Pain is a major public health issue throughout the world. Increased understanding of the different forms of pain and identification of novel susceptibility genes could contribute to improved treatments. The main aims of this thesis were to identify the underlying genetic causes of pain by studying two large families affected with migraine and pain insensitivity, respectively. Migraine is one of the most common neurovascular disorders, affecting over 12% of the western population. According to family and twin studies, the genetic contribution to migraine is about 50%. To identify novel susceptibility loci for migraine, we performed a genome-wide screen in a large family with migraine from northern Sweden. Linkage analysis revealed significant evidence of linkage (LOD = 5.41) on chromosome 6p12.2- p21.1. A predisposing haplotype spanning 10 Mb was inherited with migraine in all affected members of the pedigree. Further fine-mapping of multiple SNP markers allowed restriction of the disease-critical region to 8.5 Mb. Nine candidate genes were sequenced, revealing no disease-associated polymorphisms in SLC29A1, CLIC5, PLA2G7, IL17, SLC25A27 and TNFRSF21, but rare novel polymorphisms segregating with the disease haplotype in EFHC1, RHAG and MEP1A. EFHC1 has recently been shown to be involved in epilepsy, which is interesting considering the link between migraine and epilepsy. However, association analysis of EFHC1 revealed no difference between patients and controls, suggesting that this gene is not a risk factor for migraine. The combination of the two polymorphisms in RHAG and MEP1A could not, however, be found in any control individuals, indicating that they might be involved in genetic predisposition to migraine in this family. Disorders with reduced pain sensitivity are very rare, since pain perception is essential for survival. A number of disorders have still been identified with pain insensitivity and peripheral nerve degeneration as major clinical signs, including the hereditary sensory and autonomic neuropathies (HSAN). In order to identify novel susceptibility genes for HSAN V, we performed a genome-wide screen in a large consanguineous pedigree from a small village in northern Sweden. A homozygous region identical-by-descent was identified on chromosome 1p11.2-p13.2 in the three most severely affected patients. Subsequent analysis of candidate genes revealed a missense mutation in a conserved region of the nerve growth factor beta (NGFB) gene, causing a drastic amino acid change (R211W) in the NGF protein. NGF is important for the development and maintenance of the sympathetic and sensory nervous system and is therefore likely to be involved in disease. Functional analysis revealed that mutant NGF failed to induce neurite outgrowth and cell differentiation in PC12 cells. Furthermore, almost no mutant NGF was secreted by COS-7 cells, indicating that the processing or secretion of the protein might be disrupted. In conclusion, these findings have revealed a novel migraine locus on chromosome 6 and identification of two rare polymorphisms that may be risk factors for migraine. Furthermore, a mutation in NGFB was found to cause complete loss of deep pain perception, which represents a very interesting model system for the study of pain mechanisms.

3 TABLE OF CONTENTS

ABBREVIATIONS…………………………………………………………………….....6

PUBLICATIONS…………………………………………………………………………7

AIMS OF THIS THESIS.....……………………………………………………………..8

BACKGROUND………………………………………………………………………….9 Why study pain? What is pain? Is pain heritable?

INTRODUCTION………………………………………………………………………..10 Migraine Genetic epidemiology Prevalence Gender and age Ethnicity Types of migraine Migraine without aura (MO) Migraine with aura (MA) Familial hemiplegic migraine (FHM) Relationship between MA and MO Heritability of migraine Family studies Twin studies Susceptibility genes and risk factors Susceptibility genes in familial hemiplegic migraine Susceptibility loci in common forms of migraine Linkage studies of candidate FHM loci Linkage studies of other candidate regions Genome-wide screens of common migraine Candidate gene association studies Environmental risk factors Mechanism of disease Migraine models in mice

Pain insensitivity Types of HSAN HSAN I HSAN II HSAN III HSAN IV HSAN V The NGF-TrkA system Family of neurotrophins Neurotrophin signaling Neurotrophin processing NGF mice models Neurotrophins in human disease

4 METHODOLOGY…………………………………………………………………….…34 Patient material Migraine Pain insensitivity Genealogical studies Molecular genetic methods Genome-wide scan Candidate gene analysis Genetic markers Microsatellite markers Single nucleotide polymorphisms Functional methods Splicing assay PC12 differentiation assay

RESULTS & DISCUSSION……………………………………………………………40 Paper I: Genome-wide screen of common migraine Paper II: Fine-mapping and candidate gene analysis of migraine Paper III: Genome-wide screen of pain insensitivity Paper IV: Functional analysis of the NGFB mutation

CONCLUSIONS………………………………………………………………………...53

CONCLUDING REMARKS……………………………………………………………54

POPULÄRVETENSKAPLIG SAMMANFATTNING………………………………..55

ACKNOWLEDGEMENTS……………………………………………………………..57

REFERENCES …………………………………………………………………………59

PAPERS I-IV……………………………………………………………………………….

5 ABBREVIATIONS

bp basepair cM centimorgan CSD cortical spreading depression DNA deoxyribonucleic acid DZ dizygotic twins HSAN hereditary sensory and autonomic neuropathy FHM familial hemiplegic migraine IHS International Headache Society LOD logarithm of odds MA migraine with aura Mb megabase (one million basepairs) MO migraine without aura MZ monozygotic twins NGFB nerve growth factor beta SNP single nucleotide polymorphism

6 PUBLICATIONS

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals (I–IV).

I. Carlsson A, Forsgren L, Nylander P-O, Hellman U, Forsman-Semb K, Holmgren G, Holmberg D and Holmberg M. Identification of a susceptibility locus for migraine with and without aura on 6p12.2-p21.1. Neurology 59, 1804-1807, 2002.

II. Norberg A, Forsgren L, Holmberg D and Holmberg M. Exclusion of the juvenile myoclonic epilepsy gene EFHC1 as the cause of migraine on chromosome 6, but association to two rare polymorphisms in MEP1A and RHAG. Neurosci Lett 396, 137-142, 2006.

III. Einarsdottir E, Carlsson A, Minde J, Toolanen G, Svensson O, Solders G, Holmgren G, Holmberg D and Holmberg, M. A mutation in the nerve growth factor beta gene (NGFB) causes loss of pain perception. Hum Mol Gen 13, 799-805, 2004.

IV. Larsson E*., Kuma R*., Norberg A., Minde J., Holmberg M. Mutant Nerve Growth Factor (NGF) – responsible for Hereditary and Sensory Autonomic Neuropathy type V – is defectively secreted and accumulates as proNGF. Manuscript.

*These authors contributed equally to this work.

Paper I is reprinted with permission from Lippincott Williams & Wilkins, Paper II with permission from Elsevier, and Paper III with permission from Oxford University Press.

7 AIMS OF THIS THESIS

The main aim of this thesis was to identify the underlying genetic causes of pain, and to contribute to our knowledge of the pathways involved in pain and pain control. The strategy was to investigate two large families from northern Sweden that are affected with either migraine or pain insensitivity.

More specifically, the aims of this thesis were to:

• identify the chromosomal position and disease-causing gene for migraine with and without aura, inherited in an autosomal dominant way (Paper I).

• fine-map the migraine susceptibility region and investigate candidate genes potentially associated with migraine (Paper II).

• characterize additional migraine families related to the original family, by means of a population-based study (Paper II).

• identify the chromosomal position and disease-causing gene for pain insensitivity in a large family with recessive transmission of the disease (Paper III).

• determine the functional significance and mechanisms of the NGFB mutation identified in the family with pain insensitivity (Paper IV).

8 BACKGROUND

Why study pain? Pain is of major public concern throughout the world. Millions of people suffer from chronic or recurrent pain, of which back pain and headaches are the most common. In addition, the costs in terms of drug treatment and lost work productivity are enormous. Increased understanding of the different forms of pain and their underlying mechanisms could contribute to new and better treatments for patients with chronic pain disorders.

What is pain? According to the International Association for the Study of Pain, pain is “an unpleasant sensory and emotional experience associated with actual or potential tissue damage. Each individual learns the application of the word through experiences related to injury in early life”. Pain is a multistep process usually triggered by stimulation of peripheral nerves, which then transmit sensory signals from the periphery to the spinal cord or brain stem, where it is translated into a conscious feeling (Silberstein, 2003).

Pain most often has a physical cause, but sometimes people feel pain in the absence of tissue damage. This usually occurs for psychological reasons, and is experienced in the same way as pain caused by tissue damage. The sensation of pain is always subjective, and is influenced by the individual’s genetic composition, prior experience, physiological condition, expectations, and sociocultural environment (Turk and Okifuji, 1999). It is difficult to determine whether one person’s pain response is the same as another’s, which complicates the clinical diagnosis of pain and the evaluation of the underlying mechanisms.

Is pain heritable? Individuals show a high variability in their perception and tolerance of pain, as well as their response to analgesic drugs, and susceptibility to develop painful pathologies. Some proportion of these differences is heritable, due to allelic variants in genes that are of relevance to pain (Mogil, 1999). The heritability is usually quite strong in many diseases where pain is a major symptom, such as migraine, whereas it accounts for much less of the individual variation in nonpathological pain perception (Mogil, 1999). However, investigation of genetic influences on pain perception is complex, since they most likely reflect the interactions of multiple genetic factors and environment.

Mediation of pain is believed to be regulated by pain mechanism genes, while individual variation in responses to pain and susceptibility to pathological conditions is mediated by pain susceptibility genes. The main pain mechanism genes are highly conservative, since pain perception is essential for survival. Thus, mutations in these genes are very rare in nature. One such example is the hereditary sensory and autonomic neuropathies, where single critical mutations cause reduced

9 pain sensitivity, and consequently lead to frequent injuries and accidental self- mutilation. Most of the individual variation in pain perception is probably due to summation of many polymorphisms causing small changes in multiple pain susceptibility genes. So far, few genetic changes influencing pain perception have been identified. One of the few examples includes a rare form of familial hemiplegic migraine (Mogil et al., 2000).

The work in this thesis focuses on two different pain disorders, migraine and insensitivity to pain. In the Introduction, I will give an overview of the current literature in these fields, especially areas concerning hereditary factors and underlying molecular mechanisms of disease. In the Results and Discussion, I will summarize the main findings of the papers included in this thesis.

INTRODUCTION

Migraine

Migraine is one of the most common neurovascular disorders, characterized by recurrent attacks of disabling headache that last from a few hours to a few days. Usually, migraine patients are perfectly well between attacks. The mechanisms underlying migraine are not completely understood. Thus, a major goal with migraine research is to identify susceptibility genes for migraine, which may give clues as to the pathological mechanisms and may contribute to the development of improved therapeutic strategies.

Genetic epidemiology

Prevalence Prevalence studies of migraine have been reported from many different countries. Lifetime prevalence refers to the proportion of individuals in a population who have ever had migraine during their lives, whereas one-year prevalence means the proportion of individuals who have had migraine during the year prior to the study. The lifetime prevalence in the western world has been estimated to be about 6–8% in men and 15–25% in women (Pietrobon and Striessnig, 2003). Examples include a Danish study of 1,000 people between 25-64 years of age, where the lifetime prevalence of migraine was 7.8% in men and 25.2% in females (Rasmussen et al., 1991). In the USA, the 1-year prevalence among 4,376 people between 18 and 65 years was 6.0% in men and 17.2% in women (Lipton et al., 2002). In Sweden, the 1-year prevalence among 1,668 individuals aged 18–74 years was 9.5% in men and 16.7% in women (Dahlof and Linde, 2001).

Gender and age Most of the variation in migraine prevalence is due to differences in age and gender in the study population. Migraine is two to three times more common in women

10 than in men. At age 20, the female-male ratio is about 2:1, whereas it peaks to 3:1 at age 40. The overall prevalence for both men and women increases with age and peaks at about mid-life, although the peak is reached earlier in men than in women. Thereafter, the prevalence decreases for both genders, but remains higher in women even after menopause (Breslau and Rasmussen, 2001). The prevalence is higher in boys than in girls before the age of 12, whereas it is higher in girls after puberty (Abu-Arefeh and Russell, 1994).

Ethnicity Migraine is a greater problem in the western world than in Africa and Asia, most probably due to different genetic susceptibilities in these populations, but also to different environment and lifestyle. Examples include Saudi Arabia (Abduljabbar et al., 1996) and Ethiopia (Tekle Haimanot et al., 1995), where total migraine prevalence has been estimated to be only 2.6% and 3.0%, respectively.

Types of migraine There are no biological markers to diagnose migraine, which makes it important to have strict clinical criteria for classification that can be used throughout the world. These criteria were provided for the first time by the International Headache Society in 1988 (IHS, 1988). These criteria were updated recently in the second edition of the IHS classification of headache disorders (IHS, 2004). According to IHS, migraine can be divided into two major subtypes: migraine without aura (MO) and migraine with aura (MA).

Migraine without aura (MO) It has been estimated that MO accounts for about 2/3 of migraine cases (Russell et al., 1995). MO is characterized by attacks lasting from 4 h to three days. A typical headache has at least two of the following characteristics: unilateral location of pain, pulsating quality, moderate to severe intensity, and sensitivity to physical activity (Table 1). The attacks are usually associated with various combinations of vomiting, nausea and photo- and/or phonophobia. According to the IHS criteria, at least five attacks of MO are required in order to classify a person as affected. The frequency, duration and severity of the attacks vary substantially between patients. Migraine sufferers may have as few as one attack per year to as many as 2–3 separate attacks per week. In an epidemiological study in the USA, 68% of 568 migraine patients reported one or more attacks per month, whereas 25% reported one or more attack per week (Lipton et al., 2002). In a Swedish study, 60% of 423 migraine patients had an attack frequency of at least once per month, and the mean attack duration was 19 hr (Dahlof and Linde, 2001).

Migraine with aura (MA) MA is less common than MO, and accounts for about one-third of cases (Russell et al., 1995). MA is characterized by attacks of neurological symptoms, which usually

11 Table 1 IHS diagnostic criteria for migraine without aura (MO)

A. At least five attacks fulfilling B-D. B. Headache lasting 4-72 hr (untreated or unsuccessfully treated). C. Headache has at least two of the following characteristics: 1. unilateral location 2. pulsating quality 3. moderate or severe pain intensity 4. aggravation by routine physical activity (eg, walking or climbing stairs) D. During headache at least one of the following: 1. nausea and/or vomiting 2. photophobia and phonophobia E. Not attributed to another disorder1.

Table 2 IHS diagnostic criteria for migraine with aura (MA)

A. At least two attacks fulfilling B-D. B. Aura consisting of at least one of the following, but no motor weakness: 1. fully reversible visual symptoms including positive features (eg, flickering lights, spots or lines) and/or negative features (ie, loss of vision). 2. fully reversible sensory symptoms including positive features (ie, pins and needles) and/or negative features (ie, numbness). 3. fully reversible dysphasic speech disturbance. C. At least two of the following: 1. homonymous visual symptoms and/or unilateral sensory symptoms. 2. at least one aura symptom develops gradually over >5 minutes and/or different aura symptoms occur in succession over >5 minutes. 3. each symptom lasts >5 and <60 minutes. D. Headache begins during the aura or follows aura within 60 minutes. E. Not attributed to another disorder1.

Table 3 IHS diagnostic criteria for familial hemiplegic migraine (FHM)

A. At least two attacks fulfilling criteria B and C. B. Aura consisting of fully reversible motor weakness and at least one of the following: 1. fully reversible visual symptoms including positive features (eg, flickering lights, spots or lines) and/or negative features (ie, loss of vision). 2. fully reversible sensory symptoms including positive features (ie, pins and needles) and/or negative features (ie, numbness). 3. fully reversible dysphasic speech disturbance. C. At least two of the following: 1. at least one aura symptom develops gradually over >5 minutes and/or different aura symptoms occur in succession over >5 minutes. 2. each aura symptom lasts >5 and <24 hours. 3. headache begins during the aura or follows onset of aura within 60 minutes. D. At least one first- or second-degree relative has had attacks fulfilling these criteria A-E. E. Not attributed to another disorder1.

1history and physical and neurological examinations do not suggest another disorder, or such disorder is present but attacks do not occur for the first time in close temporal relation to the disorder.

12 develop gradually over 5 to 20 minutes and last less than an hour (Table 2). The aura is most often manifest as visual symptoms, such as zigzag patterns, spots or flashing lights. However, it can also involve sensory disturbances such as hearing hallucinations or speech difficulties or—more rarely—motor symptoms (such as loss of strength). Headache usually follows directly after the aura symptoms or after an interval of less than one hour. According to the IHS criteria, at least two attacks of MA are required in order to classify a person as affected.

Familial hemiplegic migraine (FHM) There are also other less common types of migraine, such as familial hemiplegic migraine. This is a rare and severe form of migraine with aura and it affects less than 1% of all migraine cases. FHM resembles typical MA, although the symptoms are more severe and the aura lasts longer (Table 3). The aura part of hemiplegic migraine is usually manifested as motor weakness or paralysis on one side of the body. In addition, some patients have attacks with impaired consciousness (coma), fever, and/or seizures (Ducros et al., 2001). Several genetic studies have focused on FHM, since it is the only form of migraine that has a simple Mendelian mode of inheritance. Interestingly, in some FHM families, both individuals with hemiplegic migraine and individuals with MA or MO can be found. Furthermore, FHM patients may also have attacks without hemiplegic symptoms (Ophoff et al., 1996; Ophoff et al., 1994). This suggests that there is a link between FHM and common forms of migraine. FHM is therefore a useful model for the study of the more complex genetics of common forms of migraine.

Relationship between MA and MO There is conflicting evidence as to whether MA and MO should be regarded as separate disorders or whether they represent two clinical forms of the same disease. Some studies have suggested that MO and MA may have different etiologies and should thus be regarded as distinct disorders (Russell and Olesen, 1995; Russell et al., 2002). However, several studies have shown that both subtypes of migraine can occur within the same family or in the same individual. Examples include a study of 210 Finnish migraine families, where the authors suggested the possibility of “a continuum with pure MA at the neural end of the spectrum and pure MO at the headache end, and MO plus MA lying in-between the two” (Kallela et al., 2001). According to this study, 23% of the patients within migraine families have only MO, 41% have both MA and MO attacks, 11% have only MA, 20% have unclassified aura, and 3% have aura without headache. In agreement with this, a recent study on 598 Australian migraine twin pairs indicated that MA and MO are not etiologically distinct from each other. The authors believed in the “existence of multiple causative factors contributing to migraine susceptibility and severity, with increased risk of aura associated with increased severity” (Nyholt et al., 2004). This suggests that there are common underlying genetic and/or environmental factors involved in these forms of migraine. It seems likely that the same pathophysiological mechanisms are at least partly shared in different types of migraine.

13 Heritability of migraine In very common diseases such as migraine, familial aggregation of a disorder may occur purely by chance. Thus, many studies have been performed to show the existence of genetic factors in migraine. As determined by several family and twin studies, hereditary factors contribute significantly to migraine.

Family studies Studies of families with migraine give information about the effect of genetic factors and the potential mode of inheritance. These studies measure the relative risk of migraine in first-degree relatives (i.e. parents, children or siblings) of migraine probands compared to the general population. The idea is that an increased risk of disease in first-degree relatives reflects a genetic cause for migraine, whereas an increased risk in spouses indicates an environmental cause. In one study of 378 migraine probands selected out of 4,000 Danish individuals, it was shown that first-degree relatives of probands with MO had 1.9 times the risk of MO, whereas first-degree relatives of MA probands had 3.8 times the risk of MA (Russell and Olesen, 1995). Spouses showed an increased risk of MO only. The authors suggested that MA is mainly determined by genetic factors, whereas MO is determined by a combination of genetic and environmental factors.

Twin studies Twin studies compare the concordance rates between monozygotic (MZ) and dizygotic (DZ) twins, which make it possible to separate the genetic and environmental contribution to a disease. MZ twins are genetically identical, whereas DZ twins have the same genetic similarities as first-degree relatives. If the concordance rate is higher in MZ twins than in DZ twins, this suggests that genetic factors are important for the trait. However, if the concordance rate in MZ twins is less than 100%, environmental factors also influence the trait. Proband-wise concordance rate is calculated as the proportion of affected co-twins of probands with migraine in relation to the total number of affected twins.

An example of this is a Danish twin study of 605 twin pairs, where at least one twin had migraine. The proband-wise concordance rate for MO was 40% in MZ twins and 28% in DZ twins (Gervil et al., 1999), while it was slightly higher for MA: 50% in MZ and 21% in DZ twins (Ulrich et al., 1999). In a Swedish study of 2,156 twin pairs, the proband-wise concordance rate for migraine was 48% in MZ twins and 31% in DZ twins, with an estimated heritability of 39–58% (Larsson et al., 1995). In a large study of 29,717 migraine twin pairs from several European countries and Australia, the migraine correlations in MZ twins were at least twice as high as in DZ twins. The total genetic effect of migraine ranged from 34% to 57% (Mulder et al., 2003). All studies done so far have shown that the concordance rate in MZ twins is higher than in DZ twins, suggesting that migraine has a genetic component. In addition, the genetic component seems to be higher for MA than for MO.

14 Susceptibility genes and risk factors Migraine is a complex disorder in which multiple genetic factors, lifestyle and environment interact. Only a combination of these risk factors leads to the onset of disease. The mode of inheritance for the common forms of migraine is most probably multifactorial, whereas it is Mendelian for the rare form of FHM. Furthermore, migraine is a genetically heterogeneous disorder, which means that different individuals may have different combinations of susceptibility genes. Mapping of susceptibility loci for migraine is therefore challenging. To date, three genes involved in FHM have been identified. For the common forms of migraine, four large and two small genome-wide screens have been reported, as well as several linkage and association studies investigating certain candidate loci (Figure 1) (Ducros et al., 2002; Estevez and Gardner, 2004; Wessman et al., 2004).

Susceptibility genes in familial hemiplegic migraine Chromosome 19p13 (FHM1): The first genetic mapping study of hemiplegic migraine was reported in 1993, when a susceptibility region on chromosome 19p13 was identified in two large French families (Joutel et al., 1993). More than half of all FHM families have been linked to this region. Later, this migraine syndrome was shown to be caused by mutations in the α1 subunit of a brain-specific neuronal calcium channel (CACNA1A) gene (Ophoff et al., 1996). So far, 15 different CACNA1A missense mutations have been identified in FHM1 (Kors et al., 2002), resulting in substitutions of amino acids in important functional domains of the channel. Different mutations lead to variable clinical expression among the patients, but patients with the same mutation can also show variability of symptoms, indicating that other genetic or environmental factors also influence the trait (Ducros et al., 2001). Most mutations result in a gain-of-function in the calcium channel that increases Ca2+ influx at the synaptic terminal, and subsequently cause altered release of different neurotransmitters and changes in signal processing and neural excitability (Ophoff et al., 1996; Tottene et al., 2002).

Mutations in CACNA1A have been identified in patients with spinocerebellar ataxia type 6 (SCA6) (Zhuchenko et al., 1997), and episodic ataxia type 2 (EA2) (Ophoff et al., 1996). Both of these are neurological disorders that show a phenotypic overlap with hemiplegic migraine (Terwindt et al., 1998). Typically, missense mutations in CACNA1A result in FHM1, whereas truncating mutations usually cause EA2, and expanded CAG repeats cause SCA6, although there are several deviations from this. Interestingly, a mutation in CACNA1A has been identified in a patient with absence epilepsy and episodic ataxia (Jouvenceau et al., 2001), and a case-control study reported a significant association between a polymorphism in CACNA1A and idiopathic generalized epilepsy (Chioza et al., 2001). This suggests that migraine and epilepsy may have partially overlapping mechanisms.

Chromosome 1q23 (FHM2): The second locus for familial hemiplegic migraine (FHM2) was identified on chromosome 1q21-23 in three French families (Ducros et al., 1997), and later refined to 1q23 in a large Italian family (Marconi et al.,

15 2003). Less than one-third of all FHM families are linked to this region. One study has also reported linkage of a large American FHM family to a second region near 1q31 (Gardner et al., 1997). Recently, missense mutations in the α2 subunit of a sodium/potassium pump (ATP1A2) were identified as the cause of FHM2 in two Italian families (De Fusco et al., 2003), and later in two Dutch families (Vanmolkot et al., 2003). In one of these Dutch families, the migraine syndrome partly co- segregated with benign familial infantile convulsions, demonstrating that epilepsy can be observed in FHM2 families. Similar to the CACNA1A gene, ATP1A2 is expressed in the brain. These pumps are responsible for the active transport of Na+ and K+ across cell membranes, thereby generating ion gradients that are important for regulation of cell volume and propagation of action potentials. Missense mutations in ATP1A2 result in loss of function of the pump, and impaired maintenance of the ion gradients. To date, seven different mutations in the gene have been described (De Fusco et al., 2003; Vanmolkot et al., 2003).

Chromosome 2q24 (FHM3): Recently, a third locus for familial hemiplegic migraine (FHM3) was identified on chromosome 2q24 in three German families, and shown to be caused by a missense mutation in a neuronal sodium channel (SCN1A) gene (Dichgans et al., 2005). Sodium channels are important for the generation and propagation of action potentials in the brain. The identified mutation results in an amino acid change in a part of the channel that contributes to rapid closure after opening in response to membrane depolarization. Thus, the mutated channel allows a persistent sodium influx instead of rapid closure and repolarization of the membrane after an action potential. This is believed to cause excessive firing of neurons, which results in enhanced excitability (Dichgans et al., 2005). Mutations in SCN1A have also been identified as a cause of severe myoclonic epilepsy of infancy (Claes et al., 2001) and generalized epilepsy with febrile seizures plus (Escayg et al., 2000), further strengthening the link between migraine and epilepsy. For a recent review of SCN1A mutations and epilepsy, see Mulley et al. (Mulley et al., 2005).

Susceptibility loci in common forms of migraine Linkage studies of candidate FHM loci Chromosome 19p13: Several studies have investigated whether the CACNA1A locus may be involved in common forms of migraine. Evidence of linkage to 19p13 has been reported in 28 German families (May et al., 1995) and in 36 Dutch families (Terwindt et al., 2001), indicating that this region contributes to the genetic susceptibility of common migraine. Linkage to 19p13 was also found in one of four large Australian families (Nyholt et al., 1998b), but no disease-causing mutations were revealed in this family when sequencing the coding parts of CACNA1A (Lea et al., 2001). It is possible that other genes may contribute to the linkage findings on 19p13 for the common forms of migraine. Interestingly, a linkage study in 16 American migraine families presented evidence for a new locus on 19p13, most likely distinct from the CACNA1A gene (Jones et al., 2001). This group subsequently performed a large case-control association study, where a significant

16 association was found between five SNPs in the insulin receptor (INSR) gene on 19p13 and migraine. However, the functional significance of these polymorphisms is unclear, since no changes in receptor function could be observed (McCarthy et al., 2001). Recently, the INSR gene was sequenced in affected members of one Australian family linked to 19p13, but no disease-causing mutations were found (Curtain et al., 2006). Another study investigated the role of the INSR and CACNA1AE loci in 72 Finnish families. However, none of the genotyped markers around these genes showed any evidence of linkage (Kaunisto et al., 2005). Exclusion of this region has also been reported in 4 Finnish families (Hovatta et al., 1994), in 64 Canadian families (Noble-Topham et al., 2002), and in 82 Australian families (Lea et al., 2001). Thus, 19p13 does not seem to be a major susceptibility locus for the common forms of migraine in these populations.

Chromosome 1q31: To investigate whether the reported FHM2 loci on 1q23 or 1q31 may contribute to common migraine, markers near these regions have been tested for linkage to migraine. To date, one study has been performed, in which a large Australian family was found to be linked to 1q31 (Lea et al., 2002). In addition, the authors found support for linkage at 1q31 among 82 additional Australian families (Lea et al., 2002). Nominal evidence of linkage to chromosome 1q has also been reported in one genome-wide screen of 50 Finnish MA families, but it is unclear whether this represents true linkage (Wessman et al., 2002).

Figure 1. Some of the chromosomal susceptibility loci for common forms of migraine.

17 Linkage studies of other candidate regions Chromosome Xq24-28: Since migraine is more common in women than in men, it seems likely that genetic factors on chromosome X may be involved in the disease. An Australian study showed significant linkage to Xq24-28 in two of three large migraine families, indicating the existence of a susceptibility gene in this region (Nyholt et al., 2000; Nyholt et al., 1998a). An excellent candidate gene is the serotonin receptor (5-HT2C) on Xq24, since abnormal serotonin metabolism has been observed in migraine patients, and serotonin receptors are targets for migraine medications (Johnson et al., 1998). However, when the coding regions of this gene were sequenced in all patients of the two Australian families, no mutations were found (Johnson et al., 2003).

Chromosome 15q11: Recently, a linkage study was performed with markers on chromosome 15 in 10 Italian MA families (Russo et al., 2005). This region contains three GABA-A receptor genes, which have been hypothesized to have a role in migraine. For five of the families, significant linkage was obtained on 15q11 in the region containing the candidate genes. Mutational analysis of the coding region in these genes revealed no significant mutation, only a single polymorphism (C75T) in the GABA receptor (GABRB3) gene in the affected members of one family (Russo et al., 2005). However, this polymorphism did not result in any amino acid change, and it is therefore unclear whether it contributes to migraine susceptibility.

Genome-wide screens of common migraine Chromosome 4q: The first genome-wide screen for a more common form of migraine was performed in 2002, by analyzing 50 Finnish MA families with an autosomal dominant mode of inheritance (Wessman et al., 2002). Using parametric affected-only analysis, a significant two-point LOD score of 4.20 was obtained on chromosome 4q24. This region is about 60 cM and contains over 170 known genes, including several good candidates. The locus on chromosome 4 was later confirmed in a genome-wide screen of 103 Icelandic MO families performed by the company deCODE Genetics (Bjornsson et al., 2003). Using non-parametric affected-only analysis, a multipoint LOD score of 2.05 was obtained on chromosome 4q21. When affected females only and a slight relaxed definition of MO were used, the LOD score increased to 4.08. Interestingly, these two studies indicate that the 4q locus may contribute to both MA and MO.

Chromosome 6p12.2-p21.1: The second genome-wide screen for common migraine was performed by our group in a large Swedish family with both MA and MO (Carlsson et al., 2002; see Paper I). A significant two-point LOD score of 5.41 was obtained on chromosome 6p12.2-p21.1, assuming an autosomal dominant inheritance. No other chromosomal regions produced significant evidence of linkage. The disease region spanned 10 Mb and contained over 50 known genes, of which three were excluded as the cause of disease. Recently, our group fine- mapped the disease region (Norberg et al., 2006; see Paper II), reducing it to 8.5 Mb. Six candidate genes were analyzed, in which two rare polymorphisms were identified in the meprin (MEP1A) and rhesus-associated glycoprotein (RHAG)

18 genes in migraine patients carrying the disease haplotype. However, it is unclear whether these polymorphisms have any functional significance for migraine.

Chromosome 5q21: A recent genome-wide screen was performed in 756 Australian twin families with migraine (Nyholt et al., 2005). Quantitative-trait linkage analysis obtained a multipoint LOD score of 3.70 on chromosome 5q21. Furthermore, replication of the migraine loci on 6p12.2-p21.1 and 1q21-q23 was reported. Interestingly, a locus for stroke has been identified on 5q12 in an Icelandic study (Gretarsdottir et al., 2002) and a Swedish study (Nilsson-Ardnor et al., 2005). Stroke and migraine have a complex relationship, and there are data suggesting that patients with migraine with aura have a slightly increased risk of stroke. This indicates that genes involved in stroke may be risk factors for migraine as well (Bousser and Welch, 2005).

Chromosome 11q24: This genome-wide study was performed in 43 Canadian families, segregating MA in an apparent autosomal dominant way (Cader et al., 2003). Parametric linkage analysis yielded a significant two-point LOD score of 4.24 on chromosome 11q24. No other loci were statistically significant, although the authors suggested closer evaluation of chromosomes 1 and 16 for which there was suggestive evidence of linkage. The disease region contains several good candidate genes, including the potassium channel genes KCNJ1 and KCNJ5, and the sodium channel gene SCNB2, but so far none of these have been analyzed.

Chromosome 14q21.2-q22.3: This study was performed on a single MO family originating from a restricted geographical area in northern Italy (Soragna et al., 2003). A genome-wide screen, using a parametric affected-only model, produced a maximum two-point LOD score of 3.70 on chromosome 14q21.2-q22.3, consistent with autosomal dominant inheritance. No other chromosomal loci presented statistical evidence of linkage. The disease region spans 12 Mb and contains over 45 known genes, including the prostaglandin receptors PTGDR and PTGER2, which can be considered good candidate genes in migraine pathogenesis.

Candidate gene association studies Although several linkage studies have been reported for the common forms of migraine, none of these have been successful in identifying the disease genes responsible. One reason for this may be that the mutations are subtle, and only confer a small risk to the overall phenotype. Another possibility is that the mutations occur in regulatory regions of the gene that are not well characterized. In addition to linkage studies, several association studies have been performed, where exclusion or possible association have been reported between different gene polymorphisms and migraine (Table 4). These studies differ from each other, depending on the population studied, the number of individuals analyzed, or on whether migraine has been divided into MA or MO. In addition, some studies have separated men and women in different groups, or have associated the polymorphism with certain sub-characteristics of migraine such as frequency of attacks or age at onset. Unfortunately, the results of most association studies often

19 remain controversial and need to be confirmed in independent replication studies. Furthermore, final proof of a disease gene will depend on functional studies, such as showing a biological effect of a specific variant when it is transferred to cells or disease models in animals.

Receptor and ion channel genes The fact that all FHM genes are involved in ion transport indicates that genes in similar cellular pathways are good candidates for common forms of migraine. Several association studies have therefore been performed in genes encoding different receptors or ion channels. The dopamine D2 receptor (DRD2) gene has been significantly associated with migraine in two independent studies (Del Zompo et al., 1998; Peroutka et al., 1997). Furthermore, significant associations have been reported between migraine and the dopamine beta-hydroxylase (DBH) (Lea et al., 2000) and the dopamine receptor D4 (DRD4) genes (Mochi et al., 2003). These results indicate a role for dopamine in migraine pathogenesis. In line with this, antagonists that block DRD2 are used in migraine treatment, and can relieve attacks in some patients (Peroutka et al., 1997).

Vascular and hormonal genes Genes involved in vascular function are good candidates for migraine susceptibility (Colson et al., 2006). One example is the angiotensin-converting enzyme (ACE), which is important for regulating vascular tension and blood pressure. A polymorphism in ACE has been significantly associated with migraine in two different case-control studies (Kowa et al., 2005; Paterna et al., 2000). This polymorphism accounts for about half of the variance in individual enzyme serum levels, increasing the levels in individuals homozygous for the polymorphism. Interestingly, ACE antagonists that inhibit the enzyme seem to have a prophylactic effect in migraine (Kowa et al., 2005). Furthermore, risk genes for stroke are also good candidates for migraine (Bousser and Welch, 2005). Studies have shown that plasma homocysteine levels are higher in patients with coronary artery disease and stroke. Methylene tetrahydrofolate reductase (MTHFR) catalyzes the formation of homocysteine to methionine, and a polymorphism (C677T) in this gene is known to reduce the enzymatic capacity by half (Colson et al., 2006). This polymorphism has been significantly associated with migraine in several different case-control studies (Kara et al., 2003; Kowa et al., 2000; Lea et al., 2004), and in one large population- based association study (Scher et al., 2006), suggesting a potential role of MTHFR in migraine pathogenesis.

Other examples of vascular genes that have been significantly associated with migraine are the serotonin transporter (5-HTT) (Marziniak et al., 2005; Ogilvie et al., 1998), and the endothelin type A receptor (EDNRA) (Tzourio et al., 2001). Serotonin is an important neurotransmitter involved in vascular function, especially in constriction and dilation of blood vessels. Furthermore, many migraine treatments act as antagonists that block the serotonin receptors (Johnson et al., 1998). Endothelin is a very strong vasoconstrictor that is elevated in the plasma of migraine patients during an attack, implicating this gene or its receptors in migraine

20 susceptibility (Kallela et al., 1998). Interestingly, EDNRA is located on chromosome 4q31, where two migraine loci have been mapped (Bjornsson et al., 2003; Wessman et al., 2002). Genes involved in hormonal function are also good candidates, since variations in hormone levels may influence the risk of migraine, mainly in women (Colson et al., 2006). In line with this, a significant association has been found between the estrogen receptor (ESR1) and migraine (Colson et al., 2004; Oterino et al., 2006).

Table 4. Association studies in common forms of migraine. Gene Population Sample Migraine p- Odds ratio Reference size; cases subtype value (95% CI) vs. controls DRD2 American 129 vs. 121 MA 0.005 n.d. Peroutka, 1997 DRD2 Sardinia 50 families MO1 0.004 n.d. Del Zompo, 1998 DBH Australian 177 vs. 182, MA/MO 0.019 n.d. Lea, 2000 82 families MA/MO 0.035 n.d. DRD4 Italian 194 vs. 117 MO 0.0002 2.49 (1.5-4.0) Mochi, 2003 ACE Italian 302 vs. 201 MO 0.044 n.d. Paterna, 2000 ACE Japanese 176 vs. 248 MA 0.01 5.26 (1.7-16) Kowa, 2005 MTHFR Japanese 74 vs. 261 MA 0.0001 6.5 (2.5-16.8) Kowa, 2000 MTHFR Turkish 102 vs. 136 MA/MO 0.015 5.70 (1.2-27) Kara, 2003 MTHFR Australian 270 vs. 270, MA 0.0025 2.54 (1.4-4.7) Lea, 2004 92 families MA 0.039 1.88 (1.0-3.5) MTHFR Dutch 413 vs 1212 MA 0.006 2.05 (1.2-3.4) Scher, 2005 5-HTT Danish 266 vs. 133 MO 0.031 1.68 (1.1-2.7) Ogilvie, 1998 MA 0.011 n.d. 5-HTT German 197 vs. 115 MA 0.006 2.90 (1.3-6.3) Marziniak, 2005 ESR1 Australian 224 vs. 224, MA/MO 0.003 1.96 (1.4-2.7) Colson, 2004 260 vs. 260 MA2 8 x10-6 ESR1 Spanish 367 vs. 232 MA/MO2 0.012 3.24 (1.3-8.1) Oterino, 2006 EDNRA French 140 vs 1039 MO 0.001 0.51 (0.3-0.7) Tzourio, 2001

1Association observed in a subgroup of migraine patients with enhanced dopamine sensitivity. 2Association observed in women only. n.d. = not determined.

Environmental risk factors The genetic component of the common forms of migraine has been estimated to be about 40–60%, which means that approximately one-half of the variation in migraine is explained by genetic factors. The remainder of the variance is most likely due to environmental factors (Gervil et al., 1999; Larsson et al., 1995; Ulrich et al., 1999). Many factors are suspected of triggering individual migraine attacks, although the relevance of most of these precipitating factors is not clear. The migraine triggers are highly variable and depend on the lifestyle and environment of the individual. Many migraine patients are sensitive to stress or sudden absence of stress (“weekend migraine”), lack of sleep, missed meals or even weather changes or high altitude. Migraine patients may also report sensitivity to strong

21 smells or certain foods—in particular red wine, which contains some vasodilating substances, and chocolate, cheese and coffee (Breslau and Rasmussen, 2001).

In women, hormonal changes are considered to be specific migraine triggers. The attacks may be more frequent at the time of menstruation, and commonly become less frequent during pregnancy or menopause (Colson et al., 2006). Following accurate diagnosis of migraine, effective treatment usually involves avoidance of the particular headache triggers and regulation of the individual’s lifestyle, such as regular sleep, diet, and exercise. Many patients also feel relief from migraine attacks by certain treatments, such as relaxation and physiotherapy (Goadsby, 2003).

Mechanism of disease The pathophysiology of migraine involves both neural and vascular mechanisms, but it is not completely known why and how migraine attacks are initiated. Activation of the sensory trigeminovascular system is thought to be responsible for the pain of migraine, whereas cortical spreading depression (CSD) seems to underlie the visual aura symptoms. CSD is characterized by a wave of intense neuronal activity that slowly spreads across the brain cortex, and this is followed by a long-lasting neuronal inhibition (Pietrobon and Striessnig, 2003). Animal models of migraine pathogenesis have suggested that CSD triggers activation of the nociceptive trigeminovascular system, which leads to release of various factors, such as the calcitonin gene-related peptide (CGRP), substance P, neurokinin A, and nitric oxide (NO). These molecules subsequently cause vasodilation and neurogenic inflammation of the pain-sensitive meningeal blood vessels, thus generating a headache (Bolay et al., 2002). Furthermore, the depolarization phase of CSD has been associated with an increase in cerebral blood flow.

The fact that the visual aura precedes the headache in most MA patients supports the idea that CSD is the primary event that activates the trigeminovascular system. Contrasting with this idea is the observation that some MA patients experience aura after their headache, and that MO patients experience no aura at all (Pietrobon and Striessnig, 2003). Thus, other studies have suggested the existence of a central brainstem generator that gives rise to both CSD/aura on the one hand, and vasodilation, neurogenic inflammation and headache on the other. This is referred to as the parallel concept of migraine pathogenesis (Goadsby, 2001). There is a growing body of evidence implicating brainstem activation in the generation of migraine. Recently, PET scans were used to study regional activation of different brain areas in migraine patients after induction of migraine by glyceryl trinitrate administration. It was shown that patients with right-sided pain had significant brainstem activation in the right dorsolateral pons and patients with left-sided pain had activation of the left dorsolateral pons, suggesting that the unilateral pain of migraine results from an asymmetrical brainstem dysfunction (Afridi et al., 2005).

22 In the normal situation, there is an individual threshold to having a migraine attack. Migraine risk genes probably reduce this threshold, and genetically susceptible patients are consequently more sensitive to a variety of migraine-specific triggers. It is thought that the mechanisms generating headache may be similar in healthy individuals and in migraine patients, but that headache is more easily triggered in the patients, due to hyperexcitability and increased responsiveness of the cerebral cortex to external stimuli (Pietrobon and Striessnig, 2003). However, the molecular mechanisms that lead to cortical hyperexcitability remain unknown. One theory is that abnormal release of neurotransmitters, such as increased calcium influx produced by FHM mutations, increases neuronal cortical excitability and makes the brain cortex more susceptible to CSD (Pietrobon and Striessnig, 2003).

Migraine models in mice There are some mutant mouse models that may be useful for investigation of the mechanisms of migraine, in particular mutations in any of the FHM genes identified. Knockout mice that lack the Cacna1a gene showed severe cerebellar ataxia and dyskinetic movements, as well as a rapidly progressive degeneration of the cerebellum. The null mice had balance problems when walking, and 20 days after birth they were unable to walk at all. They often fell and displayed sustained, twisting movements of the trunk and limbs. These neurological defects resulted in death of the mice at approximately 3–4 weeks after birth (Slaugenhaupt et al., 2001). There are also mouse strains that have different spontaneous missense or splice mutations in Cacna1a—such as the tottering, leaner, rolling, and rocker mice. These mice have mild to severe ataxia, and a dramatic reduction in calcium current density (Kors et al., 2002). However, these phenotypes in mice are mainly models for ataxia or epilepsy, and it is unclear whether they are also suitable models for migraine.

Recently, the first Cacna1a knock-in transgenic mouse model carrying a human FHM mutation (R192Q) was generated (van den Maagdenberg et al., 2004). The heterozygous and homozygous mice are viable and breed normally, and do not show any phenotype of epilepsy and/or ataxia. Furthermore, immunohistochemistry showed a normal cerebellar expression pattern of the calcium channel in both wild- type and homozygous mutant mice. Interestingly, the knock-in mice had multiple gain-of-function effects of the calcium channel, including increased calcium current density in cerebellar neurons, enhanced neurotransmission at the neuromuscular junction, and an increased susceptibility of the brain for cortical spreading depression. The increased calcium current density resulted in mutant channels being activated at lower voltages than wild-type channels, and they consequently opened more readily and released more neurotransmitters. The reduced threshold for cortical spreading depression in the knock-in mice was most likely a result of cortical hyperexcitability, due to excessive release of excitatory amino acids.

23 Knockout mice that lack the Atp1a2 gene are severely affected and do not survive after birth (Ikeda et al., 2003). These null mice were found to have impaired re- uptake of neurotransmitters, enhanced neural excitation, and selective neuronal cell death in the amygdala and piriform cortex. Disruption of the sodium/potassium ATPase pump may result in impaired clearance of excitatory amino acids in the extracellular space of the brain, which possibly results in enhanced spontaneous neural activity and excitotoxic neuronal apoptosis in these specific regions. Furthermore, the increased concentrations of excitatory amino acids may facilitate cortical spreading depression. The heterozygous Atp1a2+/- mice were viable and grew normally, although they had several altered features, such as increased fear/anxiety behavior and enhanced neuronal activity. To date, no knock-in mice carrying the human FHM mutations in ATP1A2 have been reported.

Pain insensitivity

Pain insensitivity is a condition in which patients display a life-long lack of response to painful stimuli. Most of these patients find it possible to distinguish between sharp and dull, or hot and cold, but sharp stimuli or extreme temperatures are not felt as painful. When individuals cannot sense acutely painful stimuli, the consequences are devastating—leading to frequent cuts, bruises, fractures, frostbitten extremities, burn wounds, and neuropathic joints. The disorder is particularly a problem in small children, since they do not feel pain from bumps and falls, and they are thus not afraid of jumping from trees and other high objects. Many children also mutilate themselves unintentionally by thumb biting or chewing on their lips and tongue (Axelrod and Hilz, 2003; Dyck et al., 1983; Thomas, 1993).

In most cases of insensitivity to pain, an underlying neuropathy leads to the inability to perceive pain. According to the International Association for the Study of Pain, neuropathy is defined as “a disturbance of function or pathological change in a nerve”. Disorders with reduced pain sensitivity are very rare, since sensory perception is of critical importance for the survival of the organism. Even so, a number of disorders have been identified with pain insensitivity as a major clinical sign, including the hereditary sensory and autonomic neuropathies (HSAN).

Types of HSAN HSAN is a genetically and clinically heterogeneous group of disorders, characterized by loss of pain sensation in combination with other sensory and autonomic abnormalities. These disorders affect the development and survival of autonomic and sensory neurons, and also cause degeneration of peripheral nerves. At least five types of HSAN have been identified, the classification of which is mainly based on their mode of inheritance, pathology, natural history, and neurophysiological and autonomic abnormalities. Although there are specific symptoms characteristic of each HSAN type, differentiation can be difficult due to

24 the rarity of the disorders and some clinical similarities. In recent years, identification of specific genetic mutations for some disorders has aided diagnosis (Axelrod and Hilz, 2003; Dyck et al., 1983; Thomas, 1993). For each of the HSAN types, penetrance is complete but there can be variable clinical expression in different patients (Axelrod and Hilz, 2003).

HSAN I HSAN I is a dominantly inherited and slowly progressive disorder, characterized by distal loss of pain and thermal sensation. The sensory loss begins in and affects the lower limbs to a greater extent than the upper limbs. The trunk, head and neck are usually not affected. Patients may feel spontaneous pain such as burning or shooting pains in the feet and lower legs. Other features of the disease are perforating ulcers of the feet, distal muscle weakness, no sweating in the distal lower limbs, and (in some families) deafness. Intelligence may also be mildly impaired. The onset of the disorder usually occurs in the second or third decade of life. Other functions, such as taste, and touch and pressure sensation are relatively normal (Axelrod and Hilz, 2003; Thomas, 1993). Sural nerve biopsies in patients have shown that there is a severe loss of peripheral unmyelinated C-fibers, and a moderate loss of small myelinated Aδ-fibers that mediate perception of warm, cold and pain (Axelrod and Hilz, 2003). There is also a progressive degeneration of dorsal root ganglia and motor neurons (Dawkins et al., 2001).

In 1996, a genome-wide screen in four Australian families mapped HSAN I to chromosome 9q22.1-q22.3 (Nicholson et al., 1996). Later, this disorder was shown to be caused by mutations in the SPTLC1 gene in 11 HSAN I families (Dawkins et al., 2001), and independently in two other HSAN I families (Bejaoui et al., 2001). SPTLC1 encodes serine palmitoyltransferase, long-chain base subunit-1, which is a biosynthetic enzyme in a lipid pathway. The mutations are associated with increased ceramide synthesis, which triggers massive cell death of neurons in the peripheral nervous system and spinal cord during development. This could thus be a possible mechanism for the neural degeneration seen in HSAN I (Dawkins et al., 2001).

HSAN II HSAN II presents in infancy or early childhood and is not progressive. The disorder is recessively inherited, and many of the cases have consanguineous parents. The worldwide prevalence of HSAN II is low, but clustering of cases in eastern Canada has been reported (Lafreniere et al., 2004). The disorder is generally defined by degeneration of peripheral sensory neurons. Symptoms involve loss of pain, touch, and temperature sensation, as well as impairment of position senses and taste sensation (Axelrod and Hilz, 2003). The sensory loss is predominantly distal, extending from the elbows to the fingertips and from the knees to the toes, but the trunk may also be involved in some patients. Many patients develop painless ulcers in the fingers and feet, which consequently lead to damage to the underlying bone and to spontaneous amputations (Lafreniere et al.,

25 2004). Painless fractures may also occur, as well as deformed joints (Charcot joints), and self-mutilating behavior starting from a few months of age. Furthermore, some patients are mildly retarded, with expressive aphasia. There are various autonomic dysfunctions, such as eating and swallowing difficulties, constipation, and episodic fever. Sural nerve biopsies from patients have revealed an almost complete loss of small myelinated Aδ-fibers, but only a slightly reduced number of unmyelinated C-fibers (Axelrod and Hilz, 2003).

In 2004, a genome-wide screen in two large consanguineous Newfoundland pedigrees mapped HSAN II to chromosome 12p13, and mutations were identified in a novel gene termed “HSN2” (Lafreniere et al., 2004). This gene consists of a single exon, which is nested within an intron of another gene, PRKWNK1. The predicted amino acid sequence identified a potential signal peptide motif, indicating that HSN2 may be a secreted protein. The authors speculated that the protein might be a novel neurotrophic factor that plays a role in the development and maintenance of peripheral sensory neurons. Further work is required, however, since there is no obvious sequence similarity between HSN2 and any known protein (Lafreniere et al., 2004).

HSAN III HSAN III, also called familial dysautonomia or Riley-Day syndrome, is an autosomal recessive and progressive degenerative disorder that begins in infancy or early childhood. The disorder is the most prevalent of the HSAN types and occurs almost exclusively in individuals of Eastern European Jewish descent (Axelrod and Hilz, 2003). In this population, the carrier frequency is as high as 1 in 30, and the incidence of live births is 1/3,600 (Slaugenhaupt et al., 2001). The disorder involves reduced (but not absent) sensitivity to pain and temperature. While bone and skin pain are poorly perceived, sensitivity to visceral pain is normal, such as esophageal irritation or menstrual cramps. Autonomic disturbances are very prominent, and include gastrointestinal dysfunction with vomiting crises and diarrhea, episodic hypertension, cardiac dysfunction, absence of overflow tears, and profuse sweating (Axelrod, 2002; Axelrod and Hilz, 2003). The earliest signs of disease include feeding difficulties in newborns due to poor oral coordination and misdirected swallowing of liquids, often leading to chronic lung disease. Somatic growth is also poor, and by 10 years of age 85% of patients have scoliosis. The disorder is fatal, and only about half of the patients reach adulthood. Sural nerve biopsies from patients have shown a severe reduction in the numbers of unmyelinated C-fibers and small myelinated Aδ-fibers, as well as a slight reduction in the number of large myelinated axons (Axelrod, 2002; Axelrod and Hilz, 2003). Furthermore, the numbers of neurons in the dorsal root and trigeminal ganglia are reduced to about half (Thomas, 1993).

In 1993, HSAN III was mapped to chromosome 9q31-q33 using 26 Eastern European Jewish families (Blumenfeld et al., 1993). Later, disease-causing mutations were found in the IKBKAP gene encoding the IκB kinase complex- associated protein (IKAP) (Anderson et al., 2001; Slaugenhaupt et al., 2001). The

26 most frequent mutation (> 99%) occurred in a splice site, resulting in a drastic reduction in correctly spliced mRNA in the brain and therefore a predicted lack of functional protein. IKAP is a member of a human Elongator complex and is believed to play a role in transcriptional elongation and in general gene-activation mechanisms. It is therefore possible that the HSAN III phenotype may be caused by abnormal expression of genes that are crucial to the development of the sensory and autonomic nervous systems (Slaugenhaupt et al., 2001).

HSAN IV HSAN IV, also referred to as congenital insensitivity to pain with anhidrosis (CIPA), is the second most common type of HSAN. The disorder has an autosomal recessive inheritance, and begins in infancy. The most prominent feature of HSAN IV is absence of, or markedly reduced sweating (anhidrosis) that leads to repeated episodes of high fever, especially in small children. Almost 20% of the children die within the first 3 years of life due to overheating (hyperpyrexia) (Rosemberg et al., 1994). Other characteristics of the disease are a profound and widespread loss of pain sensation resulting in insensitivity to superficial and deep visceral pain, severe self-mutilation of the tongue and lips in small children, multiple bone fractures, Charcot joints, and amputation of fingers or limbs as the children grow older. Temperature sensation is also reduced or absent (Axelrod and Hilz, 2003; Rosemberg et al., 1994). Furthermore, irritability, hyperactivity, and susceptibility to rage are seen in about half of the patients. There can also be severe learning problems or mild mental retardation. Skin biopsies have shown a severe reduction in the number of unmyelinated C-fibers and some loss of small myelinated Aδ- fibers. Although sweat glands can be found in the skin, there are no unmyelinated fibers innervating these glands, which accounts for the severe anhidrosis seen in the patients (Rosemberg et al., 1994).

It has been shown that mice lacking the TrkA gene display a phenotype similar to HSAN IV patients, including loss of responses to painful stimuli, and severe sensory and sympathetic neuropathy (Smeyne et al., 1994). Based on the phenotype of this mouse model, TrkA on human chromosome 1q21-q22 was investigated as a candidate gene for HSAN IV, revealing several different mutations (Indo et al., 1996). TrkA is the receptor for nerve growth factor (NGF), which is important for survival and maintenance of small sympathetic and sensory neurons. At least 37 different mutations in TrkA have been identified, all of which are distributed in the extracellular domain involved in NGF binding or in the intracellular signal- transduction domain (Indo, 2001). Interestingly, a mutation in TrkA has been identified in a patient diagnosed with HSAN V, suggesting that these two disorders may be allelic (Houlden et al., 2001). The patient had distal loss of pain and temperature sensation, as well as severe reduction of small myelinated fibers, but only modest reduction of unmyelinated fibers. It is, however, possible that the patient would have been better classified as having a mild form of HSAN IV rather than HSAN V, considering his impaired sweating, mental retardation and recurrent episodes of high fever during early childhood.

27 HSAN V HSAN V is the rarest of the HSAN types, with only a few cases described. Its phenotypic description thus remains somewhat uncertain. The disease is autosomal recessive, and symptoms can start during the first months after birth. It is characterized by loss of pain sensation and impaired temperature sensation, resulting in repeated tissue damage, painless fractures, and Charcot joints. However, responses to other sensory stimuli are normal, including those to touch, pressure and vibration. There is no impairment of motor function or neurological functions, such as sweating and intelligence. Furthermore, some patients may have ulcers or exhibit self-mutilation. Sural nerve biopsies have shown a severe reduction in small myelinated Aδ-fibers, and some loss of unmyelinated C-fibers (Axelrod and Hilz, 2003).

In 2004, our group mapped a large consanguineous Swedish HSAN V pedigree to chromosome 1p11.2-p13.2, and subsequently found that the disorder was caused by mutations in the nerve growth factor beta (NGFB) gene, encoding the NGF protein (Einarsdottir et al., 2004; see Paper III). NGF is important for the differentiation and maintenance of sensory neurons, which makes it a very interesting target for neurological disorders. The fact that HSAN IV is caused by mutations in the receptor for NGF, TrkA (Indo et al., 1996), is in line with the knowledge that the NGF-TrkA system has a crucial role in the development and function of the nociceptive system.

The NGF-TrkA system Family of neurotrophins Nerve growth factor (NGF) is the first-described member of a family of structurally related proteins known as the neurotrophins, which are required for differentiation and survival of neurons in the developing nervous system. The other members of the family are brain-derived neurotrophic factor (BDNF), NT-4/5, and NT-3 (Bibel and Barde, 2000; Chao, 2003). In addition to their functions in cell survival, the neurotrophins also mediate “higher-order” activities, such as learning, memory and behavior. The biological effects of the neurotrophins mainly depend on their availability, their binding affinities for different receptors, and which downstream signaling pathways are induced (Chao, 2003).

NGF is important for maintenance of cholinergic forebrain neurons, as well as for initiation of axon growth, synaptic rearrangement, dendritic sprouting, and survival of sensory and sympathetic neurons during development (Bibel and Barde, 2000). In the skin of mice, NGF expression begins by embryonic day 11 and continues to rise until day 14 (Davies et al., 1987). Thereafter, the concentration of NGF falls sharply, followed by the death of nearly half of the neurons in the trigeminal ganglion. This process of naturally occurring cell death was the basis for the neurotrophic hypothesis, which states that an excess of developing neurons competes for access to neurotrophic factors that are secreted in limited amounts by

28 the peripheral target tissue (e.g. skin, muscle, glands or neurons). Those neurons that obtain sufficient neurotrophic support survive, whereas those that fail die. This process is thought to serve at least two functions: to ensure that an appropriate number of synaptic contacts are made, and to eliminate inappropriate neuronal projections (Bibel and Barde, 2000; Oppenheim, 1991).

Neurotrophin signaling Different types of neurons require different neurotrophins for survival, depending on the receptors expressed on their membranes. However, the effects of neurotrophins are quite complex; some neurons switch their survival requirements from one neurotrophin to another during development, whereas others depend on several neurotrophins for survival or do not respond to any of the known factors (Johnson and Oppenheim, 1994). Neurotrophins bind to two different classes of structurally unrelated receptors, the Trk receptors and the p75 neurotrophin receptor. The Trk (tropomyosin receptor kinase) receptors belong to the family of receptor tyrosine kinases, and three trk genes have been identified in mammals. The p75 receptor binds all neurotrophins equally well, while TrkA preferentially binds to NGF, TrkB to BDNF and NT4/5, and TrkC to NT3 (Figure 2). However, these specificities are not absolute; for instance, NT3 is also a ligand for TrkA and TrkB, although with a lower affinity (Bibel and Barde, 2000; Chao, 2003). The expression of neurotrophin receptors is highly regulated, for example in subgroups of sensory neurons. P75 is coexpressed with the Trk receptors in many neuronal populations. In the central nervous system, few neurons express TrkA, whereas TrkB is widely expressed. TrkA is expressed by all peptide-containing, small- diameter nociceptive fibers, while the other Trk receptors are predominantly expressed by the large-diameter non-nociceptive afferents (Mogil et al., 2000).

Activation of these different types of receptors leads to opposite effects, in particular survival of neurons or activation of programmed cell death. Typically, Trk signaling stimulates neuronal survival, differentiation, neurite outgrowth, synaptic plasticity and myelination (Bibel and Barde, 2000; Chao, 2003). The p75 receptor has a dual role; it can both facilitate Trk-mediated neuronal survival and promote neuronal cell death. During early development, p75 refines target innervation by eliminating non-preferred neurons, and at later stages it acts as a coreceptor for Trk to enhance affinity and specificity for the neurotrophins (Carter and Lewin, 1997; Nykjaer et al., 2005). This formation of a high-affinity neurotrophin receptor complex is crucial to limit the amount of neurotrophin required for binding. Furthermore, the enhanced specificity of the complex increases ligand discrimination by the Trk receptors, which is especially important for TrkA and TrkB since they bind more than one neurotrophin (Bibel and Barde, 2000). The apoptotic function of p75 is also believed to be important for eliminating damaged cells after various injuries to the nervous system (Beattie et al., 2002).

29 NGF NT3 BDNF NT4/5

TrkA TrkC TrkB p75NTR

Apoptosis Cell survival

Figure 2. Interaction between neurotrophins and their receptors.

Neurotrophin processing Although purified neurotrophins can induce apoptosis via p75, the cellular responses are generally weak and typically require ligand concentrations greater than those known to occur in vivo. Furthermore, Trk signaling is favored in cells that coexpress p75 and Trks, since Trks bind neurotrophins with higher affinity (Chao and Bothwell, 2002; Nykjaer et al., 2005). This raises the question as to how apoptosis via p75 is induced. It has been shown that the precursor forms of neurotrophins (pro-NTs) are biologically active and bind to p75 with a higher affinity than the mature proteins, thus explaining the observations above (Lee et al., 2001). Neurotrophins are initially synthesized as proforms that can be cleaved intracellularly to release mature, secreted proteins. The mature proteins associate tightly as biologically active homodimers (Chao and Bothwell, 2002). The proforms can also be secreted and cleaved extracellularly by specific proteases. Mature NGF promotes cell survival through binding to TrkA, whereas proNGF preferentially induces apoptosis through p75 (Lee et al., 2001). However, not all p75-expressing cells respond to proNGF, which suggests that additional molecules must be involved. Recently, it was discovered that proNGF binds simultaneously to p75 and a coreceptor called sortilin, in order to induce apoptosis (Nykjaer et al., 2004). In the absence of sortilin, proNGF might be cleaved to mature NGF, and thereby stimulate TrkA-mediated neuronal survival. These data suggest that the neurotrophin family is capable of regulating multiple biological processes, depending on the cellular context and the differential processing of the proneurotrophins.

30 NGF mice models Knockout techniques have been used in mice to delete various neurotrophins and their receptors, resulting in dramatic effects on the development of the nervous system as well as on the postnatal survival of the animal. Given the high degree of sequence conservation among neurotrophins of different species, it is likely that they are essential for survival, and lethality in knockout mice is therefore not surprising (Johnson and Oppenheim, 1994). Knockout mice homozygous for Ngf (Crowley et al., 1994), TrkA (Smeyne et al., 1994) or p75 (Lee et al., 1992), all exhibit specific losses of peripheral sensory innervation as well as deficits in pain sensitivity.

Ngf null mice lack almost all of the smallest sensory and sympathetic neurons, corresponding to a loss of all unmyelinated C-fibers and about half of the small myelinated Aδ-fibers in the peripheral nervous system (Crowley et al., 1994). Not surprisingly, the cells that are lost express TrkA, whereas there is no change in the numbers of TrkB- or TrkC-expressing neurons. Mutant mice lacking NGF died within a few weeks of birth, whereas heterozygous mice grew normally and were fertile. Tests of pain sensitivity revealed almost no response to painful stimuli in the homozygous null mice. The heterozygous mice displayed a mild phenotype, with slightly reduced responsiveness to heat-induced pain and loss of neurons in the peripheral nervous system (Crowley et al., 1994). Interestingly, adult heterozygous mice also displayed significant behavioral deficits in memory and learning, as well as reduced cholinergic innervation of the hippocampus (Chen et al., 1997).

Transgenic mice overexpressing NGF in the skin have been generated to investigate the role of NGF in the development of the peripheral nervous system. These mice were found to have a phenotype opposite to that of NGF-/- mice, with increased innervation of the skin and enhanced survival of both sensory and sympathetic neurons. The transgenic mice had about double the number of cells in their trigeminal ganglia compared to the controls, suggesting that the abnormal level of NGF prevented cell death (Albers et al., 1994). Furthermore, pain sensitivity tests showed that the transgenic mice were hyperalgesic, exhibiting lower thresholds for painful mechanical and thermal stimuli (Davis et al., 1993). The probable mechanism for this is that NGF triggers acute peripheral sensitization by a direct action on sensory nerve endings, and also central sensitization by increased release of the neuropeptide substance P and calcitonin gene-related peptide (Mendell et al., 1999).

Deletion of TrkA produces a syndrome in mice that is similar to HSAN IV in children, but most mice die within one month of birth (Smeyne et al., 1994). The null mice had extensive loss of neurons in the trigeminal, sympathetic and dorsal root ganglia of the peripheral nervous system, and reduced numbers of cholinergic basal forebrain projections to the hippocampus and cortex of the central nervous system. Not surprisingly, there was also a severe loss of small-diameter sensory fibers that expressed TrkA. As a result of the severe cell loss, the null mice were

31 insensitive to painful stimuli and heat. Furthermore, after 3 to 4 weeks, most mice developed severe skin ulcerations of their paws and some digits were missing, probably due to self-mutilation.

Adult mice lacking p75 have reduced skin innervation and a massive loss of sympathetic and sensory neurons, which is consistent with a role of p75 in TrkA- mediated neuronal survival. However, newborn null mice were found to have an increased number of sympathetic neurons, suggesting that p75 promotes apoptosis rather than survival during early development. The null mice had a reduced sensitivity to noxious heat stimuli. By 4 months of age, most null mice had also developed severe ulcers in the distal extremities, leading to loss of toenails (Lee et al., 1992). They were viable and fertile, however, and thus displayed a less severe phenotype than both TrkA and Ngf null mice. Later, the p75 null mice were shown to have significant performance deficits in several behavioral tasks, indicating cognitive dysfunction or a general sensorimotor impairment (Peterson et al., 1999). Interestingly, transgenic mice overexpressing p75 show massive neuron death, which is consistent with a role of p75 in promoting apoptosis (Majdan et al., 1997). To date, no spontaneous mutations in p75 have been identified in HSAN patients.

Neurotrophins in human disease The existence of spontaneous mutations in TrkA and NGFB causing HSAN IV (Indo et al., 1996) and HSAN V (Einarsdottir et al., 2004), respectively, indicates that neurotrophins or their receptors may be involved in other complex psychiatric or neurological disorders in humans. One of the few other examples includes a common polymorphism (Val66Met) in BDNF that has been shown to affect memory and hippocampal function in humans, most likely because of abnormal sorting and secretion of BDNF (Egan et al., 2003). The same polymorphism has also been significantly associated with bipolar disorder—a severe psychiatric disease characterized by recurrent episodes of mania and depression—in 283 Canadian families (Neves-Pereira et al., 2002), and with schizophrenia in a Scottish case-control study of 321 patients and 350 controls (Neves-Pereira et al., 2005). Furthermore, an Italian case-control study of 130 sporadic patients with Alzheimer’s disease and 111 controls found that homozygosity for the BDNF Val66 allele conferred an almost twofold increased risk of developing Alzheimer’s disease (Ventriglia et al., 2002). The authors suggested that neurotrophins might be useful therapeutic agents in specific human neurodegenerative disorders.

In line with this, several studies have implicated NGF as a potential therapeutic agent in neurodegenerative disorders linked to aging, such as Alzheimer’s disease (Counts and Mufson, 2005). The reason for this is that NGF is critical for differentiation and survival of basal forebrain cholinergic neurons, the type of cells that degenerates in the aged human brain. Moreover, the precursor form of NGF has been shown to be increased in end-stage Alzheimer’s disease (Fahnestock et al., 2001). Because of lethality in Ngf-/- mice (Crowley et al., 1994), Ruberti et al. generated transgenic mice to study the action of NGF in the adult nervous system

32 (Ruberti et al., 2000). These mice expressed a neutralizing antibody against NGF that was a thousand times higher in adult mice than in newborns, effectively inhibiting the action of NGF in adult animals only. The adult mice had severely affected sympathetic and sensory neurons, greatly reduced basal forebrain cholinergic neurons, and impaired ability in spatial learning behavioral tasks. Later, the same group showed that aged transgenic mice displayed amyloid plaques, neuronal death, cholinergic deficits, and selective behavioral impairment, closely resembling the phenotypic changes found in patients with Alzheimer’s disease (Capsoni et al., 2000). Interestingly, intranasal administration of NGF to the aged transgenic mice has been shown to rescue neurodegeneration (Capsoni et al., 2002), as well as reverting the deficits in recognition memory (De Rosa et al., 2005).

33 METHODOLOGY

A summary of the main methods used in this thesis is given below. All human samples were collected with informed consent. All studies on human subjects were approved by the Medical Research Ethics Committee of Umeå University.

Patient material Migraine For the genome-wide screen of migraine in Paper I, blood samples were collected from all members of a large four-generation migraine family from Västerbotten, Sweden. Genomic DNA was extracted by a simple salting-out procedure (Miller et al., 1988). Frequency and type of headache in these individuals were assessed by a neurologist according to the classification criteria of the International Headache Society (IHS, 1988). Among these individuals, 17 (14 MO, 3 MA) fulfilled all the IHS criteria for migraine with or without aura (called class 1). Another 13 individuals (12 MO, 1 MA) fulfilled all but one of the IHS criteria for migraine with or without aura (e.i. young adults with attacks lasting less than four hours but otherwise fulfilling the criteria). These individuals were considered to be differently affected (called class 2).

In Paper II, questionnaires were sent to 1,909 sporadic migraine patients identified through healthcare centers in Västerbotten, Sweden. Selection of the patients was based on the following criteria: being over 18 years old, having been born in northern Sweden, and having been diagnosed with migraine when visiting the healthcare center. We did not distinguish between MA or MO, since the observed linkage in the original migraine family included both subtypes. Of these individuals, 759 agreed to participate, and genomic DNA was extracted from buccal cheek swabs (Walker et al., 1999). For the association study of EFHC1, a subset of 178 of these patients was analyzed. The mean age at onset in these individuals was 27 years, and about 74% had at least one first-degree relative affected with migraine. As controls when analyzing the frequencies of the rare variants in EFHC1, RHAG and MEP1A, unaffected individuals from the same geographical region were used.

Pain insensitivity For the genome-wide screen of pain insensitivity in Paper III, blood was collected from more than 80 individuals belonging to a large family in Norrbotten, Sweden. DNA was prepared by standard salting-out procedures (Miller et al., 1988). The diagnosis was established by clinical examination of the patients. Three patients were severely affected, with symptoms starting in their early years. A number of cases were mildly affected, but due to the uncertainty of their clinical disease status, they were not included in the genome-wide screen. The individuals analyzed in the genome-wide screen were the three severe cases, and also their parents and siblings.

34 Genealogical studies The ancestry of the original migraine family and the newly-identified migraine family (proband 471, see Figure 1a in Paper II) was traced using the Swedish church records. In Sweden, well-kept population registries have been maintained over centuries, with detailed information about family relationships, age, sex, civil status, birth of children and mortality. This information can be used to construct extended pedigrees reaching over several centuries. The ancestors of the two migraine families originated from different parts of Västerbotten, such as Bygdeå, Sävar, Lövånger, Nysätra, and Hörnefors, during the seventeenth and eighteenth centuries. The genealogical analysis also showed that the two migraine families were related. The program used for building the pedigrees was Cyrillic Pedigree Editor 2.0.

In Paper III, the genealogical information was obtained by Dr. Jan Minde from relatives of the family, genealogists and church records (Minde, 2006). The ancestors of the family originated from southern Finland and immigrated to the Tornio river valley in northern Sweden during the 1600s. The founder of Vittangi village stemmed from this immigrant family. Since the village was small and relatively isolated, consanguinity (marriage between relatives) was common. According to local history, earlier generations of this family had had problems with deformed bones and other neuropathic symptoms (Minde, 2006).

Molecular genetic methods Identification of susceptibility genes for a disease can be done in two main ways. Commonly, a genome-wide scan of the entire genome is performed in related individuals, and then a chromosomal disease region is located through linkage analysis. The genes located within this disease region are called positional candidate genes, and some of these might be selected for mutation analysis to identify the disease-causing variant. The advantages of genome-wide scans are that entirely novel genes can be identified, and no prior knowledge of the disease mechanisms is necessary. Alternatively, if the pathophysiology of the disease is fairly well understood, genes can be selected and tested for association with the disease phenotype based on their function. This is usually performed by a case- control association study in which the frequency of a certain gene variant is compared between patients and suitably matched controls (unaffected individuals who are similar to the patients in terms of ethnicity, sex, age, lifestyle and geographical origin).

In this thesis, genome-wide scans were performed for both migraine and pain insensitivity, followed by analysis of positional candidate genes in the disease regions identified. No association studies were performed on candidate genes in other chromosomal regions.

35 Genome-wide scan The aim of genetic mapping is to find chromosomal regions that are shared between affected relatives. This is done by using genetic markers that are evenly spaced throughout the genome, in order to find a marker that is inherited together with the disease in the pedigree. All genetic mapping methods are based on a biological phenomenon called recombination (crossing over), which is used to determine the genetic distance between two loci (usually marker vs. disease). Crossovers occur between two homologous chromosomes during meiosis. The number of recombinations depends on the distance between two loci; loci that are far apart will recombine more often than loci that are close to each other. The number of recombinations between two loci can therefore be used to derive a statistical measurement of genetic distance between them, or more specifically, to determine whether the genetic marker is close to the potential disease locus.

After genotyping a marker, the likelihood of linkage is calculated between the marker and the disease locus. Statistically, linkage between a marker and the disease locus is evaluated using LOD score analysis, which represents the logarithm of the odds that two loci are linked. The traditional threshold of LOD 3 means that the observed data is 103-fold more likely to arise under a specified hypothesis of linkage than under the null hypothesis of no linkage. According to more strict recommendations, a LOD score threshold of 3.3 corresponds to genome-wide significance (Lander and Kruglyak, 1995). Once a chromosomal region has been linked to the disease, fine-mapping and haplotype analysis is performed in order to define the disease-critical region. This is done by analyzing a large number of markers in the region and defining the minimal common haplotype that is shared by the affected individuals.

Genome-wide scans were performed for migraine in Paper I and for pain insensitivity in Paper III. For each disease, 400 polymorphic microsatellite markers with an average spacing of 10 cM were amplified by multiplex PCR according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). For fine-mapping in regions of interest, extra markers were ordered from DNA Technology (Copenhagen, Denmark). The order of markers and their distances were obtained from the database of the University of California at Santa Cruz (UCSC; http://genome.ucsc.edu/), and from the database of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). In Paper I, markers were analyzed using an ABI 377 DNA sequencer, and genotypes scored using GenoTyper and GenBase software (Applied Biosystems). In Paper III, markers were analyzed using an ABI 3100 DNA sequencer, and scored with GeneMapper 3.0.

For identification of the migraine susceptibility locus in Paper I, two-point parametric LOD scores were calculated for each marker using the LINKAGE package, version 2.51 (Terwilliger and Ott, 1994). The model assumed an autosomal dominant inheritance with 80% penetrance for individuals who fulfilled all criteria for migraine (class 1), and 60% penetrance for the differently affected

36 individuals (class 2). Due to the high population prevalence of migraine, the phenocopy rate was set at 10% for class 1 and 30% for class 2. The disease allele frequency was specified as 0.001 and the allele frequencies at marker loci were evenly distributed over the number of alleles. Children under the age of 19 years who had not developed full migraine at the time of diagnosis were considered to have unknown disease status.

For identification of susceptibility loci for pain insensitivity in Paper III, no LOD scores were calculated because of the low number of individuals genotyped. Instead, a homozygosity mapping approach was employed, where we constructed haplotypes for each chromosome and then looked for regions in the genome where the three severe cases should be homozygous for the same genetic region. The reason for this is that HSAN V is a very rare recessive disease, and the two copies of the mutation in the severe cases can be assumed to originate from the same founder (i.e. the two copies are identical-by-descent). This is further supported by the fact that the parents of the three severely affected patients are all related to each other.

Candidate gene analysis Candidate genes were analyzed after the genome-wide scans had located the chromosomal disease regions for migraine and pain insensitivity, respectively. No candidate genes located outside the disease regions were analyzed. The genes were selected based on having a function and/or expression pattern that was consistent with the disease phenotype. For all genes, the exons and exon-intron boundaries were examined for the presence of disease-associated mutations. For some genes, the promoter region was also analyzed. The exon-intron structures were obtained from the CHIP Bioinformatic Tool SNPper (http://snpper.chip.org/bio/snpper- enter). Primers were designed using the primer3 program (http://www- genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi), and ordered from DNA Technology (Copenhagen, Denmark). DNA fragments were amplified using standard PCR protocols, then labeled using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and separated on an ABI 3100 DNA sequencer (Applied Biosystems). For some genes, the fragments were analyzed by denaturing high-performance liquid chromatography (DHPLC) using the WAVE fragment analysis system (Transgenomic, Santa Clara, CA) instead of sequencing.

Candidate gene analysis for migraine in Papers I and II was performed in at least two affected patients of the family carrying the disease haplotype and two unaffected spouses. If a variant was to be considered disease-associated, it should co-segregate with the disease haplotype and be more frequent in affected individuals than in controls. Candidate gene analysis for pain insensitivity in Paper III was performed for the three severe cases, as well as for their siblings and parents. Since the disease was recessive, we searched for homozygous mutations that should be present on both chromosomes. In Paper II, a case-control association study was performed in the migraine candidate gene EFHC1 to determine whether there was a difference in frequency of the promoter variant

37 between sporadic patients and controls. In total, 178 patients and 193 controls were genotyped by sequencing on an ABI 3100 DNA sequencer (Applied Biosystems).

Genetic markers Microsatellite markers The genetic markers used in linkage analysis are most commonly microsatellite markers, which consist of a variable number of repeats of a few base pairs (2–4 nucleotides), for example dinucleotide repeats (CA)n. Each different repeat length represents a particular allele, and the microsatellites are therefore multi-allelic markers. These markers occur at known chromosomal positions and are chosen to span the genome at a density of approximately 10 to 20 cM, allowing systematic, high-throughput scanning of the entire genome. Usually, complete panels of optimized markers are commercially available. The microsatellite markers are highly polymorphic, which means that multiple alleles are present in a population (although a single individual can only have two different alleles, one on each chromosome). If a marker has many alleles (about 5-10 or more), it is said to be more informative than a marker with few alleles (about 2–4). If all individuals in a population have the same allele of a certain marker, this marker is uninformative and cannot be used for linkage analysis. In such a case, it might be necessary to replace the uninformative marker to avoid leaving a large gap in that region. Extra markers might also be added to increase the resolution in a particularly interesting region. Microsatellite markers are relatively common in the human genome, which makes fine-mapping possible after the initial genome-wide scan has been performed. However, the spacing between the microsatellites is often too large to allow high-resolution fine-mapping, and some regions in the genome have fewer microsatellites.

Single nucleotide polymorphisms One way of increasing the resolution in regions with fewer microsatellites is to use single-nucleotide polymorphisms (SNPs), which is another type of genetic marker. An SNP consists of a variation at a single nucleotide. Most SNPs are bi-allelic (i.e. only two alleles exist), and are therefore much less informative than microsatellite markers. The advantage of SNPs is that they are widely distributed throughout the genome and can be used for high-resolution mapping. It is estimated that an SNP occurs every 1,000 bp on average, but some regions might have a lower or higher number. The total number of SNPs reported in the public database NCBI (http://www.ncbi.nlm.nih.gov/) is about 3 million at present.

Functional methods Splicing assay In Paper II, an in vitro assay was performed to determine whether the intronic variant (located only 20 bp upstream of the splice site) in the migraine candidate gene MEP1A had any functional effect on splicing. Genomic DNA of an unaffected individual and of a patient carrying the mutation were PCR-amplified and cloned

38 into the mammalian splicing vector pSPL3 (Life Technologies). The fragments included exon 4, as well as 145 nucleotides of upstream intronic sequence and 124 nucleotides of downstream intronic sequence. The constructs were used to transfect monkey kidney COS7 cells using electroporation (Gene Pulser Xcell system, Bio- Rad). After 48 h, the cells were harvested, then total RNA was isolated (RNeasy Mini Kit, Qiagen) and cDNA was synthesized using the Superscript II reverse transcriptase system (Life Technologies). After reverse transcription, the reaction products were PCR-amplified and visualized using photographs of ethidium bromide-stained agarose gels.

PC12 differentiation assay In Paper III, the functional significance of the NGFB mutation was examined. The coding region of NGF was PCR-amplified from the genomic DNA of an unaffected individual and from a patient homozygous for the mutation, and cloned into the mammalian expression vector pCI-neo (Promega). The constructs were used to transfect rat pheochromocytoma PC12 cells, and the cells were photographed over four days using a digital camera (DS-U1; Nikon) attached to a phase-contrast microscope (Axiovert 135M; Zeiss). Transfection with empty vector was used as a negative control, whereas adding human recombinant NGF (100 ng/ml; Sigma) to untransfected cells was used as a positive control. The cells from four randomly selected fields were counted, and the percentage of differentiation calculated (cells bearing one or more neurites with lengths at least twice the diameter of the cell body). The experiments were repeated three times. Data are presented as the mean ± standard deviation of the mean. Statistical comparisons were made by student’s t- test, and the significance level was set at p < 0.05.

39 RESULTS AND DISCUSSION

Paper I Genome-wide screen of common migraine In this paper, we reported the results of a genome-wide screen to identify susceptibility genes for migraine with and without aura. The study was performed in a large and well-characterized family with migraine (Figure 3). Migraine is a complex disorder because of complicating factors such as multiple phenocopies, genetic heterogeneity, variable clinical expression, incomplete penetrance, polygenic inheritance and environmental risks. It is therefore hard to identify possible disease genes for migraine. The advantage of this linkage study was that the family originated from an area in northern Sweden where the population is relatively homogenous genetically, as compared to other populations in which the population has expanded mainly due to immigration (Einarsdottir, 2005). Furthermore, since we analyzed a large multi-generation family rather than several small families, we can assume that the migraine trait in this family has most likely been caused by the same genetic defects. This made it easier to identify possible susceptibility loci for migraine in this family.

MO MO MO MO MO MO MA MO MO

MO MA MO MO MO MO MO MO MO MO MA MO MO MO

MA MO MO MO MO MO MO

Figure 3. The pedigree of the Swedish migraine family. MA = migraine with aura; MO = migraine without aura; filled symbols = class 1; half-filled symbols = class 2; white symbols = unaffected.

This family had multiple members affected by MA or MO (defined in Figure 1 and Table 1, Paper I). Some studies have suggested that MA and MO should be considered as separate disorders, but the fact that both forms of migraine segregate in the same family indicate that there may be common underlying susceptibility genes involved. This is supported by several recent studies (Kallela et al., 2001; Nyholt et al., 2004). Thus, all patients of this family were included in the analysis. The patients were either classified as affected according to strict IHS criteria for

40 migraine (class 1), or as differently affected using less stringent criteria (class 2). The differently affected patients fulfilled all but one of the criteria, and were considered to have potentially important migraine symptoms. They were therefore included in the genome-wide screen, although the penetrance was assumed to be lower for this class (60%) than for class 1 (80%). The use of less stringent IHS criteria has been applied to other studies as well, including a genome-wide screen performed by deCODE Genetics (Bjornsson et al., 2003). These authors obtained significant lod scores when using an alternative model with affected females only and a slightly relaxed definition of MO (e.g. by dropping either pain criteria C or D, but not both). Furthermore, a Swedish population study reported that a considerable number of individuals had attacks lasting less than 4 h or longer than 72 h, but otherwise fulfilled all IHS criteria for migraine. The authors suggested that it might be reasonable to disregard the exact duration of migraine attacks when making diagnoses (Dahlof and Linde, 2001). Thus, classification of individuals as simply being affected or unaffected (for genetic studies) does not reflect the important clinical heterogeneity of migraine.

The disease in this family seems to follow an autosomal dominant mode of inheritance, where the migraine phenotype is passed from one generation to the next. In one case only, an unaffected individual had affected offspring. This suggests that there is reduced penetrance for the disease gene or that phenocopies (i.e. false positives) may exist, something that is to be expected in a very common disease such as migraine. In three cases, the spouse of an affected individual also reported migraine symptoms. These individuals may either have represented phenocopies or they may have had a migraine phenotype caused by other disease genes. They were, however, included in the analysis as being affected.

Since the family had a simplified inheritance pattern, we performed a 10 cM parametric genome-wide screen, assuming an autosomal dominant model with reduced penetrance (for details, see the Methods section of Paper I). The initial linkage analysis was performed by a two-point approach, since two-point analysis is less sensitive to map and genotyping errors than are multipoint methods. Significant linkage was found on chromosome 6p12.2-p21.1, with a maximum two- point LOD score of 5.41 for marker D6S452 (see Table 2, Paper I). No other chromosomal regions presented statistical evidence of linkage. After this initial linkage analysis, non-parametric two-point and multipoint parametric analyses were performed on chromosome 6, producing maximum LOD scores of 3.29 and 5.78, respectively. Furthermore, significant LOD scores of 5.09 and 4.96 were also obtained when excluding either class 2 members from the analysis or MA patients (see Tables E1 and E2 in the supplementary section of Paper I). Haplotype analysis of the family showed that a 10-Mb haplotype was inherited with migraine in all affected members of the pedigree (see Figure 1, Paper I). These data convincingly demonstrate the existence of a migraine susceptibility locus on chromosome 6. This was the second genome-wide screen for common migraine to be published, not long after the first study (Wessman et al., 2002). Interestingly, one recent genome- wide screen of 756 Australian migraine twin pairs has replicated the migraine locus

41 on 6p12.2-p21.1 (MERLIN multipoint LOD score = 1.22), further supporting the existence of a genetic factor for migraine in this region (Nyholt et al., 2005).

The 10-Mb region on chromosome 6p12.2-p21.1 contains about 50 known genes and several predicted genes with unknown function. Based on their function and expression pattern, three candidate genes in the disease region were selected for mutation analysis in Paper I, including SLC29A1, CLIC5 and PLA2G7. SLC29A1 has a role in the transport of adenosine (Coe et al., 1997; Griffiths et al., 1997), which is a powerful vasodilator that participates in the control of cerebral blood flow and could thus be involved in the pathophysiology of migraine (Guieu et al., 1998). CLIC5 is a chloride channel that has a role in the maintenance of electrolyte balance and in the control of membrane potentials (Berryman and Bretscher, 2000). Since dysfunction of ion transport is involved in familial hemiplegic migraine, CLIC5 is a good candidate gene. PLA2G7 is associated with activation of proinflammatory cells and alteration of vascular permeability (Tjoelker et al., 1995), and has been implicated in diseases such as asthma (Kruse et al., 2000), stroke (Hiramoto et al., 1997) and coronary heart disease (Packard et al., 2000). Sequence analysis of these genes did not reveal any disease-specific mutation, although we could not exclude the possibility of mutations in regulatory regions or introns. SLC29A1, however, can be excluded – solely based on its physical location, since it was found to be outside the disease region after fine-mapping in Paper II. So far, none of the other genome-wide screens for common migraine have reported mutational analysis of any candidate genes (Bjornsson et al., 2003; Cader et al., 2003; Nyholt et al., 2005; Soragna et al., 2003; Wessman et al., 2002).

To conclude, the linkage study in Paper I presents significant evidence for a new migraine susceptibility locus on chromosome 6p12.2-p21.1, as well as further genetic heterogeneity of the disease. Furthermore, three candidate genes in the disease region could be excluded as a likely cause of disease.

42 Paper II Fine-mapping and candidate gene analysis of migraine We have shown that a 10-Mb region on chromosome 6 is significantly linked to migraine with and without aura in a Swedish family (described in Paper I). Continuing this work, we next fine-mapped the disease region and analyzed additional candidate genes.

Fine-mapping was achieved by analyzing multiple SNPs in the flanking regions of the disease haplotype, thereby reducing it to 8.5 Mb. This region is still quite large for selection of candidate genes, but no further refinement could be made. The only way to reduce the disease region was to include other affected family members who could be used to identify more recombinations. Since all close relatives of the family had already been included in the study, we searched for related families in sporadic migraine cases in northern Sweden (Figure 4). These individuals were screened for the presence of two rare polymorphisms in the MEP1A and RHAG genes, segregating with the disease haplotype (see Figure 2, Paper II). Patients carrying either or both of these polymorphisms might be related to the original migraine family, and therefore might share parts of the disease haplotype.

Patients who have received migraine diagnosis when visiting healthcare centers in northern Sweden (n = 1.909)

Questionnaire sent to Patients not born in sporadic migraine patients northern Sweden or moved in northern Sweden outside Sweden (n = 1.416) (n = 493)

Correct address Unknown address (n = 1.393) (n = 23)

Informed consent given Did not want to No response and saliva sample donated participate (n = 612) (n = 759; 54%) (n = 22)

Patients with no Patients with MEP1A or polymorphisms RHAG polymorphisms (n = 755) (n = 4)

Figure 4. Overview of the steps taken in screening of the sporadic migraine patients.

43 The screening identified one patient carrying both polymorphisms on the same disease haplotype as the original migraine family, and three patients carrying only the MEP1A polymorphism on a completely different haplotype background. Genealogical analysis showed that the double carrier was closely related to the original migraine family (see Figure 1b, Paper II). An extended pedigree of these two families is shown in Figure 5. No further restriction of the disease region could be made, since no recombinations were identified in the new family.

? ? ? ? ? ? ? ? ? ?

- + - + + + - - + - - - + - - + - + + - + - + - - -

- + - - - + + - - - + ------+ + - - + - - - - + - + -

+ + - - - + + + - + + -

Figure 5. Extended pedigree of the two related migraine families, showing individuals carrying (+) or not carrying (-) the disease haplotype. The arrow indicates the double carrier in the newly identified family.

Sequence analysis of six possible migraine candidate genes in the disease region revealed several polymorphisms segregating with the disease haplotype (see Table 1, Paper II). Three of these polymorphisms were previously unknown; they were located in the promoter of EFHC1, at the 3´end of RHAG, and in intron 3 of MEP1A (see Figure 2, Paper II). Mutations in EFHC1 have been found to be the cause of juvenile myoclonic epilepsy (Suzuki et al., 2004), which had previously been mapped to the same region as migraine on chromosome 6p21.2-p11 (Liu et al., 1995). Since migraine and epilepsy share certain features, it seems likely that genes involved in epilepsy are good candidates for migraine as well (Bigal et al., 2003). EFHC1 encodes a protein containing a Ca2+-sensing EF-hand motif, the expression of which overlaps with that of a calcium channel, CACNA1E. Furthermore, there is evidence that EFHC1 may enhance calcium influx through that channel and stimulate apoptosis. This is interesting, considering that another calcium channel, CACNA1A, is involved in familial hemiplegic migraine (Ophoff et al., 1996). Subsequent analysis of the EFHC1 polymorphism in a migraine case- control material revealed no significant association (OR = 1.03; 95% CI: 0.51– 2.06), suggesting that this gene is not involved in migraine in this family. It is possible that the number of patients (n = 178) and controls (n = 193) in this cohort

44 was too low to detect any significant association. However, there was not even a tendency to association; the results were strongly non-significant statistically using several different models, such as separation of sexes or only including participants with a family history of migraine.

Analysis of the RHAG and MEP1A polymorphisms in a large control material identified no individuals carrying the MEP1A polymorphism and only one individual carrying the RHAG polymorphism, indicating that these variants are specifically associated with the disease haplotype. RHAG encodes a rhesus blood group-associated glycoprotein that is expressed in red blood cells. Defects in RHAG cause the Rh-deficiency syndrome, which is a form of chronic hemolytic anemia in which the red blood cells have an abnormal morphology, an increased osmotic fragility, and an altered ion transport system (Cherif-Zahar et al., 1996). RHAG may also be important for promoting export of ammonium ions that are accumulated in red blood cells (Westhoff et al., 2002), which is interesting considering the link between abnormal ion transport and hemiplegic migraine. MEP1A encodes the metalloendopeptidase meprin A which is expressed mainly in the kidney and intestine. This enzyme hydrolyzes peptides such as bradykinin, angiotensin, substance P and neurotensin, as well as various hormones and growth factors. Of special interest in migraine pathophysiology are substance P, since it is one of the vasoactive neuropeptides released following activation of the trigeminovascular system (Pietrobon and Striessnig, 2003), and neurotensin, since it has been shown to induce a rise in intracellular Ca2+ concentration and a long- lasting desensitization in a subset of neurons (Trudeau, 2000).

Since the intronic MEP1A variant was located only 20 bp upstream of the exon- intron junction, we hypothesized that it might affect the splicing process. Functional analysis of the MEP1A polymorphism in a cellular model, however, revealed no significant effect on splicing efficiency of this variant. Furthermore, relatives of one of the sporadic migraine patients carrying the MEP1A polymorphism did not have migraine (see Figure 1c, Paper II), suggesting that this variant alone is not enough to cause migraine. It is possible that the MEP1A and RHAG polymorphisms interact, and thereby confer an increased risk of migraine in this family. The concept of interaction of disease genes has been supported by several studies, including a recent Australian case-control study of 270 migraine patients and 270 controls. The authors showed that combining the risk genotypes for MTHFR (677 T/T) and ACE (ID/DD) increased migraine susceptibility by more than double (p=0.018) (Lea et al., 2005). An example of this in a different disease would be polymorphisms in the PPARG and PPP1R3A genes, which have been shown to confer only a modest risk when alone, but which result in severe insulin resistance when combined (Savage et al., 2002). This is often the case in complex diseases such as migraine: one cannot expect to find a single mutation in a gene that explains most of the disease. The disease is more likely to be caused by several subtle gene changes that only have clinical consequences under certain environmental triggers. Furthermore, a risk gene in one population does not

45 necessarily constitute a risk in another population, due to differences in genetic susceptibility in these populations.

To conclude, the study in Paper II refined the migraine disease region to 8.5 Mb and excluded four candidate genes as the likely cause of disease—among them the epilepsy disease gene EFHC1. Two rare polymorphisms in the MEP1A and RHAG genes were found to be specific for the disease haplotype, and it is possible that they act in combination to increase migraine susceptibility in this family.

46 Paper III Genome-wide screen of pain insensitivity In Papers I and II, we showed that an 8.5-Mb region on chromosome 6 harbors a susceptibility gene for common migraine, although the exact identity of this gene remains unknown. We then proceeded to identify additional pain-related genes by analyzing a multi-generation consanguineous family with pain insensitivity.

The family originates from the small village of Vittangi in northern Sweden, where the population is relatively homogenous genetically. The family includes three severe cases with an almost complete loss of deep pain perception and impaired temperature sensation, and multiple cases with a less severe phenotype. The severely affected patients do not feel pain from bone fractures and joints, which leads to destroyed joints (Charcot joints) and multiple fractures in childhood (Figure 6). Sural nerve biopsies have revealed a severe reduction of unmyelinated C-fibers and a moderate loss of small myelinated Aδ-fibers (Minde et al., 2004), which is similar to the pattern observed in HSAN I and HSAN IV. However, clinically, the patients fit best into the rare HSAN V category, since most of their other neurological functions, including sweating and intelligence, are normal. Furthermore, the patients have no problems with spontaneous shooting pains, episodic fevers, and self-mutilating behaviour, which are often reported in the other disorders. For more details of the severe cases, see the clinical description in Paper III or the report by Minde et al. (Minde et al., 2004).

Figure 6. Three patients homozygous for the NGFB mutation, presenting with severe neuropathic joints. Case 1 is a 39-year old man (left), case 2 is a 21-year old woman (middle), and case 3 is a 13-year old boy (right). Photographs, courtesy of Dr. Jan Minde, Department of Orthopaedics, Gällivare Hospital, Sweden.

47 We assumed that the trait in this family was most likely caused by a single gene defect, since all patients shared a common ancestor 8–10 generations back, and the inheritance seemed to be simple Mendelian. Furthermore, we assumed a recessive model of inheritance for the severe phenotype, since there was extensive consanguinity in the family and all parents of the severe cases were related (Figure 7). Due to the difficulty of assigning a correct disease status for all but the three severe cases, only these were considered affected in the genetic analysis. We performed a genome-wide screen, looking for homozygous regions identical-by- descent in the three severe cases, thereby identifying an 8.3-Mb haplotype on chromosome 1p11.2-p13.2 (see Figure 1, Paper III). This region presented several potential candidate genes, of which the most interesting was nerve growth factor beta (NGFB) because of its involvement in the differentiation and maintenance of sensory neurons. Furthermore, mutations in TrkA, the receptor for NGF, have been shown to cause HSAN IV (Indo et al., 1996).

5 6

3 1 2

Figure 7. Extended pedigree showing several consanguineous loops. Individuals 1–3, representing severe juvenile cases homozygous for the NGFB mutation, were included in the genotyping in Paper III. Individuals 4–6, representing heterozygous milder cases with an adult onset of the disease, have been described in more detail elsewhere (Minde et al., 2004).

48 Subsequent mutation analysis of NGFB revealed a missense mutation, C661T, in exon 3 of the gene (see Figure 2A, Paper III). This mutation had several features suggesting that it was pathogenic. First, the point mutation was homozygous in all severe cases, which is consistent with a recessive transmission of the disease. Secondly, the mutation was not present in 100 control individuals or unaffected members of the family, implying that it is not a neutral polymorphism. Thirdly, the mutation caused a very drastic amino acid change (R211W), replacing a basic arginine with a non-polar tryptophan at position 100 of the mature protein (see Figure 2B, Paper III). Finally, the mutated amino acid was completely conserved among different neurotrophins, and also NGFs from distantly related species (see Figure 2C, Paper III).

Interestingly, a number of milder cases in the family turned out to be heterozygous for the missense mutation, suggesting a gene dosage effect. A phenotype-genotype correlation study of three severe homozygous cases and three milder heterozygous cases in the family have been reported (Figure 7, individuals 1–3 and 4–6, respectively) (Minde et al., 2004). The symptoms of the homozygous cases appeared in early childhood and involved multiple painless fractures and joint destructions. The symptoms of the heterozygous cases started in adult life and were less severe, resulting in Charcot joints later in life. Furthermore, nerve biopsy of patient 4 revealed a less severe reduction of nerve fibers. This indicates that several individuals in the family have symptoms representing a milder expression of the disease. However, the parents of the three homozygous patients were all heterozygous carriers of the mutation, but presented no symptoms, indicating that further studies are required to determine the clinical effect in heterozygous carriers. An extensive phenotype-genotype correlation study is currently under way, in which several family members are investigated (by Minde and co-workers).

Another interesting notion is that the NGFB mutation identified in Paper III causes a less severe phenotype than mutations in the TrkA gene, since HSAN IV is characterized by profound loss of pain sensitivity, self-mutilation, widespread anhidrosis and mental retardation (Indo et al., 1996). We first suspected that the ability of mutant NGF to activate TrkA was not completely abolished, but rather that the interaction with the p75 receptor might be affected. This was supported by the fact that p75 knockout mice (Lee et al., 1992) have a less severe phenotype than mice lacking either NGF (Crowley et al., 1994) or TrkA (Smeyne et al., 1994). However, recent results in our laboratory suggest an alternative hypothesis, which is presented in Paper IV.

In conclusion, we have identified a pathological mutation in the nerve growth factor beta (NGFB) gene on chromosome 1p11.2-p13.2 that leads to a complete loss of deep pain perception in a large consanguineous family from northern Sweden. This is the first disease gene for HSAN V to be identified. It is also the first time NGF has been found to be involved in a human neurological disorder. Thus, this disease provides an interesting model for the study of pain mechanisms and development of NGF-dependent neurons in the nervous system.

49 Paper IV Functional analysis of the NGFB mutation In Paper III, we showed that a mutation in the NGFB gene on chromosome 1 causes the pain insensitivity syndrome in HSAN V patients. We then proceeded to investigate the functional consequences of this mutation and the underlying mechanisms.

To determine whether the R211W mutation would affect the biological activity of NGF, we used the well-characterized PC12 cell model for studying neuronal differentiation. These cells express both TrkA and p75, and can differentiate into sympathetic neuron-like cells in response to NGF treatment (Figure 8). After transfecting the cells with either the wild-type or mutant NGF plasmid, a significant difference in the number of differentiated PC12 cells was observed. The wild-type NGF induced differentiation of about 90% of the cells after 4 days, which was similar to the positive control. For the mutant NGF, however, less than 5% of the cells were differentiated after 4 days (see Figure 1, Paper IV). Interestingly, a tendency toward increased differentiation was seen 4 days after transfection with the mutant NGF (4.4%) compared to the negative control (2.0%), which would indicate that a small amount of the mutant NGF that is secreted is at least to some degree biologically active. Taken together, these results suggest that the mutation is a loss-of-function alteration that dramatically reduces the function of NGF.

A B

Figure 8. PC12 cells three days after transfection. (A) Differentiated cells transfected with wild-type NGF. (B) Undifferentiated cells transfected with mutant NGF.

To determine whether the inability of mutant NGF to differentiate PC12 cells was due to decreased expression, PC12 cells as well as non-neuronal COS-7 cells were transfected with the wild-type or mutant NGF plasmids, and then the expression of NGF were analyzed by western blot. We observed normal secretion of mature wild- type NGF into the medium of both PC12 and COS-7 cells, while mutant NGF was barely detectable (see Figures 2A and 2B, Paper IV). The same expression pattern

50 was also observed with ELISA, which showed that the amount of wild-type NGF secreted by PC12 cells 3 or 4 days after transfection was about 100 times higher than the amount of mutant NGF, and the amount of wild-type NGF secreted by COS-7 cells 3 days after transfection was about 125 times higher than the mutant NGF.

The western blot and ELISA results indicate that the mutation causes a defect in the synthesis/processing of the protein, resulting in reduced secretion. However, another possibility may be that the protein is unstable and thereby rapidly degraded before it can be detected. To investigate the processing and secretion pattern of wild-type and mutant NGF, a pulse-chase study was performed in COS-7 cells. The pulse-chase showed that the processing of mutant proNGF was defect, resulting in intracellular accumulation of proNGF and almost no secretion of mature NGF. In the wild-type, however, the relative amounts of cellular proNGF decreased over time, while the processed form of cellular mature NGF increased and was efficiently secreted into the medium (see Figure 3, Paper IV). These results indicate that the functional effect of the R211W mutation was defective processing, at least in the cell system we have tested.

Interestingly, a common polymorphism (Val66Met) in the neurotrophic factor BDNF affects memory and hippocampal function in humans (Egan et al., 2003), and has also been significantly associated with a number of neuropsychiatric disorders, such as schizophrenia (Neves-Pereira et al., 2005), Alzheimer’s disease (Ventriglia et al., 2002), and bipolar disorder (Neves-Pereira et al., 2002). The Met polymorphism reduces the regulated activity-dependent form of BDNF secretion in neurons, without affecting the constitutive secretion. Furthermore, Met-BDNF forms aggregates that accumulate in the cell body and may cause defects in sorting and secretion at synapses (Egan et al., 2003). NGF can be secreted via both the constitutive pathway (endogenously production of NGF) and regulatory activity- dependent pathway (requiring specific triggering mechanisms), depending on the cell type. COS-7 cells secrete NGF by the constitutive pathway, while PC12 cells and other neurons utilize both constitutive and regulated secretion. It is possible that the R211W mutation may affect either the constitutive or the regulatory pathway.

The effect of the R211W mutation is most probably defective processing/secretion. It is possible that the accumulation of mutant proNGF may also contribute to the phenotype, since proNGF is known to induce apoptosis by binding to p75 (Lee et al., 2001), and over-expression of p75 in mice results in massive neuron death (Majdan et al., 1997). Another possibility is that the low expression of mature NGF in the mutant results in a lack of survival signals and thereby increased neuronal death. This hypothesis is consistent with the situation in NGF knockout mice, which have markedly reduced numbers of small sensory and sympathetic neurons (Crowley et al., 1994). Moreover, the null mice have almost no response to painful stimuli, which is also observed in HSAN V patients. The results therefore suggest

51 that the sensory fiber loss in the patients is most probably due to partial loss of function of NGF rather than over-expression of proNGF.

Interestingly, the HSAN V patients who are homozygous for the mutation are viable and can function quite normally, while homozygous knockout mice lacking NGF die a few weeks after birth (Crowley et al., 1994). This suggests that the R211W mutation does not completely abolish the biological function of NGF, or that there must be other mechanisms in humans to compensate for the loss of NGF. As discussed above, the R211W mutation may also result in a more complex phenotype due to the accumulation of proNGF known to have biological activity. The failure of mutant NGF to induce differentiation of PC12 cells in our experiments suggests that the function of the protein is lost. However, we cannot exclude the possibility that mutant NGF might still have some activity in vivo, since low levels of secretion could be detected. We have thus initiated a project to create knock-in mice carrying the human R211W mutation. These mice will then be analyzed with respect to the development of the nervous system, perception of pain, as well as cognitive function. It will be interesting to see if these mice are viable and fertile, which would indicate that the human R211W mutation does not result in a complete loss of function of NGF.

Recently, a considerable interest has been gained for using NGF antagonists as a therapeutic approach in many pain disorders. The reason for this is that NGF has a crucial role in the generation of pain in adults, and that its expression is increased in injured and inflamed tissues. In a recent review, administration of NGF was described to provoke pain and hyperalgesia (an increased response to painful stimuli) in both animals and humans, while inhibition of NGF with anti-NGF antibodies reduced pain in animal models of many acute and chronic pain states without producing any adverse effects (Hefti et al., 2006). In view of this, the mutation we have identified in NGF is an interesting model to study the lack of pain perception in the HSAN V patients at the molecular level, as well as basic pain mechanisms.

In conclusion, we have shown that the R211W mutation results in a loss of function of NGF. This is most probably due to defective processing/secretion of mutant proNGF to NGF, resulting in accumulation of intracellular proNGF and markedly reduced secretion of mature NGF. Further studies are needed to determine whether mutant proNGF or NGF has some activity in vivo, or if there are other systems in the HSAN V patients that can compensate for a lack of NGF.

52 CONCLUSIONS

The overall aim of this thesis was to identify genetic factors involved in pain, and to gain an understanding of the different mechanisms involved in pain. The main conclusions are:

I. A genome-wide screen identified a susceptibility locus for common migraine within a 10-Mb region of chromosome 6p12.2-p21.1.

II. Fine-mapping reduced the migraine disease region to 8 Mb.

A large population screening was performed in sporadic migraine patients, allowing identification of an additional family that shared the same disease haplotype and that was closely related to the original migraine family.

The combination of two novel polymorphisms in the RHAG and MEP1A genes does not occur in any control individuals and might act in combination to increase the risk of migraine.

III. A genome-wide screen identified a susceptibility locus for HSAN V within an 8.3-Mb region of chromosome 1p11.2-p13.2.

Mutation analysis of candidate genes revealed a homozygous missense mutation in the nerve growth factor beta (NGFB) gene, replacing the amino acid arginine with tryptophan (R211W) in a highly conserved region of the protein.

IV. Mutant NGF cannot induce differentiation of PC12 cells as effectively as wild-type NGF, indicating that the mutation affects the biological function of the protein.

The mutation causes defective processing/secretion of proNGF, resulting in intracellular accumulation of proNGF and markedly reduced secretion of mature NGF.

53 CONCLUDING REMARKS

The sensation of pain acts as the body’s early warning system and is crucial in order to protect organisms from dangerous stimuli and injury. Loss of pain is devastating and occurs in several conditions of insensitivity to pain, leading to frequent injuries and accidental self-mutilation. There are also conditions, such as migraine, in which pain is maladaptive and becomes persistent and chronic. In its pathological state, pain is no longer biologically useful, but a burden to both individuals and the public welfare—with high costs in terms of lost work productivity and drain on the medical system.

The signaling systems and mechanisms that cause pain are highly complex and diverse. The outcome of pain is most probably influenced by the interaction of many genes, each with a minor effect, and environmental factors, rather than coming from a few genes with major effects. It is therefore a challenge to identify which of the over 30,000 genes in the mammalian genome are involved in the mediation of pain. Genetics, such as linkage or association studies, is a powerful tool for identification of pain-related genes. Linkage analyses follow familial inheritance patterns and can identify entirely novel pain genes, while association studies compare allele frequencies of a certain pain candidate gene in different groups of individuals, such as patients and controls. The identification of genes contributing to pain is an important step towards our understanding of the underlying biological processes generating pain. Every gene identified in different pain conditions is important, even though the disease is very rare, since this gives more insights into pain genetics and may reveal candidate genes in metabolic pathways that were not previously implicated in pain pathogenesis. This knowledge can then be used to develop new diagnostic methods and improved treatments that are hopefully more effective and produce less adverse effects than the currently used drugs. As both the population and the mean age increase, the incidence of chronic painful diseases such as cardiovascular disease, cancer, and rheumatoid arthritis will increase, increasing the need for more and better analgesic drugs (“painkillers”).

The work of this thesis will hopefully contribute to increased knowledge of the mechanisms involved in pain. For migraine, we have shown that there is a possible disease gene located on chromosome 6. For HSAN V, we have shown that the pain insensitivity is caused by a defect in the nerve growth factor beta gene. Since inhibition of NGF seems to block pain sensation, this disease provides an interesting model for the study of pain mechanisms.

54 POPULÄRVETENSKAPLIG SAMMANFATTNING

Syftet med den här avhandlingen var att identifiera ärftliga faktorer för smärta, och därmed bidra till att öka kunskapen om vilka mekanismer som leder till smärta. Smärta fungerar som kroppens varningssystem för att skydda oss från farliga faktorer och skador, som t.ex en varm spisplatta eller ett vasst föremål. Oförmåga att känna smärta är därför förödande och förekommer i sällsynta fall av smärtokänslighet, vilket leder till upprepade skador och benbrott, men även s.k. självstympning hos små barn som kan bita av sig tungan eller fingertopparna utan att känna smärta. Det finns också sjukdomar, som t.ex migrän, där smärta uppkommer utan synbar anledning, och därmed blir ett patologiskt problem. Miljontals människor över hela världen lider av kronisk eller återkommande värk, som innebär svåra konsekvenser för både individen och hans/hennes familj samt samhället i form av höga kostnader för nedsatt arbetsförmåga och olika mediciner och behandlingar. Kunskap om vilka gener som är inblandade i smärta kan på sikt användas för att utveckla bättre metoder för smärtlindring, som förhoppningsvis är mer effektiva än tidigare läkemedel och ger färre skadliga biverkningar. Det faktum att både befolkningen och medellivslängden ökar medför att kroniska sjukdomar som hjärt-kärlsjukdomar och reumatism också ökar, och detta kommer att leda till ett större behov att smärtlindrande preparat i framtiden.

De biologiska mekanismer som leder till smärta är mycket komplicerade och beror mest troligt på samverkan mellan många olika gener och miljöfaktorer. Dessutom är smärta högst subjektivt och påverkas av individens genetiska bakgrund, tidigare erfarenheter, fysiologiska tillstånd, förväntningar och kulturella miljö, vilket ytterligare komplicerar fenomenet. Därför uppvisar olika individer stor variation i sin förmåga att tolerera smärta och reagera på olika smärtstillande preparat, samt hur mottagliga de är för att utveckla kroniska smärtsjukdomar. En viss del av dessa skillnader är ärftliga, och beror på genetiska variationer i gener som har betydelse för smärta. Man tror att dessa variationer endast leder till små genförändringar, d.v.s. att ensam har en ändrad gen ingen eller liten effekt, men tillsammans med många andra förändrade gener kan det leda till sjukdom. Ärftligheten är ofta ganska hög i sjukdomar där smärta är ett framträdande symptom, som t.ex migrän där man uppskattar att 50% av sjukdomen kan förklaras av gener.

I mitt avhandlingsarbete har jag studerat två stora familjer med olika smärtsymptom, migrän samt smärtokänslighet. Migränfamiljen kommer från Västerbotten och består av 17 individer med migrän och 13 individer med en alternativ form av migrän. Migrän är en av de vanligaste kroniska sjukdomarna i västvärlden, och drabbar så många som var femte kvinna och var tionde man. Enbart i Sverige är över en miljon människor drabbade. Sjukdomen kännetecknas av återkommande migränanfall som varar från några timmar till flera dagar, med olika grad av påverkan hos olika individer. Vanliga symptom är stark känslighet för ljus och ljud, blekhet, illamående, samt hos många även aura (ögonflimmer eller synfältsbortfall). Den som har lindrig migrän kan ha 1-2 anfall per år medan en svårt drabbad kan få upp till 3-4 anfall i veckan. Vad som utlöser ett migränanfall

55 varierar från individ till individ, men faktorer som stress, oro och dålig sömn kan ligga bakom, liksom hormonella variationer (särskilt hos kvinnor) och vissa födoämnen. För att ta reda på den genetiska bakgrunden till migrän analyserade vi genetiska markörer jämnt utspridda över alla kromosomer. Om någon kromosom- region är inblandad i sjukdomen kommer den att vara överrepresenterad hos de sjuka jämfört med de friska individerna. I den första studien (Paper I) upptäckte vi att ett område på kromosom 6 var kopplat till migrän. I den andra studien (Paper II) undersökte vi detta område närmare för att försöka hitta den eller de gener som orsakade sjukdomen. Vi hittade tre genvariationer som inte tidigare var kända, och som följde sjukdomsregionen. Den första genen, EFHC1, var extra intressant eftersom den tidigare visats vara inblandad i epilepsi, en neurologisk sjukdom som har många likheter med migrän. Men vid en noggrannare undersökning av denna gen i ett stort patient-kontrollmaterial kunde vi inte se någon skillnad mellan friska och sjuka individer. Däremot fann vi att kombinationen av de två andra genvariationerna i RHAG och MEP1A inte förekom hos någon av de över 200 friska kontrollpersoner som testades, vilket tyder på att de kan ha betydelse för att öka risken för migrän. Vi undersökte också om dessa variationer förekom hos någon av 759 sporadiska migränpatienter som samlats in via vårdcentraler i Västerbotten. Endast en individ hade båda dessa variationer, och släktforskning visade att hon var nära släkt (fyrmänning) med den ursprungliga migränfamiljen.

Den andra sjukdomen som jag har studerat, smärtokänslighet av typen HSAN V, är mycket ovanlig och relativt få fall finns beskrivna. Familjen kommer från en liten by i Norrbotten, och består av tre svåra fall och flera mildare fall. Symptomen hos de svåra fallen börjar redan i barndomen, och kännetecknas av nedsatt temperaturkänslighet och oförmåga att känna djup smärta. Detta gör att individerna drabbas av många benbrott och ledskador, eftersom de felbelastar lederna och inte känner när det gör ont. För att hitta den genetiska orsaken till denna sjukdom, analyserade vi även här genetiska markörer utspridda över alla kromosomer. I den tredje studien (Paper III) hittade vi en region på kromosom 1 som delades av de tre svåra fallen. Därefter undersökte vi vilken gen som kunde vara inblandad, och upptäckte en mutation i NGFB, som orsakar att en aminosyra i en viktig del av proteinet NGF byts ut till en annan aminosyra. I den fjärde studien (Paper IV) undersökte vi hur proteinet påverkas av denna genförändring. Vi upptäckte att processningen av en pro-form av proteinet inte fungerade som det skulle, vilket ledde till att denna form ackumulerades inne i cellen och inte kunde utsöndras till omgivningen i samma grad som det normala proteinet. Detta protein, NGF (nerve growth factor), är en tillväxtfaktor som har flera viktiga funktioner i nervsystemet, bl.a. för överlevnad av nervceller under den embryonala utvecklingen, samt för att mediera smärtsignaler hos vuxna individer. Internationella studier med olika djurmodeller har visat att tillfällig inhibering av NGF kan lindra smärta, vilket gör att den mutation vi hittat är ett mycket intressant modellsystem för att studera de mekanismer som leder till smärta.

56 ACKNOWLEDGEMENTS

During my work I have come to know many people that in one way or another have contributed to this thesis.

First of all, I would like to thank my supervisor Monica Holmberg for introducing me to human genetics. Your enthusiasm for this project and encouragement for all experiments (even the ones that didn’t work out as supposed to) have truly been an inspiration. Thanks also for always having the time to discuss with me and for believing in my abilities. It has really been fun to be a part of your group.

My co-supervisor Dan Holmberg, thanks for taking me into your group when Monica was not at the University, and for all help with critical reading of my manuscripts (even though I always had to remind you to read them). My co-supervisor Lars Forsgren, thanks for your great knowledge of neurology and for always being interested whenever we have wanted to include more patients in the study. Gösta Holmgren (in memory), thanks for being a nice person and having a true interest for genetics.

Thanks to the present members of group MH, Elin Larsson and Regina Kuma, for sharing the office with many laughs and for your good sense of humour. The lab is never boring (or safe…) when you are around! Regina – remember to use a real solarium instead of the UV-lamp next time you try to get a suntan. Elin – too bad the isotope didn’t show up that Friday night… Thanks also to past members of group MH, Anna-Lena Ström and Jenni Jonasson (nowadays also a good neighbour in Holmsund) for helping me when I was new in the group.

Thanks to all past and present members in Dan Holmberg´s group for having shared the funs and agonies of being a PhD student, and for all valuable help in the lab: Elísabet Einarsdóttir – for keeping me company in the human genetics field in the old days and for nice company during many trips (why did we always end up in weird hotels, like in the middle of a festival or next to a karaoke stage…), Marie Lundholm – for your entertaining choreograph lectures for “grp DH party productions”, Ingela Wikström – for writing thesis at the same time as me and for keeping up the fika list, Anna Löfgren- Burström and Sofia Mayans for much nice baby talk, Maria Eriksson – for being a lab guru with answers about almost anything, Sofie Nilsson-Ardnor – for good company during many car rides to Holmsund, Petter Lindgren – for helping me with statistics (even though I did not always understand your explanations), Nadia Duarte, Vinicius Motta, Kurt Lackovic, Anna-Karin Nilsson, Åsa Larefalk, Maria Lindberg, Pia Osterman, Tomas Janunger, Mikael Hedlund, Johan Forssell, Ann-Sofie Johansson, and Ulrika Norén-Nyström – thank you all for good company and many nice discussions.

Thanks also to all other people at Medical Genetics or “down the hall” who have helped create a nice working place, especially during lunches, retreats and Friday´s fika: Kristina Lejon – for always being enthusiastic and helpful, and for many fun song practices, Mia Sundström, Linda Köhn, Malin Olsson and Martin Johansson – the “new people”, for keeping up the good spirit, Urban Hellman – for your sense of humour and assistance with mutation screening, Stefan Escher – for being a nice person and helping me with genealogical analysis, Susann Haraldsson – for helping me with genotyping and sharing

57 the office in the old days, Kristina Cederquist, Lisbet Lind, Lotta Nilsson, Solveig Linghult, Lisbeth Ärlestig, and Irina Golovleva – thank you all for good company.

Thanks to everyone who have helped making life easier: Birgitta Berglund – for being a nice person and helping me with lots of administrative stuff, P-A Lundström – for your many smiles and help with almost everything that has needed to be fixed or delivered, Terry Persson and Åsa Lundsten – for organizing many nice retreats/parties at the department, and the Media department – for keeping everything clean.

Thanks to my co-authors (those not already mentioned) for important contributions: Kristina Forsman-Semb (thanks also for interesting discussions at our meetings), Jan Minde, Göran Toolanen, Olle Svensson, Göran Solders and P-O Nylander.

Thanks to Kjell Asplund, Gunborg Rönnberg and Birgitta Stegmayr at department of Medicine for helping me obtain the migraine patients from the healthcare centres.

Thanks to the FGB Research School in Genomics and Bioinformatics for sponsoring my PhD work and for many nice research courses.

Thanks to the patients and their relatives for kindly donating samples for this project. I truly hope they will benefit from our research.

Thanks to all my friends in “real life”, as well as some fellow PhD students (“ingen nämnd och ingen glömd”) for lots of parties and dinners, fun corridor life in the old days, weddings, sporting (I really miss those badminton matches), travelling, science fiction- movies, baby talk and many laughs! Life would have been less fun without you!

Thanks to my “old” and “new” family for all support: my parents in law Carin and Sigvard for always making me feel welcome and for much help with babysitting (especially during thesis writing), my twin sister and best friend Pernilla and Peter for sharing much of my life in Holmsund, my brother Peter and Linda for being nice and trying to seem interested whenever I talk about my research, and my parents Barbro and Sören for much love and support over the years, and for always believing in me.

Finally, Anders and Simon, the two boys in my life – you are the sunshine in my life. I love you the most!!

58 REFERENCES

Abduljabbar, M., Ogunniyi, A., al Balla, S., Alballaa, S., and al-Dalaan, A. (1996). Prevalence of primary headache syndrome in adults in the Qassim region of Saudi Arabia. Headache 36, 385- 388.

Abu-Arefeh, I., and Russell, G. (1994). Prevalence of headache and migraine in schoolchildren. BMJ 309, 765-769.

Afridi, S. K., Matharu, M. S., Lee, L., Kaube, H., Friston, K. J., Frackowiak, R. S., and Goadsby, P. J. (2005). A PET study exploring the laterality of brainstem activation in migraine using glyceryl trinitrate. Brain 128, 932-939.

Albers, K. M., Wright, D. E., and Davis, B. M. (1994). Overexpression of nerve growth factor in epidermis of transgenic mice causes hypertrophy of the peripheral nervous system. J Neurosci 14, 1422-1432.

Anderson, S. L., Coli, R., Daly, I. W., Kichula, E. A., Rork, M. J., Volpi, S. A., Ekstein, J., and Rubin, B. Y. (2001). Familial dysautonomia is caused by mutations of the IKAP gene. Am J Hum Genet 68, 753-758.

Axelrod, F. B. (2002). Hereditary sensory and autonomic neuropathies. Familial dysautonomia and other HSANs. Clin Auton Res 12 Suppl 1, I2-14.

Axelrod, F. B., and Hilz, M. J. (2003). Inherited autonomic neuropathies. Semin Neurol 23, 381- 390.

Beattie, M. S., Harrington, A. W., Lee, R., Kim, J. Y., Boyce, S. L., Longo, F. M., Bresnahan, J. C., Hempstead, B. L., and Yoon, S. O. (2002). ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury. Neuron 36, 375-386.

Bejaoui, K., Wu, C., Scheffler, M. D., Haan, G., Ashby, P., Wu, L., de Jong, P., and Brown, R. H., Jr. (2001). SPTLC1 is mutated in hereditary sensory neuropathy, type 1. Nat Genet 27, 261-262.

Berryman, M., and Bretscher, A. (2000). Identification of a novel member of the chloride intracellular channel gene family (CLIC5) that associates with the actin cytoskeleton of placental microvilli. Mol Biol Cell 11, 1509-1521.

Bibel, M., and Barde, Y. A. (2000). Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system. Genes Dev 14, 2919-2937.

Bigal, M. E., Lipton, R. B., Cohen, J., and Silberstein, S. D. (2003). Epilepsy and migraine. Epilepsy Behav 4 Suppl 2, S13-24.

Bjornsson, A., Gudmundsson, G., Gudfinnsson, E., Hrafnsdottir, M., Benedikz, J., Skuladottir, S., Kristjansson, K., Frigge, M. L., Kong, A., Stefansson, K., and Gulcher, J. R. (2003). Localization of a gene for migraine without aura to chromosome 4q21. Am J Hum Genet 73, 986-993.

Blumenfeld, A., Slaugenhaupt, S. A., Axelrod, F. B., Lucente, D. E., Maayan, C., Liebert, C. B., Ozelius, L. J., Trofatter, J. A., Haines, J. L., Breakefield, X. O., and et al. (1993). Localization of the gene for familial dysautonomia on chromosome 9 and definition of DNA markers for genetic diagnosis. Nat Genet 4, 160-164.

59 Bolay, H., Reuter, U., Dunn, A. K., Huang, Z., Boas, D. A., and Moskowitz, M. A. (2002). Intrinsic brain activity triggers trigeminal meningeal afferents in a migraine model. Nat Med 8, 136-142.

Bousser, M. G., and Welch, K. M. (2005). Relation between migraine and stroke. Lancet Neurol 4, 533-542.

Breslau, N., and Rasmussen, B. K. (2001). The impact of migraine: Epidemiology, risk factors, and co-morbidities. Neurology 56, S4-12.

Cader, Z. M., Noble-Topham, S., Dyment, D. A., Cherny, S. S., Brown, J. D., Rice, G. P., and Ebers, G. C. (2003). Significant linkage to migraine with aura on chromosome 11q24. Hum Mol Genet 12, 2511-2517.

Capsoni, S., Giannotta, S., and Cattaneo, A. (2002). Nerve growth factor and galantamine ameliorate early signs of neurodegeneration in anti-nerve growth factor mice. Proc Natl Acad Sci U S A 99, 12432-12437.

Capsoni, S., Ugolini, G., Comparini, A., Ruberti, F., Berardi, N., and Cattaneo, A. (2000). Alzheimer-like neurodegeneration in aged antinerve growth factor transgenic mice. Proc Natl Acad Sci U S A 97, 6826-6831.

Carlsson, A., Forsgren, L., Nylander, P. O., Hellman, U., Forsman-Semb, K., Holmgren, G., Holmberg, D., and Holmberg, M. (2002). Identification of a susceptibility locus for migraine with and without aura on 6p12.2-p21.1. Neurology 59, 1804-1807.

Carter, B. D., and Lewin, G. R. (1997). Neurotrophins live or let die: does p75NTR decide? Neuron 18, 187-190.

Chao, M. V. (2003). Neurotrophins and their receptors: a convergence point for many signalling pathways. Nat Rev Neurosci 4, 299-309.

Chao, M. V., and Bothwell, M. (2002). Neurotrophins: to cleave or not to cleave. Neuron 33, 9-12.

Chen, K. S., Nishimura, M. C., Armanini, M. P., Crowley, C., Spencer, S. D., and Phillips, H. S. (1997). Disruption of a single allele of the nerve growth factor gene results in atrophy of basal forebrain cholinergic neurons and memory deficits. J Neurosci 17, 7288-7296.

Cherif-Zahar, B., Raynal, V., Gane, P., Mattei, M. G., Bailly, P., Gibbs, B., Colin, Y., and Cartron, J. P. (1996). Candidate gene acting as a suppressor of the RH locus in most cases of Rh-deficiency. Nat Genet 12, 168-173.

Chioza, B., Wilkie, H., Nashef, L., Blower, J., McCormick, D., Sham, P., Asherson, P., and Makoff, A. J. (2001). Association between the alpha(1a) calcium channel gene CACNA1A and idiopathic generalized epilepsy. Neurology 56, 1245-1246.

Claes, L., Del-Favero, J., Ceulemans, B., Lagae, L., Van Broeckhoven, C., and De Jonghe, P. (2001). De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy. Am J Hum Genet 68, 1327-1332.

Coe, I. R., Griffiths, M., Young, J. D., Baldwin, S. A., and Cass, C. E. (1997). Assignment of the human equilibrative nucleoside transporter (hENT1) to 6p21.1-p21.2. Genomics 45, 459-460.

Colson, N. J., Lea, R. A., Quinlan, S., and Griffiths, L. R. (2006). The role of vascular and hormonal genes in migraine susceptibility. Mol Genet Metab.

60 Colson, N. J., Lea, R. A., Quinlan, S., MacMillan, J., and Griffiths, L. R. (2004). The estrogen receptor 1 G594A polymorphism is associated with migraine susceptibility in two independent case/control groups. Neurogenetics 5, 129-133.

Counts, S. E., and Mufson, E. J. (2005). The role of nerve growth factor receptors in cholinergic basal forebrain degeneration in prodromal Alzheimer disease. J Neuropathol Exp Neurol 64, 263- 272.

Crowley, C., Spencer, S. D., Nishimura, M. C., Chen, K. S., Pitts-Meek, S., Armanini, M. P., Ling, L. H., McMahon, S. B., Shelton, D. L., Levinson, A. D., and et al. (1994). Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell 76, 1001-1011.

Curtain, R., Tajouri, L., Lea, R., Macmillan, J., and Griffiths, L. (2006). No mutations detected in the INSR gene in a chromosome 19p13 linked migraine pedigree. Eur J Med Genet 49, 57-62.

Dahlof, C., and Linde, M. (2001). One-year prevalence of migraine in Sweden: a population-based study in adults. Cephalalgia 21, 664-671.

Davies, A. M., Bandtlow, C., Heumann, R., Korsching, S., Rohrer, H., and Thoenen, H. (1987). Timing and site of nerve growth factor synthesis in developing skin in relation to innervation and expression of the receptor. Nature 326, 353-358.

Davis, B. M., Lewin, G. R., Mendell, L. M., Jones, M. E., and Albers, K. M. (1993). Altered expression of nerve growth factor in the skin of transgenic mice leads to changes in response to mechanical stimuli. Neuroscience 56, 789-792.

Dawkins, J. L., Hulme, D. J., Brahmbhatt, S. B., Auer-Grumbach, M., and Nicholson, G. A. (2001). Mutations in SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I. Nat Genet 27, 309-312.

De Fusco, M., Marconi, R., Silvestri, L., Atorino, L., Rampoldi, L., Morgante, L., Ballabio, A., Aridon, P., and Casari, G. (2003). Haploinsufficiency of ATP1A2 encoding the Na+/K+ pump alpha2 subunit associated with familial hemiplegic migraine type 2. Nat Genet 33, 192-196.

De Rosa, R., Garcia, A. A., Braschi, C., Capsoni, S., Maffei, L., Berardi, N., and Cattaneo, A. (2005). Intranasal administration of nerve growth factor (NGF) rescues recognition memory deficits in AD11 anti-NGF transgenic mice. Proc Natl Acad Sci U S A 102, 3811-3816.

Del Zompo, M., Cherchi, A., Palmas, M. A., Ponti, M., Bocchetta, A., Gessa, G. L., and Piccardi, M. P. (1998). Association between dopamine receptor genes and migraine without aura in a Sardinian sample. Neurology 51, 781-786.

Dichgans, M., Freilinger, T., Eckstein, G., Babini, E., Lorenz-Depiereux, B., Biskup, S., Ferrari, M. D., Herzog, J., van den Maagdenberg, A. M., Pusch, M., and Strom, T. M. (2005). Mutation in the neuronal voltage-gated sodium channel SCN1A in familial hemiplegic migraine. Lancet 366, 371-377.

Ducros, A., Denier, C., Joutel, A., Cecillon, M., Lescoat, C., Vahedi, K., Darcel, F., Vicaut, E., Bousser, M. G., and Tournier-Lasserve, E. (2001). The clinical spectrum of familial hemiplegic migraine associated with mutations in a neuronal calcium channel. N Engl J Med 345, 17-24.

Ducros, A., Joutel, A., Vahedi, K., Cecillon, M., Ferreira, A., Bernard, E., Verier, A., Echenne, B., Lopez de Munain, A., Bousser, M. G., and Tournier-Lasserve, E. (1997). Mapping of a second locus for familial hemiplegic migraine to 1q21-q23 and evidence of further heterogeneity. Ann Neurol 42, 885-890.

61 Ducros, A., Tournier-Lasserve, E., and Bousser, M. G. (2002). The genetics of migraine. Lancet Neurol 1, 285-293.

Dyck, P. J., Mellinger, J. F., Reagan, T. J., Horowitz, S. J., McDonald, J. W., Litchy, W. J., Daube, J. R., Fealey, R. D., Go, V. L., Kao, P. C., et al. (1983). Not 'indifference to pain' but varieties of hereditary sensory and autonomic neuropathy. Brain 106 (Pt 2), 373-390.

Egan, M. F., Kojima, M., Callicott, J. H., Goldberg, T. E., Kolachana, B. S., Bertolino, A., Zaitsev, E., Gold, B., Goldman, D., Dean, M., et al. (2003). The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell 112, 257-269.

Einarsdottir, E. (2005). Mapping genetic diseases in northern Sweden. Umeå, Sweden.

Einarsdottir, E., Carlsson, A., Minde, J., Toolanen, G., Svensson, O., Solders, G., Holmgren, G., Holmberg, D., and Holmberg, M. (2004). A mutation in the nerve growth factor beta gene (NGFB) causes loss of pain perception. Hum Mol Genet 13, 799-805.

Escayg, A., MacDonald, B. T., Meisler, M. H., Baulac, S., Huberfeld, G., An-Gourfinkel, I., Brice, A., LeGuern, E., Moulard, B., Chaigne, D., et al. (2000). Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS+2. Nat Genet 24, 343-345.

Estevez, M., and Gardner, K. L. (2004). Update on the genetics of migraine. Hum Genet 114, 225- 235.

Fahnestock, M., Michalski, B., Xu, B., and Coughlin, M. D. (2001). The precursor pro-nerve growth factor is the predominant form of nerve growth factor in brain and is increased in Alzheimer's disease. Mol Cell Neurosci 18, 210-220.

Gardner, K., Barmada, M. M., Ptacek, L. J., and Hoffman, E. P. (1997). A new locus for hemiplegic migraine maps to chromosome 1q31. Neurology 49, 1231-1238.

Gervil, M., Ulrich, V., Kyvik, K. O., Olesen, J., and Russell, M. B. (1999). Migraine without aura: a population-based twin study. Ann Neurol 46, 606-611.

Goadsby, P. J. (2001). Migraine, aura, and cortical spreading depression: why are we still talking about it? Ann Neurol 49, 4-6.

Goadsby, P. J. (2003). Migraine: diagnosis and management. Intern Med J 33, 436-442.

Gretarsdottir, S., Sveinbjornsdottir, S., Jonsson, H. H., Jakobsson, F., Einarsdottir, E., Agnarsson, U., Shkolny, D., Einarsson, G., Gudjonsdottir, H. M., Valdimarsson, E. M., et al. (2002). Localization of a susceptibility gene for common forms of stroke to 5q12. Am J Hum Genet 70, 593-603.

Griffiths, M., Beaumont, N., Yao, S. Y., Sundaram, M., Boumah, C. E., Davies, A., Kwong, F. Y., Coe, I., Cass, C. E., Young, J. D., and Baldwin, S. A. (1997). Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs. Nat Med 3, 89-93.

Guieu, R., Devaux, C., Henry, H., Bechis, G., Pouget, J., Mallet, D., Sampieri, F., Juin, M., Gola, R., and Rochat, H. (1998). Adenosine and migraine. Can J Neurol Sci 25, 55-58.

Hefti, F. F., Rosenthal, A., Walicke, P. A., Wyatt, S., Vergara, G., Shelton, D. L., and Davies, A. M. (2006). Novel class of pain drugs based on antagonism of NGF. Trends Pharmacol Sci 27, 85- 91.

62 Hiramoto, M., Yoshida, H., Imaizumi, T., Yoshimizu, N., and Satoh, K. (1997). A mutation in plasma platelet-activating factor acetylhydrolase (Val279- ->Phe) is a genetic risk factor for stroke. Stroke 28, 2417-2420.

Houlden, H., King, R. H., Hashemi-Nejad, A., Wood, N. W., Mathias, C. J., Reilly, M., and Thomas, P. K. (2001). A novel TRK A (NTRK1) mutation associated with hereditary sensory and autonomic neuropathy type V. Ann Neurol 49, 521-525.

Hovatta, I., Kallela, M., Farkkila, M., and Peltonen, L. (1994). Familial migraine: exclusion of the susceptibility gene from the reported locus of familial hemiplegic migraine on 19p. Genomics 23, 707-709.

IHS (1988). Headache Classification Committee of the International Headache Society. Classification and diagnostic criteria for headache disorders, cranial neuralgia and facial pain. Cephalalgia 8, 1-96.

IHS (2004). The International Classification of Headache Disorders: 2nd edition. Cephalalgia 24 Suppl 1, 9-160.

Ikeda, K., Onaka, T., Yamakado, M., Nakai, J., Ishikawa, T. O., Taketo, M. M., and Kawakami, K. (2003). Degeneration of the amygdala/piriform cortex and enhanced fear/anxiety behaviors in sodium pump alpha2 subunit (Atp1a2)-deficient mice. J Neurosci 23, 4667-4676.

Indo, Y. (2001). Molecular basis of congenital insensitivity to pain with anhidrosis (CIPA): mutations and polymorphisms in TRKA (NTRK1) gene encoding the receptor tyrosine kinase for nerve growth factor. Hum Mutat 18, 462-471.

Indo, Y., Tsuruta, M., Hayashida, Y., Karim, M. A., Ohta, K., Kawano, T., Mitsubuchi, H., Tonoki, H., Awaya, Y., and Matsuda, I. (1996). Mutations in the TRKA/NGF receptor gene in patients with congenital insensitivity to pain with anhidrosis. Nat Genet 13, 485-488.

Johnson, J., and Oppenheim, R. (1994). Neurotrophins. Keeping track of changing neurotrophic theory. Curr Biol 4, 662-665.

Johnson, K. W., Phebus, L. A., and Cohen, M. L. (1998). Serotonin in migraine: theories, animal models and emerging therapies. Prog Drug Res 51, 219-244.

Johnson, M. P., Lea, R. A., Curtain, R. P., MacMillan, J. C., and Griffiths, L. R. (2003). An investigation of the 5-HT2C receptor gene as a migraine candidate gene. Am J Med Genet B Neuropsychiatr Genet 117, 86-89.

Jones, K. W., Ehm, M. G., Pericak-Vance, M. A., Haines, J. L., Boyd, P. R., and Peroutka, S. J. (2001). Migraine with aura susceptibility locus on chromosome 19p13 is distinct from the familial hemiplegic migraine locus. Genomics 78, 150-154.

Joutel, A., Bousser, M. G., Biousse, V., Labauge, P., Chabriat, H., Nibbio, A., Maciazek, J., Meyer, B., Bach, M. A., Weissenbach, J., and et al. (1993). A gene for familial hemiplegic migraine maps to chromosome 19. Nat Genet 5, 40-45.

Jouvenceau, A., Eunson, L. H., Spauschus, A., Ramesh, V., Zuberi, S. M., Kullmann, D. M., and Hanna, M. G. (2001). Human epilepsy associated with dysfunction of the brain P/Q-type calcium channel. Lancet 358, 801-807.

Kallela, M., Farkkila, M., Saijonmaa, O., and Fyhrquist, F. (1998). Endothelin in migraine patients. Cephalalgia 18, 329-332.

63 Kallela, M., Wessman, M., Havanka, H., Palotie, A., and Farkkila, M. (2001). Familial migraine with and without aura: clinical characteristics and co-occurrence. Eur J Neurol 8, 441-449.

Kara, I., Sazci, A., Ergul, E., Kaya, G., and Kilic, G. (2003). Association of the C677T and A1298C polymorphisms in the 5,10 methylenetetrahydrofolate reductase gene in patients with migraine risk. Brain Res Mol Brain Res 111, 84-90.

Kaunisto, M. A., Tikka, P. J., Kallela, M., Leal, S. M., Papp, J. C., Korhonen, A., Hamalainen, E., Harno, H., Havanka, H., Nissila, M., et al. (2005). Chromosome 19p13 loci in Finnish migraine with aura families. Am J Med Genet B Neuropsychiatr Genet 132, 85-89.

Kors, E. E., van den Maagdenberg, A. M., Plomp, J. J., Frants, R. R., and Ferrari, M. D. (2002). Calcium channel mutations and migraine. Curr Opin Neurol 15, 311-316.

Kowa, H., Fusayasu, E., Ijiri, T., Ishizaki, K., Yasui, K., Nakaso, K., Kusumi, M., Takeshima, T., and Nakashima, K. (2005). Association of the insertion/deletion polymorphism of the angiotensin I-converting enzyme gene in patients of migraine with aura. Neurosci Lett 374, 129-131.

Kowa, H., Yasui, K., Takeshima, T., Urakami, K., Sakai, F., and Nakashima, K. (2000). The homozygous C677T mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for migraine. Am J Med Genet 96, 762-764.

Kruse, S., Mao, X. Q., Heinzmann, A., Blattmann, S., Roberts, M. H., Braun, S., Gao, P. S., Forster, J., Kuehr, J., Hopkin, J. M., et al. (2000). The Ile198Thr and Ala379Val variants of plasmatic PAF-acetylhydrolase impair catalytical activities and are associated with atopy and asthma. Am J Hum Genet 66, 1522-1530.

Lafreniere, R. G., MacDonald, M. L., Dube, M. P., MacFarlane, J., O'Driscoll, M., Brais, B., Meilleur, S., Brinkman, R. R., Dadivas, O., Pape, T., et al. (2004). Identification of a novel gene (HSN2) causing hereditary sensory and autonomic neuropathy type II through the Study of Canadian Genetic Isolates. Am J Hum Genet 74, 1064-1073.

Lander, E., and Kruglyak, L. (1995). Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 11, 241-247.

Larsson, B., Bille, B., and Pedersen, N. L. (1995). Genetic influence in headaches: a Swedish twin study. Headache 35, 513-519.

Lea, R. A., Curtain, R. P., Hutchins, C., Brimage, P. J., and Griffiths, L. R. (2001). Investigation of the CACNA1A gene as a candidate for typical migraine susceptibility. Am J Med Genet 105, 707- 712.

Lea, R. A., Dohy, A., Jordan, K., Quinlan, S., Brimage, P. J., and Griffiths, L. R. (2000). Evidence for allelic association of the dopamine beta-hydroxylase gene (DBH) with susceptibility to typical migraine. Neurogenetics 3, 35-40.

Lea, R. A., Ovcaric, M., Sundholm, J., MacMillan, J., and Griffiths, L. R. (2004). The methylenetetrahydrofolate reductase gene variant C677T influences susceptibility to migraine with aura. BMC Med 2, 3.

Lea, R. A., Ovcaric, M., Sundholm, J., Solyom, L., Macmillan, J., and Griffiths, L. R. (2005). Genetic variants of angiotensin converting enzyme and methylenetetrahydrofolate reductase may act in combination to increase migraine susceptibility. Brain Res Mol Brain Res 136, 112-117.

64 Lea, R. A., Shepherd, A. G., Curtain, R. P., Nyholt, D. R., Quinlan, S., Brimage, P. J., and Griffiths, L. R. (2002). A typical migraine susceptibility region localizes to chromosome 1q31. Neurogenetics 4, 17-22.

Lee, K. F., Li, E., Huber, L. J., Landis, S. C., Sharpe, A. H., Chao, M. V., and Jaenisch, R. (1992). Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell 69, 737-749.

Lee, R., Kermani, P., Teng, K. K., and Hempstead, B. L. (2001). Regulation of cell survival by secreted proneurotrophins. Science 294, 1945-1948.

Lipton, R. B., Scher, A. I., Kolodner, K., Liberman, J., Steiner, T. J., and Stewart, W. F. (2002). Migraine in the United States: epidemiology and patterns of health care use. Neurology 58, 885- 894.

Liu, A. W., Delgado-Escueta, A. V., Serratosa, J. M., Alonso, M. E., Medina, M. T., Gee, M. N., Cordova, S., Zhao, H. Z., Spellman, J. M., Peek, J. R., and et al. (1995). Juvenile myoclonic epilepsy locus in chromosome 6p21.2-p11: linkage to convulsions and electroencephalography trait. Am J Hum Genet 57, 368-381.

Majdan, M., Lachance, C., Gloster, A., Aloyz, R., Zeindler, C., Bamji, S., Bhakar, A., Belliveau, D., Fawcett, J., Miller, F. D., and Barker, P. A. (1997). Transgenic mice expressing the intracellular domain of the p75 neurotrophin receptor undergo neuronal apoptosis. J Neurosci 17, 6988-6998.

Marconi, R., De Fusco, M., Aridon, P., Plewnia, K., Rossi, M., Carapelli, S., Ballabio, A., Morgante, L., Musolino, R., Epifanio, A., et al. (2003). Familial hemiplegic migraine type 2 is linked to 0.9Mb region on chromosome 1q23. Ann Neurol 53, 376-381.

Marziniak, M., Mossner, R., Schmitt, A., Lesch, K. P., and Sommer, C. (2005). A functional serotonin transporter gene polymorphism is associated with migraine with aura. Neurology 64, 157-159.

May, A., Ophoff, R. A., Terwindt, G. M., Urban, C., van Eijk, R., Haan, J., Diener, H. C., Lindhout, D., Frants, R. R., Sandkuijl, L. A., and Ferrari, M. D. (1995). Familial hemiplegic migraine locus on 19p13 is involved in the common forms of migraine with and without aura. Hum Genet 96, 604-608.

McCarthy, L. C., Hosford, D. A., Riley, J. H., Bird, M. I., White, N. J., Hewett, D. R., Peroutka, S. J., Griffiths, L. R., Boyd, P. R., Lea, R. A., et al. (2001). Single-nucleotide polymorphism alleles in the insulin receptor gene are associated with typical migraine. Genomics 78, 135-149.

Mendell, L. M., Albers, K. M., and Davis, B. M. (1999). Neurotrophins, nociceptors, and pain. Microsc Res Tech 45, 252-261.

Miller, S. A., Dykes, D. D., and Polesky, H. F. (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16, 1215.

Minde, J. (2006). Norrbottnian congenital insensitivity to pain. Gällivare, Sweden.

Minde, J., Toolanen, G., Andersson, T., Nennesmo, I., Remahl, I. N., Svensson, O., and Solders, G. (2004). Familial insensitivity to pain (HSAN V) and a mutation in the NGFB gene. A neurophysiological and pathological study. Muscle Nerve 30, 752-760.

65 Mochi, M., Cevoli, S., Cortelli, P., Pierangeli, G., Soriani, S., Scapoli, C., and Montagna, P. (2003). A genetic association study of migraine with dopamine receptor 4, dopamine transporter and dopamine-beta-hydroxylase genes. Neurol Sci 23, 301-305.

Mogil, J. S. (1999). The genetic mediation of individual differences in sensitivity to pain and its inhibition. Proc Natl Acad Sci U S A 96, 7744-7751.

Mogil, J. S., Yu, L., and Basbaum, A. I. (2000). Pain genes?: natural variation and transgenic mutants. Annu Rev Neurosci 23, 777-811.

Mulder, E. J., Van Baal, C., Gaist, D., Kallela, M., Kaprio, J., Svensson, D. A., Nyholt, D. R., Martin, N. G., MacGregor, A. J., Cherkas, L. F., et al. (2003). Genetic and environmental influences on migraine: a twin study across six countries. Twin Res 6, 422-431.

Mulley, J. C., Scheffer, I. E., Petrou, S., Dibbens, L. M., Berkovic, S. F., and Harkin, L. A. (2005). SCN1A mutations and epilepsy. Hum Mutat 25, 535-542.

Neves-Pereira, M., Cheung, J. K., Pasdar, A., Zhang, F., Breen, G., Yates, P., Sinclair, M., Crombie, C., Walker, N., and St Clair, D. M. (2005). BDNF gene is a risk factor for schizophrenia in a Scottish population. Mol Psychiatry 10, 208-212.

Neves-Pereira, M., Mundo, E., Muglia, P., King, N., Macciardi, F., and Kennedy, J. L. (2002). The brain-derived neurotrophic factor gene confers susceptibility to bipolar disorder: evidence from a family-based association study. Am J Hum Genet 71, 651-655.

Nicholson, G. A., Dawkins, J. L., Blair, I. P., Kennerson, M. L., Gordon, M. J., Cherryson, A. K., Nash, J., and Bananis, T. (1996). The gene for hereditary sensory neuropathy type I (HSN-I) maps to chromosome 9q22.1-q22.3. Nat Genet 13, 101-104.

Nilsson-Ardnor, S., Wiklund, P. G., Lindgren, P., Nilsson, A. K., Janunger, T., Escher, S. A., Hallbeck, B., Stegmayr, B., Asplund, K., and Holmberg, D. (2005). Linkage of ischemic stroke to the PDE4D region on 5q in a Swedish population. Stroke 36, 1666-1671.

Noble-Topham, S. E., Dyment, D. A., Cader, M. Z., Ganapathy, R., Brown, J. D., Rice, G. P., and Ebers, G. C. (2002). Migraine with aura is not linked to the FHM gene CACNA1A or the chromosomal region, 19p13. Neurology 59, 1099-1101.

Norberg, A., Forsgren, L., Holmberg, D., and Holmberg, M. (2006). Exclusion of the juvenile myoclonic epilepsy gene EFHC1 as the cause of migraine on chromosome 6, but association to two rare polymorphisms in MEP1A and RHAG. Neurosci Lett 396, 137-142.

Nyholt, D. R., Curtain, R. P., and Griffiths, L. R. (2000). Familial typical migraine: significant linkage and localization of a gene to Xq24-28. Hum Genet 107, 18-23.

Nyholt, D. R., Dawkins, J. L., Brimage, P. J., Goadsby, P. J., Nicholson, G. A., and Griffiths, L. R. (1998a). Evidence for an X-linked genetic component in familial typical migraine. Hum Mol Genet 7, 459-463.

Nyholt, D. R., Gillespie, N. G., Heath, A. C., Merikangas, K. R., Duffy, D. L., and Martin, N. G. (2004). Latent class and genetic analysis does not support migraine with aura and migraine without aura as separate entities. Genet Epidemiol 26, 231-244.

Nyholt, D. R., Lea, R. A., Goadsby, P. J., Brimage, P. J., and Griffiths, L. R. (1998b). Familial typical migraine: linkage to chromosome 19p13 and evidence for genetic heterogeneity. Neurology 50, 1428-1432.

66 Nyholt, D. R., Morley, K. I., Ferreira, M. A., Medland, S. E., Boomsma, D. I., Heath, A. C., Merikangas, K. R., Montgomery, G. W., and Martin, N. G. (2005). Genomewide significant linkage to migrainous headache on chromosome 5q21. Am J Hum Genet 77, 500-512.

Nykjaer, A., Lee, R., Teng, K. K., Jansen, P., Madsen, P., Nielsen, M. S., Jacobsen, C., Kliemannel, M., Schwarz, E., Willnow, T. E., et al. (2004). Sortilin is essential for proNGF- induced neuronal cell death. Nature 427, 843-848.

Nykjaer, A., Willnow, T. E., and Petersen, C. M. (2005). p75NTR--live or let die. Curr Opin Neurobiol 15, 49-57.

Ogilvie, A. D., Russell, M. B., Dhall, P., Battersby, S., Ulrich, V., Smith, C. A., Goodwin, G. M., Harmar, A. J., and Olesen, J. (1998). Altered allelic distributions of the serotonin transporter gene in migraine without aura and migraine with aura. Cephalalgia 18, 23-26.

Ophoff, R. A., Terwindt, G. M., Vergouwe, M. N., van Eijk, R., Oefner, P. J., Hoffman, S. M., Lamerdin, J. E., Mohrenweiser, H. W., Bulman, D. E., Ferrari, M., et al. (1996). Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell 87, 543-552.

Ophoff, R. A., van Eijk, R., Sandkuijl, L. A., Terwindt, G. M., Grubben, C. P., Haan, J., Lindhout, D., Ferrari, M. D., and Frants, R. R. (1994). Genetic heterogeneity of familial hemiplegic migraine. Genomics 22, 21-26.

Oppenheim, R. W. (1991). Cell death during development of the nervous system. Annu Rev Neurosci 14, 453-501.

Oterino, A., Pascual, J., de Alegria, C. R., Valle, N., Castillo, J., Bravo, Y., Gonzalez, F., Sanchez- Velasco, P., Cayon, A., Leyva-Cobian, F., et al. (2006). Association of migraine and ESR1 G325C polymorphism. Neuroreport 17, 61-64.

Packard, C. J., O'Reilly, D. S., Caslake, M. J., McMahon, A. D., Ford, I., Cooney, J., Macphee, C. H., Suckling, K. E., Krishna, M., Wilkinson, F. E., et al. (2000). Lipoprotein-associated phospholipase A2 as an independent predictor of coronary heart disease. West of Scotland Coronary Prevention Study Group. N Engl J Med 343, 1148-1155.

Paterna, S., Di Pasquale, P., D'Angelo, A., Seidita, G., Tuttolomondo, A., Cardinale, A., Maniscalchi, T., Follone, G., Giubilato, A., Tarantello, M., and Licata, G. (2000). Angiotensin- converting enzyme gene deletion polymorphism determines an increase in frequency of migraine attacks in patients suffering from migraine without aura. Eur Neurol 43, 133-136.

Peroutka, S. J., Wilhoit, T., and Jones, K. (1997). Clinical susceptibility to migraine with aura is modified by dopamine D2 receptor (DRD2) NcoI alleles. Neurology 49, 201-206.

Peterson, D. A., Dickinson-Anson, H. A., Leppert, J. T., Lee, K. F., and Gage, F. H. (1999). Central neuronal loss and behavioral impairment in mice lacking neurotrophin receptor p75. J Comp Neurol 404, 1-20.

Pietrobon, D., and Striessnig, J. (2003). Neurobiology of migraine. Nat Rev Neurosci 4, 386-398.

Rasmussen, B. K., Jensen, R., Schroll, M., and Olesen, J. (1991). Epidemiology of headache in a general population--a prevalence study. J Clin Epidemiol 44, 1147-1157.

Rosemberg, S., Marie, S. K., and Kliemann, S. (1994). Congenital insensitivity to pain with anhidrosis (hereditary sensory and autonomic neuropathy type IV). Pediatr Neurol 11, 50-56.

67 Ruberti, F., Capsoni, S., Comparini, A., Di Daniel, E., Franzot, J., Gonfloni, S., Rossi, G., Berardi, N., and Cattaneo, A. (2000). Phenotypic knockout of nerve growth factor in adult transgenic mice reveals severe deficits in basal forebrain cholinergic neurons, cell death in the spleen, and skeletal muscle dystrophy. J Neurosci 20, 2589-2601.

Russell, M. B., and Olesen, J. (1995). Increased familial risk and evidence of genetic factor in migraine. BMJ 311, 541-544.

Russell, M. B., Rasmussen, B. K., Thorvaldsen, P., and Olesen, J. (1995). Prevalence and sex-ratio of the subtypes of migraine. Int J Epidemiol 24, 612-618.

Russell, M. B., Ulrich, V., Gervil, M., and Olesen, J. (2002). Migraine without aura and migraine with aura are distinct disorders. A population-based twin survey. Headache 42, 332-336.

Russo, L., Mariotti, P., Sangiorgi, E., Giordano, T., Ricci, I., Lupi, F., Chiera, R., Guzzetta, F., Neri, G., and Gurrieri, F. (2005). A new susceptibility locus for migraine with aura in the 15q11- q13 genomic region containing three GABA-A receptor genes. Am J Hum Genet 76, 327-333.

Savage, D. B., Agostini, M., Barroso, I., Gurnell, M., Luan, J., Meirhaeghe, A., Harding, A. H., Ihrke, G., Rajanayagam, O., Soos, M. A., et al. (2002). Digenic inheritance of severe insulin resistance in a human pedigree. Nat Genet 31, 379-384.

Scher, A. I., Terwindt, G. M., Verschuren, W. M., Kruit, M. C., Blom, H. J., Kowa, H., Frants, R. R., van den Maagdenberg, A. M., van Buchem, M., Ferrari, M. D., and Launer, L. J. (2006). Migraine and MTHFR C677T genotype in a population-based sample. Ann Neurol 59, 372-375.

Silberstein, S. D. (2003). Neurotoxins in the neurobiology of pain. Headache 43 Suppl 1, S2-8.

Slaugenhaupt, S. A., Blumenfeld, A., Gill, S. P., Leyne, M., Mull, J., Cuajungco, M. P., Liebert, C. B., Chadwick, B., Idelson, M., Reznik, L., et al. (2001). Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia. Am J Hum Genet 68, 598-605.

Smeyne, R. J., Klein, R., Schnapp, A., Long, L. K., Bryant, S., Lewin, A., Lira, S. A., and Barbacid, M. (1994). Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 368, 246-249.

Soragna, D., Vettori, A., Carraro, G., Marchioni, E., Vazza, G., Bellini, S., Tupler, R., Savoldi, F., and Mostacciuolo, M. L. (2003). A locus for migraine without aura maps on chromosome 14q21.2-q22.3. Am J Hum Genet 72, 161-167.

Suzuki, T., Delgado-Escueta, A. V., Aguan, K., Alonso, M. E., Shi, J., Hara, Y., Nishida, M., Numata, T., Medina, M. T., Takeuchi, T., et al. (2004). Mutations in EFHC1 cause juvenile myoclonic epilepsy. Nat Genet 36, 842-849.

Tekle Haimanot, R., Seraw, B., Forsgren, L., Ekbom, K., and Ekstedt, J. (1995). Migraine, chronic tension-type headache, and cluster headache in an Ethiopian rural community. Cephalalgia 15, 482-488.

Terwilliger, J. D., and Ott, J. (1994). Handbook of Human Genetic Linkage (Baltimore: Johns Hopkins University Press).

Terwindt, G. M., Ophoff, R. A., Haan, J., Sandkuijl, L. A., Frants, R. R., and Ferrari, M. D. (1998). Migraine, ataxia and epilepsy: a challenging spectrum of genetically determined calcium channelopathies. Dutch Migraine Genetics Research Group. Eur J Hum Genet 6, 297-307.

68 Terwindt, G. M., Ophoff, R. A., van Eijk, R., Vergouwe, M. N., Haan, J., Frants, R. R., Sandkuijl, L. A., and Ferrari, M. D. (2001). Involvement of the CACNA1A gene containing region on 19p13 in migraine with and without aura. Neurology 56, 1028-1032.

Thomas, P. K. (1993). Hereditary sensory neuropathies. Brain Pathol 3, 157-163.

Tjoelker, L. W., Wilder, C., Eberhardt, C., Stafforini, D. M., Dietsch, G., Schimpf, B., Hooper, S., Le Trong, H., Cousens, L. S., Zimmerman, G. A., and et al. (1995). Anti-inflammatory properties of a platelet-activating factor acetylhydrolase. Nature 374, 549-553.

Tottene, A., Fellin, T., Pagnutti, S., Luvisetto, S., Striessnig, J., Fletcher, C., and Pietrobon, D. (2002). Familial hemiplegic migraine mutations increase Ca(2+) influx through single human CaV2.1 channels and decrease maximal CaV2.1 current density in neurons. Proc Natl Acad Sci U S A 99, 13284-13289.

Trudeau, L. E. (2000). Neurotensin regulates intracellular calcium in ventral tegmental area astrocytes: evidence for the involvement of multiple receptors. Neuroscience 97, 293-302.

Turk, D. C., and Okifuji, A. (1999). Assessment of patients' reporting of pain: an integrated perspective. Lancet 353, 1784-1788.

Tzourio, C., El Amrani, M., Poirier, O., Nicaud, V., Bousser, M. G., and Alperovitch, A. (2001). Association between migraine and endothelin type A receptor (ETA -231 A/G) gene polymorphism. Neurology 56, 1273-1277.

Ulrich, V., Gervil, M., Kyvik, K. O., Olesen, J., and Russell, M. B. (1999). Evidence of a genetic factor in migraine with aura: a population-based Danish twin study. Ann Neurol 45, 242-246.

Walker, A. H., Najarian, D., White, D. L., Jaffe, J. M., Kanetsky, P. A., and Rebbeck, T. R. (1999). Collection of genomic DNA by buccal swabs for polymerase chain reaction-based biomarker assays. Environ Health Perspect 107, 517-520. van den Maagdenberg, A. M., Pietrobon, D., Pizzorusso, T., Kaja, S., Broos, L. A., Cesetti, T., van de Ven, R. C., Tottene, A., van der Kaa, J., Plomp, J. J., et al. (2004). A Cacna1a knockin migraine mouse model with increased susceptibility to cortical spreading depression. Neuron 41, 701-710.

Vanmolkot, K. R., Kors, E. E., Hottenga, J. J., Terwindt, G. M., Haan, J., Hoefnagels, W. A., Black, D. F., Sandkuijl, L. A., Frants, R. R., Ferrari, M. D., and van den Maagdenberg, A. M. (2003). Novel mutations in the Na+, K+-ATPase pump gene ATP1A2 associated with familial hemiplegic migraine and benign familial infantile convulsions. Ann Neurol 54, 360-366.

Ventriglia, M., Bocchio Chiavetto, L., Benussi, L., Binetti, G., Zanetti, O., Riva, M. A., and Gennarelli, M. (2002). Association between the BDNF 196 A/G polymorphism and sporadic Alzheimer's disease. Mol Psychiatry 7, 136-137.

Wessman, M., Kallela, M., Kaunisto, M. A., Marttila, P., Sobel, E., Hartiala, J., Oswell, G., Leal, S. M., Papp, J. C., Hamalainen, E., et al. (2002). A susceptibility locus for migraine with aura, on chromosome 4q24. Am J Hum Genet 70, 652-662.

Wessman, M., Kaunisto, M. A., Kallela, M., and Palotie, A. (2004). The molecular genetics of migraine. Ann Med 36, 462-473.

Westhoff, C. M., Ferreri-Jacobia, M., Mak, D. O., and Foskett, J. K. (2002). Identification of the erythrocyte Rh blood group glycoprotein as a mammalian ammonium transporter. J Biol Chem 277, 12499-12502.

69 Zhuchenko, O., Bailey, J., Bonnen, P., Ashizawa, T., Stockton, D. W., Amos, C., Dobyns, W. B., Subramony, S. H., Zoghbi, H. Y., and Lee, C. C. (1997). Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel. Nat Genet 15, 62-69.

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