Mitochondrial dysfunction has been implicated in the early pathogenesis of Type 2 Diabetes. The uncoupling proteins 2 and 3 are mitochondrial proteins found in man that have been implicated in protecting mammals from the effects of over-nutrition. Examination of the effect of genetic variation in the UCP2- UCP3 genetic cluster has so far been inconclusive. The aim of this thesis was to examine, using a genetic strategy, the hypothesis that the role of the uncoupling proteins 2 and 3 in the pathogenesis of Type 2 Diabetes is via modification of oxidative stress. In a prospective study of nearly 3000 men the risk of type 2 diabetes at 10 years was increased for both the UCP2-866AA (1.94 1.18-3.19 : p=0.009) and the UCP3-55TT (2.06 1.06-3.99 : p=0.03) homozygotes. This increased risk was not explained by the association with any measured conventional risk factors. Paradoxically, in a Europe-wide cross-sectional study of 598 subjects the UCP2-866A variant was associated with lower waist-hip ratio (GX v AA,1.00 0.06 v 0.98 0.07 p=0.003), although also associated with lower insulin secretion (42.6 24.6 v 35.6 18.6 p=0.03). The UCP3 variant was not significantly associated with any metabolic trait. The significant heritability of plasma markers of oxidative stress (TAS 0.54, TOAS 0.49) suggests anti-oxidant function is a plausible mechanism to determine Type 2 Diabetes risk. The predictors of anti-oxidant stress in a family study were examined, as was the impact of UCP2-UCP3 gene cluster variation. Genetic variation in the UCP2-UCP3 was found to increase the risk of the Type 2 diabetes. While UCP2 may modify insulin secretion directly, the mechanism of action for UCP3 is likely to involve novel risk factors for Type 2 Diabetes such as modification of mitochondrial oxidative stress. Finally, the development of a human model is described to examine genetic influences on oxidative stress burden using a meal rich in used cooking oil.

Four distinct DNA ligase activities (I-IV) have been identified within mammalian cells. Evidence has indicated DNA ligase I to be central to lagging strand DNA replication, as well as being involved in DNA repair processes. It has also been reported that DNA ligase I is essential for cell viability. A patient with altered DNA ligase I displayed a phenotype similar to Bloom's Syndrome, being immunodeficient, growth retarded, and predisposed to cancer. Fibroblasts isolated from this patient (46BR) exhibited abnormal lagging strand DNA synthesis, and repair deficiency. Gene inactivation was the first step of a 'double replacement' strategy to introduce analogous mutations into the mouse DNA ligase I gene (<I>LigI</I>). Using gene targeting in HPRT-deficient embryonic stem (ES) cells, the last 5 exons of the endogenous <I>Lig1</I> gene were replaced by an <I>HPRT</I> minigene, and subsequently DNA ligase I-deficient mice were produced. Embryos lacking DNA ligase I developed normally to mid-term, when haematopoiesis usually switches to the foetal liver. Thereupon severe disruption to foetal liver erythropoiesis occurred, leading to acute anaemia and prenatal death. <I>In vitro</I> assays indicated that erythroid-committed progenitor cells were present in the liver, but in reduced numbers. Apart from the developing liver, organogenesis was not perceptibly affected and non-erythroid haematopoietic lineages were not reduced. Injection of cell suspensions from single foetal livers were unable to rescue lethally-irradiated mice. However, injection of multiple livers into a single recipient was able to effect long term repopulation of the haematopoietic system. Animals rescued with cells lacking DNA ligase I displayed splenomegaly, anaemia coupled with reticulocytosis, and aplasia in all haematopoietic tissues.

The aim of this research was to provide empirical evidence on psychological issues relevant to the potential clinical utility of genetic tests for susceptibility to complex diseases. The results of such tests could increase motivation to change health behaviours but could also be misinterpreted, causing psychological distress on the one hand, and complacency on the other. The research comprised three studies which addressed: public interest in genetic testing the psychological impact of genetic testing and the impact of genetic testing on health behaviours. Study 1 (n= 1,960) utilised questions included in the Office for National Statistics omnibus survey to examine the level of public interest in taking genetic tests. Two thirds of respondents expressed interest in taking genetic tests for two complex diseases, cancer and heart disease, and there was greatest interest among people with intermediate levels of educational attainment. Study 2a-c (n= 1,024) was a postal questionnaire survey conducted in Oxfordshire, using a 2x2 (cancer vs heart disease x leaflet vs no leaflet) experimental design. Study 2a examined the effects of the leaflet on attitudes, and found that interest in genetic testing was higher amongst respondents who received the leaflet, as was subjective understanding of genetic testing. Study 2b provided support for the hypothesis that vulnerable people may self-select themselves out of genetic testing, by showing that anticipated reactions were associated with interest in genetic testing. Study 2c looked at responses amongst the smokers in the sample, and found that smokers who were more motivated to quit were more likely to be interested in genetic testing, and that smokers with lower understanding and educational attainment were more likely to believe that receiving a lower-risk genetic test result would make them feel that it would be safe for them to carry on smoking. The findings from these studies were used to feed into the design and interpretation of Study 3 (n=61), an exploratory study in which smokers were randomly allocated to receive genetic test results for a gene associated with lung cancer susceptibility (GSTM 1) or a control group, and followed up for two months. The results from Study 3 suggested that the process of genetic testing could increase motivation to quit smoking, but that the genetic test result itself may have relatively little impact. It is hoped that this research contributes to the current debate about how the clinical utility of emergent genetic tests for susceptibility to complex diseases might be evaluated in future research.

Parkinson's disease is a highly prevalent neurodegenerative disorder. Several genes have been shown to be associated with familial Parkinson's disease and they usually lead to Parkinson's disease due to the presence of mutations that affect protein function. It has been suggested that variations in the expression of the wild type genes may also lead to Parkinson's disease. The causes of idiopathic Parkinson's disease remain unknown. Several factors may contribute to its onset, including: susceptibility genes, environmental stress and aging. This study aimed to characterize the influence of oxidative stresses on the regulation of genes associated with Parkinson's disease. The effects of oxidative stress on a- synuclein, parkin and PINK1 were investigated in a cell culture model. Both ot- synuclein and parkin were similarly up-regulated when cells were exposed to stresses such as dopamine and l-methyl-4-phenylpyridinium (MPP+). In constrast, PINK1 levels were up-regulated only by MPP+, and were down-regulated in both dopamine and MG132 treatments. This work confirmed and extended previous reports that oxidative stresses are implicated in Parkinson's disease, and also revealed the complexity of the regulation by these stresses. A further study into the regulation of a-synuclein showed a novel interaction between the a-synuclein promoter and an Early Growth Response transcription factor family member in oxidative stress conditions. Moreover, this work demonstrated that several other neuronally expressed transcription factors influenced the regulation of a- synuclein, such as the product of the Parkinson's disease associated gene, Nurrl. The decreased expression of this gene increased a-synuclein transcription. This is of interest, as variations in the levels of either of these genes can cause Parkinson's disease and such an interaction was novel. This work further demonstrated that the POU family trancription factor, Brn3a, was involved in this pathway. Brn3a appeared to function antagonistically to Nurrl in a-synuclein regulation. In addition to studies of gene regulation, mutational and/or protein analysis were performed on Nurrl and PINK1. Studies of PINK 1 protein established the functional importance of cleavage of precursor PINK1 and also provided a better estimation of the location of the cleavage site. These genes are more recent discoveries compared to a-synuclein and parkin, thus, such studies will give important insights into their Parkinson's disease properties.

Turner Syndrome (TS) results from the complete or partial absence of one X chromosome in females. Short stature and gonadal dysgenesis are characteristic, with increased risks of cardiovascular disease, diabetes and obesity. This thesis investigates the prevalence and pathogenesis of factors contributing to cardiovascular risk. Because women with TS differ from normals in terms of their X-chromosome defect and oestrogen deficiency, they were compared to similarly-aged normal women, and a second control group of similarly-aged women with 46,XX oestrogen deficiency was also recruited. A cross-sectional study investigated the spectrum of structural disease in the heart, aorta and conduit vessels using various imaging techniques. Intima media thickness (IMT), arterial stiffness and endothelial function were also assessed. Progression of aortic dilatation was investigated in a longitudinal echocardiography study. Metabolic abnormalities in TS were investigated by measuring anthropometric parameters and serum markers of adiposity and comparing them in all three groups. Adipose tissue distribution was further investigated in a subgroup of women with TS and normal controls. A longitudinal oestrogen dose-ranging study was performed in women with TS and 46,XX primary amenorrhoea to assess oestrogen effects on various parameters pertaining to cardiovascular risk. Women with TS had greater height-adjusted arterial diameters than controls, and greater IMT, the latter amenable to reduction by increasing doses of oestrogen. The rate of aortic dilatation was greater than in the normal population. Endothelial function did not differ significantly. Women with TS have some features of the metabolic syndrome, but fasting insulin, glucose and leptin concentrations are surprisingly low, despite increased C-reactive protein and Interleukin-6 concentrations, greater central obesity and increased visceral fat than controls. Age, bicuspid aortic valve, blood pressure and oestrogen status were the most important predictors of cardiovascular disease in TS. This knowledge should aid identification of therapeutic targets to improve care in this population.

Within recent years there has been a move towards development of regression inspired methods for analysis of genomewide association studies of complex diseases. This is because multiple testing methods, such as the Bonferroni correction, tend to impose stringent significance thresholds and consequently, unless the study is very large, can reliably identify only those genomic regions with very strong signals of disease-gene association. However many complex diseases are suspected to be the result of the cumulative action of many loci, each having a small effect and there is a high probability the association signals in such studies will in fact be moderate and, furthermore, that extremely strong signals will be very rare. Although methods with higher power than the Bonferroni correction have been proposed, these tend to produce more false positive findings. This challenging problem of analysis methodology for genomewide association studies that is more efficient than existing approaches, but with false positive findings comparable with Bonferroni, is addressed in this thesis. A novel method based on nonparametric regression, capable of reliably identifying significant regions of disease-gene association in data from high dimensional genomewide studies, is developed and evaluated. The method is model-free and establishes significance thresholds that inherently account for the correlation (linkage disequilibrium) structure in the data through a tuning (bandwidth) parameter and assigned weights. A theoretically supported, computationally efficient method for obtaining the optimal tuning parameter is proposed and evaluated using simulations. Results of extensive evaluations and comparisons with existing methods show that the nonparametric approach is not only powerful but also leads to substantial reduction in false positive findings. The method is illustrated using data from the Wellcome Trust Case Control Consortium study (2007) of Crohn's disease.

The <I>WT1</I> gene was isolated by positional cloning as a candidate gene implicated to predisposition to the paediatric kidney cancer Wilms' tumour. Because of its early onset and histological resemblance to immature kidneys, Wilms' tumour was thought to arise from an aberration in the normal developmental pathway. From the amino acid sequence, <I>WT1</I> was predicted to be a transcription factor with an N-terminal proline/glutamine rich transregulatory domain and four C-terminal <I>TFIIIA</I>-like zinc fingers. Two alternative splices have been found in the transcript, resulting in the insertion of 17aa or 3aa (KTS). Functional analysis has shown that <I>WT1</I> is capable of sequence specific DNA binding and of regulating transcription. Analysis of <I>WT1</I> mRNA in the developing embryo has shown a pattern of expression consistent with an important role in nephrogenesis, specifically an involvement in mesenchymal-epithelial cell transition. <I>WT1</I> analysis in Wilms' tumours revealed that both functional copies are lost in about 10% of sporadic Wilms' tumours. This confirmed the role of <I>WT1</I> as a tumour suppressor gene in at least some cases, (following Knudson's two hit hypothesis for tumorigenesis). Predisposition to Wilms' tumour can also be associated with developmental abnormalities, including genitourinary malformations. Expression of <I>WT1</I> is seen in the earliest stages of gonad development and constitutionally heterozygous mutations in <I>WT1</I> are frequently associated with genitourinary abnormalities. The most sever abnormalities, in Denys-Drash syndrome, have been linked to heterozygous constitutional <I>WT1</I> missense mutations in the zinc fingers. The cloning of the mouse <I>WT1</I> gene revealed a very high level of similarity to the human gene, as well as a very similar pattern of expression during development.

The cause of the disease in two familial and two sporadic cases of aniridia associated with chromosome 11 rearrangements has been analysed. Since the rearrangements apparently leave the PAX6 gene intact, a cosmid contig encompassing <I>PAX6 </I>was assembled and facilitated mapping of the four breakpoints. All were found to lie distal of <I>PAX6</I> at distances of between 20 and 150kb beyond the 3' end of the gene. Phenotypically these patients are indistinguishable from other aniridia cases, suggesting the involvement of PAX6 haploinsufficiency. The site of the most distal breakpoint has been defined by isolating chromosome 11 fragments which cross it. These fragments contain regions of conservation between species and sequencing reveals potential open reading frames, raising questions about the function of the likely gene at this position. A possible position effect mechanism involving disconnection of <I>PAX6</I> from adjacent control elements has been explored. These elements may be locus-specific or more general chromatin organising elements and may be detected by DNaseI hypersensitive site mapping in appropriate cell types. Cell lines which appeared suitable for this purpose have been characterised. An alternative possibility involves disturbance of <I>PAX6 </I>expression by the incoming chromosomal region in each rearrangement, through an inappropriate chromatin conformation or the presence of negative control elements. The detailed physical map generated for the aniridia-associated breakpoint region will allow further exploration of the position effect mechanism. These aniridia-associated rearrangements contribute to a growing list of possible position effect cases in humans and other mammals. Extensive study of position effects in fruit flies and yeast shows that gene inactivation can result from a switch in chromatin environment between euchromatin and heterochromatin. In humans, the phenomenon of position effect may also involve disturbing the usual chromatin environment of a gene, reflecting the heterogeneity of the human genome in terms of chromatin structure and transcriptional permissiveness.

The aim of this study was to examine the causes of sensorineural hearing loss in the Bangladeshi population resident in East London. Almost all of this population originates from Sylhet, a province in Bangladesh. The study was conducted at a community based audiology clinic and tertiary level genetics department. One hundred and fifteen families (134 patients) were ascertained 11 families declined to participate and 4 families could not be contacted. All children of Bangladeshi ethnic origin with bilateral sensorineural hearing loss more than 40dB in the better hearing ear were included in this study. Information on all these patients was collected from their case notes. For the 67 patients in whom the cause of deafness was not clear from the records or unknown (or non- syndromic deafness), families were seen in the research clinic. The prevalence of deafness >40db in Bangladeshi children under 16 years of age in East London was calculated to be approximately 3.86 per 1000 (95%CI: 3.24, 4.47). This is nearly 2.3 times the national average. Parents were consanguineous in 35 out of 105 families (33.3%) in which this information was available. On calculating the prevalence of deafness in the Bangladeshi children belonging to non-consanguineous families, the prevalence falls to 2.72 per 1000 (95%CI: 2.10, 3.34). Genetic causes appear to be the most common cause of deafness in Bangladeshi population in 59.6% patients. Environmental causes were responsible for hearing loss in 18.5% patients and in 21.8% cases the cause of deafness was undetermined. Of the deafness due to genetic causes, 57.7% were non-syndromic, 25.3% syndromic and 16.9% were chromosomal. The single most common cause of sensorineural hearing loss in the Bangladeshi population in this study was due to mutations in the GJB2 gene (Connexin 26) in 14 of these families. The mutations in GJB2 in this population were W24X, IVS1 + 1G->A, M1V, V95M and W77X. W24X was the most common mutation seen in 40% (8/20) patients. Genetic causes are the common cause of deafness in subjects of Bangladeshi origin and 29.8% children with non-syndromic deafness were positive for mutations in GJB2.

Our global population is ageing and an ever increasing number of elderly are affected with neurodegenerative diseases, including the subjects of the studies in this work, Alzheimer's disease (AD), Parkinson's disease (PD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). On strong evidence that several genes may influence the development of sporadic neurodegenerative diseases, the genetic association approach was used in the work of this thesis to identify the multiple variants of small effect that may modulate susceptibility to common, complex neurodegenerative diseases. It has been shown that the common genetic variation of one of these susceptibility genes, MAPT, that of the microtubule associated protein, tau, is an important genetic risk factor for neurodegenerative diseases. There are two major MAPT haplotypes at 17q21.31 designated as H1 and H2. In order to dissect the relationship between MAPT variants and the pathogenesis of neurodegenerative diseases, the architecture and distribution the major haplotypes of MAPT have been assessed. The distribution of H2 haplotype is almost exclusively in the Caucasian population, with other populations having H2 allele frequencies of essentially zero. A series of association studies of common variation of MAPT in PSP, CBD, AD and PD in different populations were performed in this work with the hypothesis that common molecular pathways are involved in these disorders. Multiple common variants of the H1 haplotypes were identified and one common haplotype, H1c, showed preferential association with PSP and AD. A whole-genome association study of PD was also undertaken in this study in order to detect if common genetic variability exerts a large effect in risk for disease in idiopathic PD. Twenty six candidate loci have been found in this whole-genome association study and they provide the basis for our investigation of disease causing genetic variants in idiopathic PD.