无创DNA检测技术在产前胎儿非整倍体筛查中的应用

目的:探讨无创DNA检测技术在孕妇产前临床检测中的应用。方法:对276例孕妇进行胎儿无创DNA产前检测,包括高龄(年龄≥35岁)、唐筛结果为高风险或单项指标异常、超声软指标异常者。结果:276例中,267例检测为低风险,9例检测为高风险。高风险包括5例提示21-三体综合征、2例提示18-三体综合征、1例提示13-三体综合征、1例提示胎儿性染色体DNA含量不足。9例异常者经羊水和脐血穿刺检测证实与无创DNA结果吻合,准确率为100%。结论:无创DNA产前检测针对胎儿21-、18-、13-三体综合征筛查,具有简单安全、可靠等优点,较血清学筛查有着无可比拟的优越性。这项技术大大减少了有创产前诊断人数,将作为一种筛查技术大规模应用于临床,是未来发展的趋势。     

21三体综合症植入前诊断的临床前运用研究

探讨建立一个高效、稳定的21三体遗传病植入前诊断的方法,以染色体G显带核型分析为对照,取正常成人外周血单个淋巴细胞40枚、21三体患者外周血单个淋巴细胞40枚及单卵裂球20枚,采用荧光定量PCR技术同时扩增21号染色体上特异区域基因片段(DSCR)和12号染色体上管家基因(GAPDH)片断作内对照,结果在正常组织同时扩增二片断的有效率为95%(38/40),扩增产物的荧光强度比值为1.00±0.05;21三体患者单个淋巴细胞同时扩增二片断的有效扩增率为92.5%(37/40),DSCR/GAPDH荧光强度的比值约为1.58±0.17;单卵裂球的扩增效率为80.0%(16/20).三者实验结果与染色体核型分析结果完全一致,准确率100%.研究结果表明荧光定量PCR技术产前检测21三体综合征具有准确、快速、安全、实用等特点,有较高的临床推广应用价值.     

妊娠中期1641例产前筛查及95例产前诊断结果分析

目的:通过检测孕中期孕妇血清学甲胎蛋白(APF)、游离β-绒毛膜促性腺激素(Freeβ-hCG)、非结合雌三醇(uE3),进行唐氏综合征(Down's syndrome,DS)、18三体综合征和神经管缺陷(neural tube defect,NTD)的无创性产前筛查.方法:采用时间分辨荧光免疫分析法,对1641例16～20+6周孕妇进行血清AFP、Frees-hCG、uE3检测,结合孕妇年龄、孕周及体重等因素,经专用软件进行分析校正,计算风险率,对唐氏综合征和18三体综合征高风险孕妇,进行羊水细胞染色体核型分析.结果:1641例孕妇中筛查出高危孕妇98例,其中唐氏高风险孕妇73例,18三体综合征高风险孕妇4例,神经管畸形高风险孕妇21例;95例选择羊水细胞染色体分析的产前诊断,确诊唐氏综合征1例,18三体综合征1例.结论:孕中期血清三联筛查是可靠、有效的产前筛查方案,结合产前诊断可以有效降低缺陷儿出生,提高出生人口素质.     

无创产前DNA检测可有效筛查唐氏综合症降低出生缺陷

为了探究无创产前DNA检测筛查唐氏综合症降低出生缺陷的效果,本研究选择2012年4月至2017年12月我院收治的孕期产检6 192例孕妇作为研究对象,应用DNA测序方法对孕妇血浆中胎儿游离DNA进行染色体非整倍体检测,并检测其灵敏度和特异性,同时采用羊水核型分析两种结果。研究显示,阳性共73例,69例做了进一步检查,显示40例胎儿染色体检查核型正常继续妊娠,29例染色体异常。比较不同年龄组的孕妇血清学筛查唐氏综合征高风险情况,研究表明随着孕妇年龄的增加,DS阳性率逐渐降低(p0.05);血清学筛查后胎儿游离DNA (cffDNA)检测与染色体核型分析的阳性例数基本一致(p0.05)。本研究表明,采用产前无创检测胎儿游离DNA操作方便,检测灵敏度和特异性高,不会对胎儿的正常发育造成不良影响,且阳性检测率与羊水核染色体核型结果基本相同,是无创产前诊断唐氏综合征的重要方法,值得在临床上推广使用。     

黑龙江地区466例孕妇羊水穿刺产前诊断结果分析

目的:探讨羊水细胞染色体异常核型与各产前诊断之间的关系。方法:466例高危孕妇行羊膜腔穿刺术后羊水细胞培养及染色体核型分析。结果:异常核型66例,异常率14.16%,包括染色体数目异常27例,三体综合征22例(21-三体15例、18-三体6例、13-三体1例),占异常染色体核型的33.33%,占染色体数目异常的81.48%;染色体结构异常39例,主要包括染色体多态性、平衡易位、倒位和衍生等,占染色体异常核型的59.10%。异常核型检出率中血清学筛查高危组(14.44%)要高于高龄妊娠组(10.89%)和有不良孕产史组(11.11%)(P0.05);超声提示胎儿发育异常组(23.26%)要高于血清筛查高危组(P0.05)。结论:血清筛查高危和超声提示胎儿发育异常是黑龙江地区最主要的产前诊断指征,异常核型以21-三体综合征检出率最高。通过对高危孕妇羊水细胞染色体的核型分析可发现部分染色体疾病,从而避免此类出生缺陷儿的出生。     

美国白蛾核型多角体病毒实时荧光定量PCR检测方法的建立及应用

为了建立对昆虫核型多角体病毒（nucleopolyhedrovirus）进行定量检测方法,本研究选用美国白蛾Hyphantria cunea核型多角体病毒的polyhedrin基因为目的基因,经引物设计、PCR 扩增、目的片段与载体连接转化以及重组质粒的鉴定,并对重组质粒标准品和样品DNA进行检测,构建出美国白蛾核型多角体病毒SYBR GreenⅠ荧光定量标准曲线。统计学分析显示标准品浓度的对数值与Ct 值之间存在良好的线性关系（R=0.998）。该方法的检测灵敏度为10¹～10²拷贝/μL,线性范围达5个数量级,扩增产物形成单一的特异性熔解峰,组内组间的变异系数均小于3%。根据已建立的方法对不同感染时间段的感病幼虫进行检测,每毫克幼虫样本内病毒polyhedrin基因拷贝数增殖倍数对数值与感染时间（d）呈正相关（R=0.987）。这些结果表明,建立的美国白蛾核型多角体病毒荧光定量 PCR检测方法是可靠的,具有灵敏度高、重复性好等特点,可为昆虫核型多角体病毒体内增殖动态研究及昆虫病毒的标准化生产提供参考。     

彩色多普勒超声检查对胎儿颅内畸形筛查的应用价值及染色体异常分析

摘要 目的：探讨彩色多普勒超声检查对胎儿颅内畸形筛查的应用价值,并进行染色体异常分析。方法：选择2016年2月至2019年5月本院收治的进行胎儿颅内畸形筛查的高危孕妇120例,所有孕妇都给予彩色多普勒二维超声与三维超声筛查,对超声筛查异常者进行染色体异常分析,记录预后情况。结果：在120例孕妇中,二维超声诊断为胎儿颅内畸形12例,三维超声诊断为13例(预后都确诊为胎儿颅内畸形)。染色体核型筛查检出胎儿颅内畸形12例,其中21-三体综合征8例,18-三体综合征3例,13-三体综合征1例。确诊为胎儿颅内畸形的孕妇超声NT值都显著高于非胎儿颅内畸形孕妇,差异都有统计学意义(P<0.05)。孕妇选择终止妊娠10例,选择继续妊娠3例,继续妊娠3例胎儿都最终死亡。结论：产前彩色多普勒超声结合染色体核型在胎儿颅内畸形筛查中具有很高的价值,两者可互相补充,共同发挥诊断与预后评估价值。     

利用胎儿核酸进行产前诊断的研究进展

本文分别介绍了孕妇血浆中胎儿DNA和RNA的特性以及近年采利用胎儿DNA和胎儿RNA进行产前基因诊断技术的研究进展,并且着重介绍了其中三种产前基因诊断技术:多重连结探针扩增法(MLPA)、单碱基延伸-质谱分析(SABER-MS)和SNP-RNA等位基因比率法。综述了利用胎儿核酸进行产前诊断技术在临床上的应用。     

羊水直接PCR法快速诊断脊肌萎缩症的实验研究

目的：建立一种快速、简便的产前诊断脊肌萎缩症的检测方法。方法：基于运动神经元生存基因SMNc和SMNt的两个同源拷贝碱基的差异,对SMNt基因第7、8外显子进行缺失分析,抽取孕妇羊水后用作者自行研制的“HpH—Buffer”,以离心后羊水沉淀中的胎儿细胞为模板直接进行PCR扩增,扩增产物进行酶切限制性片断长度多态性分析,对2例有脊肌萎缩症患儿生育史的孕妇怀有的胎儿进行产前基因诊断。同时,为考察羊水直接扩增方法的效率,对羊水样本直接扩增和对羊水样本中提取的基因组DNA扩增进行了平行实验。结果：2例胎儿均有SMNt基因第7、8外显子缺失;以羊水为模板和以基因组DNA为模板进行扩增的效率相似。结论：应用“HpHBuffer”直接对羊水样本中SMN基因上外显子7、8进行扩增,结合限制性片断长度多态性分析SMNt上是否发生缺失,可缩短诊断时间,节约诊断成本,减少检测过程中可能出现的样本交叉污染,有望成为临床上产前诊断脊肌萎缩症的新方法。     

1200例孕中期产前筛查结果的回顾性分析

目的：评价孕妇血清标记物（甲胎蛋白AFP、β-绒毛膜促性腺激素β-hCG和雌三醇uE3）的孕中期三联筛查在临床中的应用价值。方法：采用酶联免疫吸附法（ELISA）对1200例孕中期（14～22周）孕妇进行血清标记物AFP、β-hCG和uE3的检测,结合孕龄、孕周、体重等因素,经专门的筛查分析软件,计算唐氏综合征,18三体及神经管缺陷（NTD）的风险率。如孕妇为高风险,则进行胎儿的超声检查和染色体核型分析的产前诊断。结果：在1200例孕妇中,筛查高风险的孕妇有73例,其中唐氏综合征,18三体,NTD高风险孕妇分别为65例,5例和3例,假阳性率为6.08%（73/1200）。其中59例接受了产前诊断,占高风险孕妇的80.8%（59/73）。共检出1例唐氏综合征儿和1例无脑儿,未发现18三体,检出率为100%（2/2）,未有漏诊的情况。妊娠不良结局在筛查高风险组和低风险组的比率分别为17.1%和1.32%,两组有显著性差异（P〈0.01）。结论：利用孕妇血清标记物（AFP、β-hCG和uE3）的孕中期无创伤性产前筛查,结合产前诊断,对减少出生缺陷儿的出生,具有重要意义,并且高风险的筛查结果对胎儿的预后有一定的提示作用。     

Chromosomal findings in fetuses with prenatally diagnosed cysts of the choroid plexus

Summary Choroid plexus cysts were diagnosed in 25 out of 823 fetuses with prenatally diagnosed abnormalities (growth retardation/malformations). Among these, 5 revealed a chromosomal disorder (4 cases with trisomy 18 and one case with a translocation trisomy 21). Additional abnormalities, such as growth retardation, holoprosencephaly, hydrocephalus and club foot, were found in 6 out of the 20 fetuses with no chromosomal abnormality. All fetuses with a chromosomal disorder revealed further typical prenatally recognizable abnormalities. Our observation indicates that prenatally diagnosed choroid plexus cysts should be considered as an indication for prenatal chromosomal diagnosis, although the risk of there being an underlying chromosomal disorder is low in cases with no additional abnormalities.     

Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for Rapid Diagnosis of Sex Chromosome Aneuploidies

Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR). Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY), five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377), one X/Y-common STR (X22), and two autosomal STRs (D13S305 and D21S11). Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.     

The combined QF-PCR and cytogenetic approach in prenatal diagnosis

In this study, the importance of quantitative fluorescence polymerase chain reaction (QF-PCR) aneuploidy diagnosis test which provides earlier and easier results were discussed. The cell cultures and DNA isolations were performed on 100 amniotic fluids. DNA isolations were made from peripheral blood samples of mothers who had blood-stained amniotic fluid samples. The reasons of references of these pregnant women to our division were increased maternal age, positive double/triple screening test and fetal anomaly history. QF-PCR applied to 19 short tandem repeat markers in the chromosomes 13, 18, 21 and genes X and Y chromosomes. All electropherogram peaks were evaluated on ABI3130. Thirty two (32 %) samples have high maternal age, seven (7 %) have fetal anomaly and the others have double/triple screening test positivity. Ninety-nine (99 %) of the 100 amniotic fluid samples were resulted, but one (1 %) of them could not examined because of the culture failure. The maternal contamination rates were determined as 3 %. Of 100 samples, 2 had trisomy 21 (2 %), 1 had trisomy 13 (1 %), 1 had structural abnormalities (1 %) and the others (97 %) have not any aneuploidy. The results of QF-PCR were in compatible with the results of cell culture and chromosome analysis. Although QF-PCR is an easier and an earlier test, it has a limitation of not to able to scan full genome. It is also sensitive for maternal contamination, so it should be tested together with maternal blood samples. QF-PCR aneuploidy test is the fastest diagnostic test for prenatal diagnosis and so it provides less stressful period for pregnant women.     

Comparative genomic hybridization in clinical cytogenetics.

We report the results of applying comparative genomic hybridization (CGH) in a cytogenetic service laboratory for (1) determination of the origin of extra and missing chromosomal material in intricate cases of unbalanced aberrations and (2) detection of common prenatal numerical chromosome aberrations. A total of 11 fetal samples were analyzed. Seven cases of complex unbalanced aberrations that could not be identified reliably by conventional cytogenetics were successfully resolved by CGH analysis. CGH results were validated by using FISH with chromosome-specific probes. Four cases representing common prenatal numerical aberrations (trisomy 21, 18, and 13 and monosomy X) were also successfully diagnosed by CGH. We conclude that CGH is a powerful adjunct to traditional cytogenetic techniques that makes it possible to solve clinical cases of intricate unbalanced aberrations in a single hybridization. CGH may also be a useful adjunct to screen for euchromatic involvement in marker chromosomes. Further technical development may render CGH applicable for routine aberration screening.     

Rapid-prenatal diagnosis through fluorescence in situ hybridization for preventing aneuploidy related birth defects

BACKGROUND AND OBJECTIVE:

Women with high-risk pregnancies are offered prenatal diagnosis through amniocentesis for cytogenetic analysis of fetal cells. The aim of this study was to evaluate the effectiveness of the rapid fluorescence in situ hybridization (FISH) technique for detecting numerical aberrations of chromosomes 13, 21, 18, X and Y in high-risk pregnancies in an Indian scenario.

MATERIALS AND METHODS:

A total of 163 samples were received for a FISH and/or a full karyotype for prenatal diagnosis from high-risk pregnancies. In 116 samples both conventional culture techniques for getting karyotype through G-banding techniques were applied in conjunction to FISH test using the AneuVysion kit (Abbott Molecular, Inc.), following standard recommended protocol to compare the both the techniques in our setup.

RESULTS:

Out of 116 patients, we got 96 normal for the five major chromosome abnormality and seven patients were found to be abnormal (04 trisomy 21, 02 monosomy X, and 01 trisomy 13) and all the FISH results correlated with conventional cytogenetics. To summarize the results of total 163 patients for the major chromosomal abnormalities analyzed by both/or cytogenetics and FISH there were 140 (86%) normal, 9 (6%) cases were abnormal and another 4 (2.5%) cases were suspicious mosaic and 10 (6%) cases of culture failure. The diagnostic detection rate with FISH in 116 patients was 97.5%. There were no false-positive and false-negative autosomal or sex chromosomal results, within our established criteria for reporting FISH signals.

CONCLUSION:

Rapid FISH is a reliable and prompt method for detecting numerical chromosomal aberrations and has now been implemented as a routine diagnostic procedure for detection of fetal aneuploidy in India.     

From karyotyping to array-CGH in prenatal diagnosis

Conventional karyotyping detects chromosomal anomalies in up to 35% of pregnancies with fetal ultrasound anomalies, depending on the number and type of these anomalies. Extensive experience gained in the past decades has shown that prenatal karyotyping is a robust technique which can detect the majority of germline chromosomal anomalies. For most of these anomalies the phenotype is known. In postnatal diagnosis of patients with congenital anomalies and intellectual disability, array-CGH/SNP array has become the first-tier investigation. The higher abnormality detection yield and its amenability to automation renders array-CGH also suitable for prenatal diagnosis. As both findings of unclear significance and unexpected findings may be detected, studies on the outcome of array-CGH in prenatal diagnosis were initially performed retrospectively. Recently, prospective application of array-CGH in pregnancies with ultrasound anomalies, and to a lesser extent in pregnancies referred for other reasons, was studied. Array-CGH showed an increased diagnostic yield compared to karyotyping, varying from 1-5%, depending on the reason for referral. Knowledge of the spectrum of array-CGH anomalies detected in the prenatal setting will increase rapidly in the years to come, thus facilitating pre- and posttest counseling. Meanwhile, new techniques like non-invasive prenatal diagnosis are emerging and will claim their place. In this review, we summarize the outcome of studies on prenatal array-CGH, the clinical relevance of differences in detection rate and range as compared to standard karyotyping, and reflect on the future integration of new molecular techniques in the workflow of prenatal diagnosis.     

Chromosomal mosaicism detected by karyotyping and chromosomal microarray analysis in prenatal diagnosis

To investigate the incidence and clinical significance of chromosomal mosaicism (CM) in prenatal diagnosis by G-banding karyotyping and chromosomal microarray analysis (CMA). This is a single-centre retrospective study of invasive prenatal diagnosis for CM. From 5758 karyotyping results and 6066 CMA results, 104 foetal cases with CM were selected and analysed further. In total, 50% (52/104) of foetal cases with CM were affected by ultrasound-detectable phenotypes. Regardless of whether they were singleton or twin pregnancies, isolated structural defects in one system (51.35%, 19/37 in singletons; 86.67%, 13/15 in twins) and a single soft marker (18.92%, 7/37 in singletons; 13.33%, 2/15 in twins) were the most common ultrasound anomalies. Mosaic autosomal trisomy (19.23%, 20/104) was the most frequent type, and its rate was higher in phenotypic foetuses (28.85%, 15/52) than in non-phenotypic foetuses (9.62%, 5/52). There was no difference in mosaic fractions between phenotypic and non-phenotypic foetuses based on specimen sources or overall classification. Discordant mosaic results were observed in 16 cases (15.38%, 16/104) from different specimens or different testing methods. Genetic counselling and clinical management regarding CM in prenatal diagnosis remain challenging due to the variable phenotypes and unclear significance. Greater caution should be used in prenatal counselling, and more comprehensive assays involving serial ultrasound examinations, different specimens or testing methods verifications and follow-up should be applied.     

Prenatal detection of aneuploidy and imbalanced chromosomal arrangements by massively parallel sequencing

Fetal chromosomal abnormalities are the most common reasons for invasive prenatal testing. Currently, G-band karyotyping and several molecular genetic methods have been established for diagnosis of chromosomal abnormalities. Although these testing methods are highly reliable, the major limitation remains restricted resolutions or can only achieve limited coverage on the human genome at one time. The massively parallel sequencing (MPS) technologies which can reach single base pair resolution allows detection of genome-wide intragenic deletions and duplication challenging karyotyping and microarrays as the tool for prenatal diagnosis. Here we reported a novel and robust MPS-based method to detect aneuploidy and imbalanced chromosomal arrangements in amniotic fluid (AF) samples. We sequenced 62 AF samples on Illumina GAIIx platform and with averagely 0.01× whole genome sequencing data we detected 13 samples with numerical chromosomal abnormalities by z-test. With up to 2× whole genome sequencing data we were able to detect microdeletion/microduplication (ranged from 1.4 Mb to 37.3 Mb of 5 samples from chorionic villus sampling (CVS) using SeqSeq algorithm. Our work demonstrated MPS is a robust and accurate approach to detect aneuploidy and imbalanced chromosomal arrangements in prenatal samples.     

Prenatal diagnosis and characteristics of chromosome aberrations in families with genetic risk

Characteristics of the chromosomal aberrations diagnosed in 959 prenatal tests in the II trimester of pregnancy is presented. Chromosomal aberrations were diagnosed in 33 tests (3.4%). Twenty one out of these aberrations (2.2%) were of labile character. Six aberrations resulted from the parental segregation, translocation or chromosomal inversion. In 12 cases fetus inherited stable aberration from one of parents. It amounted to 1.2% of all tested cases. Chromosomal aberrations were diagnosed in 2.7% cases tested due to the risk related to the mother's age. Half of them was trisomy of chromosome 21. Chromosomal aneuploidy in the progeny of families with a child with the same abnormality was diagnosed in 1.6% of cases. Chromosomal mosaicism was diagnosed in 2.2% of cases including 0.2% of cases with true mosaicism and 1.98% of cases with pseudomosaicism. Incidence and type of the diagnosed chromosomal aberrations coincided with foreseen aberrations for each group of the genetic risk.     

Trisomy 18 in neonates: prenatal diagnosis, clinical features, therapeutic dilemmas and outcome

The study aimed to analyse the clinical courses of aggressively treated neonates with cytogenetically confirmed trisomy 18, with special attention focused on the efficiency of prenatal diagnostics, associated malformations, therapeutic dilemmas and outcomes. We investigated retrospectively the data concerning 20 neonates with trisomy 18, admitted to the Neonatal Intensive Care Unit (NICU) in Katowice between January 2000 and February 2005. Their birth weights ranged from 650 g to 2400 g, mean 1812 g; gestational age ranged from 27 to 42 weeks, median 38 weeks. Intrauterine growth retardation was noticed in 90% of neonates. Trisomy 18 was suspected prenatally in 40% of cases. Most (80%) of newborns were delivered by caesarean section (92% of neonates with prenatally unrecognized chromosomal defects, 62% of neonates with trisomy 18 suspicion) and 70% of infants needed respiratory support immediately after birth. Cardiac defects were present in 95%, central nervous system malformations in 65%, severe anomalies of digestive system or abdominal wall in 25% of patients. Nine surgical operations were performed during hospitalization (4 were palliative cardiac surgeries). Six patients (30%) survived the neonatal period and were discharged from the NICU. The median survival of the neonates who died was 20 days. In 4 cases cardiac problems implicated their death; in others, deaths were attributed to multiorgan failure, prematurity and/or infection. Further improvement of efficiency of prenatal ultrasound screening for diagnosis of trisomy 18 in the fetus is necessary. A lack of prenatal diagnosis of trisomy 18 in the fetus results in a high rate of unnecessary caesarean sections in these pregnancies. Despite the aggressive treatment most neonates with trisomy 18 died during the neonatal period. The majority of deaths were attributed to cardiorespiratory and multiorgan failure. Concerning the poor prognosis, prompt karyotyping (using FISH) of clinically suspected trisomy 18 is very important, because many invasive procedures and surgeries may then be avoided.