RHOA Is a Modulator of the Cholesterol-Lowering Effects of Statin

Abstract

Although statin drugs are generally efficacious for lowering plasma LDL-cholesterol levels, there is considerable variability in response. To identify candidate genes that may contribute to this variation, we used an unbiased genome-wide filter approach that was applied to 10,149 genes expressed in immortalized lymphoblastoid cell lines (LCLs) derived from 480 participants of the Cholesterol and Pharmacogenomics (CAP) clinical trial of simvastatin. The criteria for identification of candidates included genes whose statin-induced changes in expression were correlated with change in expression of HMGCR, a key regulator of cellular cholesterol metabolism and the target of statin inhibition. This analysis yielded 45 genes, from which RHOA was selected for follow-up because it has been found to participate in mediating the pleiotropic but not the lipid-lowering effects of statin treatment. RHOA knock-down in hepatoma cell lines reduced HMGCR, LDLR, and SREBF2 mRNA expression and increased intracellular cholesterol ester content as well as apolipoprotein B (APOB) concentrations in the conditioned media. Furthermore, inter-individual variation in statin-induced RHOA mRNA expression measured in vitro in CAP LCLs was correlated with the changes in plasma total cholesterol, LDL-cholesterol, and APOB induced by simvastatin treatment (40 mg/d for 6 wk) of the individuals from whom these cell lines were derived. Moreover, the minor allele of rs11716445, a SNP located in a novel cryptic RHOA exon, dramatically increased inclusion of the exon in RHOA transcripts during splicing and was associated with a smaller LDL-cholesterol reduction in response to statin treatment in 1,886 participants from the CAP and Pravastatin Inflamation and CRP Evaluation (PRINCE; pravastatin 40 mg/d) statin clinical trials. Thus, an unbiased filter approach based on transcriptome-wide profiling identified RHOA as a gene contributing to variation in LDL-cholesterol response to statin, illustrating the power of this approach for identifying candidate genes involved in drug response phenotypes.

Transmembrane Protein 55B Is a Novel Regulator of Cellular Cholesterol Metabolism

“…Recently, we have successfully used a similar approach to identify RHOA as a novel determinant of LDL-cholesterol response to statin treatment 9 . Although cellular phenotyping of LCLs has long been employed to functionalize the molecular impact of genetic variation associated with elevated cholesterol levels 2-4, 6, 9 , to our knowledge this is the first instance of capitalizing on the inter-individual variation of a cellular phenotype within LCLs to identify novel genes involved in cellular cholesterol homeostasis.…”

“…For example, given the unclear genetic background of familial combined hyperlipidemia (FCHL), expression array analysis of LCLs from FCHL patients versus healthy controls was used to identify disease-specific transcriptomic profiles 8 . Through expression array analysis of 480 in vitro simvastatin and sham incubated LCLs derived from participants of the Cholesterol and Pharmacogenetics (CAP) statin clinical trial, we recently reported the identification of RHOA as a novel candidate gene implicated in LDL-cholesterol lowering in response to statin treatment 9 . Since HMGCR encodes the rate-limiting enzyme of the cholesterol biosynthesis pathway and is tightly regulated at the level of gene transcription by sterol response element binding factor 2 (SREBF2) in response to changes in intracellular sterol content 10 , we identified genes whose statin-induced expression level changes were most highly correlated with statin-induced changes in HMGCR .…”

SUGP1 is a novel regulator of cholesterol metabolism

“…Whole transcriptome sequencing (RNA-seq) was performed on LCLs from eight African American individuals (seven female and one male) as previously described (48). Libraries were sequenced on an Illumina GAII machine and aligned to hg19 using TopHat v1.2.0 and Bowtie v0.12.7 (49,50).…”

“…Novel junctions were validated using RT-PCR with Sanger sequencing. The effect of SUGP1 on the stability of these HMGCR transcripts was assessed in HepG2 and Huh7 cells incubated with actinomycin D, with transcript half-life calculated as previously described (48). …”

Lipids: new biology and therapeutic targets revealed through human genetics

“…In a systematic manner, this can be achieved by analyzing cells, either by quantifying functional readouts upon candidate gene knockdown and overexpression, as discussed in my own presentation [10], or by comparing functional insights obtained from patient-derived cells with their respective geno types. Following the latter strategy in lympho blasts, Ron Krauss (Children's Hospital, Oakland Research Institute, CA, USA) and Marisa Wang Medina (Children's Hospital, Oakland Research Institute) reported several novel genes for which expression correlates with that of HMGCR and is induced by statins [11], among them GATM, RHOA and HNRNPA1. Interestingly, for RHOA and HNRNPA1, distinct isoforms appear to differentially affect cellular cholesterol levels and the expression of lipid-regulatory genes, which hints at a little-explored layer of lipid metabolism regulation.…”