Published papers

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.

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

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

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

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

Background: The impact of body mass index (BMI) on reproductive health is well-established, but the effect of within-individual changes in BMI on the menstrual cycle, a valuable and accessible indicator of reproductive health, remains less studied. This study aimed to investigate the association between changes in individuals’ BMI and menstrual irregularity. Methods: This cohort study analyzed data from a widely used menstrual tracking app in Japan. Menstrual cycle logs were collected from January 2019 to March 2021, and background information from questionnaires (Wave 1: Jan-Mar 2020; Wave 2: May-Jun 2020). Long-term BMI changes were defined as changes from age 18 to Wave 2, and short-term changes were defined as changes from Wave 1 to Wave 2. BMI was categorized as underweight (15–18.4), normal (18.5–22.9), and overweight/obese (23–35). Menstrual irregularity was defined as an individual’s average cycle length ranging outside the 24–38 day range. Results: A total of 126,008 cycles from 5,444 individuals were analyzed. Individuals with overweight/obesity who became normal weight long-term had significantly lower odds of menstrual irregularity (long-term: aOR 0.39, 95% CI 0.20–0.75; short-term: aOR 0.61, 95% CI 0.18–2.02) compared to those who remained overweight/obese. Conversely, increases from normal weight to overweight/obese increased the odds (long-term: aOR 2.02, 95% CI 1.50–2.71; short-term: aOR 2.53, 95% CI 1.31–4.90) compared to those who remained normal weight. No significant association was found for individuals with underweight. Conclusion: Within-individual changes in BMI influence menstrual irregularity. Long-term, but not short-term, reductions from overweight/obese BMI to the normal BMI category were associated with lower odds of irregular cycles, whereas increases from a normal BMI to overweight/obesity raised the risk. These findings highlight the importance of sustainable weight management in enhancing reproductive health.

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

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

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

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.

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

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

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

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

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