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Wei Ru Li, Chien Ting Wu, Navneet Mander, Jie Duan, John Chiang; Comprehensive Molecular Diagnosis of Inherited Retinal Degeneration by Combining NGS and aCGH Techniques. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3291.
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Molecular diagnosis of inherited retinal degeneration has been difficult due to the following reasons: (1) locus heterogeneity (more than 200 genes involved); (2) allele heterogeneity (many private mutations); (3) overlapping clinical presentations; and (4) progressive nature of some conditions. The arrival of mass parallel sequencing: Next Generation Sequencing (NGS) provides an opportunity to revolutionize the task. At Casey Molecular Diagnostic Laboratory, we have developed a PCR based approach of sequencing the entire non-syndromic retinal degeneration genes plus some common syndromic genes (130 eye genes). Based on our initial analysis, mutation detection rate was increased after testing the 130 genes simultaneously. However, it has also become clear to us that indel mutations should be included especially for those patients with one definitive mutation identified in a recessive gene. In order to cover indel mutations, we have developed an array CGH assay for eye genes.
Direct sequencing for mutations of retinal dystrophy panel (130 eye genes) was performed by standard PCR and next generation sequencing (MiSeq Reagent Kits V2, 2X250; MiSeq, Illumina). After NGS data analysis, Sanger sequencing would be performed to fill all gaps and the low coverage (<30X) regions. All exons and exon/intron boundaries were sequenced. aCGH was performed using Cytosure Eye Plus V2 chip designed by Oxford Gene Technology (OGT, UK). The indel mutations were confirmed by either TaqMan qPCR or PCR and gel electrophoresis.
Twenty patients with one mutation identified or with no mutations identified were tested by the array. Three indel mutations in the EYS gene, an indel mutation in RPGRIP1, an indel mutation in CACNA2D4 and finally an indel mutation in ABCA4 were identified and confirmed by either TaqMan qPCR or by PCR and gel electrophoresis. Most interestingly, a patient with the clinical diagnosis of Stargardt but with no detectable mutation by sequencing in ABCA4 was found to possess a deletion of exon 20 to 22 in the ABCA4 gene.
Our initial result suggests that indel mutations should be analyzed in order to complete the search for mutations. By combining our retinal dystrophy NGS panel and aCGH analysis, a more comprehensive and precise diagnosis can be provided in a more cost efficient way.
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