Designing candidate gene and genome-wide case-control association studies

This protocol describes how to appropriately design a genetic association case-control study, either focusing on a candidate gene (CG) or region or implementing a genome-wide approach. The steps described involve: (i) defining the case phenotype in adequate detail; (ii) checking the heritability of the disease in question; (iii) considering whether a population-based study is the appropriate design for the research question; (iv) the appropriate selection of controls; (v) sample size calculations and (vi) giving due consideration to whether it is a de novo or replication study. General guidelines are given, as well as specific examples of a CG and a genome-wide association study into type 2 diabetes. Software and websites used in this protocol include the International HapMap Consortium website, Genetic Power Calculator, CaT, and SNPSpD. Running each of the programs takes only a few seconds; the rate-limiting steps involve thinking through the designs and parameters in the disease models.     

The architecture of gene regulatory variation across multiple human tissues: the MuTHER study

While there have been studies exploring regulatory variation in one or more tissues, the complexity of tissue-specificity in multiple primary tissues is not yet well understood. We explore in depth the role of cis-regulatory variation in three human tissues: lymphoblastoid cell lines (LCL), skin, and fat. The samples (156 LCL, 160 skin, 166 fat) were derived simultaneously from a subset of well-phenotyped healthy female twins of the MuTHER resource. We discover an abundance of cis-eQTLs in each tissue similar to previous estimates (858 or 4.7% of genes). In addition, we apply factor analysis (FA) to remove effects of latent variables, thus more than doubling the number of our discoveries (1,822 eQTL genes). The unique study design (Matched Co-Twin Analysis--MCTA) permits immediate replication of eQTLs using co-twins (93%-98%) and validation of the considerable gain in eQTL discovery after FA correction. We highlight the challenges of comparing eQTLs between tissues. After verifying previous significance threshold-based estimates of tissue-specificity, we show their limitations given their dependency on statistical power. We propose that continuous estimates of the proportion of tissue-shared signals and direct comparison of the magnitude of effect on the fold change in expression are essential properties that jointly provide a biologically realistic view of tissue-specificity. Under this framework we demonstrate that 30% of eQTLs are shared among the three tissues studied, while another 29% appear exclusively tissue-specific. However, even among the shared eQTLs, a substantial proportion (10%-20%) have significant differences in the magnitude of fold change between genotypic classes across tissues. Our results underline the need to account for the complexity of eQTL tissue-specificity in an effort to assess consequences of such variants for complex traits.     

The role of Helicobacter spp. in the pathogenesis of primary biliary cirrhosis and primary sclerosing cholangitis

Helicobacter species DNA has been detected in liver tissue of patients affected by primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). To investigate a potential causative relation between Helicobacter species and PBC/PSC, we compared the presence of Helicobacter species-specific DNA in liver tissue of patients with PBC/PSC (n=18/n=13) with those of a control group of patients with various liver diseases with known cause (n=29). A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from paraffin embedded liver tissue. Control patients had hepatitis-B (n=9), alcoholic cirrhosis (n=14), or non-cirrhotic metabolic liver disease (n=6). There was no significant difference between the incidence of Helicobacter spp.-specific DNA in PBC/PSC (9/31; 29%) and the control group (10/29; 34%). Sequence analysis confirmed Helicobacter spp. DNA. Because Helicobacter spp. DNA can be found in approximately one-third of all samples tested, it is unlikely that PSC and PBC are caused by Helicobacter infection.     

Lymphotoxin-α Gene and Risk of Myocardial Infarction in 6,928 Cases and 2,712 Controls in the ISIS Case-Control Study

Lymphotoxin-α (LTA) is a pro-inflammatory cytokine that plays an important role in the immune system and local inflammatory response. LTA is expressed in atherosclerotic plaques and has been implicated in the pathogenesis of atherosclerosis and coronary heart disease (CHD). Polymorphisms in the gene encoding lymphotoxin-α (LTA) on Chromosome 6p21 have been associated with susceptibility to CHD, but results in different studies appear to be conflicting. We examined the association of seven single nucleotide polymorphisms (SNPs) across the LTA gene, and their related haplotypes, with risk of myocardial infarction (MI) in the International Study of Infarct Survival (ISIS) case-control study involving 6,928 non-fatal MI cases and 2,712 unrelated controls. The seven SNPs (including the rs909253 and rs1041981 SNPs previously implicated in the risk of CHD) were in strong linkage disequilibrium with each other and contributed to six common haplotypes. Some of the haplotypes for LTA were associated with higher plasma concentrations of C-reactive protein (p = 0.004) and lower concentrations of albumin (p = 0.023). However, none of the SNPs or related haplotypes were significantly associated with risk of MI. The results of the ISIS study were considered in the context of six previously published studies that had assessed this association, and this meta-analysis found no significant association with CHD risk using a recessive model and only a modest association using a dominant model (with narrow confidence intervals around these risk estimates). Overall, these studies provide reliable evidence that these common polymorphisms for the LTA gene are not strongly associated with susceptibility to coronary disease.     

High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88–100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp.     

Variability of gene expression profiles in human blood and lymphoblastoid cell lines

Background

Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction.

Results

A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence.

Conclusion

This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.     

Data quality control in genetic case-control association studies

This protocol details the steps for data quality assessment and control that are typically carried out during case-control association studies. The steps described involve the identification and removal of DNA samples and markers that introduce bias. These critical steps are paramount to the success of a case-control study and are necessary before statistically testing for association. We describe how to use PLINK, a tool for handling SNP data, to perform assessments of failure rate per individual and per SNP and to assess the degree of relatedness between individuals. We also detail other quality-control procedures, including the use of SMARTPCA software for the identification of ancestral outliers. These platforms were selected because they are user-friendly, widely used and computationally efficient. Steps needed to detect and establish a disease association using case-control data are not discussed here. Issues concerning study design and marker selection in case-control studies have been discussed in our earlier protocols. This protocol, which is routinely used in our labs, should take approximately 8 h to complete.     

Lymphotoxin-alpha gene and risk of myocardial infarction in 6,928 cases and 2,712 controls in the ISIS case-control study

Lymphotoxin-alpha (LTA) is a pro-inflammatory cytokine that plays an important role in the immune system and local inflammatory response. LTA is expressed in atherosclerotic plaques and has been implicated in the pathogenesis of atherosclerosis and coronary heart disease (CHD). Polymorphisms in the gene encoding lymphotoxin-alpha (LTA) on Chromosome 6p21 have been associated with susceptibility to CHD, but results in different studies appear to be conflicting. We examined the association of seven single nucleotide polymorphisms (SNPs) across the LTA gene, and their related haplotypes, with risk of myocardial infarction (MI) in the International Study of Infarct Survival (ISIS) case-control study involving 6,928 non-fatal MI cases and 2,712 unrelated controls. The seven SNPs (including the rs909253 and rs1041981 SNPs previously implicated in the risk of CHD) were in strong linkage disequilibrium with each other and contributed to six common haplotypes. Some of the haplotypes for LTA were associated with higher plasma concentrations of C-reactive protein (p = 0.004) and lower concentrations of albumin (p = 0.023). However, none of the SNPs or related haplotypes were significantly associated with risk of MI. The results of the ISIS study were considered in the context of six previously published studies that had assessed this association, and this meta-analysis found no significant association with CHD risk using a recessive model and only a modest association using a dominant model (with narrow confidence intervals around these risk estimates). Overall, these studies provide reliable evidence that these common polymorphisms for the LTA gene are not strongly associated with susceptibility to coronary disease.     

Human metabolic profiles are stably controlled by genetic and environmental variation

¹H Nuclear Magnetic Resonance spectroscopy (¹H NMR) is increasingly used to measure metabolite concentrations in sets of biological samples for top‐down systems biology and molecular epidemiology. For such purposes, knowledge of the sources of human variation in metabolite concentrations is valuable, but currently sparse. We conducted and analysed a study to create such a resource. In our unique design, identical and non‐identical twin pairs donated plasma and urine samples longitudinally. We acquired ¹H NMR spectra on the samples, and statistically decomposed variation in metabolite concentration into familial (genetic and common‐environmental), individual‐environmental, and longitudinally unstable components. We estimate that stable variation, comprising familial and individual‐environmental factors, accounts on average for 60% (plasma) and 47% (urine) of biological variation in ¹H NMR‐detectable metabolite concentrations. Clinically predictive metabolic variation is likely nested within this stable component, so our results have implications for the effective design of biomarker‐discovery studies. We provide a power‐calculation method which reveals that sample sizes of a few thousand should offer sufficient statistical precision to detect ¹H NMR‐based biomarkers quantifying predisposition to disease.     

MicroRNA expression in abdominal and gluteal adipose tissue is associated with mRNA expression levels and partly genetically driven

To understand how miRNAs contribute to the molecular phenotype of adipose tissues and related traits, we performed global miRNA expression profiling in subcutaneous abdominal and gluteal adipose tissue of 70 human subjects and characterised which miRNAs were differentially expressed between these tissues. We found that 12% of the miRNAs were significantly differentially expressed between abdominal and gluteal adipose tissue (FDR adjusted p<0.05) in the primary study, of which 59 replicated in a follow-up study of 40 additional subjects. Further, 14 miRNAs were found to be associated with metabolic syndrome case-control status in abdominal tissue and three of these replicated (primary study: FDR adjusted p<0.05, replication: p<0.05 and directionally consistent effect). Genome-wide genotyping was performed in the 70 subjects to enable miRNA expression quantitative trait loci (eQTL) analysis. Candidate miRNA eQTLs were followed-up in the additional 40 subjects and six significant, independent cis-located miRNA eQTLs (primary study: p<0.001; replication: p<0.05 and directionally consistent effect) were identified. Finally, global mRNA expression profiling was performed in both tissues to enable association analysis between miRNA and target mRNA expression levels. We find 22% miRNAs in abdominal and 9% miRNAs in gluteal adipose tissue with expression levels significantly associated with the expression of corresponding target mRNAs (FDR adjusted p<0.05). Taken together, our results indicate a clear difference in the miRNA molecular phenotypic profile of abdominal and gluteal adipose tissue, that the expressions of some miRNAs are influenced by cis-located genetic variants and that miRNAs are associated with expression levels of their predicted mRNA targets.