Gotowa bibliografia na temat „Migrain candidate genes”

Background Before the genome-wide association (GWA) era, many hypothesis-driven candidate gene association studies were performed that tested whether DNA variants in genes that had been selected based on prior knowledge about migraine pathophysiology were associated with migraine. Most studies involved small sample sets without robust replication, thereby making the risk of false-positive findings high. Genome-wide marker data of thousands of migraine patients and controls from the International Headache Genetics Consortium provide a unique opportunity to re-evaluate key findings from candidate gene association studies (and other non-GWA genetic studies) in a much larger data set. Methods We selected 21 genes from published candidate gene association studies and six additional genes from other non-GWA genetic studies in migraine. Single nucleotide polymorphisms (SNPs) in these genes, as well as in the regions 500 kb up- and downstream, were inspected in IHGC GWAS data from 5175 clinic-based migraine patients with and without aura and 13,972 controls. Results None of the SNPs in or near the 27 genes, including the SNPs that were previously found to be associated with migraine, reached the Bonferroni-corrected significance threshold; neither when analyzing all migraine patients together, nor when analyzing the migraine with and without aura patients or males and females separately. Conclusion The available migraine GWAS data provide no clear evidence for involvement of the previously reported most promising candidate genes in migraine.

Some evidence indicates endometriosis and migraine have a common genetic predisposition in sex-hormone genes, which could have important implications for the treatment of these two heterogenous conditions. To date, the genes responsibility remains unknown. Based on the biological hypothesis that polymorphisms of genes involved in sex-hormone pathways may influence estrogen levels and phenotypes of both disorders, we did a literature search for candidate sex-hormone genes and genes involved in the metabolism of estradiol. The aim was to review the evidence for shared sex-hormone-related polymorphisms between endometriosis and migraine and provide an exhaustive overview of the current literature. We included case-control studies investigating associations between candidate sex-hormone-related genes and the disorders endometriosis and migraine, respectively. Results showed three overlapping sex-hormone-associated polymorphisms in estrogen receptor genes that are associated with both conditions. To confirm possible associations with other sex-hormone genes, larger studies are needed.

Background Cluster headache is the most severe primary headache disorder. A genetic basis has long been suggested by family and twin studies; however, little is understood about the genetic variants that contribute to cluster headache susceptibility. Methods We conducted a literature search of the MEDLINE database using the PubMed search engine to identify all human genetic studies for cluster headache. In this article we provide a review of those genetic studies, along with an overview of the pathophysiology of cluster headache and a brief review of migraine genetics, which have both been significant drivers of cluster headache candidate gene selection. Results The investigation of cluster headache genetic etiology has been dominated by candidate gene studies. Candidate selection has largely been driven by the pathophysiology, such as the striking rhythmic nature of the attacks, which spurred close examination of the circadian rhythm genes CLOCK and HCRTR2. More recently, unbiased genetic approaches such as genome-wide association studies (GWAS) have yielded new genetic avenues of interest including ADCYAP1R1 and MME. Conclusions The majority of candidate genes studied for cluster headache suffer from poor reproducibility. Broader genetic interrogation through larger unbiased GWAS, exome, and whole genome studies may provide more robust candidates, and in turn provide a clearer understanding of the causes of cluster headache.

Introduction Chronic migraine (CM) is at the severe end of the clinical migraine spectrum, but its genetic background is unknown. Our study searched for evidence that genetic factors are involved in the chronification process. Methods We initially selected 144 single-nucleotide polymorphisms (SNPs) from 48 candidate genes, which we tested for association in two stages: The first stage encompassed 262 CM patients, the second investigated 226 patients with high-frequency migraine (HFM). Subsequently, SNPs with p values < 0.05 were forwarded to the replication stage containing 531 patients with CM or HFM. Results Eight SNPs were significantly associated with CM and HFM in the two-stage phase. None survived replication in the third stage. Discussion We present the first comprehensive genetic association study for migraine chronification. There were no significant findings. Future studies may benefit from larger, genome-wide data sets or should use other genetic approaches to identify genetic factors involved in migraine chronification.

Статья посвящена анализу имеющихся на сегодняшний день данных генетических полиморфизмов, излучавшихся при мигрени. При мигрени гены-кандидаты мигрени сгруппированы в четыре функциональных семейства генов: гены нервной системы, гены сердечно-сосудистой системы, гормональные, воспалительные гены. Среди генов нервной системы значимость представляют гены, кодирующие структуру и функциональную активность калиевых каналов. Наибольший интерес представляют гены сердечно-сосудистой системы, которые как повышают риск мигрени, так и увеличивают риск коморбидных кардиоваскулярных заболеваний. Среди генов эндокринной системы роль в патогенезе мигрени играют полиморфизмы гена эстрогенового рецептора 1 типа. Для воспалительных генов циклооксегиназы 2, главного комплекса гистосовместимости HLA-DRB1, лимфотоксина альфа, фактора некроза опухоли альфа и бета найдены положительные ассоциации с мигренью. The article focused on analysis of currently available reports on genetic polymorphisms in migraine. The migraine candidate genes were grouped into four functional families: genes of the nervous system, genes of the cardiovascular system, hormonal genes, and inflammatory genes. Among the nervous system genes, the genes encoding the structure and functional activity of potassium channels are of a special significance. Of the greatest interest are the cardiovascular system genes, which increase the risk of both migraine and comorbid cardiovascular diseases. Among the endocrine system genes, polymorphisms of the type 1 estrogen receptor gene play a role in the pathogenesis of migraine. Positive associations with migraine were found for inflammatory genes of cyclooxygenase 2, the main histocompatibility complex HLA-DRB1, lymphotoxin alpha, and tumor necrosis factors alpha and beta.

Migraine is a common, chronic neurovascular disorder caused by a complex interaction between genetic and environmental risk factors. In the last two decades, molecular genetics of migraine have been intensively investigated. In a few cases, migraine is transmitted as a monogenic disorder, and the disease phenotype cosegregates with mutations in different genes like CACNA1A, ATP1A2, SCN1A, KCNK18, and NOTCH3. In the common forms of migraine, candidate genes as well as genome-wide association studies have shown that a large number of genetic variants may increase the risk of developing migraine. At present, few studies investigated the genotype-phenotype correlation in patients with migraine. The purpose of this review was to discuss recent studies investigating the relationship between different genetic variants and the clinical characteristics of migraine. Analysis of genotype-phenotype correlations in migraineurs is complicated by several confounding factors and, to date, only polymorphisms of the MTHFR gene have been shown to have an effect on migraine phenotype. Additional genomic studies and network analyses are needed to clarify the complex pathways underlying migraine and its clinical phenotypes.

Introduction Alternating hemiplegia of childhood (AHC) is a rare disorder mainly characterized by attacks of hemiplegia and mental retardation. It has been often associated with migraine. The CACNA1A gene on chromosome 19 is involved in familial hemiplegic migraine and other episodic cerebral disorders, but also with progressive neuronal damage. Methods We performed mutation analysis in this gene in four AHC patients, using single strand conformation polymorphism analysis. Results We found nine polymorphisms, but no mutations in any of the 47 exons. Conclusions Other cerebral ion channel genes remain candidate genes for AHC.

Objective: Genes which are involved in immune response portray possible candidate genes in migraine. One of those genes is that myelin oligodendrocyte glycoprotein (MOG) that plays an important role in mediating the complement cascade. The purpose of our study is to show the effect of MOG G511C (Val142Leu; rs2857766) polymorphism in migraine attack frequency. Materials and Methods: In the cohort of 101 Turkish migraine patients and in a control group of 101 healthy subjects, MOG Val142Leu alleles’ distribution was examined. Restriction fragment length polymorphism (RFLP) was carried out to genotype this polymorphism. Results: Although MOG Leu allele frequency was determined as under-represented in migraine patients, any significant difference between the patient and control groups’ genotype, and allele frequencies were not obtained [OR=0.47 (0.21-1.08), p=0.053 for genotypes; OR=0.50 (0.23-1.11), p=0.060 for alleles]. However, a statistically significant relationship between MOG G511C (Val142Leu) polymorphism and the decreased migraine attack frequency was determined [OR=11.71 (1.32-103.77), p=0.013]. Val/Leu genotype frequency increrased in migraine patients with two or fewer attacks per month. Conclusion: Migraine attack frequency might be related with MOG Val142Leu heterozygote genotype. So we think that MOG gene might be related to genetic susceptibility to migraine in the human leukocyte antigen (HLA) region.

Migraine, comprised of migraine with aura (MA) and migraine without aura (MO), is a painful neurovascular disease, affecting approximately 16% of the general population. It is characterised by a wide variety of symptoms including headache, nausea and vomiting, and photo- and phonophobia. The disorder is complex involving not only multiple genes, but also specific environmental factors, which can induce attacks in genetically predisposed individuals. Hyperhomocysteinaemia is a known risk factor for cerebrovascular, peripheral vascular and coronary heart disease. The Methylenetetrahydrofolate Reductase (MTHFR) enzyme is involved in homocysteine metabolism. Furthermore, it has been reported that a homozygous mutation (677C to T; Ala to Val) in the 5,10-MTHFR gene is associated with an elevation in plasma homocysteine levels (Frosst et al., 1995). This common mutation in the MTHFR gene has recently been associated with migraine with aura in a Japanese cohort (Kowa et al., 2000). The present study was designed to determine the prevalence of the MTHFR C677T mutation in Australian patients with migraine and to determine whether this mutation is associated with the disease in Caucasians. A large case-control study, consisting of 270 patients with migraine (167 with aura and 103 without aura), and 270 normal matched controls was investigated. Genotypic results indicated that the prevalence of the homozygous (T/T) genotype in migraine sufferers (15%) was higher than that of controls (9%) (P = 0.084). Furthermore, the frequency of the mutant (T/T) genotype in individuals with MA (19%) was significantly higher than in controls (9%) (P = 0.006). Interestingly, the risk of MA was ~2.5-fold higher in suffers possessing the homozygous variant (OR = 2.52, CI: 1.42 - 4.47, P = 0.0012). To confirm the MTHFR allelic association with MA, family-based tests were performed in an independent pedigrees group, where only those with MA were considered affected. Results from both the Pedigree Disequilibrium Test (PDT) and Family-Based Association Test (FBAT) analysis revealed slight, although not significant (PDT test, P = 132; and FBAT test, P = 0.390), over-transmission of the mutant allele (T) from parents to affected offspring. However, despite the MTHFR variant having a high heterozygosity (0.48), there were a limited number of informative transmissions for the MTHFR variant in the pedigree group resulting in reduced power for these tests. In conclusion, our results support the trends reported in the Japanese migraine study and suggest that the homozygous 677T gene variant causing mild hyperhomocysteinaemia, is a genetic risk factor for migraine, and indicate that further studies investigating the role of this gene are warranted. Mutations in various ion channel genes are responsible for neurovascular and other neurological disorders. Inherited ion channel mutations or "channelopathies" are increasingly found to be the cause of various neurological disorders in humans. Wittekindt and colleagues (1998) reported that the calcium-activated potassium channel (hKCa3) gene is a good candidate for schizophrenia and bipolar disorder (BD), as well as for other neurological disorders such as migraine. The hKCa3 gene is a neuronal small conductance calcium-activated potassium channel, which contains a polyglutamine tract, encoded by a polymorphic CAG repeat in the gene. The hKCa3 gene encodes a protein of 731 amino acids containing two adjacent polyglutamine sequences in its N-terminal domain separated by 25 amino acids. The C-terminal polyglutamine sequence is highly polymorphic in length (Austin et al., 1999). hKCa3 plays a critical role in determining the firing pattern of neurons via the generation of slow after-polarization pulses and the regulation of intracellular calcium channels (Kohler et al., 1996). Three distinct mutations in the a1 calcium channel gene have been shown to cause SCA-6, episodic ataxia-2 and familial hemiplegic migraine (FHM) (Ophoff et al., 1996). The hKCa3 gene contains a highly polymorphic CAG repeat that was initially mapped (Chandy et al., 1997) to a schizophrenia locus on chromosome 22 (Pulver et al., 1994). Recently Austin et al (1999) re-mapped hKCa3 and found it to reside on chromosome 1q21, a region that has been linked to FHM (Austin et al., 1999), a rare subtype of MA (Ducros et al., 1997; Gardner et al., 1998), and a region recently showing genetic linkage to typical migraine (Lea et al., 2002). The hKCa3 polymorphism results in small variations in polyglutamine number, similar to those that occur in the calcium channel a1a subunit gene (CACNA1A), which is encoded by CAG expansions and thought to cause Spinocerebellar Ataxia Type 6 via loss of channel function (Austin et al., 1999). Given the recent linkage of FHM to the region of chromosome 1q21, to which hKCa3 resides, and also linkage of typical migraine to this region, a large case-control study investigating this hKCa3 CAG marker and consisting of 270 migraine and 270 stringently matched healthy controls was undertaken. Our results indicated that there was no statistically significant difference in allele distributions for this marker between migraine and non-migraine patients (P >0.05). No significant difference in the allelic distribution was observed in the MA or MO groups when compared to controls (P >0.05) and there was no significant difference in CAG repeat length distribution between the migraine group and controls (P = 0.92), or between the MA and MO groups (P = 0.72) collectively. Hence, the CAG repeat in this gene does not show expansion in migraine. Overall, our results provide no genetic evidence to suggest that the hKCa3 CAG repeat polymorphism is involved in migraine aetiology in Australian Caucasians. Thus the involvement of the hKCa3 gene in migraine is not likely, although the hKCa3 gene remains an important candidate for other neurological disorders that may be linked to the 1q21.3 chromosomal region.

Migraine, comprised of migraine with aura (MA) and migraine without aura (MO), is a painful neurovascular disease, affecting approximately 16% of the general population. It is characterised by a wide variety of symptoms including headache, nausea and vomiting, and photo- and phonophobia. The disorder is complex involving not only multiple genes, but also specific environmental factors, which can induce attacks in genetically predisposed individuals. Hyperhomocysteinaemia is a known risk factor for cerebrovascular, peripheral vascular and coronary heart disease. The Methylenetetrahydrofolate Reductase (MTHFR) enzyme is involved in homocysteine metabolism. Furthermore, it has been reported that a homozygous mutation (677C to T; Ala to Val) in the 5,10-MTHFR gene is associated with an elevation in plasma homocysteine levels (Frosst et al., 1995). This common mutation in the MTHFR gene has recently been associated with migraine with aura in a Japanese cohort (Kowa et al., 2000). The present study was designed to determine the prevalence of the MTHFR C677T mutation in Australian patients with migraine and to determine whether this mutation is associated with the disease in Caucasians. A large case-control study, consisting of 270 patients with migraine (167 with aura and 103 without aura), and 270 normal matched controls was investigated. Genotypic results indicated that the prevalence of the homozygous (T/T) genotype in migraine sufferers (15%) was higher than that of controls (9%) (P = 0.084). Furthermore, the frequency of the mutant (T/T) genotype in individuals with MA (19%) was significantly higher than in controls (9%) (P = 0.006). Interestingly, the risk of MA was ~2.5-fold higher in suffers possessing the homozygous variant (OR = 2.52, CI: 1.42 - 4.47, P = 0.0012). To confirm the MTHFR allelic association with MA, family-based tests were performed in an independent pedigrees group, where only those with MA were considered affected. Results from both the Pedigree Disequilibrium Test (PDT) and Family-Based Association Test (FBAT) analysis revealed slight, although not significant (PDT test, P = 132; and FBAT test, P = 0.390), over-transmission of the mutant allele (T) from parents to affected offspring. However, despite the MTHFR variant having a high heterozygosity (0.48), there were a limited number of informative transmissions for the MTHFR variant in the pedigree group resulting in reduced power for these tests. In conclusion, our results support the trends reported in the Japanese migraine study and suggest that the homozygous 677T gene variant causing mild hyperhomocysteinaemia, is a genetic risk factor for migraine, and indicate that further studies investigating the role of this gene are warranted. Mutations in various ion channel genes are responsible for neurovascular and other neurological disorders. Inherited ion channel mutations or "channelopathies" are increasingly found to be the cause of various neurological disorders in humans. Wittekindt and colleagues (1998) reported that the calcium-activated potassium channel (hKCa3) gene is a good candidate for schizophrenia and bipolar disorder (BD), as well as for other neurological disorders such as migraine. The hKCa3 gene is a neuronal small conductance calcium-activated potassium channel, which contains a polyglutamine tract, encoded by a polymorphic CAG repeat in the gene. The hKCa3 gene encodes a protein of 731 amino acids containing two adjacent polyglutamine sequences in its N-terminal domain separated by 25 amino acids. The C-terminal polyglutamine sequence is highly polymorphic in length (Austin et al., 1999). hKCa3 plays a critical role in determining the firing pattern of neurons via the generation of slow after-polarization pulses and the regulation of intracellular calcium channels (Kohler et al., 1996). Three distinct mutations in the a1 calcium channel gene have been shown to cause SCA-6, episodic ataxia-2 and familial hemiplegic migraine (FHM) (Ophoff et al., 1996). The hKCa3 gene contains a highly polymorphic CAG repeat that was initially mapped (Chandy et al., 1997) to a schizophrenia locus on chromosome 22 (Pulver et al., 1994). Recently Austin et al (1999) re-mapped hKCa3 and found it to reside on chromosome 1q21, a region that has been linked to FHM (Austin et al., 1999), a rare subtype of MA (Ducros et al., 1997; Gardner et al., 1998), and a region recently showing genetic linkage to typical migraine (Lea et al., 2002). The hKCa3 polymorphism results in small variations in polyglutamine number, similar to those that occur in the calcium channel a1a subunit gene (CACNA1A), which is encoded by CAG expansions and thought to cause Spinocerebellar Ataxia Type 6 via loss of channel function (Austin et al., 1999). Given the recent linkage of FHM to the region of chromosome 1q21, to which hKCa3 resides, and also linkage of typical migraine to this region, a large case-control study investigating this hKCa3 CAG marker and consisting of 270 migraine and 270 stringently matched healthy controls was undertaken. Our results indicated that there was no statistically significant difference in allele distributions for this marker between migraine and non-migraine patients (P >0.05). No significant difference in the allelic distribution was observed in the MA or MO groups when compared to controls (P >0.05) and there was no significant difference in CAG repeat length distribution between the migraine group and controls (P = 0.92), or between the MA and MO groups (P = 0.72) collectively. Hence, the CAG repeat in this gene does not show expansion in migraine. Overall, our results provide no genetic evidence to suggest that the hKCa3 CAG repeat polymorphism is involved in migraine aetiology in Australian Caucasians. Thus the involvement of the hKCa3 gene in migraine is not likely, although the hKCa3 gene remains an important candidate for other neurological disorders that may be linked to the 1q21.3 chromosomal region.
Thesis (Masters)
Master of Philosophy (MPhil)
School of Health Sciences
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Migraine is a serious neurological disorder that affects the central nervous system causing painful attacks of headache. Attacks of head pain vary widely in intensity, frequency and duration lasting from anywhere between 4-72 hours and are often accompanied by further symptoms of nausea, vomiting, photo- and phonophobia. In 1988, a group of world leaders in the diagnosis of migraine formed the International Headache Society (IHS) and compiled and published a consensus set of diagnostic criteria known as International Classification of Headache Disorders, ICHD-I 1988. This was the first classification system and was subsequently updated in 2004, ICHD-II 2004 and more recently a 3rd Edition beta version has been released (ICHD-3rd Ed Beta Version) and is the gold standard for diagnosing headache disorders. Migraine displays two main subtypes termed migraine with or without aura (MA and MO respectively). The two forms are distinguished from each other based on the development of aura, a period of variable and diverse neurological symptoms that precede the headache phase.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Science, Environment, Engineering and Technology
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Migraine is a painful temporarily incapacitating disorder that affects an estimated 12% of the general population including 18% of adult women and 6% of adult men. The disorder involves two main subtypes termed migraine with or without aura (MA and MO respectively). Migraine can present with a variety of symptoms that vary between individuals and between episodes experienced by a single individual. This disorder causes significant social and economic burden and alarmingly is often poorly treated. A direct cause of this is a lack of understanding of the underlying pathology of migraine. Migraine is believed to be a neurogenic disorder that involves temporary disruption of pathways that receive and respond to sensory signals. While numerous environmental triggers may have been identified the exact mechanisms that cause the disruption are still largely unknown. However, familial aggregation of migraine suggests significant genetic contributors.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Typical migraine, comprised of migraine with aura (MA) and migraine without aura (MO), is a chronic, painful and debilitating neurovascular disease which is generally characterised by recurrent attacks of severe headache usually accompanied by nausea, vomiting, photo and phonophobia. Migraine has been shown to affect a large proportion of Caucasian populations with a recent comprehensive study indicating that around 25% of women and 8% of men suffer from the disease. Strong familial aggregation of typical migraine and an increased concordance for the disease in MZ twins over DZ twins, suggests that it has a significant genetic component. Heritability estimates are calculated to be between 40% and 60%, indicating that disease variation, in part, is explained by environmental determinants. The mode of transmission of typical migraine is not clear but is most likely multifactorial. Although the MA and MO subtypes exhibit some clinical heterogeneity, segregation analysis has suggested that there may be a common genetic aetiology for MA and MO, and a major gene contributing to typical migraine pathogenesis. This idea is substantiated by the fact that both subtypes of migraine can occur within the same family and even within the same individual, with up to 33% of sufferers experiencing both types of the disease. In addition, migraine prophylactics have been shown to result in similar effects in patients treated for both types of migraine. However, whether the two subtypes are truly separate entities or not remains unclear. At present, the type and number of genes involved in typical migraine is not known. Despite this, several studies into Familial Hemiplegic Migraine (FHM), a very severe subtype of MA, have led to the discovery that mutations in a brain specific calcium channel subunit gene (CACNA1A) located on chromosome 19, cause FHM in about 50% of affected families. FHM is a rare disease and is distinguished from typical migraine by its association with hemiparesis and clear autosomal dominant mode of inheritance. However, certain clinical features are common to both FHM and typical migraine including similarities in headache characteristics and triggers. Hence, FHM genetic studies provide a valuable model for investigating the genes involved in the more prevalent types of migraine with and without aura. For this reason the Genomics Research Centre has been conducting linkage studies utilising large Australian migraine pedigrees with a focus on the known FHM (CACNA1A) gene region on chromosome 19p13. Our results to date have indicated suggestive linkage to the FHM region on 19p13 in a large multigenerational pedigree (MF1) affected with typical migraine, with a maximum parametric LOD score of 1.92 (P = 0.001) obtained for a triplet repeat polymorphism situated in exon 47 of the CACNA1A gene. Expansion of this repeat was not observed, but is possible that mutations elsewhere in the CACNA1A gene may be responsible for migraine in this pedigree. To investigate this possibility, the current research involved sequencing two patients carrying the critical susceptibility haplotype surrounding the CACNA1A gene. The results of this mutation screen revealed no disease causing mutations or polymorphisms in any of the 47 exons screened. To determine whether the CACNA1A genomic region was implicated in typical migraine susceptibility in the general Caucasian population, 82 independent pedigrees and a large case-control group were also analysed using highly polymorphic microsatellite markers. There was no linkage or association detected in these groups and thus, it was concluded that if CACNA1A plays a role in typical migraine it does not confer a major effect on the disease. However, subsequent case-control studies of SNPs in the INSR gene, which is located ~15cM telomeric from CACNA1A, provided evidence of association to typical migraine. Thus, the INSR gene may now emerge as the new migraine susceptibility gene in this genomic region on chromosome 19. Family linkage studies conducted by Gardner et al have implicated an additional FHM susceptibility region on chromsome 1q31. Furthermore, independent research carried out by Ducros et al. has indicated a second FHM locus at 1q21-23, which is ~ 30cM centromeric to the region reported by Gardner et al. At this stage it is not clear whether there is a single locus, or two distinct loci, on the chromosome 1q region. This research also involved a family-based linkage and association approach to investigating the FHM susceptibility region on chromosome 1q31 for involvement in typical migraine susceptibility in affected Australian pedigrees. Initial multipoint ALLEGRO analysis provided strong evidence for linkage of Chr1q31 markers to typical migraine in a large multigenerational pedigree. The 1-LOD* unit support interval for suggestive linkage spanned ~18cM with a maximum allele sharing LOD* score of 3.36 obtained for marker D1S2782, P = 0.00004. Subsequent analysis of an independent sample of 82 affected pedigrees added support to the initial findings with a maximum LOD* of 1.24 (P = 0.008). Utilising the independent sample of 82 pedigrees we also performed a family-based association test. Results of this analysis indicated distortion of allele transmission at marker D1S249 (global c2(5) of 15.00, P = 0.010) in these pedigrees. These positive linkage and association results will need further confirmation by independent researchers, but overall they provide good evidence for the existence of a typical migraine locus near these markers on Chr1q31, and reinforce the idea that an FHM gene in this genomic region may also contribute to susceptibility to the more common forms of migraine. The serotonergic system has long been implicated in the pathophysiology of migraine. Researchers have therefore focused on the serotonin receptors and the genes that code for them when investigating this disease. Although serotonin receptor agonists have proven to be effective in the treatment of migraine, there has been little evidence of a serotonin receptor gene being associated with the disorder. However, in 1998, Ogilvie et al reported that a VNTR in the serotonin transporter gene (SERT) showed altered allelic distributions in a Danish migraine population. In addition to serotonin, there has been renewed interest in the involvement of the dopaminergic pathways in migraine. This interest has gained impetus since the study of Peroutka et al who reported an allelic association between the dopamine receptor gene DRD2 and migraine with aura. Another dopamine related gene, the dopamine beta-hydroxylase gene (DBH), has been localised to Chr 9q34 and codes for the enzyme that catalyses the conversion of dopamine to norepinephrine. It therefore plays an important role in dopaminergic and noradrenergic neurotransmission. Serum levels of DbH enzyme have been reported to be elevated in migrainous patients during the headache phase of an attack. Also, significantly increased DbH enzyme activity has been observed in migraine patients during the headache-free interval. Thus, the DBH gene is another good candidate for involvement in migraine pathophysiology and, to our knowledge, has not been previously implicated in this disease. Candidate gene studies may be useful strategies for identifying genes involved in complex diseases such as migraine, especially if the gene being examined contributes only a minor effect to the overall phenotype. This research also involved a linkage and association approach to investigating neurotransmitter related migraine candidate genes. Specifically, polymorphisms within the serotonin transporter gene (SERT), the dopamine receptor gene (DRD2) and the dopamine beta-hydroxylase (DBH) gene were tested in unrelated Caucasian migraineurs and non-migraine control individuals. In addition, an independent sample of 82 families affected with migraine were examined. Unrelated case-control association analysis of a DBH intragenic dinucleotide polymorphism indicated altered allelic distribution between migraine and control groups (c2 = 16.53, P = 0.019). Furthermore, the transmission/disequilibrium test (TDT) which was implemented on the family data also indicated distortion of allele transmission for the same DBH marker (c2 = 4.44, P = 0.035). Together, these results provide evidence for allelic association of the DBH gene with typical migraine susceptibility (Fisher's Combined P-value = 0.006) and indicate that further research into the role of the DBH gene in migraine aetiology is warranted. Nitric oxide (NO) is emerging as a key molecule affecting the pain associated with migraine. Since nitric oxide synthase (NOS) enzymes catalyse the synthesis of NO, the genes that code for these enzymes are good candidates for migraine molecular genetic analysis. This research involved investigating the role of a functionally relevant bi-allelic tetranucleotide polymorphism located in the promoter region of the human inducible nitric oxide synthase (iNOS) gene in migraine aetiology. A large group of migraine affected individuals were genotyped and compared to an age and sex matched group of unaffected controls. Results of a chi-squared analysis indicated that allele distributions for both migraine cases and controls were not significantly different (c2 = 1.93, P = 0.16). These findings offer no evidence for an allelic association of the tested iNOS polymorphism with the common forms of the disease and therefore do not support a role for this gene in migraine pathogenesis. In summary, this research involved linkage and association analysis of migraine candidate genes and genomic susceptibility regions. Whilst, the known FHM gene (CACNA1A) was excluded for significant involvement in typical migraine the adjacent INSR gene has been associated. Migraine is genetically heterogeneous and the results of this research also provide good evidence that the DBH gene is involved in disease predisposition, whilst the DRD2, SERT and INOS gene were not shown to be implicated. An additional susceptibility region for typical migraine is also likely to localise to chromosome 1q31. Overall, the results presented in this thesis have contributed valuable data to the understanding of the molecular genetics of migraine with and without aura. Future research into the molecular pathophysiological mechanisms of migraine will greatly facilitate the development of more effective diagnosis and treatment strategies.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
Full Text

Typical migraine, comprised of migraine with aura (MA) and migraine without aura (MO), is a chronic, painful and debilitating neurovascular disease which is generally characterised by recurrent attacks of severe headache usually accompanied by nausea, vomiting, photo and phonophobia. Migraine has been shown to affect a large proportion of Caucasian populations with a recent comprehensive study indicating that around 25% of women and 8% of men suffer from the disease. Strong familial aggregation of typical migraine and an increased concordance for the disease in MZ twins over DZ twins, suggests that it has a significant genetic component. Heritability estimates are calculated to be between 40% and 60%, indicating that disease variation, in part, is explained by environmental determinants. The mode of transmission of typical migraine is not clear but is most likely multifactorial. Although the MA and MO subtypes exhibit some clinical heterogeneity, segregation analysis has suggested that there may be a common genetic aetiology for MA and MO, and a major gene contributing to typical migraine pathogenesis. This idea is substantiated by the fact that both subtypes of migraine can occur within the same family and even within the same individual, with up to 33% of sufferers experiencing both types of the disease. In addition, migraine prophylactics have been shown to result in similar effects in patients treated for both types of migraine. However, whether the two subtypes are truly separate entities or not remains unclear. At present, the type and number of genes involved in typical migraine is not known. Despite this, several studies into Familial Hemiplegic Migraine (FHM), a very severe subtype of MA, have led to the discovery that mutations in a brain specific calcium channel subunit gene (CACNA1A) located on chromosome 19, cause FHM in about 50% of affected families. FHM is a rare disease and is distinguished from typical migraine by its association with hemiparesis and clear autosomal dominant mode of inheritance. However, certain clinical features are common to both FHM and typical migraine including similarities in headache characteristics and triggers. Hence, FHM genetic studies provide a valuable model for investigating the genes involved in the more prevalent types of migraine with and without aura. For this reason the Genomics Research Centre has been conducting linkage studies utilising large Australian migraine pedigrees with a focus on the known FHM (CACNA1A) gene region on chromosome 19p13. Our results to date have indicated suggestive linkage to the FHM region on 19p13 in a large multigenerational pedigree (MF1) affected with typical migraine, with a maximum parametric LOD score of 1.92 (P = 0.001) obtained for a triplet repeat polymorphism situated in exon 47 of the CACNA1A gene. Expansion of this repeat was not observed, but is possible that mutations elsewhere in the CACNA1A gene may be responsible for migraine in this pedigree. To investigate this possibility, the current research involved sequencing two patients carrying the critical susceptibility haplotype surrounding the CACNA1A gene. The results of this mutation screen revealed no disease causing mutations or polymorphisms in any of the 47 exons screened. To determine whether the CACNA1A genomic region was implicated in typical migraine susceptibility in the general Caucasian population, 82 independent pedigrees and a large case-control group were also analysed using highly polymorphic microsatellite markers. There was no linkage or association detected in these groups and thus, it was concluded that if CACNA1A plays a role in typical migraine it does not confer a major effect on the disease. However, subsequent case-control studies of SNPs in the INSR gene, which is located ~15cM telomeric from CACNA1A, provided evidence of association to typical migraine. Thus, the INSR gene may now emerge as the new migraine susceptibility gene in this genomic region on chromosome 19. Family linkage studies conducted by Gardner et al have implicated an additional FHM susceptibility region on chromsome 1q31. Furthermore, independent research carried out by Ducros et al. has indicated a second FHM locus at 1q21-23, which is ~ 30cM centromeric to the region reported by Gardner et al. At this stage it is not clear whether there is a single locus, or two distinct loci, on the chromosome 1q region. This research also involved a family-based linkage and association approach to investigating the FHM susceptibility region on chromosome 1q31 for involvement in typical migraine susceptibility in affected Australian pedigrees. Initial multipoint ALLEGRO analysis provided strong evidence for linkage of Chr1q31 markers to typical migraine in a large multigenerational pedigree. The 1-LOD* unit support interval for suggestive linkage spanned ~18cM with a maximum allele sharing LOD* score of 3.36 obtained for marker D1S2782, P = 0.00004. Subsequent analysis of an independent sample of 82 affected pedigrees added support to the initial findings with a maximum LOD* of 1.24 (P = 0.008). Utilising the independent sample of 82 pedigrees we also performed a family-based association test. Results of this analysis indicated distortion of allele transmission at marker D1S249 (global c2(5) of 15.00, P = 0.010) in these pedigrees. These positive linkage and association results will need further confirmation by independent researchers, but overall they provide good evidence for the existence of a typical migraine locus near these markers on Chr1q31, and reinforce the idea that an FHM gene in this genomic region may also contribute to susceptibility to the more common forms of migraine. The serotonergic system has long been implicated in the pathophysiology of migraine. Researchers have therefore focused on the serotonin receptors and the genes that code for them when investigating this disease. Although serotonin receptor agonists have proven to be effective in the treatment of migraine, there has been little evidence of a serotonin receptor gene being associated with the disorder. However, in 1998, Ogilvie et al reported that a VNTR in the serotonin transporter gene (SERT) showed altered allelic distributions in a Danish migraine population. In addition to serotonin, there has been renewed interest in the involvement of the dopaminergic pathways in migraine. This interest has gained impetus since the study of Peroutka et al who reported an allelic association between the dopamine receptor gene DRD2 and migraine with aura. Another dopamine related gene, the dopamine beta-hydroxylase gene (DBH), has been localised to Chr 9q34 and codes for the enzyme that catalyses the conversion of dopamine to norepinephrine. It therefore plays an important role in dopaminergic and noradrenergic neurotransmission. Serum levels of DbH enzyme have been reported to be elevated in migrainous patients during the headache phase of an attack. Also, significantly increased DbH enzyme activity has been observed in migraine patients during the headache-free interval. Thus, the DBH gene is another good candidate for involvement in migraine pathophysiology and, to our knowledge, has not been previously implicated in this disease. Candidate gene studies may be useful strategies for identifying genes involved in complex diseases such as migraine, especially if the gene being examined contributes only a minor effect to the overall phenotype. This research also involved a linkage and association approach to investigating neurotransmitter related migraine candidate genes. Specifically, polymorphisms within the serotonin transporter gene (SERT), the dopamine receptor gene (DRD2) and the dopamine beta-hydroxylase (DBH) gene were tested in unrelated Caucasian migraineurs and non-migraine control individuals. In addition, an independent sample of 82 families affected with migraine were examined. Unrelated case-control association analysis of a DBH intragenic dinucleotide polymorphism indicated altered allelic distribution between migraine and control groups (c2 = 16.53, P = 0.019). Furthermore, the transmission/disequilibrium test (TDT) which was implemented on the family data also indicated distortion of allele transmission for the same DBH marker (c2 = 4.44, P = 0.035). Together, these results provide evidence for allelic association of the DBH gene with typical migraine susceptibility (Fisher's Combined P-value = 0.006) and indicate that further research into the role of the DBH gene in migraine aetiology is warranted. Nitric oxide (NO) is emerging as a key molecule affecting the pain associated with migraine. Since nitric oxide synthase (NOS) enzymes catalyse the synthesis of NO, the genes that code for these enzymes are good candidates for migraine molecular genetic analysis. This research involved investigating the role of a functionally relevant bi-allelic tetranucleotide polymorphism located in the promoter region of the human inducible nitric oxide synthase (iNOS) gene in migraine aetiology. A large group of migraine affected individuals were genotyped and compared to an age and sex matched group of unaffected controls. Results of a chi-squared analysis indicated that allele distributions for both migraine cases and controls were not significantly different (c2 = 1.93, P = 0.16). These findings offer no evidence for an allelic association of the tested iNOS polymorphism with the common forms of the disease and therefore do not support a role for this gene in migraine pathogenesis. In summary, this research involved linkage and association analysis of migraine candidate genes and genomic susceptibility regions. Whilst, the known FHM gene (CACNA1A) was excluded for significant involvement in typical migraine the adjacent INSR gene has been associated. Migraine is genetically heterogeneous and the results of this research also provide good evidence that the DBH gene is involved in disease predisposition, whilst the DRD2, SERT and INOS gene were not shown to be implicated. An additional susceptibility region for typical migraine is also likely to localise to chromosome 1q31. Overall, the results presented in this thesis have contributed valuable data to the understanding of the molecular genetics of migraine with and without aura. Future research into the molecular pathophysiological mechanisms of migraine will greatly facilitate the development of more effective diagnosis and treatment strategies.

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.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
Full Text

Migraine is a common, debilitating neurovascular disease charactensed by severe recurrent headache, nausea and vomiting, photophobia and phonophobia. It is clinically diagnosed based on criteria specified by the International Headache Society (IHS), defining two major classes of migraine: migraine with aura (MA) and migraine without aura (MO) MA sufferers experience neurovascular disturbances that precede the headache phase of an attack. Although migraine is partly influenced by environmental determinants, there is a significant genetic component, with disease heritability estimated to be up to 60% and mode of transmission multifactorial. The disorder is common with a large Dutch study reporting lifetime prevalence estimates of 33% in women and 13.3% in men, with an earlier study estimating 24% of women and 12% of men in the overall population. Mutations in various ion channel genes are responsible for neuromuscular and other neurological disorders. Inherited ion channel mutations or 'channelopathies' are increasingly found to be the cause of various neurological disorders in humans. In familial hemiplegic migraine (FHM), a rare subtype of migraine with aura, mutations in the CACNA1A gene (localised at C19p13) have been fbund (FHM1). This gene codes for the alphalA subunit of the neuronal voltage-dependent P/Q-type calcium channel. Recently a second gene, ATP1A2 (FHM2) (localised at C1q23), was implicated in some EHM families. The ATP1A2 ion channel gene, codes for the alpha2 subunit of the Na+, K+ ion ATPase pump. These findings of mutations in these genes have focused attention on central nervous system ionic channels and helped to better understand EHM pathophysiology, where the best genetic evidence providing molecular insight into migraine still comes flom the mutations detected in the rare form of migraine with aura; FHM. Migraine family studies, at the Genomic Research Centre (GRC), have utilised linkage analysis methods in providing results that have indicated suggestive linkage to the FHM1-CACNA1A region on l9p13, in a large multigenerational family (Migraine Family 1; MEl) affected with typical migraine. Also linkage studies conducted within the GRC have implicated an additional susceptibility region on chromosome 1q31, but still not ruling out a second susceptibility region on C1q23, with the possibility of there being two distinct loci, on the chromosome lq region. The focus of research in this thesis is on two main chromosomal regions, which were tested for migraine susceptibility on chromosome 1 and chromosome 19. The research involved a cross-disciplinary approach utilising association, linkage and mutation screening approaches. Allelic candidate gene studies can provide a suitable method for locating genes of small effect that contribute to complex genetic disorders, such as migraine. Family linkage studies are useful for detection of chromosomal susceptibility regions and association studies are powerful when a plausible candidate gene and a sequence variant with potential functional relevance is examined. Mutation screening studies can indicate a direct cause of disorders such as migraine, where possible sequence variants may alter the translation of proteins in genes, causing the disease. The first gene exanted on chromosome 19 was that of the Low Density Lipoprotein Receptor (LDLR) gene. The LDLR gene is a cell surface receptor that plays an important role in cholesterol homeostasis. We investigated the (TA)n polymorphism in exon 18 of the LDLR gene on chromosome l9pl3.2 performing an association analysis in 244 typical migraine affected patients, 151 suffering from migraine with aura, 96 with migraine without aura and 244 unaffected controls. The populations consisted of Caucasians only and controls were age and sex matched. The results showed no significant difference between groups for allele frequency distributions of the (TA)n polymorphism even after separation of the migraine affected individuals into subgroups of MA and MO affected patients. This is in contradiction to Mochi et al, 2003 who found a positive association of this variant with MO. Our study discusses possible differences between the two studies and extends this research by investigating circulating cholesterol levels in a migraine affected genetically-isolated population. Another gene examined on chromosome l9pl3 was the insulin receptor gene (1NSR). The aim of this study was to investigate through direct sequencing the INSR gene in DNA samples from a migraine affected family previously showing linkage to chromosome l9pl3 in an attempt to detect disease associated mutations. The insulin receptor gene (INSR) on chromosome 19pl3.3-13.2 is a gene of interest since a number of SNPs located within the gene have been implicated in migraine with (MA) and without aura (MO). Six DNA samples obtained from non-founding migraine affected members of migraine family one (MF 1) were used in this study. Genomic DNA was sequenced for the 1NSR gene in exons 1-22 and the promoter region. In the six migraine family member samples, previously reported single nucleotide polymorphisms (SNP5) were detected within two exonic DNA coding regions of the INSR gene. These SNPs, in exon 13 and 17, do not alter the normal INSR polypeptide sequence. In addition, intron 7 also revealed a DNA base sequence variation. For the 5' untranslated promoter region of the gene, no mutations were detected. In conclusion, this study detected no INSR mutations in affected members of a chromosome 19 linked migraine pedigree. Hence, migraine linkage to this chromosomal region may involve other candidate genes. The NOTCH3 gene on C19p13.2-p13.l has previously been shown to be a gene involved in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and may also be implicated in migraine as there are some symptom similarities between the two disorders. The TNFSF7 gene localised on Cl9pl3 is homologous to the ligands of the TNF receptor family, including TNF-alpha and TNF-beta, genes that have both been previously associated with migraine. This study investigated the migraine susceptibility locus at Cl9p13 studying two genes that may be involved in the disorder. The NOTCH3 gene was analysed by sequencing all exons with known CADASIL mutations in a family (MF1) that has previously been shown to be linked to Cl9pl3. The sequencing results for affected members of this pedigree proved to be negative for all known sequence variants giving rise to mutation causing amino acid changes for CADASIL. The direct sequencing results displayed that of a normal coding sequence for the NOTCH3 gene F or the TNFSF7 gene, this was investigated through SNP association analysis using a matched case-control migraine diagnosed population. Chi-square results showed non-significant P values across all populations tested against controls except for the MO subgroup which displayed a weak association with the TNFSF7 SNP (genotype, allele analysis P = 0.036, P = 0 017 respectively). Our results suggest that common migraine is not caused by any known CADASIL mutations in the NOTCH3 gene of interest however, the TNFSF7 gene displayed signs of involvement in a MO affected population, but, further studies are needed to confirm these results and to further explore a TNF receptor - migraine potential interaction. A final examination on chromosome 19 involved a case report of an extremely rare and severe form of migraine. As stated earlier Familial Hemiplegic Migraine (FHM) is a severe rare sub-type of migraine and gene mutations on chromosome 19 have been identified in the calcium channel gene CACNA1A (Cl9pl3) fOr FHM. Recently a gene mutation (S218L) for a dramatic syndrome originating from FHM, commonly named 'migraine coma', has implicated exon 5 of the CACNA1A gene. The occurrence of trivial head trauma, in FHM patients, may also be complicated by severe, sometimes even fatal, cerebral edema and coma occurring after a lucid interval. Hemiplegic migraine has also been found to be sporadic in which both forms share a similar spectrum of clinical presentations and genetic heterogeneity. The case report presented in this study enhances the involvement of the S218L CACNA1A mutation in the extremely rare disorder of minor head trauma induced migraine coma. It not only proves to be a powerful diagnostic tool in detecting cases of FHM head trauma induced coma but also for sporadic hemiplegic migraine (SHM) coma subjects. We conclude from this case study that the S218L mutation, in the CACNA1A calcium channel subunit gene, is involved in sporadic hemiplegic migraine (SHM), delayed cerebral edema and coma after minor head trauma. This thesis also involved analysis of chromosome 1 for migraine susceptibility, where FHM studies provided a foundation fOr common migraine research on chromosome 1. Studies have suggested that mutations in the CACNA1A gene on chromosome l9p cause FHM in only approximately 50% of affected pedigrees. The CACNAIA gene has previously been tested, within the Genomics Research Centre, in the common forms of migraine; however no new mutations or the FHM mutations were detected in these MA/MO affected samples. A second FHM susceptibility locus maps to chromosome 1q23 and mutations in the ATP1A2 gene have recently been implicated in two Cl-linked FHM pedigrees. As FHM is considered a rare and severe form of MA, it is possible that the chromosome 1q23 locus, and the ATP1A2 gene, may be involved in the common forms of migraine with (MA) and possibly without aura (MO). Also, we have previously reported evidence of linkage to microsatellite markers on chromosome 1q31 in a large pedigree affected predominately with MA, which suggests the possibility that there are two distinct loci for migraine susceptibility on chromosome 1. The objectives of this study were to extend our linkage analysis of chromosome lq microsatellite markers in predominantly migraine with aura pedigrees. Also, our aim was to test the novel FHM-2 ATP1A2 gene for involvement in these migraine affected pedigrees and a previous pedigree (Migraine Family 14; MF 14) showing evidence of linkage of markers to Clq31. This was performed by a chromosome 1 scan (31 markers) in 21 multiplex pedigrees affected mainly with MA. Also, the known FHM-2 ATP1A2 gene mutations were tested, by sequencing, fOr involvement in MA and MO in these pedigrees. Mutation screening by direct sequencing was also performed throughout the coding areas of the ATP1A2 gene in 3 MA individuals fiom MF14. The results of this study detected evidence for linkage in our migraine pedigrees at chromosome 1q23, to microsatellite markers spanning the ATP1A2 (FHM-2) gene. However testing of the known ATP1A2 gene mutations (for FHM) in migraine probands of pedigrees showing excess allele sharing was negative, with no mutations detected in these migraineurs. Sequencing of the entire coding areas of the gene through 3 MA affecteds from MF14, a pedigree showing significant linkage to this region, was also negative for mutations. In conclusion, this study reported that microsatellite markers on chromosome 1q23 show evidence of excess allele sharing in MA and some MO pedigrees, suggesting linkage to the common forms of migraine and the presence of a susceptibility gene in this region. The new FHM-2 (ATPIA2 gene) mutations reported by Fusco et al, 2003 do not cause migraine in probands of affected pedigrees showing excess allele sharing to markers in this genomic region. Also no mutations were detected in all exons of the ATP1A2 gene in 3 MA affected individuals from a large pedigree (MF14) showing linkage to this region. Investigation in this thesis continued on chromosome 1, with other genes being examined on C1q23, as well as the C1q31 region for a migraine susceptibility locus or gene. Previously in our laboratory, evidence for linkage was shown to migraine at C1q31 in one family predominantly affected with MA, with microsatellite markers in this region. The initial Cl study (above; ATP1A2 gene) has also provided evidence for linkage to the chromosome 1 locus 1q23, with evidence for excess allele sharing of markers in predominantly MA affected pedigrees. To further investigate both chromosome I loci, an investigation with six candidate genes that lie within the C1q23 and 1q31 regions through association analysis was undertaken. The results from this study reported non-significant chi-square results, showing P values greater than 0.05 across all SNPs (and a CA rpt) tested. An exception was the rs704326 SNP from exon 43 of the CACNA1E gene on C1q31. P values significantly less than 0.001 were obtained in the total migraine population and the MA subgroup, with similar frequency comparisons ascertained in both genotype and allele analysis. Examination through contingency table analysis of the CACNA1E flequency data indicated that the risk allele (A) was over-represented in the migraine group compared to the control group. Further comparison of the genotype data indicated a difference in frequency distributions (P less than 0 0001). Stratified analyses of migraine subtypes indicated that this association was specifically attributed to the MA subtype group. Odds ratios produced an OR of 4.14 with a 95% CI of 2.36 - 7.26 (P less than 0.0001). The positive association results obtained within the CACNA1E gene are interesting in the fact that FHM is considered to be a rare and severe form of migraine with aura (MA) and FHM-1 is caused by mutations contained within the calcium channel gene CACNA1A (localized at Cl9p13). The idea that FHM and specifically an FHM gene in the C1q31 genomic region may also contribute to susceptibility to the more common forms of migraine i e. migraine with aura, strongly supports and reinforces the idea that a common defective gene may be influencing both FHM and typical migraine. In conclusion, this thesis undertook a cross-disciplinary approach to genetic research of a complex disorder. The research involved linkage, association and mutation analysis strategies of migraine. This research implicated a specific variant on chromosome 1 and further supported the heterogeneic nature of migraine. Future directions into migraine research should involve further investigation of this specific variant and this genomic region. Such studies may aid in the development of more precise diagnosis and treatment methods for this complex disorder.

Migraine is a common, debilitating neurovascular disease charactensed by severe recurrent headache, nausea and vomiting, photophobia and phonophobia. It is clinically diagnosed based on criteria specified by the International Headache Society (IHS), defining two major classes of migraine: migraine with aura (MA) and migraine without aura (MO) MA sufferers experience neurovascular disturbances that precede the headache phase of an attack. Although migraine is partly influenced by environmental determinants, there is a significant genetic component, with disease heritability estimated to be up to 60% and mode of transmission multifactorial. The disorder is common with a large Dutch study reporting lifetime prevalence estimates of 33% in women and 13.3% in men, with an earlier study estimating 24% of women and 12% of men in the overall population. Mutations in various ion channel genes are responsible for neuromuscular and other neurological disorders. Inherited ion channel mutations or 'channelopathies' are increasingly found to be the cause of various neurological disorders in humans. In familial hemiplegic migraine (FHM), a rare subtype of migraine with aura, mutations in the CACNA1A gene (localised at C19p13) have been fbund (FHM1). This gene codes for the alphalA subunit of the neuronal voltage-dependent P/Q-type calcium channel. Recently a second gene, ATP1A2 (FHM2) (localised at C1q23), was implicated in some EHM families. The ATP1A2 ion channel gene, codes for the alpha2 subunit of the Na+, K+ ion ATPase pump. These findings of mutations in these genes have focused attention on central nervous system ionic channels and helped to better understand EHM pathophysiology, where the best genetic evidence providing molecular insight into migraine still comes flom the mutations detected in the rare form of migraine with aura; FHM. Migraine family studies, at the Genomic Research Centre (GRC), have utilised linkage analysis methods in providing results that have indicated suggestive linkage to the FHM1-CACNA1A region on l9p13, in a large multigenerational family (Migraine Family 1; MEl) affected with typical migraine. Also linkage studies conducted within the GRC have implicated an additional susceptibility region on chromosome 1q31, but still not ruling out a second susceptibility region on C1q23, with the possibility of there being two distinct loci, on the chromosome lq region. The focus of research in this thesis is on two main chromosomal regions, which were tested for migraine susceptibility on chromosome 1 and chromosome 19. The research involved a cross-disciplinary approach utilising association, linkage and mutation screening approaches. This research implicated a specific variant on chromosome 1 and further supported the heterogeneic nature of migraine. Future directions into migraine research should involve further investigation of this specific variant and this genomic region. Such studies may aid in the development of more precise diagnosis and treatment methods for this complex disorder.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Full Text

This chapter explores anorexia nervosa as an unusually clear example of how instincts can self-organize as specific stories, and it also demonstrates a role for dreaming in the emergence of particular narratives. Human nature seems to emerge as beliefs and stories about our roles and our place in our community. Those who develop anorexia nervosa become obsessed by a story that demands that they restrict feeding and move hyperactively, which seems quite mad, because they starving. Anorexia symptoms are homogeneous and delusional and we can observe a clear correspondence between the manifestly insane cognitions and specific neurocircuit changes recently revealed by brain imaging studies. A wholescale reorganization of the brain’s response to feeding opportunities is triggered by a critical drop in the energy regulation hormone, leptin. Thus, serious weight loss in those with the genetic predisposition initiates anorexia nervosa. The symptoms of anorexia nervosa (AN) were probably selected during Paleolithic famines to help hunter-gatherers searching for better lands. Decreasing feeding, moving actively, and denying starvation would have helped individuals migrate. A bifurcation to the same behaviors has been described in starving rats and pigs. Moreover, post-pubescent girls are most vulnerable because ovarian hormones turn on the heritability of AN-related genes. This specific gene and environment interaction indicates that teenaged girls were the best candidates to search for better lands during famine, probably because they were more likely to survive to reproduce if they encountered an enemy band. Dreaming appears to help condition fear of food and consolidate anorexic beliefs. However, different neural modules also seem to use dreaming to help people to recover from anorexia nervosa. The hypothesis that anorexia nervosa is due to evolutionary adaptations to search for better lands during famine rather than learned habits or psychological issues has major treatment implications.

The long term goal of this study was to reduce caloric and fat content of beef and other red meats by means of genetic modification of the animal such that fat would not be accumulated. This was attempted by introducing into the germ line myogenic regulatory genes that would convert fat tissue to skeletal muscle. We first determined the consequences of ectopic expression of the myogenic regulatory gene MyoD1. It was found that deregulation of MyoD1 did not result in ectopic skeletal muscle formation but rather led to embryonic lethalities, probably due to its role in the control of the cell cycle. This indicated that MyoD1 should be placed under stringent control to allow survival. Embryonic lethalities were also observed when the regulatory elements of the adipose-specific gene adipsin directed the expression of MyoD1 or myogenin cDNAs, suggesting that these sequences are probably not strong enough to confer tissue specificity. To determine the specificity of the control elements of another fat specific gene (adipocyte protein 2-aP2), we fused them to the bacterial b-galactosidase reporter gene and established stable transgenic strains. The expression of the reporter gene in none of the strains was adipose specific. Each strain displayed a unique pattern of expression in various cell lineages. Most exciting results were obtained in a transgenic strain in which cells migrating from the ventro-lateral edge of the dermomyotome of developing somites to populate the limb buds with myoblasts were specifically stained for lacZ. Since the control sequences of the adipsin or aP2 genes did not confer fat specificity in transgenic mice we have taken both molecular and genetic approaches as an initial effort to identify genes important in the conversion of a multipotential cell such as C3H10T1/2 cell to adipoblast. Several novel adipocyte cell lines have been established that differ in the expression of transcription factors of the C/EBP family known to be markers for adipocyte differentiation. These studies revealed that one of the genetic programming changes which occur during 10T1/2 conversion from multipotential cell to a committed adipoblast is the ability to linduce C/EBPa gene expression. It is expected that further analysis of this gene would identify elements which regulate this lineage-specific expression. Such elements might be good candidates in future attempts to convert adipoblasts to skeletal muscle cells in vivo.

The goal of this research was to provide a better understanding of the interface between root-knot nematodes, Meloidogyne spp., and their host in order to develop rational targets for plantibodies and other novel methods of nematode control directed against the nematode surface coat (SC). Specific objectives were: 1. To produce additional monoclonal SC antibodies for use in Objectives 2, 3, and 4 and as candidates for development of plantibodies. 2. To determine the production and distribution of SC proteins during the infection process. 3. To use biochemical and immunological methods to perturbate the root-knot nematode SC in order to identify SC components that will serve as targets for rationally designed plantibodies. 4. To develop SC-mutant nematodes as additional tools for defining the role of the SC during infection. The external cuticular layer of nematodes is the epicuticle. In many nematodes, it is covered by a fuzzy material termed "surface coat" (SC). Since the SC is the outermost layer, it may playa role in the interaction between the nematode and its surroundings during all life stages in soil and during pathogenesis. The SC is composed mainly of proteins, carbohydrates (which can be part of glycoproteins), and lipids. SC proteins and glycoproteins have been labeled and extracted from preparasitic second-stage juveniles and adult females of Meloidogyne and specific antibodies have been raised against surface antigens. Antibodies can be used to gain more information about surface function and to isolate genes encoding for surface antigens. Characterization of surface antigens and their roles in different life-stages may be an important step towards the development of alternative control. Nevertheless, the role of the plant- parasitic nematode's surface in plant-nematode interaction is still not understood. Carbohydrates or carbohydrate-recognition domains (CROs) on the nematode surface may interact with CROs or carbohydrate molecules, on root surfaces or exudates, or be active after the nematode has penetrated into the root. Surface antigens undoubtedly play an important role in interactions with microorganisms that adhere to the nematodes. Polyclonal (PC) and monoclonal (MC) antibodies raised against Meloidogyne javanica, M. incognita and other plant-parasitic nematodes, were used to characterize the surface coat and secreted-excreted products of M. javanica and M. incognita. Some of the MC and PC antibodies raised against M. incognita showed cross-reactivity with the surface coat of M. javanica. Further characterization, in planta, of the epitopes recognized by the antibodies, showed that they were present in the parasitic juvenile stages and that the surface coat is shed during root penetration by the nematode and its migration between root cells. At the molecular level, we have followed two lines of experimentation. The first has been to identify genes encoding surface coat (SC) molecules, and we have isolated and characterized a small family of mucin genes from M. incognita. Our second approach has been to study host genes that respond to the nematode, and in particular, to the SC. Our previous work has identified a large suite of genes expressed in Lycopersicon esculentum giant cells, including the partial cDNA clone DB#131, which encodes a serine/threonine protein kinase. Isolation and predicted translation of the mature cDNA revealed a frame shift mutation in the translated region of nematode sensitive plants. By using primers homologous to conserved region of DB#131 we have identified the orthologues from three (nematode-resistant) Lycopersicon peruvianum strains and found that these plants lacked the mutation.