Variants of the Coagulation and Inflammation Genes Are Replicably Associated with Myocardial Infarction and Epistatically Interact in Russians

Background

In spite of progress in cardiovascular genetics, data on genetic background of myocardial infarction are still limited and contradictory. This applies as well to the genes involved in inflammation and coagulation processes, which play a crucial role in the disease etiopathogenesis.

Methods and Results

In this study we found genetic variants of TGFB1, FGB and CRP genes associated with myocardial infarction in discovery and replication groups of Russian descent from the Moscow region and the Republic of Bashkortostan (325/185 and 220/197 samples, correspondingly). We also found and replicated biallelic combinations of TGFB1 with FGB, TGFB1 with CRP and IFNG with PTGS1 genetic variants associated with myocardial infarction providing a detectable cumulative effect. We proposed an original two-component procedure for the analysis of nonlinear (epistatic) interactions between the genes in biallelic combinations and confirmed the epistasis hypothesis for the set of alleles of IFNG with PTGS. The procedure is applicable to any pair of logical variables, e.g. carriage of two sets of alleles. The composite model that included three single gene variants and the epistatic pair has AUC of 0.66 both in discovery and replication groups.

Conclusions

The genetic impact of TGFB1, FGB, CRP, IFNG, and PTGS and/or their biallelic combinations on myocardial infarction was found and replicated in Russians. Evidence of epistatic interactions between IFNG with PTGS genes was obtained both in discovery and replication groups.     

Multifactor Effects and Evidence of Potential Interaction between Complement Factor H Y402H and LOC387715 A69S in Age-Related Macular Degeneration

Background

Variants in the complement cascade genes and the LOC387715/HTRA1, have been widely reported to associate with age-related macular degeneration (AMD), the most common cause of visual impairment in industrialized countries.

Methods/Principal Findings

We investigated the association between the LOC387715 A69S and complement component C3 R102G risk alleles in the Finnish case-control material and found a significant association with both variants (OR 2.98, p = 3.75×10⁻⁹; non-AMD controls and OR 2.79, p = 2.78×10⁻¹⁹, blood donor controls and OR 1.83, p = 0.008; non-AMD controls and OR 1.39, p = 0.039; blood donor controls), respectively. Previously, we have shown a strong association between complement factor H (CFH) Y402H and AMD in the Finnish population. A carrier of at least one risk allele in each of the three susceptibility loci (LOC387715, C3, CFH) had an 18-fold risk of AMD when compared to a non-carrier homozygote in all three loci. A tentative gene-gene interaction between the two major AMD-associated loci, LOC387715 and CFH, was found in this study using a multiplicative (logistic regression) model, a synergy index (departure-from-additivity model) and the mutual information method (MI), suggesting that a common causative pathway may exist for these genes. Smoking (ever vs. never) exerted an extra risk for AMD, but somewhat surprisingly, only in connection with other factors such as sex and the C3 genotype. Population attributable risks (PAR) for the CFH, LOC387715 and C3 variants were 58.2%, 51.4% and 5.8%, respectively, the summary PAR for the three variants being 65.4%.

Conclusions/Significance

Evidence for gene-gene interaction between two major AMD associated loci CFH and LOC387715 was obtained using three methods, logistic regression, a synergy index and the mutual information (MI) index.     

Biochemical analysis of a common human polymorphism associated with age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed countries. A large number of human genetic studies have associated a common variant (Y402H) of complement factor H (CFH) with a highly significant increase in AMD risk. CFH is a modular protein with 20 homologous short consensus repeats (SCRs). The Y402H variant is located in SCR7 of both CFH and factor H-like protein 1 (FHL-1), a splice variant of CFH (containing SCR1-7) with unique biochemical properties. Because SCR7 is known to bind to heparin, C-reactive protein (CRP), and M protein from Streptococcus pyogenes, it has been hypothesized that the AMD-associated polymorphism may affect interactions with these CFH ligands. In this study, we tested this hypothesis in the context of full-length CFH (SCR1-20) and FHL-1. We systematically analyzed the interactions of the Y402 and H402 variants of CFH and FHL-1 with heparin, CRP, and several bacterial ligands: M6 protein of Streptococcus pyogenes, PspC of Streptococcus pneumoniea, and BbCRASP-1 of Borrelia burgdorferi. In comparing the Y and H variants of CFH and FHL-1, we found no significant difference in their protein secretion, cofactor activity, or interactions with heparin, BbCRASP-1, or PspC, but a significant difference in binding to CRP and M6 protein. This study reveals the fundamental properties of a common polymorphism of CFH and lays the groundwork for elucidating the role of CFH in AMD pathogenesis.     

Epistatic Module Detection for Case-Control Studies: A Bayesian Model with a Gibbs Sampling Strategy

The detection of epistatic interactive effects of multiple genetic variants on the susceptibility of human complex diseases is a great challenge in genome-wide association studies (GWAS). Although methods have been proposed to identify such interactions, the lack of an explicit definition of epistatic effects, together with computational difficulties, makes the development of new methods indispensable. In this paper, we introduce epistatic modules to describe epistatic interactive effects of multiple loci on diseases. On the basis of this notion, we put forward a Bayesian marker partition model to explain observed case-control data, and we develop a Gibbs sampling strategy to facilitate the detection of epistatic modules. Comparisons of the proposed approach with three existing methods on seven simulated disease models demonstrate the superior performance of our approach. When applied to a genome-wide case-control data set for Age-related Macular Degeneration (AMD), the proposed approach successfully identifies two known susceptible loci and suggests that a combination of two other loci—one in the gene SGCD and the other in SCAPER—is associated with the disease. Further functional analysis supports the speculation that the interaction of these two genetic variants may be responsible for the susceptibility of AMD. When applied to a genome-wide case-control data set for Parkinson's disease, the proposed method identifies seven suspicious loci that may contribute independently to the disease.     

Analysis of the possible involvement of the glutamate transporter gene EAAT2 and the glutamate receptor genes GRIA1 and GRIA2 in the pathogenesis of motor neuron disease in the Russian population

Polymorphisms of the genes of the glutamatergic system EAAT2, GRIA1, and GRIA2 have been analyzed in patients with sporadic motor neuron disease (MND) from Russia. The disease is not associated with polymorphic alleles of the genes studied, which indicates that EAAT2, GRIA1, and GRIA2 play an insignificant role in the pathogenesis of sporadic MND.     

Subgroups of Paediatric Acute Lymphoblastic Leukaemia Might Differ Significantly in Genetic Predisposition to Asparaginase Hypersensitivity

L-asparaginase (ASP) is a key element in the treatment of paediatric acute lymphoblastic leukaemia (ALL). However, hypersensitivity reactions (HSRs) to ASP are major challenges in paediatric patients. Our aim was to investigate genetic variants that may influence the risk to Escherichia coli-derived ASP hypersensitivity. Sample and clinical data collection was carried out from 576 paediatric ALL patients who were treated according to protocols from the Berlin—Frankfurt—Münster Study Group. A total of 20 single nucleotide polymorphisms (SNPs) in GRIA1 and GALNT10 genes were genotyped. Patients with GRIA1 rs4958351 AA/AG genotype showed significantly reduced risk to ASP hypersensitivity compared to patients with GG genotype in the T-cell ALL subgroup (OR = 0.05 (0.01–0.26); p = 4.70E-04), while no such association was found in pre-B-cell ALL. In the medium risk group two SNPs of GRIA1 (rs2055083 and rs707176) were associated significantly with the occurrence of ASP hypersensitivity (OR = 0.21 (0.09–0.53); p = 8.48E-04 and OR = 3.02 (1.36–6.73); p = 6.76E-03, respectively). Evaluating the genders separately, however, the association of rs707176 with ASP HSRs was confined only to females. Our results suggest that genetic variants of GRIA1 might influence the risk to ASP hypersensitivity, but subgroups of patients can differ significantly in this respect.     

Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.     

Immunohistochemical localization of complement regulatory proteins in the human retina

Age-related macular degeneration (AMD) is a complex disease. Genetic studies have found strong associations between AMD and variants of several complement pathway-associated genes. The regulation of the complement cascade seems to be critical in the pathogenesis of AMD. In 45 human donor eyes immunohistochemistry was performed using antibodies directed against major regulators of the complement system: complement factor H (CFH), decay accelerating factor (DAF/CD55), complement receptor 1 (CR1/CD35), and membrane cofactor protein (MCP/CD46). All eyes were classified in AMD and controls. 11 eyes were graded as early AMD. 34 eyes were controls. In all eyes staining was found in intercapillary pillars of choroid adjacent to Bruch's membrane for CFH, at the basal surface of RPE cells for MCP, and at the apical side of the retinal pigment epithelium for CR1. DAF immunoreactivity was increased along the inner segments of rod and cone photoreceptor cells at the level of the external limiting membrane Labeling of soft drusen was found for CFH and CR1. In addition, DAF and CR1 showed staining of ganglion cells in all eyes. CFH and particularly MCP showed decreased or absent staining in eyes with early AMD adjacent to Bruch's membrane. The overlapping expression of regulators at the level of Bruch's membrane and the retinal pigment epithelium shows the importance of this site for control of the complement system. Decreased and therefore unbalanced expression of regulators, as shown in this study for CFH and MCP, may ultimately lead to AMD.     

The molecular genetic basis of age-related macular degeneration: an overview

Age-related macular degeneration (AMD) is a complex disorder of the eye and the third leading cause of blindness worldwide. With a multifactorial etiology, AMD results in progressive loss of central vision affecting the macular region of the eye in elderly. While the prevalence is relatively higher in the Caucasian populations, it has gradually become a major public health issue among the non-Caucasian populations (including Indians) as well due to senescence, rapidly changing demographics and life-style factors. Recent genome-wide association studies (GWAS) on large case-control cohorts have helped in mapping genes in the complement cascade that are involved in the regulation of innate immunity with AMD susceptibility. Genes involved with mitochondrial oxidative stress and extracellular matrix regulation also play a role in AMD pathogenesis. Majority of the associations observed in complement (CFH, CFB, C2 and C3) and other (ARMS2 and HTRA1) genes have been replicated in diverse populations worldwide. Gene-gene (CFH with ARMS2 and HTRA1) interactions and correlations with environmental traits (smoking and body mass index) have been established as significant covariates in AMD pathology. In this review, we have provided an overview on the underlying molecular genetic mechanisms in AMD worldwide and highlight the AMD-associated-candidate genes and their potential role in disease pathogenesis.     

Whole Exome Sequencing in Patients with the Cuticular Drusen Subtype of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly people worldwide. Cuticular drusen (CD) is a clinical subtype of AMD, which typically displays an earlier age at onset, and has a strong genetic component. Genetic studies support a role for rare sequence variants in CD susceptibility, and rare sequence variants in the CFH gene have been identified in 8.8% of CD cases. To further explore the role of rare variants in CD, we performed whole exome sequencing (WES) in 14 affected members of six families and 12 sporadic cases with CD. We detected rare sequence variants in CFH and FBLN5, which previously were shown to harbor rare variants in patients with CD. In addition, we detected heterozygous rare sequence variants in several genes encoding components of the extracellular matrix (ECM), including FBLN1, FBLN3/EFEMP1, FBLN5, FBLN6/HMCN1, FBN2, and COL15A1. Two rare pathogenic variants were identified in the COL15A1 gene: one in a sporadic case and another was found to segregate in a family with six affected individuals with CD. In addition, two rare pathogenic variants were identified in the FGL1 gene in three unrelated CD cases. These findings suggest that alterations in the ECM and in the coagulation pathway may play a role in the pathogenesis of CD. The identified candidate genes require further analyses in larger cohorts to confirm their role in the CD subtype of AMD. No evidence was found of rare sequence variants in a single gene that segregate with CD in the six families, suggesting that the disease is genetically heterogeneous.     

CXCL5 polymorphisms are associated with variable blood pressure in cardiovascular disease-free adults

Age-related macular degeneration (AMD) is a complex and multifaceted disease involving contributions from both genetic and environmental influences. Previous work exploring the genetic contributions of AMD has implicated numerous genomic regions and a variety of candidate genes as modulators of AMD susceptibility. Nevertheless, much of this work has revolved around single-nucleotide polymorphisms (SNPs), and it is apparent that a significant portion of the heritability of AMD cannot be explained through these mechanisms. In this review, we consider the role of common variants, rare variants, copy number variations, epigenetics, microRNAs, and mitochondrial genetics in AMD. Copy number variations in regulators of complement activation genes (CFHR1 and CFHR3) and glutathione S transferase genes (GSTM1 and GSTT1) have been associated with AMD, and several additional loci have been identified as regions of potential interest but require further evaluation. MicroRNA dysregulation has been linked to the retinal pigment epithelium degeneration in geographic atrophy, ocular neovascularization, and oxidative stress, all of which are hallmarks in the pathogenesis of AMD. Certain mitochondrial DNA haplogroups and SNPs in mitochondrially encoded NADH dehydrogenase genes have also been associated with AMD. The role of these additional mechanisms remains only partly understood, but the importance of their further investigation is clear to elucidate more completely the genetic basis of AMD.     

ACACβ gene (rs2268388) and AGTR1 gene (rs5186) polymorphism and the risk of nephropathy in Asian Indian patients with type 2 diabetes

Patients with type 2 diabetes (T2DM) are usually obese and concurrent obesity results into activation of the renin–angiotensin-system (RAS) which is a risk factor for diabetic nephropathy (DN). Gene–gene interaction between acetyl-coenzymeA carboxylase beta (ACACβ) gene, which is involved in fatty acid metabolism and angiotensin II receptors (AGTR1) gene, which mediates RAS proteins actions on renal tissue, polymorphism with DN have not been studied earlier. The present study was designed with the aim to examine the association of an ACACβ (rs2268388) and AGTR1 (rs5186) gene polymorphism with the risk of DN in Asian Indians. 1,158 patients with T2DM belonging to two independently ascertained North Indian and one South Indian cohorts were genotyped for ACACβ (rs2268388) and AGTR1 (rs5186) polymorphism using real time PCR-based Taq-man assay and PCR–RFLP assays. In all the three cohorts, a significantly higher frequency of T allele and TT genotypes of ACACβ and C allele and CC genotypes of AGTR1 were found in patients with DN as compared to patients without nephropathy. Further, T allele of ACACβ and C allele of AGTR1 were found to be significantly associated with proteinuria, a hallmark of DN. We also found significant epistatic interactions between these two genes. TT genotypes of ACACβ gene and CC genotype of AGTR1 gene confers the risk of DN and both genes had significant epistatic interaction in Asian Indian patients with T2DM.     

Haplotypes in the complement factor H (CFH) gene: associations with drusen and advanced age-related macular degeneration

Background

Age-related macular degeneration (AMD), the leading cause of blindness in the Western world, is a complex disease that affects people over 50 years old. The complement factor H (CFH) gene has been repeatedly shown to be a major factor in determining susceptibility to the advanced form of the condition. We aimed to better understand the functional role of this gene in the AMD disease process and assess whether it is associated with earlier forms of the disease.

Methodology/Principal Findings

We genotyped SNPs at the CFH gene locus in three independent populations with AMD: (a) extended families where at least 3 family members had AMD; (b) sporadic cases of advanced AMD and (c) cases from the Age-Related Eye Disease Study (AREDS). We investigated polymorphisms and haplotypes in and around the CFH gene to assess their role in AMD. CFH is associated with early/intermediate and advanced AMD in both familial and sporadic cases. In our populations, the CFH SNP, rs2274700, is most strongly associated with AMD and when incorporated into a haplotype with the Y402H SNP and rs1061147, the strongest association is observed (p<10⁻⁹).

Conclusions/Significance

Our results, reproduced in three populations that represent the spectrum of AMD cases, provide evidence that the CFH gene is associated with drusen as well as with advanced AMD. We also identified novel susceptibility and protective haplotypes in the AMD populations.     

Detection of epistatic interactions between exotic alleles introgressed from wild barley (H. vulgare ssp. spontaneum)

The expression of a quantitative phenotype can be controlled through genotype, environment and genotype by environment interaction effects. Further, genotype effects can be attributed to major genes, quantitative trait loci (QTL) and gene by gene interactions, which are also termed epistatic interactions. The present study demonstrates that two-way epistatic interactions can play an important role for the expression of domestication-related traits like heading date, plant height and yield. In the BC₂DH population S42, carrying wild barley introgressions in the genetic background of the spring barley cultivar Scarlett, 13, 8 and 12 marker by marker interaction effects could be detected for the traits heading date, plant height and yield, respectively. Significant allelic combinations at interacting loci coincided for heading date, plant height and yield suggesting the presence of pleiotropic effects rather than several linked QTL. The mode of epistasis observed was primarily characterised by either (1) compensatory effects, where allelic combinations from the same genotype buffered the phenotype, or (2) augmented effects, where only the combination of the exotic allele at both interacting loci caused an altered phenotype. The present study shows that estimates of main effects of QTL can be confounded by interactions with background loci, suggesting that the identification of epistatic effects is important for gene cloning and marker-assisted selection. Furthermore, interaction effects between loci and putative candidate genes detected in the present study reveal potential functional relationships, which can be used to further elucidate gene networks in barley.     

Evaluating the Effect of 3′-UTR Variants in DICER1 and DROSHA on Their Tissue-Specific Expression by miRNA Target Prediction

Untranslated gene regions (UTRs) play an important role in controlling gene expression. 3′-UTRs are primarily targeted by microRNA (miRNA) molecules that form complex gene regulatory networks. Cancer genomes are replete with non-coding mutations, many of which are connected to changes in tumor gene expression that accompany the development of cancer and are associated with resistance to therapy. Therefore, variants that occurred in 3′-UTR under cancer progression should be analysed to predict their phenotypic effect on gene expression, e.g., by evaluating their impact on miRNA target sites. Here, we analyze 3′-UTR variants in DICER1 and DROSHA genes in the context of myelodysplastic syndrome (MDS) development. The key features of this analysis include an assessment of both “canonical” and “non-canonical” types of mRNA-miRNA binding and tissue-specific profiling of miRNA interactions with wild-type and mutated genes. As a result, we obtained a list of DICER1 and DROSHA variants likely altering the miRNA sites and, therefore, potentially leading to the observed tissue-specific gene downregulation. All identified variants have low population frequency consistent with their potential association with pathology progression.     

Identification of significant association and gene-gene interaction of GABA receptor subunit genes in autism

Autism is a common neurodevelopmental disorder with a significant genetic component. Existing research suggests that multiple genes contribute to autism and that epigenetic effects or gene-gene interactions are likely contributors to autism risk. However, these effects have not yet been identified. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, has been implicated in autism etiology. Fourteen known autosomal GABA receptor subunit genes were studied to look for the genes associated with autism and their possible interactions. Single-nucleotide polymorphisms (SNPs) were screened in the following genes: GABRG1, GABRA2, GABRA4, and GABRB1 on chromosome 4p12; GABRB2, GABRA6, GABRA1, GABRG2, and GABRP on 5q34-q35.1; GABRR1 and GABRR2 on 6q15; and GABRA5, GABRB3, and GABRG3 on 15q12. Intronic and/or silent mutation SNPs within each gene were analyzed in 470 white families with autism. Initially, SNPs were used in a family-based study for allelic association analysis--with the pedigree disequilibrium test and the family-based association test--and for genotypic and haplotypic association analysis--with the genotype-pedigree disequilibrium test (geno-PDT), the association in the presence of linkage (APL) test, and the haplotype family-based association test. Next, with the use of five refined independent marker sets, extended multifactor-dimensionality reduction (EMDR) analysis was employed to identify the models with locus joint effects, and interaction was further verified by conditional logistic regression. Significant allelic association was found for markers RS1912960 (in GABRA4; P = .01) and HCV9866022 (in GABRR2; P = .04). The geno-PDT found significant genotypic association for HCV8262334 (in GABRA2), RS1912960 and RS2280073 (in GABRA4), and RS2617503 and RS12187676 (in GABRB2). Consistent with the allelic and genotypic association results, EMDR confirmed the main effect at RS1912960 (in GABRA4). EMDR also identified a significant two-locus gene-gene effect model involving RS1912960 in GABRA4 and RS2351299 in GABRB1. Further support for this two-locus model came from both the multilocus geno-PDT and the APL test, which indicated a common genotype and haplotype combination positively associated with disease. Finally, these results were also consistent with the results from the conditional logistic regression, which confirmed the interaction between GABRA4 and GABRB1 (odds ratio = 2.9 for interaction term; P = .002). Through the convergence of all analyses, we conclude that GABRA4 is involved in the etiology of autism and potentially increases autism risk through interaction with GABRB1. These results support the hypothesis that GABA receptor subunit genes are involved in autism, most likely via complex gene-gene interactions.     

rs5888 Variant of SCARB1 Gene Is a Possible Susceptibility Factor for Age-Related Macular Degeneration

Major genetic factors for age-related macular degeneration (AMD) have recently been identified as susceptibility risk factors, including variants in the CFH gene and the ARMS2 LOC387715/HTRA1locus. Our purpose was to perform a case-control study in two populations among individuals who did not carry risk variants for CFHY402H and LOC387715 A69S (ARMS2), called “study” individuals, in order to identify new genetic risk factors. Based on a candidate gene approach, we analyzed SNP rs5888 of the SCARB1 gene, coding for SRBI, which is involved in the lipid and lutein pathways. This study was conducted in a French series of 1241 AMD patients and 297 controls, and in a North American series of 1257 patients with advanced AMD and 1732 controls. Among these individuals, we identified 61 French patients, 77 French controls, 85 North American patients and 338 North American controls who did not carry the CFH nor ARMS2 polymorphisms. An association between AMD and the SCARB1 gene was seen among the study subjects. The genotypic distribution of the rs5888 polymorphism was significantly different between cases and controls in the French population (p<0.006). Heterozygosity at the rs5888 SNP increased risk of AMD compared to the CC genotypes in the French study population (odds ratio (OR) = 3.5, CI95%: 1.4–8.9, p<0.01) and after pooling the 2 populations (OR = 2.9, 95% CI: 1.6–5.3, p<0.002). Subgroup analysis in exudative forms of AMD revealed a pooled OR of 3.6 for individuals heterozygous for rs5888 (95% CI: 1.7–7.6, p<0.0015). These results suggest the possible contribution of SCARB1, a new genetic factor in AMD, and implicate a role for cholesterol and antioxidant micronutrient (lutein and vitamin E) metabolism in AMD.