Epidemiological and Molecular Characterization of a Mexican Population Isolate with High Prevalence of Limb-Girdle Muscular Dystrophy Type 2A Due to a Novel Calpain-3 Mutation

Abstract

Limb-Girdle Muscular Dystrophy type 2 (LGMD2) is a group of autosomally recessive inherited disorders defined by weakness and wasting of the shoulder and pelvic girdle muscles. In the past, several population isolates with high incidence of LGMD2 arising from founder mutation effects have been identified. The aim of this work is to describe the results of clinical, epidemiologic, and molecular studies performed in a Mexican village segregating numerous cases of LGMD2. A population census was conducted in the village to identify all LGMD affected patients. Molecular analysis included genome wide homozygosity mapping using a 250K SNP Affymetrix microarray followed by PCR amplification and direct nucleotide sequencing of the candidate gene. In addition, DNA from 401 randomly selected unaffected villagers was analyzed to establish the carrier frequency of the LGMD2 causal mutation. A total of 32 LGMD2 patients were identified in the village, rendering a disease prevalence of 4.3 (CI: 2.9-5.9) cases per 1,000 habitants (1 in 232). Genome wide homozygosity mapping revealed that affected individuals shared a 6.6 Mb region of homozygosity at chromosome 15q15. The identified homozygous interval contained CAPN3, the gene responsible for LGMD2 type A (LGMD2A). Direct sequencing of this gene revealed homozygosity for a novel c.348C>A mutation (p.Ala116Asp) in DNA from all 20 affected subjects available for genetic screening, except one which was heterozygous for the mutation. In such patient, a heterozygous c.2362AG>TCATCT deletion/insertion was recognized as the second CAPN3 mutation. Western blot and autocatalytic activity analyses in protein lysates from skeletal muscle biopsy obtained from a p.Ala116Asp homozygous patient suggested that this particular mutation increased the autocatalytic activity of CAPN3. Thirty eigth heterozygotes of the p.Ala116Asp mutation were identified among 401 genotyped unaffected villagers, yielding a population carrier frequency of 1 in 11. This study demonstrates that a cluster of patients with LGMD2A in a small Mexican village arises from a novel CAPN3 founder mutation. Evidence of allelic heterogeneity is demonstrated by the recognition of an additional CAPN3 mutation in a single affected. Our study provides an additional example of genetic isolation causing a high prevalence of LGMD and of successful molecular characterization of the disease by means of homozygosity mapping. The identification of a very high carrier frequency of the LGMD2-causing mutation has implications for more rational genetic counseling in this community.

Fig 1. Variable clinical phenotype in LGMD2 patients.

(A) Severe winged scapulae are evident in a 22-year- old male patient. (B) Dorso-lumbar scoliosis is observed in a patient aged 25 years.

Fig 2. Genome-wide homozygosity analysis in LGMD2.

Genotypes obtained with the Affymetrix 250K SNPs chip were analyzed with the HomozygosityMapper software for the identification of large regions of homozygosity. Top bars indicate homozygous regions identified in pooled DNA from three affected patients. As shown in the screen shot, two chromosomal regions of maximal homozygosity were identified. The largest region (6.6 Mb) corresponded to chromosome 15q (pointed with a blue arrow), from nucleotide 39,911,352 to nucleotide 46,517,283, and includes the CAPN3 gene.

Fig 3. Partial nucleotide sequence of the CAPN3 gene.

(A) Normal sequence showing homozygosity for the wild type GCC (alanine) codon at position 116. (B) Sequence from a heterozygous (GCC/GAC) subject (C) Sequence in DNA from a homozygous mutant (GAC/GAC) LGMD2 subject. The arrow indicates the mutated nucleotide (c.348C>A).

Fig 4. In silico analyses of the p.Ala116Asp mutation in CAPN3.

(A) Pathogenicity prediction analysis by Polyphen-2 indicates that the missense substitution is a damaging mutation. (B) Phylogenetic comparison of CAPN3 proteins. Alanine 116 is strictly conserved in proteins from different species including Equus caballus (horse), Canis familiaris (dog), Sus scrofa (wild pig) Bos taurus (cow), Loxodonta africana (elephant), Oryctolagus cuniculus (rabbit), and Macaca mulatta (macaque), among many others.

Fig 5. Inmunoblot analysis of CAPN3 in muscle biopsy from a LGMD2A patient with the novel p.Ala116Asp mutation.

Western blot on skeletal muscle of normal control (C) and an affected patient (P) at 20 min post-incubation in saline solution indicates preserved autocatalytic activity of CAPN3 in both samples. However, both the absence of the 94 kDa band (top arrow) and the increased density of the degradation 60 kDa band (bottom arrow) suggest an increased autolytic activity in patient’s sample. Approximate molecular masses (kDa) are shown as a guide on the left. The range of protein degradation products is indicated. GAPDH was used as internal control.