No description available.

No description available.

Ectopic recombination is an essential process that creates gene families, causes copy number variation and generates DNA rearrangements, sometimes leading to genetic disorders. Despite its importance, little is known about the dynamics and processes of aberrant crossover in humans. The human a-globin gene cluster is a classic system for studying ectopic recombination. DNA rearrangements such as single a-globin gene deletions (-a chromosomes) arise from unequal crossover between localised homologous regions, and are most likely favoured by malaria, leading to a +-thalassaemia. The aglobin gene cluster was therefore chosen as a test system for studying how copy number variation arises in the human genome. By developing single-DNA-molecule strategies including size fractionation and single-molecule PCR, de novo -a deletions and aaa duplications were both detected for the first time. These rearrangements occur both in blood and in sperm. Analyses o f these mutants show that they are generated by two distinct mechanisms. The major pathway involves ectopic sister chromatid exchange, the frequencies of which appear to be strongly influenced by mutational mosaicism. These rearrangements were common in both blood and sperm, while meiotic exchanges between homologous chromosomes were restricted to the germ-line and with lower frequencies. There was significant reciprocity of deletion and duplication processes, with respect to ectopic exchange breakpoints, haplotype symmetries and recombination frequencies. This indicates that these mutants were most likely generated by a reciprocal intermolecular recombination pathway. However, there was also evidence for extrachromosomal circles, which might suggest the existence of an additional intramolecular pathway that plays only a minor role in generating deletions. The surprisingly high frequencies of de novo deletion and duplication suggest that significant selective forces must have acted against individuals with -a or aaa chromosomes to maintain a constant and high level o f normal a a chromosomes in most malaria-free populations, and with low frequencies of -a and aaa chromosomes.

C-C Chemokine receptor 5 (CCR5) is involved in the regulation of the inflammatory response. Abdominal aortic aneurysms (AAA) may arise as the result of a chronic inflammatory process which is influenced by genetic predisposition. The CCR5 gene is associated with a 32 base pair deletion (the Δ32 polymorphism). The aim of this study was to investigate the role of the CCR5 Δ32 polymorphism in the development of AAA. A case control study was conducted including 285 patients with AAA and 273 control subjects. A blood sample was taken from each individual and DNA extracted. CCR5 genotype was determined using the polymerase chain reaction (PCR). Flow cytometry was used to investigate the biological activity of the Δ32 polymorphism. There was no significant difference between the AAA and the control group in relation to the Δ32 allele frequency (AAA group10%, control group=12%, P=0.82, chi squared analysis). Genotype analysis revealed no significant difference between the groups (AAA vs controls, wild type homozygotes=82% vs 77% heterozygotes=16% vs 21%, vs Δ32 homozygotes= 2% and 2% respectively, P=33, chi squared analysis). The polymorphism was shown to be biologically active with the number of Δ32 alleles correlating with cell expression of CCR5 as detected with flow cytometry (P = < 0.05). This study demonstrates that the CCR5 Δ32 is a biologically active genetic polymorphism, however, there is no association between this polymorphism and AAA.

Dyggve Melchior Clausen (DMC) syndrome is an autosomal recessive skeletal dysplasia caused by mutations in the Dymeclin (DYM ) gene on chromosome 18q12-21. Affected individuals have multiple bony abnormalities and mental retardation.;The aim of this work was to elucidate the function of the DYM gene product (DYM) and determine the mechanisms by which mutation of the disease gene lead to cellular and clinical phenotype. Ten affected individuals from eight families were recruited to the study and five DYM mutations identified. These included two novel and complex genomic duplication/repetition events each predicted to result in a truncated transcript.;In-silico analyses of DYM suggest it encodes a transmembrane protein involved in protein sorting and targeting within the cell. The DYM transcript was shown by in-situ hybridization to be expressed at high levels in cartilage and brain, particularly in resting and hypertrophic chondrocytes. Sub-cellular localisation demonstrated the DYM gene product to be located within the endoplasmic reticulum.;Yeast two-hybrid analysis performed to detect DYM interacting proteins identified EGF-containing fibulin-like extracellular matrix protein (EFEMP1) and vacuolar protein sorting protein 25 (VPS25, human homologue known as EAP20). EFEMP1 is an extracellular matrix (ECM) protein and EAP20 a component of the endosomal sorting complex required for transport which acts in transport of transmembrane proteins for export and recycling.;Taken together, these findings indicate that DYM is an endoplasmic reticulum transmembrane protein required for cargo transport through the endosomal compartment. Abnormal chondrocyte differentiation and brain function occur in the absence of adequate functional DYM. Given that cartilage and brain both have substantial requirements for extracellular matrix, it is suggested that DYM contributes to the transport of components of the ECM.

No description available.

<i>Haemophilia A</i>: 44 families with haemophilia A have been studied comprising 107 males and 125 females. Of the males 53 were haemophiliacs and of these 37 had severe (VIII:C < 0.01iu/dl), 7 had moderate disease (VIII:C > 0.01 but < 0.05iu/dl) and 9 had mild disease (VII:C > 0.05iu/dl). From pedigree analysis 40 women could be established as obligatory carriers, 5 women were considered to be normal and the remaining 80 women were potential carriers. In 22 families the disease appeared to have arisen spontaneously whilst in the remaining 22 there was a family history of haemophilia. Phenotypic analysis using VIII:C vWF:Ag vWF:RCo assays and the ratios VIII:C/vWF:Ag, VIII:C/vWF:RCo were used to identify carriers and the results compared to those obtained using genotypic analysis with 3 intragenic (Bcl I, Bgl I, Xba I) and 2 extragenic (Taq I, Bgl II) restriction fragment length polymorphisms. A control group of 31 age-matched normal females and obligatory carriers were used as controls to establish values for the measurements of VIII:C/vWF:Ag and VIII:C/vWF:RCO ratios which correctly classified all the normal women. Evaluation of the VIII:C/vWF:Ag and VIII:C/vWF:RCo ratios in the control group demonstrated the VIII:C/vWF:Ag ratio to be superior, correctly classifying 77% of the obligate carrier compared to only 42% using the VIII:C/vWF:RCo ratio. Of the 40 obligate carriers 26/37 were shown to have an abnormal coagulation phenotype, 11 to have a normal phenotype and in 3 no coagulation data was available. 27 of the potential carriers had an abnormal coagulation phenotype, 47 were normal and in 6 no data was available. <i>Haemophilia B</i>: 5 families with haemophilia have been studied comprising 11 males and 12 females. Of the males 4 were haemophiliacs (IX:C > 0.01iu/dl). Of the 12 females, 3 could be classified as obligate carriers, 8 as potential carriers and 1 as a probable haemophilac female from pedigree data. Although the mean IX:C for the obligatory carriers was lower than that of the control group, there was a considerable overlap of values. Only limited phenotypic data was available but 2 of 3 obligate carriers and 3 of 4 potential carriers had IX:C assays below the lowest value obtained in a control group of 15 normal women suggesting carriership. No CRM* families were identified.

In order to map the breakpoints of the translocation, a series of somatic cell hybrids has been characterised by PCR and Southern analysis. This analysis has resulted in the determination of flanking markers of the breakpoints on both translocated chromosomes and in the production of a panel through which markers can be mapped, at high resolution, with respect to the chromosome 11 breakpoint region. To generate new markers for the region, microdissection and microcloning of the derived chromosome 1 was undertaken by others and I undertook to map the clones generated in this way. In total I have mapped 28 new microdissection markers, either to chromosome 1 or to a series of intervals on chromosome 11q. The markers which were found to map immediately distal to the chromosome 11 breakpoint were used to isolate YACs from that region. On the basis of co-hybridisation, fingerprint and pulsed field gel electrophoretic analysis, the YACs were assembled into a contig. Pulsed field gel electrophoretic analysis, of genomic DNA from an individual carrying the translocation, compared with DNA from a control individual, with a probe in the interval closest to the translocation breakpoint identified differences in three restriction fragments. This data was consistent with the translocation breakpoint being within approximately 550 kb of this marker.

Evidence exists which suggests that the expression of certain genes is associated with the development of human malignancy. The aims of the work reported in this thesis were (i) to quantitate accurately the expression (ie transcription) of selected important genes in human breast cancer, to relate this to clinical and pathological factors and to other genetic changes in the tumours and (ii) to develop a xenograft model in which to study the effect of an anticancer agent on breast tumour gene expression in vivo and thus to establish whether these approaches migh have therapeutic implications. The particular approach used to achieve these objectives was the development of molecular techniques to measure gene expression and loss of genetic material at the nucleic acid level. Gene expression was measured in primary breast cancers from 80 female patients and compared to that in normal control tissues from other patients who had surgical removal of normal breast tissue. Ribonucleic acid (RNA) was extracted from the tumours and probed with radiolabelled gene probes. Gene expression (messenger RNA) was then quantified using laser densitometry on the resultant Northern blots. Increased expression of three oncogenes (p53, c-myc, c-erbB-2), a growth factor gene (TGF-beta) and an oestrogen regulated mRNA (pS2) was detected in varying proportions of the tumours. Overexpression of individual genes was found to correlate significantly to different clinical and pathological parameters. Since the loss of specific regions of deoxyribonucleic acid (DNA) is associated with oncogenesis, DNA was also extracted from the paired venous blood and tumour tissue of each patient. Allele loss specific to the tumour DNA was then determined using Southern blots probed with radiolabelled DNA. The p53 gene is located near the tip of the short arm of chromosome 17 at 17p13 and so this region was examined for loss of genetic material. Specific DNA loss in this region was demonstrated in 64% of tumours. Loss close to, but not including, the p53 locus correlated with p53 overexpression. Using the polymerase chain reaction, exons 5 and 6 of the p53 gene were selectively amplified and subsequently sequencing suggested the overexpressed p53 may be mutant and oncogenic in at least some tumours. These results suggest that chromosome 17p is important in breast cancer. Lack of normal p53 tumour suppressor gene expression or deletion of a nearby regulatory locus resulting in overexpression of mutant p53 are proposed as key events in the pathogenesis of breast cancer. It is concluded that the use of molecular approaches, such as those employed in this work, will help not only to elucidate the pathogenesis of breast cancer, but also clarify the mechanisms by which anticancer therapy is mediated and lead to new therapeutic approaches.

RFLP analysis was used to detect loss of heterozygosity in tumour/blood pairs, from patients with cervical carcinoma, to examine the role of commonly implicated tumour suppressor genes in cervical carcinogenesis. Allele losses were detected less frequently than has been reported in many other common solid tumours. This relatively low level of allele loss was supported by the infrequent genetic alteration identified when comparison was made between tumour/blood DNA fingerprints from cervical carcinoma patients and those with cancers of non-cervical origin. No correlation was found between allele loss and HPV status when polymerase chain reaction (PCR) was used with DNA extracted from cervical carcinomas to detect HPV types commonly associated with genital lesions. The mutational status of p53 gene was examined in a series of cervical carcinomas by a method employing PCR and denaturing gradient gel electrophoresis, and comparisons were made between the HPV and p53 mutational status of these tumours. Mutation in p53 gene was detected relatively infrequently, and contrary to recent reports, was not commonly associated with HPV negative status. Mutations were characterised by sequencing. The use of p53 specific monoclonal antibodies in immunohistochemical analysis of normal, premalignant, and malignant cervical epithelium confirmed that p53 was seldom present in detectable quantities at any stage in the progression of this disease, and lent support to the finding of a low frequency of p53 mutations in this tumour type. Y13 259, a monoclonal antibody to ras p21 oncoprotein, used to compare ras expression at various stages in cervical carcinogenesis, identified differences in expression in the glandular, but not the squamous, component of malignant and non-malignant cervical epithelium.