高通量测序技术在产前诊断中的临床应用

发布时间：2018-09-17 19:07

【摘要】：目的我国是出生缺陷高发国家之一,出生缺陷给患儿家庭及整个社会带来沉重的经济负担。如何进行有效的产前诊断,降低出生缺陷,一直是产前诊断领域专家关注的热点。产前诊断是预测胎儿出生前是否患有某种遗传性疾病或先天畸形的方法,是在遗传咨询的基础上,应用现代生物学、生物化学、免疫遗传学、细胞遗传学、分子遗传学等技术,通过母体检查或者对胚胎/胎儿直接检测,诊断胎儿是否患有遗传缺陷以及先天畸形。近年来,随着孕妇优生优育意识的不断加强,我国产前诊断医疗资源严重不足的现象越发明显。寻求一种安全、简单、经济、实用的产前筛查及诊断方案,对降低出生缺陷具有重要意义。同时,随着对疾病认识的不断提高,人们对于临床疾病的认识逐渐由临床诊断到分子诊断水平。然而,目前关于产前诊断的临床诊疗还停留在细胞遗传诊断水平,远远不能满足临床需求。随着测序成本的降低,高通量测序技术(High-throughput sequencing technology)又称下一代测序技术(Next generation sequencing,NGS)迅速发展,使其广泛临床应用成为可能。高通量测序技术的迅速发展,必将导致传统医学诊疗模式的改革。探讨高通量测序技术在产前诊断中的临床应用,为建立基于高技术平台的遗传学诊疗新模式提供理论依据。目前,临床应用相对广泛的无创产前检测技术(Noninvasive prenatal genetic testing,NIPT),是基于高通量测序技术的一项产前筛查新技术。它通过检测孕妇外周血中存在胎儿游离DNA(cell-free fetal DNA,cff DNA),检测胎儿21、18、13染色体非整倍体[1]。前期实验证实该技术具有较高的灵敏度、特异度,但该项技术对于传统产前诊断技术的影响尚需要进一步证实[2]。如何将该技术应用到临床,尚需要大样本前瞻性研究。近年来,临床对流产组织以及羊水细胞遗传学检测需求越来越多。随着越来越多的染色体微缺失、微重复等染色体结构异常的临床意义被进一步证实,其可能是导致发育异常以及智力低下等重大出生缺陷的主要原因之一。全基因组拷贝数变异分析(Copy number variations,CNVs),作为一种新的遗传学标记物,在产前诊断领域临床中的应用尚在研究阶段。近期研究发现,一旦CNVs涉及重要的基因,就可能导致胚胎生理功能缺陷和相关疾病的发生[3]。染色体微结构异常检测成为近年来人们关注的热点。关于CNVs检测在流产物、羊水细胞方面研究较少,缺乏临床研究数据。随着越来越多有明确临床意义的CNVs被证实,NGS-CNVs检测有望成为临床遗传检测新技术。方法本研究选取2012年1月至2014年1月来唐山市妇幼保健院就诊的6005例高危孕妇为研究对象,依据不同的产前诊断方案分为两组:传统的产前诊断方案组(A组)和联合NIPT产前诊断方案组(B组)。比较了两种方案在产前诊断临床应用对胎儿染色体异常的检出效率;分析了孕妇选择产前诊断率,21-三体、18-三体和13-三体检出率等相关指标;分析了NIPT灵敏度、特异度、假阳性率、阳性预测值等相关指标;建立了将NIPT技术与传统的产前筛查及诊断技术相结合的联合NIPT产前诊断方案,并将该方案应用于产前筛查高危人群检测胎儿染色体异常,评价了该方案的临床应用价值。选取2015年6月至2015年12月在唐山市妇幼保健院产前诊断中心确诊为稽留流产,流产原因不明,要求遗传学检测的患者105例为研究对象,对流产物样本同时进行了传统染色体核型分析和基于高通量测序技术的CNVs检测(NGS-CNVs)。探讨了CNVs技术在流产物临床遗传学诊断中的应用价值;分析了将该技术临床应用于现有的遗传学检测流程的可行性。选取2015年6月至2015年12月因各种高危因素来唐山市妇幼保健院产前诊断中心就诊,要求羊水穿刺产前诊断的孕妇为研究对象。在对孕妇实施羊水穿刺术,细胞培养染色体核型分析的同时,留取部分羊水样本行NGS-CNVs检测,探讨了该技术在产前诊断中的临床应用价值。为建立基于高技术平台的临床诊疗新模式提供科学依据。结果1、与传统的产前诊断方案相比,联合NIPT产前诊断方案能够使更多的孕妇接受进一步的产前检测,放弃进一步产前诊断的孕妇明显减少(81.0%vs13.9%,p0.001),差异具有统计学意义;联合NIPT产前诊断方案,21-三体、18-三体和13-三体的检出率明显提高(0.4%vs1.0%,p=0.01)。NIPT检测胎儿21、18、13号染色体非整倍体具有高度敏感性、特异性。NIPT检测21-三体、18-三体和13-三体的灵敏度、特异度分别为100%、99.9%、99.9%,阳性预测值分别为100%、83.3%和50.0%,假阳性率为0%、0.04%和0.04%。2、NGS-CNVs在流产物遗传学检测中的临床应用结果表明:NGS-CNVs与绒毛细胞培养染色体核型分析技术比较,NGS-CNVs检测成功率明显高于染色体核型分析,差异有统计学意义,(p0.001)。绒毛细胞培养染色体分析能检测出更多的染色体平衡易位,差异具有统计学意义,(p0.001);临床应用NGS-CNVs技术检测染色体非整倍体,结果与绒毛细胞培养染色体核型分析结果一致。与染色体核型分析相比,NGS-CNVs检测能够发现更多的染色体微结构异常,差异具有统计学意义,(p0.001)。3、NGS-CNVs在产前诊断中的临床应用表明:NGS-CNVs与羊水细胞染色体核型分析技术相比,染色体核型分析能够检测到更多的平衡易位、臂间倒位等染色体结构异常,差异具有统计学意义,(p0.001);NGS-CNVs检测出更多的染色体微结构异常,差异具有统计学意义,(p0.001);分析CNVs阳性结果与羊水穿刺指征的关系显示,产前超声检查异常与CNVs阳性密切相关。结论1、NIPT技术能够快速、准确、非侵入性检测21、18、13号染色体非整倍体,更容易被孕妇接受。将NIPT作为传统产前诊断技术的有效补充,合理的将该技术与现有的产前诊断技术相结合,联合NIPT产前诊断方案切实可行。联合NIPT产前诊断方案,使更多的高危孕妇参与了进一步产前检测,在不影响侵入性产前诊断率的同时,检测出更多的染色体病患儿,有效的降低了出生缺陷,是较为经济实用的产前诊断方案。2、基于高通量测序的NIPT技术,对于检测胎儿21、18、13号染色体非整倍体,具有高度的准确性。NIPT对于检测21-三体、18-三体和13-三体灵敏度、特异度分别为100%、99.9%和99.9%,阳性预测值分别为100%、83.3%和50.0%,假阳性率分别为0%、0.04%和0.04%。3、NGS-CNVs检测用于流产物、羊水细胞染色体微缺失、微重复的遗传学检测切实可行,能够快速、准确的发现常见的染色体异常,显著缩短了检测周期。NGS-CNVs检测染色体非整倍体结果与染色体核型分析结果一致。NGS-CNVs能够检出不能通过常规染色体核型分析方法发现的,具有明确临床意义的染色体微结构异常,提高了遗传学检测水平。NGS-CNVs发现大量致病性未知的微失衡,其临床意义有待进一步证实。分析CNVs与羊水穿刺指征的关系显示,产前超声检查异常与CNVs阳性密切相关。4、高通量测序在产前筛查及诊断领域临床应用具有较大的发展空间,与传统的细胞培养染色体核型分析、产前超声、产前筛查及诊断技术相结合,能更好的发挥降低出生缺陷的作用。
[Abstract]:Objective China is one of the countries with high incidence of birth defects, which brings heavy economic burden to the family and the whole society. How to carry out effective prenatal diagnosis and reduce birth defects has always been a hot spot in the field of prenatal diagnosis. Based on genetic counseling and using modern biology, biochemistry, immunogenetics, cytogenetics, molecular genetics and other techniques, we can diagnose whether the fetus has genetic defects and congenital malformations by maternal examination or direct detection of the embryo/fetus. It is important to find a safe, simple, economical and practical prenatal screening and diagnosis scheme to reduce birth defects. At the same time, with the continuous improvement of the understanding of the disease, people's understanding of clinical diseases has gradually changed from clinical diagnosis to molecular diagnosis. At present, the clinical diagnosis and treatment of prenatal diagnosis is still at the level of cellular genetic diagnosis, which is far from meeting the clinical needs. To explore the clinical application of high-throughput sequencing in prenatal diagnosis and to provide theoretical basis for the establishment of a new genetic diagnosis and treatment model based on high-tech platform. E-prenatal genetic testing (NIPT) is a new prenatal screening technology based on high-throughput sequencing technology. It detects fetal aneuploidy of chromosome 21, 18 and 13 by detecting the presence of fetal free DNA (cff DNA) in maternal peripheral blood. In recent years, there is an increasing demand for cytogenetic testing of abortion tissues and amniotic fluid. With the increasing number of chromosomal microdeletions, microduplication and other chromosomal abnormalities, the clinical application of this technique needs a large sample of prospective studies. The clinical significance has been further confirmed that it may be one of the main causes of major birth defects such as developmental abnormalities and mental retardation. As a new genetic marker, whole-genome copy number variations (CNVs) are still in the research stage of clinical application in prenatal diagnosis. Once CNVs involve important genes, it may lead to embryonic physiological defects and related diseases [3]. Detection of chromosomal microstructural abnormalities has become a hot topic in recent years. It has been proved that NGS-CNVs test is expected to become a new clinical genetic testing technology. Methods 6005 high-risk pregnant women who came to Tangshan Maternal and Child Health Hospital from January 2012 to January 2014 were selected as the study subjects. According to different prenatal diagnosis schemes, they were divided into two groups: the traditional prenatal diagnosis scheme group (group A) and the combined NIPT prenatal diagnosis scheme group (group B). The detection efficiency of the two schemes in prenatal diagnosis of fetal chromosomal abnormalities was compared; the prenatal diagnosis rate, 21-trisomy, 18-trisomy and 13-trisomy detection rate were analyzed; the NIPT sensitivity, specificity, false positive rate, positive predictive value and other related indicators were analyzed; the NIPT technology and the traditional NIPT technology were established. The combination of prenatal screening and diagnostic techniques with NIPT prenatal diagnosis program was applied to prenatal screening of high-risk groups for fetal chromosomal abnormalities, and the clinical value of the program was evaluated. In this study, 105 patients with abortion were asked to undergo both conventional chromosome karyotype analysis and high-throughput sequencing-based CNVs (NGS-CNVs). The application value of CNVs in clinical genetic diagnosis of abortion was discussed, and the clinical application of this technique in current genetic testing was analyzed. Feasibility of the test procedure. From June 2015 to December 2015, pregnant women who came to Tangshan Maternal and Child Health Hospital for prenatal diagnosis due to various high-risk factors were selected as the study subjects. Results 1. Compared with the traditional prenatal diagnosis scheme, the combined NIPT prenatal diagnosis scheme can make more pregnant women accept further prenatal testing and give up further prenatal diagnosis. The detection rate of 21-trisomy, 18-trisomy and 13-trisomy in combination with NIPT was significantly increased (0.4% vs 1.0%, P = 0.01). NIPT was highly sensitive and specific in detecting fetal chromosome 21, 18, 13 aneuploidy. The specificity was 100%, 99.9%, 99.9%, the positive predictive value was 100%, 83.3% and 50.0%, the false positive rate was 0%, 0.04% and 0.04%. The difference was statistically significant (p0.001). Chromosome analysis of villous cell culture could detect more balanced chromosome translocations, and the difference was statistically significant (p0.001); NGS-CNVs technique was used to detect chromosomal aneuploidy, and the results were consistent with the karyotype analysis of villous cell culture. Compared with amniotic fluid cell karyotype analysis, NGS-CNVs can detect more balanced translocation, inter-arm inversion and other chromosomal abnormalities. There was statistical significance (p0.001); NGS-CNVs detected more chromosomal microstructural abnormalities, the difference was statistically significant (p0.001); analysis of the relationship between positive results of CNVs and amniocentesis indications showed that prenatal ultrasound abnormalities were closely related to CNVs positive. Conclusion 1, NIPT technology can be rapid, accurate, non-invasive detection of 21, 18, 13 infections. Chromosome aneuploidy is easier to be accepted by pregnant women. NIPT is an effective complement to traditional prenatal diagnosis technology. It is feasible to combine NIPT with prenatal diagnosis technology reasonably. The combination of NIPT with prenatal diagnosis program will enable more high-risk pregnant women to participate in further prenatal testing without affecting invasion. It is an economical and practical prenatal diagnosis scheme. 2. NIPT based on high-throughput sequencing has high accuracy in detecting fetal chromosome 21, 18, 13 aneuploidy. NIPT is used to detect 21-trisomy, 18-trisomy and 13-trisomy. The sensitivity, specificity, positive predictive value were 100%, 99.9% and 99.9%, respectively. The positive predictive value was 100%, 83.3% and 50.0%, false positive rate was 0%, 0.04% and 0.04% respectively. NGS-CNVs can detect chromosomal microstructural abnormalities which can not be detected by conventional chromosomal karyotype analysis methods. NGS-CNVs can improve the level of genetic detection. NGS-CNVs can detect a large number of microimbalances with unknown pathogenicity. Analysis of the relationship between CNVs and amniocentesis indicated that prenatal ultrasound abnormalities were closely related to CNVs positive. 4. High-throughput sequencing has great potential for clinical application in prenatal screening and diagnosis. Combining technology with breaking technology can play a better role in reducing birth defects.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R714.5

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