Identification and characterization of novel collagen VI non-canonical splicing mutations causing ullrich congenital muscular dystrophy

Abstract

Splicing mutations occurring outside the invariant GT and AG dinucleotides are frequent in disease genes and the definition of their pathogenic potential is often challenging. We have identified four patients affected by Ullrich congenital muscular dystrophy and carrying unusual mutations of COL6 genes affecting RNA splicing. In three cases the mutations occurred in the COL6A2 gene and consisted of nucleotide substitutions within the degenerated sequences flanking the canonical dinucleotides. In the fourth case, a genomic deletion occurred which removed the exon8-intron8 junction of the COL6A1 gene. These mutations induced variable splicing phenotypes, consisting of exon skipping, intron retention and cryptic splice site activation/usage. A quantitative RNA assay revealed a reduced level of transcription of the mutated in-frame mRNA originating from a COL6A2 point mutation at intronic position +3. At variance, the transcription level of the mutated in-frame mRNA originating from a genomic deletion which removed the splicing sequences of COL6A1 exon 8 was normal. These findings suggest a different transcriptional efficiency of a regulatory splicing mutation compared to a genomic deletion causing a splicing defect.

Collagen VI at a glance

“…It is well known that ColVI is abundantly expressed by skin fibroblasts, and indeed, human skin biopsies and in vitro primary skin fibroblast cultures have been widely used to characterize ColVI defects in Bethlem myopathy and UCMD individuals (Lamandé et al, 1999(Lamandé et al, , 2002Sasaki et al, 2000;Zhang et al, 2002;Gualandi et al, 2009;Martoni et al, 2009). Not surprisingly, several skin abnormalities, such as keloids or 'cigarette paper' scars, dry skin, striae rubrae and follicular keratosis, have been described in individuals with mutations in the COL6A1, COL6A2 or COL6A3 genes Lettmann et al, 2014).…”

Antisense-Induced Messenger Depletion Corrects a COL6A2 Dominant Mutation in Ullrich Myopathy

“…7/2009) were cultured at 37°C and 5% CO 2 as described (Martoni et al, 2009). Cells were transfected for 4 hr with either AON (200 nM) plus AON ExGen 500 polyethylenimine (PEI) (3.3 ll/lg; MBI Fermentas/Thermo Fisher Scientific, Waltham, MA) or with PEI alone, as described (Evers et al, 2011).…”

“…Cells were harvested 24 hr post-transfection, and total RNA was isolated and retrotranscribed as reported (Martoni et al, 2009). RT-PCR was performed with COL6A primers within exons 2-10 and 4-10 (sequence available on request) to quantify exon 3 skipping, and to assess the proportions of wildtype (exon 9-plus) versus mutated (exon 9-minus) COL6A2 transcripts, calculated as the ratio between each type of transcript and the sum of all transcripts, on an Agilent high-sensitivity DNA chip (Agilent Technologies, Santa Clara, CA) ( Supplementary Fig.…”

“…TaqMan expression assays (Applied Biosystems) were used to quantify COL6A2 and COL6A1 transcripts (Hs00242484_m1 Ex27/28; Hs00242448_m1 Ex20/ 21) with respect to b-actin, the endogenous control. Real-time PCR was performed as described (Martoni et al, 2009). cDNAs from untreated fibroblasts served for calibration.…”

“…Protein content was quantified with a bicinchoninic acid (BCA) protein assay kit (Pierce/Thermo Fisher Scientific, Rockford, IL). Western blotting was performed as previously reported (Martoni et al, 2009). A loading check was performed with anti-b-catenin antibody (diluted 1:1000; BD Biosciences Transduction Laboratories, Lexington, KY) for cell layer extracts and Coomassie staining for culture medium.…”