Validation of fetal DNA fraction estimation and its application in noninvasive prenatal testing for aneuploidy detection in multiple pregnancies.

OBJECTIVE: To analyze the fetal fraction, fetal sex, and chromosomal aneuploidy in multiple pregnancies using noninvasive prenatal testing (NIPT). METHOD: A total of 362 pregnant women including 203 singleton pregnancies, 69 twins, and 90 higher-order multiple pregnancies were recruited. Fetal fractions estimated by size ratio-based and Y chromosome-based approaches in singleton pregnancies with male fetus were used as source data to establish the model. The model was then applied to multiple pregnancies for fetal fraction estimation. By comparing the fetal fractions estimated by size ratio to those estimated by Y chromosome or autosomal chromosomes, fetal sex and chromosomal aneuploidy can be analyzed. RESULTS: The size ratio-based approach has been well established in estimating fetal fractions for twin and higher-order multiple pregnancies. Fetal fraction had a positive correlation with gestational age in twin and triplet pregnancies. Fetal sex was determined with accuracies of 98.6% (95% CI, 92.19%-99.96%) in twins and 97.6% (95% CI, 91.76%-99.71%) in triplet pregnancies. Four trisomy 21, one trisomy 18, and one trisomy 13 cases were detected by NIPT. Two trisomy 21 singleton pregnancies and one trisomy 21 twin pregnancy were confirmed by karyotyping. CONCLUSION: Fetal sex and chromosomal aneuploidy in multiple pregnancies can be determined using NIPT.

Noninvasive prenatal testing (NIPT) in twin pregnancies with treatment of assisted reproductive techniques (ART) in a single center.

OBJECTIVE: The objective of the study is to report the performance of noninvasive prenatal testing (NIPT) in twin pregnancies after the treatment of assisted reproductive technology (ART). METHOD: In two years period, 565 pregnant women with ART twin pregnancies were prospectively tested by NIPT for screening for trisomy 21 (T21), 18 (T18), and 13 (T13) by sequencing cell-free DNA in maternal plasma. Positive NIPT results were confirmed by karyotyping, while negative results were interviewed after delivery. Pregnant decision based on NIPT and confirmation results was discussed during post-test counseling. RESULTS: In total of 565 cases, NIPT had a failure rate of 0.9% (5/565). Four cases of T21 were identified by NIPT and confirmed by karyotyping, resulting in 100% (95%CI 39.8%-100%) positive predictive value. Among 556 cases with NIPT negative results, 506 cases (91.0%) were confirmed by follow-up of postnatal phenotypes, while 33 cases (5.9%) had adverse pregnant outcomes with unconfirmed reasons because of the lack of cytogenetic samples. The remaining 17 cases (3.1%) refused follow-up. No false negative result was reported. CONCLUSION: With apparently high positive predictive value and low false positive rate, NIPT has the potential to be used as a good alternative approach of conventional prenatal screening at the first trimester in ART twin pregnancy. © 2016 John Wiley & Sons, Ltd.

[Genetic analysis and prenatal diagnosis of Xp deletion in a family with Duchenne/Becker muscular dystrophy].

OBJECTIVE To delineate a deletional mutation of the Dystrophin gene on the short arm of chromosome X in a family affected with Duchenne/Becker muscular dystrophy. METHODS G-banded karyotyping, multiple ligation probe amplification (MLPA), array-based comparative genomic hybridization(array-CGH) and whole genome exon high-throughput sequencing were employed to delineate the mutation in the family. RESULTS GTG banding has demonstrated deletion of the terminal part of the short arm of chromosome X in the fetus. The same deletion was also found in its mother and maternal grandmother. MLPA analysis has revealed removal of exons 52 to 79 of the Dystrophin gene. A 30 Mb deletion in Xp22.33-p21.1 and a 10 Mb duplication in Xq27.2-q28 were identified by array-CGH and whole genome exon high-throughput sequencing. CONCLUSION The Xp deletion has led to deletion of exons 52 to 79 of the Dystrophin gene in the family. The female carriers also had certain features of Turner syndrome due to the same deletion.

Association between variants of PRDM1 and NDP52 and Crohn's disease, based on exome sequencing and functional studies.

BACKGROUND & AIMS: Genome-wide association studies (GWAS) have identified 140 Crohn's disease (CD) susceptibility loci. For most loci, the variants that cause disease are not known and the genes affected by these variants have not been identified. We aimed to identify variants that cause CD through detailed sequencing, genetic association, expression, and functional studies. METHODS: We sequenced whole exomes of 42 unrelated subjects with CD and 5 healthy subjects (controls) and then filtered single nucleotide variants by incorporating association results from meta-analyses of CD GWAS and in silico mutation effect prediction algorithms. We then genotyped 9348 subjects with CD, 2868 subjects with ulcerative colitis, and 14,567 control subjects and associated variants analyzed in functional studies using materials from subjects and controls and in vitro model systems. RESULTS: We identified rare missense mutations in PR domain-containing 1 (PRDM1) and associated these with CD. These mutations increased proliferation of T cells and secretion of cytokines on activation and increased expression of the adhesion molecule L-selectin. A common CD risk allele, identified in GWAS, correlated with reduced expression of PRDM1 in ileal biopsy specimens and peripheral blood mononuclear cells (combined P = 1.6 × 10(-8)). We identified an association between CD and a common missense variant, Val248Ala, in nuclear domain 10 protein 52 (NDP52) (P = 4.83 × 10(-9)). We found that this variant impairs the regulatory functions of NDP52 to inhibit nuclear factor κB activation of genes that regulate inflammation and affect the stability of proteins in Toll-like receptor pathways. CONCLUSIONS: We have extended the results of GWAS and provide evidence that variants in PRDM1 and NDP52 determine susceptibility to CD. PRDM1 maps adjacent to a CD interval identified in GWAS and encodes a transcription factor expressed by T and B cells. NDP52 is an adaptor protein that functions in selective autophagy of intracellular bacteria and signaling molecules, supporting the role of autophagy in the pathogenesis of CD.

Integration of targeted sequencing and NIPT into clinical practice in a Chinese family with maple syrup urine disease.

PURPOSE: This article demonstrates a prominent noninvasive prenatal approach to assist the clinical diagnosis of a single-gene disorder disease, maple syrup urine disease, using targeted sequencing knowledge from the affected family. METHODS: The method reported here combines novel mutant discovery in known genes by targeted massively parallel sequencing with noninvasive prenatal testing. RESULTS: By applying this new strategy, we successfully revealed novel mutations in the gene BCKDHA (Ex2_4dup and c.392A>G) in this Chinese family and developed a prenatal haplotype-assisted approach to noninvasively detect the genotype of the fetus (transmitted from both parents). CONCLUSION: This is the first report of integration of targeted sequencing and noninvasive prenatal testing into clinical practice. Our study has demonstrated that this massively parallel sequencing-based strategy can potentially be used for single-gene disorder diagnosis in the future.

Noninvasive prenatal testing of trisomies 21 and 18 by massively parallel sequencing of maternal plasma DNA in twin pregnancies.

OBJECTIVE: The objective of this study is to assess the performance of noninvasive prenatal testing for trisomies 21 and 18 on the basis of massively parallel sequencing of cell-free DNA from maternal plasma in twin pregnancies. METHOD: A double-blind study was performed over 12 months. A total of 189 pregnant women carrying twins were recruited from seven hospitals. Maternal plasma DNA sequencing was performed to detect trisomies 21 and 18. The fetal karyotype was used as gold standard to estimate the sensitivity and specificity of sequencing-based noninvasive prenatal test. RESULTS: There were nine cases of trisomy 21 and two cases of trisomy 18 confirmed by karyotyping. Plasma DNA sequencing correctly identified nine cases of trisomy 21 and one case of trisomy 18. The discordant case of trisomy 18 was an unusual case of monozygotic twin with discordant fetal karyotype (one normal and the other trisomy 18). The sensitivity and specificity of maternal plasma DNA sequencing for fetal trisomy 21 were both 100% and for fetal trisomy 18 were 50% and 100%, respectively. CONCLUSION: Our study further supported that sequencing-based noninvasive prenatal testing of trisomy 21 in twin pregnancies could be achieved with a high accuracy, which could effectively avoid almost 95% of invasive prenatal diagnosis procedures.

Feasibility study of semiconductor sequencing for noninvasive prenatal detection of fetal aneuploidy.

BACKGROUND: Noninvasive prenatal detection of common fetal aneuploidies with cell-free DNA from maternal plasma has been achieved with high-throughput next-generation sequencing platforms. Turnaround times for previously tested platforms are still unsatisfactory for clinical applications, however, because of the time spent on sequencing. The development of semiconductor sequencing technology has provided a way to shorten overall run times. We studied the feasibility of using semiconductor sequencing technology for the noninvasive detection of fetal aneuploidy. METHODS: Maternal plasma DNA from 13 pregnant women, corresponding to 4 euploid, 6 trisomy 21 (T21), 2 trisomy 18 (T18), and 1 trisomy 13 (T13) pregnancies, were sequenced on the Ion Torrent Personal Genome Machine sequencer platform with 318 chips. The data were analyzed with the T statistic method after correcting for GC bias, and the T value was calculated as an indicator of fetal aneuploidy. RESULTS: We obtained a mean of 3 524 401 high-quality reads per sample, with an efficiency rate of 77.9%. All of the T21, T13, and T18 fetuses could be clearly distinguished from euploid fetuses, and the time spent on library preparation and sequencing was 24 h. CONCLUSIONS: Semiconductor sequencing represents a suitable technology for the noninvasive prenatal detection of fetal aneuploidy. With this platform, sequencing times can be substantially reduced; however, a further larger-scale study is needed to determine the imprecision of noninvasive fetal aneuploidy detection with this system.

A method for noninvasive detection of fetal large deletions/duplications by low coverage massively parallel sequencing.

OBJECTIVE: To report the feasibility of fetal chromosomal deletion/duplication detection using a novel bioinformatic method of low coverage whole genome sequencing of maternal plasma. METHOD: A practical method Fetal Copy-number Analysis through Maternal Plasma Sequencing (FCAPS), integrated with GC-bias correction, binary segmentation algorithm and dynamic threshold strategy, was developed to detect fetal chromosomal deletions/duplications of >10 Mb by low coverage whole genome sequencing (about 0.08-fold). The sensitivity/specificity of the resultant FCAPS algorithm in detecting deletions/duplications was firstly assessed in silico and then tested in 1311 maternal plasma samples from those with known G-banding karyotyping results of the fetus. RESULTS: Deletions/duplications, ranged from 9.01 to 28.46 Mb, were suspected in four of the 1311 samples, of which three were consistent with the results of fetal karyotyping. In one case, the suspected abnormality was not confirmed by karyotyping, representing a false positive case. No false negative case was observed in the remaining 1307 low-risk samples. The sensitivity and specificity for detection of >10-Mb chromosomal deletions/duplications were100% and 99.92%, respectively. CONCLUSION: Our study demonstrated FCAPS has the potential to detect fetal large deletions/duplications (>10 Mb) with low coverage maternal plasma DNA sequencing currently used for fetal aneuploidy detection.

Newborn ornithine carbamyltransferase deficiency caused by new OTC gene mutations: a report of two cases and review of the literature on phenotype and genotype.

Background: Ornithine carbamyltransferase deficiency (OTCD) is the most common urea cycle disorder disease. Neonatal-type cases usually involve a serious condition, including the complete loss of ornithine carbamyltransferase (OTC) enzyme activities. Case Description: Case 1: A 3-day-old boy was hospitalized due to decreased muscle tone over three days, 10 hours of dyspnea, and two instances of convulsions. Diagnostic tests showed elevated blood ammonia levels, decreased citrulline concentration via mass spectrometry (MS/MS), and increased orotic acid observed by gas chromatography-mass spectrometry (GC/MS), while uracil was normal. Head ultrasound revealed abnormal brain parenchyma, indicating brain damage. Whole exon gene sequencing detected a new pathogenic mutation [c. 961T > C (p.Ser321Pro)] in the OTC gene, and the mother was identified as a carrier of this mutation. Case 2: A 3-day-old boy was admitted to our hospital because of "heart failure". The patient's blood ammonia was significantly increased; the MS/MS results showed that citrulline concentration decreased; the GC/MS results demonstrated that orotic acid was significantly increased; uracil was normal; and head magnetic resonance imaging (MRI) + diffusion-weighted imaging (DWI) + magnetic resonance spectroscopy (MRS) showed abnormal signals in the brain, with partial cystic malacia. Clinical exon gene sequencing showed that the 500 bp short interspersed nuclear elements (SINE) element may have been inserted into exon 5 of the OTC gene, which was inherited from his mother; this has not been reported previously in the literature. Although the blood ammonia of two patients decreased, they both died after giving up because of serious nervous system damage. In addition, a total of 62 neonatal OTCD patients were found in the literature. The clinical manifestations of these patients were not specific. The MS/MS results of 81% of neonatal OTCD patients showed that the concentration of citrulline was decreased. About 91% showed a significant increase in orotic acid, mutation sites were detected in 100% of the patients, and the prognosis was poor. Conclusions: This study improves our understanding of the clinical characteristics and OTC gene mutation sites of neonatal OTCD cases. c.961T>C and c.ins SINE? were identified as new mutations. The clinical manifestations of neonatal OTCD patients lack specificity, and the clinical and biochemical characteristics combined with gene detection are important for the diagnosis of OTCD.

Clinical application of massively parallel sequencing-based prenatal noninvasive fetal trisomy test for trisomies 21 and 18 in 11,105 pregnancies with mixed risk factors.

OBJECTIVE: To report the performance of massively parallel sequencing (MPS) based prenatal noninvasive fetal trisomy test based on cell-free DNA sequencing from maternal plasma in a routine clinical setting in China. METHOD: The MPS-based test was offered as a prenatal screening test for trisomies 21 and 18 to pregnant women in 49 medical centers over 2 years. A total of 11,263 participants were recruited and the MPS-based test was performed in 11,105 pregnancies. Fetal outcome data were obtained after the expected date of confinement. RESULTS: One hundred ninety cases were classified as positive, including 143 cases of trisomy 21 and 47 cases of trisomy 18. With the karyotyping results and the feedback of fetal outcome data, we observed one false positive case of trisomy 21, one false positive case of trisomy 18 and no false negative cases, indicating 100% sensitivity and 99.96% specificity for the detection of trisomies 21 and 18. CONCLUSION: Our large-scale multicenter study proved that the MPS-based test is of high sensitivity and specificity in detecting fetal trisomies 21 and 18. The introduction of this screening test into a routine clinical setting could avoid about 98% of invasive prenatal diagnostic procedures.

Noninvasive prenatal testing for ß-thalassemia by targeted nanopore sequencing combined with relative haplotype dosage (RHDO): a feasibility study.

Noninvasive prenatal testing (NIPT) for single gene disorders remains challenging. One approach that allows for accurate detection of the slight increase of the maternally inherited allele is the relative haplotype dosage (RHDO) analysis, which requires the construction of parental haplotypes. Recently, the nanopore sequencing technologies have become available and may be an ideal tool for direct construction of haplotypes. Here, we explored the feasibility of combining nanopore sequencing with the RHDO analysis in NIPT of ß-thalassemia. Thirteen families at risk for ß-thalassemia were recruited. Targeted region of parental genomic DNA was amplified by long-range PCR of 10 kb and 20 kb amplicons. Parental haplotypes were constructed using nanopore sequencing and next generation sequencing data. Fetal inheritance of parental haplotypes was classified by the RHDO analysis using data from maternal plasma DNA sequencing. Haplotype phasing was achieved in 12 families using data from 10 kb library. While data from the 20 kb library gave a better performance that haplotype phasing was achieved in all 13 families. Fetal status was correctly classified in 12 out of 13 families. Thus, targeted nanopore sequencing combined with the RHDO analysis is feasible to NIPT for ß-thalassemia.

L1CAM mutations in three fetuses diagnosed by medical exome sequencing.

OBJECTIVE: The L1 cell adhesion molecule (L1CAM) gene, encodes the L1 cell adhesion molecule, is involved in the central nervous system development. Its mutations result in L1 syndrome which is associated with brain malformation and nervous developmental delay. CASE REPORT: We presented three fetuses with hydrocephalus and agenesis of the corpus callosum detected by ultrasound, followed by medical exome sequencing (MES) test with L1CAM mutations: two known missense mutation c.551G > A (p. R184Q) and c.1354G > A (p. G452R), and a novel frameshift mutation c.1322delG which causes the early termination of translation (p. G441Afs∗72). By utilizing multiple computational analysis, all the variants were scored to be likely pathogenic. CONCLUSION: Combined use of ultrasound and MES to identify the molecular etiology of fetal anomalies may contribute to expanding our knowledge of the clinical phenotype of L1 syndrome observed in the south Chinese population.

Noninvasive prenatal diagnosis of ß-thalassemia by relative haplotype dosage without analyzing proband.

BACKGROUND: ß-thalassemia is one of the most common monogenic diseases in the world. Southeast China is a highly infected area affected by four ß-thalassemia mutation types (HBB:c.-78A>G, HBB:c.52A>T, HBB:c.126_129delCTTT, and HBB:c.316-197C>T). Relative haplotype dosage (RHDO), a haplotype-based approach, has shown promise as an application for noninvasive prenatal diagnosis (NIPD); however, additional family members (such as the proband) are required for haplotype construction. The abovementioned circumstances make RHDO-based NIPD cost prohibitive; additionally, the genetic information of the proband is not always available. Thus, it is necessary to find a practical method to solve these problems. METHODS: Targeted sequencing was applied to sequence parental genomic DNA and cell-free fetal DNA (cffDNA). Parental haplotypes were constructed with the SHAPEIT software based on the 1000 Genomes Project (1000G) Phase 3 v5 Southern Han Chinese (CHS) haplotype dataset. Single-nucleotide polymorphisms (SNPs) in the target region were called and classified, and the fetal mutation inheritance status was deduced using the RHDO method. RESULTS: Construction of the parental haplotypes and detection of the inherited parental mutations were successfully achieved in five families, despite a suspected recombination event. The status of the affected fetuses is consistent with the results of traditional reverse dot blot (RDB) diagnosis. CONCLUSION: This research introduced SHAPEIT into the classical RHDO workflow and proved that it is applicable to construct parental haplotypes without information from other family members.

A reliable, high-resolution and high-throughput genotyping method for HLA-DRB1.

Human leukocyte antigen (HLA) DNA typing is an essential part to identify a donor who may be a good match for the patients who need haematopoietic stem cells from bone marrow. Thus, fetching quickly high-resolution genotype is very important at present. However, current HLA DNA typing methods especially for HLA-DRB1 generally yielded ambiguous typing results because of high degree of heterozygosity on exome region and primer pairs design limitations, which generally amplified only exon2 of HLA-DRB1 and the position of its primer pairs is on exome region. To solve this problem, we developed a reliable, high-resolution and high-throughput (RHH) sequence based typing approach for HLA-DRB1 through massively parallel paired-end sequencing. Several primer pairs were designed to amplify three exon regions, which are part of exon1, the whole region of exon2 and exon3 of HLA-DRB1, to genotyping for HLA-DRB1 by synthesis. 94 samples were included to analyze and the highly successful genotyping ratio (98.94%) and no ambiguous genotyping result demonstrated the accurate performance of our method for HLA-DRB1 genotyping.

Effects of Maternal and Fetal Characteristics on Cell-Free Fetal DNA Fraction in Maternal Plasma.

OBJECTIVE: To study factors that influence the concentration of cell-free fetal DNA (fetal fraction) using a large clinical data set of pregnancies with male fetus. METHOD: A retrospective analysis of 23 067 pregnancies that received noninvasive prenatal testing from January 2012 to October 2013, including 22 650 normal singleton pregnancies (control group) and 417 pregnancies with aneuploidy, twin pregnancy, or various maternal conditions including preexisting hypertension, preexisting diabetes, hyperthyroidism, and carrier of the surface antigen of the hepatitis B virus (HBsAg; study group). Multiples of the median (MoM) analysis was performed in the control group to derive gestation and body mass index (BMI)-corrected fetal fraction. The effects of study group conditions on fetal fraction were examined by calculating the ratio of MoM (RMoM) values. RESULTS: Fetal fraction showed a positive correlation with gestational age (r(2) = .10, P < .001) and increased rapidly after the 21 weeks of gestation (r(2) = .26, P < .001). Negative association with maternal BMI was found with fetal fraction (r(2) = .04, P < .001). In study group, fetal fraction was higher among pregnant women with a trisomy 21 fetus (RMoM = 1.24, P < .001) and lower among trisomy 18 (RMoM = 0.84, P < .001). A 1.6-fold incensement of fetal fraction was observed in twin fetuses comparing to singleton pregnancy (RMoM = 1.62, P < .001). Women with preexisting hypertension had significantly lower fetal fraction (RMoM = 0.85, P = .02). Preexisting diabetes, hyperthyroidism, or carrier of HBsAg did not affect fetal fraction. CONCLUSION: The fetal fraction was affected by fetal aneuploidy, maternal BMI, and the number of gestation. Maternal preexisting of hypertension appeared to reduce fetal fraction.

Genetic effects of a 13q31.1 microdeletion detected by noninvasive prenatal testing (NIPT).

Microdeletions of chromosome 13q31.1 are relatively rare. These types of deletions may cause different genetic effects on genotypes and/or phenotypes. There are several ways to detect microdeletions; noninvasive prenatal testing (NIPT) is the newest detection method. In this study, we aimed to investigate the genetic effects of a 13q31.1 microdeletion detected by NIPT and to reconfirm the feasibility of this procedure in predicting sub-chromosomal copy number variations (CNVs). The 13q31.1 microdeletion, which has previously been described as a disease-associated fragment, was detected by NIPT in a pregnant woman. To validate the finding and to explain the origin of this sub-chromosomal CNV, we collected fetal amniotic fluid and parental blood samples and tested the samples using array-based comparative genomic hybridization (aCGH). Karyotype analysis was performed on all of the samples to rule out balanced or mosaic anomalies. The aCGH results confirmed the NIPT findings. We detected the same type of microdeletion in the fetus and the mother via aCGH. The mother had a normal phenotype; therefore, in a post-test genetic counseling session, we predicted a normal phenotype for the fetus. After delivery, the normal phenotype of the newborn confirmed our prediction. Based on the present study, this 13q31.1 microdeletion may be considered as a chromosomal polymorphism. This study also reconfirmed the feasibility of obtaining a molecular karyotype of a fetus via NIPT.

A prenatal case with discrepant findings between non-invasive prenatal testing and fetal genetic testings.

At 17(+4) week, non-invasive prenatal testing (NIPT) results of a 24-years-old mother showed high risk of monosomy X (45, X). Abnormally shaped head and cardiac defects were observed in prenatal ultrasound scan at 19(+3) week. Amniocentesis conducted at 19(+3) week identified karyotype 47, XX, +18, which suggested that the NIPT failed to detect trisomy 18 (T18) in this case. With a further massively parallel sequencing (MPS) of maternal blood, fetal and placental tissues, we found a confined placental mosaicism (CPM) with non-mosaic T18 fetus and multiclonal placenta with high prevalence of 45, X and low level of T18 cells. FISH and SNP-array evidence from the placental tissue confirmed genetic discrepancy between the fetus and placenta. Because the primary source of the fetal cell-free DNA that NIPT assesses is mostly originated from trophoblast cells, the level of T18 placental mosaicism may cause false negative NIPT result in this rare case of double aneuploidy.

Non-invasive fetal sex determination by maternal plasma sequencing and application in X-linked disorder counseling.

OBJECTIVE: To develop a fetal sex determination method based on maternal plasma sequencing (MPS), assess its performance and potential use in X-linked disorder counseling. METHODS: 900 cases of MPS data from a previous study were reviewed, in which 100 and 800 cases were used as training and validation set, respectively. The percentage of uniquely mapped sequencing reads on Y chromosome was calculated and used to classify male and female cases. Eight pregnant women who are carriers of Duchenne muscular dystrophy (DMD) mutations were recruited, whose plasma were subjected to multiplex sequencing and fetal sex determination analysis. RESULTS: In the training set, a sensitivity of 96% and false positive rate of 0% for male cases detection were reached in our method. The blinded validation results showed 421 in 423 male cases and 374 in 377 female cases were successfully identified, revealing sensitivity and specificity of 99.53% and 99.20% for fetal sex determination, at as early as 12 gestational weeks. Fetal sex for all eight DMD genetic counseling cases were correctly identified, which were confirmed by amniocentesis. CONCLUSIONS: Based on MPS, high accuracy of non-invasive fetal sex determination can be achieved. This method can potentially be used for prenatal genetic counseling.

Non-invasive prenatal screening of fetal Down syndrome by maternal plasma DNA sequencing in twin pregnancies.

Non-invasive prenatal screening for fetal Down syndrome (NIFTY) by maternal plasma sequencing was performed in 12 subjects with twin pregnancies, including 11 with normal fetuses and 1 with discordant fetal Trisomy 21. For every sample, it was processed, sequenced and reported as soon as it was collected as other clinical samples for singleton pregnancies. The NIFTY test was negative in the 11 pregnancies carried normal fetuses, and was positive (high risk) in the case with discordant fetal Trisomy 21. The sensitivity and specificity were both 100%. This small case series suggested the NIFTY as a screening test for fetal Trisomy 21 is feasible in twin pregnancies.