Anu Bashamboo

Mutations in NR5A1 Associated with Ovarian Insufficiency

BACKGROUNDnThe genetic causes of nonsyndromic ovarian insufficiency are largely unknown. A nuclear receptor, NR5A1 (also called steroidogenic factor 1), is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-steroidogenic axis. Mutation of NR5A1 causes 46,XY disorders of sex development, with or without adrenal failure, but growing experimental evidence from studies in mice suggests a key role for this factor in ovarian development and function as well.nnnMETHODSnTo test the hypothesis that mutations in NR5A1 cause disorders of ovarian development and function, we sequenced NR5A1 in four families with histories of both 46,XY disorders of sex development and 46,XX primary ovarian insufficiency and in 25 subjects with sporadic ovarian insufficiency. None of the affected subjects had clinical signs of adrenal insufficiency.nnnRESULTSnMembers of each of the four families and 2 of the 25 subjects with isolated ovarian insufficiency carried mutations in the NR5A1 gene. In-frame deletions and frameshift and missense mutations were detected. Functional studies indicated that these mutations substantially impaired NR5A1 transactivational activity. Mutations were associated with a range of ovarian anomalies, including 46,XX gonadal dysgenesis and 46,XX primary ovarian insufficiency. We did not observe these mutations in more than 700 control alleles.nnnCONCLUSIONSnNR5A1 mutations are associated with 46,XX primary ovarian insufficiency and 46,XY disorders of sex development.

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.

Loss-of-function mutation in GATA4 causes anomalies of human testicular development

Approximately 1 of every 250 newborns has some abnormality of genital and/or gonadal development. However, a specific molecular cause is identified in only 20% of these cases of disorder of sex development (DSD). We identified a family of French origin presenting with 46,XY DSD and congenital heart disease. Sequencing of the ORF of GATA4 identified a heterozygous missense mutation (p.Gly221Arg) in the conserved N-terminal zinc finger of GATA4. This mutation was not observed in 450 ancestry-matched control individuals. The mutation compromised the ability of the protein to bind to and transactivate the anti-Müllerian hormone (AMH) promoter. The mutation does not interfere with the direct protein–protein interaction, but it disrupts synergistic activation of the AMH promoter by GATA4 and NR5A1. The p.Gly221Arg mutant protein also failed to bind to a known protein partner FOG2 that is essential for gonad formation. Our data demonstrate the key role of GATA4 in human testicular development.

Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy

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.

Mutation Analysis of NR5A1 Encoding Steroidogenic Factor 1 in 77 Patients with 46, XY Disorders of Sex Development (DSD) Including Hypospadias

Background Mutations of the NR5A1 gene encoding steroidogenic factor-1 have been reported in association with a wide spectrum of 46,XY DSD (Disorder of Sex Development) phenotypes including severe forms of hypospadias. Methodology/Principal Findings We evaluated the frequency of NR5A1 gene mutations in a large series of patients presenting with 46,XY DSD and hypospadias. Based on their clinical presentation 77 patients were classified either as complete or partial gonadal dysgenesis (uterus seen at genitography and/or surgery, nu200a=u200a11), ambiguous external genitalia without uterus (nu200a=u200a33) or hypospadias (nu200a=u200a33). We identified heterozygous NR5A1 mutations in 4 cases of ambiguous external genitalia without uterus (12.1%; p.Trp279Arg, pArg39Pro, c.390delG, c140_141insCACG) and a de novo missense mutation in one case with distal hypospadias (3%; p.Arg313Cys). Mutant proteins showed reduced transactivation activity and mutants p.Arg39Pro and p.Arg313Cys did not synergize with the GATA4 cofactor to stimulate reporter gene activity, although they retained their ability to physically interact with the GATA4 protein. Conclusions/Significance Mutations in NR5A1 were observed in 5/77 (6.5%) cases of 46,XY DSD including hypospadias. Excluding the cases of 46,XY gonadal dysgenesis the incidence of NR5A1 mutations was 5/66 (7.6%). An individual with isolated distal hypopadias carried a de novo heterozygous missense mutation, thus extending the range of phenotypes associated with NR5A1 mutations and suggesting that this group of patients should be screened for NR5A1 mutations.

New Technologies for the Identification of Novel Genetic Markers of Disorders of Sex Development (DSD)

Although the genetic basis of human sexual determination and differentiation has advanced considerably in recent years, the fact remains that in most subjects with disorders of sex development (DSD) the underlying genetic cause is unknown. Where pathogenic mutations have been identified, the phenotype can be highly variable, even within families, suggesting that other genetic variants are influencing the expression of the phenotype. This situation is likely to change, as more powerful and affordable tools become widely available for detailed genetic analyses. Here, we describe recent advances in comparative genomic hybridisation, sequencing by hybridisation and next generation sequencing, and we describe how these technologies will have an impact on our understanding of the genetic causes of DSD.

Lack of Association between Genetic Polymorphisms in Enzymes Associated with Folate Metabolism and Unexplained Reduced Sperm Counts

Background The metabolic pathway of folate is thought to influence DNA stability either by inducing single/double stranded breaks or by producing low levels of S-adenosyl-methionine leading to abnormal gene expression and chromosome segregation. Polymorphisms in the genes encoding enzymes in the folate metabolism pathway show distinct geographic and/or ethnic variations and in some cases have been linked to disease. Notably, the gene Methylenetetrahydrofolate reductase (MTHFR) in which the homozygous (TT) state of the polymorphism c.665C>T (p.A222V) is associated with reduced specific activity and increased thermolability of the enzyme causing mild hyperhomocysteinemia. Recently several studies have suggested that men carrying this polymorphism may be at increased risk to develop infertility. Methodology/Principal Findings We have tested this hypothesis in a case/control study of ethnic French individuals. We examined the incidence of polymorphisms in the genes MTHFR (R68Q, A222V and E429A), Methionine synthase reductase MTRR; (I22M and S175L) and Cystathionine beta-synthase (CBS; G307S). The case population consisted of DNA samples from men with unexplained azoospermia (nu200a=u200a70) or oligozoospermia (nu200a=u200a182) and the control population consisted of normospermic and fertile men (nu200a=u200a114). We found no evidence of an association between the incidence of any of these variants and reduced sperm counts. In addition haplotype analysis did not reveal differences between the case and control populations. Conclusions/Significance We could find no evidence for an association between reduced sperm counts and polymorphisms in enzymes involved in folate metabolism in the French population.

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.

An ancient protein-DNA interaction underlying metazoan sex determination

DMRT transcription factors are deeply conserved regulators of metazoan sexual development. They share the DM DNA-binding domain, a unique intertwined double zinc-binding module followed by a C-terminal recognition helix, which binds a pseudopalindromic target DNA. Here we show that DMRT proteins use a unique binding interaction, inserting two adjacent antiparallel recognition helices into a widened DNA major groove to make base-specific contacts. Versatility in how specific base contacts are made allows human DMRT1 to use multiple DNA binding modes (tetramer, trimer and dimer). Chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) indicates that multiple DNA binding modes also are used in vivo. We show that mutations affecting residues crucial for DNA recognition are associated with an intersex phenotype in flies and with male-to-female sex reversal in humans. Our results illuminate an ancient molecular interaction underlying much of metazoan sexual development.

Methylation changes in mature sperm deoxyribonucleic acid from oligozoospermic men: assessment of genetic variants and assisted reproductive technology outcome

OBJECTIVEnTo characterize a potential genetic cause for methylation errors described in oligozoospermia.nnnDESIGNnAnalysis of PEG1/MEST-DMR and H19-DMR methylation level in sperm, in parallel with the study of several genes on the Y chromosome, DNMT3A, and DNMT3L. Clinical outcome was also looked at regarding PEG1/MEST-DMR and H19-DMR methylation level in sperm.nnnSETTINGnResearch and diagnostic laboratories.nnnPATIENT(S)nOne hundred nineteen normospermic and 175 oligozoospermic men consulting for couple infertility.nnnINTERVENTION(S)nWe studied PEG1/MEST-DMR and H19-DMR methylation profiles in 294 men. We searched for Y chromosome gene aberrations and for mutations in both DNMT3A and DNMT3L genes in men showing epimutations. Assisted reproductive technology (ART) outcomes were also investigated.nnnMAIN OUTCOME MEASURE(S)nSperm samples were collected from 294 volunteers for genomic DNA isolation that was used to study methylation profiles in imprinted loci and Y chromosome SMCY, DNMT3A, and DNMT3L genes. Pregnancy rate was also studied after ART treatment using sperm showing epimutations.nnnRESULT(S)nEpimutations in H19-DMR and PEG1/MEST-DMR were found in 20% and 3% of oligozoospermic men, respectively. We identified an amino acid change in DNMT3A in one case and in DNMT3L in eight men with altered methylation profiles. No mutations were detected in SMCY or in selected Y chromsome genes. No correlation between ART outcome and epimutations was found.nnnCONCLUSION(S)nWe observed epimethylations in spermatozoa of oligozoospermic individuals, but no association was found with genetic variants or in the ART outcome.