Linda S. Wood

Pain perception is altered by a nucleotide polymorphism in SCN9A

The gene SCN9A is responsible for three human pain disorders. Nonsense mutations cause a complete absence of pain, whereas activating mutations cause severe episodic pain in paroxysmal extreme pain disorder and primary erythermalgia. This led us to investigate whether single nucleotide polymorphisms (SNPs) in SCN9A were associated with differing pain perception in the general population. We first genotyped 27 SCN9A SNPs in 578 individuals with a radiographic diagnosis of osteoarthritis and a pain score assessment. A significant association was found between pain score and SNP rs6746030; the rarer A allele was associated with increased pain scores compared to the commoner G allele (P = 0.016). This SNP was then further genotyped in 195 pain-assessed people with sciatica, 100 amputees with phantom pain, 179 individuals after lumbar discectomy, and 205 individuals with pancreatitis. The combined P value for increased A allele pain was 0.0001 in the five cohorts tested (1277 people in total). The two alleles of the SNP rs6746030 alter the coding sequence of the sodium channel Nav1.7. Each was separately transfected into HEK293 cells and electrophysiologically assessed by patch-clamping. The two alleles showed a difference in the voltage-dependent slow inactivation (P = 0.042) where the A allele would be predicted to increase Nav1.7 activity. Finally, we genotyped 186 healthy females characterized by their responses to a diverse set of noxious stimuli. The A allele of rs6746030 was associated with an altered pain threshold and the effect mediated through C-fiber activation. We conclude that individuals experience differing amounts of pain, per nociceptive stimulus, on the basis of their SCN9A rs6746030 genotype.

An association study of 43 SNPs in 16 candidate genes with atorvastatin response

Variation in individual response to statin therapy has been widely studied for a potential genetic component. Multiple genes have been identified as potential modulators of statin response, but few study findings have replicated. To further examine these associations, 2735 individuals on statin therapy, half on atorvastatin and the other half divided among fluvastatin, lovastatin, pravastatin and simvastatin were genotyped for 43 SNPs in 16 genes that have been implicated in statin response. Associations with low-density lipoprotein cholesterol (LDL-C) lowering, total cholesterol lowering, HDL-C elevation and triglyceride lowering were examined. The only significant associations with LDL-C lowering were found with apoE2 in which carriers of the rare allele who took atorvastatin lowered their LDL-C by 3.5% more than those homozygous for the common allele and with rs2032582 (S893A in ABCB1) in which the two groups of homozygotes differed by 3% in LDL-C lowering. These genetic effects were smaller than those observed with the demographic variables of age and gender. The magnitude of all the differences found is sufficiently small that genetic data from these genes should not influence clinical decisions on statin administration.

Comprehensive Whole-Genome and Candidate Gene Analysis for Response to Statin Therapy in the Treating to New Targets (TNT) Cohort

Background—Statins are effective at lowering low-density lipoprotein cholesterol and reducing risk of cardiovascular disease, but variability in response is not well understood. To address this, 5745 individuals from the Treating to New Targets (TNT) trial were genotyped in a combination of a whole-genome and candidate gene approach to identify associations with response to atorvastatin treatment. Methods and Results—A total of 291 988 single-nucleotide polymorphisms (SNPs) from 1984 individuals were analyzed for association with statin response, followed by genotyping top hits in 3761 additional individuals. None was significant at the whole-genome level in either the initial or follow-up test sets for association with low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, or triglyceride response. In addition to the whole-genome platform, 23 candidate genes previously associated with statin response were analyzed in these 5745 individuals. Three SNPs in apoE were most highly associated with low-density lipoprotein cholesterol response, followed by 1 in PCSK9 with a similar effect size. At the candidate gene level, SNPs in HMGCR were also significant though the effect was less than with those in apoE and PCSK9. rs7412/apoE had the most significant association (P=6×10−30), and its high significance in the whole-genome study (P=4×10−9) confirmed the suitability of this population for detecting effects. Age and gender were found to influence low-density lipoprotein cholesterol response to a similar extent as the most pronounced genetic effects. Conclusions—Among SNPs tested with an allele frequency of at least 5%, only SNPs in apoE are found to influence statin response significantly. Less frequent variants in PCSK9 and smaller effect sizes in SNPs in HMGCR were also revealed.

Comprehensive Whole Genome and Candidate Gene Analysis for Response to Statin Therapy in TNT Cohort

Background—Statins are effective at lowering low-density lipoprotein cholesterol and reducing risk of cardiovascular disease, but variability in response is not well understood. To address this, 5745 individuals from the Treating to New Targets (TNT) trial were genotyped in a combination of a whole-genome and candidate gene approach to identify associations with response to atorvastatin treatment. Methods and Results—A total of 291 988 single-nucleotide polymorphisms (SNPs) from 1984 individuals were analyzed for association with statin response, followed by genotyping top hits in 3761 additional individuals. None was significant at the whole-genome level in either the initial or follow-up test sets for association with low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, or triglyceride response. In addition to the whole-genome platform, 23 candidate genes previously associated with statin response were analyzed in these 5745 individuals. Three SNPs in apoE were most highly associated with low-density lipoprotein cholesterol response, followed by 1 in PCSK9 with a similar effect size. At the candidate gene level, SNPs in HMGCR were also significant though the effect was less than with those in apoE and PCSK9. rs7412/apoE had the most significant association (P=6×10−30), and its high significance in the whole-genome study (P=4×10−9) confirmed the suitability of this population for detecting effects. Age and gender were found to influence low-density lipoprotein cholesterol response to a similar extent as the most pronounced genetic effects. Conclusions—Among SNPs tested with an allele frequency of at least 5%, only SNPs in apoE are found to influence statin response significantly. Less frequent variants in PCSK9 and smaller effect sizes in SNPs in HMGCR were also revealed.

Significant support for DAO as a schizophrenia susceptibility locus : Examination of five genes putatively associated with schizophrenia

BACKGROUND Schizophrenia is a complex psychiatric disorder with a strong genetic component. Past linkage studies have implicated several chromosomal regions in the etiology of schizophrenia. Within these regions, several genes have been identified via candidate gene association studies as strong schizophrenia susceptibility loci, including DAO, DAOA, DISC1, DTNBP1, and RGS4. METHODS The present study attempted to replicate these association findings by analyzing a total of 120 markers across these genes in 311 schizophrenia subjects, 140 schizoaffective subjects, and 291 control subjects. RESULTS Our study found no association for DAOA and DTNBP1 with schizophrenia. Although no association was seen with DAOA and DTNBP1, several other markers in the other genes resulted in significant association with schizophrenia (p < .05). However, after a conservative Bonferroni correction for multiple testing, only one marker, rs3918346, within DAO remained significant (odds ratio = 1.71, confidence interval = 1.32-2.22, p = 4 x 10(-5)). This significant association was concordant with previous DAO genetic findings. CONCLUSIONS Our results significantly support DAO as a susceptibility locus for schizophrenia and offer some support for the implication of both RGS4 and DISC1 in the etiology of schizophrenia. However, we see no evidence to support either DAOA or DTNBP1 as schizophrenia disease loci.

Cholesteryl ester transfer protein variants have differential stability but uniform inhibition by torcetrapib

Cholesteryl ester transfer protein (CETP) is an important modulator of high density lipoprotein cholesterol in humans and thus considered to be a therapeutic target for preventing cardiovascular disease. The gene encoding CETP has been shown to be highly variable, with multiple single nucleotide polymorphisms responsible for altering both its transcription and sequence. Examining nine missense variants of CETP, we found some had significant associations with CETP mass and high density lipoprotein cholesterol levels. Two variants, Pro-373 and Gln-451, appear to be more stable in vivo, an observation mirrored by partial proteolysis studies performed in vitro. Because these naturally occurring variant proteins are potentially present in clinical populations that will be treated with CETP inhibitors, all commonly occurring haplotypes were tested to determine whether the proteins they encode could be inhibited by torcetrapib, a compound currently in clinical trials in combination with atorvastatin. Torcetrapib behaved similarly with all variants, with no significant differences in inhibition.

High-density genotyping and functional SNP localization in the CETP gene

The cholesteryl ester transfer protein gene (CETP) has been the subject of hundreds of genetic analyses that typically focus on a small number of polymorphisms within a single ethnic group. Furthermore, the extent of DNA beyond the transcribed sequence from which single nucleotide polymorphisms (SNPs) may influence CETP expression has not been well defined. To better understand the role of natural variation in modulating CETP and high density lipoprotein-cholesterol (HDL-C) levels, dense genotyping of CETP and regions up to 15 kb on either side of the gene was carried out on >2,000 individuals. A complex, nonlinear set of linkage disequilibrium bins was found, with many bins interspersed along the DNA sequence and spread over large regions of the gene. Bins assigned based on large numbers of individuals matched the small subset of SNPs that had been assigned to bins previously with a small number of individuals. Associations of known functional SNPs with HDL-C were found, but there were suggestions that there are additional functional SNPs not characterized previously. Narrowing of the set of likely functional SNPs was accomplished by comparing associations observed in different ethnic groups. The promoter SNP most highly associated with HDL-C that is likely to be functional, position −4,502, alters a consensus transcription factor binding site.

Association between neprilysin polymorphisms and sporadic Alzheimer's disease

The deposition of amyloid beta peptide (Abeta) in the form of plaques in the brain is a hallmark of Alzheimers disease (AD). Neprilysin is the major Abeta-degradating enzyme and reduction in neprilysin activity could contribute to Alzheimers by increasing the steady-state level of Abeta. To provide further evidence for the role of neprilysin in AD we genotyped 22 polymorphisms, 21 SNPs and the GT repeat in the promoter region, across the neprilysin gene in 298 Caucasian sporadic Alzheimers patients and 298 age-matched controls. Several SNPs showed genotypic and allelic association to AD. SNP rs1836915, in linkage disequilibrium block 2, showed the greatest extent of genotypic association with AD (p=0.0076). We were unable to replicate any of the SNPs that were previously reported as putatively associated with AD. However, these novel findings add to the weight of evidence supporting the involvement of neprilysin in the aetiology of Alzheimers disease.

Genetic Association of the CCR5 Region with Lipid Levels in At-Risk Cardiovascular Patients

Background—There is mounting evidence to suggest that chemokine receptor 5 (CCR5) plays an important role in the development and progression of atherosclerosis. A naturally occurring variant of the CCR5 gene CCR532, exists at allele frequencies of typically 10% in European populations and results in a nonfunctional CCR5 receptor. Methods and Results—The CCR5&Dgr;32 deletion and 26 other variants within the chemokine receptor 2-CCR5-chemokine receptor-like protein 2 (CCRL2) gene cluster spanning 59 kilobases of chromosome 3 were genotyped in 5748 subjects from the Treating to New Targets atorvastatin trial to determine whether genetic associations could be identified with circulating lipid values and cardiovascular disease. Our results demonstrate an association between the CCR5&Dgr;32 deletion and increased plasma high-density lipoprotein cholesterol and decreased plasma triglycerides, both of which are beneficial from a cardiovascular perspective. Three single-nucleotide polymorphisms (rs1154428, rs6808835, and rs6791599) in CCRL2 in linkage disequilibrium (r2≥0.65) with CCR5&Dgr;32 and located up to 45 kilobases distal to it were associated with high-density lipoprotein cholesterol. The high-density lipoprotein cholesterol and triglycerides findings were replicated in an additional set of >6000 individuals from the Incremental Decrease in Endpoints through Aggressive Lipid Lowering atorvastatin trial. Conclusions—Our study provides evidence that a locus within the region of the genome encompassing the CCR5-CCRL2 region is associated with lipid levels and suggests that chemokine activity influences lipid levels in populations with preexisting cardiovascular disease. Clinical Trial Registration—clinicaltrials.gov. Identifier: TNT, NCT00327691; IDEAL, NCT00159835.

Frequency and function of CETP variants among individuals of Asian ancestry.

Genetic variation in CETP (cholesteryl ester transfer protein) has been clearly associated with HDL cholesterol levels but its association with cardiovascular disease and related phenotypes has been more controversial, possibly due to variability of polymorphisms and their frequencies across different ethnic populations. To see if there are undetected polymorphisms affecting protein sequence in individuals of Asian ancestry and to determine the functionality of such variants, all exons and adjacent intronic segments were resequenced in 96 individuals and the observed variants cloned and analyzed. Two novel SNPs, including one coding change, S332 to Y332, were identified. Y332 and all other reported variants in Asians were cloned for study in vitro. Secretion efficiency was determined by Western blotting of protein from cell lysates and media. Cholesteryl ester transfer activity was measured in vitro by following the extent of transfer of fluorescently labeled substrate. Y332, Q296 and G442 are all secreted less well than wild type protein but retain significant transfer activity. P151 is not secreted and no transfer activity was detected. These protein variants should all contribute to higher HDL cholesterol in individuals carrying them. Additionally, a splicing variation that causes a protein truncation and non-functional CETP that has been reported predominantly in Asians was also found in two individuals of European ancestry and was on the same haplotype background in the two populations, suggesting a common origin of this null variant. This improved understanding of CETP variation in Asians will allow a more effective comparison of studies across populations.