Objective We evaluated whether there is an association between beta-globin (HBB) pathogenic variants and fetal fraction (FF), and whether the association has a clinically relevant impact on non-invasive prenatal screening (NIPS). Method A whole-genome sequencing NIPS laboratory database was retrospectively queried for women who underwent NIPS and carrier screening of both HBB and the alpha-globin genes (HBA1/HBA2). Women affected with either condition were excluded from the study, yielding a cohort size of 15,853. A "corrected FF" was obtained via multivariable linear regression adjusted for the systematic impacts of maternal age, gestational age and BMI. Corrected FF distributions of HBB and HBA1/HBA2 carriers were each compared to non-carriers using the Kolmogorov-Smirnov test. Results In this cohort, 291 women were carriers for HBB alone, and 1016 were carriers for HBA1/HBA2 alone. The HBB carriers had a lower corrected FF when compared to non-carriers (p < 0.0001). There was no difference in corrected FF among carriers and non-carriers of HBA1/HBA2. Conclusion Carriers of pathogenic variants in the HBB gene, but not the HBA1/HBA2 genes, are more likely to have lower FF when compared to women with structurally normal hemoglobin. This decrease in FF could result in an elevated test-failure rate if FF thresholds were used.

Objective To evaluate the efficacy of three different carrier screening workflows designed to identify couples at risk for having offspring with autosomal recessive conditions. Methods Partner testing compliance, unnecessary testing, turnaround time, and ability to identify at-risk couples (ARCs) were measured across all three screening strategies (sequential, tandem, or tandem reflex). Results A total of 314,100 individuals who underwent carrier screening were analyzed. Sequential, tandem, and tandem reflex screening yielded compliance frequencies of 25.8%, 100%, and 95.9%, respectively. Among 14,595 couples tested in tandem, 42.2% of females were screen-negative, resulting in unnecessary testing of the male partner. In contrast, less than 1% of tandem reflex couples included unnecessary male testing. The median turnaround times were 29.2 days (sequential), 8 days (tandem), and 13.3 days (tandem reflex). The proportion of ARCs detected per total number of individual screens were 0.5% for sequential testing and 1.3% for both tandem and tandem reflex testing. Conclusion The tandem reflex strategy simplifies a potentially complex clinical scenario by providing a mechanism by which providers can maximize partner compliance and the detection of at-risk couples while minimizing workflow burden and unnecessary testing and is more efficacious than both sequential and tandem screening strategies.

Purpose: The American College of Obstetricians and Gynecologists (ACOG) and the American College of Medical Genetics and Genomics (ACMG) suggest carrier screening panel design criteria intended to ensure meaningful results. This study used a data-driven approach to interpret the criteria to identify guidelines-consistent panels. Methods: Carrier frequencies in >460,000 individuals across 11 races/ethnicities were used to assess carrier frequency. Other criteria were interpreted on the basis of published data. A total of 176 conditions were then evaluated. Stringency thresholds were set as suggested by ACOG and/or ACMG or by evaluating conditions already recommended by ACOG and ACMG. Results: Forty and 75 conditions had carrier frequencies of >= 1 in 100 and >= 1 in 200, respectively; 175 had a well-defined phenotype; and 165 met at least 1 severity criterion and had an onset early in life. Thirty-seven conditions met conservative thresholds, including a carrier frequency of >= 1 in 100, and 74 conditions met permissive thresholds, including a carrier frequency of >= 1 in 200; thus, both were identified as guidelines-consistent panels. Conclusion: Clear panel design criteria are needed to ensure quality and consistency among carrier screening panels. Evidence-based analyses of criteria resulted in the identification of guidelines-consistent panels of 37 and 74 conditions. (C) 2021 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics.

PURPOSE:Carrier status associates strongly with genetic ancestry, yet current carrier screening guidelines recommend testing for a limited set of conditions based on a patient's self-reported ethnicity. Ethnicity, which can reflect both genetic ancestry and cultural factors (e.g., religion), may be imperfectly known or communicated by patients. We sought to quantitatively assess the efficacy and equity with which ethnicity-based carrier screening captures recessive disease risk.METHODS:For 93,419 individuals undergoing a 96-gene expanded carrier screen (ECS), correspondence was assessed among carrier status, self-reported ethnicity, and a dual-component genetic ancestry (e.g., 75% African/25% European) calculated from sequencing data.RESULTS:Self-reported ethnicity was an imperfect indicator of genetic ancestry, with 9% of individuals having >50% genetic ancestry from a lineage inconsistent with self-reported ethnicity. Limitations of self-reported ethnicity led to missed carriers in at-risk populations: for 10 ECS conditions, patients with intermediate genetic ancestry backgrounds-who did not self-report the associated ethnicity-had significantly elevated carrier risk. Finally, for 7 of the 16 conditions included in current screening guidelines, most carriers were not from the population the guideline aimed to serve.CONCLUSION:Substantial and disproportionate risk for recessive disease is not detected when carrier screening is based on ethnicity, leading to inequitable reproductive care.

The association of low fetal-fraction (FF) in women affected by beta-chain hemoglobinopathies has previously been described. It is not clear if this holds true for unaffected beta-chain hemoglobinopathy carriers. We evaluated whether there is an association between β-globin (HBB) pathogenic variants and FF, and whether it has a clinically relevant impact on non-invasive prenatal screening (NIPS). A whole-genome sequencing NIPS laboratory database was retrospectively queried for all women with NIPS and carrier screening of the HBB gene; NIPS patients tested for α-globin (HBA1/HBA2) gene(s) were used as biological comparators. Affected women were excluded from the study, and all subjects included in the analysis had consented to deidentified research. Mutation status for both genes was classified as non-carrier or carrier. Multivariable linear regression removed the systematic impacts of maternal age, gestational age and BMI to yield a "corrected FF" for each patient. Corrected FF distributions were compared between the two mutation-status classes using the Kolmogorov-Smirnov test, and expected test-failure rates were calculated as a function of FF threshold. A total of 19,929 women had both NIPS and carrier screening involving the HBB gene, with 19,686 and 243 being non-carrier and carrier, respectively. The HBB carrier groups have lower corrected FF when compared to non-carrier (p < 0.0001; Fig. 1A). By comparison, 15,854 and 1,017 women were found to be non-carrier and carrier of HBA1/HBA2 pathogenic variants, respectively (N=16,871 patients), yet FF values did not differ among mutation carrier groups (p > 0.05; Fig. 1B). Expected test-failure rate for a fixed FF was higher in the HBB groups (Fig. 1 insets). Carriers of pathogenic variants in the HBB gene, but not the HBA1/HBA2 genes, are more likely to have lower FF when compared to women with structurally normal hemoglobin. This decrease in FF could result in an elevated test-failure rate if a fixed FF threshold were to be used.

INTRODUCTION: Obtaining carrier screening results in a timely manner is important to support reproductive decision-making. Reproductive partners are typically tested in a sequential manner: the female is tested first and if a carrier, her partner is tested. This imposes workflow challenges due to the need for a subsequent visit to draw the partner’s sample, reducing the likelihood of the partner getting screened and hampering detection of at-risk couples. We implemented a “tandem reflex” strategy wherein both partners submit samples in tandem but are tested sequentially, with the second partner’s sample tested only if the first partner was found to be a carrier. METHODS: The time to delivery of a combined report was measured before and after implementation of the “tandem reflex” strategy. Comparison to a simultaneous strategy, ie, both partners samples tested as soon as they are received, is also reported. RESULTS: Before implementing the “tandem reflex” strategy, the average time for a sequentially tested couple to receive a report was 35 days (95th percentile 76 days, N=16,785 couples). After implementation, the average time was reduced to 15 days (95th percentile 25 days, N=1,132 couples). Among 13,161 couples tested simultaneously, 42% of females were negative for all tested conditions; in this scenario, the tandem reflex strategy would have reduced unnecessary testing because it would not have triggered testing of the male partner. CONCLUSION: The “tandem reflex” strategy time to receipt of a combined report by half, allowing for more timely reproductive and pregnancy management.

AbstractBackgroundPathogenic variants in HEXA that impair β‐hexosaminidase A (Hex A) enzyme activity cause Tay‐Sachs Disease (TSD), a severe autosomal‐recessive neurodegenerative disorder. Hex A enzyme analysis demonstrates near‐zero activity in patients affected with TSD and can also identify carriers, whose single functional copy of HEXA results in reduced enzyme activity relative to noncarriers. Although enzyme testing has been optimized and widely used for carrier screening in Ashkenazi Jewish (AJ) individuals, it has unproven sensitivity and specificity in a pan‐ethnic population. The ability to detect HEXA variants via DNA analysis has evolved from limited targeting of a few ethnicity‐specific variants to next‐generation sequencing (NGS) of the entire coding region coupled with interpretation of any discovered novel variants.MethodsWe combined results of enzyme testing, retrospective computational analysis, and variant reclassification to estimate the respective clinical performance of TSD screening via enzyme analysis and NGS. We maximized NGS accuracy by reclassifying variants of uncertain significance and compared to the maximum performance of enzyme analysis estimated by calculating ethnicity‐specific frequencies of variants known to yield false‐positive or false‐negative enzyme results (e.g., pseudodeficiency and B1 alleles).ResultsIn both AJ and non‐AJ populations, the estimated clinical sensitivity, specificity, and positive predictive value were higher by NGS than by enzyme testing. The differences were significant for all comparisons except for AJ clinical sensitivity, where NGS exceeded enzyme testing, but not significantly.ConclusionsOur results suggest that performance of an NGS‐based TSD carrier screen that interrogates the entire coding region and employs novel variant interpretation exceeds that of Hex A enzyme testing, warranting a reconsideration of existing guidelines.

Expanded carrier screening (ECS) panels that use next-generation sequencing aim to identify pathogenic variants in coding and clinically relevant non-coding regions of hundreds of genes, each associated with a serious recessive condition. ECS has established analytical validity and clinical utility, meaning that variants are accurately identified and pathogenic variants tend to alter patients' clinical management, respectively. However, the clinical validity of ECS, that is, correct discernment of whether an identified variant is indeed pathogenic, has only been shown for single conditions, not for panels. Here, we evaluate the clinical validity of a >170-condition ECS panel by assessing concordance between >12 000 variant interpretations classified with guideline-based criteria to their corresponding per-variant combined classifications in ClinVar. We observe 99% concordance at the level of unique variants. A more clinically relevant frequency-weighted analysis reveals that fewer than 1 in 500 patients are expected to receive a report with a variant that has a discordant classification. Importantly, gene-level concordance is not diminished for rare ECS conditions, suggesting that large panels do not balloon the panel-wide false-positive rate. Finally, because ECS is intended to serve all reproductive-age couples, we show that classification of novel variants is feasible and scales predictably for a large population.

To determine the impact of the "tandem reflex" strategy on turnaround-time and test utilization. Expanded carrier screening aims to detect couples at risk for having children with severe and profound Mendelian disorders. As many couples are pregnant or actively trying to conceive while undergoing carrier screening, obtaining screening results in a timely manner is important. Reproductive partners are typically tested in a sequential manner: the female partner is tested first and if she is a carrier, her partner is tested for the condition(s) for which she was found to be a carrier. In current practice, this commonly necessitates a subsequent visit to a physician for submission of the partner's sample, such that the time to receive a combined couple report is roughly double the time it takes to receive an individual carrier screening report. This need for a secondary sample submission imposes workflow challenges to the clinic and patients, reduces the likelihood of the partner getting screened, and thus may hamper detection of at-risk couples. To minimize turnaround-time and maximize the detection of at-risk couples, we implemented a "tandem reflex" strategy wherein both partners submit samples in tandem, but are tested sequentially, with the second partner's sample tested only if the first partner was found to be a carrier. Retrospective data analysis. The time between a reproductive couple's submission of its first sample to the delivery of a combined report was measured before and after implementation of the "tandem reflex" strategy. Samples submitted and tested simultaneously were also analyzed for unnecessary partner testing. Before implementing the "tandem reflex" strategy, the average time for a sequentially tested couple to receive a full couple-based carrier screening report was approximately 34 days (95th percentile 70 days, N = 11,434 couples). After implementing the tandem-reflex strategy, the average wait time for a couple to receive their combined report was reduced to approximately 15 days (95th percentile 25 days, N = 383 couples), with individual reports returned within 11 days (95th percentile just under 21 days). Among 9,718 couples for which samples were submitted and tested simultaneously, 41% of females were negative for all tested conditions; in this scenario, the tandem reflex strategy would not have triggered testing of the male partner. The "tandem reflex" strategy decreased by half the turnaround-time for receiving a combined carrier screening report compared to sequential testing, resulting in the timely receipt of crucial information for reproductive and pregnancy management.

BackgroundNoninvasive prenatal screening (NIPS) of common aneuploidies using cell-free DNA from maternal plasma is part of routine prenatal care and is widely used in both high-risk and low-risk patient populations. High specificity is needed for clinically acceptable positive predictive values. Maternal copy-number variants (mCNVs) have been reported as a source of false-positive aneuploidy results that compromises specificity.MethodsWe surveyed the mCNV landscape in 87,255 patients undergoing NIPS. We evaluated both previously reported and novel algorithmic strategies for mitigating the effects of mCNVs on the screen's specificity. Further, we analyzed the frequency, length, and positional distribution of CNVs in our large dataset to investigate the curation of novel fetal microdeletions, which can be identified by NIPS but are challenging to interpret clinically.ResultsmCNVs are common, with 65% of expecting mothers harboring an autosomal CNV spanning more than 200kb, underscoring the need for robust NIPS analysis strategies. By analyzing empirical and simulated data, we found that general, outlier-robust strategies reduce the rate of mCNV-caused false positives but not as appreciably as algorithms specifically designed to account for mCNVs. We demonstrate that large-scale tabulation of CNVs identified via routine NIPS could be clinically useful: together with the gene density of a putative microdeletion region, we show that the region's relative tolerance to duplications versus deletions may aid the interpretation of microdeletion pathogenicity.ConclusionsOur study thoroughly investigates a common source of NIPS false positives and demonstrates how to bypass its corrupting effects. Our findings offer insight into the interpretation of NIPS results and inform the design of NIPS algorithms suitable for use in screening in the general obstetric population.

Purpose The recent growth in pan-ethnic expanded carrier screening (ECS) has raised questions about how such panels might be designed and evaluated systematically. Design principles for ECS panels might improve clinical detection of at-risk couples and facilitate objective discussions of panel choice. Methods Guided by medical-society statements, we propose a method for the design of ECS panels that aims to maximize the aggregate and per-disease sensitivity and specificity across a range of Mendelian disorders considered serious by a systematic classification scheme. We evaluated this method retrospectively using results from 474,644 de-identified carrier screens. We then constructed several idealized panels to highlight strengths and limitations of different ECS methodologies. Results Based on modeled fetal risks for “severe” and “profound” diseases, a commercially available ECS panel (Counsyl) is expected to detect 183 affected conceptuses per 100,000 US births. A screen’s sensitivity is greatly impacted by two factors: (i) the methodology used (e.g., full-exon sequencing finds more affected conceptuses than targeted genotyping) and (ii) the detection rate of the screen for diseases with high prevalence and complex molecular genetics (e.g., fragile X syndrome). Conclusion The described approaches enable principled, quantitative evaluation of which diseases and methodologies are appropriate for pan-ethnic expanded carrier screening.

I Supplementary Materials and Methods 2 1 CNV Deletion Calling 2 2 Census Weighting 2 3 Disease Risk Calculation 2 3.1 Additive Approximation to Disease Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 3.2 Example: Autosomal Recessive Disease Risk for one Disease . . . . . . . . . . . . . . . . . . . . . . 3 3.3 Calculating Disease Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3.4 Designing Optimal Targeted Genotyping Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4 References 4

To examine counseling experience with incidental cancer genes in expanded carrier screening (ECS). ECS identifies couples at risk for transmitting a genetic condition to their offspring. While carriers for most genes on ECS do not experience symptoms, mutations in certain genes, such as ATM and NBN, may predispose patients to an increased risk for cancer (IRC). We analyzed Counsyl's counseling experience to determine whether counseling patterns differed in patients with IRC. Our analysis included 152,973 individuals who had undergone routine carrier screening of up to 108 genes using Counsyl's results workflow: patients were contacted in accordance with our privacy policy to view their results online with tailored informational videos and request genetic counseling (GC). GCs are available on-demand or by appointment. Consult rates were compared among carriers of mutations in IRC genes (ATM and NBN) and non-IRC genes. The 13 genes with fewer than 10 observed carriers were omitted from the analysis. Carriers within at-risk couples were also excluded. IRC genes were determined based on 2017 National Comprehensive Cancer Network guidelines. ATM and NBN carriers were significantly more likely to pursue GC than carriers of non-IRC genes. Specifically, 32% of NBN and 27% of ATM carriers pursued GC as compared to the non-IRC average of 20% (p=0.0086). We observed no significant preference for on-demand vs. scheduled consultation as 45% of ATM and 56% of NBN carriers pursued on-demand GC, compared to 48% amongst non-IRC gene carriers (p > 0.05 in both cases). Furthermore, median consult times across all Counsyl-facilitated GC sessions for ATM (13 minutes) and NBN carriers (10 minutes) were not significantly different from the overall median consult time (10 minutes; p > 0.05 in both cases). Carriers of IRC genes are more likely to seek GC, suggesting that personal health implications are an important determinant of patients' desire for additional information. Clinicians should also consider having GC resources readily available to provide support for carriers with IRC.

BACKGROUND Fragile X syndrome (FXS, OMIM #300624) is an X-linked condition caused by trinucleotide repeat expansions in the 5' UTR (untranslated region) of the fragile X mental retardation 1 (FMR1) gene. FXS testing is commonly performed in expanded carrier screening and has been proposed for inclusion in newborn screening. However, because pathogenic alleles are long and have low complexity (>200 CGG repeats), FXS is currently tested by a single-plex electrophoresis-resolved PCR assay rather than multiplexed approaches like next-generation sequencing or mass spectrometry. In this work, we sought an experimental design based on nonadaptive group testing that could accurately and reliably identify the size of abnormally expanded FMR1 alleles of males and females. METHODS We developed a new group testing scheme named StairCase (SC) that was designed to the constraints of the FXS testing problem, and compared its performance to existing group testing schemes by simulation. We experimentally evaluated SC's performance on 210 samples from the Coriell Institute biorepositories using pooled PCR followed by capillary electrophoresis on 3 replicates of each of 3 pooling layouts differing by the mapping of samples to pools. RESULTS The SC pooled PCR approach demonstrated perfect classification of samples by clinical category (normal, intermediate, premutation, or full mutation) for 90 positives and 1800 negatives, with a batch of 210 samples requiring only 21 assays. CONCLUSIONS Group testing based on SC is an implementable approach to trinucleotide repeat expansion disorder testing that offers ≥10-fold reduction in assay costs over current single-plex methods.

Protein-DNA interactions are essential to constructing biological devices for synthetic gene circuits. Ideal devices should be interoperable and extensible with respect to each other. They should also exhibit minimal unwanted interactions with the host cells in which they reside and be portable between different host chassis. Here, we discuss two classes of protein-DNA devices, memory modules and transcription factors, that can be used to construct genetic circuits with novel functionalities. In addition, we describe a methodology for identifying candidate DNA targets to be used in designing artificial protein-DNA interactions. We identified 9 base-pair (bp) sites within each of six useful host organisms that were absent in individual host genomes but were unable to find any 9 bp sites that were absent from all of the genomes. Extending our search to 12 bp and 15 bp DNA sequences revealed tens of thousands and millions, respectively, of DNA sequences that were absent in all host organisms we evaluated; these sequences were targetable by publicly accessible zinc-finger methodologies. By targeting these sites, it may be possible to build interoperable, orthogonal, and portable protein-DNA devices. This work lays a foundation for future efforts to engineer novel biological devices in the emerging field of synthetic biology.