Myosin binding protein C1: a novel gene for autosomal dominant distal arthrogryposis type 1

Abstract

Distal arthrogryposis type I (DA1) is a disorder characterized by congenital contractures of the hands and feet for which few genes have been identified. Here we describe a five-generation family with DA1 segregating as an autosomal dominant disorder with complete penetrance. Genome-wide linkage analysis using Affymetrix GeneChip Mapping 10K data from 12 affected members of this family revealed a multipoint LOD(max) of 3.27 on chromosome 12q. Sequencing of the slow-twitch skeletal muscle myosin binding protein C1 (MYBPC1), located within the linkage interval, revealed a missense mutation (c.706T>C) that segregated with disease in this family and causes a W236R amino acid substitution. A second MYBPC1 missense mutation was identified (c.2566T>C)(Y856H) in another family with DA1, accounting for an MYBPC1 mutation frequency of 13% (two of 15). Skeletal muscle biopsies from affected patients showed type I (slow-twitch) fibers were smaller than type II fibers. Expression of a green fluorescent protein (GFP)-tagged MYBPC1 construct containing WT and DA1 mutations in mouse skeletal muscle revealed robust sarcomeric localization. In contrast, a more diffuse localization was seen when non-fused GFP and MYBPC1 proteins containing corresponding MYBPC3 amino acid substitutions (R326Q, E334K) that cause hypertrophic cardiomyopathy were expressed. These findings reveal that the MYBPC1 is a novel gene responsible for DA1, though the mechanism of disease may differ from how some cardiac MYBPC3 mutations cause hypertrophic cardiomyopathy.

Figure 1.

Distal limb contractures in affected individuals from family 5432. (A) Photograph of proband with treated bilateral congenital clubfoot and his great aunt with unilateral left-sided clubfoot before and after multiple corrective surgeries. (B) Hand contractures consisting of camptodactyly and ulnar deviation of the fingers in three affected individuals of family 5432. The final photo shows fourth finger extension contractures present in two individuals of this family.

Figure 2.

Haplotype analysis indicating markers on chromosome 12q common to all affected individuals with distal arthrogryposis type I. Common haplotype is shown in red. Arrow indicates proband. + Symbol indicates individuals with hand contractures; VT indicates individuals with vertical talus. All other affected individuals have clubfoot. Haplotypes were created with GENEHUNTER and viewed on Haplopainter (55).

Figure 3.

MYBPC1 missense mutations in two families with distal arthrogryposis type 1. Chromatograms showing missense mutations in proband of family 5432 (A) and 8039 (B). Both mutations result in amino acid substitutions at highly conserved residues as shown by multiple species alignment of MYBPC1 (skeletal muscle, slow-twitch) and MYBPC2 (skeletal muscle, fast-twitch) and MYBPC3 (cardiac). Also shown in (B) is a pedigree of family 8039 and clinical photographs of hand and foot contractures (congenital vertical talus) occurring in the proband.

Figure 4.

Location of two myosin binding protein C1 (MYBPC1) mutations in two patients with DA1. The W236R mutation is located within the MyBP-C unique motif between the C1 and C2 immunoglobulin domains. This region has been shown to interact with myosin S2 (42). The Y856H mutation is within the C8 repeat, a region known to mediate interactions with LMM portion of myosin (45,46) and titin (47). Two MYBPC3 mutations found in patients with hypertrophic cardiomyopathy, R326Q and E334K, corresponding to MYBPC1 K240Q and E248K, are shown in italics. Immunoglobulin domains are shown as circles and fibronectin type III domains as squares.

Figure 5.

Histochemistry of muscle biopsy of the proband with MYBPC1 W236R mutation showing small type I fibers. (A) Hematoxylin and eosin staining demonstrating variability in fiber size as well as the presence of centrally localized basophilia in some larger fibers (arrows). Scale bar equals 18 µm. (B) Higher magnification of hematoxylin and eosin stained image shown in (A). (C) ATPase 9.4 staining highlighting smaller type I fibers (lightly stained). Scale bar equals 19 µm.

Figure 6.

MYBPC1 W236R and Y856H mutant proteins localize to the sarcomeric C-zone similar to wild-type protein. (A) Immunoblot of transiently transfected U20S cells expressing WT, DA1 mutants W236R or Y856H or the homologous MYBCP3 mutations E248K or K240Q using a GFP antibody. Note that all proteins express an intact full-length protein. The location of full-length MYBPC1 protein is shown with an arrowhead. (B) Fluorescent image of mouse epitrochlearis muscle fibers after electroporation with GFP WT MYBPC1-GFP showing localization of the protein to the C zones of the A band. Schematic illustration shows three sarcomeres with thin (actin) and thick (myosin) filaments oriented horizontally and Z lines oriented vertically. (C) Fluorescent images of mouse epitrochlearis muscle fibers after electroporation with GFP-tagged constructs. The unfused GFP construct, containing only green fluorescent protein, shows diffuse cytoplasmic staining of single skeletal muscle fibers. High-powered images (at right) show the characteristic doublet pattern of MYBPC1 expression in the C-zone of the sarcomere with the expression of wild-type (WT) MYBPC1, W236R and Y856H mutant GFP-tagged proteins. However, expression of two MYBPC1–GFP constructs (K240Q and E248K) containing the corresponding MYBPC3 mutations (R326Q and E334K) identified in hypertrophic cardiomyopathy patients show poor or absent sarcomeric localization and instead show diffuse cytoplasmic staining.