An association of genotype with DCM has long been suspected but has not been clearly established 2–4, 24
. In this study, we demonstrated that genotype is a determinant of the age of DCM manifestations in BMD and XLDCM patients. The large sample size (78 patients) and stringent selection criteria including only patients with small deletions (11 exons) enabled us to capture informative dystrophin protein domains. This allowed patient grouping based on dystrophin mutations affecting a single rather than multiple adjacent functional protein domains, thus increasing the statistical power of the study. We also provided novel evidence of a strong association of cardiomyopathy with specific structural alterations of the dystrophin rod domain. Prior studies reported contradictory findings on the potential link of deletions including exons 48 and/or 49 with severity and occurrence of DCM 2–4, 24
. Analysis of our 56 patients in Group 2 showed that DCM in this region is more sensitive to altered phasing of the spectrin repeats rather than absence or presence of any one individual exon. Our analysis of the effects of specific deletions on the three-dimensional structure of dystrophin and their correlation with cardiac phenotype highlights the importance of integrating protein structure information in genotype-phenotype studies.
Our results also indicate that the rod domain of dystrophin may not be as permissive to alterations as previously thought. This study suggests that preservation of phasing delays the onset of DCM by about a decade. This is likely not due to a difference in expression of cardiac dystrophin protein since Arbustini et al. 20
reported similar amount and distribution of dystrophin in cardiac biopsy samples from BMD patients with either in-phase or out-of-phase mutations. Rather, our modeling suggests that the alterations caused by out-of-phase mutations extend beyond the spectrin repeat unit and may lead to a severely altered configuration of the rod domain, ultimately affecting the entire dystrophin protein. This major structural change is likely a main determinant of early onset DCM. Interestingly, most Group 2 BMD patients have late-onset skeletal muscle symptoms and mild disease progression, irrespective of the effects of their mutation on phasing. This is in agreement with studies in dystrophin-null mdx
mice expressing a mini-dystrophin construct that lacks the exon 45 to 49 region but has an intact hinge 3 domain. In these mice only a partial restoration of cardiac function was achieved in spite of a complete rescue of the skeletal muscle pathology 25
. Thus cardiac dystrophin may be particularly sensitive to structural disruptions of the exon 45 to 49 region compared to skeletal muscle dystrophin. The reasons for this disparity are currently unknown but highlight the importance of mapping domains of dystrophin essential for cardiac function to improve upon current treatment approaches relying on exon skipping or gene replacement with mini-/micro-dystrophin constructs.
This study provides expected median ages of onset of DCM associated with three distinct regions of the dystrophin protein and with specific re-arrangements of its rod-domain. The deletion mutations studied here are among the most frequent, rendering our findings relevant to most BMD and XLDCM patients. Of interest, within groups, XLDCM patients did not have an earlier age of cardiomyopathy compared to BMD patients. Instead, the earliest age of DCM is associated with mutations affecting the amino-terminal domain of the protein (early 20’s), and out-of-phase mutations in the exon 45 to 49 region of the dystrophin rod domain (mid 20’s). While cardiac expression of dystrophin has been confirmed in several out-of-phase Group 2 patients, such information is not available for Group 1 patients. The best studied mutations affecting the 5′ region of the dystrophin gene (including the muscle promoter, exon 1, or intronic regions that alter exon splicing 26–28
) lead to a selective lack of cardiac dystrophin. While none of our Group 1 patients had mutations affecting non-coding regions or exon 1, we cannot exclude the possibility that the early DCM onset in Group 1 patients reflects a selective absence of cardiac dystrophin. Of note, the median age of XLDCM patients with lack of cardiac dystrophin (24 years for 6 independent families, Supplemental Table 1
) is very similar to that of Group 1patients (23 years). Further studies are needed to determine the mechanism(s) by which deletion mutations in the amino-terminal region lead to earlier onset of cardiomyopathy compared to mutations affecting other regions. A greater awareness of the value of cardiac tissue sampling at the time of cardiac transplantation and the design of transgenic mdx
mice mimicking human mutations could yield important information on cardiac-specific mechanisms regulating this region of dystrophin at a transcriptional and protein level.
Significantly later DCM onset is associated Group 2 in-phase (mid 30’s) and Group 3 mutations (mid 40’s). The cardio-protective effect of hinge 3 deletion seen in Group 3 patients mirrors findings reported for skeletal muscle in both mice and humans 6, 17
. However, while loss of hinge 3 delays onset of cardiomyopathy, it correlated with slower disease progression in skeletal muscle but had no effect on age of onset 6
. Due to the small number of Group 3 patients with identical deletions, we could not determine whether this partially protective effect is associated with a specific structural alteration of the dystrophin protein backbone. Further studies are needed to explain the significant cardio-protective effect conferred by the loss of hinge 3.
The median age of cardiac involvement for each patient group reported here is currently the best approximation available for this patient population. This information is valuable because cardiac involvement in BMD patients is often asymptomatic in its initial stages and can therefore be underestimated. Since genotyping has become a more common practice, the median ages reported here may prove valuable for individualized risk assessment, and for timely cardiac evaluation and intervention. An important next step is to conduct a large-scale longitudinal study to further refine the age of DCM onset associated with the dystrophin domains identified here. This information underscores the importance of genotype information in the cardiac care of BMD patients and bears relevance to the design of therapies aimed at the myocardium in BMD, XLDCM and DMD patients.