Relationship Between Gene-Phenotype and Clinical Manifestations of Chromosomal Copy Number Variations Indicated by Non-Invasive Prenatal Testing

Objective:To analyze the clinical value of non-invasive prenatal testing(NIPT)in detecting chromosomal copy number variations(CNVs)and to explore the relationship between gene expression and clinical manifestations of chromosomal copy number variations.Methods:3551 naturally conceived singleton pregnant women who underwent NIPT were included in this study.The NIPT revealed abnormalities other than sex chromosome abnormalities and trisomy 13,18,and 21.Pregnant women with chromosome copy number variations underwent genetic counseling and prenatal ultrasound examination.Interventional prenatal diagnosis and chromosome microarray analysis(CMA)were performed.The clinical phenotypes and pregnancy outcomes of different prenatal diagnoses were analyzed.Additionally,a follow-up was conducted by telephone to track fetal development after birth,at six months,and one year post-birth.Results:A total of 53 cases among 3551 cases showed chromosomal copy number variation.Interventional prenatal diagnosis was performed in 36 cases:27 cases were negative and 8 were consistent with the NIPT test results.This indicates that NIPT’s positive predictive value(PPV)in CNVs is 22.22%.Conclusion:NIPT has certain clinical significance in screening chromosome copy number variations and is expected to become a routine screening for chromosomal microdeletions and microduplications.However,further interventional prenatal diagnosis is still needed to identify fetal CNVs.

Comparison of next generation sequencing-based and methylated DNA immunoprecipitation-based approaches for fetal aneuploidy non-invasive prenatal testing

Over the past few years, many researchers have attempted to develop non-invasive prenatal testing methods in order to investigate the genetic status of the fetus. The aim is to avoid invasive procedures such as chorionic villus and amniotic fluid sampling, which result in a significant risk for pregnancy loss. The discovery of cell free fetal DNA circulating in the maternal blood has great potential for the development of non-invasive prenatal testing(NIPT) methodologies. Such strategies have been successfully applied for the determination of the fetal rhesus status and inherited monogenic disease but the field of fetal aneuploidy investigation seems to be more challenging. The main reason for this is that the maternal cell free DNA in the mother's plasma is far more abundant, and because it is identical to half of the corresponding fetal DNA. Approaches developed are mainly based on next generation sequencing(NGS) technologies and epigenetic genetic modifications, such as fetal-maternal DNA differential methylation. At present, genetic services for non-invasive fetal aneuploidy detection are offered using NGS-based approaches but, for reasons that are presented herein, they still serve as screening tests which are not readily accessed by the majority of couples. Here we discuss the limitations of both strategies for NIPT and the future potential of the methods developed.

Discrepancy between non-invasive prenatal testing result and fetal karyotype caused by rare confined placental mosaicism: A case report

BACKGROUND Confined placental mosaicism(CPM)is one of the major reasons for discrepancies between the results of non-invasive prenatal testing(NIPT)and fetal karyotype analysis.CASE SUMMARY We encountered a primiparous singleton pregnant woman with a rare CPM consisting of 47,XY,+21;47,XXY;and 46,XY,who obtained a false-positive result on NIPT with a high risk for trisomy 21.Copy-number variation sequencing on amniotic fluid cells,fetal tissue,and placental biopsies showed that the fetal karyotype was 47,XXY,while the placenta was a rare mosaic of 47,XY,+21;47,XXY;and 46,XY.CONCLUSION The patient had a rare CPM consisting of 47,XY,+21;47,XXY;and 46,XY,which caused a discrepancy between the result of NIPT and the actual fetal karyotype.It is important to remember that NIPT is a screening test,not a diagnostic test.Any positive result should be confirmed with invasive testing,and routine ultrasound examination is still necessary after a negative result.

Non-invasive Prenatal Gene Diagnosis: Progress through Cell-free Fetal DNA and RNA in Maternal Plasma and Urine

Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also prevent tangential illness in fetuses and thus, reduce the incidence of diseases. Moreover, it is non-invasive prenatal gene diagnosis that prevents potential threaten and danger to both mothers and fetuses. Therefore, it is welcomed by clinical gynecologist and obstetrian, researchers of medical genetics, and especially, pregnancies. This review article touches briefly on the advanced development of using cell-free DNA, RNA in maternal plasma and urine for non-invasive prenatal gene diagnosis.

Two approaches for calculating female fetal DNA fraction in noninvasive prenatal testing based on size analysis of maternal DNA fragments

The concentration of cell-free fetal DNA fragments should be detected before noninvasive prenatal testing(NIPT).The fetal DNA molecules have significant clinical potential in determining the overall performance of NIPT and clinical interpretation.It is important to measure fetal DNA fraction before NIPT.However,there is still little research on how to calculate the concentration of female fetuses.Two estimation approaches were proposed to calculate fetal DNA fraction,including the fragments size-based approach,aneuploid-based approach,which are all approaches based on chromosome segments.Based on high-throughput sequencing data,two approaches to calculate the DNA fraction of male fetuses were tested and obtained the experiment values,which were close to the actual values.The correlation coefficient of fragments size-based approach was 0.9243(P<0.0001)and the aneuploid-based approach reached 0.9339(P<0.0001).We calculated the concentration of female fetuses and obtained remarkable experimental results.We came up with two approaches for calculating the fetal DNA fraction of female fetuses.It provides an important theoretical basis for the detection of female fetal concentration in future clinical diagnosis.

Non-invasive Prenatal Diagnosis of Trisomy 21 by Dosage Ratio of Fetal Chromosome-specific Epigenetic Markers in Maternal Plasma

This study examined the methylation difference in AIRE and RASSF1A between maternal and placental DNA, and the implication of this difference in the identification of free fetal DNA in maternal plasma and in prenatal diagnosis of trisomy 21. Maternal plasma samples were collected from 388 singleton pregnancies, and placental or chorionic villus tissues from 112 of them. Methylation-specific PCR （MSP） and methylation-sensitive restriction enzyme digestion followed by fluorescent quantitative PCR （MSRE ＋ PCR） were employed to detect the maternal-fetal methylation difference in AIRE and RASSF1A. Diagnosis of trisomy 21 was established according to the ratio of fetal-specific AIRE to RASSF1A in maternal plasma. Both methods confirmed that AIRE and RASSF1A were hypomethylated in maternal blood cells but hypermethylated in placental or chorionic villus tissues. Moreover, the differential methylation for each locus could be seen during the whole pregnant period. The positive rates of fetal AIRE and RASSF1A in maternal plasma were found to be 78.1% and 82.1% by MSP and 94.8% and 96.9% by MSRE ＋ PCR. MSRE ＋ PCR was superior to MSP in the identification of fetal-specific hypermethylated sequences （P〈0.05）. Based on the data from 266 euploidy pregnancies, the 95% reference interval of the fetal AIRE/RASSF1A ratio in maternal plasma was 0.33-1.77, which was taken as the reference value for determining the numbers of fetal chromosome 21 in 102 pregnancies. The accu-racy rate in 98 euploidy pregnancies was 96.9% （95/98）. Three of the four trisomy 21 pregnancies were confirmed with this method. It was concluded that hypermethylated AIRE and RASSF1A may serve as fetal-specific markers for the identification of fetal DNA in maternal plasma and may be used for noninvasive prenatal diagnosis of trisomy 21.

Noninvasive Prenatal Testing for Fetal Chromosomal Abnormalities Using Massively Parallel Sequencing: Clinical Experience from 7910 Korean Pregnancies

Objective: The purpose of this study is to review the clinical experience and performance of noninvasive prenatal testing (NIPT) method, using cell-free DNAto detect chromosomes 21, 18, 13, X, and Y abnormalities in over 7910 clinical samples from South Korean population. Method: Pregnant women between 1st of November 2015 to 18th of February 2018, with obstetric clinical findings participated in the study. NIPT was performed based on masivelly parallel sequencing with 0.3× low coverage paired-end sequencing using cell-free DNA in maternal plasma. Further invasive prenatal testing was recommended for pregnant women with positive NIPT results. Results: Of the total 7910 participants, 7890 (99.75%) were tested for NIPT and the remaining 20 (0.25%) were below the Quality Control (QC) standards. T13, T18, XXX, XXY and XYY had 100% of sensitivity, specificity, positive predictive values (PPV) and accuracy. The overall sensitivity was 100% and specificity, PPV and accuracy of all chromosomal abnormalities with further validation were 99.92%, 94.25%, and, 99.92% respectively. Conclusion: Our NIPT results showed high positive predictive value for the detection of autosomal trisomies and sex chromosome aneuploidies in our sample cohort.

Multiple z-Score Based Method for Noninvasive Prenatal Test Using Cell-Free DNA in Maternal Plasma

Objective: To improve the detecting accuracy of chromosomal aneuploidy of fetus by non-invasive prenatal testing (NIPT) using next generation sequencing data of pregnant women’s cell-free DNA. Methods: We proposed the multi-Z method which uses 21 z-scores for each autosomal chromosome to detect aneuploidy of the chromosome, while the conventional NIPT method uses only one z-score. To do this, mapped read numbers of a certain chromosome were normalized by those of the other 21 chromosomes. Average and standard deviation (SD), which are used for calculating z-score of each sample, were obtained with normalized values between all autosomal chromosomes of control samples. In this way, multiple z-scores can be calculated for 21 autosomal chromosomes except oneself. Results: Multi-Z method showed 100% sensitivity and specificity for 187 samples sequenced to 3 M reads while the conventional NIPT method showed 95.1% specificity. Similarly, for 216 samples sequenced to 1 M reads, Multi-Z method showed 100% sensitivity and 95.6% specificity and the conventional NIPT method showed a result of 75.1% specificity. Conclusion: Multi-Z method showed higher accuracy and robust results than the conventional method even at low coverage reads.

DNA无创产前检测及彩色多普勒超声检查在高危孕妇胎儿 染色体异常筛查中的应用价值

目的探讨DNA无创产前检测(NIPT)及彩色多普勒超声(简称彩超)检查在高危孕妇胎儿染色体异常筛查中的应用价值。方法选取2020年1月至2022年12月于该院接受产前检查的5862例高危孕妇作为研究对象,均接受NIPT、彩超检查,以羊水穿刺结果或分娩结局作为诊断胎儿染色体异常的金标准,比较NIPT、彩超检查及二者联合检查对高危孕妇胎儿染色体异常的诊断效能。结果5862例高危孕妇中共检出167例胎儿染色体异常,检出率为2.85%。167例胎儿染色体异常中胎儿染色体数目异常161例,构成比为96.41%;胎儿染色体结构异常6例,构成比为3.59%。彩超检查共诊断出119例孕妇胎儿染色体异常,经一致性分析,彩超检查诊断胎儿染色体异常的灵敏度为0.713,特异度为0.884,准确率为87.96%,Kappa=0.215,P<0.05。NIPT共诊断出133例孕妇胎儿染色体异常,经一致性分析,NIPT诊断胎儿染色体异常的灵敏度为0.796,特异度为0.945,准确率为94.05%,Kappa=0.408,P<0.05。彩超检查联合NIPT共诊断出158例孕妇胎儿染色体异常,经一致性分析,二者联合检查诊断胎儿染色体异常的灵敏度为0.946,特异度为0.986,准确率为98.50%,Kappa=0.775,P<0.05。结论NIPT与彩超检查用于筛查高危孕妇胎儿染色体异常均具有一定价值,二者联合检查可获得更高的灵敏度、特异度和准确率,能有效降低漏诊及误诊风险。

游离DNA最新研究进展及法医学应用展望

近年来,随着DNA提取和检测技术的不断进步,游离DNA(cell-free DNA,cfDNA)已经在生命科学领域得到了广泛应用,在法医学鉴定领域中的潜在应用价值也越来越明显。本文回顾了cfDNA概念、形成机制与分类等,并阐述了cfDNA在法医学现场接触检材的个体识别和无创产前亲缘关系鉴定应用中的最新研究进展,同时总结了cfDNA在损伤推断中的应用潜力,并探讨了常用cfDNA分析方法和技术的优缺点及应用展望,为cfDNA在法医学领域的广泛应用提供新思路。

母体循环中胎儿游离DNA在单基因病产前诊断的研究进展

单基因病是引起胎儿出生缺陷的主要原因之一,其种类多,病情复杂,缺乏有效治疗方法,且预后差。目前,无创产前检测(NIPT)已成为筛查胎儿非整倍体的主要手段,在临床上广泛应用。基于母体循环中胎儿游离DNA(cffDNA)的发现及高通量测序技术的发展,单基因病的NIPT成为研究热点,且检测技术及其检测的疾病种类也在不断增加,但是单基因病的检测存在难度大、成本高等问题。目前胎儿单基因病的NIPT仍然处于研究阶段,尚未在临床应用。因此,深入研究母体循环中cffDNA的NIPT对单基因病产前诊断具有重要意义。

无创胎儿游离DNA血型检测在产前诊断中的研究进展

胎儿游离DNA(cell-free fetal DNA,cff-DNA)存在于孕妇妊娠期外周血中,其为携带胎儿相关遗传信息的DNA片段,可以进行胎儿染色体、基因相关疾病筛查。因其操作风险低、无副作用,目前已被广泛用于无创产前诊断检测(non-invasive prenatal testing,NIPT)。无创cff-DNA血型检测是运用分子生物学检测cff-DNA血型相关基因,得出胎儿血型结果。以此在妊娠期即可检测出胎母血型是否一致,判断胎儿有无发生血型不合所致胎儿新生儿溶血病(hemolytic disease of the fetus and newborn,HDFN)的风险。

应用孕妇血浆中胎儿游离DNA进行产前筛查的临床研究进展

无创产前检测(NIPT)是采用二代测序技术对孕妇血浆中胎儿游离DNA(cffDNA)片段进行检测,通过生物信息学分析进行常见的胎儿染色体非整倍体异常的筛查。因其具有较高的检出率、敏感度和特异度以及较低的假阳性率,成为目前广泛应用的染色体非整倍体的产前筛查技术。通过NIPT筛查高风险孕妇选择性进行有创产前诊断,可避免大量不必要的有创性产前诊断。母体及胎儿因素可影响NIPT检测结果。临床应用时应充分考虑不同孕妇的个体情况。本文就应用孕妇血浆中cffDNA进行NIPT的临床研究进展及其影响因素进行综述,为个体化、规范化应用NIPT技术提供参考。

高通量无创DNA产前检测技术对胎儿染色体疾病的诊断价值

目的探讨高通量无创DNA产前检测(NIPT)技术在胎儿染色体疾病中的应用价值。方法孕12～26+6周的孕妇3 477例,抽取其外周血行NIPT检查,对NIPT检出染色体非整倍体高风险孕妇行羊水细胞核型分析,对比NIPT检测结果与羊水细胞核型分析结果的差异。结果 (1) 3 477例孕妇,NIPT检出染色体非整倍体高风险34例(0. 98%),其中21-三体高风险10例,18-三体高风险2例,13号染色体高风险2例,性染色体异常20例,不同年龄段孕妇染色体异常阳性检出率比较,差异有统计学意义(P <0. 05);> 40岁组的异常染色体阳性检出率高于35～40岁、<35岁组(P <0. 05)。(2) 34例高风险孕妇中,有31例行羊水穿刺细胞核型分析检查,结果确诊胎儿染色体异常20例,NIPT诊断假阳性11例。(3) NIPT检测21-三体、18-三体、47,XXX/XXY和47,XYY类型的胎儿染色体非整倍体异常的灵敏度均为100%,特异度均大于99%。结论 NIPT检测对21-三体(除嵌合体)、18-三体以及性染色体三体型(47,XXX/XXY/XYY)有较高的灵敏度和特异度,但对性染色体(45,XO)异常检测的准确性仍有待从技术上进一步提高,NIPT结果异常的孕妇仍需羊水穿刺行核型分析确诊。

外周血胎儿游离DNA检测在无创性产前诊断中的价值

目的:探讨孕妇外周血胎儿游离DNA无创性产前检测在诊断胎儿非整倍体中的价值。方法:选择2017年1月—2017年12月本院行外周血胎儿游离DNA无创产前检测和羊水穿刺胎儿染色体核型分析的孕妇616例,以羊水穿刺结果为金标准,判断无创产前检测的准确率。结果:胎儿游离DNA无创性产前检出胎儿染色体非整倍体高风险11例,其中21-三体高风险5例,18-三体高风险3例,13-三体高风险1例;性染色体非整倍体高风险2例。羊水染色体核型分析共检出染色体非整倍体12例,其中21-三体5例,18-三体3例,13-三体1例,性染色体非整倍体3例,总体符合率91.7%。胎儿游离DNA无创性产前检测胎儿非整倍体的灵敏度、特异度、阳性预测值、阴性预测值、准确度分别为91.7%、100.0%、100.0%、99.8%、99.8%、95.0%,ROC曲线下面积为0.958(95%CI:0.867～1.000)。结论:无创性胎儿游离DNA检测可有效检出胎儿染色体非整倍体,是一种可靠的产前检测方法。但不排除存在假阳性及假阴性的可能。

血浆游离DNA低深度测序技术对胎儿性染色体异常筛查价值的初步探讨

背景染色体异常是导致出生缺陷的重要因素,目前无针对性染色体非整倍体的产前筛查方法。无创产前基因检测(noninvasive prenatal test,NIPT)应用于临床除了能常规检测目标疾病外,同时也能检测出性染色体异常,包括微缺失微重复。目的探讨血浆游离DNA低深度测序技术对胎儿性染色体异常的筛查价值。方法选取2018年6月-2021年7月于解放军总医院第一医学中心妇产科实验室行NIPT孕妇11239例,年龄17~46岁,检测时孕周为12~31周。对其中42例有产前诊断报告的性染色体异常病例进行回顾性分析。结果11239例标本中NIPT筛查出性染色体异常51例,阳性率为0.45%。42例(42/51)接受羊膜腔穿刺,确诊性染色体异常22例,阳性预测值(positive predictive value,PPV)为52%(22/42);18例性染色体数目偏少(XO)确诊8例(44%),21例性染色体数目偏多(XXX、XXY、XYY)确诊12例(57%),3例性染色体其他复杂异常(合并5号染色体微缺失)确诊2例(67%)。9例(9/51)拒绝接受产前诊断。结论NIPT检测技术应用于筛查胎儿性染色体异常有一定的临床意义,可发现性染色体罕见核型及微缺失微重复,但总体假阳性率偏高。

年龄及孕周对双胎妊娠孕妇胎儿游离DNA浓度的影响

目的回顾性分析筛查年龄及采样孕周对双胎妊娠孕妇外周血胎儿游离DNA浓度的影响。方法以同期进行无创产前检测的单胎妊娠孕妇为对照,对双胎妊娠孕妇无创产前检测结果及数据进行分析,评价双胎妊娠孕妇的筛查年龄和采样孕周对胎儿游离DNA浓度的影响。结果双胎妊娠孕妇NIPT平均筛查年龄及采样孕周均小于单胎,二者筛查年龄分别为31.72±3.45岁和33.14±3.73岁(P<0.01);采样孕周分别为15.30±1.94周和16.19±2.94周(P<0.01)。各年龄组双胎和单胎妊娠孕妇的胎儿游离DNA浓度差异无统计学意义(P>0.05)。双胎妊娠孕妇胎儿游离DNA浓度在孕16~17周及18~19周显著高于单胎(P<0.05)。双胎妊娠孕妇首次采血建库检测成功率低于单胎,二者成功率分别为97.13%和99.07%(P<0.05)。结论双胎妊娠孕妇cfDNA个体差异较大,可根据其临床指征,对双胎妊娠孕妇的NIPT筛查年龄及孕周做出适宜选择,适当提前NIPT筛查年龄、延后采样孕周,以减少因cfDNA浓度不足造成NIPT的检测失败。

基于胎儿游离DNA和高通量测序的地中海贫血无创产前检测的研究进展

地中海贫血是由于珠蛋白基因缺陷导致珠蛋白链合成障碍而引起的遗传性溶血性疾病.重型地中海贫血是流行区域出生缺陷的主要病因,已成为影响社会和谐发展的公共卫生问题.产前诊断是避免重型地中海贫血胎儿出生的唯一有效途径,通过筛查流行区域地中海贫血基因携带者,对可能生育重型患儿的高危夫妇实施基因诊断和胎儿产前基因诊断,可达到干预的目的.目前国内外对地中海贫血主要采用有创产前诊断技术,但取材不可避免会对母体或胎儿造成伤害,因此安全有效的无创产前诊断方法和技术一直是遗传性疾病产前诊断的方向和目标.本文综述基于孕妇外周血游离胎儿DNA的无创产前诊断应用和基于高通量测序的无创产前检测技术的最新进展,并展望其发展方向.

男性胎儿DNA含量低的孕妇的无创产前检测

目的探讨外周血中胎儿DNA含量低的孕妇的无创产前检测(NIPT)。方法回顾分析2015年4月至2016年3月间在佛山市妇幼保健院行NIPT的3 240例孕妇实验数据与随访资料,根据其Y染色体z值,筛选出胎儿为男性且胎儿DNA含量低于8%的样品共150例,利用琼脂糖凝胶电泳富集法提高胎儿DNA含量,进行NIPT,对比非整倍体筛查结果的准确率。结果琼脂糖凝胶电泳富集法将样品中的胎儿DNA含量从平均5%提高到9.2%。对比胎儿DNA含量提高前后的NIPT结果是一致的。结论当胎儿DNA含量高于5%时,胎儿DNA含量不会影响NIPT结果的准确率。

无创DNA阳性结果的验证和分析

目的探讨无创DNA产前检测在产前诊断中的应用及价值。方法对2012年1月至2014年12月在西京医院妇产科做羊水穿刺的病人资料回顾性分析,发现因无创阳性做羊水穿刺的孕妇84例,用羊水染色体核型分析和FISH对其结果进行验证,并对两者不一致的结果进行电话随访。结果无创DNA84例阳性结果中21-三体48例,经羊水穿刺后确认为假阳性的有2例;18-三体11例,经羊水穿刺后确认为假阳性3例;13-三体4例,经羊水穿刺验证2例为假阳性;性染色体异常21例,经验证6例假阳性。结论孕妇外周血中游离胎儿DNA检测对21-三体检测准确率达95.8%,18-三体准确率73%,性染色体检出准确率71%,13-三体准确率50%。无创DNA是产前筛查21-三体的有效方法,对其他染色体异常的检测还需要进一步提高现有检测技术。