Tibial muscular dystrophy is a titinopathy caused by mutations in TTN, the gene encoding the giant skeletal-muscle protein titin

Abstract

Tibial muscular dystrophy (TMD) is an autosomal dominant late-onset distal myopathy linked to chromosome 2q31. The linked region includes the giant TTN gene, which encodes the central sarcomeric protein, titin. We have previously shown a secondary calpain-3 defect to be associated with TMD, which further underscored that titin is the candidate. We now report the first mutations in TTN to cause a human skeletal-muscle disease, TMD. In Mex6, the last exon of TTN, a unique 11-bp deletion/insertion mutation, changing four amino acid residues, completely cosegregated with all tested 81 Finnish patients with TMD in 12 unrelated families. The mutation was not found in 216 Finnish control samples. In a French family with TMD, a Leu-->Pro mutation at position 293,357 in Mex6 was discovered. Mex6 is adjacent to the known calpain-3 binding site Mex5 of M-line titin. Immunohistochemical analysis using two exon-specific antibodies directed to the M-line region of titin demonstrated the specific loss of carboxy-terminal titin epitopes in the TMD muscle samples that we studied, thus implicating a functional defect of the M-line titin in the genesis of the TMD disease phenotype.

Figure  1

Structure of skeletal-muscle titin. The location of calpain-3 binding sites and the site of Mex6 mutations are indicated. Lower schematic picture shows the 3′/C-terminal modular structure of titin, providing a more detailed illustration of ligand and antibody binding sites.

Figure  2

The TMD-associated 11-bp mutation at positions 293,268–293,280 in exon Mex6 of TTN. A, Nucleotide sequence with borders of mutated area indicated by arrows. B, SSCP analysis of exon Mex6 of TTN, including the associated mutation, from patients with TMD and unaffected control individuals. The migration of PCR products matches the haplotypes and phenotypes of the patients. Bands indicating the mutation are marked by arrows. Samples are denoted “1”–“16.” w/w = Wild-type sequence from an unaffected control individual; w/m = TMD heterozygote; m/m = TMD homozygote; L = 100-bp DNA ladder.

Figure  3

The French TMD-associated base substitution CTG→CCG at position 293,357, causing a Leu→Pro change in exon Mex6 of TTN. Nucleotide sequence with the mutation indicated by an arrow. w/w = Wild-type sequence from healthy relative; w/m = heterozygous affected relative.

Figure  4

Muscle sections from an unaffected control individual (A), from a heterozygous patient with TMD (B), and from a homozygous patient with TMD and LGMD (C), immunostained with titin M8/M9 antibody, and sections from an unaffected control individual (D), from a heterozygous patient with TMD (E), and from a homozygous patient with TMD and LGMD (F), immunostained with titin A169/A170 antibody and detected by FITC.