P.26 - COL6A genes transcriptomic by RNAseq and fluidic card tools

Abstract

Mutations in collagen VI A1, A2, and A3 genes cause collagen VI related myopathies, ranging from the severe Ullrich congenital muscular dystrophy (UCMD) to the milder Bethlem myopathy (BM). COL6A1 gene produces a single transcript. COL6A2 gene is transcribed in multiple alternatively spliced mRNAs that differ in the 5'-untranslated region as well as in the 3'-coding and noncoding sequences. COL6A3 transcription is even more complex with various N-terminal exons subjected to multiple alternative splicing. The pattern of isoforms representation in muscle and their functional role has been fairly investigated. Furthermore, COL6A genes mutations affecting splicing are a frequent cause of UCMD-BM phenotypes (around 20% of cases), with genotype-phenotype correlations often unclear. In these cases, non-sense mediated decay of aberrant transcript represents a frequent event, often hampering the detection of mutated transcripts. We have compared two different approaches, RNAseq and a TaqMan® low-density array for Real-Time transcription analysis, for the ability to profile COL6A genes isoforms and to detected mutated transcripts resulting for splicing mutations. RNA-seq was performed on three COL6A mutated patients, carrying either a small in-frame deletion, a mutation expected to induce NMD (homozygous nonsense change) and a canonical splicing mutation. In parallel, the ability of the fluidic card tool to detect splicing mutations and to profile COL6A genes isoforms was also tested in patients and control muscle samples. The RNA-seq turned out to be the optimal strategy, able to pick-up low quantity COL6A genes mRNAs derived both from physiological alternative splicing and from pathogenic mutations. We constructed a RNA-seq based map of COL6A genes isoforms expression in normal and affected muscles.