XY sex reversal associated with a nonsense mutation in SRY.

Sex determination in humans is mediated through the expression of a testis-determining gene on the Y chromosome. In humans, a candidate gene for the testis-determining factor (TDF) that encodes a protein with a putative DNA-binding motif and has been isolated is termed SRY. Here we describe an XY sex-reversed female with pure gonadal dysgenesis who harbors a de novo nonsense mutation in the SRY open reading frame (SRY-orf). This single-basepair substitution results directly in the formation of a termination codon in the putative SRY DNA-binding motif, presumably leading to a nonfunctional gene product. This brings the number of reported XY sex-reversed females with de novo mutations in the known SRY-orf to three, each occurring in the putative DNA-binding domain. This provides further evidence to support SRY being TDF in humans and also indicates the functional importance of the putative DNA-binding domain of the SRY protein.     

Evidence for increased prevalence of SRY mutations in XY females with complete rather than partial gonadal dysgenesis.

The Y chromosome gene SRY (sex-determining region, Y gene) has been equated with the mammalian testis-determining factor. The SRY gene of five subjects with 46,XY complete gonadal dysgenesis (46,XY karyotype, completely female external genitalia, normal Müllerian ducts, and streak gonads) was evaluated for possible mutations in the coding region by using both single-strand conformation polymorphism (SSCP) assay and DNA sequencing. Mutations were identified in three subjects, of which two gave altered SSCP patterns. Two of them were point mutations causing amino acid substitutions, and the third was a single-base deletion causing a frameshift. All three mutations caused alterations in the putative DNA-binding region of the SRY protein. Genomic DNA was obtained from the fathers of two of the three mutant patients: one mutation was demonstrated to be de novo, and the other was inherited. The presence of SRY mutations in three of five patients suggests that the frequency of SRY mutations in XY females is higher than current estimates.     

A novel double nucleotide substitution in the HMG box of the SRY gene associated with Swyer syndrome

We describe a novel double nucleotide substitution in the SRY gene of a 46,XY female with gonadal dysgenesis or Swyer syndrome. The SRY sequence was analysed by both the single-strand conformational polymorphism assay and direct DNA sequencing of products from the polymerase chain reaction. A double nucleotide substitution was identified at codon 18 of the conserved HMG box motif, causing an arginine to asparagine amino-acid substitution. The altered residue is situated in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. Since the mutation abolishes one HhaI recognition site, the results were confirmed by HhaI restriction mapping. No other mutations were found in the remaining regions of the gene. The corresponding DNA region from the patient’s brother was analysed and found to be normal. We conclude that the SRY mutation in the reported XY female occurred de novo and is associated with sex reversal. Received: 16 December 1996 / Accepted: 5 May 1997     

Familial case with sequence variant in the testis-determining region associated with two sex phenotypes.

The human Y chromosome encodes a testis-determining factor (TDF) which is responsible for initiating male sex determination. Recently a region of the Y chromosome (SRY) was identified as part of the TDF gene. We have identified a three-generation family (N) in which all XY individuals have a single base-pair substitution resulting in a conservative amino acid change in the conserved domain of the SRY open reading frame. Three individuals are XY sex-reversed females, and two are XY males. Several models are proposed to explain association between a sequence variant in SRY and two sex phenotypes.     

46,XY女性性反转患者SRY基因的两个新突变类型

Y染色体上的性别决定区域——SRY基因作为睾丸决定因子,可以调控男性性别发育过程。SRY基因是一种转录因子,属于带有高迁移率族蛋白家族,该家族成员包含能与DNA结合的HMG盒基序。已知SRY基因的缺失和点突变是造成XY女性性反转的病因之一。通过筛查10位中国46,XY女性性反转病人SRY基因的开放阅读框区域,探寻新的突变类型。用标准方法从外周血中抽提gDNA,通过聚合酶链式反应扩增SRY基因中部的609bp的DNA片段。扩增后的PCR片段被克隆到pUCm-T载体中,在ABI377-3自动测序仪上完成测序。运用限制性内切酶酶切分析的方法验证DNA测序的结果。结果表明,在两个患者的SRY基因中分别发现了新的核苷酸点突变,并都导致氨基酸替代。一个突变发生在SRY基因的5’端HMG盒外的核苷酸第113位腺嘌呤(A)被鸟嘌呤(G)取代,并导致谷氨酸被甘氨酸替换;另一个突变是第387位核苷酸发生T被A替换,该突变引起第129位的酪氨酸变成终止密码,她父亲的SRY序列被证明是正常的野生型。通过查询文献和人类基因突变数据库(HGMD),这两个突变都是以前未见报道过的新型SRY基因突变,并使因核苷酸替换引起SRY基因突变总数增加到45。     

46,XY女性患者SRY基因启动子区域的突变分析

大约15%的46,XY女性患者中发现SRY基因编码区突变,其他患者可能是SRY基因的调节区, 包括启动子区域发生了突变,或者其他相关基因发生突变所致。本文采用限制性酶切、PCR-SSCP及银染检测技术,对7例患者SRY基因的启动子区域进行了突变筛查, 结果未发现异常,提示这些患者的病因与SRY基因启动子区域本身无关,结合对患者SRY基因HMG基序DNA的突变分析结果,表明除SRY基因异常外还存在其他导致46,XY女性性反转综合征的遗传机制。 Abstract:Using restriction endonuelease digestion and PCR-SSCP with silver staining,we analyzed the promotor region of SRY gene in seven 46,XY femalcs.The results showed no abnormality,thus ruling out the mutations in the promotor region of the SRY gene as a possible cause of sex reversal in these XY females.In view with the absence of the mutations in the HMG regions of the SRY genes of several patients,it is suggested that SRY gene is not the only gene responsible for testicular development but is one of many hierarchical genes involved in a genetic cascade for sexual differentiation.     

Two novel SRY missense mutations reducing DNA binding identified in XY females and their mosaic fathers.

Two novel mutations in the sex-determining gene SRY were identified by screening DNA from 30 sex-reversed XY females by using the SSCP assay. Both point mutations lead to an amino acid substitution in the DNA-binding high-mobility-group domain of the SRY protein. The first mutation, changing a serine at position 91 to glycine, was found in a sporadic case. The second mutation, leading to replacement of a highly conserved proline at position 125 with leucine, is shared by three members of the same family, two sisters and a half sister having the same father. The mutant SRY proteins showed reduced DNA-binding ability in a gel-shift assay. Analysis of lymphocyte DNA from the respective fathers revealed that they carry both the wild-type and the mutant version of the SRY gene. The fact that both fathers transmitted the mutant SRY copy to their offspring implies that they are mosaic for the SRY gene in testis as well as in blood, as a result of a mutation during early embryonic development.     

True hermaphroditism in a 46,XY individual, caused by a postzygotic somatic point mutation in the male gonadal sex-determining locus (SRY): molecular genetics and histological findings in a sporadic case.

Recently, the gene for the determination of maleness has been identified in the sex-determining region on the short arm of the Y chromosome (SRY) between the Y-chromosomal pseudoautosomal boundary (PABY) and the ZFY gene locus. Experiments with transgenic mice confirmed that SRY is a part of the testis-determining factor (TDF). We describe a sporadic case of a patient with intersexual genitalia and the histological finding of ovotestes in the gonad, which resembles the mixed type of gonadal tissue without primordial follicle structures. The karyotype of the patient was 46,XY. By PCR amplification, we tested for the presence of PABY, SRY, and ZFY by using DNA isolated from peripheral blood leukocytes and for the presence of SRY by using DNA obtained from histological gonadal slices. The SRY products of both DNA preparations were further analyzed by direct sequencing. All three parts of the sex-determining region of the Y chromosome could be amplified from leukocytic DNA. The patient's and the father's SRY sequences were identical with the published sequence. In the SRY PCR product of gonadal DNA, the wild-type and two point mutations were present in the patient's sequence, simulating a heterozygous state of a Y-chromosomal gene: one of the mutations was silent, while the other encoded for a nonconservative amino acid substitution from leucine to histidine. Subcloning procedures showed that the two point mutations always occurred together. The origin of the patient's intersexuality is a postzygotic mutation of the SRY occurring in part of the gonadal tissue. This event caused the loss of the testis-determining function in affected cells.     

Gene SRY et anomalies de la determination genetique du sexe chez l’homme

Normal sexual development in man is the consequence of a complex process. This review focuses on the translation of genedal sex (XX or XY karyotype) into gonadal sex (testis or ovary). During the last three years attempts to identify and clone the testis determining factor (TDF) have exploited detailed maps of the Y chromosome established by geneticists over the last decade. A candidate gene, named SRY (sex determining region, Y) located at the tip of the short arm of the Y chromosome, shows many characteristics in common with TDF in that it is the sole element of the Y chromosome required for male development. The discovery of TDF led us to analyse sex-reversed individuals, i.e. XX males and XY females, with the aim of constructing a model for the processes regulating the development of an organ as complex as the testis. This SRY gene is now the subject of intense molecular biological effort by various groups, effort which we hope will elucidate the mechanism(s) of sex determination.     

Pathology of 46, XY pure gonadal dysgenesis: absence of testis differentiation associated with mutations in the testis-determining factor

Abstract. Individuals with 46, XY pure gonadal dysgenesis present with a completely female phenotype. These individuals develop bilateral streak gonads and have normal Müllerian structures. The apparent absence of testicular tissue in these individuals suggests a mutation in the initial steps of the male sex-determining pathway. A candidate gene for the primary signal in this pathway was recently cloned ( SRY ) which encodes a protein with a DNA-binding capacity. In a study of 14 XY females with pure gonadal dysgenesis harbouring SRY , we analysed the histology of the gonads and compared it to the presence or absence of mutations in the SRY open reading frame ( SRY -orf). The histological analysis revealed two distinct groups of streak gonads. In the first group, the gonad was composed of exclusively ovarian-like stroma, with sclero-hyaline nodules in some areas. No tubules were observed. The gonads in the second group were composed of undifferentiated stroma harbouring either tubules or a rete structure. This suggests that in the latter group some differentiation (towards testis formation) has occurred, whereas in the first group ovarian differentiation has been interrupted. Individuals with mutations in the SRY -orf were found to have streak gonads of the first group, whereas most of the remaining XY females without detectable mutation in the SRY -orf had streak gonads belonging to the second group.
On the basis of histology, it may be possible to distinguish between mutations in the sex-determining or sex-differentiation pathways. We suggest that SRY may play a role in rete testis formation. We also present arguments favouring the mesonephros as the origin of testicular somatic cells in humans.     

Molecular biology of disorders of sex differentiation.

Sexual ambiguity can be a difficult and sometimes confusing diagnostic problem in children. Recent developments in molecular biology have provided the opportunity to analyze the gene responsible for testicular determination, SRY, the androgen receptor gene and the gene encoding the cP450 enzyme specific for 21-hydroxylation, CYP21B, whose defects are responsible for congenital adrenal hyperplasia. Southern-blotting studies and PCR analyses of SRY, androgen receptor and CYP21B genes can be routinely used for the direct diagnosis of gonadal dysgenesis, androgen insensitivity syndromes and congenital adrenal hyperplasia, respectively. In sex-reversed XY females, several de novo mutations or deletions in the SRY gene have been reported. Defects in the human androgen receptor cause a spectrum of defects in male phenotypic sexual development associated with abnormalities in the receptor protein. Analyses of the androgen receptor gene structure have identified the causative mutation in some families: mutations that result in large-scale alterations of the structure of the androgen receptor, mRNA or gene mutations that alter the primary structure of the androgen receptor protein and mutations that alter the level of mRNA. The diversity of clinical phenotypes, apparent in 21-hydroxylase deficiency, is paralleled by a considerable degree of mutational heterogeneity in the CYP21 gene locus. Various changes causing severe 21-hydroxylase deficiency have been reported: point mutations, gene conversions and gene deletions. In conclusion, substantial progress has been made elucidating genetic defects causing sex reversal in XY females, the androgen insensitivity syndrome and congenital adrenal hyperplasia. Molecular genetics can also be applied for carrier identification and prenatal diagnosis.     

A novel missense mutation (S18N) in the 5′ non-HMG box region of the SRY gene in a patient with partial gonadal dysgenesis and his normal male relatives

Mutations in the sex-determining region of the Y chromosome (the SRY gene) have been reported in low frequency in patients with 46,XY gonadal dysgenesis. We investigated 21 Brazilian 46,XY sex-reversed patients, who presented either complete or partial gonadal dysgenesis or embryonic testicular regression syndrome. Using Southern blotting, polymerase chain reaction, denaturing gradient gel electrophoresis and direct sequencing, we analyzed deletions and point mutations in the SRY gene. We found a missense mutation at codon 18 upstream of the 5′ border of the HMG box of the SRY gene in one patient with partial gonadal dysgenesis. This variant sequence was also found in DNA obtained from blood and sperm cells of his father and in blood cells of his normal brother. The S18N mutation was not found in 50 normal males, ruling out the possibility of a common polymorphism. We identified a novel familial missense mutation (S18N) in the 5’ non-HMG box of the SRY gene in 1 of 21 patients with 46,XY sex reversal. Received: 6 May 1997 / Accepted: 2 October 1997     

Sex-reversing mutations affect the architecture of SRY-DNA complexes.

The testis determining factor, SRY, is a DNA binding protein that causes a large distortion of its DNA target sites. We have analysed the biochemical properties of the DNA binding domains (HMG-boxes) of mutant SRY proteins from five patients with complete gonadal dysgenesis. The mutant proteins fall into three categories: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to male progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence and thus adds another level of discrimination in target site recognition. These data suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY is essential for sex determination.     

No evidence of mutations in four candidate genes for male sex determination/differentiation in sex-reversed XY females with campomelic dysplasia.

Campomelic dysplasia (Cd) occurs combined with sex reversal resulting in XY females. The recent identification of candidate genes for sex determination/differentiation and of a sex determining region on the human Y chromosome prompted the authors to study these genes for mutations in patients with Cd and sex reversal. In a total of five cases, no evidence for a mutation in the genes SRY, ZFY, ZFX, MEA and some anonymous Y-linked sequences was found. In addition to Southern analysis, gene expression of ZFY, ZFX and MEA was found to be normal as well. It is concluded that sex reversal in this condition is due to mutation in a so far unidentified gene which may act secondary to the testis-determining factor (TDF).     

一个46,XY"女性"不育症家系的遗传学分析

运用常规的染色体G带分析和基因分析技术对-46,XY男性女性化家系进行遗传学分析,发现：先证者及其妹妹的染色体核型为46,XY,其母亲和父亲的核型正常;对睾丸决定基因（SRY）和雄激素受体基因（AR）进行突变检测,在SRY基因的整个编码区中没有发现突变,而AR基因的第7个外显子的第840个密码子由CGT（编码精氨酸）变为CAT（编码组氨酸）,这一改变可能是导致核型为46,XY女性化而发生不育。     

Screening for Pax8 mutations in patients with congenital hypothyroidism in South-West Germany

AIMS: To study the frequency of mutations in the Pax8 gene in a cohort of patients with congenital hypothyroidism (CH) in South West Germany. METHODS: A cohort of 95 patients with CH (60 females, 35 males), identified in our newborn screening program, was analyzed for mutations in Pax8 by single-stranded conformational polymorphism (SSCP) and DNA sequencing. RESULTS: SSCP analysis and direct sequencing of exon 3 of a female patient with a hypoplastic thyroid gland revealed two heterozygous mutations in Pax8 resulting in a transition of T to C (codon 34) and G to A (codon 35), replacing isoleucine by threonine and valine by isoleucine. Using allele-specific PCR we could demonstrate that both mutations are located on the same allele. Furthermore, a polymorphism was documented in 24 patients with thyroid hypoplasia in intron 6 at nucleotide +51 (CC, GG, CG). Comparison of the polymorphisms between hypothyroid patients and controls revealed no significant differences suggesting that this polymorphism does not play a role in the pathogenesis of hypothyroidism. No further mutations or polymorphisms were found in the cohort. CONCLUSIONS: These findings confirm the contribution of mutations in the Pax8 gene to the etiology of thyroid dysgenesis with a variable penetrance, but also demonstrate the rare overall incidence in CH.     

Sexual maldevelopment and sex reversal, chromosomal causes

The SRY gene on the Y chromosome is the testis determining factor (TDF). It is therefore the initial male determining factor. However, phenotypic sex determination includes a cascade of genes located on autosomes as well as sex chromosomes. Aberrations of these genes may cause sexual maldevelopment or sex reversal. Abnormalities may include single gene mutations and gene loss or gain-changes may involve only sex organs or may be part of syndromes. These changes may also arise as chromosome abnormalities involving contiguous genes. Eight cases with chromosomal abnormalities involving different causative mechanisms are described herein. The most common cause is nondisjunction, including loss or gain of sex chromosomes. Less common causes are mispairing and crossing over in meiosis, chromosome breaks with repair, nonhomologous pairing due to low copy repeats and crossing over, and translocation (familial or de novo) with segregation. Cases include: [see: text].     

Mutational Analysis of Patients with Neurofibromatosis 2

Neurofibromatosis 2 (NF2) is a genetic disorder characterized by the development of multiple nervous-system tumors in young adulthood. The NF2 gene has recently been isolated and found to encode a new member of the protein 4.1 family of cytoskeletal associated proteins, which we have named merlin. To define the molecular basis of NF2 in affected individuals, we have used SSCP analysis to scan the exons of the NF2 gene from 33 unrelated patients with NF2. Twenty unique SSCP variants were seen in 21 patients; 10 of these individuals were known to be the only affected person in their kindred, while 7 had at least one other known affected relative. In all cases in which family members were available, the SSCP variant segregated with the disease; comparison of sporadic cases with their parents confirmed the de novo variants. DNA sequence analysis revealed that 19 of the 20 variants observed are predicted to lead to a truncated protein due to frameshift, creation of a stop codon, or interference with normal RNA splicing. A single patient carried a 3-bp deletion removing a phenylalanine residue. We conclude that the majority of NF2 patients carry an inactivating mutation of the NF2 gene and that neutral polymorphism in the gene is rare.