Identification of a new nonsense mutation (Tyr129Stop) of the SRY gene in a newborn infant with XY sex-reversal

Point mutations and deletions of SRY gene have been described in several cases of XY gonadal dysgenesis. To date, most of these mutations affect the HMG domain of SRY which plays a central role in DNA binding activity of SRY. We report on a non-mosaic XY sex-reversed newborn girl (completely female external genitalia). The direct sequencing of SRY showed a new nonsense mutation in a codon of SRY gene flanking the 3' end of the HMG domain: a thymine is replaced by a guanine at position +387 in codon 129, resulting in the replacement of the amino acid tyrosine (TAT) by a stop codon (TAG). The new mutation of this patient provides further evidence to support the functional importance of the putative DNA binding activity of the HMG-box domain.     

Biochemical defects in eight SRY missense mutations causing XY gonadal dysgenesis

Sex determining Region of the Y chromosome (SRY) is the Y-borne gene required for male sex determination. Many XY females with complete gonadal dysgenesis carry SRY mutations. We describe here the effects of eight clinically isolated point mutations on the DNA-binding and -bending functions of SRY. We found that the seven mutations in the HMG domain affected the protein's DNA-binding and -bending activities to varying degrees, although all cause complete gonadal dysgenesis. DNA binding was abolished by the R75N and L94P mutations, severely disrupted by the F67V mutation and reduced by the M64R (6-fold), R76P (4-fold), A113T (3-fold), and M78T (1.7-fold) mutations. Of these, variant M64R showed no DNA-bending activity, while M78T caused a mild reduction in DNA bending. The S18N mutation, a familial mutation that lies outside the HMG domain and caused partial gonadal dysgenesis in one patient, had minimal effect on DNA binding and bending. Analysis of the NMR solution structure of the SRY HMG domain bound to DNA suggests that mutations disrupt the protein's conformation (helicity, packing), or interactions at the DNA interface. The degree to which mutations causing complete gonadal dysgenesis affect the DNA-binding activity varies. We propose that there is a threshold level of SRY activity or expression required for testis determination, as we observe that familial mutations have the least effect on SRY activity.     

A new de novo mutation (A113T) in HMG box of the SRY gene leads to XY gonadal dysgenesis.

We describe a new point mutation in the SRY gene of a Chinese XY female with gonadal dysgenesis (Swyer syndrome). Using the double stranded DNA cycle sequencing method, a single nucleotide substitution of G-->A was identified at codon 113 of the patient's SRY gene, resulting in a conservative amino acid change from alanine (A) to threonine (T) at a residue that lies within the putative DNA binding motif. With this mutation, one MnlI recognition site is abolished and a new BsmAI site is present in the DNA sequence of the SRY gene; therefore, it is easily detected by analysis of the digestion of the amplified SRY DNA fragment on an electrophoretic agarose gel. In situ hybridisation to the XY female's chromosomes showed that her mutant SRY gene was indeed located on the short arm of her Y chromosome. The SRY mutation in the XY female reported here occurred de novo, as sequence analysis showed that it was not present in her father or other family members.     

Identification and molecular modelling of a novel familial mutation in the SRY gene implicated in the pure gonadal dysgenesis

SRY gene is responsible for initiating male sexual differentiation. The protein encoded by SRY contains a homeobox (HMG) domain, which is a DNA-binding domain. Mutations of the SRY gene are reported to be associated with XY pure gonadal dysgenesis. The majority of these are de novo mutations affecting only one individual in a family. Only a small subset of mutations is shared between the father and one or more of his children. Most of these familial mutations are localized within the HMG box and only two are at the N-terminal domain of the SRY protein. Herein, we describe a young girl with pure gonadal dysgenesis and her father carrying a novel familial mutation in the SRY gene at codon number 3. This mutation is resulting in a serine (S) to leucine (L) substitution. The secondary structure of the SRY protein was carried out by protein modelling studies. This analysis suggests, with high possibility, that the N-terminal domain of the SRY protein, where we found the mutation, could form an alpha-helix from amino acid in position 2 to amino acid in position 13. The secondary structure prediction and the chemical properties of serine to leucine substitution stands for a potential disruption of this N-terminal alpha-helix in the SRY protein. This mutation could have some role in impeding the normal function of the SRY protein.     

Novel mutations affecting SRY DNA-binding activity: the HMG box N65H associated with 46,XY pure gonadal dysgenesis and the familial non-HMG box R30I associated with variable phenotypes

The SRY gene (sex-determining region of the Y chromosome) initiates the process of male sex differentiation in mammalians. In humans mutations in the SRY gene have been reported to account for 10-15% of the XY sex reversal cases. We describe here two novel missense mutations in the SRY gene after the screening of 17 patients, including 3 siblings, with 46,XY gonadal dysgenesis and 4 true hermaphrodites. One of the mutations, an A to C transversion within the HMG box, causes the N65H substitution and it was found in a patient presenting 46,XY pure gonadal dysgenesis. The Escherichia coli expressed SRY(N65H) protein did not present DNA-binding activity in vitro. The other mutation, a G to T transversion, causes the R30I substitution. This mutation was found in affected and nonaffected members of a family, including the father, two siblings with partial gonadal dysgenesis, a phenotypic female with pure gonadal dysgenesis, and three nonaffected male siblings. The G to T base change was not found in the SRY sequence of 100 normal males screened by ASO-PCR. The R30I mutation is located upstream to the HMG box, within the (29)RRSSS(33) phosphorylation site. The E. coli expressed SRY(R30I) protein was poorly phosphorylated and consequently showed reduced DNA-binding capacity in vitro.     

一例46,XY女性患者SRY基因的无义突变分析

采用ＰＣＲ－ＳＳＣＰ检测及ＤＮＡ测序技术，对一例４６，ＸＹ女性患者ＳＲＹ基因的ＨＭＧ基序进行了突变分析。结果发现在该基因的Ｓｐ引物扩增区域内存在突变。进一步的ＤＮＡ序列分析证实该突变为第６９９位碱基Ｃ被Ｔ置换，导致产生终止密码子的无义突变。结合临床表型可以认为本例患者的性反转是由于该突变所致。这一研究结果为ＳＲＹ基因是ＴＤＦ最佳候选基因的假说提供了一直接证据，有助于进一步阐明４６，ＸＹ女性发病的分     

Isodicentric Y (p11.32) chromosome in an infant with mixed gonadal dysgenesis

Among the structural abnormalities affecting the human Y chromosome, dicentric chromosomes are the most common. A wide spectrum of phenotypes of patients with a dicentric Y chromosome exists, ranging from almost males through mixed gonadal dysgenesis to females with Turner syndrome. Here, we describe an infant with mixed gonadal dysgenesis and mosaic karyotype 45,X/46,X,idic(Y)(qter-->p11.32:p11.32-->qter)/47,X,+2idic(Y) (qter-->p11.32:p11.32-->qter)/47,XYY. This was demonstrated by fluorescence in situ hybridization (FISH) analysis with whole Y chromosome painting (WCP-Y) probe. Molecular studies were performed on genomic DNA extracted from peripheral blood lymphocytes. To examine the sex determined region (SRY), azoospermia factor (AZF) region and deletion in azoospermia gene (DAZ), polymerase chain reaction (PCR) analyses were done with sequence-tagged site (STS) primers of 20 loci along the Y chromosome (SRY, DYS271, DYS148, DYS273, KALY, DYS212, SMCY, DYS215, DYS218, DYS219, DYS221, DYS223, DYS224, DYF51S1, DYS236, DAZ, DYS240), and all tested loci were found positive. Because of the possibility of a mutation in the SRY gene, we analyzed the PCR fragment by DNA sequencing and did not observe any mutation or nucleotide alteration. We present detailed molecular-cytogenetic characterization of a patient with idic(Y)(p11.32), and results are discussed with the previously described patients. As far as we know, this is the fifth report of a 46,X, idic(Y)(p11.32) karyotype and the first presentation with mixed gonadal dysgenesis and isodicentric Y. Since the correlation between phenotype and karyotype is not yet well defined, the clinical reports will be helpful in defining the phenotypic range of this chromosomal abnormality.     

Localization of SRY by primed in situ labeling in XX and XY sex reversal

Primed in situ labeling (PRINS) can be used to localize DNA segments too small to be detected by fluorescence in situ hybridization. By PRINS we identified the SRY gene in two XX males, a woman with XY gonadal dysgenesis, and an azoospermic male with Xp-Yp interchange. Because PRINS has been used generally in the study of repetitive sequences, we modified the technique for study of the single copy 2. 1-kb SRY sequence. SRY signals were identified at band Yp11.31p11.32 in normal XY males and in the woman with XY gonadal dysgenesis. SRY signals were identified on Xp22 in one XX male but not in the other. They were identified in the corresponding region (Xp22) of the der(X) in the azoospermic male with Xp-Yp interchange. SRY signals were not observed in normal XX females. Presence of SRY in DNA samples from the various subjects was confirmed by polymerase chain reaction. We conclude that PRINS is ideal for rapid localization of single copy genes and small DNA segments in general.     

6例性别异常患者的SRY基因分析

本文采用ＰＣＲ扩增、琼脂糖凝胶电泳方法，检测了６例性别异常患者的ＳＲＹ基因。结果表明，３例４６，ＸＹ女性患者中，１例ＳＲＹ基因阴性，２例呈阳性；１例如，ＸＸ男性，ＳＲＹ基因呈阳性；另２例两性畸形患者ＳＲＹ基因呈阳性。分析认为，４６，ＸＹ女性性反转，是由于ＳＲＹ基因丢失或突变所致；４６，ＸＸ男性性反转，是由于ＸＰ－ＹＰ易位引起；而两性畸形的发生则与ＳＲＹ以外的其它性别决定基因有关。     

SRY mutation and tumor formation on the gonads of XP pure gonadal dysgenesis patients.

We report three patients with XY pure gonadal dysgenesis. Two of these patients developed gonadoblastoma and associated dysgerminoma. Molecular analyses were undertaken to investigate the relationship between the formation of these tumors and Y chromosome aberrations. Deletion analyses were performed by polymerase chain reaction (PCR) amplification of Y chromosome-specific DNA sequences (PABY, SRY, DYS250, DYS254, and DYZ1). A cryptic deletion of the short arm of the Y chromosome that included the PABY, SRY, DYS250, and DYS254 loci was observed in one of the patients (22-years-old) with an associated tumor. In the other two patients who did not demonstrate such a deletion, the sequence of the SRY open reading frame was determined by the dideoxynucleotide method. Two nucleotide substitutions followed by a seven nucleotide deletion were observed in the 3' end of HMG (high mobility group)-box in the other patient (15-years-old) with an associated tumor. The patient (22-years-old) without an associated tumor did not have the cryptic deletion or mutation of SRY. A Y chromosome specific sequence (DYZ1) was demonstrated by PCR amplification of microdissected tumor tissues from these two patients. These results suggest that SRY may play a role in the formation of gonadal tumors, especially dysgerminoma.     

46,XY女性性反转患者分子遗传学诊断

目的探讨46,XY女性性反转患者的发病机制.方法应用PCR扩增SRY基因;PCR产物通过限制性酶切-单链构象多态性方法检测未知突变;基因测序以进一步明确突变.结果11例46,XY女性患者中检出2例SRY基因缺失,9例46,XY女性患者PCR扩增出现609bp特异性片段,通过限制性酶切-单链构象多态性方法检测发现其中1例患者单链电泳带发生变异;基因克隆测序分析表明HMG盒内第244位核苷酸的位置存在点突变(A→T),导致第82位密码子发生错义突变,由原来编码天冬酰胺Asn(AAT)变为编码酪氨酸Tyr(TAT).结论SRY基因的研究有助于阐明46,XY女性性反转的发病机理,为临床确诊提供科学依据.     

Gene dosage imbalances in patients with 46,XY gonadal DSD detected by an in-house-designed synthetic probe set for multiplex ligation-dependent probe amplification analysis

The development of a testis requires the proper spatiotemporal expression of the SRY gene and other genes that act in a dosage-sensitive manner. Mutations in the SRY gene account for only 10–15% of patients with 46,XY gonadal disorder of sex development (DSD). To enable the diagnostics of deletions and duplications of genes known to be involved in different forms of DSD, we developed a synthetic probe set for multiplex ligation-dependent probe amplification (MLPA) analysis. Here, we report the results from the analysis of 22 patients with 46,XY gonadal DSD. The analysis with the DSD probe set has led to the identification of two copy number variations, an 800-kb NR0B1 ( DAX1 ) locus duplication on Xp21 in a patient with isolated partial gonadal dysgenesis and a duplication of the SRD5A2 gene that represents a rare normal variant. The described MLPA kit represents an optimal complement to DNA sequence analysis in patients with DSD, enabling screening for deletions and duplications of several genes simultaneously. Furthermore, the second identification of an NR0B1 locus duplication in a patient with isolated gonadal dysgenesis, without dysmorphic features and/or mental retardation, highlights the importance of evaluating NR0B1 duplication in patients with gonadal dysgenesis.     

A novel SRY missense mutation affecting nuclear import in a 46,XY female patient with bilateral gonadoblastoma

Patients with disorders of sex development (DSD), especially those with gonadal dysgenesis and hypovirilization, are at risk of developing the so-called type II germ cell tumors (GCTs). Both carcinoma in situ and gonadoblastoma (GB) can be the precursor lesion, resulting in a seminomatous or non-seminomatous invasive cancer. SRY mutations residing in the HMG domain are found in 10–15% of 46,XY gonadal dysgenesis cases. This domain contains two nuclear localization signals (NLSs). In this study, we report a unique case of a phenotypical normal woman, diagnosed as a patient with 46,XY gonadal dysgenesis, with an NLS missense mutation, on the basis of the histological diagnosis of a unilateral GB. The normal role of SRY in gonadal development is the upregulation of SOX9 expression. The premalignant lesion of the initially removed gonad was positive for OCT3/4, TSPY and stem cell factor in germ cells, and for FOXL2 in the stromal component (ie, granulosa cells), but not for SOX9. On the basis of these findings, prophylactical gonadectomy of the other gonad was performed, also showing a GB lesion positive for both FOXL2 (ovary) and SOX9 (testis). The identified W70L mutation in the SRY gene resulted in a 50% reduction in the nuclear accumulation of the mutant protein compared with wild type. This likely explains the diminished SOX9 expression, and therefore the lack of proper Sertoli cell differentiation during development. This case shows the value of the proper diagnosis of human GCTs in identification of patients with DSD, which allows subsequent early diagnosis and prevention of the development of an invasive cancer, likely to be treated by chemotherapy at young age.     

Discordant phenotypes and 45,X/46,X,idic(Y).

Mosaicism introduces wide variability into the clinical expression of numerical and unbalanced structural chromosomal abnormalities. The phenotypic range of variability of 45,X/46,XY mosaicism extends from Turner syndrome to mixed gonadal dysgenesis to normal males. The specific phenotype is primarily dependent on the chromosomal constitution of the developing gonad. Similar phenotypic variability is observed with mosaicism for 45,X and a second cell line with an abnormal sex chromosome. This report describes a patient with Turner syndrome and a patient with mixed gonadal dysgenesis who have identical karyotypes, namely 45,X/46,X,idic(Y)(p11.2). While mosaicism alone might have accounted for the phenotypic differences, by PCR analysis the Turner syndrome patient was SRY and ZFY negative and the mixed gonadal dysgenesis patient was SRY and ZFY positive.     

Evidence that WT1 mutations in Denys -- Drash syndrome patients may act in a dominant-negative fashion

The triad of nephropathy, partial gonadal dysgenesis and Wilms'tumour (WT) is known as Denys — Drash syndrome (DDS).The WT predisposition gene WT1, which plays a vital role inboth genital and renal development, is known to be mutated inDDS patients. The WT1 mutations in these patients are constitutionalpoint mutations clustered in the zinc finger (ZF) encoding exons,particularly the exons encoding ZF2 and ZF3. The predicted functionalalteration in WT1 is thought to underlie DDS aetiology eitherby abolishing binding of the WT1 ZF domain to its normal targetDNA binding site(s), perhaps blocking the binding of the wildtype WT1 present (dominant negative mutation), and/or by conferringthe ability to recognise novel but inappropriate DNA bindingsites (dominant mutation). We report here on the analysis ofWT1 in a further five cases of DDS. In each case a constitutionalpoint mutation was detected in either ZF2 or ZF3. Three of thesemutations are novel, with two affecting the conserved histidineand cysteine residues crucial for ZF tertiary structure. Theprotein product of the third is predicted to lack ZF2, 3 and4 as a result of a chain termination mutation, and is presumablyincapable of binding DNA. However since the DDS phenotype isonly elicited by mutations which lead to loss or alterationof ZF function (presumably DNA binding) while the N-terminalupstream portion of the gene remains intact, we suggest thata dominant negative mechanism is at work here.     

Analysis of the SRY gene in 22 sex-reversed XY females identifies four new point mutations in the conserved DNA binding domain

The open reading frame of the SRY gene has been examined ina series of 22 XY females with clinically defined pure gonadaldysgenesis by direct sequencing of biotinylated PCR productbound to streptavidin coated beads. Amongst the 22 XY femalesexamined, five (two of whom are sisters) were found to havesingle base changes all within the highly conserved DNA binding(or HMG box) domain. In the remaining 17 cases, the SRY genesequence was indistinguishable from that found in normal males.In three of the XY females with point mutations, the alteredamino acids occur in highly conserved positions leading to non-conservativechanges (Arg to Gly at position 5, Met to Thr at position 21and Arg to Trp at position 76). Examination of the SRY genefrom the father's Y chromosome has shown that the mutationsat position 5 in patient SHM60 and position 21 in patient HN31have arisen de novo. In the case of the two sibs, both haveidentical mutations where a C to T transition in codon 17 hascreated a TAG termination signal, thus suggesting that the deceasedfather is likely to be a gonadal mosaic for the mutation. Inthe case of the mutations at positions 17 and 76, the fathersare not available for investigation and so it has not been possibleto determine whether the changes are de novo. These data indicatethat the majority of XY females with pure gonadal dysgenesisowe their sex-reversed phenotype to mutations in as yet uncharacterisedsegments of the SRY gene, or, at other loci acting early inthe sex-determining pathway.     

A heterozygous mutation in the desert hedgehog gene in patients with mixed gonadal dysgenesis

Aetiology of mixed gonadal dysgenesis (MGD) has not been completely elucidated. Molecular analyses have failed to demonstrate the presence of mutations in sex-determining region on Y chromosome (SRY); it has been suggested that these individuals may bear mutations in other genes involved in the testis-determining pathway. Desert hedgehog's (DHH) importance regarding male sex differentiation has been demonstrated in various studies we describe here, for the first time, two cases of MGD in which a monoallelic single base deletion in DHH is associated with the disorder. Genomic DNA was isolated from paraffin-embedded gonad tissue from 10 unrelated patients with MGD and three controls; in addition to, DNA from peripheral blood leukocytes in 100 controls. Coding sequence abnormalities in DHH were assessed by exon-specific PCR, single-stranded conformation polymorphism (SSCP) and direct sequencing. In two patients, a heterozygous 1086delG in exon 3 was found. Comparing previously described mutations in DHH to the one observed in this study, we can affirm that the phenotypic spectrum of patients with gonadal dysgenesis due to mutations in DHH is variable. This study continues to demonstrate the importance that DHH has in mammalian male sexual differentiation, providing extended evidence that DHH constitutes a key gene in gonadal differentiation.     

Tandem duplication and copy number polymorphism of the SRY gene in patients with sex chromosome anomalies and males exposed to natural background radiation

Mutations in the SRY gene encompassing the HMG box have been well characterized in gonadal dysgenesis, male infertility and other types of sex chromosome related anomalies (SCRA). However, no information is available on copy number status of this gene under such abnormal conditions. Employing 'Taqman Probe Assay' specific to the SRY gene, we screened 16 DNA samples from patients with SCRA and 36 samples from males exposed to high levels of natural background radiation (HNBR). Patients with SCRA showed 2-16 copies of the SRY gene of which, one, Oxen (49, XYYYY) had eight copies with sequences different from one another. Of the 36 HNBR samples, 12 had one copy whereas 24 harboured 2-8 copies of the SRY gene. A HNBR male 33F had one normal and one mutated copy of this gene. Analysis of 25 DNA samples from blood and semen of normal males showed only one copy of this gene. Despite multiple copies in affected males, fluorescence in-situ hybridization (FISH) with SRY probe detected a single signal on the Y chromosome in HNBR males suggesting its possible localized tandem duplication. Copy number status of the other Y-linked loci is envisaged to augment DNA diagnostics facilitating genetic counselling to affected patients.