Impact of mosaicism ratio on positive predictive value of cfDNA screening.

OBJECTIVE: To examine the relationship between the fraction of cell-free DNA (cfDNA) affected by aneuploidy compared to the overall fetal fraction of a prenatal screening specimen and its effect on positive predictive value (PPV). METHOD: CfDNA specimens positive for trisomy 13, 18, and 21 with diagnostic outcomes were analysed over a 22-month period in one clinical laboratory. For each positive specimen, a "mosaicism ratio" (MR) was calculated by dividing the fraction of cfDNA affected by aneuploidy by the overall fetal fraction of the specimen. PPVs were calculated and analyzed based on various MR ranges. RESULTS: Trisomy 13 was the aneuploidy most commonly seen in mosaic form, followed by trisomy 18 and trisomy 21. Significant differences in positive predictive values were noted for all three trisomies between samples with an MR in the "mosaic" versus "non-mosaic" range, as well as between results classified as "low-mosaic" versus "high-mosaic." CONCLUSION: PPVs may be influenced, in part, by the mosaicism ratio associated with a particular result. The data generated from this study may be useful in providing more personalized risk assessments for patients with positive cfDNA screening results.

Deletion rescue resulting in segmental homozygosity: A mechanism underlying discordant NIPT results.

With the increasing capabilities of non-invasive prenatal testing (NIPT), detection of sub-chromosomal deletions and duplications are possible. This case series of deletion rescues resulting in segmental homozygosity helps provide a biological explanation for NIPT discrepancies and adds to the dearth of existing literature surrounding segmental UPD cases and their underlying mechanisms. In the three cases presented here, NIPT reported a sub-chromosomal deletion (in isolation or as part of a complex finding). Diagnostic testing, however, revealed segmental homozygosity or UPD for the region reported deleted on NIPT. Postnatal placental testing was pursued in two cases and confirmed the NIPT findings. This discordance between the screening and diagnostic testing is suggestive of a corrective post-zygotic event, such as telomere capture and/or deletion rescue, ultimately resulting in segmental homozygosity and fetoplacental mosaicism. Imprinted chromosomes and autosomal recessive disease genes make homozygosity an important clinical consideration. Amniocentesis with SNP microarray is particularly useful in determining both copy number and UPD issues alike.

Cell-free DNA screening in twin pregnancies: A more accurate and reliable screening tool.

OBJECTIVE: Outcome data from cell-free DNA (cfDNA) screening in twin gestations are limited. This study adds an appreciable number of confirmed outcomes to the literature, and assesses performance of cfDNA screening in twins over a 4.5-year period at one large clinical laboratory. METHOD: Prenatal cytogenetic and SNP microarray results were cross-referenced with cfDNA results for twin pregnancies, yielding 422 matched cases. Using diagnostic results as truth, performance of cfDNA screening in this population was assessed. RESULTS: Of the 422 twin pregnancies with both cfDNA and diagnostic results, 3 specimens failed amniocyte analysis, and 48 samples (11.5%) were nonreportable from the initial cfDNA draw. Analysis of the 371 reportable samples demonstrated a collective sensitivity of 98.7% and specificity of 93.2% for trisomies 21/18/13. Positive predictive values (PPVs) in this study population, which is enriched for aneuploidy, were 78.7%, 84.6%, and 66.7% for trisomy 21, 18, and 13, respectively. CONCLUSION: CfDNA screening in a cohort of twin pregnancies with matched diagnostic results showed superior performance compared to traditional serum biochemical screening in twins. This study adds to a growing body of evidence suggesting that cfDNA is an accurate and reliable screening tool for the major trisomies in twin pregnancies.

Incidental Detection of Maternal Neoplasia in Noninvasive Prenatal Testing.

BACKGROUND: Noninvasive prenatal testing (NIPT) uses cell-free DNA (cfDNA) as an analyte to detect copy-number alterations in the fetal genome. Because maternal and fetal cfDNA contributions are comingled, changes in the maternal genome can manifest as abnormal NIPT results. Circulating tumor DNA (ctDNA) present in cases of maternal neoplasia has the potential to distort the NIPT readout to a degree that prevents interpretation, resulting in a nonreportable test result for fetal aneuploidy. METHODS: NIPT cases that showed a distortion from normal euploid genomic representation were communicated to the caregiving physician as nonreportable for fetal aneuploidy. Follow-up information was subsequently collected for these cases. More than 450000 pregnant patients who submitted samples for clinical laboratory testing >3 years are summarized. Additionally, in-depth analysis was performed for >79000 research-consented samples. RESULTS: In total, 55 nonreportable NIPT cases with altered genomic profiles were cataloged. Of these, 43 had additional information available to enable follow-up. A maternal neoplasm was confirmed in 40 of these cases: 18 malignant, 20 benign uterine fibroids, and 2 with radiological confirmation but without pathological classification. CONCLUSIONS: In a population of pregnant women who submitted a blood sample for cfDNA testing, an abnormal genomic profile not consistent with fetal abnormalities was detected in about 10 out of 100000 cases. A subset of these observations (18 of 43; 41.9%) was attributed to maternal malignant neoplasms. These observational results suggest the need for a controlled trial to evaluate the potential of using cfDNA as an early biomarker of cancer.

Genome-wide cfDNA screening: clinical laboratory experience with the first 10,000 cases.

PurposeInvasive diagnostic prenatal testing can provide the most comprehensive information about the genetic status of a fetus. Noninvasive prenatal screening methods, especially when using cell-free DNA (cfDNA), are often limited to reporting only on trisomies 21, 18, and 13 and sex chromosome aneuploidies. This can leave a significant number of chromosomal and subchromosomal copy-number variations undetected. In 2015, we launched a new genome-wide cfDNA screening test that has the potential to narrow this detection gap.MethodsHere, we review the results from the first 10,000 cases submitted to the Sequenom clinical laboratory for genome-wide cfDNA screening.ResultsThe high-risk indication for this cohort differed compared with standard cfDNA screening. More samples were submitted with ultrasound indications (25% compared with 13% for standard cfDNA screening) and fewer for advanced maternal age (51% for genome-wide screening versus 68% for standard cfDNA screening). A total of 554 positive calls were made, of which 164 were detectable only via genome-wide analysis.ConclusionThis reports indicates a difference in utilization compared with standard cfDNA screening, where positivity rates are higher and a large subset of positive calls could not have been made using standard cfDNA screening.

Detection of fetal subchromosomal abnormalities by sequencing circulating cell-free DNA from maternal plasma.

BACKGROUND: The development of sequencing-based noninvasive prenatal testing (NIPT) has been largely focused on whole-chromosome aneuploidies (chromosomes 13, 18, 21, X, and Y). Collectively, they account for only 30% of all live births with a chromosome abnormality. Various structural chromosome changes, such as microdeletion/microduplication (MD) syndromes are more common but more challenging to detect. Recently, several publications have shown results on noninvasive detection of MDs by deep sequencing. These approaches demonstrated the proof of concept but are not economically feasible for large-scale clinical applications. METHODS: We present a novel approach that uses low-coverage whole genome sequencing (approximately 0.2×) to detect MDs genome wide without requiring prior knowledge of the event's location. We developed a normalization method to reduce sequencing noise. We then applied a statistical method to search for consistently increased or decreased regions. A decision tree was used to differentiate whole-chromosome events from MDs. RESULTS: We demonstrated via a simulation study that the sensitivity difference between our method and the theoretical limit was <5% for MDs ≥9 Mb. We tested the performance in a blinded study in which the MDs ranged from 3 to 40 Mb. In this study, our algorithm correctly identified 17 of 18 cases with MDs and 156 of 157 unaffected cases. CONCLUSIONS: The limit of detection for any given MD syndrome is constrained by 4 factors: fetal fraction, MD size, coverage, and biological and technical variability of the event region. Our algorithm takes these factors into account and achieved 94.4% sensitivity and 99.4% specificity.

Factors affecting levels of circulating cell-free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing.

OBJECTIVE: Sufficient fetal DNA in a maternal plasma sample is required for accurate aneuploidy detection via noninvasive prenatal testing, thus highlighting a need to understand the factors affecting fetal fraction. METHOD: The MaterniT21™ PLUS test uses massively parallel sequencing to analyze cell-free fetal DNA in maternal plasma and detect chromosomal abnormalities. We assess the impact of a variety of factors, both maternal and fetal, on the fetal fraction across a large number of samples processed by Sequenom Laboratories. RESULTS: The rate of increase in fetal fraction with increasing gestational age varies across the duration of the testing period and is also influenced by fetal aneuploidy status. Maternal weight trends inversely with fetal fraction, and we find no added benefit from analyzing body mass index or blood volume instead of weight. Strong correlations exist between fetal fractions from aliquots taken from the same patient at the same blood draw and also at different blood draws. CONCLUSION: While a number of factors trend with fetal fraction across the cohort as a whole, they are not the sole determinants of fetal fraction. In this study, the variability for any one patient does not appear large enough to justify postponing testing to a later gestational age.

Noninvasive prenatal detection of sex chromosomal aneuploidies by sequencing circulating cell-free DNA from maternal plasma.

OBJECTIVE: Whole-genome sequencing of circulating cell free (ccf) DNA from maternal plasma has enabled noninvasive prenatal testing for common autosomal aneuploidies. The purpose of this study was to extend the detection to include common sex chromosome aneuploidies (SCAs): [47,XXX], [45,X], [47,XXY], and [47,XYY] syndromes. METHOD: Massively parallel sequencing was performed on ccf DNA isolated from the plasma of 1564 pregnant women with known fetal karyotype. A classification algorithm for SCA detection was constructed and trained on this cohort. Another study of 411 maternal samples from women with blinded-to-laboratory fetal karyotypes was then performed to determine the accuracy of the classification algorithm. RESULTS: In the training cohort, the new algorithm had a detection rate (DR) of 100% (95%CI: 82.3%, 100%), a false positive rate (FPR) of 0.1% (95%CI: 0%, 0.3%), and nonreportable rate of 6% (95%CI: 4.9%, 7.4%) for SCA determination. The blinded validation yielded similar results: DR of 96.2% (95%CI: 78.4%, 99.8%), FPR of 0.3% (95%CI: 0%, 1.8%), and nonreportable rate of 5% (95%CI: 3.2%, 7.7%) for SCA determination CONCLUSION: Noninvasive prenatal identification of the most common sex chromosome aneuploidies is possible using ccf DNA and massively parallel sequencing with a high DR and a low FPR.

Intron retention facilitates splice variant diversity in calcium-activated big potassium channel populations.

We report that the stress axis-regulated exon (STREX)-containing calcium-activated big potassium (BKCa) channel splice variant expression and physiology are regulated in part by cytoplasmic splicing and intron retention. NextGen sequencing of the mRNA complement of pooled hippocampal dendrite samples found intron 17a (i17a), the intron immediately preceding STREX, in the BKCa mRNA. Further molecular analyses of i17a revealed that the majority of i17a-containing BKCa channel mRNAs associate with STREX. i17a siRNA treatment followed by STREX protein immunocytochemistry demonstrated both reduced levels and altered subcellular distribution of STREX-containing BKCa channel protein. Selective reduction of i17a-BKCa or STREX-BKCa mRNAs induced similar changes in the burst firing properties of hippocampal neurons. Collectively, these data show that STREX splice variant regulation via cytoplasmic splicing and intron retention helps generate STREX-dependent BKCa current diversity in hippocampal neurons.

Spatiotemporal patterns and transcription kinetics of induced RNA in single bacterial cells.

Bacteria have a complex internal organization with specific localization of many proteins and DNA, which dynamically move during the cell cycle and in response to changing environmental stimuli. Much less is known, however, about the localization and movements of RNA molecules. By modifying our previous RNA labeling system, we monitor the expression and localization of a model RNA transcript in live Escherichia coli cells. Our results reveal that the target RNA is not evenly distributed within the cell and localizes laterally along the long cell axis, in a pattern suggesting the existence of ordered helical RNA structures reminiscent of known bacterial cytoskeletal cellular elements.

Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting.

OBJECTIVE: We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA. STUDY DESIGN: Sample multiplexing and cost-optimized reagents were evaluated as improvements to a noninvasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed. RESULTS: In all, 480 prospectively collected samples were obtained from our third-party storage site; 13 of these were removed due to insufficient quantity or quality. Eighteen samples failed prespecified assay quality control parameters. In all, 449 samples remained: 39 trisomy 21 samples were correctly classified; 1 sample was misclassified as trisomy 21. The overall classification showed 100% sensitivity (95% confidence interval, 89-100%) and 99.7% specificity (95% confidence interval, 98.5-99.9%). CONCLUSION: Extending the scope of previous reports, this study demonstrates that plasma DNA sequencing is a viable method for noninvasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.

Application of mosaicism ratio to multifetal gestations.

Mosaicism ratio, or MR, is a laboratory metric that can be calculated using massively parallel sequencing data from cell-free DNA (cfDNA) screening. MR compares the amount of cfDNA present from a particular chromosome or chromosomal region to the overall fetal fraction of the specimen. In singleton gestations, MR may be used to refine the positive predictive value of an abnormal cfDNA screening result by identifying cases that could be impacted by various biological factors, such as placental mosaicism or prior co-twin demise. The current study was designed to examine the behavior of mosaicism ratio (MR) in multifetal gestations. Multifetal cfDNA specimens with positive results for trisomies 21, 18, or 13 and confirmed diagnostic outcomes were compiled to examine MR of the aneuploid chromosome based on the number of affected fetuses/placentas. A second multifetal cohort was assembled to analyze the MR of the Y chromosome in cases with at least one male fetus. For aneuploid cases, the average MR of affected singletons (used as a biological proxy for two affected twins) was significantly higher than the average MR for twins in which one fetus was affected. The average MR of the aneuploid chromosome for one affected twin was 52%, 42%, and 48% of that of singleton gestations for trisomy 21, 18, and 13 cases, respectively. MR cutoffs of 0.7 for trisomy 21, and 0.5 for trisomies 18 and 13 may help predict whether one versus both twins are affected with aneuploidy when clinical concern arises. For male cases, the Y MR of XX/XY gestations was 48% of the Y MR for XY/XY gestations. Using a Y MR cutoff of 0.8 allowed determination of XX/XY versus XY/XY gestations with 92.3-94.9% accuracy. Based on the data presented, MR may have utility in the analysis and interpretation of cfDNA data from multifetal gestations.

A new era in aneuploidy screening: cfDNA testing in >30,000 multifetal gestations: Experience at one clinical laboratory.

Since introducing cell-free DNA screening, Sequenom Laboratories has analyzed over 1 million clinical samples. More than 30,000 of these samples were from multifetal gestations (including twins, triplets and higher-order multiples). The clinical laboratory experience with the first 30,000 multifetal samples will be discussed. Maternal plasma samples from multifetal gestations were subjected to DNA extraction and library preparation followed by massively parallel sequencing. Sequencing data were analyzed to identify autosomal trisomies and other subchromosomal events. Fetal fraction requirements were adjusted in proportion to fetal number. Outcome data, when voluntarily received from the ordering provider, were collected from internal case notes. Feedback was received in 50 cases. The positivity rate in multifetal samples for trisomy 21 was 1.50%, 0.47% for trisomy 18, and 0.21% for trisomy 13. Average total sample fetal fraction was 12.2% at a mean gestational age of 13 weeks 6 days. Total non-reportable rate was 5.95%. Estimated performance based on ad hoc clinical feedback demonstrates that possible maximum sensitivity and specificity meet or exceed the original performance from clinical validation studies. Cell-free DNA (cfDNA) screening provides certain advantages over that of conventional screening in multifetal gestations and is available in higher-order multiples.

High-throughput alternative splicing quantification by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Alternative splicing is a significant contributor to transcriptome diversity, and a high-throughput experimental method to quantitatively assess predictions from expressed sequence tag and microarray analyses may help to answer questions about the extent and functional significance of these variants. Here, we describe a method for high-throughput analysis of known or suspected alternative splicing variants (ASVs) using PCR, primer extension and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Reverse-transcribed mRNA is PCR amplified with primers surrounding the site of alternative splicing, followed by a primer extension reaction designed to target sequence disparities between two or more variants. These primer extension products are assayed on a MALDI-TOF mass spectrometer and analyzed automatically. This method is high-throughput, highly accurate and reproducible, allowing for the verification of the existence of splicing variants in a variety of samples. An example given also demonstrates how this method can eliminate potential pitfalls from ordinary gel electrophoretic analysis of splicing variants where heteroduplexes formed from different variants can produce erroneous results. The new method can be used to create alternative variant profiles for cancer markers, to study complex splicing regulation, or to screen potential splicing therapies.

Non-invasive prenatal chromosomal aneuploidy testing--clinical experience: 100,000 clinical samples.

OBJECTIVE: As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT) for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT. STUDY DESIGN: The study includes plasma samples collected from patients with high-risk pregnancies in our CLIA-licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting. RESULTS: NIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age ≥ 35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5%) with 2.61% yielding a positive NIPT result. CONCLUSION: NIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the current standard of care.

The suitability of matrix assisted laser desorption/ionization time of flight mass spectrometry in a laboratory developed test using cystic fibrosis carrier screening as a model.

We designed a laboratory developed test (LDT) by using an open platform for mutation/polymorphism detection. Using a 108-member (mutation plus variant) cystic fibrosis carrier screening panel as a model, we completed the last phase of LDT validation by using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Panel customization was accomplished via specific amplification primer and extension probe design. Amplified genomic DNA was subjected to allele specific, single base extension endpoint analysis by mass spectrometry for inspection of the cystic fibrosis transmembrane regulator gene (NM_000492.3). The panel of mutations and variants was tested against 386 blinded samples supplied by "authority" laboratories highly experienced in cystic fibrosis transmembrane regulator genotyping; >98% concordance was observed. All discrepant and discordant results were resolved satisfactorily. Taken together, these results describe the concluding portion of the LDT validation process and the use of mass spectrometry to detect a large number of complex reactions within a single run as well as its suitability as a platform appropriate for interrogation of scores to hundreds of targets.

RNA visualization in live bacterial cells using fluorescent protein complementation.

We describe a technique for the detection and localization of RNA transcripts in living cells. The method is based on fluorescent-protein complementation regulated by the interaction of a split RNA-binding protein with its corresponding RNA aptamer. In our design, the RNA-binding protein is the eukaryotic initiation factor 4A (eIF4A). eIF4A is dissected into two fragments, and each fragment is fused to split fragments of the enhanced green fluorescent protein (EGFP). Coexpression of the two protein fusions in the presence of a transcript containing eIF4A-interacting RNA aptamer resulted in the restoration of EGFP fluorescence in Escherichia coli cells. We also applied this technique to the visualization of an aptamer-tagged mRNA and 5S ribosomal RNA (rRNA). We observed distinct spatial and temporal changes in fluorescence within single cells, reflecting the nature of the transcript.