Andrew J.P. Smith

Cytokine and cytokine receptor gene polymorphisms and their functionality.

Cytokines, signaling proteins produced by a variety of cell types, are essential for the development and functioning of both innate and adaptive immune response. Cytokine gene expression is tightly regulated, and aberrant expression from environmental and genetic polymorphism has been implicated in a range of diseases, susceptibility to infections, and responses to treatment. This review concentrates on the functionality of cytokine and cytokine receptor gene polymorphisms; it is through these variants that genuine disease-associations are based. Several mechanisms for single nucleotide polymorphism (SNP) functionality are present within cytokine genes including: amino acid changes (IL-6R, IL-13, IL-1alpha), exon skipping (IL-7Ralpha), proximal promoter variants (IL-1beta, IL-Ra, IL-2, IL-6, IL-10, IL-12, IL-13, IL-16, TNF, IFN-gamma, TGF-beta), distal promoter variants (IL-6, IL-18) and intronic enhancer variants (IL-8).

Interleukin-6 receptor pathways in abdominal aortic aneurysm

Methods We conducted a systematic review and meta-analysis of studies reporting circulating IL-6 in AAA, and new investigations of the association between a common non-synonymous functional variant (Asp358Ala) in the IL-6R gene (IL6R) and AAA, followed the analysis of the variant both in vitro and in vivo. Inflammation may play a role in the development of abdominal aortic aneurysms (AAA). Interleukin-6 (IL-6) signalling through its receptor (IL-6R) is one pathway that could be exploited pharmacologically. We investigated this using a Mendelian randomization approach. Results Up to October 2011, we identified seven studies (869 cases, 851 controls). Meta-analysis demonstrated that AAA cases had higher levels of IL-6 than controls [standardized mean difference (SMD) = 0.46 SD, 95% CI = 0.25–0.66, I2 = 70%, P = 1.1 × 10–5 random effects]. Meta-analysis of five studies (4524 cases/15 710 controls) demonstrated that rs7529229 (which tags the non-synonymous variant Asp358Ala, rs2228145) was associated with a lower risk of AAA, per Ala358 allele odds ratio 0.84, 95% CI: 0.80–0.89, I2 = 0%, P = 2.7 × 10–11). In vitro analyses in lymphoblastoid cell lines demonstrated a reduction in the expression of downstream targets (STAT3, MYC and ICAM1) in response to IL-6 stimulation in Ala358 carriers. Conclusions A Mendelian randomization approach provides robust evidence that signalling via the IL-6R is likely to be a causal pathway in AAA. Drugs that inhibit IL-6R may play a role in AAA management.

LARGE SCALE META-ANALYSIS OF INTERLEUKIN-1 BETA AND INTERLEUKIN-1 RECEPTOR ANTAGONIST POLYMORPHISMS ON RISK OF RADIOGRAPHIC HIP AND KNEE OSTEOARTHRITIS AND SEVERITY OF KNEE OSTEOARTHRITIS

OBJECTIVE To clarify the role of common genetic variation in the Interleukin-1β (IL1B) and Interleukin-1R antagonist (IL1RN) genes on risk of knee and hip osteoarthritis (OA) and severity of knee OA by means of large-scale meta-analyses. METHODS We searched PubMed for articles assessing the role of IL1B and IL1RN polymorphisms/haplotypes on the risk of hip and/or knee OA. Novel data were included from eight unpublished studies. Meta-analyses were performed using fixed- and random-effects models with a total of 3595 hip OA and 5013 knee OA cases, and 6559 and 9132 controls respectively. The role of ILRN haplotypes on radiographic severity of knee OA was tested in 1918 cases with Kellgren-Lawrence (K/L) 1 or 2 compared to 199 cases with K/L 3 or 4. RESULTS The meta-analysis of six published studies retrieved from the literature search and eight unpublished studies showed no evidence of association between common genetic variation in the IL1B or IL1RN genes and risk of hip OA or knee OA (P>0.05 for rs16944, rs1143634, rs419598 and haplotype C-G-C (rs1143634, rs16944 and rs419598) previously implicated in risk of hip OA). The C-T-A haplotype formed by rs419598, rs315952 and rs9005, previously implicated in radiographic severity of knee OA, was associated with reduced severity of knee OA (odds ratio (OR)=0.71 95%CI 0.56-0.91; P=0.006, I(2)=74%), and achieved borderline statistical significance in a random-effects model (OR=0.61 95%CI 0.35-1.06 P=0.08). CONCLUSION Common genetic variation in the Interleukin-1 region is not associated with prevalence of hip or knee OA but our data suggest that IL1RN might have a role in severity of knee OA.

INSIG2 gene polymorphism is not associated with obesity in Caucasian, Afro-Caribbean and Indian subjects.

A common polymorphism, rs7566605, 10 kb upstream of the insulin-induced gene 2 transcription start site has been associated with obesity in Caucasian and African-American populations, with the hypothesis that an alteration in gene expression results in elevated plasma triglyceride levels. The goal of this study was to verify the findings in a cohort of 2721 healthy Caucasian men (second Northwick Park Heart Study), and a separate study of 747 type 2 diabetic patients from Caucasian, Afro-Caribbean and Indian groups (University College London Diabetes and Cardiovascular Disease Study).The rs7566605 single-nucleotide polymorphism (SNP) was not related to plasma triglyceride levels in either study, and we found no association with body mass index or obesity in either cohort, despite having the power to detect the previously reported effect. This suggests that, at the least, the true size of the effect on obesity of this SNP is likely to be considerably less than reported previously.

The functional interaction on in vitro gene expression of APOA5 SNPs, defining haplotype APOA5*2, and their paradoxical association with plasma triglyceride but not plasma apoAV levels

Plasma triglyceride (TG) and apoAV levels are reported to be positively correlated, yet SNPs defining haplotype APOA52 have consistently shown association with elevated plasma triglyceride (TG) but not plasma apoAV levels. We previously reported that individually -1131T>C, -3A>G and +1891T>C did not influence luciferase activity or in vitro translation efficiency. To investigate the combined effect of these SNPs additional constructs were examined. Compared to the wildtype -1131T/-3A/+1891T (TAT), the triple rare allele construct -1131C/-3G/+1891C (CGC) conferred 46% lower luciferase activity (p<0.0001), showing these SNPs are acting co-operatively. Although only these two combinations occur in vivo, we experimentally altered the TAT construct one site at a time; -3G (TGT) had the largest effect (94% lower luciferase), with lesser effects from CAT (-77%) and TAC (-70.3%) (all p<0.0001). Deletion constructs excluding one site at a time showed that -3G/1891C ( -GC) in combination, compared to -AT, was having the largest effect on luciferase activity (-59%, p=0.055). Using sequence homology and EMSA analysis no transcription factor binding at -1131 or +1891 was identified, though +1891 lies within a putative mRNA stability motif. Taken together, these data identify -3A>G in the Kozak sequence as functional, affecting translation initiation and driving the haplotype effects, while showing interaction with +1891T>C and to a lesser extent -1131T>C. A paradox arises since these results predict that APOA52 will lead to reduced apoAV with concomitant reduced LPL activation or lipoprotein-receptor interaction, resulting in higher plasma TG levels. We conclude that APOA5 expression, and not circulating plasma apoAV levels, is causatively associated with plasma TG levels.

Characterization of DNA-Binding Proteins Using Multiplexed Competitor EMSA

We describe a low-cost high-throughput technique to characterize nuclear protein DNA-binding interactions. This technique, known as Multiplexed Competitor Electrophoretic Mobility Shift Assay, uses a series of multiplexed oligonucleotide DNA consensus competitors, in combination with a standard electrophoretic mobility shift assay procedure, to efficiently characterize DNA-binding proteins. We show utility for the method to identify a previously unreported hepatocyte nuclear factor-3 site created in intron 8 of the lipoprotein lipase gene by a common single-nucleotide polymorphism (rs327).

Cortical ependymoma or monomorphous angiocentric glioma

Ependymoma is the third most common childhood intracranial tumor after medulloblastoma and pilocytic astrocytoma. Most ependymomas occur in the posterior fossa and spinal cord but only five cases confined to the cerebral cortex have been reported. The current case is a 5‐year‐old boy with a somewhat ill‐defined cortical tumor diagnosed as pilocytic astrocytoma on biopsy, and treated with radiotherapy. Nine years later, resection of the essentially unaltered tumor was performed for treatment of intractable seizures. Histologically, the tumor had some areas with the typical appearance of ependymoma as well other areas which contained piloid cells. There was also evidence of focal infiltrative growth. These findings bore resemblance to a recently described entity monomorphous angiocentric glioma/angiocentric neuroepithelial tumor, which combines features of ependymoma with pilocytic and diffuse astrocytomas. Both cortical ependymomas and angiocentric monomorphous glioma/angiocentric neuroepithelial tumor appear to be low‐grade tumors although their rarity makes accurate prognosis problematic. The current case has features of both entities, suggesting they may be closely related.

Application of statistical and functional methodologies for the investigation of genetic determinants of coronary heart disease biomarkers: lipoprotein lipase genotype and plasma triglycerides as an exemplar

Genome-wide association studies have proved very successful in identifying novel single-nucleotide polymorphisms (SNPs) associated with disease or traits, but the related, functional SNP is usually unknown. In this paper, we describe a methodology to locate and validate candidate functional SNPs using lipoprotein lipase (LPL), a gene previously associated with triglyceride levels, as an exemplar. Two thousand seven hundred and eighty-six healthy middle-aged men from the NPHSII UK prospective study (with up to six measures of plasma lipid levels) were genotyped for 20 LPL tagging (t)SNPs using Illumina Bead technology. Using model-selection procedures and haplotypes, we identified eight SNPs that consistently maximized the fit of the model to the phenotype. Fifteen SNPs in high linkage disequilibrium with these were identified, and functional assays were carried out on all 23 SNPs. Electrophoretic mobility shift assay (EMSA) was used to identify SNPs that had the potential to alter DNA–protein interactions, reducing the number to eight possible candidate SNPs. These were examined for ability to alter expression using a luciferase reporter assay, and two regulatory SNPs, showing genotype differences, rs327 and rs3289, were identified. Finally, multiplexed-competitor-EMSA (MC-EMSA) and supershift EMSA identified FOXA2 to rs327T, and CREB-binding protein (CBP) and CCAAT displacement protein (CDP) to rs3289C as the factors responsible for transcription binding. We have identified two novel candidate functional SNPs in LPL and presented a procedure aimed to efficiently detect SNPs potentially causal to genetic association. We believe that this methodology could be successfully applied to future re-sequencing data.

A functional mutation in the LDLR promoter (-139C > G) in a patient with familial hypercholesterolemia

A novel sequence change in repeat 3 of the promoter of the low-density lipoprotein receptor (LDLR) gene, −139C>G, has been identified in a patient with familial hypercholesterolemia (FH). LDLR -139G has been passed to one offspring who also shows an FH phenotype. Transient transfection studies using luciferase gene reporter assays revealed a considerable reduction (74±1.4% SEM) in reporter gene expression from the −139G variant sequence compared to the wild-type sequence, strongly suggesting that this change is the basis for FH in these patients. Analysis using electrophoretic mobility shift assay demonstrated the loss of Sp1 binding to the variant sequence in vitro, explaining the reduction of transcription.

Novel genetic approach to investigate the role of plasma secretory phospholipase A2 (sPLA2)-V isoenzyme in coronary heart disease: modified Mendelian randomization analysis using PLA2G5 expression levels.

Background—Secretory phospholipase A2 (sPLA2) enzymes are considered to play a role in atherosclerosis. sPLA2 activity encompasses several sPLA2 isoenzymes, including sPLA2-V. Although observational studies show a strong association between elevated sPLA2 activity and CHD, no assay to measure sPLA2-V levels exists, and the only evidence linking the sPLA2-V isoform to atherosclerosis progression comes from animal studies. In the absence of an assay that directly quantifies sPLA2-V levels, we used PLA2G5 mRNA levels in a novel, modified Mendelian randomization approach to investigate the hypothesized causal role of sPLA2-V in coronary heart disease (CHD) pathogenesis. Methods and Results—Using data from the Advanced Study of Aortic Pathology, we identified the single-nucleotide polymorphism in PLA2G5 showing the strongest association with PLA2G5 mRNA expression levels as a proxy for sPLA2-V levels. We tested the association of this SNP with sPLA2 activity and CHD events in 4 prospective and 14 case–control studies with 27 230 events and 70 500 controls. rs525380C>A showed the strongest association with PLA2G5 mRNA expression (P=5.1×10−6). There was no association of rs525380C>A with plasma sPLA2 activity (difference in geometric mean of sPLA2 activity per rs525380 A-allele 0.4% (95% confidence intervals [−0.9%, 1.6%]; P=0.56). In meta-analyses, the odds ratio for CHD per A-allele was 1.02 (95% confidence intervals [0.99, 1.04]; P=0.20). Conclusions—This novel approach for single-nucleotide polymorphism selection for this modified Mendelian randomization analysis showed no association between rs525380 (the lead single-nucleotide polymorphism for PLA2G5 expression, a surrogate for sPLA2-V levels) and CHD events. The evidence does not support a causal role for sPLA2-V in CHD.