Comprehensive SNP-chip for retinitis pigmentosa-Leber congenital amaurosis diagnosis: new mutations and detection of mutational founder effects

Abstract

Fast and efficient high-throughput techniques are essential for the molecular diagnosis of highly heterogeneous hereditary diseases, such as retinitis pigmentosa (RP). We had previously approached RP genetic testing by devising a chip based on co-segregation analysis for the autosomal recessive forms. In this study, we aimed to design a diagnostic tool for all the known genes (40 up to now) responsible for the autosomal dominant and recessive RP and Leber congenital amaurosis (LCA). This new chip analyzes 240 single nucleotide polymorphisms (SNPs) (6 per gene) on a high-throughput genotyping platform (SNPlex, Applied Biosystems), and genetic diagnosis is based on the co-segregation analysis of SNP haplotypes in independent families. In a single genotyping step, the number of RP candidates to be screened for mutations is considerably reduced, and in the most informative families, all the candidates are ruled out at once. In a panel of RP Spanish pedigrees, the disease chip became a crucial tool for selecting those suitable for genome-wide RP gene search, and saved the burdensome direct mutational screening of every known RP gene. In a large adRP family, the chip allowed ruling out of all but the causative gene, and identification of an unreported null mutation (E181X) in PRPF31. Finally, on the basis of the conservation of the SNP haplotype linked to this pathogenic variant, we propose that the E181X mutation spread through a cohort of geographically isolated families by a founder effect.

Mutations in Splicing Factor Genes Are a Major Cause of Autosomal Dominant Retinitis Pigmentosa in Belgian Families

“…Overall, mutations in splicing factor genes are the second most common cause of adRP [2]. We identified heterozygous mutations in adRP splicing genes ( SNRNP200 , PRPF8 , PRPF31 ) in 17 probands (Table 1) [2,26,28,47–49]. …”

HSG/Mfn2 Gene Polymorphism and Essential Hypertension: a Case-Control Association Study in Chinese

“…This genotyping kit, TaqMan ® PCR Master Mix, No AmpErase ® UNG and approximately 5 ng genomic DNA were employed in a final volume of 5 L. A 7300 Real-Time PCR System was used for amplification, and the first step of the thermocycle was initial denaturation and activation at 95 for 10 minutes, followed by 40 cycles of 95 for 15 seconds and 60 for 1 minute. SNP kits for C_26207636_10 (rs4240897), C_1267235_20 (rs1474868), C_11461995_10 (rs2336384), C_8861262_10 ( r s 8 7 3 4 5 8 ) , C _ 1 1 4 6 1 9 9 6 _ 2 0 ( r s 8 7 3 4 5 7 ) , C_1267226_10 (rs4846085) were used and the genotypes detected following the instructions supplied by Applied Biosystems 12) .…”

Autozygome-guided exome sequencing in retinal dystrophy patients reveals pathogenetic mutations and novel candidate disease genes

“…The resequencing chip theoretically circumvents this limitation, but the prerequisite step of amplifying all known RD genes represents a major challenge (Booij et al 2011). We and others have shown that the autozygome approach can be very effective in guiding the mutation analysis (Aldahmesh et al 2009;Pomares et al 2010). Interestingly, this approach was also used successfully in populations where consanguinity is uncommon (Hildebrandt et al 2009;Collin et al 2011;Hagiwara et al 2011;Schuurs-Hoeijmakers et al 2011).…”