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.