Françoise Audran

Phenotypical, Biological, and Molecular Heterogeneity of 5α-Reductase Deficiency: An Extensive International Experience of 55 Patients

CONTEXT In 46,XY disorders of sex development, 5α-reductase deficiency is rare and is not usually the first-intention diagnosis in newborn ambiguous genitalia, contrary to partial androgen insensitivity syndrome. Yet the cause of ambiguous genitalia may guide sex assignment, and rapid, precise diagnosis of 5α-reductase deficiency is essential. OBJECTIVE The aim of the study was to describe relevant data for clinical diagnosis, biological investigation, and molecular determination from 55 patients with srd5A2 mutations identified in our laboratory over 20 yr to improve early diagnosis. SETTING The study was performed at Montpellier University Hospital. PATIENTS We studied a cohort of 55 patients with srd5A2 gene mutations. MAIN OUTCOME MEASURE(S) Genetic analysis of srd5A2 was conducted. RESULTS Clitoromegaly (49.1%) and microphallus with various degrees of hypospadias (32.7%) were frequent phenotypes. Female external genitalia (7.3%) and isolated micropenis (3.6%) were rare. Seventy-two percent of patients were initially assigned to female gender; five of them (12.5%) switched to male sex in peripuberty. Over 72% of patients were considered for 5α-reductase deficiency diagnosis when the testosterone/dihydrotestosterone cutoff was 10. In 55 patients (with 20 having a history of consanguinity), we identified 33 different mutations. Five have never been reported: p.G32S, p.Y91H, p.G104E, p.F223S, and c.461delT. Homozygous mutations were present in 69.1% of cases, compound heterozygous mutations in 25.5%, and compound heterozygous mutations alone with the V89L polymorphism in 5.4%. Exons 1 and 4 were most affected, with 35.8 and 21.7% mutant alleles per exon, respectively. CONCLUSIONS In the largest cohort to date, we demonstrate a wide spectrum of phenotypes and biological profiles in patients with 5α-reductase deficiency, whatever their geographical or ethnic origins.

Mutations of CXorf6 are associated with a range of severities of hypospadias

OBJECTIVE Mutations in chromosome X open reading frame 6 (CXorf6), a recently described candidate gene involved in the development of male genitalia, have been found in patients with complex 46,XY disorders of sexual development (46,XY DSD) including micropenis, bifid scrotum, and penoscrotal hypospadias. The objective of this work was to identify genomic variants of CXorf6 in patients with isolated hypospadias, severe or non-severe. DESIGN AND METHODS Forty-one patients with glandular to perineal hypospadias and thirty controls were studied. Direct sequencing for coding exons 3-6 of CXorf6 and their flanking splice sites was performed on DNA extracted from foreskin collected from surgery. Secondary and tertiary structures of the protein were predicted using NNpredict and Protein Homology/analogY Recognition Engine engines. RESULTS Four mutations (9.7% of cases) were identified. One missense mutation (1295T>C, V432A) and two deletions (325delG, predicted to cause a stop codon L121X) occurred in patients with penoscrotal and proximal hypospadias. One patient with subcoronal hypospadias had CAG-repeat amplification in the second polyglutamine domain of CXorf6. Secondary structure prediction indicated that this insertion occurred in a helix element of the protein. The tertiary structure prediction showed an alteration of the shape of the protein and crowding between domains. CONCLUSION CXorf6 mutations are associated with isolated hypospadias of varying severity. However, the pathophysiology of these mutations and the function of the CXorf6 gene product remain to be investigated.

Steroidogenic factor-1 (SF-1) gene mutation as a frequent cause of primary amenorrhea in 46,XY female adolescents with low testosterone concentration

BackgroundPrimary amenorrhea due to 46,XY disorders of sex differentiation (DSD) is a frequent reason for consultation in endocrine and gynecology clinics. Among the genetic causes of low-testosterone primary amenorrhea due to 46,XY DSD, SRY gene is reported to be frequently involved, but other genes, such as SF1 and WT1, have never been studied for their prevalence.MethodsWe directly sequenced SRY, SF1 and WT1 genes in 15 adolescent girls with primary amenorrhea, low testosterone concentration, and XY karyotype, to determine the prevalence of mutations. We also analyzed the LH receptor gene in patients with high LH and normal FSH concentrations.ResultsAmong the 15 adolescents with primary amenorrhea and low testosterone concentration, we identified two new SRY mutations, five new SF1 mutations and one new LH receptor gene mutation. Our study confirms the 10-15% prevalence of SRY mutations and shows the high prevalence (33%) of SF1 abnormalities in primary amenorrhea due to 46,XY DSD with low plasma testosterone concentration.ConclusionsThe genetic analysis of low-testosterone primary amenorrhea is complex as several factors may be involved. This work underlines the need to systematically analyze the SF1 sequence in girls with primary amenorrhea due to 46,XY DSD and low testosterone, as well as in newborns with 46,XY DSD.

Is Hypospadias Associated with Prenatal Exposure to Endocrine Disruptors? A French Collaborative Controlled Study of a Cohort of 300 Consecutive Children Without Genetic Defect

BACKGROUND Numerous studies have focused on the association between endocrine-disrupting chemicals (EDCs) and hypospadias. Phenotype variability, the absence of representative comparison groups and concomitant genetic testing prevent any definitive conclusions. OBJECTIVE To identify the role of occupational and environmental exposures to EDCs in nongenetic isolated hypospadias. DESIGN, SETTING, AND PARTICIPANTS A total of 408 consecutive children with isolated hypospadias and 302 normal boys were prospectively included (2009-2014) in a multi-institutional study in the south of France, the area of the country with the highest prevalence of hypospadias surgery. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS In patients without AR, SRD5A2, and MAMLD1 mutations, parental occupational and professional exposures to EDCs were evaluated based on European questionnaire QLK4-1999-01422 and a validated job-exposure matrix for EDCs. Environmental exposure was estimated using the zip code, the type of surrounding hazards, and distance from these hazards. Multivariate analysis was performed. RESULTS Fetal exposure to EDCs around the window of genital differentiation was more frequent in the case of hypospadias (40.00% vs 17.55%, odds ratio 3.13, 95% confidence interval 2.11-4.65). The substances were paints/solvents/adhesives (16.0%), detergents (11.0%), pesticides (9.0%), cosmetics (5.6%), and industrial chemicals (4.0%). Jobs with exposure were more frequent in mothers of hypospadiac boys (19.73% vs 10.26%, p=0.0019), especially cleaners, hairdressers, beauticians, and laboratory workers. Paternal job exposure was more frequent in the cases of hypospadias (40.13% vs 27.48%, p=0.02). Industrial areas, incinerators, and waste areas were more frequent within a 3-km radius for mothers of hypospadiac boys (13.29% vs. 6.64%, p<0.00005). Association of occupational and environmental exposures increases this risk. CONCLUSIONS This multicenter prospective controlled study with a homogeneous cohort of hypospadiac boys without genetic defects strongly suggests that EDCs are a risk factor for hypospadias through occupational and environmental exposure during fetal life. The association of various types of exposures may increase this risk. PATIENT SUMMARY Our multi-institutional study showed that parental professional, occupational, and environmental exposures to chemical products increase the risk of hypospadias in children.

Complete androgen insensitivity syndrome is frequently due to premature stop codons in exon 1 of the androgen receptor gene: an international collaborative report of 13 new mutations.

OBJECTIVE To confirm the clinical diagnosis of complete androgen insensitivity syndrome (CAIS) by molecular genetic analysis and to determine the prevalence of exon 1 mutations in the androgen receptor (AR) transactivation defects of a large series of CAIS patients. DESIGN International retrospective study. SETTING University Hospital of Montpellier, Department of Hormonology. PATIENT(S) 105 patients with normal female external genitalia, bilateral intra-abdominal or inguinal testis, normal breast development, absent or sparse pubic hair, normal or high endogenous testosterone production, hypoplastic or absent wolffian structures, and 46,XY karyotype. INTERVENTION(S) Sequencing of the AR gene. MAIN OUTCOME MEASURE(S) Prevalence of AR exon 1 mutations. RESULT(S) Over a 10-year period (1997 to 2007), we identified 78 AR gene mutations in 105 patients with CAIS; 21 of them were located in exon 1, and 13 of these were new mutations. We report 13 new mutations in the AR gene. All but one were stop codons, and the last was a splicing abnormality. CONCLUSION(S) The finding that 27% of the mutations in our cohort were localized in exon 1 versus 15% in previous works justifies the sequencing of this exon in patients with CAIS.

High prevalence of micropenis in 2710 male newborns from an intensive‐use pesticide area of Northeastern Brazil

Exposure to endocrine-disrupting chemicals (EDCs) has been suggested to contribute to the increasing trends of external genital malformation in male newborns. In Northeastern Brazil, the poor sanitary conditions found in the favelas encourage the widespread use of pesticides. This 2-year study of a total birth cohort of full-term male newborns in the regional hospitals of Campina Grande (Paraíba, Brazil) sought to (1) accurately establish for the first time the incidences of neonatal male genital malformations, (2) investigate the endocrine and genetic aetiologies of these malformations, and (3) evaluate their associations with possible prenatal exposure to EDCs. A total of 2710 male newborns were explored for cryptorchidism, hypospadias and micropenis. Cases were referred to the Pediatric Endocrine Clinic for endocrine and genetic investigations, and all parents were interviewed about their environmental/occupational exposure to EDCs before/during pregnancy by paediatric endocrinologists using a detailed questionnaire. We observed 56 cases of genital malformation (2.07%), including 23 cryptorchidism (0.85%), 15 hypospadias (0.55%), and 18 micropenis (0.66%). All cases exhibited normal/subnormal testosterone production and none presented androgen receptor or 5α-reductase gene mutation. More than 92% of these newborns presented foetal contamination by EDCs, as their mothers reported daily domestic use of pesticides (i.e., DDT) and other EDCs. Most of these undervirilized male newborns presented additional EDC contamination, as 80.36% of the mothers and 58.63% of the fathers reported paid or unpaid work that entailed the use of pesticides and other EDCs before/during pregnancy for the mothers and around the time of fertilization for the fathers. The high rate of micropenis in our population associated with an elevated percentage of parental environmental/occupational exposure to EDCs before/during pregnancy indicates that foetal contamination may be a risk factor for the development of male external genital malformation.

Predominant Sertoli cell deficiency in a 46,XY disorders of sex development patient with a new NR5A1/SF-1 mutation transmitted by his unaffected father

OBJECTIVE To further investigate the molecular mechanism by which NR5A1/SF-1 mutation led to gonadal dysgenesis with predominant Sertoli cell defect. DESIGN Genetic and functional mutation study. SETTING University hospital. PATIENT(S) Genetic analysis of an XY newborn with hypospadias and micropenis. Puberty developed spontaneously with a rise in T levels and normal LH contrasting with high FSH and low inhibin B concentrations, revealing a Sertoli cell defect. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) NR5A1/SF-1 gene molecular analysis. RESULT(S) Genetic analysis identified a new NR5A1/SF-1 mutation, c.842G>C (p.Arg281Pro). In vitro functional studies showed that the p.Arg281Pro mutant mainly altered Sertoli cell function, as observed in vivo with a high FSH level and low inhibin B concentration contrasting with normal LH concentration. The mutation was found in the fathers DNA at a low copy number through direct sequencing and high-resolution melting assay, suggesting mosaicism. CONCLUSION(S) We describe a new heterozygous NR5A1/SF-1 mutation that mainly altered Sertoli cell function. However, this 46,XY disorders of sex development (DSD) boy had no Müllerian derivatives, suggesting normal Sertoli cell function during fetal life. During puberty, Sertoli cell deficiency became more apparent. This is the first report of a progressive and predominant Sertoli cell defect in an XY patient with testicular dysgenesis owing to NR5A1/SF-1 mutation.

Screening of MAMLD1 Mutations in 70 Children with 46,XY DSD: Identification and Functional Analysis of Two New Mutations

More than 50% of children with severe 46,XY disorders of sex development (DSD) do not have a definitive etiological diagnosis. Besides gonadal dysgenesis, defects in androgen biosynthesis, and abnormalities in androgen sensitivity, the Mastermind-like domain containing 1 (MAMLD1) gene, which was identified as critical for the development of male genitalia, may be implicated. The present study investigated whether MAMLD1 is implicated in cases of severe 46,XY DSD and whether routine sequencing of MAMLD1 should be performed in these patients. Seventy children with severe non-syndromic 46,XY DSD of unknown etiology were studied. One hundred and fifty healthy individuals were included as controls. Direct sequencing of the MAMLD1, AR, SRD5A2 and NR5A1 genes was performed. The transactivation function of the variant MAMLD1 proteins was quantified by the luciferase method. Two new mutations were identified: p.S143X (c.428C>A) in a patient with scrotal hypospadias with microphallus and p.P384L (c.1151C>T) in a patient with penile hypospadias with microphallus. The in vitro functional study confirmed no residual transactivating function of the p.S143X mutant and a significantly reduced transactivation function of the p.P384L protein (p = 0.0032). The p.P359S, p.N662S and p.H347Q variants are also reported with particularly high frequency of the p.359T- p.662G haplotype in the DSD patients. Severe undervirilization in XY newborns can reveal mutations of MAMLD1. MAMLD1 should be routinely sequenced in these patients with otherwise normal AR, SRD5A2 and NR5A1genes.

Apparent male gender identity in a patient with complete androgen insensitivity syndrome.

Clinicians universally agree on female sex of rearing inpatients with complete androgen insensitivity syndrome(CAIS).Thesepatientshaveafemalephenotypethoughtheyhave an XY karyotype and testis. Long-term studies haveshown an overall patient satisfaction with the assigned fe-male sex, female gender identity, and heterosexual prefer-encesinpatientswithCAIS(Hines,Ahmed,HMazur,2005;Wisniewskietal.,2000).WereporthereacaseofCAIS(rearedasafemaleuntilage11 years)whopresentedwith a desire to live as a male at age 11 years.This 11 year, 3 month old girl was the youngest of fivesiblings (one brother and three sisters). She had undergonesurgery for bilateral inguinal swellings one year prior at alocal hospital. Testicular tissue was found on exploration.The wound was closed without any surgical manipulationand the patient was referred to the endocrine department ofthis hospital. The child was accompanied by father andbrotheratthefirstvisit.ThiswasaHindufamilythathadbeenresiding in a village in Haryana. The father, around 45 yearsof age, was a farmer. The father and brother expressed theirdesire for masculinizing genitoplasty to rear the child as amale.Theyreportedthattheyhadobservedboy-likebehaviorin the child from early childhood and the revelation of thetesticular tissue had made them all convinced about the truemale identity of the child. She had been the most aggressiveamong all the female siblings and her play preferences anddressingpatternweresimilartoherelderbrother.Thepatientreturned with her mother in the subsequent visit (1 monthlater).Motherwasahousewife.Duringthesevisits,thischildwas dressed like a boy, expressed a desire to go to a boy’sschool as a boy, and said that she preferred boy’s company.Oncarefulquestioning,motherrevealedthat,duringthebirthofthischild,bothparentshadnursedastrongdesireforaboyafter three girls were born. She had always preferred a malepattern of dressing for the child and took pleasure in seeingmale appropriate behavior in her. Examination revealed acomplete female phenotype with no development of secon-darysexualcharacters(A1B1P

Isolated micropenis reveals partial androgen insensitivity syndrome confirmed by molecular analysis

Partial androgen insensitivity syndrome (PAIS) is the milder variant of androgen receptor (AR) defects. The subtle effects of AR mutations present in a patient with micropenis, peno-scrotal hypospadias, infertility, clitoromegaly and posterior labial fusion. We studied the association of isolated micropenis with the genetic defects resulting in androgen resistance, that is, AR gene defects and 5-alpha reductase type 2 (SRD5A2) deficiency. We describe two cases of isolated micropenis: one in a 14-year-old boy and the other in a 3-year-old boy who was followed until he was 10 years old. There were no findings of hypospadias, cryptorchidism or gynecomastia in either of these patients. Serum gonadotrophin and androgen levels were obtained and karyotyping was done. Human chorionic gonadotropin (hCG) stimulation testing assessed the functional capacity of the testes. DNA was extracted from peripheral leukocytes, and all exons of the SRD5A2 and AR genes were amplified by polymerase chain reaction and sequenced. In both patients, baseline testosterone (T) level was low and the values were elevated after hCG testing. The sequence of the SRD5A2 gene was normal in patient 1, and a heterozygous polymorphism, V89L, was found in patient 2. Two known mutations, P390S and A870V, were identified in patients 1 and 2, respectively. Mutations in the AR gene can be associated with isolated micropenis without other features of PAIS, such as hypospadias or gynecomastia. This underlines the importance of including AR gene analysis in the evaluation of isolated micropenis with normal plasma T to ensure proper management of the patient and appropriate genetic counseling for the family.