Newborn Screening Quality Assurance Program for CFTR Mutation Detection and Gene Sequencing to Identify Cystic Fibrosis

Abstract

All newborn screening laboratories in the United States and many worldwide screen for cystic fibrosis. Most laboratories use a second-tier genotyping assay to identify a panel of mutations in the CF transmembrane regulator (CFTR) gene. Centers for Disease Control and Prevention’s Newborn Screening Quality Assurance Program houses a dried blood spot repository of samples containing CFTR mutations to assist newborn screening laboratories and ensure high-quality mutation detection in a high-throughput environment. Recently, CFTR mutation detection has increased in complexity with expanded genotyping panels and gene sequencing. To accommodate the growing quality assurance needs, the repository samples were characterized with several multiplex genotyping methods, Sanger sequencing, and 3 next-generation sequencing assays using a high-throughput, low-concentration DNA extraction method. The samples performed well in all of the assays, providing newborn screening laboratories with a resource for complex CFTR mutation detection and next-generation sequencing as they transition to new methods.

Investigation of newborns with abnormal results in a newborn screening program for four lysosomal storage diseases in Brazil

“…Therefore, molecular analysis of the IDUA gene was performed, with the identification of the variant c.251G>C [p.(Gly84Ala)] and the variant NM_000203.4(IDUA):c.246C>G (p.His82Gln). The variant p.(Gly84Ala) was a recently reported variant, predicted as possibly pathogenic by in silico analysis and located at the same codon where two pathogenic variants were already described [18]. The variant p.His82Gln was previously described as benign and possibly leading to pseudodeficiency, resulting to low in vitro enzyme activity in normal subjects [28], [29], [30].…”

Assessing the Performance of Dried-Blood-Spot DNA Extraction Methods in Next Generation Sequencing

“…Sanger sequencing was performed on all exons, intron/exon borders and a region of interest in intron 22 known to contain the CF-causing variant, 3849+10kbC>T (c.3717+12191C>T), according to the method described in Hendrix et al [16].…”

“…Data from Sanger sequencing was analyzed using SeqScape software (version 3) (Thermo Fisher Scientific) with GenBank CFTR genomic reference sequence NG_016465 according to the method described in Hendrix et al [16]. All analyzed NGS data were then compared for concordance against the Sanger sequence data.…”

“…The Accel-Amplicon™ CFTR panel is a commercially available NGS library preparation that is optimized to work with a variety of lower quality or quantity sample types including DBS-extracted DNA, with the optimal recommended DNA input between 10-25 ng [16]. Twenty samples were extracted…”

“…CDC's Newborn Screening Quality Assurance Program (NSQAP), part of the Newborn Screening and Molecular Biology Branch (NSMBB), is mandated by Congress under the Newborn Screening Saves Lives Act to provide proficiency-testing (PT) and quality-assurance services for NBS laboratories [14]. For the molecular detection of CF, NSQAP provides a PT program to detect pathogenic variants in the CFTR gene that can result in CF [15,16]. CDC's CF DNA DBS repository is made from anonymous donor-CF-patient and family-donor blood samples and contains a diverse number of CFTR pathogenic variants including the 23 recommended by the American College of Medical Genetics (ACMG), as well as 58 expanded pathogenic variants.…”