染色体核型分析联合产前BoBs技术进行羊水产前诊断的应用价值研究

目的探讨传统染色体核型分析技术联合基于细菌人工染色体标记微球的产前BACs-on-Beads(BoBs)技术在产前诊断中的临床应用价值。方法对2017年2月16日至2018年12月20日来陕西省宝鸡市妇幼保健院产前门诊就诊的有产前诊断指征的1113位孕妇行B超引导下经腹羊膜腔穿刺术,联合应用传统核型分析与产前BoBs技术对羊水样本进行遗传学产前诊断。结果1113例羊水样本核型分析和产前BoBs检测成功率为100%。其中1031例样本应用两种检测方法结果均为正常。产前BoBs共检出29例21-三体综合征、5例18-三体综合征和8例性染色体数目异常,与核型分析结果符合率为100%。此外,产前BoBs还检出了2例微重复和1例微缺失,这2例微重复异常的核型分析结果均为正常,1例10号染色体的微缺失产前BoBs与核型分析同时检出。另有37例样本核型分析结果异常(包括染色体易位5例、倒位7例、标记染色体2例,大Y染色体5例和其他染色体多态现象18例),不在产前BoBs设计的目标染色体探针检测范围之内。结论产前BoBs可以快速准确地检出13号、18号、21号染色体和X、Y染色体的非整倍体变异和9种微缺失/微重复综合征,尤其在目标染色体数目异常检测方面,此技术与核型分析结果一致性好,与传统核型分析技术联合使用能提高胎儿常见染色体异常及微缺失综合征的检出率和检测效率,又能缓解受检者等待的焦虑情绪,具有很高的临床应用价值。     

未培养羊水荧光原位杂交快速检测60例胎儿常见染色体数目异常

目的 应用细菌人工染色体(bacterial artificial chromosome,BAC)克隆自行制备荧光探针,对胎儿常见染色体数目异常(13,18,21,X,Y)行快速产前诊断.方法 利用中国医学遗传学国家重点实验室BAC库中相应染色体特异位点克隆,自行制备荧光探针.经外周血淋巴细胞染色体杂交验证后,用于胎儿未培养羊水的快速荧光原位杂交fluorescence in situ hybridization,FISH)检测,已检测60例羊水标本.结果 所有探针特异性均为100%,杂交成功率为97.86%;FISH结果与常规核型分析一致,检出21三体2例,18三体1例.有两例涉及其它染色体的结构异常未能检出.结论 自制探针用于未培养羊水快速FISH,使用标本量少、快速、简便,可有效检出上述5种染色体的数目异常,但本法不能检出其他染色体的数目异常和结构异常,其应用仍有一定局限性.     

Rapid prenatal diagnosis of congenital Toxoplasma infection by using polymerase chain reaction and amniotic fluid.

Infection of pregnant women with Toxoplasma gondii places the developing fetus at risk for congenital infection. We report a prospective study of 43 documented cases of acute maternal Toxoplasma infections acquired during gestation in which the polymerase chain reaction (PCR) was evaluated for diagnosis of fetal infection and compared with the current standard methods. On the basis of direct lysis of pelleted amniotic fluid cells followed by amplification of a gene sequence specific for T. gondii, PCR correctly identified the presence of T. gondii in five of five samples of amniotic fluid from four proven cases of congenital infection. PCR also detected three of five positive cases from a nonprospective group. The two diagnostic methods of comparable speed, detection of specific immunoglobulin M from fetal blood and and inoculation of amniotic fluid into tissue culture, correctly identified only 3 and 4 of the 10 positive samples, respectively. The considerably more time-consuming methods of mouse inoculation of amniotic fluid and fetal blood both detected 7 of 10 positive samples. There were no false-positive diagnoses by any of the methods. Therefore, detection of T. gondii by PCR appears to be the most promising method for prenatal diagnosis of congenital Toxoplasma infection, since it is both extremely rapid and highly sensitive.     

Mutation analysis for heterozygote detection and the prenatal diagnosis of cystic fibrosis

The cystic fibrosis gene was recently cloned, and a three-base deletion removing phenylalanine 508 from the coding region was identified as the mutation on the majority of cystic fibrosis chromosomes. We used the polymerase chain reaction and hybridization with allele-specific oligonucleotides to analyze the presence or absence of this mutation on 439 cystic fibrosis chromosomes and 433 normal chromosomes from non-Ashkenazic white families. This mutation was present on 75.8 percent of the cystic fibrosis chromosomes. Using the DNA markers XV-2c and KM-19, we found that 96 percent of cystic fibrosis chromosomes with the mutation had a single DNA haplotype that occurs frequently with cystic fibrosis chromosomes. This haplotype was also found on 54 percent of the cystic fibrosis chromosomes without the three-base deletion. The three-base deletion was found on only 30.3 percent of cystic fibrosis chromosomes from Ashkenazic families, although the common cystic fibrosis haplotype was present on 97 percent of cystic fibrosis chromosomes from Ashkenazic families. The ability to detect the common mutation causing cystic fibrosis represents a major improvement in prenatal diagnosis and heterozygote detection, particularly in families in which no DNA sample is available from the affected child, and provides an improved method of testing for spouses of carriers of cystic fibrosis. Mutation analysis introduces the possibility of population-based screening programs for carriers, which on the basis of the sample in this study, would currently identify about 57 percent of the non-Ashkenazic white couples at risk.     

产前诊断266例胎儿脐带血染色体检测结果分析

目的探讨脐带血染色体分析在产前诊断中的应用和临床意义。方法对符合产前诊断指征的266例孕妇在妊娠16-38周行脐血穿刺术,抽取脐血1-2ml进行细胞培养,G显带分析胎儿染色体核型。结果 266例脐带血中培养成功261例,成功率达98.12%。在培养成功的脐血中,发现染色体异常20例（7.66%）,以三体型最多见,共16例,占染色体异常的80.0%;染色体多态性14例（5.36%）,以Y染色体异染色区增长最常见,共9例,占多态性的64.29%。结论通过脐血细胞培养染色体核型分析,可以实现胎儿染色体病的产前诊断,减少染色体异常患儿出生,对减轻家庭和社会的经济负担以及提高出生人口素质具有重要意义。     

应用间期荧光原位杂交技术进行快速产前诊断

选用１８、Ｘ着丝粒探针及２１号染色体特异重复序列探针分别对未培养的羊水和绒毛细胞进行了间期荧光原位杂交。结果表明，常染色体探针出现３个及１个信号点的比例约为１％和６％。Ｘ染色体探针出现３点的比例也在１％左右，可为常染色体单体或三体以及Ｘ三体或多体提供诊断依据。同传统的细胞遗传学方法比较，间期荧光原位杂交具有简便、迅速、灵敏等优点，有一定临床应用价值     

羊水细胞原位培养与产前诊断镶嵌体分析

目的了解载片培养瓶对羊水细胞原位培养的效果,并探讨产前诊断羊水染色体中镶嵌体不同分级水平与临床意义。方法使用载片培养瓶（研究组）与载片培养皿（对照组）对1274例有产前诊断指征的孕妇进行羊水细胞原位培养并G显带分析。结果研究组与对照组培养时间平均为6.7和8.9d,细胞克隆数平均为11.5和6.0个,两组培养天数和细胞克隆数比较差异有统计学意义（χ2=1814.4,P〈0.01;χ2=2049.04,P〈0.01）。羊水镶嵌体检出34例,Ⅰ级水平25例,Ⅱ级水平7例,Ⅲ级水平2例,其中真性镶嵌体3例。结论载片培养瓶对羊水细胞原位培养优于载片培养皿,羊水原位培养镶嵌体分级水平的研究对产前诊断有非常重要的临床意义,Ⅱ级水平不能认为全是假性镶嵌体。     

Microarray comparative genomic hybridization (CGH)-based prenatal diagnosis for chromosome abnormalities using cell-free fetal DNA in amniotic fluid

Cell-free fetal DNA (cffDNA) in the supernatant of amniotic fluid, which is usually discarded, can be used as a sample for prenatal diagnosis. For rapid prenatal diagnosis of frequent chromosome abnormalities, for example trisomies 13, 18, and 21, and monosomy X, using cffDNA, we have developed a targeted microarray-based comparative genomic hybridization (CGH) panel on which BAC clones from chromosomes 13, 18, 21, X, and Y were spotted. Microarray-CGH analysis was performed for a total of 13 fetuses with congenital anomalies using cffDNA from their uncultured amniotic fluid. Microarray CGH with cffDNA led to successful molecular karyotyping for 12 of 13 fetuses within 5 days. Karyotypes of the 12 fetuses (one case of trisomy 13, two of trisomy 18, two of trisomy 21, one of monosomy X, and six of normal karyotype) were later confirmed by conventional chromosome analysis using cultured amniocytes. The one fetus whose molecular-karyotype was indicated as normal by microarray CGH actually had a balanced translocation, 45,XY,der(14;21)(q10;q10). The results indicated that microarray CGH with cffDNA is a useful rapid prenatal diagnostic method at late gestation for chromosome abnormalities with copy-number changes, especially when combined with conventional karyotyping of cultured amniocytes.     

染色体病产前诊断方法的研究进展

产前诊断是预防严重遗传性疾病或先天性缺陷胎儿出生、提高人口质量的重要保障之一,是优生优育的基础。染色体检查是诊断遗传性疾病的可靠指标,染色体异常的产前诊断占产前诊断的40%-50%。因此,开展染色体病产前诊断具有重要意义。目前,染色体病产前诊断方法很多,各有其实用范围,本文就近年的研究进展进行了综述。     

Direct prenatal chromosome diagnosis of a malignancy

A fetal tumor was suspected at 31 weeks of gestation. The occurrence of polyhydramnios led to an ultrasound examination, which revealed deformation of the fetal head, face, eye, and neck. This was confirmed by computerized tomography. Amniocentesis yielded cells with an inverted duplication of chromosome #1. This abnormality of chromosome #1 marked the malignant teratoma cells in the amniotic fluid. Cytogenetic analysis of tumor tissue and of normal tissue obtained postnatally confirmed that the abnormality of chromosome #1 observed in amniotic fluid cells was confined to the tumor. The constitutional karyotype was normal. To our knowledge, this is the first report of the direct chromosomal detection of malignancy before birth.     

417例孕妇产前染色体多态性分布频率和形态学特征分析

目的分析产前诊断标本的染色体多态性分布频率和形态学特征,为鉴别染色体多态性和染色体异常积累经验。方法对各种高危因素和超声软指标阳性的孕妇按孕周取绒毛,羊水,脐血进行染色体核型分析。结果6262例产前诊断标本染色体多态性有417例,次缢痕的增减148例（2．36％）,D／G组随体变化174例（2．78％）,臂间倒位95例（1．52％）。结论染色体多态性在临床上没有明显的不良效应,应区别于染色体异常。     

产前诊断中羊水细胞嵌合体的回顾性分析

目的通过对产前诊断中羊水细胞嵌合体发生率,真假嵌合体的鉴别以及不同类型嵌合体胎儿的妊娠结局的回顾性分析,从而对羊水细胞嵌合体病例的产前咨询和处理做一个更好的临床指引。方法收集2001年1月至2008年12月,因各种产前诊断指征来我院行羊膜腔穿刺术及产前染色体检查检出为嵌合体的病例共26例。统计嵌合体的检出率、检出类型、相应的脐血染色体分析结果。并对所有病例进行出生后随访。结果嵌合体发生率为0.89%。其中,性染色体XX/XY嵌合体5例(占19.23%);数目异常嵌合体9例(占34.62%);结构异常嵌合体12例(占46.15%)。经脐血染色体检查,确诊为真性嵌合体的仅2例,均终止妊娠;诊为假性嵌合体的16个病例中,除1例失访外,余均正常分娩,出生随访未见异常。未行进一步脐血检查的8个病例中,3例失访,3例终止妊娠,余2例均正常分娩,出生随访未见异常。结论产前诊断羊水细胞嵌合体多为假性嵌合体,各种类型的假嵌合体一般预后均良好。当发现羊水嵌合体时,首先应正确诊断真假嵌合体;并可根据嵌合体的类型进行预后评估。     

早孕期胎儿颈部囊性淋巴瘤的产前诊断探讨

目的探讨早孕期超声探测胎儿颈部透明层厚度(nuchal translucency,NT)在诊断胎儿颈部囊性淋巴瘤(Fetal nuchal cystic hygrom as,NCH)的作用以及早孕期诊断NCH的胎儿预后。方法前瞻性分析我院2004年9月~2006年9月两年间,孕10~14w开展胎儿NT检查孕妇共520例,胎儿NT≥3mm者,介入性穿刺查胎儿染色体或基因,胎儿病理。结果520例中,胎儿NT≥3mm者6例,阳性率为1.17%。其中2例为巴氏水肿胎儿,均在孕16w超声介导下行“羊膜腔穿刺术”,羊水α地中海贫血基因诊断确诊。父母均在婚检确诊为轻型α地中海贫血。4例(1例双胎)为NCH,NCH的检出率为0.78%。NCH超声图像:胎儿颈部小囊肿8.5mm×5.8mm至9.1mm×7.5mm,NT为2.5mm~4.0mm。绒毛及羊水染色体检查1例,为46,XY,Yqh-。6例均行引产。胎儿病理:颈部囊状淋巴瘤4例,分别合并先天性上肢关节屈曲畸形、先天性胸腺发育不全、先天性胸腺缺如和Ⅱ型多囊肾。巴氏水肿胎儿2例。结论早孕期超声探测胎儿NT可以早期诊断NCH、水肿胎儿和其它染色体异常胎儿,早孕期发现的NCH多伴发多发畸形。     

Early prenatal diagnosis of congenital toxoplamosis using amniotic fluid samples and tissue culture

The presence of Toxoplasma gondii in amniotic fluid was demonstrated using tissue culture in four of nine cases of congenital toxoplasmosis, whereas Toxoplasma gondii antigen was undetectable using a sensitive enzyme immunoassay technique. Parasites were identified in monolayers four days after inoculation using an indirect immunofluorescence assay. Since tissue culture may provide evidence of infection within a few days, this method is proposed for early prenatal diagnosis of congenital toxoplasmosis.     

Rapid prenatal diagnosis of aneuploidy by quantitative fluorescent PCR on fetal samples from mothers at high risk for chromosome disorders

We report the results of a prospective study using quantitative fluorescent polymerase chain reaction (QF-PCR) and small tandem repeat markers (STR) for the rapid prenatal detection of aneuploidies in a group of pregnant women at increased risk of having fetuses with numerical chromosome disorders. Amniotic fluid samples (n = 52) were collected from mothers undergoing prenatal invasive testing for fetal abnormalities on ultrasonographic examination or abnormal maternal serum aneuploidy screening results. All samples were tested by cytogenetic analysis, but rapid diagnoses of aneuploidies were offered and performed using QF-PCR analysis with several STRs specific for chromosomes 21, 18, 13 and X. All cases with numerical chromosome aberrations involving chromosomes 21, 18 and 13 (n = 8) were correctly diagnosed. Three gonosomal aneuplodies (one 47,XXY and two 45,X) were not detected because they were uninformative for the X markers. Another sample with a deletion (46,XX,7q-), that the present assay was not designed to detect, was not identified. One sample was heavily contaminated with maternal blood and the results of the QF-PCR assays were uninformative. The remaining samples from normal fetuses provided QF-PCR patterns disomic for chromosomes 21, 18, 13 and X. Our study demonstrates that QF-PCR is a rapid method for the detection of common numerical chromosome disorders and it may play an important role in prenatal diagnosis for women at high risk for fetal aneuploidy.     

Rapid prenatal diagnosis of common chromosome aneuploidies by QF-PCR. Assessment on 18,000 consecutive clinical samples

The quantitative fluorescent PCR (QF-PCR) assay, introduced during the last few years, allows prenatal diagnoses of common chromosome aneuploidies in a few hours after sampling. We report the first assessment of QF-PCR performed on a large cohort of 18,000 consecutive clinical specimens analysed in two different Centres. All samples were analysed by QF-PCR using several selected STR markers together with amelogenin and, occasionally, SRY for fetal sexing. Results were compared with those obtained by conventional cytogenetic analysis. In 17,129 tests, normal fetuses were detected by QF-PCR. No false positives were observed. All 732 cases of trisomy 21, 18, 13, triploidies, double trisomies as well as all but one fetuses with X and Y aneuploidies were correctly diagnosed. Chromosome mosaicism could also be suspected in several samples. In some cases of in vitro culture failures, QF-PCR was the only evidence of fetal X, Y, 21, 18 and 13 chromosome complement. QF-PCR proved to be efficient and reliable in detecting major numerical chromosome disorders. The main advantages of the molecular assay are its very low cost, speed and automation enabling a single operator to perform up to 40 assays per day. QF-PCR relieves anxiety of most parents within 24 h from sampling and accelerates therapeutic interventions in the case of an abnormal result. In countries where large scale conventional cytogenetics is hampered by its high cost and lack of technical expertise, QF-PCR may be used as the only prenatal diagnostic test.     

Confirmation of prenatal diagnosis of sex chromosome mosaicism

Prenatal diagnosis of mosaicism causes problems in interpretation and in genetic counselling. Part of the difficulty with any prenatal diagnosis of mosaicism is interpretation of results without knowing the exact origin, embryonic or extraembryonic, of the abnormal cell line. To confuse the issue in cases of prenatal diagnosis of 45,X/46,XY mosaicism is the recent demonstration that a diagnosis of 45,X/46,XY made prenatally is not necessarily associated with the same phenotype as when diagnosed postnatally. We present two cases of prenatal diagnosis of sex chromosome mosaicism (45,X/46,XY and 45,X/47,XYY). Posttermination examination of the phenotypically normal male fetuses and their placentas established that the placenta was the most likely source of the 45,X cell line. An approach to confirming the prenatal diagnosis of sex chromosome mosaicism and establishing its origin utilizing detailed cytogenetic examination of both fetus and placenta is suggested.