Risk factors for open angle glaucoma; a clinical and molecular genetic study

Glaucoma, optic neuropathy with excavation in the optic nerve head and corresponding visual field defect, is one of the leading causes for blindness worldwide. However, visual disability can often be avoided or delayed if the disease is diagnosed at an early stage. Therefore, recognising the risk factors for development and progression of glaucoma may prevent further damage. The purpose of the present study was to evaluate factors associated with visual disability caused by glaucoma and the genetic features of two risk factors, exfoliation syndrome (ES) and a positive family history of glaucoma. The present study material consisted of three study groups 1) deceased glaucoma patients from the Ekenäs practice 2) glaucoma families from the Ekenäs region and 3) population based families with and without exfoliation syndrome from Kökar Island. For the retrospective study, 106 patients with open angle glaucoma (OAG) were identified. At the last visit, 17 patients were visually impaired. Blindness induced by glaucoma was found in one or both eyes in 16 patients and in both eyes in six patients. The cumulative incidence of glaucoma caused blindness for one eye was 6% at 5 years, 9% at 10 years, and 15% at 15 years from initialising the treatment. The factors associated with blindness caused by glaucoma were an advanced stage of glaucoma at diagnosis, fluctuation in intraocular pressure during treatment, the presence of exfoliation syndrome, and poor patient compliance. A cross-sectional population based study performed in 1960-1962 on Kökar Island and the same population was followed until 2002. In total 965 subjects (530 over 50 years) have been examined at least once. The prevalence of exfoliation syndrome (ES) was 18% among subjects older than 50 years. Seventy-five of all 78 ES-positives belonged to the same extended pedigree. According to the segregation and family analysis, exfoliation syndrome seemed to be inherited as an autosomal dominant trait with reduced penetrance. The penetrance was more reduced for males, but the risk for glaucoma was higher in males than in females. To find the gene or genes associated with exfoliation syndrome, a genome wide scan was performed for 64 members (28 ES affected and 36 controls) of the Kökar pedigree. A promising result was found: the highest two-point LOD score of 3.45 (θ=0.04) in chromosome18q12.1-21.33. The presence of mutations in glaucoma genes TIGR/MYOC (myocilin) and OPTN (optineurin) was analysed in eight glaucoma families from the Ekenäs region. An inheritance pattern resembling autosomal dominant mode was detected in all these families. Primary open angle glaucoma or exfoliation glaucoma was found in 35% of 136 family members and 28% were suspected to have glaucoma. No mutations were detected in these families.

Genetic analysis of chromosomal regions 2q33, 7q32 and 19q13 in multiple sclerosis susceptibility

Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system (CNS) affecting 0.1-0.2% of Northern European descent population. MS is considered to be a multifactorial disease, both environment and genetics play a role in its pathogenesis. Despite several decades of intense research, the etiological and pathogenic mechanisms underlying MS remain still largely unknown and no curative treatment exists. The genetic architecture underlying MS is complex with multiple genes involved. The strongest and the best characterized predisposing genetic factors for MS are located, as in other immune-mediated diseases, in the major histocompatibility complex (MHC) on chromosome 6. In humans MHC is called human leukocyte antigen (HLA). Alleles of the HLA locus have been found to associate strongly with MS and remained for many years the only consistently replicable genetic associations. However, recently other genes located outside the MHC region have been proposed as strong candidates for susceptibility to MS in several studies. In this thesis a new genetic locus located on chromosome 7q32, interferon regulatory factor 5 (IRF5), was identified in the susceptibility to MS. In particular, we found that common variation of the gene was associated with the disease in three different populations, Spanish, Swedish and Finnish. We also suggested a possible functional role for one of the risk alleles with impact on the expression of the IRF5 locus. Previous studies have pointed out a possible role played by chromosome 2q33 in the susceptibility to MS and other autoimmune disorders. The work described here also investigated the involvement of this chromosomal region in MS predisposition. After the detection of genetic association with 2q33 (article-1), we extended our analysis through fine-scale single nucleotide polymorphism (SNP) mapping to define further the contribution of this genomic area to disease pathogenesis (article-4). We found a trend (p=0.04) for association to MS with an intronic SNP located in the inducible T-cell co-stimulator (ICOS) gene, an important player in the co-stimulatory pathway of the immune system. Expression analysis of ICOS revealed a novel, previously uncharacterized, alternatively spliced isoform, lacking the extracellular domain that is needed for ligand binding. The stability of the newly-identified transcript variant and its subcellular localization were analyzed. These studies indicated that the novel isoform is stable and shows different subcellular localization as compared to full-length ICOS. The novel isoform might have a regulatory function, but further studies are required to elucidate its function. Chromosome 19q13 has been previously suggested as one of the genomic areas involved in MS predisposition. In several populations, suggestive linkage signals between MS predisposition and 19q13 have been obtained. Here, we analysed the role of allelic variation in 19q13 by family based association analysis in 782 MS families collected from Finland. In this dataset, we were not able to detect any statistically significant associations, although several previously suggested markers were included to the analysis. Replication of the previous findings on the basis of linkage disequilibrium between marker allele and disease/risk allele appears notoriously difficult because of limitations such as allelic heterogeneity. Re-sequencing based approaches may be required for elucidating the role of chromosome 19q13 with MS. This thesis has resulted in the identification of a new MS susceptibility locus (IRF5) previously associated with other inflammatory or autoimmune disorders, such as SLE. IRF5 is one of the mediators of interferons biological function. In addition to providing new insight in the possible pathogenetic pathway of the disease, this finding suggests that there might be common mechanisms between different immune-mediated disorders. Furthermore the work presented here has uncovered a novel isoform of ICOS, which may play a role in regulatory mechanisms of ICOS, an important mediator of lymphocyte activation. Further work is required to uncover its functions and possible involvement of the ICOS locus in MS susceptibility.

SPECT-aided diagnostics and genetic risk factors in Parkinson's disease

Hypokinesia, rigidity, tremor, and postural instability are the cardinal symptoms of Parkinson s disease (PD). Since these symptoms are not specific to PD the diagnosis may be uncertain in early PD. Etiology and pathogenesis of PD remain unclear. There is no neuroprotective therapy. Genetic findings are expected to reveal metabolic routes in PD pathogenesis and thereby eventually lead to therapeutic innovations. In this thesis, we first aimed to study the usefulness and accuracy of 123I-b-CIT SPECT in the diagnosis of PD in a consecutive clinic-based material including various movement disorders. We subsequently a genetic project to identify genetic risk factors for sporadic PD using a candidate gene approach in a case-control setting including 147 sporadic PD patients and 137 spouse controls. Dopamine transporter imaging by 123I-b-CIT SPECT could distinguish PD from essential tremor, drug-induced parkinsonism, dystonia and psychogenic parkinsonism. However, b-CIT uptake in Parkinson plus syndromes (PSP and multiple system atrophy) and dementia with Lewy bodies was not significantly different from PD. 123I-b-CIT SPECT could not reliably differentiate PD from vascular parkinsonism. 123I-b-CIT SPECT was 100% sensitive and specific in the diagnosis of PD in patients younger than 55 years but less specific in older patients, due to differential distribution of the above conditions in the younger and older age groups. 123I-b-CIT SPECT correlated with symptoms and detected bilateral nigrostriatal defect in patients whose PD was still in unilateral stage. Thus, in addition to as a differential diagnostic aid, 123I-b-CIT SPECT may be used to detect PD early, even pre-symptomatically in at-risk individuals. 123I-b-CIT SPECT was used to aid in the collection of patients to the genetic studies. In the genetic part of this thesis we found an association between PD and a polymorphic CAG-repeat in POLG1 gene encoding the catalytic subunit of mitochondrial polymerase gamma. The CAG-repeat encodes a polyglutamine tract (polyQ), the two most common lengths of which are 10Q (86-90%) and 11Q. In our Finnish material, the rarer non-10Q or non-11Q length variants (6Q-9Q, 12Q-14Q, 4R+9Q) were more frequent in patients than in spouse controls (10% vs. 3.5 %, p=0.003), or population controls (p=0.001). Therefore, we performed a replication study in 652 North American PD patients and 292 controls. Non-10/11Q alleles were more common in the US PD patients compared to the controls but the difference did not reach statistical significance (p=0.07). This larger data suggested our original definition of variant length allele might need reconsideration. Most previous studies on phenotypic effects of POLG1 polyQ have defined 10Q as the only normal allele. Non-10Q alleles were significantly more common in patients compared to the controls (17.3% vs. 12.3 %, p= 0.005). This association between non-10Q length variants and PD remained significant when compared to a larger set of 1541 literature controls (p=0.00005). In conclusion, POLG1 polyQ alleles other than 10Q may predispose to PD. We did not find association between PD and parkin or DJ-1, genes underlying autosomal recessive parkinsonism. The functional Val158Met polymorphism, which affects the catalytic effect of COMT enzyme, and another coding polymorphism in COMT were not associated with PD in our patient material. The APOE e2/3/4 polymorphism modifies risk for Alzheimer s disease and prognosis of for example brain trauma. APOE promoter and enhancer polymorphisms 219G/T and +113G/C, and APOE e3 haplotypes, have also been shown to modify the risk of Alzheimer s disease but not reported in PD. No association was found between PD and APOE e2/3/4 polymorphism, the promoter or enhancer polymorphisms, or the e3 haplotypes.

Genotype-Phenotype Relationships in Long QT Syndrome: Role of Mental Stress, Adrenergic Activity and a Common KCNH2 Polymorphism

Long QT syndrome is a congenital or acquired arrhythmic disorder which manifests as a prolonged QT-interval on the electrocardiogram and as a tendency to develop ventricular arrhythmias which can lead to sudden death. Arrhythmias often occur during intense exercise and/or emotional stress. The two most common subtypes of LQTS are LQT1, caused by mutations in the KCNQ1 gene and LQT2, caused by mutations in the KCNH2 gene. LQT1 and LQT2 patients exhibit arrhythmias in different types of situations: in LQT1 the trigger is usually vigorous exercise whereas in LQT2 arrhythmia results from the patient being startled from rest. It is not clear why trigger factors and clinical outcome differ from each other in the different LQTS subtypes. It is possible that stress hormones such as catecholamines may show different effects depending on the exact nature of the genetic defect, or sensitivity to catecholamines varies from subject to subject. Furthermore, it is possible that subtle genetic variants of putative modifier genes, including those coding for ion channels and hormone receptors, play a role as determinants of individual sensitivity to life-threatening arrhythmias. The present study was designed to identify some of these risk modifiers. It was found that LQT1 and LQT2 patients show an abnormal QT-adaptation to both mental and physical stress. Furthermore, as studied with epinephrine infusion experiments while the heart was paced and action potentials were measured from the right ventricular septum, LQT1 patients showed repolarization abnormalities which were related to their propensity to develop arrhythmia during intense, prolonged sympathetic tone, such as exercise. In LQT2 patients, this repolarization abnormality was noted already at rest corresponding to their arrhythmic episodes as a result of intense, sudden surges in adrenergic tone, such as fright or rage. A common KCNH2 polymorphism was found to affect KCNH2 channel function as demonstrated by in vitro experiments utilizing mammalian cells transfected with the KCNH2 potassium channel as well as QT-dynamics in vivo. Finally, the present study identified a common β-1-adrenergic receptor genotype that is related a shorter QT-interval in LQT1 patients. Also, it was discovered that compound homozygosity for two common β-adrenergic polymorphisms was related to the occurrence of symptoms in the LQT1 type of long QT syndrome. The studies demonstrate important genotype-phenotype differences between different LQTS subtypes and suggest that common modifier gene polymorphisms may affect cardiac repolarization in LQTS. It will be important in the future to prospectively study whether variant gene polymorphisms will assist in clinical risk profiling of LQTS patients.

Identification of genetic susceptibility loci for migraine

Migraine is the common cause of chronic episodic headache, affecting 12%-15% of the Caucasian population (41 million Europeans and some half a million Finns), and causes considerable loss of quality of life to its sufferers, as well as being linked to increased risk for a wide range of conditions, from depression to stroke. Migraine is the 19th most severe disease in terms of disability-adjusted life years, and 9th among women. It is characterized by attacks of headache accompanied by sensitivity to external stimuli lasting 4-72 hours, and in a third of cases by neurological aura symptoms, such as loss of vision, speech or muscle function. The underlying pathophysiology, including what triggers migraine attacks and why they occur in the first place, is largely unknown. The aim of this study was to identify genetic factors associated with the hereditary susceptibility to migraine, in order to gain a better understanding of migraine mechanisms. In this thesis, we report the results of genetic linkage and association analyses on a Finnish migraine patient collection as well as migraineurs from Australia, Denmark, Germany, Iceland and the Netherlands. Altogether we studied genetic information of nearly 7,000 migraine patients and over 50,000 population-matched controls. We also developed a new migraine analysis method called the trait component analysis, which is based on individual patient responses instead of the clinical diagnosis. Using this method, we detected a number of new genetic loci for migraine, including on chromosome 17p13 (HLOD 4.65) and 10q22-q23 (female-specific HLOD 7.68) with significant evidence of linkage, along with five other loci (2p12, 8q12, 4q28-q31, 18q12-q22, and Xp22) detected with suggestive evidence of linkage. The 10q22-q23 locus was the first genetic finding in migraine to show linkage to the same locus and markers in multiple populations, with consistent detection in six different scans. Traditionally, ion channels have been thought to play a role in migraine susceptibility, but we were able to exclude any significant role for common variants in a candidate gene study of 155 ion transport genes. This was followed up by the first genome-wide association study in migraine, conducted on 2,748 migraine patients and 10,747 matched controls followed by a replication in 3,209 patients and 40,062 controls. In this study, we found interesting results with genome-wide significance, providing targets for future genetic and functional studies. Overall, we found several promising genetic loci for migraine providing a promising base for future studies in migraine.

Genetic background of bipolar disorder and related cognitive impairments

Bipolar disorder (BP) is a complex psychiatric disorder characterized by episodes of mania and depression. BP affects approximately 1% of the world’s population and shows no difference in lifetime prevalence between males and females. BP arises from complex interactions among genetic, developmental and environmental factors, and it is likely that several predisposing genes are involved in BP. The genetic background of BP is still poorly understood, although intensive and long-lasting research has identified several chromosomal regions and genes involved in susceptibility to BP. This thesis work aims to identify the genetic variants that influence bipolar disorder in the Finnish population by candidate gene and genome-wide linkage analyses in families with many BP cases. In addition to diagnosis-based phenotypes, neuropsychological traits that can be seen as potential endophenotypes or intermediate traits for BP were analyzed. In the first part of the thesis, we examined the role of the allelic variants of the TSNAX/DISC1 gene cluster to psychotic and bipolar spectrum disorders and found association of distinct allelic haplotypes with these two groups of disorders. The haplotype at the 5’ end of the Disrupted-in-Schizophrenia-1 gene (DISC1) was over-transmitted to males with psychotic disorder (p = 0.008; for an extended haplotype p = 0.0007 with both genders), whereas haplotypes at the 3’ end of DISC1 associated with bipolar spectrum disorder (p = 0.0002; for an extended haplotype p = 0.0001). The variants of these haplotypes also showed association with different cognitive traits. The haplotypes at the 5’ end associated with perseverations and auditory attention, while the variants at the 3’ end associated with several cognitive traits including verbal fluency and psychomotor processing speed. Second, in our complete set of BP families with 723 individuals we studied six functional candidate genes from three distinct signalling systems: serotonin-related genes (SLC6A4 and TPH2), BDNF -related genes (BDNF, CREB1 and NTRK2) and one gene related to the inflammation and cytokine system (P2RX7). We replicated association of the functional variant Val66Met of BDNF with BP and better performance in retention. The variants at the 5’ end of SLC6A4 also showed some evidence of association among males (p = 0.004), but the widely studied functional variants did not yield any significant results. A protective four-variant haplotype on P2RX7 showed evidence of association with BP and executive functions: semantic and phonemic fluency (p = 0.006 and p = 0.0003, respectively). Third, we analyzed 23 bipolar families originating from the North-Eastern region of Finland. A genome-wide scan was performed using the 6K single nucleotide polymorphism (SNP) array. We identified susceptibility loci at chromosomes 7q31 with a LOD score of 3.20 and at 9p13.1 with a LOD score of 4.02. We followed up both linkage findings in the complete set of 179 Finnish bipolar families. The finding on chromosome 9p13 was supported (maximum LOD score of 3.02), but the susceptibility gene itself remains unclarified. In the fourth part of the thesis, we wanted to test the role of the allelic variants that have associated with bipolar disorder in recent genome-wide association studies (GWAS). We could confirm findings for the DFNB31, SORCS2, SCL39A3, and DGKH genes. The best signal in this study comes from DFNB31, which remained significant after multiple testing corrections. Two variants of SORCS2 were allelic replications and presented the same signal as the haplotype analysis. However, no association was detected with the PALB2 gene, which was the most significantly associated region in the previous GWAS. Our results indicate that BP is heterogeneous and its genetic background may accordingly vary in different populations. In order to fully understand the allelic heterogeneity that underlies common diseases such as BP, complete genome sequencing for many individuals with and without the disease is required. Identification of the specific risk variants will help us better understand the pathophysiology underlying BP and will lead to the development of treatments with specific biochemical targets. In addition, it will further facilitate the identification of environmental factors that alter risk, which will potentially provide improved occupational, social and psychological advice for individuals with high risk of BP.

Genetic studies on Finnish lupus erythematosus patients with cutaneous manifestations

Lupus erythematosus (LE) is a chronic, heterogeneous autoimmune disorder with abnormal immune responses, including production of autoantibodies and immune complexes. Clinical presentations of the disease range from mild cutaneous manifestations to a more generalised systemic involvement of internal organs. Cutaneous (CLE) forms are further subclassified into discoid LE (DLE), subacute cutaneous LE (SCLE) and acute cutaneous lupus erythematosus (ACLE), and may later progress to systemic disease (SLE). Both genetic and environmental factors contribute to the disease, although the precise aetiology is still elusive. Furthermore, complex gene-gene or gene-environment interactions may result in different subphenotypes of lupus. The genetic background of CLE is poorly known and only a few genes are confirmed, while the number of robust genetic associations in SLE exceeds 30. The aim of this thesis was to characterise the recruited patients clinically, and identify genetic variants conferring susceptibility to cutaneous variants of LE. Given that cutaneous and systemic disease may share underlying genetic factors, putative CLE candidate genes for genotyping were selected among those showing strong evidence of association in SLE. The correlation between relevant clinical manifestations and risk genotypes was investigated in order to find specific subphenotype associations. In addition, epistatic interactions in SLE were studied. Finally, the role of tissue degrading matrix metalloproteinases (MMP) in LE tissue injury was explored. These studies were conducted in Finnish case-control and family cohort, and Swedish case-control cohort. The clinical picture of the patients in terms of cutaneous, haematological and immunological findings resembled that described in the contemporary literature. However, the proportion of daily smokers was very high supporting the role of smoking in disease aetiology. The results confirmed that, even though clinically distinct entities, CLE and SLE share predisposing genetic factors. For the first time it was shown that known SLE susceptibility genes IRF5 and TYK2 also increase the risk of CLE. A tendency toward gene-gene interaction between these genes was found in SLE. As a remarkable novel finding, it was observed that ITGAM polymorphisms associated even more strongly to DLE than SLE, and the risk estimates were substantially higher than those reported for SLE. Several other recently identified SLE susceptibility genes showed signs of good or modest association especially in DLE. Subphenotype analyses indicated possible associations to clinical features, but marginally significant results reflected lack of sufficient power for these studies. Thorough immunohistochemical analyses of several MMPs demonstrated a role in epidermal changes and dermal tissue remodelling in diseased skin, and suggested that targeted action using selective MMP inhibitors may reduce lupus-induced damage in inflamed tissues. In conclusion, the results provide an insight into the genetics of CLE and demonstrate that genetic predisposition is at least in part shared between cutaneous and systemic variants of LE. This doctoral study has contributed IRF5, TYK2, ITGAM and several other novel genes to the so far short list of genes implicated in CLE susceptibility. Detailed examination of the function of these genes in CLE pathogenesis warrants further studies. Furthermore, the results support the need of subphenotype analysis with sample sizes large enough to reveal possible specific disease associations in order to better understand the heterogeneous nature and clinical specificities of the disease. Comprehensive analysis of clinical data suggests that smoking is an environmental triggering factor.

Timing of puberty : Genetic regulation

In the general population, the timing of puberty is normally distributed. This variation is determined by genetic and environmental factors, but the exact mechanisms underlying these influences remain elusive. The purpose of this study was to gain insight into genetic regulation of pubertal timing. Contributions of genetic versus environmental factors to the normal variation of pubertal timing were explored in twins. Familial occurrence and inheritance patterns of constitutional delay of growth and puberty, CDGP (a variant of normal pubertal timing), were studied in pedigrees of patients with this condition. To ultimately detect genes involved in the regulation of pubertal timing, genetic loci conferring susceptibility to CDGP were mapped by linkage analysis in the same family cohort. To subdivide the overall phenotypic variance of pubertal timing into genetic and environmental components, genetic modeling based on monozygous twins sharing 100% and dizygous twins sharing 50% of their genes was used in 2309 girls and 1828 boys from the FinnTwin 12-17 study. The timing of puberty was estimated from height growth, i.e. change in the relative height between the age when pubertal growth velocity peaks in the general population and adulthood. This reflects the percentage of adult height achieved at the average peak height velocity age, and thus, pubertal timing. Boys and girls diagnosed with CDGP were gathered through medical records from six pediatric clinics in Finland. First-degree relatives of the probands were invited to participate by letter; altogether, 286 families were recruited. When possible, families were extended to include also second-, third-, or fourth-degree relatives. The timing of puberty in all family members was primarily assessed from longitudinal growth data. Delayed puberty was defined by onset of pubertal growth spurt or peak height velocity taking place 1.5 (relaxed criterion) or 2 SD (strict criterion) beyond the mean. If growth data were unavailable, pubertal timing was based on interviews. In this case, CDGP criteria were set as having undergone pubertal development more than 2 (strict criterion) or 1.5 years (relaxed criterion) later than their peers, or menarche after 15 (strict criterion) or 14 years (relaxed criterion). Familial occurrence of strict CDGP was explored in families of 124 patients (95 males and 29 females) from two clinics in Southern Finland. In linkage analysis, we used relaxed CDGP criteria; 52 families with solely growth data-based CDGP diagnoses were selected from all clinics. Based on twin data, genetic factors explain 86% and 82% of the variance of pubertal timing in girls and boys, respectively. In families, 80% of male and 76% of female probands had affected first-degree relatives, in whom CDGP was 15 times more common than the expected (2.5%). In 74% (17 of 23) of the extended families with only one affected parent, familial patterns were consistent with autosomal dominant inheritance. By using 383 multiallelic markers and subsequently fine-mapping with 25 additional markers, significant linkage for CDGP was detected to the pericentromeric region of chromosome 2, to 2p13-2q13 (multipoint HLOD 4.44, α 0.41). The findings of the large twin study imply that the vast majority of the normal variation of pubertal timing is attributed to genetic effects. Moreover, the high frequency of dominant inheritance patterns and the large number of affected relatives of CDGP patients suggest that genetic factors also markedly contribute to constitutional delay of puberty. Detection of the locus 2p13-2q13 in the pericentromeric region of chromosome 2 associating with CDGP is one step towards unraveling the genes that determine pubertal timing.