Publications by authors named "Satoshi Narumi"

108 Publications

Hereditary paraganglioma presenting with atypical symptoms: Case report.

Medicine (Baltimore) 2021 Nov;100(46):e27888

Department of Pediatrics, St. Luke's International Hospital, Tokyo, Japan.

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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http://dx.doi.org/10.1097/MD.0000000000027888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601346PMC
November 2021

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

Biochem Biophys Res Commun 2021 Oct 27;583:29-34. Epub 2021 Oct 27.

Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo, 160-8402, Japan. Electronic address:

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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http://dx.doi.org/10.1016/j.bbrc.2021.10.055DOI Listing
October 2021

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

Clin Pediatr Endocrinol 2021 1;30(4):179-185. Epub 2021 Oct 1.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.

We previously performed next-generation sequencing-based genetic screening in patients with autoantibody-negative type 1 diabetes, and identified the p.Leu168Pro mutation in 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 (POU) 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 mutation, and the destabilization is likely due to the structural changes involving the hydrophobic core of POU. The disease-causing Leu168Pro mutation is a loss-of-function mutation without a dominant-negative effect.
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http://dx.doi.org/10.1297/cpe.30.179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8481079PMC
October 2021

Acquired uniparental disomy of chromosome 7 in a patient with MIRAGE syndrome that veiled a pathogenic variant.

Clin Pediatr Endocrinol 2021 1;30(4):163-169. Epub 2021 Oct 1.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.

Gain-of-function variants in , 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 heterozygous variant (p.Ala1479Ser). In this study, we confirmed the pathogenicity of Ala1479Ser-SAMD9 . 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 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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http://dx.doi.org/10.1297/cpe.30.163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8481078PMC
October 2021

Phase separation and toxicity of C9orf72 poly(PR) depends on alternate distribution of arginine.

J Cell Biol 2021 11 9;220(11). Epub 2021 Sep 9.

Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan.

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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http://dx.doi.org/10.1083/jcb.202103160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8438627PMC
November 2021

Role of Liquid-Liquid Separation in Endocrine and Living Cells.

J Endocr Soc 2021 Oct 19;5(10):bvab126. Epub 2021 Jul 19.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 157-8535 Tokyo, Japan.

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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http://dx.doi.org/10.1210/jendso/bvab126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358989PMC
October 2021

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

Hum Genome Var 2021 Jul 12;8(1):27. Epub 2021 Jul 12.

Division of Pediatrics & Perinatology, Tottori University Faculty of Medicine, Yonago, Japan.

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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http://dx.doi.org/10.1038/s41439-021-00158-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8275606PMC
July 2021

Mutation Screening for 117 Patients with Kallmann Syndrome.

J Endocr Soc 2021 Jul 30;5(7):bvab056. Epub 2021 Mar 30.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 157-8535 Tokyo, Japan.

Introduction: Kallmann syndrome (KS) is a genetically heterogeneous condition characterized by hypogonadotropic hypogonadism (HH) and olfactory dysfunction. Although , 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 variants as the cause of KS remains uncertain.

Patients And Methods: A total of 117 patients with KS underwent mutation screening of and 14 other causative genes for KS/HH. Rare variants were subjected to in silico and in vitro analyses. We also examined clinical data of the patients and their parents with variants.

Results: Sequence analysis identified 2 heterozygous variants of (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 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 haploinsufficiency accounts for a small percentage of KS cases. 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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http://dx.doi.org/10.1210/jendso/bvab056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8170842PMC
July 2021

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

Clin Epigenetics 2021 05 26;13(1):119. Epub 2021 May 26.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan.

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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http://dx.doi.org/10.1186/s13148-021-01106-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8157728PMC
May 2021

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

Clin Pediatr Endocrinol 2021 3;30(2):99-104. Epub 2021 Apr 3.

Department of Endocrinology and Metabolism, Aichi Children's Health and Medical Center, Aichi, Japan.

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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http://dx.doi.org/10.1297/cpe.30.99DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8022032PMC
April 2021

Clinical and Immunological Analyses of Ten Patients with MIRAGE Syndrome.

J Clin Immunol 2021 04 9;41(3):709-711. Epub 2021 Jan 9.

Department of Pediatrics, National Defense Medical College, Saitama, Japan.

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http://dx.doi.org/10.1007/s10875-020-00964-7DOI Listing
April 2021

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.

Am J Med Genet A 2021 04 5;185(4):1067-1075. Epub 2021 Jan 5.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.

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 (Sox9 ), 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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http://dx.doi.org/10.1002/ajmg.a.62063DOI Listing
April 2021

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

J Clin Endocrinol Metab 2021 03;106(3):718-723

Division of Diabetes and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.

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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http://dx.doi.org/10.1210/clinem/dgaa893DOI Listing
March 2021

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

J Clin Endocrinol Metab 2021 01;106(1):e265-e272

Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan.

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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http://dx.doi.org/10.1210/clinem/dgaa772DOI Listing
January 2021

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

NPJ Digit Med 2020 21;3:138. Epub 2020 Oct 21.

First Ascent Inc., Tokyo, 104-0061 Japan.

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 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;  < 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;  < 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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http://dx.doi.org/10.1038/s41746-020-00345-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578091PMC
October 2020

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

Clin Pediatr Endocrinol 2020 3;29(4):173-178. Epub 2020 Oct 3.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.

Mutations in , the gene for a thyroid-specific transcription factor, causes congenital hypothyroidism (CH) with autosomal dominant inheritance. All previously detected 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 T level (0.4 ng/dL). Ultrasonography revealed that the proband had thyroid hypoplasia. Importantly, he had a family history of CH, , his mother also had CH and hypoplasia. Next generation sequencing-based mutation screening revealed a novel heterozygous 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 promoter-luciferase reporter. In conclusion, we identified and described a novel loss-of-function mutation in a family with thyroid hypoplasia. Patients with dominantly inherited CH and no extrathyroidal abnormalities could have mutations.
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http://dx.doi.org/10.1297/cpe.29.173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534521PMC
October 2020

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

Mol Reprod Dev 2020 11 6;87(11):1124-1125. Epub 2020 Oct 6.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.

This study provides supporting evidence for the association between SOX9 and liquid-liquid phase separation. We show that SOX9 colocalized with a paraspeckle protein NONO in many, but not all, of the immortalized and primary murine Sertoli cells examined. In addition, we confirmed that SOX9 has structural characteristics of intrinsically disordered proteins.
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http://dx.doi.org/10.1002/mrd.23425DOI Listing
November 2020

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

Obstet Gynecol 2020 10;136(4):666-674

Department of Pediatrics, Perinatal and Maternal Medicine (Ibaraki), Graduate School, Tokyo Medical and Dental University, the Department of Social Medicine and the Division of Maternal Medicine, Center for Maternal-Fetal, Neonatal and Reproductive Medicine, National Center for Child Health and Development, the Department of Obstetrics and Gynecology, Tokyo Saiseikai Central Hospital, and the Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.

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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http://dx.doi.org/10.1097/AOG.0000000000003910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505142PMC
October 2020

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

Int J Endocrinol 2020 30;2020:9132372. Epub 2020 Aug 30.

Tokyo Medical University, Department of Diabetes, Metabolism and Endocrinology, Tokyo 160-0023, Japan.

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 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 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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http://dx.doi.org/10.1155/2020/9132372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477596PMC
August 2020

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

J Clin Endocrinol Metab 2020 11;105(11)

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.

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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http://dx.doi.org/10.1210/clinem/dgaa584DOI Listing
November 2020

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

BMC Nephrol 2020 08 12;21(1):340. Epub 2020 Aug 12.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.

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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http://dx.doi.org/10.1186/s12882-020-02011-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424677PMC
August 2020

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

Br J Haematol 2020 12 7;191(5):835-843. Epub 2020 Aug 7.

Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, Tokyo, Japan.

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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http://dx.doi.org/10.1111/bjh.17006DOI Listing
December 2020

Predicting the pathogenicity of and variants with in silico bioinformatic tools.

Authors:
Satoshi Narumi

Clin Pediatr Endocrinol 2020 11;29(3):123-126. Epub 2020 Jul 11.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.

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http://dx.doi.org/10.1297/cpe.29.123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348628PMC
July 2020

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

Clin Pediatr Endocrinol 2020 11;29(3):99-103. Epub 2020 Jul 11.

Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan.

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 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 mutation (c.1163G>A, p.Arg388Gln). We performed 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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http://dx.doi.org/10.1297/cpe.29.99DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348635PMC
July 2020

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

Clin Epigenetics 2020 06 16;12(1):86. Epub 2020 Jun 16.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan.

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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http://dx.doi.org/10.1186/s13148-020-00865-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298762PMC
June 2020

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

Hum Genome Var 2020 5;7. Epub 2020 Mar 5.

Department of Gastroenterology, Nutrition, and Endocrinology, Osaka Women's and Children's Hospital, Osaka, Japan.

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 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 gene (c.4435 G > T; p.Ala1479Ser).
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http://dx.doi.org/10.1038/s41439-020-0091-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057985PMC
March 2020

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

J Clin Invest 2020 02;130(2):641-654

Division of Diabetes and Endocrinology, Department of Internal Medicine, and.

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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http://dx.doi.org/10.1172/JCI127378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994153PMC
February 2020

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

Clin Pediatr Endocrinol 2019 19;28(4):147-153. Epub 2019 Oct 19.

Division of Endocrinology and Metabolism, Tokyo Metropolitan Children's Medical Center, Tokyo, Japan.

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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http://dx.doi.org/10.1297/cpe.28.147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801359PMC
October 2019

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

J Clin Endocrinol Metab 2019 12;104(12):6229-6237

Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan.

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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http://dx.doi.org/10.1210/jc.2019-00657DOI Listing
December 2019
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