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STUDY QUESTIONCan whole-exome sequencing (WES) reveal a shared pathogenic variant responsible for primary gonadal failure in both male and female patients from a consanguineous family?SUMMARY ANSWERPatients with primary ovarian insufficiency (POI) and non-obstructive azoospermia (NOA) were homozygous for the rare missense variant p. S754L located in the highly conserved MSH4 MutS signature motif of the ATPase domain. An oligozoospermic patient was heterozygous for the variant.WHAT IS KNOWN ALREADYMSH4 is a meiosis-specific protein expressed at a certain level in the testes and ovaries. Along with its heterodimer partner MSH5, it is responsible for double-strand Holliday junction recognition and stabilization, to ensure accurate chromosome segregation during meiosis. Knockout male and female mice for Msh4 and Msh5 are reportedly infertile due to meiotic arrest. In humans, MSH4 is associated with male and female gonadal failure, with distinct variations in the MutS domain V.STUDY DESIGN, SIZE, DURATIONThis was a retrospective genetics study of a consanguineous family with multiple cases of gonadal failure in both genders. The subject family was recruited in Iran, in 2018.PARTICIPANTS/MATERIALS, SETTING, METHODSThe proband who is affected by POI, an NOA brother, a fertile sister and their parents were subjected to WES. The discovered variant was validated in these individuals, and the rest of the family was also genotyped by Sanger sequencing. The variant was not detected in 800 healthy Iranian individuals from the Iranome database nor in 30 sporadic NOA and 30 sporadic POI patients. Suggested effect in aberrant splicing was studied by RT-PCR. Moreover, protein homology modeling was used to further investigate the amino acid substitution in silico.MAIN RESULTS AND THE ROLE OF CHANCEThe discovered variant is very rare and has never been reported in the homozygous state. It occurs in the ATPase domain at Serine 754, the first residue within the highly conserved MutS signature motif, substituting it with a Leucine. All variant effect prediction tools indicated this variant as deleterious. Since the substitution occurs immediately before the Walker B motif at position 755, further investigations based on protein homology were conducted. Considering the modeling results, the nature of the substituted amino acid residue and the distances between p. S754L variation and the residues of the Walker B motif suggested the possibility of conformational changes affecting the ATPase activity of the protein.LARGE SCALE DATAWe have submitted dbSNP entry rs377712900 to ClinVar under SCV001169709, SCV001169708 and SCV001142647 for oligozoospermia, NOA and POI, respectively.LIMITATIONS, REASONS FOR CAUTIONStudies in model organisms can shed more light on the role of this variant as our results were obtained by variant effect prediction tools and protein homology modeling.WIDER IMPLICATIONS OF THE FINDINGSIdentification of variants in meiotic genes should improve genetic counseling for both male and female infertility. Also, as two of our NOA patients underwent testicular sperm extraction (TESE) with no success, ruling out the existence of pathogenic variants in meiotic genes in such patients prior to TESE could prove useful.STUDY FUNDING/COMPETING INTEREST(S)This study was financially supported by Royan Institute in Tehran, Iran, and Institut Pasteur in Paris, France. The authors declare no competing interests.TRIAL REGISTRATION NUMBERN/A

International audience; In 46,XY men, testis is determined by a genetic network(s) that both promotes testis formation and represses ovarian development. Disruption of this process results in a lack of testis-determination and affected individuals present with 46,XY gonadal dysgenesis (GD), a part of the spectrum of Disorders/Differences of Sex Development/Determination (DSD). A minority of all cases of GD are associated with pathogenic variants in key players of testis-determination, SRY, SOX9, MAP3K1 and NR5A1. However, most of the cases remain unexplained. Recently, unbiased exome sequencing approaches have revealed new genes and loci that may cause 46,XY GD. We critically evaluate the evidence to support causality of these factors and describe how functional studies are continuing to improve our understanding of genotype-phenotype relationships in genes that are established causes of GD. As genomic data continues to be generated from DSD cohorts, we propose several recommendations to help interpret the data and establish causality.

International audience; Objective: SRY-negative 46,XX testicular and ovotesticular disorders/differences of sex development (T/OTDSD) represent a very rare and unique DSD condition where testicular tissue develops in the absence of a Y chromosome. To date, very few studies have described the phenotype, clinical and surgical management and long-term outcomes of these patients. Particularly, early blockade of the gonadotropic axis in patients raised in the female gender to minimize postnatal androgenization has never been reported.Design: Retrospective description of sixteen 46,XX T/OTDSD patients.Results: Sixteen 46,XX SRY-negative T/OTDSD were included. Most (12/16) were diagnosed in the neonatal period. Sex of rearing was male for six patients and female for ten, while the clinical presentation varied, with an external masculinization score from 1 to 10. Five patients raised as girl were successfully treated with GnRH analog to avoid virilization during minipuberty. Ovotestes/testes were found bilaterally for 54% of the patients and unilaterally for the others (with a contralateral ovary). Gonadal surgery preserved appropriate tissue in the majority of cases. Spontaneous puberty occurred in two girls and one boy, while two boys required hormonal induction of puberty. One of the girls conceived spontaneously and had an uneventful pregnancy. DNA analyses (SNP-array, next-generation sequencing and whole-exome sequencing) were performed. A heterozygous frameshit mutation in the NR2F2 gene was identified in one patient.Conclusions: This study presents a population of patients with 46,XX SRY-negative T/OTDSD. Early blockade of gonadotropic axis appears efficient to reduce and avoid further androgenization in patients raised as girls.

International audience; Disorders/differences of sex development (DSD) comprise a group of congenital disorders that affect the genitourinary tract and usually involve the endocrine and reproductive system. The aim of this work was to identify genetic variants responsible for disorders of human urogenital development in a cohort of Egyptian patients. This three-year study included 225 patients with various DSD forms, referred to the genetic DSD and endocrinology clinic, National Research Centre, Egypt. The patients underwent thorough clinical examination, hormonal and imaging studies, detailed cytogenetic and fluorescence in situ hybridization analysis, and molecular sequencing of genes known to commonly cause DSD including AR, SRD5A2, 17BHSD3, NR5A1, SRY, and WT1. Whole exome sequencing (WES) was carried out for 18 selected patients. The study revealed a high rate of sex chromosomal DSD (33%) with a wide array of cytogenetic abnormalities. Sanger sequencing identified pathogenic variants in 33.7% of 46,XY patients, while the detection rate of WES reached 66.7%. Our patients showed a different mutational profile compared with that reported in other populations with a predominance of heritable DSD causes. WES identified rare and novel pathogenic variants in NR5A1, WT1, HHAT, CYP19A1, AMH, AMHR2, and FANCA and in the X-linked genes ARX and KDM6A. In addition, digenic inheritance was observed in two of our patients and was suggested to be a cause of the phenotypic variability observed in DSD.

Pituitary stalk interruption syndrome is a rare disorder characterized by an absent or ectopic posterior pituitary, interrupted pituitary stalk and anterior pituitary hypoplasia, as well as in some cases, a range of heterogeneous somatic anomalies. A genetic cause is identified in only around 5% of all cases. Here, we define the genetic variants associated with PSIS followed by the same pediatric endocrinologist. Exome sequencing was performed in 52 (33 boys and 19 girls), including 2 familial cases single center pediatric cases, among them associated 36 (69.2%) had associated symptoms or syndromes. We identified rare and novel variants in genes (37 families with 39 individuals) known to be involved in one or more of the following—midline development and/or pituitary development or function (BMP4, CDON, GLI2, GLI3, HESX1, KIAA0556, LHX9, NKX2-1, PROP1, PTCH1, SHH, TBX19, TGIF1), syndromic and non-syndromic forms of hypogonadotropic hypogonadism (CCDC141, CHD7, FANCA, FANCC, FANCD2, FANCE, FANCG, IL17RD, KISS1R, NSMF, PMM2, SEMA3E, WDR11), syndromic forms of short stature (FGFR3, NBAS, PRMT7, RAF1, SLX4, SMARCA2, SOX11), cerebellum atrophy with optic anomalies (DNMT1, NBAS), axonal migration (ROBO1, SLIT2), and agenesis of the corpus callosum (ARID1B, CC2D2A, CEP120, CSPP1, DHCR7, INPP5E, VPS13B, ZNF423). Pituitary stalk interruption syndrome is characterized by a complex genetic heterogeneity, that reflects a complex phenotypic heterogeneity. Seizures, intellectual disability, micropenis or cryptorchidism, seen at presentation are usually considered as secondary to the pituitary deficiencies. However, this study shows that they are due to specific gene mutations. PSIS should therefore be considered as part of the phenotypic spectrum of other known genetic syndromes rather than as specific clinical entity.

Significance Sex development involves a precise spatiotemporal expression and interactions of numerous genetic factors, including the WT1 (Wilms tumor 1) gene. Complete and partial loss-of-function WT1 variants are associated with 46,XY disorders/differences of sex development (DSD). Some 46,XX individuals develop testis in absence of the testis-determining gene SRY. We describe a genotype/phenotype association where variants impacting the C-terminal zinc finger (ZF4) of WT1 cause testis development in 46,XX individuals. XX mice carrying a pathogenic variant of ZF4 display masculinization of the fetal gonads. Testis formation may be due to inappropriate interaction between the mutated WT1 and an essential ovarian determinant β-CATENIN. These data show that variants affecting a specific domain of a developmental transcription factor can switch organ fate., Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms’ tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10−6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10−4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor β-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.

International audience; Mammalian sex determination is controlled by the antagonistic interactions of two genetic pathways: The SRY-SOX9-FGF9 network promotes testis determination partly by opposing proovarian pathways, while RSPO1/WNT-β-catenin/FOXL2 signals control ovary development by inhibiting SRY-SOX9-FGF9. The molecular basis of this mutual antagonism is unclear. Here we show that ZNRF3, a WNT signaling antagonist and direct target of RSPO1-mediated inhibition, is required for sex determination in mice. XY mice lacking ZNRF3 exhibit complete or partial gonadal sex reversal, or related defects. These abnormalities are associated with ectopic WNT/β-catenin activity and reduced Sox9 expression during fetal sex determination. Using exome sequencing of individuals with 46,XY disorders of sex development, we identified three human ZNRF3 variants in very rare cases of XY female presentation. We tested two missense variants and show that these disrupt ZNRF3 activity in both human cell lines and zebrafish embryo assays. Our data identify a testis-determining function for ZNRF3 and indicate a mechanism of direct molecular interaction between two mutually antagonistic organogenetic pathways.

International audience; PURPOSE:XY individuals with disorders/differences of sex development (DSD) are characterized by reduced androgenization caused, in some children, by gonadal dysgenesis or testis regression during fetal development. The genetic etiology for most patients with 46,XY gonadal dysgenesis and for all patients with testicular regression syndrome (TRS) is unknown.METHODS:We performed exome and/or Sanger sequencing in 145 individuals with 46,XY DSD of unknown etiology including gonadal dysgenesis and TRS.RESULTS:Thirteen children carried heterozygous missense pathogenic variants involving the RNA helicase DHX37, which is essential for ribosome biogenesis. Enrichment of rare/novel DHX37 missense variants in 46,XY DSD is highly significant compared with controls (P value = 5.8 × 10-10). Five variants are de novo (P value = 1.5 × 10-5). Twelve variants are clustered in two highly conserved functional domains and were specifically associated with gonadal dysgenesis and TRS. Consistent with a role in early testis development, DHX37 is expressed specifically in somatic cells of the developing human and mouse testis.CONCLUSION:DHX37 pathogenic variants are a new cause of an autosomal dominant form of 46,XY DSD, including gonadal dysgenesis and TRS, showing that these conditions are part of a clinical spectrum. This raises the possibility that some forms of DSD may be a ribosomopathy.

International audience; A 5,6 Mb de novo 19q12-q13.12 interstitial deletion was diagnosed prenatally by array-comparative genomic hybridization in a 26 weeks male fetus presenting with intra-uterine growth retardation, left clubfoot, atypical genitalia and dysmorphic features. Autopsic examination following termination of pregnancy identified a severe disorder of sex development (DSD) including hypospadias, micropenis, bifid scrotum and right cryptorchidism associated with signs of ectodermal dysplasia: scalp hypopigmentation, thick and frizzy hair, absence of eyelashes, poorly developed nails and a thin skin with prominent superficial veins. Other findings were abnormal lung lobation and facial dysmorphism.This new case of DSD with a 19q12q13 deletion expands the phenotypic spectrum associated with this chromosomal rearrangment and suggests that WTIP is a strong candidate gene involved in male sex differentiation.

International audience; Background: Several studies have shown a high rate of consanguinity and endogamy in North African populations. As a result, the frequency of autosomal recessive diseases is relatively high in the region with the co-occurrence of two or more diseases.Methods: We report here on a consanguineous Libyan family whose child was initially diagnosed as presenting Fanconi anemia (FA) with uncommon skeletal deformities. The chromosome breakage test has been performed using mitomycin C (MMC) while molecular analysis was performed by a combined approach of linkage analysis and whole exome sequencing.Results: Cytogenetic analyses showed that the karyotype of the female patient is 46,XY suggesting the diagnosis of a disorder of sex development (DSD). By looking at the genetic etiology of FA and DSD, we have identified p.[Arg798*];[Arg798*] mutation in FANCJ (OMIM #605882) gene responsible for FA and p.[Arg108*];[Arg1497Trp] in EFCAB6 (Gene #64800) gene responsible for DSD. In addition, we have incidentally discovered a novel mutation p.[Gly1372Arg];[Gly1372Arg] in the ERCC6 (CSB) (OMIM #609413) gene responsible for COFS that might explain the atypical severe skeletal deformities.Conclusion: The co-occurrence of clinical and overlapping genetic heterogeneous entities should be taken into consideration for better molecular and genetic counseling

Mutation of the Wilms tumor suppressor gene (WT1) has been recognized as one of the etiologies of steroid-resistant nephrotic syndrome (SRNS). The mutation is also responsible for gonadal dysgenesis in 46,XY individuals. Early recognition of the presence of Y chromosome is of particular importance because of the high risk of gonadal tumor. We present here three cases of steroid-resistant nephrotic syndrome with WT1 mutation and 46,XY karyotype. Patient 1 and 2 have intron splice site (IVS9+5G A) mutation. Patient 3 has c.1301GA (p. R434H) mutation. All cases had normal female external genitalia at birth and eluded the diagnosis of gonadal dysgenesis until later in life. We suggest that chromosomal analysis should be promptly performed in female patients with early-onset steroid-resistant nephrotic syndrome. .

International audience; Differences of sex development are conditions with discrepancies between chromosomal, gonadal and phenotypic sex. In congenital hypogonadotropic hypogonadism, a lack of gonadotropin activity results primarily in the absence of pubertal development with prenatal sex development being (almost) unaffected in most patients. To expedite progress in the care of people affected by differences of sex development and congenital hypogonadotropic hypogonadism, the European Union has funded a number of scientific networks. Two Actions of the Cooperation of Science and Technology (COST) programmes - DSDnet (BM1303) and GnRH Network (BM1105) - provided the framework for ground-breaking research and allowed the development of position papers on diagnostic procedures and special laboratory analyses as well as clinical management. Both Actions developed educational programmes to increase expertise and promote interest in this area of science and medicine. In this Perspective article, we discuss the success of the COST Actions DSDnet and GnRH Network and the European Reference Network for Rare Endocrine Conditions (Endo-ERN), and provide recommendations for future research.

International audience; R-spondin proteins are secreted agonists of canonical WNT/β-catenin signaling. Homozygous RSPO1 mutations cause a syndrome of 46,XX disorder of sexual development (DSD), palmoplantar keratoderma (PPK), and predisposition to squamous cell carcinoma. We report exome sequencing data of two 46,XX siblings, one with testicular DSD and the other with suspected ovotesticular DSD. Both have PPK and hearing impairment and carried a novel homozygous mutation c.332G>A (p.Cys111Tyr) located in the highly conserved furin-like cysteine-rich domain-2 (FU-CRD2). Cysteines in the FU-CRDs are strictly conserved, indicating their functional importance in WNT signaling through interaction with the leucine-rich repeat-containing G-protein-coupled receptors. This is the first RSPO1 missense mutation reported in association with human disease.

SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n=274) and two independent cohorts of women with primary ovarian insufficiency (POI; n=153 and n=104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P=4.5×10-5) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI. Fil: Portnoi, Marie France. Inserm; Francia. Sorbonne Université. Faculté de Medecine; Francia. Hôpital Armand Trousseau; Francia Fil: Dumargne, Marie Charlotte. Instituto Pasteur; Francia Fil: Rojo, Sandra. Instituto Pasteur; Francia Fil: Witchel, Selma F.. University of Pittsburgh; Estados Unidos Fil: Duncan, Andrew J.. Great Ormond Street Hospital for Children; Reino Unido Fil: Eozenou, Caroline. Instituto Pasteur; Francia Fil: Bignon Topalovic, Joelle. Instituto Pasteur; Francia Fil: Yatsenko, Svetlana A.. University of Pittsburgh; Estados Unidos Fil: Rajkovic, Aleksandar. University of Pittsburgh; Estados Unidos Fil: Reyes Mugica, Miguel. University of Pittsburgh; Estados Unidos Fil: Almstrup, Kristian. Rigshospitalet; Dinamarca Fil: Fusee, Leila. Instituto Pasteur; Francia Fil: Srivastava, Yogesh. Chinese Academy of Sciences; República de China Fil: Chantot Bastaraud, Sandra. Hôpital Armand Trousseau; Francia Fil: Hyon, Capucine. Hôpital Armand Trousseau; Francia. Inserm; Francia Fil: Louis Sylvestre, Christine. Institut Mutualiste Montsouris; Francia Fil: Validire, Pierre. Institut Mutualiste Montsouris; Francia Fil: de Malleray Pichard, Caroline. Hôpital Cochin; Francia Fil: Ravel, Celia. Centre Hospitalier Universitaire de Rennes; Francia Fil: Christin Maitre, Sophie. Inserm; Francia. Hôpital Saint-Antoine; Francia Fil: Brauner, Raja. Universite de Paris; Francia Fil: Rossetti, Raffaella. Università degli Studi di Milano; Italia. Istituto Auxologico Italiano; Italia Fil: Persani, Luca. Istituto Auxologico Italiano; Italia. Università degli Studi di Milano; Italia Fil: Charreau, Eduardo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Administracion Nacional de Laboratorios E Institutos de Salud "dr. Carlos G. Malbran". Instituto Nacional de Epidemiologia. Departamento de Investigacion.; Argentina Fil: Dain, Liliana Beatriz. Administracion Nacional de Laboratorios E Institutos de Salud "dr. Carlos G. Malbran". Instituto Nacional de Epidemiologia. Departamento de Investigacion.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Chiauzzi, Violeta Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Administracion Nacional de Laboratorios E Institutos de Salud "dr. Carlos G. Malbran". Instituto Nacional de Epidemiologia. Departamento de Investigacion.; Argentina Fil: Mazen, Inas. National Research Centre ; Egipto Fil: Rouba, Hassan. Institut Pasteur du Maroc; Marruecos Fil: Schluth Bolard, Caroline. Hôpital Femme Mère Enfant; Francia Fil: Mac Gowan, Stuart. University of Dundee; Reino Unido Fil: Mc Lean, W. H. Irwin. University of Dundee; Reino Unido Fil: Patin, Etienne. Instituto Pasteur; Francia Fil: Rajpert De Meyts, Ewa. Rigshospitalet; Dinamarca Fil: Jauch, Ralf. Chinese Academy of Sciences; República de China Fil: Achermann, John C.. Great Ormond Street Hospital for Children; Reino Unido Fil: Siffroi, Jean Pierre. Hôpital Armand Trousseau; Francia Fil: Mc Elreavey, Ken. Instituto Pasteur; Francia Fil: Bashamboo, Anu. Inserm; Francia. Instituto Pasteur; Francia

International audience; Emerging evidence from murine studies suggests that mammalian sex determination is the outcome of an imbalance between mutually antagonistic male and female regulatory networks that canalize development down one pathway while actively repressing the other. However, in contrast to testis formation, the gene regulatory pathways governing mammalian ovary development have remained elusive. We performed exome or Sanger sequencing on 79 46,XX SRY-negative individuals with either unexplained virilization or with testicular/ovotesticular disorders/differences of sex development (TDSD/OTDSD). We identified heterozygous frameshift mutations in NR2F2, encoding COUP-TF2, in three children. One carried a c.103_109delGGCGCCC (p.Gly35Argfs∗75) mutation, while two others carried a c.97_103delCCGCCCG (p.Pro33Alafs∗77) mutation. In two of three children the mutation was de novo. All three children presented with congenital heart disease (CHD), one child with congenital diaphragmatic hernia (CDH), and two children with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). The three children had androgen production, virilization of external genitalia, and biochemical or histological evidence of testicular tissue. We demonstrate a highly significant association between the NR2F2 loss-of-function mutations and this syndromic form of DSD (p = 2.44 × 10-8). We show that COUP-TF2 is highly abundant in a FOXL2-negative stromal cell population of the fetal human ovary. In contrast to the mouse, these data establish COUP-TF2 as a human "pro-ovary" and "anti-testis" sex-determining factor in female gonads. Furthermore, the data presented here provide additional evidence of the emerging importance of nuclear receptors in establishing human ovarian identity and indicate that nuclear receptors may have divergent functions in mouse and human biology.

WT1 gene mutations have been described in 46,XY patients with ambiguous genitalia or complete gonadal dysgenesis with or without Wilms' tumor, nephropathy, gonadoblastoma, and other defects, e.g., cryptorchidism or hypospadias. p.R462W is a hot spot mutation in exon 9 and is the most common mutation in patients with Denys-Drash syndrome. However, in this study we report an Egyptian patient with a novel phenotype carrying the p.R462W mutation. We also review the heterogeneity of phenotypes of previously reported patients with the p.R462W (previously referred to as Arg394Trp) mutation.

Disorders of sex development (DSD) are defined as 'congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical' [Lee et al., Pediatrics 2006;118:e488-e500]. Studies conducted in Western countries, with low rates of consanguinity, show that truly ambiguous genitalia have an estimated incidence of 1:5,000 births. There are indications that the prevalence of DSD is higher in endogamous communities. The incidence of ambiguous genitalia in Saudi Arabia has been estimated at 1:2,500 live births; whilst in Egypt, it has been estimated at 1:3,000 live births. This may be due in part to an increase in disorders of androgen synthesis associated with 46,XX DSD. There is clearly a need for further studies to address the frequency of DSD in communities with high levels of consanguinity. This will be challenging, as an accurate diagnosis is difficult and expensive even in specialized centres. In developing countries with high levels of consanguinity, these limitations can be compounded by cultural, social and religious factors. Overall there is an indication that consanguinity may lead to an increase in incidences of both 46,XY and 46,XX DSD, and a co-ordinated study of populations with higher incidences of consanguinity/endogamy is needed to resolve this.

Temozolomide is an oral alkylating agent with proven efficacy in recurrent high-grade glioma. The antitumour activity of this molecule is attributed to the inhibition of replication through DNA methylation. However, this methylation may also perturb other DNA-dependent processes, such as spermatogenesis. The ability to father a child may be affected by having this treatment. Here we report a pregnancy and a baby born after 6 cures of temozolomide.The quality of gametes of the father has been studied through these cures and after the cessation of treatment. Sperm parameters, chromosomal content and epigenetic profiles of H19, MEST and MGMT have been analysed.Sperm counts decrease significantly and hypomethylation of the H19 locus increase with time even staying in the normal range.This is the first report of an epigenetic modification in sperm after temozolomide treatment suggesting a potential risk for the offspring. A sperm cryopreservation before the initiation of temozolomide treatment should be recommended.

International audience; Aromatase deficiency (AD) is a very rare disorder resulting from mutations in the CYP19A1 gene encoding aromatase, a cytochrome P450 enzyme that plays a pivotal role in androgen conversion to estrogens. AD is inherited in an autosomal recessive trait, and to date only 35 cases have been described in the literature. Herein, we depict a new patient reared as a male, who presented at the age of 21 years with no palpable testis, hypoplastic scrotum, penis-like phallus (3 cm), and penoscrotal hypospadias. The patient was born to consanguineous parents, his karyotype was 46,XX, and SRY was negative. Pelvic sonar showed a small hypoplastic uterus, and no testis could be identified. Serum testosterone was within the reference range of females along with high gonadotropins. Pathology of gonadal biopsy showed ovarian stroma negative for oocytic follicle consistent with streak gonads. All these data were suggestive of AD, which was subsequently confirmed by molecular investigation of the CYP19A1 gene. A homozygous splice site mutation in the donor splice site of exon 9 was identified, c.1263 + 1G>T. This is the first report of such a rare disorder in an Egyptian patient. Our results reinforce the importance of considering AD in patients with 46,XX disorders of sex development after ruling out congenital adrenal hyperplasia.

Cell lineages of the early human gonad commit to one of the two mutually antagonistic organogenetic fates, the testis or the ovary. Some individuals with a 46,XX karyotype develop testes or ovotestes (testicular or ovotesticular disorder of sex development; TDSD/OTDSD), due to the presence of the testis-determining gene, SRY. Other rare complex syndromic forms of TDSD/OTDSD are associated with mutations in pro-ovarian genes that repress testis development (e.g. WNT4); however, the genetic cause of the more common non-syndromic forms is unknown. Steroidogenic factor-1 (known as NR5A1) is a key regulator of reproductive development and function. Loss-of-function changes in NR5A1 in 46,XY individuals are associated with a spectrum of phenotypes in humans ranging from a lack of testis formation to male infertility. Mutations in NR5A1 in 46,XX women are associated with primary ovarian insufficiency, which includes a lack of ovary formation, primary and secondary amenorrhoea as well as early menopause. Here, we show that a specific recurrent heterozygous missense mutation (p.Arg92Trp) in the accessory DNA-binding region of NR5A1 is associated with variable degree of testis development in 46,XX children and adults from four unrelated families. Remarkably, in one family a sibling raised as a girl and carrying this NR5A1 mutation was found to have a 46,XY karyotype with partial testicular dysgenesis. These unique findings highlight how a specific variant in a developmental transcription factor can switch organ fate from the ovary to testis in mammals and represents the first missense mutation causing isolated, non-syndromic 46,XX testicular/ovotesticular DSD in humans.

In this review we will consider the gene mutations responsible for the non-syndromic forms of disorders of sex development (DSD) and how recent genetic findings are providing insights into the mechanism of sex determination. High-throughput sequencing technologies are having a major impact on our understanding of the genetic basis of rare human disorders, including DSD. The study of human DSD is progressively revealing subtle differences in the genetics of the sex-determining system between the mouse and the human. This plasticity of the sex-determining pathway is apparent in (a) the difference in phenotypes in human and mouse associated with the same gene, (b) the different gene regulatory mechanisms between human and mouse, and finally (c) the different and unexpected reproductive phenotypes seen in association with mutations in well-studied sex-determining genes.

Background The mechanism that initiates the onset of puberty is largely unknown but the age of onset is mainly under genetic control and influenced by environmental factors including nutrition. The coexistence in the same family of central precocious puberty and advanced puberty, both representing early puberty, suggests that they may represent a clinical spectrum of the same trait due to early activation of the GnRH pulse generator. We therefore evaluated the mode of inheritance of early puberty in a large series of familial cases. Methods A retrospective, single center study was carried out on 154 probands (116 girls and 38 boys), from 139 families seen for idiopathic central precocious puberty (onset before 8 years in girls and 9–10 years in boys, n = 93) and/or advanced puberty (onset between 8 and 10 years in girls and 10 and 11 years in boys, n = 61) seen over a period of 8 years. Results Of the 139 families, 111 (80.4 %) had at least one affected 1st degree relatives, 17 (12 %) had only 2nd, 5 (3.6 %) only 3rd and 3 (2.2 %) had both 2nd and 3rd degree affected individuals. In the two remaining families, the unaffected mother had affected girls from two unaffected fathers. In the majority of families the inheritance of the phenotype was consistent with autosomal dominant mode of transmission with incomplete penetrance. An exclusively maternal mode of transmission could be observed or inferred in 83 families, paternal in only 2 families (p

The phenotypic spectrum of patients carrying NR5A1 mutations ranges from 46,XY gonadal dysgenesis to male infertility. Phenotypic variability could be due to digenic or oligogenic inheritance of pathogenic variants in other testis-determining genes. Here, exome sequencing identified 2 pathogenic de novo NR5A1 mutations in 2 patients with 46,XY gonadal dysgenesis, p.Q206Tfs*20 and p.Arg313Cys. The latter patient also carried a missense mutation in MAP3K1. Our data extend the number of NR5A1 gene mutations associated with gonadal dysgenesis. The combination of an NR5A1 mutation with a MAP3K1 variant may explain the phenotypic variability associated with NR5A1 mutations.

Background The mechanism that initiates the onset of puberty is largely unknown but the age of onset is mainly under genetic control and influenced by environmental factors including nutrition. Familial forms of constitutional delay of puberty (CDP) suggest the involvement of genetic factors. The purpose of this study is to describe the presentation and the mode of inheritance of CDP in a series of familial cases. Methods A retrospective, single center study was carried out over 10 years on 48 probands (14 girls and 34 boys) from 48 families seen for CDP with a familial component. Results Of the 48 probands, 46 (96 %) had at least one affected 1st degree relatives and 2 (4 %, 2 boys) had only 2nd degree relatives affected. In girls, 11 families (79 %) exhibited exclusive maternal inheritance, 1 (7 %) paternal inheritance and 2 (14 %) both maternal and paternal inheritance. In boys, 14 families (41 %) exhibited exclusive maternal inheritance, 12 (35 %) paternal inheritance and 8 (24 %) both maternal and paternal inheritance. In the boys with bilineal inheritance, the ages at onset of puberty (16 ± 1.41 years) and at evaluation (16.05 ± 2.47 years) were higher than in those with unilineal inheritance (15.25 ± 0.35 and 15.1 ± 0.42 years respectively), but the difference was not significant. Conclusions In girls exclusive maternal inheritance seems to be the major mode of inheritance whereas for boys the mode of inheritance was almost equally maternal, paternal or bilineal. Clinical phenotype of boys with bilineal inheritance seems to be more severe, but the difference did not reach statistical significance, perhaps because of the small sample size. This greater severity of the phenotype in boys with bilineal inheritance is likely due to inheriting different puberty timing genes from each parent. Future research should be directed at identifying such genes.

Mullerian agenesis, also termed the Mayer–Rokitansky–Kuster–Hauser syndrome (MRKHS) is a disorder with an incidence of approximately 1 in 4500 newborn girls. This study screened 12 patients with MRKHS for mutations in two genes, LAMC1 and DLGH1 , involved in the development of Mullerian structures and found 10 previously described variants and no novel variants in the coding sequence. It is highly unlikely that these variants are pathological since these are common in the general population. It is the first time that an extensive study of LAMC1 and DLGH1 has been undertaken in patients with MRKHS. The data support the notion that mutations in the coding sequence of LAMC1 and DLGH1 may not be associated with MRKHS.

Primary ovarian insufficiency (POI) is defined as cessation of menstruation with associated elevation of gonadotropin levels as a result of decreased ovarian function before the age of 40. The incidence of POI is 1% in women prior to age 40, and 0.1% prior to age 30. There is evidence of a strong genetic component associated with POI. However, the gene mutations/variations influencing POI still remain uncharacterized. NR5A1, a member of the nuclear receptor superfamily, is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-gonadal steroidogenic axis. Newborn mice deficient in NR5A1 lack both gonads and adrenal glands and have impaired expression of pituitary gonadotrophins. NR5A1 is also expressed in multiple cell types in the fetal, postnatal, prepubertal and mature ovary. Until 2008, 18 NR5A1 mutations were described in the human. Three of these were identified in individuals with adrenal insufficiency, two associated with 46,XY disorders of sex development (DSD) and the third a 46,XX female with conserved ovarian function. Other mutations were associated with various anomalies of testis development with no evidence of adrenal failure. We have identified further 19 mutations in NR5A1 including mutations in four familial cases having individuals with 46,XY DSD as well as POI. A further analysis of 25 sporadic cases of POI revealed two additional mutations. Functional analysis revealed that each mutant protein had altered transactivational properties on gonadal promoters. These data reveal novels insights into the role of NR5A1 in ovarian developmental and function and indicate that mutations of the NR5A1 gene may be a significant cause of human ovarian insufficiency.

We screened 100 individuals with anomalies of testicular development or function for mutations in the TSPYL1 gene. A 46,XY female with complete gonadal dysgenesis carried a p.K320R mutation in the highly conserved NAP domain, and a 46,XY male with idiopathic azoospermia harbored a p.R89H mutation, and this data supports the hypothesis that mutations in TSPYL1 may contribute to anomalies of testicular development/function.

The aim of this study is to determine if Müllerian agenesis has a genetic basis linked to the WNT genes. Genomic DNA analyses for mutations in the coding sequences of four members of this family in a series of 11 women with Mayer-Rokitansky-Kuster-Hauser syndrome found four variants in the coding sequence of these genes, but causal mutations were not observed. This supports the hypothesis that mutations in the coding sequence of WNT4, WNT5A, WNT7A, and WNT9B are not responsible for the Mayer-Rokitansky-Kuster-Hauser syndrome.

Primary ovarian insufficiency, also known as premature ovarian failure, is characterized by loss of function of the ovaries before the age 40. Observational evidence of familial associations suggests a genetic basis for ovarian insufficiency. Despite extensive research, specific genetic causes have not been identified. A nuclear receptor, NR5A1, also called Ad4 binding protein or steroidogenic factor 1, is a protein that regulates the transcription of genes involved in the hypothalamic―pituitary―steroidogenic axis that play a key role in sexual development and reproduction. NR5A1 I is expressed in multiple ovarian cell types during fetal development, postnatal and prepubertal growth, and at maturity. In this study, the investigators tested the hypothesis that mutations in NR5A1 are associated with disorders of ovarian development and function. NR5A1 was sequenced in affected study subjects who were members of 4 families with a history of 46,XY disorders of sex development and 46,XX ovarian insufficiency and 25 women with 46,XX sporadic ovarian insufficiency. A panel of 1465 subjects of various ancestral origins who did not carry NRSA1 mutations served as controls. There was no clinical evidence of adrenal insufficiency among any of the affected patients. Mutations in the NR5A1 gene were found among members of each of the 4 families and 2 of the 25 subjects with isolated ovarian insufficiency. Analysis of the NRSA1 gene revealed a series of in-frame, frame-shift and missense mutations. Testing the effect of each of the NRSA1 mutations on protein function revealed a severe quantitative impairment of NRSA1 transactivational activity. A range of ovarian anomalies, including 46,XY gonadal dysgenesis and 46,XX primary ovarian insufficiency, were associated with the mutations. None of these mutations were found in any of the unaffected control subjects. These findings indicate that mutations in NRSA1 are associated with a number of ovarian anomalies characterized by loss of ovarian reproductive capacity.

Context Pituitary stalk interruption syndrome (PSIS) is characterized by a thin or absent pituitary stalk usually in association with an ectopic posterior pituitary and hypoplasia/aplasia of the anterior pituitary. Associated phenotypes include varied ocular anomalies, hypoglycemia, micropenis/cryptorchidism, growth failure, or combined pituitary hormone deficiencies. Although genetic causes have been identified, they explain only around 5% of PSIS cases. Objective To identify genetic causes of PSIS by exome sequencing. Design Exon enrichment was performed using the Agilent SureSelect Human All Exon V4. Paired-end sequencing was performed on the Illumina HiSeq2000 platform with an average sequencing coverage of ×50. Patients Patients with unexplained PSIS were included in the study. Results In five cases of unexplained PSIS including two familial cases, we identified a novel heterozygous frameshift and nonsense and missense mutations in the ROBO1 gene (p.Ala977Glnfs*40, two affected sibs; p.Tyr1114Ter, sporadic case, and p.Cys240Ser, affected child and paternal aunt) that controls embryonic axon guidance, and branching in the nervous system. Interestingly, four of the five cases of PSIS also presented with ocular anomalies, including hypermetropia with strabismus as well as ptosis. Conclusions These data suggest that mutations in ROBO1 contribute to PSIS and associated ocular anomalies.

The role of a genetically impaired epidermal barrier as a major predisposing factor in the pathogenesis of atopic disorders is currently under closer investigation. Variants on three candidate genes (SPINK5, KLK7 and FLG) have been associated with atopic dermatitis. A functional relevance has already been established for filaggrin variants, but not for SPINK5 and KLK7 polymorphisms. The objectives of this study were to confirm the association between SPINK5, KLK7, FLG variants and atopic dermatitis and to assess how variants influence selected phenotypic traits. This cross-sectional study was carried out over 20 months in 99 children and adults with atopic dermatitis (median age 7 years). The following items were analysed: SCORAD, TEWL, ichthyosis vulgaris, presence of asthma, total IgE serum levels. The SPINK5 E420K SNP, the KLK7 4bp insertion polymorphism and the filaggrin mutants (R510X and 2282del4) were analysed as described previously. The control group for genetic analysis was recruited in an ethnically matched, phenotypically anonymous cohort (n=102). The allelic frequencies were 0.525 for SPINK5, 0.26 for KLK7 polymorphisms, 0.101 and 0.075 for 2282del4 and R501X FLG mutants, respectively. The association of atopic dermatitis with filaggrin variants was confirmed, but not that of SPINK5 or KLK7 polymorphisms. SCORAD and TEWL measurements were not influenced by any of the variants. The SPINK5 polymorphism was associated with high IgE serum levels (p=0.011). Abnormal barrier genes do not influence the severity of atopic dermatitis. The SPINK5 gene polymorphism may modulate systemic immune effects favouring the IgE response to atopens. TEWL does not allow the characterization of subsets of patients with or without abnormal barrier genes.

In 46, XY individuals, the testicular development is initiated by the expression of SRY, which sets into motion a chain of signal transduction events leading to development of the male external and internal genitalia. In the absence of SRY, ovarian development occurs, associated with a female phenotype [1]. Disorders of Sex Development (DSD) are a range of congenital conditions of atypical gonadal development leading to discordance between chromosomal, gonadal and phenotypic sex. According to the Chicago consensus [2], DSD are divided in three categories based on karyotype: 46, XX DSD, 46, XY DSD and sex chromosome DSD. Although there have been considerable advances in our understanding of the genetic factors and mechanisms involved in gonadal differentiation in the last 10 years [3], a molecular diagnosis is made in only around 20 % of cases of DSD, except in cases where the biochemical profile indicates a specific steroidogenic block [2]. In 46, XY DSD with disorders of gonadal development, individuals with complete gonadal dysgenesis are phenotypically females with streak gonads and are often diagnosed because of primary amenorrhea whereas, in individuals with partial gonadal dysgenesis, the phenotype is more ambiguous. Approximately 15 % of all cases of gonadal dysgenesis carry inactivating mutations in SRY, with the majority localized within the HMG-domain [4]. Rare cases of gonadal dysgenesis with small interstitial deletions outside the SRY-open reading frame have been described [5]. When SRY function is normal, other genes are known to be involved in the occurrence of 46, XY DSD gonadal dysgenesis, with a loss-of-function (for example SOX9, WT1, NR5A1/SF1, DMRT1, MAP3K1) or with a gain-of-function (for example WNT4 and NR0B1/DAX1) [1, 6]. Nevertheless, the underlying genetic basis of complete gonadal dysgenesis is unknown in about 50 % of the patients [7]. As many of the genes involved in gonadal development have a dosage effect, DSD may be caused by copy-number variations (CNVs) corresponding to deletions or duplications of these genes [8]. Conventional karyotype may not be adequate to detect these changes but array-CGH has been emerging as a powerful tool to identify submicroscopic CNVs on a genome wide scale.

BACKGROUND: The Tyr978X BRCA1 mutation is a founder mutation detected in high-risk Iraqi-Iranian Jewish families as well as in the general non-Ashkenazi population. The same mutation was also reported in non-Jewish high-risk women from Canada. Its occurrence in non-Jewish individuals from Iran has never been tested. OBJECTIVE: Assess the occurrence rate of Tyr978X BRCA1 germline mutation in the general population of Iranian non- Jewish individuals and compare the BRCA1-linked haplotype of Jewish and non-Jewish mutation carriers. METHODS: PCR amplification of the relevant fragment of the BRCA1 gene, followed by restriction enzyme digestion that differentiates wild type from mutant allele. For haplotyping, 7 BRCA1-linked markers were used. The tested population included 442 apparently healthy Iranian non-Jewish individuals, and 17 mutation carriers from Israel and Canada. RESULTS: The Tyr978X BRCA1 mutation was not detected in any Iranian non-Jewish individual. The intragenic haplotype of all Jewish Israeli mutation carriers was identical, but differed from that of Canadian non-Jews in two intragenic markers. CONCLUSIONS: The Tyr978X BRCA1 mutation which is a founder mutation in Jews, may be a hot spot in non-Jewish high risk women, and probably does not represent a rare sequence variant in Iranian non-Jews.

A mosaic karyotype consisting of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome is relatively common and is associated with a wide spectrum of clinical phenotypes. The aim of this study was to investigate patients with such a mosaic karyotype for Y chromosome material loss and then study the possible association of the absence of these regions with the phenotype, diagnosis, and Y-chromosome instability. We studied 17 clinically well-characterized mosaic patients whose karyotype consisted of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome. The presence of the Y chromosome centromere was verified by fluorescence in situ hybridization (FISH) and was then characterized by 44 Y-chromosome specific-sequence tagged site (STS) markers. This study identifies a high frequency of Yq chromosome deletions (47%). The deletions extend from interval 5 to 7 sharing a common deleted interval (6F), which overlaps with the azoospermia factor region (AZF) region. This study finds no association between Y-chromosome loci hosting genes other than SRY, and the phenotypic sex, the diagnosis, and the phenotype of the patients. Furthermore, this study shows a possible association of these deletions with Y-chromosome instability.

The southwestern and Central Asian corridor has played a pivotal role in the history of humankind, witnessing numerous waves of migration of different peoples at different times. To evaluate the effects of these population movements on the current genetic landscape of the Iranian plateau, the Indus Valley, and Central Asia, we have analyzed 910 mitochondrial DNAs (mtDNAs) from 23 populations of the region. This study has allowed a refinement of the phylogenetic relationships of some lineages and the identification of new haplogroups in the southwestern and Central Asian mtDNA tree. Both lineage geographical distribution and spatial analysis of molecular variance showed that populations located west of the Indus Valley mainly harbor mtDNAs of western Eurasian origin, whereas those inhabiting the Indo-Gangetic region and Central Asia present substantial proportions of lineages that can be allocated to three different genetic components of western Eurasian, eastern Eurasian, and south Asian origin. In addition to the overall composite picture of lineage clusters of different origin, we observed a number of deep-rooting lineages, whose relative clustering and coalescent ages suggest an autochthonous origin in the southwestern Asian corridor during the Pleistocene. The comparison with Y-chromosome data revealed a highly complex genetic and demographic history of the region, which includes sexually asymmetrical mating patterns, founder effects, and female-specific traces of the East African slave trade.

A variant in steroidogenic factor-1 (SF-1, encoded by the gene NR5A1), p.Arg92Trp, has recently been reported in multiple families with 46,XX ovotesticular or testicular disorders of sex development (DSD). This amino acid change impacts the DNA-binding domain and perturbs gonadal differentiation pathways.Whole-exome sequencing was performed on a 46,XX subject with ovotesticular DSD.Exome results identified a heterozygous NR5A1 variant, p.Arg92Gln, in the 46,XX ovotesticular DSD proband. This arginine-to-glutamine change has been previously reported in the homozygous state in a 46,XY patient with gonadal and adrenal dysgenesis, though 46,XY and 46,XX heterozygous carriers of this variant have not been previously reported to have any clinical phenotype.The NR5A1 p.Arg92Gln variant, which has thus far only been seen in a family with 46,XY DSD, most likely contributes to the ovotesticular DSD in this case. In light of the recent reports of unrelated 46,XX subjects with testicular or ovotesticular DSD with the NR5A1 variant p.Arg92Trp, it appears that other mutations in the DNA binding domain have the potential to impact the factors determining testicular and ovarian differentiation. This case demonstrates the variability of phenotypes with the same genotype and broadens our understanding of the role of SF-1 in gonadal differentiation.

Testicular dysgenesis syndrome encompasses low sperm quality, hypospadias, cryptorchidism and testicular cancer. Epidemiological studies and genetic data from familial cases suggest that testicular dysgenesis syndrome has a common etiology. The Y chromosome is known to encode genes that are involved in germ cell development or maintenance. We have therefore investigated if different classes of Y chromosomes in the general population (Y chromosome haplogroups) are associated with aspects of the testicular dysgenesis syndrome. We defined the Y chromosome haplogroups in individuals from different European counties who presented with either (i) oligo- or azoospermia associated with a Y chromosome microdeletion, (ii) unexplained reduced sperm counts (20 x 10(6)/ml) or (iii) testicular cancer. We failed to find Y chromosome haplotype associations with either microdeletion formation or testicular cancer. However, in a study of the Danish population, we found that a specific Y chromosome haplogroup (hg26) is significantly overrepresented in men with unexplained reduced sperm counts compared with a Danish control population. The factors encoded by genes on this class of Y chromosome may be particularly susceptible to environmental influences that cause testicular dysgenesis syndrome. Our current data highlight the need for further analyses of clinically well-defined patient groups from a wide range of ethnic and geographic origins.

A locus on distal 9p has been reported to be deleted in patients with mental retardation, craniofacial dysmorphic traits, notably trigonocephaly, and a high frequency of genital and/or gonadal anomalies (Alfi's syndrome).1 More recently, some cases of 46,XY male to female sex reversal without dysmorphic traits have been described in association with distal deletions of 9p.2–5 46,XY patients described so far have had gonadal anomalies ranging from streak gonads, to partially masculinised dysgenetic gonads, to normal testes. A family of genes containing a unique zinc finger DNA binding domain, which has been called DM, as it is contained in two proteins involved in invertebrate sex determination, doublesex and mab-3, has recently been recognised. Three genes belonging to this family have been identified on 9p24.3, immediately proximal to the centromeric boundary of the minimal region, which is required in two copies for testis development.5 The first gene identified was termed DMRT1 and the two additional DM gene family members are placed in tandem more distally to the breakpoint of the minimal deleted region.6,7 DMRT1 is transcribed only in the embryonic gonads of both sexes and in fetal and adult testis, and is required for postnatal differentiation of both somatic and germ cells in the mouse testis.8 Therefore, this gene is a good candidate to explain the 46,XY sex reversal associated with 9p deletions. Recently, a 1 year old 46,XX female patient with premature ovarian failure and deletion of 9p was reported.9 This led to the suggestion of a role for a gene located on 9p in both male and female gonadal development. DMRT1 represents a good candidate for a function in both sexes as it is expressed in the genital ridge in both sexes in mouse and humans.7,8 Here, we report …

Recent advances in the evolutionary genetics of sex determination indicate that DMRT1 may be a vertebrate equivalent of the Drosophila melanogaster master sex regulator gene, doublesex. The role of DMRT1 seems to be confined to some aspects of male sex differentiation, whereas in Drosophila, doublesex has wider developmental effects in both sexes. This suggests other homologs of doublesex may exist in the vertebrate genome and encode sex-specific functions not displayed by DMRT1. We identified and characterized five novel human DM genes, distinct from previously described family members. Human DM genes map to three well-defined regions of chromosomes 1, 9, and 19 (one gene on chromosome 19 having an additional homolog on chromosome X). We collated data indicating these chromosomal regions harbor multiple syntenic genes sharing highly specific paralogy relations, suggesting that they arose early during vertebrate evolution. The 9p21-p24.3 bands represent the ancestral copy and harbor closely linked DM genes that may reflect the overall diversity of the fruit fly DM gene family. The human genome contains a small number of potential doublesex homologs that may be involved in human sexual development. Identifying highly conserved chromosomal regions, such as distal 9p, is an important tool to trace complex ancient evolutionary processes inaccessible by other approaches.

Objective To assess genetic mutations and associated somatic anomalies in a series of patients with 46,XY gonadal dysgenesis (GD). Design Single center retrospective study. Setting University pediatric hospital. Patient(s) Fourteen patients with 46,XY GD. Intervention(s) None. Main Outcome Measure(s) Genotype-phenotype relationship. Result(s) The presenting symptom was disorders of sex development (6 patients), primary amenorrhea (2 patients), discordance between 46,XY karyotype and female external genitalia (3 patients), discovery of Mullerian structures at surgery (2 patients), or diagnosed in the evaluation of a gonadal tumor (1 patient). Mullerian structures were shown by ultrasound evaluation in 7 of 13 patients, genitography in 3 of 6 patients and/or surgery in 8 of 10 patients (3 not seen at imaging), or only by histologic examination (1 patient). Three patients had gonadoblastoma and/or seminoma. A mutation was found in 7 patients of whom 2 had family history of reproductive problems and 5 had associated somatic anomalies. The mutations were FOG2/ZFPM2 (1 patient), SRY (2 patients), WT1 (1 patient), or deletions of distal chromosome 9p (3 patients). Among the three other patients with associated anomalies and no mutation, two had ectodermal dysplasia and one had leukemia. Conclusion(s) Mutations were observed in half of the patients with 46,XY GD with Mullerian structures. We also describe for the first time the association between GD and ectodermal dysplasia. Mullerian structures can be found in some cases only by histologic examination, which should be coupled to preventive gonadectomy because of the risk of tumor formation.

The cause of the primary ovarian insufficiency (POI) remains unknown in the majority of cases. A retrospective study was carried out in 17 girls with POI and normal 46,XX karyotype evaluated before 20 years of age. The etiology of POI was determined in eight girls (group 1) and remained idiopathic in nine girls (group 2). In group 1, five patients had a medical history: cerebellar ataxia due to congenital disorder of glycosylation (CDG) 1 in three cases, mitochondrial disease in one case, and autoimmune deficiencies in one case. The diagnosis of POI was made on pubertal delay or primary amenorrhea in these five patients, whilst the others presented with clitoral hypertrophy at birth or short stature and pubertal delay in two cases with NR5A1 mutation or with short stature and learning difficulties in one case with mitochondrial disease. In group 2, associated diseases were arthrogryposis malformative, gut, and bladder malformations and kidney failure or parieto-occipital tumor. The genes tested (NR5A1, BMP15, GDF9, and NOBOX) showed no mutation.The frequency of defined etiologies (47%) is high. This is probably because of the recruitment of the cases at the pediatric center, where other somatic anomalies can lead to the accurate determination of the etiology.

Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical features of ciliopathies. Here we identify mutations in the genes encoding PLK4 kinase, a master regulator of centriole duplication, and its substrate TUBGCP6 in individuals with microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy, thereby extending the human phenotypic spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenotypes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.

In recent years, considerable advances have been made in our understanding of genetics of mammalian gonad development; however, the underlying genetic aetiology in the majority of patients with 46,XY disorders of sex development (DSD) still remains unknown. Based on mouse models, it has been hypothesized that haploinsufficiency of the Friend of GATA 2 (FOG2) gene could lead to 46,XY gonadal dysgenesis on specific inbred genetic backgrounds. Using whole exome sequencing, we identified independent missense mutations in FOG2 in two patients with 46,XY gonadal dysgenesis. One patient carried a non-synonymous heterozygous mutation (p.S402R), while the other patient carried a heterozygous p.R260Q mutation and a homozygous p.M544I mutation. Functional studies indicated that the failure of testis development in these cases could be explained by the impaired ability of the mutant FOG2 proteins to interact with a known regulator of early testis development, GATA4. This is the first example of mutations in the coding sequence of FOG2 associated with 46,XY DSD in human and adds to the list of genes in the human known to be associated with DSD.

The etiology of most cases of 46,XY gonadal dysgenesis in the absence of extragenital anomalies is not accounted for by mutations in the genes known to date to be involved in sex determination. We have investigated the possibility that mutations in the gene LHX9, whose murine ortholog causes isolated gonadal agenesis when inactivated, might be responsible for gonadal dysgenesis and agenesis in humans. We isolated a human LHX9 complementary DNA (cDNA), mapped the gene to the long arm of human chromosome 1, and determined its genomic structure. We found that LHX9 is highly conserved between species, sharing in particular over 98% amino acid identity. A mutational screen was performed in a sample of patients with a range of gonadal maldevelopment, including bilateral gonadal agenesis in two sisters with an opposite sex karyotype. We did not detect mutations in the open reading frame of LHX9 in the patients studied. However, the extent of between-species structural conservation suggests that LHX9 deserves further consideration as a determinant of gonadal function in humans.

Distal chromosome 9p contains a locus that, when deleted, is a cause of 46,XY gonadal dysgenesis in the absence of extragenital anomalies. This locus might account for the frequently observed cases of 46,XY pure gonadal dysgenesis who do not harbor mutations in SRY, the sex master regulator gene found in mammalian species. The genomic organization of 9p positional candidate genes is currently being studied and mutational screens are ongoing. Among other positional candidates, including two additional doublesex-related genes, the evidence to support a role for the gene DMRT1 in vertebrate male sexual development is accumulating. Although formal proof of the requirement of DMRT1 in gonadal sex fate choice has not been obtained so far, the particular interest in this gene and perhaps other doublesex-related genes identified in vertebrates lies in that they may provide an entry point to a conserved mechanism of sex determination across animal phyla. We discuss recent results and emerging views on the genetics of sex determination, while stressing that the majority of cases of 46,XY gonadal dysgenesis remain unexplained. The latter is likely to be efficiently addressed by positional cloning efforts, particularly by considering the wealth of sequence data provided by the Human Genome Project.

Deletions of 9p have been associated with 46,XY gonadal dysgenesis, and the smallest region of overlap has been mapped to the tip of chromosome 9. Two candidate genes (DMRT1 and 2) have been found in the region. Despite intensive mutation searches, no mutations have been detected in these genes. To gain insights into the genomics of the region and to isolate other candidate genes for the phenotype, we have constructed a P1 artificial chromosome (PAC)/bacterial artificial chromosome (BAC) contig spanning over 500 kb and covering the consensus critical region. We have analyzed the expression pattern of several ESTs mapped or sublocalized within the framework of the contig. In addition, a sample shotgun sequencing of a PAC containing the mentioned DM genes led to the detection of novel transcripts displaying an expression pattern specific to testis and kidney, consistent with a role in the development of the urogenital system. One of them, expressed in adult testis and human embryos aged 4-5 weeks, encodes a potential polypeptide and is located immediately downstream of a sequence capable of encoding a novel DM domain. The region was partially screened for mutations in sex-reversed patients by Southern blot, sequencing, and FISH. No mutations were found. Our results suggest that the critical region on 9p involved in male-to-female sex reversal displays greater gene density and genomic complexity than previously anticipated. Future investigations will include functional and mutational studies of the novel transcripts mapped or sublocalized within the critical region by this study as well as cloning efforts to isolate additional candidate genes.

DNA analysis is making a valuable contribution to the understanding of human evolution [1]. Much attention has focused on mitochondrial DNA (mtDNA) [2] and the Y chromosome [3] and [4], both of which escape recombination and so provide information on maternal and paternal lineages, respectively. It is often assumed that the polymorphisms observed at loci on mtDNA and the Y chromosome are selectively neutral and, therefore, that existing patterns of molecular variation can be used to deduce the histories of populations in terms of drift, population movements, and cultural practices. The coalescence of the molecular phylogenies of mtDNA and the Y chromosome to recent common ancestors in Africa [5] and [6], for example, has been taken to reflect a recent origin of modern human populations in Africa. An alternative explanation, though, could be the recent selective spread of mtDNA and Y chromosome haplotypes from Africa in a population with a more complex history [7]. It is therefore important to establish whether there are selective differences between classes (haplotypes) of mtDNA and Y chromosomes and, if so, whether these differences could have been sufficient to influence the distributions of haplotypes in existing populations. A precedent for this hypothesis has been established for mtDNA in that one mtDNA background increases susceptibility to Leber hereditary optic neuropathy [8]. Although studies of nucleotide diversity in global samples of Y chromosomes have suggested an absence of recent selective sweeps or bottlenecks [9], selection may, in principle, be very important for the Y chromosome because it carries several loci affecting male fertility [10] and [11] and as many as 5% of males are infertile [11] and [12]. Here, we show that one class of infertile males, PRKX/PRKY translocation XX males, arises predominantly on a particular Y haplotypic background. Selection is, therefore, acting on Y haplotype distributions in the population.

The recent transposition to the Y chromosome of the autosomal DAZL1 gene, potentially involved in germ cell development, created a unique opportunity to study the rate of Y chromosome evolution and assess the selective forces that may act upon such genes, and provided a new estimate of the male-to-female mutation rate (alpham). Two different Y-located DAZ sequences were observed in all Old World monkeys, apes and humans. Different DAZ copies originate from independent amplification events in each primate lineage. A comparison of autosomal DAZL1 and Y-linked DAZ intron sequences gave a new figure for male-to-female mutation rates of alpham = 4. It was found that human DAZ exons and introns are evolving at the same rate, implying neutral genetic drift and the absence of any functional selective pressures. We therefore hypothesize that Y-linked DAZ plays little, or a limited, role in human spermatogenesis. The two copies of DAZ in man appear to be due to a relatively recent duplication event (55 000-200 000 years). A worldwide survey of 67 men from five continents representing 19 distinct populations showed that most males have both DAZ variants. This implies a common origin for the Y chromosome consistent with a recent 'out of Africa' origin of the human race.

Sex is determined in mammals by the SRY gene, which is located on the non-recombining region of the Y chromosome. Although the presence of mutations in SRY associated with male to female sex reversal clearly indicates that this gene is essential for testis formation, little is known concerning other genes in this process or how the expression of SRY itself is controlled. This is mainly due to the absence of an appropriate in vitro cellular model. Previous studies have indicated that SRY coding sequences are undergoing rapid evolution in mammals. In this study, we cloned and compared a Y chromosome-specific region immediately 5' to the SRY open reading frame in several primate species. The divergence of the region 5' to SRY between primates was found to be comparable with that described for autosomal sequences. An alignment of sequences within the primate lineage, together with sequences from the cow and pig, revealed the presence of several highly conserved motifs. These domains may have a function in the control of SRY expression during fetal development.

The condition termed 46,XY complete gonadal dysgenesis is characterized by a completely female phenotype and streak gonads. In contrast, subjects with 46,XY partial gonadal dysgenesis and those with embryonic testicular regression sequence usually present ambiguous genitalia and a mix of Müllerian and Wolffian structures. In 46,XY partial gonadal dysgenesis gonadal histology shows evidence of incomplete testis determination. In 46,XY embryonic testicular regression sequence there is lack of gonadal tissue on both sides. Various lines of evidence suggest that embryonic testicular regression sequence is a variant form of 46,XY gonadal dysgenesis. The sex-determining region Y chromosome gene (SRY) encodes sequences for the testis-determining factor. To date germ-line mutations in SRY have been reported in approximately 20% of subjects with 46,XY complete gonadal dysgenesis. However, no germ-line mutations of SRY have been reported in subjects with the partial forms. We studied 20 subjects who presented either 46,XY partial gonadal dysgenesis or 46,XY embryonic testicular regression sequence. We examined the SRY gene and the minimum region of Y-specific DNA known to confer a male phenotype. The SRY-open reading frame (ORF) was normal in all subjects. However a de novo interstitial deletion 3' to the SRY-ORF was found in one subject. Although it is possible that the deletion was unrelated to the subject's phenotype, we propose that the deletion was responsible for the abnormal gonadal development by diminishing expression of SRY. We suggest that the deletion resulted either in the loss of sequences necessary for normal SRY expression or in a position effect that altered SRY expression. This case provides further evidence that deletions of the Y chromosome outside the SRY-ORF can result in either complete or incomplete sex reversal.