Les virus adéno-associés (AAVs) sont des virus à ADN simple brin, nonenveloppés, considérés comme des candidats de choix pour la thérapie génique. Pour augmenter les chances de succès des thérapies géniques basées sur l’AAV, des efforts sont actuellement faits pour développer de nouvelles capsides virales, qui seraient plus résistantes à l’immunité préexistante, plus spécifiques de certains tissus, et compatibles avec une production à grande échelle. L’un des défis posés par le développement de nouveaux vecteurs consiste à comprendre comment conférer de nouvelles fonctions biologiques aux capsides d’AAVs, sans compromettre leur intégrité structurale. Pour ce faire, il est nécessaire d’améliorer notre compréhension des mécanismes gouvernant la métastabilité des capsides d’AAVs. L’objectif de cette thèse était d’étudier la thermostabilité des AAVs, ses liens avec leurs propriétés biologiques, ainsi que ses applications dans le domaine du contrôle qualité des préparations d’AAVs recombinants Dans un premier temps, nous étendons les travaux existants à l’étude de virus AAVs ancestraux (AncAAVs), reconstruits in silico. Nous montrons que Anc80, l’ancêtre commun prédit d’AAV1, 2, 8 et 9, est plus thermostable que ses descendants (ΔT = 15-20°C). Nous identifions ensuite, par une analyse de type phénotype-phylogénie, 12 acides aminés jouant potentiellement un rôle important dans la stabilisation des capsides virales. Nous montrons ensuite que la thermostabilité des capsides d’AAVs, mesurée par fluorimétrie différentielle à balayage (DSF), est utile pour déterminer, à l’échelle protéique, l’identité des préparations de vecteurs viraux, une opération requise par les agences réglementaires. Pour finir, nous appliquons ce test d’identité à l’étude de l’homogénéité structurale des librairies d’AAVs. Ces travaux de thèse pourraient s’avérer utiles pour développement et le manufacturing de nouveaux AAVs recombinants pour la thérapie génique. / Adeno-associated virus (AAV) vectors have emerged as promising gene delivery vehicles for gene therapy. To improve the probability of success of AAV-based therapeutic strategies, efforts are currently being made to engineer novel capsids able to produce and purify well, escape pre-existing immunity, and target specific cell populations more efficiently. One challenge in AAV vector engineering is to understand how to confer new functions to the viral capsid without altering its structural integrity. To do so, there is a critical need to gain further knowledge on the mechanisms steering AAV capsid metastability. The objective of this thesis is to investigate the thermal stability of AAVs, its impact on AAV biology, and applications to quality control of AAV preparations. First, we extend existing thermal stability studies to in silico reconstructed ancestral AAV particles (AncAAVs), and show that, Anc80, the common putative ancestor of AAV1, 2, 8 and 9, is 15-20°C more thermostable than its contemporary homologs. Using phenotype-tophylogeny mapping, we also identify a set of 12 residues potentially playing a key role in capsid metastability. Second, we demonstrate that capsid thermal stability, as measured by Differential Scanning Fluorimetry (DSF), can be used for identification of AAV preparations at the protein level, a requirement of regulatory agencies. Last, we apply this identity assay to the study of capsid mosaic formation in AAV library preparations. This work will help guide the engineering and manufacturing of improved AAV vectors for gene therapy.

Neurodegenerative diseases are fatal disorders in which disease pathogenesis results in the progressive degeneration of the central and/or the peripheral nervous systems. These diseases currently affect -2% of the population but are expected to increase in prevalence as average life expectancy increases. The majority of these diseases have a complex genetic basis. The work presented in this thesis aimed to investigate the genetic basis of two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and the human prion diseases kuru and sporadic Creutzfeldt-Jakob disease (sCJD), using novel complex genetic approaches. ALS is a fatal neurodegenerative disease in which motor neurons are seen to degenerate. It is a complex disease with 10% of individuals having a family history and the remaining 90% of non-familial cases having some genetic component. The gene DYNC1H1 is involved in retrograde axonal transport and is a good candidate for ALS. In this thesis the genetic architecture of DYNC1H1 was elucidated and a mutation screen of exons 8, 13 and 14 was undertaken in familial forms of ALS and other motor neuron diseases. No mutations were found. A linkage disequilibrium (LD) based association study was conducted using two tagging single nucleotide polymorphisms (tSNPs) which were identified as sufficient to represent genetic variation across DYNC1HI. These tSNPs were tested for an association with sporadic ALS (SALS) in 261 cases and 225 matched controls but no association was identified. Kuru is a devastating epidemic prion disease which affected a highly geographically restricted area of the Papua New Guinea highlands, predominantly affected adult women and children. Its incidence has steadily declined since the cessation of its route of transmission, endocannibalism, in the late 1950's. Kuru imposed strong balancing selection on codon 129 of the prion gene (PRNP). Analysis of kuru-exposed and unexposed populations showed significant deviations from Hardy-Weinberg equilibrium (HWE) consistent with the known protective effect of codon 129 heterozygosity. Signatures of selection were investigated in the surviving populations, such as deviations from HWE and an increasing cline in codon 129 valine allele frequency, which covaried with disease exposure. A novel PRNP G127V polymorphism was detected which, while common in the area of highest kuru incidence, was absent from kuru patients and unexposed population groups. Genealogical analysis revealed that the heterozygous PRNP G127V genotype confers strong prion disease resistance, which has been selected by the kuru epidemic. Finally, PRNP copy number was investigated as a possible genetic mechanism for susceptibility to kuru and sCJD. No conclusive copy number changes were identified.

BEST1 encodes bestrophin-1, an integral membrane protein expressed in the basolateral membrane of retinal pigment epithelial (RPE) cells. Bestrophin-1 is hypothesised to function as a calcium-activated chloride channel (CaCC) (Sun et al. 2002) and/or a modulator of voltage-dependant calcium channels (VDCC) (Rosenthal et al. 2006) in the RPE. Mutations in BEST1 have previously been linked to a number of retinal dystrophies including Best disease (Marquardt et al. 1998; Petrukhin et al. 1998), ADVIRC (Yardley et al. 2004) and ARB (Burgess et al. 2008b). The aim of this work was to advance understanding of the spectrum of ocular disorders associated with BEST1 mutations. Novel BEST1 mutations were identified in Best disease and ARB patients. The identification of eleven ARB patients from eight separate families with either proven or presumed biallelic mutations in BEST1 supports previous data that ARB is caused by biallelic mutations in BEST1. The identification of homozygous nonsense mutation in BEST1 in a patient with ARB has greatly strengthened the hypothesis that ARB represents the null bestrophin-1 phenotype in humans. The work presented here also demonstrates that a greater range of retinal dystrophies, including concentric RP and severe early onset retinal dystrophies are associated with mutations in BEST1 expanding the clinical heterogeneity known to be associated with BEST1 mutations. Functional assays have been used to investigate the pathogenic effects of disease-associated BEST1 mutations. Whole-cell patch-clamp analysis has shown that ARB-associated BEST1 mutants inhibit the CaCC activity of wildtype bestrophin-1 to a lesser extent than Best disease-associated mutants, confirming the recessive and dominant inheritance patterns of the two diseases. The study of bestrophin-1 and disease-associated mutants in a polarised cell line reveals that many mutant isoforms have disrupted localisation and stability in vitro. The pathogeniCmechanisms contributing to bestrophinopathies therefore may, in many instances, be attributed to aberrant protein folding, stability and localisation Notably, ARB-associated bestrophin-1 isoforms were shown to be rapidly degraded via a proteasomaldependant mechanism. Rapid degradation of bestrophin-1 isoforms leading to reduced levels of functional bestrophin-1, unable to sustain normal physiological function may explain the loss of function phenotype displayed by ARB patients with biallelic missense mutations in BEST1. In conclusion the data presented here has advanced understanding about the spectrum of retinal dystrophies associated with mutations in BEST1 in addition to elucidating pathogenic mechanism that contribute to the bestrophinopathies.

<I>WT1 </I>identified as a gene repeatedly disrupted in Wilms' tumour, a common paediatric renal malignancy. This study is one of many which sought to confirm and understand some of the roles of <I>WT1 </I>in both normal development and tumorigenesis. Mutation analysis of <I>WT1 </I>in samples from patients with sporadic, bilateral and syndrome-associated Wilms' tumour has produced a pattern of results consistent with the findings of other groups. No mutations were detected in the sporadic and bilateral tumours, however exonic point mutations were detected in 7 out of 9 syndrome-associated Wilms' tumour samples that were analysed in detail. Studies examining parental <I>WT1 </I>status, genomic imprinting and allele loss distal to the <I>WT1 </I>locus are detailed for the syndrome-associated Wilms' tumour samples. <I>p16</I> was identified not only as a cell cycle regulator, but also as a gene repeatedly deleted in tumour cell lines. Previous studies showed that tumours of various types, including primary malignant mesothelioma, often contain deletions spanning the <I>p16</I> locus. In this study detailed deletion analysis is presented for <I>p16</I> and the adjacent related gene, <I>p15, </I>in Wilms' tumour and malignant mesothelioma samples. Both primary tumours and tumour cell lines were analysed. Deletions of <I>p16</I> were not detected in Wilms' tumour and derived tumour cell lines. Deletions of <I>p16</I> were however detected in all malignant mesothelioma cell lines analysed, and in the majority of these (14 out of 15) the deletions extended to the <I>p15</I> locus. Analysis of <I>p16 </I>and <I>p15 </I>in primary malignant mesothelioma samples did not identify deletions of either gene. The contribution, resulting effects and complementary nature of <I>WT1 </I>and <I>p16 </I>mutations in Wilms' tumour and malignant mesothelioma is discussed.

The incidence of the reciprocal translocation t(14;18))(q32;q21) in follicular lymphoma varies from 50% to 90% in American series. In the largest series from Europe, the translocation was present in only 21/51 (41&37) of cases. The wide variety in incidence may in part be due to the method of detection used - usually cytogenetics alone or a combination of Southern blotting and polymerase chain amplification (PCR) of DNA extracted from malignant tissue. The shortcomings of each technique suggest that a combination of all three may best determine the true incidence of the translocation. The study describes and discusses the cytogenetic analysis of lymph nodes from seventy two patients with follicular lymphoma. Forty nine patients also had DNA extracted from diagnostic specimens for molecular analysis using both Southern blotting and the polymerase chain reaction (PCR) to detect t(14;18). The translocation was found in 76% using a combination of all three methods. The most common other cytogenetic abnormalities included + 7, + der(18), + 18, + 21, + X and &43 8. Cytogenetics proved the most comprehensive method of detecting t(14;18) and Southern blotting was superior to PCR. The numerous additional karyotypic abnormalities present contributed little to the management of patients with follicular lymphoma. However, molecular methods detected t(14;18) in samples where karyotyping was unsuccessful and the stability of t(14;18) as a marker of disease was confirmed by the finding of molecular evidence of the translocation after analysis of sequential samples from several patients. The increased sensitivity of PCR allowed detection of t(14;18) bearing cells in samples of marrow and peripheral blood stem cell harvests from patients who were thought by conventional criteria to be in clinical remission. As treatment of follicular lymphoma becomes more aggressive molecular detection of t(14;18) will become more important in monitoring of treatment and detection of early relapse.

No description available.

No description available.

The identification of people at increased genetic risk of colorectal cancer and the provision of appropriate clinical screening represents one approach to the prevention of colorectal cancer in the Scottish population. This thesis aims to contribute to current knowledge regarding the available tools for identifying those at increased genetic risk in a population, namely genetic testing and family history assessment. Key issues relating to the use of family history in this context were addressed through the analysis of a unique data set, comprising family history information reported by a colorectal cancer case or control subject at interview and the results of record linkage of this data to the Scottish Cancer Registry. Retrospective family history case-control analysis showed that individuals with an affected first-degree relative were at increased risk of developing colorectal cancer (OR_MH 2.14, 95% CI = 1.11, 4.14). Prevalence of such a family history in control subjects was 9.4% (95% CI = 4.9, 13.9). Substantial under-reporting of family history was evident, with sensitivity of interview as a means of determining a history of colorectal cancer in a first-degree relative being approximately 0.55 for both cases and controls. These studies illustrate the potential advantages of targeting people with a family history, but also highlight some of the limitations of such an approach. The genetic epidemiology of the mismatch repair genes hMLH1 and hMSH2 and their association with colorectal cancer were considered in a systematic literature review. Although conventional epidemiological studies are lacking, there is compelling evidence to implicate mutations in these genes in the aetiology of a sub-set of colorectal cancers, with penetrance of approximately 80% in males and 40% in females. This review indicates that carriers of mismatch repair gene mutations merit particular consideration in the context of colorectal cancer prevention through targeting people at increased genetic risk.

The application of molecular techniques for the identification of cellular damage in response to environmental exposure is presented in this thesis. Using the <SUP>32</SUP>P-postlabelling assay (<SUP>32</SUP>PPL) buccal mucosa has been shown to represent an easily obtainable, alternative tissue for human biomonitoring. Relative adduct levels (RAL) in oral biopsies and buccal mucosa, taken from smokers and non-smokers, were analysed. Mean RAL for non-smokers were not statistically different in the two tissues: 2.1 X 10<SUP>-7</SUP> in buccal mucosa and 1.66 X 10<SUP>-7</SUP> in oral biopsies (p=0.72). Likewise no statistical difference between the two tissues was observed for smokers: 6.73 X 10<SUP>-7</SUP> and 6.16 X 10<SUP>-7</SUP> (p=0.75) respectively. Statistically higher levels of damage were seen in smokers compared to non-smokers. The p53 mutation spectra of oral tumours, obtained from smokers and non-smokers, was assessed using PCR amplification and Sanger sequencing. Four mutations were found: two in exon 7 at codon 247 (AAC to AC); one in exon 8 at codon 294 (GAG to GAAG) and one in codon 299 (CTG CCC to CTG AA CCC). No relationship was found between cigarette smoking and p53 mutation. A small study was carried out to assess persons living in an industrial area of South Wales. Using the butanol and nuclease P<SUB>1</SUB> enhanced <SUP>32</SUP>PPL methods to measure DNA adducts in WBC revealed the mean RAL to be statistically higher in the exposed group than the control group (p=<0.05). Analysis of buccal mucosa samples by butanol <SUP>32</SUP>PPL revealed no statistical difference between RAL in the exposed and control groups (p=>0.05). The p53 gene in exposed persons was analysed for mutations. WBC were analysed using the Restriction Site Mutation assay. No mutations were found.

Extensive regions of copy number variation (CNV) located throughout the genome play a significant role in common disease. This thesis describes the study of a structural variation on chromosome 12p13.31, and its involvement in susceptibility to complex disease, specifically the autoimmune disorder rheumatoid arthritis (RA). Methods of studying CNV include oligo-array Comparative Genomic Hybridisation (oaCGH), for which we developed a relatively optimised protocol on an in-house customisable microarray platform. In parallel, studies of the 12p13.31 locus using PCR-based methods revealed a large novel tandem duplication. Using quantitative assays we detected copy number variation within this duplication which occurs at a frequency of ~4% in European populations. At least two distinct points of recombination have been identified, supporting our theory that CNV in this region initiated from NAHR between the two units of the tandem duplication. We assessed copy number of sequences within the tandem duplication in a Swedish RA cohort, and revealed that a low copy number within this region occurs at a significantly higher rate in control samples compared to cases (p=0.001, OR=2.3 (95% CI 1.4-3.9)), suggesting a protective role for this variant. This was replicated in a UK cohort (p=0.036, OR=1.90 (95% CI 0.93-3.82)). We believe that the size of this effect is as large as any previously reported impact of CNV on common disease. An association was also detected in a Swedish psoriasis cohort (p=0.013, OR=2.16 (95% CI 1.2-4.1)). Future investigations into the effect of CNV at 12p13.31 on gene and protein expression may provide an insight into mechanisms of RA susceptibility and development. Given the tendency for autoimmune disease loci to share susceptibility regions, as well as the biological importance of genes located with the tandem duplication, we consider it likely that this region may also play a role in other complex disorders.