Joelle Bignon-Topalovic

Human Male Infertility Associated with Mutations in NR5A1 Encoding Steroidogenic Factor 1

One in seven couples worldwide are infertile, and male factor infertility accounts for approximately 30%-50% of these cases. Although many genes are known to be essential for gametogenesis, there are surprisingly few monogenic mutations that have been conclusively demonstrated to cause human spermatogenic failure. A nuclear receptor, NR5A1 (also called steroidogenic factor 1), is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-steroidogenic axis, and it is expressed in the steroidogenic tissue of the developing and adult human gonad. Mutations of NR5A1 have been reported in 46,XY disorders of sex development and in 46,XX primary ovarian insufficiency. To test the hypothesis that mutations in NR5A1 cause male infertility, we sequenced NR5A1 in 315 men with idiopathic spermatogenic failure. We identified seven men with severe spermatogenic failure who carried missense mutations in NR5A1. Functional studies indicated that these mutations impaired NR5A1 transactivational activity. We did not observe these mutations in more than 4000 control alleles, including the entire coding sequence of 359 normospermic men and 370 fertile male controls. NR5A1 mutations are found in approximately 4% of men with otherwise unexplained severe spermatogenic failure.

Mutations in the FOG2/ZFPM2 gene are associated with anomalies of human testis determination

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.

A recurrent p.Arg92Trp variant in steroidogenic factor-1 (NR5A1) can act as a molecular switch in human sex development

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.

A Nonsense Mutation in the Hedgehog Receptor CDON Associated With Pituitary Stalk Interruption Syndrome.

BACKGROUND Pituitary stalk interruption syndrome (PSIS) and holoprosencephaly (HPE) are congenital midline defects. Rare mutations in the sonic hedgehog (SHH) signaling gene CDON have recently been reported in patients with HPE. OBJECTIVE To report a unique case of PSIS with a maternally inherited nonsense mutation in the SHH signaling protein CDON. METHOD We performed exome sequencing on a case of PSIS. Control databases (1000 Genomes, dbSNP, Exome Variant Server, ExAC Browser) and an ancestry-matched control panel were screened upon identification of CDON mutation. RESULTS We identified a novel heterozygous nonsense mutation (c.2764T>C, Glu922Ter) in a case of PSIS without HPE who presented with neonatal hypoglycemia and cholestasis associated with GH, TSH, and ACTH deficiencies. This mutation was absent in all control databases and from 400 healthy ancestry-matched control subjects. The mutation was inherited from the patients mother, who was operated on in childhood for strabismus. The absence of this variant in control samples suggests that it is likely to be responsible for the phenotype. CONCLUSION We report for the first time a mutation in the CDON gene associated with PSIS.

Identification of NR5A1 Mutations and Possible Digenic Inheritance in 46,XY Gonadal Dysgenesis.

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.

Polymorphisms in DLGH1 and LAMC1 in Mayer–Rokitansky–Kuster–Hauser syndrome

Müllerian 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 Müllerian 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.

Homozygous Mutation of the FGFR1 Gene Associated with Congenital Heart Disease and 46,XY Disorder of Sex Development

Congenital heart diseases (CHDs) are the most common cause of all birth defects and account for nearly 25% of all major congenital anomalies leading to mortality in the first year of life. Extracardiac anomalies including urogenital aberrations are present in ∼30% of all cases. Here, we present a rare case of a 46,XY patient with CHD associated with ambiguous genitalia consisting of a clitoris-like phallus and a bifid scrotum. Exome sequencing revealed novel homozygous mutations in the FGFR1 and STARD3 genes that may be associated with the phenotype.

Longitudinal Evaluation of the Hypothalamic-Pituitary- Testicular Function in 8 Boys with Adrenal Hypoplasia Congenita (AHC) Due to NR0B1 Mutations

Background Boys carrying mutations in the NR0B1 gene develop adrenal hypoplasia congenita (AHC) and impaired sexual development due to the combination of hypogonadotropic hypogonadism (HH) and primary defects in spermatogenesis. Methods We analysed the evolution of hypothalamic-pituitary-testicular function of 8 boys with AHC due to NR0B1 mutations. Our objective was to characterize and monitor the progressive deterioration of this function. Results The first symptoms appeared in the neonatal period (n = 5) or between 6 months and 8.7 years (n = 3). Basal plasma adrenocorticotrophic hormone (ACTH) concentrations increased in all boys, whilst cortisol levels decreased in one case. The natremia was equal or below 134 mmol/L and kaliemia was over 5 mmol/L. All had increased plasma renin. In 3 of 4 patients diagnosed in the neonatal period and evaluated during the first year, the basal plasma gonadotropins concentrations, and their response to gonadotropin releasing hormone (GnRH) test (n = 2), and those of testosterone were normal. The plasma inhibin B levels were normal in the first year of life. With the exception of two cases these concentrations decreased to below the normal for age. Anti-Müllerian hormone concentrations were normal for age in all except one case, which had low concentrations before the initiation of testosterone treatment. In 3 of the 8 cases the gene was deleted and the remaining 5 cases carried frameshift mutations that are predicted to introduce a downstream nonsense mutation resulting in a truncated protein. Conclusions The decreases in testosterone and inhibin B levels indicated a progressive loss of testicular function in boys carrying NR0B1 mutations. These non-invasive examinations can help to estimate the age of the testicular degradation and cryopreservation of semen may be considered in these cases as investigational procedure with the aim of restoring fertility.

Genetic mutations and somatic anomalies in association with 46,XY gonadal dysgenesis

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 Müllerian structures at surgery (2 patients), or diagnosed in the evaluation of a gonadal tumor (1 patient). Müllerian 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 Müllerian structures. We also describe for the first time the association between GD and ectodermal dysplasia. Müllerian 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.

Mutations in the Human ROBO1 Gene in Pituitary Stalk Interruption Syndrome

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.