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.

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.

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.

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.

Enrichment of Fetal Nucleated Red Blood Cells by Multi-core Magnetic Composite Particles for Non-invasive Prenatal Diagnosis

A novel kind of multi-core magnetic composite particles, the surfaces of which were respectively mo- dified with goat-anti-mouse IgG and antitransferrin receptor（anti-CD71）, was prepared. The fetal nucleated red blood cells（FNRBCs） in the peripheral blood of a gravida were rapidly and effectively enriched and separated by the mo- dified multi-core magnetic composite particles in an external magnetic field. The obtained FNRBCs were used for the identification of the fetal sex by means of fluorescence in situ hybridization（FISH） technique. The results demonstrate that the multi-core magnetic composite particles meet the requirements for the enrichment and speration of FNRBCs with a low concentration and the accuracy of detetion for the diagnosis of fetal sex reached to 95%. Moreover, the obtained FNRBCs were applied to the non-invasive diagnosis of Down syndrome and chromosome 3p21 was de- tected. The above facts indicate that the novel multi-core magnetic composite particles-based method is simple, relia- ble and cost-effective and has opened up vast vistas for the potential application in clinic non-invasive prenatal diag- nosis.

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.

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。

Application of Fetal DNA in Maternal Plasma in Noninvasive Prenatal Diagnosis

To explore the application of fetal DNA in maternal plasma for noninvasive prenatal diagnosis, the DNA template was extracted by hydroxybenzene chloroform from 44 maternal (7-41 weeks) plasma. The Fetus derived Y sequence DYZ 1 gene (149bp) was chosen to be amplified by PCR. The fragment was identified in all the plasma of male bearing pregnant women with the diagnostic accordance rate being 100.00 %. Two of the 22 female bearing pregnant women had false positive results. Among the 44 pregnant women, the diagnostic accordance rate was 88.89 % at early pregnant stage, 100.00 % at medium pregnant stage, and 96.55 % at late stage respectively. The final accuracy of 95.45 % was obtained in all cases. It was concluded that by means of hydroxybenzene chloroform extraction the authors of this article promoted the concentration and purity of the DNA template, and diagnosed more accurately. The results showed that free fetal DNA in the maternal plasma could be regarded as the gene resource for noninvasive prenatal diagnosis.

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.

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。

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.

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。

18045例无创产前筛查游离胎儿DNA比例分析

目的:探讨游离胎儿DNA比例(FF)的影响因素及其与染色体异常疾病的相关性。方法:回顾分析孕妇年龄、孕周、体质量指数以及NIPS提示高风险胎儿与FF的关系。结果:FF主要分布范围是4%~25%。FF与孕周呈正相关,与孕妇年龄和体质量指数呈负相关。单胎胎儿的FF高于双/多胎妊娠胎儿,自然妊娠胎儿的FF高于辅助生殖妊娠胎儿,差异均有统计学意义(P<0.05)。高风险胎儿的FF高于低风险胎儿,差异均有统计学意义(P<0.05)。21、18和13-三体综合征的Z值与FF呈正相关。性染色体组真阳性和假阳性胎儿样本的FF比较,差异均有统计学意义(P<0.05)。微缺失组真阳性和假阳性胎儿样本的FF比较,差异均有统计学意义(P<0.05);微重复组真阳性和假阳性胎儿样本的FF无显著差异。结论:孕妇血浆cffDNA水平受母体和胎儿特征共同影响。临床实践中,应根据孕妇特征和FF进行NIPS的遗传咨询。

基于胎儿游离DNA的地中海贫血无创产前诊断的研究进展

重型地中海贫血(地贫)目前尚无有效治疗措施,通过产前诊断阻止重型患儿出生是国内外公认的首选预防措施。目前,地贫的产前诊断仍依赖于有创的方法,即绒毛活检、羊水穿刺和脐静脉血穿刺,存在一定的宫内感染和流产风险,安全、无创、快速和准确的地贫产前诊断方法对高发区意义重大。孕妇外周血胎儿游离DNA(cffDNA)的发现,下一代测序(NGS)的快速发展与应用,使无创产前DNA检测(NIPT)成为现实。目前,利用cffDNA检测胎儿13、18和21号染色体非整倍体的NIPT技术已经成熟应用于临床,但地贫的NIPT仍处于研究探索阶段。文章主要基于cffDNA和NGS的地贫NIPT研究进展进行综述。

无创产前筛查在双胎妊娠染色体非整倍体筛查中的应用及胎儿游离DNA浓度分析

目的评估无创产前筛查(non-invasive prenatal testing,NIPT)在双胎妊娠染色体非整倍体的应用价值,并对胎儿游离DNA浓度进行分析。方法收集2018年1月至2023年5月在广州市花都区妇幼保健院接受NIPT检测的双胎样本617例(辅助生殖双胎284例,自然妊娠双胎333例)为研究对象,同期12688例单胎妊娠样本作为对照,采用高通量测序方法进行检测,对染色体非整倍体高风险样本行核型分析,根据Y染色体唯一比对条数对男胎进行胎儿游离DNA浓度计算,使用基于浅深度的母亲血浆DNA测序方法(SeqFF)对女胎进行胎儿游离DNA浓度计算。结果双胎妊娠检出2例T21和1例T18,未检出T13,T21、T18的阳性预测值分别为100%、0,阴性预测值均为100%;单胎妊娠T21、T18、T13的阳性预测值分别为88.89%、45.45%、25%,阴性预测值均为100%。双胎妊娠胎儿游离DNA浓度为10.48%,较单胎妊娠(11.35%)稍低;双胎妊娠首次建库成功率为99.03%,较单胎妊娠(99.99%)低,差异有统计学意义(χ^(2)=104.105,P=0.00)。结论NIPT在双胎妊娠染色体非整倍体筛查有一定应用价值,T21筛查效率高,胎儿游离DNA浓度影响NIPT检测的成败和结果准确性。

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.

Non-invasive genetic analysis indicates low population connectivity in vulnerable Chinese gorals:concerns for segregated population management

Detailed information on the size and genetic structure of wildlife populations is critical for developing effective conservation strategies, especially for those species that have suffered population decline and fragmentation due to anthropogenic activities. In the present study, we used a non-invasive approach combining fecal pellet sampling with mitochondrial DNA and nuclear DNA microsatellite marker analysis to monitor and compare the population structure of the Chinese goral (Naemorhedus griseus) in Beijing and northeast Inner Mongolia in China. Of the 307 fecal samples confirmed to be from N. griseus, 15 individuals (nine females and six males) were found in the Beijing population and 61 individuals (37 females and 24 males) were found in the Inner Mongolian population. Among these 76 individuals, we identified eight haplotypes and 13 nucleotide polymorphic sites from mtDNA and 45 alleles from 10 microsatellite loci. Spatially structured genetic variation and a significant level of genetic differentiation were observed between the two populations. In both populations, the sex ratios were skewed toward females, indicating high reproductive potential, which is crucial for population recovery and conservation of this patchily distributed vulnerable species. We suggest that managing the two populations as evolutionarily significant units with diverse genetic backgrounds could be an effective solution for present population recovery, with the possible relocation of individuals among different groups to help ensure future goral species prosperity.

胎儿染色体异常应用无创DNA与羊水细胞染色体检查对比

目的探讨羊水产前诊断和无创DNA产前检测对胎儿染色体异常检查的应用价值。方法收集2020年1月—2022年12月在南平市市妇幼保健院接受产前检查中超声软指标异常的200例孕妇资料,200例孕妇分别应用羊水穿刺检查与无创DNA检查,对比两种检查结果差异性。结果两种检查胎儿染色体异常检出率比较,差异有统计学意义(P<0.05)。结论产前诊断中应用无创基因检测可以提高染色体异常检出率,但是对于具体异常类型的诊断范围相对受限,而且无创阳性者需再进行羊水染色体核型分析,所以羊水产前诊断依旧具有无法取缔的优势。

孕早期颈项透明层检查联合孕中期无创DNA检测在高龄孕妇唐氏综合征筛查中的临床应用价值

目的探讨孕早期颈项透明层(NT)检查联合孕中期无创DNA检测在高龄孕妇唐氏综合征筛查中的临床应用价值。方法回顾性分析2021年6月至2022年12月上饶市立医院行产前筛查的60例高龄孕妇的临床资料。所有孕妇于孕早期进行NT检查,孕中期进行无创DNA检测,并在孕中期进行羊水穿刺,分析羊水细胞染色体核型。以羊水穿刺结果为金标准,分析NT检查和无创DNA检测对唐氏综合征诊断的灵敏度与特异度。结果一致性分析显示,NT检查与金标准羊水穿刺对唐氏综合征的筛查具有较高的一致性(Kappa=0.756),无创DNA检测与金标准羊水穿刺对唐氏综合征的筛查几乎完全一致(Kappa=0.928)。NT检查的灵敏度为86.84%,特异度为90.91%;无创DNA检测的灵敏度为97.37%,特异度为95.45%。两种方法联合检测的灵敏度为100%,特异度为90.91%。结论孕早期NT检查联合孕中期无创DNA检测在高龄孕妇唐氏综合征筛查中具有较高的准确性,可作为主要筛查手段。