PurposeTo define the molecular basis of retinal degeneration in consanguineous Pakistani pedigrees with early onset retinal degeneration.MethodsA cohort of 277 individuals representing 26 pedigrees from the Punjab province of Pakistan was analyzed. Exomes were captured with commercial kits and sequenced on an Illumina HiSeq 2500. Candidate variants were identified using standard tools and analyzed using exomeSuite to detect all potentially pathogenic changes in genes implicated in retinal degeneration. Segregation analysis was performed by dideoxy sequencing and novel variants were additionally investigated for their presence in ethnicity-matched controls.ResultsWe identified a total of nine causal mutations, including six novel variants in RPE65, LCA5, USH2A, CNGB1, FAM161A, CERKL and GUCY2D as the underlying cause of inherited retinal degenerations in 13 of 26 pedigrees. In addition to the causal variants, a total of 200 variants each observed in five or more unrelated pedigrees investigated in this study that were absent from the dbSNP, HapMap, 1000 Genomes, NHLBI ESP6500, and ExAC databases were identified, suggesting that they are common in, and unique to the Pakistani population.ConclusionsWe identified causal mutations associated with retinal degeneration in nearly half of the pedigrees investigated in this study through next generation whole exome sequencing. All novel variants detected in this study through exome sequencing have been cataloged providing a reference database of variants common in, and unique to the Pakistani population.

While more than 250 genes are known to cause inherited retinal degenerations (IRD), nearly 40-50% of families have the genetic basis for their disease unknown. In this study we sought to identify the underlying cause of IRD in a family by whole genome sequence (WGS) analysis. Clinical characterization including standard ophthalmic examination, fundus photography, visual field testing, electroretinography, and review of medical and family history was performed. WGS was performed on affected and unaffected family members using Illumina HiSeq X10. Sequence reads were aligned to hg19 using BWA-MEM and variant calling was performed with Genome Analysis Toolkit. The called variants were annotated with SnpEff v4.11, PolyPhen v2.2.2, and CADD v1.3. Copy number variations were called using Genome STRiP (svtoolkit 2.00.1611) and SpeedSeq software. Variants were filtered to detect rare potentially deleterious variants segregating with disease. Candidate variants were validated by dideoxy sequencing. Clinical evaluation revealed typical adolescent-onset recessive retinitis pigmentosa (arRP) in affected members. WGS identified about 4 million variants in each individual. Two rare and potentially deleterious compound heterozygous variants p.Arg281Cys and p.Arg487* were identified in the gene ATP/GTP binding protein like 5 (AGBL5) as likely causal variants. No additional variants in IRD genes that segregated with disease were identified. Mutation analysis confirmed the segregation of these variants with the IRD in the pedigree. Homology models indicated destabilization of AGBL5 due to the p.Arg281Cys change. Our findings establish the involvement of mutations in AGBL5 in RP and validate the WGS variant filtering pipeline we designed.

Inherited retinal dystrophies are a group of genetically heterogeneous conditions with broad phenotypic heterogeneity. We analyzed a large five-generation pedigree with early-onset recessive retinal degeneration to identify the causative mutation. Linkage analysis and homozygosity mapping combined with exome sequencing were carried out to map the disease locus and identify the p.G178R mutation in the asparaginase like-1 gene (ASRGL1), segregating with the retinal dystrophy phenotype in the study pedigree. ASRGL1 encodes an enzyme that catalyzes the hydrolysis of L-asparagine and isoaspartyl-peptides. Studies on the ASRGL1 expressed in Escherichia coli and transiently transfected mammalian cells indicated that the p.G178R mutation impairs the autocatalytic processing of this enzyme resulting in the loss of functional ASRGL1 and leaving the inactive precursor protein as a destabilized and aggregation-prone protein. A zebrafish model overexpressing the mutant hASRGL1 developed retinal abnormalities and loss of cone photoreceptors. Our studies suggest that the p.G178R mutation in ASRGL1 leads to photoreceptor degeneration resulting in progressive vision loss.