the syndrome remains to be elucidated. METHODS AND RESULTS: We describe two unrelated patients who showed manifestations compatible with MIRAGE syndrome, with the exception of haematological features. Leucocyte genomic DNA samples were analysed with next-generation sequencing and Sanger sequencing, revealing the patients to have two de novoSAMD9 mutations on the same allele (patient 1 p.[Gln695*; Ala722Glu] and patient 2 p.[Gln39*; Asp769Gly]). In patient 1, p.Gln695* was absent in genomic DNA extracted from hair follicles, implying that the non-sense mutation was acquired somatically. In patient 2, with the 46,XX karyotype, skewed X chromosome inactivation pattern was found in leucocyte DNA, suggesting monoclonality of cells in the haematopoietic system. In vitro expression experiments confirmed the growth-restricting capacity of the two missense mutant SAMD9 proteins that is a characteristic of MIRAGE-associated SAMD9 mutations. CONCLUSIONS: Acquisition of a somatic nonsense SAMD9 mutation in the cells of the haematopoietic system might revert the cellular growth repression caused by the germline SAMD9 mutations (ie, second-site reversion mutations). Unexpected lack of haematological features in the two patients would be explained by the reversion mutations.

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Poor outcome with hematopoietic stem cell transplantation for bone marrow failure and MDS with severe MIRAGE syndrome phenotype.

Sarthy J, Zha J, Babushok D, Shenoy A, Fan JM, Wertheim G, Himebauch A, Munchel A, Taraseviciute A, Yang S, Shima H, Narumi S, Meshinchi S, Olson TS

Blood advances 2 （ 2 ） 120 - 125 2018年01月

査読有り, ISSN 2473-9529

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Comment on: Acquired monosomy 7 myelodysplastic syndrome in a child with clinical features of dyskeratosis congenita and IMAGe association.

Wilson DB, Bessler M, Ferkol TW, Shenoy S, Amano N, Ishii T, Shima H, Narumi S

Pediatric blood & cancer （WILEY） 65 （ 1 ） e26747 2018年01月

査読有り, ISSN 1545-5009

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[Association between SAMD9/SAMD9L and hematological malignancies].

Narumi S, Hasegawa T

[Rinsho ketsueki] The Japanese journal of clinical hematology 59 （ 11 ） 2475 - 2480 2018年

ISSN 0485-1439

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Molecular and Clinical Analyses of Two UPD(16)Mat Patients Detected by Screening of 94 Silver-Russell Syndrome Patients Without Known Etiology

Inoue Takanobu, Yagasaki Hideaki, Nishioka Junko, Nakamura Akie, Matsubara Keiko, Narumi Satoshi, Nakabayashi Kazuhiko, Yamazawa Kazuki, Fuke Tomoko, Oka Akira, Ogata Tsutomu, Fukami Maki, Kagami Masayo

HORMONE RESEARCH IN PAEDIATRICS 90 122 - 123 2018年

査読有り, ISSN 1663-2818

MIRAGE syndrome is a rare cause of 46,XY DSD born SGA without adrenal insufficiency.

Shima H, Hayashi M, Tachibana T, Oshiro M, Amano N, Ishii T, Haruna H, Igarashi M, Kon M, Fukuzawa R, Tanaka Y, Fukami M, Hasegawa T, Narumi S

PloS one 13 （ 11 ） e0206184 2018年

査読有り

BACKGROUND: MIRAGE syndrome, a congenital multisystem disorder due to pathogenic SAMD9 variants, describes a constellation of clinical features including 46,XY disorders of sex development (DSD), small for gestational age (SGA) and adrenal insufficiency (AI). It is poorly understood whether SAMD9 variants underlie 46,XY DSD patients born SGA (46,XY DSD SGA) without AI. This study aimed to define the frequency and phenotype of SAMD9 variants in 46,XY DSD SGA without AI. METHODS: Forty-nine Japanese patients with 46,XY DSD SGA (Quigley scale, 2 to 6; gestational age-matched birth weight percentile, <10) without history of AI were enrolled. The single coding exon of SAMD9 was PCR-amplified and sequenced for each patient. Pathogenicity of an identified variant was verified in vitro. Placenta tissues were obtained from the variant-carrying patient, as well as from another previously described patient, and were analyzed histologically. RESULTS: In one 46,XY DSD SGA patient, a novel heterozygous SAMD9 variant, p.Phe1017Val, was identified. Pathogenicity of the mutant was experimentally confirmed. In addition to DSD and SGA, the patient had neonatal thrombocytopenia, severe postnatal grow restriction, chronic diarrhea and susceptibility to infection, all features consistent with MIRAGE, leading to premature death at age 14 months. The patient did not have any manifestations or laboratory findings suggesting AI. Placenta tissues of the two variant-carrying patients were characterized by maldevelopment of distal villi without other findings of maternal underperfusion. CONCLUSIONS: MIRAGE syndrome is a rare cause of 46,XY DSD SGA without AI. This study exemplifies that AI is a common feature of MIRAGE syndrome but that the absence of AI should not rule out a diagnosis of the syndrome.

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Methylome analysis of thyroid ectopy shows no disease-specific DNA methylation signature.

Narumi S, Matsubara K, Ishii T, Hasegawa T

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 27 （ 4 ） 235 - 238 2018年

査読有り, ISSN 0918-5739

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Identification of compound heterozygous TSHR mutations (R109Q and R450H) in a patient with nonclassic TSH resistance and functional characterization of the mutant receptors.

Sugisawa C, Abe K, Sunaga Y, Taniyama M, Hasegawa T, Narumi S

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 27 （ 3 ） 123 - 130 2018年

査読有り, ISSN 0918-5739

Genetic defects of the TSH receptor (TSHR) signaling pathway cause a form of congenital hypothyroidism (CH) known as TSH resistance. Consistent with the physiological understanding that thyroidal iodine uptake is up-regulated by TSHR signaling, most patients with TSH resistance have low to normal thyroidal 123I uptake representing the classic TSH resistance. However, paradoxically high 123I uptake was reported in four molecularly-confirmed patients indicating nonclassic TSH resistance. Here, we report the fifth patient with the nonclassic phenotype. He was a 12-yr-old CH patient and treated with levothyroxine. At the age 11 yr, he showed slightly small thyroid gland and elevated thyroidal 123I uptake. Genetic analysis showed that he was compound heterozygous for two known missense mutations (Arg109Gln and Arg450His) in the TSHR gene. Further, the signal transduction of Arg109Gln-TSHR was defective in both Gs- and Gq-coupled pathways, while Arg450His-TSHR showed Gq-dominant defect. 123I uptake was evaluated earlier in 16 patients with TSH resistance, and a correlation between TSH levels and 123I uptake was shown in patients with specific genotypes (Arg450His or Leu653Val). Collectively, we have re-confirmed that the emergence of the nonclassic phenotype requires two factors: mutant TSHR with Gq-dominant coupling defect and relatively high levels of serum TSH.

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Functional Characterization of a Novel KLF11 Mutation Identified in a Family with Autoantibody-Negative Type 1 Diabetes

Ushijima Kikumi, Kawamura Tomoyuki, Ogata Tsutomu, Yokota Lchiro, Sugihar Shigetaka, Narumi Satoshi, Fukami Maki

HORMONE RESEARCH IN PAEDIATRICS 90 84 - 85 2018年

査読有り, ISSN 1663-2818

Fetal Goitrous Hypothyroidism and Polyhydramnios in a Patient with Compound Heterozygous DUOXA2 Mutations.

Tanase-Nakao K, Miyata I, Terauchi A, Saito M, Wada S, Hasegawa T, Narumi S

Hormone research in paediatrics 90 （ 2 ） 132 - 137 2018年

査読有り, ISSN 1663-2818

BACKGROUND: Fetal goiter is only rarely observed in pregnant women without autoimmune thyroid disorders, and there is no epidemiological data on its pathophysiology. Dual oxidase maturation factor 2 (DUOXA2), together with dual oxidase 2, serves pivotal roles in thyroid hormone biosynthesis. To date, all reported patients with DUOXA2 mutations were diagnosed postnatally through newborn screening for congenital hypothyroidism. CASE REPORT: The mother of a male fetus presented at 33 + 4 gestational weeks (GW) with a fetal goiter and polyhydramnios. Cordocentesis revealed fetal hypothyroidism (TSH 253.4 mU/L, FT4 0.29 ng/dL). Intra-amniotic levothyroxine injections were performed at GW 34 + 3 and 35 + 3. The patient was born after spontaneous vaginal delivery at 35 + 6 GW without obstetrical complications. He was treated with levothyroxine until the age of 6 years when reevaluation of his thyroid functions showed normal results (TSH 1.32 mU/L, FT4 1.81 ng/dL). Eleven causative genes of CH, including DUOXA2, were analyzed with use of a next-generation sequencing technique. RESULTS: A next-generation sequencing-based mutation screen led us to find that he was compound heterozygous for 2 previously reported nonsense DUOXA2 mutations (p.[Tyr138*];[Tyr246*]). CONCLUSION: The present case not only illustrates the phenotypic diversity of DUOXA2 mutation carriers but also implies that DUOXA2 is important in prenatal thyroid hormone production.

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Novel compound heterozygous mutations identified by whole exome sequencing in a Japanese patient with geroderma osteodysplastica.

Takeda R, Takagi M, Shinohara H, Futagawa H, Narumi S, Hasegawa T, Nishimura G, Yoshihashi H

European journal of medical genetics （ELSEVIER SCIENCE BV） 60 （ 12 ） 635 - 638 2017年12月

査読有り, ISSN 1769-7212

Geroderma osteodysplastica (GO) is a subtype of cutis laxa syndrome characterized by congenital wrinkly skin, a prematurely aged face, extremely short stature, and osteoporosis leading to recurrent fractures. GO exhibits an autosomal recessive inheritance pattern and is caused by loss-of-function mutations in GORAB, which encodes a protein important for Golgi-related transport. Using whole exome sequencing, we identified novel compound heterozygous nonsense mutations in the GORAB in a GO patient.
The patient was a 14-year-old Japanese boy. Wrinkled skin and joint laxity were present at birth. At 1 year of age, he was clinically diagnosed with cutis laxa syndrome based on recurrent long bone fractures and clinical features, including wrinkled skin, joint laxity, and a distinctive face. He did not show retarded gross motor and cognitive development. At 11 years of age, he was treated with oral bisphosphonate and vitamin D owing to recurrent multiple spontaneous fractures of the vertebral and extremity bones associated with a low bone mineral density (BMD). Bisphosphonate treatment improved his BMD and fracture rate. Whole exome sequencing revealed two novel compound heterozygous nonsense mutations in the GORAB gene (p. Arg60* and p. Gln124*), and the diagnosis of GO was established. GO is a rare connective tissue disorder. Approximately 60 cases have been described to date, and this is the first report of a patient from Japan. Few studies have reported the effects of bisphosphonate treatment in GO patients with recurrent spontaneous fractures. Based on this case study, we hypothesize that oral bisphosphonate and vitamin D are effective and safe treatment options for the management of recurrent fractures in GO patients. It is important to establish a precise diagnosis of GO to prevent recurrent fractures and optimize treatment plans. (C) 2017 Elsevier Masson SAS. All rights reserved.

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PTEN胚細胞変異を同定した4歳発症の機能性甲状腺結節の1例

長崎啓祐, 入月浩美, 鳴海覚志, 小飼貴彦, 菱沼昭

日本内分泌学会雑誌（(一社)日本内分泌学会） 93 （ 4 ） 1126 - 1126 2017年12月

ISSN 0029-0661

Mild thyroid peroxidase deficiency caused by TPO mutations with residual activity: Correlation between clinical phenotypes and enzymatic activity.

Narumi S, Fox LA, Fukudome K, Sakaguchi Z, Sugisawa C, Abe K, Kameyama K, Hasegawa T

Endocrine journal （JAPAN ENDOCRINE SOC） 64 （ 11 ） 1087 - 1097 2017年11月

査読有り, ISSN 0918-8959

Thyroid peroxidase (TPO) deficiency, caused by biallelic TPO mutations, is a well-established genetic form of congenital hypothyroidism (CH). More than 100 patients have been published, and the patients have been diagnosed mostly in the frame of newborn screening (NBS) programs. Correlation between clinical phenotypes and TPO activity remains unclear. Here, we report clinical and molecular findings of two unrelated TPO mutation-carrying mildly hypothyroid patients. The two patients were born at term after an uneventful pregnancy and delivery, and were NBS negative. They sought medical attention due to goiter at age 8 years. Evaluation of the thyroid showed mild elevation of serum TSH levels, normal or slightly low serum T-4 levels, high serum T-3 to T-4 molar ratio, high serum thyroglobulin levels, and high thyroidal I-123 uptake. We performed next-generation sequencing-based genetic screening, and found that one patient was compound heterozygous for two novel TPO mutations (p.Asp224del; c.820-2A>G), and the other was homozygous for a previously known mutation (p.Trp527Cys). In vitro functional analyses using HEK293 cells showed that the two amino acid-altering mutations (p.Asp224del and p.Trp527Cys) caused partial loss of the enzymatic activity. In conclusion, we report that TPO mutations with residual activity are associated with mild TPO deficiency, which is clinically characterized by marked goiter, mild TSH elevation, high serum T-3 to T-4 molar ratio, and high serum thyroglobulin levels. Our findings illuminate the hitherto under-recognized correlation between clinical phenotypes and residual enzymatic activity among patients with TPO deficiency.

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Next generation sequencing-based mutation screening of 86 patients with idiopathic short stature.

Hattori A, Katoh-Fukui Y, Nakamura A, Matsubara K, Kamimaki T, Tanaka H, Dateki S, Adachi M, Muroya K, Yoshida S, Ida S, Mitani M, Nagasaki K, Ogata T, Suzuki E, Hata K, Nakabayashi K, Matsubara Y, Narumi S, Tanaka T, Fukami M

Endocrine journal 64 （ 10 ） 947 - 954 2017年10月

査読有り, ISSN 0918-8959

Although mutations in ACAN, FGFR3, NPR2, and SHOX typically lead to skeletal dysplasia, and mutations in GHRHR, GH1, GHR, STAT5B, IGF1, IGFALS, and IGF1R usually underlie hormonal defects of the growth hormone (GH)-insulin-like growth factor 1 (IGF1) axis, such mutations have also been identified in patients with idiopathic short stature (ISS). Of these, SHOX abnormalities are known to account for a certain percentage of ISS cases, whereas the frequency of mutations in the other 10 genes in ISS cohorts remains unknown. Here, we performed next-generation sequencing-based mutation screening of the 10 genes in 86 unrelated Japanese ISS patients without SHOX abnormalities. We searched for rare protein-altering variants. The functional significance of the identified variants was assessed by in silico analyses. Consequently, we identified 18 heterozygous rare variants in 19 patients, including four probable damaging variants in ACAN, six pathogenicity-unknown variants in FGFR3, GHRHR, GHR, and IGFALS, and eight possible benign variants. Pathogenic variants in NPR2, GH1, and IGF1 were absent from our cohort. Unlike previously reported patients with ACAN mutations, our four patients with ACAN variants manifested non-specific short stature with age-appropriate or mildly delayed bone ages, and had parents of normal stature. These results indicate that ACAN mutations can underlie ISS without characteristic skeletal features, and that such mutations are possibly associated with de novo occurrence or low penetrance. In addition, our data imply that mutations in FGFR3, NPR2, and GH-IGF1 axis genes play only limited roles in the etiology of ISS.

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Paradoxical gain-of-function mutant of the G-protein-coupled receptor PROKR2 promotes early puberty

Maki Fukami, Erina Suzuki, Yoko Izumi, Tomohiro Torii, Satoshi Narumi, Maki Igarashi, Mami Miyado, Momori Katsumi, Yasuko Fujisawa, Kazuhiko Nakabayashi, Kenichiro Hata, Akihiro Umezawa, Yoichi Matsubara, Junji Yamauchi, Tsutomu Ogata

JOURNAL OF CELLULAR AND MOLECULAR MEDICINE （WILEY） 21 （ 10 ） 2623 - 2626 2017年10月

査読有り, ISSN 1582-4934

The human genome encodes -750 G-protein-coupled receptors (GPCRs), including prokineticin receptor 2 (PROKR2) involved in the regulation of sexual maturation. Previously reported pathogenic gain-of-function mutations of GPCR genes invariably encoded aberrant receptors with excessive signal transduction activity. Although in vitro assays demonstrated that an artificially created inactive mutant of PROKR2 exerted paradoxical gain-of-function effects when co-transfected with wild-type proteins, such a phenomenon has not been observed in vivo. Here, we report a heterozygous frameshift mutation of PROKR2 identified in a 3.5-year-old girl with central precocious puberty. The mutant mRNA escaped nonsense-mediated decay and generated a GPCR lacking two transmembrane domains and the carboxyl-terminal tail. The mutant protein had no in vitro signal transduction activity; however, cells co-expressing the mutant and wild-type PROKR2 exhibited markedly exaggerated ligand-induced Ca2+ responses. The results indicate that certain inactive PROKR2 mutants can cause early puberty by enhancing the functional property of coexisting wild-type proteins. Considering the structural similarity among GPCRs, this paradoxical gain-of-function mechanism may underlie various human disorders.

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Paradoxical gain-of-function mutant of the G-protein-coupled receptor PROKR2 promotes early puberty.

Fukami M, Suzuki E, Izumi Y, Torii T, Narumi S, Igarashi M, Miyado M, Katsumi M, Fujisawa Y, Nakabayashi K, Hata K, Umezawa A, Matsubara Y, Yamauchi J, Ogata T

Journal of cellular and molecular medicine 21 （ 10 ） 2623 - 2626 2017年10月

ISSN 1582-1838

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Paradoxical gain-of-function mutant of the G-protein-coupled receptor PROKR2 promotes early puberty.

Journal of cellular and molecular medicine （Wiley-Blackwell） 21 （ 10 ） 2623 - 2626 2017年10月

査読有り, ISSN 1582-1838

The human genome encodes ~750 G-protein-coupled receptors (GPCRs), including prokineticin receptor 2 (PROKR2) involved in the regulation of sexual maturation. Previously reported pathogenic gain-of-function mutations of GPCR genes invariably encoded aberrant receptors with excessive signal transduction activity. Although in vitro assays demonstrated that an artificially created inactive mutant of PROKR2 exerted paradoxical gain-of-function effects when co-transfected with wild-type proteins, such a phenomenon has not been observed in vivo. Here, we report a heterozygous frameshift mutation of PROKR2 identified in a 3.5-year-old girl with central precocious puberty. The mutant mRNA escaped nonsense-mediated decay and generated a GPCR lacking two transmembrane domains and the carboxyl-terminal tail. The mutant protein had no in vitro signal transduction activity; however, cells co-expressing the mutant and wild-type PROKR2 exhibited markedly exaggerated ligand-induced Ca2+ responses. The results indicate that certain inactive PROKR2 mutants can cause early puberty by enhancing the functional property of coexisting wild-type proteins. Considering the structural similarity among GPCRs, this paradoxical gain-of-function mechanism may underlie various human disorders.

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Homozygous DUOXA2 mutation (p.Tyr138(*)) in a girl with congenital hypothyroidism and her apparently unaffected brother: Case report and review of the literature.

Sugisawa C, Higuchi S, Takagi M, Hasegawa Y, Taniyama M, Abe K, Hasegawa T, Narumi S

Endocrine journal （JAPAN ENDOCRINE SOC） 64 （ 8 ） 807 - 812 2017年08月

査読有り, ISSN 0918-8959

Mutations in DUOXA2, encoding dual oxidase maturation factor 2, is a rare genetic cause of congenital hypothyroidism. Only four biallelic DUOXA2 mutation carriers have been described to date. This study was conducted to report the clinical and genetic findings of a DUOXA2 mutation-carrying family, and to review the previously reported cases. The proband was a 4-year-old girl, who was diagnosed as having congenital hypothyroidism in the frame of newborn screening. She had a high serum TSH level (138 mU/L) and a low free T4 level (0.4 ng/dL). Ultrasonography revealed goiter. She was immediately treated with levothyroxine. At age 3 years, reevaluation of her thyroid function showed a slightly elevated serum TSH level (11.0 mU/L) with normal free T4 level. Screening of the eleven congenital hypothyroidism-related genes demonstrated a previously reported nonsense DUOXA2 mutation (p.Tyr138*) in the homozygous state. Unexpectedly, we also found that the elder brother of the proband, who had no significant past medical history, had the identical homozygous mutation. Using expression experiments with HEK293 cells, we confirmed that p. Tyr138* was a loss-of-function mutation. In the literature, clinical courses of three patients were described, showing characteristic age-dependent improvement of the thyroid function. In conclusion, The proband showed comparable clinical phenotype to previously reported cases, while her brother was unaffected. The phenotypic spectrum of DUOXA2 mutations could be broader than currently accepted.

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SOX2 nonsense mutation in a patient clinically diagnosed with non-syndromic hypogonadotropic hypogonadism.

Shima H, Ishii A, Wada Y, Kizawa J, Yokoi T, Azuma N, Matsubara Y, Suzuki E, Nakamura A, Narumi S, Fukami M

Endocrine journal （JAPAN ENDOCRINE SOC） 64 （ 8 ） 813 - 817 2017年08月

査読有り, ISSN 0918-8959

Hypogonadotropic hypogonadism (HH) is a genetically heterogeneous condition that occurs either as an isolated disorder or as a component of congenital malformation syndromes. SOX2 is a causative gene of syndromic HH characterized by anophthalmia, microphthalmia, or coloboma and other neurological defects such as epilepsy. To date, the causal relationship between SOX2 abnormalities and non-syndromic HH remains speculative. Here, we identified a nonsense mutation of SOX2 in a male patient clinically diagnosed with non-syndromic HH. The patient had epilepsy but no additional clinical features. Ophthalmological examination revealed no abnormalities except for decreased thickness of the retinal nerve fiber layer. Audiometry showed mild sensorineural hearing impairment of both ears. Hormonal evaluation suggested isolated gonadotropin deficiency. Next-generation sequencing-based mutation screening of 13 major causative genes for HH identified a p.Lys35* mutation in SOX2 and excluded pathogenic mutations in other tested genes. The p.Lys35* mutation appeared to encode a non-functioning SOX2 protein that lacks 283 of 317 amino acids. The SOX2 mutation was absent in the maternal DNA sample, while a paternal sample was unavailable for sequence analysis. These results expand the clinical consequences of SOX2 haploinsufficiency to include non-syndromic HH. Systematic mutation screening using a next-generation sequencer and detailed evaluation of nonspecific ocular/neurological features may help identify SOX2 mutation-positive individuals among HH patients.

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Genetic defects in pediatric-onset adrenal insufficiency in Japan.

Amano N, Narumi S, Hayashi M, Takagi M, Imai K, Nakamura T, Hachiya R, Sasaki G, Homma K, Ishii T, Hasegawa T

European journal of endocrinology 177 （ 2 ） 187 - 194 2017年08月

査読有り, ISSN 0804-4643

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部分型TPO異常症新生児マススクリーニングが陰性であったTPO変異の2例

鳴海覚志, 阿部清美, Fox Larry A., 福留啓祐, 坂口善市, 深見真紀, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 93 （ 1 ） 261 - 261 2017年04月

ISSN 0029-0661

A novel de novo germline mutation Glu40Lys in AKT3 causes megalencephaly with growth hormone deficiency.

Takagi M, Dobashi K, Nagahara K, Kato M, Nishimura G, Fukuzawa R, Narumi S, Hasegawa T

American journal of medical genetics. Part A （WILEY） 173 （ 4 ） 1071 - 1076 2017年04月

査読有り, ISSN 1552-4825

Germline or somatic gain-of-function mutations in the v-akt murine thymoma viral oncogene homolog 3 (AKT3) have been reported to cause syndromic megalencephaly. We describe a novel germline mutation, p.Glu40Lys, in AKT3. Phenotypically, the patient presented with megalencephaly with hypotonia, apparent connective tissue laxity, and growth hormone (GH) deficiency. To our knowledge, this is the first instance of a patient with megalencephaly with GH deficiency, harboring a germline de novo mutation in AKT3. (C) 2017 Wiley Periodicals, Inc.

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Luscan-Lumish症候群による巨人症の機序の解析

隅田健太郎, 福岡秀規, 井口元三, 小武由紀子, 吉田健一, 松本隆作, 坂東弘教, 西澤衡, 高橋路子, 鳴海覚志, 小川渉, 長谷川奉延, 高橋裕

日本内分泌学会雑誌（(一社)日本内分泌学会） 93 （ 1 ） 263 - 263 2017年04月

ISSN 0029-0661

同胞間で異なる経過をたどる先天性甲状腺機能低下症病型診断結果と遺伝子診断結果

小泉美紀子, 川井正信, 大沼真輔, 中長摩利子, 庄司保子, 阿部清美, 佐藤武志, 鳴海覚志, 長谷川奉延, 惠谷ゆり, 位田忍

日本内分泌学会雑誌（(一社)日本内分泌学会） 93 （ 1 ） 343 - 343 2017年04月

ISSN 0029-0661

新規遺伝子変異を同定したアンドロゲン不応症の1例

岩橋めぐみ, 小澤綾子, 佐藤武志, 鳴海覚志, 長谷川奉延, 宮田市郎

日本内分泌学会雑誌（(一社)日本内分泌学会） 93 （ 1 ） 381 - 381 2017年04月

ISSN 0029-0661

サイログロブリン遺伝子異常による先天性甲状腺機能低下症4例の臨床的比較検討

川北葵, 山本幸代, 久保和泰, 江口真美, 齋藤玲子, 後藤元秀, 川越倫子, 河田泰定, 鳴海覚志, 長谷川奉延, 楠原浩一

日本内分泌学会雑誌（(一社)日本内分泌学会） 93 （ 1 ） 261 - 261 2017年04月

ISSN 0029-0661

A novel heterozygous intronic mutation in POU1F1 is associated with combined pituitary hormone deficiency.

Takagi M, Kamasaki H, Yagi H, Fukuzawa R, Narumi S, Hasegawa T

Endocrine journal （JAPAN ENDOCRINE SOC） 64 （ 2 ） 229 - 234 2017年02月

査読有り, ISSN 0918-8959

POU class 1 homeobox 1 (POUlFi) regulates pituitary cell- specific gene expression of somatotropes, lactotropes, and thyrotropes. In humans, two POUlF1 isoforms (long and short isoform), which are generated by the alternative use of the splice acceptor site for exon 2, have been identified. To date, more than 30 POU1F1 mutations in patients with combined pituitary hormone deficiency (CPHD) have been described. All POU1F1 variants reported to date affect both the short and long isoforms of the POUlF I protein; therefore, it is unclear at present whether a decrease in the function of only one of these two isoforms is sufficient for disease onset in humans. Here, we described a sibling case of CPHD carrying a heterozygous mutation in intron 1 of POU1F1. In vitro experiments showed that this mutation resulted in exon 2- skipping of only in the short isoform of POU1F1, while the long isoform remained intact. This result strongly suggests the possibility, for the first time, that isolated mutations in the short isoform of POU1Fi could be sufficient for induction of POU1Fl- related CPHD. This finding improves our understanding of the molecular mechanisms, and developmental course associated with mutations in POU1F1.

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TSH受容体細胞内ドメインの系統的変異導入研究どのようなナンセンス/フレームシフトバリアントが機能低下をきたすか?

杉澤千穂, 阿部清美, 谷山松雄, 長谷川奉延, 鳴海覚志

日本内分泌学会雑誌（(一社)日本内分泌学会） 92 （ 3 ） 607 - 607 2017年01月

ISSN 0029-0661

A de novo 50-bp GNAS Intragenic Duplication in a Patient with Pseudohypoparathyroidism Type 1a.

Suzuki E, Bo R, Sue K, Awano H, Ogata T, Narumi S, Kagami M, Sano S, Fukami M

Cytogenetic and genome research 153 （ 3 ） 125 - 130 2017年

査読有り, ISSN 1424-8581

Germline intragenic mutations in the GNAS locus result in pseudohypoparathyroidism type 1a (PHP1a) and related conditions. Nearly half of the previously reported GNAS intragenic mutations were structural variants, including 3 tandem duplications of 12-25 bp. However, the precise mutation spectrum and the genomic basis of GNAS structural variants remain to be clarified. Here, we report a de novo 50-bp tandem duplication in GNAS (c.723_772dup50, p.Glu259Leufs*29) identified in a patient with typical clinical features of PHP1a. The mutant transcript was predicted to undergo mRNA decay or encode a nonfunctional protein. The 2 breakpoints of the duplication shared a 1-bp microhomology but were not associated with long homology or nucleotide stretches. We also examined the breakpoint structures of 3 previously reported GNAS duplications and found that 1 had a structure similar to that of our case, while the remaining 2 had blunt-ended breakpoints without microhomologies. In silico analyses revealed that the GNAS-flanking region was not enriched with repeats, palindromes, noncanonical DNA motifs, or GC content. This study expands the mutation spectrum of GNAS and provides the first indication that GNAS intragenic structural variants are induced by multiple processes, including nonhomologous end-joining and/or microhomology-mediated break-induced replication, independently of known rearrangement-inducing DNA features.

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PROTEIN-TRUNCATING MUTATION IN "GENE A" IN A GIRL WITH CENTRAL PRECOCIOUS PUBERTY: IMPLICATIONS FOR A NOVEL GAIN-OF-FUNCTION MECHANISM OF G-PROTEIN COUPLED RECEPTORS

Fukami Maki, Suzuki Erina, Izumi Yoko, Torii Tomohiro, Igarashi Maki, Miyado Mami, Narumi Satoshi, Katsumi Momori, Yamauchi Junji, Fujisawa Yasuko, Ogata Tsutomu

HORMONE RESEARCH IN PAEDIATRICS 88 547 2017年

査読有り, ISSN 1663-2818

A MIRAGE SYNDROME PATIENT WITHOUT HEMATOLOGICAL PHENOTYPES: INACTIVATION OF A GERMLINE ACTIVATING SAMD9 MUTATION BY A SOMATICALLY ACQUIRED NONSENSE MUTATION IN HEMATOPOIETIC CELLS

Shima Hirohito, Nomura Yumiko, Sugimoto Kazuhiko, Satoh Akira, Ogata Tsutomu, Fukami Maki, Narumi Satoshi

HORMONE RESEARCH IN PAEDIATRICS 88 49 - 50 2017年

査読有り, ISSN 1663-2818

Syndromic disorder of sex development due to a novel hemizygous mutation in the carboxyl-terminal domain of ATRX.

Takagi M, Yagi H, Fukuzawa R, Narumi S, Hasegawa T

Human genome variation 4 17012 - 17012 2017年

ISSN 2054-345X

Alpha-thalassemia/mental retardation syndrome X-linked (ATRX; OMIM #301040), which is caused by mutations in the ATRX gene, is characterized by alpha-thalassemia, distinct dysmorphic facies, psychomotor development delay and genital abnormalities. Here, we describe a neonatal case of syndromic disorder of sex development, harboring a novel hemizygous mutation, p.Asp2352fs*1 in the carboxyl-terminal domain of ATRX. Our study provides additional evidence that deletion of the carboxyl terminus of ATRX is associated with severe genital anomalies.

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Xp22.31 Microdeletion due to Microhomology-Mediated Break-Induced Replication in a Boy with Contiguous Gene Deletion Syndrome.

Nagai K, Shima H, Kamimura M, Kanno J, Suzuki E, Ishiguro A, Narumi S, Kure S, Fujiwara I, Fukami M

Cytogenetic and genome research （S. Karger AG） 151 （ 1 ） 1 - 4 2017年

査読有り, ISSN 1424-8581

The Xp22.31 region is characterized by a low frequency of interspersed repeats and a low GC content. Submicroscopic deletions at Xp22.31 involving STS and ANOS1 (alias KAL1) underlie X-linked ichthyosis and Kallmann syndrome, respectively. Of the known microdeletions at Xp22.31, a common approximately 1.5-Mb deletion encompassing STS was ascribed to nonallelic homologous recombination, while 2 ANOS1-containing deletions were attributed to nonhomologous end-joining. However, the genomic bases of other microdeletions within the Xp22.31 region remain to be elucidated. Here, we identified a 2,735,696-bp deletion encompassing STS and ANOS1 in a boy with X-linked ichthyosis and Kallmann syndrome. The breakpoints of the deletion were located within Alu repeats and shared 2-bp microhomology. The fusion junction was not associated with nucleotide stretches, and the breakpoint-flanking regions harbored no palindromes or noncanonical DNA motifs. These results indicate that microhomology-mediated break-induced replication (MMBIR) can cause deletions at Xp22.31, resulting in contiguous gene deletion syndrome. It appears that interspersed repeats without other known rearrangement-inducing DNA features or high GC contents are sufficient to stimulate MMBIR at Xp22.31.

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Three-Quarters Adrenalectomy for Infantile-Onset Cushing Syndrome due to Bilateral Adrenal Hyperplasia in McCune-Albright Syndrome.

Itonaga T, Goto H, Toujigamori M, Ohno Y, Korematsu S, Izumi T, Narumi S, Hasegawa T, Ihara K

Hormone research in paediatrics （S. Karger AG） 88 （ 3-4 ） 285 - 290 2017年

査読有り, ISSN 1663-2818

Background: Bilateral adrenalectomy is performed in cases with infantile-onset Cushing syndrome due to bilateral adrenal hyperplasia in McCune-Albright syndrome (MAS) because severe Cushing syndrome with heart failure and liver dysfunction can have a lethal outcome. This procedure can completely ameliorate hypercortisolism, although lifetime steroid replacement therapy and steps to prevent adrenal crisis are necessary. Recently, the efficacy of unilateral adrenalectomy has been reported in adult cases of bilateral macronodular adrenal hyperplasia, but there is no consensus regarding the appropriate surgical treatment for bilateral adrenal hyperplasia in MAS. Objective: A 6-month-old girl presented with café-au-lait spots, short stature, central obesity, a moon face, and hypertension. Endocrinological tests and imaging studies led to the diagnosis of ACTH-independent Cushing syndrome due to bilateral adrenal hyperplasia induced by MAS. "Three-quarters adrenalectomy", namely right-sided total adrenalectomy and left-sided half adrenalectomy, was carried out. An activating mutation of the GNAS1 gene (p.Arg201Cys) was identified in the adrenal tissues. Since the operation, our patient has been in a state of clinical remission for more than 2 years. Conclusion: Our original surgical intervention, three-quarters adrenalectomy, may be a new treatment option for Cushing syndrome associated with MAS.

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A novel dominant negative mutation in the intracellular domain of GHR is associated with growth hormone insensitivity.

Takagi M, Shinohara H, Nagashima Y, Hasegawa Y, Narumi S, Hasegawa T

Clinical endocrinology 85 （ 4 ） 669 - 71 2016年10月

ISSN 0300-0664

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A patient with pseudohypoaldosteronism type II complicated by congenital hypopituitarism carrying a KLHL3 mutation.

Mitani M, Furuichi M, Narumi S, Hasegawa T, Chiga M, Uchida S, Sato S

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology （Jeff Corporation Co. Ltd） 25 （ 4 ） 127 - 134 2016年10月

査読有り, ISSN 0918-5739

Pseudohypoaldosteronism type II (PHA II) is a renal tubular disease that causes hyperkalemia, hypertension, and metabolic acidosis. Mutations in four genes (WNK4, WNK1, KLHL3, and CUL3) are known to cause PHA II. We report a patient with PHA II carrying a KLHL3 mutation, who also had congenital hypopituitarism. The patient, a 3-yr-old boy, experienced loss of consciousness at age 10 mo. He exhibited growth failure, hypertension, hyperkalemia, and metabolic acidosis. We diagnosed him as having PHA II because he had low plasma renin activity with normal plasma aldosterone level and a low transtubular potassium gradient. Further investigations revealed defective secretion of GH and gonadotropins and anterior pituitary gland hypoplasia. Genetic analyses revealed a previously known heterozygous KLHL3 mutation (p.Leu387Pro), but no mutation was detected in 27 genes associated with congenital hypopituitarism. He was treated with sodium restriction and recombinant human GH, which normalized growth velocity. This is the first report of a molecularly confirmed patient with PHA II complicated by congenital hypopituitarism. We speculate that both GH deficiency and metabolic acidosis contributed to growth failure. Endocrinological investigations will help to individualize the treatment of patients with PHA II presenting with growth failure.

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SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7

Satoshi Narumi, Naoko Amano, Tomohiro Ishii, Noriyuki Katsumata, Koji Muroya, Masanori Adachi, Katsuaki Toyoshima, Yukichi Tanaka, Ryuji Fukuzawa, Kenichi Miyako, Saori Kinjo, Shouichi Ohga, Kenji Ihara, Hirosuke Inoue, Tadamune Kinjo, Toshiro Hara, Miyuki Kohno, Shiro Yamada, Hironaka Urano, Yosuke Kitagawa, Koji Tsugawa, Asumi Higa, Masakazu Miyawaki, Takahiro Okutani, Zenro Kizaki, Hiroyuki Hamada, Minako Kihara, Kentaro Shiga, Tetsuya Yamaguchi, Manabu Kenmochi, Hiroyuki Kitajima, Maki Fukami, Atsushi Shimizu, Jun Kudoh, Shinsuke Shibata, Hideyuki Okano, Noriko Miyake, Naomichi Matsumoto, Tomonobu Hasegawa

Nature Genetics （Nature Publishing Group） 48 （ 7 ） 792 - 797 2016年07月

ISSN 1546-1718

Adrenal hypoplasia is a rare, life-threatening congenital disorder. Here we define a new form of syndromic adrenal hypoplasia, which we propose to term MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) syndrome. By exome sequencing and follow-up studies, we identified 11 patients with adrenal hypoplasia and common extra-adrenal features harboring mutations in SAMD9. Expression of the wild-type SAMD9 protein, a facilitator of endosome fusion, caused mild growth restriction in cultured cells, whereas expression of mutants caused profound growth inhibition. Patient-derived fibroblasts had restricted growth, decreased plasma membrane EGFR expression, increased size of early endosomes, and intracellular accumulation of giant vesicles carrying a late endosome marker. Of interest, two patients developed myelodysplasitc syndrome (MDS) that was accompanied by loss of the chromosome 7 carrying the SAMD9 mutation. Considering the potent growth-restricting activity of the SAMD9 mutants, the loss of chromosome 7 presumably occurred as an adaptation to the growth-restricting condition.

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SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7

NATURE GENETICS （NATURE PUBLISHING GROUP） 48 （ 7 ） 792 - + 2016年07月

査読有り, ISSN 1061-4036

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SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7.

Narumi S, Amano N, Ishii T, Katsumata N, Muroya K, Adachi M, Toyoshima K, Tanaka Y, Fukuzawa R, Miyako K, Kinjo S, Ohga S, Ihara K, Inoue H, Kinjo T, Hara T, Kohno M, Yamada S, Urano H, Kitagawa Y, Tsugawa K, Higa A, Miyawaki M, Okutani T, Kizaki Z, Hamada H, Kihara M, Shiga K, Yamaguchi T, Kenmochi M, Kitajima H, Fukami M, Shimizu A, Kudoh J, Shibata S, Okano H, Miyake N, Matsumoto N, Hasegawa T

Nature genetics 48 （ 7 ） 792 - 7 2016年07月

ISSN 1061-4036

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新生児マス・スクリーニングで発見され、DUOX2遺伝子変異が確認された一過性甲状腺機能低下症の1例

石田倫也, 松浦信夫, 白井宏幸, 石井正浩, 鳴海覚志, 阿部清美

神奈川医学会雑誌（(公社)神奈川県医師会） 43 （ 2 ） 315 - 315 2016年07月

ISSN 0285-0680

A novel mutation in HESX1 causes combined pituitary hormone deficiency without septo optic dysplasia phenotypes.

Takagi M, Takahashi M, Ohtsu Y, Sato T, Narumi S, Arakawa H, Hasegawa T

Endocrine journal （Japan Endocrine Society） 63 （ 4 ） 405 - 10 2016年04月

査読有り, ISSN 0918-8959

Heterozygous and/or homozygous HESX1 mutations have been reported to cause isolated growth hormone deficiency (IGHD) or combined pituitary hormone deficiency (CPHD), in association with septo optic dysplasia (SOD). We report a novel heterozygous HESX1 mutation in a CPHD patient without SOD phenotypes. The propositus was a oneyear- old Japanese girl. Shortly after birth, she was found to be hypoglycemic. She was diagnosed with central adrenal insufficiency based on low cortisol and ACTH at a time of severe hypoglycemia. Further endocrine studies indicated that the patient also had central hypothyroidism and growth hormone deficiency. Using a next-generation sequencing strategy, we identified a novel heterozygous HESX1 mutation, c.326G&gt
A (p.Arg109Gln). Western blotting and subcellular localization revealed no significant difference between wild type and mutant HESX1. Electrophoretic mobility shift assays showed that the mutant HESX1 abrogated DNA-binding ability. Mutant HESX1 was unable to repress PROP1-mediated activation. In conclusion, this study identified Arg109 as a critical residue in the HESX1 protein and extends our understanding of the phenotypic features, molecular mechanism, and developmental course associated with mutations in HESX1. When multiple genes need to be analyzed for mutations simultaneously, targeted sequence analysis of interesting genomic regions is an attractive approach.

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複合型下垂体前葉機能低下症と診断されていたIGSF1異常症の1例

朝倉由美, 阿部清美, 花川純子, 島田綾, 室谷浩二, 鳴海覚志, 長谷川奉延, 安達昌功

日本内分泌学会雑誌（(一社)日本内分泌学会） 92 （ 1 ） 221 - 221 2016年04月

ISSN 0029-0661

Stippled calcification in an infant with a recurrent SRCAP gene mutation.

Yagi H, Takagi M, Narumi S, Hasegawa T, Nishimura G, Hasegawa Y

American journal of medical genetics. Part A 170A （ 4 ） 1088 - 91 2016年04月

ISSN 1552-4825

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ヒストンメチル基転移酵素SETD2遺伝子変異を認めた巨人症の一例

隅田健太郎, 鳴海覚志, 福岡秀規, 井口元三, 小武由紀子, 吉田健一, 松本隆作, 坂東弘教, 西沢衡, 高橋路子, 小川渉, 長谷川奉延, 高橋裕

日本内分泌学会雑誌（(一社)日本内分泌学会） 92 （ 1 ） 301 - 301 2016年04月

ISSN 0029-0661

Transient congenital hypothyroidism caused by compound heterozygous mutations affecting the NADPH-oxidase domain of DUOX2

Atsuko Yoshizawa-Ogasawara, Kiyomi Abe, Sayaka Ogikubo, Satoshi Narumi, Tomonobu Hasegawa, Mari Satoh

JOURNAL OF PEDIATRIC ENDOCRINOLOGY & METABOLISM （WALTER DE GRUYTER GMBH） 29 （ 3 ） 363 - 371 2016年03月

査読有り, ISSN 0334-018X

Here, we describe three cases of loss-of-function mutations in the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase (NOX) domain of dual oxidase 2 (DUOX2) occurring along with concurrent missense mutations in thyroid peroxidase (TPO), leading to transient congenital hypothyroidism (CH). Three Japanese boys with nonconsanguineous parents were diagnosed with CH during their neonatal screenings. All patients presented with moderate-to-severe neonatal hypothyroidism and were diagnosed with transient CH after re-evaluation of thyroid function. Two siblings were compound heterozygous for p.[R1110Q]+[Y1180X] in DUOX2; one of them was also heterozygous for p.[R361L] in TPO. The third patient was compound heterozygous for p.[L1160del]+[R1334W] in DUOX2 and heterozygous for p.[P883S] in TPO. This is the first report of a de novo L1160del mutation affecting the DUOX2 gene and of the novel mutations Y1180X in DUOX2 and R361L in TPO. R1110Q and L1160del were found to reduce H2O2 production (5%-9%, p < 0.01), while Y1180X, which introduces a premature stop codon, did not confer detectable H2O2 production (-0.7%+/- 0.6%, p < 0.01). Moreover, R1334W, a missense mutation possibly affecting electron transfer, led to reduced H2O2 production (24%+/- 0.9%, p < 0.01) in vitro, and R1110Q and R1334W resulted in reduced protein expression. Y1180X was detected in a 120 kDa truncated form, whereas L1160del expression was maintained. Further, R361L, a novel missense mutation in TPO, caused partial reduction in peroxidase activity (20.6%+/- 0.8%, p = 0.01), whereas P883S, a missense variant, increased it (133.7%+/- 2.8%, p = 0.02). The protein expression levels in the case of R361L and P883S were maintained. In conclusion, we provide clinical and in vitro demonstrations of different functional defects and phenotypic heterogeneity in the same thyroid hormonogenesis pathway.

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Whole exome sequencing identified a novel COL2A1 mutation that causes mild Spondylo-epiphyseal dysplasia mimicking autosomal dominant brachyolmia.

Takagi M, Shimizu M, Suzuki E, Shinohara H, Narumi S, Hasegawa T, Nishimura G, Hasegawa Y

American journal of medical genetics. Part A （WILEY-BLACKWELL） 170 （ 3 ） 795 - 8 2016年03月

査読有り, ISSN 1552-4825

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A novel mutation of the THRB gene in a Japanese family with resistance to thyroid hormone.

Ito J, Narumi S, Nishizawa K, Kamimaki T, Hori N, Hasegawa T

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 25 （ 1 ） 19 - 22 2016年01月

査読有り, ISSN 0918-5739

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Complex X-Chromosomal Rearrangements in Two Women with Ovarian Dysfunction: Implications of Chromothripsis/Chromoanasynthesis-Dependent and -Independent Origins of Complex Genomic Alterations.

Suzuki E, Shima H, Toki M, Hanew K, Matsubara K, Kurahashi H, Narumi S, Ogata T, Kamimaki T, Fukami M

Cytogenetic and genome research （KARGER） 150 （ 2 ） 86 - 92 2016年

査読有り, ISSN 1424-8581

Our current understanding of the phenotypic consequences and the molecular basis of germline complex chromosomal rearrangements remains fragmentary. Here, we report the clinical and molecular characteristics of 2 women with germline complex X-chromosomal rearrangements. Patient 1 presented with nonsyndromic ovarian dysfunction and hyperthyroidism; patient 2 exhibited various Turner syndromeassociated symptoms including ovarian dysfunction, short stature, and autoimmune hypothyroidism. The genomic abnormalities of the patients were characterized by arraybased comparative genomic hybridization, high-resolution karyotyping, microsatellite genotyping, X-inactivation analysis, and bisulfite sequencing. Patient 1 carried a rearrangement of unknown parental origin with a 46, X, der(X)(pter -> p22.1:: p11.23 -> q24:: q21.3 -> q24:: p11.4 -> pter) karyotype, indicative of a catastrophic chromosomal reconstruction due to chromothripsis/chromoanasynthesis. Patient 2 had a paternally derived isochromosome with a 46, X, der(X)(pter. p22.31:: q22.1 -> q10:: q10 -> q22.1:: p22.31 -> pter) karyotype, which likely resulted from 2 independent, sequential events. Both patients showed completely skewed X inactivation. CpG sites at Xp22.3 were hypermethylated in patient 2. The results indicate that germline complex X-chromosomal rearrangements underlie nonsyndromic ovarian dysfunction and Turner syndrome. Disease-causative mechanisms of these rearrangements likely include aberrant DNA methylation, in addition to X-chromosomal mispairing and haplo-insufficiency of genes escaping X inactivation. Notably, our data imply that germline complex X-chromosomal rearrangements are created through both chromothripsis/chromoanasynthesis-dependent and - independent processes. (C) 2017 S. Karger AG, Basel

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NR0B1 Frameshift Mutation in a Boy with Idiopathic Central Precocious Puberty.

Shima H, Yatsuga S, Nakamura A, Sano S, Sasaki T, Katsumata N, Suzuki E, Hata K, Nakabayashi K, Momozawa Y, Kubo M, Okamura K, Kure S, Matsubara Y, Ogata T, Narumi S, Fukami M

Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation 10 （ 4 ） 205 - 209 2016年

査読有り, ISSN 1661-5425

NR0B1 is the causative gene for X-linked adrenal hypoplasia congenita, characterized by adrenal insufficiency, hypogonadotropic hypogonadism, and infertility. We identified an NR0B1 frameshift mutation in a boy with precocious puberty who had no signs of adrenal insufficiency. Blood examination revealed elevated testosterone levels and gonadotropin hyperresponses to gonadotropin releasing hormone (GnRH) stimulation, together with normal adrenal hormone levels. GnRH analog treatment partially ameliorated his clinical features. Molecular analysis identified a p.Glu3fsAla*16 in NR0B1. These results expand the clinical manifestations of NR0B1 mutations to include central precocious puberty without adrenal insufficiency. NR0B1 mutations likely underlie androgen overproduction via GnRH-dependent and -independent mechanisms.

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Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice.

Kotajima-Murakami H, Narumi S, Yuzaki M, Yanagihara D

PloS one 11 （ 11 ） e0166144 2016年

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偽性副甲状腺機能低下症の視床下部下垂体甲状腺軸の特徴

阿部清美, 鳴海覚志, 三井俊賢, 長崎啓祐, 中村明枝, 田島敏広, 湯野暁子, 臼井健, 室谷浩二, 朝倉由美, 安達昌功, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 91 （ 2Suppl. ） 74 - 74 2015年11月

ISSN 0029-0661

本邦初のDUOXA2変異による先天性甲状腺機能低下症の1例

杉澤千穂, 樋口真司, 高木優樹, 長谷川行洋, 長崎啓祐, 谷山松雄, 阿部清美, 鳴海覚志, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 91 （ 2Suppl. ） 75 - 75 2015年11月

ISSN 0029-0661

MIRAGE症候群副腎皮質機能低下症を含む多彩な症状で新生児期に発症する新規疾患概念確立と責任遺伝子同定

長谷川奉延, 木原美奈子, 北川陽介, 津川浩二, 河野美幸, 浦野博央, 豊島勝昭, 釼持学, 北島博之, 比嘉明日美, 宮脇正和, 奥谷貴弘, 井上普介, 金城唯宗, 井原健二, 天野直子, 鳴海覚志, 石井智弘

日本新生児成育医学会雑誌（(公社)日本新生児成育医学会） 27 （ 3 ） 531 - 531 2015年09月

ISSN 2189-7549

Pseudodominant inheritance in a family with nonautoimmune hypothyroidism due to biallelic DUOX2 mutations.

Abe K, Narumi S, Suwanai AS, Hamajima T, Hasegawa T

Clinical endocrinology （WILEY-BLACKWELL） 83 （ 3 ） 394 - 8 2015年09月

査読有り, ISSN 0300-0664

ObjectivesMutations in the dual oxidase 2 gene (DUOX2) is the most common genetic cause of congenital hypothyroidism (CH) in Japan. All previously described DUOX2 mutation-carrying families have followed autosomal recessive inheritance. We report a nonconsanguineous Japanese family harbouring biallelic DUOX2 mutations, which presented an apparently dominant inheritance of nonautoimmune hypothyroidism.
Design and methodsThe proband and her two sisters had been diagnosed as having CH on newborn screening and were treated with levothyroxine. Their mother had subclinical hypothyroidism. We sequenced DUOX2 in the proband and her family members. Pathogenicity of the identified novel mutation (p.Y1347C) was verified in vitro.
ResultsWe found that the proband and her sisters were compound heterozygous for a novel DUOX2 mutation p.Y1347C and a previously reported functional variant p.H678R. Unexpectedly, we found that the mother was homozygous for p.H678R. Expression experiments showed that the p.Y1347C mutant had reduced H2O2-producing activity, although there was no significant difference in the level of protein expression or localization, between wild type and p.Y1347C.
ConclusionsWe report a DUOX2 mutation-carrying pedigree presenting pseudodominant inheritance of nonautoimmune hypothyroidism. We speculate that the relatively high frequency of DUOX2 mutations could lead to pseudodominant inheritance in Japan.

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個体のヨウ素感受性を規定する遺伝因子の探索

鳴海覚志, 長谷川奉延, 阿部清美, 安達昌功, 荒田尚子

成長科学協会研究年報（(公財)成長科学協会）（ 38 ） 55 - 56 2015年08月

ISSN 0386-7617

Severe osteogenesis imperfecta caused by double glycine substitutions near the amino-terminal triple helical region in COL1A2.

Takagi M, Shinohara H, Narumi S, Nishimura G, Hasegawa Y, Hasegawa T

American journal of medical genetics. Part A （WILEY-BLACKWELL） 167 （ 7 ） 1627 - 31 2015年07月

査読有り, ISSN 1552-4825

Most cases of osteogenesis imperfecta (OI) are caused by heterozygous mutations in COL1A1 or COL1A2, the genes encoding the two type I procollagen alpha chains, proa1 (I) and proa2 (I). We report on a unique case of severe OI, a long term survivor of lethal type II OI, rather than progressively deforming type III, due to double substitutions of glycine residues in COL1A2 (p.Gly208Glu and p.Gly235Asp), located on the same allele. To the best of our knowledge, this is the first example of a patient with double COL1A2 glycine substitution mutations on the same allele. We show for the first time that double COL1A2 glycine substitution mutations located near the amino-terminal triple helical region, which individually are likely to result in mild OI, cause severe OI in combination. (C) 2015 Wiley Periodicals, Inc.

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先天性甲状腺機能低下症マススクリーニングの現状の問題点と今後の展望病型診断と遺伝子検査について

鳴海覚志, 阿部清美, 室谷浩二, 朝倉由美, 安達昌功, 長谷川奉延

日本マス・スクリーニング学会誌（(一社)日本マススクリーニング学会） 25 （ 2 ） 164 - 164 2015年07月

ISSN 0917-3803

新生児マス・スクリーニング検査でみつかったTPO異常症の2例

石津桂, 森川俊太郎, 鳴海覚志, 長谷川奉延, 田島敏広

日本マス・スクリーニング学会誌（(一社)日本マススクリーニング学会） 25 （ 2 ） 227 - 227 2015年07月

ISSN 0917-3803

A case with neonatal hyperinsulinemic hypoglycemia: It is a characteristic complication of sotos syndrome

Yoshie Nakamura, Masaki Takagi, Hiroshi Yoshihashi, Masaru Miura, Satoshi Narumi, Tomonobu Hasegawa, Yoshishige Miyake, Yukihiro Hasegawa

American Journal of Medical Genetics, Part A （Wiley-Liss Inc.） 167 （ 5 ） 1171 - 1174 2015年05月

査読有り, ISSN 1552-4833

Sotos syndrome (SoS, OMIM #117550) is an overgrowth syndrome. Deletions or intragenic mutations of the NSD1 , which is located at chromosome 5q35, are responsible for more than 75% of SoS. Conventionally, neonatal hypoglycemia was reported briefly as one of the infrequent symptoms of SoS. However, Matsuo et al. published a report describing five patients with SoS who presented with transient hyperinsulinemic hypoglycemia (HIH) in the neonatal period. We report on an additional patient of SoS, who presented transient HIH in the neonatal period. All of this patient and previous patients have microdeletions at the 5q35 chromosome. Therefore, we examined the following three in considering the possibility that other factor than NSD1 caused HIH. 1) This patient had no mutation of four currently known HIH related genes, ABCC8, KCNJ11, GLUD1, and GCK. 2) He had no further deletion than commonly observed region encompassing NSD1 by comparative genomic hybridization to DNA microarrays. 3) He had no mutation in the 5q35 region in the non-deleted chromosome using exsome sequence analysis. In conclusion, our patient supported that HIH could be one of the characteristic symptoms of SoS in the neonatal period, and could be useful for early diagnosis.

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T4補充療法中の先天性甲状腺機能低下症患者におけるFT3、FT4、FT3/FT4比病型別検討

阿部清美, 鳴海覚志, 石井智弘, 井ノ口美香子, 天野直子, 柴田浩憲, 安達昌功, 室谷浩二, 朝倉由美, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 91 （ 1 ） 307 - 307 2015年04月

ISSN 0029-0661

生化学的評価で原因特定が困難な原発性副腎機能低下症における包括的遺伝子解析

天野直子, 鳴海覚志, 林美恵, 蜂屋瑠見, 佐々木悟郎, 本間桂子, 石井智弘, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 91 （ 1 ） 290 - 290 2015年04月

ISSN 0029-0661

一過性甲状腺機能低下症の同朋発生により判明したDUOX2異常症の1家系

西門優一, 井澤雅子, 濱島崇, 阿部清美, 鳴海覚志, 長谷川奉延

日本小児科学会雑誌（(公社)日本小児科学会） 119 （ 3 ） 652 - 652 2015年03月

ISSN 0001-6543

Congenital hypothyroidism due to PAX8 mutations

Satoshi Narumi, Tomonobu Hasegawa

Thyroid Diseases in Childhood: Recent Advances from Basic Science to Clinical Practice （Springer International Publishing） 65 - 74 2015年01月

査読有り

PAX8 is one of the best characterized thyroid-specific transcription factors that play a role in development and physiology of the thyroid gland. PAX8 causes, when mutated, congenital hypothyroidism in both mice and humans, although mode of inheritance is different among the two species (mice, recessive
humans, dominant). Genetically engineered PAX8-deficient mice show abnormalities in early proliferation of thyroid precursor cells, resulting in markedly severe thyroid hypoplasia. In humans, clinical phenotypes of mutation carriers are highly variable, ranging from severe congenital hypothyroidism with thyroid hypoplasia to normal thyroid function with a morphologically normal gland. Several genetic epidemiology studies have revealed that PAX8 mutation is a relatively rare cause of human congenital hypothyroidism, observed in about 2 % of cases. Nonetheless, PAX8 mutation is the leading genetic cause of congenital hypothyroidism showing dominant inheritance. Thus, clinicians should consider PAX8 mutations when he/she encounters a patient with positive family history of congenital hypothyroidism. This review focuses on the molecular genetics of human congenital hypothyroidism due to PAX8 mutations.

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Combined pituitary hormone deficiency with unique pituitary dysplasia and morning glory syndrome related to a heterozygous PROKR2 mutation.

Asakura Y, Muroya K, Hanakawa J, Sato T, Aida N, Narumi S, Hasegawa T, Adachi M

査読有り, ISSN 0918-5739

Recent reports have indicated the role of the prokineticin receptor 2 gene (PROKR2) in the etiology of congenital hypopituitarism, including septo-optic dysplasia and Kallmann syndrome. In the present study, using next-generation targeted sequencing, we identified a novel heterozygous PROKR2 variant (c.742C&gt
T
p.R248W) in a female patient who had combined pituitary hormone deficiency (CPHD), morning glory syndrome and a severely malformed pituitary gland. No other mutation was present in 27 genes related to hypogonadotropic hypogonadism, pituitary hormone deficiency and optic nerve malformation. The substituted amino acid was located on the third intracellular loop of the PROKR2 protein, which is a G protein-coupled receptor. Computational analyses with two programs (SIFT and PolyPhen-2) showed that the substitution was deleterious to PROKR2 function. The p.R248W mutation was transmitted from the patient’s mother, who had a slightly delayed menarche. Collectively, we provide further genetic evidence linking heterozygous PROKR2 mutations and the development of CPHD.

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Combined Growth Hormone and Thyroid-Stimulating Hormone Deficiency in a Japanese Patient with a Novel Frameshift Mutation in IGSF1

Yumi Asakura, Kiyomi Abe, Koji Muroya, Junko Hanakawa, Yuji Oto, Satoshi Narumi, Tomonobu Hasegawa, Masanori Adachi

HORMONE RESEARCH IN PAEDIATRICS （KARGER） 84 （ 5 ） 349 - 354 2015年

査読有り, ISSN 1663-2818

Background: Recent reports have indicated that loss-of-function mutations in the immunoglobulin superfamily member 1 gene (IGSF1, OMIM 300888) cause congenital central hypothyroidism with macroorchidism. Methods: We conducted a next-generation sequencing-based comprehensive mutation screening for pituitary hormone deficiencies to elucidate molecular mechanisms other than anatomical abnormalities of the pituitary that might be responsible for multiple anterior hormone deficiency in a male patient who originally visited our institute complaining of short stature. He was born large for gestational age (4,370 g, + 3.0 SD) after an obstructed labour. Endocrinological evaluation revealed growth hormone and thyroid-stimulating hormone deficiency. Magnetic resonance imaging showed a discontinuity of the pituitary stalk with an ectopic posterior lobe and a hypoplastic anterior lobe, likely explaining multiple anterior pituitary hormone deficiency. Result: We identified a novel hemizygous IGSF1 mutation (c.1137_1138delCA, p.Asn380Glnfs*6) in the patient. In reviewing the literature, we noticed that all reported Japanese male IGSF1 mutation carriers were born larger than mean standards for gestational age (mean birth weight SD score of +2.0, 95% confidence interval 1.0-3.0). Conclusion: This case suggests that more attention should be paid to intrauterine growth and birth history when patients are suspected of having an IGSF1 mutation. (C) 2015 S. Karger AG, Basel

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Human Chorionic Gonadotropin Stimulation Test in Prepubertal Children with Micropenis Can Accurately Predict Leydig Cell Function in Pubertal or Postpubertal Adolescents.

Ishii T, Matsuo N, Sato S, Ogata T, Tamai S, Anzo M, Kamimaki T, Sasaki G, Inokuchi M, Hori N, Amano N, Narumi S, Shibata H, Hasegawa T

Hormone research in paediatrics （KARGER） 84 （ 5 ） 305 - 10 2015年

査読有り, ISSN 1663-2818

Background/Aim: To evaluate the accuracy of the human chorionic gonadotropin (hCG) stimulation test in children with micropenis in predicting later Leydig cell function. Methods: We conducted a retrospective investigation of testosterone response to a 3-day hCG test (3,000 IU/m(2)/day) in prepuberty to indicate the need for hormone replacement therapy (HRT) in adolescence. Results: Fifty Japanese boys (range, 0.8-15.4 years of age; median, 8.9) with micropenis were enrolled. Thirty-four spontaneously developed puberty and preserved the ability of testosterone production (group 1), while 16 did not develop any pubertal signs without HRT (group 2). Serum testosterone levels after the hCG test (post-hCG T) in group 2 (range, <0.05-1.1 ng/ml; median, 0.24) were significantly lower than in group 1 (range, 0.5-8.7 ng/ml; median, 2.4; p < 0.0001). Based on true positives who required continuous HRT, the area under the receiver-operating characteristics curve for post-hCG T was 0.983 [95% confidence interval (CI), 0.90-1.00]. The post-hCG T cut-off level corresponding to the Youden index was 1.1 ng/ml (95% CI, 1.0-1.1), with a sensitivity of 100.0% (95% CI, 79.4-100.0) and a specificity of 94.1% (95% CI, 80.3-99.3). Conclusions: The hCG test in prepubertal children with micropenis can be useful for predicting Leydig cell function in pubertal or postpubertal adolescents. The post-hCG T cutoff level of 1.1 ng/ml is recommended to screen for those who will likely require HRT for pubertal development. (C) 2015 S. Karger AG, Basel

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TSH resistance revisited.

Narumi S, Hasegawa T

Endocrine journal （JAPAN ENDOCRINE SOC） 62 （ 5 ） 393 - 8 2015年

査読有り, ISSN 0918-8959

Genetic defects of hormone receptors are the most common form of end-organ hormone resistance. One example of such defects is TSH resistance, which is caused by biallelic inactivating mutations in the TSH receptor gene (TSHR). TSH, a master regulator of thyroid functions, affects virtually all cellular processes involving thyroid hormone production, including thyroidal iodine uptake, thyroglobulin iodination, reuptake of iodinated thyroglobulin and thyroid cell growth. Resistance to TSH results in defective thyroid hormone production from the neonatal period, namely congenital hypothyroidism. Classically, clinical phenotypes of TSH resistance due to inactivating TSHR mutations were thought to vary depending on the residual mutant receptor activity. Nonfunctional mutations in the two alleles produce severe thyroid hypoplasia with overt hypothyroidism (uncompensated TSH resistance), while hypomorphic mutations in at least one allele produce normal-sized thyroid gland with preserved hormone-producing capacity (compensated TSH resistance). More recently, a new subgroup of TSH resistance (nonclassic TSH resistance) that is characterized by paradoxically high thyroidal iodine uptake has been reported. In this article, the pathophysiology and clinical features of TSH resistance due to inactivating TSHR mutations are reviewed, with particular attention to the nonclassic form.

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Comprehensive next-generation sequencing analyses of hypoparathyroidism: identification of novel GCM2 mutations.

Mitsui T, Narumi S, Inokuchi M, Nagasaki K, Nakazawa M, Sasaki G, Hasegawa T

The Journal of clinical endocrinology and metabolism （ENDOCRINE SOC） 99 （ 11 ） E2421 - 8 2014年11月

査読有り, ISSN 0021-972X

Context: In most patients with hypoparathyroidism (HP), the etiology is not defined clinically. Eight genes (AIRE, CASR, CLDN16, GATA3, GCM2, PTH, TBCE, and TRPM6) are known to be responsible genes associated with HP; however, no previous study has screened the eight responsible genes comprehensively in HP patients.
Objectives: This study was conducted to determine the genetic defect in HP patients. We also described clinical and molecular findings of two HP patients with novel GCM2 mutations.
Subjects and Methods: We enrolled 20 nonconsanguineous Japanese patients with child-onset permanent HP without 22q11 deletion. Mutations and genomic rearrangements involving the eight genes were screened by targeted next-generation sequencing (NGS). We also screened genetic rearrangements by array comparative genomic hybridization (aCGH) in the mutation-negative patients. A putative deletion, which was suspected by NGS, was additionally analyzed by droplet digital PCR (ddPCR) and junction PCR. Identified novel nucleotide-level GCM2 mutants were characterized in vitro.
Results: We identified seven patients with a single gene disorder, including a CASR mutation, GATA3 mutations, and novel GCM2 mutations (R367Tfs*15, T370M, and the deletion encompassing exon 1). This submicroscopic deletion, which had been suspected by NGS, could not be detected by aCGH and was confirmed by ddPCR and junction PCR. Functional studies of R367Tfs*- and T370M-GCM2 demonstrated a reduction of target gene transactivation in both.
Conclusions: Using comprehensive NGS analyses, we identified the genetic defect in 35% of HP patients in our cohort and discovered novel GCM2 mutations including submicroscopic deletion that was undetectable by aCGH.

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遷延する妊娠甲状腺中毒症に検出されたTSH受容体遺伝子変異

谷山松雄, 杉澤千穂, 百渓尚子, 井上ゆか子, 阿部清美, 鳴海覚志

日本内分泌学会雑誌（(一社)日本内分泌学会） 90 （ 2 ） 506 - 506 2014年09月

ISSN 0029-0661

変異TSH受容体V711FfsX18の機能解析 1塩基欠失によるたんぱく質不安定化シグナルの獲得

鳴海覚志, 杉澤千穂, 阿部清美, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 90 （ 2 ） 528 - 528 2014年09月

ISSN 0029-0661

奇異性123I摂取率高値を認めた両アリル性TSHR変異の一男児例世界で5例目の非古典型TSH不応症

杉澤千穂, 鳴海覚志, 阿部清美, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 90 （ 2 ） 507 - 507 2014年09月

ISSN 0029-0661

TPO遺伝子異常による甲状腺腫性クレチン症の8歳女児例

福留啓祐, 坂口善市, 冨田理絵, 藤岡啓介, 阿部容子, 木下ゆき子, 市原朋子, 藤井笑子, 幸山洋子, 関口隆憲, 大原克明, 鳴海覚志, 阿部清美, 長谷川奉延

日本小児科学会雑誌（(公社)日本小児科学会） 118 （ 7 ） 1126 - 1126 2014年07月

ISSN 0001-6543

サイログロブリン著明高値、甲状腺腫大を契機に診断したDUOX2異常症の2例

後藤元秀, 荒木俊介, 久保和泰, 川越倫子, 山本幸代, 楠原浩一, 齋藤玲子, 河田泰定, 阿部清美, 鳴海覚志, 長谷川奉延

日本小児科学会雑誌（(公社)日本小児科学会） 118 （ 6 ） 1016 - 1016 2014年06月

ISSN 0001-6543

新生児マススクリーニング(MS)で発見されなかった先天性甲状腺機能低下症の3例

上村美季, 鳴海覚志, 梅木郁美, 箱田明子, 菅野潤子, 長谷川奉延, 藤原幾磨

日本小児科学会雑誌（(公社)日本小児科学会） 118 （ 5 ） 875 - 875 2014年05月

ISSN 0001-6543

4世代・世界最大のPAX8変異陽性家系からみえた甲状腺表現型の多様性高齢PAX8変異保有者の臨床像

西岡淳子, 阿部清美, 鳴海覚志, 長谷川奉延, 八ツ賀秀一, 佐々木孝子, 片山幸樹, 久野建夫, 古賀靖敏

日本内分泌学会雑誌（(一社)日本内分泌学会） 90 （ 1 ） 296 - 296 2014年04月

ISSN 0029-0661

IGSF1変異による中枢性先天性甲状腺機能低下症の3男児例

阿部清美, 鳴海覚志, 杉澤千穂, 室谷浩二, 朝倉由美, 安達昌功, 沼倉周彦, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 90 （ 1 ） 271 - 271 2014年04月

ISSN 0029-0661

Identification and functional characterization of two novel NPR2 mutations in Japanese patients with short stature.

Amano N, Mukai T, Ito Y, Narumi S, Tanaka T, Yokoya S, Ogata T, Hasegawa T

The Journal of clinical endocrinology and metabolism （ENDOCRINE SOC） 99 （ 4 ） E713 - 8 2014年04月

査読有り, ISSN 0021-972X

Context: C-type natriuretic peptide-natriuretic peptide receptor B (NPR-B) signaling is critical for endochondral ossification, which is responsible for longitudinal growth in limbs and vertebrae. Biallelic NPR2 mutations cause acromesomelic dysplasia, type Maroteaux, which is bone dysplasia characterized by severe short stature and short limbs. A monoallelic NPR2 mutation has been suggested to mildly impair long bone growth.
Objective: The goal of this study was to identify and characterize NPR2 mutations among Japanese patients with short stature.
Subjects and Methods: We enrolled 101 unrelated Japanese patients with short stature. NPR2 and NPPC were sequenced, and the identified variants were characterized in vitro.
Results: In two subjects, we identified two novel heterozygous NPR2 mutations (R110C and Q417E) causing a loss of C-type natriuretic peptide-dependent cGMP generation capacities and having dominant-negative effects. R110C was defective in trafficking from the endoplasmic reticulum to the Golgi apparatus. In contrast, Q417E showed clear cell surface expression.
Conclusions: We identified heterozygous NPR2 mutations in 2% of Japanese patients with short stature. Our in vitro findings indicate that NPR2 mutations have a dominant negative effect, and their dominant-negative mechanisms vary corresponding to the molecular pathogenesis of the mutations.

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A novel KAL1 mutation is associated with combined pituitary hormone deficiency.

Takagi M, Narumi S, Hamada R, Hasegawa Y, Hasegawa T

Human genome variation （Nature Publishing Group） 1 14011 2014年

ISSN 2054-345X

Using a next-generation sequencing strategy, we identified a novel KAL1 missense mutation (p.His568Gln) in a patient with combined pituitary hormone deficiency, right microphthalmia, right renal aplasia and severe developmental delay. Our findings will provide additional evidence that KAL1 mutations are associated with hypopituitarism, in addition to luteinizing hormone, and follicle-stimulating hormone deficiencies, and improve our understanding of the phenotypic features and developmental course associated with KAL1 mutations.

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A novel mutation in SOX2 causes hypogonadotropic hypogonadism with mild ocular malformation.

Takagi M, Narumi S, Asakura Y, Muroya K, Hasegawa Y, Adachi M, Hasegawa T

Hormone research in paediatrics （KARGER） 81 （ 2 ） 133 - 8 2014年

査読有り, ISSN 1663-2818

Background: Heterozygous SOX2 mutations have been reported to cause isolated hypogonadotropic hypogonadism (HH) in addition to ocular and brain abnormalities. Objective: We report a novel missense SOX2 (Y110C) mutation in an HH patient with mild ocular malformation. Patients: The 20-year-old male was referred because of typical signs of complete hypogonadism, with small intrascrotal testes (2 ml), no pubic hair (P1), and a micropenis. Hormone assays revealed very low plasma testosterone levels and very low levels of plasma gonadotropin. He was found to have retinal detachment in his right eye and surgery was performed at the age of 14 years. Results: Using a next-generation sequencing strategy, we identified a novel heterozygous SOX2 mutation, c. 329A>G (p.Y110C). Y110C SOX2 had reduced transactivation and no dominant negative effect. Subcellular localization revealed no significant difference between wild-type and mutant SOX2. EMSA experiments showed that the Y110C SOX2 abrogated DNA-binding ability. Conclusion: The Y110C mutation affects a critical residue in the SOX2 protein. This study extends our understanding of the phenotypic features, molecular mechanism, and developmental course associated with mutations in SOX2. When multiple genes need to be analyzed for mutations simultaneously, targeted sequence analysis of interesting genomic regions is an attractive approach. (C) 2014 S. Karger AG, Basel

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新生児マス・スクリーニング検査でみつかったTPO異常症の2例

石津桂, 森川俊太郎, 鳴海覚志, 長谷川奉延, 田島敏広

日本内分泌学会雑誌（(一社)日本内分泌学会） 89 （ 3 ） 977 - 977 2013年12月

ISSN 0029-0661

IGSF1遺伝子変異による中枢性甲状腺機能低下症の2家系家族検索とPRL分泌能

松浦信夫, 橘田一輝, 阿部清美, 小池明美, 椿淳子, 鳴海覚志, 長谷川奉延, 横田行史, 大津成之, 田久保憲行, 柴山啓子, 大山宜秀

日本内分泌学会雑誌（(一社)日本内分泌学会） 89 （ 2 ） 463 - 463 2013年09月

ISSN 0029-0661

PAX8変異陽性甲状腺機能低下症の世界最大家系甲状腺表現型の多様性

阿部清美, 鳴海覚志, 八ツ賀秀一, 西岡淳子, 久野建夫, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 89 （ 2 ） 474 - 474 2013年09月

ISSN 0029-0661

A report of two novel NR5A1 mutation families: possible clinical phenotype of psychiatric symptoms of anxiety and/or depression.

Suwanai AS, Ishii T, Haruna H, Yamataka A, Narumi S, Fukuzawa R, Ogata T, Hasegawa T

Clinical endocrinology （WILEY-BLACKWELL） 78 （ 6 ） 957 - 65 2013年06月

査読有り, ISSN 0300-0664

Objective NR5A1 or steroidogenic factor 1 is a nuclear receptor that plays important roles in the hypothalamuspituitarysteroidogenic axis. The clinical phenotype of most 46,XY mutation carriers includes disorders of sex development (DSD) without adrenal insufficiency, whereas 46,XX mutation carriers have phenotypes ranging from no symptoms to ovarian insufficiency. Although genetically engineered ventromedial hypothalamus-specific Nr5a1 knockout mice show anxiety behaviour, no psychiatric symptoms have been reported in human NR5A1 mutation carriers. We report clinical and molecular findings for individuals (from two families) with NR5A1 mutations, showing psychiatric symptoms. Design and methods We screened for NR5A1 mutations in a cohort of 34 patients with 46,XY DSD using PCR-based sequencing. Psychiatric symptoms were assessed using mental health assessment tools and structured clinical interviews. Functional properties of detected mutant NR5A1s were studied in silico and in vitro, including three-dimensional (3D) mutation models, subcellular NR5A1 protein localization and transactivation assays. Results We found 2 (46,XY) patients with NR5A1 heterozygous novel mutations (p.D257fs and p.V424del), which were transmitted from their respective mothers. The patients' clinical findings indicated DSD without adrenal insufficiency. Both mothers showed psychiatric symptoms, including excessive anxiety and/or depression. The mother and grandmother of one patient had premature ovarian insufficiency. Functional studies showed altered 3D models of p.V424del and normal subcellular NR5A1 localization and impaired transcriptional activation without dominant-negative effects in both mutations. Conclusions We found 2 (46,XX) NR5A1 mutation carriers with excessive anxiety and/or depression. These results suggest that excessive anxiety and/or depression are possible clinical phenotypes of 46,XX NR5A1 mutations.

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Congenital hypothyroidism caused by a novel mutation of the dual oxidase 2 (DUOX2) gene

Atsuko Yoshizawa-Ogasawara, Sayaka Ogikubo, Mari Satoh, Satoshi Narumi, Tomonobu Hasegawa

JOURNAL OF PEDIATRIC ENDOCRINOLOGY & METABOLISM （WALTER DE GRUYTER GMBH） 26 （ 1-2 ） 45 - 52 2013年02月

査読有り, ISSN 0334-018X

The dual oxidase 2 (DUOX2) mutation results in an impairment of the hydrogen peroxidase-generating system and is identified as a dyshormonogenic cause of congenital hypothyroidism (CH). Here, we describe two unrelated Japanese girls with CH due to a novel DUOX2 mutation. They had high serum thyrotropin levels and low free thyroxine/thyroxine concentrations during the neonatal period. A novel missense mutation with a transversion of G to A at position 1462 in exon 12 of the DUOX2 gene that caused a replacement of glycine (G) with arginine (R) at codon 488 of the protein (c.1462G>A, p.[G488R]) was identified. One patient was a compound heterozygote forp. [L479SfsX3] + [G488R]. The other was homozygous for p.[G488R]. This p. G488R substitution occurred in a highly conserved glycine residue of the mammalian DUOX2 protein. The two patients had different haplotypes, suggesting that the p. G488R alleles were the result of independent, recurrent mutations. Later in life, both patients were still euthyroid even after discontinuing thyroid hormone therapy. We conclude that this p. G488R missense mutation in the DUOX2 gene of the patients is associated with thyroid dysfunction that presents during the neonatal period.

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Delayed onset congenital hypothyroidism in a patient with DUOX2 mutations and maternal iodine excess

Toshihiko Kasahara, Satoshi Narumi, Keisuke Okasora, Ryuzo Takaya, Hiroshi Tamai, Tomonobu Hasegawa

AMERICAN JOURNAL OF MEDICAL GENETICS PART A （WILEY） 161A （ 1 ） 214 - 217 2013年01月

査読有り, ISSN 1552-4825

Congenital hypothyroidism (CH), one of the most common congenital endocrine disorders, causes irreversible intellectual disability in untreated patients. Today, the vast majority of patients receive early diagnosis and treatment in the context of newborn screening for CH, and achieve satisfactory cognitive development. However, a subset of patients with delayed onset are undetectable by newborn screening, and miss benefit from early intervention. Here, we report on a delayed-onset CH patient that had two contributing factors in the pathogenesis of CH simultaneously, i.e., a genetic defect and iodine excess. The patient was exposed to excessive iodine in utero because her mother consumed massive amounts of seaweed during pregnancy. Surprisingly, the patient had a negative result in newborn screening, but developed overt CH at age 3 months. She received thyroxine supplementation until when normalization of the thyroid function was confirmed at age 3 years (i.e., transient CH). Mutation screening for DUOX2, a causative gene for transient CH, showed biallelic mutations (p.[E327X]?+?[H678R]). This report provides a new example of environmental modification of phenotypes of CH due to a genetic defect, which can potentially distort screening results. (c) 2012 Wiley Periodicals, Inc.

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A study of the etiology of transient congenital hypothyroidism in Niigata Prefecture, Japan.

Nagasaki K, Narumi S, Abe K, Asami T, Sato H, Ogawa Y, Kikuchi T, Hasegawa T, Saitoh A

International journal of pediatric endocrinology 2013年

査読有り

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Quantitative and sensitive detection of GNAS mutations causing mccune-albright syndrome with next generation sequencing.

Narumi S, Matsuo K, Ishii T, Tanahashi Y, Hasegawa T

PloS one （PUBLIC LIBRARY SCIENCE） 8 （ 3 ） e60525 2013年

査読有り, ISSN 1932-6203

Somatic activating GNAS mutations cause McCune-Albright syndrome (MAS). Owing to low mutation abundance, mutant-specific enrichment procedures, such as the peptide nucleic acid (PNA) method, are required to detect mutations in peripheral blood. Next generation sequencing (NGS) can analyze millions of PCR amplicons independently, thus it is expected to detect low-abundance GNAS mutations quantitatively. In the present study, we aimed to develop an NGS-based method to detect low-abundance somatic GNAS mutations. PCR amplicons encompassing exons 8 and 9 of GNAS, in which most activating mutations occur, were sequenced on the MiSeq instrument. As expected, our NGS-based method could sequence the GNAS locus with very high read depth (approximately 100,000) and low error rate. A serial dilution study with use of cloned mutant and wildtype DNA samples showed a linear correlation between dilution and measured mutation abundance, indicating the reliability of quantification of the mutation. Using the serially diluted samples, the detection limits of three mutation detection methods (the PNA method, NGS, and combinatory use of PNA and NGS [PNA-NGS]) were determined. The lowest detectable mutation abundance was 1% for the PNA method, 0.03% for NGS and 0.01% for PNA-NGS. Finally, we analyzed 16 MAS patient-derived leukocytic DNA samples with the three methods, and compared the mutation detection rate of them. Mutation detection rate of the PNA method, NGS and PNA-NGS in 16 patient-derived peripheral blood samples were 56%, 63% and 75%, respectively. In conclusion, NGS can detect somatic activating GNAS mutations quantitatively and sensitively from peripheral blood samples. At present, the PNA-NGS method is likely the most sensitive method to detect low-abundance GNAS mutation.

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Functional characterization of four novel PAX8 mutations causing congenital hypothyroidism: new evidence for haploinsufficiency as a disease mechanism.

Narumi S, Araki S, Hori N, Muroya K, Yamamoto Y, Asakura Y, Adachi M, Hasegawa T

European journal of endocrinology （BIOSCIENTIFICA LTD） 167 （ 5 ） 625 - 32 2012年11月

査読有り, ISSN 0804-4643

Background: Individuals carrying a heterozygous inactivating PAX8 mutation are affected by congenital hypothyroidism (CH), although heterozygous Pax8 knockout mice are not. It has remained unclear whether CH in PAX8 mutation carriers is caused by haploinsufficiency or a dominant negative mechanism.
Objective: To report clinical and molecular findings of four novel PAX8 mutations, including one early-truncating frameshift mutation.
Subjects and methods: Four probands were CH patients. Two had family history of congenital or childhood hypothyroidism. Three probands were diagnosed in the frame of newborn screening for CH, while one had a negative result in screening but was diagnosed subsequently. Three had thyroid hypoplasia and one had a slightly small thyroid with low echogenicity. For these probands and their family members, we sequenced PAX8 using a standard PCR-based method. Pathogenicity of identified mutations was verified in vitro.
Results: We found four novel heterozygous PAX8 mutations in the four probands: L16P, F20S, D46SfsX24, and R133Q. Family studies showed four additional mutation carriers, who were confirmed to have high serum TSH levels. Expression experiments revealed that three mutations (L16P, F20S, and R133Q) had defects in target DNA binding, while D46fs had protein instability that was rescued by the proteasome inhibitor MG132. All four mutations had reduced transactivation on the thyroglobulin promoter, supporting that they were inactivating mutations.
Conclusion: D46fs is the first PAX8 mutation with confirmed protein instability. Our clinical and in vitro findings together suggest that pure PAX8 haploinsufficiency can cause CH in humans.

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両アレル性DUOX2変異例におけるヨード有機化能の検討

長崎啓祐, 鳴海覚志, 阿部清美, 佐藤英利, 小川洋平, 浅見直, 長谷川奉延, 菊池透

日本内分泌学会雑誌（(一社)日本内分泌学会） 88 （ 2 ） 497 - 497 2012年09月

ISSN 0029-0661

次世代シーケンシングによる先天性甲状腺機能低下症の包括的遺伝子解析

鳴海覚志, 阿部清美, 天野直子, 石井智弘, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 88 （ 2 ） 498 - 498 2012年09月

ISSN 0029-0661

先天性甲状腺機能低下症発症におけるPathway Burden仮説の提唱

阿部清美, 鳴海覚志, 天野直子, 石井智弘, 室谷浩二, 朝倉由美, 安達昌功, 佐々木悟郎, 長崎啓祐, 阿部貴行, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 88 （ 2 ） 543 - 543 2012年09月

ISSN 0029-0661

Acceleration of diabetes development in CXC chemokine receptor 3 (CXCR3)-deficient NOD mice.

Yamada Y, Okubo Y, Shimada A, Oikawa Y, Yamada S, Narumi S, Matsushima K, Itoh H

Diabetologia 55 （ 8 ） 2238 - 45 2012年08月

ISSN 0012-186X

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新生児マス・スクリーニングの震災対応はどうあるべきか東日本大震災1年を経過して東日本大震災が新生児マス・スクリーニングシステムに及ぼした影響内分泌疾患を中心に

南谷幹史, 皆川真規, 伊藤順庸, 鬼形和道, 鈴木順造, 高橋明雄, 長崎啓祐, 鳴海覚志, 難波範行, 原田正平, 藤原幾磨, 堀川玲子, 水野晴夫, 横谷進

日本マス・スクリーニング学会誌（日本マススクリーニング学会） 22 （ 2 ） 158 - 158 2012年07月

ISSN 0917-3803

東日本大震災の新生児マススクリーニングに対する影響調査

南谷幹史, 皆川真規, 伊藤順庸, 鬼形和道, 鈴木順造, 高橋明雄, 長崎啓祐, 鳴海覚志, 難波範行, 原田正平, 藤原幾磨, 堀川玲子, 水野晴夫, 横谷進

日本内分泌学会雑誌（(一社)日本内分泌学会） 88 （ 1 ） 308 - 308 2012年04月

ISSN 0029-0661

東日本大震災の新生児マス・スクリーニングに対する影響調査

南谷幹史, 皆川真規, 鈴木順造, 高橋明雄, 長崎啓祐, 鳴海覚志, 原田正平, 藤原幾磨, 堀川玲子, 横谷進, 日本小児内分泌学会震災小児甲状腺PT

日本小児科学会雑誌（(公社)日本小児科学会） 116 （ 2 ） 278 - 278 2012年02月

ISSN 0001-6543

A family of pseudohypoparathyroidism type Ia with an 850-kb submicroscopic deletion encompassing the whole GNAS locus.

Mitsui T, Nagasaki K, Takagi M, Narumi S, Ishii T, Hasegawa T

American journal of medical genetics. Part A （WILEY-BLACKWELL） 158A （ 1 ） 261 - 4 2012年01月

査読有り, ISSN 1552-4825

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Gradual loss of ACTH due to a novel mutation in LHX4: comprehensive mutation screening in Japanese patients with congenital hypopituitarism.

Takagi M, Ishii T, Inokuchi M, Amano N, Narumi S, Asakura Y, Muroya K, Hasegawa Y, Adachi M, Hasegawa T

PloS one （PUBLIC LIBRARY SCIENCE） 7 （ 9 ） e46008 2012年

査読有り, ISSN 1932-6203

Mutations in transcription factors genes, which are well regulated spatially and temporally in the pituitary gland, result in congenital hypopituitarism (CH) in humans. The prevalence of CH attributable to transcription factor mutations appears to be rare and varies among populations. This study aimed to define the prevalence of CH in terms of nine CH-associated genes among Japanese patients. We enrolled 91 Japanese CH patients for DNA sequencing of POU1F1, PROP1, HESX1, LHX3, LHX4, SOX2, SOX3, OTX2, and GLI2. Additionally, gene copy numbers for POU1F1, PROP1, HESX1, LHX3, and LHX4 were examined by multiplex ligation-dependent probe amplification. The gene regulatory properties of mutant LHX4 proteins were characterized in vitro. We identified two novel heterozygous LHX4 mutations, namely c.249-1G>A, p.V75I, and one common POU1F1 mutation, p.R271W. The patient harboring the c.249-1G>A mutation exhibited isolated growth hormone deficiency at diagnosis and a gradual loss of ACTH, whereas the patient with the p.V75I mutation exhibited multiple pituitary hormone deficiency. In vitro experiments showed that both LHX4 mutations were associated with an impairment of the transactivation capacities of POU1F1 and alpha GSU, without any dominant-negative effects. The total mutation prevalence in Japanese CH patients was 3.3%. This study is the first to describe, a gradual loss of ACTH in a patient carrying an LHX4 mutation. Careful monitoring of hypothalamic-pituitary -adrenal function is recommended for CH patients with LHX4 mutations.

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【小児内分泌学の進歩2012】骨代謝・副甲状腺 GNAS変異陰性-偽性副甲状腺機能低下症IaにおけるGNAS遺伝子領域の全てを含む欠失の同定

三井俊賢, 長崎啓祐, 高木優樹, 鳴海覚志, 石井智弘, 長谷川奉延

ホルモンと臨床（(有)医学の世界社） 59 （ 12 ） 1085 - 1087 2011年12月

ISSN 0045-7167

非古典型TSH不応症奇異性123I摂取率高値を特徴とする先天性甲状腺機能低下症の新しい病型の提唱

鳴海覚志, 長崎啓祐, 石井智弘, 室谷浩二, 朝倉由美, 安達昌功, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 87 （ 2 ） 516 - 516 2011年09月

ISSN 0029-0661

DUOX2変異による偽性優性遺伝を示した先天性甲状腺機能低下症の一家系

阿部清美, 濱島崇, 鳴海覚志, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 87 （ 2 ） 509 - 509 2011年09月

ISSN 0029-0661

A new rapid protocol for eyeblink conditioning to assess cerebellar motor learning.

Emi K, Kohda K, Kakegawa W, Narumi S, Yuzaki M

Neurochemical research 36 （ 7 ） 1314 - 22 2011年07月

ISSN 0364-3190

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当院で経験した自律性機能性甲状腺結節(AFTN)の1男児例

神原亜紀子, 小山さとみ, 志村直人, 鳴海覚志, 長谷川奉延, 有阪治

日本小児科学会雑誌（(公社)日本小児科学会） 115 （ 2 ） 434 - 434 2011年02月

ISSN 0001-6543

一過性甲状腺機能低下症におけるDUOX2遺伝子解析の検討

長崎啓祐, 鳴海覚志, 小川洋平, 菊池透, 浅見直, 長谷川奉延, 内山聖

日本内分泌学会雑誌（(一社)日本内分泌学会） 86 （ 2 ） 266 - 266 2010年09月

ISSN 0029-0661

甲状腺形成異常におけるCopy number variation異常解析

鳴海覚志, 室谷浩二, 朝倉由美, 安達昌功, 長崎啓祐, 天野直子, 石井智弘, 石井美穂, 江見充, 長谷川奉延

日本内分泌学会雑誌（(一社)日本内分泌学会） 86 （ 2 ） 267 - 267 2010年09月

ISSN 0029-0661

One Novel and Two Recurrent THRB Mutations Associated with Resistance to Thyroid Hormone: Structure-based Computational Mutation Prediction.

Narumi S, Cho H, Tamada I, Kozu Y, Tsuchiya T, Nagai T, Hasegawa T

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 19 （ 4 ） 91-99 - 9 2010年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0918-5739

Inactivating mutations of THRB, which encodes the thyroid hormone receptor β (TRβ), cause resistance to thyroid hormone (RTH
OMIM 190160). To date, more than 100 THRB mutations have been reported among RTH patients. Most mutations substitute a single amino-acid residue in the ligand-binding domain. In this report, we describe clinical and molecular findings of three families with RTH. Three families harbored one novel (p.I431M) and two recurrent (p.R320H and p.R383C) THRB mutations. To examine the pathogenicity of identified mutations, we introduced a novel computational mutation prediction method based on three-dimensional structure data of TRβ-T3 complex. First, to define the accuracy of our prediction system, we evaluated ten previously reported 'positive control' mutations, as well as 30 seemingly benign sequence variations observed among vertebral species as 'negative controls'. We found that our system had a sensitivity of 80% and a specificity of 93%. We then analyzed three mutations detected in the present study and found that all three mutations are predicted to be deleterious. Our data suggest that our structure-based prediction system would be a prompt, inexpensive and feasible method for evaluating the pathogenicity of missense THRB mutations. © 2010 by The Japanese Society for Pediatric Endocrinology.

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A patient with Pendred syndrome whose goiter progressed with normal serum thyrotropin and iodine organification

Asakura Y, Narumi S, Muroya K, Fujita K, Aida N, Hasagawa T, Adachi M.

American Journal of Medical Genetics Part A （WILEY-LISS） 152A （ 7 ） 1793-1797 - 1797 2010年07月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1552-4825

Biallelic mutations of SLC26A4 (encoding pendrin) cause Pendred syndrome (PS), an autosomal recessive genetic disorder with deafness and goiter. The mechanism underlying the development of the goiter is unknown. Here, we report clinical and molecular findings of a patient with PS. This 27-year-old woman was born to nonconsanguineous healthy parents. She was seen at our hospital due to hearing loss at age 3 years, and subsequently developed goiter at age 10 years. From age 15 years, her thyroid gland showed progressive enlargement accompanied by elevation of serum thyroglobulin reaching 10-fold the normal amount. Thyroidal iodine uptake was also increased during goiter progression ((123)I uptake at 24 hr: 20.2% at age 17 years; 69.4% at age 24 years; reference, 8-40), while serum thyrotropin (TSH) levels and iodine organification (examined by the per-chrolate or thiocyanate discharge test) remained normal. We sequenced SLC26A4 using standard PCR-based technique, and found one novel (p.T537P) and one recurrent (p.H723R) mutations in a compound heterozygous state. Expression experiments using COS-7 cells showed that the two mutants were entrapped in the endoplasmic reticulum and were poorly localized at the plasma membrane. In summary, a molecularly confirmed PS patient showed goiter progression accompanied by elevated serum thyroglobulin and increased thyroidal iodine uptake, but normal serum TSH levels and normal iodine organification. This implies that some pendrin mutations may involve direct stimulation of thyroid cell proliferation with no TSH hyperstimulation and no iodine organification defect. (C) 2010 Wiley-Liss, Inc.

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Cbln1 is a ligand for an orphan glutamate receptor delta2, a bidirectional synapse organizer.

Matsuda K, Miura E, Miyazaki T, Kakegawa W, Emi K, Narumi S, Fukazawa Y, Ito-Ishida A, Kondo T, Shigemoto R, Watanabe M, Yuzaki M

Science (New York, N.Y.) 328 （ 5976 ） 363 - 8 2010年04月

ISSN 0036-8075

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Transcription factor mutations and congenital hypothyroidism: systematic genetic screening of a population-based cohort of Japanese patients.

Narumi S, Muroya K, Asakura Y, Adachi M, Hasegawa T

The Journal of clinical endocrinology and metabolism （ENDOCRINE SOC） 95 （ 4 ） 1981-1985 - 5 2010年04月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0021-972X

Context: Gene mutations of transcription factors that are predominantly expressed in the thyroid gland cause congenital hypothyroidism (CH). The prevalence of CH due to transcription factor mutations remains undetermined.
Objective: This study was designed to define the prevalence of CH due to mutations of PAX8, NKX2-1 [encoding thyroid transcription factor (TTF)-1], FOXE1 (encoding TTF-2), and NKX2-5 among patients with permanent primary CH and in the general population in Japan.
Subjects and Methods: We enrolled 102 CH patients that represent 353,000 newborns born in Kanagawa prefecture from October 1979 to June 2006. We sequenced PAX8, NKX2-1, FOXE1, and NKX2-5 using PCR-based methods. Additionally, deletion/duplication of PAX8, NKX2-1, and FOXE1 was screened by multiplex ligation-dependent probe amplification. Molecular functions of putative mutations were verified in vitro.
Results: We identified a novel small duplication of PAX8 (p.K80_A84dup) in two half-sibling patients with thyroid hypoplasia. We also found a novel NKX2-1 variation (p.H60W) in a sporadic nonsyndromic CH patient. In vitro experiments showed that K80_A84dup PAX8 had impaired transactivation of the thyroglobulin promoter. H60W TTF-1 exhibited a comparable transactivating capacity with wild-type TTF-1, suggesting a benign variation. We estimate the prevalence of PAX8 mutations to be 2.0% (two in 102) among Japanese CH patients and one in 176,000 (two in 353,000) in the general Japanese population.
Conclusions: Using a population-based sample, we confirmed that a minor subset of CH patients has transcription factor mutations, but they are rare. In our cohort, PAX8 mutations were the leading cause of such a rare condition. (J Clin Endocrinol Metab 95: 1981-1985, 2010)

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Various types of LRP5 mutations in four patients with osteoporosis-pseudoglioma syndrome: identification of a 7.2-kb microdeletion using oligonucleotide tiling microarray.

Narumi S, Numakura C, Shiihara T, Seiwa C, Nozaki Y, Yamagata T, Momoi MY, Watanabe Y, Yoshino M, Matsuishi T, Nishi E, Kawame H, Akahane T, Nishimura G, Emi M, Hasegawa T

American journal of medical genetics. Part A （WILEY-LISS） 152A （ 1 ） 133-140 - 40 2010年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1552-4825

Osteoporosis-pseudoglioma syndrome (OPS; OMIM 259770) is an autosomal-recessive genetic disorder characterized by severe osteoporosis and visual disturbance from childhood. Biallelic mutations in the low-density lipoprotein receptor-related protein 5 gene (LRP5) have been frequently detected, while a subset of patients had only one or no detectable mutation. We report on the clinical and molecular findings of four unrelated Japanese patients with the syndrome. The four patients had typical skeletal and ocular phenotypes of OPS, namely severe juvenile osteoporosis and early-onset visual disturbance, with or without mental retardation. We undertook standard PCR-based sequencing for LRP5 and found four missense mutations (p.L145F, p.T244M, p.P382L, and p.T552M), one nonsense mutation (p.R1534X), and one splice site mutation (c.1584+1G>A) among four OPS patients. Although three patients had two heterozygous mutations, one had only one heterozygous splice site mutation. In this patient, RT-PCR from lymphocytic RNA demonstrated splice error resulting in 63-bp insertion between exons 7 and 8. Furthermore, the patient was found to have only mutated RT-PCR fragment, implying that a seemingly normal allele did not express LRP5 mRNA. We then conducted custom-designed oligonucleotide tiling microarray analyses targeted to a 600-kb genome region harboring LRP5 and discovered a 7.2-kb microdeletion encompassing exons 22 and 23 of LRP5. We found various types of LRP5 mutations, including an exon-level deletion that is undetectable by standard PCR-based mutation screening. Oligonucleotide tiling microarray seems to be a powerful tool in identifying cryptic structural mutations. (C) 2009 Wiley-Liss, Inc.

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Ruvalcaba syndrome revisited

Masanori Adachi, Koji Muroya, Yumi Asakura, Kenji Kurosawa, Gen Nishimura, Satoshi Narumi, Tomonobu Hasegawa

American Journal of Medical Genetics, Part A （Wiley-Liss Inc.） 152 （ 7 ） 1854 - 1857 2010年

査読有り, ISSN 1552-4833

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【小児内分泌学の進歩2009】家族性先天性甲状腺機能低下症の臨床的・分子遺伝学的検討

長崎啓祐, 鳴海覚志, 浅見直, 小川洋平, 菊池透, 長谷川奉延, 内山聖

ホルモンと臨床（(有)医学の世界社） 57 （ 12 ） 1001 - 1005 2009年12月

ISSN 0045-7167

病型別の家族性先天性甲状腺機能低下症(CH)の頻度、家族性CHの臨床的・分子遺伝学的特徴の解明を目的に、CHおよび一過性甲状腺機能低下症の患者264名について検討した。明確な家族歴を認めない例にも積極的に検索した。その結果、CHの家族歴を認めたのは17家系33名(12.5%)で、同胞例12家系、いとこを含む同胞例3家系、親子例1家系、罹患患者7名を含む家系1家系であった。甲状腺形態別の家族歴の頻度は、異所性甲状腺、甲状腺無形成の45名では0%で、正所性CH(低形成、正常大、腫大)174名中32名(18.4%)であった。家族性CHのうち14名の病型診断の結果、臨床的TSH不応症5家系、合成障害3家系、合成障害疑い6家系で、大半は軽症であった。遺伝子解析を行った9家系のうち、遺伝子変異を認めたのは1家系であった。以上より、CHの遺伝性の予測には甲状腺形態、TSH不応症の有無が有用な情報で、軽症CH発生は未知の遺伝子変異、環境要因の関与を示唆した。

TSHR mutations as a cause of congenital hypothyroidism in Japan: a population-based genetic epidemiology study.

Narumi S, Muroya K, Abe Y, Yasui M, Asakura Y, Adachi M, Hasegawa T

The Journal of clinical endocrinology and metabolism （ENDOCRINE SOC） 94 （ 4 ） 1317-1323 - 23 2009年04月

研究論文（学術雑誌）, 共著, ISSN 0021-972X

Context: The prevalence of congenital hypothyroidism (CH) associated with mutations in the TSH receptor gene (TSHR) has not been established.
Objective: We examined the frequency of TSHR mutations among patients with permanent primary CH and in the general population in Japan.
Subjects and Methods: We enrolled 102 patients with permanent primary CH [70 with "moderate to severe CH" (TSH, >= 10 mU/liter) and 32 with "mild CH" (TSH, 5-10 mU/liter)], who were identified through newborn screening among 353,000 newborns born in Kanagawa prefecture from October 1979 to June 2006. These subjects were tested for TSHR mutations by PCR-based direct sequencing. We further characterized molecular functions of identified mutant TSHRs in vitro.
Results: We found three patients with moderate to severe CH who had biallelic mutations in TSHR and three patients with mild CH who had monoallelic mutations. Observed mutations included one previously characterized mutation (p.R450H) and three uncharacterized mutations (p.G132R, p.A204V, and p.D403N). In vitro experiments confirmed loss of functions of these four mutants. Among four mutations, p.R450H was particularly frequent: six of nine mutant alleles harbored p.R450H. All six alleles with p.R450H commonly carried a minor single nucleotide polymorphism, suggesting a founder effect. We estimated the prevalence of biallelic TSHR mutations to be 4.3% (three in 70) in Japanese patients with moderate to severe CH, and 1 in 118,000 (three in 353,000) in the general Japanese population.
Conclusions: In Japan, TSHR mutations are relatively common among patients with CH, and a founder mutation (p.R450H) accounts for about 70% of mutants. (J Clin Endocrinol Metab 94: 1317-1323, 2009)

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The role of the CXCL10/CXCR3 system in type 1 diabetes.

Shimada A, Oikawa Y, Yamada Y, Okubo Y, Narumi S

The review of diabetic studies : RDS 6 （ 2 ） 81 - 4 2009年

ISSN 1613-6071

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【小児内分泌学の進歩2008】先天性甲状腺機能低下症におけるTSH不応症の臨床的および遺伝学的検討

長崎啓祐, 鳴海覚志, 浅見直, 菊池透, 長谷川奉延, 内山聖

ホルモンと臨床（(有)医学の世界社） 56 （ 12 ） 1213 - 1216 2008年12月

ISSN 0045-7167

小児科で先天性甲状腺機能低下症(CH)マススクリーニングのガイドラインに従い病型診断を行った症例のうち、臨床的にTSH不応症と診断した16例を対象に、臨床像と分子遺伝的特徴を検討した。全例にTRH負荷試験、血清Tg測定、甲状腺エコー、123I摂取率測定(摂取率20%以下の場合はパークロレイト放出試験)を行った。偽性副甲状腺機能低下症Ia型と臨床診断した1例ではGNAS1遺伝子解析を行い、残り15例にはTSH受容体遺伝子(TSHR)、PAX8、TITF1、FOXE1、NKX2-5の遺伝子解析を行った。いずれの遺伝子も翻訳領域をPCR-直接シーケンス法で解析した。その結果、TSH高値・fT4正常範囲の軽症11例、TSH高値・fT4低値の顕性5例で、重症度はさまざまであった。血清Tg値は1例を除き高値、甲状腺サイズは正常8例、低形成8例、123I摂取率は5例が40%以上と高値であった。7例にCHの家族歴があった。遺伝子解析では、TSHR複合へテロ接合性変異を1例、TSHRへテロ接合性変異を1例、TITF1へテロ接合性変異を1例に認め、偽性副甲状腺機能低下症Ia型の症例ではGNAS1遺伝子変異は認めなかった。

Mutation of a gene for thyroid transcription factor-1 (TITF1) in a patient with clinical features of resistance to thyrotropin.

Nagasaki K, Narumi S, Asami T, Kikuchi T, Hasegawa T, Uchiyama M.

Endocrine Journal （JAPAN ENDOCRINE SOC） 55 （ 5 ） 875-878 - 878 2008年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0918-8959

Resistance to TSH (RTSH [MIM 275200]) is a heterogeneous condition defined by variable degree of insensitivity to biologically active TSH. While this condition is classically caused by loss-of-function mutations of the TSH receptor gene (TSHR), several patients have exhibited RTSH-like phenotype in the apparent absence of TSHR mutations, and some of them have mutations of PAX8 or GNAS1. We identified a Japanese boy with congenital hypothyroidism who suffered from recurrent lower respiratory infection during infancy and choreoathetosis at a later age. At 14 years of age, lie was diagnosed as having RTSH, on the basis of compensated hypothyroidism (TSH, 30.2 mU/L; FT4, 1.2 ng/dl), disproportionate increments of thyroid hormones and TSH during a TRH test (Delta FT3, 0.4 pg/ml; Delta T3, 13 ng/dl; and Delta TSH, 88.3 mU/L), and normal ultrasound thyroid image and radioactive iodine uptakes. Molecular analysis for TITF1 revealed a novel de novo heterozygous deletion/insertion mutation (c.470_479delinsGCG,) that is predicted to lose the entire homeodomain and the NK2-specific domain. We suggest that a heterozygous loss-of-function TITF1 mutation can also cause RTSH-compatible phenotype.

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先天性甲状腺機能低下症におけるTSH不応症の臨床像及び遺伝学的検討

長崎啓祐, 鳴海覚志, 浅見直, 小川洋平, 菊池透, 長谷川奉延, 内山聖

日本内分泌学会雑誌（(一社)日本内分泌学会） 84 （ 2 ） 414 - 414 2008年09月

ISSN 0029-0661

Radiological evolution in IMAGe association: a case report.

Amano N, Naoaki H, Ishii T, Narumi S, Hachiya R, Nishimura G, Hasegawa T

American journal of medical genetics. Part A 146A （ 16 ） 2130 - 3 2008年08月

査読有り, ISSN 1552-4825

IMAGe association is a recently recognized multi-system disorder of unknown etiology. IMAGe is a mnemonic acronym that stands for Intrauterine growth retardation, Metaphyseal dysplasia, adrenal hypoplasia congenita, and Genital anomalies (OMIM 300290). Suspicion for the disorder is readily raised by the distinctive clinical and endocrinological constellation, and radiological identification of metaphyseal dysplasia is crucial for the diagnosis. However, knowledge of the onset, evolution, severity, and variation of the metaphyseal dysplasia is currently limited. We illustrate the radiological evolution of an affected girl from her premature birth to early childhood. Her initial skeletal changes included thin ribs, delayed ossification of the juxtatruncal bones, and delayed epiphyseal ossification. The former two became less conspicuous during infancy. Metaphyseal dysplasia was not discerned at birth. However, mild metaphyseal cupping, sclerosis and longitudinal striations became manifest in late infancy, and then progressed with age. It is thought that the skeletal alterations in IMAGe association encompass retarded endochondral ossification normalized later on and mild metaphyseal dysplasia of postnatal onset. © 2008 Wiley-Liss, Inc.

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Correspondence between computed tomography and endoscopy in Menetrier's disease

Narumi S, Omori T, Mitamura K, Yamashita Y, Nakao A, Cho H.

Pediatrics International （BLACKWELL PUBLISHING） 50 （ 2 ） 245-247 - 247 2008年04月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1328-8067

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A novel mutation of androgen receptor gene in complete androgen insensitivity syndrome.

Narumi S, Amano N, Hachiya R, Ishii T, Hasegawa T

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 16 （ 2 ） 59-61 - 61 2007年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0918-5739

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Growth failure in an infant with congenital nephrogenic diabetes insipidus during sodium restriction.

Sasaki G, Ishii T, Amano N, Hachiya R, Narumi S, Hasegawa T

Clinical pediatric endocrinology : case reports and clinical investigations : official journal of the Japanese Society for Pediatric Endocrinology 16 （ 4 ） 95 - 8 2007年

査読有り, ISSN 0918-5739

Congenital nephrogenic diabetes insipidus (CNDI) is an inherited disorder characterized by renal tubular insensitivity to antidiuretic hormone, resulting in an inability to concentrate urine. We report on an infant boy with CNDI who showed growth failure during treatment with sodium restriction. At the age of 4 mo, he was diagnosed as having CNDI, judging from fever with hypernatremia (serum Na 153 mEq/L), diluted urine (urine osmolarity 193 mOsm/kg), high antidiuretic hormone (plasma antidiuretic hormone 53 pg/mL), and normal renal function (serum creatinine 0.3 mg/dL). His length and weight were mean +0.4 and -1.1 SD, respectively, at that time. He was treated with sodium restriction (sodium intake
0.53 mEq/kg/day) using low sodium formula in addition to trichlormethiazide, spironolactone, and mefenamic acid. Growth failure developed: his length and weight were mean -2.4 and -3.3 SD, respectively, at the age of 10 mo. After withdrawal of sodium restriction to 1.5 mEq/kg/day of sodium intake without any change of caloric intake and medication, catch-up growth was observed. At the age of 39 mo, the patient's height and weight were mean -0.8 and -0.6 SD, respectively. We conclude that excessive sodium restriction can cause growth failure in infants with CNDI. Copyright© 2007 by The Japanese Society for Pediatric Endocrinology.

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[11Beta-hydroxylase deficiency].

Narumi S, Hasegawa T

Nihon rinsho. Japanese journal of clinical medicine Suppl 1 699 - 701 2006年05月

ISSN 0047-1852

Growth-chart-based qualitative evaluation of height growth after hematopoietic stem cell transplantation.

Narumi S, Shimada H, Shimasaki N, Takahashi T, Hasegawa T, Mori T

Pediatric transplantation （BLACKWELL PUBLISHING） 10 （ 1 ） 26-31 - 31 2006年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1397-3142

Growth failure is one of the most common late complications in children undergoing hematopoietic stem cell transplantation (SCT). The present report describes a qualitative method of evaluating height growth after SCT, using a growth chart. The patients were divided into three groups according to the shape of their growth chart: the normal growth chart group, the early-onset growth retardation group (E-group), in which a decreased growth rate was seen during the first year after SCT, and the late-onset growth retardation group (L-group), in which a decreased growth rate was seen more than 1 yr after the SCT. In the E-group, total body irradiation and prolonged steroid therapy were thought to contribute to the growth failure, whereas in the L-group, impaired pubertal development was thought to be responsible. The growth pattern in the L-group may, therefore, be of particular clinical importance, because the final stature of the subjects in this group can be improved by pharmacological adjustment of pubertal onset. Although limited by the small size and heterogeneous nature of the sample, our results suggest that growth-chart-based evaluation may provide important information to stratify subjects showing inadequate growth after SCT into two groups whose follow-up and treatment should be individualized.

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Significance of serum CXCL10/IP-10 level in type 1 diabetes.

Shigihara T, Oikawa Y, Kanazawa Y, Okubo Y, Narumi S, Saruta T, Shimada A

Journal of autoimmunity 26 （ 1 ） 66 - 71 2006年02月

ISSN 0896-8411

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CXCL10 DNA vaccination prevents spontaneous diabetes through enhanced beta cell proliferation in NOD mice.

Shigihara T, Shimada A, Oikawa Y, Yoneyama H, Kanazawa Y, Okubo Y, Matsushima K, Yamato E, Miyazaki J, Kasuga A, Saruta T, Narumi S

Journal of immunology (Baltimore, Md. : 1950) 175 （ 12 ） 8401 - 8 2005年12月

ISSN 0022-1767

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CXC chemokine ligand 10 neutralization suppresses the occurrence of diabetes in nonobese diabetic mice through enhanced beta cell proliferation without affecting insulitis.

Morimoto J, Yoneyama H, Shimada A, Shigihara T, Yamada S, Oikawa Y, Matsushima K, Saruta T, Narumi S

Journal of immunology (Baltimore, Md. : 1950) 173 （ 11 ） 7017 - 24 2004年12月

ISSN 0022-1767

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Two cases of "fulminant" type 1 diabetes suggesting involvement of autoimmunity.

Nakagawa Y, Shimada A, Oikawa Y, Irie J, Shigihara T, Tsumura K, Narumi S, Saruta T

Annals of the New York Academy of Sciences 1005 359 - 61 2003年11月

ISSN 0077-8923

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T-cell function in anti-GAD65(+)diabetes with residual beta-cell function.

Suzuki R, Shimada A, Maruyama T, Funae O, Morimoto J, Kodama K, Oikawa Y, Kasuga A, Matsubara K, Saruta T, Narumi S

Journal of autoimmunity 20 （ 1 ） 83 - 90 2003年02月

ISSN 0896-8411

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Elevated serum IP-10 levels observed in type 1 diabetes.

Shimada A, Morimoto J, Kodama K, Suzuki R, Oikawa Y, Funae O, Kasuga A, Saruta T, Narumi S

Diabetes care 24 （ 3 ） 510 - 5 2001年03月

ISSN 0149-5992

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MIRAGE Syndrome.

Adam MP, Feldman J, Mirzaa GM, Pagon RA, Wallace SE, Amemiya A, Tanase-Nakao K, Olson TS, Narumi S

（University of Washington, Seattle） 1993年

CLINICAL CHARACTERISTICS: MIRAGE syndrome is an acronym for the major findings of myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy. Cytopenias are typically seen soon after birth; Thrombocytopenia is the most common followed by anemia and pancytopenia. Recurrent infections from early infancy include pneumonia, urinary tract infection, gastroenteritis, meningitis, otitis media, dermatitis, subcutaneous abscess, and sepsis. Reported genital phenotypes in those with 46,XY karyotype included hypospadias, microphallus, bifid shawl scrotum, ambiguous genitalia, or complete female genitalia. Hypoplastic or dysgenetic ovaries have been reported in females. Gastrointestinal complications include chronic diarrhea and esophageal dysfunction. Moderate-to-severe global developmental delay is reported in most affected individuals. Autonomic dysfunction and renal dysfunction are also reported. DIAGNOSIS/TESTING: The diagnosis of MIRAGE syndrome is established in a proband with suggestive findings and a heterozygous germline gain-of-abnormal-function pathogenic variant in SAMD9 identified by molecular genetic testing. MANAGEMENT: Treatment of manifestations: Individuals with severe anemia and thrombocytopenia due to bone marrow failure should be treated with standard transfusion approaches; bacterial infection prevention including antibiotic prophylaxis and fever precautions in individuals with severe neutropenia. Individuals with severe neutropenia and chronic transfusion requirements, along with individuals who develop monosomy 7 myelodysplastic syndrome should be considered for hematopoietic stem cell transplantation. Standard treatment of infections with antibiotics, antiviral or antifungal agents as needed; consider prophylactic intravenous immunoglobulin if endogenous immunoglobulin levels are low; management by nutritionist to ensure adequate caloric intake and to assist with elemental diet for chronic diarrhea; hydrocortisone and fludrocortisone as needed for adrenal hypoplasia; surgical removal of dysgenetic gonads or surgical intervention may be considered for those with external genital anomalies; consider duodenal tube feeding in those with recurrent aspiration pneumonia; early intervention with occupational, physical, speech and feeding therapy for developmental delay; artificial tear solutions and treatment per ophthalmologist for ocular manifestations of autonomic dysfunction such as hypolacrima; management of ambient temperature for those with temperature instability; management of renal dysfunction per nephrologist. Surveillance: Complete blood count with differential every four to six months; annual bone marrow aspirate and biopsy (with analysis for somatic alterations including chromosome 7 abnormalities) in those with cytopenias including anemia, thrombocytopenia, or neutropenia; at least annual assessment of height, weight, head circumference, physical examination for features of adrenal hypoplasia, and measurement of serum sodium, potassium, glucose, cortisol, and ACTH. Assess for diarrhea, feeding issues, and esophageal dysfunction as needed; monitor developmental milestones every three to six months in the first year of life and at least annually thereafter; assess for keratoconjunctivitis sicca, corneal ulcer, and temperature instability as needed; at least annual measurement of serum creatinine, blood urea nitrogen, and urinalysis to evaluate renal function. Evaluation of relatives at risk: It is appropriate to clarify the genetic status of apparently asymptomatic at-risk relatives of an affected individual in order to identify as early as possible those who would benefit from evaluation for myelodysplasia. GENETIC COUNSELING: MIRAGE syndrome is an autosomal dominant disorder typically caused by a de novo pathogenic variant. Rarely, individuals diagnosed with MIRAGE syndrome have the disorder as the result of a variant inherited from a heterozygous parent with no apparent features of MIRAGE syndrome. If the proband has an SAMD9 pathogenic variant that is not detected in the leukocyte DNA of either parent, the recurrence risk to sibs is slightly greater than that of the general population because of the possibility of parental germline mosaicism or the possibility of a false negative result in a parent due to preferential loss of the chromosome with the SAMD9 pathogenic variant. Once the SAMD9 pathogenic variant has been identified in an affected family member, prenatal and preimplantation genetic testing are possible.

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