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.

孕妇血中胎儿微小RNA等核酸在唐氏综合征无创产前诊断中的应用

唐氏综合征是最常见的染色体非整倍体遗传病,出生干预是预防该病的有效措施。传统的产前诊断具有创伤等缺陷,无创产前诊断是未来发展的需求。孕妇血胎儿细胞、胎儿游离DNA、胎儿游离RNA及胎儿游离microRNA的分析是新近发展的4种唐氏综合征无创产前诊断技术。母血胎儿游离miRNA有足够的稳定性、特异性和准确性,是最具临床应用价值的生物标志物。

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.

Prenatal diagnosis of Down syndrome using cell-free fetal DNA in amniotic fluid by quantitative fluorescent polymersase chain reaction

Backgroud Amniotic fluid （AF） supernatant contains cell-free fetal DNA （cffDNA） fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simple quantitative fluorescent polymerase chain reaction （QF-PCR） to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.Methods AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes.Peripheral blood samples of the parents were collected at the same time.Short tandem repeat （STR） fragments on chromosome 21 were amplified by QF-PCR.Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.Results The sensitivity of the assay for the aneuploid was 93％ （26/28; confidence interval,CI：77％-98％） and the specificity was 100％ （26/26; CI：88％-100％）.The determination rate of the origin of the extra chromosome was 69％.The sensitivity and the specificity of the assay in the euploid were 100％ （27/27）.Conclusions Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant.This karyotype analysis method greatly reduces the requirement for the specimen size.It will be a benefit for early amniocentesis and could avoid pregnancy complications.The method may become an ancillary method for prenatal diagnosis of trisomy 21.

母血中胎儿遗传物质与无创性产前诊断

经过10余年的发展,利用母体外周血中胎儿遗传物质进行无创性产前诊断的技术正逐步面向临床。本文综述了目前较为广泛被研究的母血中的胎儿遗传物质,着重阐述了使用无创的方法获得母血中胎儿遗传物质的方法及其优缺点。

Emerging blood-based biomarkers for detection of gastric cancer

Early detection and efficient monitoring of tumor dynamics are prerequisites for reducing disease burden and mortality, and for improving the management of patients with gastric cancer(GC). Blood-based biomarker assays for the detection of early-stage GC could be of great relevance both for population-wide or risk groupbased screening programs, while circulating biomarkers that reflect the genetic make-up and dynamics of the tumor would allow monitoring of treatment efficacy, predict recurrences and assess the genetic heterogeneity of the tumor. Recent research to identify blood-based biomarkers of GC has resulted in the identification of a wide variety of cancer-associated molecules, including various proteins, autoantibodies against tumor associated antigens, cell-free DNA fragments, m RNAs and various non-coding RNAs, circulating tumor cells and cancer-derived extracellular vesicles. Each type of these biomarkers provides different information on the disease status, has different advantages and disadvantages, and distinct clinical usefulness. In the current review, we summarize the recent developments in blood-based GC biomarker discovery, discuss the origin of various types of biomarkers and their clinical usefulness and the technological challenges in the development of biomarker assays for clinical use.

表观遗传调控在胎源性精神疾病中的研究进展

宫内不良环境可导致胎儿某些分子、组织结构发生永久性改变即编程效应,使其成年后对内外环境变化的反应性产生异常,进而导致其患某些疾病的风险增加,而这其中的机制尚未阐明。近年来研究发现,表观遗传修饰如DNA甲基化、组蛋白乙酰化、micro RNA转录调节等可能是产生编程效应的重要机制之一。本文就表观遗传学机制在胎源性精神疾病中的研究进展进行综述,以期待对部分胎儿起源的成年疾病发生、治疗带来新的启示。

孕妇外周血中游离胎儿核酸的研究进展

非创伤性产前诊断一直是人类遗传学发展的长期目标 ,从非细胞成分中如母血浆和血清中检测胎儿DNA为非创伤性产前诊断的开展提供了可能。实时定量PCR分析技术的发展 ,使母血浆和血清中胎儿DNA检测的敏感性和特异性接近 10 0 % 。

Reappraising the Value of Fetal First-Trimester Ultrasonography

In the last few years,the introduction of cell-free DNA has rapidly altered prenatal screening regimens and is increasingly offered as the second- or,at times,even the first-tier screening test.Should an early anomaly scan also be part of an up-to-date screening policy? This paper reappraises the value of fetal first-trimester ultrasonography.The primary aims of the first-trimester scan are to establish gestational age based on the measurement of fetal crown-rump length,to detect multiple pregnancy and chorionicity,and to measure fetal nuchal translucency thickness as part of a combined screening test for chromosomal abnormalities.With recent advancements in ultrasound technology,there is compelling evidence that a majority of fetuses with major structural abnormalities and almost half of them without chromosomal abnormalities can be detected in the first trimester.We focused on the first-trimester screening of fetal major defects,especially including fetal congenital heart disease and cleft lip and palate by ultrasound markers and views.Moreover,it is critical to highlight that after a detailed anomaly scan in the first trimester without major structural anomalies and positive genetic tests,the residual chance of favorable outcome in fetuses with isolated increased nuchal translucency is relatively high.The discussion on the role of cell-free DNA in prenatal screening is still ongoing.Even in the event of it becoming a first-line screening test for aneuploidies,the importance of a first-trimester fetal scan,including assessment of markers for other anomalies,remains undisputed.

母体外周血游离RNA分子特性及其在无创产前检测中的潜在应用

胎儿游离DNA在母体外周血血浆中的发现颠覆性地改变现有的无创产前检测方法,例如基于孕妇外周血血浆DNA对胎儿染色体异常的无创检测在全球范围内快速得到了认可和广泛应用。受到胎儿游离DNA研究成果的鼓舞,科学家也陆续展开了关于孕妇外周血中胎儿游离RNA的研究。现将集中讨论血浆RNA的特性及其潜在的应用。

Non-invasive prenatal molecular detection of a fetal point mutation for congenital adrenal hyperplasia using co-amplification at lower denaturation temperature PCR

Conventional prenatal diagnosis relies on invasive chorionic biopsy or amniocentesis, which increases the risk of miscarriage, and is undertaken at 11-20 weeks gestation.1 The discovery of cell-free fetal DNA in maternal plasma has, however, offered a new strategy for non-invasive prenatal diagnosis.2 Cell-free fetal DNA in maternal plasma has been used for the determination of fetal gender3 and RHD status4 as well as testing certain monogenic diseases such as 13-thalassemia5 and cystic fibrosis.6 However,

无创性产前诊断胎儿染色体异常的研究进展

利用孕妇外周血和宫颈黏液中胎儿来源物质,对胎儿染色体病进行诊断,一直是产前诊断领域的研究热点。本文对近年来用孕妇外周血和宫颈黏液中胎儿细胞和孕妇外周血中游离胎儿DNA/RNA,进行无创性产前监测胎儿染色体非整倍体异常的研究,综述如下。

Two factors affecting the success rate of the second non-invasive prenatal screening after initial no-call result: experience from a single tertiary center in China

Background:One inevitable shortcoming of non-invasive prenatal screening(NIPS)/cell-free DNA(cfDNA)sequencing is the uninterpretable(“no-call”)result,which is mainly caused by an insufficient fetal fraction.This study was performed to investigate the factors associated with a successful second NIPS in these cases and determine the optimal management for women with initial no-call results.Methods:We retrospectively analyzed the data of women who underwent NIPS with initial no-call results due to an insufficient fetal fraction from 2017 to 2019 in our center.We compared these women's maternal and pregnancy information with the data of women who had attained a successful second NIPS result and women who had received no-call results for a second time.Results:Among the 33,684 women who underwent NIPS,137 with a no-call result underwent a retest.Comparison between the 87(63.50%)women with a successful retest and the other 50(36.50%)women showed a significant difference in both the initial fetal fraction and maternal body mass index(BMI),whereas the other factors showed no significant differences.In addition,with an initial fetal fraction of<2.00%,the retest success rate was very limited.Conclusions:We identified two major factors associated with a successful NIPS retest:the initial fetal fraction and the maternal BMI.These findings suggest the need for specialized management for this subset of women and would be instructional for the counseling for these women.

应用胎儿细胞游离核酸物质诊断唐氏综合征研究进展

唐氏综合征（Downsyndrome，DS）是一种人类常见的染色体异常疾病，又名21-三体、先天愚型，主要表现为非进行性智力低下并伴有心脏和其他器官的先天畸形，活产儿发病率约为1／800。随人类产前保健意识及优生优育观念的增强，唐氏综合征患儿的出生率虽有所降低，但针对它的传统产前诊断方法（中孕期绒毛膜取样、羊膜腔或脐血穿刺）因有创操作等局限性的存在不能被孕妇普遍接受，

唐氏综合征的无创产前诊断研究进展

唐氏综合征是最常见的染色体非整倍体遗传病,该病尚无有效治疗手段,出生干预是预防该病的有效措施。传统的产前筛查与产前诊断均具有一定的缺陷,无创产前诊断是未来发展的趋势。本文从母血胎儿细胞、母血胎儿游离DNA、母血胎儿游离RNA三个角度对目前唐氏综合征的无创产前诊断研究作一综述,以期对相关领域的研究发展有所帮助。