An assessment of the apex microarray technology in genotyping patients with Leber congenital amaurosis and early-onset severe retinal dystrophy.
OBJECTIVE
Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy (EOSRD) are genetically heterogeneous, with 11 genes currently implicated. The LCA chip may be used to interrogate many variants in one hybridization reaction. The purpose of this study was to assess the utility of this technology.


METHODS
One hundred fifty-three patients with LCA and EOSRD were screened using an array (Asper Ophthalmics, Tartu, Estonia) containing 344 published disease-causing variants and polymorphisms in eight genes: AIPL1, GUCY2D, CRB1, CRX, RPGRIP1, RPE65, MERTK, and LRAT. One hundred thirty-six probands underwent bidirectional sequencing of the full coding region of the RPE65 gene. The same technique was also used to confirm CRB1 and AIPL1 mutations initially identified with the Apex chip (Asper Ophthalmics). Single nucleotide polymorphism (SNP) analysis within control populations was performed for two variants, P701S and W21R, on the chip for GUCY2D.


RESULTS
Of the possible 109,392 interrogations, 3,346 (3.06%) failed on one strand whereas 259 (0.47%) failed on both. The chip reported mutations in 68 (44%) patients; 26 patients had two alleles identified (17%). Direct sequencing of RPE65 showed no discrepancies, whereas sequencing of AIPL1 and CRB1 revealed seven samples called erroneously. The SNP analysis of both GUCY2D variants revealed equal prevalence in the EOSRD panel and the normal population. Subsequent reanalysis, after excluding these polymorphisms, revealed one (18.3%) or two (11.7%) mutations identified in 46 patients. When evaluated by diagnosis, 46% of patients with LCA had one or two mutations identified, compared with 24% of patients with EOSRD.


CONCLUSIONS
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.
Moorfields Eye Hospital, London, United Kingdom.
Robert H
RH
Henderson
Naushin
N
Waseem
Rowan
R
Searle
Jacqueline
J
van der Spuy
Isabelle
I
Russell-Eggitt
Shomi S
SS
Bhattacharya
Dorothy A
DA
Thompson
Graham E
GE
Holder
Michael E
ME
Cheetham
Andrew R
AR
Webster
Anthony T
AT
Moore
D048868:Adaptor Proteins, Signal Transducing; D001766:Blindness / Q000151:congenital / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D003598:Cytoskeletal Proteins; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling; D005838:Genotype; D006162:Guanylate Cyclase / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011506:Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics; D012162:Retinal Degeneration / Q000151:congenital / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics; D000076202:c-Mer Tyrosine Kinase; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006162:Guanylate Cyclase; D018398:Homeodomain Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020794:Receptor Protein-Tyrosine Kinases; D011956:Receptors, Cell Surface; D015534:Trans-Activators; D000076202:c-Mer Tyrosine Kinase; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C403625:AIPL1 protein, human; C448939:CRB1 protein, human; C415903:RPGRIP1 protein, human; C109288:cone rod homeobox protein; C110715:guanylate cyclase 1; C097072:MERTK protein, human