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).

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.

Obesity and no call results: optimal timing of cell-free DNA testing and redraw.

BACKGROUND: Fetal fraction of cell-free DNA decreases with increasing maternal weight. Consequently, cell-free DNA screening for fetal aneuploidy has higher screen failures or "no call" rates in women with obesity owing to a low fetal fraction. The optimal timing of testing based on maternal weight is unknown. OBJECTIVE: This study aimed to identify the optimal timing of initial cell-free DNA testing based on maternal weight and to identify the optimal timing of repeat cell-free DNA testing in cases with an initial screen failure. STUDY DESIGN: This was a retrospective cohort study of women undergoing cell-free DNA for fetal aneuploidy screening between 9 and 18 weeks through a single laboratory over 1 year from 2018 to 2019. Fetal fraction change per week was calculated, and generalized linear models were used to calculate relative risk and 95% confidence interval of a no call result at given maternal weights and gestational ages. RESULTS: The vast majority of samples (99.22%) received a test result. The risk of a no call result owing to a low fetal fraction was higher with increasing maternal weight. At 9 to 12 weeks, the rate of a no call result owing to a low fetal fraction in women who weighed <150 lb was 0.14% compared with 17.39% in women weighing >400 lb. Fetal fraction increased with increasing gestational age, although the incremental increase in fetal fraction over time is inversely proportional to maternal weight. At 13 to 18 weeks' gestation, 6.45% of women weighing >400 lb received a no call result owing to a low fetal fraction. In women in the highest weight category, >400 lb, fetal fraction increased 0.5% with each week of gestation. CONCLUSION: Although the risk of a no call result increases with maternal weight, cell-free DNA screening should be offered to all women at 9 to 12 weeks' gestation, allowing the option to have chorionic villus sampling after a positive test result. Pretest counseling for women with obesity should include the increased chance for a test failure. Most women weighing less than 400 lb will receive a test result and more than 80% of women with a weight of >400 lb will receive a test result at 9 to 12 weeks' gestation. Data regarding the expected increase in cell-free DNA fetal fraction per week may help guide the timing of a redraw to optimize test success.

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.

Not all low fetal fraction cell-free DNA screening failures are at increased risk for aneuploidy.

OBJECTIVE: To evaluate cell-free DNA (cfDNA) redraws and pregnancy outcomes following low fetal fraction (FF) cfDNA failures, as it has been suggested that a failed cfDNA screen due to insufficient FF carries increased risk for fetal aneuploidy. METHODS: Here >200,000 consecutive samples were reviewed and >1,100 patients were identified with a failed cfDNA due to low FF using genome-wide massively parallel sequencing. Redraw results following the initial low FF failure were analyzed, as well as pregnancy outcomes for patients with repeated low FF failure on redraw. RESULTS: Upon redraw 84.2% of samples yielded a reportable result with no enrichment of aneuploidy observed (p = 0.332). Higher maternal weights and multifetal pregnancy rates were observed in samples with insufficient FF. In patients with repeated low FF failure on redraw, almost all pregnancies resulted in apparently healthy liveborns. CONCLUSION: Insufficient FF was not an indicator of aneuploidy risk or adverse pregnancy outcomes in this study. Caution should be taken in generalizing aneuploidy risk to all low FF cfDNA failures. Redrawing may be an appropriate next step, as proceeding directly with diagnostic testing for aneuploidy may be unwarranted for most patients.

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.