染色体13/21α卫星探针用于产前诊断21三体综合征

目的探讨应用染色体13/21α卫星探针荧光原位杂交（FISH）技术行产前诊断21三体综合征的价值。方法选择10例经产前细胞遗传学检查证实为孕正常胎儿孕妇的羊水细胞（对照组）、3例证实为孕21三体胎儿孕妇的羊水细胞（观察组）,用13/21α卫星探针对未经培养的羊水细胞间期核进行FISH杂交。结果两组总杂交率分别为36.7%和38.6%,差异无显著性（P>0.05）。对照组和观察组含4个杂交信号的核平均百分比分别为36.5%和3.9%,含5个杂交信号的核平均百分比分别为4.0%和36.1%,差异有极显著性（P<0.01）,含5个信号的核百分比＜36.1%可作为21三体综合征的诊断标准。结论 13/21α卫星探针间期FISH 用于未培养的羊水细胞可以快速、准确地在产前诊断21三体综合征。     

产前行妊娠相关血浆蛋白A筛查联合FISH诊断Down综合征

目的 :应用单项妊娠相关血浆蛋白A(PAPP A)筛查与羊水间期荧光原位杂交 (FISH)产前诊断相结合预防妊娠Down综合征胎儿。方法 :采用酶联免疫方法 (ELISA)对孕周分别为 6～ 2 7周的 1839例孕妇进行母血PAPP A单项筛查 ,以低于同一孕周的中位数时视为可能妊娠Down综合征胎儿的高风险孕妇。取高风险孕妇羊水细胞直接进行间期FISH产前诊断并同时用部分羊水细胞遗传学检查作对照。结果 :检出 1例孕 7周孕妇其PAPP A值为 0 0 5 1U/L ,低于同一孕周中位数 2 0多倍。羊水细胞间期FISH结果显示 ,含 5个杂交信号的核占所有杂交核的 38 5 % ,与细胞遗传学分析的 2 1三体核型完全一致。结论 :单项PAPP A筛查与羊水间期细胞FISH相结合是早期防治妊娠Down综合征有效可行的方法。     

妊娠早期应用荧光原位杂交技术快速诊断唐氏综合征

目的：评价产前应用２１号染色体特异性探针荧光原位杂交技术快速诊断胎儿唐氏综合征的可行性。方法：应用２１号染色体区域特异性探针对３０例未经培养的早孕期绒毛细胞进行原位杂交，并同时行常规细胞遗传学分析以对比诊断。结果：正常染色体核型标本中，只有约１％（０％￣５％）的间期核呈现３个杂交信号，而在２１，三体型标本中，平均８６％（７８％￣９１％）的细胞核呈现３个杂交信号。结论：应用荧光原位杂交技术在妊娠早期     

2837例妊娠中期孕妇羊水间期细胞荧光原位杂交检测胎儿染色体异常的残余风险

目的 评估妊娠中期羊水间期细胞荧光原位杂交(fluorescence in situ hybridization,FISH)检测后漏诊非13、18、21、X或Y染色体非整倍体异常核型的残余风险. 方法 对中国医学科学院北京协和医学院北京协和医院2010年1月1日至2013年7月31日2 837例妊娠中期行羊膜腔穿刺术同时行间期细胞FISH检测和传统核型分析的病例及其结果进行回顾性分析.采用GLP 13/GLP21/CSP 18/CSPX/CSPY探针针对13、18、21、X或Y染色体进行间期细胞FISH检测.计算具有三大产前诊断指征(孕妇高龄、母体血清学筛查18或21-三体高风险、妊娠23周前超声发现胎儿有结构异常)的孕妇间期细胞FISH检测的检出率以及残余风险. 结果 2 837例标本中,共85例(3.0％)羊水细胞染色体核型分析异常.85例中的73例为13、18、21、X或Y染色体非整倍体的异常核型,间期细胞FISH结果均为阳性,其中21-三体共43例(包括2例嵌合体)、18-三体1 5例、13-三体l例、性染色体非整倍体14例(包括4例嵌合体).12例(14.1％)异常结果为非13、1 8、21、X或Y染色体的非整倍体,包括平衡性结构重排6例、非平衡性结构重排5例、其他染色体非整倍体1例.这12例中,间期细胞FISH结果阳性3例,均涉及21号染色体的不平衡性结构异常;阴性9例,其中4例遗传性平衡性重排和2例新生平衡性重排.2 837例病例间期细胞FISH分析对染色体异常的检出率为89.4％(76/85),异常核型漏诊率为14.1％(12/85),残余风险为0.43％(12/2 761). 结论 间期细胞FISH检测是核型分析的有效补充,但不能取代核型分析,单纯间期细胞FISH检测进行产前诊断可能漏诊部分染色体结构异常.在产前遗传咨询时,应向患者解释间期细胞FISH的检出率和残余风险,合理选择产前诊断方法.     

用荧光原位杂交技术检测唐氏综合征

目的: 探讨用荧光原位杂交技术在检测唐氏综合征中的应用价值。方法: 以Biotin标记的DSCR21q22.3探针与经处理的20例唐氏综合征患者标本外周血中期染色体及其间期细胞核进行原位杂交,统计杂交信号数量。结果: 14例唐氏综合征患者出现3个杂交信号的细胞,染色体和间期细胞核杂交平均出现率分别为98.79％和98.46％,与染色体检测的结果一致;其余染色体核型检测为嵌合体的6例患者,染色体和间期细胞核中3个杂交信号细胞平均出现率分别为75.33％和7 3.50％, 2个杂交信号细胞平均出现率分别为22.67％和21.33％。结论: 荧光原位杂交技术检测唐氏综合征具有快速、敏感度高、信号强、背景低、直观安全等优点,故FISH技术在临床遗传病检测领域中具有重要的应用价值和发展前景。     

超声检查在出生缺陷诊断中的应用：21-三体综合征和18-三体综合征的产前超声诊断

21-三体综合征（又称Down综合征）是最常见的常染色体畸形，35岁以上孕妇发生率为1／300，35岁以下为1／800，是能够通过产前羊水或绒毛染色体核型分析明确诊断的一种疾病。但由于羊水或绒毛检查属于存在一定风险性的介入性方法，不可能应用于所有孕妇。因此．首先通过其他安全有效的手段筛选出高危妊娠者进行羊水或绒毛检查，才能保证最大程度地检出21-三体综合征胎儿。超声检查能够直观显示胎儿生长发育情况，是目前敏感性和特异性最高的一种筛查手段，也是染色体异常胎儿筛查的热点研究领域。     

荧光原位杂交技术在检测假性肥大型肌营养不良症缺失型携带者中的应用

目的:应用荧光原位杂交(FISH)筛查技术检测假性肥大型肌营养不良症(DMD/BMD)缺失型携带者。方法:以外显子特异Cosmid DNA为探针(含18个外显子),采用中期和间期单色FISH技术,对9例正常男、女性及来自不同缺失型DMD/BMD家系的5例女性外周血标本、来自健康孕妇的2例羊水和2例绒毛标本进行分析。结果:72～100％外周血淋巴细胞中期相或间期核、60～70％羊水细胞间期核、95～99％绒毛细胞间期核显示预期信号。FISH检出1名、排除2名缺失型携带者。结论:充分利用FISH技术优点,结合现有其它技术,可有效筛查DMD/BMD缺失型携带者,并为女性胎儿DMD/BMD缺失型携带者产前诊断奠定基础。     

国产探针荧光原位杂交技术用于产前诊断未培养羊水细胞染色体异常的研究

目的 探讨国产探针荧光原位杂交(FISH)技术用于产前诊断未培养羊水细胞染色体非整倍体异常的临床价值,评价国产探针的性能.方法 应用FISH技术埘全旧37家省级及地区级医院产前诊断中心就诊的孕16～24周的1369例孕妇的未培养羊水细胞进行快速产前诊断;应用多色FISH技术对5条染色体(21、13、18、X和Y)进行检测.同时将羊水细胞接种、培养,行常规细胞染色体核型分析,作为FISH检测结果的对照.结果 被检测的1369份样本中,1361例未培养羊水细胞获得诊断结果,检测成功率99.42%(1361/1369).共检出异常核型35例,异常核型枪出率为2.57%(35/1361),其中包括21三体22例;13三体4例;18三体6例;18二倍体、X0 1例;18二倍体、XXY 2例.FISH榆测结果与常规细胞染色体核型分析结果一致.结论 应用国产探针FISH技术检测未培养羊水细胞染色体数目异常具有快速、简便、所用样本量少的优势,结果准确可靠.     

荧光原位杂交技术在产前诊断中的应用价值

目的探讨荧光原位杂交(fluorescence in situ hybridization,FISH)技术在产前诊断中应用价值。方法对2009年11月至2012年8月在华中科技大学同济医学院附属同济医院进行产前诊断的1148例孕妇,利用FISH技术对羊水间期核细胞进行染色体数目检测。结果 1148例孕妇中确诊胎儿为21-三体综合征28例;18-三体综合征7例;13-三体综合征2例;45,XO(特纳综合征)1例;47,XXY1例;47,XYY1例;共检测出染色体数目异常患儿40例。结论 FISH技术应用于产前诊断染色体数目异常,成功率及准确率较高,结果可靠。     

FISH技术在未培养羊水间期细胞产前诊断1例先天愚型的研究

目的 本文报告1例曾生育1胎同源罗式易位先天愚型儿的中孕妇女。用双色荧光原位杂交(FISH)检测未培养羊水细胞，产前诊断先天愚型的研究。方法 (1)选择LSI21(红色标记)及CEP18(绿色标记)DNA探针，对未培养羊水行双色FISH，同时作常规细胞培养。(2)对引产胎儿实体组织直接制备间期细胞，行21号染色体单色FISH分析。结果 (1)96％羊水细胞显示3红、2绿荧光信号。(2)羊水培养胎儿核型为46，xx，-21，t(21；21)，与第一胎基本类同。(3)引产物90％以上细胞有3个红色荧光信号。以上三项检测结果一致。结论 FISH是一种新型产前诊断失天愚型的有效方法。直接制备胎儿实体组织间期细胞行FISH分析，验证产前诊断准确性方法快速、简便。本例二次生育同源罗式易位先天愚型，病例实属罕见。     

Mosaic trisomy 12 at amniocentesis: Prenatal diagnosis and molecular genetic analysis

ObjectiveThis study is aimed at prenatal diagnosis of mosaic trisomy 12 and reviewing the literature.Materials and MethodsA 34-year-old woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Cytogenetic analysis of cultured amniocytes revealed a karyotype of 47,XX,+12[9]/46,XX[14]. She was referred to the hospital for genetic counseling. Repeated amniocentesis was performed at 22 weeks of gestation. Array comparative genomic hybridization (aCGH), interphase fluorescence in situ hybridization (FISH) and quantitative fluorescent polymerase chain reaction (QF-PCR) were applied on uncultured amniocytes, and conventional cytogenetic analysis was applied on cultured amniocytes.ResultsThe aCGH analysis on uncultured amniocytes revealed a small genomic gain in chromosome 12. Interphase FISH analysis on uncultured amniocytes using a 12q11-q12-specific probe of RP11-496H24 (green spectrum) showed three green signals in 17.8% (8/45 cells) of uncultured amniocytes. QF-PCR analysis on uncultured amniocytes using chromosome 12-specific microsatellite markers excluded uniparental disomy 12. Cytogenetic analysis of cultured amniocytes revealed a karyotype of 47,XX,+12[5]/46,XX[25]. The parents decided to continue the pregnancy. A healthy 3270 g female baby was delivered at 39 weeks of gestation, with no phenotypic abnormalities. Cytogenetic analysis of the cord blood revealed a karyotype of 46,XX in 40/40 cultured lymphocytes. The neonate was normal in growth and psychomotor development at 6 months of age. Interphase FISH analysis on uncultured urinary cells revealed 5% (1/20 cells) mosaicism for trisomy 12.ConclusionPrenatal diagnosis of mosaic trisomy 12 at amniocentesis should alert a clinically significant aneuploidy. Interphase FISH and aCGH on uncultured amniocytes are useful for rapid confirmation of low-level trisomy 12 mosaicism at repeated amniocentesis.     

Mosaic trisomy 21 at amniocentesis associated with a favorable fetal outcome and perinatal progressive decrease of the trisomy 21 cell line

ObjectiveWe present mosaic trisomy 21 at amniocentesis associated with a favorable fetal outcome and perinatal progressive decrease of the trisomy 21 cell line.Case reportA 33-year-old woman underwent elective amniocentesis at 17 weeks of gestation because of anxiety, and the karyotype of cultured amniocytes was 47,XX,+21[4]/46,XX[13]. In 17 colonies of cultured amniocytes, four colonies had 47,XX,+21, while the other 13 colonies had 46,XX. Simultaneous array comparative genomic hybridization (aCGH) analysis on uncultured amniocytes revealed the result of arr (21) × 3 [0.32] consistent with 32% mosaicism for trisomy 21. Repeat amniocentesis performed at 25 weeks of gestation revealed 47,XX,+21[4]/46,XX[24] with four colonies of 47,XX,+21 and 24 colonies of 46, XX on cultured amniocytes, and arr 21q11.2q22.3 × 2.25 by aCGH, 19.2% mosaicism for trisomy 21 (20/104 cells) by interphase fluorescence in situ hybridization (FISH), and no uniparental disomy (UPD) 21 by quantitative fluorescence polymerase chain reaction (QF-PCR) on uncultured amniocytes. The parental karyotypes were normal, and prenatal ultrasound was unremarkable. A phenotypically normal 2815-g female baby was delivered at 38 weeks of gestation. Cytogenetic analysis on the cord blood, umbilical cord and placenta revealed the karyotype of 47,XX,+21[10]/46,XX[30]. 47,XX,+21[5]/46,XX[35] and 47,XX,+21[38]/46,XX[2], respectively. QF-PCR analysis on the DNA extracted from parental bloods, uncultured amniocytes, cord blood, umbilical cord and placenta confirmed a paternal origin of trisomy 21. When follow-up at age two months, the neonate was phenotypically normal, the peripheral blood had a karyotype of 47,XX,+21[6]/46,XX[34], and no trisomy 21 signals by interphase FISH was found on 100 buccal mucosal cells. When follow-up at age 13 months, the neonate was phenotypically normal, and the peripheral blood had a karyotype of 47,XX,+21[3]/46,XX[37].ConclusionMosaic trisomy 21 at amniocentesis can be a transient and benign condition, and the abnormal trisomy 21 cell line may decrease and disappear after birth.     

应用荧光原位杂交技术快速诊断胎儿染色体数目异常

目的 探讨荧光原位杂交（ｆｌｕｏｒｅｃｅｎｔ ｉｎ ｓｉｔｕ ｈｙｂｒｉｄｉｚａｔｉｏｎ,ＦＩＳＨ）技术在快速产前诊断胎儿染色体数目异常中的价值。方法 对２０例孕１６￣３６周,有产前诊断指征者,在Ｂ超引导下经腹抽取羊水后,应用Ｘ、Ｙ、１８号染色体着丝粒探针１３ｑ１４－ｑ２１和２１ｑ１１特异性探讨,对未培养的羊水间期细胞进行ＦＩＳＨ,然后用荧光显微镜进行观察,并用Ａｐｐｌｉｅｄ ｉｍａｇｉｎｇ染色体     

Relative efficiency of FISH on metaphase and interphase nuclei from non-mosaic trisomic or triploid fibroblast cultures

Chromosome specific probes that are used in interphase fluorescence in situ hybridization (FISH) analysis are usually tested on disomic control samples. When used for preimplantation or prenatal diagnosis the aim is to detect aneuploidy, most frequently trisomy. In this study, skin fibroblast cultures from non-mosaic trisomic and triploid fetuses were analysed by FISH to assess probe efficiency regarding interphase detection of trisomy. Skin fibroblast cultures were used because they are considered to be stable in culture. FISH experiments were performed using centromeric probes for chromosomes X, Y, 18 and locus specific probes for chromosomes 13 and 21. In metaphase nuclei, the expected signals were found in 100% of at least 30 metaphases counted on each sample and this also confirmed non-mosaicism in agreement with conventional karyotyping of the fetuses. On interphase nuclei, however, only 80-89% of nuclei per population displayed the expected signals for autosomal probes and 90% for probes for the sex chromosomes. For each probe, a range of percentages was obtained that can be regarded as indicative of non-mosaic trisomy in uncultured specimens. In the case of prenatal samples, the expected presence of maternal cells may lead to a lowering of the threshold for a trisomic diagnosis. In the case of preimplantation diagnosis, the accuracy can be improved by the use of two probes per chromosome or by the analysis of two cells from each embryo.     

Mosaic trisomy 2 at amniocentesis: Prenatal diagnosis and molecular genetic analysis

ObjectiveThis study aims at presenting prenatal diagnosis of mosaic trisomy 2 and reviewing the literature.Materials, Methods, and ResultsA 32-year-old woman underwent amniocentesis at 21 weeks of gestation because of abnormal maternal serum biochemistry. Amniocentesis revealed a karyotype of 47,XY,+2[1]/46,XY[21] in in situ cultures. The single colony with trisomy 2 had two metaphase cells, and both had the karyotype of 47,XY,+2. Repeated amniocentesis was performed at 23 weeks of gestation. Interphase fluorescence in situ hybridization (FISH) analysis on uncultured amniocytes using a 2q11.1-specific probe RP11-468G5 (spectrum green) showed three green signals in 11 of 47 uncultured amniocytes, indicating 23.4% mosaicism for trisomy 2. The cultured amniocytes had a karyotype of 46,XY[20 colonies]. Polymorphic DNA marker analysis excluded uniparental disomy 2. The woman underwent the third amniocentesis at 25 weeks of gestation. Interphase FISH analysis on uncultured amniocytes revealed 9.4% (5/53 cells) mosaicism for trisomy 2. The cultured amniocytes had a karyotype of 46,XY[30 colonies]. Prenatal ultrasound was normal. The parents decided to continue the pregnancy to term, and a 3316-g baby was delivered with no phenotypic abnormalities. Cord blood had a karyotype of 46,XY[40 cells]. Interphase FISH analysis on uncultured urinary cells revealed 8.2% (4/49 cells) mosaicism for trisomy 2. The neonate was normal in growth and psychomotor development at 6 months of age.ConclusionPrenatal diagnosis of a single colony with two or more cells with trisomy 2 at amniocentesis should alert a clinically significant aneuploidy, and interphase FISH on uncultured amniocytes is useful for rapid confirmation of low-level trisomy 2 mosaicism at amniocentesis. The abnormal cell line of trisomy 2 may disappear after long-term amniocyte cultures.     

Rapid prenatal diagnosis of trisomy 21 in 5049 consecutive uncultured amniotic fluid samples by fluorescence in situ hybridisation (FISH).

OBJECTIVES: This was a retrospective study on the results of interphase fluorescence in situ hybridization (FISH), performed routinely for chromosome 21 and on ultrasonographic indications for chromosomes 13, 18, X and Y in a series of 5049 amniotic fluid samples. METHODS: Interphase FISH for chromosome 21 was performed in 5049 consecutive amniotic fluid samples for the rapid prenatal diagnosis of Down syndrome. Aneuploidy for four other chromosomes (13, 18, X and Y) was tested following ultrasonographic indications. Karyotypes from standard cytogenetic analysis were compared to the FISH results. RESULTS: Using conventional cytogenetics 3.6% (183/5049) chromosomal anomalies were detected. After exclusion of familial chromosome rearrangements, i.e. balanced autosomal reciprocal or Robertsonian translocations (30/5049) and inversions (19/5049), 2.65% chromosomal anomalies (134/5049) were diagnosed. Of this group 0.18% (9/5049) were chromosomal rearrangements not detectable by FISH and 2.47% (125/5049) were numerical chromosomal anomalies detectable by interphase FISH for chromosomes 13, 18, 21, X and Y. With routine interphase FISH for chromosome 21 and FISH on echographic indication for the other four chromosomes we detected 107/125 of these numerical chromosomal anomalies, i.e. 85.6%. All 70 cases of trisomy 21 were detected by FISH and confirmed with conventional cytogenetics (sensitivity=100%) and there were no false-positive results (specificity=100%). Maternal cell contamination of amniotic fluid samples occurred in 1.27% (64/5049) of samples; 0.26% (13/5049) of these samples were uninformative by FISH due to maternal cell contamination (12/5049) or absence of nuclei in one sample (1/5049). CONCLUSION: In this group of 5049 samples we found that FISH is a reliable technique for the rapid prenatal diagnosis of trisomy 21. The number of uninformative cases due to maternal cell contamination was low. The strategy to perform FISH for chromosome 21 in all samples and only on ultrasonographic indication for the four other chromosomes (13, 18, X and Y) followed by standard cytogenetics is effective.     

Role of FISH on uncultured amniocytes for the diagnosis of aneuploidies in the presence of fetal anomalies

OBJECTIVE: To assess the accuracy of fluorescent in situ hybridization (FISH) on amniocytes in fetuses affected by structural malformations suggestive of chromosomal anomalies. METHODS: FISH of uncultured amniotic fluid cells and conventional cytogenetic analysis were performed on 48 pregnancies with ultrasonographic (US) evidence of fetal anomalies. The AneuVysion assay (Vysis) with specific probes for chromosomes 13, 18, 21, X and Y, was used. Amniotic fluid samples were obtained between the 14th and 34th weeks of gestation. RESULTS: In cases with a single abnormal US finding (n = 15), 5 aneuploidies were detected (1 case of trisomy 13 and 4 of trisomy 21). In the group with two or more malformations (n = 33) there were 15 aneuploidies (9 cases of trisomy 18, 2 of trisomy 21, 2 monosomy X, 1 trisomy 13, and 1 triploidy). In this group, conventional cytogenetic analysis revealed two additional chromosomal anomalies not detectable by FISH (1 trisomy 16 mosaic, and a terminal deletion 4p). No sex aneuploidies were observed. CONCLUSIONS: The lack of false-positive diagnosis in the FISH analysis in our sample prompts us to consider interphase FISH as a useful tool in pregnancies at high risk for chromosomal aneuploidies. When FISH analysis is normal, the overall risk of chromosomal abnormalities is significantly reduced. However, the finding of two chromosomal anomalies undetectable by AneuVysion assay confirms the need for conventional chromosome analysis to complement FISH results. Moreover, the results collected here, in agreement with those already reported in the literature, indicate that FISH analysis on uncultured amniocytes can play an important role in counselling and decision-making, especially in cases at risk for aneuploidies, such as those with structural abnormalities at US.     

Trisomy 7 mosaicism at amniocentesis: interphase FISH, QF-PCR, and aCGH analyses on uncultured amniocytes for rapid distinguishing of true mosaicism from pseudomosaicism

ObjectiveTo present prenatal diagnosis of true trisomy 7 mosaicism.Materials, Methods and ResultsA 36-year-old woman underwent amniocentesis at 18 weeks of gestation. Amniocentesis revealed a karyotype of 47,XY,+7[20]/46,XY[9]. The parental karyotypes were normal. Repeated amniocentesis was performed at 20 weeks of gestation. Array comparative genomic hybridization (aCGH) analysis on uncultured amniocytes manifested a genomic gain in chromosome 7. Quantitative fluorescent polymerase chain reaction (QF-PCR) analysis on uncultured amniocytes showed a biparental diallelic pattern with a dosage increase in the maternal allele. Interphase fluorescence in situ hybridization (FISH) on uncultured amniocytes revealed three 7q-specific signals in 13 of 50 (26%) of the cells. The cultured amniocytes had a karyotype of 47,XY,+7[12]/46,XY[14]. The ultrasound findings were unremarkable. The pregnancy was subsequently terminated, and a fetus was delivered with facial dysmorphisms. Postnatal tissue samplings revealed the mosaic trisomy 7 level of 37.5% (15/40), 30% (12/40), 42.5% (17/40), 82.5% (33/40), 52.5% (21/40), and 27.5% (11/40) in skin, liver, lungs, placenta, membrane, and cord, respectively. The cord blood had a karyotype of 46,XY. PEG1/MEST methylation-sensitive high-resolution melting PCR assay of cord blood showed no uniparental disomy for chromosome 7.ConclusionInterphase FISH, QF-PCR, and aCGH analyses on uncultured amniocytes are useful for rapid distinguishing of true mosaicism from pseudomosaicism for trisomy 7 at amniocentesis. Cord blood sampling for confirmation of fetal trisomy 7 mosaicism is not practical.