Several patients with previously reported titin gene (TTN) mutations causing tibial muscular dystrophy (TMD) have more complex, severe or unusual phenotypes. This study aimed to clarify the molecular cause of the variant phenotypes in eight patients of seven European families. Clinical, histopathological and muscle imaging data of patients and family members was reanalyzed. The titin protein was analyzed by Western blotting and TTN gene by RT-PCR and Sanger sequencing. Western blotting showed more pronounced C-terminal titin abnormality than expected for heterozygous probands, suggesting the existence of additional TTN mutations. RT-PCR indicated unequal mRNA expression of the TTN alleles in biopsies of six patients, three with an LGMD2J phenotype. Novel frameshift mutations were identified in five patients. A novel A-band titin mutation, c.92167C>T (p.P30723S), was found in one patient, and one Portuguese patient with a severe TMD phenotype proved to be homozygous for the previously reported Iberian TMD mutation. The unequal expression levels of TTN transcripts in five probands suggested severely reduced expression of the frameshift mutated allele, probably through nonsense-mediated decay, explaining the more severe phenotypes. The Iberian TMD mutation may cause a more severe TMD rather than LGMD2J when homozygous. The Finnish patient compound heterozygous for the FINmaj TMD mutation and the novel A-band titin missense mutation showed a phenotype completely different from previously described titinopathies. These results further expand the complexity of muscular dystrophies caused by TTN mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism explaining phenotype variability.
