An Assessment of the Apex Microarray Technology in Genotyping Patients with Leber Congenital Amaurosis and Early-Onset Severe Retinal Dystrophy

Henderson, Robert H.; Waseem, Naushin; Searle, Rowan; Spuy, Jacqueline van der; Russell‐Eggitt, Isabelle; Bhattacharya, Siladitya; Thompson, Dorothy; Holder, Graham E.; Cheetham, Michael E.; Webster, Andrew R.; Moore, Anthony T.

doi:10.1167/iovs.07-0207

Cited by 53 publications

(41 citation statements)

References 27 publications

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“…The first part of this study was mainly aimed at validation of the enrichment protocol (proof of concept, patients 1-10), whereas the second part of this study consisted of a blind screening of 12 prescreened mutation-negative patients with LCA (patients [11][12][13][14][15][16][17][18][19][20][21][22]. enrichment of LcA disease genes qPCR was used to target all exons from 16 LCA disease genes.…”

Section: Resultsmentioning

confidence: 99%

“…Of these, p.Arg768Trp was a missed call on the LCA chip, for which ~3.5% of interrogations fail. 13 Moreover, MPS revealed a second inconsistency of the LCA chip, namely a homozygous instead of heterozygous call for c.907-16_907-14del. Patients 13 and 17 are homozygous for the novel mutations p.Leu59Arg (RDH12) and p.Ser296ProfsX10 (AIPL1), respectively.…”

Section: Discussionmentioning

confidence: 99%

“…For the second lane, libraries were prepared from a 200-300-bp size-selected fraction, and library quantification was performed using qPCR following manufacturer's protocols (lane 2, patients [11][12][13][14][15][16][17][18][19][20][21][22]. In total, 120 µl of a pool of the 12 normalized libraries (concentration of 10 pmolar) was subjected to paired-end sequencing of 2 × 45 cycles.…”

Section: Sequencing On the Illumina Genome Analyzer Iixmentioning

confidence: 99%

“…This study included 17 patients in which previous Sanger sequencing of six genes and/or LCA chip analysis did not identify causal mutations (lane 1: patients 1-3, 7, and 8; lane 2: patients [11][12][13][14][15][16][17][18][19][20][21][22]. Given the exclusion of 3 of 107 known variants by the in 12.7% of the Yoruba population (1000Genomes Project, pilot_1_YRI_low_coverage_panel) points to a polymorphism rather than a mutation (rs61748445).…”

Section: Validation Of the Protocol Using Previously Identified Variamentioning

confidence: 99%

“…12 Although this technique represents a good first-pass screening, it fails in detecting new mutations, is expensive in a routine context, and has a variable, population-dependent detection rate. [13][14][15] Secondary genetic tests comprise denaturing high-performance liquid chromatography and Sanger sequencing, mostly of a subset of genes because of excessive costs and workload. 1,16,17 Hence, an urgent need exists for an effective approach able to identify mutations in all currently known LCA genes.…”

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confidence: 99%

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Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

Coppieters

Wilde

Lefever

et al. 2012

Genetics in Medicine

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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

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Sequencing On the Illumina Genome Analyzer Iixmentioning

confidence: 99%

Section: Validation Of the Protocol Using Previously Identified Variamentioning

confidence: 99%

mentioning

confidence: 99%

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Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

Coppieters

Wilde

Lefever

et al. 2012

Genetics in Medicine

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Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes

Casteels²,

et al. 2010

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Leber Congenital Amaurosis (LCA), the most severe inherited retinal dystrophy, is genetically heterogeneous, with 14 genes accounting for 70% of patients. Here, 91 LCA probands underwent LCA chip analysis and subsequent sequencing of 6 genes (CEP290, CRB1, RPE65, GUCY2D, AIPL1and CRX), revealing mutations in 69% of the cohort, with major involvement of CEP290 (30%). In addition, 11 patients with early-onset retinal dystrophy (EORD) and 13 patients with Senior-Loken syndrome (SLS), LCA-Joubert syndrome (LCA-JS) or cerebello-oculo-renal syndrome (CORS) were included. Exhaustive re-inspection of the overall phenotypes in our LCA cohort revealed novel insights mainly regarding the CEP290-related phenotype. The AHI1 gene was screened as a candidate modifier gene in three patients with the same CEP290 genotype but different neurological involvement. Interestingly, a heterozygous novel AHI1 mutation, p.Asn811Lys, was found in the most severely affected patient. Moreover, AHI1 screening in five other patients with CEP290-related disease and neurological involvement revealed a second novel missense variant, p.His758Pro, in one LCA patient with mild mental retardation and autism. These two AHI1 mutations might thus represent neurological modifiers of CEP290-related disease. © 2010 Wiley-Liss, Inc.

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CRB1 mutations in inherited retinal dystrophies

et al. 2011

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Mutations in the CRB1 gene are associated with variable phenotypes of severe retinal dystrophies, ranging from Leber Congenital Amaurosis (LCA) to rod-cone dystrophy (also called retinitis pigmentosa (RP)). Moreover, retinal dystrophies resulting from CRB1 mutations may be accompanied by specific fundus features: preservation of the para-arteriolar retinal pigment epithelium (PPRPE) and retinal telangiectasia with exudation (also referred to as Coats-like vasculopathy). In this publication we report seven novel mutations and classify over 150 reported CRB1 sequence variants that were found in more that 240 patients. The data from previous reports was used to analyse a potential correlation between CRB1 variants and the clinical features of respective patients. This meta-analysis suggests that the differential phenotype of patients with CRB1 mutations is due to additional modifying factors rather than particular mutant allele combination.

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An Assessment of the Apex Microarray Technology in Genotyping Patients with Leber Congenital Amaurosis and Early-Onset Severe Retinal Dystrophy

Abstract: This approach is a rapid and reasonably low-cost technique for identifying both previously identified mutations and common polymorphisms. The addition of further genes and mutations to the chip will improve its utility, though it is advised that all results be checked by direct sequencing.

Cited by 53 publications

References 27 publications

Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes

CRB1 mutations in inherited retinal dystrophies

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