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

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

Underweight in young Japanese women over time: a longitudinal retrospective study of the change in body mass index from ages 6 to 20 years.

BACKGROUND: The high prevalence of underweight in young women has become a serious health problem in Japan. When and how young women reach a low body mass index (BMI) has not been clarified. AIM: To clarify the characteristics of BMI standard deviation scores (BMI SDS) trajectory of young Japanese women with underweight. SUBJECTS AND METHODS: A total of 601 Japanese female university students aged 20 years were classified into underweight and healthy weight groups. Their school health check-up data were available from the ages of 6 to 20 years. We evaluated the estimated mean values of BMI SDS at each age and differences in BMI SDS (ΔBMI SDS) from 6 years to each age using a mixed-effects model and compared between the two groups at each age. RESULTS: In the underweight group, the BMI SDS at every age (-1.67 to -0.91) and the ΔBMI SDS after 16 years of age (-0.76 to -0.38) were significantly lower than those in the healthy weight group (-0.41 to -0.13, -0.07 to 0.04), respectively. CONCLUSION: Young Japanese women with underweight have at least two characteristics of BMI SDS trajectory: being constitutionally underweight and shifting their weight status from baseline towards underweight in their late teens.

Development of sexual dimorphism of skeletal muscles through the adrenal cortex, caused by androgen-induced global gene suppression.

The zona fasciculata (zF) in the adrenal cortex contributes to multiple physiological actions through glucocorticoid synthesis. The size, proliferation, and glucocorticoid synthesis characteristics are all female biased, and sexual dimorphism is established by androgen. In this study, transcriptomes were obtained to unveil the sex differentiation mechanism. Interestingly, both the amount of mRNA and the expressions of nearly all genes were higher in females. The expression of Nr5a1, which is essential for steroidogenic cell differentiation, was also female biased. Whole-genome studies demonstrated that NR5A1 regulates nearly all gene expression directly or indirectly. This suggests that androgen-induced global gene suppression is potentially mediated by NR5A1. Using Nr5a1 heterozygous mice, whose adrenal cortex is smaller than the wild type, we demonstrated that the size of skeletal muscles is possibly regulated by glucocorticoid synthesized by zF. Taken together, considering the ubiquitous presence of glucocorticoid receptors, our findings provide a pathway for sex differentiation through glucocorticoid synthesis.

RMRP-related short stature: A report of six additional Japanese individuals with cartilage hair hypoplasia and literature review.

Biallelic pathogenic variants in RMRP, the gene encoding the RNA component of RNase mitochondrial RNA processing enzyme complex, have been reported in individuals with cartilage hair hypoplasia (CHH). CHH is prevalent in Finnish and Amish populations due to a founder pathogenic variant, n.71A > G. Based on the manifestations in the Finnish and Amish individuals, the hallmarks of CHH are prenatal-onset growth failure, metaphyseal dysplasia, hair hypoplasia, immunodeficiency, and other extraskeletal manifestations. Herein, we report six Japanese individuals with CHH from four families. All probands presented with moderate short stature with mild metaphyseal dysplasia or brachydactyly. One of them had hair hypoplasia and the other immunodeficiency. By contrast, the affected siblings of two families showed only mild short stature. We also reviewed all previously reported 13 Japanese individuals. No n.71A > G allele was detected. The proportions of Japanese versus Finnish individuals were 0% versus 70% for birth length < -2.0 SD, 84% versus 100% for metaphyseal dysplasia and 26% versus 88% for hair hypoplasia. Milder manifestations in the Japanese individuals may be related to the difference of genotypes. The mildest form of CHH phenotypes is mild short stature without overt skeletal alteration or extraskeletal manifestation and can be termed "RMRP-related short stature".

Genotype-Phenotype Correlations in Thirty Japanese Patients with Congenital Hypothyroidism Attributable to TG Defects.

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

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

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

A MinION-based Long-Read Sequencing Application With One-Step PCR for the Genetic Diagnosis of 21-Hydroxylase Deficiency.

CONTEXT: Recently developed long-read sequencing (LRS) technology has been considered an option for CYP21A2 analysis. However, the clinical use of LRS for CYP21A2 analysis is limited. OBJECTIVE: This study's objective is to develop an efficient and low-cost LRS system for CYP21A2 screening. METHODS: A DNA fragment library was prepared in a single polymerase chain reaction (PCR) that covers the entire CYP21A2 gene and all known junctions caused by TNXB gene structural rearrangements, yielding a single 8-kb product of CYP21A2 or CYP21A1P/CYP21A2 chimera. After barcoding, the PCR products were sequenced on a MinION-based platform with Flongle Flow Cell R9.4.1 and R10.4.1. RESULTS: The reference genotypes of 55 patients with 21-hydroxylase deficiency (21OHD) were established using the conventional method with multiplex ligation-dependent probe amplification (MLPA) and nested PCR. LRS using Flongle Flow Cell R9.4.1 yielded consistent results. Additionally, the recently updated LRS "duplex" analysis with Flongle flow cell R10.4.1 was tested to reveal an advantage of accurately sequencing a variant located on the homopolymer region. By introducing a barcode system, the cost was reduced to be comparable to that of conventional analysis. A novel single-nucleotide variation was discovered at the acceptor site of intron 7, c.940-1G > C. We also identified a subtype of the classical chimeric junction CH2, "CH2a," in the region from the latter part of intron 5 to exon 6. CONCLUSION: We successfully established a novel low-cost and highly accurate LRS system for 21OHD genetic analysis. Our study provides insight into the feasibility of LRS for diagnosing 21OHD and other genetic diseases caused by structural rearrangements.

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

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

Incidence and Risk Factors for Adrenal Crisis in Pediatric-onset Adrenal Insufficiency: A Prospective Study.

PURPOSE: To determine the incidence and risk factors for adrenal crisis (AC) in patients with pediatric-onset adrenal insufficiency (AI). MATERIALS AND METHODS: This multicenter, prospective cohort study conducted in Japan enrolled patients diagnosed with AI at ≤ 15 years of age. The incidence of AC was calculated as events per person-year (PY), and risk factors for AC were assessed using Poisson regression multivariable analysis. RESULTS: The study population comprised 349 patients (164 male, 185 female) with a total follow-up of 961 PY. The median age at enrollment was 14.3 years (interquartile range [IQR] 8.5-21.2 years), and the median follow-up was 2.8 years (IQR 2.2-3.3 years). Of these patients, 213 (61%) had primary AI and 136 (39%) had secondary AI. Forty-one AC events occurred in 31 patients during the study period. The calculated incidence of AC was 4.27 per 100 PY (95% confidence interval [CI] of 3.15-5.75). Poisson regression analysis identified younger age at enrollment (relative risk [RR] 0.93 [95% CI 0.89-0.97]) and increased number of infections (RR 1.17 [95% CI 1.07-1.27]) as significant risk factors. Female sex (RR 0.99 [95% CI 0.53-1.86]), primary AI (RR 0.65 [95 % CI 0.30-1.41]), or equivalent dosage of hydrocortisone per square meter of body area (RR 1.02 [95% CI 0.96-1.08]) was not a significant risk factor. CONCLUSION: A substantial proportion of patients with pediatric-onset AI experience AC. Younger age and an increased number of infections are independent risk factors for developing AC in these patients.

Two Japanese Infants With Hypothyroidism Following Exposure to Iodinated Contrast Media.

We report 2 Japanese infants with hypothyroidism requiring levothyroxine (LT4) replacement therapy following exposure to iodinated contrast media (ICM). Patient 1 was born at 32 weeks gestation. He had congenital heart disease and underwent contrast-enhanced computed tomography (CT) on day 22 (estimated amount of iodine: 600 mg/kg/dose). The newborn mass screening showed normal thyrotropin (thyroid-stimulating hormone; TSH) levels at day 4, but high TSH and low free thyroxine levels on retest at day 44. LT4 replacement therapy was administered on days 46 to 74. No hypothyroidism requiring LT4 replacement therapy was observed afterward. The ultrasonography showed a hypoplastic thyroid gland. Patient 2 was born full-term. She had congenital heart disease and underwent contrast-enhanced CT on day 52 (estimated amount of iodine: 1500 mg/kg/dose). The newborn mass screening showed normal TSH levels on day 4, but high TSH levels on retest on day 62. LT4 replacement therapy was administered from day 65 to 3 years of age. Genetic analysis showed a heterozygous variant of DUOX2. Exposure to ICM can result in hypothyroidism, requiring LT4 replacement therapy. The severity of hypothyroidism may depend on risk factors, such as genetic predisposition, preterm birth, thyroid hypoplasia, or early exposure to ICM.