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Elite individual sports in which success depends on power, speed, or endurance are conventionally divided into male and female events using traditional binary definitions of sex. Male puberty creates durable physical advantages due to the 20- to 30-fold increase in circulating testosterone producing a sustained uplift in men's muscle, bone, hemoglobin, and cardiorespiratory function resulting from male puberty and sustained during men's lives. These male physical advantages provide strong justification for a separate protected category of female events allowing women to achieve the fame and fortune from success they would be denied if competing against men. Recent wider social acceptance of transgender individuals, together with the less recognized involvement of intersex individuals, challenge and threaten to defeat the sex classifications for elite individual female events. This can create unfair advantages if seeking inclusion into elite female events of unmodified male-bodied athletes with female gender identity who have gained the physical advantages of male puberty. Based on reproductive physiology, this paper proposes a working definition of sport sex based primarily on an individual's experience of male puberty and can be applied to transgender and various XY intersex conditions. Consistent with the multidimensionality of biological sex (chromosomal, genetic, hormonal, anatomical sex), this definition may be viewed as a multistrand cable whose overall strength survives when any single strand weakens or fails, rather than as a unidimensional chain whose strength is only as good as its weakest link. [ABSTRACT FROM AUTHOR]

Differences/disorders of sex development (DSDs) in individuals with a 46, XY karyotype are a group of congenital disorders that manifest as male gonadal hypoplasia or abnormalities of the external genitalia. Approximately 50% of patients with 46, XY DSDs cannot obtain a molecular diagnosis. The aims of this paper were to review the most common causative genes and rare genes in patients with 46, XY DSDs, analyze global molecular diagnostic cohorts for the prevalence and geographic distribution of causative genes, and identify the factors affecting cohort detection results. Although the spectrum of genetic variants varies across regions and the severity of the clinical phenotype varies across patients, next-generation sequencing (NGS), the most commonly used detection method, can still reveal genetic variants and aid in diagnosis. A comparison of the detection rates of various sequencing modalities revealed that whole-exome sequencing (WES) facilitates a greater rate of molecular diagnosis of the disease than panel sequencing. Whole-genome sequencing (WGS), third-generation sequencing, and algorithm advancements will contribute to the improvement of detection efficiency. The most commonly mutated genes associated with androgen synthesis and action are AR, SR5A2, and HSD17B3, and the most commonly mutated genes involved in gonadal formation are NR5A1 and MAP3K1. Detection results are affected by differences in enrollment criteria and sequencing technologies. [ABSTRACT FROM AUTHOR]

Introduction: One of the common causes of 46,XY differences in sex development (DSD) cases is androgen insensitivity syndrome. This X-linked recessive inherited condition is associated with pathological variations of the AR gene, leading to defects in androgen action. Affected 46,XY infants or individuals experience variable degrees of undervirilization and those with severe form will have female-like external genitalia. Therefore, they were more likely assigned and reared as females. The confirmatory molecular test is often needed due to similar clinical manifestations with other conditions causing 46,XY DSD. Since in our country, the molecular test for the AR gene is lacking, the study is conducted as a preliminary study to elaborate on the possibility of developing a molecular test for the AR gene in 46,XY DSD cases. Methods: Archived DNAs of 13 46,XY DSD cases were analyzed using polymerase chain reaction and direct sequencing for molecular defects in the AR gene. Clinical and hormonal data were collected and analyzed. Results: The study successfully amplified and visualized the eight exons of the AR gene and revealed two subjects carrying AR gene variants at exon 7. In the first case, 1.2-year-old boy carried heterozygous p.Gln825Arg, which has never been reported elsewhere, and the second subject, a 2.1-year-old girl with heterozygous p.Arg841His. Both subjects presented with severe undervirilization of external genitalia with external genitalia masculinization scores (EMS) of 1.5 and 3. Conclusion: In this series, two of 13 46,XY DSD cases carried variants at the AR gene, resulting in complete androgen insensitivity syndrome. [ABSTRACT FROM AUTHOR]

Introduction: NR5A1 and NR5A2 belong to an orphan nuclear receptor group, and approximately 60% of their amino acid sequences are conserved. Transcriptional regulation of NR5A receptors depends on interactions with co-factors or unidentified ligands. Purpose and methods: We employed in vitro and in silico analysis for elucidating the pathophysiology of a novel variant in the ligand-binding domain of NR5A1, p.R350W which was identified from a 46,XY patient with atypical genitalia. Results: In the study, [1] reporter assays demonstrated that R350 is essential for NR5A1; [2] 3D model analysis predicted that R350 interacted with endogenous ligands or unknown cofactors rather than stabilizing the structure; [3] R350 is not conserved in NR5A2 but is specifically required for NR5A1; and [4] none of the 22 known missense variants of the ligand binding domain satisfied all the previous conditions [1]-[3], suggesting the unique role of R350 in NR5A1. Conclusion: Our data suggest that NR5A1 has unidentified endogenous ligands or co-activators that selectively potentiate the transcriptional function of NR5A1 in vivo. [ABSTRACT FROM AUTHOR]

Objective: The literature regarding gonadoblastoma risk in exonic Wilms’ tumor suppressor gene (WT1) pathogenic variants is sparse. The aim of this study is to describe the phenotypic and genotypic characteristics of Asian–Indian patients with WT1 pathogenic variants and systematically review the literature on association of exonic WT1 pathogenic variants and gonadoblastoma. Design: Combined retrospective–prospective analysis. Methods: In this study, 46,XY DSD patients with WT1 pathogenic variants detected by clinical exome sequencing from a cohort of 150 index patient s and their affected relatives were included. The PubMed database was searched for the literature on gonadoblastoma with exonic WT1 pathogenic variants. Results: The prevalence of WT1 pathogenic variants among 46,XY DSD index patients was 2.7% (4/150). All the four patients had atypical genitalia and cryptorchidism. None of them had Wilms’ tumor till the last follow-up, whereas one patient had late-onset nephropathy. 11p13 deletion was present in one patient with aniridia. The family with p.Arg458Gln pathogenic variant had varied phenotypic spectrum of Frasier syndrome; two siblings had gonadoblastoma, one of them had growing terato ma syndrome (first to report with WT1). On literature review, of >100 exonic point pathogenic variants, only eight variants (p.Arg462Trp, p.Tyr177*, p.Arg434His, p.Met410Arg, p.Gln142*, p.Glu437Lys, p.Arg458*, and p.Arg458Gln) in WT1 were associated with gonadoblastoma in a total of 15 cases (including our two cases). Conclusions: WT1 alterations account for 3% of 46,XY DSD patients in our cohort. 46,XY DSD patients harboring exonic WT1 pathogenic variants carry a small but definitive risk of gonadoblastoma; hence, these patients require a gonadoblastoma surveillance with a more stringent surveillance in those harboring a gonadoblastoma-associated variant.

A girl presenting with delayed puberty and elevated gonadotropins may have a range of conditions such as Turner syndrome (TS), primary ovarian insufficiency (POI), and 46,XY disorders of sexual development (DSD). An organized and measured approach to investigation can help reach a timely diagnosis. Management of young people often requires specialist multidisciplinary input to address the endocrine and nonendocrine features of these complex conditions, as well as the psychological challenges posed by their diagnosis. Next-generation sequencing within the research setting has revealed several genetic causes of POI and 46,XY DSD, which may further facilitate an individualized approach to care of these young people in the future. Pubertal induction is required in many and the timing of this may need to be balanced with other issues specific to the condition (eg, allowing time for information-sharing in 46,XY DSD, optimizing growth in TS). Shared decision-making and sign-posting to relevant support groups from the outset can help empower young people and their families to manage these conditions. We describe 3 clinical vignettes of girls presenting with delayed puberty and hypergonadotropic amenorrhea and discuss their clinical management in the context of current literature and guidelines. [ABSTRACT FROM AUTHOR]

BACKGROUND:Disorders of sex development (DSD) have an estimated frequency of 0.5% of live births encompassing a variety of urogenital anomalies ranging from mild hypospadias to a discrepancy between sex chromosomes and external genitalia. In order to identify the underlying genetic etiology, we had performed exome sequencing in a subset of DSD cases with 46,XY karyotype and were able to identify the causative genetic variant in 35% of cases. While the genetic etiology was not ascertained in more than half of the cases, a large number of variants of unknown clinical significance (VUS) were identified in those exomes. METHODS:To investigate the relevance of these VUS in regards to the patient's phenotype, we utilized a mouse model in which the presence of a Y chromosome from the poschiavinus strain (Y POS ) on a C57BL/6J (B6) background results in XY undervirilization and sex reversal, a phenotype characteristic to a large subset of human 46,XY DSD cases. We assessed gene expression differences between B6-Y B6 and undervirilized B6-Y POS gonads at E11.5 and identified 515 differentially expressed genes (308 underexpressed and 207 overexpressed in B6-Y POS males). RESULTS:We identified 15 novel candidate genes potentially involved in 46,XY DSD pathogenesis by filtering the list of human VUS-carrying genes provided by exome sequencing with the list of differentially expressed genes from B6-Y POS mouse model. Additionally, we identified that 7 of the 15 candidate genes were significantly underexpressed in the XY gonads of mice with suppressed Sox9 expression in Sertoli cells suggesting that some of the candidate genes may be downstream of a well-known sex determining gene, Sox9. CONCLUSION:The use of a DSD-specific animal model improves variant interpretation by correlating human sequence variants with transcriptome variation.

Investigation of disorders of sex development (DSD) has resulted in the discovery of multiple sex-determining genes. MAP3K1 encodes a signal transduction regulator in the sex determination pathway and is emerging as one of the more common genes responsible for 46,XY DSD presenting as complete or partial gonadal dysgenesis. Clinical assessment, endocrine evaluation, and genetic analysis were performed in six individuals from four unrelated families with 46,XY DSD. All six individuals were found to have likely pathogenic MAP3K1 variants. Three of these individuals presented with complete gonadal dysgenesis, characterized by bilateral streak gonads with typical internal and external female genitalia, while the other three presented with partial gonadal dysgenesis, characterized by incomplete testicular development, resulting in clitoral hypertrophy with otherwise typical female external genitalia. Testing for MAP3K1 variants should be considered in patients with 46,XY complete or partial gonadal dysgenesis, particularly in families with multiple members affected with 46,XY DSD. Identification of a MAP3K1 variant should prompt an evaluation for DSD in female siblings of the proband.

Context There are more than 100 pathogenic variants in CYP17A1 that have been identified in patients with 17α-hydroxylase/17,20-lyase deficiency (17OHD). Objective We aimed to describe 46,XY patients with 17OHD from our center and review the literature. Methods We retrospectively analyzed genetically proven index cases of 17OHD from our 46,XY disorders of sex development cohort and reviewed similar cases from the literature (n = 150). Based on the phenotype, 17OHD probands were classified into combined severe deficiency (n = 128) and combined partial deficiency (n = 16). Additionally, patients with the apparent isolated 17,20-lyase deficiency (n = 7, from 6 families) were noted. Residual enzyme activities with the observed mutant enzymes were divided in 2 categories as < 1% and ≥ 1%, each for hydroxylase and lyase. Results We present 4 index cases of 46,XY 17OHD with a complete spectrum of undervirilization and 2 novel variants in CYP17A1. In the review, the combined severe deficiency was the most common form, with more frequent female sex of rearing, hypertension, hypokalemia, suppressed renin, higher plasma corticotropin, lower serum cortisol, and androgens. Immunoassay-measured serum aldosterone was frequently (68.2%) unsuppressed (>5 ng/dL). Elevated serum progesterone had high sensitivity for diagnosis of combined 17OHD, even in combined partial deficiency (83.3%). Among patients with clinical phenotype of combined severe deficiency, 11.5% had partial 17α-hydroxylase and complete 17,20-lyase deficiency (>1%/<1%) and had significantly higher serum cortisol than those with < 1%/<1% activity. Conclusion We report the first monocentric case series of Asian Indian 46,XY patients with 17OHD. We propose that a phenotype of severe undervirilization with milder cortisol deficiency may represent a distinct subtype of combined severe 17OHD with residual 17α-hydroxylase activity but severe 17,20-lyase deficiency (>1%/<1%), which needs further validation. [ABSTRACT FROM AUTHOR]

Rafael Loch Batista, Berenice Bilharinho Mendonca Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular/LIM42, Hospital das Clínicas, Disciplina de Endocrinologia, do Departamento de Clínica Médica, Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilCorrespondence: Berenice Bilharinho Mendonca Eneas Carvalho de Aguiar, 255, São Paulo, SP 05403-000, BrazilTel +55 1126617512Email beremen@usp.brIntroduction: The conversion of testosterone into dihydrotestosterone is catalyzed by the 5α-reductase type 2 enzyme which plays a crucial role in the external genitalia virilization. It is encoded by the SRD5A2 gene. Allelic variants in this gene cause a 46,XY DSD with no genotype–phenotype relationship. It was firstly reported in the early 70s from isolated clusters. Since then, several cases have been reported. Putting together, it will expand the knowledge on the molecular bases of androgen milieu.Methods: We searched for SRD5A2 allelic variants (AV) in the literature (PubMed, Embase, MEDLINE) and websites (ensembl, HGMD, ClinVar). Only cases with AV in both alleles, either in homozygous or compound heterozygous were included. The included cases were analyzed according to ethnicity, exon, domain, aminoacid (aa) conservation, age at diagnosis, sex assignment, gender reassignment, external genitalia virilization and functional studies. External genitalia virilization was scored using Sinnecker scale. Conservation analysis was carried out using the CONSURF platform. For categorical variables, we used X2 test and Cramer’s V. Continuous variables were analyzed by t test or ANOVA. Concordance was estimated by Kappa.Results: We identified 434 cases of 5ARD2 deficiencies from 44 countries. Most came from Turkey (23%), China (17%), Italy (9%), and Brazil (7%). Sixty-nine percent were assigned as female. There were 70% of homozygous allelic variants and 30% compound heterozygous. Most were missense variants (76%). However, small indels (11%), splicing (5%) and large deletions (4%) were all reported. They were distributed along with all exons with exon 1 (33%) and exon 4 (25%) predominance. Allelic variants in the exon 4 (NADPH-binding domain) resulted in lower virilization (p< 0.0001). The codons 55, 65, 196, 235 and 246 are hotspots making up 25% of all allelic variants. Most of them (76%) were located at conserved aa. However, allelic variants at non-conserved aa were more frequently indels (28% vs 6%; p< 0.01). The overall rate of gender change from female to male ranged from 16% to 70%. The lowest rate of gender change from female to male occurred in Turkey and the highest in Brazil. External genitalia virilization was similar between those who changed and those who kept their assigned gender. The gender change rate was significantly different across the countries (V=0.44; p< 0.001) even with similar virilization scores.Conclusion: 5ARD2 deficiency has a worldwide distribution. Allelic variants at the NADPH-ligand region cause lower virilization. Genitalia virilization influenced sex assignment but not gender change which was influenced by cultural aspects across the countries. Molecular diagnosis influenced on sex assignment, favoring male sex assignment in newborns with 5α-reductase type 2 deficiency.Keywords: SRD5A2, 46XY DSD, differences of sexual development, atypical genitalia, dihydrotestosterone, 5α-reductase type 2 deficiency

Aim: This study examines the distribution of genital abnormalities based on physical examination of our patients with 46, XY disorders of sex development (46, XY DSD), and aims to define severity and frequency of hypospadias in 46, XY DSD. Hypospadias is a relatively prevalent congenital anomaly. Although genetic, environmental and hormonal factors are considered to be responsible, etiology is not clarified in several hypospadias cases. Materials and Methods: Clinical, laboratory and genetic records of all cases with 46, XY DSD, who were evaluated by the sex determination monitoring board were retrospectively reviewed. In the diagnosis, hypospadias cases were examined in terms of the place of hypospadias and coexisting other external genital findings. Results: There were 72 patients with 46, XY DSD. 5-α reductase deficiency [n=32 (44.4%)] was the most commonly encountered diagnosis followed by androgen insensitivity syndrome [n=26, (36.1%)]. Proximal hypospadias were presented in 44.4% (n: 32) of the cases and only 6 of them (18.8%) were isolated hypospadias. In 81.2% of these cases, at least one of the anomalies such as cordi, bifid scrotum, undescended testis and micropenis accompanied proximal hypospadias. None of the distal hypospadias cases were referral clinic finding. Conclusions: 46 XY DSD is a heterogeneous group of patients with a varying age of presentation and a diverse clinical profile. It can be stated that proximal hypospadias is the most common referral clinic finding of 46, XY DSD, and the risk of 46, XY DSD increases with the intensifying degree of hypospadias and the presence of coexisting genital abnormalities such as cordi, bifid scrotum, undescended testis and micropenis. [ABSTRACT FROM AUTHOR]

Missense variants in the RNA-helicase DHX37 are associated with either 46,XY gonadal dysgenesis or 46,XY testicular regression syndrome (TRS). DHX37 is required for ribosome biogenesis, and this subgroup of XY DSD is a new human ribosomopathy. In a cohort of 140 individuals with 46,XY DSD, we identified 7 children with either 46,XY complete gonadal dysgenesis or 46,XY TRS carrying rare or novel DHX37 variants. A novel p.R390H variant within the RecA1 domain was identified in a girl with complete gonadal dysgenesis. A paternally inherited p.R487H variant, previously associated with a recessive congenital developmental syndrome, was carried by a boy with a syndromic form of 46,XY DSD. His phenotype may be explained in part by a novel homozygous loss-of-function variant in the NGLY1 gene, which causes a congenital disorder of deglycosylation. Remarkably, a homozygous p.T477H variant was identified in a boy with TRS. His fertile father had unilateral testicular regression with typical male genital development. This expands the DSD phenotypes associated with DHX37. Structural analysis of all variants predicted deleterious effects on helicase function. Similar to all other known ribosomopathies, the mechanism of pathogenesis is unknown. [ABSTRACT FROM AUTHOR]

Context Little is known about the physical health of individuals with 46,XY disorders of sex development (DSD). Objective To assess physical and reported subjective health of individuals with XY DSD. Methods As part of the dsd-LIFE study, patients with an XY DSD condition were analyzed in different diagnosis groups for metabolic parameters, comorbidities, metabolic syndrome, bone outcomes, and reported subjective health. Findings were evaluated by descriptive statistics. Results A total of 222 patients with XY DSD were included with a mean age of 28.8 ± 12.2 years, mean height of 175.3 ± 7.7 cm, mean weight of 74.3 ± 20.0 kg, and mean body mass index of 24.1 ± 6.0 kg/m2. Obesity rate was not increased when descriptively compared with Eurostat data. Fourteen patients had metabolic syndrome (14/175; 8.0%). In descriptive comparison with data from the DECODE study and World Health Organization, subjects fared better in the categories waist circumference, glucose, triglyceride, cholesterol, and high-density lipoprotein. Of participants with available bone health data, 19/122 (15.6%) patients had a Z-score ≤ –2.0 at lumbar spine indicating lowered bone mineral density (BMD). Mostly gonadectomized individuals with complete androgen insensitivity syndrome (CAIS) and no estrogen therapy had lowered BMD at lumbar spine. Individuals with XY DSD performed poorly in the category subjective health in descriptive comparison with Eurostat data. Conclusion Participants reported a lower subjective health status than Eurostat data but their overall metabolic health status was good. Decreased BMD at lumbar spine was especially present in gonadectomized individuals with CAIS and no estrogen therapy. [ABSTRACT FROM AUTHOR]

46,XY differences and/or disorders of sex development (DSD) are clinically and genetically heterogeneous conditions. Although complete androgen insensitivity syndrome has a strong genotype–phenotype correlation, the other types of 46,XY DSD are less well defined, and thus, the precise diagnosis is challenging. This study focused on comparing the relationship between clinical assessment and genetic findings in a cohort of well-phenotyped patients with 46,XY DSD. The study was an analysis of clinical investigations followed by genetic testing performed on 35 patients presenting to a single center. The clinical assessment included external masculinization score (EMS), endocrine profiling and radiological evaluation. Array-comparative genomic hybridization (array-CGH) and sequencing of DSD-related genes were performed. Using an integrated approach, reaching the definitive diagnosis was possible in 12 children. The correlation between clinical and genetic findings was higher in patients with a more severe phenotype (median EMS 2.5 vs 6; P = 0.04). However, in 13 children, at least one variant of uncertain significance was identified, and most times this variant did not correspond to the original clinical diagnosis. In three patients, the genetic studies guided further clinical assessment which resulted in a reclassification of initial clinical diagnosis. Furthermore, we identified eight patients harboring variants in more than one DSD genes, which was not seen in controls (2.5%; P = 0.0003). In summary, taking into account potential challenges in reaching the definitive diagnosis in 46,XY DSD, only integrated approach seems to be the best routine practice.

Non-syndromic 46,XY DSD (disorders of sex development) represent a phenotypically diversiform group of disorders. We focus on the association between gene variants and the most frequent types of non-syndromic 46,XY DSD, options of molecular genetic testing which has surely taken its place in diagnostics of DSD in the past couple of years. We emphasize the need of molecular genetic testing in individuals with non-syndromic 46,XY DSD in Slovak Republic.

Background5α-reductase type 2 deficiency (5αRD) is an autosomal recessive hereditary disease of the group of 46, XY disorders of sex development (DSD).ObjectiveTo study the growth pattern in Chinese pediatric patients with 5αRD.SubjectsData were obtained from 141 patients with 5αRD (age: 0–16 years old) who visited eight pediatric endocrine centers from January 2010 to December 2017.MethodsIn this retrospective cohort study, height, weight, and other relevant data were collected from the multicenter hospital registration database. Baseline luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), and dihydrotestosterone (DHT) after human chorionic gonadotropin (HCG) stimulation test were measured by enzyme enhanced chemiluminescence assay. Bone age (BA) was assessed using the Greulich-Pyle (G-P) atlas. Growth curve was constructed based on λ-median-coefficient of variation method (LMS).ResultsThe height standard deviation scores (HtSDS) and weight standard deviation scores (WtSDS) in 5αRD children were in the normal range as compared to normal boys. Significantly higher HtSDS was observed in patients with 5αRD who were

Background: 5α-reductase type 2 deficiency (5αRD) is an autosomal recessive hereditary disease of the group of 46, XY disorders of sex development (DSD). Objective: To study the growth pattern in Chinese pediatric patients with 5αRD. Subjects: Data were obtained from 141 patients with 5αRD (age: 0–16 years old) who visited eight pediatric endocrine centers from January 2010 to December 2017. Methods: In this retrospective cohort study, height, weight, and other relevant data were collected from the multicenter hospital registration database. Baseline luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), and dihydrotestosterone (DHT) after human chorionic gonadotropin (HCG) stimulation test were measured by enzyme enhanced chemiluminescence assay. Bone age (BA) was assessed using the Greulich-Pyle (G-P) atlas. Growth curve was constructed based on λ-median-coefficient of variation method (LMS). Results: The height standard deviation scores (HtSDS) and weight standard deviation scores (WtSDS) in 5αRD children were in the normal range as compared to normal boys. Significantly higher HtSDS was observed in patients with 5αRD who were <1 year old (t = 3.658, 2.103, P = 0.002, 0.048, respectively), and higher WtSDS in those <6 months old (t = 2.756, P = 0.012). Then HtSDS and WtSDS decreased gradually and fluctuated near the median of the same age until 13 years. WtSDS in 5αRD children from northern China were significantly higher than those from the south (Z = -2.670, P = 0.008). The variation tendency of HtSDS in Chinese 5αRDs was consistent with the trend of stimulating T. HtSDS and stimulating T in the external masculinization score (EMS) <7 group were slightly higher than those in EMS ≥ 7 group without significant difference. Additionally, the ratio of BA over chronological age (BA/CA) was significantly <1 in children with 5αRD. Conclusion: Children with 5αRD had a special growth pattern that was affected by high levels of T, while DHT played a very small role in it. Their growth accelerated at age <1 year, followed by slowing growth and fluctuating height near normal median boys' height. The BA was delayed in 5αRD children. Androgen treatment, which may be considered anyway for male 5αRD patients with a micropenis, may also be beneficial for growth. [ABSTRACT FROM AUTHOR]

Keywords: gonadal dysgenesis; haematological chimerism; sex-discordant; twins; XY DSD EN gonadal dysgenesis haematological chimerism sex-discordant twins XY DSD 487 489 3 04/30/20 20200501 NES 200501 46,XY Disorders of sex differentiation (DSD) such as complete gonadal dysgenesis, disorders of androgen synthesis and action, ovotesticular DSD can present with unambiguous or minimally virilized female external genitalia. Genotype analysis on lymphocyte DNA from proband's twin brother showed the presence of a female genotype, which was identical to proband's genotype. Genotype analysis and karyotyping of the proband's twin brother's lymphocytes showed a genotype profile similar to the proband and a 46,XY [98%]/46,XX [2%] karyotype. [Extracted from the article]

Empty follicle syndrome (EFS) is a disorder associated with female infertility and presents as a complete failure to retrieve oocytes during ART cycles despite normal follicle development and careful aspiration. To date, only two EFS cases have been reported with homozygous missense mutations in the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) gene, and both cases showed normal estradiol (E2) production during ovulation induction. The molecular genetic mechanisms of EFS remain unknown. Herein, we report two novel homozygous inactivating LHCGR mutations, c.736 C>T (p.Q246*) and c.846dupT (p.R283*), in two female EFS patients from unrelated consanguineous families. The probands had impaired E2 production during the ART process, which differs from previously reported EFS cases. The inactivating mutations not only led to EFS in the two female probands, but also resulted in 46, XY disorder of sex development (46, XY DSD) in their male siblings. As far as we know, this is the first report of LHCGR mutations leading to both EFS and 46, XY DSD within the same pedigree. Our findings provide researchers and clinicians with a better understanding of phenotype-genotype correlations between EFS and 46, XY DSD and the LHCGR gene. [ABSTRACT FROM AUTHOR]

Disorders of sex development (DSD) consist of a wide range of conditions involving numerous genes. Nevertheless, about half of 46,XY individuals remain genetically unsolved. GATA4 gene variants, mainly related to congenital heart defects (CHD), have also been recently associated with 46,XY DSD. In this study, we characterized three individuals presenting with 46,XY DSD with or without CHD and GATA4 variants in order to understand the phenotypical variability. We studied one patient presenting CHD and 46,XY gonadal dysgenesis, and two patients with a history of genetically unsolved 46,XY DSD, also known as male primary hypogonadism. Mutation analysis was carried out by candidate gene approach or targeted gene panel sequencing. Functional activity of GATA4 variants was tested in vitro on the CYP17 promoter involved in sex development using JEG3 cells. We found two novel and one previously described GATA4 variants located in the N-terminal zinc finger domain of the protein. Cys238Arg variant lost transcriptional activity on the CYP17 promoter reporter, while Trp228Cys and Pro226Leu behaved similar to wild type. These results were in line with bioinformatics simulation studies. Additional DSD variations, in the LRP4 and LHCGR genes, respectively, were identified in the two 46,XY individuals without CHD. Overall, our study shows that human GATA4 mutations identified in patients with 46,XY DSD may or may not be associated with CHD. Possible explanations for phenotypical variability may comprise incomplete penetrance, variable sensitivity of partner genes, and oligogenic mechanisms. [ABSTRACT FROM AUTHOR]

Background: Disorders of sex development (DSD) have an estimated frequency of 0.5% of live births encompassing a variety of urogenital anomalies ranging from mild hypospadias to a discrepancy between sex chromosomes and external genitalia. In order to identify the underlying genetic etiology, we had performed exome sequencing in a subset of DSD cases with 46,XY karyotype and were able to identify the causative genetic variant in 35% of cases. While the genetic etiology was not ascertained in more than half of the cases, a large number of variants of unknown clinical significance (VUS) were identified in those exomes. Methods: To investigate the relevance of these VUS in regards to the patient's phenotype, we utilized a mouse model in which the presence of a Y chromosome from the poschiavinus strain (YPOS) on a C57BL/6J (B6) background results in XY undervirilization and sex reversal, a phenotype characteristic to a large subset of human 46,XY DSD cases. We assessed gene expression differences between B6-YB6 and undervirilized B6-YPOS gonads at E11.5 and identified 515 differentially expressed genes (308 underexpressed and 207 overexpressed in B6-YPOS males). Results: We identified 15 novel candidate genes potentially involved in 46,XY DSD pathogenesis by filtering the list of human VUS-carrying genes provided by exome sequencing with the list of differentially expressed genes from B6-YPOS mouse model. Additionally, we identified that 7 of the 15 candidate genes were significantly underexpressed in the XY gonads of mice with suppressed Sox9 expression in Sertoli cells suggesting that some of the candidate genes may be downstream of a well-known sex determining gene, Sox9. Conclusion: The use of a DSD-specific animal model improves variant interpretation by correlating human sequence variants with transcriptome variation. [ABSTRACT FROM AUTHOR]

Objective Disorders of sex development ( DSD) are a heterogeneous group of conditions affecting the differentiation and development of the internal and external genitalia. Here, we aimed at identifying the genetic cause of DSD in two 46, XY sisters from a consanguineous family. Design We performed a whole-exome sequencing of two 46, XY female individuals. Sanger sequencing was used to validate the most likely candidate variant, affecting the desert hedgehog ( DHH) gene. Molecular dynamics simulations were performed to get insights into the impact of the variant on protein structure and on its interaction with the protein partner BOC (brother of CDO/cell adhesion molecule, downregulated by oncogenes). Patients The index patient presented with a female phenotype, primary amenorrhoea (low oestradiol and testosterone and high FSH and LH). She also had an apparent absence of intra-abdominal gonads and uterus, facial dysmorphy, psychomotor retardation and neuropathy. Her sister displayed a similar gonadal and endocrinological picture, without dysmorphy or psychomotor retardation. Results Whole-exome sequencing revealed a homozygous variant in DHH leading to the p.Trp173Cys substitution. The relevant Trp residue is conserved, and its alteration was predicted to be deleterious. Molecular dynamics simulations showed that the mutation increases the conformational flexibility of the protein and potentially alters its interaction with BOC, a positive regulator of Hedgehog signalling. We do not exclude an interference of the mutation with DHH-intein-mediated auto-processing. Conclusions This report increases the number of described homozygous DHH variants and highlights the importance of advanced bioinformatic tools to better understand the pathogenicity of human variants. [ABSTRACT FROM AUTHOR]

Study Question: What is the likelihood of identifying genetic or endocrine abnormalities in a group of boys with 46, XY who present to a specialist clinic with a suspected disorder of sex development (DSD)?Summary Answer: An endocrine abnormality of the gonadal axis may be present in a quarter of cases and copy number variants (CNVs) or single gene variants may be present in about half of the cases.What Is Known Already: Evaluation of 46, XY DSD requires a combination of endocrine and genetic tests but the prevalence of these abnormalities in a sufficiently large group of boys presenting to one specialist multidisciplinary service is unclear.Study, Design, Size, Duration: This study was a retrospective review of investigations performed on 122 boys.Participants/materials, Setting, Methods: All boys who attended the Glasgow DSD clinic, between 2010 and 2015 were included in the study. The median external masculinization score (EMS) of this group was 9 (range 1-11). Details of phenotype, endocrine and genetic investigations were obtained from case records.Main Results and the Role Of Chance: An endocrine abnormality of gonadal function was present in 28 (23%) with a median EMS of 8.3 (1-10.5) whilst the median EMS of boys with normal endocrine investigations was 9 (1.5-11) (P = 0.03). Endocrine abnormalities included a disorder of gonadal development in 19 (16%), LH deficiency in 5 (4%) and a disorder of androgen synthesis in 4 (3%) boys. Of 43 cases who had array-comparative genomic hybridization (array-CGH), CNVs were reported in 13 (30%) with a median EMS of 8.5 (1.5-11). Candidate gene analysis using a limited seven-gene panel in 64 boys identified variants in 9 (14%) with a median EMS of 8 (1-9). Of the 21 boys with a genetic abnormality, 11 (52%) had normal endocrine investigations.Limitations, Reasons For Caution: A selection bias for performing array-CGH in cases with multiple congenital malformations may have led to a high yield of CNVs. It is also possible that the yield of single gene variants may have been higher than reported if the investigators had used a more extended gene panel.Wider Implications Of the Findings: The lack of a clear association between the extent of under-masculinization and presence of endocrine and genetic abnormalities suggests a role for parallel endocrine and genetic investigations in cases of suspected XY DSD.Study Funding/competing Interest(s): RN was supported by the James Paterson Bursary and the Glasgow Children's Hospital Charity Summer Scholarship. SFA, RM and EST are supported by a Scottish Executive Health Department grant 74250/1 for the Scottish Genomes Partnership. EST is also supported by MRC/EPSRC Molecular Pathology Node and Wellcome Trust ISSF funding. There are no conflicts of interest.Trial Registration Number: None. [ABSTRACT FROM AUTHOR]

The majority of patients with DSD will be found to carry an XY karyotype and be assigned male gender. From a phenotypical standpoint, most will present with proximal hypospadias ± cryptorchidism. In this review article, the authors present the current status of reconstruction of the male genitalia in this setting. The report addresses the following topics: surgical input in the evaluation of the newborn with an undervirilized external genitalia, including gender assignment considerations; controversies surrounding timing and indication for hypospadias surgery in proximal cases as well as use of testosterone; surgical techniques and decision-making process for one- vs. two-stage repairs; complications of hypospadias surgery based on technique used for repair; and long-term follow-up. The high complication rates observed in the treatment of proximal hypospadias attest to its challenging nature. Concentration of experience, tracking carefully identified patient-centered outcomes and long-term follow-up of this patient population are recommended. [ABSTRACT FROM AUTHOR]

The 46, XY disorder of sex development (DSD) is the main cause of birth defects; however, as it is a group of highly heterogeneous diseases, >50% of cases are not accurately diagnosed. Identification of more cases will improve understanding of the relationship between genotype and phenotype for DSD. The present study conducted a systematic analysis of the clinical characteristics of a proband with 46, XY DSD, applied genetic analysis by whole-exome sequencing to this pedigree and performed bioinformatics analysis of the identified variant. The proband presented with a short penis, lack of testicles and partial growth hormone (GH) deficiency at 1 year old. Histopathological examination revealed there were oviduct, epididymis and fibrous vascular tissue on both sides of the abdomen. The last follow-up at 5 years of age revealed that the patient exhibited restricted growth, a 1.5-cm penis and lack of testicles. Notably, a novel pathogenic mitogen-activated protein kinase kinase kinase 1 (MAP3K1) variant (c.3020A>G) was identified in the proband, resulting in a change in the 1,007th amino acid (glutamine) of the encoded protein. This variant caused the uncharged neutral glutamine to be replaced by a positively charged basic arginine. p.Gln1007 in MAP3K1 was confirmed to be conserved across various species. Pathogenicity analysis using bioinformatics tools suggested that this MAP3K1 variant may cause functional defects. In conclusion, the present study identified a novel MAP3K1 variant that was the cause of 46, XY DSD and partial GH deficiency. The present findings extend the mutation spectrum of MAP3K1 and provide novel characteristics of 46, XY DSD. [ABSTRACT FROM AUTHOR]

Inactivating mutations of the 5 α-steroid reductase type-2 ( SRD5A2) gene result in a broad spectrum of masculinization defects, ranging from a male phenotype with hypospadias to a female phenotype with Wolffian structures. Molecular studies of the SRD5A2 revealed a new heterozygous gene variant within the coding region that results in phenotypic expression. A c.92C>T transition changing serine to phenylalanine at codon 31 of exon 1 (p.Ser31Phe) was identified in a patient with 46,XY disorder of sexual development who displayed glandular hypospadias with micropenis and bilateral cryptorchidism. The restoration of the p.Ser31Phe mutation by site-directed mutagenesis and transient expression assays using cultured HEK-293 cells showed that this novel substitution does not abolish but does deregulate the catalytic efficiency of the enzyme. Thus, the maximum velocity ( Vmax) value was higher for the mutant enzyme (22.5 ± 6.9 nmol DHT mg protein−1 h−1) than for the wild-type enzyme (9.8 ± 2.0 nmol DHT mg protein−1 h−1). Increased in vitro activity of the p.Ser31Phe mutant suggested an activating effect. This case provides evidence that heterozygous missense mutations in SRD5A2 may induce the abnormal development of male external genitalia. [ABSTRACT FROM AUTHOR]

At least one genetic defect in each reaction of the classical androgen biosynthesis pathway has been described. For some steps, such as the conversion of cholesterol to pregnenolone and the 17,20-lyase reaction, two or three genetic defects cause similar disorders with overlapping phenotypes and biochemical profiles. The elucidation of the molecular basis for these diseases has helped to define the pathways, essential genes, and enzymatic steps required to make androgens, and this knowledge is being exploited to develop better treatments of androgen-dependent diseases. Furthermore the description of nonclassical lipoid CAH and the protean manifestations of P450 oxidoreductase (POR) deficiencies has expanded the spectrum of human disease caused by disordered steroidogenesis. Finally, the recognition of the backdoor pathway to DHT has added a new dimension to our understanding of how steroid flux is maintained in normal and pathologic states. The traditional view of male external genital development has been that fetal testicular testosterone is converted to DHT by 5α-reductase Type 2 in genital skin, which then acts in a paracrine fashion to stimulate fusion of the labio-scrotal folds and phallic growth. This view is consistent with the incomplete external genital development in persons with severe deficiencies of 5α-reductase type 2. The new observations concerning AKR1C2/4 and the backdoor pathway indicate that DHT produced in the testis via the backdoor pathway also acts as a hormone to induce labio-scrotal fusion. Thus, both the classic and backdoor pathways are needed, and DHT acts in male genital development as both a paracrine factor and as a hormone. These surprising findings are revising our understanding of the mechanisms by which male sexual differentiation occurs, and illustrate the importance of detailed studies of rare patients with 46,XY DSD. [ABSTRACT FROM AUTHOR]

MAMLD1 (mastermind-like domain containing 1) is a recently discovered causative gene for 46,XY disorders of sex development (DSD), with hypospadias as the salient clinical phenotype. To date, microdeletions involving MAMLD1 have been identified in six patients, and definitive mutations (nonsense and frameshift mutations that are predicted to undergo nonsense mediated mRNA decay [NMD]) have been found in six patients. In addition, specific MAMLD1 cSNP(s) and haplotype may constitute a susceptibility factor for hypospadias. Furthermore, in vitro studies have revealed that (1) the mouse homolog is expressed in fetal Sertoli and Leydig cells around the critical period for sex development; (2) transient Mamld1 knockdown results in significantly reduced testosterone production primarily because of compromised 17α-hydroxylation and Cyp17a1 expression in Murine Leydig tumor cells; (3) MAMLD1 localizes to the nuclear bodies and transactivates the promoter activity of a non-canonical Notch target gene hairy/enhancer of split 3, without demonstrable DNA-binding capacity; and (4) MAMLD1 is regulated by steroidogenic factor 1 (SF1). These findings suggest that the MAMLD1 mutations cause 46,XY DSD primarily because of compromised testosterone production around the critical period for sex development. Further studies will provide useful information for the molecular network involved in fetal testosterone production. [ABSTRACT FROM AUTHOR]

The molecular steps in normal sexual development were largely discovered by studying patients and animal models with disorders of sexual development (DSD). Although several types of DSD have been reported in the cat and dog, which are often strikingly similar to human DSD, these have been infrequently utilized to contribute to our knowledge of mammalian sexual development. Canine and feline cases of DSD with sufficient evidence to be considered as potential models are summarized in this report. The consensus DSD terminology, and reference to previous terminology, is used to foster adoption of a common nomenclature that will facilitate communication and collaboration between veterinarians, physicians, and researchers. To efficiently utilize these unique resources as molecular tools continue to improve, it will be helpful to deposit samples from valuable cases into repositories where they are available to contribute to our understanding of sexual development, and thus improve human and animal health. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The article discusses research on the presence of androgen receptor (AR) and SRD5A2 genes in patient suffering from 46, XY Disorders of Sexual Development (DSD). Twenty-nine patients were involved in the study which used polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). It found that patients with SRD5A2 gene defect usually have p.R246Q mutation.

17α-Hydroxylase deficiency (17OHD) is a rare form of congenital adrenal hyperplasia caused by mutations in the CYP17A1 gene. This condition shows considerable clinical and biochemical variation. Molecular characterization of novel mutations in the CYP17A1 gene and detailed study of their structural, enzymatic, and clinical consequences are required to fully understand enzyme behavior. Here, we present the first molecular characterization of two novel mutations in CYP17A1 in a 15-year-old female Mexican mestizo 46,XY female with primary amenorrhea and lack of pubertal development and severe hypertension that manifested only after surgery. A complete clinical and biochemical evaluation was compatible with 17OHD. Structural anomalies in the CYP17A1 gene were discovered by direct automated sequencing, which revealed a novel compound heterozygous K110X/R362H mutation that leads to a complete lack of enzyme activity. Immunohistochemical analyses performed to determine protein expression and localization showed that cytochrome P450 17A1 was completely absent in the patient’s testicular tissue. Studies of novel mutations, such as those described here, provide important information that allows us to better understand the effect of a given mutation on enzyme function and to observe the impact of the mutation on clinical phenotype. [ABSTRACT FROM AUTHOR]

Dihydrotestosterone is crucial for normal development of external genitalia and prostate in the male embryo. Autosomal recessive mutations in the 5α-reductase type 2 (SRD5A2) gene disrupt the synthesis of dihydrotestosterone in the urogenital tract and give rise to genetic males with undervirilized external genitalia that may be female-like or ambiguous. In this study, three unrelated 46,XY children (0.5, 3, and 8 years old) who presented severe undermasculinization at birth were examined for genetic abnormalities in the SRD5A2 gene. Coding sequence abnormalities were ascertained by exon-specific polymerase chain reaction (PCR), single-stranded conformational polymorphism (SSCP), and sequencing analysis. Functional properties of the mutant alleles were investigated by means of site-directed mutagenesis assays. DNA molecular studies showed that all three patients were compound heterozygotes for SRD5A2 mutations. Patient 1 had a point mutation 547G → A in exon 3 (G183S) and a novel dinucleotidic mutation 634,635CC → TG in exon 4 (P212X). This double change results in premature termination signal (TGA) at codon 212, which predicts the expression of a truncated 211-amino acid protein. Patient 2 was the carrier of mutations G115D in exon 3 and S210F in exon 4. Patient 3 had two substitution mutations in exon 1, including a novel G → C transversion at nucleotide 169 (E57Q) and a G → A transition at nucleotide 254 (G85D). In transitory transfection assays, the recombinant cDNAs harboring mutations E57Q and G85D showed residual 5α-reductase activity, whereas those with mutations G115D, S210F, and P212X were devoided of activity. In contrast, the G183S substitution affected the catalytic activity of the enzyme by decreasing its affinity for testosterone substrate. We describe six different mutations of the SRD5A2 gene detected in three children with genital ambiguity. These genotypes are consistent with the clinical phenotype of steroid 5α-reductase 2 deficiency. Our data suggest that the combined gene variants (E57Q/G85D, G115D/S210F, and G183S/P212X) result in subfunctional or nonfunctional enzymes, causing masculinization defects in these patients. This further underscores that exon 4 of SRD5A2 may be a site prone to inactivating mutations. [ABSTRACT FROM AUTHOR]

Deficiency of 17β-hydroxysteroid dehydrogenase type3 (17β-HSD3) isoenzyme which catalyzes the synthesis of testosterone from Δ4-androstenedione, is the cause of 46, XY disorders of sex development (DSD). 17β-HSD3 deficiency is a rare autosomal recessive disorder, which is caused by mutations in the HSD17B gene found on chromosome 9q22. Up to now, almost 33 mutations in the HSD17B3 gene have been reported. Here, we report a patient with a novel mutation in HSD17B3 gene leading to 17β-HSD3 deficiency. The patient was admitted because of primary amenorrhea and signs of virilization at puberty. The chromosome analysis showed a 46, XY karyotype. Hormonal evaluation revealed a high Δ4-androstenedione level with a low serum testosterone/androstenedione (T/A) ratio. Sequence analysis of HSD17B3 gene revealed the presence of a homozygous missense mutation in exon 11 resulting in a premature stop codon (p.Y287). Gonadectomy was performed after the molecular diagnosis and estrogen replacement therapy was initiated. With this report, we emphasize that 17β-HSD3 deficiency should be considered in virilized female patients at puberty if the T/A ratio is less than 0.8, and the molecular analysis should be performed for the precise diagnosis and genetic counseling. [ABSTRACT FROM AUTHOR]

Objective: The literature regarding gonadoblastoma risk in exonic Wilms' tumor suppressor gene (WT1) pathogenic variants is sparse. The aim of this study is to describe the phenotypic and genotypic characteristics of Asian-Indian patients with WT1 pathogenic variants and systematically review the literature on association of exonic WT1 pathogenic variants and gonadoblastoma., Design: Combined retrospective-prospective analysis., Methods: In this study, 46,XY DSD patients with WT1 pathogenic variants detected by clinical exome sequencing from a cohort of 150 index patients and their affected relatives were included. The PubMed database was searched for the literature on gonadoblastoma with exonic WT1 pathogenic variants., Results: The prevalence of WT1 pathogenic variants among 46,XY DSD index patients was 2.7% (4/150). All the four patients had atypical genitalia and cryptorchidism. None of them had Wilms' tumor till the last follow-up, whereas one patient had late-onset nephropathy. 11p13 deletion was present in one patient with aniridia. The family with p.Arg458Gln pathogenic variant had varied phenotypic spectrum of Frasier syndrome; two siblings had gonadoblastoma, one of them had growing teratoma syndrome (first to report with WT1). On literature review, of >100 exonic point pathogenic variants, only eight variants (p.Arg462Trp, p.Tyr177*, p.Arg434His, p.Met410Arg, p.Gln142*, p.Glu437Lys, p.Arg458*, and p.Arg458Gln) in WT1 were associated with gonadoblastoma in a total of 15 cases (including our two cases)., Conclusions: WT1 alterations account for 3% of 46,XY DSD patients in our cohort. 46,XY DSD patients harboring exonic WT1 pathogenic variants carry a small but definitive risk of gonadoblastoma; hence, these patients require a gonadoblastoma surveillance with a more stringent surveillance in those harboring a gonadoblastoma-associated variant.

• A female patient with a classic picture of complete androgen insensitivity syndrome. • A novel mutation in the AR gene was identified by direct sequencing: p.R856L. • Functional studies were performed to investigate the functional properties of p.R856L. • p.R856L mutation strongly affected both transactivation capacity and N/C interaction. • Functional studies confirmed the pathogenicity of p.R856L mutation. Androgens are critical for male sex differentiation. Their actions are mediated by the androgen receptor (AR). Mutations disrupting AR function result in the androgen insensitivity syndrome (AIS). In this study, we identified in a patient with complete AIS, a novel AR mutation p.R856L. To investigate the functional properties of p.R856L, we performed functional studies. In comparison, we have characterized two already described mutations: p.R856H and p.R856C. We used a model composed of two different promoters fused to a reporter gene, two cell lines, and showed that all mutations were able to transactivate the (ARE) 2 -TATA promoter expressed in CHO cells more highly. Moreover, we confirmed the pathogenicity of the p.R856L and p.R856C mutations, and their associations with complete AIS. In contrast, the p.R856H mutation, which is associated with a spectrum of AIS phenotypes, showed less severe transcriptional constraints. Altogether, our studies allowed us to better characterize arginine residue at p.R856 position. [ABSTRACT FROM AUTHOR]