Masafumi Kon

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.

Early Discontinuation of Antibiotic Prophylaxis in Patients with Persistent Primary Vesicoureteral Reflux Initially Detected during Infancy: Outcome Analysis and Risk Factors for Febrile Urinary Tract Infection

PURPOSE We retrospectively assessed the incidence of and risk factors for febrile urinary tract infection in children during active surveillance after early discontinuation of antibiotic prophylaxis. MATERIALS AND METHODS We retrospectively evaluated 9 females and 61 uncircumcised males diagnosed with primary vesicoureteral reflux before age 1 year who had persistent reflux on followup voiding cystourethrogram and were subsequently followed under active surveillance without continuous antibiotic prophylaxis. Patients with secondary vesicoureteral reflux or associated urological abnormality were excluded. Clinical outcomes, including incidence of febrile urinary tract infection and new scar formation, were evaluated. Risk factors for febrile urinary tract infection were also analyzed. RESULTS Mean age at stopping continuous antibiotic prophylaxis was 21 months, and mean followup was 61 months. During active surveillance 21 patients had febrile urinary tract infection, and the 5-year infection-free rate under active surveillance was 67.5%. One or 2 foci of minimal new scarring developed in 4 of 16 patients who underwent followup dimercapto-succinic acid scan after febrile urinary tract infection. On multivariate analysis dilated vesicoureteral reflux on followup voiding cystourethrogram was the only significant risk factor for febrile urinary tract infection. CONCLUSIONS This study revealed that about two-thirds of patients with persistent vesicoureteral reflux were free of febrile urinary tract infection during 5 years of active surveillance. Those with dilated vesicoureteral reflux on followup voiding cystourethrogram are at significantly greater risk for febrile urinary tract infection. Accordingly active surveillance, especially in patients with nondilated vesicoureteral reflux on followup voiding cystourethrogram, seems to be a safe option even in children who have not yet been toilet trained.

Fertility preservation in a family with a novel NR5A1 mutation.

The common phenotype of nuclear receptor superfamily 5, group A, member 1 (NR5A1) gene mutations in 46,XY is gonadal dysgenesis without adrenal deficiency. Though the phenotype of gonadal dysgenesis is variable, ranging from complete female to normal male genitalia, an asymptomatic 46,XY male is rare. Preserved fertility has so far been described in only three affected 46,XY males with different mutations, but no functional analysis of these mutations has been performed. Here, we report on male siblings with hypospadias and their asymptomatic father in whom we identified a heterozygous NR5A1 mutation of c.910G>A, p.E304K. Western blotting and subcellular localization revealed no significant difference between the wild type (WT) and E304K. Electrophoretic mobility shift assay experiments showed that E304K abrogated DNA-binding ability. E304K reduced transactivation and had no dominant negative effect. In conclusion, we report on a novel hypomorphic NR5A1 mutation, which may be associated with the phenotype of the family.

Submicroscopic copy-number variations associated with 46,XY disorders of sex development

BackgroundMutations in known causative genes and cytogenetically detectable chromosomal rearrangements account for a fraction of cases with 46,XY disorders of sex development (DSD). Recent advances in molecular cytogenetic technologies, including array-based comparative genomic hybridization (aCGH) and multiplex ligation-dependent probe amplification (MLPA), have enabled the identification of copy-number variations (CNVs) in individuals with apparently normal karyotypes.FindingsThis review paper summarizes the results of 15 recent studies, in which aCGH or MLPA were used to identify CNVs. Several submicroscopic CNVs have been detected in patients with 46,XY DSD. These CNVs included deletions involving known causative genes such as DMRT1 or NR5A1, duplications involving NR0B1, deletions involving putative cis-regulatory elements of SOX9, and various deletions and duplications of unknown pathogenicity.ConclusionsThe results of recent studies highlight the significance of submicroscopic CNVs as the genetic basis of 46,XY DSD. Molecular cytogenetic analyses should be included in the diagnostic workup of patients with 46,XY DSD of unknown origin. Further studies using aCGH will serve to clarify novel causes of this condition.

Prevalence and Chronological Changes of Testicular Microlithiasis in Isolated Congenital Undescended Testes Operated On at Less Than 3 Years of Age

OBJECTIVE To clarify the prevalence and chronological changes of testicular microlithiasis in isolated congenital undescended testes, retrospective chart review was performed. MATERIALS AND METHODS Among children with palpable isolated undescended testes who underwent orchiopexy at less than 3 years of age between January 2009 and May 2016, those who had preoperative testicular ultrasonography were enrolled. Testicular microlithiasis was classified as limited or classic. RESULTS Sixty-five patients (54 unilateral undescended testes and 11 bilateral undescended testes) were enrolled. Preoperative evaluation demonstrated limited testicular microlithiasis in only 2 undescended testes in 2 patients (1 with unilateral undescended testes and 1 with bilateral undescended testes). Of these patients, 1 with unilateral undescended testes had limited testicular microlithiasis and the other with bilateral undescended testes had classic testicular microlithiasis after surgery. Among 53 unilateral undescended testes without microlithiasis preoperatively, limited and classic testicular microlithiasis was found in 1 and 6 testes, respectively, during follow-up. Testicular microlithiasis was identified in 2 on the contralateral descended testis of unilateral undescended testes postoperatively. Among 10 patients with bilateral undescended testes without microlithiasis preoperatively, limited testicular microlithiasis was detected in 4 during follow-up. Testicular microlithiasis was not diminished or resolved during follow-up. The overall prevalence of testicular microlithiasis in undescended testes (21.1%) was significantly higher than that in the contralateral descended testis in patients with unilateral undescended testes (3.7%) (P < .01). CONCLUSION Most testicular microlithiasis was identified postoperatively and never improved. The prevalence of testicular microlithiasis in isolated congenital undescended testes increased with time even if operated on early in life.

Impact of laparoscopy for diagnosis and treatment in patients with disorders of sex development

OBJECTIVE To review laparoscopy in patients with disorders of sex development (DSD) in order to clarify its usefulness in diagnosis, devising subsequent therapeutic strategies and managing patients with various conditions. PATIENTS AND METHODS Between April 1992 and December 2012, 29 laparoscopic surgeries were performed in 25 DSD patients. Among them, ten were diagnostic laparoscopy including gonadal biopsy, and 19 were therapeutic laparoscopy. Surgical procedures and complications were evaluated. RESULTS For diagnostic laparoscopy, laparoscopic gonadal biopsy was performed in three patients. Inspection, with or without open gonadal biopsy, was performed on four out of seven patients with 46XY DSD or mixed gonadal dysgenesis (MGD). Additional surgery was planned and performed based on diagnostic laparoscopic findings in six out of seven patients. In the three patients with ovotesticular DSD, the gonadal pathology was diagnosed as: testis/ovary in one, testis/ovotestis in one and ovary/ovotestis in one--this was from the laparoscopic inspection and/or gonadal biopsy. However, the final diagnoses were bilateral ovotestis in two patients and ovary/ovotestis in one patient. For therapeutic laparoscopy, surgical procedures were: gonadectomy in 17 patients (bilateral in 13, unilateral in three, partial in two); hysterectomy in two patients; orchiopexy in one; and sigmoid vaginoplasty in one patient (included multiple procedures). There were no severe perioperative complications. In the four patients with a history of diagnostic laparoscopy, no severe intra-abdominal adhesions that would disturb therapeutic laparoscopic surgery were observed. CONCLUSION While diagnostic laparoscopy was helpful in devising a therapeutic surgical strategy in most of the patients with DSD who were suspected as having complex gonadal status or Müllerian duct derivatives, attention must be paid to precisely diagnosing the gonadal status in ovotesticular DSD. On the other hand, therapeutic laparoscopic surgeries were valuable procedures in treating DSD patients, even with a history of previous diagnostic laparoscopy.

Impact of Preoperative Ultrasonographic Evaluation for Detection of a Viable Testis in Patients With a Unilateral Nonpalpable Testis: Ultrasonographic Evaluation of a Unilateral Nonpalpable Testis

To investigate the impact of preoperative ultrasonography (US) for detecting a viable testis in patients with a unilateral nonpalpable testis.

Somatically Acquired Isodicentric Y and Mosaic Loss of Chromosome Y in a Boy with Hypospadias

Isodicentric Y chromosome [idic(Y)] represents a relatively common subtype of Y chromosomal rearrangements in the germline; however, limited evidence supports the postzygotic occurrence of idic(Y). Here, we report a boy with hypospadias and somatically acquired idic(Y). The 3.5-year-old boy has been identified in our previous study for patients with hypospadias. In the present study, cytogenetic analysis including FISH revealed a 45,X[5]/46,X,idic(Y)[7]/46,XY[8] karyotype. MLPA showed a mosaic deletion involving PPP1R12BP1 and RBMY2DP. The idic(Y) was likely to have been formed through aberrant recombination between P1 palindromes and subsequently underwent mosaic loss. The patients phenotype was attributable to deletion of some Y chromosomal genes and/or mosaic loss of chromosome Y (mLOY). The results suggest that idic(Y) can originate in postzygotic cells via palindrome-mediated crossovers. Moreover, our data indicate that somatically acquired idic(Y) can trigger mLOY, which usually appears as an aging-related phenomenon in elderly men.

GATA4 mutations are uncommon in patients with 46,XY disorders of sex development without heart anomaly

Eggers et al.9 identified two likely pathogenic GATA4 variants (p.P407Q and p.W228C), together with two variants of uncertain significance, in six patients with 46,XY DSD without heart anomalies. These findings suggest that GATA4 mutations can underlie 46,XY DSD without heart malformations. However, there has been no further report of GATA4 sequencing analyses for large 46,XY DSD cohorts, and therefore, the clinical significance of GATA4 mutations as the cause of 46,XY DSD remains uncertain. Here, we performed GATA4 mutation screening for 119 patients with 46,XY DSD without heart anomalies. Patients with additional congenital anomalies, cytogenetically detectable chromosomal abnormalities, or pathogenic mutations in the major known causative genes for 46,XY DSD, androgen receptor (AR), chromobox 2 (CBX2), desert hedgehog (DHH), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), NR5A1, SOX9, SRY, steroid 5 alpha-reductase 2 (SRD5A2), Wilms tumor 1 (WT1), and ZFPM2,3 were excluded from the study group. Detailed methods are described in Supplementary Information. This study was approved by the Institutional Review Board Committee at the National Center for Child Health and Development, Tokyo, Japan, and performed after obtaining written informed consent. Of the 119 patients, 111 manifested male-type external genitalia with micropenis, cryptorchidism, and/or hypospadias, while the remaining eight presented with ambiguous genitalia. All patients were of Japanese origin. The coding region and splice sites of GATA4 were analyzed using next-generation sequencers. We selected variants whose allele frequencies in Exome Aggregation Consortium (ExAC) Browser (http://exac.broadinstitute.org/; last accessed on 25 September 2017) and the 1000 Genomes Browser (http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/; last accessed on 25 September 2017) were both <1.0%. All variants of interest were confirmed by Sanger sequencing and subjected to in silico functional prediction. Furthermore, the transactivating activity of each variant protein was examined by in vitro reporter assays using the Amh promoter. In these assays, the activities of the variants were compared to that of p.G221R, a mutation previously identified in three patients with 46,XY DSD.8 To clarify the frequency of each variant in the Japanese general population, we sequenced DNA samples from 100 unaffected Japanese males. We also analyzed familial samples of variant-positive patients, when possible. Dear Editor, Disorders of sex development (DSDs) are a group of conditions in which chromosomal, gonadal, or anatomical sex is atypical.1 DSD in genetic males (46,XY DSD) primarily results from impaired androgen production or action or perturbed genital morphogenesis.1 The current understanding of the genetic basis of 46,XY DSD remains fragmentary. For example, although more than 15 genes have been implicated in the development of nonsyndromic hypospadias,2 one of the most common forms of 46,XY DSD,1 mutations in these genes account for <20% of the cases.3 GATA-binding protein 4 (GATA4) is a transcription factor involved in cardiac and sexual development.4–7 GATA4 harbors two zinc finger domains which mediate the interaction with various proteins such as friend of GATA2/zinc finger protein, multitype 2 (FOG2/ZFPM2) and nuclear receptor subfamily 5, group A, member 1 (NR5A1).4,5 Known target genes of GATA4 include sex-determining region Y (SRY), SRY box-9 (SOX9), and anti-Mullerian hormone (AMH) involved in testicular or genital development.4,5 GATA4 was initially reported as a causative gene for congenital heart anomalies.6 To date, heterozygous GATA4 mutations have been identified in more than 140 patients with ventricular septal defect, tetralogy of Fallot, or other cardiac malformations.7 In 2011, Lourenço et al.8 identified a heterozygous GATA4 missense mutation (p.G221R) in three 46,XY DSD patients from one family. Two of the three patients manifested ambiguous external genitalia with hypospadias and intra-abdominal gonads, and the remaining one patient exhibited male-type genitalia with micropenis and intra-abdominal gonads. The proband had no heart anomaly, whereas the other affected males and two female family members showed various cardiac abnormalities including tetralogy of Fallot. In vitro assays confirmed that the p.G221R mutant protein failed to bind to FOG2 and did not transactivate the Amh promoter. Subsequently, LETTER TO THE EDITOR

Prevalence and risk factors of symptomatic urinary tract infection after endoscopic incision for the treatment of ureterocele in children

To clarify the impact of endoscopic incision (EI) for ureterocele as an initial procedure, by performing a retrospective chart review, focusing on the prevalence of and risk factors for symptomatic urinary tract infection (UTI) after EI.