Abstract
The goal of this study was to identify stop-gain variants in FLG and closely related genes in African Americans with AD from the Pediatric Eczema Elective Registry (PEER; Margolis et al., 2012 ). From this cohort we randomly selected 60 subjects for whole-exome sequencing to ensure sufficient power to detect variants with a minor allelic frequency (MAF) of greater than 3%. Sequencing was performed by Ambry Genetics (Aliso Viejo, CA) using whole exome–targeted enrichment by Agilent SureSelectXT Human All Exon 50Mb kit. Quality assessment revealed that most samples were above 50% on target and mean coverage per gene was excellent. The libraries were indexed using 100 base paired ends and processed using Illumina HiSeq2000 at × 100 coverage per exon. Data were assessed using a pipeline generated at the University of Pennsylvania based on the best practices protocol from the Broad Institute (Cambridge, MA). This report focused on stop-gain mutations of exon 3 (i.e., loss-of-function mutations) in the SFTP genes because of their likely functional relevance (Marenholz et al., 2011 Brown and McLean, 2012 ; Henry et al., 2012). Taqman allelic discrimination assays were created for any newly identified FLG loss-of-function mutations, which were then used to genotype an additional random sample of 100 African-American PEER children.