Which Parent Decides the Gender of the Baby? Genes Sry Dax Wnt4 X_linked

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

Disorders of sex development (DSD) are defined as congenital weather 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 activity reversal [Lee et al., 2006]. These terms were ofttimes confusing, both to clinicians and patients as well as to other family members. This umbrella definition of DSD provides a rational basis for the nomenclature of a range of conditions, merely more than importantly, information technology 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 activeness. Errors in testis decision may manifest as either consummate gonadal dysgenesis (CGD) or partial gonadal dysgenesis (PGD). 46,XY CGD is characterized by completely female external ballocks, well-adult Müllerian structures, and a gonad composed of a streak of gristly tissue, whereas 46,XY PGD is characterized by partial testis formation, ordinarily a mixture of Wolffian and Müllerian ducts, and varying degrees of masculinization of the external genitalia. Embryonic testicular regression sequence tin can as well exist 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 cryptic external or internal ballocks. Gonad tissue is absent on i 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 likewise raises the concept that nosotros are dealing with a continuum of phenotypes rather than clearly distinct and unrelated categories of atypical testicular formation. The genetic arguments too 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 fractional 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 centrality is compromised. In 46,XY individuals CHH is characterized by delayed or absent sexual evolution and infertility associated with inappropriately depression gonadotropin (LH and FSH) and testosterone levels. Male patients oftentimes show nether-androgenisation with micropenis and cryptorchidism observed at nascency. When anomalies of smell, hyposmia or anosmia, is associated with hypogonadotropic hypogonadism, in 60% of patients the disease is chosen Kallmann syndrome. This combination of phenotypes is explained by the mutual embryonic origins and developmental pathways of GnRH and olfactory neurons. More than xx 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 46,20 DSD are due to built adrenal hyperplasia (CAH). The almost common form of CAH is due to deficiency of 21-hydroxylase, which is acquired by mutations in the 21-hydroxylase cistron (CYP21A2) and accounts for xc-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 pocket-size 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, normally ovotestes but less usually a testis (or ovotestis) on one side and an ovary on the other [Ergun-Longmire et al., 2005]. The external genitalia are ordinarily cryptic or feminine, with the caste 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-similar 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,Ten/46,XY (mixed gonadal dysgenesis or OTDSD), and 46,Xx/46,XY (chimerism or OTDSD). 45,10/46,XY mosaicism is one of the about common causes of DSD and also ane of the about hard for predicting genotype-phenotype correlations. In several studies, no correlation was establish betwixt 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 testify balmy intellectual inability or signs of autism.

A large number of syndrome associations of DSD have been described including cloacal anomalies, Robinow, Aarskog, Paw-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 sexual activity conclusion.

Incidence of Disorders of Sex Development

There is very limited information available on the precise incidence of DSD. This reflects both the rarity of some of these conditions equally well as the challenge of achieving a definitive clinical diagnosis. In the newborn, truly ambiguous ballocks that may pose a trouble for binary gender consignment has an estimated incidence of 1:4,500-5,500 births [Thyen et al., 2006; Sax, 2002]. Overall, around fifty% 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 effectually 1:100,000 births [Lee et al., 2016]. TDSD/OTDSD are estimated to occur in i:100,000 births [Sax, 2002]. More commonly, developmental anomalies of the external genitalia may be 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). All the same, most of this published data on the incidence of DSD is bachelor only from Western countries; therefore, the worldwide prevalence of DSD is unclear. A German study indicated that the incidence of cryptic genitalia in infants of non-German language background was 4× higher compared to the full 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. At that place is some evidence to support the hypothesis that there is a higher charge per unit of DSD in societies with a college rate of consanguinity. The incidence of ambiguous genitalia in Kingdom of saudi arabia has been estimated at i:2,500 live births, whilst in Egypt information technology has been estimated at one:3,000 live births [Abdullah et al., 1991; Mazen et al., 2008], which is higher than the reported frequency of one:4,500-one:5,500 in European countries. Another hindrance in defining the prevalence of DSD is the lack of an authentic or even any diagnosis in many cases. In the German language written report almost one-half of the children did non have a definitive diagnosis by the age of 6 months [Thyen et al., 2006]. Excluding cases where the biochemical contour indicates a specific mistake in steroidogenesis, information technology has been estimated that a specific molecular diagnosis is obtained in most twenty% of cases of DSD and that only fifty% 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 sexual activity determination and therefore DSD.

Factor Mutations and 46,XY Gonadal Dysgenesis

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 pocket-size interstitial deletions v′ and 3′ to the SRY factor, as well equally within the SRY promoter itself [McElreavey et al., 1992, 1996; Assumpção et al., 2005]. In dissimilarity to the mouse, the human being SRY protein is expressed in Sertoli cells and germ cells from the moment of testis determination until adulthood. The function, if any, of SRY in germ cells is unknown since SRY mutations are associated with a failure of testis development. SRY mutations are unremarkably de novo, only some are inherited from a fertile begetter. Functional studies suggest that these inherited mutant proteins retain partial biological activity, and the incomplete penetrance could be acquired 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 straight target of SRY is SOX9, another HMG-box containing protein. Sox9 plays both an essential office 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 besides as establishing Sertoli cell identity in the developing testis immediately post-obit the expression of SRY. Many mutations take been reported in the SOX9 gene and upstream and downstream flanking regions associated with campomelic dysplasia (CD). At that place is a variable severity of testicular dysgenesis in about 75% of affected XY individuals [Foster et al., 1994]. Notwithstanding, recently ii 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 equally having spina bifida. Both of these mutations are located in the C-concluding PQS-rich domain of SOX9 that is involved in protein-protein interactions with factors such equally 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 aforementioned phenotypic continuum.

Besides the bespeak mutations, rearrangements involving the SOX9 locus are as well known to upshot in not-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.vi 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 easily/feet, hyponychia or anonychia) [Kurth et al., 2009]. Pierre Robin sequence is a craniofacial disorder characterized past micrognathia, cleft palate, and macroglossia that is associated with normal testis evolution and is acquired by either a 75-kb deletion located one.38 Mb upstream or a deletion located one.56 Mb downstream of SOX9 [Benko et al., 2009]. In mice, a testis-specific enhancer element has been mapped to a 1.iv-kb region termed Tesco that is located 13 kb upstream of Sox9 [Sekido and Lovell-Bluecoat, 2008]. Both Sry and Nr5a1 bind to the Tesco enhancer sequence in vivo, maybe through a direct physical interaction to upregulate Sox9 gene expression. Once Sox9 poly peptide levels accomplish a disquisitional threshold, several positive regulatory loops are initiated for its maintenance, including autoregulation of its own expression and formation of feed-forrard loops via Ffg9 or Pgd2 signaling. Other cofactors are likely to be involved in this procedure simply have not nonetheless been fully characterized. Mutations involving the human being TESCO element accept not been reported equally 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 example 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 two predicted enhancer motifs [Hyon et al., 2015]. There is besides information suggesting that deletions of an immediately adjacent and not-overlapping region are associated with 46,XY gonadal dysgenesis [Kim et al., 2015]. In our feel, about 10% of cases of 46,Twenty TDSD/OTDSD and 46,XY gonadal dysgenesis take rearrangements involving the RevSex locus [unpubl. data].

NR5A1

Approximately 15% of all cases of 46,XY DSD are acquired past mutations involving the cistron NR5A1. NR5A1 belongs to the subfamily of transcription factors known equally nuclear receptor subfamily five (group A, fellow member 1), which is highly conserved in vertebrates [Morohashi et al., 1992]. The protein consists of a Deoxyribonucleic acid-binding motif equanimous of 2 zinc-chelating modules that coordinate the interaction between the receptor and hormone response element [El-Khairi and Achermann, 2012]. NR5A1 binds Dna every bit a monomer, with Dna binding stabilized via a xxx amino acid extension termed the A-box. The C-concluding ligand-binding domain (LBD) is required for maximal biological action 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 information technology 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 master 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 report 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 human being with azoospermia was suggestive of a mild course of testicular dysgenesis rather than Sertoli-prison cell-simply syndrome, once again reinforcing the idea that errors in testis determination can manifest as dissimilar 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 cardinal gene in human sexual practice determination, NR5A1, are associated with either male or female infertility establishes a link between human being sex determination and fertility.

The mechanism behind the phenotype variability associated with NR5A1 mutations is however 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 comport novel or rare variants in other genes involved in sexual evolution that may influence the severity of the phenotype. We reported 2 individuals with the aforementioned missense mutation p.Arg313Cys in NR5A1 [Allali et al., 2011; Mazen et al., 2016]. In the first example information technology was associated with mild hypospadias [Allali et al., 2011], but in the 2nd case it was associated with 46,XY gonadal dysgenesis [Mazen et al., 2016], and in both cases the mutation was de novo. The more than severe phenotype may be explained by digenic inheritance since the patient carried a missense mutation in the MAP3K1 gene (see beneath). In other cases of DSD carrying mutations in NR5A1 the severity of the phenotype may be a consequence of the specific amino acrid involved (see NR5A1 p.R92W below). A homozygous p.R103Q NR5A1 mutation was reported in a kid with severe 46,XY DSD and absent spleen. The mutation decreased the ability of the mutated NR5A1 protein to transactivate TLX1, a transcription cistron that is essential for spleen evolution [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 vest to a course of evolutionarily conserved lineage-limited zinc finger transcription factors characterized by the presence of 2 conserved type Iv zinc finger domains that participate in jail 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 conclusion and differentiation [Viger et al., 2008]. Male mice lacking Gata4 prove partially descended pocket-sized testis with irregular cords, are infertile, and lack expression of Dmrt1 during embryogenesis [Manuylov et al., 2011]. Gata4 ^ki mice, which carry a p.Val217Gly mutation in the N-concluding zinc finger domain of the poly peptide 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 prison cell function, for example a Gata4/Mef2 circuitous regulates Star factor 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 cistron accept hypospadias and bilateral cryptorchidism [Wat et al., 2009]. We identified a familial case of 46,XY DSD and congenital eye 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 Gata4 ^ki mutation in the Due north-terminal zinc finger domain [Lourenço et al., 2011]. In functional studies the p.Gly221Arg variant failed to demark 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 even so to exist established, nosotros have identified other heterozygous missense mutations in GATA4 in 46,XY DSD patients who have no evidence of center disease [unpubl. data].

FOG2 (likewise 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 cardinal genes involved in testis determination such equally Sry and Sox9 is dramatically reduced, it suggests that Fog2 is involved in the early stages of testis conclusion. The bear witness that FOG2 may be involved specifically in homo testis determination was suggested by ii cases of 46,XY gonadal dysgenesis in association with plain balanced translocations that included the FOG2 locus on chromosome eight (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 circuitous somatic anomalies were reported. Using exome sequencing, we identified 2 independent cases, of 46,XY gonadal dysgenesis each with missense mutations in the FOG2 cistron [Bashamboo et al., 2014]. In that location 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 exist 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

CBX2 encodes for chromobox homolog two, a component of the polycomb grouping (PcG) complex of regulatory proteins. In mammals, PcG proteins are associated with 2 master families of complexes, referred to as polycomb repressive complex one (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 defective 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 Twenty 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 ii 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.five years revealed apparently normal ovaries. Although polycomb group proteins are traditionally regarded as transcriptional repressors, there is testify that at to the lowest degree 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 private. Recently, several potential downstream targets of CBX2 that are relevant to testis decision take 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 function of CBX2 (and by implication the PcG circuitous) in sex determination is unknown, although both the human and mouse phenotypes suggest that it is acting at an early stage of gonad formation.

MAP3K1

The mitogen-activated protein kinases (MAPKs) are activated through an evolutionarily conserved three-component signal transduction pour equanimous 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 to the lowest degree 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 filibuster in Sry expression [Gierl et al., 2012]. Furthermore, the absence of both the p38a and p38b MAPK isoforms results in XY sexual activity reversal associated with reduced Sry expression [Warr et al., 2012]. Recent data also propose that Map2k6 is required for the normal spatiotemporal expression contour of Sry [Warr et al., 2016]. Therefore, at to the lowest degree in mice, available data are consistent with a GADD45γ/MAP3K4/p38 pathway which is required for the appropriate timing of Sry expression in sex decision.

Nineteen MAP3Ks are present in mammals, though their precise biological roles are non fully understood. MAP3K1 (also known equally MEKK1) plays a office 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 activeness, a caspase-iii 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, prison cell shape disturbance, pro-inflammatory cytokines, and many other physiological stresses [Yujiri et al., 1998]. In humans, mutations in MAP3K1 accept been identified in cases of 46,XY DSD [Pearlman et al., 2010; Loke et al., 2014]. The mechanism whereby MAP3K1 mutations crusade 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 non been identified, and considering the fact that MAP3K1 has a widespread expression pattern these more than severe mutations may be embryonic lethal. Available data suggest that mutations observed in DSD cases may exist 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 defective Map3K1 are viable and fertile only with an increased embryonic gonadal length [Warr et al., 2011]. XY Map3k1 ^mPHD/+ 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]. Some other Map3k1 mouse model, goya, exhibits a severe hearing loss [Parker et al., 2015]. This information propose that in testis determination either the MAP kinase signaling pathways in human being or mouse have diverged or the difference in phenotype is caused by an intrinsic deviation in the type of mutation. In our experience nigh ten% 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 man 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 on stages of testis conclusion in humans, too.

DMRT1, an Evolutionary Conserved Sex-Determining Gene

Deletions of final 9p are associated with monosomy 9p syndrome, which is characterized by intellectual inability together with a distinctive series of somatic anomalies, and in approximately seventy% of 46,XY individuals anomalies of testis development are seen that range from a completely female person phenotype to a male person 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 sexual practice fate option in Drosophila, and mab-3 is necessary to confer male traits in C. elegans. In mice, Dmrt1 is not required for testis decision, nevertheless, its continuous expression in the adult testis is required to maintain organ identity, because forced attenuation of Dmrt1 expression in developed testis results in transdifferentiation of the testis to an ovary [Matson et al., 2011]. Although deletions of 9p24 advise that DMRT1 hemizygosity is sufficient in some individuals to lead to a failure of testicular development, these deletions ordinarily remove other genes, including the evolutionary related DMRT2 and DMRT3 genes [Ottolenghi and McElreavey, 2000]. Formally, the phenotype could exist due to haploinsufficiency of i, 2, or all 3 of these DMRT genes. Show to signal that the key player in human testis determination is DMRT1 came through the identification of a de novo missense mutation in the functionally of import DM-Deoxyribonucleic acid-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 testify of testicular cloth, 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 poly peptide, it altered the stoichiometry of the wild-type poly peptide [White potato 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 action. This ascertainment may besides 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 accept only identified a unmarried mutation associated with the phenotype. The residual variation intolerance score (RVIS) ranks human genes by their deviation from the genome-broad average number of nonsynonymous mutations institute in genes with a like amount of global mutational burden (http://genic-intolerance.org/). Mendelian disease causing genes are less tolerant to coding variations than other genes. Put just, genes known to behave few common functional variants in healthy individuals may be considered to be more than probable to cause sure diseases, such equally 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 information within the NHLBI-ESP6500 information fix. A skilful instance is the cistron SOX9, which has an RVIS score of 14.4% and a arrears in loss-of-function variants (%ExAC_RVIS) among the nine.81% lowest of the human genome. Equivalent %ExAC_RVIS figures for NR5A1, MAP3K1 and CBX2 are 15.76, 6.58 and 11.63%, respectively. All the same, 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 salubrious individuals than the other genes mentioned above. This suggests that for a DMRT1 mutation to exist pathogenic (or penetrant) resulting in a failure of testis determination, information technology may exist required to prove either dominant negative action on the wild-type allele or modify the normal interactions of the protein.

Gonadal anomalies may non 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, just the expression of DMRT1 was constitute to be higher in lung macrophages from men with various lung diseases [Schieck et al., 2016].

46,20 Testicular and Ovotesticular DSD

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 sexual practice determination may upshot in testicular evolution in a chromosomal 20 female. Although most cases of 46,XX TDSD/OTDSD are acquired 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 being, SOX3 loss-of-part mutations are associated with mental retardation and growth hormone deficiency [Laumonnier et al., 2002]. However, three 46,XX SRY-negative testicular DSD patients take been reported who conduct rearrangements at the SOX3 locus on the X chromosome. Ane patient carried 2 microduplications, one of ∼123 kb that spanned the entire SOX3 cistron and some other of 85 kb that was located 350 kb proximal to SOX3. A 2d patient carried a single 343-kb microdeletion immediately upstream of SOX3, and a 3rd Xx male person with multiple built anomalies carried a vi-Mb duplication including SOX3 and at least xviii 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 farther duplications of the SOX3 gene take been reported in Xx males [Moalem et al., 2012; Vetro et al., 2015; Grinspon et al., 2016].

Consummate or partial duplications of chromosome 22 in 46,Twenty-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 past 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], all the same, 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 man ovary development. In 20 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 full general, RSPOs stimulate WNT signaling by bounden to the leucine-rich repeat-containing K protein-coupled receptors LGR4, LGR5, and LGR6 [Wang et al., 2013]. RSPOs tin can also bind to two negative feedback regulators of the WNT signaling pathway, the RING-blazon 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 past XX testicular DSD, palmoplantar hyperkeratosis, and predisposition to squamous prison cell carcinoma of the peel [Parma et al., 2006]. Mutations involving RSPO1 accept not been reported in not-syndromic cases of testicular and ovotesticular DSD [unpubl. data].

The absenteeism of Wnt4 in XX mice results in a fractional 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 aberrant evolution 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 unit with an embryonic lethal syndrome of 46,XX testicular DSD and dysgenesis of kidneys, adrenals, and lungs (SERKAL syndrome; Sexual activity Reversion, Kidneys, Adrenal and Lung dysgenesis) [Mandel et al., 2008].

NR5A1 p.Arg92Trp and 46,Twenty DSD

Primary ovarian insufficiency, also termed premature ovarian failure, is defined past the arrest of normal ovarian role before the historic period of 40 years and includes premature menopause, primary and secondary amenorrhea as well every bit ovarian dysgenesis. In 2007, when nosotros were analyzing cases of 46,XX primary ovarian insufficiency for mutations in the NR5A1 gene, a patient (sporadic example ii; Lourenco et al. [2009]) was clinically investigated at 4 months of age considering of a hypertrophy of the clitoris. The daughter 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 factor. We rapidly discovered a minor family with 2 virilized sibs who carried a p.Arg92Trp mutation. The unaffected mother likewise carried this mutation. At the time, this observation was interesting but could just have been explained every bit a take a chance finding - a rare genetic variant unrelated to the phenotype. This view was supported by our screen of further forty cases of 46,XX DSD that did not reveal any other NR5A1 mutations. In 2015, nosotros were contacted by different groups who had also identified the same NR5A1 p.Arg92Trp mutation in association with 46,Twenty testicular DSD [Bashamboo et al., 2016]. Two families had a unmarried affected child who carried a de novo NR5A1 p.Arg92Trp mutation, and this formally excluded a founder mutation. A fourth family unit was identified with 2 afflicted sibs, one with 46,XY gonadal dysgenesis and raised equally a girl and the other a boy with 46,Twenty testicular DSD. They both carried the mutation. A number of mutations have been described in NR5A1 in 46,20 individuals in association with ovarian insufficiency, merely we are unaware of any other amino acid changes in NR5A1 except p.Arg92Trp that is associated with virilization in 46,20 individuals. Interestingly, a homozygous p.Arg92Gln alter 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. one). The mutation involves a highly conserved amino acid residue located in the A-box motif of the protein. The A-box consists of a thirty amino acrid basic region carboxyl-terminal to the DNA binding domain. NR5A1 binds to DNA as a monomer with the A-box recognizing Deoxyribonucleic acid sequences 5′ to the NR5A1 consensus motif in the small-scale groove. The p.Arg92Trp mutation is predicted to disrupt Deoxyribonucleic acid bounden and this is what we observed using the consensus bounden motif CCAAGGTCA as a target. In contrast the p.Arg92Gln mutant has essentially wild-type binding activity. However, this difference in DNA bounden activity cannot explicate why testis determination is occurring due to the 92Trp commutation. 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 evolution 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 information technology does not explain testis formation in an XX background. Nosotros and then assayed the ability of the 92Trp and 92Gln mutations to influence the pro-ovary canonical WNT signaling pathway. β-Catenin tin 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-ane) cistron on the Ten 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 exist due to contradistinct regulation of the expression of pro-ovarian genes that normally suppress testis development (fig. two). 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 menstruation of ovarian germination between E13.v-E16.five [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 betwixt the molecular mechanisms involved in gonad conclusion 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 private, NR5A1 synergizes with SRY to upregulate male-specific gene expression (east.g., SOX9) leading to testis germination. In 46,Xx individuals, NR5A1 synergizes with β-catenin to upregulate the expression of anti-testis genes (e.g., DAX-one/NR0B1) and peradventure pro-ovarian genes. In the 46,XY DSD instance, the p.Arg92Trp mutant shows a reduced ability to upregulate SOX9 gene expression leading to a lack of testis germination. 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. 2

The molecular and genetic events in mammalian sex conclusion 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, maybe at least in human, by interacting with NR5A1 [Bashamboo et al., 2016]. In one case Sox9 levels reach a critical threshold, several positive regulatory loops are initiated, including autoregulation of its own expression and formation of feed-frontward 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 principal sexual practice-determining switch in birds. In mice it is not involved in male person primary sexual practice determination, but in that location is evidence to betoken that information technology is important in man testis determination [Murphy et al., 2015].

Conclusions

The last few years have seen a considerable number of new genes involved in human sex conclusion 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 (due east.grand. NR5A1, MAP3K1, GATA4, FOG2), whereas gene mutations leading to testis formation in a chromosomal XX female person background are mainly associated with dysregulation of the expression of SOX genes (e.g. SOX9, SOX3, SOX10). In the latter, indicate mutations in coding genes are ordinarily associated with syndromic forms of DSD involving the RSPO/WNT signaling pathway. Overall, the genetic etiology in the bulk 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 volition reveal rare genetic mutations that are responsible for these phenotypes; nonetheless, if we are to fully found the causality of these mutations and understand the mechanisms, a series of gratuitous combinatorial studies using different model systems are required.

Acknowledgements

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

Disclosure Statement

The authors have no conflicts of involvement to declare.

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