Variability in Severe Acute Respiratory Syndrome Coronavirus 2 IgG Antibody Affinity to Omicron and Delta Variants in Convalescent and Community mRNA-Vaccinated Individuals.

The emergence of the omicron and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has begun a number of discussions regarding breakthrough infection, waning immunity, need and timing for vaccine boosters, and whether existing mRNA vaccines for the original SARS-CoV-2 strain are adequate. Our work leverages a biosensor-based technique to evaluate the binding efficacy of SARS-CoV-2 S1-specific salivary Abs to the omicron and delta variants using a cohort of mRNA-vaccinated (n = 109) and convalescent (n = 19) subjects. We discovered a wide range of binding efficacies to the variant strains, with a mean reduction of 60.5, 26.7, and 14.7% in measurable signal to the omicron strain and 13.4, 2.4, and -6.4% mean reduction to the delta variant for convalescent, Pfizer-, and Moderna-vaccinated groups, respectively. This assay may be an important tool in determining susceptibility to infection or need for booster immunization as the pandemic evolves.

Variability in SARS-Cov-2 IgG Antibody Affinity To Omicron and Delta Variants in Convalescent and Community mRNA Vaccinated Individuals.

The emergence of Omicron and Delta variants of SARS-CoV-2 has begun a number of discussions regarding breakthrough infection, waning immunity, need and timing for vaccine boosters and whether existing mRNA vaccines for the wildtype strain are adequate. Our work leverages a biosensor-based technique to evaluate the binding efficacy of SARS-CoV-2 S1 specific salivary antibodies to the Omicron and Delta variants using a cohort of mRNA vaccinated (n=109) and convalescent (n=19) subjects. We discovered a wide range of binding efficacies to the variant strains, with a mean reduction of 60.5%, 26.7%, and 14.7% in measurable signal to the Omicron strain and 13.4%, 2.4%, and âˆ'6.4% percent mean reduction to the Delta Variant for convalescent, Pfizer, and Moderna vaccinated groups respectively. This assay may be an important tool in determining susceptibility to infection or need for booster immunization as the pandemic evolves. Key Points: AMPERIAL assay developed to quantify salivary SARS-CoV-2 S1 IgG antibodies to Omicron and Delta variantsThere was a reduction in affinity to both Delta and Omicron VariantsThe reduction in affinity was more pronounced to Omicron than for Delta VariantsThere was a significant difference between IgG affinities in Individuals vaccinated with Pfizer versus Moderna Vaccines.

The Kinetics of COVID-19 Vaccine Response in a Community-Vaccinated Population.

We used a noninvasive electrochemical quantitative assay for IgG Abs to SARS-CoV-2 S1 Ag in saliva to investigate the kinetics of Ab response in a community-based population that had received either the Pfizer or Moderna mRNA-based vaccine. Samples were received from a total of 97 individuals, including a subset of 42 individuals who collected samples twice weekly for 3 mo or longer. In all, >840 samples were collected and analyzed. In all individuals, salivary SARS-CoV-2 S1 IgG Ab levels rose sharply in the 2-wk period after their second vaccination, with peak Ab levels seen at 10-20 d after vaccination. We observed that 20%, 10%, and 2.4% of individuals providing serial samples had a 90%, 95%, and 99% drop, respectively, from peak levels during the duration of monitoring, and in two patients, Abs fell to prevaccination levels (5%). The use of noninvasive quantitative salivary Ab measurement can allow widespread, cost-effective monitoring of vaccine response.

Development and validation of a quantitative, non-invasive, highly sensitive and specific, electrochemical assay for anti-SARS-CoV-2 IgG antibodies in saliva.

Amperial™ is a novel assay platform that uses immobilized antigen in a conducting polymer gel followed by detection via electrochemical measurement of oxidation-reduction reaction between H2O2/Tetrametylbenzidine and peroxidase enzyme in a completed assay complex. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARS-CoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination.

Development and validation of a highly sensitive and specific electrochemical assay to quantify anti-SARS-CoV-2 IgG antibodies to facilitate pandemic surveillance and monitoring of vaccine response.

Amperial™ is a novel assay platform that uses immobilized antigen in a conductive polymer gel followed by an electrochemical detection. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARSCoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination.

Electric Field-Induced Release and Measurement (EFIRM): Characterization and Technical Validation of a Novel Liquid Biopsy Platform in Plasma and Saliva.

Electric field-induced release and measurement (EFIRM) is a novel, plate-based, liquid biopsy platform capable of detecting circulating tumor DNA containing EGFR mutations directly from saliva and plasma in both early- and late-stage patients with non-small-cell lung cancer. We investigated the properties of the target molecule for EFIRM and determined that the platform preferentially detects single-stranded DNA molecules. We then investigated the properties of the EFIRM assay and determined the linearity, linear range, precision, and limit of detection for six different EGFR variants (the four most common g.Exon19del variants), p.T790M, and p.L858R). The limit of detection was in single-digit copy number for the latter two mutations, and the limit of detection for Exon19del was 5000 copies. Following these investigations, technical validations were performed for four separate EFIRM liquid biopsy assays, qualitative and quantitative assays for both saliva and plasma. We conclude that EFIRM liquid biopsy is an assay platform that interrogates a biomarker not targeted by any other extant platform (namely, circulating single-stranded DNA molecules). The assay has acceptable performance characteristics in both quantitative and qualitative assays on both saliva and plasma.

Electric Field-Induced Release and Measurement Liquid Biopsy for Noninvasive Early Lung Cancer Assessment.

Previously, we detected circulating tumor DNA that contained two EGFR mutations (p.L858R and exon19 del) in plasma of patients with late-stage non-small-cell lung carcinoma (NSCLC) using the electric field-induced release and measurement (EFIRM) platform. Our aim was to determine whether EFIRM technology can detect these mutations in patients with early-stage NSCLC. Prospectively, 248 patients with radiographically determined pulmonary nodules were recruited. Plasma was collected before biopsy and histologic examination of the nodule. Inclusion criteria were histologic diagnosis of benign nodule (control) and stage I or II adenocarcinoma harboring either p.L858R or exon19 delEGFR mutations. Plasma samples were available from 44 patients: 23 with biopsy-proven benign pulmonary nodules and 21 with stage I or II adenocarcinoma (12 p.L858R and 9 exon19 delEGFR variants). Samples were analyzed for the EGFR mutations using the EFIRM platform. Assay sensitivity was 92% for p.L858R (11 of 12 samples positive) and 77% for exon19 del (7 of 9 samples positive). Specificity was 91% with two false-positive results in 23 patients with EGFR-positive nodules and 95% for the entire 44-patient series. Concordance was 100% with identical mutations discovered in plasma and nodule biopsy. The EFIRM platform is able to noninvasively detect two EGFR mutations in individuals with early-stage NSCLC.

A Multigene Test Could Cost-Effectively Help Extend Life Expectancy for Women at Risk of Hereditary Breast Cancer.

BACKGROUND: The National Comprehensive Cancer Network recommends that women who carry gene variants that confer substantial risk for breast cancer consider risk-reduction strategies, that is, enhanced surveillance (breast magnetic resonance imaging and mammography) or prophylactic surgery. Pathogenic variants can be detected in women with a family history of breast or ovarian cancer syndromes by multigene panel testing. OBJECTIVES: To investigate whether using a seven-gene test to identify women who should consider risk-reduction strategies could cost-effectively increase life expectancy. METHODS: We estimated effectiveness and lifetime costs from a payer perspective for two strategies in two hypothetical cohorts of women (40-year-old and 50-year-old cohorts) who meet the National Comprehensive Cancer Network-defined family history criteria for multigene testing. The two strategies were the usual test strategy for variants in BRCA1 and BRCA2 and the seven-gene test strategy for variants in BRCA1, BRCA2, TP53, PTEN, CDH1, STK11, and PALB2. Women found to have a pathogenic variant were assumed to undergo either prophylactic surgery or enhanced surveillance. RESULTS: The incremental cost-effectiveness ratio for the seven-gene test strategy compared with the BRCA1/2 test strategy was $42,067 per life-year gained or $69,920 per quality-adjusted life-year gained for the 50-year-old cohort and $23,734 per life-year gained or $48,328 per quality-adjusted life-year gained for the 40-year-old cohort. In probabilistic sensitivity analysis, the seven-gene test strategy cost less than $100,000 per life-year gained in 95.7% of the trials for the 50-year-old cohort. CONCLUSIONS: Testing seven breast cancer-associated genes, followed by risk-reduction management, could cost-effectively improve life expectancy for women at risk of hereditary breast cancer.

Improving the Positive Predictive Value of Non-Invasive Prenatal Screening (NIPS).

We evaluated performance characteristics of a laboratory-developed, non-invasive prenatal screening (NIPS) assay for fetal aneuploidies. This assay employs massively parallel shotgun sequencing with full automation. GC sequencing bias correction and statistical smoothing were performed to enhance discrimination of affected and unaffected pregnancies. Maternal plasma samples from pregnancies with known aneuploidy status were used for assay development, verification, and validation. Assay verification studies using 2,085 known samples (1873 unaffected, 69 trisomy 21, 20 trisomy 18, 17 trisomy 13) demonstrated complete discrimination between autosomal trisomy (Z scores >8) and unaffected (Z scores <4) singleton pregnancies. A validation study using 552 known samples (21 trisomy 21, 10 trisomy 18, 1 trisomy 13) confirmed complete discrimination. Twin pregnancies showed similar results. Follow-up of abnormal results from the first 10,000 clinical samples demonstrated PPVs of 98% (41/42) for trisomy 21, 92% (23/25) for trisomy 18, and 69% (9/13) for trisomy 13. Adjustment for causes of false-positive results identified during clinical testing (eg, maternal duplications) improved PPVs to 100% for trisomy 21 and 96% for trisomy 18. This NIPS test demonstrates excellent discrimination between trisomic and unaffected pregnancies. The PPVs obtained in initial clinical testing are substantially higher than previously reported NIPS methods.

BRCA Share: A Collection of Clinical BRCA Gene Variants.

As next-generation sequencing increases access to human genetic variation, the challenge of determining clinical significance of variants becomes ever more acute. Germline variants in the BRCA1 and BRCA2 genes can confer substantial lifetime risk of breast and ovarian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share™, a database created by a unique international alliance of academic centers and commercial testing laboratories. By integrating the content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of two large commercial laboratories, Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA Share™ has assembled one of the largest publicly accessible collections of BRCA variants currently available. Although access is available to academic researchers without charge, commercial participants in the project are required to pay a support fee and contribute their data. The fees fund the ongoing curation effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share™ databases can therefore be considered as models of successful data sharing between private companies and the academic world.

Frequency and Complexity of De Novo Structural Mutation in Autism.

Genetic studies of autism spectrum disorder (ASD) have established that de novo duplications and deletions contribute to risk. However, ascertainment of structural variants (SVs) has been restricted by the coarse resolution of current approaches. By applying a custom pipeline for SV discovery, genotyping, and de novo assembly to genome sequencing of 235 subjects (71 affected individuals, 26 healthy siblings, and their parents), we compiled an atlas of 29,719 SV loci (5,213/genome), comprising 11 different classes. We found a high diversity of de novo mutations, the majority of which were undetectable by previous methods. In addition, we observed complex mutation clusters where combinations of de novo SVs, nucleotide substitutions, and indels occurred as a single event. We estimate a high rate of structural mutation in humans (20%) and propose that genetic risk for ASD is attributable to an elevated frequency of gene-disrupting de novo SVs, but not an elevated rate of genome rearrangement.

A Standardized DNA Variant Scoring System for Pathogenicity Assessments in Mendelian Disorders.

We developed a rules-based scoring system to classify DNA variants into five categories including pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign. Over 16,500 pathogenicity assessments on 11,894 variants from 338 genes were analyzed for pathogenicity based on prediction tools, population frequency, co-occurrence, segregation, and functional studies collected from internal and external sources. Scores were calculated by trained scientists using a quantitative framework that assigned differential weighting to these five types of data. We performed descriptive and comparative statistics on the dataset and tested interobserver concordance among the trained scientists. Private variants defined as variants found within single families (n = 5,182), were either VUS (80.5%; n = 4,169) or likely pathogenic (19.5%; n = 1,013). The remaining variants (n = 6,712) were VUS (38.4%; n = 2,577) or likely benign/benign (34.7%; n = 2,327) or likely pathogenic/pathogenic (26.9%, n = 1,808). Exact agreement between the trained scientists on the final variant score was 98.5% [95% confidence interval (CI) (98.0, 98.9)] with an interobserver consistency of 97% [95% CI (91.5, 99.4)]. Variant scores were stable and showed increasing odds of being in agreement with new data when re-evaluated periodically. This carefully curated, standardized variant pathogenicity scoring system provides reliable pathogenicity scores for DNA variants encountered in a clinical laboratory setting.

Development and Validation of a Next-Generation Sequencing Assay for BRCA1 and BRCA2 Variants for the Clinical Laboratory.

The objective of this study was to design and validate a next-generation sequencing assay (NGS) to detect BRCA1 and BRCA2 mutations. We developed an assay using random shearing of genomic DNA followed by RNA bait tile hybridization and NGS sequencing on both the Illumina MiSeq and Ion Personal Gene Machine (PGM). We determined that the MiSeq Reporter software supplied with the instrument could not detect deletions greater than 9 base pairs. Therefore, we developed an alternative alignment and variant calling software, Quest Sequencing Analysis Pipeline (QSAP), that was capable of detecting large deletions and insertions. In validation studies, we used DNA from 27 stem cell lines, all with known deleterious BRCA1 or BRCA2 mutations, and DNA from 67 consented control individuals who had a total of 352 benign variants. Both the MiSeq/QSAP combination and PGM/Torrent Suite combination had 100% sensitivity for the 379 known variants in the validation series. However, the PGM/Torrent Suite combination had a lower intra- and inter-assay precision of 96.2% and 96.7%, respectively when compared to the MiSeq/QSAP combination of 100% and 99.4%, respectively. All PGM/Torrent Suite inconsistencies were false-positive variant assignments. We began commercial testing using both platforms and in the first 521 clinical samples MiSeq/QSAP had 100% sensitivity for BRCA1/2 variants, including a 64-bp deletion and a 10-bp insertion not identified by PGM/Torrent Suite, which also suffered from a high false-positive rate. Neither the MiSeq nor PGM platform with their supplied alignment and variant calling software are appropriate for a clinical laboratory BRCA sequencing test. We have developed an NGS BRCA1/2 sequencing assay, MiSeq/QSAP, with 100% analytic sensitivity and specificity in the validation set consisting of 379 variants. The MiSeq/QSAP combination has sufficient performance for use in a clinical laboratory.

Discordant noninvasive prenatal testing and cytogenetic results: a study of 109 consecutive cases.

PURPOSE: Recent published studies have demonstrated the incremental value of the use of cell-free DNA for noninvasive prenatal testing with 100% sensitivity for trisomies 21 and 18 and a specificity of ≥99.7% for both. Data presented by two independent groups suggesting positive results by noninvasive prenatal testing were not confirmed by cytogenetic studies. METHODS: Concordance of results among cases with noninvasive prenatal testing referred for cytogenetic prenatal and/or postnatal studies by karyotyping, fluorescence in situ hybridization, and/or oligo-single-nucleotide polymorphism microarray was evaluated for 109 consecutive specimens. RESULTS: Cytogenetic results were positive for trisomy 21 in 38 of the 41 noninvasive prenatal testing-positive cases (true-positive rate: 93%) and for trisomy 18 in 16 of the 25 noninvasive prenatal testing-positive cases (true-positive rate: 64%). The true-positive rate was only 44% (7/16 cases) for trisomy 13 and 38% (6/16 cases) for sex chromosome aneuploidy. CONCLUSION: These findings raise concerns about the limitations of noninvasive prenatal testing and the need for analysis of a larger number of false-positive cases to provide true positive predictive values for noninvasive testing and to search for potential biological or technical causes. Our data suggest the need for a careful interpretation of noninvasive prenatal testing results and cautious transmission of the same to providers and patients.

Regions of homozygosity identified by oligonucleotide SNP arrays: evaluating the incidence and clinical utility.

Copy neutral segments with allelic homozygosity, also known as regions of homozygosity (ROHs), are frequently identified in cases interrogated by oligonucleotide single-nucleotide polymorphism (oligo-SNP) microarrays. Presence of ROHs may be because of parental relatedness, chromosomal recombination or rearrangements and provides important clues regarding ancestral homozygosity, consanguinity or uniparental disomy. In this study of 14 574 consecutive cases, 832 (6%) were found to harbor one or more ROHs over 10 Mb, of which 651 cases (78%) had multiple ROHs, likely because of identity by descent (IBD), and 181 cases (22%) with ROHs involving a single chromosome. Parental relatedness was predicted to be first degree or closer in 5%, second in 9% and third in 19%. Of the 181 cases, 19 had ROHs for a whole chromosome revealing uniparental isodisomy (isoUPD). In all, 25 cases had significant ROHs involving a single chromosome; 5 cases were molecularly confirmed to have a mixed iso- and heteroUPD15 and 1 case each with segmental UPD9pat and segmental UPD22mat; 17 cases were suspected to have a mixed iso- and heteroUPD including 2 cases with small supernumerary marker and 2 cases with mosaic trisomy. For chromosome 15, 12 (92%) of 13 molecularly studied cases had either Prader-Willi or Angelman syndrome. Autosomal recessive disorders were confirmed in seven of nine cases from eight families because of the finding of suspected gene within a ROH. This study demonstrates that ROHs are much more frequent than previously recognized and often reflect parental relatedness, ascertain autosomal recessive diseases or unravel UPD in many cases.

Characterization of a complex chromosomal rearrangement using chromosome, FISH, and microarray assays in a girl with multiple congenital abnormalities and developmental delay.

Complex chromosomal rearrangements (CCRs) are balanced or unbalanced structural rearrangements involving three or more cytogenetic breakpoints on two or more chromosomal pairs. The phenotypic anomalies in such cases are attributed to gene disruption, superimposed cryptic imbalances in the genome, and/or position effects. We report a 14-year-old girl who presented with multiple congenital anomalies and developmental delay. Chromosome and FISH analysis indicated a highly complex chromosomal rearrangement involving three chromosomes (3, 7 and 12), seven breakpoints as a result of one inversion, two insertions, and two translocations forming three derivative chromosomes. Additionally, chromosomal microarray study (CMA) revealed two submicroscopic deletions at 3p12.3 (467 kb) and 12q13.12 (442 kb). We postulate that microdeletion within the ROBO1 gene at 3p12.3 may have played a role in the patient's developmental delay, since it has potential activity-dependent role in neurons. Additionally, factors other than genomic deletions such as loss of function or position effects may also contribute to the abnormal phenotype in our patient.

Submicroscopic deletion of 5q involving tumor suppressor genes (CTNNA1, HSPA9) and copy neutral loss of heterozygosity associated with TET2 and EZH2 mutations in a case of MDS with normal chromosome and FISH results.

Advances in genome-wide molecular cytogenetics allow identification of novel submicroscopic DNA copy number alterations (aCNAs) and copy-neutral loss of heterozygosity (cnLOH) resulting in homozygosity for known gene mutations in myeloid neoplasms. We describe the use of an oligo-SNP array for genomic profiling of aCNA and cnLOH, together with sequence analysis of recurrently mutated genes, in a patient with myelodysplastic syndrome (MDS) presenting with normal karyotype and FISH results. Oligo-SNP array analysis revealed a hemizygous deletion of 896 kb at chromosome 5q31.2, representing the smallest 5q deletion reported to date. The deletion involved multiple genes, including two tumor suppressor candidate genes (CTNNA1 and HSPA9) that are associated with MDS/AML. The SNP-array study also detected 3 segments of somatic cnLOH: one involved the entire long arm of chromosome 4; the second involved the distal half of the long arm of chromosome 7, and the third encompassed the entire chromosome 22 (UPD 22). Sequence analysis revealed mutations in TET2 (4q), EZH2 (7q), ASXL1 (20q11.21), and RUNX1 (21q22.3). Coincidently, TET2 and EZH2 were located at segments of cnLOH resulting in their homozygosity. Loss of heterozygosity affecting these two chromosomes and mutations in TET2 and EZH2 are indicative of a myelodysplastic syndrome with a poor prognosis. Deletion of the tumor suppressor genes CTNNA1 and HSPA9 is also likely to contribute to a poor prognosis. Furthermore, the original cnLOHs in multiple chromosomes and additional cnLOH 14q in the follow-up study suggest genetic evolution of the disease and poor prognosis. This study attests to the fact that some patients with a myelodysplastic syndrome who exhibit a normal karyotype may have underlying genetic abnormalities detectable by chromosomal microarray and/or targeted mutation analyses.

Clinical experience and follow-up with large scale single-nucleotide polymorphism-based noninvasive prenatal aneuploidy testing.

OBJECTIVE: We sought to report on laboratory and clinical experience following 6 months of clinical implementation of a single-nucleotide polymorphism-based noninvasive prenatal aneuploidy test in high- and low-risk women. STUDY DESIGN: All samples received from March through September 2013 and drawn ≥9 weeks' gestation were included. Samples that passed quality control were analyzed for trisomy 21, trisomy 18, trisomy 13, and monosomy X. Results were reported as high or low risk for fetal aneuploidy for each interrogated chromosome. Relationships between fetal fraction and gestational age and maternal weight were analyzed. Follow-up on outcome was sought for a subset of high-risk cases. False-negative results were reported voluntarily by providers. Positive predictive value (PPV) was calculated from cases with an available prenatal or postnatal karyotype or clinical evaluation at birth. RESULTS: Samples were received from 31,030 patients, 30,705 met study criteria, and 28,739 passed quality-control metrics and received a report detailing aneuploidy risk. Fetal fraction correlated positively with gestational age, and negatively with maternal weight. In all, 507 patients received a high-risk result for any of the 4 tested conditions (324 trisomy 21, 82 trisomy 18, 41 trisomy 13, 61 monosomy X; including 1 double aneuploidy case). Within the 17,885 cases included in follow-up analysis, 356 were high risk, and outcome information revealed 184 (51.7%) true positives, 38 (10.7%) false positives, 19 (5.3%) with ultrasound findings suggestive of aneuploidy, 36 (10.1%) spontaneous abortions without karyotype confirmation, 22 (6.2%) terminations without karyotype confirmation, and 57 (16.0%) lost to follow-up. This yielded an 82.9% PPV for all aneuploidies, and a 90.9% PPV for trisomy 21. The overall PPV for women aged ≥35 years was similar to the PPV for women aged <35 years. Two patients were reported as false negatives. CONCLUSION: The data from this large-scale report on clinical application of a commercially available noninvasive prenatal test suggest that the clinical performance of this single-nucleotide polymorphism-based noninvasive prenatal test in a mixed high- and low-risk population is consistent with performance in validation studies.