Biology subjects

Comander, J.

Publications and source records attributed to Comander, J..

2 recordsLinked to original sources

bioRxiv2019

Copy-number variation contributes 9% of pathogenicity in the inherited retinal degenerations

PurposeCurrent sequencing strategies can genetically solve 55-60% of inherited retinal degeneration (IRD) cases, despite recent progress in sequencing. This can partially be attributed to elusive pathogenic variants (PVs) in known IRD genes, including copy number variations (CNVs), which we believe are a major contributor to unsolved IRD cases.\n\nMethodsFive hundred IRD patients were analyzed with targeted next generation sequencing (NGS). The NGS data was used to detect CNVs with ExomeDepth and gCNV and the results were compared to CNV detection with a SNP-Array. Likely causal CNV predictions were validated by quantitative (q)PCR.\n\nResultsLikely disease-causing single nucleotide variants (SNVs) and small indels were found in 55.8% of subjects. PVs in USH2A (11.6%), RPGR (4%) and EYS (4%) were the most common. Likely causal CNVs were found in an additional 8.8% of patients. Of the three CNV detection methods, gCNV showed the highest accuracy. Approximately 30% of unsolved subjects had a single likely PV in a recessive IRD gene.\n\nConclusionsCNV detection using NGS-based algorithms is a reliable method that greatly increases the genetic diagnostic rate of IRDs. Experimentally validating CNVs helps estimate the rate at which IRDs might be solved by a CNV plus a more elusive variant.

genomics↗

bioRxiv2019

Characterizing variants of unknown significance inrhodopsin: a functional genomics approach

Characterizing the pathogenicity of DNA sequence variants of unknown significance (VUS) is a major bottleneck in human genetics, and is increasingly important in determining which patients with inherited retinal diseases could benefit from gene therapy. A library of 210 rhodopsin (RHO) variants from literature and in-house genetic diagnostic testing was created to efficiently detect pathogenic RHO variants that fail to express on the cell surface. This study, while focused on RHO, demonstrates a streamlined, generalizable method for detecting pathogenic VUS. A relatively simple next generation sequencing (NGS)-based readout was developed so that a flow cytometry-based assay could be performed simultaneously on all variants in a pooled format, without the need for barcodes or viral transduction. The resulting dataset characterized surface expression of every RHO library variant with a high degree of reproducibility (Z=0.94, R2=0.92-0.95), recategorizing 37 variants. For example, three retinitis pigmentosa pedigrees were solved by identifying VUS which showed low expression levels (G18D, G101V, P180T). Results were validated across multiple assays and correlated with clinical disease severity. This study presents a parallelized, higher-throughput cell-based assay for the functional characterization of VUS in rhodopsin, and can be applied more broadly to other inherited retinal disease genes and other disorders.

genetics↗