Gene Copy Number Quantification of SHOX, VAMP7, and SRY for the Detection of Sex Chromosome Aneuploidies in Neonates.

Aims: To explore the feasibility of detecting sex chromosome aneuploidies (SCAs) by means of gene copy number quantification of short stature homeobox (SHOX), vesicle-associated membrane protein 7 (VAMP7), and SRY in newborns. Materials and Methods: Gene doses of SHOX, VAMP7, and SRY were determined by quantitative polymerase chain reaction (qPCR) using DNA obtained from dried blood samples from newborns. Relative quantification values were obtained. An aneuploidy profile was established according to cutoff values. Samples with ≥2 gene doses (out of range) were reanalyzed, and those with aneuploidy profiles were confirmed by karyotyping. Sensitivity, specificity, and positive and negative predictive values were obtained. Results: A total of 10,033 samples were collected (4945 females and 5088 males). Of 244 (2.43%) samples with ≥2 gene doses that were retested, 20 cases were confirmed. The overall incidence of SCAs was 1 in 500 live newborns. There were six cases of Turner syndrome (1/824), 3 cases of XXX (1/1648), 7 cases of Klinefelter syndrome (1/726), and 4 cases of of XYY (1/1272). The sensitivity was 0.952 (95.42%); the specificity was 0.975 (97.56%); the positive predictive value was 0.909 (90.91%) and the negative predictive value was 0.987 (98.77%). Conclusions: Gene copy number analyses of the VAMP7, SHOX, and SRY genes by qPCR from blood samples spotted onto filter paper is a highly reliable method for the early detection of male and female SCAs.

Putative contributions of the sex chromosome proteins SOX3 and SRY to neurodevelopmental disorders.

The male-biased prevalence of certain neurodevelopmental disorders and the sex-biased outcomes associated with stress exposure during gestation have been previously described. Here, we hypothesized that genes distinctively targeted by only one or both homologous proteins highly conserved across therian mammals, SOX3 and SRY, could induce sexual adaptive changes that result in a differential risk for neurodevelopmental disorders. ChIP-seq/chip data showed that SOX3/SRY gene targets were expressed in different brain cell types in mice. We used orthologous human genes in rodent genomes to extend the number of SOX3/SRY set (1,721). These genes were later found to be enriched in five modules of coexpressed genes during the early and mid-gestation periods (FDR < 0.05), independent of sexual hormones. Genes with differential expression (24, p < 0.0001) and methylation (40, p < 0.047) between sexes were overrepresented in this set. Exclusive SOX3 or SRY target genes were more associated with the late gestational and postnatal periods. Using autism as a model sex-biased disorder, the SOX3/SRY set was enriched in autism gene databases (FDR ≤ 0.05), and there were more de novo variations from the male autism spectrum disorder (ASD) samples under the SRY peaks compared to the random peaks (p < 0.024). The comparison of coexpressed networks of SOX3/SRY target genes between male autism and control samples revealed low preservation in gene modules related to stress response (99 genes) and neurogenesis (78 genes). This study provides evidence that while SOX3 is a regulatory mechanism for both sexes, the male-exclusive SRY also plays a role in gene regulation, suggesting a potential mechanism for sex bias in ASD.

Gonadoblastoma in patients with Ullrich-Turner syndrome.

Ullrich-Turner syndrome (UTS) is a common chromosomal abnormality caused by partial or complete X chromosome monosomy. One half of the patients have a 45,X karyotype, whereas the remaining patients display other X chromosome anomalies. In 6% to 11% of UTS, a normal or partly deleted Y chromosome has been found. A 10% to 30% risk of developing gonadoblastoma was found in the latter patients. The aim of this study was to evaluate the prevalence of Y chromosome-derived material, the occurrence of gonadoblastoma, and the incidence of possible neoplasms in patients with UTS. Of 217 patients studied with UTS and chromosome analysis of peripheral-blood lymphocytes, Y chromosome material was found in 20 patients. Fluorescence in situ hybridization (FISH) testing was performed to characterize the structurally abnormal Y chromosome in 13 cases. Molecular analysis of the SRY gene could only be performed in 20 patients with 45,X karyotype. Two patients had the SRY genomes. Of the 20 patients with Y chromosome-derived material, 17 underwent gonadectomy. The incidence of gonadoblastoma development in our series was 35.5%. Furthermore, 1 patient also showed a pure dysgerminoma, and another showed a mixed dysgerminoma and embryonal carcinoma. We emphasize the importance of complete processing of the gonadectomy specimen, including step sections, molecular studies, and FISH, in addition to the classic cytogenetic searching for Y chromosome sequences, in patients who present with a nonmosaic 45,X karyotype. Finally, we propose to routinely collect a sample for storage in the tumor bank for future studies.

Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program.

Preimplantation genetic diagnosis (PGD) allows identifying genetic traits in early embryos. Because in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied (i) the effect on pregnancy rates of holding biopsied embryos for 7 to 10 hours in holding medium at 32 °C before transfer, (ii) the effect on pregnancy rates of using embryos of different sizes for biopsy, and (iii) the efficiency of amplification by heating biopsies before polymerase chain reaction. Equine embryos were classified by size (≤300, 300-1000, and >1000 µm), biopsied, and transferred 1 to 2 or 7 to 10 hours after flushing. Some of the biopsy samples obtained were incubated for 10 minutes at 95 °C and the rest remained untreated. Pregnancy rates were recorded at 25 days of gestation; fetal gender was determined using ultrasonography and compared with PGD results. Holding biopsied embryos for 7 to 10 hours before transfer produced pregnancy rates similar to those for biopsied embryos transferred within 2 hours (63% and 57%, respectively). These results did not differ from pregnancy rates of nonbiopsied embryos undergoing the same holding times (50% for 7-10 hours and 63% for 1-2 hours). Pregnancy rates for biopsied and nonbiopsied embryos did not differ between size groups or between biopsied and nonbiopsied embryos within the same size group (P > 0.05). Incubating biopsy samples for 10 minutes at 95 °C before polymerase chain reaction significantly increased the diagnosis rate (78.5% vs. 45.5% for treated and nontreated biopsy samples respectively). Gender determination using incubated biopsy samples matched the results obtained using ultrasonography in all pregnancies assessed (11/11, 100%); untreated biopsy samples were correctly diagnosed in 36 of 41 assessed pregnancies (87.8%), although the difference between treated and untreated biopsy samples was not significant. Our results demonstrated that biopsied embryos can remain in holding medium before being transferred, until gender diagnosis by PGD is complete (7-10 hours), without affecting pregnancy rates. This simplifies the management of an embryo transfer program willing to incorporate PGD for gender selection, by transferring only embryos of the desired sex. Embryo biopsy can be performed in a clinical setting on embryos of different sizes, without affecting their viability. Additionally, we showed that pretreating biopsy samples with a short incubation at 95 °C improved the overall efficiency of embryo sex determination.

Combined genetic and imaging diagnosis for two large Chinese families affected with Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a rare X-linked recessive disorder characterized by nystagmus, impaired motor development, ataxia, and progressive spasticity. Genetically defective or altered levels of proteolipid protein (PLP1) or gap-junction alpha protein 12 gene have been found to be a common cause. Here we report on two large Han Chinese families affected with this disease. The probands of both families had produced sons featuring cerebral palsy that had never been correctly diagnosed. PMD was suspected after careful analysis of family history and clinical features. Three rounds of molecular testing, including RT-PCR, genetics linkage and SRY sequence analyses, in combination with fetal ultrasound and magnetic resonance imaging, confirmed the diagnosis. In Family 1, in addition to two patients, three carriers were identified, including one who was not yet married. Genetic testing indicated that a fetus did not have the disease. A healthy girl was born later. In Family 2, two patients and two carriers were identified, while a fetus was genetically normal. A healthy girl was born later. We concluded that by combining genetic testing and imaging, awareness of the symptoms of PMD and understanding of its molecular biology, there is great benefit for families that are at risk for producing offspring affected with this severe disease.

Molecular detection of cryptic Y-chromosomal material in patients with Turner syndrome.

A systematic search for a hidden Y-chromosome mosaicism, in Turner syndrome (TS) patients is justified by the evaluation of the risk of development of germ cell tumors. In this study, we analyzed cryptic Y-chromosome derivatives by polymerase chain reaction (PCR) coupled with fluorescence in situ hybridization (FISH) using Y-specific sequences in patients with TS, and validated this methodology. Unrelated patients with TS (n=32) of Mexican mestizo ethnic origin were diagnosed using cytogenetic analysis. Clinical assessment, endocrine evaluation, karyotyping, FISH and PCR analysis of the Y-chromosomal loci were performed. We found that 9.4% (3 out of 32) patients with TS had Y-chromosome material. Two patients showed Y-chromosome by conventional cytogenetics. One patient had no Y-chromosome by initial karyotyping (45, X) but was positive by lymphocyte PCR DNA analysis of the Y-sequence-specific sex-determining region Y (SRY) gene. Our results suggest that the detection of the Y-chromosome material using sensitive methods, such as PCR coupled with FISH, should be carried out in all patients with TS and should not be limited to TS patients with cytogenetically identifiable Y-chromosome and/or virilization.

Identification of sex-determining region Y (SRY) in maternal plasma after paternal lymphocyte immunization: is it possible?

PROBLEM: Women treated with allogeneic immunization using paternal lymphocytes often request laboratory molecular tests using cell-free fetal DNA in maternal plasma. There is concern whether the treatment can interfere with its results. This study evaluated the applicability of fetal sex determination using fragments of sex-determining region Y (SRY) in the plasma of women submitted to paternal lymphocyte immunization. METHOD OF STUDY: Non-pregnant women blood samples were collected at two different moments: prior to paternal lymphocyte immunization and after three doses of the immunotherapy, in a prospective study. For women who became pregnant, another sample was collect during the first trimester. Amplification of the fragment of the Y chromosome (SRY) was performed using real-time PCR. RESULTS: The SRY gene was not identified in any of the plasma samples of the 50 non-pregnant women submitted to paternal lymphocyte immunization at either of the two moments evaluated. For the 26 pregnant women, the results of the identification of sex -determining in maternal plasma were completely in agreement with the infant sex. CONCLUSION: Paternal lymphocyte immunization does not affect the results of SRY fragment investigation in the plasma of women submitted to paternal lymphocyte immunization therapy.

Primed in situ labeling for detecting single-copy genes.

In order to analyze male sterility caused by deletion of SRY and DAZ, we examined the accuracy and cost-effectiveness of a modified primed in situ labeling (PRINS) technique for detection of single-copy genes. Peripheral blood samples were collected from 50 healthy men; medium-term cultured lymphocytes from these samples were suspended in fixative solution and then spread on clean slides. We used four primers homologous to unique regions of the SRY and DAZ regions of the human Y-chromosome and incorporated reagents to increase polymerase specificity and to enhance the hybridization signal. PRINS of SRY and DAZ gave bands at Yp11.3 and Yq11.2, respectively, in all 50 metaphase spreads. The PRINS SRY signals were as distinct as those obtained using traditional fluorescence in situ hybridization (FISH). This new method is ideal for rapid localization of single-copy genes or small DNA segments, making PRINS a cost-effective alternative to FISH. Further enhancement of PRINS to increase its speed of implementation may lead to its wide use in the field of medical genetics.

The role of SRY mutations in the etiology of gonadal dysgenesis in patients with 45,X/46,XY disorder of sex development and variants.

BACKGROUND: The potential involvement of SRY in abnormal gonadal development in 45,X/46,X,der(Y) patients was proposed following the identification of SRY mutations in a few patients with Turner syndrome (TS). However, its exact etiological role in gonadal dysgenesis in patients with Y chromosome mosaicisms has not yet been clarified. AIMS: it was the aim of this study to screen for allelic variation in SRY in a large cohort of patients with disorders of sex development due to chromosomal abnormalities with 45,X/46,X,der(Y) karyotype. PATIENTS: twenty-seven patients, 14 with TS and 13 with mixed gonadal dysgenesis (MGD), harboring 45,X/46,X,der(Y) karyotypes were selected. METHODS: Genomic DNA was extracted from peripheral blood leukocytes of all patients and from gonadal tissue in 4 cases. The SRY coding region was PCR amplified and sequenced. RESULTS: We identified only 1 polymorphism (c.561C→T) in a 45,X/46,XY MGD patient, which was detected in blood and in gonadal tissue. CONCLUSION: our results indicate that mutations in SRY are rare findings in patients with Y chromosome mosaicisms. Therefore, a significant role of mutated SRY in the etiology of gonadal dysgenesis in patients harboring 45,X/46,XY karyotype and variants seems very unlikely.

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.

46,XX/SRY-negative true hermaphrodite.

OBJECTIVE: To describe genetic evaluation and response to surgery and letrozole therapy of a 46,XX/SRY-negative true hermaphrodite. DESIGN: Case report. SETTING: University Medical Center. PATIENT(S): Nineteen-year-old male with penile hypospadias, micropenis, and crytorchidism at the time of birth. INTERVENTION(S): Unilateral gonadectomy, and contralateral conservative gonadal surgery, followed by therapy with letrozole. MAIN OUTCOME MEASURE(S): Histopathologic, genetic and hormonal studies. RESULT(S): Genetic analysis showed that the subject was 46,XX/SRY-negative. Gonadectomy of the left gonad was performed at 16 years. The gonad resected was an ovotestes. The patient's estradiol was high (492±25 pmol/L), whereas the testosterone was low (4.2±0.5 nmol/L). Nineteen months later, conservative gonadal surgery of the contralateral gonad was performed to resect ovarian tissue, and treatment with letrozole was started. During letrozole treatment, testosterone was significantly increased (8±0.7 nmol/L), but estradiol was not changed (323±118 pmol/L). After letrozole withdrawal, testosterone did not decreased significantly (6.9±0.4 nmol/L), estradiol showed an oscillating pattern and a gonadal ultrasound showed an ovoid structure, which appeared to correspond to a follicle. At that time, estradiol was elevated (393 pmol/L). CONCLUSION(S): We present the case of a 46,XX/SRY-negative phenotypic male with bilateral ovotestes. Conservative gonadal surgery should be performed only when all ovarian tissue can be resected. Our results suggest that letrozole is not an adequate treatment for 46,XX true hermaphrodite males with ovotestes.

Mix gonadal dysgenesis associated with ring Y chromosome mosaics in a phenotypic male.

Ring chromosomes are present in 1 in 25,000 human fetuses; 99% arise de novo while less than 1% of rings are inherited. This chromosomal rearrangement may arise through a cytogenetic mechanism involving breaks in chromosome arms and fusion of the proximal broken ends, leading to a loss of distal material. Most patient Y ring chromosomes are present in a 45,X/46,X,r(Y) mosaic karyotype; molecular analyses of infertile men have shown that it is not rare to find r(Y) in these patients. However, the clinical spectrum in those cases with a 45,X cell line is broad and depends on the percentage of the monosomic cell line in different tissues. Y chromosome abnormalities and 45,X mosaic karyotypes are often associated with disorders of sex determination. Here, we report a male patient with hypospadias, cryptorchidism and a mosaic karyotype containing a low proportion of 45,X monosomic cells and multiple ring Y chromosomes in peripheral blood. Clinical, surgical, and molecular evidence was sufficient for a diagnosis of mixed gonadal dysgenesis. We suggest that a detailed cytogenetic and molecular analysis should be done in all males with bilateral descended testes and infertility.

Male infertility related to an aberrant karyotype, 46,XY,9ph,9qh+.

OBJECTIVE: To report a man with primary infertility and variant karyotype. DESIGN: Case report. SETTING: Private practice. PATIENT(S): A 37-year-old man with 4 years of primary infertility due to oligoasthenozoospermia. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): G- and C-banding. Polymerase chain reaction for SRY, DBY, RBMY, DAZ, AMELX, and AMELY. RESULT(S): G-band analysis of the proband revealed a 46,XY,9ph,9qh+ karyotype. C-banding confirmed increase in the heterochromatin in one chromosome 9 and inversion in the other. CONCLUSION(S): The morphologic difference between the homologous chromosomes 9 may have been responsible for an error in crossing-over, leading to aberrant spermatozoa and consequently to infertility.

DNA binding activity studies and computational approach of mutant SRY in patients with 46, XY complete pure gonadal dysgenesis.

Mutations of SRY are the cause of 46,XY complete pure gonadal dysgenesis (PGD) in 10-15% of patients. In this study, DNA was isolated and sequenced from blood leukocytes and from paraffin-embedded gonadal tissue in five patients with 46,XY complete PGD. DNA binding capability was analyzed by three different methods. The structure of the full length SRY and its mutant proteins was carried out using a protein molecular model. DNA analysis revealed two mutations and one synonymous polymorphism: in patient #4 a Y96C mutation, and a E156 polymorphism; in patient #5 a S143G mosaic mutation limited to gonadal tissue. We demonstrated, by all methods used, that both mutant proteins reduced SRY DNA binding activity. The three-dimensional structure of SRY suggested that besides the HMG box, the carboxy-terminal region of SRY interacts with DNA. In conclusion, we identified two SRY mutations and a polymorphism in two patients with 46,XY complete PGD, demonstrating the importance of the carboxy-terminal region of SRY in DNA binding activity.

Full-length SRY protein is essential for DNA binding.

SRY directs testicular development. It has been suggested that the only high-mobility group (HMG) box of the SRY is important for the function of this protein; however, other studies have suggested that the N- and C-terminal regions are also involved in this process. Herein, we analysed and compared in vitro the DNA-binding activity of the full-length SRY and three mutants (HMG box alone, N-terminal less and C-terminal less SRY proteins). DNA-binding capability was analysed by mobility shift assays, optical density and dissociation constant by using pure non-fusion SRY proteins. The structure of the full-length SRY was carried out using a protein molecular model. The HMG box SRY alone and C-terminal less SRY proteins had a statistically diminished DNA binding in comparison with the full-length SRY. In contrast, the affinity for DNA of the N-terminal less SRY was relatively similar to the full-length SRY. Likewise, three-dimensional structure of the full-length SRY suggested that some residues of the C-terminal region of the SRY interact with DNA. We demonstrate the importance that full-length SRY has, particularly the C-terminal region of the protein, in DNA binding in vitro. Likewise, the affinity of the HMG box alone is clearly reduced when compared with the full-length SRY.

XX Maleness and XX true hermaphroditism in SRY-negative monozygotic twins: additional evidence for a common origin.

CONTEXT: Differentiation of testicular tissue in 46,XX individuals is seen either in XX males, the majority of them with SRY gene, or in individuals, usually SRY(-), with ovotesticular disorder of sex development (OT-DSD). Although they are sporadic cases, there are some reports on familial recurrence, including coexistence of XX maleness and OT-DSD in the same family. OBJECTIVE: We report on a case of SRY(-) 46,XX monozygotic twins with genital ambiguity. METHODS: Hormonal evaluation included testosterone, FSH, and LH measurements. SRY gene was investigated by PCR and two-step PCR in peripheral leukocytes and gonadal tissues, respectively. Direct DNA sequencing of the DAX-1 coding sequence was performed. Real-time PCR for SOX9 region on chromosome 17 was obtained. RESULTS: Both twins had a 46,XX karyotype. Twin A had a 1-cm phallus with chordee, penoscrotal hypospadias, and palpable gonads. Serum levels of FSH (2.34 mIU/ml), LH (8.8 mIU/ml), and testosterone (1.6 ng/ml) were normal, and biopsies revealed bilateral testes. Twin B had a 0.5-cm phallus, perineal hypospadias, no palpable gonad on the right, and a left inguinal hernia. Hormonal evaluation revealed high FSH (8.2 mIU/ml) and LH (15 mIU/ml) and low testosterone (0.12 ng/ml). Upon herniotomy, a right testis (crossed ectopia) and a small left ovotestis were found. SRY gene was absent in both peripheral leukocytes and gonadal tissue samples. Neither DAX-1 mutations nor SOX9 duplication was identified. CONCLUSIONS: This case provides evidence that both XX maleness and XX OT-DSD are different manifestations of the same disorder of gonadal development.

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.

Mutations in SRY and WT1 genes required for gonadal development are not responsible for XY partial gonadal dysgenesis.

The WT1 transcription factor regulates SRY expression during the initial steps of the sex determination process in humans, activating a gene cascade leading to testis differentiation. In addition to causing Wilms' tumor, mutations in WT1 are often responsible for urogenital defects in men, while SRY mutations are mainly related to 46,XY pure gonadal dysgenesis. In order to evaluate their role in abnormal testicular organogenesis, we screened for SRY and WT1 gene mutations in 10 children with XY partial gonadal dysgenesis, 2 of whom with a history of Wilms' tumor. The open reading frame and 360 bp of the 5' flanking sequence of the SRY gene, and the ten exons and intron boundaries of the WT1 gene were amplified by PCR of genomic DNA. Single-strand conformation polymorphism was initially used for WT1 mutation screening. Since shifts in fragment migration were only observed for intron/exon 4, the ten WT1 exons from all patients were sequenced manually. No mutations were detected in the SRY 5' untranslated region or within SRY open-reading frame sequences. WT1 sequencing revealed one missense mutation (D396N) in the ninth exon of a patient who also had Wilms' tumor. In addition, two silent point mutations were found in the first exon including one described here for the first time. Some non-coding sequence variations were detected, representing one new (IVS4+85A>G) and two already described (-7ATG T>G, IVS9-49 T>C) single nucleotide polymorphisms. Therefore, mutations in two major genes required for gonadal development, SRY and WT1, are not responsible for XY partial gonadal dysgenesis.

Screening for mutations in the SRY gene in patients with mixed gonadal dysgenesis or with Turner syndrome and Y mosaicism.

OBJECTIVE: To investigate the presence of mutations in the open reading frame (ORF), as well as on the 5' and 3', flanking regions of the SRY gene in patients with mixed gonadal dysgenesis (MGD) or with Turner syndrome (TS) and Y mosaicism. STUDY DESIGN: We studied 13 patients with MGD and three patients with TS and Y mosaicism. DNA was isolated from blood leukocytes for subsequent polymerase chain reaction (PCR) and direct sequencing were performed in the ORF, as well as from the 5' and 3' flanking regions of the SRY gene. RESULTS: No mutations were present in any of the patients studied. CONCLUSION: The absence of mutations in these regions indicated that mutations were an unlikely cause of MGD or TS with Y mosaicism and suggested that there are others genes playing an important role in sex development.