Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

Abstract

Purpose: Leber congenital amaurosis (LCA) is a rare congenital retinal dystrophy associated with 16 genes. Recent breakthroughs in LCA gene therapy offer the first prospect of treating inherited blindness, which requires an unequivocal and early molecular diagnosis. While present genetic tests do not address this due to a tremendous genetic heterogeneity, massively parallel sequencing (MPS) strategies might bring a solution. Here, we developed a comprehensive molecular test for LCA based on targeted MPS of all exons of 16 known LCA genes. methods:We designed a unique and flexible workflow for targeted resequencing of all 236 exons from 16 LCA genes based on quantitative PCR (qPCR) amplicon ligation, shearing, and parallel sequencing of multiple patients on a single lane of a short-read sequencer. Twenty-two prescreened LCA patients were included, five of whom had a known molecular cause.Results: Validation of 107 variations was performed as proof of concept. In addition, the causal genetic defect and a single heterozygous mutation were identified in 3 and 5, respectively, of 17 patients without previously identified mutations. conclusion:We propose a novel targeted MPS-based approach that is suitable for accurate, fast, and cost-effective early molecular testing in LCA, and easily applicable in other genetic disorders.Genet Med 2012:14(6):576-585

Identity-by-descent–guided mutation analysis and exome sequencing in consanguineous families reveals unusual clinical and molecular findings in retinal dystrophy

“…In the first family (F1, LCA), a novel homozygous nonsense mutation was identified in RDH12: c.912G>A (p.Trp304*). 7 This mutation is located in the last exon and thus not expected to induce nonsense-mediated decay. The proband was diagnosed with LCA at the age of 2 years and 3 months based on severe visual deficiency from birth, congenital nystagmus, hyperopia, and night blindness.…”

Expanding the clinical, allelic, and locus heterogeneity of retinal dystrophies

“…This homozygous variant is absent in 615 Saudi exomes and in the ExAC browser (Supplementary Figure S2 online). ABCA4 [13] TULP1 [10] MERTK [8] CRB1 [7] RPE65 [7] RPGRIP1 [7] I MPG2 [6] KCNV2 [6] NR2E3 [6] RP1 [6] ALMS1 [5] CNGA3 [5] RDH12 [5] AIPL1 [4] CNGB1 [4] CRX [4] GUCY2D [4] SPATA7 [4] BBS2 [3] BBS4 [3] C8orf37 [3] CDHR1 [3] FAM161A [3] PDE6C [3] RLBP1 [3] USH2A [3] BBS10 [2] BBS5 [2] BBS7 [2] CERKL [2] CNNM4 [2] GRP179 [2] LRP5 [2] PDE6B [2] P ROM1 [2] PRPH2 [2] RP2 [2] SNRNP200 [2] ADAM9 [1] ADAMTS18 [1] ARL6 [1] BBS9 [1] BEST1 [1] C2ORF71 [1] CACNA1F [1] CEP290 [1] CHM [1] EFEMP1 [1] EYS …”

“…This technology is particularly suited for such a genetically and clinically heterogeneous condition as RD, and this has been demonstrated by several studies. [7][8][9][10][11][12][13] In this study, we describe the application of a next-generation sequencing-based gene panel strategy to study a very large cohort of RD patients in the diagnostic phase. In the discovery phase, we genotyped all familial cases that tested negative on the assay and identified three that mapped to a single locus and subjected them to whole-exome sequencing, which led us to identify AGBL5 and CDH16 as two novel RD candidate disease genes.…”

Clinical application of massively parallel sequencing in the molecular diagnosis of glycogen storage diseases of genetically heterogeneous origin

“…[8][9][10] For clinically and genetically heterogeneous diseases caused by a group of genes involving a common metabolic pathway, MPS can also be used for simultaneous sequencing of the group of candidate genes. [11][12][13] To facilitate Table S1 online). This approach eliminates the hurdle of having to prioritize multiple candidate genes for step-wise sequencing.…”