荧光原位杂交技术的妇产科应用进展

荧光原位杂交（FISH）技术是一门新兴的分子细胞学遗传技术,具有快速、安全、简便、高灵敏度、高特异性及多色等优点,已经广泛应用于分子细胞遗传学检测、间期细胞遗传学分析以及DNA片段的染色体定位等研究中。推动了临床细胞遗传学的发展。随着细胞遗传学研究技术的不断深入,在FISH技术上建立了比较基因组杂交技术（CGH）、微阵列比较基因组杂交技术（arrayCGH）。目前FISH技术在产前诊断、植入前遗传学诊断、妇科肿瘤相关基因诊断和分析中展现出更加广阔的应用前景。从FISH技术探针的类型、标记方法及在妇产科领域的应用做简要综述。     

唐氏综合征的产前诊断分子技术研究进展

唐氏综合征是人类常见的染色体疾病，经典的细胞遗传学方法是诊断唐氏综合征的金标准，但其局限性不适合大规模的产前诊断。随着分子细胞遗传学技术发展，荧光原位杂交技术（FISH）、定量荧光PCR（QF-PCR）、微阵列-比较基因组杂交（Array CGH）、多重连接探针扩增技术（MIJPA）、引物原位标记技术（PRINs）等被用于唐氏综合征快速产前诊断，各种方法各有优劣，改进后会大力推进唐氏综合征的产前诊断速度和准确性。     

先天性疾病研究及产前诊断中Array CGH的应用

染色体微缺失、重复、扩增和非整倍体等基因组DNA失衡是导致胎儿发育迟缓、畸形、死胎、流产和其他先天性疾病的内在原因。微阵列－比较基因组杂交技术（Array CGH）利用基因芯片取代了传统比较基因组杂交技术的正常中期染色体作为杂交靶标,与分别采用不同荧光标记的待测DNA和参照DNA杂交,通过比较两种荧光强度的比率,检测出染色体基因拷贝数变化。Array CGH已成为一个重要的细胞遗传学研究工具,用于产前诊断和先天性疾病诊断染色体亚显微结构异常。     

孕妇血浆中胎儿DNA在产前诊断中的应用

目的依据孕妇血浆中存在游离胎儿DNA的理论,寻找一种无创性产前基因诊断的新方法。方法提取42例孕14~40周的产妇血浆中游离胎儿DNA,采用引物延伸预扩增(primerextensionpreamplification,PEP)后行PCR,特异性扩增其Y染色体重复序列(DYZ3)基因。同时采用9个短串联重复序列(shorttandemrepeat,STR)多态性位点的多重扩增方法以检出血浆中胎儿DNA。结果DYZ3-PCR中,32例妊娠男性胎儿孕妇中有28例血浆中出现基因扩增带,检出率为87.5%,10例妊娠女性胎儿孕妇血浆有2例假阳性结果。性别总符合率为85.7%,STR-PCR中,检测出孕妇血浆中父源性胎儿DNA的存在。结论应用孕妇血浆中胎儿DNA作产前诊断敏感性和特异性较高,是一种无创性产前基因诊断方法,具有广泛的临床应用前景。     

先天性疾病研究及产前诊断中Array CGH的应用

染色体微缺失、重复、扩增和非整倍体等基因组DNA失衡是导致胎儿发育迟缓、畸形、死胎、流产和其他先天性疾病的内在原因。微阵列-比较基因组杂交技术（Array CGH）利用基因芯片取代了传统比较基因组杂交技术的正常中期染色体作为杂交靶标,与分别采用不同荧光标记的待测DNA和参照DNA杂交,通过比较两种荧光强度的比率,检测出染色体基因拷贝数变化。Array CGH已成为一个重要的细胞遗传学研究工具,用于产前诊断和先天性疾病诊断染色体亚显微结构异常。     

分子生物学技术在植入前诊断中的应用

植入前诊断是产前诊断非常早的一种方法，目的是放弃携带严重遗传病的胚胎，将健康胚胎植入母体。两种主要的方法是聚合酶链反应（PCR）和荧光原位杂交（FISH）。PCR用于单基因病诊断，FISH用于染色体异常诊断。临床主要应用于存在遗传风险的患者如携带单基因病和染色体易位的患者。随着分子生物学技术的飞速发展，如比较基因组（CGH），全基因组扩增技术（WGA），引物延伸预扩增（PEP），间期核转换技术及DNA芯片技术（DNAchip）等PGD先进检测手段的应用，单细胞用于诊断单基因或多基因突变及染色体疾病，为期不远。     

FISH技术在产前诊断中的应用研究

目的探讨FISH技术在产前诊断中的应用特点。方法采用着丝粒、专一序列探针(13、18、21、X、Y)对产前诊断孕妇的羊水细胞进行FISH检测,并与常规细胞遗传学方法检测结果进行对比。结果孕中期产前诊断的6 617例孕妇,FISH技术发现195例胎儿染色体异常,其中21-三体111例,18-三体26例,13-三体3例,三倍体1例,47,XXY 13例,45,XO 6例,47,XXX 9例,47,XYY 10例,嵌合体16例,在着丝粒、专一序列探针FISH技术检测的范围内,其结果与常规细胞遗传学方法检测结果基本一致,在FISH技术检测出的1例低比例45,X/46,XX嵌合病例中,常规细胞遗传学方法检测未见异常。另外,常规细胞遗传学方法检测出169例染色体结构异常及13、18、21、X、Y染色体以外的数目异常的病例,FISH技术未能发现。结论FISH技术在产前诊断中具有快速简便,准确性高,可靠性强的特点,在孕周偏大羊水培养成功率较低的病例中,尤其在嵌合体病例的诊断中具有优势。但是,目前商品化的产前诊断着丝粒、专一序列FISH探针对染色体结构异常无法检测。因此,FISH技术具有一定的局限性,产前诊断中选择FISH技术要有相应使用指征。     

1例嵌合型45,X/46,X,r(Y)患者的遗传学分析

目的:应用细胞遗传学和分子生物学技术分析1例嵌合型45,X/46,X,r(Y)患者的核型。方法:应用常规染色体标本制备方法进行G-显带和C-显带;并应用CEPX(DXZ1,Xp11.1-q11.1,Spectrum Green,Vysis)探针、LSI SRY(Yp11.3,Spectrum Orange,Vysis)探针和CEP18(D18Z1,18p11.1-q11.1,Spectrum Aqua,Vysis)与患者的中期分裂相进行荧光原位杂交(fluorescence in situ hybridization,FISH);同时应用PCR技术对患者进行Y染色体微缺失检测。结果:结合G-显带、C-显带、FISH检测结果和Y染色体微缺失的检测结果,确定该患者核型为46,X,r(Y)(p11.3q12)[85]/45,X[15]。Yq11区生精基因微缺失检测未显示该患者存在缺失。结论:细胞遗传学检测结合FISH可以诊断复杂的染色体异常,为患者提供正确的遗传咨询和生育指导。     

荧光原位杂交技术的妇产科应用进展

荧光原位杂交(FISH)技术是一门新兴的分子细胞学遗传技术,具有快速、安全、简便、高灵敏度、高特异性及多色等优点.已经广泛应用于分子细胞遗传学检测、间期细胞遗传学分析以及DNA片段的染色体定位等研究中,推动了临床细胞遗传学的发展.随着细胞遗传学研究技术的不断深入,在FSH技术上建立了比较基因组杂交技术(CGH)、微阵列比较基因组杂交技术(array CGH).目前FISH技术在产前诊断、植入前遗传学诊断、妇科肿瘤相关基因诊断和分析中展现出更加广阔的应用前景.从FISH技术探针的类型、标记方法及在妇产科领域的应用做简要综述.     

46,XX男性和46，XX／45，X病例分析

目的 探讨性别决定区基因(SRY)在性分化和发育中的作用。方法 细胞遗传学核型分析以及PCR技术检测外周血SRY基因。结果 病例1的核型为46，XX，SRY( )。诊断为46，XX男性性反转综合征。病例2的核型为46，XX／45，X，SRY(-)。诊断为Turner嵌合型。结论 SRY基因检测比Y染色体更能预示睾丸组织的存在，是诊断性别发育异常患者的重要手段。性腺的病理取决于性腺组织的染色体核型和SRY基因。除SRY基因外，还存在多个参与性别决定和分化的基因，性分化异常表现高度遗传异常性。     

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.     

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.     

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.     

Complex rearrangements of Y chromosome suggest RPS4Y1 as lymphedema candidate gene

ObjectiveCystic hygromas are frequently encountered in fetus with Turner syndrome (TS). Nevertheless, identification of genetic loci responsible for the cystic hygroma has been problematic. Here, we tried to elucidate the candidate gene for cystic hygroma through a rare case of complex Y chromosomal rearrangements involving duplication of partial Yq and monosomy of partial Yp.Case reportA 30-year-old woman, gravida 1 para 0, was diagnosed with fetal cystic hygroma at 12 weeks of gestation. The genetic analysis of the product of conception revealed complex rearrangement of Y chromosome: microdeletion in Yp11.2p11.31 and microduplicatin in Yq11.223q11.23. The deleted region spans about 6.25 Mb and includes 76 genes, including SRY. The duplicated region spans about 4.76 Mb and includes 145 genes.ConclusionFrom this rare case with non-mosaic complex Y-chromosome rearrangements, we could narrow down Turner stigmata critical region to Yp11.2～p11.3. We also propose RPS4Y1 as lymphedema candidate gene.     

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.     

Evaluation of sex chromosome aneuploidies in women with Turner's syndrome by G-banding and FISH. A serial case study

OBJECTIVE: To evaluate sex chromosome aneuploidies in patients with Turner's syndrome using two cytogenetic techniques. STUDY DESIGN: A sample of 35 women with a clinical suspicion of Turner syndrome was examined in the Hospital of Obstetrics and Gynecology, Instituto Mexicano del Seguro Social, Monterrey, Mexico. They were subjected to a conventional cytogenetic technique with G-banding and to fluorescence in situ hybridization (FISH) using a specific alpha satellite X chromosome (DXZ1) and specific alpha satellite Y chromosome (DYZ1). RESULTS: Using both techniques, 17 cases (48.57%) showed the same karyotype. Using FISH: (1) in 8 cases the presence of the Y chromosome was confirmed, (2) in 18 cases (51.43%) a new cell line was identified, (3) in 2 cases (5.71%) the derivative X was clarified, and (4) in 3 cases (8.57%) the origin of the chromosome markers (1 of X chromosome and 2 of Y chromosome) was delineated. FISH highlighted the differences between the initial diagnosis, based on G-banding, and the final diagnosis, determined by specific probes for the X and Y chromosomes. CONCLUSION: FISH is a useful tool in the detection of low-frequency cell lines and identification of the nature and origin of derivative chromosomes and unknown chromosome markers that have important implications for the treatment of patients with Turner's syndrome.     

JAK3突变致严重联合免疫缺陷 1例报告并文献复习

目的    报道我国首例JAK3突变致严重联合免疫缺陷（SCID）患儿资料并文献复习。方法    2011-04-25重庆医科大学附属儿童医院对1例疑似SCID 患儿血标本（患儿、患儿父母及外祖母）进行JAK3基因PCR扩增、基因测序、单核苷酸多态性（SNPs）及TCR Vβ亚家族克隆多态性分析。结果    患儿免疫表型符合T-B+NK-，主要表现为反复呼吸道、消化道感染。JAK3基因突变为复合杂合突变，两个等位基因均为错义突变，分别来自父系第9外显子（1308G＞A：R403H）和母系的第24外显子（3354G＞A：R1085Q），患儿父母和外祖母分别为上述突变携带者。患儿T细胞抗原受体库未能检出。查阅文献截止2007年，国内外共35例患儿30种JAK3基因突变在JAK3 base注册，免疫学表型均符合T-B+NK- SCID，但临床表现从经典SCID到基本正常均有报道。结论    JAK3缺陷患者外周血T细胞及NK细胞严重减少，B细胞数量正常或稍减少但存在功能障碍，免疫球蛋白水平明显降低，故临床表现为反复细菌和病毒感染，对临床症状及实验室检查高度怀疑JAK3缺陷的患者，可通过STAT5磷酸化分析、基因测序、蛋白印迹或流式测定JAK3蛋白含量等技术进一步明确诊断。主要治疗方式为干细胞移植，若不及时进行免疫重建多数死于婴儿期。     

Y-specific sequences in Turner syndrome]

Turner Syndrome (TS) is the only one monosomy that occurrs+ in humans. The cytogenetics of TS is very well known from years. It has been estimated that almost 98-99% of TS foetuses end in abortion. It was suggested that the monosomy arises relatively late during embryonal development and survived TS individuals could be mosaics. It has been proved that mosaic karyotype mos 45,X/46X, + mar(Y) occurrs++ in 2% to 11% of TS patients. The patients having additional cell line containing der(Y) are at increased risk of gonadoblastoma development. In these cases gonadectomy should be considered. Therefore detection of mosaic and establishing the origin of marker chromosome (specially containing Y-specific sequences) is of special importance. The aim of present study was to detect the small mosaics, containing mar(Y) in TS patients, by using PCR and FISH techniques. Eight Y sequences for the PCR analyses as well as bicolor in situ hybridisation with painting probes for Y and X chromosomes have been applied. The positive amplification for Y-specific sequences has been detected in 7% of TS patients. Our results support the thesis that searching for the Y sequences should be introduced to routine genetic TS diagnosis.