In this review we will consider the gene mutations responsible for the non-syndromic forms of disorders of sex development (DSD) and how recent genetic findings are providing insights into the mechanism of sex determination. High-throughput sequencing technologies are having a major impact on our understanding of the genetic basis of rare human disorders, including DSD. The study of human DSD is progressively revealing subtle differences in the genetics of the sex-determining system between the mouse and the human. This plasticity of the sex-determining pathway is apparent in (a) the difference in phenotypes in human and mouse associated with the same gene, (b) the different gene regulatory mechanisms between human and mouse, and finally (c) the different and unexpected reproductive phenotypes seen in association with mutations in well-studied sex-determining genes.

Disorders of sex development (DSD) are defined as congenital conditions with discordant development of chromosomal and gonadal/anatomical sex. Just over 10 years ago, at the Chicago Consensus conference, the term DSD was coined to include previous descriptions such as intersex, pseudohermaphroditism, hermaphroditism, and sex reversal [Lee et al., 2006]. These terms were often confusing, both to clinicians and patients as well as to other family members. This umbrella definition of DSD provides a rational basis for the classification of a range of conditions, but more importantly, it avoids confusion with terms such as transgender, gender dysphoria, or homosexuality.

46,XY DSD includes aberrant testis determination or undermasculinization of an XY male due to errors in either androgen synthesis or action. Errors in testis determination may manifest as either complete gonadal dysgenesis (CGD) or partial gonadal dysgenesis (PGD). 46,XY CGD is characterized by completely female external genitalia, well-developed Müllerian structures, and a gonad composed of a streak of fibrous tissue, whereas 46,XY PGD is characterized by partial testis formation, usually a mixture of Wolffian and Müllerian ducts, and varying degrees of masculinization of the external genitalia. Embryonic testicular regression sequence can also be regarded as part of the clinical spectrum of 46,XY gonadal dysgenesis [Marcantonio et al., 1994]. Affected individuals have a 46,XY karyotype and usually present with ambiguous external or internal genitalia. Gonad tissue is absent on one or both sides. Patients with this condition are considered to have incomplete testicular determination with the loss of gonad material early in gestation before testis differentiation is complete [Marcantonio et al., 1994]. This also raises the concept that we are dealing with a continuum of phenotypes rather than clearly distinct and unrelated categories of atypical testicular formation. The genetic arguments also favor this, since in some families with 46,XY DSD, the affected individuals can present as girls with either CGD or PCG or as men with isolated hypospadias and/or cryptorchidism [Le Caignec et al., 2003]. Other forms of 46,XY DSD are disorders in androgen synthesis or action. This includes androgen biosynthesis defects such as 17-hydroxysteroid dehydrogenase deficiency, 5α reductase deficiency, and StAR mutations. Defects in androgen action include complete or partial androgen insensitivity associated with mutations in the androgen receptor. Recessive LH receptor mutations result in Leydig cell hypoplasia or aplasia. Central hypogonadotropic hypogonadism (CHH) occurs when the physiologic function of the hypothalamic-pituitary-gonadal axis is compromised. In 46,XY individuals CHH is characterized by delayed or absent sexual development and infertility associated with inappropriately low gonadotropin (LH and FSH) and testosterone levels. Male patients frequently show under-androgenisation with micropenis and cryptorchidism observed at birth. When anomalies of smell, hyposmia or anosmia, is associated with hypogonadotropic hypogonadism, in 60% of patients the disease is called Kallmann syndrome. This combination of phenotypes is explained by the common embryonic origins and developmental pathways of GnRH and olfactory neurons. More than 20 genes are known to cause CHH [Marino et al., 2014].

46,XX DSD includes overvirilization or masculinization of an XX individual due to androgen excess, and the vast majority of cases of 46,XX DSD are due to congenital adrenal hyperplasia (CAH). The most common form of CAH is due to deficiency of 21-hydroxylase, which is caused by mutations in the 21-hydroxylase gene (CYP21A2) and accounts for 90-95% of all cases [White et al., 1984; Arlt and Krone, 2007]. The much rarer forms are 46,XX testicular DSD (TDSD) and 46,XX ovotesticular DSD (OTDSD). Individuals with TDSD are males with small and azoospermic testis [de la Chapelle, 1972] and a normal male habitus. 46,XX OTDSD refers to individuals that have both ovarian and testicular tissue in the gonads, usually ovotestes but less commonly a testis (or ovotestis) on one side and an ovary on the other [Ergun-Longmire et al., 2005]. The external genitalia are usually ambiguous or feminine, with the degree of masculinization broadly correlating with the amount of testicular tissue present. In both TDSD and OTDSD the histological examination of the gonads shows distinct tubule structures in the testicular-like tissue and the presence of follicles in the ovarian-like tissue, in the case of OTDSD.

Sex chromosome DSD includes 47,XXY (Klinefelter syndrome and variants), 45,X (Turner syndrome and variants), 45,X/46,XY (mixed gonadal dysgenesis or OTDSD), and 46,XX/46,XY (chimerism or OTDSD). 45,X/46,XY mosaicism is one of the most common causes of DSD and also one of the most difficult for predicting genotype-phenotype correlations. In several studies, no correlation was found between the proportion of the 45,X/46,XY cell lines in the blood or the fibroblasts of the patient and the phenotype. Furthermore, some of these cases show mild intellectual disability or signs of autism.

A large number of syndrome associations of DSD have been described including cloacal anomalies, Robinow, Aarskog, Hand-Foot-Genital, popliteal pterygium and Serkel syndrome, and Müllerian duct aplasia. This review will focus on recent developments in our understanding of the non-syndromic forms of DSD associated with errors in sex determination.

There is very limited data available on the precise incidence of DSD. This reflects both the rarity of some of these conditions as well as the challenge of achieving a definitive clinical diagnosis. In the newborn, truly ambiguous genitalia that may pose a problem for binary gender assignment has an estimated incidence of 1:4,500-5,500 births [Thyen et al., 2006; Sax, 2002]. Overall, around 50% of all cases of DSD with truly ambiguous genitalia are due to either CAH or 46,XY mixed gonadal dysgenesis caused by a 45,X/46,XY mosaicism [Thyen et al., 2006]. The incidence of 46,XY DSD is estimated to be 1:20,000 births and of 46,XY gonadal dysgenesis around 1:100,000 births [Lee et al., 2016]. TDSD/OTDSD are estimated to occur in 1:100,000 births [Sax, 2002]. More commonly, developmental anomalies of the external genitalia may exist in 1 in 300 newborn infants [Nordenvall et al., 2014]. These include undescended testis or anomalies of the opening of the urethra on the penis (hypospadias). However, most of this published data on the incidence of DSD is available only from Western countries; therefore, the worldwide prevalence of DSD is unclear. A German study indicated that the incidence of ambiguous genitalia in infants of non-German background was 4× higher compared to the general population [Thyen et al., 2006], which they attributed to an increase in autosomal recessive forms of DSD due to higher rates of consanguinity in the migrant populations. There is some evidence to support the hypothesis that there is a higher rate of DSD in societies with a higher rate of consanguinity. The incidence of ambiguous genitalia in Saudi Arabia has been estimated at 1:2,500 live births, whilst in Egypt it has been estimated at 1:3,000 live births [Abdullah et al., 1991; Mazen et al., 2008], which is higher than the reported frequency of 1:4,500-1:5,500 in European countries. Another hindrance in defining the prevalence of DSD is the lack of an accurate or even any diagnosis in many cases. In the German study almost half of the children did not have a definitive diagnosis by the age of 6 months [Thyen et al., 2006]. Excluding cases where the biochemical profile indicates a specific error in steroidogenesis, it has been estimated that a specific molecular diagnosis is obtained in about 20% of cases of DSD and that only 50% of 46,XY children with DSD will receive a definitive clinical diagnosis [Lee et al., 2006]. The detailed genetic analyses of individuals with DSD have been a powerful tool in the identification of genes involved in sex determination and therefore DSD.

SRY and SOX9

Approximately 15% of 46,XY CGD patients carry mutations in the testis-determining gene SRY, with the majority of these mutations localized within the functional DNA-binding HMG-domain [McElreavey and Fellous, 1999] of the protein. Both CGD and PGD are also associated with small interstitial deletions 5′ and 3′ to the SRY gene, as well as within the SRY promoter itself [McElreavey et al., 1992, 1996; Assumpção et al., 2005]. In contrast to the mouse, the human SRY protein is expressed in Sertoli cells and germ cells from the moment of testis determination until adulthood. The role, if any, of SRY in germ cells is unknown since SRY mutations are associated with a failure of testis development. SRY mutations are usually de novo, but some are inherited from a fertile father. Functional studies suggest that these inherited mutant proteins retain partial biological activity, and the incomplete penetrance could be caused by stochastic effects around a threshold level of biological activity required for testis formation [Phillips et al., 2011]. Although SRY mutations are usually associated with gonadal dysgenesis, a 46,XY woman with premature menopause was reported to carry a de novo p.Gln2Ter mutation [Brown et al., 1998].

The direct target of SRY is SOX9, another HMG-box containing protein. Sox9 plays both an essential role in the specification and differentiation of mesenchymal cells toward the chondrogenic lineage through transcriptional modulation of Col2a1, the major matrix protein of the mature cartilage as well as establishing Sertoli cell identity in the developing testis immediately following the expression of SRY. Many mutations have been reported in the SOX9 gene and upstream and downstream flanking regions associated with campomelic dysplasia (CD). There is a variable severity of testicular dysgenesis in about 75% of affected XY individuals [Foster et al., 1994]. However, recently 2 patients with male external genitalia, unpalpable testis, and either hypospadias or micropenis have been reported who carried p.Arg394Gly and p.Arg437Cys heterozygous missense mutations [Katoh-Fukui et al., 2015]. Neither of the boys had signs of CD, although one boy carrying the p.Arg394Gly mutation was reported as having spina bifida. Both of these mutations are located in the C-terminal PQS-rich domain of SOX9 that is involved in protein-protein interactions with factors such as CREB-binding protein and p300, both of which positively regulate gene expression. The boy carrying the p.Arg437Cys mutation presented with testicular regression sequence, reinforcing the hypothesis that both gonadal dysgenesis and testicular regression sequence are part of the same phenotypic continuum.

Besides the point mutations, rearrangements involving the SOX9 locus are also known to result in non-syndromic 46,XY or 46,XX DSD. The developmental timing and tissue-specific transcriptional regulation of SOX9 is highly complex and involves multiple elements located in flanking regions of at least 1 Mb upstream and 1.6 Mb downstream. Upstream of SOX9, translocations and inversion breakpoints associated with CD fall within 2 clusters located about 400 kb apart [Leipoldt et al., 2007]. Large (>1 Mb) duplications 5′ to SOX9 are associated with brachydactyly-anonychia (symmetric brachydactyly of the hands/feet, hyponychia or anonychia) [Kurth et al., 2009]. Pierre Robin sequence is a craniofacial disorder characterized by micrognathia, cleft palate, and macroglossia that is associated with normal testis development and is caused by either a 75-kb deletion located 1.38 Mb upstream or a deletion located 1.56 Mb downstream of SOX9 [Benko et al., 2009]. In mice, a testis-specific enhancer element has been mapped to a 1.4-kb region termed Tesco that is located 13 kb upstream of Sox9 [Sekido and Lovell-Badge, 2008]. Both Sry and Nr5a1 bind to the Tesco enhancer sequence in vivo, possibly through a direct physical interaction to upregulate Sox9 gene expression. Once Sox9 protein levels reach a critical threshold, several positive regulatory loops are initiated for its maintenance, including autoregulation of its own expression and formation of feed-forward loops via Ffg9 or Pgd2 signaling. Other cofactors are likely to be involved in this process but have not yet been fully characterized. Mutations involving the human TESCO element have not been reported as a cause of DSD, however, rearrangements involving another regulatory element, termed RevSex, located 600 kb upstream of SOX9, are associated with both XY and XX DSD. Five cases of 46,XX testicular or ovotesticular DSD that carried duplications of this region and a familial case of 46,XY DSD that carried a deletion of the element have been reported [Benko et al., 2011; Cox et al., 2011; Vetro et al., 2011; Hyon et al., 2015]. The minimal region associated with 46,XX-SRY negative DSD has been narrowed down to a 40.7-41.9-kb element, which contains 2 predicted enhancer motifs [Hyon et al., 2015]. There is also data suggesting that deletions of an immediately adjacent and non-overlapping region are associated with 46,XY gonadal dysgenesis [Kim et al., 2015]. In our experience, about 10% of cases of 46,XX TDSD/OTDSD and 46,XY gonadal dysgenesis have rearrangements involving the RevSex locus [unpubl. data].


Approximately 15% of all cases of 46,XY DSD are caused by mutations involving the gene NR5A1. NR5A1 belongs to the subfamily of transcription factors known as nuclear receptor subfamily 5 (group A, member 1), which is highly conserved in vertebrates [Morohashi et al., 1992]. The protein consists of a DNA-binding motif composed of 2 zinc-chelating modules that coordinate the interaction between the receptor and hormone response element [El-Khairi and Achermann, 2012]. NR5A1 binds DNA as a monomer, with DNA binding stabilized via a 30 amino acid extension termed the A-box. The C-terminal ligand-binding domain (LBD) is required for maximal biological activity with co-activators such as NCOA1. Posttranslational modification plays an important role in modulating NR5A1 activation and repressor functions. Phosphorylation of Ser203 within the LBD enhances the positive interaction of regulatory cofactors, whereas strong transcriptional repression requires sumoylation of the lysines Lys119 and Lys194. Sumoylation plays an important role in NR5A1 function. If it is eliminated from the mouse Nr5a1 protein, the mutant mice exhibit marked endocrine abnormalities and changes in cell fate that reflect an inappropriate activation of hedgehog signaling [Lee et al., 2011].

In humans, mutations involving NR5A1 are associated with a wide range of reproductive anomalies including 46,XY gonadal dysgenesis with or without adrenal insufficiency, ambiguous genitalia, hypospadias, micropenis, spermatogenic failure with normal genitalia, and primary ovarian insufficiency [reviewed by El-Khairi and Achermann, 2012]. Familial cases have been described where both 46,XY gonadal dysgenesis and 46,XX ovarian insufficiency are present in the same family [Fabbri et al., 2014]. In a study of 315 men with spermatogenic failure, we identified heterozygous missense mutations in NR5A1 in 7 men with either azoospermia or severe oligozoospermia [Bashamboo et al., 2010]. Testis histology in one man with azoospermia was suggestive of a mild form of testicular dysgenesis rather than Sertoli-cell-only syndrome, again reinforcing the idea that errors in testis determination can manifest as different human reproductive phenotypes. In all cases, the men carrying the NR5A1 mutations had normal development of the external genitalia [Bashamboo et al., 2010]. The observation that mutations involving a key gene in human sex determination, NR5A1, are associated with either male or female infertility establishes a link between human sex determination and fertility.

The mechanism behind the phenotype variability associated with NR5A1 mutations is yet to be explained, including variability associated with the same NR5A1 mutation. For example, male infertility, female infertility, or 46,XY DSD are associated with the variants p.Gly123Ala and p.Pro129Leu. It is possible that some patients with NR5A1 mutations carry novel or rare variants in other genes involved in sexual development that may influence the severity of the phenotype. We reported 2 individuals with the same missense mutation p.Arg313Cys in NR5A1 [Allali et al., 2011; Mazen et al., 2016]. In the first case it was associated with mild hypospadias [Allali et al., 2011], but in the second case it was associated with 46,XY gonadal dysgenesis [Mazen et al., 2016], and in both cases the mutation was de novo. The more severe phenotype may be explained by digenic inheritance since the patient carried a missense mutation in the MAP3K1 gene (see below). In other cases of DSD carrying mutations in NR5A1 the severity of the phenotype may be a consequence of the specific amino acid involved (see NR5A1 p.R92W below). A homozygous p.R103Q NR5A1 mutation was reported in a child with severe 46,XY DSD and absent spleen. The mutation decreased the ability of the mutated NR5A1 protein to transactivate TLX1, a transcription factor that is essential for spleen development [Zangen et al., 2014]. The R103Q mutation impaired activation of steroidogenic genes without affecting the synergistic NR5A1/SRY co-activation of SOX9.

GATA4 and the Cofactor FOG2

GATA4 belong to a class of evolutionarily conserved lineage-limited zinc finger transcription factors characterized by the presence of 2 conserved type IV zinc finger domains that participate in cell fate determination, proliferation, and maturation [Zaytouni et al., 2011]. Both GATA4 and GATA6 are expressed in the somatic tissues of the embryonic testis [Ketola et al., 1999]. GATA4 cooperatively interacts with NR5A1 to regulate the expression of genes critical for testis determination and differentiation [Viger et al., 2008]. Male mice lacking Gata4 show partially descended small testis with irregular cords, are infertile, and lack expression of Dmrt1 during embryogenesis [Manuylov et al., 2011]. Gata4ki mice, which carry a p.Val217Gly mutation in the N-terminal zinc finger domain of the protein that abrogates the physical interaction of Gata4 with the cofactor Fog2, present with severe testicular dysgenesis [Molkentin et al., 1997; Crispino et al., 2001; Bouma et al., 2007]. Gata4 also plays a pivotal role in Leydig cell function, for example a Gata4/Mef2 complex regulates Star gene expression in mouse Leydig cells [Bergeron et al., 2015; Daems et al., 2015; Schrade et al., 2015]. Although mutations in human GATA4 are associated with congenital heart anomalies, a proportion of XY males carrying deletions of 8p23.1 that includes the GATA4 gene have hypospadias and bilateral cryptorchidism [Wat et al., 2009]. We identified a familial case of 46,XY DSD and congenital heart disease that affected both 46,XX and 46,XY individuals [Lourenço et al., 2011]. This family carried a heterozygous missense mutation (p.Gly221Arg) located immediately adjacent to the mouse p.Val217Gly Gata4ki mutation in the N-terminal zinc finger domain [Lourenço et al., 2011]. In functional studies the p.Gly221Arg variant failed to bind to DNA, did not to transactivate the AMH promoter in a transient gene activation assay, and lacked the ability to bind to its protein partner FOG2. Although the incidence of GATA4 mutations in association with DSD has yet to be established, we have identified other heterozygous missense mutations in GATA4 in 46,XY DSD patients who have no evidence of heart disease [unpubl. data].

FOG2 (also known as ZFPM2) is a zinc finger cofactor that modulates the activity of GATA4 by binding to the N-terminal zinc finger [Zaytouni et al., 2011]. XY Fog2-/- mice fail to develop testis, and since the expression of key genes involved in testis determination such as Sry and Sox9 is dramatically reduced, it suggests that Fog2 is involved in the early stages of testis determination. The evidence that FOG2 may be involved specifically in human testis determination was suggested by 2 cases of 46,XY gonadal dysgenesis in association with apparently balanced translocations that included the FOG2 locus on chromosome 8 (t(8;10)(q23.1;q21.1) and t(8;18)(q22;q21)) [Finelli et al., 2007; Tan et al., 2012]. In both cases other complex somatic anomalies were reported. Using exome sequencing, we identified 2 independent cases, of 46,XY gonadal dysgenesis each with missense mutations in the FOG2 gene [Bashamboo et al., 2014]. There was no history of cardiac anomalies in either the patients or their families. 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 GATA4. These studies established GATA4 and FOG2 mutations as causes of 46,XY DSD.


CBX2 encodes for chromobox homolog 2, a component of the polycomb group (PcG) complex of regulatory proteins. In mammals, PcG proteins are associated with 2 main families of complexes, referred to as polycomb repressive complex 1 (PRC1) and PRC2. These complexes catalyze mono-ubiquitination of histone H2A on lysine 119 and tri-methylation of histone H3 on lysine 27, respectively. Human CBX2 exists in 2 isoforms, a 532 amino acid isoform termed CBX2.1 and a second shorter 211 amino acid isoform termed CBX2.2. Mice lacking Cbx2 display posterior transformation of the vertebral columns and sternal ribs, failure of T cell expansion, and XY mice show male-to-female sex reversal whereas XX animals have either absent or smaller ovaries [Katoh-Fukui et al., 1998].

A single patient with 46,XY gonadal dysgenesis and mutations in the human CBX2 gene has been reported. This was a 46,XY girl who carried 2 independent mutations in CBX2 - a paternally inherited p.Pro98Leu mutation and a maternally inherited p.Arg443Pro mutation [Biason-Lauber et al., 2009]. Histology of the gonads at 4.5 years revealed apparently normal ovaries. Although polycomb group proteins are traditionally regarded as transcriptional repressors, there is evidence that at least in some cellular or promoter contexts CBX2 acts as a transcriptional activator of NR5A1 and SRY expression [Biason-Lauber et al., 2009]. In the patient described above, the presence of apparently normal ovaries suggests that CBX2 actively represses fetal ovarian development in an XY individual. Recently, several potential downstream targets of CBX2 that are relevant to testis determination have been reported using a DamID-NGS approach [Eid et al., 2015]. The gene targets of CBX2 include many factors that are known or suspected to be involved in sex development including SOX9, MAMLD1, SOX3, FGFR2, ATRX, TEX10, EXO1, TBX2, TSPYL4, WTAP, and MTM1 [Eid et al., 2015]. The exact role of CBX2 (and by implication the PcG complex) in sex determination is unknown, although both the human and mouse phenotypes suggest that it is acting at an early stage of gonad formation.


The mitogen-activated protein kinases (MAPKs) are activated through an evolutionarily conserved three-component signal transduction cascade composed of a mitogen-activated protein kinase kinase kinase 1 (MAP3K1), a MAP2K and a MAPK. Historically, these were considered to be cellular housekeeping factors, and it was a surprise to find that mutations in at least some of these factors could generate gonad-specific phenotypes.

In mice, XY embryos lacking functional Map3k4 on a predominantly C57BL/6J background exhibit embryonic gonadal XY sex reversal associated with a failure to transcriptionally upregulate Sry [Bogani et al., 2009]. Mice lacking Gadd45g, which encodes a protein that interacts with Map3k4, also show a lack of testis determination that is associated with a delay in Sry expression [Gierl et al., 2012]. Furthermore, the absence of both the p38a and p38b MAPK isoforms results in XY sex reversal associated with reduced Sry expression [Warr et al., 2012]. Recent data also suggest that Map2k6 is required for the normal spatiotemporal expression profile of Sry [Warr et al., 2016]. Therefore, at least in mice, available data are consistent with a GADD45γ/MAP3K4/p38 pathway which is required for the appropriate timing of Sry expression in sex determination.

Nineteen MAP3Ks are present in mammals, though their precise biological roles are not fully understood. MAP3K1 (also known as MEKK1) plays a role in lymphocyte differentiation and function [Suddason and Gallagher, 2016], vasculature remodeling [Li et al., 2005], cardiogenesis [Minamino et al., 2002], as well as injury repair [Deng et al., 2006]. The MAP3K1 protein contains an amino terminal plant homeodomain (PHD) motif that has E3 Ub ligase activity, a caspase-3 cleavage site, and a conserved kinase domain. MAP3K1 is the only MAP3K that has a PHD motif. MAP3K1 is activated in response to a number of different stimuli. These include growth factors, hyperosmolarity, microtubule disruption, cell shape disturbance, pro-inflammatory cytokines, and many other physiological stresses [Yujiri et al., 1998]. In humans, mutations in MAP3K1 have been identified in cases of 46,XY DSD [Pearlman et al., 2010; Loke et al., 2014]. The mechanism whereby MAP3K1 mutations cause a failure of testis determination is unclear. The mutations are heterozygous and are either missense, splice site, or in-frame deletions. A clearly disruptive mutation, such as nonsense or frameshift mutation, has not been identified, and considering the fact that MAP3K1 has a widespread expression pattern these more severe mutations may be embryonic lethal. Available data suggest that mutations observed in DSD cases may be subtle gain-of-function variants that result in the increased phosphorylation of the downstream MAPK proteins p38 MAPK and ERK1/2 [Pearlman et al., 2010; Loke et al., 2014]. Patients carrying these mutations show no other apparent phenotypic anomalies other than 46,XY gonadal dysgenesis [Le Caignec et al., 2003; unpubl. data].

Mice lacking Map3K1 are viable and fertile but with an increased embryonic gonadal length [Warr et al., 2011]. XY Map3k1mPHD/+ mice that are heterozygous for an inactive PHD motif have a significantly enlarged testes but with a reduced number of Leydig cells [Charlaftis et al., 2014]. Another Map3k1 mouse model, goya, exhibits a severe hearing loss [Parker et al., 2015]. This data suggest that in testis determination either the MAP kinase signaling pathways in human or mouse have diverged or the difference in phenotype is caused by an intrinsic difference in the type of mutation. In our experience about 10% of 46,XY DSD cases with gonadal dysgenesis carry rare or novel variants in the MAP3K1 gene that could potentially contribute to the phenotype. Although the MAP kinase signaling pathway involved in mouse testis determination is becoming clearer, the MAP3K1 pathway or the targets of MAP Kinase signaling in human testis determination are unknown. The phenotype associated with MAP3K1 mutations is 46,XY CGD, a phenotype that is also associated with mutations in the SRY gene. This suggests that MAP3K1 signaling, like the equivalent pathway in the mouse, is required for the early stages of testis determination in humans, too.

DMRT1, an Evolutionary Conserved Sex-Determining Gene

Deletions of terminal 9p are associated with monosomy 9p syndrome, which is characterized by intellectual disability together with a distinctive series of somatic anomalies, and in approximately 70% of 46,XY individuals anomalies of testis development are seen that range from a completely female phenotype to a male phenotype with hypospadias and/or cryptorchidism [Ottolenghi and McElreavey, 2000]. Two DMRT genes, DMRT1 and DMRT3 (DMRTA3), which are orthologues of the doublesex (dsx) of Drosophila and mab-3 of Caenorhabditis elegans, are located within the minimal recurrently deleted region [Raymond et al., 1998]. Dsx controls the terminal switch of the pathway leading to sex fate choice in Drosophila, and mab-3 is necessary to confer male traits in C. elegans. In mice, Dmrt1 is not required for testis determination, however, its continuous expression in the adult testis is required to maintain organ identity, because forced attenuation of Dmrt1 expression in adult testis results in transdifferentiation of the testis to an ovary [Matson et al., 2011]. Although deletions of 9p24 suggest that DMRT1 hemizygosity is sufficient in some individuals to lead to a failure of testicular development, these deletions usually remove other genes, including the evolutionary related DMRT2 and DMRT3 genes [Ottolenghi and McElreavey, 2000]. Formally, the phenotype could be due to haploinsufficiency of 1, 2, or all 3 of these DMRT genes. Evidence to indicate that the key player in human testis determination is DMRT1 came through the identification of a de novo missense mutation in the functionally important DM-DNA-binding domain in a patient with 46,XY CGD [Murphy et al., 2015]. There were no other somatic anomalies in this healthy girl. The histology of the gonad was similar to that of an SRY mutation and showed no evidence of testicular material, suggesting that the mutation was indeed impacting on primary testis determination. In vitro studies indicated that the mutant protein had reduced DNA affinity, altered sequence specificity and when mixed with wild-type protein, it altered the stoichiometry of the wild-type protein [Murphy et al., 2015]. This suggests that the lack of testis determination seen in this patient is due to a combination of haploinsufficiency and dominant negative activity. This observation may also explain, at least in part, the absence of mutations that have been identified thus far in cases of 46,XY gonadal dysgenesis. In a screen of over 100 cases of XY gonadal dysgenesis, we have only identified a single mutation associated with the phenotype. The residual variation intolerance score (RVIS) ranks human genes by their deviation from the genome-wide average number of nonsynonymous mutations found in genes with a similar amount of global mutational burden ( Mendelian disease causing genes are less tolerant to coding variations than other genes. Put simply, genes known to carry few common functional variants in healthy individuals may be considered to be more likely to cause certain diseases, such as rare forms of DSD, than genes known to carry many functional variants. The intolerance score is based upon allele frequency obtained from whole exome sequence data within the NHLBI-ESP6500 data set. A good example is the gene SOX9, which has an RVIS score of 14.4% and a deficit in loss-of-function variants (%ExAC_RVIS) among the 9.81% lowest of the human genome. Equivalent %ExAC_RVIS figures for NR5A1, MAP3K1 and CBX2 are 15.76, 6.58 and 11.63%, respectively. However, DMRT1 has an RVIS score of 25.56% and a deficit in loss-of-function variants among the 40% lowest of the human genome. This indicates that DMRT1 is more tolerant to genetic variation in healthy individuals than the other genes mentioned above. This suggests that for a DMRT1 mutation to be pathogenic (or penetrant) resulting in a failure of testis determination, it may be required to show either dominant negative activity on the wild-type allele or alter the normal interactions of the protein.

Gonadal anomalies may not be the only phenotype associated with mutations involving DMRT1. Recently, genome-wide association studies indicated a variant in the putative promoter of DMRT1 for sex-specific asthma [Schieck et al., 2016]. The role, if any, of DMRT1 in the regulation of allergic immune responses is unclear, but the expression of DMRT1 was found to be higher in lung macrophages from men with various lung diseases [Schieck et al., 2016].

SOX Gene Mutations

In recent years it has become evident that the ectopic expression of HMG-box containing proteins in the urogenital ridge at the moment of sex determination may result in testicular development in a chromosomal XX female. Although most cases of 46,XX TDSD/OTDSD are caused by the presence of the SRY gene, usually on the X chromosome, the remaining cases stay unexplained. As compared to 46,XY DSD, there are relatively fewer known causes of 46,XX DSD. In human, SOX3 loss-of-function mutations are associated with mental retardation and growth hormone deficiency [Laumonnier et al., 2002]. However, three 46,XX SRY-negative testicular DSD patients have been reported who carry rearrangements at the SOX3 locus on the X chromosome. One patient carried 2 microduplications, one of ∼123 kb that spanned the entire SOX3 gene and another of 85 kb that was located 350 kb proximal to SOX3. A second patient carried a single 343-kb microdeletion immediately upstream of SOX3, and a third XX male with multiple congenital anomalies carried a 6-Mb duplication including SOX3 and at least 18 other genes [Sutton et al., 2011]. 46,XX testicular DSD has also been reported in association with a 774-kb insertion translocation from chromosome 1 into a palindromic sequence 82 kb distal to SOX3 [Haines et al., 2015]. Three further duplications of the SOX3 gene have been reported in XX males [Moalem et al., 2012; Vetro et al., 2015; Grinspon et al., 2016].

Complete or partial duplications of chromosome 22 in 46,XX-SRY negative individuals are associated with various degrees of masculinization [Nicholl et al., 1994; Aleck et al., 1999; Seeherunvong et al., 2004]. Further delimitation of the minimal region was demonstrated by a de novo duplication of 22q11.2q13 in a 46,XX SRY-negative male with mild hypospadias, dysmorphic features, and hypotonia [Polanco et al., 2010]. Human SOX10 maps to 22q13.1 and may be responsible for the phenotype. Loss-of-function heterozygous mutations in SOX10 are associated with Waardenburg-Shah and Waardenburg-Hirschsprung disease [Pingault et al., 1998; Touraine et al., 2000], however, in the mouse, transgenic expression of Sox10 in the gonads of XX mice results in testis formation [Polanco et al., 2010].

RSPO1/WNT4/β-Catenin Signaling

Little is known about the genetic pathway(s) involved in human ovary development. In XX individuals, activation of the β-catenin signaling pathway by the proteins RSPO1 and WNT4 is necessary for granulosa cell differentiation leading to ovarian development. Stabilization of β-catenin by the RSPO1/WNT4 pathway results in transcription of its target genes. The mechanism by which RSPO1 stimulates WNT4 in the developing ovary is unknown. In general, RSPOs stimulate WNT signaling by binding to the leucine-rich repeat-containing G protein-coupled receptors LGR4, LGR5, and LGR6 [Wang et al., 2013]. RSPOs can also bind to 2 negative feedback regulators of the WNT signaling pathway, the RING-type E3 ubiquitin ligases ZNRF3 or RNF43, leading to their clearance and resulting in enhanced WNT signaling [Jiang et al., 2015]. Mutations involving RSPO1 and WNT4 are associated with exceptionally rare syndromic forms of 46,XX testicular/ovotesticular DSD. Human homozygous RSPO1 mutations are associated with a rare recessive syndrome, which is characterized by XX testicular DSD, palmoplantar hyperkeratosis, and predisposition to squamous cell carcinoma of the skin [Parma et al., 2006]. Mutations involving RSPO1 have not been reported in non-syndromic cases of testicular and ovotesticular DSD [unpubl. data].

The absence of Wnt4 in XX mice results in a partial masculinization of the gonad including the differentiation of some Leydig-like cells. In human, 4 dominant heterozygous missense mutations in WNT4 have been reported in 46,XX women with various degrees of virilization including androgen excess and abnormal development of Müllerian ducts [Baison-Lauber et al., 2004, 2007; Philibert et al., 2008, 2011]. A single homozygous WNT4 mutation was reported in a consanguineous family with an embryonic lethal syndrome of 46,XX testicular DSD and dysgenesis of kidneys, adrenals, and lungs (SERKAL syndrome; SEx Reversion, Kidneys, Adrenal and Lung dysgenesis) [Mandel et al., 2008].

NR5A1 p.Arg92Trp and 46,XX DSD

Primary ovarian insufficiency, also termed premature ovarian failure, is defined by the arrest of normal ovarian function before the age of 40 years and includes premature menopause, primary and secondary amenorrhea as well as ovarian dysgenesis. In 2007, when we were analyzing cases of 46,XX primary ovarian insufficiency for mutations in the NR5A1 gene, a patient (sporadic case 2; Lourenco et al. [2009]) was clinically investigated at 4 months of age because of a hypertrophy of the clitoris. The girl had high levels of FSH indicating ovarian insufficiency and carried an NR5A1 mutation. The clitoral hypertrophy suggested that the girl had been exposed to androgens in utero. Therefore, we decided to screen idiopathic cases of 46,XX TDSD/OTDSD for mutations in the NR5A1 gene. We rapidly discovered a small family with 2 virilized sibs who carried a p.Arg92Trp mutation. The unaffected mother also carried this mutation. At the time, this observation was interesting but could simply have been explained as a chance finding - a rare genetic variant unrelated to the phenotype. This view was supported by our screen of further 40 cases of 46,XX DSD that did not reveal any other NR5A1 mutations. In 2015, we were contacted by different groups who had also identified the same NR5A1 p.Arg92Trp mutation in association with 46,XX testicular DSD [Bashamboo et al., 2016]. Two families had a single affected child who carried a de novo NR5A1 p.Arg92Trp mutation, and this formally excluded a founder mutation. A fourth family was identified with 2 affected sibs, one with 46,XY gonadal dysgenesis and raised as a girl and the other a boy with 46,XX testicular DSD. They both carried the mutation. A number of mutations have been described in NR5A1 in 46,XX individuals in association with ovarian insufficiency, but we are unaware of any other amino acid changes in NR5A1 except p.Arg92Trp that is associated with virilization in 46,XX individuals. Interestingly, a homozygous p.Arg92Gln change has been observed in a 46,XX girl with no evidence of virilization [Guran et al., 2015]. This suggests that the p.Arg92Trp mutation specifically results in testis formation in a chromosomal female background (fig. 1). The mutation involves a highly conserved amino acid residue located in the A-box motif of the protein. The A-box consists of a 30 amino acid basic region carboxyl-terminal to the DNA binding domain. NR5A1 binds to DNA as a monomer with the A-box recognizing DNA sequences 5′ to the NR5A1 consensus motif in the minor groove. The p.Arg92Trp mutation is predicted to disrupt DNA binding and this is what we observed using the consensus binding motif CCAAGGTCA as a target. In contrast the p.Arg92Gln mutant has essentially wild-type binding activity. However, this difference in DNA binding activity cannot explain why testis determination is occurring due to the 92Trp substitution. The p.Arg92Trp mutation could be associated either with inappropriate activation of testis-specific pathways in the ovary or with disruption of pathways that oppose testis development and maintain ovarian integrity. Transient transfection assays demonstrated that the p.Arg92Trp NR5A1 mutant had reduced activation of several minimal promoters involved in testis development as well as the Sox9 Tesco enhancer. This is consistent with a 46,XY PGD phenotype, but it does not explain testis formation in an XX background. We then assayed the ability of the 92Trp and 92Gln mutations to influence the pro-ovary canonical WNT signaling pathway. β-Catenin can interact functionally with NR5A1 to modulate target gene expression [Gummow et al., 2003; Hossain and Saunders, 2003; Jordan et al., 2003; Kennell et al., 2003; Mizusaki et al., 2003; Parakh et al., 2006; Salisbury et al., 2007; Ehrlund et al., 2012]. Specifically, the Nr5a1 and β-catenin proteins physically interact to upregulate the expression of the Nr0b1(Dax-1) gene on the X chromosome [Mizusaki et al., 2003]. In 46,XY individuals, 2 copies of NR0B1 result in a failure of testis determination and are associated with 46,XY gonadal dysgenesis, indicating that NR0B1 is an anti-testis gene [Muscatelli et al., 1994]. In contrast to the p.Arg92Gln mutant, we found that the p.Arg92Trp NR5A1 mutant showed loss of synergy with β-catenin to activate target gene expression. The phenotype in the 46,XX children with the p.Arg92Trp variant may be due to altered regulation of the expression of pro-ovarian genes that normally suppress testis development (fig. 2). In humans, NR5A1 is expressed in the granulosa cells of the early developing ovary, whereas in the mouse the expression of Nr5a1 in the embryonic gonad is sexually dimorphic. It is continuously expressed in the mouse embryonic testis, but Nr5a1 transcripts are absent during the period of ovarian formation between E13.5-E16.5 [Ikeda et al., 1994]. Therefore in this case, the mouse model may not be informative for elucidation of the mechanism. This further reiterates the differences between the molecular mechanisms involved in gonad determination in mouse and human and emphasizes the need to analyze human cases of DSD to establish novel causes of 46,XX and 46,XY DSD.

Fig. 1

NR5A1 may regulate anti-testis gene expression in the ovary. In a 46,XY individual, NR5A1 synergizes with SRY to upregulate male-specific gene expression (e.g., SOX9) leading to testis formation. In 46,XX individuals, NR5A1 synergizes with β-catenin to upregulate the expression of anti-testis genes (e.g., DAX-1/NR0B1) and possibly pro-ovarian genes. In the 46,XY DSD case, the p.Arg92Trp mutant shows a reduced ability to upregulate SOX9 gene expression leading to a lack of testis formation. In a 46,XX child with TDSD/OTDSD (shown on the right), the same mutant shows reduced ability to synergize with β-catenin to upregulate the expression of anti-testis genes. As a consequence of this lack of repression, the expression of pro-testis genes (e.g., SOX9) leads to testis formation.

Fig. 1

NR5A1 may regulate anti-testis gene expression in the ovary. In a 46,XY individual, NR5A1 synergizes with SRY to upregulate male-specific gene expression (e.g., SOX9) leading to testis formation. In 46,XX individuals, NR5A1 synergizes with β-catenin to upregulate the expression of anti-testis genes (e.g., DAX-1/NR0B1) and possibly pro-ovarian genes. In the 46,XY DSD case, the p.Arg92Trp mutant shows a reduced ability to upregulate SOX9 gene expression leading to a lack of testis formation. In a 46,XX child with TDSD/OTDSD (shown on the right), the same mutant shows reduced ability to synergize with β-catenin to upregulate the expression of anti-testis genes. As a consequence of this lack of repression, the expression of pro-testis genes (e.g., SOX9) leads to testis formation.

Close modal
Fig. 2

The molecular and genetic events in mammalian sex determination and differentiation. In the XY gonad the activation of SRY expression, possibly initiated by CBX2/WT1/GATA4/FOG2/NR5A1, leads to the upregulation of Sox9 expression via a synergy with Nr5a1 at a Sox9 enhancer such as Tesco [Sekido and Lovell-Badge, 2008]. In the XX gonad the supporting cell precursors accumulate β-catenin in response to RSPO1/WNT4 signaling, which either directly or indirectly represses SOX9 expression, perhaps at least in human, by interacting with NR5A1 [Bashamboo et al., 2016]. Once Sox9 levels reach a critical threshold, several positive regulatory loops are initiated, including autoregulation of its own expression and formation of feed-forward loops via FGF9 or PGD2 signaling. At later stages, Foxl2 may repress Sox9 expression to maintain ovarian identity [Uhlenhaut et al., 2009]. In the testis, Sox9, together with other Sox proteins including Sox8, promotes the testis pathway by for example stimulating Amh expression, and it also probably represses the ovarian genes Wnt4 and Foxl2 [Uhlenhaut et al., 2009]. DMRT1 controls sex determination in some species of fish and may be the master sex-determining switch in birds. In mice it is not involved in male primary sex determination, but there is evidence to indicate that it is important in human testis determination [Murphy et al., 2015].

Fig. 2

The molecular and genetic events in mammalian sex determination and differentiation. In the XY gonad the activation of SRY expression, possibly initiated by CBX2/WT1/GATA4/FOG2/NR5A1, leads to the upregulation of Sox9 expression via a synergy with Nr5a1 at a Sox9 enhancer such as Tesco [Sekido and Lovell-Badge, 2008]. In the XX gonad the supporting cell precursors accumulate β-catenin in response to RSPO1/WNT4 signaling, which either directly or indirectly represses SOX9 expression, perhaps at least in human, by interacting with NR5A1 [Bashamboo et al., 2016]. Once Sox9 levels reach a critical threshold, several positive regulatory loops are initiated, including autoregulation of its own expression and formation of feed-forward loops via FGF9 or PGD2 signaling. At later stages, Foxl2 may repress Sox9 expression to maintain ovarian identity [Uhlenhaut et al., 2009]. In the testis, Sox9, together with other Sox proteins including Sox8, promotes the testis pathway by for example stimulating Amh expression, and it also probably represses the ovarian genes Wnt4 and Foxl2 [Uhlenhaut et al., 2009]. DMRT1 controls sex determination in some species of fish and may be the master sex-determining switch in birds. In mice it is not involved in male primary sex determination, but there is evidence to indicate that it is important in human testis determination [Murphy et al., 2015].

Close modal

The last few years have seen a considerable number of new genes involved in human sex determination that when mutated cause non-syndromic DSD. A failure of testis determination, 46,XY gonadal dysgenesis is mainly associated with point mutations in coding sequences (e.g. NR5A1, MAP3K1, GATA4, FOG2), whereas gene mutations leading to testis formation in a chromosomal XX female background are mainly associated with dysregulation of the expression of SOX genes (e.g. SOX9, SOX3, SOX10). In the latter, point mutations in coding genes are usually associated with syndromic forms of DSD involving the RSPO/WNT signaling pathway. Overall, the genetic etiology in the majority of cases of these extreme forms of DSD is unknown and reflects our poor knowledge of the genetic pathways that are involved. High throughput sequencing will reveal rare genetic mutations that are responsible for these phenotypes; however, if we are to fully establish the causality of these mutations and understand the mechanisms, a series of complimentary combinatorial studies using different model systems are required.

A.B. is funded in part by the program Actions Concertees Interpasteuriennes (ACIP). A.B. and K.McE. are funded by a research grant from the EuroDSD in the European Community's Seventh Framework Programme FP7/2007-2013 under grant agreement No. 201444 as well as grant No. 295097 entitled GM_NCD_in_Co - Reinforcing IPT capacities in Genomic Medicine, Non Communicable Diseases Investigation and international cooperation as part of the EU call FP7-INCO-2011-6. The work is also funded by a Franco-Egyptian AIRD-STDF grant and the Agence Nationale de la Recherche (Laboratoire d'Excellence Revive).

The authors have no conflicts of interest to declare.

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