Development of a diagnostic genetic test for simplex and autosomal recessive retinitis pigmentosa.
OBJECTIVE
Retinitis pigmentosa (RP) causes hereditary blindness in adults (prevalence, approximately 1 in 4000). Each of the more than 30 causative genes identified to date are responsible for only a small percentage of cases. Genetic diagnosis via traditional methods is problematic, and a single test with a higher probability of detecting the causative mutation would be very beneficial for the clinician. The goal of this study therefore was to develop a high-throughput screen capable of detecting both known mutations and novel mutations within all genes implicated in autosomal recessive or simplex RP.


METHODS
Evaluation of diagnostic technology.


METHODS
Participants were 56 simplex and autosomal recessive RP patients, with 360 population controls unscreened for ophthalmic disease.


METHODS
A custom genechip capable of resequencing all exons containing known mutations in 19 disease-associated genes was developed (RP genechip). A second, commercially available arrayed primer extension (APEX) system was used to screen 501 individual previously reported variants. The ability of these high-throughput approaches to identify pathogenic variants was assessed in a cohort of simplex and autosomal recessive RP patients.


METHODS
Number of mutations and potentially pathogenic variants identified.


RESULTS
The RP genechip identified 44 sequence variants: 5 previously reported mutations; 22 known single nucleotide polymorphisms (SNPs); 11 novel, potentially pathogenic variants; and 6 novel SNPs. There was strong concordance with the APEX array, but only the RP genechip detected novel variants. For example, identification of a novel mutation in CRB1 revealed a patient, who also had a single previously known CRB1 mutation, to be a compound heterozygote. In some individuals, potentially pathogenic variants were discovered in more than one gene, consistent with the existence of disease modifier effects resulting from mutations at a second locus.


CONCLUSIONS
The RP genechip provides the significant advantage of detecting novel variants and could be expected to detect at least one pathogenic variant in more than 50% of patients. The APEX array provides a reliable method to detect known pathogenic variants in autosomal recessive RP and simplex RP patients and is commercially available. High-throughput genotyping for RP is evolving into a clinically useful genetic diagnostic tool.


BACKGROUND
The author(s) have no proprietary or commercial interest in any materials discussed in this article.
Clark
Graeme Richard
GR
Centre for Vision and Vascular Science, Queen's University, Belfast, Northern Ireland.
Crowe
Paul
P
Muszynska
Dorota
D
O'Prey
Dominic
D
O'Neill
Justin
J
Alexander
Sharon
S
Willoughby
Colin Eric
CE
McKay
Gareth John
GJ
Silvestri
Giuliana
G
Simpson
David Arthur
DA
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D004596:Electroretinography; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D005808:Genes, Recessive; D005820:Genetic Testing; D005838:Genotype; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010375:Pedigree; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012174:Retinitis Pigmentosa / Q000175:diagnosis* / Q000235:genetics*; D014792:Visual Acuity
D005136:Eye Proteins