Microsatellite loci are important for genetic studies due to the many desirable qualities they possess. However, speculation continues about the types of mutation that act on microsatellites. In particular, whether different motif types for stable, non-coding loci have different rates of mutation is not clear. This thesis addresses three aspects of the mutation problem. First, an investigation of the types of mutation acting on microsatellites is presented. The models compared are the single-step stepwise mutation model and the infinite alleles model. Second, two models of allele size distributions under a non-symmetric, single-step SMM model are proposed. Third, an indirect estimate of mutation rates for di- and tetranucleotide loci is obtained.

Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism. Severe enzymatic deficiency results in metabolic disease with occasional early demise. Mild deficiency is due to a common polymorphism (A222V) which increases risk for several common disorders. Pharmacogenetic studies of the antifolate methotrexate on Mthfr heterozygous knockout mice (Mthfr +/-), a model of the mild human deficiency, revealed significantly higher apoptosis rates in several tissues compared to wildtype mice (Mthfr +/+). These results suggest that mild MTHFR deficiency is a risk factor for methotrexate toxicity in mice and possibly in humans. A new medication, metafolin, was administered to Mthfr +/- mothers to assess survival of their Mthfr -/- pups. Survival rate of pups at 5 weeks of age from treated mothers was significantly higher than that from untreated mothers (54% versus 20%). The results of this study suggest that metafolin could be useful for treatment of severe MTHFR deficiency in humans.

Background. Susceptibility to a complex disease such as tuberculosis generally involves interactions among several genes and environmental factors. Several association studies have been conducted to examine the association between candidate genes and tuberculosis. However, the genetic risk factors are not fully understood. / Objective. To examine the effect of several candidate genes, including natural resistance associated macrophage protein 1 (NRAMP1), vitamin D receptor (VDR), surfactant proteins (SFTPA1), and mannose-binding lectin (MBL), and also to assess the effect of several risk factors on their association with tuberculosis. The other objectives were to test for mode of inheritance and also to estimate the relative risks of disease for different genotypes. / Methods. A prospective case-parental control study was conducted. Ninety-five nuclear families were selected from an existing database of families with tuberculosis in Ethiopia. Each family consisted of one affected child and two parents. The primary outcome was transmission/nontransmission of alleles from parents to affected offspring. / Results. The transmission disequilibrium test showed that marker SFTPA1-294 was significantly associated with the outcome (chi 2 = 4.297; p = 0.038). When other risk factors such as age, sex, ethnicity, certain symptoms or other genes were allowed to modify the transmission probabilities in a logistic regression model, several other markers were found to be significantly associated with the outcome. / Conclusions. Despite the limitations of this study, this thesis provided evidence for inheritance of susceptibility to tuberculosis in Hadiayan families in Ethiopia. To confirm the findings in this thesis, it would be useful to conduct similar research in populations with different ethnic origins, where genetic and environmental exposures can be examined and compared.

To define the role of mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5; mGPD) in energy balance and intermediary metabolism, we studied female transgenic mice lacking the mGPD gene (mGPD-/-). These mice had higher serum glycerol and triglycerides; lower body weight, blood glucose, and energy expenditure (QO2); and higher glycerol-3-phosphate and lactate/pyruvate ratio in muscle than controls with wild type genotype (WT). When given a high fat/low carbohydrate diet, mGPD-/- mice gain more weight than WT, without the genotype differentially affecting QO2 or calorie intake. On a low fat/high carbohydrate diet, mGPD-/- mice failed to increase QO2 as the WT and gained more weight. After a 30-hour fasting or food restriction to 70% for 10 days, WT lost significantly more weight than mGPD-/- mice, but these latter had lower body temperature and QO2. Thus, mGPD-/- mice exhibit a thrifty phenotype largely resulting from reduced obligatory thermogenesis.

The principal dose-limiting toxicity of most chemotherapeutic agents is myelosuppression. In this study we evaluated the chemoprotection conferred against alkylators by a bicistronic retroviral vector expressing the chemoresistance genes GSTA3 and DHFRL22Y. As reported herein, GID-transduced murine hematopoietic cells exhibited greater survival compared to control cells in presence of chlorambucil concentrations ranging from 5 x 10-6 M to 1 x 10-4M. After increasing the fraction of GID-expressing cells via chemoselection with TMTX and NBMPR, the level of resistance was increased by up to 2.9-fold at 5 x 10-5 M chlorambucil. GID-transduced hematopoietic cell enrichment by ex vivo chemoselection was limited, under our experimental conditions, by a marked loss of hematopoietic reconstitution ability, while in vivo chemoselection was shown to hold greater potential to selectively enrich GID-expressing blood cells. The presence of replication competent virus in the experiments makes it difficult to reach to conclusive interpretations of results. Notwithstanding the unfortunate outcome of murine leukemia that did not allow for the evaluation of GID-mediated hematopoietic chemoprotection in vivo, the promising in vitro experimental results reported here warrant further investigation of this approach.

Cellular ATP is synthesized by the mitochondrial oxidative phosphorylation (OXPHOS) system, composed of five enzyme complexes (Complexes I--V), which consist collectively of over 80 subunits. The majority of these subunits are encoded by nuclear genes, and 13 of them, by mitochondrial DNA (mtDNA). OXPHOS deficiencies resulting in mitochondrial disorders can be caused by either nuclear or mitochondrial mutations; however, most pathogenic mutations reported in adults occur in mtDNA. Such mutations often impair mitochondrial translation and are associated with a characteristic muscle pathology consisting of a mosaic pattern of normal fibers interspersed with fibers displaying mitochondrial proliferation and decreased OXPHOS activity. In this thesis, the molecular basis for a severe mitochondrial myopathy in two adult patients was investigated. All patient muscle fibers showed mitochondrial proliferation and barely detectable Complex IV activity (a measure of OXPHOS activity), a pattern never before reported. Biochemical studies demonstrated decreased activities of Complexes I and IV (5% of control) and Complex II+III (41% of control) in patient muscle. Immunoblot analysis of nuclear and mitochondrial subunits of Complexes 1, III and IV showed a greater than 90% decrease in the steady-state level of these subunits in mature muscle, but no change in nuclear-encoded subunits of Complexes II and V. A generalized mitochondrial translation defect was identified by pulse-label experiments in myotubes, but not in myoblasts cultured from both patients. This defect moved with the nucleus in patient cybrid cells. Myoblasts from one patient transplanted into the muscle bed of SCID mice differentiated into mature muscle fibers that displayed a defect similar to that seen in the patient muscle. Mapping of the defective gene in this patient was attempted using a functional complementation approach. Microcell-mediated transfer of mouse chromosomes in patient

The androgen receptor plays a critical role in both normal and neoplastic prostate growth. The first exon of the androgen receptor contains a polymorphic CAG repeat region that has been implicated in the development of prostate cancer. Epidemiologic studies suggest that shorter CAG repeats are associated with an increased risk of prostate cancer. Interestingly, the length of the CAG repeat region in the androgen receptor is inversely correlated with the transactivational competence of the receptor. This provides a biologically plausible mechanism by which the more active androgen receptors with shorter CAG repeats can contribute to prostate cancer. However, the effect of variation in CAG repeat length is unknown in the late-stages of the disease. / The primary goal of the work presented in this thesis was to determine the effects of different AR CAG repeat lengths in a late-stage prostate cancer environment where androgen receptor mutations are more common and may override the functional attributes of short CAG repeats. To that end, we have assessed the effects of CAG repeat lengths in a common prostate cancer mutant androgen receptor Thr877Ala. Our results suggest that the Thr877Ala mutant exhibits different transactivation trends than the wild-type receptor with respect to CAG repeat length. These data may indicate that, although CAG length may have a significant effect in wild-type receptors and in early disease, the activity of mutant receptors found in later stages of prostate cancer may limit the CAG effect.

Genetic predisposition to insulin-dependent diabetes mellitus (IDDM) involves one or more loci within the class II region of the major histocompatibility complex (MHC). The strongest MHC-linked determinants of disease susceptibility have been associated with polymorphisms in both the $ alpha$ and $ beta$ chain subunits of HLA-DQ (human) and I-A (mouse) class II molecules. Specifically, protection against IDDM development is associated with the presence of aspartic acid at position 57 of the $ beta$ chain while non-aspartic acid residues are found in diabetogenic $ beta$ chains. In humans, the greatest risk of disease development is observed in individuals with Arg 52$ rm sp+ alpha/Asp 57 sp- beta$ heterodimers. The BB rat is a useful system in which one can identify and characterize the genes necessary for the onset of IDDM. Although sequence data have been reported for various alleles of these class II genes, the effect of polymorphisms in the rat MHC is not nearly as well characterized as it is in humans or the NOD mouse. The rat MHC (RT1) contains two expressed class II loci, RT1.B and RT1.D, each encoding two class II molecules: RT1.B $ alpha$ and $ beta,$ and RT1.D $ alpha$ and $ beta.$ This study determined the nucleotide sequences of relevant regions from the RT1.B and RT1.D $ alpha/ beta$ genes in five rat strains (ACI, BB, Buffalo, Lewis, and Wistar-Furth) of varying susceptibility to IDDM to examine the role of specific polymorphisms in predisposition to disease. The data show that BB and Wistar-Furth rats (RT1$ sp{ rm u}$ haplotype) have identical class II sequences at all of the regions examined. Although no unique $ alpha$ chain sequences were found to associate with IDDM, we did confirm the association of aspartic acid at position 57 of RT1.B$ beta$ with susceptibility to diabetes. (Abstract shortened by UMI.)

Cytogenetic studies to identify chromosomal aberrations in ovarian tumors allow for a better understanding of tumor biology. Fifteen ovarian tumors (1 benign, 7 borderline, and 7 malignant tumors) and one normal control were successfully characterized using conventional cytogenetic methods. Among simple karyotypic changes, trisomy 10, not previously reported in ovarian cancer, was confirmed with fluorescence in situ hybridization (FISH). This latter may be considered as a single causative event or as one among many other chromosomal alterations, being both associated with ovarian tumorigenesis and tumor progression. Among complex karyotypic abnormalities, one cell one (OV90) was further characterized using combined GTG banding and fluorescence in situ hybridization (FISH) techniques with painting probes from all the chromosomes, and chromosome specific centromeric probes, and a 10p telomeric probe derived from a half-YACs. The short arm of one chromosome 3 was deleted and replaced by a homogeneous staining region (HSR) which was demonstrated to be originated from amplification of yet unidentified genes on chromosome 22. We also identified a complex chromosome rearrangement (CCR) involving chromosome 9, 10 and 17. A dicentric chromosome, dic(9;17)(p11;p11), and a derivative chromosome, der(10)del(10)(pter)t(10;17)(p15;p11.1p11.2 or p13) were demonstrated, with a deletion in the short arm of chromosome 9, and a partial deletion of chromosome 17p. A translocation of chromosome 10p telomere to chromosome 1p was also observed. In addition, partial trisomy of chromosome 13q14-qter was also characterized and considered to result from a duplication and a pericentric inversion. These chromosomal aberrations were. identified in all cells analyzed and in each cell culture despite long-term cell culture, repeated freezing and thawing. These abnormalities are thus probably responsible for tumorigenesis, tumor evolution or in vitro survival of cancer cells. Further characteriz

We identified two large French-Canadian (FC) pedigrees with idiopathic partial epilepsy. Family studies of over 500 members from these two families revealed over 63 individuals reported to have seizures or seizure-like histories. Pedigree analysis confirmed an autosomal dominant inheritance with reduced penetrance. Most of the affected individuals shared a number of clinical characteristics compatible with the definition of a novel genetic form of epilepsy syndrome. We termed it as familial partial epilepsy with variable foci (FPEVF), which was recently described by Scheffer et al. (1998) in one Australian family with eight possibly affected individuals. The most prominent clinical feature of FPEVF is the presence of variable seizure foci in different affected individuals within the same family. Most affected individuals in the two FC families present infrequent, brief, non-clustering, nocturnal, partial seizures. Age of onset is between 5 and 25 years. Affected individuals are neurologically intact without any detectable pathological lesion(s). / These two families were then subjected to a full-scale molecular genetic study. No linkage to any known loci for idiopathic partial epilepsy was detected, including the suggested chromosome (Chr) 2q locus for FPEVF in the Australian FPEVF family, the Chr 20q13.2 and 15q24 loci for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and the Chr 10q locus for autosomal dominant temporal lobe epilepsy with auditory symptoms (ADTLEAS). Significant linkage (LOD score = 8.60) was detected with markers on Chr 22 q12 during a genome scan. Collaborating with a group from Australia, we confirmed another two FC families and two non-FC families with compatible linkage to the same locus and a cumulative LOD score of 14.79 with marker D22S689. The minimum candidate region of FPEVF has been finally defined to an interval of 3.8 cM between markers D22S 1163 and D22S 1686 on Chr 22q12. We proceeded to search for the gene mutation by directly sequencing the coding regions of all candidate genes within the FPEVF region, an interval of 5.04 Mb harboring approximately 100 known or predicted genes. So far, 77 candidate genes have been sequenced, and no mutation has yet been found. Since there is no known ion channel gene mapped to this region, FPEVF is probably the first non-ion channel gene causing human idiopathic epilepsy*. / *After submission of this thesis for examination, Kalachikov et al. (2002) identified LGI1 (leucine-rich gene, glioma inactivated), a possible tumor suppressor, as the gene for ADTLEAS. This is the first evidence for a non-ion channel gene causing idiopathic epilepsy in humans.