Noninvasive Prenatal Detection Research Articles

Enhancing fetal outcomes in GCK-MODY pregnancies: a precision medicine approach via non-invasive prenatal GCK mutation detection.

Mutations in the GCK gene cause Maturity Onset Diabetes of the Young (GCK-MODY) by impairing glucose-sensing in pancreatic beta cells. During pregnancy, managing this type of diabetes varies based on fetal genotype. Fetuses carrying a GCK mutation can derive benefit from moderate maternal hyperglycemia, stimulating insulin secretion in fetal islets, whereas this may cause macrosomia in wild-type fetuses. Modulating maternal glycemia can thus be viewed as a form of personalized prenatal therapy, highly beneficial but not justifying the risk of invasive testing. We therefore developed a monogenic non-invasive prenatal diagnostic (NIPD-M) test to reliably detect the transmission of a known maternal GCK mutation to the fetus. A small amount of fetal circulating cell-free DNA is present in maternal plasma but cannot be distinguished from maternal cell-free DNA. Determining transmission of a maternal mutation to the fetus thus implies sequencing adjacent polymorphisms to determine the balance of maternal haplotypes, the transmitted haplotype being over-represented in maternal plasma. Here we present a series of such tests in which fetal genotype was successfully determined and show that it can be used to guide therapeutic decisions during pregnancy and improve the outcome for the offspring. We discuss several potential hurdles inherent to the technique, and strategies to overcome these. Our NIPD-M test allows reliable determination of the presence of a maternal GCK mutation in the fetus, thereby allowing personalized in utero therapy by modulating maternal glycemia, without incurring the risk of miscarriage inherent to invasive testing.

NanoNIPT: Short-fragment nanopore sequencing of cell-free DNA for non-invasive prenatal testing of fetal aneuploidies and sex chromosome aberrations.

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Droplet Digital PCR for Non-Invasive Prenatal Detection of Fetal Single-Gene Point Mutations in Maternal Plasma.

Non-invasive prenatal testing (NIPT) is based on the detection and characterization of circulating cell-free fetal DNA (ccffDNA) in maternal plasma and aims to identify genetic abnormalities. At present, commercial NIPT kits can detect only aneuploidies, small deletions and insertions and some paternally inherited single-gene point mutations causing genetic diseases, but not maternally inherited ones. In this work, we have developed two NIPT assays, based on the innovative and sensitive droplet digital PCR (ddPCR) technology, to identify the two most common β thalassemia mutations in the Mediterranean area (β+IVSI-110 and β039), maternally and/or paternally inherited, by fetal genotyping. The assays were optimized in terms of amplification efficiency and hybridization specificity, using mixtures of two genomic DNAs with different genotypes and percentages to simulate fetal and maternal circulating cell-free DNA (ccfDNA) at various gestational weeks. The two ddPCR assays were then applied to determine the fetal genotype from 52 maternal plasma samples at different gestational ages. The diagnostic outcomes were confirmed for all the samples by DNA sequencing. In the case of mutations inherited from the mother or from both parents, a precise dosage of normal and mutated alleles was required to determine the fetal genotype. In particular, we identified two diagnostic ranges for allelic ratio values statistically distinct and not overlapping, allowing correct fetal genotype determinations for almost all the analyzed samples. In conclusion, we have developed a simple and sensitive diagnostic tool, based on ddPCR, for the NIPT of β+IVSI-110 and β039 mutations paternally and, for the first time, maternally inherited, a tool, which may be applied to other single point mutations causing monogenic diseases.

Segmental duplication as potential biomarkers for non-invasive prenatal testing of aneuploidies.

BackgroundSegmental duplication (SD) regions are distinct targets for aneuploidy detection owing to the virtual elimination of amplification bias. The difficulty of searching SD sequences for assay design has hampered their applications.MethodsWe developed a computational program, ChAPDes, which integrates SD searching, refinement, and design of specific PCR primer/probe sets in a pipeline to remove most of the manual work. The generated primer/probe sets were first tested in a multiplex multicolour melting curve analysis for the detection of five common aneuploidies. The primer/probe sets were then tested in a digital PCR assay for the detection of trisomy 21. Finally, a digital PCR protocol was established to quantify maternal plasma DNA sequences for the non-invasive prenatal detection of fetal trisomy 21.FindingsChAPDes could output 21,772 candidate primer/probe sets for trisomy 13, 18, 21 and sex chromosome aneuploidies within 2 working days. Clinical evaluation of the multiplex multicolour melting curve analysis involving 463 fetal genomic DNA samples revealed a sensitivity of 100% and specificity of 99.64% in comparison with the reference methods. Using the established digital PCR protocol, we correctly identified two trisomy 21 fetuses and thirteen euploid foetuses from the maternal plasma samples.InterpretationThe combination of ChAPDes with digital PCR detection could facilitate the use of SD as potential biomarkers for the non-invasive prenatal testing of fetal chromosomal aneuploidies.

The yield of chromosomal microarray in pregnancies with congenital cardiac defects and normal noninvasive prenatal screening

Evidence comparing the yield of chromosomal microarray analysis to noninvasive prenatal screening in pregnancies with congenital heart anomalies is currently limited. This study aimed to examine the residual risk of clinically significant chromosomal microarray analysis results in fetuses with congenital heart defects by its various subtypes following a normal noninvasive prenatal screening. Using a population-based, countrywide computerized database, we retrieved the reports of all pregnancies undergoing chromosomal microarray analysis because of congenital heart defects through the years 2013-2019. We examined the risk of clinically significant (pathogenic and likely pathogenic) chromosomal microarray analysis results and compared it with the results of a local cohort of low-risk pregnancies. Of 5541 fetuses, 78 (1.4%) showed abnormal results. The residual risk of abnormal chromosomal microarray analysis results was calculated using several options-trisomies 21, 18, and 13; sex chromosome aneuploidies; 22q11.2 deletion, and deletions and duplications of at least 10 MB in size (genome-wide noninvasive prenatal screening)-following the exclusion of theoretically detectable noninvasive prenatal screening anomalies. Of the 1728 fetuses with congenital heart defects, 93 (5.4%) showed clinically significant chromosomal microarray analysis results (relative risk, 2.7; 95% confidence interval, 2.3-3.1). The result of pregnancies with fetuses with congenital heart defects was compared with the results of the control population. Unique variants were found in 15 pregnancies (16.1%). The detection rate of noninvasive prenatal screening in isolated congenital heart defects varied from 1.0% (aimed at 3 common trisomies) to 2.2% (aimed at 5 common aneuploidies and 22q11.2 deletion) using noninvasive prenatal screening. In nonisolated congenital heart defects, the noninvasive prenatal screening detection rates ranged from 7.8% (aimed at common autosomal trisomies) to 9.2% using genome-wide noninvasive prenatal screening. The residual risk of clinically significant chromosomal microarray analysis results following normal noninvasive prenatal screening ranged from 2.0% to 2.8% in isolated congenital heart defects and 4.5% to 5.9% in nonisolated cases and was significantly higher than those of the control cohort in all noninvasive prenatal screening options. In addition, the residual risk following noninvasive prenatal screening aimed at chromosomes 13, 18, 21, X, and Y was significantly higher than those of the control cohort for most specific congenital heart defect subtypes, except for ventricular septal defects and aberrant right subclavian artery. The residual risk of clinically significant chromosomal microarray analysis results in pregnancies with fetuses with congenital heart defects following normal noninvasive prenatal screening was higher than those in pregnancies with normal ultrasound in most isolated and nonisolated congenital heart defect subtypes. This information should be taken into account by obstetricians and genetic counselors when considering the option of diagnostic testing.

Non-invasive prenatal detection of ocutaneous albinism type I based on cfDNA barcode-enabled single-molecule test

To assess the value of non-invasive prenatal testing based on cfDNA barcode-enabled single-molecule test (cfBEST) for the prenatal diagnosis of oculocutaneous albinism type I in a family. Prenatal genetic diagnosis was carried out by using the cfBEST-based method as well as invasive prenatal diagnosis through amniocentesis. The outcome of the pregnancy was followed up. Non-invasive prenatal testing based on cfBEST showed a fetal DNA concentration of 6.6%, with the proportion of c.929_930insC (p.Arg311Lysfs*7) and c.1037-7T>A mutations being 45.7% and 0%, respectively. The posterior frequency of the negative results was 1, suggesting that the fetus carried neither of the two mutations. The result was consistent with that of invasive prenatal diagnosis, and the follow-up found that the fetus was normal. Non-invasive prenatal testing based on cfBEST can be used to detect maternal and fetal genotypes in maternal cell-free DNA, which is clinically feasible.

Noninvasive prenatal detection of fetal sex chromosome abnormalities using the semiconductor sequencing platform (SSP) in Southern China

BackgroundNoninvasive prenatal testing (NIPT) has been widely used to screen for fetal aneuploidies, including fetal sex chromosome aneuploidies (SCAs). However, there is less information on the performance of NIPT in detecting SCAs.MethodsA cohort of 47,800 pregnancies was recruited to review the high-risk NIPT results for SCAs. Cell-free fetal DNA (cffDNA) was extracted and sequenced. All NIPT high-risk cases were recommended to undergo invasive prenatal diagnosis for karyotyping analysis and chromosome microarray analysis (CMA).ResultsA total of 238 high-risk cases were detected by NIPT, including 137 cases of 45,X, 27 cases of 47,XXX, and 74 cases of 47,XYY/47,XXY. Prenatal diagnosis, including karyotyping analysis and CMA, was available in 170 cases. The positive predictive value (PPV) was 30.00% for 45,X, 70.58% for 47,XXX, and 81.13% for 47,XYY/47,XXY. In addition, 13 cases of sex chromosome mosaicism and 9 cases of sex chromosome CNVs were incidentally found in this study.ConclusionOur study showed that NIPT was reliable for screening SCAs based on a large sample, and it performed better in predicting sex chromosome trisomies than monosomy X. Our study will provide an important reference for clinical genetic counseling and further processing of the results.

Segmental Duplication as Potential Biomarkers for Non-Invasive Prenatal Testing of Aneuploidies

Background: Segmental duplication (SD) regions are distinct targets for aneuploidy detection owing to the virtual elimination of amplification bias. The difficulty of searching SD sequences for assay design has hampered their applications. Methods:We developed a computational program, ChAPDes, which integrates SD searching, refinement, and design of specific PCR primer/probe sets in a pipeline to remove most of the manual work. The generated primer/probe sets were first tested in a multiplex multicolor melting curve analysis for the detection of five common aneuploidies. The primer/probe sets were then tested in a digital PCR assay for the detection of trisomy 21. Finally, a digital PCR protocol was established to quantify maternal plasma DNA sequences for the non-invasive prenatal detection of fetal trisomy 21. Findings: ChAPDes could output 21,772 candidate primer/probe sets for trisomy 13, 18, 21 and sex chromosome aneuploidies within 2 working days. Clinical evaluation of the multiplex multicolour melting curve analysis involving 463 fetal genomic DNA samples revealed a sensitivity of 100% and specificity of 99.64% in comparison with the reference methods. Using the established digital PCR protocol, we correctly identified two trisomy 21 fetuses and thirteen euploid foetuses from the maternal plasma samples. Interpretation: The combination of ChAPDes with digital PCR detection could facilitate the use of SD as potential biomarkers for the non-invasive prenatal testing of fetal chromosomal aneuploidies. Funding Information: This work was supported by the National Key Research & Developmental Program of China (2018YFC1003601), the National Natural Science Foundation of China (8167210 to Q.H.), the Science and Technology Project of Xiamen City (3502Z20183013), and the Guangxi Key Laboratory Thalassemia Research Project (20-065-20). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The collection of specimens and associated clinical data used in this study were approved by the Affiliated Suzhou Hospital of Nanjing Medical University and Suzhou Municipal Hospital. All patients who participated in this study provided written informed consent prior to enrollment and specimen collection. This study was performed in accordance with the principles of the Helsinki Declaration.

Nondestructive Identification of Rare Trophoblastic Cells by Endoplasmic Reticulum Staining for Noninvasive Prenatal Testing of Monogenic Diseases.

Noninvasive prenatal detection of monogenic diseases based on cell‐free DNA is hampered by challenges in obtaining a sufficient fraction and adequate quality of fetal DNA. Analyzing rare trophoblastic cells from Papanicolaou smears carrying the entire fetal genome provides an alternative method for noninvasive detection of monogenic diseases. However, intracellular labeling for identification of target cells can affect the quality of DNA in varying degrees. Here, a new approach is developed for nondestructive identification of rare fetal cells from abundant maternal cells based on endoplasmic reticulum staining and linear discriminant analysis (ER‐LDA). Compared with traditional methods, ER‐LDA has little effect on cell quality, allowing trophoblastic cells to be analyzed on the single‐cell level. Using ER‐LDA, high‐purity of trophoblastic cells can be identified and isolated at single cell resolution from 60 pregnancies between 4 and 38 weeks of gestation. Pathogenic variants, including –SEA/ deletion mutation and point mutations, in 11 fetuses at risk for α‐ or β‐thalassemia can be accurately detected by this test. The detection platform can also be extended to analyze the mutational profiles of other monogenic diseases. This simple, low‐cost, and noninvasive test can provide valuable fetal cells for fetal genotyping and holds promise for prenatal detection of monogenic diseases.

Silica microbeads capture fetal nucleated red blood cells for noninvasive prenatal testing of fetal ABO genotype.

ABO hemolytic disease of the newborn (ABO-HDN), which may cause neonatal jaundice and polycythemia, or even stillbirth or neonatal death, is widespread in China. Prenatal testing for the fetal ABO blood group can reduce unnecessary concerns or ensure prompt treatment. Herein, we presented a method to employ high-density silica microbeads (SiO2 MBs) for capturing fetal nucleated red blood cells (fnRBCs) in maternal peripheral blood, and we detected the ABO genotype of the fetus using these captured cells. We evaluated 52 patients using the SiO2 MBs. Among 26 pregnant women with type O blood, 8 (30.8%) of the fetuses had type A blood, 5 (19.2%) had type B blood, and 13 (50%) had type O blood. SRY genes were detected in all 27 male fetuses. This study represents a simple and effective method for noninvasive prenatal detection of the fetal ABO genotype. We believe that this method has great potential for noninvasive prenatal testing of the fetal Rh blood group and other fetal diseases as well.

Noninvasive Prenatal Testing and Detection of Occult Maternal Malignancies.

Since the introduction of noninvasive prenatal testing (NIPT)7 in prenatal diagnostics in 2011, incidental findings of an occult maternal malignancy following a “false-positive” NIPT test have been reported repeatedly. In these cases, NIPT, originally designed to interrogate placenta-derived “fetal” cell-free DNA (cfDNA) in the maternal circulation, also detected the presence of tumor-derived cfDNA. This freely circulating tumor DNA is characterized by multiple chromosomal imbalances—a hallmark of human neoplasms—that skew the signals and give rise to an aberrant NIPT result (Fig. 1). Worldwide controversy exists about disclosing such NIPT results as suggestive of a maternal cancer. At the same time, these incidental findings prompted retrospective investigations into the association between a deviating NIPT outcome and an occult maternal malignancy. However, because these studies were not designed to evaluate the ability of NIPT to detect cancer, deductions about the test's specificity and sensitivity for cancer detection could not be made. In a recent publication, Ji and colleagues collected over 1.9 million NIPT data and retrospectively evaluated the performance of a novel bioinformatics algorithm to detect occult maternal malignancies in pregnant women possessing multiple chromosomal aneuploidies in NIPT results (1). Their work confirmed that the presence of multiple reproducible chromosomal aneuploidies in plasma cfDNA of asymptomatic pregnant women was indeed an indicator of cancer risk. In their hands, the positive predictive value (PPV) of detecting multiple chromosomal aneuploidies for the identification of a maternal malignancy was 7.6%. To increase performance, Ji and colleagues further developed their cancer detection algorithm by introducing a new parameter, that is, the “fraction of significant (sub)chromosomal copy number alterations (CNAs), detected across the entire genome” (called the “FCNV score”). FCNV scores were compared among the purported “cancer group,” the “noncancer group,” and the group of control participants for whom no multiple chromosomal aneuploidies were detected. Implementation of …

Advances in non-invasive prenatal gene detection technologies

Non-invasive prenatal testing is a novel detection method which uses sequencing of maternal plasma cell-free DNA in order to screen genetic disease for pregnant women. Compared to traditional prenatal pregnancy tests and disease diagnosis techniques, this technology is non-invasive, convenient, accurate and so on. Since the discovery of cell-free DNA in the peripheral maternal plasma in 1997, non-invasive prenatal genetic testing has been widely used in clinical pregnancy tests and related medical research with the development of high-throughput gene sequencing,it has meaningful achievements in the detection of fetal aneuploidy and enormous challenges in the detection of complex genetic diseases. This article reviews the development of non-invasive prenatal genetic testing technology. Key words: Genetic testing; Prenatal diagnosis; Chromosome aberrations; Genetic diseases,inborn

Fast Temperature-Gradient COLD PCR for the enrichment of the paternally inherited SNPs in cell free fetal DNA; an application to non-invasive prenatal diagnosis of β-thalassaemia

ObjectiveTo develop a sensitive, specific, simple, cost-effective and reproducible platform for the non-invasive prenatal detection of paternally inherited alleles for β-thalassaemia. The development of such an assay is of major significance in order to replace currently-applied invasive methods containing inherent fetal loss risks.MethodsWe present a fast Temperature-Gradient Co-amplification at Lower Denaturation Temperature Polymerase Chain Reaction (fast TG COLD PCR) methodology for the detection of the paternally-inherited fetal alleles in maternal plasma. Two single-nucleotide polymorphisms (SNPs), rs7480526 (G/T) and rs968857 (G/A) that are located on the β-globin gene cluster and exhibit a high degree of heterozygosity in the Cypriot population were selected for evaluation. Seventeen maternal plasma samples from pregnancies at risk for β-thalassemia were analysed for the selected SNPs using the novel fast TG COLD PCR assay.ResultsUsing fast TG COLD PCR, the paternally inherited allele in cell free fetal DNA was correctly determined for all the 17 maternal plasma samples tested, showing full agreement with the Chorionic Villus Sampling (CVS) analysis.ConclusionsOur findings are encouraging and demonstrate the efficiency and sensitivity of fast TG COLD PCR in detecting the minor paternally-inherited fetal alleles in maternal plasma for the development of a NIPD assay for β-thalassaemia.

Prenatal detection of thalassemia by cell-free fetal DNA (cffDNA) in maternal plasma using surface enhanced Raman spectroscopy combined with PCR.

Thalassemias are widely occurring genetic hemoglobin disorders; patients with severe thalassemia often require regular blood transfusions for survival. Prenatal detection of thalassemia is currently invasive and carries the risk of miscarriage and infection. A polymerase chain reaction (PCR)-based surface enhanced Raman spectroscopy (SERS) technique was investigated in this paper for the purpose of detecting prenatal α-thalassemia Southeast Asian (SEA) type deletion using maternal plasma. Couples with the same SEA thalassemia (-SEA/αα) were selected, and the quantification of SEA and wild type (WT) alleles in the maternal plasma sample predicted the fetal genotype. PCR was performed using two pairs of fluorescence tag-labeled primers to produce tag-labeled PCR products for both the SEA (labeled with R6G) and WT (labeled with Cy3) alleles. Then, the labeled PCR products containing the two fluorescence tags were measured by SERS. The ratios between the R6G and Cy3 tags were obtained using multiple linear regressions (MLR), and these ratios corresponded with the physical ratio of WT and SEA concentrations in maternal plasma. After verifying this technique on DNA mixtures with known SEA and WT ratios, the plasma from 24 pregnant women was screened. An accuracy of 91.7% was achieved for detecting the fetal genotypes of Hb Bart's, alpha-trait, and normal trait. The results indicated that the simple PCR-SERS method may be sensitive enough for use on cell free fetal DNA (cffDNA) in maternal plasma for non-invasive prenatal detection (NIPD).

Non-invasive prenatal detection for copy number variation

Aim: To assess the technology of non-invasive prenatal testing (NIPT) and the robust mathematical model fetal copy-number analysis through maternal plasma sequencing (FCAPS) detecting large fetal deletions or duplications. Materials and Methods: Peripheral venous blood were taken from three pregnant women with high risk, and maternal plasma DNA were extracted and detected by NIPT and FCAPS. The results were validated through Array-CGH or karyotyping with amniotic fluid or umbilical cord blood obtained from the patients. Results: One out of three cases was positive by NIPT, but all were found with abnormalities by FCAPS. The results were further confirmed using array-CGH or karyotyping. Discussion: This study provides novel insights into noninvasive prenatal diagnosis using low-coverage maternal plasma sequencing to detect large fetal deletions or duplications, as well as correlations between fetal genotypes and phenotypes.

Predicting fetoplacental chromosomal mosaicism during non-invasive prenatal testing.

Non-invasive prenatal detection of aneuploidies can be achieved with high accuracy through sequencing of cell-free maternal plasma DNA in the maternal blood plasma. However, false positive and negative non-invasive prenatal testing (NIPT) results remain. Fetoplacental mosaicism is the main cause for false positive and false negative NIPT. We set out to develop a method to detect placental chromosomal mosaicism via genome-wide circulating cell-free maternal plasma DNA screening. Aneuploidy detection was combined with fetal fraction determination to enable the detection of placental mosaicism. This pipeline was applied to whole genome sequencing data derived from 19735 plasma samples. Following an abnormal NIPT, test results were validated by conventional invasive prenatal or postnatal genetic testing. Respectively 3.2% (5/154), 12.8% (5/39), and 13.3% (2/15) of trisomies 21, 18, and 13 were predicted and confirmed to be mosaic. The incidence of other, rare autosomal trisomies was ~0.3% (58/19,735), 45 of which were predicted to be mosaic. Twin pregnancies with discordant fetal genotypes were predicted and confirmed. This approach permits the non-invasive detection of fetal autosomal aneuploidies and identifies pregnancies with a high risk of fetoplacental mosaicism. Knowledge about the presence of chromosomal mosaicism in the placenta influences risk estimation, genetic counseling, and improves prenatal management.

Analysis of non-invasive prenatal screening detection in fetal chromosome aneuploidy

Objective: To evaluate the efficacy of non-invasive prenatal screening (NIPS) in the detection of fetal aneuploidies. Methods: Cell free DNA was sequenced in 5 566 pregnant women to identify the fetal aneuploidies in the First Affiliated Hospital of Zhengzhou University from January 1(st), 2015 to March 15(th), 2016. Among them, 5 230 (93.96%, 5 230/5 566) were singleton pregnancies and 336 (6.04%, 336/5 566) were twin pregnancies. In singleton pregnancies, 1 809 (34.59%, 1 809/5 230) were women with advanced maternal age, and 3 421 (65.41%, 3 421/5 230) were young women. The positive results of NIPS were validated by karyotyping through invasive procedures and neonatal outcomes were followed up by telephone. Results: Among the 5 566 women, 69 (1.24%, 69/5 566) got positive NIPS results, with 66 in singleton pregnancies and 3 in twin pregnancies. Two were monochorionic diamniotic twins and 1 was dichorionic twin pregnancy. The positive predictive value of NIPS for trisomy 21, 18 and 13 were 100.0%, 90.9% and 100.0%, and was 55.6% for sex chromosome aneuploidies. There was no false negative case found during the follow-up. In the advanced maternal age group and young women group, the prevalence rates of fetal chromosomal aneuploidies were 1.11%(20/1 809) and 0.94%(32/3 421), respectively. In the young women with soft markers in fetal ultrasound, the prevalence of fetal chromosomal aneuploidies was 1.44% (7/487), and in serum high risk women, it was 0.94% (7/747). In women with the serum screening risk with cut-off value, 0.89%(9/1 016) had fetal aneuploidies, and the prevalence was 0.77%(9/1 171) in volunteers. There was no statistically significant difference among these groups (P=0.636). Conclusions: There is no difference in the detection rate of fetal aneuploidies between high-risk women in serum screening and volunteers in NIPS. NIPS is more suitable as a first line screening test for women without fetal ultrasound abnormalities. It should be used carefully when there is ultrasound abnormalities.

A Pilot Study of Noninvasive Prenatal Diagnosis of Alpha- and Beta-Thalassemia with Target Capture Sequencing of Cell-Free Fetal DNA in Maternal Blood.

Thalassemia is a dangerous hematolytic genetic disease. In south China, ∼24% Chinese carry alpha-thalassemia or beta-thalassemia gene mutations. Given the fact that the invasive sampling procedures can only be performed by professionals in experienced centers, it may increase the risk of miscarriage or infection. Thus, most people are worried about the invasive operation. As such, a noninvasive and accurate prenatal diagnosis is needed for appropriate genetic counseling for families with high risks. Here we sought to develop capture probes and their companion analysis methods for the noninvasive prenatal detection of deletional and nondeletional thalassemia. Two families diagnosed as carriers of either beta-thalassemia gene or Southeast Asian deletional alpha-thalassemia gene mutation were recruited. The maternal plasma and amniotic fluid were collected for prenatal diagnosis. Probes targeting exons of the genes of interest and the highly heterozygous SNPs within the 1Mb flanking region were designed. The target capture sequencing was performed with plasma DNA from the pregnant woman and genomic DNA from the couples and their children. Then the parental haplotype was constructed by the trios-based strategy. The fetal haplotype was deduced from the parental haplotype with a hidden Markov model-based algorithm. The fetal genotypes were successfully deduced in both families noninvasively. The noninvasively constructed haplotypes of both fetuses were identical to the invasive prenatal diagnosis results with an accuracy rate of 100% in the target region. Our study demonstrates that the effective noninvasive prenatal diagnosis of alpha-thalassemia and beta-thalassemia can be achieved with the targeted capture sequencing and the haplotype-assisted analysis method.

Noninvasive Prenatal Detection of Trisomy 21 by Targeted Semiconductor Sequencing: A Technical Feasibility Study

Objective: To develop an alternate noninvasive prenatal testing method for the assessment of trisomy 21 (T21) using a targeted semiconductor sequencing approach. Methods: A customized AmpliSeq panel was designed with 1,067 primer pairs targeting specific regions on chromosomes 21, 18, 13, and others. A total of 235 samples, including 30 affected with T21, were sequenced with an Ion Torrent Proton sequencer, and a method was developed for assessing the probability of fetal aneuploidy via derivation of a risk score. Results: Application of the derived risk score yields a bimodal distribution, with the affected samples clustering near 1.0 and the unaffected near 0. For a risk score cutoff of 0.345, above which all would be considered at “high risk,” all 30 T21-positive pregnancies were correctly predicted to be affected, and 199 of the 205 non-T21 samples were correctly predicted. The average hands-on time spent on library preparation and sequencing was 19 h in total, and the average number of reads of sequence obtained was 3.75 million per sample. Conclusion: With the described targeted sequencing approach on the semiconductor platform using a custom-designed library and a probabilistic statistical approach, we have demonstrated the feasibility of an alternate method of assessment for fetal T21.

Single-Stranded DNA Library Preparation Preferentially Enriches Short Maternal DNA in Maternal Plasma.

Recent studies have suggested that single-stranded DNA (ssDNA) library preparation can enrich short DNA species from the plasma of healthy individuals, cancer patients, and transplant recipients. Based on previous observations that fetal DNA molecules in the maternal plasma are shorter than maternal DNA molecules, ssDNA library preparation may potentially enrich fetal DNA and provide substantial improvement in noninvasive prenatal testing. We tested this hypothesis by comparing the maternal plasma DNA sequencing results using 2 types of ssDNA library preparation methods and a standard double-stranded DNA (dsDNA) library method using samples from first- and third-trimester pregnancies. We also evaluated the performance of ssDNA and dsDNA library methods in the noninvasive prenatal detection of trisomy 21 from maternal plasma. Short DNA species were significantly enriched in ssDNA libraries. However, contrary to previous speculation, no significant enrichment was observed in the overall fetal fraction in maternal plasma collected in the first trimester. Our use of an ssDNA library did not reduce the variation in chromosomal representation when compared with a standard dsDNA library in the first-trimester plasma samples. ssDNA libraries also showed inferior performance in the noninvasive prenatal detection of trisomy 21 from maternal plasma. Detailed fetal fraction analysis using size-fractionated Y chromosome sequences and fetal-specific single-nucleotide polymorphisms (SNPs) revealed an unexpected finding that short maternal DNA was preferentially enriched over short fetal DNA in an ssDNA library irrespective of GC content. Our findings have shown that ssDNA library preparation preferentially enriches short maternally derived DNA in maternal plasma.