Targeted Next-Generation Sequencing Reveals Mutations in Non-coding Regions and Potential Regulatory Sequences of Calpain-3 Gene in Polish Limb-Girdle Muscular Dystrophy Patients.

Limb-girdle muscular dystrophy type R1 (LGMDR1) is caused by mutations in and is the most common type of recessive LGMD. Even with the use of whole-exome sequencing (WES), only one mutant allele of is found in a significant number of LGMDR patients. This points to a role of non-coding, intronic or regulatory, sequence variants in the disease pathogenesis. Targeted sequencing of the whole gene including not only intronic, 3' and 5' UTRs but also potential regulatory regions was performed in 27 patients suspected with LGMDR1. This group included 13 patients with only one mutated allele detected previously with exome sequencing. A second rare variant in the non-coding part of was found in 11 of 13 patients with previously identified single mutation. Intronic mutations were found in 10 cases, with c.1746-20C>G variant present in seven patients. In addition, a large deletion of exons 2-8 was found in one patient. In the patients with no causative mutation previously found, we detected rare variants in 5 out of 10 patients and in two of them in a compound heterozygous state. Rare variants within putative regulatory sequences distant from the gene were found in 15 patients, although in 11 of these cases, other variants are deemed causative. The results indicate that intronic mutations are common in Polish LGMDR patients, and testing for non-coding mutations in should be performed in apparently single heterozygous patients.