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A6323 - Multiplexed Direct Genomic Selection to Identify Non-Coding and Copy Number Variation in ABCA3 Among Neonates with Respiratory Failure and Children with Interstitial Lung Disease (chILD) with Monoallelic ABCA3 Variants
Author Block: J. Wambach1, J. B. Tam-Williams2, D. J. Wegner3, D. M. Alvarado4, A. Hamvas5, F. Cole3, L. M. Nogee6; 1Pediatrics, Washington University School of Medicine, St. Louis, MO, United States, 2Pediatrics, Children's Mercy Hospital, Kansas City, MO, United States, 3Pediatrics, Washington University School of Medicine, Saint Louis, MO, United States, 4Orthopedics, Washington University School of Medicine, Saint Louis, MO, United States, 5Division of Neonatology, Northwestern University Feinberg School of Medicine, Chicago, IL, United States, 6Johns Hopkins Childrens Ctr, Baltimore, MD, United States.
Introduction: ABCA3 deficiency is a recessive disorder characterized by progressive neonatal respiratory failure (NRF) or childhood interstitial lung disease (chILD). Infants and children with ABCA3 deficient phenotypes but only a monoallelic ABCA3 variant have been reported. Large deletions involving the ABCA3 locus that may be missed by commonly used sequencing strategies have been reported among symptomatic individuals and may account for ABCA3 deficiency. Multiplexed direct genomic selection (MDiGS) uses bacterial artificial chromosomes (BAC) to capture and sequence genomic regions for detection of exonic, intronic and copy number variants (CNVs).
Objective: To use MDiGS with BAC capture to identify non-coding and copy number variants of the ABCA3 locus among infants and children with ABCA3 deficient phenotypes but only a monoallelic ABCA3 variant identified by Sanger sequencing.
Methods: We identified 48 infants and children with severe respiratory distress syndrome (RDS) that resolved (n=20), progressive NRF (n=13) or chILD (n=15) and a monoallelic ABCA3 variant. We included 2 samples with previously identified CNVs in ABCA3 as positive controls. After shearing DNA by sonication, we performed next generation library construction using unique sequence indexes for each sample and pooled all samples. We used 2 biotinylated BACs to capture the ABCA3 locus as well as noncoding regions 177kb upstream and 94kb downstream. We performed next generation sequencing in a single run with a MiSeq instrument and aligned data to the human genome (hg19) using NovoAlign. Single nucleotide variants (SNVs) and small insertion/deletions were identified with SamTools. We used read depth analysis, 5’ paired end analysis, and split read analysis to identify CNVs.
Results: We detected all previously identified ABCA3 exonic variants and the 2 previously identified CNVs. We identified an additional exonic variant in an infant with NRF not previously identified with Sanger sequencing: a maternally-inherited missense variant (p.G378R) in trans with a paternally-inherited 21 base pair insertion. The DECIPHER database (http://decipher.sanger.ac.uk) of ~25,000 individuals includes 4 individuals with large deletions (40kb-372kb), 25 individuals with large duplications (538kb-20Mb) involving ABCA3 (0.1% of individuals) and no common large CNVs involving ABCA3.
Conclusions: MDiGS reliably detects SNVs and CNVs involving ABCA3. In this symptomatic cohort and a large adult cohort, large CNVs involving ABCA3 are rare. Parental samples inform variant orientation and may improve diagnostic yield for infants and children with a monoallelic ABCA3 variant and NRF or chILD. Additional genetic, epigenetic or environmental factors likely influence disease expression in these individuals.