Turner综合征Y染色体物质嵌合分子遗传学研究

摘要：目的　分析Turner综合征（TS）患儿Y染色体物质及衍生物嵌合发生的情况，为TS患儿诊断后的监测提供科学建议，改善国内TS监测和保健管理的现状。方法　选取2006年2月至2007年8月在重庆医科大学附属儿童医院诊断为TS患儿30例，进行基因组DNA 的Y编码睾丸特异性蛋白基因（TSPY）、 Y染色体中心着丝粒DYZ3重复序列（DYZ3 ）和Y性别决定区域（SRY）3个Y染色体特异序列多聚酶链反应（PCR）检测，反应结果阳性的病例补充SRY探针原位荧光杂交（FISH）分析。结果　基因组DNA 的PCR结果显示，3例患儿的TSPY、 DYZ3扩增均为阳性（10.00%），其中只有1例 SRY 扩增阳性（3.33%）；3例Y染色体物质阳性病例进一步进行FISH研究，结果显示3例SRY杂交信号均为阳性。结论　运用3个Y染色体特异序列的分子遗传学研究，证实Y染色体物质嵌合在TS不少见，每一个TS患儿都应在诊断后进行Y染色体物质的分子遗传学监测。     

Y chromosome mosaicism and occurrence of gonadoblastoma in cases of Turner syndrome and amenorrhoea

In the present study, 73 cases with a clinical diagnosis of Turner syndrome, or with primary or secondary amenorrhoea without frank Turner phenotype, were evaluated for presence of low level Y chromosome mosaicism using molecular methods. Fluorescence in-situ hybridization for centromere and q arm of the Y chromosome and nested polymerase chain reaction for the sex determining region on Y (SRY) gene were performed in peripheral blood, buccal cells and gonadal biopsies. The overall frequency of Y chromosome mosaicism was found to be 18% (13/73 cases). Four cases (16%) of Turner syndrome had Y chromosome mosaicism, seven cases (28%) with primary amenorrhoea and two cases (9%) with secondary amenorrhoea had Y chromosome mosaicism. Histologically detectable gonadoblastoma was observed in one of seven cases (14%) that had Y chromosome mosaicism. This frequency is lower than that reported previously, underscoring the need for large prospective investigations to determine the frequency of Y chromosome mosaicism and occurrence of gonadoblastoma in cases of Turner syndrome and other forms of amenorrhoea.     

A novel sex-determining region on Y (SRY) nonsense mutation identified in a 45,X/47,XYY female.

OBJECTIVE: To determine whether an SRY mutation participated in the phenotypic outcome in the case of a 45,X/47,XYY female. DESIGN: Analysis of genomic DNA for mutations in SRY. SETTING: An academic teaching hospital. PATIENT(S): A family that included one phenotypic female with 45,X/47,XYY mosaicism. INTERVENTION(S): Extraction of DNA, polymerase chain reaction analysis, nucleotide sequencing, and restriction enzyme analysis. MAIN OUTCOME MEASURE(S): Comparison of control and subject DNA sequences. RESULT(S): The patient demonstrated one nucleotide (thymine, T) deletion at position 422, leading to a frame-shift mutation. This mutation changes the codon for Tyr (TAT) to a stop codon (TAG) within the open reading frame just upstream of a conserved DNA-binding motif. Neither other mutations nor nucleotide mosaicisms were found in the remaining regions of the gene. This mutation was not present in the patient's normal father. CONCLUSION(S): The mutant SRY may be assumed to induce a nonfunctional SRY-coded protein that lacks a DNA-binding motif. These results explain the phenotypic female and the gonadal dysgenesis in the 45,X/ 47,XYY sex-reversed offspring.     

Y chromosome heterochromatin of differing lengths in two cell populations of the same individual

OBJECTIVE: To present a prenatal diagnosis report on a case where G-banding analysis of fetal metaphase chromosomes showed populations of cells with two different Y chromosomes; one with a short block of heterochromatin (Yqh-) and one with a longer block of heterochromatin (Yqh+). METHODS: These two populations of the Y chromosome were studied using fluorescent quinacrine banding and fluorescent in situ hybridization (FISH). A chromosome paint corresponding to the euchromatic region of the Y chromosome, and probes corresponding to the SRY, DYZ1, and DYZ3 regions were used for this study. RESULTS: Both Y chromosomes appeared to be structurally normal by these analyses. Subsequent ultrasound examination at 20 weeks' gestation revealed normal male genitalia. Follow-up with a neonatal blood sample analysis confirmed the above findings. CONCLUSIONS: This study reports a direct prenatal diagnosis case of two populations of the Y chromosome in the same individual. This apparent mosaicism may be explained by a postzygotic simple deletion or unequal crossover event between sister chromatids in the DYZ region.     

46,XY monozygotic twins with discordant sex phenotype

Objective: To analyze male and female sex differentiation in monozygotic twins.

Design: Retrospective study.

Setting: Multiple academic centers.

Patient(s): A pair of monozygotic twins.

Intervention(s): Skin and blood samples were obtained for DNA analysis and karyotyping.

Main Outcome Measure(s): Mutation within the SRY gene was analyzed by the polymerase chain reaction–single-stranded conformation polymorphism test. Monozygosity was ascertained by short tandem repeat analysis. Karyotypes were studied in blood and skin fibroblasts.

Result(s): SRY was present in both twins, but no mutations were detected in the SRY conserved motif. Monozygosity was confirmed by the use of short tandem repeat analysis in four loci: c-fms, thyroid peroxidase, von Willebrand factor, and tyrosine hydroxylase. The karyotype was 46,XY uniformly in both twins.

Conclusion(s): Monozygotic twins can develop discordant male and female phenotypes despite the presence of a common karyotype and despite the presence of intact testis-determining genes. In the present case, this could be due to mutation or to mosaicism involving occult 45,X cell lines in the dysgenetic gonads.     

Delineation of an isodicentric Y chromosome in a mosaic 45,X/46,X,idic(Y)(qter-p11.3::p11.3-qter) fetus by SRY sequencing, G-banding, FISH, SKY and study of distribution in different tissues.

Many factors such as genetic, developmental and hormonal are involved in mammalian sex determination. The relative importance and the mutual interactions among those factors are obscure. Study of cytogenetic mosaicism involving sex chromosomes may help to further unravel the mysterious process. We report a fetus with a mosaic karyotype, 45,X/46,X,idic(Y)(qter-p11.3::p11.3-qter), with unambiguous male external genitalia and a defect in the interventricular septum of the heart. Genotype of this fetus was extensively studied by technologies including sequencing of SRY (sex-determining region on the Y chromosome) gene, G-banding, FISH (fluorescence in situ hybridization) and SKY (spectral karyotyping). A markedly higher percentage of Y-containing cells was observed in the gonads (55%) than in the amniotic fluid (17%) and placental villi (11%), which was considered to be the major reason why the fetus did not have ambiguous genitalia.     

Phase II clinical trial of capecitabine in the treatment of advanced, persistent or recurrent squamous cell carcinoma of the cervix with translational research: a gynecologic oncology group study

PURPOSE: To evaluate the anti-tumor activity and adverse events of capecitabine in advanced, persistent or recurrent squamous cell carcinoma of the cervix, and to explore biomarkers with the potential to predict capecitabine response and toxicity. EXPERIMENTAL DESIGN: Eligible, consenting patients were treated with a starting dose of 2500 mg/m(2)/day or 1800 mg/m(2)/day (divided into two doses given every 12 h) for 14 days of each 21-day cycle. Prior chemotherapy was allowed only in the context of radiation "sensitization". Genotyping in the 5' and 3' ends of thymidylate synthase (TS) was performed in DNA from pretreatment blood. Relative gene expression of TS, dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) was quantified in RNA extracted from paraffin-embedded tumor. RESULTS: All patients had prior radiotherapy and 22 received a radiation sensitizer. A partial response was observed in 4 of 26 (15%) evaluable patients. An additional 35% of patients achieved stable disease while 42% experienced increasing disease. The most common serious non-hematological toxicities were gastrointestinal and dermatologic. Exploratory analyses suggested that: a germline polymorphism in the 3' or the 5' end of TS was not associated with TS gene expression, relative tumor expression of TS, DPD and TP were not correlated, and relative tumor expression of TP may predict severe anemia. CONCLUSIONS: Based on the modest response rate, this trial was closed without a second stage of accrual; single agent capecitabine was not selected for further study in advanced persistent or recurrent squamous cell carcinoma of the cervix previously treated with radiation or chemoradiation.     

Absence of the testicular determining factor gene SRY in XX true hermaphrodites and presence of this locus in most subjects with gonadal dysgenesis caused by Y aneuploidy.

OBJECTIVES: The purpose of our study was to discover whether the testicular determining factor gene SRY (sex-determining region on Y) is present or absent in XX true hermaphrodites and in subjects with gonadal dysgenesis caused by Y aneuploidy. STUDY DESIGN: We screened five XX true hermaphrodites and 24 subjects with gonadal dysgenesis caused by Y aneuploidy for the presence or absence of SRY. With the polymerase chain reaction technique, the sequence coding the 80 amino acid-conserved motif was amplified. The 0.9 kb Hincll pY53.3 subclone, which covers the open reading frame of SRY, serves as a probe for Southern blot analysis. RESULTS: Test results for all five XX true hermaphrodites were negative for SRY. Conversely, 22 of the 24 individuals with 45,X/46,XY gonadal dysgenesis were positive for SRY, including the 10 subjects with only bilateral streak gonads. CONCLUSIONS: The absence of SRY in XX true hemaphrodites and the presence of SRY in 10 subjects with 45,X/46,XY constitution who harbored only bilateral streak gonads seem to indicate that multiple genes are involved in gonadal differentiation.     

Molecular identification of chromosome Y sequences in Brazilian patients with Turner syndrome

The investigation of Y-specific sequences in patients with Turner Syndrome (TS) with karyotype 45,X or mosaic, has a fundamental role in the clinical management of these patients. The relationship between the presence of Y chromosome fragments and a higher risk of gonadoblastoma in TS has already been established. The aim of the study was to investigate the presence of Y-chromosome fragments in a population of 42 female Brazilian patients with TS from Mato Grosso state. Cytogenetic analysis has shown the karyotypes 45,X in 27 of them (64.3%) and mosaic in 15 (35.7%). The presence of the Y-primers SRY, DYZ3, ZFY, DYZ1, DYS1 and PABY was investigated in all patients. These markers were amplified by polymerase chain reaction (PCR) technique, using DNA genomic from peripheral blood lymphocytes. None of these patients had shown any Y-chromosome fragments when they were analysed only by the classic cytogenetic technique. The PCR analysis with the Y-specific sequences ZFY and DYZ3 were identified in two different patients (4.8%), both with karyotype 45,X. It was concluded that PCR is efficient in the investigation of hidden Y-fragments in TS patients. Therefore, this method should be included in the routine assistance of these patients.     

Absence of microdeletions in the Y chromosome in patients with a history of cryptorchidism and azoospermia or oligospermia

OBJECTIVE: To determine if cryptorchidism is associated with microdeletions of interval 6 of the Y chromosome, we evaluated this locus in men with a history of cryptorchidism with and without azoospermia or oligospermia and in a control group. DESIGN: Clinical study. SETTING: Academic research environment. PATIENT(S): Men in whom surgical treatment of cryptorchidism had been performed in childhood and healthy control male subjects. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genotyping of interval 6 of the Y chromosome. RESULT(S): Analysis of semen obtained from men treated for cryptorchidism in childhood showed azoospermia or oligospermia in 14 of 38 (37%) men. No microdeletions were identified with polymerase chain reaction amplification of 17 distinct sequence tagged sites located on the long arm of the Y chromosome and the sex determining region on Y (SRY) gene. CONCLUSION(S): Microdeletions of interval 6 of the Y chromosome were not detected in either the formerly cryptorchid or in the healthy subjects. Although we cannot exclude the possibility of point mutations, we conclude that cryptorchidism or cryptorchidism associated with azoospermia or oligospermia is not due to microdeletions involving interval 6 of the Y chromosome.     

Molecular analysis of defects in the CFTR gene and AZF locus of the Y chromosome in male infertility

OBJECTIVE: To investigate the frequency and potential impact of mutations and polymorphisms in the CFTR gene and deletions in AZF locus of the Y chromosome in patients with azoospermia (AZOO), cryptozoospermia (CRYPTO) or oligoasthenoteratozoospermia (OAT) who were to be included in an assisted reproductive technologies (ART) program. STUDY DESIGN: A total of 188 infertile men were enrolled in the study: 100 patients with AZOO, 38 with CRYPTO and 50 with OAT. RESULTS: The CFTR gene mutations or IVS8-5T variant in at least 1 allele was identified with similar frequencies among the AZOO (33%) and CRYPTO (21%) patients; 55% of the AZOO patients with normal spermatogenesis (NS) had mutations in 1 or 2 alleles. The novel R810G mutation in exon 13 was identified in 1 NS patient. The OAT or AZOO patients with Sertoli cell only syndrome (SCO) had mutations in the CFTR gene with similar frequencies to that in the general Polish population. The deletions in the AZF locus were detected in 20% of SCO patients, 11.5% of AZOO patients with maturation arrest and in 5% of CRYPTO patients. The other groups (NS, OAT) did not carry deletions in the region studied. CONCLUSION: Molecular diagnosis of the CFTR gene, Y chromosome deletion analysis and genetic counseling are necessary diagnostic elements for patients with male infertility, especially if the are included in an ART program.     

Cytogenetic analysis of 179 Iranian women with premature ovarian failure

Abstract

The importance of chromosomal abnormalities in etiology of premature ovarian failure (POF) is well known but in many cases, POF still remains idiopathic. We investigated the frequency and type of chromosomal aberrations in Iranian women diagnosed with idiopathic POF. Standard cytogenetic analysis was carried out in a total of 179 patients. Karyotype analysis of these patients revealed that 161 (89.95%) patients had normal female karyotype and 18 (10.05%) patients had abnormal karyotypes. The abnormal karyotypes included sex reverse sex determining region Y (SRY) negative (five Cases), X chromosome mosaicism (five cases), abnormal X chromosomes (three cases), abnormal autosomes (three cases) and X-autosome translocation (two cases). The overall prevalence of chromosomal abnormalities was 10.05% in this first large-scale report of chromosomal aberrations in Iranian women with POF. The results confirm previous observations and emphasis on the critical role of X chromosome abnormalities as one of the possible etiologies for POF.     

Phase II clinical trial of capecitabine in ovarian carcinoma recurrent 6-12 months after completion of primary chemotherapy, with exploratory TS, DPD, and TP correlates: a Gynecologic Oncology Group study

PURPOSE: A phase II trial was conducted to evaluate the anti-tumor activity and adverse effects of capecitabine in women with measurable platinum-sensitive ovarian cancer or platinum-sensitive primary peritoneal cancer and to explore the ability of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and thymidine phosphorylase (TP) to predict response and toxicities. EXPERIMENTAL DESIGN: Patients were treated with a daily starting dose of 2500 mg/m2/day (divided in two doses given every 12 h) for 14 days of each 21-day cycle. Genotyping in the 5' and 3' ends of TS was performed in DNA from 23/23 pre-treatment blood specimens. Relative gene expression of TS, DPD, and TP was quantified in 18/21 paraffin-embedded tumor specimens. RESULTS: Of the 27 patients enrolled on study, 2 were never treated leaving 25 patients evaluable. Two patients (8.0%) achieved a partial response, 13 (52%) exhibited stable disease, 5 (20%) displayed increasing disease, and response could not be assessed in 5 (20%). The median time to progression and survival was 3.9 and 21.2 months, respectively. The most common serious toxicities were nausea/vomiting, gastrointestinal, and dermatological. There was one treatment-related death. TS expression was associated with severe nausea/vomiting (P = 0.039), but not with other severe toxicities. TS genotype or expression of DPD or TP was not associated with any of the severe toxicities. CONCLUSIONS: Based on the low response rate, this trial was closed after the first stage of accrual, the drug was not selected for further study in this patient population, and biomarker associations with response could not be assessed.     

特发性无精症和严重少精症患者Y染色体微缺失的分子检测

目的 :研究特发性无精症和严重少精症患者与 Y染色体微缺失的关系 ,建立无精症和严重少精症患者 Y染色体微缺失的分子检测方法。方法 :应用 PCR技术对 1 0 0例无精症和严重少精症患者 (其中无精症 72例 ,严重少精症 2 8例 )进行 Y染色体 AZFa、AZFb、AZFc/DAZ、SRY的微缺失检测。结果 :1 2例患者 (1 2 % )有 AZFc的微缺失 (其中无精症 8例 ,占 1 1 .1 % ;严重少精症 4例 ,占 1 4.3% ) ,且其中 1例无精症患者为 AZFb、AZFc双重缺失 ;所有病例未发现有 AZFa的缺失 ;SRY基因 PCR扩增均为阳性。6 0例已有生育的正常男性均无 AZFa、AZFb、AZFc、SRY微缺失。结论 :Y染色体微缺失 ,特别是 AZFc/DAZ的缺失是引起无精和严重少精、造成男性不育的重要原因之一 ,在进行遗传咨询和行卵细胞质内注入精子术 (ICSI)时 ,有必要对不明原因的不育男性患者进行 Y染色体微缺失的分子检测     

A 46,XX SRY-negative man with complete virilization and infertility as the main anomaly

OBJECTIVE: To report a case of a 46,XX SRY-negative man with a male phenotype and azoospermia. DESIGN: Case report. SETTING: Molecular and Cytogenetic Unit in a University Hospital. PATIENT(S): A 35-year-old man with complete masculinization who referred to our institution because of a history of several years of infertility. INTERVENTION(S): Lymphocytic karyotype and genetic counseling. MAIN OUTCOME MEASURE(S): Peripheral blood metaphases were analyzed by standard G-banding and Q-banding. Fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) analyses were performed. RESULT(S): Semen analysis showed azoospermia. Chromosome analysis revealed a 46,XX karyotype; molecular and cytogenetic analyses excluded the presence of SRY (the sex-determining region of the Y chromosome) gene. CONCLUSION(S): This case is one of the rare patients reported in the literature in whom testicular differentiation and a complete virilization in a 46,XX chromosomal constitution does not account for a translocation of the SRY gene to the X chromosome or to the autosomes. This finding suggests that other genes downstream from SRY, not yet identified, play an important role in sex determination.     

Earliest gestational age for fetal sexing in cell-free maternal plasma

OBJECTIVES: To evaluate at what gestational age fetal DNA can reliably be detected at the earliest in maternal plasma. METHODS: We performed consecutive blood sampling in the first trimester of pregnancy in 17 women who were pregnant after in vitro fertilization (IVF) or intrauterine insemination (IUI). DNA was isolated and the Y-chromosome specific SRY was amplified by real-time polymerase chain reaction (PCR).We likewise studied 31 women prior to invasive prenatal diagnosis procedures for test validation purposes. All test results were compared to cytogenetic sex or sex at birth. RESULTS: The earliest SRY detection was at a gestational age of 5 weeks and 2 days. In none of 4 pregnancies ending in a miscarriage was SRY detected. We detected SRY in maternal plasma in 1 of 2 patients (50%) carrying a male fetus at a gestational age of 5 weeks, in 4 of 5 (80%) at a gestational age of 7 weeks, in 4 of 4 (100%) at a gestational age of 9 weeks. In all 7 women pregnant with a male fetus, the correct fetal sex was detected by 10 weeks. In none of the 6 patients who delivered a girl was SRY detected. In the validation group, SRY was detected in 13 of the 13 male, and none of the 18 female fetuses. CONCLUSIONS: We conclude that real-time PCR of the SRY gene promises to be a reliable technique for early fetal sexing in maternal plasma.     

Molecular mechanisms in sex determination: from gene regulation to pathology

Testis determination is the complex process by which the bipotential gonad becomes a normal testis during embryo development. As a consequence, this process leads to sexual differentiation corresponding to the masculinization of both genital track and external genitalia. The whole phenomenon is under genetic control and is particularly driven by the presence of the Y chromosome and by the SRY gene, which acts as the key initiator of the early steps of testis determination. However, many other autosomal genes, present in both males and females, are expressed during testis formation in a gene activation pathway, which is far to be totally elucidated. All these genes act in a dosage-sensitive manner by which quantitative gene abnormalities, due to chromosomal deletions, duplications or mosaicism, may lead to testis determination failure and sex reversal.     

Swyer syndrome with SRY + Y chromosome and rudimentary internal genitalia demonstrating temporary action of antimüllerian hormone in utero: a case report

BACKGROUND: XY gonadal dysgenesis is characterized by streak gonads in phenotypic females without somatic abnormalities. This case demonstrated a hypoplastic uterus, an unlikely finding for the syndrome, suggesting insufficient function of antimüllerian hormone prenatally. CASE: A 20-year-old, female virgin was first seen 2 years earlier complaining of primary amenorrhea. She was 168 cm tall, and secondary sexual characteristics, such as breast development and pubic and axillary hair, were absent on physical examination. Chromosome analysis with fluorescence in situ hybridization revealed 46,XY, and a molecular investigation was undertaken to assess the possibility of a mutation in SRY through DNA sequencing. SRY mutations were absent. Bilateral laparoscopic removal of dysgenetic gonads was performed at another medical center immediately after genetic confirmation for an increased risk of malignancy. When the patient was seen 1 year later, we performed ultrasonography because of no menstrual outflow. Pelvic ultrasonography revealed a hypoplastic uterus (26 x 12 mm) with a rudimentary cervix. CONCLUSION: Clinical phenotypes of different mutations of the Y chromosome, particularly on SRY, may cause Swyer syndrome patients to have a uterus with fertility potential after oocyte donation.     

Clinical, cytogenetic, and molecular analysis with 46,XX male sex reversal syndrome: case reports

Purpose

To investigate the clinical characteristics of different categories of sex-reversed 46,XX individuals and their relationships with chromosomal karyotype and the SRY gene.

Methods

Chromosome karyotyping for peripheral blood culture and multi-PCR and FISH were performed.

Results

Endocrinological data showed that their endocrine hormone levels were similar to that observed for Klinefelter syndrome, with higher FSH and LH levels and lower T levels. Chromosome karyotyping for peripheral blood culture revealed 46, XX complement for 11 males. Molecular studies showed that there were locus deletions at SY84, SY86, SY127, SY134, SY254 and SY255 in AZF on chromosome Y in 9 cases, with the SRY gene present at the terminus of the X chromosome short arm. In one case, besides 6 locus deletions in AZF, there was also SRY gene deletion. In another case, there were locus deletions only at SY254 and SY255, with SY84, SY86, SY127 SY134 loci and SRY present.

Conclusions

The majority (10/11) of 46,XX males were SRY positive, with the SRY gene translocated into the terminus of the X chromosome short arm. These patients were caused mainly by an X/Y chromosomal inter-change during paternal meiosis, leading to the differentiation of primary gonads into testes. Only a single patient (1/11) was SRY-negative, in which there might be some unknown downstream genes involved in sex determination.     

Pure non-gestational ovarian choriocarcinoma in a 45,XO/46,XX SRY-negative true hermaphrodite

Non-gestational ovarian choriocarcinoma (NGCO) is an extremely rare malignant tumor with a poor prognosis and is difficult to distinguish from gestational choriocarcinoma. True hermaphrodite (TH) is genetically a heterogenous condition causing ovarian and testicular tissue development in the same individual. We report here the first case of pure NGCO in the right ovotestis of a 23-year-old 45,XO/46X,X sex-determining region Y chromosome (SRY)-negative TH. The diagnosis of non-gestational origin was confirmed by testing five short tandem repeats (STR). The patient responded well to radical surgery with bleomycin, etoposide, cisplatin (BEP) regimen. We also hypothesize that some mutations of an X-linked or autosomal gene lead to testicular determination in SRY-negative TH patients.