Bharat Thyagarajan, Ph.D. - US grants

DESCRIPTION (provided by applicant): Although early detection and removal of colorectal adenomas, the precursors of most colorectal cancers, has significantly reduced morbidity and mortality due to colorectal cancer, the disease remains the second leading cause of cancer mortality in the United States. There is increasing evidence that oxidative stress plays an important role in the pathogenesis of colorectal cancer. We recently found that increased circulating concentrations of oxidative stress biomarkers are associated with increased colorectal adenoma risk. Mitochondria (Mt) are the predominant source of intracellular reactive oxygen species (ROS) and a major cause of endogenous oxidative stress. Mitochondrial variations (mitochondrial DNA (mtDNA) copy number and mitochondrial polymorphisms) have been associated with increased ROS production. Despite the central role for mitochondria in ROS production, their role in pathogenesis of colorectal adenomas has not been evaluated. We hypothesize that mtDNA copy number and germline variations in the mitochondrial genome will be associated with colorectal adenoma risk. We propose to evaluate this hypothesis by measuring mtDNA copy number using real time polymerase chain reaction and genotyping mitochondrial tagging single nucleotide polymorphisms (tagSNPs) and D-loop polymorphisms in 392 colorectal adenoma cases and 565 controls collected from three methodologically very similar, colonoscopy-based case-control studies of incident, sporadic colorectal adenomas. We will use unconditional logistic regression to analyze the association between mtDNA copy number, mitochondrial polymorphisms and colorectal adenoma risk. This innovative epidemiologic study seeks to translate our understanding of mitochondrial biology into discovery of novel mitochondrial biomarkers that can inform strategies for the primary prevention of colorectal adenoma. Findings from this exploratory study will provide preliminary data to design a prospective study with adequate sample size to confirm the findings from this study and comprehensively evaluate the role of mitochondria in colorectal cancer etiology. PUBLIC HEALTH RELEVANCE: This application aims to evaluate the association between mitochondrial variants (mitochondrial DNA copy number and mitochondrial polymorphisms) and risk of colorectal adenoma is an existing dataset of 392 colorectal adenoma cases and 565 controls. The long-term goal of the proposed project is to further our understanding of the complex interactions between genetic and environmental/lifestyle factors that affect risk for incident, sporadic colorectal cancer.

DESCRIPTION (provided by applicant): Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) may have a key role in the pathogenesis of several cancers, including colorectal cancer. Extensive studies have shown mitochondria to be the predominant endogenous source of ROS in cells. Increased ROS exposure induces oxidative DNA damage in mitochondrial and nuclear DNA as well as the activation of apoptotic and inflammation pathways. Through these activities, ROS exposure can lead to malignant transformation of cells. The extent of oxidative damage and malignant transformation is influenced by the activity of the ROS detoxification system. Subtle changes in ROS production and detoxification are influenced by environmental factors and genetic variation between individuals. While these activities are well established in cellular and animal studies, few human studies have evaluated the relationship between mitochondrial dysfunction and colorectal cancer risk. We will prospectively evaluate the association between several aspects of mitochondrial dysfunction, genetic variation, oxidative damage and the ROS detoxification system and colorectal cancer in a frequency matched nested case-control study of 640 colorectal cancer cases and 1280 controls within a well-established prospective cohort, the Singapore Chinese Health Study. Indicators of mitochondrial dysfunction include mitochondrial DNA copy number, mitochondria DNA damage as measured by quantitative real time polymerase chain reaction and oxidative damage as measured by urinary F2-isoprostanes and plasma fluorescent oxidation products. The mitochondrial genome and nuclear genes related to mitochondrial biogenesis will be evaluated for genetic susceptibility to oxidative stress through the measurement of polymorphisms and haplogroups. Additional major determinants of oxidative stress that will be measured include antioxidant enzymes of the ROS detoxification system, dietary intake of fruits and vegetables and more specifically by plasma carotenoids. Together, these measures provide a multi-pronged and integrated approach that addresses major aspects of mitochondrial dysfunction and oxidative stress that can be analyzed in humans. It will provide valuable information on the role of mitochondrial dysfunction and the mitochondrial related genetic variation in the risk of colorectal cancer, which will aid in the identification of high risk indivduals and may provide novel opportunities for prevention of colorectal cancer.

The Long Life Family Study (LLFS) has enrolled 4,953 participants in 539 pedigrees in the USA and Denmark that are enriched for exceptional longevity, and has measured them longitudinally in two extensive in-home visits measuring key healthy aging phenotypes in all of the major domains of the aging process. We have demonstrated through many publications that selecting on longevity in the first (proband) generation, results in the second (offspring) generation being much healthier than average in many key phenotypes. However, the pedigrees are heterogeneous by phenotype, with different families showing familial clustering of protection in cognition, grip strength, pulmonary function, blood pressure, etc. Further, comprehensive linkage analysis of the LLFS sample identifies extremely strong genetic linkage peaks for cross-sectional as well as longitudinal trajectory rates of change phenotypes for a wide variety of healthy aging domains such as exceptional cognitive performance and lack of Alzheimer?s disease. These peaks are NOT explained by GWAS SNPs (or those that can be imputed by GWAS). Pedigree specific LODs and preliminary deep sequencing suggests that these peaks are driven by rare, protective variants running in selected pedigrees. We propose to do Whole Genome Sequencing on this unique cohort, to identify the rare protective variants driving these strong linkage peaks. We propose to continue longitudinal assessment of the cohort with a third in-person visit, which will allow us to assess potential non-linear patterns of aging, and adding formal assessment of dementia diagnosis for Alzheimer?s Disease and other dementia types, which will increase specificity and power to discover and follow- up on protective variants against Alzheimer?s Disease and other dementia diagnoses. For pedigrees driving multiple strong linkage peaks, we also propose to phenotypically measure the third generation (grandchildren), as these are likely to carry more copies of the rare protective alleles running in these families, which will exponentially increase our power to resolve them. Preliminary evidence from the Danish Medical Registry suggests that, at least in Denmark, the protection persists into this third generation, with significantly lower rates of medical conditions across the disease spectrum. We also propose to do extensive transcriptomics, methylomics, and proteomics on these selected high linkage pedigrees, to begin to move from ?statistically associated variants/loci? to the biological genes of action, since we expect most of the driving variants will be regulatory and non-coding. It is critical to find the modes of action of these rare protective variants. We also propose to do metabolomics on the entire LLFS cohort, longitudinally, with the goal of identifying novel biomarkers of healthy aging and resistance to diseases such as Alzheimer?s in this unusually heathy cohort. Combined with a systems biology/network approach to data integration of the proposed ?Big Data?, such biomarkers would improve our power to detect even more novel protective genetic variants and identify the genetic signatures and pathways of genes conferring protection in this unique cohort to prevent onset of major diseases such as diabetes, cardiovascular disease, cancer and Alzheimer?s Disease and other dementia types.

The Long Life Family Study (LLFS) has enrolled 4,953 participants in 539 pedigrees in the USA and Denmark that are enriched for exceptional longevity, and has measured them longitudinally in two extensive in-home visits measuring key healthy aging phenotypes in all of the major domains of the aging process. We have demonstrated through many publications that selecting on longevity in the first (proband) generation, results in the second (offspring) generation being much healthier than average in many key phenotypes. However, the pedigrees are heterogeneous by phenotype, with different families showing familial clustering of protection in cognition, grip strength, pulmonary function, blood pressure, etc. Further, comprehensive linkage analysis of the LLFS sample identifies extremely strong genetic linkage peaks for cross-sectional as well as longitudinal trajectory rates of change phenotypes for a wide variety of healthy aging domains such as exceptional cognitive performance and lack of Alzheimer?s disease. These peaks are NOT explained by GWAS SNPs (or those that can be imputed by GWAS). Pedigree specific LODs and preliminary deep sequencing suggests that these peaks are driven by rare, protective variants running in selected pedigrees. We propose to do Whole Genome Sequencing on this unique cohort, to identify the rare protective variants driving these strong linkage peaks. We propose to continue longitudinal assessment of the cohort with a third in-person visit, which will allow us to assess potential non-linear patterns of aging, and adding formal assessment of dementia diagnosis for Alzheimer?s Disease and other dementia types, which will increase specificity and power to discover and follow- up on protective variants against Alzheimer?s Disease and other dementia diagnoses. For pedigrees driving multiple strong linkage peaks, we also propose to phenotypically measure the third generation (grandchildren), as these are likely to carry more copies of the rare protective alleles running in these families, which will exponentially increase our power to resolve them. Preliminary evidence from the Danish Medical Registry suggests that, at least in Denmark, the protection persists into this third generation, with significantly lower rates of medical conditions across the disease spectrum. We also propose to do extensive transcriptomics, methylomics, and proteomics on these selected high linkage pedigrees, to begin to move from ?statistically associated variants/loci? to the biological genes of action, since we expect most of the driving variants will be regulatory and non-coding. It is critical to find the modes of action of these rare protective variants. We also propose to do metabolomics on the entire LLFS cohort, longitudinally, with the goal of identifying novel biomarkers of healthy aging and resistance to diseases such as Alzheimer?s in this unusually heathy cohort. Combined with a systems biology/network approach to data integration of the proposed ?Big Data?, such biomarkers would improve our power to detect even more novel protective genetic variants and identify the genetic signatures and pathways of genes conferring protection in this unique cohort to prevent onset of major diseases such as diabetes, cardiovascular disease, cancer and Alzheimer?s Disease and other dementia types.