Exploration of the pathogenesis for a SRY-negative male with 46,XX disorder of sex development / 中华医学遗传学杂志

OBJECTIVE@#To explore the pathogenesis for a SRY-negative male with 46,XX disorder of sex development (DSD).@*METHODS@#Peripheral blood samples of the patient and his family members were subjected to chromosomal karyotyping, routine PCR, real-time fluorescence quantitative PCR, whole exome sequencing and whole genome sequencing. The data was analyzed with NextGENe software.@*RESULTS@#Both the proband and his brother presented a 46,XX karyotype with negative SRY gene, while their father presented normal phenotype and karyotype with positive SRY gene. No pathogenic variant associated with sex development was detected by whole exome sequencing, while a 243 kb duplication was detected by whole genome sequencing in the 5' upstream region of the SOX9 gene in the proband, his brother and father. The same duplication was not found in his sister and mother.@*CONCLUSION@#The 243 kb duplication at the 5' upstream of the SOX9 gene may predispose to the 46,XX DSD in this family. It is speculated that there exist an unknown core regulatory element in the upstream of the SOX9, and its duplication may trigger expression of SOX9 and initiate testicular differentiation in the absence of SRY gene.

Genetic analysis of a case of 46, XX, SRY－ male syndrome / 中华男科学杂志

<p><b>Objective</b>To identify the etiology of chromosome abnormality in an infertile man and analyze the correlation between the genotype and phenotype.</p><p><b>METHODS</b>We analyzed the karyotype of an infertile male using the routine G-banding technique and then the chromosome abnormality of the patient by Illumina Human CytoSNP-12 Beadchip array.</p><p><b>RESULTS</b>Negative results were found in the examination of the sex-determining region Y (SRY) gene and the STR locus in the AZF zone of the patient. The karyotype of the patient was 46, XX. SNP array showed a 1.05 Mb 19p12 duplication and a 0.93 Mb Xq27.1 duplication.</p><p><b>CONCLUSIONS</b>The patient was confirmed as a case of 46,XX male syndrome. The increased copies of the FGF13 gene may be the major causes of abnormal sex determination and testis development.</p>

Genetic analysis for 2 females carrying idic(Y)(p) and with sex development disorders / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the phenotype-genotype association of isodicentromere Y chromosome by analysis of two female patients carrying the chromosome with sexual development disorders.</p><p><b>METHODS</b>The karyotypes of the two patients were determined by application of conventional G banding of peripheral blood samples and fluorescence in situ hybridization (FISH). PCR was applied to detect the presence of SRY gene.</p><p><b>RESULTS</b>Conventional karyotype analysis showed case 1 to be a mosaic: mos.45,X[38]/46,X,+mar[151]/47,XY,+mar[5]/47,X,+mar × 2[2]/46,XY[4], FISH showed that 12 different cell lines were presented in the karyotype of case 1 and partial cell lines with SRY gene, the marker is an isodicentromere Y chromosome [idic(Y)(p)]. No mutation was found in the SRY gene. The karyotype of case 2 was mos.45,X[25]/46,X,+mar[35]. FISH showed the marker to be an idic(Y)(p) without the SRY gene.</p><p><b>CONCLUSION</b>The karyotype of patients carrying idic(Y)(p) seems unstable, and female patients have the characteristics of short stature and secondary sexual hypoplasia. Karyotype analysis combined with FISH analysis can accurately determine the breakpoint of idic(Y) and identify the types of complex mosaic, which may facilitate genetic counseling and prognosis.</p>

Clinical characteristics, cytogenetic and molecular findings in patients with disorders of sex development / 华中科技大学学报（医学）（英德文版）

The clinical characteristics of patients with disorders of sex development (DSD), and the diagnostic values of classic cytogenetic and molecular genetic assays for DSD were investigated. In the enrolled 56 cases, there were 9 cases of 46,XY DSD, 6 cases of Turner syndrome (TS), one case of Super female syndrome, 25 cases of Klinefelter syndrome, 14 cases of 46,XX DSD, and one case of autosomal balanced rearrangements with hypospadias. The diagnosis of sex was made through physical examination, cytogenetic assay, ultrasonography, gonadal biopsy and hormonal analysis. PCR was used to detect SRY, ZFX, ZFY, DYZ3 and DYZ1 loci on Y and X chromosomes respectively. The DSD patients with the same category had similar clinical characteristics. The karyotypes in peripheral blood lymphocytes of all patients were identified. PCR-based analysis showed presence or absence of the X/Y-linked loci in several cases. Of the 9 cases of 46,XY DSD, 6 were positive for SRY, 9 for ZFX/ZFY, 9 for DYZ3 and 8 for DYZ1 loci. Of the 6 cases of TS, only 1 case with the karyotype of 45,X,/46,XX/46,XY was positive for all 5 loci. Of the 25 cases of Klinefelter syndrome, all were positive for all 5 loci. In one case of rare Klinefelter syndrome variants azoospermia factor (AZF) gene detection revealed the loss of the AZFa+AZFb region. In 14 cases of 46,XX DSD, 7 cases were positive for SRY, 14 for ZFX, 7 for ZFY, 7 for ZYZ3, and 5 for DYZ1. PCR can complement and also confirm cytogenetic studies in the diagnosis of sex in cases of DSD.

SRY gene-testing in the diagnosis of disorders of sex development among children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the value of direct sequencing of sex-determining region Y (SRY) gene, as well as peripheral blood karyotype analysis, in the diagnosis of disorders of sex development (DSD) among children and adolescents with ambiguous genitalia.</p><p><b>METHODS</b>The karyotypes of 20 children and adolescents with ambiguous genitalia were determined by conventional G-banding analysis. PCR amplification was used to detect SRY gene in these patients, and direct sequencing was used to judge whether there was SRY gene mutation.</p><p><b>RESULTS</b>Of the 20 cases, 17 were positive for SRY gene, and 3 were negative for SRY gene. Direct sequencing revealed no SRY gene mutation in the positive cases, however karyotype analysis found 4 special karyotypes in these patients: 46, XY, del(Y) (q12)/45, X; 46, XY, add(Y) (p11); 46, XY, r(9); 46, XY, 9qh+.</p><p><b>CONCLUSIONS</b>SRY gene detection can help determine the type of DSD among children and has the advantage of quick detection. Used together with G-banding analysis, it is helpful for primary diagnosis of DSD among children.</p>

SRY sequence in maternal plasma: Implications for non-invasive prenatal diagnosis: First report from India.

AIM: The presence of circulatory cell-free fetal DNA in maternal plasma has found new applications in non-invasive risk-free prenatal diagnosis. MATERIALS AND METHODS: We made use of a size separation approach along with real time polymerase chain reaction (PCR) to evaluate the use of fetal DNA in the detection of the sex of the fetus. Cell-free fetal DNA was isolated from the plasma of 30 women (10–20 weeks gestation) using a size separation approach. We made use of Taq Man Chemistry and real time PCR using primers and probes for GAPDH and SRY. RESULTS: Only 24 cases could be studied as there was no amplification in six cases. Fetal sex was accurately determined in all of the 24 cases wherein 19 women were carrying male fetuses and five women were carrying female fetuses. An increase in the amount of fetal DNA was observed with an increase in the gestational age. CONCLUSIONS: Real time PCR analysis is a highly sensitive and accurate tool for non-invasive prenatal diagnosis, allowing detection of the sex of the fetus as early as 10 weeks of gestation. Non-invasive prenatal diagnosis eliminates the risk of fetal loss associated with the invasive procedure.

Varón 46 XX, un desorden del desarrollo sexual testicular: a propósito de un caso clínico / 46 XX disturbance of testicular sexual development: report of one case

We report a previously healthy child that consulted for the first time at the age of 11 years for short stature. At that moment, his height was 138 cm, with a mid-parental target height of 175 cm. He was in an initial pubertal stage with a Tanner II pubic hair and a testicular volume of 4 ml. Initial laboratory examination was normal and the child had a concordant bone age. He consulted again at 16 years of age, with a height of 162.4 cm (percentile 5 for age), a bone age of 18 years and a Tanner IV pubic hair, but the testicular volume persisted at 4 ml. A genetic study disclosed a 46 XX karyogram and a fluorescence in situ hybridization (FISH) for chromosomes X and Y that showed a positive sex determining region Y (SRY) in X chromosome.

Screening of sexual differentially expressed genes in the chicken early embryonic gonads using DNA microarray / 生物工程学报

Using Affymetrix's Chicken Genome Array, we used total RNA isolated from the gonads of male and female chicks at embryonic day 9 to identify the genes differentially expressed between male and female. Statistical results show 19 493 genes expressed in male chick's embryonic gonads and 19 368 genes expressed in female. There were 145 genes specificity expressed in male and 189 genes in female. The gene ontology classification (GOC) indicated these differentially expressed genes were mainly involved in cellular component, cell process and molecular banding, a part of genes were involved in organelle component, metabolic process, biologic process, catalytic activity and signal transducer activity. Some genes had reported for sex determination and differentiation in birds, such as avian sex-specific avian sex-specific W-linked, chomodomain-helicase-DNA-binding protein 1 and sex determining region Y-box 9. In addition, we also found several genes or hypothetical proteins were unknown function for the gonad differentiation and development, focus to their biological function and expression pattern in further works would provide a beneficial reference for understand the mechanism of sex differentiation and determination in birds.

Validation of the non-cellular metastasis hypothesis of malignant tumors in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To test the hypothesis that malignant tumor metastasis is mediated also through a non-cellular, essentially molecular, mechanism in addition to the cellular pathway.</p><p><b>METHODS</b>The sex-determining region on the Y chromosome was detected as the marker of the primary tumors using PCR in Lewis lung carcinoma (LLC) in vitro and in female C57BL/6 mice bearing LLC with spontaneous metastasis. The macroscopic and microscopic metastases in the tumor-bearing mice were examined for SRY expression by PCR and in situ hybridization, using the tissues from male and female mice as the positive or negative controls.</p><p><b>RESULTS AND CONCLUSION</b>Positive SRY gene expression was detected in the metastatic foci in the LLC-bearing female mice, suggesting the origination of these tumor cells from the primary tumor foci. We have failed to verify the non-cellular metastasis hypothesis in this animal experiment, but given the limitations of this experiment, we consider further investigation still necessary for verification of this hypothesis using other methods.</p>

Genes related with male gonadal morphogenesis in mammals / 中华男科学杂志

Gene expressions are sex-specific in the sex development of mammals. Different genes express in different phases and tend to change with the time. The functions of some genes, such as SRY, SOX9, SOX8, DAX1, and FGF9, have already been defined in male gonadal morphogenesis. This paper presents a review of the genes involved in the formation of the male gonad in mammals.

A Case of a 46,XX Male with SRY Gene

46,XX male is a rare sex constitution characterized by the development of bilateral testis in persons who lack a Y chromosome. Manifestations of 46,XX males are usually hypogonadism, gynecomastia, azoospermia, and hyalinations of seminiferous tubules. The incidence of XX male reversal is approximately 1 in 20,000 male neonates. The SRYgene is located at the short arm of the Y chromosome(Yp11.31) and codes for testis determining factor in humans. Here, the patient, who presented with a normal male phenotype, was referred for azoospermia. Conventional cytogenetic analysis showed a 46,XX karyotype. Quantitative fluorescent polymerase chain reaction(QF-PCR) and Multiplex PCR studies identified SRY gene. And, Fluorescence In Situ Hybridization(FISH) confirmed the SRY gene on the distal short arm of chromosome X. We identified the SRY gene on the distal short arm of chromosome X by molecular cytogenetic and molecular analyses. Therefore, molecular-cytogenetics and molecular studies were proved to be clinically useful adjunctive tool to conventional prenatal cytogenetic analysis.

Isolation and sequencing of seven Sox genes from the lacertid lizard Eremias breuchleyi

The Sox family of genes shares a high sequence similarity with the HMG box region of the human Y chromosomal gene, SRY. We used highly degenerate primers to clone and sequence seven Eremias breuchleyi Sox genes (EbSox2, EbSox3, EbSox4, EbSox11, EbSox12, EbSox14 and EbSox21). A database search for the cloned sequences revealed the following percentage identity with the homologous human SOX genes: EbSox2 = 96 percent, EbSox3 = 88 percent, EbSox4 = 94 percent, EbSox11 = 99 percent, EbSox12 = 96 percent, EbSox14 = 98 percent, EbSox21 = 97 percent. Cluster analysis indicates that they seem to belong to group B and group C of Sox gene family, respectively.

Analysis of SRY gene in 8 cases of sex abnormality / 华中科技大学学报（医学）（英德文版）

In order to investigate the relationship between sex dysplasia and sex-determining region Y (SRY) gene, 8 patients with sexual abnormality were analyzed by cytogenetic and molecular genetic methods. Fluorescence in situ hybridization (FISH) using PY3.4, X alpha satellite, and SRY probes was performed in each case to analyze the sex chromosome translocation and gene translocation. SRY gene was amplified by polymerase chain reaction (PCR) and its mutation was detected by direct sequencing. The results showed that among 8 patients, 5 were positive for SRY and the remaining negative for SRY. In the patients positive for SRY genes, 3 presented testes and the left 2 streak ovaries. In the patients negative for SRY, only one case presented testes, while 2 ovaries. Direct sequencing demonstrated that all SRY genes were normal in the patients positive for SRY genes. FISH technique demonstrated that SRY genes translocated from Ypter to Xpter in 2 46,XX phenotypic males positive for SRY genes. It was concluded that SRY gene is strongly involved in male sex determination, while a sequence of other genes may be taken into account in sexual development.

Analysis of SRY gene in 8 cases of sex abnormality / 华中科技大学学报（医学）（英德文版）

In order to investigate the relationship between sex dysplasia and sex-determining region Y (SRY) gene, 8 patients with sexual abnormality were analyzed by cytogenetic and molecular genetic methods. Fluorescence in situ hybridization (FISH) using PY3.4, X alpha satellite, and SRY probes was performed in each case to analyze the sex chromosome translocation and gene translocation. SRY gene was amplified by polymerase chain reaction (PCR) and its mutation was detected by direct sequencing. The results showed that among 8 patients, 5 were positive for SRY and the remaining negative for SRY. In the patients positive for SRY genes, 3 presented testes and the left 2 streak ovaries. In the patients negative for SRY, only one case presented testes, while 2 ovaries. Direct sequencing demonstrated that all SRY genes were normal in the patients positive for SRY genes. FISH technique demonstrated that SRY genes translocated from Ypter to Xpter in 2 46,XX phenotypic males positive for SRY genes. It was concluded that SRY gene is strongly involved in male sex determination, while a sequence of other genes may be taken into account in sexual development.

Detection of fetal SRY gene in maternal plasma by real-time fluorescence quantitative PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To isolate fetal DNA from maternal plasma and examine its fetal origin.</p><p><b>METHODS</b>Fetal DNA in maternal plasma was isolated from 150 samples in the first trimester and mid-trimester of pregnancy, respectively. Real-time fluorescence quantitative polymerase chain reaction PCR (FQ-PCR) was used to determine sex-determining region Y (SRY) gene on Y chromosome.</p><p><b>RESULTS</b>Eighty-two women in the first trimester and 90 women in the mid-trimester carried male fetuses,70 and 90 samples of them were positive, respectively. The mean concentrations were (58.82+/-20.90) copies/ml and (152.08+/-62.61) copies/ml. The results of FQ-PCR were negative in the women who carried female fetuses.</p><p><b>CONCLUSION</b>The results show that fetal SRY gene can be found at a time as early as 42 days of gestation in maternal plasma by the use of FQ-PCR. The number of fetal DNA increases with gestational age. The real-time FQ-PCR is of great value in the non-invasive prenatal diagnosis.</p>

Molecular Analysis of the Y Chromosome in a 46,XY Female Phenotype

This is a case report of 46,XY female phenotype (46,XY karyotype, no pubic hair, blind vagina and absence of uterus)in an 18-year-old patient. To confirm whether a Y chromosome has a structural abnormality, fluorescent in situ hybridization (FISH) with the chromosome X/Y cocktail probe was simultaneously performed, and the six loci [PABY, RPS4Y(sy16, sy17), ZFY, DYS14] on the short arm, one locus (DYZ3) on the centromere and one locus (DYZ1) on the long arm were amplified by polymerase chain reaction (PCR). The probes used FISH hybridized to centromere of the X chromosome and heterochromatin region (Yq12) of the Y chromosome, and all PCR related Y chromosome showed positive band like normal male. From the results obtained, it seemed that the Y chromosome from the 46,XY female was structurely normal. Especially, the SRY gene has been equated with the mammalian testis-determining factor, and absence or point mutation in the SRY gene causes XY female. To detect the point mutations of SRY sequencesn, single-strand conformation polymorphism (SSCP) assay was used. Our results confirm that this patient has no mutation in the SRY gene on the Y chromosome.

A Case of 46 , XX Male / 대한산부인과학회잡지

46, XX male is a rare sex cluomasomal constitution characterized by the development of bilateral testis in persons who lack a Y chomosome. The majority of affected persons have normal external genitalia and usually seek medical advice due to infertility, hypogonadism and/or gynecomastia in adulthood. Although Y chromosomsl sequences can be detected in the majority of male subjects with 46, XX karyotype, several studies have shown thst approximately 10 % of patients lack Y chromosomal material including the SRY ( sex-determining region of Y-chromosome) gene. Several hypothesis have been proposed to explain the etiology of this constitution. 1. Translocation of the testis-determining factor (TDF) fiom the Y to the X chromosome or autosome. 2. Acquisition of Y chromosome function by a mutant autosomal or X-linked gene. 3. Undetected mosaicism with Y-bearing cell line 4. Loss of Y chmmosome hom the XXY Klinefelter zygote. We experienced a case of 46, XX male who was 30 years old. We report a case with review of the literature.

Analysis of SRY Gene in Korean Patients with Swyer Syndrome and their Family Members / 대한산부인과학회잡지

Individuals affected with Swyer syndrome are phenotypic females with 46, XY karyotype, sexual infantilism, mullerina derivatives, and bilateral streak gonads that may undergo neoplastic transformation. The pathogenesis of this syndrome is uncertain, but may be related to a defect in the regulation or expression of the testicular determining factor which is believed to be located on the short arm of the Y chromosome. Recently, a region termed "SRY", a single copy gene of the Y chromosome was identified as belonging to a testis-determining gene. This gene is Y-specific, highly conserved among mammals, and transcribed only in testis. The predicted amino acid sequence of SRY shares homology with DNA-binding domains of transcription factors such as chromatinassociated, nonhistone proteins HMG 1 and HMG 2. Hence, it was thought that there may be some change in SRY gene of the patients with Swyer syndrome. And it was reported in some cases that there was deletion or mutation in the gene, but no abnormality of SRY gene was observed in other cases. So, it is a new approach to understand the mechanism of the human sexual differentiation to investigate this syndrome in terms of DNA sequence of SRY gene. To verify the presence or absence of SRY, or the change in SRY, we performed polymerase chain reaction and DNA sequencing of the conserved region of SRY gene from four patients with Swyer syndrome and their family members. The results are as follows. 1) Four patients with Swyer syndrome, their father, and two normal male control were positive whereas two female control were negative for the band that represents the coding sequence of SRY. 2) The DNA sequences of SRY gene from four patients with Swyer syndrome, their father, and two normal male control were the same, and there was no deletion or mutation in the gene. In conclusion, there may be complex sex determining cascade including other genes not only on the Y chromosome, but also on the X chromosome or even autosome in human sexual differentiation.

Reliable sex determination of mouse preimplantation embryos by PCR amplification of male-specific genes in single blastomeres.

OBJECTIVE: To assess the reliability of sex determination in mouse preimplantation embryos using the two-step polymerase chain reaction method. SETTING: Division of Immunology, Department of Microbiology and Division of Reproductive Medicine, Department of OB/GYN. METHODS: The Sry and Zfy genes, known to be present in the sex-determining region of mouse Y chromosome, were selected for Y-specific target sequences and DXNds 3 locus located on mouse X chromosome was served as the internal control sequence. DNAs extracted from heart blood of male and female mice were used to test the correctness and specificity of the selected primers using the two-step PCR method. The same experimental conditions were then used to amplify the single copy genes in single mouse blastomeres with two pairs of primers for each of the target sequences. The sex-determined embryos were transferred to the uteri of pseudopregnant recipients to test the consistency of the assay system. RESULTS: All male and female blood DNA sample results confirmed the correct sex identification of the origin (100%). Nineteen of 20 single blastomeres showed the accurate diagnosis when compared with theirs 7/8 embryos. The sex of 36 of 37 mouse pups born from biopsied male and female embryos agreed with the predicted sex. CONCLUSION: The reliable genetic analysis of sex chromosome- specific sequences in single cell is possible by the two-step PCR method and could be applied for diagnosis of defective genes of human preimplantation embryos derived from the in vitro fertilization program.