While the EYS
gene was originally reported as an eye-specific gene20,21
, our finding of expression in the spinal cord is consistent with findings in Drosophila
/spam expression occurs in the photoreceptor cells of the eye, the central nervous system and peripheral sensory ganglia 31
. Two different functions of Drosophila EYS
/spam have been identified by genetic screens. Spam is essential for the formation of a matrix-filled intra-rhabdomeral space within ommatidia of the Drosophila
. This function is consistent with the finding that mutations in human EYS
result in retinitis pigmentosa 20,21
. Second, Drosophila
spam has been identified in a screen for a heat-sensitive loss of locomotor function 33
. Extracellular spam was shown to protect mechanoreceptor neurons and to be capable of protecting other cell types from hyper-osmotic shock. Cell damage of motor neurons and/or skeletal muscle cells by hyper-osmotic shock is a possible mechanism in the pathology of exercise intolerance and statin-induced myopathy, i.e. human spam could protect motor neurons and/or skeletal muscle cells from exercise-induced damage.
The sequence similarity of the EGF-like repeats in the N-terminal region of spam to the EGF-like repeats of Notch1 suggests a role in the Notch signaling pathway, since proteins containing similar clusters of EGF-like domains in the Notch pathway have been shown to bind to each other. Members of the Notch family and their ligands play fundamental roles in development, 34
and mutations within these genes are responsible for a number of human diseases including aortic valve disease and the most common form of hereditary stroke disorder (CADASIL) 35–37
. The sequence similarity of spam to Notch1, along with the observed expression of spam in spinal cord, is intriguing in view of the involvement of the Notch signaling pathway in satellite cell differentiation to myoblasts and regeneration of muscle tissue after exercise 38
. A reduction in the ability of satellite cells to regenerate due to reduced Notch signaling has been specifically proposed as the reason for the decline of regenerative potential in aging muscle 38,39
Laminin G domains bind to a wide variety of ligands including proteins, carbohydrates and steroids 40–42
. The three agrin laminin G domains, which in human sequence comparisons are most similar to spam LG1-5, are known to specifically bind at least four different proteins 43
. Structure/function studies with laminin G domains of agrin suggest that binding to α-dystroglycan concentrates agrin at the muscle surface facilitating the activation of muscle-specific tyrosine-protein kinase receptor 30
. It is therefore possible that motor neuron-derived spam could bind α-dystroglycan and allow its specific interaction with an as yet unidentified specific receptor to be expressed by muscle cells. Similarly, α-dystroglycan binding by spam could serve to localize the N-terminal Notch-like ligand to the extracellular matrix in proximity to satellite cells where it could act as a positive regulator of the Notch pathway.
The significant SNP-myopathy associations we identified within intron 26 of the EYS gene near the first laminin G domain (LG1) are located 38–85 kb from exon 27, and it is therefore difficult to predict a functional effect. It is possible that one of these or a more strongly associating variant within this region has long range regulatory effects on EYS expression or splicing that result in a loss or alteration of function in neuromuscular tissue. Splicing variations could potentially prevent the laminin G multidomain region from binding to its specific receptor or interfere with α-dystroglycan binding which would impair extracellular localization of spam and indirectly prevent specific receptor binding by either the laminin G or Notch-like domains. Loss-of-function sequence variations in EYS could thus result in a diminished capacity for muscle regeneration, and this would be consistent with the symptomatic manifestations of statin-induced myopathy.
A strong association of polymorphic variants in the SLCO1B1
gene only with high dose (80 mg) simvastatin has recently been found with a genome-wide scan of a different group of statin myopathy patients19
. We did not genotype the reported SNPs rs4149056 or rs4363657 in our pooled GWAS, but we did interrogate rs2900478 which is in strong LD with the two aforementioned SNPs. The resulting statistic was not significant in our group of severe statin myopathy patients. The SLCO1B1
product, a transporter protein (OATP1B1), is predicted to be involved in simvastatin clearance. When tested for an association with atorvastatin, the recent STRENGTH study showed that there was an increased risk in SLCO1B1
carriers taking atorvastatin, however, the finding was not statistically significant44
. Pravastatin was the only other cholesterol-lowering drug tested, and there was no association found in carriers of the SLCO1B1
variant. No other agents (rosuvastatin, lovastatin, or fluvastatin) have been evaluated as yet for an association with the SLCO1B1
. Therefore, the association we describe with EYS
and severe statin myopathy across a variety of statins and dosages is the first that provides possible insight into a mechanism for statin myopathy.
The possibility of population stratification cannot be ruled out, since we did not explicitly control for it in the study design. In this study, where cases are rare in the population and obtained from multiple centers, it was particularly difficult to obtain a hospital-based control group that perfectly represents the source population. We considered the use of statin-tolerant controls to at least be superior to using unexposed subjects from the general population.
The results of this GWAS, replication and fine mapping study provide the first reported suggestive evidence that genetic variants (rs1337512, 3857532 and 9342288), within the EYS gene, are associated with severe statin myopathy and suggest that only one copy of the variant is necessary to confer this risk. Our study also demonstrates that EYS gene products are complex, resulting from numerous alternative splicing patterns, and relatively abundant in the spinal cord in addition to the retina. A proposed mechanism for EYS gene involvement in severe statin myopathy is that loss of function variants may result in diminished capacity for the regeneration of damaged muscle. This biologic plausibility supports the EYS gene as a candidate gene for susceptibility to statin-induced myopathy that warrants further replication and functional study.