EP377: Enabling single-platform testing and carrier screening of the FMR1, SMN1/2, and CFTR gene trio

Abstract

Carrier screening can provide valuable information for couples to help guide their reproductive decision-making. Screening studies have shown that as many as 1 in 20 individuals has pathogenic variants associated with at least 1 of 3 conditions: spinal muscular atrophy (SMA), fragile X syndrome (FXS), and cystic fibrosis (CF). Many of these conditions cause severe symptoms, and SMA and CF can lead to premature death. Early detection can both inform reproductive choices and offer early therapeutic intervention to help mitigate the most severe symptoms if implemented early enough in the course of disease progression. The three genes associated with SMA, FXS and CF (SMN1, FMR1 and CFTR, respectively) each present unique technical challenges and currently require distinct molecular diagnostic methods and analysis platforms. As a result, a simple, cost-effective, and unified screening system is not yet available for this trio. Here we demonstrate the feasibility of a common, scalable workflow for analyzing combinations of PCR products from all 3 genes on a single capillary electrophoresis (CE) instrument. DNA samples were PCR amplified using AmplideX PCR/CE SMN1/2 Plus, FMR1, and CFTR kits (Asuragen) and electrophoresed on the Applied Biosystems 3500 and 3730 Series Genetic Analyzers (Thermo Fisher Scientific). PCR products from each of the 3 assays were generated according to kit instructions, and combined onto a single CE plate for injection using harmonized conditions. A collection of 48 unique genomic DNA samples isolated from human cell lines or whole blood were tested in singleton with each assay. Samples were designed to cover the breadth of possible genotypes detected by each kit, representing 0 to ≥4 copies of SMN1 or SMN2, FMR1 categorical genotypes of normal, premutation, intermediate, or full mutation [GJL1], and at least 23 unique CFTR variants representing the minimum recommended CFTR carrier screening variant panel from ACMG. Data were processed using AmplideX Reporter Software. Samples were analyzed using a single CE plate for all 3 assays injected with a single set of injection conditions, then compared to the same amplicons injected on separate CE plates for each assay using the conditions recommended for each assay. The overall percent agreement between the genotype calls of the co-injected samples and their individually injected counterparts was >98% for SMN1/2, FMR1, and CFTR on both the 3500 and 3730 CE instruments. Concordance was similar when compared to known reference results for each sample. The co-injection of SMN1/2, FMR1, and CFTR products did not alter the genotype category or copy number calls when compared to their respective, individually assessed results. Using this approach, comprehensive trio genotypes were generated within a single day; further, the system has the potential to process up to 1000 samples per day using a single 3730 CE instrument. The co-injection of PCR products of SMN1/2, FMR1, and CFTR genes can be achieved using commonly available high throughput CE instruments. This approach consolidates existing workflows and improves run and cost efficiencies, with the flexibility to accommodate a few samples at a time or up to 1000 per day. Our study demonstrates the potential for a unified single-instrument, single-software analysis of SMN1/2, FMR1, and CFTR using an accessible, easy-to-run system that runs on equipment that labs already have and can adapt to meet the increasing needs of carrier screening. This versatility can translate to significant cost savings for screening labs that typically rely on 2, 3, or even 4 unique methodologies (with reflexes) spanning different platform requirements, workflows, and analysis tools. The workflow similarities among the 3 PCR/CE assays also simplify their implementation in labs, as the protocols are largely identical.