Large‐scale Molecular Analysis of a 34 Mb Interval on Chromosome 6q: Major Refinement of the RP25 Interval

Abstract

SummaryA large scale bioinformatics and molecular analysis of a 34 Mb interval on chromosome 6q12 was undertaken as part of our ongoing study to identify the gene responsible for an autosomal recessive retinitis pigmentosa (arRP) locus, RP25. Extensive bioinformatics analysis indicated in excess of 110 genes within the region and we also noted unfinished sequence on chromosome 6q in the Human Genome Database, between 58 and 61.2 Mb. Forty three genes within the RP25 interval were considered as good candidates for mutation screening. Direct sequence analysis of the selected genes in 7 Spanish families with arRP revealed a total of 244 sequence variants, of which 67 were novel but none were pathogenic. This, together with previous reports, excludes 60 genes within the interval (∼ 55%) as disease causing for RP. To investigate if copy number variation (CNV) exists within RP25, a comparative genomic hybridization (CGH) analysis was performed on a consanguineous family. A clone from the tiling path array, chr6tp-19C7, spanning ∼ 100-Kb was found to be deleted in all affected members of the family, leading to a major refinement of the interval. This will eventually have a significant impact on cloning of the RP25 gene.

Identification of a 2 Mb Human Ortholog of Drosophila eyes shut/spacemaker that Is Mutated in Patients with Retinitis Pigmentosa

“…The EYS gene is located on chromosome 6q12 and resides within the 15 Mb RP25 locus. 3,4 Recently, a~100 kb clone from a tiling-path array located within the RP25 interval was found to be deleted in all affected members of a Spanish family linked to RP25, 8 suggesting that genes residing within this deletion might be underlying RP in families linked to RP25. On the basis of the array-CGH data, the total length of the deletion was predicted to be 100-200 kb in size, spanning EYS exons 14-19.…”

“…[5][6][7][8][9][10][11][12][13][14][15] Recently, the RP25 locus was significantly reduced by linkage studies in additional Spanish families and the identification of a 100-200 kb deletion in one of the linked families, but the causative gene has not yet been identified. 3,8 Homozygosity mapping has proven to be an effective approach in the search for genes [16][17][18] and in the discovery of mutations in known arRP genes. 19 The purpose of this study was to identify retinal dystrophy genes, utilizing homozygosity mapping with SNP microarray technology.…”

EYS, encoding an ortholog of Drosophila spacemaker, is mutated in autosomal recessive retinitis pigmentosa

“…In parallel, a high-throughput screening for deletions was undertaken using array comparative genomic hybridization (array CGH). Notably, we found that a ~100-kb clone (chr6tp-19C7) was deleted in all affected members of one of the originally linked families (RP5)6. This suggested that the deleted clone could contain or overlap with the disease-associated gene (provisionally referred to here as the RP25 gene).…”

“…A detailed review of the RP25 interval revealed information on 110 genes, emphasizing the extent of the work required to identify the causative gene. We therefore adopted the approach of (i) exclusion of 15 candidate genes, such as GABRR1 , GABRR2 , MYO6 , EEF1A1 , ELOVL4 , RIMS1 , IMPG1 and LCA5 ( C6ORF52 )5, on the basis of their known retina-related function or their involvement in other retinal degenerations overlapping with RP25; and (ii) systematic screening of a further 45 genes, leading to the exclusion of 60 of the original 110 genes6. At this stage, mapping of five additional families at the RP25 locus helped refine the disease interval to a 2.67-cM region between D6S257 and D6S1557, thereby focusing our search for the disease-associated gene to a much smaller interval7.…”