Identification and Functional Analysis of Genetic Variants Causing Neurodevelopment Disorders and Diamond Blackfan Anemia

Abstract

Next generation sequencing has the potential to detect the entire set of variations in a genome, however, due to challenges in interpretation, a large number of patients with neurodevelopmental disorders (NDDs) and some rare blood disorders are still awaiting a definitive diagnosis. To meet this challenge, data from genetic and functional approaches were combined to improve diagnostic yield and mechanistic understanding. Whole exome sequencing was employed to identify splice site variants (RPS7: c.508- 3T>G; PNKP: c.1299 -3C>G; LAMA1: c.7196 -7 G>A; and CDH1: c.1009-3C>G) underlying rare genetic disorders in four unrelated families characterized with different neurodevelopmental disorders and Diamond Blackfan Anemia. The effect of candidate splice site variants on mRNA processing was assessed by in silico predications, patient’s mRNA analysis and a genomic DNA based RNA splicing assay. Mini-gene splicing assays showed abnormal splicing for RPS7 and PNKP splice variants; LAMA1 splicing remained intact. Furthermore, assessment of mRNA processing using RNA obtained from a CDH1 patient showed aberrant splicing. These findings explain the pathogenicity of splice variants and pinpoint the significance of functional verification of in silico findings using in vitro system. Independent of splice variants analysis, current work also generated in vitro cellular models for ten randomly selected genes associated with NDDs in humans. Induced pluripotent stem cells (iPSCs) derived from healthy individuals were used to generate null KO lines by CRISPR/Cas9 double gRNA technology. Phenotypic characterization and assessment of pluripotency validated the genomic integrity and ability to form different body cells for IKBKG and NCDN KO lines. Additionally, phenotypic evaluation of NCDN KO SH-SY5Y line showed functionally relevant phenotypes, including abnormal neurogenesis, using in vitro neurite growth assay. In another consanguineous family with mild intellectual disability and recurrent seizures, WES identified a rare deleterious variant in NCDN gene. Three additional families with NCDN mutations and overlapping clinical features were identified through a public data sharing platform, GeneMatcher. In vitro complementation assay in SH-SY5Y cells confirmed pathogenicity of these mutations. Altogether, these findings highlight the xiv utility of combinatorial approaches to assess the pathogenicity of gene variants with unknown significance, improve our understanding of disease gene function and provide useful cellular models for prospective patho-mechanism and therapeutic intervention.