Congenital Hypothyroidism with thyroid in situ: a case report with NKX2-1 and DUOX2 hypomorphic variants.

INTRODUCTION: NK2 homeobox 1 (NKX2-1) encodes a transcription factor NKX2-1 that is expressed in the thyroid gland, lung and brain. Dual oxidase 2 (DUOX2) encodes an enzyme which generates hydrogen peroxide and is involved in the thyroid hormone synthesis. Cases of congenital hypothyroidism (CH) with dyshormonogenesis showing two or more genetic variants are increasingly reported. We describe the first case of transient dyshormonogenesis who had experimentally-verified a loss of function NKX2-1 variant and DUOX2 variants. CASE PRESENTATION: The proband was a 15-year-old female patient with CH who was diagnosed in the frame of newborn screening for CH. She had a mildly elevated serum TSH level (14.56 mU/L), a low free thyroxine level (0.87 ng/dL), and a high thyroglobulin (Tg) level (>800 ng/mL). Ultrasonography revealed goiter. She was followed clinically without levothyroxine treatment, and showed normal growth and development. She had slightly high Tg levels throughout the clinical course. Next-generation sequencing-based genetic analysis revealed that the patient was heterozygous for an NKX2-1 variant (p.Ile228Ser), a nonsense DUOX2 variant (p.[Lys530*;His678Arg]), and a functional DUOX2 polymorphism (p.His678Arg). NKX2-1 p.Ile228Ser showed about 50% reduced residual activity on the Tg-promoter. CONCLUSION: A partial loss-of-function NKX2-1 variant with a monoallelic nonsense DUOX2 variant and a DUOX2 functional polymorphism can cause transient CH with high serum Tg levels.

Exome-wide benchmark of difficult-to-sequence regions using short-read next-generation DNA sequencing.

Next-generation DNA sequencing (NGS) in short-read mode has recently been used for genetic testing in various clinical settings. NGS data accuracy is crucial in clinical settings, and several reports regarding quality control of NGS data, primarily focusing on establishing NGS sequence read accuracy, have been published thus far. Variant calling is another critical source of NGS errors that remains unexplored at the single-nucleotide level despite its established significance. In this study, we used a machine-learning-based method to establish an exome-wide benchmark of difficult-to-sequence regions at the nucleotide-residue resolution using 10 genome sequence features based on real-world NGS data accumulated in The Genome Aggregation Database (gnomAD) of the human reference genome sequence (GRCh38/hg38). The newly acquired metric, designated the 'UNMET score,' along with additional lines of structural information from the human genome, allowed us to assess the sequencing challenges within the exonic region of interest using conventional short-read NGS. Thus, the UNMET score could provide a basis for addressing potential sequential errors in protein-coding exons of the human reference genome sequence GRCh38/hg38 in clinical sequencing.

(Epi)genetic and clinical characteristics in 84 patients with pseudohypoparathyroidism type 1B.

OBJECTIVE: Pseudohypoparathyroidism type 1B (PHP1B) caused by methylation defects of differentially methylated regions (DMRs) on the GNAS locus can be categorized into groups according to etiologies and methylation defect patterns of the DMRs. The aim of this study was to clarify the clinical characteristics of each group. DESIGN: Comprehensive molecular analyses consisting of methylation, copy number, and microsatellite analyses. METHODS: Eighty-four patients with PHP1B were included in this study. We classified them into 5 groups, namely, autosomal dominant inheritance-PHP1B (Group 1, G1), sporadic-PHP1B (G2), and atypical-PHP1B (G3-G5), based on the methylation defect patterns in 4 DMRs on the GNAS locus and etiologies and evaluated the clinical findings in each group and compared them among the groups. RESULTS: G2 had the youngest age and the highest serum intact parathyroid hormone levels among the 5 groups at the time of diagnosis. The most common symptoms at the time of diagnosis were tetany in G1, and seizures or loss of consciousness in G2. Albright's hereditary osteodystrophy and PHP-suggestive features were most frequently observed in the G2 proband. Nine patients had neurodevelopmental disorders (NDs) consisting of mild to borderline intellectual disability and/or developmental delay. There were no significant correlations between the average methylation ratios of 7 CpG sites in the GNAS-A/B:TSS-DMR and hormonal and biochemical findings. CONCLUSION: This study revealed the differences in some clinical characteristics, particularly clinical features, and ages at the time of diagnosis between G2 and other groups and detailed NDs observed in some patients with PHP1B.

Variations in gender identity and sexual orientation of university students.

Background: Previous studies have shown that a small percentage of people in the general population have atypical gender identity and/or sexual orientation. Aim: This study aimed to explore variations in gender identity and sexual orientation in university students and determine genetic factors associated with these variations. Methods: Deviations from complete gender congruence and exclusive heterosexual orientation in 736 Japanese university students were quantitatively assessed with self-assessment questionnaires. Next, we conducted genetic tests for 80 participants who showed relatively low gender identity scores and/or atypical sexual orientation. These genetic tests consisted of repeat number analysis of the androgen receptor gene (AR) and a SKAT-O: an optimal unified sequence kernel association test, which is an exome-based rare variant association study. The results of the genetic tests were compared with the Japanese reference data and the results of our 637 control samples. Outcomes: We calculated the gender identity and sexual orientation scores of all participants and analyzed the molecular data of 80 selected participants. Results: The gender identity scores of 736 participants were broadly distributed: only ~15% of natal males and ~5% of natal females had the maximum score that corresponds to complete gender congruence. The sexual orientation scores also varied: ~80% of natal males and ~60% of natal females showed exclusive heterosexual orientation. We found no association between gender characteristics and AR repeat numbers. The SKAT-O showed that rare damaging variants of TDRP and 3 other genes were more common in the 80 participants than in the control group. Clinical Implications: Our data support the view that gender is a phenotypic continuum rather than a binary trait. Strength and Limitations: This study quantitatively assessed the gender characteristics of a large cohort of university students. Moreover, we conducted systematic screening for genetic factors associated with gender variations. The weaknesses of the study were the limited analytic power of the questionnaires, the relatively small sample for molecular analyses, and incomplete clinical information and relatively advanced ages of the control group. Conclusion: This study revealed significant variations in gender identity and sexual orientation in university students, which may be partly associated with variants in TDRP or other genes.

Rare sequence variants associated with the risk of non-syndromic biliary atresia.

AIM: The etiology of non-syndromic biliary atresia (BA) remains largely unknown. In this study, we performed genome-wide screening of genes associated with the risk of non-syndromic BA. METHODS: We analyzed exome data of 15 Japanese patients with non-syndromic BA and 509 control individuals using an optimal sequence kernel association test (SKAT-O), a gene-based association study optimized for small-number subjects. Furthermore, we examined the frequencies of known BA-related single-nucleotide polymorphisms in the BA and control groups. RESULTS: SKAT-O showed that rare damaging variants of MFHAS1, a ubiquitously expressed gene encoding a Toll-like receptor-associated protein, were more common in the BA group than in the control group (Bonferroni corrected p-value = 0.0097). Specifically, p.Val106Gly and p.Arg556Cys significantly accumulated in the patient group. These variants resided within functionally important domains. SKAT-O excluded the presence of other genes significantly associated with the disease risk. Of 60 known BA-associated single-nucleotide polymorphisms, only eight were identified in the BA group. In particular, p.Ile3421Met of MYO15A and p.Ala421Thr of THOC2 were more common in the BA group than in the control group. However, the significance of these two variants is questionable, because MYO15A has been linked to deafness, but not to BA, and the p.Ala421Thr of THOC2 represents a relatively common single-nucleotide polymorphism in Asia. CONCLUSIONS: The results of this study indicate that rare damaging variants in MFHAS1 may constitute a risk factor for non-syndromic BA, whereas the contribution of other monogenic variants to the disease predisposition is limited.

Risk assessment of assisted reproductive technology and parental age at childbirth for the development of uniparental disomy-mediated imprinting disorders caused by aneuploid gametes.

BACKGROUND: Our previous study suggested that assisted reproductive technology (ART) may be a possible risk factor for the development of epimutation-mediated imprinting disorders (epi-IDs) for mothers aged ≥ 30 years. However, whether ART or advanced parental age facilitates the development of uniparental disomy-mediated IDs (UPD-IDs) has not yet been investigated. RESULTS: We enrolled 130 patients with aneuploid UPD-IDs including various IDs confirmed by molecular studies and obtained ART data of the general population and patients with epi-IDs from a robust nationwide database and our previous report, respectively. We compared the proportion of ART-conceived livebirths and maternal childbearing age between patients with UPD-IDs and the general population or patients with epi-IDs. The proportion of ART-conceived livebirths in patients with aneuploid UPD-IDs was consistent with that in the general population of maternal age ≥ 30 years and was lower than that in the patients with epi-IDs, although there was no significant difference. The maternal childbearing age of patients with aneuploid UPD-IDs was skewed to the increased ages with several cases exceeding the 97.5th percentile of maternal childbearing age of the general population and significantly higher than that of patients with epi-IDs (P < 0.001). In addition, we compared the proportion of ART-conceived livebirths and parental age at childbirth between patients with UPD-IDs caused by aneuploid oocytes (oUPD-IDs) and that by aneuploid sperm (sUPD-IDs). Almost all ART-conceived livebirths were identified in patients with oUPD-IDs, and both maternal age and paternal age at childbirth were significantly higher in patients with oUPD-IDs than in patients with sUPD-IDs. Because maternal age and paternal age were strongly correlated (rs = 0.637, P < 0.001), higher paternal age in oUPD-IDs was explained by the higher maternal age in this group. CONCLUSIONS: Different from the case of epi-IDs, ART itself is not likely to facilitate the development of aneuploid UPD-IDs. We demonstrated that advanced maternal age can be a risk factor for the development of aneuploid UPD-IDs, particularly oUPD-IDs.

Perinatal and neonatal characteristics of Prader-Willi syndrome in Japan.

BACKGROUND: Prader-Willi syndrome (PWS) is suspected at birth if extreme hypotonia, difficulty in feeding, hypogonadism, and failure to thrive are present. Genetic diagnosis of PWS can generally be made within the first few months of life; however, a delayed diagnosis of PWS is frequently reported. Although the clinical characteristics of perinatal and neonatal patients with PWS have been reported, there are no such reports on the clinical characteristics of these patients in Japan. METHODS: This retrospective, single-center study involved 177 Japanese patients with PWS and their medical data regarding the perinatal and neonatal periods were evaluated. RESULTS: The median maternal age at birth was 34 years; 12.7% of the mothers had a history of assisted reproductive technology (ART). Of the mothers, 13.5% reported polyhydramnios and 4.3% had oligohydramnios. Decreased fetal movement during pregnancy was reported by 76% of the mothers. A total of 60.5% of patients were born by cesarean section. Genetic subtypes included deletions (66.1%), uniparental disomy (31.0%), imprinting defects (0.6%), and other or unknown subtypes (2.3%). The median birth length was 47.5 cm and the median birthweight was 2476 g. Of the 160 patients, 14 (8.8%) were classified as small for gestational age. Most patients had hypotonia (98.8%), and 89.3% required gavage feeding at birth. Breathing problems, congenital heart disease, and undescended testis were noted in 33.1%, 7.0%, and 93.5% of patients, respectively. CONCLUSION: In our study, higher rates of ART, polyhydramnios, decreased fetal movements, cesarean section, hypotonia, feeding difficulties, and undescended testis were observed in PWS.

Exome-based genome-wide screening of rare variants associated with the risk of polycystic ovary syndrome.

Purpose: Genetic factors associated with the risk of polycystic ovary syndrome (PCOS) remain largely unknown. Here, we conducted an optimal sequence kernel association test (SKAT-O), an exome-based rare variant association study, to clarify whether rare variants in specific genes contribute to the development of PCOS. Methods: SKAT-O was performed using exome data of 44 Japanese patients with PCOS and 301 control women. We analyzed frequencies of rare probably damaging variants in the genome. Results: Rare variants of GSTO2 were more commonly identified in the patient group than in the control group (6/44 vs. 1/301; Bonferroni-corrected p-value, 0.028), while the frequencies of variants in other genes were comparable between the two groups. The identified GSTO2 variants were predicted to affect the function, structure, stability, hydrophobicity, and/or the formation of intrinsically disordered regions of the protein. GSTO2 encodes a glutathione transferase that mediates the oxidative stress response and arsenic metabolism. Previously, common variants in GSTO2 and its paralog GSTO1 were associated with the risk of PCOS. Conclusions: The results indicate that there are no genes whose rare variants account for a large fraction of the etiology of PCOS, although rare damaging variants in GSTO2 may constitute a risk factor in some cases.

A nationwide survey of Schaaf-Yang syndrome in Japan.

Schaaf-Yang syndrome (SYS) is a congenital disorder characterized by developmental delay, autism spectrum disorder and congenital joint contractures. In this study, a nationwide epidemiological questionnaire-based survey of SYS in the Japanese population was conducted to establish patient numbers, clinical features and genetic information. In the primary survey, we investigated the number of SYS patients. In the secondary survey, we obtained and analyzed detailed clinical and genetic information of SYS patients. This survey collected information on 25 genetically-confirmed patients. The major clinical symptoms included neonatal hypotonia (96% of the patients), poor suck in infancy (82%), developmental delay (100%) and joint contractures (83%). Other main symptoms and findings included characteristic facial features (100%), small hands (92%), eye abnormalities (92%) and short stature (79%). Based on the information collected on activities of daily living, 71% of patients were unable to walk, while 67%, 71%, and 81% of patients required full assistance with eating, toileting and bathing, respectively. Regarding inheritability, the genetic analysis of 21 patients revealed that 14 (67%) carried de novo truncating variants in the melanoma antigen L2 (MAGEL2) gene and seven (33%) had inherited truncating variants from their fathers who were carriers. This survey revealed the clinical and genetic features in Japanese SYS patients. The majority of SYS patients required assistance in many aspects of daily living, and there were a certain number of carriers of the imprinting disorder.

Pathogenic Copy Number and Sequence Variants in Children Born SGA With Short Stature Without Imprinting Disorders.

CONTEXT: Children born small-for-gestational-age with short stature (SGA-SS) is associated with (epi)genetic defects, including imprinting disorders (IDs), pathogenic copy number variants (PCNVs), and pathogenic variants of genes involved in growth. However, comprehensive studies evaluating these 3 factors are very limited. OBJECTIVE: To clarify the contribution of PCNVs and candidate pathogenic variants to SGA-SS. DESIGN: Comprehensive molecular analyses consisting of methylation analysis, copy number analysis, and multigene sequencing. METHODS: We enrolled 140 patients referred to us for genetic testing for SGA-SS. Among them, we excluded 42 patients meeting Netchine-Harbison clinical scoring system criteria for Silver-Russell syndrome and 4 patients with abnormal methylation levels of the IDs-related differentially methylated regions. Consequently, we conducted copy number analysis and multigene sequencing for 86 SGA-SS patients with sufficient sample volume. We also evaluated clinical phenotypes of patients with PCNVs or candidate pathogenic variants. RESULTS: We identified 8 (9.3%) and 11 (12.8%) patients with PCNVs and candidate pathogenic variants, respectively. According to the American College of Medical Genetics standards and guidelines, 5 variants were classified as pathogenic and the remaining 6 variants were classified as variants of unknown significance. Genetic diagnosis was made in 12 patients. All patients with PCNVs or candidate pathogenic variants did not correspond perfectly to characteristic clinical features of each specific genetic cause. CONCLUSION: We clarified the contribution of PCNVs and pathogenic variants to SGA-SS without IDs. Comprehensive molecular analyses, including copy number analysis and multigene sequencing, should be considered for patients with unknown SGA-SS etiology.

Frequency and clinical characteristics of distinct etiologies in patients with Silver-Russell syndrome diagnosed based on the Netchine-Harbison clinical scoring system.

Silver-Russel syndrome (SRS) is a representative imprinting disorder (ID) characterized by growth failure and diagnosed by clinical features. Recently, international consensus has recommended using the Netchine-Harbison clinical scoring system (NH-CSS) as clinical diagnostic criteria. Loss of methylation of H19/IGF2:intergenic differentially methylated region (H19LOM) and maternal uniparental disomy chromosome 7 (UPD(7)mat) are common etiologies of SRS; however, other IDs, pathogenic variants (PVs) of genes, and pathogenic copy number variants (PCNVs) have been reported in patients meeting NH-CSS. To clarify the frequency and clinical characteristics of each etiology, we conducted (epi)genetic analysis in 173 patients satisfying NH-CSS. H19LOM and UPD(7)mat were identified in 34.1%. PCNVs, other IDs, and PVs were in 15.0%. Patients with all six NH-CSS items were most frequently observed with H19LOM and UPD(7)mat. This study confirmed the suitability of NH-CSS as clinical diagnostic criteria, the (epi)genetic heterogeneity of SRS, and showed the necessity of further discussion regarding the "SRS spectrum".

A boy with overgrowth caused by multi-locus imprinting disturbance including hypomethylation of MEST:alt-TSS-DMR.

Most imprinting disorders (IDs) entail growth abnormalities. Some patients with IDs caused by epimutation have multi-locus imprinting disturbance (MLID) showing aberrant methylation patterns in multiple differentially methylated regions (DMRs). Patients with MLID often have typical ID-specific symptoms. However, certain MLID cases have only non-specific symptoms, and it is necessary to clarify the association between their clinical features and the affected DMRs. We report a case of MLID presenting with overgrowth and temporarily impaired glucose tolerance. Genome-wide methylation analysis for the DMRs revealed hypomethylation of PLAGL1:alt-TSS-DMR, MEST:alt-TSS-DMR, and other DMRs. Because no MEST expression and increased PLAGL1 expression cause growth failure and transient neonatal diabetes mellitus, hypomethylation of MEST:alt-TSS-DMR and PLAGL1:alt-TSS-DMR may have caused overgrowth and temporary impaired glucose tolerance in our case. In cases with multiple non-specific ID-related symptoms, such as growth abnormalities, psychomotor developmental delay, and mild glucose metabolic disorders, multi-locus methylation analysis needs to be considered.

Collection of 2429 constrained headshots of 277 volunteers for deep learning.

Deep learning has rapidly been filtrating many aspects of human lives. In particular, image recognition by convolutional neural networks has inspired numerous studies in this area. Hardware and software technologies as well as large quantities of data have contributed to the drastic development of the field. However, the application of deep learning is often hindered by the need for big data and the laborious manual annotation thereof. To experience deep learning using the data compiled by us, we collected 2429 constrained headshot images of 277 volunteers. The collection of face photographs is challenging in terms of protecting personal information; we therefore established an online procedure in which both the informed consent and image data could be obtained. We did not collect personal information, but issued agreement numbers to deal with withdrawal requests. Gender and smile labels were manually and subjectively annotated only from the appearances, and final labels were determined by majority among our team members. Rotated, trimmed, resolution-reduced, decolorized, and matrix-formed data were allowed to be publicly released. Moreover, simplified feature vectors for data sciences were released. We performed gender and smile recognition by building convolutional neural networks based on the Inception V3 model with pre-trained ImageNet data to demonstrate the usefulness of our dataset.

Achievement of Developmental Milestones Recorded in Real Time: A Mobile App-Based Study.

OBJECTIVE: To clarify the characteristics of milestone achievement of Japanese infants using data assessed and recorded in real time via a Web-based app by caregivers. STUDY DESIGN: Between 2014 and 2019, the achievement of developmental milestones of 16 627 Japanese infants were recorded via the mobile app Papatto Ikuji along with pertinent information including sex, birth date, anthropometric measurements, and information on feeding. The milestones consisted of 20 items belonging to 4 domains: personal-social, fine motor-adaptative, language, and gross motor. The distribution of age at milestone achievement was compared with 4 conventional normative data. Subgroup analyses according to sex, season of birth, and feeding methods were performed with restricted mean survival time analysis. RESULTS: Age distributions of milestone achievement were generally similar in the app-based and conventional data. The exception was "Smile responsively," for which the age at achievement seemed higher in the app-based data. Subgroup analyses showed female-dominant sex differences in the achievement of person-social and language milestones. The results also showed seasonality in achievement of gross motor milestones, with earlier achievement in infants born in winter compared with those born in summer. CONCLUSIONS: We describe mobile app-based data on developmental milestone achievements that were recorded in real time. The app-based data were generally comparable with conventional normative data, suggesting that its potential to assess the development of children in real time. This could be used complementarily with the current well-child visits at scheduled time points.

Psychiatric behavioral effect and characteristics of type 2 diabetes mellitus on Japanese patients with Prader-Willi syndrome: a preliminary retrospective study.

OBJECTIVES: In recent years, research on behavioral and psychiatric problems of adults with Prader-Willi syndrome (PWS) has gained attention. However, no report is available regarding the relationship between psychiatric illness and type 2 diabetes mellitus (T2DM) in patients with PWS. Therefore, we evaluated a behavioral assessment to address the lack of data on the association between psychiatric behavior and T2DM. METHODS: This was a retrospective single-center study of patients with PWS. Patients with PWS whose blood tests were performed in our hospital between January 2018 and December 2019 and aged >10 years were included. We evaluated the data, including the behavioral patterns of Japanese PWS patients with T2DM. RESULTS: Overall, 114 patients were evaluated; 33 patients (28.9%) developed T2DM. The age of T2DM onset was 18.0 years (interquartile range [IQR], 14.6-21.4 years). The median body mass index at T2DM onset was 33.7 kg/m2 (IQR, 30.0-37.4 kg/m2). Between-group comparisons of the intelligence quotient, Food-Related Problem Questionnaire (FRPQ), and Japanese versions of the Short Sensory Profile and Aberrant Behavior Checklist showed a significant difference only in FRPQ scores (p=0.003). CONCLUSIONS: The occurrence of T2DM among Japanese patients with PWS remains high. Only the FRPQ was significantly different between the T2DM and the non-T2DM group.

Female-dominant estrogen production in healthy children before adrenarche.

OBJECTIVE: Ultra-sensitive hormone assays have detected slight sex differences in blood estradiol (E2) levels in young children before adrenarche. However, the origin of circulating E2 in these individuals remains unknown. This study aimed to clarify how E2 is produced in young girls before adrenarche. DESIGN: This is a satellite project of the Japan Environment and Children's Study organized by the National Institute for Environmental Studies. METHODS: We collected blood samples from healthy 6-year-old Japanese children (79 boys and 71 girls). Hormone measurements and data analysis were performed in the National Institute for Environmental Studies and the Medical Support Center of the Japan Environment and Children's Study, respectively. RESULTS: E2 and follicle stimulating hormone (FSH) levels were significantly higher in girls than in boys, while dehydroepiandrosterone sulfate (DHEA-S) and testosterone levels were comparable between the two groups. Girls showed significantly higher E2/testosterone ratios than boys. In children of both sexes, a correlation was observed between E2 and testosterone levels and between testosterone and DHEA-S levels. Moreover, E2 levels were correlated with FSH levels only in girls. CONCLUSIONS: The results indicate that in 6-year-old girls, circulating E2 is produced primarily in the ovary from adrenal steroids through FSH-induced aromatase upregulation. This study provides evidence that female-dominant E2 production starts several months or years before adrenarche. The biological significance of E2 biosynthesis in these young children needs to be clarified in future studies.

Methylation status of genes escaping from X-chromosome inactivation in patients with X-chromosome rearrangements.

BACKGROUND: X-chromosome inactivation (XCI) is a mechanism in which one of two X chromosomes in females is randomly inactivated in order to compensate for imbalance of gene dosage between sexes. However, about 15% of genes on the inactivated X chromosome (Xi) escape from XCI. The methylation level of the promoter region of the escape gene is lower than that of the inactivated genes. Dxz4 and/or Firre have critical roles for forming the three-dimensional (3D) structure of Xi. In mice, disrupting the 3D structure of Xi by deleting both Dxz4 and Firre genes led to changing of the escape genes list. To estimate the impact for escape genes by X-chromosome rearrangements, including DXZ4 and FIRRE, we examined the methylation status of escape gene promoters in patients with various X-chromosome rearrangements. RESULTS: To detect the breakpoints, we first performed array-based comparative genomic hybridization and whole-genome sequencing in four patients with X-chromosome rearrangements. Subsequently, we conducted array-based methylation analysis and reduced representation bisulfite sequencing in the four patients with X-chromosome rearrangements and controls. Of genes reported as escape genes by gene expression analysis using human hybrid cells in a previous study, 32 genes showed hypomethylation of the promoter region in both male controls and female controls. Three patients with X-chromosome rearrangements had no escape genes with abnormal methylation of the promoter region. One of four patients with the most complicated rearrangements exhibited abnormal methylation in three escape genes. Furthermore, in the patient with the deletion of the FIRRE gene and the duplication of DXZ4, most escape genes remained hypomethylated. CONCLUSION: X-chromosome rearrangements are unlikely to affect the methylation status of the promoter regions of escape genes, except for a specific case with highly complex rearrangements, including the deletion of the FIRRE gene and the duplication of DXZ4.

Growth hormone treatment and bone mineral density in pediatric patients with Prader-Willi syndrome.

OBJECTIVES: Previous reports indicate that growth hormone (GH) treatment for Prader-Willi syndrome (PWS) improves bone mineral density (BMD) only when initiated at a young age and not when initiated in adulthood. However, there are no data on BMD during long-term GH treatment of Japanese children and adolescents with PWS. Thus, this study aimed to investigate BMD changes among patients with PWS, who were undergoing GH treatment from childhood to adolescence. METHODS: Sixty-seven pediatric patients with PWS who had GH treatment initiated during childhood between January 2003 and June 2020 were evaluated. To avoid underestimation, we used total body BMD, which was evaluated using dual-X-ray absorptiometry adjusted for the BMD z-score using patient height, sex, and age. RESULTS: In both sexes, age was negatively correlated with the BMD-standard deviation score (SDS) (male: r=-0.156 [p=0.042]; female: r=-0.197 [p=0.043]), which started to decrease in childhood. CONCLUSIONS: The BMD-SDS of patients with PWS decreases gradually despite GH treatment. As there are no clear recommendations about monitoring of bone health in patients with PWS, further studies are needed to improve the guidelines for screening of BMD and treatment of patients with PWS.

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

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.

A patient with Silver-Russell syndrome with multilocus imprinting disturbance, and Schimke immuno-osseous dysplasia unmasked by uniparental isodisomy of chromosome 2.

Silver-Russell syndrome (SRS) is a congenital disorder characterized by prenatal and postnatal growth failure and craniofacial features. Hypomethylation of the H19/IGF2:IG-differential methylated region (H19LOM) is observed in 50% of SRS patients, and 15% of SRS patients with H19LOM had multilocus imprinting disturbance (MLID). Schimke immuno-osseous dysplasia (SIOD), characterized by spondyloepiphyseal dysplasia and nephropathy, is an autosomal recessive disorder caused by mutations in SMARCAL1 on chromosome 2. We report a patient with typical SRS-related features, spondyloepiphyseal dysplasia, and severe nephropathy. Molecular analyses showed H19LOM, paternal uniparental isodisomy of chromosome 2 (iUPD(2)pat), and a paternally inherited homozygous frameshift variant in SMARCAL1. Genome-wide methylation analysis showed MLID in this patient, although it showed no MLID in another patient with SIOD without SRS phenotype. These results suggest that iUPD(2)pat unmasked the recessive mutation in SMARCAL1 and that the SMARCAL1 gene mutation may have no direct effect on the patient's methylation defects.