Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

[摘要] Purpose: Mutations in IDH3B, an enzymeparticipating in the Krebs cycle, have recently been found to causeautosomal recessive retinitis pigmentosa (arRP). The MDH1 genemaps within the RP28 arRP linkage interval and encodescytoplasmic malate dehydrogenase, an enzyme functionally related toIDH3B. As a proof of concept for candidate gene screening to beroutinely performed by ultra high throughput sequencing (UHTs), weanalyzed MDH1 in a patient from each of the two familiesdescribed so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, weamplified all introns and exons of the MDH1 gene(23.4 kb). PCR products were then sequenced by short-read UHTswith no further processing. Computer-based mapping of the reads andmutation detection were performed by three independent softwarepackages. Results: Despite the intrinsiccomplexity of human genome sequences, reads were easily mapped andanalyzed, and all algorithms used provided the same results. The twopatients were homozygous for all DNA variants identified in the region,which confirms previous linkage and homozygosity mapping results, buthad different haplotypes, indicating genetic or allelic heterogeneity.None of the DNA changes detected could be associated with the disease. Conclusions: The MDH1 gene isnot the cause of RP28-linked arRP. Our experimental strategyshows that long-range genomic PCR followed by UHTs provides anexcellent system to perform a thorough screening of candidate genes forhereditary retinal degeneration.

[学科分类] 生物化学/生物物理