Published papers

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/β-catenin signaling. R-spondin 1 (RSPO1) enhances Wnt/β-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/β-catenin signaling. Cell-based functional assays with the TCF-LEF reporter showed that RSPO1-dependent activation of Wnt/β-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/β-catenin signaling by ΔEx2-ZNRF3.

A hypothyroid mother, due to thyroid stimulation-blocking antibody (TSBAb), gave birth three times. Although her TSBAb levels remained almost 100%, her TSH receptor antibody (TRAb) levels before delivery varied as follows: 315.0, 88.2 and 34.9 IU/L in the first, second, and third pregnancies, respectively. The first baby had a hypoplastic thyroid gland and developed severe hypothyroidism three days after birth. The second and third babies only showed latent hypothyroidism. Apparent neonatal hypothyroidism due to maternal TSBAb occurs when TRAb levels are very high, and extremely high TSBAb activity might impair not only the fetal thyroid function, but also its development.

Following the LEAP and PETIT studies demonstrating that early allergen introduction can prevent food allergies, Japan’s Ministry of Health, Labor and Welfare revised its weaning guideline in 2019. The updated guideline recommended earlier egg introduction and noted that delaying specific foods may increase allergy risk. This study examined trends in the timing of solid food introduction among Japanese infants before and after this revision. We conducted a retrospective analysis using data from the parenting application Papatto Ikuji collected between 2014 and 2023. After excluding outliers and errors, 59,433 infants were analyzed. The age at solid food introduction was examined by birth year. From 2014 to 2018, the age distribution showed two peaks at 5 and 6 months. After 2019, the 6-month peak declined, leaving a single 5-month peak by 2021. These findings suggest that national guideline revisions substantially influenced caregivers’ feeding behaviors, leading to earlier solid food introduction in Japan.

Germline gain-of-function variants in SAMD9 have been associated with a high risk of mortality and a newly recognized constellation of symptoms described by the acronym MIRAGE: Myelodysplasia, Infection, Restriction of growth, Adrenal insufficiency, Genital phenotypes, and Enteropathy. Here, we describe two additional patients currently living with the syndrome, including one patient with a novel de novo variant for which we provide functional data supporting its pathogenicity. We discuss features of dysmorphology, contrasting with previously described patients as well as drawing attention to additional clinical features, dysautonomia and hearing loss that have not previously been reported. We detail both patients’ courses following diagnosis, with attention to treatment plans and recommended specialist care. Our patients are the oldest known with arginine-substituting amino acid variants, and we conclude that early diagnosis and multidisciplinary management may positively impact outcomes for this vulnerable group of patients.

Aim: To quantify the impact of the COVID-19 pandemic on paediatric care use compared to adult care and identify affected subdomains. Methods: Data from the National Database of Health Insurance Claims and Specific Health Checkups of Japan (NDB) for six fiscal years (FY2016-2021) were analysed. Pre-pandemic (FY2016-FY2019) and pandemic years (FY2020, FY2021) were compared to delineate changes in medical services for paediatric and adult patients. Virtually all individuals who received medical care in Japan during this period were included. Results: In FY2019, healthcare utilisation by the paediatric population (under 15 years) accounted for 24% of 245 million first outpatient visits, 7% of 1.2 billion follow-up visits, and 2.6% of 297 million patient-days hospitalisations. In FY2020, outpatient visits of those aged 0–4 years decreased by 44% (first visit) and 24% (follow-up visit) compared to FY2019, and hospitalisations decreased by 47%, with the largest declines observed in community medical institutions across all age groups. Conclusion: The COVID-19 pandemic led to a substantial reduction in paediatric healthcare utilisation in Japan, varying by age and type of care. These findings may inform the rational allocation of healthcare resources during public health emergencies.

Introduction: Atrophic autoimmune thyroiditis (AAT) is a form of autoimmune hypothyroidism characterized by the absence of a goiter. Thyroid stimulation-blocking antibody (TSBAb) has been detected in a subset of pediatric AAT cases. Although the disappearance of TSBAb has been related with the recovery of thyroid function in adult AAT cases, similar outcomes have not been documented in pediatric cases. Case Presentation: A 2-year-old Japanese boy presented for evaluation of stunted growth from1 year 10 months of age. Tests for congenital hypothyroidism were negative on newborn screening, and he had no significant medical history. However, he showed symptoms of hypothyroidism (inactiveness, hair loss, dry skin), and primary hypothyroidism was confirmed by blood test (serum TSH level, 818 mU/L; serum free T4 level, <0.40 ng/ dL). The patient exhibited a unique antibody profile: positive for TSH receptor antibody (TRAb) and TSBAb and negative for anti-thyroglobulin antibody (TgAb) and antiperoxidase antibody (TPOAb). He was treated with levothyroxine, after which his growth was normalized. During the 8-year follow-up, the patient's TSBAb levels decreased, allowing for the discontinuation of levothyroxine therapy. Conclusion: We reported the case of a 2-year-old boy diagnosed with AAT who presented with a characteristic antibody profile, negative for TgAb and TPOAb, but positive for TRAb and TSBAb. During 8 years of follow-up, TSBAb seroconversion to negative was observed, leading to treatment discontinuation at age 10 years. This case suggests that monitoring of TSBAb after a diagnosis of AAT may be used to determine treatment discontinuation even in children.

Context: Primary adrenal insufficiency (PAI) is a rare, life-threatening condition, and at times is associated with differences of sexual differentiation (DSD). Cytochrome P450 enzymes, which are essential for steroidogenesis in adrenals and gonads, have heme in their active center. CPOX encodes an enzyme coproporphyrinogen oxidase (CPOX) that is involved in the synthesis of heme. Objective: This study aims to report the identification of biallelic inactivating CPOX variants in 3 unrelated patients with PAI and their clinical characteristics. Methods: We report 3 patients with childhood-onset PAI, including 2 with 46,XY DSD. All 3 had adrenal hypoplasia. Additionally, they commonly had severe neonatal jaundice; 2 developed skin blisters in the areas exposed to phototherapy and 2 showed severe neonatal anemia requiring transfusions. Exome sequencing was performed to explore the genetic basis of the patients. The pathogenicity of the identified variants was confirmed with targeted mRNA and proteomic analyses of the patient-derived peripheral blood cells. Results: We identified biallelic rare CPOX variants in each patient, including c.2T > G, p.Arg426*, c.1277G > A, and p.Tyr429Cysfs33*. The 3 patients commonly had the start codon-altering c.2T > G variant. Analysis of the mRNA and proteome of peripheral blood cells from 1 patient (c.2T > G and p.Arg426*) showed that CPOX mRNA expression was comparable to controls; however, CPOX protein expression was significantly decreased to 1%. Conclusion: We provided genetic evidence linking CPOX deficiency and PAI with 46,XY DSD, suggesting that the heme synthesis pathway plays an important role in human steroidogenesis.

INTRODUCTION: Variants affecting a microsatellite on the non-coding region of chromosome 15q26.1 are associated with familial non-autoimmune thyroid abnormalities characterized by mild congenital hypothyroidism (CH) with elevated thyroglobulin (Tg) levels. Some individuals who carry these variants may develop multinodular goiter (MNG) if left untreated. Although these variants have been recognized as genetic etiologies of CH, nongoitrous, 3 (CHNG3), the associated severity of CH remains unclear. METHODS: A cohort of 63 participants diagnosed with CH at Tohoku University underwent screening for genetic variants on 15q26.1. We then analyzed the clinical phenotypes of the variant-carrying participants. RESULTS: We identified five 15q26.1 variant carriers from four families among the cohort. Family histories of thyroid abnormalities were documented in three of these five cases. The variant carriers had mild CH phenotypes, with two discontinuing levothyroxine treatment and the others requiring relatively low doses (1.33-1.89 µg/kg/day) at their final visit. During levothyroxine treatment, serum thyroglobulin and thyroid-stimulating hormone (TSH) levels were within the reference ranges at the majority of the evaluation points. Three of the five participants continued treatment into adulthood, whereas the other two discontinued it and maintained serum TSH levels within the reference range. All five participants exhibited normal intellectual development and stature. CONCLUSION: These findings provide further evidence supporting the role of 15q26.1 variants as a common genetic etiology of CH, with clinical phenotypes including transient CH. Early genetic evaluation may facilitate the identification of 15q26.1 variant carriers among patients who are diagnosed with CH.

Context 46,XY differences of sex development (DSD) are heterogeneous in etiology. The detailed phenotypes of 46,XY DSD patients with monogenic disorders have not been fully elucidated. Objectives To estimate the proportion of monogenic disorders in 46,XY DSD and to characterize the clinical phenotypes of patients with monogenic disorders. Methods A total of 185 Japanese patients (178 families) were enrolled. We sequenced 25 genes related to 46,XY DSD and assessed the pathogenicity of the identified sequence variants according to the American College of Medical Genetics and Genomics guidelines, supplemented by in silico and/or in vitro analyses. We analyzed the clinical phenotypes of patients with monogenic disorders, with particular attention to the external or internal genitalia. Results We identified 51 patients (47 families) with any monogenic disorders (26%), who possessed pathogenic variants in AR (11%), SRD5A2 (4.5%), NR5A1 (4.0%), SRY (2.8%), WT1 (1.1%), STAR (1.1%), CYP17A1 (0.56%), HSD3B2 (0.56%), or MAP3K1 (0.56%). The proportion of monogenic disorders was significantly higher in subjects with detected Müllerian derivatives (57%) than in those undetected (26%) (P =. 029), and in subjects with female-typical genitalia (91%) than in those with ambiguous genitalia (19%) (P <. 001). Conclusion The proportion of monogenic disorders in Japanese 46,XY DSD patients was approximately 26%. Monogenic disorders were frequent among patients with severe undermasculinization of the external or internal genitalia.

Objective: To assess the background, psychosocial, and menstrual factors associated with premenstrual syndrome (PMS) and premenstrual dysphoric disorder (PMDD) in a community-based cohort. Methods: A prospective cohort study was conducted using data from users of a menstrual tracking application. Participants were classified into control, PMS, or PMDD groups based on responses to the Japanese version of the premenstrual symptoms screening tool (PSST). Primary outcomes included background factors (e.g., healthcare literacy, stress coping), psychosocial measures (psychological distress, sleep quality, and work–home balance), and menstrual characteristics (average cycle length [CL], CL variability, abnormal cycle length, and menstrual symptoms). Age-adjusted multinomial logistic regression models were used to examine associations. Results: A total of 7227 individuals were included; 23% met criteria for PMS and 10% for PMDD. Both PMS and PMDD were associated with younger age, smoking history (current and past), more adverse childhood experiences, lower healthcare literacy, and higher maladaptive coping scores. PMDD was additionally linked to being married and co-residing with children, while PMS was associated with alcohol consumption. Both groups reported higher levels of psychological distress, poor sleep quality, and negative work–home balance. Although the prevalence of abnormal cycle length did not differ between groups, menstrual symptom burden was greater in both PMS and PMDD. Conclusion: Premenstrual disorders are associated with a wide range of sociodemographic and psychosocial difficulties. Although menstrual irregularities were similar across groups, menstrual symptom burden was markedly higher. These findings underscore the need for targeted, multidisciplinary support.

Head shape undergoes rapid changes during infancy, but the age-dependent changes in cranial asymmetry remain inadequately characterized. This study aims to analyze factors associated with these parameters, and to establish reference curves in Japanese infants. Two indices of head shape, the cranial vault asymmetry index (CVAI) and cranial index (CI) were collected from 127,605 Japanese infants via a smartphone app. We performed univariate and multivariate analyses to identify factors associated with CVAI and CI. Using 72,726 data derived from infants born via spontaneous vaginal delivery with normal birth weight, reference curves for CVAI and CI were constructed. CVAI exhibited a peak at 3–4 mo of age, followed by a gradual decline, whereas CI increased until approximately 6 mo of age. Boys had significantly higher CVAI than girls. Low birth weight was associated with low CI. Reference curves revealed distinct age-specific patterns, with CVAI peaking at 3–4 mo and declining thereafter, and CI increasing until around 6 mo. In conclusion, this study provides the first reference curves for CVAI and CI that can be applied for Japanese infants. The findings highlight the natural course of cranial asymmetry and proportionality, emphasizing the importance of age-specific assessment.

Craniosynostosis is the premature fusion of cranial sutures, which interferes with normal brain development and requires surgical treatment. By contrast, positional cranial deformity primarily results from external pressure exerted by the sleeping position and is usually managed conservatively. Accurately differentiating between these 2 conditions is essential. In Japan, the use of helmet therapy to treat positional cranial deformities has rapidly expanded, particularly among pediatricians. However, diagnoses based solely on visual inspection and palpation are limited. The aim of this study was to evaluate the diagnostic ability of pediatricians and physicians who treat craniosynostosis and to clarify the current status of diagnostic accuracy. Using computed tomography (CT) data from 10 patients with craniosynostosis and 1 patient with positional cranial deformity treated at Keio University Hospital between 2018 and 2025, 11 physical cranial models were created with 3-dimensional printing and covered with silicone to simulate soft tissue. These models were exhibited at academic meetings, and a questionnaire survey was conducted among pediatricians, craniofacial surgeons, and pediatric neurosurgeons to assess the accuracy of differentiating positional cranial deformities. Three hundred thirty-nine pediatricians and 49 craniofacial and pediatric neurosurgeons responded. The overall correct answer rate among pediatricians was 16.6%, with unilateral lambdoid craniosynostosis being the most frequently selected diagnosis (26.3%). By contrast, craniofacial and pediatric neurosurgeons had a correct answer rate of 49.0%, with unilateral lambdoid craniosynostosis being the second most frequent diagnosis in this group (21.6%). Many respondents indicated that visual inspection alone was insufficient for differentiation and that radiographic imaging was necessary. Pediatricians’ diagnostic accuracy for infant cranial deformities is lower than that of craniofacial surgeons/pediatric neurosurgeons, suggesting that craniosynostosis may be overlooked. To ensure accurate diagnosis and appropriate treatment, clinical systems, including specialist referral and the appropriate use of imaging, need improvement.

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 characterise active enhancers. Temporal expression profile changes in mouse WT1-positive ovarian cells were obtained from cap analysis of gene expression (CAGE) 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. Further, 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.

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.

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.