Affiliations: [a] Neuromuscular and Neurogenetic Disorders of Childhood Section, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA
Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
| [c] Department of Pediatric Neurology, Goryeb Children’s Hospital, Morristown, NJ, USA
| [d] Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA
| [e] Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| [f] Department of Genetics, Yale University School of Medicine, New Haven, CT
Correspondence to: Carsten G. Bönnemann, MD Chief, Neuromuscular and Neurogenetic Disorders of Childhood Section Neurogenetics Branch National Institute of Neurological Disorders and Stroke/NIH 35 Convent Drive, Bldg 35, Room 2A-116 Bethesda, MD 20892-3705, USA. Tel.: +1 301 594 5496; Fax: +1 301 480 3365; E-mail: email@example.com.
Abstract: Calpainopathy, also known as limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) or LGMD R1 Calpain3-related, is one of the most common genetically characterized forms of limb-girdle muscular dystrophy with a wide range of phenotypic severity. We evaluated a consanguineous family with a clinical phenotype consistent with calpainopathy in whom conventional sequencing did not detect any mutations in the CAPN3 gene. Using whole exome sequencing paired with haplotype analysis, we identified a homozygous deep intronic single base pair deletion in CAPN3 (c.946-29delT). Familial segregation studies were consistent with recessive inheritance. Immunoblotting of muscle tissue from the patient showed complete absence of calpain 3. In silico analysis predicted the deletion to disrupt the branch point and subsequently alter splicing of exon 7. Studies of patient fibroblasts and muscle tissue confirmed altered splicing, resulting in an inclusion of a 389-bp intronic sequence upstream of exon 7, originating from a cryptic splice acceptor site in intron 6. This out-of-frame insertion results in a premature stop codon, leading to an apparent absence of protein likely due to degradation of the transcript via nonsense-mediated decay. We then designed phosophorodiamidate morpholino oligomers (PMOs) as splice modulators to block the new splice acceptor site. This approach successfully prevented the aberrant splicing – reverting the majority of the splice to the wildtype transcript. These results confirm the pathogenicity of this novel deep intronic mutation and provide a mutation-specific therapeutic strategy. Thus, deep intronic mutations in CAPN3 may be pathogenic and should be considered in the appropriate clinical setting. The identification of mutations which may be missed by traditional Sanger sequencing is essential as they may be excellent targets for individualized therapeutic strategies using RNA-directed splice modulation.
Keywords: Calpainopathy, LGMD2A, deep intronic mutation, phosophorodiamidate morpholino oligomer (PMO)