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Clinical Genetic Aspects of Consanguinity
Hum Hered 2014;77:108–117 DOI: 10.1159/000360763
Consanguinity and Disorders of Sex Development
Anu Bashamboo Ken McElreavey
Human Developmental Genetics, Institut Pasteur, Paris , France
Introduction
Disorders of sex development (DSD) are defined as ‘congenital conditions in which the development of chro- mosomal, gonadal, or anatomical sex is atypical’ [1] . DSD has recently been coined to encompass terms such as in- tersex, pseudohermaphroditism, hermaphroditism and sex-reversal, which can be confusing to clinicians, pa- tients and parents. The definition provides a rational ba- sis for designating each phenotype. 46,XY DSD includes errors of testis determination or undermasculinization of an XY male due to errors in either androgen synthesis or androgen action. 46,XY gonadal dysgenesis is an error of testis determination that is either complete (complete go- nadal dysgenesis; CGD) or partial (partial gonadal dys- genesis; PGD). 46,XY CGD is characterized by complete- ly female external genitalia, well-developed Müllerian structures and a gonad composed of a streak of fibrous tissue. On the other hand, 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. 46,XX DSD includes overviril- ization or masculinization of an XX individual due to an- drogen excess, and the vast majority of cases of 46,XX DSD are associated with congenital adrenal hyperplasia (CAH) [2] . The much rarer 46,XX testicular DSD refers to a male with testes and a normal male habitus, whereas 46,XX ovotesticular DSD refers to individuals that have
Key Words Consanguinity · Disorders of sex development
Abstract Disorders of sex development (DSD) are defined as ‘congen- ital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical’ [Lee et al., Pediatrics 2006; 118:e488–e500]. Studies conducted in Western coun- tries, with low rates of consanguinity, show that truly am- biguous genitalia have an estimated incidence of 1: 5,000 births. There are indications that the prevalence of DSD is higher in endogamous communities. The incidence of am- biguous 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. This may be due in part to an increase in disorders of androgen synthesis associated with 46,XX DSD. There is clearly a need for further studies to address the fre- quency of DSD in communities with high levels of consan- guinity. This will be challenging, as an accurate diagnosis is difficult and expensive even in specialized centres. In devel- oping countries with high levels of consanguinity, these lim- itations can be compounded by cultural, social and religious factors. Overall there is an indication that consanguinity may lead to an increase in incidences of both 46,XY and 46,XX DSD, and a co-ordinated study of populations with higher incidences of consanguinity/endogamy is needed to resolve this. © 2014 S. Karger AG, Basel
Published online: July 29, 2014
Anu Bashamboo Human Developmental Genetics, Institut Pasteur 25, Rue du Dr. Roux FR–75724 Paris Cedex 15 (France) E-Mail anu.bashamboo @ pasteur.fr
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both ovarian and testicular tissue in the gonads, usually as ovotestes but also less commonly as separate gonads [1] .
There is very limited data available on the incidence rate and prevalence of DSD. In the newborn, truly am- biguous genitalia have an estimated incidence rate of 1: 4,500–5,500 births [3, 4] . However, a concern about the development of the external genitalia may exist in 1: 300 newborn infants. This includes undescended testes or anomalies of the opening of the urethra on the penis (hy- pospadias). Most of the published data on the incidence of DSD is available from Western countries with low rates of consanguinity, and it may not be a true reflection of the worldwide prevalence. In a study conducted in Germany, the incidence of ambiguous genitalia in infants of non- German background was four times higher compared to the general population [3] . This was considered to be due to higher rates of consanguinity in migrant populations, since several conditions in DSD have an autosomal reces- sive inheritance. One hindrance in defining the preva- lence of DSD is the lack of an accurate and sometimes even any diagnosis. In the German study, almost half of the children did not have a definitive diagnosis by the age of 6 months [3] . Indeed, excluding cases where the bio- chemical profile indicates a specific error in steroidogen- esis, it has been estimated that a specific molecular diag- nosis is obtained in about 20% of cases of DSD and that only 50% of 46,XY DSD children will ever receive a de- finitive clinical diagnosis [1] . In this review, we will de- scribe the biology of sex determination and the condi- tions associated with errors in these processes along with the role that consanguinity may play in the incidence of atypical gonad development.
Genetic and Hormonal Control of Sexual Development The urogenital ridge, the common precursor of the
urinary and genital systems, develops at approximately 4 weeks’ fertilization in the human embryo as a thickening of the mesodermic mesonephros covered by coelomic ep- ithelium [5] . At 5 weeks, the human gonadal ridge is bi- potential and must choose between two mutually oppos- ing fates, developing either into an ovary or a testis. At 7 weeks in the XY gonad, SRY is expressed in pre-Sertoli cells and this, in synergy with NR5A1, results in the up- regulation of SOX9 expression beyond the critical thresh- old, leading to the initiation of definitive Sertoli cell dif- ferentiation [6] . Sertoli cells act as the organizing centre of the testis. SOX9 functions by regulating the production of anti-Müllerian hormone from Sertoli cells and repress- es (directly or indirectly) genetic pathways involved in
ovarian development. Once Sertoli cells are formed, they induce the development of fetal Leydig cells, via a hedge- hog signalling pathway. At 8–9 weeks of development, Leydig cells produce androgens and insulin-like factor 3. Testosterone and anti-Müllerian hormone cause the re- gression of Müllerian structures and differentiation of the Wolffian duct into the epididymis, vas deferens and sem- inal vesicles, whereas insulin-like factor 3 is required for testicular descent. Testosterone is the most abundant an- drogen in serum [7] . Approximately 97% of testosterone is bound to albumen and sex-hormone-binding globulin, the remaining 3% are free and biologically active. In male fetuses, it stimulates the differentiation of the Wolffian duct into male internal genitalia (epididymis, vas defer- ens and seminal vesicles) and at puberty it stimulates the libido, enlargement of the vocal cords, skeletal muscles, penis and scrotum and the initiation of spermatogenesis [7] . Intracellular testosterone is converted into dihy- drotestosterone, the preferred ligand for androgen recep- tor transactivation, by the enzyme 5α reductase. Upon ligand binding and transactivation, the dihydrotestoster- one-androgen receptor complex moves from the cyto- plasm to the nucleus and activates the transcription of androgen regulated genes [7] .
In the XX gonad, the absence of SRY results in the in- ability of SOX9 expression to reach a critical threshold. This, together with the expression of factors such as RSPO1/WNT4 and FOXL2, leads to the formation of the ovary, at least in part through suppressing the activity of ‘testis’ genes [8] . Similarly, at least in the mouse, the Ser- toli cells’ fate is constantly reinforced since the loss of the testis-determining gene Dmrt1 in the Sertoli cells of the adult testis allows their transformation into female gran- ulosa cells, which are the female equivalent of Sertoli cells [9] . Thus, the development and maintenance of the mam- malian gonad is regulated by a double repressive system, where equilibrium of mutually antagonistic pathways must be attained for the normal development of either the testis or ovaries [10] .
Consanguinity and Genetic Errors in Testis Determination
There has been a considerable increase in our under- standing of the genetic networks controlling testis deter- mination, but a surprising number of cases of 46,XY go- nadal dysgenesis still remain unexplained [11] . 46,XY CGD and PGD have been reported in association with mutations involving a number of genes that are impor-
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tant for the Sertoli cells’ development, including SRY , SOX9 , NR5A1 , GATA4 , FOG2 , WT1 , CBX2 , ATRX , MAP3K1 and TSPYL1 genes [11] . Recessive mutations involving these genes are rare. In the vast majority of cas- es, the mutations associated with the phenotype are het- erozygous or hemizygous (SRY , ATRX) . This indicates that testis formation is sensitive to the gene dosage of autosomal transcription factors. NR5A1 (also known as SF-1) has originally been identified as a master-regulator of steroidogenic enzymes in the early 1990s which con- trols many key aspects of adrenal and reproductive func- tion [12] . Mutations involving NR5A1 are one of the most common causes of 46,XY DSD and they are associated with a range of phenotypes including 46,XY gonadal dys- genesis and adrenal failure, 46,XY DSD with apparently normal adrenal function, 46,XY infertile males and 46,XX females with ovarian insufficiency [12–15] . Although the vast majority of reported mutations are heterozygous, a few rare homozygous mutations have been identified. A homozygous p.R92Q mutation has been reported in as- sociation with gonadal dysgenesis and adrenal insuffi- ciency [16] . The effects of this homozygous change are variable in functional assays, but the mean functional ac- tivity was in the order of 30–40% of wild-type. A further homozygous mutation (p.D293N) has been reported in association with 46,XY PGD [15] . In this case, the mutant protein retained about half the biological activity of the wild-type NR5A1 protein. In both of these examples, the residual activity of the mutated protein in the homozy- gous state was around 50% of wild-type, a situation simi- lar to heterozygous mutants that have a complete loss of function. A limited number of studies have described mutations involving NR5A1 in North African communi- ties that have a relatively high degree of consanguinity, but these are heterozygous with endogamy having no ap- parent influence on the phenotype [14, 17] .
Approximately 80% of 46,XX individuals with testicu- lar DSD and 10% with ovotesticular DSD carry SRY in their genome that may explain the phenotype [18] . The remaining SRY -negative cases may be explained by either hidden mosaicism of a 46,XY cell line in the gonad, muta- tions in a sex-determining gene downstream from SRY or neomorphic mutations. Rare cases of 46,XX SRY-negative testicular or ovotesticular DSD are due to rearrangements of the regulatory regions of the SOX9 or SOX3 genes [11] . The majority of cases appear to be sporadic, but familial cases have been described that are indicative of a mono- factorial recessive mode of inheritance [19–21] . Many of these pedigrees have been reported in families of North African/Middle Eastern origin, suggesting that there is a
recessive deleterious allele in these populations [ 19–21 , and unpubl. data]. 46,XX SRY -negative ovotesticular DSD is also relatively common in South Africa. As many as 51% of South African patients investigated for ambiguous gen- italia are 46,XX ovotesticular DSD [22, 23] . The mode of inheritance of the phenotype and the genetic cause have not yet been established in this population.
Rare recessive mutations in two genes that function at least in part by repressing the testis pathway have been described. Two Italian families have been identified that carry homozygous mutations in RSPO1 associated with a rare recessive syndrome characterized by XX testicular DSD, palmoplantar hyperkeratosis and a predisposition to squamous cell carcinoma of the skin [24] ; and a further case of 46,XX ovotesticular DSD has been described with a splice-site mutation involving RSPO1 [25] . Four hetero- zygous mutations in WNT4 have been reported in asso- ciation with androgen excess, abnormal development of Müllerian ducts but normal female genitalia [26] . A ho- mozygous WNT4 mutation has been identified in a con- sanguineous family of Arab Muslim origin with an em- bryonic lethal syndrome encompassing 46,XX testicular DSD and the dysgenesis of kidneys, adrenals and lungs (SERKAL syndrome) [27] .
Consanguinity and Disorders of Androgen Synthesis or Action
Anomalies of Leydig Cell Formation and Differentiation Several genes are known to be involved in the specifica-
tion and differentiation of Leydig cells, including the X- linked aristaless-related homeobox gene (ARX), desert hedgehog (DHH) and platelet-derived growth factor re- ceptor alpha (PDGFX). Umehara et al. [28] reported a ho- mozygous missense mutation at the initiating codon, which resulted in failure of translation of the DHH gene in a patient presenting with 46,XY PGD associated with minifascicular neuropathy. In this case of Japanese ances- try, the parents were first cousins. Four other independent homozygous mutations have been reported in association with CGD from India and Mexico without details about consanguinity or the families/communities [29, 30] .
The human LH/CG receptor, a member of the G-pro- tein-coupled receptor family, has two natural ligands: lu- teinizing hormone (LH) and human chorionic gonado- tropin. Human chorionic gonadotropin induces fetal Leydig cell differentiation and testosterone production, whereas LH promotes testosterone production by Leydig
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cells [31] . Homozygous or compound heterozygous inac- tivating mutations of the LHCGR gene encoding the LH receptor can cause Leydig cell hypoplasia in 46,XY indi- viduals [32] . These mutations are rare and the phenotype ranges from completely female external genitalia, a lack of breast development, low testosterone and high LH lev- els and primary amenorrhoea (type I) to normal male sex differentiation with hypospadias or a micropenis (type II). The most severe form is caused by mutations that completely inactivate the LHCGR and thereby block tes- tosterone production. In both forms (type I and II), the internal genitalia of an affected 46,XY individual typical- ly consists of small undescended testes with seminiferous tubules but no Leydig cells. The uterus and fallopian tubes are usually absent. Homozygous and compound hetero- zygous mutations of the LHCGR gene have been identi- fied, but these may explain only around 50% of cases, sug- gesting a contribution of other genetic factors to the phe- notype [33] . Although consanguinity has been widely reported in association with Leydig cell hypoplasia, par- ticularly in cases from Brazil and North Africa, this re- mains a very rare phenotype with an incidence rate of around 1: 1,000,000 individuals [34–36] .
Cholesterol Synthesis Defects Smith-Lemli-Opitz syndrome (SLOS) is characterized
by distinctive facial features, microcephaly, intellectual disability and behavioural problems [37] . Malformations of the heart, lungs, kidneys, gastrointestinal tract and genitalia are also common. SLOS is a recessive choles- terol deficiency syndrome caused by mutations in the DHCR7 gene. The enzyme DHCR7 catalyzes the reduc- tion of 7-dehydrocholesterol to cholesterol. Although the phenotype is due, at least in part, to deficiencies in hedge- hog signalling (as cholesterol is an activator of sonic hedgehog), the gonadal phenotype is due to a reduced testosterone production. The first step of testosterone biosynthesis is the uptake of cholesterol from the extra- cellular space and/or the endogenous synthesis of choles- terol by Leydig cells. A blockage or reduction in this can lead to undervirilization of the genitalia in an XY male. 25% of 46,XY SLOS patients have female external genita- lia, with a further 50% presenting with cryptorchidism and/or hypospadias [38] . SLOS is most prevalent in pop- ulations of Northern and Central European descent and rare in populations of African or Asian descent. Popula- tion surveys have found that the carrier frequency of the most common SLOS mutation (a splice-site mutation; c.964–1G>C) is approximately 1% [39, 40] . Given that this mutation represents <30% of alleles in surveys of af- fected individuals, the carrier rate for SLOS mutations may be as high as 1: 30 [39] . This carrier rate predicts a hypothetical SLOS rate in live births of 1: 2,500–1: 4,500, whereas the recorded SLOS incidence is 1: 10,000– 1: 16,000 births. This discrepancy between the calculated and observed incidence rates could be due to undiag- nosed mild cases, misdiagnosed severe cases, death prior to diagnosis or fetal loss. The high carrier frequencies for DHCR7 mutations suggest the possibility of a heterozy- gote advantage. These include increased vitamin D levels, providing protection against vitamin D-deficient rickets and autoimmune diseases, or increased resistance to cer- tain infections resulting from an increased 7-dehydro- cholesterol concentration in cell membranes. In North- ern European populations, heterozygosity for a null DHCR7 mutation may have provided protection against the development of vitamin D-deficient rickets [41] . Lipoid CAH StAR (the steroidogenic acute regulatory protein) is required for the transport of substrate cholesterol from the cytosol to the inner membrane of the mitochondria, where the steroid biosynthesis is initiated [42] . Lipoid CAH is a rare autosomal recessive DSD characterized by greatly reduced or absent synthesis of all adrenal and go- nadal steroids [43] . The resultant adrenal deficiency of mineralocorticoid and glucocorticoid hormones usually presents during the first weeks of life with significant fail- ure to thrive [43] . Affected individuals are phenotypic fe- males or rarely have slightly virilized external genitalia with or without cryptorchidism. Lipoid CAH caused by mutations in StAR is most frequent in East Asian and Pal- estinian communities with evidence of founder muta- tions, and it is relatively rare in Europe and America. The Q258X mutation accounts for 70% of mutations in StAR observed in Japanese and Korean cohorts [44, 45] . In the latter population, the carrier frequency has been estimat- ed at 1: 250 and may represent a founder mutation [45] . A p.R182H mutation has been reported in 5 apparently un- related families from Eastern Saudi Arabia and in a pa- tient from Qatar [46] . Interestingly, the patients were homozygous for the p.R182H mutation and showed substantial variation in disease severity and their age at symptom onset (1–14 months). The cause of the dramat- ic spectrum of clinical presentations in the same ethnic group with the same mutation is unclear. In 4 apparently unrelated Palestinian families with lipoid CAH, a homo- zygous c.201_202delCT has been detected that was pre- dicted to result in the premature truncation of the protein [47] . Informative markers at the StAR locus indicated Bashamboo/McElreavey Hum Hered 2014;77:108–117 DOI: 10.1159/000360763 112 that the c.201_202delCT mutation is a Palestinian found- er mutation. Although the 4 consanguineous families were not aware of any common ancestry, they identified their ancestral backgrounds to Hebron, Jerusalem and Ramallah. Congenital Adrenal Hyperplasia One of the most commonly inherited metabolic disor- ders is CAH. The most common form of CAH is due to a deficiency of 21-hydroxylase, which is caused by muta- tions in the 21-hydroxylase gene (CYP21A2) , and ac- counts for 90–95% of all cases [48, 49] . 21-hydroxylase- deficiency-induced CAH represents the most frequent cause of 46,XX DSD. The condition can be divided into classic (severe salt wasting and less severe simple viriliz- ing) and non-classic (or mild/late onset) forms with hy- perandrogenism being the most common clinical mani- festation [49] . Classic 21-hydroxylase deficiency is char- acterized by an impaired cortisol production leading to the accumulation of metabolic intermediates (progester- one and 17-hydroxyprogesterone) that results in an ex- cessive androgen production leading to varying degrees of virilization. In the classic form, progressive postnatal virilization can be seen that may include progressive pe- nile or clitoral enlargement, precocious pubic hair and hirsutism. The non-classic form of CAH results from a mild deficiency of 21-hydroxylase that does not induce ambiguity of the external genitalia at birth in females, but may postnatally result in varying degrees of symptoms associated with androgen excess [49] . The CYP21A2 gene is located on chromosome 6p21.3, 30 kb from the CYP21A1P pseudogene. Both CYP21A2 and CYP21A1P show sequence identity of 98% between exons and 96% between introns. The high sequence iden- tity and close proximity of CYP21A2 and CYP21A1P may result in unequal crossing-over events during meiosis or gene conversion, thus predisposing the locus to numerous rearrangements resulting in the transfer of deleterious mu- tations from the pseudogene to CYP21A2 [50] . De novo mutations in CYP21A2 associated with CAH are rare (3– 5%), with the vast majority of these mutations identified either in single families or small populations [50] . The overall prevalence at birth of the classic form of CAH in the USA is estimated at 1: 20,800 and in the UK at 1: 18,000 [51, 52] . However, the distribution of the con- dition varies with ethnicity. For example, in the UK 25% of all cases concerned individuals of Asian ethnic origin [52] . The highest frequencies of CAH are found in iso- lated populations. The highly endogamous Yupik Eski- mos of Southwestern Alaska carry an A>G transition in
the second intron of CYP21A2, and the frequency of CAH in this population is 1: 282 [53] . The frequency of the clas- sic form amongst the people of the island of La Reunion, France, is 1: 2,100 [54] . Differences in the incidence rates of the classic form are highlighted by African Americans, who have a lower rate than the white US population (1: 42,000 vs. 1: 15,500 [55] ). Lower incidence rates have also been reported in Asia [56] . In Western Australia, the overall incidence of classic CAH was estimated to be similar to figures for white Americans at around 1: 15,000; however, the ratio of the Aboriginal rate com- pared with the non-Aboriginal rate for CAH was 2.45 [57] . The basis for this increased rate is unknown since no genetic data is available [57] . In a Tunisian cohort, a sin- gle Q318X mutation in CYP21A2 had a large prevalence of 35.3% [58] . This is likely to be a founder mutation, since linkage disequilibrium was found between this mu- tation and a CYP21 gene polymorphism in 83.3% of al- leles [58] . Consanguinity was high in this study cohort; it was present in 31 families (60.8%), absent in 15 and un- known in 5. The consanguineous families were unrelated and originated from different regions of Tunisia [58] .
The estimated incidence rate of the non-classic form is about 1: 1,000 in the general Caucasian population, but it might be much more frequent in certain ethnic groups such as East European Jews that carry ancestry-specific mutations [59] . In one study, 63% of Ashkenazi Jews with non-classic CAH carried the V281L (1685G>T) mutation in CYP21A2 [60] .
CAH due to 11β-hydroxylase deficiency is an autoso- mal recessive disorder of corticosteroid biosynthesis re- sulting in androgen excess, virilization and hypertension [50] . It accounts for approximately 5–8% of all cases worldwide; however, the incidence rate can be much higher depending on the study population. In affected XX individuals, the phenotypes vary from an enlarged clitoris in the mildest forms to a severely hypertrophied clitoris with penile urethra and fused labial-scrotal folds [49] . Pa- tients with this disorder are unable to convert 11-deoxy- cortisol to cortisol. Elevated levels of adrenocorticotropic hormone (ACTH) cause steroid precursors to accumu- late proximal to the blocked step which are then shunted into the pathway for androgen biosynthesis as occurs in 21-hydroxylase deficiency [49] .
There are considerable differences in the ethnic distri- bution of 11β-hydroxylase deficiency. 11β hydroxylase has been noted at high frequencies in Israeli families of Moroccan, Tunisian, Turkish and Iranian origin [61] . The incidence in Jews of Moroccan origin is estimated to be 1: 5,000–1: 7,000 births [62] , whereas it is estimated to
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be 1: 100,000 in Caucasians [63] . White et al. [64] identi- fied a homozygous p.R448H mutation in 6 independent Israeli families of Moroccan ancestry and postulated that this was a potential founder mutation in this relatively endogamous community with a carrier rate of 1: 40. How- ever, further studies have suggested a much lower carrier frequency, suggesting that other gene mutations may be involved [65] . The R448H mutation may not be limited to Jews of Moroccan ancestry, since a Caucasian patient has been reported homozygous for this mutation [66] . Furthermore an Iranian and a Turkish patient were each found to be homozygous for a different mutation at the same codon, R448C, suggesting that this codon may be a mutational hotspot [66, 67] .
In 15 unrelated Tunisian patients with classic 11β-hydroxylase deficiency, two homozygous mutations have been detected in the CYP11B1 gene: p.Q356X (26.6%) and p.G379V (73.3%) [68] . Consanguinity was reported for 12 of the 15 apparently unrelated families from different parts of the country. Similar to the situa- tion with CYP21A2 mutations in Tunisia, these muta- tions are considered to be founder mutations [68] .
Recessive or compound heterozygous mutations in CYP11A1 and HSD3B2 (3β-hydroxysteroid dehydroge- nase type 2 deficiency) are relatively rare causes of CAH [50] . There is a lack of data regarding population-based incidence rates of mutations in these genes. The CYP17A1 gene encodes steroid 17α hydroxylase, an enzyme in- volved in cortisol and sex steroid synthesis in the adrenal glands and gonads. Mutations in CYP17A1 result in mild to severe undervirilization of 46,XY individuals. Although there is a lack of evidence for founder mutations or muta- tional hotspots within CYP17A1 , in the Brazilian popula- tion, p.W406R and p.R362C represent 50 and 32% of mutations, respectively [69] . However, the majority of all affected individuals carrying the p.W406R or p.R362C mutations are of Spanish or Portuguese ancestry, respec- tively, therefore indicating a founder effect [69] . Recur- ring mutations have also been described in unrelated in- dividuals of Friesian origin from the Netherlands. A 4-base duplication within exon 8 of the CYP17A1 gene has been identified in 6 families with 17α-hydroxylase/17,20- lyase deficiency residing in Dutch Friesland [70] . The same duplication has been found in 2 unrelated Canadian Mennonite individuals [71] , who derive their name from Menno Simons, an early leader of a sect in Friesland. This indicates that this 4-base duplication may represent a founder effect originating in Friesland.
CAH due to P450 oxidoreductase deficiency is an au- tosomal recessive disorder, due to mutations in the P450
oxidoreductase (POR) gene [53] . In contrast to all other forms of CAH, POR deficiency (PORD) results in severe sexual ambiguity in both sexes (46,XX and 46,XY DSD). Affected girls may present with significant virilization of the external genitalia, indicating prenatal exposure to an- drogen excess. Affected boys show varying degrees of un- dervirilization ranging from a borderline micropenis to severe perineoscrotal hypospadias. On the other hand, normal genital development in both 46,XX and 46,XY in- dividuals has also been reported in cases of PORD [72] . Affected children can also present with bone malforma- tions, similar to the pattern seen in patients with Antley- Bixler syndrome. POR is the crucial electron donor to all microsomal cytochrome P450 (CYP) enzymes including the steroidogenic enzymes CYP21A2, CYP17A1 and P450 aromatase (CYP19A1). The mutations in POR re- sult in deficiencies in steroid synthesis and thus in the observed phenotype. The incidence rate of PORD in the general population is unknown as the phenotype shows considerable variability and can be difficult to diagnose accurately [50, 72] .
In PORD patients of Caucasian origin, the mutation p.A287P is most frequently observed [72] . In one study on a large cohort of PORD patients, this mutation was observed in 23 of 54 alleles (43%) [72] . The frequency of this variant (rs121912974) in the general population is 4: 8,410 alleles in the European American population (NHL- BI Exome Sequencing Project: http://evs.gs.washington. edu/EVS/). In Japanese patients with PORD, a p.R457H variant has been observed in 47 of 70 alleles studied (67%) [73] . No POR patient carrying non-sense mutations on both alleles has been described so far, suggesting incom- patibility of such genotype with postnatal life. Major loss- of-function mutations on one of the affected alleles are associated with severe malformations, whereas homozy- gosity or compound heterozygosity for missense muta- tions is associated with a mild to moderate phenotype. Importantly, homozygosity for the most common muta- tion in Caucasians, p.A287P, allows for prediction of the genital phenotype and is associated with mild to moder- ate malformations [72] .
Consanguinity and Disorders of Androgen Function
5α-Reductase Deficiency In Salinas, a remote village in the Dominican Republic,
2% of live births in the 1970s were male pseudohermaph- rodites, known locally as ‘guevedoche’ or ‘guevedoce’ from the Spanish phrase ‘huevos a las doce’ meaning ‘eggs
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at twelve’. These children, who appeared to be female or male with ambiguous genitalia at birth, developed a pe- nis, testicles and all of the typical male physical character- istics at the time of puberty. Most were found to be de- scendants of a single common ancestor: Altagracia Car- rusco [74, 75] . Studies showed that these individuals carried a mutation in the 5α-reductase type 2 (SRD5A2) gene that converts testosterone to dihydrotestosterone; the androgen with the highest affinity for the androgen receptor [74] . Other independent mutations have been reported in the SRD5A2 gene in affected individuals from the Dominican Republic suggesting multiple founder mutations [76] .
SRD5A2 deficiency is a rare condition and apart from the studies in the Dominican Republic the exact inci- dence rate is largely unknown, although consanguinity has been observed in around 40% of all reported cases [77] . A cluster of male pseudohermaphrodites in the Sim- bari Anga linguistic group in the Eastern Highlands of Papua New Guinea [78] and large consanguineous fami- lies with affected members have been reported in Turkey and Egypt [79, 80] . These and other studies are case re- ports on limited numbers of consanguineous families or patient clusters from the same geographical area and/or ethnic background that resulted in the belief that SRD5A2 mutations were present in specific and isolated popula- tions. However, recently 5α-reductase deficiency has also been identified in populations not considered at risk such as those from Belgium, France, Italy, Poland, Spain or Quebec [81] .
Conclusions
Data on the actual prevalence of DSD in developing countries with high rates of consanguinity or endogamy is largely unavailable. The limited numbers of studies that are published describe, for the most part, small series of patients and may not be reflective of a population as a whole. There is, however, some evidence for 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 [82, 83] . Both figures are higher than the reported frequency of 1: 4,500–1: 5,500 in European countries, indicating that consanguinity may be a contributing factor to an increase in the incidence rate of DSD. In one recent retrospective study from Sudan, of 122 DSD cases, 69 had 46,XX DSD and 45 had 46,XY DSD. The most common cause of 46,XX DSD was CAH
21-hydroxylase deficiency (52 of 62 cases with a proven aetiology), whereas androgen insensitivity was the most frequent cause of DSD in 46,XY individuals. 70% of all 122 cases were born to first-cousin marriages [84] . A further small study of 26 DSD cases from Sudan observed paren- tal consanguinity in 70% of cases, 10% reported no con- sanguinity, whilst 20% did not provide sufficient family history to ascertain consanguinity [85] . This frequency of consanguinity is higher than in individuals of Arab de- scent in Khartoum, which was estimated at around 52% [86] . Similarly, consanguinity has been proposed as the cause of a high frequency of 46,XX DSD (CAH in 65.4% of all DSD cases) in one referral centre in Saudi Arabia over a 20-year period [87] . Other studies from Saudi Ara- bia have reported similar findings [88] . However, Mazen et al. [83] suggest a somewhat different relationship be- tween consanguinity and DSD. They reported on consan- guinity and parental origins of 208 DSD patients from Egypt; the consanguinity rate was 62.8% for all DSD cases (72% in 46,XX DSD and 59.5% in 46,XY DSD) [83] . How- ever, in contrast to other studies of Arab populations, 46,XY DSD was more common than 46,XX DSD, consti- tuting 65.9% of the total cases [83] . The higher rates of 46,XY DSD may reflect an unidentified selection bias or may represent regional differences in the general inci- dence and nature of pathogenic mutations that could give rise to DSD. The rate of consanguinity associated with DSD (62.8%) is also higher than in the general Egyptian population, where studies have reported that consanguin- ity rates during the last 40 years have been ranging be- tween 29 and 39% with considerable differences between urban and rural populations [89–92] . Although some of this increase may be due to the CAH cases, it is more dif- ficult to explain the high degree of consanguinity in 46,XY DSD cases, unless there are novel recessive mutations re- sponsible for the phenotype in this population.
There is clearly a need for further studies to address the prevalence of DSD in communities with high levels of consanguinity. This will be challenging for several rea- sons, many of which could apply equally to communities with low levels of consanguinity. Until very recently, there has been a multitude of terms used to describe DSD and, consequently, in many older publications the precise clinical phenotype is confusing. This makes it very diffi- cult to accurately assess older datasets. As mentioned ear- lier, only around 50% of all 46,XY DSD children will ever receive a definitive clinical diagnosis even at a specialized clinical centre [93–95] . Although a number of diagnostic algorithms exist, no single evaluation protocol is suitable for all circumstances and some basic tests, such as hor-
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mone measurements, are not only expensive but need specialized interpretation of the specific assay character- istics compared to normal values for gestational and chronological age. In some cases serial measurements may be needed to enable an accurate interpretation of lab- oratory tests. In developing countries with high levels of consanguinity, these limitations of diagnostic and treat- ment facilities are compounded by cultural, social and re- ligious factors that can affect the management of patients. In many developing societies, female infertility precludes marriage, which also affects employment prospects, and this drives the DSD patient’s family to choose the male sex. Indeed, several studies have indicated the preference of the male sex of rearing despite the severity of genital ambiguity [83, 96, 97] . Furthermore in some traditional communities, parents feel that their child’s condition is spiritual rather than medical and do not seek detailed clinical evaluation of the condition [98] .
There are indications that consanguinity may lead to an increase in the incidence rates of both 46,XY and 46,XX DSD, depending on the population studied. A co- ordinated study of worldwide or region-wide populations with a higher incidence of consanguinity/endogamy may provide a definitive answer.
Acknowledgements
A.B. is funded in part by the program Actions Concertées Interpasteuriennes (ACIP). A.B. and K.M. are funded by a re- search grant from the EuroDSD in the European Commu- nity’s 7th 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 Medi- cine, Non Communicable Diseases Investigation and interna- tional cooperation – as part of the EU call FP7-INCO-2011-6. The work is also funded by a Franco-Egyptian AIRD-STDF grant.
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