Дисертації з теми "Genetic risk factor"

The aim of my work was to identify additional genetic and non-genetic factors influencing female reproductive ageing in humans. Although approximately 50% of population variation in age at menopause is due to genetics, less than 3% of variation had been accounted for by common genetic variants. Of non-genetic risk factors, only smoking had consistently been found to have a strong effect on age of menopause. In the wider context of female reproduction, our understanding of the role of genetics in determining sex hormone levels was limited. By combining the results of research in these different areas, I hoped to improve our knowledge of the biology of female reproductive ageing. Chapter 1 is an introduction in which I discuss the biology of menopause, describe relationships with health and present current knowledge regarding non-genetic and genetic risk factors influencing menopause age. Chapter 2 is an analysis of the associations between non-genetic risk factors occurring in early life with early menopause. We identified an association between multiple births and early menopause, connecting events pre-birth, when the oocyte pool is formed, with reproductive ageing in later life. Chapter 3 is a genome-wide association study to identify genetic variants associated with levels of nine sex hormone related phenotypes. We highlighted loci of relevance to reproductive function, which suggested overlaps in the genetic basis of hormone regulation. Chapter 4 is a genome-wide association study of menstrual cycle length. We showed that a common genetic variant related to follicle stimulating hormone levels and age at menopause is associated with several reproductive traits including length of menstrual cycle. Chapter 5 is an investigation of the relationship between differences in length of normal FMR1 triplet repeat alleles and timing of menopause. We found no association between the length of normal FMR1 alleles and timing of menopause, contradicting the results of smaller studies and replicating a null result in another large study. Chapter 6 is large genome-wide meta-analysis to identify common and low-frequency genetic variants associated with age at menopause. We identified 44 regions containing 54 independent common signals and two rare missense alleles of large effect. Finally, in Chapter 7 I evaluate how this work has benefitted our knowledge of female reproductive ageing and describe directions for future research.

" Alzheimer’s disease (AD) was identified more than a century ago. Yet, there is still no cure and the mechanisms behind the most common form of AD (late-onset, LOAD) are still an open question. BIN1 was the second gene most frequently associated with LOAD. Bin1 depletion has been linked with AD earliest pathomechanisms: increased beta-amyloid (Aβ) accumulation, endosomal abnormalities, and synaptic defects. Sequencing of BIN1 genomic locus identified regulatory and coding variants of BIN1, indicating that Bin1 correct levels and function are essential for a healthy brain. Two coding variants associated with LOAD (rs754834233, Bin1-PL, and rs138047593, Bin1-KR) lead to missense mutations in Bin1 protein. This work aimed to understand whether LOAD mutations in BIN1 result in Bin1 loss of function and contribute to LOAD early mechanisms. "
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Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2016.
The early detection of individuals who are at risk of developing type 2 diabetes mellitus (T2DM) would decrease the morbidity and mortality associated with this disease. DNA methylation, the most widely studied epigenetic mechanism, offers unique opportunities in this regard. Aberrant DNA methylation is associated with disease pathogenesis and is observed during the asymptomatic stage of disease. DNA methylation has therefore attracted increasing attention as a potential biomarker for identifying individuals who have an increased risk of developing T2DM. The identification of high risk biomarkers for T2DM could facilitate risk stratification and lifestyle interventions, which could ultimately lead to better ways to prevent, manage and control the T2DM epidemic that is rampant worldwide. The aim of the study was to investigate global DNA methylation as a potential risk factor for T2DM by studying the association between the global DNA methylation levels and hyperglycaemic states. A cross-sectional, quantitative study design, involving 564 individuals of mixed ancestry descent, residing in Bellville South, South Africa was used. Participants were classified as normal, pre-diabetic (impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT)) or diabetic (screen detected diabetic and known diabetics) according to WHO criteria of 1998. DNA was extracted from whole blood using the salt extraction method. The percentage global DNA methylation was measured by an enzyme-linked immunosorbent assay (ELISA). The association between global DNA methylation and hyperglycaemia, as well as other biochemical markers of T2DM was tested in a robust linear regression analysis adjusted for age, gender and smoking.

Retina is a unique site in the human body where the microcirculation can be imaged directly and non-invasively, allowing us to study in vivo the structure and pathology of the human microcirculation. Retinal images can be quantitatively assessed with computerized imaging techniques, enabling us to measure several different quantitative traits derived from the retinal vasculature. Arterial and venular calibres are the most extensively studied traits of the retinal microvasculature and numerous epidemiological studies demonstrated promising associations with systemic and ocular diseases as well as with disease markers. However, there has been a lack of research into pathophysiological processes leading to retinal vascular signs, and how they link retinal microcirculation with coronary and cerebral microvasculature change. Information about genetic determinants underlying retinal vascular structure is therefore important for understanding the processes leading to microvascular pathophysiology. Two genome wide association studies have been published so far revealing four loci associated with retinal venular calibre and one locus with arteriolar calibre. Here the results from the genome-wide association analysis of 10 different retinal vessel traits in two population based cohorts are presented. Retinal images were measured in non-mydriatic fundus images from 808 subjects in the Orkney Complex Disease Study (ORCADES) and 390 subjects from the Croatian island of Korcula, using the semi-automated retinal vasculature measurement programme SIVA and VAMPIRE. Using pairwise estimates of kinship based on genomic sharing, heritability was calculated for each trait. Estimates of tortuosity measure and fractal dimensions present first published reports of heritability estimates for those traits. In addition correlation analysis with systemic risk factor was also completed, confirming already published results as well as revealing some new associations. A genome wide association analysis of retinal arteriolar width revealed a genome wide significant hit (1.8x10-7) in a region of chromosome 2q32 (within TTN gene). Replication was sought in a further independent Scottish population (LBC) and additional 400 retinal images were graded. The result did not replicate, however the direction of the effect was consistent and a larger sample size is required. Analysis of the remaining traits did not yield genome wide significant result,s and will also require larger sample sizes. Genetic analysis of a binary retinal trait was also explored in a case control study of retinal detachment, which is an important cause of vision loss. A two-stage genetic association discovery phase followed by a replication phase in a combined total of 2,833 RRD cases and 7,871 controls was carried out. None of the SNPs tested in the discovery phase reached the threshold for association. Further testing was carried out in independent case-control series from London (846 cases) and Croatia (120 cases). The combined meta-analysis identified one association reaching genome-wide significance for rs267738 (OR=1.29, p=2.11x10-8), a missense coding SNP and eQTL for CERS2 encoding the protein ceramide synthase 2. Additional genetic risk score, pathway analysis and genetic liability analysis were also carried out.

Abstract Advances over the past few decades in ante- and neonatal care have led to the survival of a growing number of premature infants of extremely low gestational age. However, the occurrence of serious diseases, particularly those affecting the most immature infants, remains high. Bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants, is one such disease. Our current understanding of the molecular pathogenesis of BPD is incomplete; consequently, there are few preventive and therapeutic options for BPD. Moreover, it is challenging to predict the risk of BPD. Previous studies of BPD in twins revealed that the heritability of BPD is quite high. However, the individual genes that predispose premature infants to BPD are largely unknown. The aim of this study was to identify and study genes associated with BPD in order to investigate its pathogenesis. An additional aim was to add to knowledge of the risk of BPD in newborn premature infants, with an emphasis on twins. A candidate gene study found no consistent association between common polymorphisms of vascular endothelial growth factor receptor 2 and BPD. A second candidate gene study noted an association between the gene encoding Kit ligand and BPD. A genome-wide association study found a suggestive association between a locus close to the gene encoding C-reactive protein (CRP) and BPD, and in subsequent analyses, plasma levels of CRP during the first week of life predicted BPD. Finally, a nationwide register study found that the risk of BPD was lower in twins than in singletons. The results of this study add to what is known of the genetics and pathogenesis of BPD. They also provide new data on the risk of BPD, which may be used to improve early identification of infants for whom the risk of developing BPD is high
Tiivistelmä Ennenaikaisen syntymän ja keskoslasten hoidon kehittymisen myötä yhä useammat huomattavan epäkypsinä syntyneet lapset jäävät henkiin. Samalla erityisesti juuri näitä lapsia uhkaavien sairauksien esiintyvyys on pysynyt korkeana. Bronkopulmonaalinen dysplasia (BPD, keskosen krooninen keuhkosairaus) on yksi näistä sairauksista. BPD:n molekyylitasoinen tautimekanismi on vielä osin tuntematon, eikä BPD:tä tehokkaasti estävää tai siitä parantavaa hoitoa ole. Myös BPD riskin arvioiminen vastasyntyneen keskoslapsen kohdalla on vaikeaa. BPD on huomattavan perinnöllinen tauti. BPD:lle altistavista geeneistä on kuitenkin vasta vähän tietoa. Tämän tutkimuksen tavoitteena oli lisätä tietoa BPD:n tautimekanismista tutkimalla BPD:lle altistavia geenejä. Lisäksi tutkimuksessa tarkasteltiin BPD:n esiintyvyyttä ja syntymää edeltäviä riskitekijöitä erityisesti kaksosten osalta. Ehdokasgeenitutkimuksessa verisuonten endoteelikasvutekijää koodaava geeni ei assosioitunut toistuvasti BPD:hen. Kit ligandia koodaava geeni sen sijaan assosioitui. Koko genomin assosiaatiotutkimuksessa C-reaktiivista proteiinia (CRP) koodaavan geenin lähistöltä löydettiin BPD:hen mahdollisesti assosioituva alue. Lisäksi ensimmäisen viikon CRP-arvojen osoitettiin ennakoivan myöhemmin kehittyvää BPD:tä. BPD-riskin todettiin olevan matalampi kaksi- kuin yksisikiöisistä raskauksista syntyneillä lapsilla. Tutkimuksen tulokset lisäävät tietoa BPD:n perinnöllisyydestä ja sitä kautta BPD:n tautimekanismista. Tutkimus toi myös uutta tietoa BPD:n riskitekijöistä parantaen vastasyntyneen keskoslapsen BPD-riskin arviota

We carried out the first genome-wide linkage analysis in British families for VWF levels. ABO and VWF loci were specifically examined. The results showed that VWF levels are highly heritable 42% ( P>0.000001) with age and C-reactive protein (CRP) the main covariates. The ABO locus was strongly associated with VWF levels ( P=2x10-18), but linkage was modest (LOD = 1.6). VWF gene marker showed no significant association or linkage with phenotype. Genome-wide linkage analysis, conditioned on age and ABO genotypes, revealed regions with potential linkage. Highlighted regions were on chromosomes 2 and 3 (LOD = 1.78 & 1.08 respectively) and two areas on chromosome 12 (LOD = 1.5 & 1.3). Inflammatory biomarkers concentrations were also investigated. Heritabilities of CRP and tumour necrosis factor-alpha (TNF-alpha) were significantly high 38% and 47% respectively, while interleukin-6 was not heritable. VWF levels showed significant genetic correlation with CRP. As ABO group has a major role in determining VWF levels, if the VWF stroke association is causal, blood groups should be associated with stroke (Mendelian Randomisation). ABO genotype frequencies in stroke patients were investigated by a case-control study (503 objectively diagnosed cases and 327 controls). Non-O groups were not significantly over-represented in stroke cases (OR 1.1, CI95 0.83--1.47). Monte Carlo method, taking into account ABO effect on VWF levels, showed no association between ABO genotypes and stroke risk. This was reinforced by the findings of the systematic review we conducted on ABO groups and stroke. In contrast to results obtained from venous TE systematic review, where almost a two fold increased risk was found with non-O groups vs. O group.

Aim The aim of this study was to investigate the most commonly used approaches to measure individual genome-wide heterozygosity (IGWH) and to investigate whether IGWH can be considered as a health risk factor or a protective factor in humans. Methods This study was based on two samples from isolated communities of Croatian Adriatic islands, with a total of 1,930 adult examinees from Islands of Vis (N=986) and Korcula (N=944). Examinees were genotyped with a total of 302,662 single nucleotide polymorphisms. Heterozygosity was estimated using five commonly calculated methods. Results Correlation coefficients between different heterozygosity methods were generally in the range of 0.7-0.8. A worsening in some phenotypic traits, including cholesterol and triglycerides as well as increased odds for osteoporosis and metabolic syndrome was recorded in cases of IGWH reduction. Nevertheless, in these cases heterozygosity explained a relatively low amount of variance, generally in range of 0.4-0.6% of total trait variance. Conclusion However, these results were significant in Vis Island sample, while in the replication sample, Korcula Island, most of the associations were not significant, possibly due to the overall lower amount of inbreeding and higher heterozygosity in Korcula Island sample. The results warrant further research in order to provide more information on the extent and importance of individual genome-wide heterozygosity, which might have an important role in communities which experience consanguinity on a greater scale. Two main shortcomings of the study include possible lack of power to detect inbreeding depression and the need to replicate the results in other populations.

This thesis systematically investigated molecular risk and protective markers of Posttraumatic Stress Disorder (PTSD). PTSD poses a significant health and societal burden in Australia, especially in at-risk groups such as military personnel. The molecular aetiology of the disorder is poorly understood and it is unclear why many people recover quickly after trauma exposure while others continue to suffer. The thesis significantly contributes to the field of stress and resilience research by identifying novel markers for replication and adding to the knowledge-base of molecular markers for resilience, a burgeoning research field. This area of research is important to develop prevention and early intervention strategies.

Background: Lipoprotein(a) [Lp(a)] is composed of a low density-lipoprotein (LDL) particle and a glycoprotein molecule known as apolipoprotein(a) [apo(a)]. Apo(a) exists in several differently-sized isoforms and is responsible for the unique properties of Lp(a). Although Lp(a) has been known for the past 40 years its relationship with coronary heart disease (CHD) has not been characterized in sufficient detail. Whether Lp(a) causes CHD is not clear. Furthermore, the role of apo(a) isoform variation and other sources of Lp(a) heterogeneity (e.g., level of oxidized phospholipids) in Lp(a)-disease association has not been determined. Objectives: To characterize in detail the association of circulating Lp(a) levels with the risk CHD To assess the nature of Lp(a)-CHD association using an integrative genetic study To explore the role of Lp(a) heterogeneity in its association with CHD Data sources: 1. The Emerging Risk Factors Collaboration (ERFC) database (36 studies, 127,000 participants) 2. The European Prospective Investigation of Cancer – Norfolk (EPIC-Norfolk) study (2200CHD cases, 2200 controls) 3. The Pakistani Risk of Myocardial Infarction Study (PROMIS) (1800 MI cases and 1800 controls) 4. Systematic quantitative reviews of published epidemiological studies Results: ERFC data - Analyses of cross-sectional data on up to 127,000 participants (predominantly of European descent) demonstrated that Lp(a) is generally not strongly correlated with known CHD risk factors. Weakly positive correlations were observed with LDL-cholesterol, apolipoprotein B100 and fibrinogen. Levels were over 2-fold higher in Blacks compared to Whites. Analyses of available data on repeat measurements in 6600 participants demonstrated that Lp(a) values have very high long-term within-person consistency (regression dilution ratio ~ 0.9). Outcome data involved 9300 incident CHD events, 1900 ischaemic strokes and 8100 nonvascular deaths. The risk ratio for CHD per 1SD higher Lp(a) concentration, adjusted for age, sex, lipids and other conventional vascular risk factors, was 1.13 (95% CI, 1.09-1.18). The corresponding risk ratios for ischaemic stroke and nonvascular death were 1.10 (1.02 – 1.18) and 1.01 (0.98-1.05), respectively. Data were too limited to assess association in nonwhites. PROMIS data – the adjusted odds ratio for MI in South Asians was comparable to that of Europeans. EPIC-Norfolk genetic data - The odds ratio for CHD per 1-SD higher Lp(a) concentration, after adjustment for cardiovascular risk factors, was 1.37 (1.20-1.56). Tagging SNPs rs10455872 and rs11751605 (minor allele frequency: 8% and 18%, respectively) were associated with 207% (95% CI, 188 - 227%) and 38% (31 - 46%) higher Lp(a) concentrations per copy of minor allele, respectively. These SNPs accounted for 35% and 5% of the variation in circulating Lp(a) levels, respectively, and were associated with an odds ratio for CHD of 1.34 (1.14-1.58) and 1.17 (1.04-1.33), respectively. The observed SNP-CHD associations were consistent with expected odds ratios corresponding to the Lp(a) effect of the SNPs. Systematic reviews – meta-analysis of published data from 40 studies (11,300 cases, 47,000 controls) demonstrated that people with smaller apo(a) isoforms have about a 2-fold higher risk of CHD or ischemic stroke than those with larger isoforms. Meta-analysis of published data from 10 studies (1500 cases, 10,200 controls) showed that people in the top third of baseline distribution of oxidized LDL levels have a 1.8-fold higher risk of CHD than those in bottom third. EPIC-Norfolk biomarker data – Levels of oxidized phospholipids were strongly correlated with Lp(a) concentration (r = 0.7, p-value < 0.0001). One SD higher concentration of oxidized phospholipids was associated with an adjusted odds ratio for CHD of 1.31 (1.15-1.49). The risk ratio was no longer significant after adjustment for Lp(a) concentration (1.08; 95% CI, 0.91-1.29). Conclusion: Lp(a) concentration is specifically, continuously and independently associated with the risk of ischaemic vascular outcomes. Available evidence supports the causal role of the particle in CHD. Lp(a) appears to induce vascular damage through causal mechanisms that involve apo(a) isoforms and oxidized phospholipids. A comprehensive study of markers of Lp(a) heterogeneity should help to understand the full impact of Lp(a) on cardiovascular diseases. In addition, further study is needed in nonwhites to assess the relevance of the factor to vascular disease risk in these populations.

Thesis (Ph.D)--University of Tennessee Health Science Center, 2008.
Title from title page screen (viewed on January 6, 2009). Research advisor: Ann K. Cashion, Ph.D. Document formatted into pages (x, 102 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 81-88).

Psychiatric genetics is a basic science field that has potential for practical application and effective translation. To date, translational frameworks utilized by this field have been linear (e.g., sequential) in nature, focusing on molecular genetic information. It is proposed that non-linear (e.g., socio-ecological) frameworks are a better way to immediately translate non-molecular genetic information. This dissertation explored the translation of psychiatric genetic information in two ways. First, a survey was sent to academic stakeholders to assess the state of the science regarding the translation of genetic information to the clinical care of mental health disorders. Findings from this indicate a translation-genetic competence gap whereby genetic knowledge reinforces linear frameworks and genetic competence is needed to achieve effective translation in this content area. Second, a new risk factor model for social anxiety was created that incorporated genetic, environmental, and neurophysiological risk factors (behavioral inhibition, parental bonding, emotion reactivity). Findings indicate that genetic etiology is more informative knowledge that can influence risk factor models and possibly prevention and intervention efforts for social anxiety. Overall this dissertation paves the way for examining the translational capacity of psychiatric genetics in a clinical setting. It constitutes the first examination of barriers to and a potential solution for the most effective translation of psychiatric genetic information.

TDP-43, a RNA-binding protein (RBP) involved in different steps of RNA metabolism, forms pathological aggregates in affected tissues of the majority of amyotrophic lateral sclerosis (ALS) patients and of a subset of frontotemporal dementia (FTLD) cases. Our group has recently demonstrated that TDP-43 regulates the alternative splicing of several pre-mRNAs related to neuronal metabolism, including TNIK, encoding for a Ser/Thr kinase highly expressed in the brain. TDP-43 promotes the skipping of TNIK alternative exon 15, which encodes for a 29-amino-acid sequence in a region of the protein with unknown function. The TNIK gene was found to be genetically associated to psychiatric disorders, such as schizophrenia, as a risk factor, and to intellectual disability for null-mutations. In neurons, TNIK is involved in regulating different processes, including synapse formation, dendrite arborization and neurogenesis. In this study, our first aim was to investigate the regulation of TNIK exon 15 alternative splicing during neuronal differentiation in order to better characterize the role played by the ubiquitously expressed and ALS/FTLD-associated TDP-43 splicing factor in a neuronal context. Our second aim was to investigate if the specific expression of TNIK exon15-cointaining (TNIKex15) protein isoforms may be important to maintain TNIK physiological function in neurons, mainly focusing on its activity in neurite development. We first observed that TNIK exon 15 alternative splicing was differently regulated in human adult tissues and TNIKex15 isoforms were exclusively expressed in brain, spinal cord and skeletal muscle. Given the prevalent expression of TNIKex15 isoforms in the central nervous system, we further investigated this alternative splicing event in in vitro models to evaluate its regulation during the neuronal differentiation process. We found a significant increase of TNIKex15 transcripts in both SK-N-BE cells treated with retinoic acid and in human iPSCs differentiated into neurons. Moreover, TNIKex15 protein isoforms were specifically expressed in neuron-differentiated cells showing a prevalent perinuclear distribution in immunofluorescence analyses. Since we previously showed that TNIK exon 15 inclusion increases upon TDP-43 knock-down, we measured TDP-43 protein content during neuronal differentiation in vitro but we found no changes. We therefore investigated the possible involvement of the neuron-specific splicing factor NOVA-1, specifically expressed in our neuron-differentiated cells, in regulating TNIK processing. In HEK293T cells, NOVA-1 over-expression increased TNIK exon 15 inclusion without negatively affecting TDP-43 protein content compared to mock-transfected cells. Furthermore, by minigene splicing assays we evaluated the NOVA-1 interplay with TDP-43 in regulating TNIK exon 15 splicing. In this analysis, we also included hnRNPA2/B1, an ubiquitous RBP that co-operates with TDP-43 in regulating its splicing activity and that we found to promote TNIK exon 15 skipping, similarly to TDP-43. In competition assay with TDP-43 and hnRNPA2/B1, NOVA-1 completely abrogated their exon skipping activity on TNIK gene interacting with TDP-43 and hnRNPA2/B1 proteins in a RNA-dependent manner. As our results suggested a neuronal relevance for TNIKex15 protein isoforms, we further investigated the specific function of these isoforms in neurite development in murine primary cortical neurons. TNIKex15 over-expression negatively affected neurite development, reducing neurite number, as already describe in literature. When we analyzed TNIKex15-expressing neurons, we observed reduced filopodia number at growth cones, reduced soma area and filamentous actin (F-actin) levels compared to control cells. In contrast, upon TNIK exon 15-deleted (TNIKΔ15) over-expression, we found similar neurite development, soma and growth cones morphology and F-actin levels to control cells and also to TNIK KM, a TNIK mutant with a defective kinase activity. Moreover, TNIKex15 over-expression affected cell spreading in HEK293T cells that showed a prevalent round morphology compared to control cells. In contrast, TNIKΔ15-expressing cells presented an intermediate phenotype. In conclusion, our study has demonstrated that TNIKex15 isoforms are specifically expressed in neuronal tissues and during neuronal differentiation in vitro and that these isoforms show a specific function in regulating neurite development, influencing F-actin organization. Moreover, the neuron-specific splicing factor NOVA-1, probably promoting the formation of new ribonucleoprotein complex(es) with the ubiquitous splicing factors TDP-43 and hnRNPA2B1, acts as “silencer” of their splicing inhibitory activity on TNIK pre-mRNA. These results suggest that investigating NOVA-1/TDP-43 competitive mechanism also for other TDP-43 splicing targets may further help the understanding of TDP-43 splicing activity in a neuronal environment and in ALS/FTD diseases.

Introduction: Amyotrophic lateral sclerosis (ALS) is a deadly, progressive neuromuscular disease that affects individuals all over the world. About 10% of the patients have a familial predisposition (FALS) while the remainder of cases are isolated or sporadic (SALS) and of unknown cause. To date, the principal recognized risk factors for ALS are higher age, male gender, slim figure (BMI<23) and a family history of ALS. In 1993, Rosen et al. observed that some FALS cases were associated with mutations in the gene encoding the CuZn superoxide dismutase enzyme (SOD1). Since then, several mutations in the SOD1 gene have been discovered, and mutations in more than 18 other genes have been associated with causing ALS. The aim of this thesis was to identify new mutations associated with ALS pathogenesis, and by comparing patients from different countries, were we also able to identify population-specific genetic variations. The studies are referred to as I–V. Methods: With written informed consent and adhering to the tenets of the Declaration of Helsinki, through a national network of ALS clinicians´, venous blood samples were collected from ALS patients and healthy subjects in Europe and the USA. The patients were diagnosed according to the El Escorial criteria, and as having FALS according to the criteria of Byrne et al. (2011). The DNA variations were amplified by various PCR techniques. (I, III and IV) The amplicons of ataxin 2 (ATXN2), profilin 1 (PFN1), and vesicle-associated membrane protein type B (VAPB) were characterised by direct sequencing. (II) After quantitative PCR, a genotype-phenotype correlation was performed to assess whether the survival motor neuron gene (SMN) modulates the phenotype of ALS. (V) The amplicons of the 50 base pair deletion in the SOD1 promotor (50 bp) were separated by electrophoresis on agarose. Results: (I) We observed a significant association between CAG expansions in the ATXN2 gene and ALS in a European cohort. (II) Abnormal copy number of the SMN1 gene was identified as a risk factor in France, but not in Sweden. Homozygosity of the SMN2 deletion prolonged survival among Swedish ALS patients, compared to French patients. (III) We identified two mutations in the PFN1 gene, the novel p.Thr109Met mutation and the p.Gln117Gly mutation, in two unrelated FALS patients. (IV) In our cohort, we identified five VAPB mutations p.Asp130Glu, p.Ser160del, p.Asp162Glu, p.Met170Ile, and p.Arg184Trp, two of which are novel. (V) The 50 bp deletion upstream of the SOD1 gene was found in equal frequencies in both the patient and control cohorts. The 50 bp deletion did not affect SOD1 enzymatic activity. Furthermore, we found no differences in age of onset or disease duration in relation to the 50 bp deletion genotype.VI Conclusions: (I) Our findings indicate that ATXN2 plays an important role in the pathogenesis of ALS, and that CAG expansions in ATXN2 are a significant risk factor for the disease. (II) We suggest that abnormal SMN1 gene copynumber cannot be considered a universal genetic susceptibility factor for ALS. We also propose that the effect of abnormal SMN2 gene copy number on ALS phenotype may differ between populations. (III) This work provides evidence that PFN1 mutations can cause ALS as a Mendelian dominant trait. The novel p.Thr109Met mutation also shows that disturbance of actin dynamics can cause motor neuron degeneration. (IV) We find it unlikely that the VAPB mutations cause ALS in our cohorts. (V) We find it unlikely that the 50 bp region contains important regulatory elements for SOD1 expression. This thesis supports the theory that ALS is a multigenetic disease, but there appears to be great genetic variation among apparently identical populations. These studies emphasise the importance of continuous genetic screening, to identify further mutations and genes involved in ALS disease, but it also highlights the importance of cooperation and comparison between countries.
On the aetiology of ALS: A comprehensive genetic study

The age range at which type 2 diabetes develops has recently expanded. Thus chronological age and age of onset are now important variables in this already heterogeneous disease. The increasing prevalence of early-onset diabetes in particular raises clinical and societal concerns. Such individuals have a longer life-time disease duration and potentially can develop more diabetes related complications, at a relatively young age, perhaps during the most productive periods of their lives. The determinants of, and impact on outcome of age of onset as a clinical variable are unclear. Therefore studies using both clinical data and molecular techniques are employed to answer questions in this area. Whether the factors that impact on the development of type 2 diabetes are specifically different for those with younger onset as compared to older onset disease is not clear. By way of three studies, the first part of this thesis explores the general question of ‘what determines the age of type 2 diabetes onset?’ The specific impact of the metabolic syndrome, insulin resistance and body weight on the age of type 2 diabetes onset is examined in two different ethnic groups. This is in recognition of the paucity of specific data in this area and the differing prevalence of the metabolic syndrome in different ethnicities. This is examined in Chapter 2. The ‘accelerators’ of disease onset were quite different, dependent on ethnicity. Weight, insulin resistance and a high prevalence of the metabolic syndrome are associated with early-onset disease in Anglo-Celtics, but not so in Chinese. The mechanistic and public health implications of these observations are discussed.

Type 2 Diabetes (T2D) disproportionally affects ethnic minorities in the United States. The development of T2D involves complex interaction between environmental factors and genetic predisposition. The genetic associations of six single nucleotide polymorphisms (SNPs) in TCF7L2 gene with the risk of T2D were evaluated in three high risk minority populations: Cuban Americans, Haitian Americans, and African Americans. For Cuban Americans, four SNPs (rs7901695, rs4506565, rs7903146 and rs11225537) were significantly associated with the risk of T2D after multivariable adjustment (p=0.018, p=0.016, p=0.014, and p=0.0008, respectively). Among controls, risk allele carriers of SNPs rs7901695, rs4506565 and rs7903146 had significantly higher fasting glucose level, compared to non-risk allele carriers. Additionally, a significant interaction between dietary fiber intake and SNP rs7903146 for the risk of T2D (p= 0.044) was found in Cuban Americans. Similarly, for SNP rs7901695, significant interaction was also found for fiber intake (p=0.014) as well as glycemic load (p=0.040). Subgroup analysis revealed that significant associations were only found within higher intake groups of dietary factors for both SNPs. For Haitian Americans, SNPs rs11196205 (p=0.059) and rs7895340 (p=0.069) showed marginal significance for the risk of T2D. After stratification by gender, SNPs with marginal significance from the gender-combined analysis became statistically significant with the same trend for the risk of T2D when analysis were done in males: rs11196205 (p=0.034) and rs7895340 (p=0.024). For African Americans, SNP rs7903146 (p=0.065) showed a marginal significance with the risk of T2D in gender-combined analysis and a statistical significance (p=0.013) in males. Two additional SNPs rs7901695 and rs4506565 were found to be significantly associated with the risk of T2D in males. Risk allele carriers of these two SNPs had significantly higher mean level of the fasting glucose level, compared to non-risk allele carriers in controls. T2D related quantitative trait analysis also demonstrated that in controls, compared to non-minor allele carriers of SNP rs12255372, minor allele carriers had significantly higher means of BMI, diastolic blood pressure, numbers of components of Metabolic Syndrome, significantly lower mean values of HDL-cholesterol and adiponectin. Taken together, our studies demonstrated ethno-specific genetic associations between TCF7L2 gene and the risk of T2D and related phenotypes.

To identify the risk factors being associated with aetiology of amyotrophic lateral sclerosis (ALS), a series of systematic reviews based on existing observational epidemiological studies identified through searching of bibliographic databases were conducted. Associations between ALS and a number of genetic and environmental risk factors were examined using meta-analysis. Specifically we found that previous exposure to lead, pesticides, solvents, experience of trauma and electric shock were associated with relative increased risks of developing ALS of 86% [odds ratio (OR) =1.86, 95% CI: 1.39-2.48], 57% (OR=1.57,95% CI: 1.19-2.08), 47% (OR=1.47, 95%CI: 1.13-1.80), 64% (OR=1.64; 95%CI: 1.36-1.98), and 2.27% (OR=3.27, 95%CI:1.87-5.73) respectively, compared to their corresponding controls. The presence of intermediate CAG repeat expansion in the ATXN2 gene was associated with a 4.4 -fold increase in the risk of ALS (OR=4.44, 95%CI: 2.91-6.76). However, the attributable risk associated with each identified risk factor was estimated to be less than 5% of all ALS cases. These results confirm that ALS is a rare multifactorial degenerative condition of motor-neurons.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer accounting for 80% of childhood leukemia. The uncontrolled proliferation of lymphoid progenitors in the bone marrow and the accumulation of malignant lymphoblasts in peripheral blood characterize the disease. The molecular analysis of common genetic alterations in lymphoblastic cells has strongly contributed to the comprehension of ALL pathogenesis. Different gene polymorphisms (most of them SNPs) play an important role in the susceptibility to childhood ALL which probably derives from a combination and relation of genetic and environmental factors. Folic acid and the pool of folate of the one carbon-metabolic-pathway are key elements involved in several processes including DNA synthesis and methylation. Polymorphisms in genes coding for enzymes of the folate metabolic pathway can alter the intracellular folate status or distribution and sub-optimal/anomalous folate levels/distribution increase the risk of developing several neoplasias. The two main enzymes involved in cyclization of folate isoforms are DHFR and MTHFR. The first one is responsible for the conversion of dihydrofolate to tetrahydrofolate whilst the second one catalyzes the reduction of 5,10-methylene-THF to 5-methyl-THF. Studies have demonstrated that subjects with the homozygous DD-genotype have higher DHFR mRNA levels that may be responsible for storage of THF and other isoforms within the cell; reduced enzyme activity instead, has been associated to MTHFR 677TT homozygotes. This condition is responsible for an underutilization of methylene-THF in the cell with storage of folate reduced isoforms. Several studies have demonstrated that DHFR and MTHFR polymorphisms may be protective against hematological cancers such as ALL. It is to note that current treatment regimens achieve levels about 80% in overall survival (OS). Unfortunately, the side effects derived from the chemotherapeutic agents used can be severe, especially for high-risk patients. Therefore, the identification of additional markers which can improve risk stratification and individual tailored therapy regimens would be a great goal, in order to avoid over-treatment which can increase long-term adverse side effects. The aim of the present study was, therefore, to investigate whether common polymorphisms (i.e. MTHFR C677T and A1298C in addition to DHFR 19 bp INS/DEL and Bcl-2 -938 C>A) might influence the risk of childhood ALL. After a single analysis we can ascribe to MTHFR C677T gene polymorphism a protective significant role against childhood ALL (P=0.046), whilst Bcl-2 -938 C>A gene polymorphism seems to be a risk factor for the susceptibility to the disease (P=0.049). Then, considering parameters such as disease onset and therapy duration we can observe a significant higher mean age onset disease for homozygotes MTHFR 1298-CC (P=0.05). From the analysis of the therapy duration we found a significant association for MTHFR A1298C and Bcl-2 -938 C>A polymorphisms: MTHFR 1298CC homozygotes showed a slight higher mean therapy duration (P=0.05), as well as Bcl-2 -938AA homozygotes (P=0.03). Finally, in an exploratory way, to validate the proposed model we evaluated among healthy PBL cells harvested from subjects with opposite genotype condition considering DFHR and MTHFR genes (respectively, WW/CC and DD/TT), possible differences in base-line cellular viability and under MTX treatment. The pharmacological induced restriction in folate availability (MTX) yields to results in favor of WW/CC, whilst the base-line cell viability yields comparable results among genotypes. This is in line with the hypothesis that a higher MTX level could be present in DD/TT cells, prone to storage either natural folate isoforms (useful for the cell viability) or synthetic toxic analogue (MTX). This observation argues us into hypothesizing that also MTX being itself a synthetic folate analogue follows the same handling process. Now, inside the cell it should result in an elevated toxicity level, being responsible for an elevated death.

Background: Multiple lines of evidence implicate the insulin-like growth factor(IGF) group of proteins in human type 2 diabetes. The actions of IGF-I and IGF-IIare modulated through their interaction with IGF binding proteins. A holisticapproach to study the IGF system is preferable to analyses of individual proteininteractions as the inter-relationships between these proteins are complex. Inparticular, the associations of IGF-II and its associated binding proteins withcardiovascular risk have been inadequately studied. This study aimed to study indetail the genotype and phenotype interactions of the IGF system with longitudinalcardiovascular risk factor trends and phenotypic outcomes in type 2 diabetes.Methods: 1000 subjects of predominantly Caucasian origin from the SalfordDiabetes Cohort were studied. Measurements of IGF proteins (IGF-I, IGF-II,IGFBP-1, IGFBP-2 and IGFBP-3) were performed in 554 of these patients. 991Caucasian subjects were successfully genotyped for 76 single nucleotidepolymorphisms (SNPs) related to ten genes in the IGF system. In this project weanalysed associations of the studied SNPs with the measured IGF proteins as well aslongitudinal risk factor trends. In addition, the baseline concentrations of themeasured proteins were studied for associations with cardiovascular risk factortrends and vascular outcomes.Results: This project demonstrates for the first time that high serum IGF-IIconcentration at baseline predicts longitudinal increases in high-density lipoproteincholesterol. High baseline IGF-II was also observed to predict longitudinal weightloss. High baseline concentration of IGFBP-2 (which has a preferential associationof IGF-II over IGF-I) was associated with a number of favourable longitudinalcardiovascular risk trends like increased HDL cholesterol and decreased diastolicblood pressure. However high IGFBP-2 was also associated with deterioration inrenal function and increased all-cause and cardiovascular mortality. The IGF2 geneand the genes encoding IGFBP-2 and IGFBP-5 (proteins with IGF-II bindingaffinity) were also associated with longitudinal trends in renal function, bloodpressure and cholesterol concentration.Discussion: This study is the most detailed exploration to date of the genotype andphenotype interactions of the IGF system in a Caucasian population with type 2diabetes. Results from this study strongly hint that changes in IGF-II bioavailabilitymay influence inter-individual variations in cardiovascular risk. The precisebiological role of IGF-II merits clarification in future expression studies in renal,adipose and vascular tissues. Replication of significant results in an independentdiabetes cohort and measurement of other IGF binding proteins will be performed inthe next stage of this study.

Genetic variants and traits in metabolic signaling pathways may interact with lifestyle factors such as obesity, physical activity, and exogenous estrogen (E), influencing postmenopausal colorectal cancer (CRC) risk, but these interrelated pathways are not fully understood. In this case-cohort study, we examined 33 single-nucleotide polymorphisms (SNPs) in genes related to insulin-like growth factor-I (IGF-I)/insulin resistance (IR) traits and signaling pathways, using data from 704 postmenopausal women in Women's Health Initiative Observation ancillary studies. Stratifying by the lifestyle modifiers, we assessed the effects of IGF-I/IR traits (fasting total and free IGF-I, IGF binding protein-3, insulin, glucose, and homeostatic model assessment-insulin resistance) on CRC risk as a mediator or influencing factor. Six SNPs in the INS, IGF-I, and IGFBP3 genes were associated with CRC risk, and those associations differed between non-obese/active and obese/inactive women and between E nonusers and users. Roughly 30% of the cancer risk due to the SNP was mediated by IGF-I/IR traits. Likewise, carriers of 11 SNPs in the IRS1 and AKT1/2 genes (signaling pathway-related genetic variants) had different associations with CRC risk between strata, and the proportion of the SNP-cancer association explained by traits varied from 30% to 50%. Our findings suggest that IGF-I/IR genetic variants interact with obesity, physical activity, and exogenous E, altering postmenopausal CRC risk, through IGF-I/IR traits, but also through different pathways. Unraveling gene-phenotype-lifestyle interactions will provide data on potential genetic targets in clinical trials for cancer prevention and intervention strategies to reduce CRC risk.

Variantes genéticas trombogênicas podem aumentar o risco de eventos adversos em pacientes com coronariopatia crônica. Estudos prévios demonstraram que o Haplótipo H2 do gene do receptor P2Y12 apresenta uma maior agregação plaquetária e está associado com a presença de isquemia arterial periférica. A metaloprotease ADAMTS13 é responsável pela clivagem do fator de von Willebrand e recentemente foi associada com doença isquêmica coronariana. O objetivo deste trabalho foi avaliar o efeito das variantes genéticas funcionais trombogênicas dos Haplótipos H1 e H2 do receptor plaquetário P2Y12 e dos polimorfismos C1342G (Q448E), C1852G (P618A) e C2699T (A900V) da metaloprotease ADAMTS13 em 611 pacientes com doença arterial coronariana multiarterial com função ventricular preservada, acompanhados por um período de 05 anos no ensaio clínico do projeto MASS II (Medical, Angioplasty, or Surgery Study II) em relação aos eventos morte, infarto agudo do miocárdio, angina refratária necessitando um novo procedimento e acidente vascular cerebral. Neste estudo, a avaliação dos Haplótipos H1 e H2 nos pacientes do MASS II não encontrou diferença entre estes haplótipos e os eventos estudados. A análise dos polimorfismos da ADAMTS13 não encontrou associação entre os polimorfismos e os eventos estudados, exceto para a variante genética T2699 (Val900) que está associada com o evento morte (OR: 1,67 95%IC: 1-2,78, p= 0,049) e morte por causa cardiovascular (OR: 2,23 95%IC: 1,2-3,94, p=0,004) e apresenta uma diminuição na sobrevida livre de morte por causa cardíaca para os portadores do genótipo TT relacionado à este polimorfismo. A análise dos haplótipos e das combinações alélicas destes polimorfismos não apresentou associação com eventos ou com a sobrevida livre dos eventos nestes pacientes.
Thrombotic genetic variants could improve the risk of adverse events related to coronary arterial disease (CAD). P2Y12 platelet receptor H2 haplotype showed higher aggregation index and a positive association was described between such genetic variant and peripheral artery disease. DAMTS13 is a metaloprotease responsible to von Willebrand factor cleavage recently found correlated to CAD. We tested the genetic variants P2Y12 receptor H1 and H2 haplotypes and ADAMTS13 polymorphisms C1342G (Q448E), C1852G (P618A) and C2699T (A900V) in a group of 611 patients enrolled in the Medical, Angioplasty, or Surgery Study II (MASS II), a randomized trial comparing treatments for patients with coronary artery disease (CAD) and preserved left ventricular function in a follow up period of 05 years. The incidence of the end points of death and death from cardiac causes, myocardial infarction, refractory angina requiring revascularization and cerebrovascular accident was determined for P2Y12 H1 and H2 haplotypes and ADAMTS polymorphisms. In our study, we did not disclose any association between H1 or H2 haplotype groups regarding the incidence of any of the studied cardiovascular end-points. The association of ADAMTS13 genotypes and cardiovascular events did not showed any association between C1342G (Q448E), C1852G (P618A) variants and cardiovascular end points. Our date provide a strong association between T2699 variant and increased risk to death (OR: 1,67 CI: 1-2,78, p= 0,049) and cardiac death (OR: 2,23 CI: 1,2-3,94, p=0,004) in a population with CAD. The allelic combinations and haplotypes obtained from ADAMTS13 polymorphisms were not associated to cardiac end points and survival differences between MASS II patients.

Oncogenic human papillomaviruses (HPVs) are implicated in 99.7 % of cervical cancer cases but require the co-operation of other factors. To investigate potential genetic risk factors we have typed the HLA class II DRB1 and DQB1 loci in 478 women diagnosed with cervical carcinoma in situ and in 608 age-matched controls. Quantitative measurements of HPV 16, HPV 18/45 and HPV 31 were obtained. The DRB1*1501 and DQB1*0602 alleles were found to increase the risk of HPV 16 infection. Carriers of DRB1*1501 and DQB1*0602 were also shown to have an increased risk of a higher viral load compared to non-carriers. The DRB1*1301 and DQB1*0603 alleles were found to protect from HPV 18/45 and 31 infections as well as resulting in a lower viral load in carriers compared to non-carriers. Women with a high HPV 16, 18/45 or 31 viral load were more prone to long-term infections and women with a low HPV 16 viral load were more prone to short-term infections. Carriers of DRB1*1501 and DQB1*0602 alleles were also shown to have an increased risk of long-term infections compared to short-term infections. We also tested if an HPV susceptibility locus found for epidermodysplasia verruciformis (EV) was also linked to HPV susceptibility in cervical cancer. We did not find any linkage to this locus in a set of 77 families, each with at least three cases diagnosed with cervical carcinoma in situ. Other potential risk factors tested were HPV 16 E6 variants together with a p53 codon 72 polymorphism and HLA class II alleles. We found an association between the E6 L83V variant and the HLA DR4-DQ3 haplotype, as well as an increased frequency of Arg homozygosity of p53 in women infected with the L83V variant. These results show that alleles at HLA class II loci represents risk factors for persistent HPV infection and thereby also contribute to the risk of development of cervical carcinoma in situ.

Abstract Low back pain (LBP) is the leading cause of years lived with disabilities worldwide. Intervertebral disc (IVD) degeneration is a strong contributing factor to LBP. Recent studies have shown that genetic determinants contribute markedly to IVD degeneration but knowledge about the actual genes involved as well as their roles is still limited. The aim of this thesis work was to study genetic factors that may predispose to IVD degeneration. Using both family and case-control association study designs, variants in five genes showed association with IVD degeneration on magnetic resonance imaging (MRI) in a population-based sample and among patients with sciatica due to lumbar disc herniation (LDH). We performed a candidate gene association study of the known variants implicated in IVD degeneration in a Finnish cohort of 538 young individuals with a moderate degree of lumbar IVD degeneration on MRI. We were able to confirm the associations of variants in the IL6, SKT, and CILP genes, which provides further evidence for true associations. Based on our earlier linkage study in Finnish sciatica families, we performed a candidate gene analysis and identified IL17F as a potential candidate gene. To the best of our knowledge this is the first study to observe an association between this gene and discogenic sciatica. Both IL-6 and IL-17 are pro-inflammatory cytokines with elevated expression levels in herniated tissues, which suggest a role in IVD degeneration. Study of the role of genes coding for inflammatory mediators is of interest as it may contribute to the understanding of the overall inflammatory response of the disc. In addition, we reported on the involvement of SKT in the etiology of lumbar disc herniation (LDH) both in Japanese and Finnish case-control samples. Experimental studies in mice have shown that Skt homozygous mutants exhibit disc abnormalities resulting in a kinky tale phenotype. We hypothesized that the human homolog SKT could have long-term importance in the onset of IVD degeneration by making the discs more vulnerable. Finally, through linkage studies and in the subsequent association analyses, the role of CHST3 as a novel risk factor for IVD degeneration was identified. CHST3 encodes an enzyme that catalyzes the sulfation of chondroitin, and mutations in this gene are associated with spondylepiphyseal dysplasia and humerospinal dysostosis. In our study, we identified this gene using genome –wide linkage based on data from a Southern Chinese family and speculated that mild CHST3 reduction caused by the reported susceptibility SNP could result in disc degeneration in adults in conjunction with other risk factors. This thesis provides new information about the genetic background of IVD degeneration and new insights into the etiology of the disease. The specific roles of these genes in the IVD function and pathogenesis of sciatica are not clear however, and need to be elucidated
Tiivistelmä Alaselkäkipu on yksi yleisimmistä sairauksista ja johtava syy työkyvyttömyyteen. Välilevyrappeuma myötävaikuttaa merkittävästi alaselän kipuun. Vaikka aiemmat tutkimukset ovat osoittaneet, että perintötekijöillä on vahva osuus välilevyrappeumaan, altistavat geenit ja niiden rooli tunnetaan huonosti. Tämän tutkimuksen tavoitteena oli arvioida tiettyjen perintötekijöiden osuutta välilevyrappeumassa ja tunnistaa taudille altistava geeni perheaineistossa aiemmin havaitulta kromosomialueelta. Aineistoina tutkimuksessa olivat perheaineistot sekä laajat potilas-kontrolliaineistot suomalaisesta ja aasialaisista väestöistä. Tutkimuksessa osoitimme, että perimän vaihtelut viidessä tutkitussa geenissä altistivat erilaisille välilevyrappeuman taudin muodoille. Tutkimus, jossa analysoimme aiemmin tunnistettuja alttiusgeenejä, vahvisti IL6, SKT ja CILP geenien vaihteluiden osuuden taudin alttiustekijöinä. Tutkimusaineistona oli pohjoissuomalainen syntymäkohortti, jossa välilevyrappeuma oli määritetty magneettikuvauksella (MRI). Suomalaisessa perheaineistossa tehdyn kokogenomin laajuisen kartoituksen pohjalta analysoimme IL17F geenin mahdollisena uutena alttiusgeeninä oireiselle välilevytaudille. Kahdesta geenin variantista koostuva haplotyyppi assosioitui tautiin merkitsevästi. Lisäksi osoitimme, että SKT-geenin tietty muutos altistaa välilevyn pullistumille sekä japanilaisessa että suomalaisessa potilasaineistossa. Hiirikokeissa on havainnoitu, että SKT-geenin homotsygootti mutaatio johtaa välilevy-poikkeamaan, joka edelleen aiheuttaa hiiren poikkeavan häntäilmiasun-. Hypoteesimme oli, että ihmisen SKT -geeni voi myötävaikuttaa välilevypullistuman kehittymiseen altistamalla välilevyt rappeumalle. Edelleen, laajassa usean populaation aineiston käsittävässä tutkimuksessa osoitimme CHST3-geenin muutoksen altistavan välilevyrappeumalle. Peittyvästi periytyvät muutokset tässä geenissä aiheuttavat perinnöllisiä harvinaisia luusairauksia. Tämä väitöstutkimus tarjoaa uutta tietoa välilevyrappeuman geneettisestä taustasta ja auttaa taudin syiden tutkintaa. Geenien rooli välilevyn toiminnassa ja muutosten vaikutus taudin kulkuun vaativat kuitenkin vielä lisätutkimuksia

Includes bibliographical references.
Carpal tunnel syndrome (CTS) is a common occupational injury that is caused by an increase in pressure within the carpal tunnel structure which, in turn, causes compression of the median nerve. Although several factors are believed to be associated with increased risk of CTS, the direct causes of this injury remain unknown and it is generally accepted that CTS, with the exception of acutely caused CTS, is a multifactorial condition. Although it is generally accepted that an increase in pressure within the carpal tunnel structure, which contains nine flexor tendons, causes compression of the median nerve, the involvement of these tendons and other connective tissue structures in the aetiology of CTS cannot be excluded. In support of this, pathology of these connective structures have been proposed as being comorbid conditions or a precursor of CTS, cause CTS and/or can lead to an increase in carpal tunnel pressure. Several studies have suggested that specific non-occupational risk factors, such as anatomical, systemic and chronic factors as well as mostly repetition- and force-related occupational risk factors are associated with CTS. Although genetic influences in the aetiology of CTS have been proposed, this area has received little attention. Common DNA sequence variants on the other hand have previously been reported to associate with common exercise-associated tendon, such as chronic Achilles tendinopathy, and ligament injuries. The aim of this thesis was to determine whether common DNA sequence variants within several genes that have been associated or implicated in the aetiology of exercise-related musculoskeletal soft tissue injuries, are associated with altered risk of CTS by using a genetic association case-control study approach.

The present study evaluated genetic and metabolic factors influencing the risk of acute cerebrovascular disease (CVD) and internal carotid artery stenosis (ICA stenosis) in a Swedish community. The threonine (T) containing protein of the FABP2 A54T gene polymorphism has a greater affinity for long chain fatty acids (FFAs) than the alanine (A) containing protein. This altered affinity for FFAs has been shown to affect the intestinal absorption of fatty acids and consequently the fatty acid composition of serum lipids, in particularly postprandially. Endothelium derived NO is a potent vasodilator and antiatherogenic agent. Asymmetric dimethyl arginine (ADMA) is an endogenous competitive inhibitor of endothelial nitric oxide synthase (eNOS). ADMA has been shown to be involved in the pathogenesis of atherosclerotic disease, and ADMA inhibits eNOS by displacement of L-arginine from the enzyme, which in turn is believed to affect the amount of NO available within the endothelium. The FABP2 A54T gene polymorphism was analyzed in 407 patients with acute CVD and also in a subset of these patients whose carotids had been evaluated with ultrasound. Both the FABP2 polymorphism and a common polymorphism of the eNOS gene, Glu298Asp, were analyzed in a different population consisting of 54 matched pairs of patients with ICA stenosis and controls. ADMA levels were measured in both study populations. We found that the T54 allele was more frequent in patients with transient ischaemic attacks (TIA), and that the TT genotype was more prevalent in young, non-smoking patients with CVD than in controls. Increased concentrations of ADMA were observed in cardio-embolic infarction and TIA, but not significantly in non-cardio-embolic infarction nor in haemorrhagic stroke. In multivariate logistic regression models, CVD increased across quartiles of ADMA in all subgroups, but this association was only significant in the TIA group. A decreased arginine/ADMA ratio, a measure of NO availability was associated with CVD in the entire study population. Patients with severe carotid stenosis had significantly higher ADMA levels than the controls. Allele and genotype frequencies of the FABP2 and eNOS polymorphisms did not differ between patients with ICA stenosis and controls. Our results indicate that ADMA is a strong marker for TIA and severe ICA stenosis, and that relative defiency of arginine, measured as L-arginine/ADMA, is present in acute CVD. We also conclude that a common polymorphism of the FABP2 gene increases susceptibility to ischaemic stroke and TIA.
Figure 4 on page 17 is publshed with kind permisson from The Journal of Physiology (http://jp.physoc.org/).

La present tesi és una aportació a el coneixement de nous factors de risc per al tumor testicular de cèl·lules germinals (TTCG). El TTCG presenta una etiologia multifactorial, atribuïble a un retard en la diferenciació dels gonocitos fetals. El TTCG és més freqüent en homes amb una espermatogènesi alterada, suggerint una possible etiopatogènia comuna. El cromosoma I conté gens essencials per a una correcta espermatogènesi, les regions de l'factor d'azoospèrmia (AZF). La regió AZF més dinàmica és la regió AZFc que presenta punts fràgils que predisposa a reordenaments. El reordenament parcial més rellevant des del punt de vista clínic de la regió AZFc és la deleció gr / gr. S'ha relacionat la delació gr / gr amb un major risc de desenvolupar un TTCG, però la falta d'informació sobre els paràmetres seminals dels pacients no ha permès d'aclarir si l'associació observada està relacionada amb l'espermatogènesi alterada o si és un factor de risc independent. A més, encara queda per establir si altres tipus de delecions i duplicacions de la regió AZFc presenten relació amb el TTCG. La primera part d'aquesta tesi s'enfoca en l'estudi dels reordenaments parcials de la regió AZFc al TTCG. S'han analitzat 497 pacients amb TTCG i 2030 controls sense TTCG. Un 3.8% dels pacients amb TTCG presentaven algun tipus de deleció parcial de la regió AZFc respecte a l'2.5% de el grup control (p = 0.078). La deleció parcial més freqüent va ser la deleció gr / gr, mentre que els altres tipus de delecions parcials de la regió AZFc van resultar ser molt rares. Segons el fenotip seminal, es va observar un major risc de TTCG en pacients normozoospérmicos portadors de delecions parcials de la regió AZFc respecte als controls normozoospérmicos. No hi va haver diferències significatives entre pacients i controls segons les duplicacions parcials de la regió AZFc. Es va mostrar que les alteracions en la dosi de el gen DAZ confereixen un major risc de TTCG. Aquests resultats confirmen que un dèficit de l'contingut gènic de la regió AZFc juga un paper important en la etiopatogènesi de l'TTCG. En particular, la deleció gr / gr confereix un risc significatiu per al desenvolupament de l'TTCG independentment dels paràmetres seminals. Els factors ambientals també estan involucrats en la etiopatogènesi de l'TTCG, especialment si interfereixen en un període específic de el desenvolupament testicular, en el denominat ""masculinization programming window"" (MPW). Un desequilibri hormonal en aquest període compromet a la correcta funció de les cèl·lules fetals de Sertoli i Leydig, originant la síndrome de disgenèsia testicular (SDT). La distància anogenital (DAG) és considerada un biomarcador de l'acció dels andrògens durant el MPW. La DAG més curta ha estat relacionada amb tots els components de l'SDT, excepte amb el TTCG. La segona part d'aquesta tesi valora l'associació entre la DAG i el TTCG. A més avalua el paper de l'polimorfisme CAG de el gen AR en el desenvolupament de l'TTCG i la DAG. Es van analitzar a 156 pacients amb TTCG i 110 controls sans normozoospérmicos. Es va observar una distància anopeneana (DAGap) i una distància anoescrotal (dagues) significativament més curta en els TTCG respecte als controls. Es van definir uns punt de tall (DAGap: 130mm; dagues: 53mm) que indiquen un major risc de TTCG en aquells individus que es trobin per sota d'aquests valors. No s'ha trobat relació entre el polimorfisme CAG i el TTCG o la longitud de la DAG. En conclusió, les dades revelen que els pacients amb una DAG més curta presenten un major risc de TTCG, recolzant la teoria sobre la influència de l'desequilibri androgènic durant el desenvolupament fetal en l'etiopatogènia de l'TTCG.
La presente tesis es una aportación al conocimiento de nuevos factores de riesgo para el tumor testicular de células germinales (TTCG). El TTCG presenta una etiología multifactorial, atribuible a un retraso en la diferenciación de los gonocitos fetales. El TTCG es más frecuente en varones con una espermatogénesis alterada, sugiriendo una posible etiopatogenia común. El cromosoma Y contiene genes esenciales para una correcta espermatogénesis, las regiones del factor de azoospermia (AZF). La región AZF más dinámica es la región AZFc que presenta puntos frágiles que predispone a reordenamientos. El reordenamiento parcial más relevante desde el punto de vista clínico de la región AZFc es la deleción gr/gr. Se ha relacionado la delación gr/gr con un mayor riesgo de desarrollar un TTCG, pero la falta de información sobre los parámetros seminales de los pacientes no ha permitido de clarificar si la asociación observada está relacionada con la espermatogénesis alterada o si es un factor de riesgo independiente. Además, aún queda por establecer si otros tipos de deleciones y duplicaciones de la región AZFc presentan relación con el TTCG. La primera parte de esta tesis se enfoca en el estudio de los reordenamientos parciales de la región AZFc en el TTCG. Se han analizado 497 pacientes con TTCG y 2030 controles sin TTCG. Un 3.8% de los pacientes con TTCG presentaban algún tipo de deleción parcial de la región AZFc respecto al 2.5% del grupo control (p= 0.078). La deleción parcial más frecuente fue la deleción gr/gr, mientras que los otros tipos de deleciones parciales de la región AZFc resultaron ser muy raras. Según el fenotipo seminal, se observó un mayor riesgo de TTCG en pacientes normozoospérmicos portadores de deleciones parciales de la región AZFc respecto a los controles normozoospérmicos. No hubo diferencias significativas entre pacientes y controles según las duplicaciones parciales de la región AZFc. Se mostró que las alteraciones en la dosis del gen DAZ confieren un mayor riesgo de TTCG. Estos resultados confirman que un déficit del contenido génico de la región AZFc juega un papel importante en la etiopatogénesis del TTCG. En particular, la deleción gr/gr confiere un riesgo significativo para el desarrollo del TTCG independientemente de los parámetros seminales. Los factores ambientales también están involucrados en la etiopatogénesis del TTCG, especialmente si interfieren en un periodo específico del desarrollo testicular, en el denominado "masculinization programming window" (MPW). Un desequilibrio hormonal en este periodo compromete la correcta función de las células fetales de Sertoli y Leydig, originando el síndrome de disgenesia testicular (SDT). La distancia anogenital (DAG) es considerada un biomarcador de la acción de los andrógenos durante el MPW. La DAG más corta ha sido relacionada con todos los componentes del SDT, excepto con el TTCG. La segunda parte de esta tesis valora la asociación entre la DAG y el TTCG. Además evalúa el papel del polimorfismo CAG del gen AR en el desarrollo del TTCG y la DAG. Se analizaron a 156 pacientes con TTCG y 110 controles sanos normozoospérmicos. Se observó una distancia anopeneana (DAGap) y una distancia anoescrotal (DAGas) significativamente más corta en los TTCG respecto a los controles. Se definieron unos punto de corte (DAGap: 130mm ;DAGas: 53mm) que indican un mayor riesgo de TTCG en aquellos individuos que se encuentren por debajo de estos valores. No se encontró relación entre el polimorfismo CAG y el TTCG o la longitud de la DAG. En conclusión, los datos revelan que los pacientes con una DAG más corta presentan un mayor riesgo de TTCG, apoyando la teoría sobre la influencia del desequilibrio androgénico durante el desarrollo fetal en la etiopatogenia del TTCG.
This thesis is a contribution to the knowledge of new risk factors for testicular germ cell tumor (TTCG). TTCG has a multifactorial etiology, attributable to a delay in the differentiation of fetal gonocytes. TTCG is more frequent in men with altered spermatogenesis, suggesting a possible common etiopathogenesis. The Y chromosome contains essential genes for correct spermatogenesis, the azoospermia factor (AZF) regions. The most dynamic AZF region is the AZFc region that presents fragile points that predispose to rearrangements. The most clinically relevant partial rearrangement of the AZFc region is the gr / gr deletion. gr / gr cheating has been associated with an increased risk of developing TTCG, but the lack of information on the seminal parameters of the patients has not made it possible to clarify whether the observed association is related to altered spermatogenesis or if it is a factor of independent risk. Furthermore, it remains to be established whether other types of deletions and duplications of the AZFc region are related to TTCG. The first part of this thesis focuses on the study of partial rearrangements of the AZFc region in the TTCG. 497 patients with TTCG and 2030 controls without TTCG have been analyzed. 3.8% of the patients with TTCG presented some type of partial deletion of the AZFc region compared to 2.5% of the control group (p = 0.078). The most frequent partial deletion was the gr / gr deletion, while the other types of partial deletions of the AZFc region were found to be very rare. According to the seminal phenotype, a higher risk of TTCG was observed in normozoospermic patients carrying partial deletions of the AZFc region compared to normozoospermic controls. There were no significant differences between patients and controls according to the partial duplications of the AZFc region. Alterations in the dose of the DAZ gene were shown to confer an increased risk These results confirm that a deficit in the gene content of the AZFc region plays an important role in the etiopathogenesis of TTCG. In particular, the gr / gr deletion confers a significant risk for the development of TTCG regardless of seminal parameters. Environmental factors are also involved in the aetiopathogenesis of TTCG, especially if they interfere in a specific period of testicular development, in the so-called "masculinization programming window" (MPW). A hormonal imbalance in this period compromises the correct function of the fetal Sertoli and Leydig cells, causing the testicular dysgenesis syndrome (TDS). The anogenital distance (DAG) is considered a biomarker of the action of androgens during MPW. The shorter DAG has been related to all components of the SDT, except the TTCG. The second part of this thesis assesses the association between the DAG and the TTCG. It also evaluates the role of the CAG polymorphism of the AR gene in the development of TTCG and DAG. 156 patients with TTCG and 110 healthy normozoospermic controls were analyzed. A significantly shorter anopeneal distance (DAGap) and anoscrotal distance (DAGas) were observed in TTCG compared to controls. Cut-off points were defined (DAGap: 130mm; DAGas: 53mm) that indicate a greater risk of TTCG in those individuals who are below these values. No relationship was found between the CAG polymorphism and the TTCG or the length of the DAG. In conclusion, the data reveal that patients with a shorter DAG have a higher risk of TTCG, supporting the theory about the influence of androgen imbalance during fetal development on the etiopathogenesis of TTCG.

Migraine manifests itself episodically with incidence ranging from one attack in a lifetime to one almost every day. Most migraineurs suffer from typical migraine with or without aura, that is inherited in a complex manner. A small number of migraineurs suffer from FHM, a condition that exhibits Mendelian inheritance. BFNC is another rare episodic disorder that exhibits Mendelian inheritance. In a four generational family the BFNC phenotype was linked to the KCNQ2 gene on chromosome 20q13.3. Blood samples and epidemiological information were collected from 214 migraine probands in the Grampian region. An Access database was created to hold these data which were subsequently input. The database was utilised to extract epidemiological information about the cohort for subsequent analysis. These data were compared to other studies to characterise the severity of the cohort. A total of six polymorphisms were analysed by association analysis for involvement in migraine. The first polymorphism to be analysed was the -141C Ins/Del polymorphism in the DRD2 gene on chromosome 11q22. This polymorphism was analysed by restriction digest. Statistical analysis excluded this polymorphism and the regions encompassed by linkage disequilibrium in the aetiology of migraine. The second and third polymorphisms were located in intron 1 of the ERa gene on chromosome 6q25. These two polymorphisms were identified by restriction digest. Individual and haplotype analysis excluded the involvement of both polymorphisms in the aetiology of migraine. The fourth, fifth and sixth polymorphisms were located in exon 47, exon 23 and exon 8 of the CACNA1A gene on chromosome 19p13. The polymorphisms were analysed by capillary electrophoresis, restriction digest and DASH, respectively. All three polymorphisms were excluded from the aetiology of migraine. A total of 12 hot spot exons were sequenced in the CACNA1A gene in one individual afflicted by FHM and two by HM. No mutations were presented in the exons sequenced.

Includes abstract.
Includes bibliographical references (p. 197-215).
The primary aim of this thesis was to identify candidate genes that may be associated with ACL ruptures, and then use a genetic association approach following a case-control study design to identify specific sequence variants (single nucleotide polymorphisms, SNPs) within these candidate genes which may predispose individuals to ACL ruptures. Candidate genes (COL1A1, COL5A1 and COL12A1) were selected based on the biological function of their encoded proteins (type I, type V and type XII collagen respectively) within the basic structural and functional unit of ligaments, namely the collagen microfibril.

Parkinson’s disease (PD) is the second most common neurodegenerative disease. Motor symptoms typically do not manifest until significant neuronal loss has already occurred, highlighting the need for early detection and prevention. In this dissertation, we sought to improve our understanding of PD epidemiology by studying associations between potential modifiable risk factors, including antioxidant vitamins, dairy products, and urate, and PD risk. We conducted prospective analyses within three large cohort studies: the Nurses’ Health Study, the Health Professionals Follow-up Study, and the Cancer Prevention Study II Nutrition Cohort. Across all analyses, PD cases were identified via biennial questionnaires and confirmed through medical record review by neurologists specializing in movement disorders. In our first two aims, we used Cox proportional hazards models to calculate relative risks of PD according to cumulative average intakes of foods and nutrients of interest. In aim 1, we found no associations between intake of vitamin E, vitamin C, or carotenoids and risk of PD. In our second aim, we found that low fat dairy intake was associated with increased PD risk, and that this association appeared to be driven by an increased risk of PD associated with skim and low-fat milk intake. The results of a meta-analysis including previously conducted prospective investigations of milk intake and PD risk suggested a relative risk of PD comparing extreme milk intake levels of 1.80 (95% CI 1.44-2.25). In our third aim, although a large body of research suggests that higher urate levels could be protective against PD risk and progression, we found that genetic variants in the SLC2A9 gene that influence circulating urate levels were not associated with risk of PD. Our analyses suggest that while antioxidant vitamins are unlikely to alter PD risk, dairy products may represent an important modifiable PD risk factor. Whether dairy products also alter rates of PD progression or conversion from premotor PD to clinical PD are important, answerable questions. Finally, the results of our third analysis suggest that genetic variants associated with plasma urate levels are not associated with PD risk; however, larger studies are needed to confirm these results.

Abstract Parkinson’s disease and Huntington’s disease are progressive neurodegenerative movement disorders that typically manifest in adulthood. In this study, genetic risk factors contributing to these two movement disorders were investigated in Finnish patients. Patients with early-onset or late-onset Parkinson’s disease as well as population controls were examined. The p.L444P mutation in GBA was found to contribute to the risk of Parkinson’s disease. POLG1 compound heterozygous mutations were detected in two patients with Parkinson’s disease and rare length variants in POLG1 were associated with Parkinson’s disease. Variants in SMPD1, LRRK2 or CHCHD10, previously detected in other populations, were not detected, suggesting that they are rare or even absent in the Finnish population. Patients with Huntington’s disease were investigated for HTT gene haplotypes as well as whether these haplotypes alter the stability of the elongated CAG repeat. Haplogroup A was less common in Finns than in other European populations, whereas it was significantly more common in patients with Huntington’s disease than in the general population. Certain HTT haplotypes as well as the parental gender were found to affect the repeat instability. We found that compound heterozygous mutations in POLG1 were causative of Parkinson’s disease, rare length variants in POLG1 were associated with Parkinson’s disease and GBA p.L444P was significantly more frequent in patients than in the controls, which suggests that these mutations are associated with the development of Parkinson’s disease. The low prevalence of Huntington’s disease in Finland correlates with the low frequency of the disease-associated HTT haplogroup A. Paternal inheritance combined with haplotype A1 increased the risk of repeat expansion. Movement disorders in Finland were found to share some of the same genetic risk factors found in other European populations, but some other recognized genetic variants could not be detected
Tiivistelmä Parkinsonin tauti ja Huntingtonin tauti ovat hermostoa rappeuttavia eteneviä liikehäiriösairauksia, jotka tyypillisesti ilmenevät aikuisiällä. Tässä tutkimuksessa selvitettiin näiden kahden liikehäiriösairauden geneettisiä riskitekijöitä suomalaisilla potilailla. Tutkimme potilaita, joilla oli varhain alkava Parkinsonin tauti tai myöhään alkava Parkinsonin tauti sekä väestökontrolleja. GBA-geenin p.L444P mutaation havaittiin lisäävän Parkinsonin taudin riskiä. Kaksi Parkinsonin tautia sairastavaa potilasta oli yhdistelmäheterotsygootteja haitallisten POLG1-geenin varianttien suhteen ja harvinaiset POLG1 CAG toistojaksovariantit assosioituivat Parkinsonin tautiin. Tutkittuja variantteja SMPD1-, LRRK2- ja CHCHD10-geeneissä ei löydetty tästä aineistosta lainkaan, mikä viittaa siihen, että ne puuttuvat suomalaisesta väestöstä tai ovat harvinaisia. Huntingtonin tautia sairastavilta potilailta tutkittiin HTT-geenin haploryhmiä ja niiden vaikutusta Huntingtonin tautia aiheuttavan pidentyneen toistojakson epästabiiliuteen. Haploryhmä A oli suomalaisessa väestössä harvinainen verrattuna eurooppalaiseen väestöön ja se oli huomattavasti yleisempi Huntingtonin tautipotilailla kuin väestössä. Toistojakson epästabiiliuteen vaikuttivat tietyt HTT-geenin haplotyypit samoin kuin sen vanhemman sukupuoli, jolta pidentynyt toistojakso periytyy. POLG1 yhdistelmäheterotsygoottien katsottiin aiheuttavat Parkinsonin tautia ja harvinaisten POLG1 CAG toistojaksovarianttien todettiin assosioituvan Parkinsonin tautiin Suomessa. GBA p.L444P mutaatio merkittävästi yleisempi Parkinsonin tautipotilailla kuin kontrolleilla, mikä viittaa siihen, että se on Parkinsonin taudin riskitekijä. Huntingtonin tautiin assosioituvan haploryhmä A:n matala frekvenssi selittää taudin vähäistä esiintyvyyttä Suomessa. Paternaalinen periytyminen ja haplotyyppi A1 lisäsivät HTT-geenin toistojakson pidentymisen riskiä. Liikehäiriösairauksilla todettiin Suomessa osittain samanlaisia riskitekijöitä kuin muualla Euroopassa, mutta kaikkia tutkittuja variantteja emme havainneet

Preeclampsia is a complex and variable maternal disturbance that ranges from a dramatic onset at early gestation to slowly developing symptoms towards term. Hypertension and renal involvement with proteinuria are cardinal signs, which are often accompanied by fluid retention, blood-clotting dysfunction, and reduced organ perfusion. HELLP (haemolysis, elevated liver enzymes, and low platelet count) syndrome is regarded as a variant of preeclampsia, and the fulminante disease, eclampsia, includes convulsions. Preeclampsia is the main cause of maternal and fetal morbidity and mortality in western countries (1, 2), and in Nordic countries, 17 percent of maternal deaths have been ascribed to preeclampsia (2). Antenatal care in Norway includes on average 12 doctor/midwife consultations per pregnancy (3), and since blood pressure monitoring and urinary testing are main aims of the consultations, preeclampsia is a pregnancy complication that also generates substantial societal costs.

Paper II, III, IV and V reproduced with permission of Elsevier, sciencedirect.com