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A.R. Webster, N. Waseem, S.A. Jenkins, S.S. Bhattacharya, A.T. Moore, R.H. H. Henderson; The Utility of Parallel Genotyping With DNA Arrays in the Molecular Diagnosis of Early–Onset Retinal Dystrophy . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1689.
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© ARVO (1962-2015); The Authors (2016-present)
Early onset retinal dystrophy (EORD), in which patients present with reduced retinal function before the age of 5 years, shows high genetic heterogeneity; 9 identified genes being responsible for approximately 50% of cases. This complexity hinders efficient molecular diagnosis. One approach is to use DNA arrays to interrogate many distinct variants in one hybridisation reaction.
100 unrelated patients with EORD were screened using an array containing 344 published sequence variants in eight genes – AIPL1, GUCY2D, CRB1, CRX, RPGRIP1, RPE65, MERTK and LRAT (AsperBio©). A detailed clinical examination of all probands was performed. DNA was PCR–amplified and hybridised to the array prior to an arrayed–primer–extension–reaction (APEX©). To test the accuracy of the technique, all probands underwent bidirectional sequencing of the whole coding region of the RPE65 gene.
Of the possible 68,900 interrogations on the 100 samples, 1094 (1.6%) failed on one strand whilst 178 (0.26%) failed on both. 3/344 variants gave consist low calls (n>10). Likely disease–associated variants were identified in 50 alleles of 36 patients. 12 patients had two alleles identified, whilst 23 patients had one. In one patient three alleles in two genes were detected. The number of patients identified with mutations were: CRB1 (11) GUCY2D (8) RPE65 (3) and AILP1 (8), RPGRIP1 (5) & CRX (1). No mutations were found in LRAT or MERTK. The C948Y CRB1 allele and P701S GUCY2D allele were especially prevalent (9 and 8 alleles respectively). An SNP assay for the P701S change in 378 normal control alleles found 12 heterozygotes and this allele frequency did not significantly differ from that in patients (8/200 v 12/378). Direct sequencing of RPE65 showed only 1 discrepancy (a heterozygote for D167Y on the array with normal sequence) out of 82 variants of 100 samples interrogated. A further 6 (2 homozygous & 4 heterozygous) likely disease–causing variants, not on the array, in 6 patients, were identified in RPE65.
This technology is highly accurate and robust at the molecular level. A definitive molecular diagnosis was determined in 12% patients. Caution is required in assigning a diagnosis to those with one likely mutation detected (24%). There remains further allelic heterogeneity at least in the RPE65 gene, not accounted for on the chip. GUCY2D P701S is unlikely to be disease–causing.
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