荧光原位杂交技术在胎儿染色体数目异常诊断中的应用

目的探讨荧光原位杂交（FISH）技术在未培养羊水细胞染色体数目异常诊断中的应用价值。方法选择30例孕16－27周、有产前诊断指征的孕妇,采用21、13染色体位点特异性探针和18、X、Y染色体着丝粒探针,用FISH技对孕妇未培养羊水间期细胞进行检测;同时对所有受检者的羊水标本进行细胞培养,然后行常规染色体核型分析。结果30例标本均获得诊断结果,发现染色体异常1例（为标准型21号染色体三体）,且FISH检测结果与常规核型分析结果完全一致。结论FISH技术用于产前诊断胎儿染色体数目异常简便、快速、准确。     

荧光原位杂交技术在快速诊断胎儿染色体数目异常中的应用

目的探讨荧光原位杂交(FISH)技术在快速产前诊断胎儿染色体数目异常中的价值。方法采用13、18、21、X和Y染色体特异性DNA探针,对123例高危孕妇的羊水间期细胞进行FISH检测,同时行常规染色体核型分析平行诊断,以此监测对比荧光原位杂交结果的准确性。结果所测123份标本的13、18、21、X、Y染色体数目均与常规染色体核型分析的结果相符,其中,检测结果显示染色体数目正常的为120例,染色体数目异常的为3例,分别为21-三体2例,18-三体1例;正、异常与常规染色体核型分析结果符合率均为100%。结论荧光原位杂交技术检测过程简单、快捷,特异性较强,且灵敏度较高,是一种可用于临床的快速产前诊断方法。     

荧光原位杂交技术产前诊断先天性心脏病22q11.2微缺失应用价值

目的:探讨荧光原位杂交技术(FISH)检测先天性心脏病22q11.2微缺失产前诊断的临床应用价值。方法:选择70例有高危妊娠指征的孕妇羊水细胞作为病例组,30例临床诊断指征正常、剖宫产分娩的孕妇羊水细胞作为对照组,抽取16～27w孕妇羊水细胞利用21、13染色体位点和18、X、Y染色体着丝粒及22q11.2微缺失3组探针,用FISH技术对未培养羊水细胞进行检测;同时对所有受检者的羊水细胞进行培养,行染色体核型分析。结果:70例病例组FISH检测获得诊断结果,检出10例异常结果,其中5例染色体非整倍体及5例22q11.2微缺失;同时行羊水细胞染色体核型分析,只检出5例染色体非整倍体改变;与5例染色体非整倍体改变FISH检测结果相符;而应用FISH技术检出5例22q11.2微缺失胎儿与其病例的引产前影像学检查结果、尸体解剖结果完全相符。结论:FISH技术检出22q11.2微缺失病例,可明显提高先天性心脏病产前诊断的检出率,作为重要辅助性检查项目,有其临床推广价值。     

羊水细胞高通量测序与染色体核型分析在产前诊断中的应用研究

目的通过高通量测序技术(即下一代测序技术,next generation sequencing,NGS)检测孕妇羊水细胞DNA,与染色体核型分析进行对比,探索NGS在羊水细胞产前诊断中的应用价值。方法选取孕龄在18~24周之间的高龄妊娠、唐氏综合征生化筛查高风险和(或)彩超显示胎儿异常并同意产前诊断的孕妇101例,抽取孕妇羊水,提取羊水细胞DNA,制备测序文库,应用Ion Proton测序仪检测,所得的DNA序列与人类DNA参考数据库比对并作统计分析,并与同一样本染色体核型分析进行对照分析。结果 101例羊水样本处理后经NGS技术检测判定2例染色体数目异常,37例染色体片段缺失/重复;羊水细胞培养检出2例染色体数目异常,2例9号染色体臂间倒位,2例多态性。结论利用高通量测序技术检测孕妇羊水中DNA诊断胎儿染色体数目异常,其特异性与染色体核型分析技术具有较高的一致性,并可检测出缺失/重复。染色体核型分析技术与高通量测序技术相结合在检测出生缺陷上具有较好的临床实际应用价值,并可进一步展开对疾病候选基因的研究。     

FISH技术对自然流产患者胎盘绒毛染色体数目的检测结果分析

目的观察自然流产患者胎盘绒毛染色体数目及荧光原位杂交（FISH）技术在染色体数目检测中的应用情况。方法收集42例不明原因自然流产患者的胎盘绒毛,采用FISH技术以GLPl3／GLP21、GLP16／GLP22、CSPl8／CSPX／CSPY三组探针进行染色体数目检测。结果42例中检测出染色体数目异常18例（42．85％）,其中三体型8例（19．04％）、三倍体3例（7．14％）、四倍体2例（4．76％）、单倍体5例（11．91％）;29例首次流产和13例复发性流产者中分别有11例（37．93％）、7例（53．85％）检测出染色体数目异常,两者比较,P〈0．05（x2=5．153）。结论自然流产者胎盘绒毛染色体数目异常率较高（尤以复发性流产者为著）,FISH技术检测染色体数目有助于某些疾病的产前诊断。     

92例多发性骨髓瘤的临床与细胞遗传学分析

目的探讨多发性骨髓瘤（MM）细胞染色体异常的临床意义。方法采用骨髓细胞直接法、24h短期培养法制备染色体标本,用R显带技术进行核型分析。结果92例MM中,常规细胞遗传学（CC）发现异常核型24例,发生率26．09％。部分病例在CC基础上应用荧光原位杂交（FISH）技术,异常检出率大为提高。结论FISH技术深入了MM的细胞遗传学研究,伴有14q32异常是MM患者最为常见的染色体核型异常,伴有多条染色体异常MM患者其临床预后差。     

基因组测序技术应用于产前诊断胎儿染色体异常的研究

目的探讨基因组测序技术在产前诊断胎儿染色体异常中的价值。方法选取2013-12~2014-05在广西壮族自治区人民医院就诊,孕龄在18~24周的高龄妊娠、唐氏综合征生化筛查高风险和(或)彩超显示胎儿异常并同意产前诊断的孕妇60例,抽取孕妇羊水,提取羊水DNA,制备测序文库,应用Ion Proton测序仪检测,所得的基因序列与人类的参考基因组比对并作统计分析。并与同一样本经细胞培养后进行染色体核型分析进行对照分析。结果 60例羊水样本处理后经大规模平行基因组测序技术检测判定3例为染色体拷贝数异常,57例无明显异常;以羊水细胞染色体核型分析为对照,检出6例异常结果。结论利用大规模平行基因组测序技术检测孕妇羊水中DNA诊断胎儿染色体异常,其特异性与染色体核型分析技术具有较高的一致性。该技术具有高准确性、高通量、高灵敏度和低成本等优点,具有临床实际应用价值。     

41例急性髓细胞白血病患者的细胞遗传学分析

目的了解急性髓细胞白血病的细胞遗传学特征及其与预后的关系。方法对41例以FAB分类标准确诊的AML初发患者的细胞遗传学资料进行回顾性分析,应用骨髓细胞短期培养法制备染色体标本,以R显带技术进行核型分析。结果共检出异常核型16例（39．0％）,单纯数目异常的有2例,染色体结构异常的12例,其余2例同时有数目及染色体结构异常。本组中最常见的结构异常为t（15;17）,t（8;21）。结论细胞遗传学对急性髓细胞白血病的诊断具有重要的意义,特别是与FAB分型具有特异性的t（15;17）,t（8;21）染色体异常对急性白血病的诊断具有决定意义。     

沈阳地区962例孕妇羊水细胞染色体异常产前诊断的分析

目的分析在羊水中染色体异常核型与产前诊断指征的关系。方法回顾性分析该院产前诊断中心2012-01~2012-12共962例孕妇的羊水穿刺产前诊断指征和染色体异常结果资料。结果羊水染色体培养成功率99.7%。发现异常核型45例(4.6%),其中常染色体三体(13-三体、18-三体、21-三体等)11例(1.1%);性染色体5例(0.5%),结构异常20例(2.1%),嵌合体9例(0.1%),正常染色体核型中发现染色体多态性改变56例(5.8%)。结论沈阳地区羊水染色体异常核型检出率较高,具有产前诊断指征的孕妇行胎儿羊水染色体诊断对防止先天缺陷有实用性价值。     

孕早期产前筛查在染色体异常胎儿诊断中的应用

目的 评价孕早期产前筛查在降低染色体异常胎儿出生率中的应用价值.方法 采用时间分辨荧光免疫技术对2 806例单胎孕妇外周血妊娠相关蛋白-A (PAPP-A)和游离人绒毛膜促性腺激素β亚基(β-HCG)含量进行检测,综合超声测量胎儿颈项透明膜厚度(NT)及孕妇年龄、体质量和孕周,用LifeCycle3.0软件计算出胎儿患21-三体和18-三体的风险率,高风险孕妇经绒毛或羊水细胞染色体核型分析确诊.结果 在2 806例孕妇中共筛查出高风险孕妇115例,阳性率为4.1％,其中83例高风险孕妇经绒毛或羊水细胞染色体核型分析发现异常核型14例,异常发生率为16.9％,其中包括6例21-三体,1例21-三体嵌合体,3例18-三体,2例Turner综合征,1例三倍体和1例染色体结构异常核型.结论 孕早期产前筛查可以有效筛查出染色体异常胎儿.     

Prenatal diagnosis of I-cell disease in the first and second trimesters

First trimester prenatal diagnosis of I-cell disease (1 case) was based on demonstration of profound deficiency of N-acetylglucosamine 1-phosphotransferase in chorionic villi and in cultured trophoblasts derived from the chorionic villus specimen. Deficiency of this enzyme in cultured amniotic fluid cells obtained via amniocentesis was the basis for prenatal diagnosis of I-cell disease in the second trimester (2 cases). In both procedures, the diagnosis was corroborated by the finding of intracellular deficiency and extracellular elevation of multiple lysosomal enzymes in the fetal cell cultures (trophoblasts and amniotic fluid cells), as well as a significant increase in several lysosomal enzyme activities in the maternal serum.     

Prenatal diagnosis of thalassemia in Songklanagarind Hospital in southern Thailand

A thalassemia screening program for pregnant women has been established in Songklanagarind Hospital since 1992. After genetic counseling, a total of 5078 pregnant women accepted entry into a screening program for thalassemia. Couples at risk who should receive prenatal diagnosis were 2.8%. Total cases who accepted prenatal diagnosis were 135. Total clinical cases were 40 (29.6%) with achievement by prenatal diagnosis of 33 cases (82.5%). Genetic amniocentesis is the most acceptable method for prenatal diagnosis. Five cases (12.5%) were misdiagnosed due to contamination of maternal blood cells in amniotic fluid cases. Questionable results were reported in 2 cases (5%). Abortion occurred in one case (0.7%). Improvement of surgical technic in prenatal diagnosis reduced the complications and contamination of maternal cells. This program shows the feasibility of prevention and control of thalassemia disease in southern Thailand.     

Prenatal diagnosis of I-cell disease by measuring altered α-Mannosidase activity in amniotic fluid

Prenatal diagnoses of I-cell disease were carried out by examination of the amniotic fluid and cultured amniotic cells in four cases of high-risk pregnancy in three different families. Three of the four fetuses were diagnosed as having I-cell disease, on the basis of observation of abnormally increased activity of lysosomal acid hydrolases in the amniotic fluid and their decreased activity in cultured amniotic cells, thus leading to therapeutic abortion. α-Mannosidase in the amniotic fluid of the fetuses with I-cell disease exhibited significant alterations in pH profile,K _m value, thermal stability and isoelectric focusing pattern, compared with the enzyme in normal controls. The results indicate that prenatal diagnosis of I-cell disease may be accomplished by demonstration of altered enzymological characteristics of α-mannosidase in the supernatant of amniotic fluid. The significance of the alteration of α-mannosidase in the amniotic fluid is not known.     

Prenatal diagnosis of Non-ketotic hyperglycinaemia

Summary Non-ketotic hyperglycinaemia (NKH) is a devastating neurological disease for which there is no effective therapy. Consequently, most couples with a pregnancy known to be at risk for NKH request prenatal diagnosis. We have applied the combination of chorionic villus (CVS) assay for glycine cleavage enzyme activity and determination of amniotic fluid glycine concentration to increase the reliability of prenatal diagnosis for this disorder beyond that of each of these methods alone. All 15 of the at-risk pregnancies monitored had CVS glycine cleavage assay and five also had amniotic fluid glycine measurements. Two cases had no detectable cleavage activity in CVS and one gave uninterpretable enzyme results. Amniotic fluid glycine concentration was increased in all three and NKH was confirmed by abortus tissue assays for cleavage activity and amino acids. The remaining 12 case had activity in CVS (two also had normal amniotic fluid glycine levels) and delivered unaffected infants. Four of these 12 cases had cleavage activities below or at the low end of the normal range, perhaps indicating carrier status. We believe that the combination of CVS glycine cleavage assay and amniotic fluid glycine measurement is currently the best approach to the prenatal diagnosis of NKH.     

Newborn screening, prenatal diagnosis, and prenatal treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency can be detected by newborn screening. Benefits of screening for affected newborns include the prevention of severe adrenal crisis, its sequelae, and progressive signs of androgen excess. First-trimester prenatal diagnosis is possible by HLA typing and/or DNA analysis of genes within the HLA complex of chorionic villus cells; for second-trimester diagnosis, hormonal measurement o f amniotic fluid and HLA typing or DNA analysis of amniotic cells are used. Results of prenatal treatment of CAH have varied, and the efficacy of prenatal treatment by maternal glucocorticoid therapy requires further investigation.     

Feasibility of DNA diagnosis of haemoglobinopathies on coelocentesis

At 5–12 weeks of gestation the amniotic sac is surrounded by celomic fluid, which contains cells of fetal origin. This fluid can be sampled by celocentesis, which involves the ultrasound‐guided insertion of a needle through the vagina. The aim of this study was to examine the feasibility of prenatal diagnosis of haemoglobinopathies from the celomic fluid using a specific protocol. Celocentesis was performed at 7–9 weeks gestation in 26 singleton pregnancies at risk for haemoglobinopathies. In 25 cases more than 30 fetal cells were recovered from the celomic fluid and in all these cases molecular analysis for haemoglobinopathies was possible and the results were confirmed by subsequent chorionic villus sampling or amniocentesis. The results of this study suggest that reliable diagnosis of thalassemia syndromes can be performed from 7 weeks gestation by celocentesis. Further work is necessary to demonstrate the safety of celocentesis before widespread use.     

Citrullinaemia: The possibility of prenatal diagnosis

Argininosuccinate synthetase activity in amniotic fluid cells from a fetus at risk for citrullinaemia was low compared to the activity in amniotic fluid cells from a normal fetus, but five times the activity in fibroblasts from a patient with citrullinaemia. These enzyme values indicated a normal or heterozygous fetus. Chromosome analysis of the amniotic fluid cells from the fetus at risk, however, showed an unusual X/20 translocation. As we could not guarantee the delivery of a normal child, the parents chose to have a therapeutic abortion. Argininosuccinate synthetase activity in the liver and kidney of the aborted fetus was in the normal and heterozygous range respectively, confirming the prenatal diagnosis. The activity in the father's fibroblasts was low, less than 10% of normal. The difficulty of interpreting the results of prenatal diagnosis in such a family and the importance of studying parental cells are discussed.     

Citrullinaemia: the possibility of prenatal diagnosis

Argininosuccinate synthetase activity in amniotic fluid cells from a fetus at risk for citrullinaemia was low compared to the activity in amniotic fluid cells from a normal fetus, but five times the activity in fibroblasts from a patient with citrullinaemia. These enzyme values indicated indicated a normal or heterozygous fetus. Chromosome analysis of the amniotic fluid cells from the fetus at risk, however, showed an unusual X/20 translocation. As we could not guarantee the delivery of a normal child, the parents chose to have a therapeutic abortion. Argininosuccinate synthetase activity in the liver and kidney of the aborted fetus was in the normal and heterozygous range respectively, confirming the prenatal diagnosis. The activity in the father's fibroblasts was low, less than 10% of normal. The difficulty of interpreting the results of prenatal diagnosis in such a family and the importance of studying parental cells are discussed.     

Pterins analysis in amniotic fluid for the prenatal diagnosis of GTP cyclohydrolase deficiency

Summary Hyperphenylalaninaemia due to tetrahydrobiopterin deficiency is a group of rare and severe diseases. Prenatal diagnosis of dihydropteridine reductase and pyruvoyltetrahydropterin synthetase deficiencies can be achieved by enzyme assay in cultured fluid cells and/or fetal blood. In contrast, prenatal diagnosis of GTP cyclohydrolase deficiency can only rely on the measurement of pterin metabolites in the amniotic fluid.A pregnancy at risk for GTP cyclohydrolase deficiency was investigated. HPLC analysis of amniotic fluid pterins revealed neopterin and biopterin concentrations below the lowest limit of normal age-matched gestations. The mother refused abortion. The early follow-up of the child confirmed the diagnosis of GTP cyclohydrolase deficiency (hyperphenylalaninaemia, abnormal profile of urinary pterins and neurological deterioration).