Novel mutations in the sarcomeric protein myopalladin in patients with dilated cardiomyopathy

Abstract

Recently, missense mutations in titin-associated proteins have been linked to the pathogenesis of dilated cardiomyopathy (DCM). The objective of this study was to search for novel disease-associated mutations in the two human titin-binding proteins myopalladin and its amino-terminal-interacting partner cardiac ankyrin-repeat protein (CARP). In a cohort of 255 cases with familial and sporadic DCM, we analyzed the coding regions and all corresponding intron flanks located in the MYPN and CARP-encoding ANKRD1 gene. Two heterozygous missense mutations were detected in the MYPN gene (p.R955W and p.P961L), but neither of these mutations was found in 300 healthy controls. Both mutations were located in the α-actinin-binding region of myopalladin. Endomyocardial biopsies from the p.R955W carrier showed normal subcellular localization of myopalladin and α-actinin in cardiac myocytes, while their regular sarcomeric staining pattern was significantly disrupted in the p.P961L carrier, indicating that disturbed myofibrillogenesis and altered sarcomere assembly are the cause of the disease. In the ANKRD1 gene, we identified synonymous base exchanges (c.108T>C and c.-79C>T, respectively), but no non-synonymous mutations. In summary, we have identified novel missense mutations in the third immunoglobulin-like domain of myopalladin, which have either no or profound effects on the molecular composition of the sarcomere. According to our epidemiological data, the prevalence of ANKRD1 mutations seems to be lower than that of its binding partner myopalladin, indicating the clinical significance of myopalladin for the functional integrity of the sarcomeric apparatus and the protection against DCM.

Figures

Figure 1

Two missense mutations in the MYPN gene detected in a population of patients with DCM. (a–c) Identification of the myopalladin mutation p.R955W in a 44-year-old male DCM patient. DNA sequencing demonstrated the presence of a heterozygous nucleotide substitution in exon 13, resulting in an amino-acid exchange in position 955 (a). The p.R955W mutation was confirmed by means of denatured gradient gel electrophoresis (DGGE, b) and restriction fragment length polymorphism analysis (RFLP) using MspI (c). (d–f) Genetic analyses demonstrating the presence of a p.P961L mutation in a 33-year-old male DCM patient. The C>T nucleotide exchange in the corresponding codon shown in an electropherogram (d) was confirmed using DGGE (e) and RFLP with BfaI (f). As controls, DNA samples from other DCM patients were included in the respective gels, with the index patient marked with an arrow.

Figure 2

Cardiac myocytes from the carrier of the p.P961L mutation showed a disrupted sarcomeric distribution of myopalladin and α-actinin. Endomyocardial biopsies obtained from three DCM patients expressing either wild-type or mutant myopalladin (p.R955W and p.P961L) were immunohistochemically stained with either a polyclonal antibody directed against myopalladin or a monoclonal α-actinin antibody. Specific immunoreactivity was detected by incubating the samples with Cy3-labeled secondary antibodies, while nuclei were stained with Hoechst dye.

Figure 3

Nucleotide exchange in exon 2 of the CARP-encoding ANKRD1 gene as detected in a 65-year-old male DCM patient. (a) Electropherogram demonstrated the heterozygous nucleotide exchange c.108 T>C in codon 36. (b) SSCP analysis showed the presence of an additional, lower migrating band corresponding to the index patient (in lane 3 marked with an arrow), which was absent in other DCM patients.

Figure 4

(a) Amino-acid residues R955 and P961 in the coding sequence of myopalladin are conserved between vertebrate species. Aligned wild-type sequences of the third immunoglobulin-like domain of myopalladin from different species and the mutant variants in DCM patients detected in our study sample (marked in red when mutated and in blue when conserved) are shown. (b) Cartoon structure of the third immunoglobulin-like domain of palladin as created by PyMOL, including the side chain of the prolyl residue 1017, which is homologous to P961 in the myopalladin sequence. (c) Peptide sequence alignment of the five immunoglobulin-like domains of myopalladin including third immunoglobulin-like domain of palladin. Mutants identified in this study and their corresponding wild-type residues are marked with red and blue, respectively.