Eric A. Evans

An empirical estimate of carrier frequencies for 400+ causal Mendelian variants: results from an ethnically diverse clinical sample of 23,453 individuals

Purpose:Recent developments in genomics have led to expanded carrier screening panels capable of assessing hundreds of causal mutations for genetic disease. This new technology enables simultaneous measurement of carrier frequencies for many diseases. As the resultant rank-ordering of carrier frequencies impacts the design and prioritization of screening programs, the accuracy of this ranking is a public health concern.Methods:A total of 23,453 individuals from many obstetric, genetics, and infertility clinics were referred for routine recessive disease carrier screening. Multiplex carrier screening was performed and results were aggregated for this study.Results:Twenty-four percent of individuals were identified as carriers for at least one of 108 disorders, and 5.2% were carriers for multiple disorders. We report tabulations of carrier frequency by self-identified ethnicity and disease.Conclusion:To our knowledge, this study of a large, ethnically diverse clinical sample provides the most accurate measurements to date of carrier frequencies for hundreds of recessive alleles. The study also yields information on the clinical considerations associated with routine use of expanded panels and provides support for a pan-ethnic screening paradigm that minimizes the use of “racial” categories by the physician, as recommended by recent guidelines.Genet Med 2013:15(3):178–186

Systematic Classification of Disease Severity for Evaluation of Expanded Carrier Screening Panels

Professional guidelines dictate that disease severity is a key criterion for carrier screening. Expanded carrier screening, which tests for hundreds to thousands of mutations simultaneously, requires an objective, systematic means of describing a given diseases severity to build screening panels. We hypothesized that diseases with characteristics deemed to be of highest impact would likewise be rated as most severe, and diseases with characteristics of lower impact would be rated as less severe. We describe a pilot test of this hypothesis in which we surveyed 192 health care professionals to determine the impact of specific disease phenotypic characteristics on perceived severity, and asked the same group to rate the severity of selected inherited diseases. The results support the hypothesis: we identified four “Tiers” of disease characteristics (1–4). Based on these responses, we developed an algorithm that, based on the combination of characteristics normally seen in an affected individual, classifies the disease as Profound, Severe, Moderate, or Mild. This algorithm allows simple classification of disease severity that is replicable and not labor intensive.

Design and validation of a next generation sequencing assay for hereditary BRCA1 and BRCA2 mutation testing

Hereditary breast and ovarian cancer syndrome, caused by a germline pathogenic variant in the BRCA1 or BRCA2 (BRCA1/2) genes, is characterized by an increased risk for breast, ovarian, pancreatic and other cancers. Identification of those who have a BRCA1/2 mutation is important so that they can take advantage of genetic counseling, screening, and potentially life-saving prevention strategies. We describe the design and analytic validation of the Counsyl Inherited Cancer Screen, a next-generation-sequencing-based test to detect pathogenic variation in the BRCA1 and BRCA2 genes. We demonstrate that the test is capable of detecting single-nucleotide variants (SNVs), short insertions and deletions (indels), and copy-number variants (CNVs, also known as large rearrangements) with zero errors over a 114-sample validation set consisting of samples from cell lines and deidentified patient samples, including 36 samples with BRCA1/2pathogenic germline mutations.

Systematic design and comparison of expanded carrier screening panels

PurposeThe 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.MethodsGuided 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.ResultsBased 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).ConclusionThe described approaches enable principled, quantitative evaluation of which diseases and methodologies are appropriate for pan-ethnic expanded carrier screening.

Development and validation of a 36-gene sequencing assay for hereditary cancer risk assessment

The past two decades have brought many important advances in our understanding of the hereditary susceptibility to cancer. Numerous studies have provided convincing evidence that identification of germline mutations associated with hereditary cancer syndromes can lead to reductions in morbidity and mortality through targeted risk management options. Additionally, advances in gene sequencing technology now permit the development of multigene hereditary cancer testing panels. Here, we describe the 2016 revision of the Counsyl Inherited Cancer Screen for detecting single-nucleotide variants (SNVs), short insertions and deletions (indels), and copy number variants (CNVs) in 36 genes associated with an elevated risk for breast, ovarian, colorectal, gastric, endometrial, pancreatic, thyroid, prostate, melanoma, and neuroendocrine cancers. To determine test accuracy and reproducibility, we performed a rigorous analytical validation across 341 samples, including 118 cell lines and 223 patient samples. The screen achieved 100% test sensitivity across different mutation types, with high specificity and 100% concordance with conventional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). We also demonstrated the screen’s high intra-run and inter-run reproducibility and robust performance on blood and saliva specimens. Furthermore, we showed that pathogenic Alu element insertions can be accurately detected by our test. Overall, the validation in our clinical laboratory demonstrated the analytical performance required for collecting and reporting genetic information related to risk of developing hereditary cancers.

Noninvasive prenatal screening at low fetal fraction: comparing whole‐genome sequencing and single‐nucleotide polymorphism methods

Performance of noninvasive prenatal screening (NIPS) methodologies when applied to low fetal fraction samples is not well established. The single‐nucleotide polymorphism (SNP) method fails samples below a predetermined fetal fraction threshold, whereas some laboratories employing the whole‐genome sequencing (WGS) method report aneuploidy calls for all samples. Here, the performance of the two methods was compared to determine which approach actually detects more fetal aneuploidies.

Detection of carriers in the Ashkenazi Jewish Population: An objective comparison of high-throughput genotyping versus gene-by-gene testing

BACKGROUND High-throughput genotyping allows rapid identification of targeted mutations at a fraction of the cost of current gene-by-gene testing methodologies. An objective comparison of the two methodologies allows providers to assess the clinical validity/utility of high-throughput carrier screening and establish a comfort level with new genomic technologies. AIM To verify that high-throughput genotyping accurately determines patient carrier status, DNA samples from previously identified carriers (n=31) of Ashkenazi Jewish genetic diseases were anonymized and submitted for retesting by high-throughput genotyping. RESULTS The results were 100% concordant (95% CI: 0.998-1), demonstrating that high-throughput genotyping assays accurately identify carriers of targeted mutations in the Ashkenazi Jewish population. In addition, carrier status for diseases and mutations not previously tested was uncovered using the high-throughput assay. CONCLUSIONS High-throughput genotyping is a cost-effective and clinically valid approach to carrier screening. The use of a broader screen for Ashkenazi Jewish individuals increases the detection of carriers in this population.

Smith–Lemli–Opitz syndrome carrier frequency and estimates of in utero mortality rates

To tabulate individual allele frequencies and total carrier frequency for Smith–Lemli–Opitz syndrome (SLOS) and compare expected versus observed birth incidences.

Development and validation of an expanded carrier screen that optimizes sensitivity via full-exon sequencing and panel-wide copy-number-variant identification

Purpose By identifying pathogenic variants across hundreds of genes, expanded carrier screening (ECS) enables prospective parents to assess risk of transmitting an autosomal recessive or X-linked condition. Detection of at-risk couples depends on the number of conditions tested, the diseases’ respective prevalences, and the screen’s sensitivity for identifying disease-causing variants. Here we present an analytical validation of a 235-gene sequencing-based ECS with full coverage across coding regions, targeted assessment of pathogenic noncoding variants, panel-wide copy-number-variant (CNV) calling, and customized assays for technically challenging genes. Methods Next-generation sequencing, a customized bioinformatics pipeline, and expert manual call review were used to identify single-nucleotide variants, short insertions and deletions, and CNVs for all genes except FMR1 and those whose low disease incidence or high technical complexity precludes novel variant identification or interpretation. Variant calls were compared to reference and orthogonal data. Results Validation of our ECS data demonstrated >99% analytical sensitivity and >99% specificity. A preliminary assessment of 15,177 patient samples reveals the substantial impact on fetal disease-risk detection attributable to novel CNV calling (13.9% of risk) and technically challenging conditions (15.5% of risk), such as congenital adrenal hyperplasia. Conclusion Validated, high-fidelity identification of different variant types—especially in diseases with complicated molecular genetics—maximizes at-risk couple detection.

Abstract 5690: Optimized molecular barcoding enables accurate targeted mutation detection in circulating cell-free DNA (cfDNA)

The evaluation of cfDNA allows novel approaches to noninvasive detection of actionable alterations, resistance mechanisms, and tumor monitoring in patients with cancer. Importantly, tumor-specific DNA fragments represent a small minority of the cfDNA and can be obscured by false positive (FP) variants introduced by chemical damage and sequencer error. To address this, we improved key processes in the design of NGS libraries, including a new molecular barcoding approach, that maximize molecular recovery while eliminating spurious variants. We engineered a set of Illumina sequencing chemistry compatible adaptors incorporating unique molecular identifiers (barcodes) enabling reconstruction of the sequence of both strands of the original DNA molecule. These barcodes incorporate a number of key design improvements as compared to published methodologies, which enhance sequencer cluster density, thereby increasing library diversity and molecular recovery. Our new design identified both chemical and sequencer errors, reducing incorrect base calls to rates below 5e-7. We validated our methodology for use in cfDNA using both dilution experiments and patient blood samples with known oncogenic alterations via a custom capture panel targeting actionable genomic alterations in a 55kb region. By identifying the molecular origin of each read, we found that the sensitivity of detection obtained from barcoded libraries followed ideal binomial sampling expectations. We obtained an average molecular depth of 1,000 molecules per site from the plasma extracted from a single blood collection tube, which corresponded to an 80% sensitivity of detection of known oncogenic single-nucleotide and indel mutations at 0.15% mutant allele frequency (MAF) in cfDNA with no FP calls. Furthermore, we successfully detected known gene-fusions at 0.5%, and amplifications (>10 copies) down to 1% MAF. We designed and validated a custom-engineered error-correcting sequencing adapters, ideal for broad range of applications requiring high accuracy detection of ultra-low frequency alterations. Note: This abstract was not presented at the meeting. Citation Format: Carlo G. Artieri, Kyle A. Beauchamp, Valentina S. Vysotskaia, Noah C. Welker, Eric A. Evans, Clement Chu, Haluk Tezcan, Imran S. Haque. Optimized molecular barcoding enables accurate targeted mutation detection in circulating cell-free DNA (cfDNA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5690. doi:10.1158/1538-7445.AM2017-5690