Clinical outcomes of screen-positive genome-wide cfDNA cases for trisomy 20: results from the global expanded NIPT Consortium.

Trisomy 20 has been shown to be one of the most frequent rare autosomal trisomies in patients that undergo genome-wide noninvasive prenatal testing. Here, we describe the clinical outcomes of cases that screened positive for trisomy 20 following prenatal genome-wide cell-free (cf.) DNA screening. These cases are part of a larger cohort of previously published cases. Members of the Global Expanded NIPT Consortium were invited to submit details on their cases with a single rare autosomal aneuploidy following genome-wide cfDNA screening for retrospective analysis. Clinical details including patient demographics, test indications, diagnostic testing, and obstetric pregnancy outcomes were collected. Genome-wide cfDNA screening was conducted following site-specific laboratory procedures. Cases which screened positive for trisomy 20 (n = 10) were reviewed. Clinical outcome information was available for 90% (9/10) of our screen-positive trisomy 20 cases; the case without diagnostic testing ended in a fetal demise. Of the nine cases with outcome information, one was found to have a mosaic partial duplication (duplication at 20p13), rather than a full trisomy 20. Only one case in the study cohort had placental testing; therefore, confined placental mosaicism could not be ruled out in most cases. Adverse pregnancy outcomes were seen in half of the cases, which could suggest the presence of underlying confined placental mosaicism or mosaic/full fetal trisomy 20. Based on our limited series, the likelihood of true fetal aneuploidy is low but pregnancies may be at increased risk for adverse obstetric outcomes and may benefit from additional surveillance.

Prenatal cfDNA Screening for Emanuel Syndrome and Other Unbalanced Products of Conception in Carriers of the Recurrent Balanced Translocation t(11;22): One Laboratory's Retrospective Experience.

Prenatal cell-free DNA screening (cfDNA) can identify fetal chromosome abnormalities beyond common trisomies. Emanuel syndrome (ES), caused by an unbalanced translocation between chromosomes 11 and 22, has lacked a reliable prenatal screening option for families with a carrier parent. A cohort of cases (n = 46) sent for cfDNA screening with indications and/or results related to ES was queried; diagnostic testing and pregnancy outcomes were requested and analyzed. No discordant results were reported or suspected; there were ten true positives with diagnostic confirmation, six likely concordant positives based on known translocations and consistent cfDNA data, and twenty-six true negatives, by diagnostic testing or birth outcomes. For cases with parental testing, all affected ES cases had maternal translocation carriers. Expanded cfDNA may provide reassurance for t(11;22) carriers with screen negative results, and screen positive results appear to reflect a likely affected fetus, especially with a known maternal translocation. Current society guidelines support the use of expanded cfDNA screening in specific circumstances, such as for translocation carriers, with appropriate counseling. Diagnostic testing is recommended for prenatal diagnosis of ES and other chromosome abnormalities in pregnancy. To our knowledge, this cohort is the largest published group of cases with prenatal screening for carriers of t(11;22).

Laboratory performance of genome-wide cfDNA for copy number variants as compared to prenatal microarray.

BACKGROUND: Noninvasive prenatal testing (NIPT) allows for screening of fetal aneuploidy and copy number variants (CNVs) from cell-free DNA (cfDNA) in maternal plasma. Professional societies have not yet embraced NIPT for fetal CNVs, citing a need for additional performance data. A clinically available genome-wide cfDNA test screens for fetal aneuploidy and CNVs larger than 7 megabases (Mb). RESULTS: This study reviews 701 pregnancies with "high risk" indications for fetal aneuploidy which underwent both genome-wide cfDNA and prenatal microarray. For aneuploidies and CNVs considered 'in-scope' for the cfDNA test (CNVs ≥ 7 Mb and select microdeletions), sensitivity and specificity was 93.8% and 97.3% respectively, with positive and negative predictive values of 63.8% and 99.7% as compared to microarray. When including 'out-of-scope' CNVs on array as false negatives, the sensitivity of cfDNA falls to 48.3%. If only pathogenic out-of-scope CNVs are treated as false negatives, the sensitivity is 63.8%. Of the out-of-scope CNVs identified by array smaller than 7 Mb, 50% were classified as variants of uncertain significance (VUS), with an overall VUS rate in the study of 2.29%. CONCLUSIONS: While microarray provides the most robust assessment of fetal CNVs, this study suggests that genome-wide cfDNA can reliably screen for large CNVs in a high-risk cohort. Informed consent and adequate pretest counseling are essential to ensuring patients understand the benefits and limitations of all prenatal testing and screening options.

Positive cfDNA screening results for 22q11.2 deletion syndrome-Clinical and laboratory considerations.

Introduction: Non-invasive prenatal screening (NIPS) via cell-free DNA (cfDNA) screens for fetal chromosome disorders using maternal plasma, including 22q11.2 deletion syndrome (22q11.2DS). While it is the commonest microdeletion syndrome and has potential implications for perinatal management, prenatal screening for 22q11.2DS carries some inherent technical, biological, and counseling challenges, including varying deletion sizes/locations, maternal 22q11.2 deletions, confirmatory test choice, and variable phenotype. Materials and methods: This study addresses these considerations utilizing a retrospective cohort of 307 samples with screen-positive 22q11.2 NIPS results on a massively parallel sequencing (MPS) platform. Results: Approximately half of the cases reported ultrasound findings at some point during pregnancy. In 63.2% of cases with diagnostic testing, observed positive predictive values were 90.7%-99.4%. cfDNA identified deletions ranging from <1 Mb to 3.55 Mb, with significant differences in confirmed fetal versus maternal deletion sizes; estimated cfDNA deletion size was highly concordant with microarray findings. Mosaicism ratio proved useful in predicting the origin of a deletion (fetal versus maternal). Prediction of deletion size, location, and origin may help guide confirmatory testing. Discussion: The data shows that MPS-based NIPS can screen for 22q11.2DS with a high PPV, and that collaboration between the laboratory and clinicians allows consideration of additional metrics that may guide diagnostic testing and subsequent management.

Multisite assessment of the impact of cell-free DNA-based screening for rare autosomal aneuploidies on pregnancy management and outcomes.

Cell-free (cf) DNA screening is a noninvasive prenatal screening approach that is typically used to screen for common fetal trisomies, with optional screening for sex chromosomal aneuploidies and fetal sex. Genome-wide cfDNA screening can screen for a wide variety of additional anomalies, including rare autosomal aneuploidies (RAAs) and copy number variants. Here, we describe a multi-cohort, global retrospective study that looked at the clinical outcomes of cases with a high-risk cfDNA screening result for a RAA. Our study cohort included a total of 109 cases from five different sites, with diagnostic outcome information available for 68% (74/109) of patients. Based on confirmatory diagnostic testing, we found a concordance rate of 20.3% for presence of a RAA (15/74) in our study population. Pregnancy outcome was also available for 77% (84/109) of cases in our cohort. Many of the patients experienced adverse pregnancy outcomes, including intrauterine fetal demise, fetal growth restriction, and preterm birth. These adverse outcomes were observed both in patients with fetal or placental confirmation of the presence of a RAA, as well as patients that did not undergo fetal and/or placental diagnostic testing. In addition, we have proposed some suggestions for pregnancy management and counseling considerations for situations where a RAA is noted on a cfDNA screen. In conclusion, our study has shown that genome-wide cfDNA screening for the presence of rare autosomal aneuploidies can be beneficial for both patients and their healthcare practitioners. This can provide a possible explanation for an adverse pregnancy outcome or result in a change in pregnancy management, such as increased monitoring for adverse outcomes.

Genome-wide cell-free DNA screening: a focus on copy-number variants.

PURPOSE: Of 86,902 prenatal genome-wide cell-free DNA (cfDNA) screening tests, 4,121 were positive for a chromosome abnormality. This study examines 490 cases screen-positive for one or more subchromosomal copy-number variants (CNV) from genome-wide cfDNA screening. METHODS: Cases positive for one or more subchromosomal CNV from genome-wide cfDNA screening and diagnostic outcomes were compiled. Diagnostic testing trends were analyzed, positive predictive values (PPVs) were calculated, and the type of chromosomal abnormalities ultimately confirmed by diagnostic testing were described. RESULTS: CNVs were identified in 0.56% of screened specimens. Of the 490 cases screen-positive for one or more CNV, diagnostic outcomes were available for 244 cases (50%). The overall PPV among the cases with diagnostic outcomes was 74.2% (95% CI: 68.1-79.5%) and 71.8% (95% CI: 65.5-77.4%) for "fetal-only" events. Overall, isolated CNVs showed a lower PPV of 61.0% (95% CI: 52.5-68.8%) compared to complex CNVs at 93.9% (95% CI: 86.6-97.5%). Isolated deletions/duplications and unbalanced structural rearrangements were the most common diagnostic outcomes when isolated and complex CNVs were identified by cfDNA screening, respectively. CONCLUSION: Genome-wide cfDNA screening identifies chromosomal abnormalities beyond the scope of traditional cfDNA screening, and the overall PPV associated with subchromosomal CNVs in cases with diagnostic outcomes was >70%.

Three years of clinical experience with a genome-wide cfDNA screening test for aneuploidies and copy-number variants.

PURPOSE: Pregnant women have unprecedented choices for prenatal screening and testing. Cell-free DNA (cfDNA) offers the option to screen for aneuploidy of all chromosomes and genome-wide copy-number variants (CNVs), expanding screening beyond the common trisomies ("traditional" cfDNA). We sought to review the utilization trends and clinical performance characteristics of a commercially available genome-wide cfDNA test, with a subset having available diagnostic testing outcomes. METHODS: Retrospective analysis of 55,517 samples submitted for genome-wide cfDNA screening at a commercial laboratory, assessing indications, demographics, results, and performance. The cohort was broken into three "testing years"' to compare trends. RESULTS: Indications shifted over time, with a decrease in referrals for ultrasound findings (22.0% to 12.0%) and an increase in no known high-risk indication (3.0% to 16.6%). Of the positive results, 25% would be missed with traditional cfDNA screening. High sensitivity and specificity were observed with a positive predictive value (PPV) of 72.6% for genome-wide CNVs and 22.4% for rare autosomal trisomies (RATs). CONCLUSION: A broader patient population is utilizing genome-wide cfDNA, yet positivity rates and the contribution of genome-wide events have remained stable at approximately 5% and 25%, respectively. Test performance in a real-world clinical population shows high PPVs in those CNVs tested, with diagnostic outcomes in over 40% of positive cases.

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.

Hydranencephaly in a newborn with a FLVCR2 mutation and prenatal exposure to cocaine.

BACKGROUND: Hydranencephaly is a relatively rare but severe structural brain abnormality that often results in perinatal death. Although several factors including infection and multiple births have been reported to be associated with this birth defect, the underlying etiology is not well understood. Recently, FLVCR2 gene mutations have been implicated in a subset of hydranencephaly cases, following an autosomal recessive pattern of inheritance. CASE: We report a male infant with hydranencephaly found to have a previously unreported six amino acid deletion in one copy of the FLVCR2 gene following a pregnancy complicated by poor prenatal care and maternal cocaine use. Although our patient currently presents with developmental delays, he is showing progress and gaining some skills. CONCLUSION: We discuss the possibility of a synergistic effect between the FLVCR2 genetic mutation and environmental cocaine exposure, creating a susceptible brain, as an explanation for this infant's phenotype. This case demonstrates the potential clinical utility of testing for mutations in FLVCR2 for patients with hydranencephaly after other possible etiologies, such as congenital infection, have been reasonably eliminated. Current literature on FLVCR2 is relatively sparse; identifying additional patients with similar mutations will aid in defining the clinical significance of a gene mutation and the contribution to the etiology of hydranencephaly.