Exome sequencing identified compound heterozygous mutations in the SRD5A2 gene in a case of 46,XY ambiguous genitalia

The disorders of sexual development (DSD) represent an array of phenotypes with ambiguous genitalia. The present case had microphallus with fused and bifid scrotum and was initially assigned androgen insensitivity syndrome; however, sequencing of the complete coding region of the androgen receptor gene failed to identify a causative mutation. We undertook whole exome sequencing for identification of the pathogenic mutation. The most promising pathogenic variants were genotyped using Sanger sequencing to confirm the genotypes. We found compound heterozygous mutations, c.169G>T and c.586G>A in the SRD5A2 gene in this case, resulting in a nonsense (p.Glu57Ter) and a nonsynonymous substitution (p.Gly196Ser), respectively. While the nonsense mutation would result in a truncated protein, p.Gly196Ser substitution has been previously reported to be pathogenic. The mutations were confirmed by Sanger sequencing. Sequencing of 96 normal male individuals did not show the above mutations, suggesting their pathogenic nature. In conclusion, we identified compound heterozygous pathogenic mutations, c.169G>T (p.Glu57Ter) and c.586G>A (p.Gly196Ser), in the SRD5A2 gene in a case of ambiguous genitalia. p.Glu57Ter is a novel mutation, which in compound heterozygote combination with Gly196Ser causes 5a reductase deficiency.     

Molecular investigation of mutations in androgen receptor and 5‐alpha‐reductase‐2 genes in 46,XY Disorders of Sex Development with normal testicular development

In this study, we aimed to determine androgen receptor (AR) and SRD5A2 gene mutations in 45 patients characterised by 46,XY Disorders of Sex Differentiation (DSD) signs with normal testicular development referred to the Children's Medical Center from February 2015 to September 2017. Karyotype and sex hormone analyses were performed. Cytogenetic investigation showed that seven patients were 46,XX DSD, six patients were chromosomal DSD and 32 patients were 46,XY DSD. Eight exons of the AR gene and five exons of the SRD5A2 gene were amplified. Two cases were affected with androgen insensitivity syndrome (AIS) (missense mutation on exon 7, position c.3637 G>A: p.R841H and position c.3610 G>A: p.R832Q), one case was affected with 5‐alpha‐reductase deficiency type 2 (missense mutation at c.578A>G: p.N193S on exon 4), and 22 cases (88%) did not demonstrate AIS or 5α‐RD2 gene abnormality. Due to the great impact of these disorders on human lifestyle, evaluation of genes involved can improve genetic counselling and therapeutic management. We focused on the AR and SRD5A2 genes in patients with 46,XY DSDs with normal testicular development referred to the Children's Medical Center from all over the country to eventually culminate in a reliable prenatal diagnosis protocol at this major referral centre giving service to a great number of families with consanguineous marriages.     

Screening of the COL2A1 mutation in idiopathic osteonecrosis of the femoral head

Idiopathic osteonecrosis of the femoral head (idiopathic ONFH) is an ischemic disorder resulting in necrosis of the subchondral bone of the femoral head. COL2A1 mutations, including c.3508G>A, have been reported to be involved in its etiology. However, the etiological role of COL2A1 mutations in idiopathic ONFH remains controversial, because the pathology of idiopathic ONFH is ischemic necrosis, not epiphyseal dysplasia usually seen in the diseases caused by COL2A1 mutations. The purpose of this study is to examine whether COL2A1 mutations have causal relation with idiopathic ONFH or not. We recruited 1,451 Japanese patients with idiopathic ONFH, including steroid‐, alcohol‐, and neither steroid nor alcohol‐associated (neither‐associated) ONFH. The diagnosis was based on the criteria of the Japanese Research Committee on idiopathic ONFH of the Ministry of Health, Labour and Welfare. By whole‐exome sequencing, entire COL2A1 coding regions and flanking introns were analyzed in 49 neither‐associated ONFH patients. In addition, the c.3508G>A mutation of COL2A1 was checked in all idiopathic ONFH patients using the invader assay. Whole‐exome sequencing did not detect any COL2A1 mutations in the 49 patients. The c.3508G>A mutation was not found in any of the 1,451 patients. In conclusion, COL2A1 is unlikely to cause idiopathic ONFH. Epiphyseal dysplasia of the femoral head caused by COL2A1 mutations may radiographically mimic idiopathic ONFH. COL2A1 mutations should prompt clinical re‐evaluation of the patient's phenotype. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:768–774, 2017.

     

Identification of two novel mutations in three Chinese families with Kallmann syndrome using whole exome sequencing

Kallmann syndrome (KS) is a rare developmental disorder that manifests as congenital hypogonadotropic hypogonadism with anosmia. More than 19 genes have been found to be associated with KS. However, approximately 70% of the causes of KS remain unclear. Here, we studied seven KS patients, from three families, who had delayed puberty and olfactory bulb dysplasia. However, the families of these patients showed a range of other unique clinical features, including hearing loss, anosmia (to varying degrees) and unilateral renal agenesis. We performed whole exome sequencing and copy number variation (CNV) sequencing on samples acquired from these patients. We identified two novel mutations (c.844delC in ANOS1, c.475C>T in SOX10) and a novel trigenic pattern, PROKR2/CHD7/FEZF1 (c.337T>C in PROKR2, c.748C>G in FEZF1, c.8773G>A in CHD7). The c.844delC mutation in the ANOS1 gene was predicted to generate a truncated form of the anosmin-1 protein. SIFT and PolyPhen-2 predicted that the c.475C>T mutation in SOX10 had a damaging effect. The PROKR2 mutation (c.337T>C) was previously reported as harmful. No pathogenic copy number alterations were detected. Our study expands the genotypic and phenotypic spectrum of KS, a disease that shows considerable clinical and genetic heterogeneity. The application of whole exome sequencing could facilitate our understanding of the pathogenesis of KS.     

Molecular diagnosis of 5α-reductase-2 gene mutation in two Indian families with male pseudohermaphroditism

Aim: To identify the genotype of two Indians with male pseudohermaphroditism. Methods: Standard radioimmu-noassay procedure was used for estimating hormonal levels. Conventional cytogenetic analysis was carried out for diagnosing the genetic sex in these subjects with genital ambiguity. Molecular analysis was carried out by standard polymerase chain reaction procedure using different sets of primers and reaction conditions specific for the 5α-reductase type 2 gene (SRD5A2) gene. Direct sequencing was carried out using the ABI Prism dye terminator sequencing kit and the ABI 310 sequencing apparatus. Results: We found an SRD5A2 gene mutation in exon 5, where arginine is substituted with glutamine (R246Q), in two males with pseudohermaphroditism and ambiguous genitalia from unrelated families. This is the first time this mutation has been reported in individuals from India. Conclusion: Identification of the R246Q mutation of the SRD5A2 gene from two unrelated Indian families possibly extends the founder gene effect.     

Monoallelic mutations in SLCO2A1 cause autosomal dominant primary hypertrophic osteoarthropathy

Primary hypertrophic osteoarthropathy (PHO) is a rare disease inherited as a recessive or irregular dominant trait and characterized by digital clubbing, pachydermia, and periostosis. Biallelic mutations in HPGD and SLCO2A1, disturbing prostaglandin E₂ (PGE₂) catabolism and leading to increased circulating PGE₂ level, cause PHO autosomal recessive 1 (PHOAR1) and PHO autosomal recessive 2 (PHOAR2), respectively. However, no causative genes have been reported for PHO autosomal dominant (PHOAD). Here, we performed Sanger sequencing and whole-genome sequencing (WGS) on DNA samples from seven Chinese PHOAD families; after excluding other single-nucleotide variants (SNVs), structural variations (SVs), and copy number variations (CNVs) in the genomes, we reported six SLCO2A1 monoallelic mutations (c.1660G>A [p.G554R], c.664G>A [p.G222R], c.1106G>A [p.G369D], c.1065dupA [p.Q356TfsX77], c.1293delT [p.S432AfsX48], and c.1807C>T [p.R603X]) in the probands and affected family members. Then, in five other PHO families with probands carrying SLCO2A1 biallelic mutations, we verified that parents with SLCO2A1 monoallelic mutations also displayed PHO manifestations, which further confirmed the pathogenicity of SLCO2A1 monoallelic mutations and illustrated the allelic nature of PHOAD and PHOAR2. Subsequently, through comparison of seven PHOAD probands and 50 PHOAR2 patients, we found onset age in puberty and skewed penetrance rate were similar in both PHO types, but symptoms and signs of PHOAD were milder, including less severe pachydermia (p = .027) and periostosis (p = .005), and less frequent cutis verticis gyrata (p = .011), acne (p = .005), arthralgia (p = .037), and anemia (p = .023). The median urinary PGE₂ level in PHOAD probands was almost half that in PHOAR2 patients (PHOAD 277.58 ng/mmoL creatinine, PHOAR2 473.19 ng/mmoL creatinine; p = .038). Moreover, through the 3-month trial of oral administration of etoricoxib, an effective response similar to that we reported previously in PHOAR2 patients was observed in PHOAD probands. In conclusion, our findings confirm that SLCO2A1 monoallelic mutations are the cause of PHOAD and broaden phenotypic spectrum of PHO. © 2021 American Society for Bone and Mineral Research (ASBMR).     

A Recurrent Mutation c.617G>A in the ACVR1 Gene Causes Fibrodysplasia Ossificans Progressiva in Two Chinese Patients

Fibrodysplasia ossificans progressiva (FOP; OMIM 135100) is a rare heritable disorder of connective tissue characterized by congenital malformations of the great toes and recurrent episodes of painful soft tissue swelling that lead to heterotopic ossifications. Recent studies have shown that the ACVR1 (activin A receptor, type I; OMIM 102576) gene, which encodes the BMP type I receptor protein, is responsible for this disease. We observed two Chinese patients who suffered from progressive pain and ankylosis of major joints with congenital bilateral hallus valgus malformation, neck stiffness, and several posttraumatic ossified lesions on the head and dorsum. Both patients were diagnosed as having FOP. This study aimed to investigate the ACVR1 gene mutation in Chinese FOP patients. Direct sequence analysis of genomic DNA and restriction enzyme digestion demonstrated the presence of a single heterozygous c.617G>A (p.R206H) mutation in the ACVR1 gene in both patients. This mutation is first reported in Chinese patients with FOP and it was de novo in both affected families.     

Molecular diagnosis of 5alpha-reductase-2 gene mutation in two Indian families with male pseudohermaphroditism.

Aim: To identify the genotype of two Indians with male pseudohermaphroditism. Methods: Standard radioimmunoassay procedure was used for estimating hormonal levels. Conventional cytogenetic analysis was carried out for diagnosing the genetic sex in these subjects with genital ambiguity. Molecular analysis was carried out by standard polymerase chain reaction procedure using different sets of primers and reaction conditions specific for the 5alpha-reductase type 2 gene (SRD5A2) gene. Direct sequencing was carried out using the ABI Prism dye terminator sequencing kit and the ABI 310 sequencing apparatus. Results: We found an SRD5A2 gene mutation in exon 5, where arginine is substituted with glutamine (R246Q), in two males with pseudohermaphroditism and ambiguous genitalia from unrelated families. This is the first time this mutation has been reported in individuals from India. Conclusion: Identification of the R246Q mutation of the SRD5A2 gene from two unrelated Indian families possibly extends the founder gene effect.     

SLC4A2 Deficiency Causes a New Type of Osteopetrosis

Osteopetrosis is a group of rare inherited skeletal disorders characterized by a marked increase in bone density due to deficient bone resorption. Pathogenic variants in several genes involved in osteoclast differentiation and/or function have been reported to cause osteopetrosis. Solute carrier family 4 member 2 (SLC4A2, encoding anion exchanger 2) plays an important role in osteoclast differentiation and function by exchange of Cl⁻ with HCO₃⁻. Biallelic Slc4a2 loss-of-function mutations in mice and cattle lead to osteopetrosis with osteoclast deficiency; however, pathogenic SLC4A2 variants in humans have not been reported. In this study, we describe a patient with autosomal recessive osteopetrosis due to biallelic pathogenic variants in SLC4A2. We identified novel compound heterozygous variants in SLC4A2 (NM_003040.4: c.556G>A [p.A186T] and c.1658T>C [p.V553A]) by exome sequencing. The measurement of intracellular Cl⁻ showed that the variants decrease the anion exchange activity of SLC4A2. The impact of the variants on osteoclast differentiation was assessed by a gene knockout-rescue system using a mouse macrophage cell line, RAW 264.7. The Slc4a2-knockout cells show impaired osteoclastogenesis, which was rescued by the wild-type SLC4A2, but not by the mutant SLC4A2s. Immunofluorescence and pit assay revealed that the mutant SLC4A2s leads to abnormal podosome belt formation with impaired bone absorption. This is the first report on an individual affected by SLC4A2-associated osteopetrosis (osteopetrosis, Ikegawa type). With functional studies, we prove that the variants lead to SLC4A2 dysfunction, which altogether supports the importance of SLC4A2 in human osteoclast differentiation. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Exclusion of homozygous PLCE1 (NPHS3) mutations in 69 families with idiopathic and hereditary FSGS

Focal and segmental glomerulosclerosis (FSGS) is the most common glomerular cause of end-stage kidney disease (ESKD). Although the etiology of FSGS has not been fully elucidated, recent results from the positional cloning of genes mutated in nephrotic syndromes are now beginning to provide insight into the pathogenesis of these diseases. Mutations in PLCE1/NPHS3 have recently been reported as a cause of nephrotic syndrome characterized by diffuse mesangial sclerosis (DMS) histology. One single family with a missense mutation had late onset of the disease that was characterized by FSGS. To further define the role of PLCE1 mutations in the etiology of FSGS, we performed mutational analysis in 69 families with FSGS. A total of 69 families with 231 affected individuals were examined. The median age of disease onset was 26 years (range 1–66 years). Onset of ESKD was at a median age of 35.5 years. Seven variants leading to non-synonymous changes were found, of which only two are new variants (exon 4 c.1682 G>A R561Q, exon 31 c.6518A>G K2173R). No known disease-causing mutations were identified in the families screened. PLCE1/NPHS3 mutations are not a cause of FSGS in this cohort. The absence of mutations in PLCE1/NPHS3 in this study indicates that there are additional genetic causes of FSGS and that hereditary FSGS is a heterogeneous disease. Kindreds appropriate for genome-wide screening are currently being subjected to analysis with the aim of identifying other genetic causes of FSGS.     

Genetic contribution of HIST1H1T regulatory region alternations to human nonobstructive azoospermia

HIST1H1T encodes H1T, a testicular variant of histone H1, which is expressed during spermatogenesis especially in primary spermatocytes and facilitates histone to protamine exchanges during maturation of spermatozoa. The goal of the conducted research was to evaluate four genetic variations of HIST1H1T in men with nonobstructive azoospermia. This case–control study was conducted among a total number of 200 men, including 100 nonobstructive azoospermic (NOA) infertile men. In this study, three single-nucleotide polymorphisms, including c.-54C>T (rs72834678), c.-912A>C (rs707892) and c.-947A>G (rs74293938) in regulatory region as well as one SNP c.40G>C (rs198844) in coding region were identified using PCR sequencing. According to statistical analysis, none of those SNPs in regulatory regions showed significant differences in case and control groups. For SNP (c.40G>C), a significantly higher frequency of C allele in the case group was observed compared to the control group (p-value: .044). In conclusion, according to statistical analysis it seems that the polymorphism of c.40G>C is not associated with nonobstructive azoospermia.     

Three Novel Mutations of the PHEX Gene in Three Chinese Families with X-linked Dominant Hypophosphatemic Rickets

X-linked dominant hypophosphatemia (XLH, OMIM307800), the most prevalent form of inherited rickets in humans, is a dominant disorder of phosphate homeostasis characterized by growth retardation, rachitic and osteomalacic bone disease, hypophosphatemia, and renal phosphate wasting. The gene responsible for XLH was identified by positional cloning and designated PHEX (formerly PEX) to depict a phosphate-regulating gene homologous with endopeptidases on the X chromosome. Recently, extensive mutation analysis of the PHEX gene has revealed a wide variety of gene defects in XLH. The ethnic distribution of the mutations is very widespread but only a few mutations in Chinese have been reported. To analyze the molecular basis in three unrelated Chinese families with XLH, we determined the nucleotide sequence of the PHEX gene and fibroblast growth factor 23 (FGF23) gene of affected members. The serum FGF23 concentrations of these patients with XLH were also measured. Three different novel mutations were observed in these three families: one deletion mutation c.264delG causing p.W88 X; one missense mutation c.1673C>G causing p.P558A; one nonsense mutation c.1809G>A causing p.W603 X. Serum concentration of FGF23 in XLH patients of these three families was significantly higher than normal. The results suggest that PHEX gene mutations were responsible for XLH in these patients and these mutations may contribute to a higher serum FGF23 level.     

Mutations in Profilin 1 Cause Early-Onset Paget's Disease of Bone With Giant Cell Tumors

Paget's disease of bone (PDB) is a late-onset chronic progressive bone disease characterized by abnormal activation of osteoclasts that results in bone pain, deformities, and fractures. PDB is very rare in Asia. A subset of PDB patients have early onset and can develop malignant giant cell tumors (GCTs) of the bone (PDB/GCTs), which arise within Paget bone lesions; the result is a significantly higher mortality rate. SQSTM1, TNFRSF11A, OPG, VCP, and HNRNPA2B1 have been identified as pathogenic genes of PDB, and ZNF687 is the only confirmed gene to date known to cause PDB/GCT. However, the molecular mechanism underlying PDB/GCT has not been fully elucidated. Here, we investigate an extended Chinese pedigree with eight individuals affected by early-onset and polyostotic PDB, two of whom developed GCTs. We identified a heterozygous 4-bp deletion in the Profilin 1 (PFN1) gene (c.318_321delTGAC) by genetic linkage analysis and exome sequencing for the family. Sanger sequencing revealed another heterozygous 1-bp deletion in PFN1 (c.324_324delG) in a sporadic early-onset PDB/GCT patient, further proving its causative role. Interestingly, a heterozygous missense mutation of PFN1 (c.335 T > C) was identified in another PDB/GCT family, revealing that not only deletion but also missense mutations in PFN1 can cause PDB/GCT. Furthermore, we established a Pfn1-mutated mouse model (C57BL/6J mice) and successfully obtained Pagetic phenotypes in heterozygous mice, verifying loss of function of PFN1 as the cause of PDB/GCT development. In conclusion, our findings reveal mutations in PFN1 as the pathological mechanism in PDB/GCT, and we successfully established Pfn1-mutated mice as a suitable animal model for studying PDB-associated pathological mechanisms. The identification of PFN1 mutations has great diagnostic value for identifying PDB individuals predisposed toward developing GCTs. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Influence of the IL17A and IL17F gene polymorphisms on the long‐term kidney allograft function and return to dialysis after kidney transplantation

The immune response after allogenic transplantation is a complex phenomenon involving cytokines, chemokines, and other mediators of inflammation. The aim of this study was to evaluate the influence of the IL17A and IL17F gene polymorphisms on long‐term kidney allograft function, graft function loss/return to dialysis, and mortality after kidney transplantation. This study enrolled 269 Caucasian deceased donor renal transplant recipients. The rs2275913:G>A (−197G>A) polymorphism within the IL17A gene promoter and rs2397084:T>C (Glu126Gly), rs11465553:G>A (Val155Ile), and rs763780:T>C (His167Arg) polymorphisms within the IL17F gene were genotyped. Creatinine concentrations 12, 24, 36, 48, and 60 months after transplantation were significantly higher in recipients with the rs2275913:A>G IL17A GG genotype (GG vs. GA + AA: p = 0.03, p = 0.004, p = 0.006, p = 0.03, p = 0.04, respectively). Moreover, the GG genotype was statistically significantly associated with increased risk of delayed graft function. This association remained significant in multivariate regression analysis adjusted for recipients' age and sex. In the case of the rs11465553, IL17F univariate Cox regression analysis showed statistically significant association of GA genotype with higher risk of graft loss/return to dialysis (GA vs. GG: HR = 2.795, 95%CI = 1.031–7.579, p = 0.04). The results of our study suggest that the GG genotype of the rs2275913 IL17A gene promoter polymorphism is associated with significantly impaired long‐term kidney allograft function, whereas the GA genotype of the rs11465553 IL17F gene polymorphism may be associated with a significantly higher risk of graft function loss and return to dialysis after kidney transplantation.     

How Far Should We Explore Hypospadias? Next-generation Sequencing Applied to a Large Cohort of Hypospadiac Patients

BackgroundNext-generation sequencing (NGS) is generally used for patients with severe disorders of sex development (DSD). However, NGS has not been applied extensively for patients with hypospadias only, and most affected children do not benefit from an etiological diagnosis.ObjectiveTo evaluate the clinical usefulness of NGS for patients with hypospadias, regardless of severity.Design, setting, and participantsProspective multicenter research included 293 children with glandular to penoscrotal hypospadias (no undescended testis and no micropenis). After excluding likely pathogenic androgen receptor (AR) variants by Sanger sequencing, an NGS panel tested 336 genes including unexplored candidates in 284 patients.Outcome measurements and statistical analysisThe rate of pathogenic and likely pathogenic variants was assessed using REVEL, ClinVar, and in-house tools (Captain-ACHAB, MobiCNV, and MobiDetails).Results and limitationsLikely pathogenic variants were identified in 16 (5.5%) patients with both Sanger sequencing and NGS taken into account. Some genes were related to DSD (AR, NR5A1, HSD17B3, and MAMLD1), but reverse phenotyping revealed two syndromic disorders with midline defects (MID1) and alteration in the retinoic acid signaling pathway (RARA). Coverage analysis revealed an 18q deletion. Identification of likely pathogenic variants increased with hypospadias severity. Other variants of unknown significance (VUSs) in genes implicated in hypogonadotropic hypogonadism, Noonan syndrome, and genital tubercle development were also identified. Genetic study mainly focused on exonic variants, and most cases remain unexplained.ConclusionsNGS reveals minor forms of DSD, undiagnosed syndromes, or candidate rare variants in new genes, indicating that even patients with mild hypospadias benefit from advanced sequencing techniques. Early molecular diagnosis would help improve follow-up at puberty and medical counseling for initially undiagnosed syndromes. Future studies will improve the diagnosis by investigating the contribution of VUSs.Patient summaryNext-generation sequencing enables simultaneous testing of numerous genes and should not be limited to disorders of sex development cases. Even patients with mild hypospadias would benefit from early diagnosis of a genetic defect implicated in sex development or other syndromes.     

Robust Genetic Diagnosis of Split Hand–Foot Malformation by Exome Sequencing

Purpose The present study aimed to evaluate the genetic diagnostic yield and accuracy of exome sequencing in Chinese patients with split hand–foot malformation(SHFM),a severe heterogeneous congenital anomaly characterized by hypodevelopment of the central ray of the hands and feet.Methods A cohort of seven families and five sporadic patients with SHFM was investigated.Genomic DNA was prepared from the peripheral blood of affected as well as unaffected individuals.Whole exome sequencing(WES)was performed to identify the pathogenic mutations.Array-based comparative genomic hybridization(aCGH),CytoScan,quantitative polymerase chain reaction(qPCR),and Sanger sequencing were performed to validate the findings of WES.WES data of an additional cohort of 24 patients with non-SHFM congenital hand anomalies were analyzed as the control.Results Pathogenic variants of TP63,c.G956A p.R319H,and c.T602A:p.L201H,were identified in two families by WES.In the remaining patients,copy number analysis of the WES data by XHMM software identified pathogenic 10q 24 duplication in five individuals from three families,which was further validated via CytoScan and qPCR;however,WES could not detect duplication in 10q24 in an additional cohort of 24 individuals with non-SHFM congenital hand anomaly.Importantly,qPCR analysis of the 10q24 region copy number revealed a definite consistency with WES data in all individuals.Genotype–phenotype analysis did not present any unique feature that could differentiate between the families with TP63 mutation and 10q24 duplication.Conclusions Our study demonstrated that WES is an accurate and sensitive method to detect the pathogenic 10q24 duplication.Collectively,with TP63 mutation,a single WES testing could yield a diagnosis rate of about 40%(5/12)for the SHFM patients,at least in our cohort.As the genotype–phenotype correlation remains unclear,WES could be used as a cost-effective method for the genetic diagnosis of SHFM.     

A novel mutation in steroidogenic factor (SF1/NR5A1) gene in a patient with 46 XY DSD without adrenal insufficiency

Steroidogenic factor‐1 (SF‐1), also known as nuclear receptor subfamily 5 group A member 1 (NR5A1), is a member of orphan receptor subfamily and located on chromosome 9 (9q33). In 46, XY individuals with mutation of SF‐1 gene, adrenal failure, testis dysgenesis, androgen synthesis defects, hypospadias and anorchia with microphallus, infertility can occur from severe to mild. We report a case of a 20‐day‐old male who is admitted to our clinic due to ambiguous genitalia. In this report, we describe a novel heterozygous c.814A > C (p. T272P) NR5A1 mutation in a patient with 46, XY DSD without adrenal insufficiency. We describe a novel missense mutation c.814A > C (p. T272P) in NR5A1 gene which had not previously been reported. Also this report highlights that the potential diagnostic utility of next‐generation sequencing is an effective strategy versus Sanger sequencing to identify genetic mosaicism in clinical practice.     

Analysis of Mutations in the Urate Transporter 1 (URAT1) Gene of Japanese Patients with Hypouricemia in Northern Japan and Review of the Literature

Background. Renal hypouricemia is an autosomal recessive disorder resulting from inactivating mutations in the urate transporter 1 (URAT1) encoded by SLC22A12. To date, 10 mutations have been identified and W258X in the URAT1 gene is the predominant cause in middle to southwestern Japan. However, it is still unclear whether there is a regional specific distribution of mutations in northern Japan. In this study, we analyzed mutations in the URAT1 gene of five Japanese patients with renal hypouricemia in northern Japan. Methods. Peripheral blood mononuclear cells were isolated from patients with hypouricemia and healthy control subjects. A mutation analysis of the URAT1 gene was performed completely by direct automated sequencing of polymerase chain reaction-amplified DNA products. Results. We identified two mutations. These mutations [c.269G>A (R90H) and c.774G>A (W258X)] have been reported in Japanese patients. Two of five patients were homozygotes (W258X), two carried single heterozygous mutations (W258X), and the remaining one was a compound heterozygote (R90H and W258X). Conclusions. Our study suggests that there is no regional different distribution of the URAT1 genetic mutations in Japanese with renal hypouricemia.     

Two-hit mechanism in cerebral cavernous malformation? A case of monozygotic twins with a CCM1/KRIT1 germline mutation

Cerebral cavernous malformations are focal vascular abnormalities that show recurrent intralesional microhemorrhage and may cause focal deficits or seizures in affected patients. These lesions occur in both sporadic and inherited autosomal dominant form. Germline mutations in three different genes have been identified yet. One explanation for the unpredictable individual clinical course with wide variability of the number of developing cerebral cavernous malformations (CCMs) and their rate of progression within CCM families is thought to be based upon a “two-hit” mechanism. However, the direct influence of a heterozygous underlying germline mutation in combination with secondary somatic mutations on a patient’s individual clinical course is hard to investigate in vivo. In this context, we present a rare and interesting case of monozygotic twins heterozygous for the CCM1 germline mutation c.730-1G>A and discuss their similar age and type of disease manifestation and their beginning divergent clinical course.