Molecular basis of non-syndromic hypospadias: systematic mutation screening and genome-wide copy-number analysis of 62 patients.

STUDY QUESTION: What percentage of cases with non-syndromic hypospadias can be ascribed to mutations in known causative/candidate/susceptibility genes or submicroscopic copy-number variations (CNVs) in the genome? SUMMARY ANSWER: Monogenic and digenic mutations in known causative genes and cryptic CNVs account for >10% of cases with non-syndromic hypospadias. While known susceptibility polymorphisms appear to play a minor role in the development of this condition, further studies are required to validate this observation. WHAT IS KNOWN ALREADY: Fifteen causative, three candidate, and 14 susceptible genes, and a few submicroscopic CNVs have been implicated in non-syndromic hypospadias. STUDY DESIGN, SIZE, DURATION: Systematic mutation screening and genome-wide copy-number analysis of 62 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study group consisted of 57 Japanese and five Vietnamese patients with non-syndromic hypospadias. Systematic mutation screening was performed for 25 known causative/candidate/susceptibility genes using a next-generation sequencer. Functional consequences of nucleotide alterations were assessed by in silico assays. The frequencies of polymorphisms in the patient group were compared with those in the male general population. CNVs were analyzed by array-based comparative genomic hybridization and characterized by fluorescence in situ hybridization. MAIN RESULTS AND THE ROLE OF CHANCE: Seven of 62 patients with anterior or posterior hypospadias carried putative pathogenic mutations, such as hemizygous mutations in AR, a heterozygous mutation in BNC2, and homozygous mutations in SRD5A2 and HSD3B2. Two of the seven patients had mutations in multiple genes. We did not find any rare polymorphisms that were abundant specifically in the patient group. One patient carried mosaic dicentric Y chromosome. LIMITATIONS, REASONS FOR CAUTION: The patient group consisted solely of Japanese and Vietnamese individuals and clinical and hormonal information of the patients remained rather fragmentary. In addition, mutation analysis focused on protein-altering substitutions. WIDER IMPLICATIONS OF THE FINDINGS: Our data provide evidence that pathogenic mutations can underlie both mild and severe hypospadias and that HSD3B2 mutations cause non-syndromic hypospadias as a sole clinical manifestation. Most importantly, this is the first report documenting possible oligogenicity of non-syndromic hypospadias. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology; by the Grant-in-Aid from the Japan Society for the Promotion of Science; by the Grants from the Ministry of Health, Labour and Welfare, from the National Center for Child Health and Development and from the Takeda Foundation. The authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Abnormal basiocciput development in CHARGE syndrome.

BACKGROUND AND PURPOSE: The causative gene of the common congenital malformation referred to as CHARGE syndrome is CHD7. Affected individuals often undergo head and neck imaging to assess abnormalities of the olfactory structures, hypothalamus-pituitary axis, and inner ear. We encountered a few children with severe hypoplasia of the basiocciput during a radiologic assessment of patients with CHARGE syndrome. To our knowledge, this anomaly has not been reported. Our purpose was to evaluate the incidence and severity of this anomaly in this syndrome. MATERIALS AND METHODS: Sagittal MR images of 8 patients with CHARGE syndrome were retrospectively reviewed by 2 radiologists who consensually evaluated the status of the basiocciput of the patients with CHARGE syndrome, as either normal or hypoplastic; and associated anomalies, which include basilar invagination, Chiari type I malformation, and syringomyelia, as either present or absent. The length between the basion (Ba) and the endo-sphenobasion (Es) and between the basion and the exo-sphenobasion (Xs) was measured on midsagittal MR images of the 8 patients and 70 age-matched controls. We searched for trends related to age in the length of Ba-Es and Ba-Xs of the control children by using a matched t test. RESULTS: Basioccipital hypoplasia was identified in 7 of the 8 patients with CHARGE syndrome and was severe in 6. Of those, 5 had associated basilar invagination and 1 had Chiari type I malformation with syringomyelia. CONCLUSIONS: Basioccipital hypoplasia and basilar invagination are prevalent in patients with CHARGE syndrome.

Androgen receptor gene and male genital anomaly.

Male sexual differentiation, testicular descent, and spermatogenesis require androgens. Their action is mediated through the androgen receptor (AR), which binds to the androgen responsive element on DNA and regulates gene transcription. No mutations in any of the AR gene exons 1-8 are detected in males with isolated cryptorchidism, hypospadias, micropenis, or idiopathic male infertility. In addition, the CAG repeat length in exon 1 of the AR gene does not expand in males with isolated cryptorchidism, hypospadias, micropenis, or idiopathic male infertility. These facts indicate that an alteration of the AR gene is rare in these males. However, further studies will permit a better clarification on the relevance of the AR gene abnormalities to the development of isolated cryptorchidism, hypospadias, micropenis, or impaired spermatogenesis.

The proportion of cells with functional X disomy is associated with the severity of mental retardation in mosaic ring X Turner syndrome females.

Turner syndrome females (45,X) do not have mental retardation (MR), whereas some mosaic ring X Turner syndrome females, with 45,X/46,X,r(X), have severe MR. The MR is believed to be caused by a failure of X chromosome inactivation (XCI) of the small ring X chromosome, which leads to functional X disomy (FXD), To explore this hypothesis, we examined the proportion of FXD cells in the peripheral blood of four ring X Turner syndrome females with various levels of MR, using two newly developed XCI assays based on DNA methylation of X-linked genes. As a result, the two patients with extremely severe MR showed complete FXD patterns, whereas the remaining two patients with relatively milder MR showed partial FXD patterns. These results indicate that the proportion of FXD cells may be associated with the severity of MR in mosaic ring X Turner syndrome females, although this association should be confirmed by examining brain cells during development. One of the cases with severe MR and a complete FXD pattern neither lacked the XIST gene nor had uniparental X isodisomy, and we discuss the mechanism of the failure of XCI in this case.

Micropenis and the AR Gene: mutation and CAG repeat-length analysis.

Various mutations of the AR gene and expanded CAG repeats at exon 1 of that gene have been reported in patients with hypospadias or genital ambiguity. However, the role of the AR gene has not been systemically studied in those with isolated micropenis lacking hypospadias or genital ambiguity. We studied 64 Japanese boys with isolated micropenis (age, 0-14 yr; median, 7 yr), whose stretched penile lengths were between -2.5 and -2.0 SD (borderline micropenis) in 31 patients (age, 0-13 yr; median, 8 yr) and below -2.5 SD (definite micropenis) in 33 patients (age, 0-14 yr; median, 6 yr). Mutation analysis of the AR gene was performed for exons 1-8 and their flanking introns, except for the CAG and GGC repeat regions at exon 1, by denaturing HPLC and direct sequencing, identifying a substitution of cytosine to thymine at a position -3 in the 3' splice site of intron 1 in a patient with definite micropenis. CAG repeat length at exon 1 was determined by electrophoresis with internal size markers and direct sequencing, revealing no statistically significant difference in the distribution of CAG repeat lengths [median (range) and mean +/- SE: total patients with isolated micropenis, 24 (14-34) and 23.5 +/- 0.38; patients with borderline micropenis, 24 (15-29) and 23.5 +/- 0.53; patients with definite micropenis, 23 (14-34) and 23.5 +/- 0.56; and 100 control males, 23 (16-32) and 23.5 +/- 0.29] or in the frequency of long CAG repeats (percentage of CAG repeats > or =26 and > or =28: total patients with isolated micropenis, 17.2 and 4.7%; patients with borderline micropenis, 19.4 and 6.5%; patients with definite micropenis, 15.2 and 3.0%; and 100 control males, 21.0 and 10.0%). These results suggest that an AR gene mutation is rare and that CAG repeat length is not expanded in children with isolated micropenis.

Turner syndrome and Xp deletions: clinical and molecular studies in 47 patients.

Although clinical features of Turner syndrome have primarily been explained by the dosage effects of SHOX (short stature homeobox-containing gene) and the putative lymphogenic gene together with chromosomal effects leading to nonspecific features, several matters remain to be determined, including modifying factors for the effects of SHOX haploinsufficiency, chromosomal location of the lymphogenic gene, and genetic factors for miscellaneous features such as multiple pigmented nevi. To clarify such unresolved issues, we examined clinical findings in 47 patients with molecularly defined Xp deletion chromosomes accompanied by the breakpoints on Xp21-22 (group 1; n = 19), those accompanied by the breakpoints on Xp11 (group 2; n = 16), i(Xq) or idic(X)(p11) chromosomes (group 3; n = 8), and interstitial Xp deletion chromosomes (group 4; n = 4). The deletion size of each patient was determined by fluorescence in situ hybridization and microsatellite analyses for 38 Xp loci including SHOX, which was deleted in groups 1-3 and preserved in group 4. The mean GH-untreated adult height was -2.2 SD in group 1 and -2.7 SD in group 2 (GH-untreated adult heights were scanty in group 3). The prevalence of spontaneous breast development in patients aged 12.8 yr or more (mean +/- 2 SD for B2 stage) was 11 of 11 in group 1, 7 of 12 in group 2, and 1 of 7 in group 3. The prevalence of wrist abnormality suggestive of Madelung deformity was 8 of 18 in group 1 and 2 of 23 in groups 2 and 3, and 9 of 18 in patients with spontaneous puberty and 1 of 23 in those without spontaneous puberty. The prevalence of short neck was 1 of 19 in group 1 and 7 of 24 in groups 2 and 3. Soft tissue and visceral anomalies were absent in group 1 preserving the region proximal to Duchenne muscular dystrophy and were often present in groups 2 and 3 missing the region distal to monoamine oxidase A (MAOA). Multiple pigmented nevi were observed in groups 1-3, with the prevalence of 0 of 7 in patients less than 10 yr of age and 15 of 36 in those 10 yr or older regardless of the presence or absence of spontaneous puberty. Turner phenotype was absent in group 4, including a fetus aborted at 21 wk gestation who preserved the region distal to MAOA. The results provide further support for the idea that clinical features in X chromosome aberrations are primarily explained by haploinsufficiency of SHOX and the lymphogenic gene and by the extent of chromosome imbalance in mitotic cells and pairing failure in meiotic cells. Furthermore, it is suggested that 1) expressivity of SHOX haploinsufficiency in the limb and faciocervical regions is primarily influenced by gonadal function status and the presence or absence of the lymphogenic gene, respectively; 2) the lymphogenic gene for soft tissue and visceral stigmata is located between Duchenne muscular dystrophy and MAOA; and 3) multiple pigmented nevi may primarily be ascribed to cooperation between a hitherto unknown genetic factor and an age-dependent factor other than gonadal E.

Female gonadal development in XX patients with distal 9p monosomy.

OBJECTIVE: A sex determining gene(s) has been mapped to a approximately 700 kb region distal to the exons of DMRT1 on 9p. The aim of this study was to examine gonadal developmental status in XX patients hemizygous for the 9p sex determining region. DESIGN: Clinical and molecular studies were performed in an 8-year-old girl with 46,XX,del(9)(p22) (case 1) and in a 2-year-old girl with 46,XX,del(9)(p23) (case 2). METHODS: Ovarian function status was assessed by gonadotrophin-releasing hormone (GnRH) tests. Hemizygosity for the sex determining region was examined by fluorescence in situ hybridisation and microsatellite analyses for a total of 17 loci on distal 9p. RESULTS: GnRH tests indicated mild gonadotrophin hyper responses in both cases (case 1: follicle stimulating hormone 9.2-->22.7 IU/l, luteinising hormone 0.7 --> 16.6 IU/l; case 2: follicle stimulating hormone 7.6 --> 38.2 IU/l, luteinising hormone 0.6 --> 9.4 IU/l). Molecular studies showed hemizygosity for the 9p sex determining region in both cases. CONCLUSIONS: The results, in conjunction with previous reports describing sex development in XX and XY patients hemizygous for the 9p sex determining region, imply that haploinsufficiency of the 9p sex determining gene(s) primarily hinders the formation of the indifferent gonad, leading to a wide range of testicular or ovarian development.

Impaired urinary water excretion in a three-generation family.

We report on a three-generation family (daughter, mother, and maternal grandmother) with a syndrome of inappropriate secretion of antidiuretic hormone (SIADH)-like condition in the absence of inappropriate ADH secretion. In the three females, a water load test showed severely reduced urinary water excretion, with the ratio of urine volume to the loaded water being 10-33% (normal value: 70.2 +/- 7.8%). Urinary AQP2 excretion was normal, as was the DNA sequence of AVPR2 and AQP2. The results suggest the presence of a new dominantly inherited disorder for tubular water resorption.

Screening for mutations of the androgen receptor gene in patients with isolated cryptorchidism.

OBJECTIVE: To evaluate the occurrence of mutations of androgen receptor (AR) gene in patients with isolated cryptorchidism. DESIGN: Controlled clinical study. SETTING: Yamagata University Hospital, Yamagata and Tokyo Electric Power Company Hospital, Tokyo, Japan. PATIENT(S): Patients with isolated cryptorchidism (n = 48) and a control group of men with normal phenotype (n = 20). INTERVENTION(S): Blood (lymphocyte DNA). MAIN OUTCOME MEASURE(S): Screening of the AR gene in exons 1-8 using single-strand conformational polymorphism analysis. RESULT(S): No abnormal band patterns were detected in patients with cryptorchidism or in control subjects within the eight exons of the AR gene. CONCLUSION(S): AR gene alterations appear to be an unlikely cause of isolated cryptorchidism.

CAG repeat length analysis and mutation screening of the androgen receptor gene in Japanese men with idiopathic azoospermia.

Because androgens are required for normal spermatogenesis, we are investigating abnormalities in the androgen receptor as a possible cause of impaired spermatogenesis in patients with idiopathic male infertility. The CAG repeat length in exon 1 and mutations of the androgen receptor gene were studied in 30 men with idiopathic azoospermia and in 51 fertile men. In men with azoospermia, plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels were measured and testicular biopsies were performed. The CAG repeat length ranged from 19 to 30 (mean 23.4 +/- 2.9) and from 17 to 28 (mean 23.7 +/- 3.2) in men with azoospermia and in controls, respectively. There was no significant difference between the 2 groups. In men with azoospermia, the Johnsen testicular biopsy score negatively correlated with plasma FSH (P < .01). However, the Johnsen testicular biopsy score did not correlate with plasma LH and testosterone levels. The CAG repeat length did not correlate with the Johnsen testicular biopsy score, or with plasma concentrations of LH, FSH, and testosterone. No abnormalities in the androgen receptor gene were detected. These facts suggest that the CAG repeat length and alterations in the androgen receptor gene are not associated with the etiology of idiopathic azoospermia.

Mutation screening and CAG repeat length analysis of the androgen receptor gene in Klinefelter's syndrome patients with and without spermatogenesis.

BACKGROUND: Mutations of the androgen receptor (AR) gene give rise to a wide array of phenotypic abnormalities. A systematic analysis of the AR gene in patients with 47,XXY has not previously been performed. METHODS: Mutations of the AR gene and expansion of the CAG repeats in exon 1 of the AR gene were studied in 13 patients with Klinefelter's syndrome either with (n = 1) or without (n = 12) spermatogenesis. RESULTS: No abnormalities in the AR gene were detected by single strand conformational polymorphism analysis. The CAG lengths ranged from 17 to 27 (mean +/- SD 22.8 +/- 3.3, median 23) for Klinefelter patients or from 17 to 28 (mean +/- SD 23.2 +/- 2.6, median 23) for control subjects. X-inactivation analysis for the methylation status of the AR gene was performed in seven patients who were heterozygous for CAG repeats of different length, showing that the longer CAG repeat alleles underwent random but more frequent inactivation in five patients and skewed inactivation in two. CONCLUSIONS: An AR gene abnormality does not constitute an important factor for impaired spermatogenesis in patients with Klinefelter's syndrome.

Undermasculinized genitalia in a boy with an abnormally expanded CAG repeat length in the androgen receptor gene.

We report an 11-year-old boy with undermasculinized genitalia and an abnormally expanded CAG repeat length at exon 1 of the androgen receptor (AR) gene. He had microphallus and scrotal hypospadias with chordee, and underwent urethroplasty at 4 years of age. At 11 years of age, a gonadotropin releasing hormone (GnRH) test yielded a relatively high leutinizing hormone (LH) response (0.7-->20.4 IU/l) and a relatively low follicle-stimulating hormone (FSH) response (1.7-->4.8 IU/l), and an human chorionic gonadotropin (hCG) test showed sufficient responses of testosterone (0.7-->23.0 nmol/l) and dihydrotestosterone (0.38-->2.95 nmol/l). The CAG repeat length was 44 for the boy and ranged from 12 to 32 for 100 control males. The DNA sequences of the AR gene were normal for the exons 1-8 and for the splice donor, splice acceptor and branch sites. The markedly expanded CAG repeat length appears to be relevant to the undermasculinized genitalia of this boy, because such an expandsion, which has previously been reported only in spinal and bulbar muscular atrophy, is known to reduce AR function.

GATA3 abnormalities and the phenotypic spectrum of HDR syndrome.

We report on GATA3 analysis and the phenotypic spectrum in nine Japanese families with the HDR syndrome (hypoparathyroidism, sensorineural deafness, and renal dysplasia) (MIM 146255). Fluorescence in situ hybridisation and microsatellite analyses showed heterozygous gross deletions including GATA3 in four families. Sequence analysis showed heterozygous novel mutations in three families: a missense mutation within the first zinc finger domain at exon 4 (T823A, W275R), an unusual mutation at exon 4 (900insAA plus 901insCCT or C901AACCCT) resulting in a premature stop at codon 357 with loss of the second zinc finger domain, and a nonsense mutation at exon 6 (C1099T, R367X). No GATA3 abnormalities were identified in the remaining two families. The triad of HDR syndrome was variably manifested by patients with GATA3 abnormalities. The results suggest that HDR syndrome is primarily caused by GATA3 haploinsufficiency and is associated with a wide phenotypic spectrum.

Hypospadias and the androgen receptor gene: mutation screening and CAG repeat length analysis.

We report on mutation screening and CAG repeat length analysis of the androgen receptor (AR) gene in 21 patients with hypospadias. The urethral meatus was located at the glandular region in six patients (glandular type), at the penile shaft in seven patients (penile type), and at the scrotal/perineal region in eight patients (scrotal/perineal type). Mutation screening was performed for exons 1-8 and their flanking introns (except for the CAG and GGC repeat regions at exon 1) by the heteroduplex detection method and showed no abnormal chromatograms. The CAG repeat length analysis was carried out using 50 normal boys and 50 fertile males as controls, and demonstrated no statistically significant difference in the median of CAG repeat lengths or in the frequency of long CAG repeats (> or = 26 or > or = 28) between the controls and the patients with the three different types of hypospadias. The results suggest that AR gene abnormalities do not constitute a major factor in the development of hypospadias.

47,XXX male: A clinical and molecular study.

We report a 53-year-old Japanese male with a 47,XXX karyotype. His clinical features included hypoplastic scrotal testes (4 ml bilaterally), normally formed small penis (3.8 cm), relatively poor pubic hair development (Tanner stage 3), gynecomastia, age-appropriate male height (159.1 cm), and mental retardation (verbal IQ of 56). Serum testosterone was markedly reduced (0.6 nmol/L). A needle biopsy showed severe testicular degeneration. FISH analysis revealed complex mosaicism consisting of (1) 47,XXX cells with a single copy of SRY (n = 177), two copies of SRY (n = 3), and no SRY (n = 1); (2) 46,XX cells with a single copy of SRY (n = 9) and no SRY (n = 3); (3) 45,X cells with no SRY (n = 5); and (4) 48,XXXX cells with a single copy of SRY (n = 1) and two copies of SRY (n = 1). PCR analysis showed the presence of Yp portion with the breakpoint between DYS264 and AMELY. Microsatellite analysis demonstrated three alleles for DMD and AR. X-inactivation analysis for the methylation status of the AR gene showed random inactivation of the three X chromosomes. The results suggest that this 47,XXX male has resulted from abnormal X-Y interchange during paternal meiosis and X-X nondisjunction during maternal meiosis. Complex mosaicism may be due to the age-related increase in mitotic nondisjunction which is prone to occur in rapidly dividing lymphocytes and to the presence of two randomly inactivated X chromosomes which may behave asynchronously during mitosis, and clinical features of this male would primarily be explained by the genetic information on the SRY (+) der(X) chromosome and his advanced age.

Genetic evidence for a novel gene(s) involved in urogenital development on 10q26.

BACKGROUND: Although the frequent association between distal 10q monosomy and urogenital anomalies suggests the presence of a gene(s) for urogenital development on distal 10q, molecular deletion mapping has not been performed for the putative gene(s). In this study, we examined genotype-phenotype correlations in patients with distal 10q monosomy. METHODS: This study consisted of six karyotypic males (cases 1 through 6) and four karyotypic females (cases 7 through 10) with 10q26 monosomy. Cases 3 through 5 and 7 through 10 had urinary anomalies such as vesicoureteral reflux and hypoplastic kidney, and cases 1 through 6, 8, and 9 exhibited genital anomalies such as micropenis, hypospadias, cryptorchidism, and hypoplastic labia majora. Fluorescence in situ hybridization (FISH) for 10q telomere, whole chromosome 10 painting, and microsatellite analysis for 35 loci on distal 10q were performed in cases 1 through 8. RESULTS: FISH and whole chromosome painting confirmed distal 10q monosomy in cases 1 through 8. Microsatellite analysis revealed that hemizygosity for the region distal to D10S186 was shared by cases with urinary anomalies and that for the region distal to D10S1248 was common to cases with genital anomalies. Furthermore, it was indicated that PAX2, GFRA1, and EMX2 on distal 10q, in which the deletions could affect urinary and/or genital development, were present in two copies in cases 1 through 8. CONCLUSIONS: The results suggest that a novel gene(s) for urinary development and that for genital development reside in the approximately 20 cM region distal to D10S186 and in the approximately 10 cM region distal to D10S1248, respectively, although it remains to be determined whether the two types of genes are identical or different.