We used candidate-gene re-sequencing and other methods to search comprehensively for known, novel, and rare genetic variants that might underlie cerivastatin-associated rhabdomyolysis. We identified two variants with significant associations: rs2819742 in the RYR2 gene and rs4149056 in the SLCO1B1 gene. Functional studies corroborated the SLCO1B1 finding. For the other candidate genes, CYP2C8 and the UGTs, there was little evidence for an association with rhabdomyolysis. The modest associations observed in this study suggest that the manner in which genetic factors affect rhabdomyolysis may be complex, involving multiple genes, interactions, or rare variants outside the candidate pharmacokinetic genes.
Though our findings support the hypothesis that underlying genetic factors contribute to extreme adverse drug reactions, we had anticipated finding a number of rare or disabling variants that affected drug-metabolizing enzymes and drug transporters. The CYP2C8
gene is predominantly responsible for the first pass oxidative metabolism of cerivastatin [8
] and contains variant alleles (CYP2C8*2
, and *4)
which alter the function of the enzyme for some substrates [19
]. The UGT1A1
glucuronidation is an alternate to the CYP-oxidation pathway for cerivastatin metabolism. Gemfibrozil, which inhibits the cerivastatin oxidation and glucuronidation pathways, was strongly associated with cerivastatin-associated rhabdomyolysis; yet in this study, neither known nor novel genetic variation in UGT1A1
was associated with the risk of rhabdomyolysis and the CYP2C8*2
, and *4
alleles were less common in cases than controls, though not significantly so. For cerivastatin, these excellent candidate genes did not explain the high incidence of rhabdomyolysis.
OATP1B1, the product of SLCO1B1,
is a transporter that mediates the hepatic uptake of cerivastatin as well as several other statins [10
]. The nonsynonymous coding variant rs4149056 variant slows the uptake of other statins in vitro
]. In the STRENGTH study, rs4149056 was associated with mild statin induced side effects (including muscle symptoms) among users of simvastatin, atorvastatin and pravastatin [28
]. A GWA study of myopathy among high-dose simvastatin users [29
] found an additional copy of the minor allele of rs4149056 was associated with a 4.5 fold increase in the risk of myopathy (95% CI 2.6 to 7.7). Our study results extend the SLCO1B1
rs4149046 findings to cerivastatin-associated rhabdomyolysis, and our functional studies provide additional support for a causal association.
Our study results suggest there may be an interaction between SLCO1B1
rs4149056 and gemfibrozil use (p=0.02). Analyses limited to subjects using gemfibrozil were null with wide confidence intervals while analyses excluding subjects who used gemfibrozil showed a strong association between rs4149056 and the risk of rhabdomyolysis (OR: 2.45; 95% CI 1.61 to 3.75). While it's difficult to draw strong conclusions about the association between rs4149056 and rhabdomyolysis among gemfibrozil users because there were so few controls using gemfibrozil (n=5), we suspect the exceedingly large effect of gemfibrozil obscured the much smaller genetic association. In our study, gemfibrozil use was associated with a 278 fold increased risk of rhabdomyolysis (95% CI: 106-733). Another study suggested that combined cerivastatin-gemfibrozil use relative to statin monotherapy was associated with a 1411 fold increased risk of rhabdomyolysis [7
]. The role of SLCO1B1
variants was minor relative to the risk associated with the drug-drug interaction.
gene encodes the ryanodine receptor type 2. The ryanodine receptors are intracellular calcium release channels that are expressed in diverse tissues [30
]. Three genes encode the different isoforms. RYR1
is expressed predominantly in skeletal muscles where it contributes to Ca2+
signaling and muscle contraction. Variants in RYR1
are associated with malignant hyperthermia and central core disease [31
but not RYR1
expression was found to be upregulated in the skeletal muscle of patients with statin-associated structural muscle injury. The expression of RYR2
was not investigated [32
is expressed in cardiac muscle and mutations in RYR2
are associated with arrhythmogenic right ventricular cardiomyopathy type 2 and stress induced polymorphic ventricular tachycardia [33
is also expressed in the brain [36
] and neonatal skeletal muscle [33
]. A study in rabbits showed that RYR2
expression is upregulated and RYR1
expression is downregulated in conditioned muscle [33
]. In mouse cardiomyocytes, RYR2
splice variants can modulate apoptosis, with certain variants reducing Ca2+
release and preventing apoptosis [38
]. An apoptotic effect of cerivastatin on skeletal muscle through Ca2+
] in the presence of genetic variants that either alter RYR2
expression or disrupt splice variants suggests potential mechanisms by which RYR2
variation might contribute to cerivastatin-associated rhabdomyolysis.
Our study had several strengths. We successfully recruited subjects with a rare adverse event. The two control groups had complementary strengths: The CHS controls, like the cases, were geographically diverse; the HVH controls had an age distribution similar to that of the cases. We used multiple approaches--resequencing, tag-SNPs, and functional SNPs--to search for genetic variants of importance in the candidate genes. The resequencing effort had greater than 90% power to detect variants with a minor allele frequency of 1% in the rhabdomyolysis cases and 80% power to detect variants with a minor allele frequency of 0.65% [41
]. Additionally, we used genome wide scans to identify genetic risk factors outside of the candidate genes.
The number of rhabdomyolysis cases, though large for a GWA study of an adverse event [42
], is nonetheless small and provided limited power to detect associations. To our knowledge, no comparable group of rhabdomyolysis cases was available for replication. Our pre-specified replication plan for the candidate genes was in vitro functional studies () [43
]. In one case,a novel CYP2C8
variant discovered in the re-sequencing effort introduced a frame shift (which changed the sequence of the last 22 amino acids of the CYP2C8 C-terminus and introduced an additional three amino acids), and this variant likely produced a non-functional protein [43
]. Recombinant proteins containing two other non-synonymous variants discovered in the CYP2C8
re-sequencing had in- vitro
kinetic values that were similar to wild type protein. Indeed, recombinant CYP2C8*3 and *4 proteins actually increased the clearance of cerivastatin relative to wild type as has been observed for some other substrates [21
]. The findings of these functional studies may account for the observation that these variants occurred less frequently in cases than controls (ORs, 0.64 to 0.73, ).
There were other limitations. Most of our controls did not use cerivastatin. The small number of cerivastatin users precluded a detailed examination of an interaction between cerivastatin dose and genotype on rhabdomyolysis risk. The cases selected for this study were not a random sample of rhabdomyolysis patients, but a select population of severe cases who went to litigation. Within this select population, the proportion participating was low. Insofar as the cases included in the study differ genetically or phenotypically from cases that were not, results may generalize poorly. We observed one finding from the GWA study at a p-value threshold at which one false positive is expected. Additional studies will be necessary to corroborate the RYR2
finding. The GWA study provided information about common, but not about rare variants, outside the candidate genes. If statin-induced rhabdomyolysis resembles a heterogeneous Mendelian disorder that has several forms caused by genetic variants at many loci in multiple genes - similar to malignant hyperthermia associated with inhalation anesthetics [44
] or clinical phenylketonuria and dietary phenylalanine [45
] - we would not have detected it. The focus of this paper was on genetic risk factors for rhabdomyolysis but other environmental exposures, including concomitant medication use, represent additional risk factors of interest.
For the SLCO1B1 rs4149056 variant, our results extend the findings for high-dose simvastatin-induced myopathy to cerivastatin-associated rhabdomyolysis. Despite a small sample size for the GWA study, our findings also suggest that disruptions in calcium signaling may be associated with the risk of cerivastatin-associated rhabdomyolysis. RYR2 is an interesting candidate for future investigations of statin-related muscle symptoms.