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Review articles

Disorders of sex development

new genetic factors and impact of medical genomics

Bashamboo, Anu; McElreavey, Ken

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Middle East Journal of Medical Genetics: January 2012 - Volume 1 - Issue 1 - p 5-11
doi: 10.1097/01.MXE.0000407742.94579.b1
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Disorder of sex development (DSD) is defined as ‘congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical’ (Hughes et al., 2006). DSD therefore covers a very wide spectrum of human phenotypes, and it is divided into a series of subgroups. 46, XY DSD includes 46, XY complete or partial gonadal dysgenesis, or undervirilisation or undermasculinization of an XY male, due to defects in androgen synthesis or action. 46, XX DSD includes gonadal dysgenesis, or more commonly overvirilisation or masculinization of an XX female, due to androgen excess. Ovotesticular DSD refers to an individual with both ovarian and testicular material present in the same or different gonads, and 46, XX testicular DSD refers to an XX male with testes. Other forms of DSD include cloacal exstrophy, severe hypospadias, vaginal atresia, and as part of other conditions such as the Mayer–Rokitansky–Kuster–Hauser syndrome, the Smith–Lemli–Opitz syndrome, or the genito–palato–cardiac syndrome (Porter, 2008; Sultan et al., 2009; Greenberg et al., 1987).

There has been a considerable advance in our understanding of the genetic factors involved in gonadal differentiation in the last 20 years since the SRY gene was first identified as the mammalian testis-determining gene (Fig. 1). However, the identification of new genetic causes of human DSD has not improved substantially in recent years. It has been estimated that a molecular diagnosis is made in only around 20% of DSD, except in cases where the biochemical profile indicates a specific steroidogenic block (Hughes et al., 2006). There is clearly an urgent need to identify new genetic factors involved in DSD. This would not only deepen our knowledge on the etiopathology of these conditions, but it will also lead to accurate and comprehensive molecular diagnosis of hitherto untreatable conditions that are difficult to diagnose.

Figure 1
Figure 1:
A summary of the genetic pathways involved in human and mouse gonad formation and differentiation. The bipotential genital ridge is established by several genes including Nr5a1 (Wilhelm et al., 2007 ; Sekido and Lovell-Badge, 2009). In the XY gonad, the presence of the Y chromosome and Sry leads to the upregulation of Sox9 expression by a synergy with Nr5a1 (Sekido and Lovell-Badge, 2008, 2009). In the XX gonad, the supporting cell precursors accumulate β-catenin in response to Rspo1/Wnt4 signaling and repress Sox9 activity (Schlessinger et al., 2010). Once Sox9 levels reach a critical threshold, several positive regulatory loops are initiated, including autoregulation of Sox9 expression (Sekido and Lovell-Badge, 2009). In the later stages of differentiation and possibly in the adult gonad, Foxl2 may repress Sox9 expression (Uhlenhaut et al., 2009). In the testis, Sox9 promotes the testis pathway, including anti-Müllerian Hormone (AMH) activation, and it also probably represses the expression of the ovarian genes Wnt4 and Foxl2 (Sekido and Lovell-Badge, 2009 ; Uhlenhaut et al., 2009 ; Schlessinger et al., 2010). DMRT1 controls sex determination in some species of fish and may be the master sex-determining switch in birds, but its role in mammalian sexual development is unclear (Wilhelm et al., 2007). Mutations in most of the genes in this pathway have been described in humans.

Disorders of sex development also represent a major burden on public health services. They represent a group of both common (hypospadias) and rare (gonadal dysgenesis) disorders, and in many cases, they require costly and long-term surgical treatment options. For the patients and families with DSD, there are additional concerns. A failure to understand the genetic cause or to offer a correct diagnosis can lead to premature and inappropriate clinical decisions that can lead to irreversible surgical interventions. Sex identity can also be a major concern for patients and families. This review will focus on recent genetic advances in one subset of DSD. These are the pathologies that are due to errors in human sex determination, the choice between the gonad developing as a testis or an ovary, that is, 46, XY complete or partial gonadal dysgenesis and 46, XX testicular or ovotesticular DSD.

Genetic model of human gonad development

Our current knowledge of the genes involved in gonadal development has come mainly from rodent studies and the molecular analysis of human cases of DSD. These have shown that several genes, including Lhx9, NR5A1, Wt1, GATA4, and Fog2 are required for the development of the early genital ridge in the mouse (Fig. 1; Sekido and Lovell-Badge, 2009). Absence of these genes leads to a failure of gonadal development in both female and male embryos. Rodent studies indicate that in the male, SRY acts during a restricted window of time during embryonal development to activate the downstream effector Sox9 (Sekido and Lovell-Badge, 2008). Sertoli cells are one of the first somatic cell lineages to differentiate during testis formation and produce anti-Müllerian Hormone (AMH). SRY expression ensures the differentiation of sufficient Sertoli cells to initiate testicular development by positively regulating Sox9 expression. Once formed, the Sertoli cells coordinate the cellular and morphogenetic events leading to sex determination. This includes the development of Leydig cells, which synthesize and produce the male hormone testosterone, and which differentiate after the appearance of Sertoli cells (Sekido and Lovell-Badge, 2008, 2009). In contrast, ovarian somatic sex determination appears to be relatively labile, with a potential to switch to a testis state. Complete or partial XX male sex-reversal is observed in humans and mice with loss-of-function mutations of the genes WNT4, RSPO1, and Foxl2 (Sekido and Lovell-Badge, 2009). In XX Wnt4−/− mutant embryos, the gonads are partially masculinized, with transient expression of male-specific genes (Sox9) and the presence of Leydig-like cells producing testosterone, and a testis-specific vasculature (Vainio et al., 1999). RSPO1 mutations give an almost identical gonadal phenotype (Tomizuka et al., 2008). In the presence of Wnt, RSPO1 binds to LRP6, a coreceptor of the Wnt receptor frizzled, to stabilize β-catenin. RSPO1 therefore amplifies Wnt signaling, indicating that β-catenin may repress the testis pathway (Fig. 1; Schlessinger et al., 2010). SRY may inhibit β-catenin and promote Sox9 transcription. In mice lacking Foxl2, the expression of several early testis-determining genes including Sox9 are dysregulated, suggesting that it acts as an antitestis gene (Schlessinger et al., 2010). In murine XY ovotestes, the somatic cells express either Sox9 or Foxl2 but never both in the same cell, suggesting that Sox9 suppresses the ovarian pathway in Sertoli cell precursors (Uhlenhaut et al., 2009; Wilhelm et al., 2009). Mammalian sex-determination may therefore involve a battle of the sexes with antagonism between the male-promoting Sox9 pathway and the female-promoting (or male-supressing) Wnt4/Foxl2 pathway(s) in the somatic cell lineage (Fig. 1).

Recent advances in our understanding of the genetic causes of 46, XY gonadal dysgenesis

Complete 46, XY gonadal dysgenesis is characterized by the absence of testicular tissue in gonads, which consists of a streak of fibrous tissue. The phenotype is female, with normal appearance of the external genitalia. These individuals are candidates for carrying mutations in genes involved in the early events of gonadal sex determination. Partial 46, XY gonadal dysgenesis is characterized by the presence of some testicular tissue in the gonads. The external genitalia may be partially masculinized depending on the quantity of testicular material present in the gonads. Although these individuals form distinct clinical groups, we argue that some cases of 46, XY ambiguous external genitalia and infertile male with apparently normal testis may be part of the clinical spectrum of gonadal dysgenesis. The reason for this is two-fold: (a) several pedigrees have been described where the phenotype ranges from 46, XY complete gonadal dysgenesis to simple hypospadias. In some pedigrees, the causative genetic mutation is known and the variation in the severity of the phenotype may be due to other genetic modifiers (Jawaheer et al., 2003; Dumic et al., 2008). (b) Mutations in the same gene, or occasionally the same amino acid change, can be associated with each of the following: 46, XY complete or partial gonadal dysgenesis, 46, XY male with hypospadias, and 46, XY infertile male.

NR5A1, a key player in human gonad development

The gene NR5A1, which encodes steroidogenic factor-1, is a pivotal transcriptional regulator of genes involved in the hypothalamic–pituitary–steroidogenic axis (Morohashi et al., 1992; Luo et al., 1994). During early male development, NR5A1 positively regulates the expression of two key genes involved in male sex determination and differentiation: SOX9 (Sry-box 9) and AMH (De Santa Barbara et al., 1998; Sekido and Lovell-Badge, 2008). NR5A1 also modulates the expression of many factors involved in cholesterol mobilization and steroid hormone biosynthesis, including HMG-CoA synthase, steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, and several cytochrome P450 steroid hydroxylase enzymes (Lin and Achermann, 2008). The NR5A1 protein consists of a DNA-binding domain of two zinc fingers (ZFs), a hinge region, a ligand-binding domain, and two activation function domains (Hoivik et al., 2010). NR5A1 is expressed in the Sertoli and Leydig cells of the developing testis and in Sertoli cells of the prepubertal and adult testis (Morohashi et al., 1994; Hanley et al., 1999). Mice lacking NR5A1 exhibit both gonadal and adrenal agenesis (Luo et al., 1994). Human NR5A1 mutations were first reported in association with 46, XY DSD and adrenal insufficiency, and in a 46, XX girl with adrenal insufficiency (Lin and Achermann, 2008). More recently, the range of phenotypes that are associated with NR5A1 mutations has broadened and includes 46, XY complete and partial gonadal dysgenesis, penoscrotal hypospadias, micropenis with anorchidia, and 46, XX primary ovarian insufficiency (Lin and Achermann, 2008; Köhler et al., 2009; Lourenço et al., 2009).

In a mouse NR5A1 Leydig cell-specific knockout, mice had hypoplastic testes where the lumens of the seminiferous tubules failed to open and spermatogonia never developed into mature sperm (Jeyasuria et al., 2004). These mice also showed reduced expression of two key genes in testosterone biosynthesis: cytochrome P450 steroid hydroxylase 11a and steroidogenic acute regulatory protein (Jeyasuria et al., 2004). We decided to investigate whether NR5A1 mutations could also be associated with some cases of male infertility. Infertility is a major public health burden, with one in seven couples worldwide having problems conceiving (Skakkebaek et al., 2006). In recent years, there has been increasing concern about a possible decline in the reproductive health, and this trend is paralleled by an increasing demand for infertility treatments. In the majority of the cases, the underlying cause of male infertility is unknown although familial clustering of male subfertility and families with multiple infertile or subfertile men, where an autosomal recessive or dominant mutation with sex-limited expression is likely to be present, indicates a substantial genetic contribution (Gianotten et al., 2004). In a mutation screen, we identified mutations in NR5A1 coding sequences in 4% of men with severe spermatogenic failure (azoospermia or severe oligospermia) in otherwise healthy men with normal external genitalia (Bashamboo et al., 2010). In these cases, other known causes of infertility were excluded. This observation is important for several reasons: (a) the data therefore support the hypothesis of Skakkebaek et al. (2006) that a subset of men with spermatogenic failure have a mild form of gonadal dysgenesis (Wohlfahrt-Veje et al., 2009). The gonadal histology in one of our reported case supports this hypothesis (Bashamboo et al., 2010). (b) Our data suggest that this subset of men with severe spermatogenic may also be at risk of endocrine dysfunction and failing testosterone with increasing age. This highlights a need for careful clinical investigation of men presenting with infertility and incongruous testosterone and gonadotropin levels. (c) We demonstrate that approximately 4% of men with otherwise unexplained severe spermatogenic failure carry mutations in the NR5A1 gene, which makes this the most common single gene defect associated with nonobstructive infertility (Bashamboo et al., 2010 and unpublished observations). The data also suggest that some forms of male infertility may be an indicator of mild testicular dysgenesis.

Our data on the role of NR5A1 in a range of gonadal anomalies is particularly informative. We see that the range of phenotypes is large. In the familial cases, we observe normal, apparently healthy men and women carrying mutations that are pathogenic in other individuals (Lourenço et al., 2009). The pathogenic phenotypes range in an XY individual from infertile men with normal appearance of the external genitalia to XY complete gonadal dysgenesis with female external genitalia. In an XX individual, we observed primary amenorrhea and premature ovarian failure. In several patients, the same amino acid change is associated with different phenotypes in different individuals (Lourenço et al., 2009; Bashamboo et al., 2010; unpublished data). This obviously presents a serious challenge in genetic counseling, and it is not yet possible to predict genotype/phenotype correlations. This also highlights the importance of establishing the complete family history in each case of DSD. Other family members with infertility (but apparently normal development of the gonad and external genitalia) may be a sign of a familial case of DSD that warrants further investigation.


GATA4 belongs to the evolutionarily conserved Gata family of sex tissue and organ-specific vertebrate transcriptional regulators, consisting of two ZFs (Patient and McGhee, 2002). The C-terminal ZF is required for recognition and DNA binding, whereas the N-terminal ZF contributes to the stability of this binding.

ZFs are also crucial for protein–protein interactions with other transcription cofactors. In humans, GATA4 is strongly expressed in the somatic cell population of the developing gonad before and during the time of sex determination (Viger et al., 1998). GATA4 cooperatively interacts with several proteins to regulate the expression of the sex-determining genes Sry, Sox9 and Amh, and key steroidogenic factors (Miyamoto et al., 2008; Nishida et al., 2008; Viger et al., 2008). Mice lacking GATA4 die in utero because of profound abnormalities in ventral morphogenesis and heart tube formation (Kuo et al., 1997; Molkentin et al., 1997). In humans, mutations in GATA4 are associated with congenital heart defects (CHD), including atrial septal defects, ventricular septal defects, pulmonary valve thickening, or insufficiency of the cardiac valves (Garg et al., 2003; Hirayama-Yamada et al., 2005; Nemer et al., 2006; Tomita-Mitchell et al., 2007). In all of the cases of CHD associated with mutations in GATA4, other organs were reported as normal.

The critical role of GATA4 in gonadal development is highlighted by Gata4ki mice that have a p.Val217Gly mutation in the N-terminal ZF domain (Bouma et al., 2007). This knock-in mutation abrogates the interaction of GATA4 with the cofactor Fog2, and these animals display severe anomalies in testis development (Crispino et al., 2001; Tevosian et al., 2002). Fog2 may act as a transcriptional repressor or activator, depending on the cellular and promoter context. Mice lacking Fog2 exhibit a block in gonadogenesis, and a translocation involving Fog2 in humans is reported to be associated with male hypergonadotropic hypogonadism (Crispino et al., 2001; Tevosian et al., 2002; Finelli et al., 2007). In vitro, Fog2 represses Gata4-dependent transcription of AMH in primary Sertoli cell cultures (Tremblay et al., 2001). Although the mechanism of Fog2 and GATA4 interaction in the gonad is not well defined, it is essential that a direct physical interaction between GATA4 and Fog2 be maintained, as abrogation of the same results in abnormal testis development in mice (Crispino et al., 2001; Tevosian et al., 2002). We identified a missense mutation in the GATA4 gene (p.Gly221Arg) associated with a familial case of 46, XY DSD and CHD (Fig. 2; Lourenço et al., 2011). The mutation showed altered biological activity. It compromised the ability of the protein to bind to and transactivate the AMH promoter. The mutation did not interfere with the direct protein–protein interaction, but it disrupted synergistic activation of the AMH promoter by GATA4 and NR5A1. The p.Gly221Arg mutant protein also failed to bind to Fog2. Our data demonstrate the key role of GATA4 in human testicular development. This familial case shows that GATA4 mutations may be associated with both syndromic and nonsyndromic forms of 46, XY DSD and that the same GATA4 amino acid change may be associated with more or less severe gonadal phenotypes, suggesting a role of genetic modifiers. However, the overall incidence of GATA4 mutations in 46, XY DSD remains to be determined.

Figure 2
Figure 2:
(a) Pedigree of a familial case with 46, XY disorders of sex development and congenital heart disease. The family is of French ethnic origin. Squares represent male family members and circles represent female family members. Solid squares represent affected 46, XY disorders of sex development individuals who were raised as boys. Striped circles and partially striped squares indicate individuals with cardiac anomalies. Symbols containing a black dot represent apparently unaffected carriers of the mutation. The index patient is indicated with an arrow. The asterisk indicates individuals who were screened for mutations in the GATA4 gene. Individuals I.2, II.2, and II.3 had implantation of a cardiac pacemaker. The index case (IV.2) was referred at birth for ambiguous external genitalia. At 1 year, he was operated on for cryptorchidism. At 20 years, he is a sexually active male with azoospermia. There was no clinical evidence of heart anomalies by echocardiogram assessment. His brother (IV.1) had a decreased phallus length and inguinal hernias at birth. He was operated on at 2.8 years for bilateral inguinal hernias, and a minor systolic murmur was noted, suggesting an atrial septal defect. Case IV.3 was referred at 26 days for ambiguous external genitalia. A minor systolic murmur was noted that did not require medical intervention. At 7 months, the gonads were removed and histological examination revealed bilateral dysgenetic testes. Case IV.4 was diagnosed at birth with tetralogy of Fallot and received corrective surgery. She is now 10 years and in good health. Case III.11 was noted at birth to have congenital cyanotic heart disease. This was well tolerated and medical intervention was not required. She has two healthy daughters and one boy with no history of medical intervention. (b) A schematic representation of the functional domains of the GATA4 protein is shown with an arrow indicating the position of the p.Gly221Arg mutation. The N-terminus transcription activation domains (TAD) and the DNA-binding domain containing two zinc-finger (ZN) motifs are indicated. The nuclear localization signal (NLS) lies distal to the second ZN.


In mammalian cells, a variety of extracellular stimuli generate intracellular signals that activate the mitogen-activated protein kinase cascade (Robinson and Cobb, 1997). Recently, XY sex reversal was described in association with a murine Map3k4 mutation, suggesting a conserved MAP kinase pathway for testis formation (Bogani et al., 2009). Several proteins involved in human sex development, including SRY, are known to be phosphorylated by an mitogen-activated protein kinase-dependent pathway, and in the case of NR5A1 and GATA4, phosphorylation increases their transcriptional activation potential (Desclozeaux et al., 1998; Charron et al., 2001; Desclozeaux et al., 2002). We previously reported a large familial case of 46, XY DSD (Jawaheer et al., 2003). The phenotype in this family shows an autosomal-dominant mode of inheritance, and linkage analysis indicated that the mutation responsible for the phenotype is located in the proximal chromosome 5q (Jawaheer et al., 2003). Further analyses revealed that this family carried a splice donor site mutation in MAP3K1, and that other, unrelated cases of 46, XY DSD, one familial and two sporadic, also carried mutations in the gene (Pearlman et al., 2010). Remarkably, for a gene product with multiple functions, the affected XY individuals had no other obvious phenotype and female carriers were normal. The incidence of MAP3K1 mutations in DSD was estimated to be around 18%.

Genetic causes of 46, XX testicular or ovotesticular disorder of sex development

Approximately 80% of XX testicular DSD (formerly termed XX male) and 10% of ovotesticular DSD (formerly XX true hermaphrodite) carry the SRY gene in their genome, usually present on the X chromosome (McElreavey et al., 1992; McElreavey and Cortes, 2001). The presence of SRY is sufficient in these cases to result in the testicular tissue being present in the gonads (McElreavey and Cortes, 2001). Considerable research attention is currently focused on understanding the genetic causes of SRY-negative cases. Loss-of-function mutations have been described in RSPO1 and WNT4 associated with syndromic forms of these phenotypes (RSPO1, XX male, palmoplantar hyperkeratosis, and predisposition to squamous cell carcinoma of the skin; Parma et al., 2006; WNT4, SERKAL syndrome, Mandel et al., 2008). In the nonsyndromic forms, the genetic cause is unclear. Rearrangements involving either a duplication of Sox9 or deletions in the regulatory elements of the related SOX9 gene have been described (Cox et al., 2011; Sutton et al., 2011), but to date, mutations involving coding sequences of a gene have not been reported. A number of familial cases of XX testicular or ovotesticular DSD have been reported from the Middle East, and these represent an important resource for mutation identification in the future (Jarrah et al., 2000).

Impact of medical genomics

In our experience, we find pathogenic mutations in approximately 20% of cases of 46, XY DSD by screening a relatively limited number of candidate genes. In the case of Sry negative XX DSD, the incidence of disease-causing mutations is much less. The identification of genes involved in these pathologies has been difficult to achieve by classical approaches in human genetics. As a key developmental process, it is surprising that the genetic factors involved in sex determination are not well conserved in evolution. The study of sex development in model organisms such as Drosophila or Caenorhabditis elegans has, for the most part, not provided major insights into mammalian sex-determination. Sex-determination is not well conserved even in vertebrates. Sequencing of candidate genes has also not been as fruitful as one would have anticipated. The other classical approach is to use familial cases to map the mutation. Familial cases of gonadal dysgenesis or XX testicular DSD are not widely reported in the literature, although in our experience, many sporadic cases may in fact be unrecognized familial forms of DSD. These difficulties can be overcome using next-generation sequencing (NGS) approaches. The advent of NGS technologies has tremendously reduced the sequencing cost and the experimental complexity involved in large-scale sequencing experiments. NGS technologies now offer excellent template coverage, rendering sequencing-based genome analysis more readily available and useful to individual, small laboratories. These sequencing methods use massively parallel approaches to sequence several hundred thousand to millions of reads simultaneously (Ansorge, 2009; Metzker, 2010). For a review of the technologies and strategies used, see Bashamboo et al. (2010).

NGS technologies offer substantial cost reductions when compared with the Sanger method, but sequencing the whole human genome remains expensive for most laboratories, and the data analyses involved can be challenging. An alternative approach involves targeting specific regions of the genome that are enriched for NGS sequencing: for example, sequencing all of the exons in the genome; sequencing specific gene families of interest; or focusing just on large chromosome regions or entire chromosomes that are implicated in disease. Recently, whole-exome sequencing has become a highly efficient and cost-effective strategy to selectively sequence the coding regions of the human genome to identify novel genes associated with rare and common Mendelian disorders. The goal of this approach is to identify the functional variant that is responsible for the disease without the high costs associated with either whole-genome sequencing or time-consuming sequencing of many potential candidate genes. It is estimated that the protein-coding regions of the human genome constitute about 85% of the disease-causing mutations. With this in mind, exome sequencing has becoming the method of choice in medical genetics for identifying pathogenic mutations in medical genetics with no prior knowledge of the genes involved (Bilgüvar et al., 2010; Johnson et al., 2010; Krawitz et al., 2010; Otto et al., 2010; Pierce et al., 2010; Caliskan et al., 2011; Kalay et al., 2011). Exome capture approaches are available for targeting approximately 50 Mb or ∼180 000 coding exons across the human genome. Many platforms also offer the capture of miRNAs and noncoding RNA sequences. Selective genomic enrichment of the human exome offers an attractive option for devising new experimental designs aimed at quick identification of potential disease-associated genetic variants. Human exome capture methods are currently based on either custom-designed oligonucleotide microarrays or solution-based hybridization strategies. In addition to generating vast amounts of sequence information, the recent NGS applications not only have the ability to generate huge amounts of sequence data but they also reveal structural variation, thereby bypassing the need for CGH analyses (McKernan et al., 2009).

NGS approaches will have a major impact on the understanding and diagnosis of DSD. However, even in 46, XY underandrogenized individuals with a testis, who are suspected to have an androgen receptor defect, a pathogenic mutation is found only in less than half of the cases (Audi et al., 2010). In cases of simple hypospadias or cryptorchidism, a genetic cause is rarely detected despite epidemiological evidence, suggesting a major genetic contribution to these phenotypes (Fukami et al., 2006; Köhler et al., 2009).

Large-scale genomic sequencing approaches also offer the opportunity to identify genetic factors that influence the severity or penetrance of the phenotype. Familial cases of both 46, XY and 46, XX DSD often show considerable variation in the expression of the phenotype, including families where the underlying genetic cause has been identified (Temel et al., 2007; Lourenço et al., 2009). In a pedigree with hypogonadotropic hypogonadism, a compound heterozygous GNRHR and a heterozygous FGFR1 mutation were identified (Pitteloud et al., 2007).


Conflicts of interest

There are no conflicts of interest.


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ambiguous genitalia; disorders of sex development; exome sequencing; gonadal dysgenesis; next generation sequencing

© 2012 Middle East Journal of Medical Genetics