研究者詳細 - 鳴海　覚志

論文 - 鳴海　覚志

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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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