The Endless Quest for Sex Determination Genes

Clin Genet 2004: 67: 15–25 Copyright # Blackwell Munksgaard 2004 Printed in Singapore. All rights reserved CLINICAL GENETICS doi: 10.1111/j.1399-0004.2004.00376.x Developmental Biology The endless quest for sex determination genes

a a,b,c Fleming A, Vilain E. The endless quest for sex determination genes. A Fleming and E Vilain Clin Genet 2004: 67: 15–25. # Blackwell Munksgaard, 2004 aDepartment of Human Genetics, bDepartment of Pediatrics, and Disorders in human sex determination cause defects in gonadal function cDepartment of Urology, David Geffen and can result in a spectrum of abnormalities in the internal and external School of Medicine at UCLA, Los genitalia, ranging from relatively mild sexual ambiguities to complete Angeles, CA, USA sex reversal. Several genes involved in sex determination have been Key words: sex determination – gonadal validated in humans, and activities of their gene products are being dysgenesis – gonads – development – elucidated, particularly in mouse models. However, how these genes sexual ambiguity – genetics interact in an overall process remains far from clear, and it is probable Corresponding author: Eric Vilain, MD, that many additional genes are involved. Management of patients with PhD, Departments of Human Genetics pathologies in sex determination and subsequent differentiation is and Pediatrics, UCLA School of currently under debate, but will require not only an understanding of the Medicine, Gonda Center, Suite 6357, 695 multiple definitions of an individual’s sex but also an increased Charles Young Drive South, Los Angeles, knowledge of the molecular mechanisms involved in sex determination. CA 90095-7088, USA. Tel.: þ1 310 267 2455; fax: þ1 310 794 5446; e-mail: [email protected] Received 23 September 2004, revised and accepted for publication 23 September 2004

Defining an individual’s sex is not simple. Geno- regress and Mu¨llerian ducts become the upper typic sex refers to genetic make-up, where males part of the vagina, uterus, and Fallopian tubes. (typically 46,XY) or females (46,XX) are defined Pathologies of sexual development are quite by presence or absence of a Y chromosome (1). varied in humans. Sex differentiation pathologies Phenotypic sex is characterized by the primary and are relatively common (about one in 100) and secondary sex characteristics of an individual. Sex- better understood than those of sex determin- ual identity includes a person’s sense of self (gender ation. Here, gonads develop normally, but defects identity) and his/her attraction to others. These in subsequent gene and/or hormone function aspects of sex are not always in concordance in a result in abnormalities of the internal or external given individual, and consideration of all three genitalia, with a wide range of type and severity. aspects is critical and complex – and currently the These abnormalities are usually variations on the subject of much debate – in the clinical setting. norm, such as a small penis, undescended testes Physiological sexual development includes both (cryptorchidism), an enlarged clitoris, or abnor- sex determination and sex differentiation, a some- mal positioning of the urethral opening (hypo- what arbitrary but useful distinction. In mam- spadias). Some, however, are severe enough to mals, gonads are indistinguishable (indifferent) be termed sexual ambiguities. early in their development; sex determination – Sex determination pathologies are compara- the decision to develop as testes or ovaries – tively rare (estimated at one in 20,000). They begins midway through gonadogenesis and ends result from duplications, mutations, or deletions with formation of the gonads. Sex differentiation of sex-determining genes and involve abnormal follows in accordance: in males, testicular hor- gonadal development (dysgenesis) and, typically, mones cause the regression of Mu¨llerian ducts a discrepancy between genotypic and phenotypic and development of Wolffian ducts into the epi- sex. XX males often have small, azoospermic didymis, vas deferens and seminiferous tubules, testes, normal male genitalia, and no Mu¨llerian- and testicular descent; in females, Wolffian ducts derived structures, but can also have hypospadias

15 Fleming and Vilain or sexual ambiguity. XX true hermaphrodites have default state must have a molecular basis has gonads with both testicular and ovarian tissue, spurred research to explore female mechanisms ambiguous genitalia with persistence of some as well. Finally, because clinical management Mu¨llerian structures. XY females with complete does not always fall easily into the categories out- or pure gonadal dysgenesis have fibrous (streak) lined above, we also include figures (Figs 1 and 2) gonads and normal female genitalia, including a that summarize the major disorders of sexual uterus. XY females with partial dysgenesis may development, including both determination and have sexual ambiguities. Although the focus of differentiation. much research, these pathologies are not yet well understood and can be difficult to diagnose. Genes involved in testis development Here, we present information about each of SRY the genes known to be involved in human sex determination (Table 1). Although many genes Around the early 1990s, a series of elegant experi- involved in sex determination have been identi- ments found SRY to be the once elusive mamma- fied in animal models, their functions are not yet lian testis-determining gene. Positional cloning fully understood, and the majority of those genes located a 35-kb fragment of the Y chromosome have not been validated in humans. Male- translocated onto the X chromosome of XX male determining genes have been more thoroughly and true hermaphrodite patients (3, 4); sequence studied to date, perhaps an outgrowth of Jost’s analysis and gene mapping defined a conserved experiments in the 1940s (2) that showed testis sequence – SRY – within that fragment (5, 6). determination to be synonymous with sex deter- Murine Sry gene expression profiles revealed a mination, leaving ovarian determination to be the male-specific increase in transcript consistent default state. Recently, understanding that even a with earliest divergence of male and female

Table 1. Known genes involved in sex determination (2, 3) Validated Gain-of-function Gene in humans Human locus Putative function Loss-of-function phenotype phenotype

Testis determining SRY X Yp11.3 Transcription factor XY female (human, mouse) XX male (human, mouse) SOX9 X 17q24 Transcription factor XY female þ campomelic XX male dysplasia (human) (human, mouse) SF1 X 9q33 Transcription factor XY female þ adrenal insufficiency (human); genital and adrenal developmental blockage (KO) WT1 X 11p13 Transcription factor XY female þ kidney defects, Wilms’ tumor (human); absent gonads, blockage in kidney and adrenal development (KO) DAX1 X Xp21.3 Transcription factor Adrenal hypoplasia congenita; XY female hypogonadism (human); (human, mouse) spermatogenesis block (mouse) DMRT1 X 9p24.3 Transcription factor XY female (human)a; postnatal loss of Sertoli and germ cells (KO) ATRX X Xq13.3 Transcription factor XY female þ mental retardation, a-thalassemia (human) M33 17q25 Transcription factor XY female, Sox9 reduced (KO) FGF9 13q11–13 Signaling molecule XY female (variable phenotype) þ lung defects (KO) Ovary determining WNT4 X 1p35 Signaling molecule XX testosterone XY female synthesis þ Wolffian duct (human) development (mice) FOXL2 Xb 3q23 Transcription factor Premature ovarian failure þ BPES (humans); XX male (goats)

KO, mouse knock out; BPES, Blepharophimosis Ptosis Epicanthus Inversus Syndrome. aDMRT1 is one gene in a large sex-reversing deletion. bNot validated for sex reversal in humans.

16 Sex determination genes

Fig. 1. Simplifieddiagnosticflowchart of the major disorders of sexual development in feminized boys.

gonadal development (7, 8). XX and XYpos are not yet clear, but several factors have been (a weak allele of Sry tending to sex reverse on a implicated, including SP1, WT1, SF1, GATA4, C57BL/6 background) mice transgenic for a FOG2 (12), and three members of the insulin 14-kb fragment containing Sry developed testes receptor tyrosine kinase family (19). (9, 10). Deletions and mutations in SRY/Sry Despite certainty that SRY is the testis- often result in XY female sex reversal [in mice: determining gene, it is still not known what (11) and (6); in humans: reviewed in (12)]. downstream targets SRY regulates and whether SRY, a single-exon gene on the Y chromosome, it acts as an activator or repressor (20), although is an apparent transcription factor (13). Its gene it is widely thought to up-regulate SOX9 expres- product contains a conserved High Mobility sion. In addition, sequence analysis of SRY does Group (HMG) motif that binds and bends its not explain all pathologies of sex determination. target DNA (14) and is flanked by two nuclear Genetic studies have shown that SRY is not localization signals (NLSs) (15). Almost all sex- detected in 8% of unambiguous XX males, 91% reversing mutations in SRY are found in the of XX males with sexual ambiguities, and 84% of HMG/NLS domain (12) and affect its DNA- XX true hermaphrodites (21), and that mutations binding affinity (13), its binding/bending of target in the SRY gene have been found in only 15% of DNA (16), or its nuclear localization (17, 18). The XY females (12). These data indicate that add- precise mechanisms regulating expression of SRY itional genes are involved in testis determination.

Fig. 2. Simplifieddiagnosticflowchart of the major disorders of sexual development in masculinized girls.

17 Fleming and Vilain SOX9 gonads, malformation of the hypothalamus, and abnormal function of the gonadotropes (41). SOX9, also involved in male sex determination, is In the indifferent gonad, SF1 is expressed one such gene. It was discovered in patients with equally in males and females and is required to campomelic dysplasia (CD), a sporadic autosomal maintain the developing gonad. Following Sry dominant disorder with severe skeletal dysplasia, expression, SF1 is up-regulated in developing accompanied by male-to-female sex reversal in testes (and down-regulated in ovaries), where it about 75% of XY CD patients (22, 23). Abnor- activates genes including Dax1 (42, 43), Amh (41), malities found on chromosome 17 subsequently and steroidogenic enzymes required to synthesize lead to identification of the SOX9 locus (24, 25). testosterone. Interestingly, recent studies suggest Like SRY, SOX9 contains an HMG domain that SF1 may also participate in regulation of Sry with two flanking NLSs (15). In addition, SOX9 (44, 45). In vitro evidence indicates that Sox9 contains two transactivation domains (26–28), a takes part in the male-specific up-regulation of nuclear export signal (29), and a dimerization SF1 during testes differentiation (34). domain (30, 31). Mutations in SOX9 can affect To date, only four patients have been described its DNA-binding affinity, DNA-binding/-bending with mutations in SF1. Three of these patients ability, transactivation, nuclear import (27, 28, showed adrenal insufficiency, corroborating a 32), and nuclear export (29). SOX9 binds DNA critical role for SF1 in human adrenal develop- as a dimer in transactivation during normal bone ment and/or function. One such patient presented development but as a monomer in sex deter- at 14 months as a phenotypically normal (46,XX) mination; mutation in its dimerization domain female with a heterozygous single de novo results in acampomelic dysplasia without sex sequence transversion in SF1 (46). Functional reversal (30, 31). studies showed the mutated protein binds DNA Murine Sox9 is expressed at low levels early in and transactivates less efficiently. It appears that the male and female genital ridge. Shortly after Sry the decreased activity of SF1, while insufficient expression begins, Sox9 expression is up-regulated for normal adrenal development, is sufficient for in the male genital ridge, and Sox9 protein moves normal development of the ovary. Two add- into the nucleus of developing Sertoli cells; it is itional patients with adrenal insufficiency were down-regulated in females at the same time (33). 46,XY females with mutations that affect the This timing and localization suggest that Sry transactivational ability of SF1 in a haploinsuffi- up-regulates Sox9 expression in males. Sox9 is cient (47) or autosomal recessive (48) manner. involved in transcriptional regulation of SF1 (34), Interestingly, XY mice heterozygous for SF1 discussed below, and Amh (35), which causes have adrenal abnormalities but are not sex regression of Mu¨llerian (female) ducts in the reversed, although the testes are small (49). developing male (35, 36). The fourth patient was 46,XY with gonadal Evidence suggests that SOX9 could have a con- agenesis but without adrenal insufficiency (50). served role in male sex determination. Its sequence Sequence analysis revealed an 8-bp deletion, is conserved across vertebrates, while Sry is found causing a frameshift that truncates translation of only in mammals (37). Sox9 and Sry have sequence SF1 upstream of a transcriptional activation and expression profile similarities. XX mice expres- motif. Studies of this mutation indicated a slight sing Sox9 ectopically develop testes (38, 39). decrease in DNA-binding ability and a dominant Finally, an XX male patient was found to have a negative effect in a mouse Leydig cell line. Con- large duplication containing SOX9 (40). versely, in a human adrenocortical cell line (though not in a mouse adrenocortical line), the SF1 mutation appeared to slightly increase transactivation. SF1 (Ad4BP; NR5A1), an orphan member of the Together, findings from these patients strongly nuclear receptor family of transcription factors, support a dosage effect of SF1 in humans – was first identified in mice as a regulator of many different from that of mice – and cell- or tissue- genes involved in steroidogenesis in the gonad specific requirements for SF1 function. and adrenal, and subsequently found to developmentally regulate gene expression at all levels of the reproductive axis – the hypothalamus, WT1 pituitary, adrenal, and gonad (41). Homozygous disruption of the SF1 gene in mice causes male- Wilms’ tumor suppressor gene (WT1)isan to-female sex reversal of internal and external apparent zinc finger transcription factor that genitalia, complete agenesis of adrenals and can act either as a repressor or an activator (51).

18 Sex determination genes It was identified by positional cloning as a critical pure gonadal dysgenesis had a duplication of part gene in Wilms’ tumor disease (52–54). WT1- of the short arm of the X chromosome, resulting disrupted mice lack gonads and kidneys, and in a double (female) dose of genes therein development of the adrenals, heart, and spleen is (dosage-sensitive sex reversal or DSS) (67). They blocked (55). During gonadogenesis, expression also noted that XY individuals with deletions in of WT1 begins early in the genital ridge and this region were not sex reversed. Subsequently, becomes localized to the granulosa and epithelial DAX1 was identified within this region and cells of the ovary and the Sertoli cells of the testes found, when mutated, to cause adrenal hypo- (56). plasia congenita (AHC), often accompanied by The WT1 locus is complex and produces mul- hypogonadotropic hypogonadism but not sex tiple isoforms. Those isoforms containing the reversal (68). In mice, a duplication of Dax1 tripeptide lysine-threonine-serine between zinc results in complete XY sex reversal only in a fingers 3 and 4 (called þKTS) have different background with weak or delayed expression of binding affinities for both proteins and nucleic Sry (69). Furthermore, XX mice with a Dax1 acids than those lacking the tripeptide (–KTS) deficiency develop as females, while XY mice (57). In the nucleus, þKTS isoforms colocalize with this deficiency have impaired spermatogen- with splice factors, suggesting a role in RNA esis and, in a weak or delayed Sry background, splicing, while –KTS isoforms primarily localize show male-to-female sex reversal (70, 71). Taken in a diffuse pattern typical of transcription fac- together, these findings suggest that Dax1 is tors. The ratio of þKTS and –KTS isoforms is essential for testes development in a dose – too constant within cell types (58). Mouse models much or too little impairs testes development – suggest that WT1 þKTS isoforms are necessary and time sensitive manner (72). Alternatively, for male sex determination, while –KTS isoforms Dax1 could have several roles in gonadogenesis, are required more for maintenance and growth of perhaps in different cell lineages (73). the bipotential gonad (59). Dax1 is expressed developmentally in the WT1 expression is up-regulated in vitro by Sry gonad, adrenal, hypothalamus, and pituitary in (60). In turn, WT1 has been implicated by func- a pattern similar to that of SF1 (42, 74–76), which tional studies in the regulation of many genes, suggests a possible interaction. Indeed, it now including several involved in sexual development. appears that SF1 up-regulates Dax1 expression WT1 þKTS isoforms appear necessary for (42, 77, 78) in synergy with b-catenin (79), and up-regulation of Sry (59). In addition, WT1 may that this action opposed by chicken ovalbumin up-regulate SF1 and Dax1 expression (61, 62), be upstream promoter-transcription factor (78). In involved in regulation of Amh (63), participate turn, Dax1 represses SF1 transcriptional activity with Sry in gene activation (51), and compete by antagonizing synergistic cofactors of SF1 (64, with Dax1 for modifying SF1 activity (64). 80) and/or by recruiting corepressors to SF1 (81). In humans, Wilms’ tumor disease and muta- The preponderance of evidence indicates that tions or deletions of WT1 are often associated Dax1 binds SF1 in these transactions, and all with three larger syndromes, all with some degree DAX1 missense mutations identified in AHC lie of genitourinary abnormality. WAGR syndrome in the ligand-binding domain, which is essential is caused by deletions in chromosome 11 that for protein–protein interaction (72). contain the WT1 locus (55). Denys–Drash A recent article has identified a previously syndrome, with more severe genitourinary unknown alternate transcript of DAX1, named abnormalities, results from dominant negative DAX1a (82). It will be interesting to hear whether missense mutations in the zinc finger region of this transcript is expressed developmentally and, WT1 that affect DNA binding (55). Frasier syn- if so, whether it sheds light on, or adds confusion drome includes XY male-to-female sex reversal to, the already complex story of DAX1. and is due to point mutations that decrease the þKTS isoforms of WT1 and the þKTS to –KTS ratio (65, 66). DMRT1 Casting DMRT1 as a male sex-determining gene in mammals has gone in and out of favor over DAX1 time. In humans, DMRT1 was identified among DAX1, a gene located on the X chromosome in several genes in a region of chromosome 9p (83) mammals, represses genes involved in steroido- that, when deleted, often results in abnormal gen- genesis and sex determination. Bardoni et al. italia and, in some cases, XY sex reversal (84–86). (1994) found that some XY female patients with The discovery of DMRT1 in this context was of

19 Fleming and Vilain great interest because DMRT1 contains a domain tal phenotypes cluster there (97). ATRX is widely common to male sex-determining genes as distant expressed and its cell-specific function is likely con- as the male isoform of Doublesex in Drosophila ferred by different protein partners (95), cell-cycle- (87) and Mab-3 in Caenorhabditis (88), thereby dependent phosphorylation (98), and alternative named the DM domain (83). This was the first splicing (99). potential human sex-determining gene to show such far-reaching, non-vertebrate conservation (89). DMRT1 has now been found in many verte- Genes involved in ovarian development brates, and its gonad-specific expression is WNT4 up-regulated in the testis of several species around the time of differential sexual development (90). WNT4, the first signaling molecule to be identified However, generation of a DMRT1-deleted in the study of sex determination, has been shown mouse model shed doubt on the role of DMRT1 in mice to regulate female sexual development as a vertebrate sex determinant. DMRT1–/– males through both negative and positive functions. were found to have postnatal defects in Sertoli Wnt4 represses male reproductive tract formation and germ cells similar to some humans with and steroid synthesis (seen relatively early in male deletions in chromosome 9p, but no sex reversal gonadogenesis). Targeted disruption of Wnt4 (91). In humans, although the DMRT1 locus is results in development of Wolffian ducts – and located close to a known 9p-deletion break point consequent male genitalia – and ectopic expression in one sex-reversed XY patient (92), there is no of at least two steroidogenic enzymes in female direct evidence for its involvement in this pheno- mice,buthasnoaffectonmaledevelopment type. In addition, several other DM domain genes (100). Wnt4 also supports early development of are known to lie within the chromosome 9p Mu¨llerian ducts in females. Although specification deletion and could act as primary or redundant of Mu¨llerian precursor cells appears not to be regulators of male sexual development. At pres- under Wnt4 control (101), Mu¨llerian structures ent, DMRT1 is considered a likely regulator of fail to develop in both XX and XY Wnt4-mutant male sex differentiation but probably not deter- mice (100). Based on their findings, Vainio et al. mination (90). suggested that Wnt4 suppresses Leydig cell differentiation in normal females. Two groups have found that transgenic overexpression of Wnt4 in male mice results in ATRX decreased testosterone synthesis and abnormal ATRX appears to be involved in male sexual vasculature but not sex reversal (102, 103). development, but its specific functions are not Furthermore, organ coculture studies showed yet known. Mutations in the gene that encodes that steroidogenic cells migrated into XX ATRX cause X-linked mental retardation, often Wnt4–/–, but not wild type, gonads from adjacent associated with a-thalassemia and with gonadal adrenal primordial tissue (103). Based on these abnormalities ranging from undescended testicles and additional data, two alternative proposals for to testicular dysgenesis and ambiguous or female Wnt4 function relative to steroid synthesis were external genitalia (93). The ATR-X syndrome has proposed: Wnt4 suppression of steroidogenesis in only been identified in males; skewed X-inacti- females could be effected either by repressing vation appears to eliminate mutant ATRX SF1 function (102) or by inhibiting migration function in females that carry an affected allele (94). of steroidogenic precursor cells into the Some roles for ATRX have been proposed. developing ovary (103). Evidence suggests that ATR-X patients with Male patients with duplications in WNT4 male pseudohermaphroditism have testicular dys- exhibit varying degrees of sexual phenotype ran- genesis resulting in reduced testosterone synth- ging from cryptorchidism (104, 105) to XY sex esis, and some Leydig cell abnormalities have reversal with ambiguities (106). Quite recently, been seen (95), indicating a role for ATRX in the first female patient carrying a mutation Leydig cell development. Sequence analysis of in WNT4 – a heterozygous substitution in exon the 36-exon ATRX gene shows that it is likely to 5 – was reported (107). Her symptomatology be a chromatin-remodeling transcription factor, included clinical excess of androgen and lack of possibly a repressor, with multiple protein–pro- Mu¨llerian-derived structures. In transfected cells, tein and protein–DNA interactive domains (95, this mutation appears to interfere with a fatty 96). The C-terminus may be specifically involved acylation modification of the WNT4 protein, in urogenital development or function, as most which then fails to repress ectopic expression of mutations associated with severe ATR-X urogeni- steroidogenic enzymes and androgens (107).

20 Sex determination genes

FOXL2 Table 2. Chromosomal abnormalities associated with sex reversal, with no known causative gene (4–6) FOXL2 is a putative transcription factor involved in ovarian development. By positional cloning, Chromosomal abnormalities Phenotype mutations in this gene were found to cause the autosomal dominant Blepharophimosis dup 22q XX true hermaphrodite Ptosis Epicanthus Inversus Syndrome (BPES), a dup 10q26-qter XY female del 2q31 XY female, mental retardation complex disorder of the eyelid associated in del 12q24.31–q24.33 XY ambiguous, mental retardation BPES type 1 with premature ovarian failure (108). Expression studies found Foxl2 to be highly conserved in species with diverse sex-determining humans with sex determination pathologies that mechanisms, where it is present and female- resulted in sex reversal. Several additional chromo- enriched from the earliest stages of ovarian dif- somal abnormalities associated with sex reversal ferentiation (109). In developing mouse ovaries, it are known (Table 2), although causal genes within is expressed in granulosa and germ cells (108, these abnormalities have not yet been isolated. 109). Expression is first seen in female gonads at Another approach is based on linkage studies, E12.5 – just as cords become visible in the male – but this method is slow, requiring location of a and continues in pregranulosa cells through number of affected families, and has not been formation of primordial and early follicles (110). successful thus far in identifying specific sex- Testis development is unaffected in BPES male determining genes. patients. Animal (particularly mouse) models have been The BPES type 1 phenotype has been recapitu- invaluable in defining the specific molecular lated in a Foxl2–/– mouse (111) and a homozy- mechanisms and cell biology of sex-determining gous Foxl2-mutant mouse (110). Granulosa cells genes and have helped identify a few genes that fail to pass from the squamous to cuboidal phase are now validated in humans, including SF1 and that precedes further proliferation of granulosa Wnt4. Large-scale screens for sex-determining cells and oocyte growth (110, 111). Two known genes in mice have recently identified a large inhibitors of primordial follicle activation, number of new genes potentially involved in this Activin-ba and Amh, are diminished or absent process (115–120). Results from these screens in Foxl2 homozygous mutated mice (110). In raise exciting possibilities. However, validation addition, Foxl2 has been shown to repress of identified genes in humans will be difficult. human steroidogenic acute regulatory gene Serious complications arise when trying to promoter activity in cell culture (112). Foxl2 compare animal models to human function. Sex- expression in pregranulosa cells may thus be crit- determining genes are evolving at a surprisingly ical in granulosa cell-directed determination of rapid rate and differences in gene function have other ovarian cell lineage fates (111). been seen even among primates (121, 122). As we This said, is FOXL2 a female sex-determining have seen above, dosage effects, timing of expres- gene? It does not appear to be in chickens (109). sion, and threshold requirements for each gene Mice are not sex-reversed even with the homo- interplay with those of other genes in what zygous absence or mutation of Foxl2 (110, 111), increasingly appears to be a finely tuned, complex nor are FOXL2 haploinsufficient BPES females. balance – that varies even within species. However, goats carrying the polled XX intersex Available molecular information regarding sex syndrome (PIS) have a deletion that affects determination and differentiation has already transcription of Foxl2 and one other gene (113), allowed improved clinical diagnosis and manage- raising the possibility of dosage or background ment of sexual pathologies, reflected in Figs 1 and effects masking a sex-determining role for Foxl2 2. As we identify additional sex-determining in non-goat vertebrates. genes in humans and discover their specific functions, we will be able to provide more rapid diagnoses, better categorization of patients, and Conclusions improved prediction of outcomes. Progress has been slow in identifying genes involved in human sex determination. The under- References lying cause of sex reversal still cannot be explained in approximately 75% of patients (114), indicating 1. Goodfellow PN, Darling SM. Genetics of sex determination in man and mouse. Development 1988: 102: 251–258. that there are many as yet unknown genes in this 2. Jost A, Vigier B, Prepin J, Perchellet JP. Studies on sex process. Most of the genes described here were first differentiation in mammals. Recent Prog Horm Res 1973: identified through positional cloning studies of 29: 1–41.

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