Tomonobu Hasegawa

Gain-of-function mutations in RIT1 cause Noonan syndrome, a RAS/MAPK pathway syndrome.

RAS GTPases mediate a wide variety of cellular functions, including cell proliferation, survival, and differentiation. Recent studies have revealed that germline mutations and mosaicism for classical RAS mutations, including those in HRAS, KRAS, and NRAS, cause a wide spectrum of genetic disorders. These include Noonan syndrome and related disorders (RAS/mitogen-activated protein kinase [RAS/MAPK] pathway syndromes, or RASopathies), nevus sebaceous, and Schimmelpenning syndrome. In the present study, we identified a total of nine missense, nonsynonymous mutations in RIT1, encoding a member of the RAS subfamily, in 17 of 180 individuals (9%) with Noonan syndrome or a related condition but with no detectable mutations in known Noonan-related genes. Clinical manifestations in the RIT1-mutation-positive individuals are consistent with those of Noonan syndrome, which is characterized by distinctive facial features, short stature, and congenital heart defects. Seventy percent of mutation-positive individuals presented with hypertrophic cardiomyopathy; this frequency is high relative to the overall 20% incidence in individuals with Noonan syndrome. Luciferase assays in NIH 3T3 cells showed that five RIT1 alterations identified in children with Noonan syndrome enhanced ELK1 transactivation. The introduction of mRNAs of mutant RIT1 into 1-cell-stage zebrafish embryos was found to result in a significant increase of embryos with craniofacial abnormalities, incomplete looping, a hypoplastic chamber in the heart, and an elongated yolk sac. These results demonstrate that gain-of-function mutations in RIT1 cause Noonan syndrome and show a similar biological effect to mutations in other RASopathy-related genes.

CXorf6 is a causative gene for hypospadias

46,XY disorders of sex development (DSD) refer to a wide range of abnormal genitalia, including hypospadias, which affects ∼0.5% of male newborns. We identified three different nonsense mutations of CXorf6 in individuals with hypospadias and found that its mouse homolog was specifically expressed in fetal Sertoli and Leydig cells around the critical period for sex development. These data imply that CXorf6 is a causative gene for hypospadias.

Molecular cytogenetic analysis of eight inversion duplications of human chromosome 13q that each contain a neocentromere.

Neocentromeres are fully functional centromeres that have arisen in previously noncentromeric chromosomal locations on rearranged chromosomes. The formation of neocentromeres results in the mitotic stability of chromosomal fragments that do not contain endogenous centromeres and that would normally be lost. Here we describe a unique collection of eight independent patient-derived cell lines, each of which contains a neocentromere on a supernumerary inversion duplication of a portion of human chromosome 13q. Findings in these patients reveal insight into the clinical manifestations associated with polysomy for portions of chromosome 13q. The results of FISH and immunofluorescent analysis of the neocentromeres in these chromosomes confirm the lack of alpha-satellite DNA and the presence of CENtromere proteins (CENP)-C, -E, and hMAD2. The positions of the inversion breakpoints in these chromosomes have been placed onto the physical map of chromosome 13, by means of FISH mapping with cosmid probes. These cell lines define, within chromosome 13q, at least three distinct locations where neocentromeres have formed, with five independent neocentromeres in band 13q32, two in band 13q21, and one in band 13q31. The results of examination of the set of 40 neocentromere-containing chromosomes that have thus far been described, including the 8 neocentromere-containing chromosomes from chromosome 13q that are described in the present study, suggest that chromosome 13q has an increased propensity for neocentromere formation, relative to some other human chromosomes. These neocentromeres will provide the means for testing hypotheses about sequence requirements for human centromere formation.

Novel and recurrent EBP mutations in X-linked dominant chondrodysplasia punctata

Chondrodysplasia punctata (CDP) is a heterogeneous group of skeletal dysplasias characterized by stippled epiphyses. A subtype of CDP, X-linked dominant chondrodysplasia punctata (CDPX2), known also as Conradi-Hünermann-Happle syndrome, is a rare skeletal dysplasia characterized by short stature, craniofacial defects, cataracts, ichthyosis, coarse hair, and alopecia. The cause of CDPX2 was unknown until recent identification of mutations in the gene encoding Delta(8),Delta(7) sterol isomerase emopamil-binding protein (EBP). Twelve different EBP mutations have been reported in 14 patients with CDPX2 or unclassified CDP, but with no evidence of correlation between phenotype and nature of the mutation. To characterize additional mutations and investigate possible phenotype-genotype correlation, we sequenced the entire EBP gene in 8 Japanese individuals with CDP; 5 of them presented with a CDPX2 phenotypes. We found EBP mutations in all 5 CDPX2 individuals, but none in non-CDPX2 individuals. Three of these CDPX2 individuals carried novel nonsense mutations in EBPand the other two, separate missense mutations that had been reported also in different ethnic groups. Our results, combined with previous information, suggest all EBP mutations that produce truncated proteins result in typical CDPX2, whereas the phenotypes resulted from missense mutations are not always typical for CDPX2. Patients with nonsense mutations showed abnormal sterol profiles consistent with a defect in Delta(8), Delta(7) sterol isomerase. X-inactivation patterns of the patients showed no skewing, an observation that supports the assumption that inactivation of the EBP gene occurs at random in affected individuals.

Transcription Factor Mutations and Congenital Hypothyroidism: Systematic Genetic Screening of a Population-Based Cohort of Japanese Patients

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.

Cytochrome P450 oxidoreductase deficiency: identification and characterization of biallelic mutations and genotype-phenotype correlations in 35 Japanese patients.

CONTEXT Cytochrome P450 oxidoreductase (POR) deficiency is a rare autosomal recessive disorder characterized by skeletal dysplasia, adrenal dysfunction, disorders of sex development (DSD), and maternal virilization during pregnancy. Although multiple studies have been performed for this condition, several matters remain to be clarified, including the presence of manifesting heterozygosity and the underlying factors for clinical variability. OBJECTIVE The objective of the study was to examine such unresolved matters by detailed molecular studies and genotype-phenotype correlations. PATIENTS Thirty-five Japanese patients with POR deficiency participated in the study. RESULTS Mutation analysis revealed homozygosity for R457H in cases 1-14 (group A), compound heterozygosity for R457H and one apparently null mutation in cases 15-28 (group B), and other combinations of mutations in cases 29-35 (group C). In particular, FISH and RT-PCR sequencing analyses revealed an intragenic microdeletion in one apparent R457H homozygote, transcription failure of apparently normal alleles in three R457H heterozygotes, and nonsense mediated mRNA decay in two frameshift mutation-positive cases examined. Genotype-phenotype correlations indicated that skeletal features were definitely more severe, and adrenal dysfunction, 46,XY DSD, and pubertal failure were somewhat more severe in group B than group A, whereas 46,XX DSD and maternal virilization during pregnancy were similar between two groups. Notable findings also included the contrast between infrequent occurrence of 46,XY DSD and invariable occurrence of 46,XX DSD and pubertal growth pattern in group A mimicking that of aromatase deficiency. CONCLUSIONS The results argue against the heterozygote manifestation and suggest that the residual POR activity reflected by the R457H dosage constitutes the underlying factor for clinical variability in some features but not other features, probably due to the simplicity and complexity of POR-dependent metabolic pathways relevant to each phenotype.

Standardized centile curves of body mass index for Japanese children and adolescents based on the 1978–1981 national survey data

Background: The prevalence of overweight among Japanese children and adolescents has steadily increased during the last 20 years. Thus, we utilized the 1978–1981 data collected by the Japanese Ministry of International Trade and Industry to construct reference curves of body mass index (BMI) for contemporary Japanese children and adolescents. Methods: BMI reference values were derived using the LMS method as based on height and weight data from the cross-sectional national survey of Japanese children and adolescents conducted in 1978–1981 (14 012 boys and 13 781 girls, aged 1.5–18.5 years). Results: The Japanese BMI reference curves were constructed for clinical use. The centile values at the upper end of the spectrum apparently differed in British, Dutch, Japanese, and US children and adolescents. In contrast, the centile values at the lower end of the spectrum nearly overlapped with each other in the four populations. Conclusions: Overweight is concentrated in a subgroup of children and does not occur across the entire population of British, Dutch, Japanese, and US children, indicating a subgroup of genetically and/or environmentally more susceptible children in each country. Résumé. Arrière plan: La prévalence du surpoids chez les enfants et adolescents japonais, s’est régulièrement accrue au cours des vingt dernières années. C’est pourquoi nous avons utilisé les données recueillies par le Ministère du Commerce International et de l’Industrie entre 1978 et 1981, afin de construire ds courbes de référence de l’indice de masse corporelle (IMC) des enfants et adolescens japonais contemporains. Méthodes: Les valeurs d’IMC de référence ont été obtenues par la méthode des moindres carrés à partir de données transversales de poids et de stature récoltées entre 1978 et 1981 sur 14012 garçons et 13781 filles âgés de 1,5 à 18,5 ans. Résultats: Les courbes de référence de l’IMC des japonais ont été construites en vue d’un usage clinique. Les valeurs les plus élevées des centiles diffèrent apparemment de celles des enfants et adolescents britanniques, néerlandais et des Etats–Unis. Au contraire, les valeurs de centiles les plus basses se chevauchent presque dans les quatre populations. Conclusions: Le surpoids est concentré dans un sous groupe d’enfants et ne s’observe pas dans l’ensemble de la population des enfants britanniques, néerlandais, japonais et des Etats–Unis, ce qui signale l’existence d’un sous groupe plus susceptible dans chaque pays, que ce soit génétiquement et/ou pour des causes environnementales. Zusammenfassung. Hintergrund: Die Prävalenz von Übergewicht bei Japanischen Kindern und Jugendlichen ist in den letzten 20 Jahren stetig gestiegen. Aus diesem Grunde verwendeten wir Daten, die in den Jahren 1978--1981 vom Japanischen Ministerium für Internationalen Handel und Industrie (Japanese Ministry of International Trade and Industry) gesammelt worden waren, um BMI-Referenzkurven für heutige Japanische Kinder und Jugendlichen zu konstruieren. Methoden: BMI-Referenzwerte wurden mit LMS-Methode basierend auf Körperhöhen- und Gewichtsdaten der nationalen Querschnittsstudie an Japanischen Kindern und Jugendlichen der Jahre 1978--1981 (14012 Knaben und 13781 Mädchen im Alter von 1,5–18,5 Jahren) entwickelt. Ergebnisse: Die Japanischen BMI-Referenzkurven wurden für den klinischen Gebrauch konstruiert. Die Perzentilwerte aus dem oberen Ende des Spektrums unterschieden sich offensichtlich von Britischen, Niederländischen, Japanischen und US-Amerikanischen Kindern und Jugendlichen. Dagegen überlappten sich die Perzentilen weitgehend am unteren Ende des Wertespektrums mit den denen der anderen vier Populationen. Zusammenfassung: Übergewicht findet sich vorwiegend in einer Untergruppe von Kindern und kommt nicht in der gesamten Population von Britischen, Niederländischen, Japanischen und US-Amerikanischen Kindern vor, was darauf hindeutet, dass es eine Untergruppe von genetisch und/oder umweltbedingt empfindlicheren Kindern in jedem dieser Länder gibt. Resumen. Antecedentes: La prevalencia de sobrepeso entre los niños y adolescentes japoneses ha aumentado de manera continua durante los últimos 20 años. Por ello, se han empleado los datos recogidos entre 1978--1981 por el Ministerio Japonés de Comercio Internacional e Industria, para construir curvas de referencia del índice de masa corporal (IMC) para los niños y adolescentes actuales. Métodos: Los valores de referencia del IMC se obtuvieron mediante el método LMS, utilizando datos de la estatura y peso procedentes de la encuesta nacional transversal de niños y adolescentes japoneses, realizada entre 1978--1981 (14.012 chicos y 13.781 chicas, de entre 1,5 y 18,5 años de edad). Resultados: Las curvas de referencia del IMC de los japoneses se construyeron para uso clínico. Los valores centilares en el extremo superior del espectro diferían aparentemente entre los niños y adolescentes británicos, holandeses, japoneses y estadounidenses. Por el contrario, los valores centilares en el extremo inferior del espectro se solapaban en las cuatro poblaciones. Conclusiones: El sobrepeso se concentra en un subgrupo de niños y no se encuentra en toda la población de niños británicos, holandeses, japoneses y estadounidenses, lo que apunta a la existencia de un subgrupo de niños más susceptible genética y/o ambientalmente en cada país.

SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7

Adrenal hypoplasia is a rare, life-threatening congenital disorder. Here we define a new form of syndromic adrenal hypoplasia, which we propose to term MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) syndrome. By exome sequencing and follow-up studies, we identified 11 patients with adrenal hypoplasia and common extra-adrenal features harboring mutations in SAMD9. Expression of the wild-type SAMD9 protein, a facilitator of endosome fusion, caused mild growth restriction in cultured cells, whereas expression of mutants caused profound growth inhibition. Patient-derived fibroblasts had restricted growth, decreased plasma membrane EGFR expression, increased size of early endosomes, and intracellular accumulation of giant vesicles carrying a late endosome marker. Of interest, two patients developed myelodysplasitc syndrome (MDS) that was accompanied by loss of the chromosome 7 carrying the SAMD9 mutation. Considering the potent growth-restricting activity of the SAMD9 mutants, the loss of chromosome 7 presumably occurred as an adaptation to the growth-restricting condition.

Heterozygous Mutations in Natriuretic Peptide Receptor-B (NPR2) Gene as a Cause of Short Stature in Patients Initially Classified as Idiopathic Short Stature

CONTEXT Based on the stature observed in relatives of patients with acromesomelic dysplasia, type Maroteaux, homozygous for mutations in natriuretic peptide receptor B gene (NPR2), it has been suggested that heterozygous mutations in this gene could be responsible for the growth impairment observed in some children with idiopathic short stature (ISS). OBJECTIVE The objective of the study was to investigate the presence of NPR2 mutations in a group of patients with ISS. PATIENTS AND METHODS The NPR2 coding region was directly sequenced in 47 independent patients with ISS. The functional consequences of NPR2 nonsynonymous variations were established using in vitro cell-based assays. RESULTS Three novel heterozygous NPR2 mutations were identified: c.226T>C (p.Ser76Pro), c.788G>C (p.Arg263Pro), and c.2455C>T (p.Arg819Cys). These allelic variants were not found in our controls or in the 1000 Genomes database. In silico analysis suggested that the three missense mutations are probably damaging. All of them were selected for in vitro functional evaluation. Cells transfected with the three mutants failed to produce cyclic GMP after treatment with C-type natriuretic peptide. Cells cotransfected with mutant and wild-type-NPR-B (1:1) showed a significant decrease in cGMP levels after C-type natriuretic peptide stimulation in comparison with cells cotrasnfected with empty vector and wild type, suggesting a dominant-negative effect. These three mutations segregated with short stature phenotype in an autosomal dominant pattern (height SD score ranged from -4.5 to -1.7). One of these patients and two relatives have disproportionate short stature, whereas in another patient a nonspecific skeletal abnormality was observed. All three of these patients were treated with recombinant human GH (33-50 μg/kg · d) without significant height SD score change during therapy. CONCLUSIONS We identified heterozygous NPR2 mutations in 6% of patients initially classified as ISS. Affected patients have mild and variable degrees of short stature without a distinct phenotype. Heterozygous mutations in NPR2 could be an important cause of nonsyndromic familial short stature.

Backdoor pathway for dihydrotestosterone biosynthesis: Implications for normal and abnormal human sex development

We review the current knowledge about the “backdoor” pathway for the biosynthesis of dihydrotestosterone (DHT). While DHT is produced from cholesterol through the conventional “frontdoor” pathway via testosterone, recent studies have provided compelling evidence for the presence of an alternative “backdoor” pathway to DHT without testosterone intermediacy. This backdoor pathway is known to exist in the tammar wallaby pouch young testis and the immature mouse testis, and has been suggested to be present in the human as well. Indeed, molecular analysis has identified pathologic mutations of genes involved in the backdoor pathway in genetic male patients with undermasculinized external genitalia, and urine steroid profile analysis has argued for the relevance of the activated backdoor pathway to abnormal virilization in genetic females with cytochrome P450 oxidoreductase deficiency and 21‐hydroxylase deficiency. It is likely that the backdoor pathway is primarily operating in the fetal testis in a physiological condition to produce a sufficient amount of DHT for male sex development, and that the backdoor pathway is driven with a possible interaction between fetal and permanent adrenals in pathologic conditions with increased 17‐hydroxyprogesterone levels. These findings provide novel insights into androgen biosynthesis in both physiological and pathological conditions. Developmental Dynamics 242:320–329, 2013.