Integrating functional scoring and regulatory data to predict the effect of non-coding SNPs in a complex neurological disease.

Most SNPs associated with complex diseases seem to lie in non-coding regions of the genome; however, their contribution to gene expression and disease phenotype remains poorly understood. Here, we established a workflow to provide assistance in prioritising the functional relevance of non-coding SNPs of candidate genes as susceptibility loci in polygenic neurological disorders. To illustrate the applicability of our workflow, we considered the multifactorial disorder migraine as a model to follow our step-by-step approach. We annotated the overlap of selected SNPs with regulatory elements and assessed their potential impact on gene expression based on publicly available prediction algorithms and functional genomics information. Some migraine risk loci have been hypothesised to reside in non-coding regions and to be implicated in the neurotransmission pathway. In this study, we used a set of 22 non-coding SNPs from neurotransmission and synaptic machinery-related genes previously suggested to be involved in migraine susceptibility based on our candidate gene association studies. After prioritising these SNPs, we focused on non-reported ones that demonstrated high regulatory potential: (1) VAMP2_rs1150 (3' UTR) was predicted as a target of hsa-mir-5010-3p miRNA, possibly disrupting its own gene expression; (2) STX1A_rs6951030 (proximal enhancer) may affect the binding affinity of zinc-finger transcription factors (namely ZNF423) and disturb TBL2 gene expression; and (3) SNAP25_rs2327264 (distal enhancer) expected to be in a binding site of ONECUT2 transcription factor. This study demonstrated the applicability of our practical workflow to facilitate the prioritisation of potentially relevant non-coding SNPs and predict their functional impact in multifactorial neurological diseases.

A genetic interaction of NRXN2 with GABRE, SYT1 and CASK in migraine patients: a case-control study.

BACKGROUND: Migraine is a multifactorial disorder that is more frequent (two to four times) in women than in men. In recent years, our research group has focused on the role of neurotransmitter release and its regulation. Neurexin (NRXN2) is one of the components of the synaptic vesicle machinery, responsible for connecting intracellular fusion proteins and synaptic vesicles. Our aim was to continue exploring the role and interaction of proteins involved in the control and promotion of neurotransmission in migraine susceptibility. METHODS: A case-control study was performed comprising 183 migraineurs (148 females and 35 males) and 265 migraine-free controls (202 females and 63 males). Tagging single nucleotide polymorphisms of NRXN2 were genotyped to assess the association between NRXN2 and migraine susceptibility. The χ2 test was used to compare allele frequencies in cases and controls and odds ratios were estimated with 95% confidence intervals. Haplotype frequencies were compared between groups. Gene-gene interactions were analysed using the Multifactor Dimensionality Reduction v2.0. RESULTS: We found a statistically significant interaction model (p = 0.009) in the female group between the genotypes CG of rs477138 (NRXN2) and CT of rs1158605 (GABRE). This interaction was validated by logistic regression, showing a significant risk effect [OR = 4.78 (95%CI: 1.76-12.97)] after a Bonferroni correction. Our data also supports a statistically significant interaction model (p = 0.011) in the female group between the GG of rs477138 in NRXN2 and, the rs2244325's GG genotype and rs2998250's CC genotype of CASK. This interaction was also validated by logistic regression, with a protective effect [OR = 0.08 (95%CI: 0.01-0.75)]. A weak interaction model was found between NRXN2-SYT1. We have not found any statistically significant allelic or haplotypic associations between NRXN2 and migraine susceptibility. CONCLUSIONS: This study unravels, for the first time, the gene-gene interactions between NRXN2, GABRE - a GABAA-receptor - and CASK, importantly it shows the synergetic effect between those genes and its relation with migraine susceptibility. These gene interactions, which may be a part of a larger network, can potentially help us in better understanding migraine aetiology and in development of new therapeutic approaches.

Going Deep into Synaptic Vesicle Machinery Genes and Migraine Susceptibility - A Case-Control Association Study.

OBJECTIVE: A number of observations, including among our study population, have implicated variants in the syntaxin-1A, a component of the synaptic vesicles, in migraine susceptibility. Therefore, we hypothesize that variants in other components of the vesicle machinery are involved in migraine. BACKGROUND: Migraine is a common and complex neurologic disorder that affects approximately 15-18% of the general population. The exact cause of migraine is unknown; however, genetic studies have made possible substantial progress toward the identification of underlying molecular pathways. Neurotransmitters have been for long considered to have a key role in migraine pathophysiology; so we investigated common variants in genes involved in the synaptic vesicle machinery and their impact in migraine susceptibility. METHODS: We performed a case-control study comprising 188 unrelated patients with headache and 286 healthy controls in a population from the north of Portugal. Benefiting from the presence of linkage disequilibrium, we selected and genotyped 119 tagging single-nucleotide polymorphisms in 18 genes. RESULTS: We found significant associations between single-nucleotide variants and migraine in 7 genes, SYN1, SYN2, SNAP25, VAMP2, STXBP1, STXBP5, and UNC13A, either conferring an increased risk or protection of migraine. Due to SYN1 X-chromosomal location, we performed the statistical analysis separated by gender and, in the female group, the C allele of rs5906435 increased the risk for migraine susceptibility (P = .021; OR = 1.69; 95% CI: 1.21-2.34). In contrast, the TT genotype of the same variant emerged as a potential protective factor (P = .003; OR = 0.45; 95% CI: 0.27-0.74). The SYN2 analysis supported the rs3773364's G allele (P = .014) as a risk factor for migraine, and although not statistically significant after correction, the AG genotype (P = .006; OR = 1.86; 95% CI: 1.20-2.90) reinforced the allelic findings. Additionally, we found the SNAP25-rs363039's CT genotype (P = .001; OR = 2.14; 95% CI: 1.36-3.34), the STXBP5-rs1765028's T allele (P = .041; OR = 1.46; 95% CI: 1.13-1.90), and the UNC13B-rs7851161's TT genotype (P = .001; OR = 2.14; 95% CI: 1.36-3.34) as statistically significant risk factors for migraine liability. VAMP2-rs1150's G allele revealed a risk association to migraine, not statistically significant after correction (P = .068). Additionally, we found haplotypes in SYN1, SYN2, STXBP1, and UNC13B to be associated with migraine. CONCLUSIONS: Overall, this study provides a new insight into migraine liability, identifying possible starting points for functional studies.

Interaction between γ-aminobutyric acid A receptor genes: new evidence in migraine susceptibility.

Migraine is a common neurological episodic disorder with a female-to-male prevalence 3- to 4-fold higher, suggesting a possible X-linked genetic component. Our aims were to assess the role of common variants of gamma-aminobutyric acid A receptor (GABAAR) genes, located in the X-chromosome, in migraine susceptibility and the possible interaction between them. An association study with 188 unrelated cases and 286 migraine-free controls age- and ethnic matched was performed. Twenty-three tagging SNPs were selected in three genes (GABRE, GABRA3 and GABRQ). Allelic, genotypic and haplotypic frequencies were compared between cases and controls. We also focused on gene-gene interactions. The AT genotype of rs3810651 of GABRQ gene was associated with an increased risk for migraine (OR: 4.07; 95% CI: 1.71-9.73, p=0.002), while the CT genotype of rs3902802 (OR: 0.41; 95% CI: 0.21-0.78, p=0.006) and GA genotype of rs2131190 of GABRA3 gene (OR: 0.53; 95% CI: 0.32-0.88, p=0.013) seem to be protective factors. All associations were found in the female group and maintained significance after Bonferroni correction. We also found three nominal associations in the allelic analyses although there were no significant results in the haplotypic analyses. Strikingly, we found strong interactions between six SNPs encoding for different subunits of GABAAR, all significant after permutation correction. To our knowledge, we show for the first time, the putative involvement of polymorphisms in GABAAR genes in migraine susceptibility and more importantly we unraveled a role for novel gene-gene interactions opening new perspectives for the development of more effective treatments.