GENETICS IN ENDOCRINOLOGY: Approaches to molecular genetic diagnosis in the management of differences/disorders of sex development (DSD): position paper of EU COST Action BM 1303 'DSDnet'

The differential diagnosis of differences or disorders of sex development (DSD) belongs to the most complex fields in medicine. It requires a multidisciplinary team conducting a synoptic and complementary approach consisting of thorough clinical, hormonal and genetic workups. This position paper of EU COST (European Cooperation in Science and Technology) Action BM1303 'DSDnet' was written by leading experts in the field and focuses on current best practice in genetic diagnosis in DSD patients. Ascertainment of the karyotpye defines one of the three major diagnostic DSD subclasses and is therefore the mandatory initial step. Subsequently, further analyses comprise molecular studies of monogenic DSD causes or analysis of copy number variations (CNV) or both. Panels of candidate genes provide rapid and reliable results. Whole exome and genome sequencing (WES and WGS) represent valuable methodological developments that are currently in the transition from basic science to clinical routine service in the field of DSD. However, in addition to covering known DSD candidate genes, WES and WGS help to identify novel genetic causes for DSD. Diagnostic interpretation must be performed with utmost caution and needs careful scientific validation in each DSD case.

Anomalies in human sex determination provide unique insights into the complex genetic interactions of early gonad development.

Human sex determination (SD) involves complex mutually antagonistic genetic interactions of testis- and ovary-determining pathways. For many years, both male and female SD were considered to be regulated by a linear cascade of pro-male and pro-female genes, respectively; however, it has become clear that male and female development is achieved through the repression of the alternative state. A gene determining the formation of a testis may function by repressing the female state and vice versa. Uniquely in development, SD is achieved by suppression of the alternate fate and maintained in adulthood by a mutually antagonistic double-repressive pathway. Here, we review genetic data generated through large-scale sequencing approaches that are changing our view of how this system works, including the recently described recurrent NR5A1 p.R92W mutation associated with testis development in 46,XX children. We also review some of the unique challenges in the field to establish that mutations, such as this are pathogenic. The impending surge of new genetic data on human SD from sequencing projects will create opportunities for the development of mechanistic models that will clarify how the system operates and importantly provide data to understand how selection and developmental processes interact to direct the evolution of SD across species.

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 patient's 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.

Sperm global DNA methylation level: association with semen parameters and genome integrity.

Sperm DNA methylation abnormalities have been detected in oligozoospermic men. However, the association between sperm DNA methylation defects, sperm parameters and sperm DNA, and chromatin integrity remains poorly understood. This study was designed to clarify this issue. We recruited a cohort of 92 men (62 normozoospermic and 30 oligoasthenozoospermic) presenting for infertility evaluation during a 1-year period. Sperm global DNA methylation was evaluated by an ELISA-like method, DNA fragmentation was evaluated by flow cytometry-based terminal transferase dUTP nick end-labeling (TUNEL) assay (reported as DNA fragmentation index or DFI), and sperm denaturation was evaluated by aniline blue staining (reported as sperm denaturation index or SDI, a marker of chromatin compaction). We found a significant positive association between sperm global DNA methylation level and conventional sperm parameters (sperm concentration and motility), supported by the results of methylation analysis on H19-DMR. We also identified significant inverse relationships between sperm global DNA methylation, and, both DFI and SDI. However, sperm global DNA methylation level was not related to sperm vitality or morphology. Our findings suggest that global sperm DNA methylation levels are related to conventional sperm parameters, as well as, sperm chromatin and DNA integrity.

Effect of temozolomide on male gametes: an epigenetic risk to the offspring?

INTRODUCTION: 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. METHODS: 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. RESULTS: Sperm counts decrease significantly and hypomethylation of the H19 locus increase with time even staying in the normal range. CONCLUSION: 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.

Understanding the genetic aetiology in patients with XY DSD.

BACKGROUND: Disorders of sex development (DSD) consist of a wide range of disorders and are commoner in those with an XY karyotype. In over half of these cases who have a 46,XY karyotype and who are raised as boys, the underlying aetiology remains unclear. AREAS OF AGREEMENT: Identification of the underlying genetic abnormality may predict long-term outcome. However, genetic abnormalities that are associated with XY DSD manifest themselves with a wide range of phenotype. To understand the aetiology as well as the phenotypic variation, there is a need to harness the advanced genetic technology that is now available. AREAS OF CONTROVERSY: The point at which genetic analysis should be undertaken in the course of investigations is unclear. In addition, there is little agreement on the most effective approach for genetic analysis that will be of clinical benefit to the patient. AREAS TIMELY FOR DEVELOPING RESEARCH: There is a need to understand and improve the clinical utility of genetic analysis in the clinical setting of the patient with a suspected DSD. This will be even more important when parallel gene sequencing identifies variations in multiple genes.

Gene mutations associated with anomalies of human gonad formation.

Here, we discuss recent progress on our understanding of the genetic anomalies that impact directly on the specification and development of the somatic cell compartment of the human gonad. Several new genes and pathways have been identified in the last 5 years associated with human disorders of sex development (DSD). New methods and analytical approaches, including comparative genomic hybridization and next-generation sequencing technologies, are beginning to provide deeper insights into the complexities and alterations of the genetic architecture that are associated with human DSD. The challenges as well as the research opportunities for the future are highlighted as efforts are made to bridge the gap between an increasing quantity of genetic information and the underlying biology.

Biological assessment of abnormal genitalia.

Biological assessment of abnormal genitalia is based on an ordered sequence of endocrine and genetic investigations that are predicated on knowledge obtained from a suitable history and detailed examination of the external genital anatomy. Investigations are particularly relevant in 46,XY DSD where the diagnostic yield is less successful than in the 46,XX counterpart. Advantage should be taken of spontaneous activity of the pituitary-gonadal axis in early infancy rendering measurements of gonadotrophins and sex steroids by sensitive, validated assays key to assessing testicular function. Allied measurement of serum anti-Müllerian hormone completes a comprehensive testis profile of Leydig and Sertoli cell function. Genetic assessment is dominated by analysis of a plethora of genes that attempts to delineate a cause for gonadal dysgenesis. In essence, this is successful in up to 20% of cases from analysis of SRY and SF1 (NR5A1) genes. In contrast, gene mutation analysis is highly successful in 46,XY DSD due to defects in androgen synthesis or action. The era of next generation sequencing is increasingly being applied to investigate complex medical conditions of unknown cause, including DSD. The challenge for health professionals will lie in integrating vast amounts of genetic information with phenotypes and counselling families appropriately. How tissues respond to hormones is apposite to assessing the range of genital phenotypes that characterise DSD, particularly for syndromes associated with androgen resistance. In vitro methods are available to undertake quantitative and qualitative analysis of hormone action. The in vivo equivalent is some assessment of the degree of under-masculinisation in the male, such as an external masculinisation score, and measurement of the ano-genital distance. This anthropometric marker is effectively a postnatal readout of the effects of prenatal androgens acting during the masculinisation programming window. For investigation of the newborn with abnormal genitalia, a pragmatic approach can be taken to guide the clinician using appropriate algorithms.

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.

NR5A1/SF-1 and development and function of the ovary.

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.

Mutations in the protamine locus: association with spermatogenic failure?

The protamine locus consists of a 28.5 kb region with a linear array of the protamine (PRM)1, PRM2, PRM3 and transition nuclear protein (TNP)2 genes. Several studies indicate an abnormal expression pattern of protamine genes associated with male infertility, although the molecular mechanism underlying this observation is unclear. Here, we determined the spectrum of DNA variants present in all four genes in men with unexplained infertility compared with an ancestry-matched fertile/normospermic population. A total of 160 control individuals and at least 125 infertile men with either idiopathic azoospermia or oligozoospermia were sequenced for the open reading frame of PRM1, PRM2, PRM3 and TNP2 genes. All individuals carried an apparently intact Y chromosome. Of the 28 variants identified, 21 were previously described in the literature. The novel variants that were observed only in the infertile cohort included the SNP c.65G>A mutation which resulted in an amino acid change at the codon 22 (p.Ser22Asn) in the PRM1 gene, a mutation in the promoter region of PRM2 (-67C>T) and a nonsense mutation in the PRM3 gene. These data are consistent with that of previous studies which have indicated that mutations in the protamine locus may be an infrequent cause of male infertility.

Sons conceived by assisted reproduction techniques inherit deletions in the azoospermia factor (AZF) region of the Y chromosome and the DAZ gene copy number.

BACKGROUND: Deletions in the azoospermia factor (AZF) region of the Y chromosome are frequent in infertile men. The clinical consequences and the mode of inheritance of these deletions are not yet clear. METHODS: Y chromosome deletion mapping and quantitative PCR analysis of the DAZ-gene copy number, supplemented with haplogroup typing in deleted patients, were performed, in combination with clinical assessments in 264 fathers and their sons conceived by assisted reproduction techniques (ART), and in 168 fertile men with normal sperm concentration. RESULTS: In the ART fathers group, a complete AZFc deletion was detected in 0.4% (1/264). AZFc rearrangements/polymorphisms were found in 6.8% (18/264; 95% CI: 4.4-10.5), which was significantly more frequent (P = 0.021) than in the controls (3/168; 1.8%, 95% CI: 0.6-5.1). All deletions were transmitted to the sons, without any clinical symptoms in early childhood. In the fathers, there was no significant correlation between the DAZ copy number and the severity of spermatogenic failure. CONCLUSIONS: AZFc rearrangements/polymorphisms are transmitted to sons and may represent a risk factor for decreased testis function and male subfertility, which needs confirmation in further studies in larger cohorts. However, deletions of two DAZ gene copies are compatible with normal spermatogenesis and fertility.

Mutations in NALP12 cause hereditary periodic fever syndromes.

NALP proteins, also known as NLRPs, belong to the CATERPILLER protein family involved, like Toll-like receptors, in the recognition of microbial molecules and the subsequent activation of inflammatory and immune responses. Current advances in the function of NALPs support the recently proposed model of a disease continuum bridging autoimmune and autoinflammatory disorders. Among these diseases, hereditary periodic fevers (HPFs) are Mendelian disorders associated with sequence variations in very few genes; these variations are mostly missense mutations whose deleterious effect, which is particularly difficult to assess, is often questionable. The growing number of identified sporadic cases of periodic fever syndrome, together with the lack of discriminatory clinical criteria, has greatly hampered the identification of new disease-causing genes, a step that is, however, essential for appropriate management of these disorders. Using a candidate gene approach, we identified nonambiguous mutations in NALP12 (i.e., nonsense and splice site) in two families with periodic fever syndromes. As shown by means of functional studies, these two NALP12 mutations have a deleterious effect on NF-kappaB signaling. Overall, these data identify a group of HPFs defined by molecular defects in NALP12, opening up new ways to manage these disorders. The identification of these first NALP12 mutations in patients with autoinflammatory disorder also clearly demonstrates the crucial role of NALP12 in inflammatory signaling pathways, thereby assigning a precise function to this particular member of an emerging family of proteins whose putative biological properties are currently inferred essentially through in vitro means.

Association of deletion 9p, 46,XY gonadal dysgenesis and autistic spectrum disorder.

Deletions of distal chromosome 9p24 are often associated with 46,XY gonadal dysgenesis and, depending on the extent of the deletion, the monosomy 9p syndrome. We have previously noted that some cases of 46,XY gonadal dysgenesis carry a 9p deletion and exhibit behavioural problems consistent with autistic spectrum disorder. These cases had a small terminal deletion of 9p with limited or no somatic anomalies that are characteristic of the monosomy 9p syndrome. Here, we present a new case of 46,XY partial gonadal dysgenesis and autistic spectrum disorder associated with a de novo deletion of 9p24 that was detected by ultra-high resolution oligo microarray comparative genomic hybridization. The deletion included the candidate sex-determining genes in the region DMRT1 and DMRT3. These data suggest that a gene responsible for autistic spectrum disorder is located within 9p24. It remains to be determined if the gonadal dysgenesis and autistic spectrum disorder are caused by a single gene or if they are caused by distinct genetic entities at 9p24.

Mutations in the protamine 1 gene associated with male infertility.

In elongating spermatids, human sperm chromatin undergoes a complex compaction in which the transition proteins are extensively replaced by the protamine proteins. Several human studies demonstrate that expression of the protamine proteins is altered in some men with male infertility. For this study, we screened the PRM1 (protamine 1) gene for mutations in a large cohort of 281 men seeking infertility treatment. We identified the c.102G>T transversion that results in an p.Arg34Ser amino acid change in two men. One of these patients presented with oligozoospermia associated with increased sperm DNA fragmentation. The second individual was normospermic but together with his partner sought treatment for idiopathic couple infertility. We also identified a novel missense mutation (c.119G>A, p.Cys40Tyr) in a man with oligoasthenozoospermia. These mutations were not observed in control populations. Interestingly, we also detected variants both 5' and 3' to the PRM1 open-reading frame specifically in infertile individuals. Four individuals with unexplained severe oligozoospermia were heterozygote for a c.-107G>C change that is located at -15 bp from the transcription initiation site of the gene. This mutation may influence PRM1 expression. In addition, a c.*51G>C variant was detected in the 3'UTR of PRM1 specifically in a man with severe oligoasthenozoospermia.

Sperm-FISH analysis in a pericentric chromosome 1 inversion, 46,XY,inv(1)(p22q42), associated with infertility.

No phenotypic effect is observed in most inversion heterozygotes. However, reproductive risks may occur in the form of infertility, spontaneous abortions or chromosomally unbalanced children as a consequence of meiotic recombination between inverted and non-inverted chromosomes. An odd number of crossovers within the inverted segment results in gametes bearing recombinant chromosomes with a duplication of the region outside of the inversion segment of one arm and a deletion of the terminal segment of the other arm [dup(p)/del(q) and del(p)/dup(q)]. Using fluorescence in-situ hybridization (FISH), the chromosome segregation of a pericentric inversion of chromosome 1 was studied in spermatozoa of a inv(1)(p22q42) heterozygous carrier. Three-colour FISH was performed on sperm samples using a probe mixture consisting of chromosome 1p telomere-specific probe, chromosome 1q telomere-specific probe and chromosome 18 centromere-specific alpha satellite DNA probe. The frequency of the non-recombinant product was 80.1%. The frequencies of the two types of recombinants carrying a duplication of the short arm and a deletion of the long arm, and vice versa, were respectively 7.6 and 7.2%, and these frequencies were not statistically significant from the expected ratio of 1:1. Sperm-FISH allows the further understanding of segregation patterns and their effect on reproductive failure and allows an accurate genetic counselling.

[Molecular anomalies of the Y chromosome: Consequences on male fertility]. / Polymorphismes du chromosome Y et fertilité masculine.

Molecular anomalies of the Y chromosome leading to male infertility are mainly microdeletions of the long arm of the Y chromosome. Three recurrently deleted portions of the long arm are the AZFa, AZFb and AZFc (AZF: Azoospermia Factor) regions. Complete deletions of the AZFc region are found in 10% of cases of severe male infertility. In addition to the AZF deletions, certain classes of Y chromosome (haplogroups) may also predispose to male infertility and could be transmitted to future male descents by various Assisted Reproductive Techniques (ART). Since the first discovery of microdeletions, the sequence of the Y chromosome has become available, revealing the mechanisms underlying deletion formation and also resulting in a coherent screening strategy. Recently, partial deletions of the AZF regions have been described. The significance of these deletions in the clinical context remains to be defined.

Haplotypes, mutations and male fertility: the story of the testis-specific ubiquitin protease USP26.

Recently, mutations in the X-linked ubiquitin protease 26 (USP26) gene have been proposed to be associated with male infertility. In particular a 371insACA, 494T>C and 1423C>T haplotype, which results in a T123-124ins, L165S and H475Y amino acid change respectively, has been reported to be associated with Sertoli cell-only syndrome (SCOS) and an absence of sperm in the ejaculate. Here, we demonstrate that two of these changes actually correspond to the ancestral sequence of the gene and that the USP26 haplotype is present in significant frequencies in sub-Saharan African and South and East Asian populations, including in individuals with known fertility. This indicates that the allele is not associated with infertility. The pattern of frequency distribution of the derived haplotype (371delACA, 494T), which is present at high frequencies in most non-African populations could be interpreted as either a result of migration followed by simple genetic drift or alternatively as positive selection acting on the derived alleles. The latter hypothesis seems likely, because there is evidence of strong positive selection acting on the USP26 gene.