Prospective clinical study on non-invasive prenatal testing for severe thalassemia using tag-labeled targeted capture sequencing combined with a modified Bayesian model

The link between cell-free DNA, inflammation and the initiation of spontaneous labor at term

A new opinion from the American College of Obstetricians and Gynecologists (ACOG) and the Society for Maternal-Fetal Medicine (SMFM) says obstetricians can offer noninvasive prenatal testing (NIPT) to women at high risk of delivering babies with Down syndrome and trisomies 13 and 18.

American Journal of Medical Genetics Part AVolume 170, Issue 3 p. 557-557 the AJMG SEQUENCE: Decoding News and Trends for the Medical Genetics Community by Deborah LevensonFree Access Noninvasive prenatal testing spots duchenne muscular dystrophy First published: 17 February 2016 https://doi.org/10.1002/ajmg.a.37591AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat For the first time, researchers have employed a haplotype-assisted method using cell-free fetal DNA circulating in mothers' blood to prenatally diagnose the X-linked disorder Duchenne muscular dystrophy (DMD). Noninvasive prenatal tests (NIPTs) that use cell-free fetal DNA are now used clinically to screen for extra fetal copies of chromosomes 13, 18, and 21. However, using these tests to prenatally screen for single-gene disorders has been challenging because most of the DNA in the maternal plasma is derived from the mother. Writing in Genetics in Medicine, researchers from China and Denmark describe how their haplotype-assisted strategy combined deep sequencing of multiple blood samples (Xu et al., 2015). Samples were obtained from both fathers and their sons diagnosed with DMD. Samples were also taken from maternal plasma so that the fetal haplotype could be deduced using a mathematical model with the assistance of the parental haplotypes deduced from the boys' samples. On average, results from eight families showed an accuracy of 99.98% for the total inferred maternal single nucleotide polymorphisms (SNPs). With a mean depth of 30× achieved in the 10 Mb target region of each sample, the researchers achieved diagnostic accuracy consistent with amniocentesis used to confirm results. While most studies have focused on demonstrating the feasibility of NIPT for single-gene disorders in case reports, this study is the first to perform NIPT in parallel with invasive prenatal diagnosis in a small pilot sample, say researchers. With further improvements in accuracy, this haplotype- based strategy could be feasible with NIPT for DMD or other X-linked single-gene disorders, the researchers conclude. Reference Xu Y, Li X, Ge HJ, Xiao B, Zhang YY, Ying XM, Pan XY, Wang L, Xie WW, Ni L, Chen SP, Jiang WT, Liu P, Ye H, Cao Y, Zhang JM, Liu Y, Yang ZJ, Chen YW, Chen F, Jiang H, Ji X. 2015. Haplotype-based approach for noninvasive prenatal tests of Duchenne muscular dystrophy using cell-free fetal DNA in maternal plasma. Genet Med 17(11): 889– 896. Volume170, Issue3March 2016Pages 557-557 ReferencesRelatedInformation

Down syndrome (DS), also known as trisomy 21 (T21), is the most common genetic disorder associated with intellectual disability. There are two methods commonly used for prenatal testing of DS: serum screening (SS) for biomarkers in maternal serum and noninvasive prenatal testing (NIPT) for aneuploidy by cell-free DNA (cfDNA) in maternal plasma. However, cost-effectiveness analyses of these two methods are mostly based on data derived from simulations with various models, with theoretical values calculated. In this study, we statistically analyzed clinical DS screening data and pregnancy outcomes during the follow-up of pregnant women in Zhuhai City, China. The economics of the two mainstream prenatal DS screening methods was evaluated from a public health perspective. A retrospective analysis was performed on the data of 17,363 pregnant women who received SS and NIPT during gestation in Zhuhai from 2018 to 2019, and a cost-effectiveness analysis was performed with four screening strategies. In strategy I, all pregnant women received SS, and those with T21 risk ≥1/270 had invasive prenatal diagnosis (IPD). In strategy II, all pregnant women received SS, those with T21 risk ≥ 1/270 had IPD, and those with 1/270 > T21 risk ≥ 1/1,000 had NIPT; then, women at high risk based on NIPT also had IPD. In strategy III, all pregnant women received SS, and those with T21 risk ≥1,000 had NIPT; then, women at high risk based on NIPT results had IPD. In strategy IV, all pregnant women received NIPT and those at high risk based on NIPT results had IPD. Finally, to assess the cost and effectiveness of DS screening, the total costs were calculated as the sum of screening and diagnosis as well as the direct and indirect economic burden during the average life cycle of DS patients. A total of 22 of the 17,363 (1/789) pregnant women had DS, of which only one woman was over 35 years of age. SS detected 1,024 cases at high risk of T21 (≥1/270), 8 cases were true positive, with a positive predictive value of 0.78% and a detection rate of 36.4%. NIPT detected 27 cases at high risk of T21 (Z ≥ 3) and 22 cases of DS, with a positive predictive value of 81.5% and a detection rate of 100%. Strategy I had the largest total cost of 65.54 million CNY, strategy II and III had similar total costs of 40 million CNY, and strategy IV had the lowest total cost of 14.91 million CNY. By comparison, the screening strategy with NIPT alone had the highest health economic value for DS. SS was greatly affected by nuchal translucency and the accuracy of gestational age measured by ultrasonography. Unstandardized ultrasonography was an important reason for the low DS detection rate with SS. The influence of interfering factors on NIPT was much lower than in SS. NIPT can be used as an alternative to SS and as a primary screening strategy of prenatal DS screening for secondary prevention and control of birth defects. NIPT greatly decreased the frequency of IPD and the miscarriages associated with IPD, saved the limited medical and health resources, and greatly increased DS detection rate. Therefore, NIPT has great social and economic benefits.

BackgroundDown syndrome (DS), also known as trisomy 21 (T21), is the most common genetic disorder associated with intellectual disability. There are two methods commonly used for prenatal testing of DS: serum screening (SS) for biomarkers in maternal serum and noninvasive prenatal testing (NIPT) for aneuploidy by cell-free DNA (cfDNA) in maternal plasma. However, cost-effectiveness analyses of these two methods are mostly based on data derived from simulations with various models, with theoretical values calculated. In this study, we statistically analyzed clinical DS screening data and pregnancy outcomes during the follow-up of pregnant women in Zhuhai City, China. The economics of the two mainstream prenatal DS screening methods was evaluated from a public health perspective.MethodsA retrospective analysis was performed on the data of 17,363 pregnant women who received SS and NIPT during gestation in Zhuhai from 2018 to 2019, and a cost-effectiveness analysis was performed with four screening strategies. In strategy I, all pregnant women received SS, and those with T21 risk ≥1/270 had invasive prenatal diagnosis (IPD). In strategy II, all pregnant women received SS, those with T21 risk ≥ 1/270 had IPD, and those with 1/270 > T21 risk ≥ 1/1,000 had NIPT; then, women at high risk based on NIPT also had IPD. In strategy III, all pregnant women received SS, and those with T21 risk ≥1,000 had NIPT; then, women at high risk based on NIPT results had IPD. In strategy IV, all pregnant women received NIPT and those at high risk based on NIPT results had IPD. Finally, to assess the cost and effectiveness of DS screening, the total costs were calculated as the sum of screening and diagnosis as well as the direct and indirect economic burden during the average life cycle of DS patients.ResultsA total of 22 of the 17,363 (1/789) pregnant women had DS, of which only one woman was over 35 years of age. SS detected 1,024 cases at high risk of T21 (≥1/270), 8 cases were true positive, with a positive predictive value of 0.78% and a detection rate of 36.4%. NIPT detected 27 cases at high risk of T21 (Z ≥ 3) and 22 cases of DS, with a positive predictive value of 81.5% and a detection rate of 100%. Strategy I had the largest total cost of 65.54 million CNY, strategy II and III had similar total costs of 40 million CNY, and strategy IV had the lowest total cost of 14.91 million CNY. By comparison, the screening strategy with NIPT alone had the highest health economic value for DS.ConclusionsSS was greatly affected by nuchal translucency and the accuracy of gestational age measured by ultrasonography. Unstandardized ultrasonography was an important reason for the low DS detection rate with SS. The influence of interfering factors on NIPT was much lower than in SS. NIPT can be used as an alternative to SS and as a primary screening strategy of prenatal DS screening for secondary prevention and control of birth defects. NIPT greatly decreased the frequency of IPD and the miscarriages associated with IPD, saved the limited medical and health resources, and greatly increased DS detection rate. Therefore, NIPT has great social and economic benefits.

With the discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma, noninvasive prenatal testing became possible. However, analysis of low-level cffDNA against high background maternal DNA remains complicated and challenging. To circumvent this limitation, selective amplification of cffDNA was used in this study. Two kinds of compound markers (namely DIP-STR and SNP-STR), both based on selective amplification, were used here for targeting fetal DNA. By designing two allele-specific forward primers for DIP-STR and SNP-STR, DNA fragments with different DIP/SNP alleles can be selectively amplified. When analyzing maternal plasma DNA, these markers can selectively target paternally inherited fetal alleles whose DIP/SNP allele was not shared with the mother. In this study, 21 families were studied with six DIP-STRs and 11 SNP-STRs. Fetal DNA was successfully detected across plasma samples for at least one marker. Detection rate varied between DIP-STR and SNP-STR markers, and DIP-STR outperforms SNP-STR. Fetal alleles obtained from maternal plasma were double confirmed by genotyping paternal genomic DNA and fetal genomic DNA from amniocentesis. This study demonstrated that selective amplification strategy can be used to target cffDNA in maternal plasma, which will be a promising method for noninvasive prenatal paternity testing.

Non-invasive prenatal testing using massively parallel sequencing of maternal plasma DNA: from molecular karyotyping to fetal whole-genome sequencing

Many countries and regions carry out prenatal screening for certain fatal and disabling serious genetic diseases with high incidence. Prenatal genetic screening is beneficial to the development of genetic counseling and prenatal diagnosis of genetic diseases. This section will focus on noninvasive prenatal screening based on high-throughput sequencing technology. Noninvasive prenatal testing (NIPT), based on cell-free fetal DNA (cffDNA) in maternal peripheral plasma, uses maternal peripheral blood as a sample to determine the extent to which the fetus is likely to have certain genetic diseases through high-throughput sequencing and bioinformatics analysis. NIPT was initially performed only for fetal trisomy 21. Later, it is extended to common aneuploidies, such as trisomy 18 and trisomy 13. Subsequently, the targets of NIPT screening are gradually extended to all aneuploidies and chromosomal structural abnormalities of the fetus thanks to the development of technology and the improvement of detection resolution. Currently, there are three sources of cffDNA: apoptosis of placental syncytiotrophoblast cells, which is the main source; apoptosis of fetal hematopoietic cells entering the maternal circulation; and direct transfer of fetal DNA molecules into maternal plasma through the placenta. cffDNA level from the placenta increases with the progression of pregnancy and the degree of placental apoptosis. cffDNA, as it is released by apoptotic cells, also has the characteristics of apoptotic cells, that is, fragmentation of genomic DNA. More than 99% of fetal DNA fragments are below 313 bp in length. For pregnant women in the first trimester, cffDNA can be detected in the peripheral blood, accounting for up to 20% of the total free DNA in maternal plasma. NIPT is generally performed after 12 weeks of gestation. In addition, cffDNA is rapidly degraded after birth and is not affected by the previous fetus.

The performance of noninvasive prenatal testing (NIPT) in monochorionic diamniotic twin pregnancies with treatment of assisted reproductive techniques (ART)

Noninvasive prenatal testing (NIPT) for monogenic disorders has been developed in recent years; however, there are still significant technical and analytical challenges for clinical use. The clinical feasibility of NIPT for methylmalonic acidemia cblC type (cblC type MMA) was investigated using our circulating single-molecule amplification and re-sequencing technology (cSMART). Trios molecular diagnosis was performed in 29 cblC type MMA-affected children and their parents by traditional Sanger sequencing. In the second pregnancy, invasive prenatal diagnosis (IPD) of the pathogenic MMACHC gene was used to determine fetal genotypes, and NIPT was performed using a novel MMACHC gene–specific cSMART assay. Maternal–fetal genotypes were deduced based on the mutation ratio in maternal plasma DNA. Concordance of fetal genotypes between IPD and NIPT, and the sensitivity and specificity of NIPT were determined. After removing two cases with a low P value or reads, the concordance ratio for NIPT and IPD was 100.00% (27/27), and the sensitivity and specificity were 100.00% (54.07–100.00%) and 100.00% (83.89–100.00%), respectively. This study demonstrates that NIPT using the cSMART assay for cblC type MMA was accurate in detecting fetal genotypes. cSMART has a potential clinical application as a prenatal diagnosis and screening tool for carrier and low-risk genotypes of cblC type MMA and other monogenic diseases.

To assess the clinical value of non-invasive prenatal testing (NIPT) for the screening of trisomy and copy number variations (CNVs) of chromosomes 21, 18 and 13. From January 2015 to December 2019, 40 628 pregnant women underwent NIPT testing using high-throughput sequencing and bioinformatics analysis to test the cell-free fetal DNA in maternal plasma. High-risk pregnant women underwent invasive prenatal diagnosis, while low-risk ones were followed up by telephone. The three most common indications included intermediate risk of serological screening, high risk of serological screening and advanced maternal age. Among all pregnant women, 257 cases were detected as trisomy 21, 18 and 13 (170, 49 and 38 cases, respectively). 227 cases chose invasive prenatal diagnosis, with respectively 122, 28 and 10 cases confirmed. The positive predictive value (PPV) was 81.33% (122/150), 65.12% (28/43), 29.41% (10/34), respectively. Two false negative cases of trisomy 18 were found during follow-up. Meanwhile, NIPT has detected 46 cases (15, 16 and 15 cases, respectively) CNVs on chromosomes 21, 18 and 13, among which 37 cases underwent invasive prenatal diagnosis. There were 5, 3 and 5 positive cases, which yielded a PPV of 41.67% (5/12), 25%(3/12) and 33.33%(5/15), respectively. Two other chromosome CNVs were accidentally discovered among the false positive samples. The incidence of chromosomal abnormalities in the serological screening high-risk group was 52.02%, which was significantly higher than other groups. NIPT has a high sensitivity and specificity for the screening of trisomies 21, 18 and 13, while its accuracy for detecting CNVs of chromosomes 21, 18 and 13 needs to be improved. As a screening method, NIPT has a great clinical value, though there are still limitations of false positive and false negative results.Comprehensive pre- and post-test genetic counseling should be provided to the patients.

Non-invasive prenatal testing (NIPT) has revolutionized prenatal diagnostics by providing a safer alternative to invasive techniques such as amniocentesis and chorionic villus sampling. NIPT detects chromosomal abnormalities through the analysis of cell-free fetal DNA (cffDNA) in maternal plasma. One of the critical factors influencing accuracy of NIPT is the fetal fraction (FF) - the proportion of fetal cell-free DNA relative to total cell-free DNA in maternal plasma. This study investigates the potential of using telomere length measurements as a novel biomarker for fetal fraction prediction in NIPT. Telomere-derived fragments, which differ between maternal and fetal DNA, may serve as a measure of FF due to the distinct telomere length. Specifically, deviations from the expected shorter telomere lengths of maternal DNA toward longer lengths could be more pronounced at higher FF levels. Various models incorporating telomere content and features selected by Ordinary Least Squares (OLS) were evaluated to enhance fetal fraction prediction. Our results showed that telomere content also works as an independent predictor (with Pearson correlation 0.23), yielding a small improvement in prediction precision when combined with traditional models.

Context: Prenatal testing aims to identify fetal chromosomal and genetic disorders prior to delivery. Current invasive procedures such as amniocentesis and chorionic villus sampling (CVS) pose a risk to mother and fetus and such diagnostic procedures are available only to high-risk pregnancies, which limits aneuploidy detection rate. The identification of cell-free fetal DNA (cffDNA) in maternal circulation has made noninvasive prenatal testing (NIPT) possible. This review seeks to highlight the necessity of investing in NIPT and briefly summarizes the technical aspects of the NIPT and application of this method in clinical practice. Evidence Acquisition: PubMed, OVID, SCOPUS, and the Cochrane database were searched for relevant articles published between 1995 and 2014, using appropriate keywords including prenatal screening, noninvasive testing, prenatal diagnosis, cell free fetal DNA, maternal circulation, chromosomal aneuploidies, trisomy, and sex determination. Results were restricted to systematic reviews, randomized clinical trials, meta-analysis, and observational studies. Results: The importance of prenatal diagnosis and risks associated with current invasive techniques makes NIPT research morally and commercially beneficial. The outstanding advantages of NIPT over current prenatal diagnosis techniques include increasing detection rate, enabling earlier diagnosis, and eliminating iatrogenic fetal loss and risk to the mother due to invasive procedures. At present, two major techniques for isolating cffDNA, namely digital PCR and massively parallel sequencing (MPS), have enabled the successful implementation of NIPT into clinical practice such as fetal sex determination, RhD genotyping, and fetal chromosomal aneuploidy detection. Conclusions: The advent of new NIPT using cffDNA has been regarded as a revolution in prenatal testing and has attracted significant commercial interest in the field. It is not overoptimistic to predict that NIPT will supplement or replace existing screening and diagnostic tools.

In Hong Kong, universal combined first-trimester screening for Down's syndrome was started as a 'free service' in July 2010. Non-invasive prenatal testing was available as a self-financed item in August 2011. This study aimed to determine whether the introduction of non-invasive prenatal testing as a contingent approach influenced the indications for invasive prenatal diagnosis and the consequent prenatal detection of Down's syndrome. This historical cohort study was conducted at the Prenatal Diagnosis Clinic of Queen Elizabeth Hospital in Hong Kong. We compared the indications for invasive prenatal diagnosis and prenatal detection of Down's syndrome in singleton pregnancies 1 year before and 2 years following the availability of non-invasive prenatal testing as a contingent test after a positive aneuploidy test. All pregnant women who attended our hospital for counselling about universal Down's syndrome screening between August 2010 and July 2013 were recruited. A total of 16 098 women were counselled. After the introduction of non-invasive prenatal testing, the invasive prenatal diagnosis rate for a positive aneuploidy screening reduced from 77.7% in 2010-11 to 68.8% in 2012-13. The new combined conventional plus non-invasive prenatal testing strategy was associated with a lower false-positive rate (6.9% in 2010-11 vs 5.2% in 2011-12 and 4.9% in 2012-13). There was no significant increase in invasive prenatal diagnosis for structural anomalies over the years. There was no significant trend in the overall prenatal detection rate of Down's syndrome (100% 1 year before vs 89.1% 2 years after introduction of non-invasive prenatal testing). Four (2.6%) of 156 women who underwent non-invasive prenatal testing for a screen-positive result had a high-risk result for trisomy 21, which was subsequently confirmed by invasive prenatal diagnosis. There were no false-negative cases. The introduction of non-invasive prenatal testing as a contingent approach reduced the invasive prenatal diagnosis rate for a positive aneuploidy screening without affecting the invasive prenatal diagnosis rate for structural anomalies or the overall detection rate of fetal Down's syndrome.

BackgroundTo evaluate the clinical significance of noninvasive prenatal testing (NIPT) for detecting fetal sex chromosome aneuploidies (SCAs) in Korean pregnant women.MethodsWe retrospectively analyzed NIPT data from 9,176 women with singleton pregnancies referred to the CHA Biotech genome diagnostics center. Cell-free fetal DNA (cffDNA) was extracted from maternal peripheral blood, and high-throughput massively parallel sequencing was conducted. Subsequently, the positive NIPT results for SCA were validated via karyotype and chromosomal microarray analyses.ResultsOverall, 46 cases were SCA positive after NIPT, including 20, 12, 8, and 6 for Turner, triple X, Klinefelter, and Jacob syndromes, respectively. Among 37 women with invasive prenatal diagnosis, 19 had true positive NIPT results. The overall positive predictive value (PPV) of NIPT for detecting SCAs was 51.35%. The PPV was 18.75% for Turner, 88.89% for triple X, 71.43% for Klinefelter, and 60.00% for Jacob’s syndromes. NIPT accuracy for detecting sex chromosome trisomies was higher than that for sex chromosome monosomy (P = 0.002). No significant correlation was observed between fetal SCA incidence and maternal age (P = 0.914), except for the borderline significance of Jacob’s syndrome (P = 0.048). No significant differences were observed when comparing NIPT and karyotyping validation for fetal SCA according to pregnancy characteristics.ConclusionOur data suggest that NIPT can reliably screen for SCAs, and it performed better in predicting sex chromosome trisomies compared with monosomy X. No correlation was observed between maternal age and fetal SCA incidence, and no association was observed between different pregnancy characteristics. The accuracy of these findings requires improvements; however, our study provides an important reference for clinical genetic counseling and further management. Larger scale studies, considering confounding factors, are required for accurate evaluation.