Characterization of a novel deep intronic COL6A1 c.930+176C>T splice activation variant causing COL6-related dystrophy

Topic:

Other

Poster Number: 116 S

Author(s):

Sandra Donkervoort, MS, CGC, National Institutes of Health, Sarah Silverstein, NIH, Rotem Orbach, MD, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Vijay Ganesh, MD, PhD, Broad Institute, Payam Mohassel, MD, Department of Neurology, Johns Hopkins University School of Medicine, Katherine R Chao, BS, Broad Insitute, Ying Hu, MS, Neuromuscular and Neurogenetic Disorders of Childhood Section, NINDS, National Institutes of Health, Lynn Pais, MS, Broad Institute of MIT and Harvard, A. Reghan Foley, MD, MD(Res), Neuromuscular and Neurogenetic Disorders of Childhood Section, NINDS, National Institutes of Health, Randal Richardson, MD, MMS, Gillette Childrens, Véronique Bolduc, PhD, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Manu Jokela, MD, PhD, Division of Clinical Neurosciences, Turku University Hospital and University of Turku, Carsten G. Bönnemann, MD, habil., Neuromuscular and Neurogenetic Disorders of Childhood Section, NINDS, National Institutes of Health

Collagen 6-related dystrophies (COL6-RD) are a group of disorders that manifests with a phenotypic spectrum, ranging from severe Ullrich congenital muscular dystrophy, intermediate phenotypes, to the milder Bethlem myopathy. Recently, COL6A1 c.930+189C>T was identified as a common recurrent deep intronic variant in patients with severe COL6-RD. Here we identified a novel heterozygous COL6A1 c.930+176C>T variant in the same intron in five patients from two independent families with dominantly inherited COL6-RD. Patients presented with child- to adulthood onset muscle weakness of mild to moderate progression, variable degrees of respiratory involvement, with distal joint hyperlaxity and minimal joint contractures. Muscle MRI imaging revealed a myopathic appearance with a ‘central cloud’ pattern of increased T1 signal in the rectus femoris muscle, as has been well-described in COL6-RD. Consistent with a dominant-negative COL6-RD mutational disease mechanism, collagen VI immunofluorescence studies in patient muscle revealed mislocalization of collagen VI expression, with intracellular retention noted in patient cultured fibroblasts. RNA sequencing on cycloheximide (CHX) treated and untreated fibroblast showed that COL6A1 c.930+176C>T creates a dominantly acting in-frame 78 bp pseudoexon insertion with a pseudoexon percent spliced (PSI) of 1.8% in untreated, and of 1.6% in CHX treated fibroblasts. Notably, this 78 bp pseudoexon transcript was not identifiable on patient muscle RNA sequencing. This highlights that even low levels of pseudo-exon sequence in the mutant collagen α1 chain, impairs normal collagen VI matrix assembly, manifesting clinically with COL6-RD, albeit milder. These findings add COL6A1 c.930+176C>T to the emerging list of pathogenic deep intronic COL6A1 variants in COL6-RD that may be amenable for splice-modulating therapeutic interventions and extends the clinical phenotypic spectrum to now also include milder Bethlem myopathy.