Improving Genetic Diagnosis Yield in Congenital Muscular Dystrophy through Cryptic Splicing Analysis



Poster Number: 179


Kyle Flannery, MS, Rutgers Robert Wood Johnson Medical School, Sylvia Safwat, MD, Medical Research Institute, Alexandria University, Eric Hoffman, Binghamton University, Chiara Manzini, PhD, Rutgers

Cryptic splice variants are estimated to comprise approximately one-tenth of disease-causing variants, yet this class of genetic variation is largely understudied in standard whole exome sequencing analyses. In this study, we maintained a database of 155 patients with congenital muscular dystrophy (CMD) and achieved a genetic diagnosis rate in 65% through standard whole exome sequencing (WES) analysis of missense, nonsense, and frameshift mutations. In the remaining unsolved cases, WES data was annotated with splice prediction scores from SpliceAI and SQUIRLS and filtered for high-confidence predictions. Three patients with merosin-deficient CMD were found to harbor intronic variants in LAMA2 that were strongly predicted to disrupt splicing (c.3556-13T>A; c.4960-17C>A; c.5234+9A>G) and in heterozygosity with another pathogenic variant. A homozygous deep intronic variant in POMT2 (c.817-394A>G) was also strongly predicted to disrupt splicing in one patient with Walker Warburg syndrome. In addition to intronic variants, two synonymous variants predicted to disrupt splicing were identified in our analyses: one in POMGNT1 (c.570C>T) in a patient with Muscle Eye Brain disease in heterozygosity with a splice donor variant, and one homozygous variant in LAMA2 (c.1884G>A) in a patient presenting with features of both dystroglycanopathy and merosin-deficient CMD who had previously been diagnosed as having a deleterious missense variant in B3GALNT2. Interestingly, LAMA2:c.3556-13T>A and POMGNT1:c.570C>T have been previously validated through cDNA analysis by other studies. Minigene splicing assays are in progress to experimentally confirm the consequence of each of the four remaining variants. We believe that further expansion of cryptic splicing analysis through whole genome sequencing and RNA-seq will aid in identification of additional cryptic splice variants in unsolved cases, thus further improving genetic diagnosis yield in this cohort.