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The 5α-reductase type 2 enzyme catalyzes the conversion of testosterone into dihydrotestosterone, playing a crucial role in male development. This enzyme is encoded by the SRD5A2 gene, which maps to chromosome 2 (2p23), consists of 5 exons and 4 introns, and encodes a 254 amino acid protein. Disruptions in this gene are the molecular etiology of a subgroup of differences of sex development (DSD) in 46,XY patients. Affected individuals present a large range of external genitalia undervirilization, ranging from almost typically female external genitalia to predominantly typically male external genitalia with minimal undervirilization, including isolated micropenis. This is an updated review of the implication of the SRD5A2 gene in 5α-reductase type 2 enzyme deficiency. For that, we identified 451 cases from 48 countries of this particular 46,XY DSD from the literature with reported variants in the SRD5A2 gene. Herein, we present the SRD5A2 mutational profile, the SRD5A2 polymorphisms, and the functional studies related to SRD5A2 variants to detail the molecular etiology of this condition., (© 2022 S. Karger AG, Basel.)

Hypospadias is a common congenital disorder of male genital formation. Children born small for gestational age (SGA) present a high frequency of hypospadias of undetermined etiology. No previous study investigated the molecular etiology of hypospadias in boys born SGA using massively parallel sequencing. Our objective is to report the genetic findings of a cohort of patients born SGA with medium or proximal hypospadias. We identified 46 individuals with this phenotype from a large cohort of 46,XY DSD patients, including 5 individuals with syndromic features. DNA samples from subjects were studied by either whole exome sequencing or target gene panel approach. Three of the syndromic patients have 5 main clinical features of Silver-Russell syndrome (SRS) and were first studied by MLPA. Among the syndromic patients, loss of DNA methylation at the imprinting control region H19/IGF2 was identified in 2 individuals with SRS clinical diagnosis. Two novel pathogenic variants in compound heterozygous state were identified in the CUL7 gene establishing the diagnosis of 3M syndrome in one patient, and a novel homozygous variant in TRIM37 was identified in another boy with Mulibrey nanism phenotype. Among the non-syndromic subjects, 7 rare heterozygous variants were identified in 6 DSD-related genes. However, none of the variants found can explain the phenotype by themselves. In conclusion, a genetic defect that clarifies the etiology of hypospadias was not found in most of the non-syndromic SGA children, supporting the hypothesis that multifactorial causes, new genes, and/or unidentified epigenetic defects may have an influence in this condition., (© 2021 S. Karger AG, Basel.)

Extra-genital congenital anomalies are often present in cases of hypospadias, but it is unclear whether they have an association with the outcome of hypospadias surgery. The aim of this study was to review all hypospadias cases that had surgery between 2009 and 2015 at a single centre and identify clinical determinants of the surgical outcome. An extra-genital congenital anomaly was reported in 139 (22%) boys and 62 (10%) had more than 1 anomaly. Of the 626 boys, 54 (9%), including 44 with proximal hypospadias, had endocrine as well as limited genetic evaluation. Of these, 10 (19%) had a biochemical evidence of hypogonadism and 5 (9%) had a molecular genetic abnormality. At least 1 complication was reported in 167 (27%) patients, with 20% of complications (most frequently fistula) occurring after 2 years of surgery. The severity of hypospadias and the existence of other anomalies were clinical factors that were independently associated with an increased risk of complications (p < 0.001). In conclusion, complications following surgery are more likely in those cases that are proximal or who have additional extra-genital anomalies. To understand the biological basis of these complications, there is a greater need to understand the aetiology of such cases., (© 2019 S. Karger AG, Basel.)

Knowledge of the molecular background of disorders of sex development (DSD) in dogs with normal sets of XY chromosomes (XY DSD) is very scarce. However, extensive studies have been carried out in humans, showing that polymorphisms and mutations of numerous genes, including SRY, MAMLD1, SRD5A2, and AR, are associated with or responsible for XY DSD. In this study, we analyzed the entire coding sequence of these genes in 7 dogs (78,XY) with ambiguous external genitalia (hypospadias, cryptorchidism, bifid scrotum, or rudimentary penis). The most common disorder was hypospadias (6 cases), followed by cryptorchidism (4 cases). The co-occurrence of both abnormalities was observed in 3 dogs. Polymorphisms were found in MAMLD1 (3 SNPs), SRD5A2 (5 SNPs), and AR (2 STRs and 1 SNP), while SRY was monomorphic. However, the distribution of the polymorphic variants in the DSD dogs and 11 control XY dogs did not differ significantly. Our study suggests that an association between the polymorphisms of the studied candidate genes and hypospadias or cryptorchidism is unlikely in dogs. We thus support the recent suggestion that hypospadias is not rare in this species, and moreover, we show that co-occurrence of hypospadias and cryptorchidism can be quite frequent., (© 2019 S. Karger AG, Basel.)

A heterozygous intragenic duplication within the repeated area (CTGCAGCTG)×2 of the NR5A1 gene was found in a 15-year-old 46,XY DSD (disorders/differences of sex development) patient with micropenis and severe proximal hypospadias. This heterozygous duplication has already been described twice in boys with a similar phenotype, whereas a deletion of 3 amino acids at the same position in the protein SF-1 has been described in a 46,XX patient with primary ovarian failure and short stature. These data suggest that this region within the NR5A1 gene has an important role for SF-1 protein function in gonads and is a hotspot for intragenic rearrangements., (© 2018 S. Karger AG, Basel.)

Mutations in the NR5A1 gene, which encodes the steroidogenic factor 1 (SF1), are responsible for different phenotypes of disorders of sex development (DSD), such as bilateral anorchia and hypospadias. Furthermore, they can be associated with primary amenorrhea, premature ovarian failure, male infertility, adrenal tumors, and endometriosis. Direct sequencing of the 7 NR5A1 exons including ∼1,000 bp of the 5'-upstream and 3'-downstream regions and all intron-exon boundaries was performed in patients with DSD. Three different in silico tools were used to assess the consequences of a splice site mutation. As a result, 3 novel NR5A1 mutations were identified in 3 patients with 46,XY partial gonadal dysgenesis: p.Lys38* and p.Leu80Trpfs*8 lead to premature translation termination codons within the SF1 DNA-binding domain, and the intronic nucleotide substitution c.1138+1G>T at the intron 6 donor splice site is considered to modify correct splicing. We assume that the anomalous mRNA produced as a result of p.Lys38* and p.Leu80Trpfs*8 will be degraded by nonsense-mediated mRNA decay even before translation, leading to SF1 haploinsufficiency. The c.1138+1G>T mutation is expected to produce a truncated protein. Heterozygous SF1 loss-of-function mutations in these cases resulted in mild DSD manifestations, such as dysgenetic testes, spontaneous puberty, and preserved adrenal function., (© 2016 S. Karger AG, Basel.)

We investigated the frequency of copy number variations (CNVs) in the Y chromosome of Japanese children with hypospadias. We analyzed the copy number of the azoospermia factor (AZF) region and SRY, using multiplex ligation-dependent probe amplification. Four AZF-linked CNVs, including one novel simple duplication, were identified in 39 of 89 patients, at a frequency comparable to that of those in unaffected individuals. SRY-linked CNVs were absent in our patients. The results imply that CNVs in the AZF region and SRY are not associated with the risk of hypospadias in the Japanese population, although the pathogenicity of the AZF-linked simple duplication remains to be elucidated., (© 2016 S. Karger AG, Basel.)

45,X/46,XY mosaicism is a rare sex chromosome disorder of sex development. Short stature is a main feature of boys with this condition. Different causes likely contribute to growth impairment. Growth hormone (GH) has been administered to treat short stature in boys with 45,X/46,XY mosaicism, but conflicting data are available. Here, spontaneous growth patterns as well as short- and long-term follow-up studies during GH therapy in these patients are reviewed. Short- and mid-term data showed an improvement of the growth pattern in GH-treated boys, mainly when hormonal therapy was started early, while long-term follow-up demonstrated similar adult heights in GH-treated and untreated patients. Individual biological factors (e.g. different chromosome constitution, different mosaicism among various tissues, impaired pubertal growth spurt), non-homogeneous GH doses and different ages at start of therapy may contribute to the variable results. Thus, early GH therapy at pharmacological doses may improve the growth pattern of short boys with 45,X/46,XY mosaicism, but data on adult height are disappointing. Evaluation of larger patient samples treated by homogeneous doses and long-term follow-up studies assessing adult height and safety are needed to reach definitive conclusions on GH therapy in boys with 45,X/46,XY mosaicism., (© 2015 S. Karger AG, Basel.)

MAMLD1 is a causative gene for disorders of sex development. Several MAMLD1 mutations have been shown to cause hypospadias by generating dysfunctional proteins and/or unstable mRNAs. Here, we identified an intronic mutation of MAMLD1 (g.IVS4-2A>G) in 1 of 180 hypospadias patients. RT-PCR of the patient's skin sample showed normal expression of full-length MAMLD1 and markedly reduced expression of a known splice variant lacking exon 4. A hitherto unreported splice variant that lacks exon 5 was similarly identified in samples of the patient and control individuals. The full-length transcript of the patient contained mutant mRNA lacking the first 10 nucleotides of exon 5 (c.1822&#95;1831delACTCATGTAG, p.K609fsX1070). In vitro assays using cells expressing the full-length wild-type and mutant proteins revealed reduced expression of the mutant. The expression of the wild-type and mutant MAMLD1 showed parallel changes upon treatment with a proteasome inhibitor and a translation inhibitor. The mutant-expressing cells exerted low transactivation activity for the Hes3 promoter, which reflected limited expression of the mutant protein. These results imply that the pathogenic events resulting from MAMLD1 mutations include splice errors. Furthermore, this study raises the possibility of translation failure of MAMLD1 mutants, which deserves further investigation., (© 2015 S. Karger AG, Basel.)

Hypospadias results from a failure of urethral closure in the male phallus and affects 1 in 200-300 boys. It is thought to be due to a combination of genetic and environmental factors. The development of the penis progresses in 2 stages: an initial hormone-independent phase and a secondary hormone-dependent phase. Here, we review the molecular pathways that contribute to each of these stages, drawing on studies from both human and mouse models. Hypospadias can occur when normal development of the phallus is disrupted, and we provide evidence that mutations in genes underlying this developmental process are causative. Finally, we discuss the environmental factors that may contribute to hypospadias and their potential immediate and transgenerational epigenetic impacts., (© 2015 S. Karger AG, Basel.)

Hypospadias is a congenital malformation and a milder form of 46,XY disorder of sexual development (DSD). In the present study, we investigated 13 haplotype tagging single nucleotide polymorphisms (SNPs) covering the steroid-5-alpha reductase (SRD5A2) and androgen receptor(AR) gene region, respectively, in a cohort consisting of 260 individuals with mild hypospadias and 77 with severe disease, in addition to 471 healthy male controls. The investigated genes are known to have an important role in the hormone-dependent stage of sexual development. Our study revealed one novel marker located in the AR gene region (rs5919436; g.67024320C>G) to be significantly associated with an increased risk of severe hypospadias (adjusted p value: 0.02; odds ratio: 2.98). In concordance with this finding, we detected an association of a haplotype tagged by the minor allele of rs5919436 (adjusted p value: 0.04). We further detected no association between the investigated disease and the haplotype tagging polymorphisms covering the SRD5A2 gene, which is of importance considering the conflicting results reported previously. In conclusion, our data implicate that the AR rs5919436 (g.67024320C>G) polymorphism may act as a novel genetic marker for increased susceptibility to severe hypospadias in Caucasians., (Copyright © 2013 S. Karger AG, Basel.)

Hypospadias is a frequent congenital malformation in boys and is characterized by incomplete fusion of the urethral folds. The steroidogenic factor-1 (SF-1, NR5A1) gene plays a key role in hypothalamic-pituitary-steroidogenic organ development, and has previously been reported to be mutated in individuals with 46,XY disorder of sex development. Here, we investigated the role of SF-1 in hypospadias, a milder form of 46,XY disorder of sex development. We performed direct sequencing analysis of the SF-1 gene in 2 male Caucasian twins exhibiting very severe hypospadias, and in 95 Caucasian boys with mild and severe hypospadias. We further extended the analysis by investigating 332 mild and severe hypospadias cases and 422 male controls using TaqMan assays. Our sequencing revealed a novel heterozygous p.R313H (c.938G>A) missense mutation in each twin, and no mutations in the 95 Caucasian cases. Instead, a missense p.G146A (c.437G>C), and a silent known p.P125P (c.375C>T) polymorphism, respectively, was found in several of the latter cases. Further investigation of the 2 polymorphisms in the larger material of cases and controls showed no significant genotypic or allelic association. In conclusion, the SF-1 gene may not play a significant role in the development of hypospadias in Caucasians., (Copyright © 2012 S. Karger AG, Basel.)

Abnormalities of sexual development causing infertility in horses have been investigated since the early 1970's. Conventional cytogenetic analysis by karyotyping has been the primary tool used to investigate these horses. Abnormalities have a broad range, from a phenotypically normal mare with gonadal dysgenesis to a horse with ambiguous external genitalia and internal male and female organs. Cytogenetic analysis can determine genetic sex but cannot identify mutations or deletions of genes involved in the sex determination pathway. Molecular technologies have been developed to confirm cytogenetic results and to aid in identifying the genetic causes of abnormal sex determination in horses. In this paper, we review the historical development of methods used to understand abnormal sexual development in the horse as well as summarize cases reported over the last 40-50 years., (Copyright © 2011 S. Karger AG, Basel.)

Hypospadias is rarely reported in dogs. In this study we pre-sent 2 novel cases of this disorder of sexual development and, in addition, a case of hereditary sex reversal in a female with an enlarged clitoris. The first case was a male Moscow watchdog with a normal karyotype (78,XY) and the presence of the SRY gene. In this dog, perineal hypospadias, bilateral inguinal cryptorchidism and testes were observed. The second case, representing the Cocker spaniel breed, had a small penis with a hypospadic orifice of the urethra, bilateral cryptorchidism, testis and a rudimentary gonad inside an ovarian bursa, a normal female karyotype (78,XX) and a lack of the SRY gene. This animal was classified as a compound sex reversal (78,XX, SRY-negative) with the hypospadias syndrome. The third case was a Cocker spaniel female with an enlarged clitoris and internally located ovotestes. Cytogenetic and molecular analyses revealed a normal female karyotype (78,XX) and a lack of the SRY gene, while histology of the gonads showed an ovotesticular structure. This case was classified as a typical hereditary sex reversal syndrome (78,XX, SRY-negative). Molecular studies were focused on coding sequences of the SRY gene (case 1) and 2 candidates for monogenic hypospadias, namely MAMLD1 (mastermind-like domain containing 1) and SRD5A2 (steroid-5-alpha-reductase, alpha polypeptide 2). Sequencing of the entire SRY gene, including 5'- and 3'-flanking regions, did not reveal any mutation. The entire coding sequence of MAMLD1 and SRD5A2 was analyzed in all the intersexes, as well as in 4 phenotypically normal control dogs (3 females and 1 male). In MAMLD1 2 SNPs, including 1 missense substitution in exon 1 (c.128A>G, Asp43Ser), were identified, whereas in SRD5A2 7 polymorphisms, including 1 missense SNP (c.358G>A, Ala120Thr), were found. None of the identified polymorphisms cosegregated with the intersexual phenotype, thus, we cannot confirm that hypospadias may be associated with polymorphism in the coding sequence of the studied genes., (Copyright © 2011 S. Karger AG, Basel.)

A 4-year-old male horse of Friesian breed with normal body conformation, development and libido, and showing an evident ventral penis deviation with hypospadias, underwent both cytogenetic and genetic investigation. Although the karyotype showed normal male arrangement (2n = 64,XY), one telomere of horse (ECA) chromosome 1 was shorter than both the other one and those of a normal horse (control), as revealed by CBA- and RBA-banding, and by Ag-NOR and FISH-mapping techniques using telomere PNA probes. Genetic investigation of the SRY and MAMLD1 coding sequences revealed a normal SRY sequence and a mutation in the MAMLD1 gene sequence: a homozygous change (C>A) was found, leading to the synthesis of an isoleucine, instead of a leucine. Although it is difficult to find a strict correlation between hypospadias and the genetic defects revealed by this investigation, this study is the first to be performed in a hypospadic horse using both cytogenetic and genetic investigation., (Copyright © 2010 S. Karger AG, Basel.)

MAMLD1 (mastermind-like domain containing 1), also known as CXorf6 (chromosome X open reading frame 6) has been shown to be a causative gene for hypospadias. This is primarily based on the identification of nonsense mutations (E124X, Q197X, and R653X) which undergo nonsense mediated mRNA decay in patients with penoscrotal hypospadias. Subsequent molecular studies have shown that (1) the mouse homolog is transiently expressed in fetal Sertoli and Leydig cells around the critical period for sex development; (2) CXorf6 protein shares homology to mastermind-like 2 (MAML2) protein that functions as a co-activator in canonical Notch signaling; (3) CXorf6 localizes to the nuclear bodies and transactivates the promoter activity of a non-canonical Notch target gene hairy/enhancer of split 3(Hes3) without demonstrable DNA binding capacity; (4) transient knockdown of CXorf6 results in significantly reduced testosterone production in murine Leydig tumor cells; and (5) CXorf6 can be regulated by steroidogenic factor 1 (SF-1). These findings suggest that the CXorf6 mutations cause hypospadias primarily because of testicular dysfunction and resultant compromised testosterone production around that period, and provide useful information for the molecular network involved in fetal testosterone production., ((c) 2008 S. Karger AG, Basel.)