Transcriptional and translational effects of intronic CAPN3 gene mutations

Abstract

Variants of unknown significance in the CAPN3 gene constitute a significant challenge for genetic counselling. Despite the frequency of intronic nucleotide changes in this gene (15-25% of all mutations), so far their pathogenicity has only been inferred by in-silico analysis, and occasionally, proven by RNA analysis. In this study, 5 different intronic variants (one novel) that bioinformatic tools predicted would affect RNA splicing, underwent comprehensive studies which were designed to prove they are disease-causing. Muscle mRNA from 15 calpainopathy patients was analyzed by RT-PCR and splicing-specific-PCR tests. We established the previously unrecognized pathogenicity of these mutations, which caused aberrant splicing, most frequently by the activation of cryptic splicing sites or, occasionally, by exon skipping. The absence or severe reduction of protein demonstrated their deleterious effect at translational level. We concluded that bioinformatic tools are valuable to suggest the potential effects of intronic variants; however, the experimental demonstration of the pathogenicity is not always easy to do even when using RNA analysis (low abundance, degradation mechanisms), and it might not be successful unless splicing-specific-PCR tests are used. A comprehensive approach is therefore recommended to identify and describe unclassified variants in order to offer essential data for basic and clinical geneticists.

Figure 1

Splicing analysis of the c. 1524+1 G>C variant. Panel A. Schematic representation of the splicing patterns generated by this variant: D1, carrying the deletion of the last 99bp of exon 11 (gray box), and D2, with the retention of 212bp of intron 11 (dashed box), resulting from the use of the alternative donor splice sites D1 and D2, respectively. The arrows indicate the localization of the primers used. Panel B. Sequences from PCR amplification of muscle cDNA from a heterozygous mutant patient, showing the co-amplification of the wild-type (WT) and the alternately spliced mRNA (D1), and from splicing-specific PCR amplification, which selectively amplifies the allele carrying the mutation (D2) in the same patient. Panel C. RT-PCR analysis of normal control (C) and a heterozygous mutant patient (Pt.) who shows the WT product and two additional low-abundant products corresponding to the alternately spliced mRNA (D1 and D2). Calpain-3 western blot shows that this mutation caused a severe reduction of protein (Pt.) corresponding to about 5% of control (C) after myosin normalization.

Figure 2

Splicing analysis of the c. 1992+1 G>T variant. Panel A. Schematic representation of the two splicing patterns generated by this variant: WTD, carrying the skipping of exon 17, and D1, with the retention of 31 bp of intron 17 (dashed box), resulting from the use of the alternative donor splice site D1. The arrows indicate the localization of the primers used. Panel B. Sequences from splicing-specific PCR amplification of muscle cDNA from a heterozygous mutant patient, showing the two aberrant transcripts (WTD and D1) generated by this variant. Panel C. Splicing-specific PCR amplification showing the selective amplification of the aberrant transcripts generated by this variant (WTD and D1) only in the heterozygous mutant patient (Pt.). Calpain-3 western blot in a patient (Pt.) shows that this mutation produced absent protein.

Figure 3

Splicing analysis and translational effect of the c. 1193+6 T>A variant. Panel A. Schematic representation of the aberrant splicing product (D1) generated by this variant, carrying the insertion of 31bp of intron 9 (dashed box) and resulting from the use of the alternative donor splice site D1. The arrows indicate the localization of the primers used. Panel B. Sequence from PCR amplification of muscle cDNA from a heterozygous mutant patient, showing the co-amplification of the WT and the alternately spliced mRNA (D1). Panel C. RT-PCR analysis of normal control (C) and a heterozygous mutant patient (II-1) who shows the WT product and one additional low-abundance product corresponding to the alternately spliced mRNA (D1). Family pedigree (case n. 7652) and western blot show that this intronic mutation (filled symbol) produced a reduction of calpain-3 protein of about 50% of control (C) after myosin normalization, as assessed in the muscle biopsy from both the heterozygous father (I-1) and his affected daughter (II-1), who was a compound heterozygote for a second missense mutation (p.E435K, dashed symbol).

Figure 4

Splicing analysis and translational effect of the c. 1746-20 C>G variant. Panel A. Schematic representation of the 5 splicing patterns generated by this variant, resulting from the use of the cryptic acceptor splice sites A1, A2, A3, A4, carrying the retention of 19, 86, 122 and 305 bp of intron 13 (dashed boxes), respectively, and WTA13, with the retention of the entire intron 13 (dashed box). The arrows indicate the localization of the primers used. Panel B. Splicing-specific PCR (designed to potentially amplify all the 5 aberrant transcripts) showing that these transcripts are variably expressed and not always detectable in the heterozygous mutant patients (Pt. 1, II-1, and Pt.3). The WTA transcript is expressed also in normal control (C). Panel C. Family pedigree (case n. 6211) and western blot showing that this intronic mutation (filled symbol) produced a deleterious effect at protein level, as demonstrated by the complete loss of calpain-3 protein in the muscle from one affected patient (II-1) who was a compound heterozygote for a second missense mutation that has a deleterious effect as well (p.L204V, dashed symbol).