Noncanonical Splice Site and Deep Intronic FRMD7 Variants Activate Cryptic Exons in X-linked Infantile Nystagmus

Abstract

Purpose We aim to report noncoding pathogenic variants in patients with FRMD7 -related infantile nystagmus (FIN). Methods Genome sequencing ( n = 2 families) and reanalysis of targeted panel next generation sequencing ( n = 2 families) was performed in genetically unsolved cases of suspected FIN. Previous sequence analysis showed no pathogenic coding variants in genes associated with infantile nystagmus. SpliceAI, SpliceRover, and Alamut consensus programs were used to annotate noncoding variants. Minigene splicing assay was performed to confirm aberrant splicing. In silico analysis of exonic splicing enhancer and silencer was also performed. Results FRMD7 intronic variants were identified based on genome sequencing and targeted next-generation sequencing analysis. These included c.285-12A>G (pedigree 1), c.284+63T>A (pedigrees 2 and 3), and c. 383-1368A>G (pedigree 4). All variants were absent in gnomAD, and the both c.285-12A>G and c.284+63T>A variants were predicted to enhance new splicing acceptor gains with SpliceAI, SpliceRover, and Alamut consensus approaches. However, the c.383-1368 A>G variant only had a significant impact score on the SpliceRover program. The c.383-1368A>G variant was predicted to promote pseudoexon inclusion by binding of exonic splicing enhancer. Aberrant exonizations were validated through minigene constructs, and all variants were segregated in the families. Conclusions Deep learning–based annotation of noncoding variants facilitates the discovery of hidden genetic variations in patients with FIN. This study provides evidence of effectiveness of combined deep learning–based splicing tools to identify hidden pathogenic variants in previously unsolved patients with infantile nystagmus. Translational Relevance These results demonstrate robust analysis using two deep learning splicing predictions and in vitro functional study can lead to finding hidden genetic variations in unsolved patients.

Genetic spectrum and characteristics of autosomal optic neuropathy in Korean: Use of next-generation sequencing in suspected hereditary optic atrophy

“…Pooled libraries were sequenced using NextSeq 550 for the targeted sequencing panel and NovaSeq6000 (Illumina, San Diego, CA, USA) for ES or GS. NGS data analysis was performed primarily through our custom pipeline ( 9 , 15 ). The interpretation of variants was done according to the 5-tier classification system recommended by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology using a step-by-step approach ( Supplementary Figure 1 ) ( 16 ).…”