Decomposition of $\rm R\times C$ contingency table chi -square: Application to binned DNA fragment size data and population structure analysis

The purpose of this research is to develop a new statistical method to determine the minimum set of rows (R) in a R x C contingency table of discrete data that explains the dependence of observations. The statistical power of the method will be empirically determined by computer simulation to judge its efficiency over the presently existing methods. The method will be applied to data on DNA fragment length variation at six VNTR loci in over 72 populations from five major racial groups of human (total sample size is over 15,000 individuals; each sample having at least 50 individuals). DNA fragment lengths grouped in bins will form the basis of studying inter-population DNA variation within the racial groups are significant, will provide a rigorous re-binning procedure for forensic computation of DNA profile frequencies that takes into account intra-racial DNA variation among populations. ^

ABERRATIONS OF A PUTATIVE TUMOR SUPPRESSOR GENE SEL1L IN PANCREATIC DUCTAL ADENOCARCINOMA

Introduction: Pancreatic cancer is the fourth leading cause of cancer-related death among males and females in the United States. Sel-1-like (SEL1L) is a putative tumor suppressor gene that is downregulated in a significant proportion of human pancreatic ductal adenocarcinoma (PDAC). It was hypothesized that SEL1L expression could be down-modulated by somatic mutation, loss of heterozygosity (LOH), CpG island hypermethylation and/or aberrantly expressed microRNAs (miRNAs). Material and methods: In 42 PDAC tumors, the SEL1L coding region was amplified using reverse transcription polymerase chain reaction (RT-PCR), and analyzed by agarose gel electrophoresis and sequenced to search for mutations. Using fluorescent fragment analysis, two intragenic microsatellites in the SEL1L gene region were examined to detect LOH in a total of 73 pairs of PDAC tumors and normal-appearing adjacent tissues. Bisulfite DNA sequencing was performed to determine the methylation status of the SEL1L promoter in 41 PDAC tumors and 6 PDAC cell lines. Using real-time quantitative PCR, the expression levels of SEL1L mRNA and 7 aberrantly upregulated miRNAs that potentially target SEL1L were assessed in 42 PDAC tumor and normal pairs. Statistical methods were applied to evaluate the correlation between SEL1L mRNA and the miRNAs. Further the interaction was determined by functional analysis using a molecular biological approach. Results: No mutations were detected in the SEL1L coding region. More than 50% of the samples displayed abnormally alternate or aberrant spliced transcripts of SEL1L. About 14.5% of the tumors displayed LOH at the CAR/CAL microsatellite locus and 10.7% at the RepIN20 microsatellite locus. However, the presence of LOH did not show significant association with SEL1L downregulation. No methylation was observed in the SEL1L promoter. Statistical analysis showed that SEL1L mRNA expression levels significantly and inversely correlated with the expression of hsa-mir-143, hsa-mir-155, and hsa-mir-223. Functional analysis indicated that hsa-mir-155 acted as a suppressor of SEL1L in PL18 and MDAPanc3 PDAC cell lines. Discussion: Evidence from these studies suggested that SEL1L was possibly downregulated by aberrantly upregulated miRNAs in PDAC. Future studies should be directed towards developing a better understanding of the mechanisms for generation of aberrant SEL1L transcripts, and further analysis of miRNAs that may downregulate SEL1L.

Molecular events associated with trisomy of chromosome 7 in mouse skin chemical carcinogenesis

The primary objective of this study has been to investigate the effects at the molecular level of trisomy of mouse chromosome 7 in chemically induced skin tumors. It was previously proposed that the initiation event in the mouse skin carcinogenesis model is a heterozygous mutation of the Ha-ras-1 gene, mapped to chromosome 7. Previous studies in this laboratory identified trisomy 7 as one of the primary nonrandom cytogenetic abnormalities found in the majority of severely dysplastic papillomas and squamous cell carcinomas induced in SENCAR mice by an initiation-promotion protocol. Therefore, the first hypothesis tested was that trisomy 7 occurs by specific duplication of the chromosome carrying a mutated Ha-ras-1 allele. Results of a quantitative analysis of normal/mutated allelic ratios of the Ha-ras-1 gene confirmed this hypothesis, showing that most of the tumors exhibited overrepresentation of the mutated allele in the form of 1/2, 0/3, and 0/2 (normal/mutated) ratios. In addition, histopathological analysis of the tumors showed an apparent association between the degree of malignancy and the dosage of the mutated Ha-ras-1 allele. To determine the mechanism for loss of the normal Ha-ras-1 allele, found in 30% of the tumors, a comparison of constitutional and tumor genotypes was performed at different informative loci of chromosome 7. By combining Southern blot and polymerase chain reaction fragment length polymorphism analyses of DNAs extracted from squamous cell carcinomas, complete loss of heterozygosity was detected in 15 of 20 tumors at the Hbb locus, and in 5 of 5 tumors at the int-2 locus, both distal to Ha-ras-1. In addition, polymerase chain reaction analysis of DNA extracted from papillomas indicated that loss of heterozygosity occurs in late-stage lesions exhibiting a high degree of dysplasia and areas of microinvasion, suggesting that this event may be associated to the acquisition of the malignant phenotype. Allelic dosage analysis of tumors that had become homozygous at Hbb but retained heterozygosis at Ha-ras-1, indicated that loss of heterozygosity on mouse chromosome 7 occurs by a mitotic recombination mechanism. Overall, these findings suggest the presence of a putative tumor suppressor locus on the 7F1-ter region of mouse chromosome 7. Thus, loss of function by homozygosis at this putative suppressor locus may complement activation of the Ha-ras-1 gene during tumor progression, and might be associated with the malignant conversion stage of mouse skin carcinogenesis. ^

Genes to blood pressure: The effects of variation in genes of the renin-angiotensin system on the hormonal and renal control of blood pressure

Objective. Essential hypertension affects 25% of the US adult population and is a leading contributor to morbidity and mortality. Because BP is a multifactorial phenotype that resists simple genetic analysis, intermediate phenotypes within the complex network of BP regulatory systems may be more accessible to genetic dissection. The Renin-Angiotensin System (RAS) is known to influence intermediate and long-term blood pressure regulation through alterations in vascular tone and renal sodium and fluid resorption. This dissertation examines associations between renin (REN), angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) gene variation and interindividual differences in plasma hormone levels, renal hemodynamics, and BP homeostasis.^ Methods. A total of 150 unrelated men and 150 unrelated women, between 20.0 and 49.9 years of age and free of acute or chronic illness except for a history of hypertension (11 men and 7 women, all off medications), were studied after one week on a controlled sodium diet. RAS plasma hormone levels, renal hemodynamics and BP were determined prior to and during angiotensin II (Ang II) infusion. Individuals were genotyped by PCR for a variable number tandem repeat (VNTR) polymorphism in REN, and for the following restriction fragment length polymorphisms (RFLP): AGT M235T, ACE I/D, and AT1 A1166C. Associations between clinical measurements and allelic variation were examined using multiple linear regression statistical models.^ Results. Women homozygous for the AT1 1166C allele demonstrated higher intracellular levels of sodium (p = 0.044). Men homozygous for the AGT T235 allele demonstrated a blunted decrement in renal plasma flow in response to Ang II infusion (p = 0.0002). There were no significant associations between RAS gene variation and interindividual variation in RAS plasma hormone levels or BP.^ Conclusions. Rather than identifying new BP controlling genes or alleles, the study paradigm employed in this thesis (i.e., measured genes, controlled environments and interventions) may provide mechanistic insight into how candidate genes affect BP homeostasis. ^

Matrix metalloproteinase-1 as a prognosticator in malignant glioma

The overall purpose of this study was to assess the relationship between the promoter region polymorphism (-2607 1G/2G) of matrix metalloproteinase-1 (MMP-1) polymorphism and outcome in brain tumor patients diagnosed with a primary brain tumor between 1994 and 2000 at The University of Texas M. D. Anderson Cancer Center. The MMP-1 polymorphism was genotyped for all brain tumor patients who participated in the Family Brain Tumor Study and for whom blood samples were available. Relevant covariates were abstracted from medical records for all cases from the original protocol, including information on demographics, tumor histology, therapy and outcome was obtained. The hypothesis was that brain tumor patients with the 2G allele have a poorer prognosis and shorter survival than brain tumor patients with the 1G allele. ^ Experimental Design: Genetic variants for the MMP-1 enzyme were determined by a polymerase chain reaction-restriction fragment length polymorphism assay. Comparison was made between the overall survival for cases with the 2G polymorphism and overall survival for cases with the 1G polymorphism using multivariable Cox Proportional-Hazard analysis, controlling for age, sex, Karnofsky Performance Scale (KPS), extent of surgery, tumor histology and treatment received. Kaplan-Meier and Cox Proportional-Hazard analyses were utilized to assess if the MMP-1 polymorphisms were related to overall survival. Results: Overall survival was not statistically significantly different between the 2G allele brain tumor patients and the 1G allele patients and there was no statistically significant difference between tumor types. ^ Conclusions: No association was found between MMP-1 polymorphisms and survival in patients with malignant gliomas. ^