A DNA Pooling Approach to Investigate Candidate Genes in Migraine

Abstract

Migraine is a common debilitating primary headache disorder with significant mental, physical and social health implications. Dichotomised into migraine without aura (MO) and migraine with aura (MA), migrainous episodes can generally be characterised by a very intense, pulsating headache. The intensity of head pain generally increases in direct proportion with the duration of head pain, is often unilateral and is also often accompanied by nausea, vomiting and heightened sensitivities to sound and/or light. The familial inheritance of migraine headache is evidenced by the findings of first degree relatives of MO and MA probands having a greater risk of developing MO and MA, respectively, than within the general population. Identified to be involved in nociceptive pathways, the brain neurotransmitter 5-hydroxytryptamine's (5-HI; serotonin) involvement in the pathophysiology of migraine has been substantiated by increased levels of 5-HI metabolites associated with migrainous episodes and reduction of 5-HI plasma levels at the onset of and during migraine attack. Employing genetic linkage, genetic association and DNA sequencing strategies, the initial part of this study investigated the potential role of an open reading flame (ORE) and 3' untranslated region (3'UIR) variants within the human serotonin receptor 2C (5-H12c) gene in migraine predisposition. Assessment of the tested 5-H12c gene variants found no indication of linkage in a multigenerational pedigree cohort and genetic association analyses of the same two loci did not indicate significant allele frequency preponderance in a migraine case control cohort. Scanning the coding regions of the 5-HT2c gene failed to identify the presence of sequence mutations and none of the migrainous individuals tested contained the ORE variant. Hence linkage, association and sequence analysis results of this gene did not support a role for 5-HT2c in migraine aetiology. Ubiquitously expressed, the messenger molecule nitnc oxide (NO) has been implicated in the aetiological mechanisms of migraine where it has a fundamental physiological role in neurotransmission, smooth muscle motility and mediation of nociception. Within neuronal tissue, NO is endogenously synthesised by its neuronal nitric oxide synthase (nNOS) isoform, where it is also abundantly present in vasodilatory nerves encasing cerebral blood vessels. The second stage of this study investigated a potential regulatory 3'UTR repeat sequence and a potential mRNA diversifying 3'UTR repeat sequence within the human nNOS gene. Using a genetic association study design investigating the frequencies of these markers in case control cohorts, it was concluded that neither of these sequence repeat variations within the nNOS gene played a role in migraine susceptibility. It was the objective of the third stage of this study to investigate genetic variants within biosynthetic and metabolic enzymes governing the rate of 5-111 activity. Specifically, relationships between the human tryptophan hydroxylase (IPH), amino acid decarboxylase (AADC) and monoamine oxidase A (MAOA) genes and migraine susceptibility were assessed. This objective was undertaken using a novel a high throughput DNA pooling approach which proved to be a very accurate, sensitive and specific measure of estimating allele fiequencies. This study showed that the assessment of SNP, STR, insertion deletion and VNTR loci using DNA pooling is a very successful high throughput genetic locus-screening tool. Despite further development of this locus-screening method, negative association results of screened loci within the TPH, AADC and MAOA genes, did not support their role in migraine susceptibility. The final aspect of this research project developed and utilised a high throughput real-time PCR assay for genotyping individual DNA samples using a locked nucleic acid (LNA) nucleotide analogue. This method of genotyping was found to be highly sensitive and specific in the discrimination of a single nucleotide polymorphic variant residing within the promoter region of the TPH gene. The sensitivity and accuracy of this real-time PCR SNP genotyping assay was further enhanced by the application of a novel set of mathematical criteria to validate ambiguous genotypes outputted from the real-time PCR assay. In summary, the results of this thesis did not implicate the involvement of the tested variants within the 5-HT2c, nNOS, TPH, AADC or MAOA genes, in predisposition to migraine. However, the highly successful application of two novel high throughput genotyping techniques (DNA pooling and real-time PCR SNP genotyping) will greatly increase the efficiency at which further potential migraine candidate loci can be screened for their involvement in this disorder. Increasing genetic throughput mechanisms to investigate migraine should greatly enhance the chance of identifying the genetic determinants of this widely distributed debilitating disorder.