Jinying Zhao, Ph.D. - US grants

DESCRIPTION (provided by applicant): This mentored research scientist career development award (KOI) proposal is a four-year plan to enable the candidate to develop into an independent investigator in the field of genetic epidemiology for human age-related disorders, in particular cardiovascular disease (CVD). The candidate has been very successful in the area of statistical genetics. However, she lacks formal training in epidemiology, aging biology and clinical cardiology, three crucial components for an outstanding genetic epidemiologist in human chronic disorders. This grant provides a unique opportunity for extensive development of skills in epidemiology, cardiovascular medicine and aging mechanism. These short term career goals will be accomplished through formal course work, extensive mentorship in a collaborative environment, and implementation of a research plan that will form the basis of a larger study aimed at investigating the role of mitochondrial gene polymorphisms in biological aging and CVD. The candidate is currently covered under a Master of Science in Clinical Research (MSCR-KL2) Program which provides her one year didactic training in epidemiological study design, confounding, clinical database management, and chronic disease epidemiology. During the first year of this KOI, she will continue formal training in clinical trials, epidemiological data analysis, grant writing, biology of CVD and aging as well as clinical cardiology. This didactic training will be complemented by the proposed research project, which proposes for the first time that mitochondrial-related genetic variants underlie the biological links among vascular aging, coronary artery disease (CAD) and major adverse cardiac events. This project will take advantage of a large well- characterized patient cohort for coronary angiography (1,000 patients with significant CAD and 1,000 matched controls) that has been compiled and maintained under the direction of her two mentors. The specific aims are 1) To examine whether mitochondrial-related variants are implicated in biological aging measured by telomere length;and 2) To determine whether mitochondrial-related polymorphisms are associated with CAD and major adverse cardiac events. This K0I award will significantly enhance the candidate's growth and maturation into an independent genetic epidemiologist in human aging disorders, in particular cardiovascular disease. RELEVANCE: Coronary artery disease (CAD), a typical aging disorder, is the leading cause of death and disability worldwide. Identification of the link between biological aging and CAD will not only provide novel insights into the pathophysiology of aging and CAD, but may also identify new biomarkers for aging and atherogenesis, which may, ultimately, improve prediction, prevention and treatment of a wide range of age-related disorders.

DESCRIPTION (provided by applicant): Cardiovascular disease (CVD) and depression are two complex diseases that often co-occur. However, the potential mechanisms linking depression and CVD remain unclear. Recent studies suggest that common genetic vulnerability may explain the comorbidity of these two disorders. Because studies in human and animal models have consistently reported the role of hypothalamic-pituitary-adrenal (HPA) axis dysfunction in the risk of both depression and CVD, genetic abnormalities in the HPA system may thus represent an important pathophysiological mechanism that contributes to the co-occurrence of depression and CVD. The overall objective of this project is to test the hypothesis that genetic variants within the HPA-related pathways are key determinants for the vulnerability to both depression and CVD. We will genotype 19 key candidate genes involved in the HPA axis and related pathways using a twin sample including 640 middle-aged male twins from the Vietnam Era Twin Registry (VETR). All these twins were extensively phenotyped in recent studies, including information on depressive symptoms, subclinical CVD, detailed measurements of stress and other psychological variables, as well as behavioral and social-demographic variables. The proposed study using twins provides a unique opportunity to tease out gene-environment effects that are usually confounded by other factors in classical genetic studies. Findings from this study may not only open new windows into the mechanisms underlying these two common disorders but in the future may also provide guidance for optimal therapeutic treatments particularly for genetically susceptible individuals. PUBLIC HEALTH RELEVANCE: This study proposes to identify common genetic polymorphisms in the HPA axis and related biological pathways for the comorbidity of depression and cardiovascular disease using a well-phenotyped twin database from the Vietnam Era Twin Registry. Results will provide valuable data for deciphering the genetic basis of cardiovascular and psychiatric diseases.

DESCRIPTION (provided by applicant): Telomeres are specialized DNA sequences at the end of each chromosome. Telomere length shortens progressively during each round of cell cycle and declines with aging, and thus has emerged as a valuable biomarker for biological aging and age-related disorders. Shorter telomeres have been associated with increased risk for type 2 diabetes and its related phenotypes. These associations, however, were primarily based on cross-sectional data, and therefore raise an important question as to whether shorter telomeres are a cause or a consequence of diabetes, or whether it is simply an epiphenomenon. Diabetes disproportionately affects American Indians. The prevalence of type 2 diabetes is, on average, 2-4 times higher than that in other ethnic groups. The objectives of this study are to delineate the prospective impact of telomere attrition on diabetes risk, and to determine genetic, behavioral and psychosocial predictors for accelerated telomere loss. Leukocyte telomere length will be measured by quantitative PCR in 4,565 DNA samples collected by the Strong Heart Family Study at two clinical visits (900 subjects examined at both visits, 2,765 examined at the second visit only). All DNA samples, well-characterized clinical data including follow-up data through December 2009 and data from a 10cM genome scan are already available for the proposed analyses. Specific aims: 1) To determine whether telomere attrition is associated with diabetes and its related phenotypes; 2) To identify genetic loci related to telomeric variation by a genome-wide linkage scan; 3) To determine behavioral, socioeconomic and psychosomatic predictors for accelerated telomere shortening in relation to diabetes risk. This is the first study to investigate prospectively the associations of telomere length and of telomere attrition rate with diabetes risk, and is also the first study to determine genetic, behavioral and psychosocial predictors for accelerated telomere erosion in this underserved population. If the proposed aims are achieved, we will be able to provide valuable information regarding a causal role of accelerated telomere loss in the pathogenesis of diabetes, thereby providing evidence for telomere length as a biomarker for diabetes and its associated disorders. The results will also provide important information for risk stratification in American Indians and other ethnic groups as well. Furthermore, this study will provide valuable information for lifestyle/behavioral interventions for diabetic risk reduction. We expect that this study will open new lines of research, and could potentially lead to critical discoveries that will accelerate the field of aging and diabetes as well as a wide range of metabolic disorders, and thus is of great significance.

DESCRIPTION (provided by applicant): Major depressive disorder (MDD) is a devastating psychiatric disorder that affects millions of Americans. Despite substantial research, no specific risk factor has yet been identified as having a causal role in MDD. Epigenetic modifications, especially DNA methylation, are increasingly being recognized as a key mechanism involved in the pathogenesis of depression. However, the biological pathways linking aberrant methylation to depression remain poorly understood. This uncertainty greatly hampers our ability to implement early diagnosis, prevention and treatment for this debilitating disorder. The objective of this study is to identify functional epigenetic determinants for MDD. Our central hypothesis is that aberrant DNA methylation and resulting alterations in gene expression are associated with MDD. The rationale for the proposed research is that: once we know the epigenetic determinants for depression, we will be able to develop novel epigenetic markers and therapeutic targets for risk assessment, prevention and treatment of MDD and related psychiatric conditions. We proposed three specific aims: (1) Identify differentially methylated regions (DMRs) associated with MDD. This aim is to conduct an epigenome-wide DNA methylation analysis to identify epigenetic variations contributing to MDD in monocytes DNA from 100 monozygotic (MZ) discordant twin pairs from the University of Washington Twin Registry (UWTR), a large community-based twin registry in the U.S. (2) Replicate the top 50 ranked genes from Aim 1 in two independent samples, including 80 MZ discordant twin pairs recruited from the same registry and 36 postmortem brain tissue of well-characterized MDD patients and matched controls. (3) Determine the functional importance of the positive methylation findings in both blood and brain by profiling gene expression levels in each of the four brain regions (frontal cortex, hippocampus, amygdala, and cingulate cortex). Differential expressed genes related to MDD will be identified. Integrative analyses will be performed to elucidate the connections between DNA methylation patterns and gene expression of cognate genes in relation to MDD. The proposed study is the only one of its kind to identify functional epigenetic determinants for MDD in a well- matched MZ discordant twin sample, followed by replication in postmortem brain tissue, the affected organ in MDD. The work proposed here is expected to have an important positive impact, because genes with both differential methylation and expression are highly likely to provide novel epigenetic targets for prevention, intervention and treatment for depression and its related psychiatric conditions in addition to fundamentally advancing the fields of psychiatric genetics.

DESCRIPTION (provided by applicant): Major depressive disorder (MDD) is a devastating psychiatric disorder that affects millions of Americans. Despite substantial research, no specific risk factor has yet been identified as having a causal role in MDD. Epigenetic modifications, especially DNA methylation, are increasingly being recognized as a key mechanism involved in the pathogenesis of depression. However, the biological pathways linking aberrant methylation to depression remain poorly understood. This uncertainty greatly hampers our ability to implement early diagnosis, prevention and treatment for this debilitating disorder. The objective of this study is to identify functional epigenetic determinants for MDD. Our central hypothesis is that aberrant DNA methylation and resulting alterations in gene expression are associated with MDD. The rationale for the proposed research is that: once we know the epigenetic determinants for depression, we will be able to develop novel epigenetic markers and therapeutic targets for risk assessment, prevention and treatment of MDD and related psychiatric conditions. We proposed three specific aims: (1) Identify differentially methylated regions (DMRs) associated with MDD. This aim is to conduct an epigenome-wide DNA methylation analysis to identify epigenetic variations contributing to MDD in monocytes DNA from 100 monozygotic (MZ) discordant twin pairs from the University of Washington Twin Registry (UWTR), a large community-based twin registry in the U.S. (2) Replicate the top 50 ranked genes from Aim 1 in two independent samples, including 80 MZ discordant twin pairs recruited from the same registry and 36 postmortem brain tissue of well-characterized MDD patients and matched controls. (3) Determine the functional importance of the positive methylation findings in both blood and brain by profiling gene expression levels in each of the four brain regions (frontal cortex, hippocampus, amygdala, and cingulate cortex). Differential expressed genes related to MDD will be identified. Integrative analyses will be performed to elucidate the connections between DNA methylation patterns and gene expression of cognate genes in relation to MDD. The proposed study is the only one of its kind to identify functional epigenetic determinants for MDD in a well- matched MZ discordant twin sample, followed by replication in postmortem brain tissue, the affected organ in MDD. The work proposed here is expected to have an important positive impact, because genes with both differential methylation and expression are highly likely to provide novel epigenetic targets for prevention, intervention and treatment for depression and its related psychiatric conditions in addition to fundamentally advancing the fields of psychiatric genetics.

? DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a devastating neurodegenerative disorder for which there is no cure or effective treatment. A thorough understanding of its molecular mechanisms is a prerequisite for discovering novel diagnostic and therapeutic strategies against AD. DNA methylation at the fifth position of cytosine (5mC) is a well-studied epigenetic mark that is implicated in AD. The newly discovered 5-hydroxymethylcytosine (5hmC) is an oxidative product of 5mC that is essential for DNA demethylation. 5hmC is particularly enriched in the brain, accumulates with aging process, and is dynamically regulated by life experiences. 5hmC exhibits distinctive genomic distribution as compared to 5mC, and altered 5hmC influences gene expression. These findings suggest that 5hmC represents a new dimension of epigenetic regulation for brain function and neurodegeneration. However, there is little research examining the genome-wide pattern of 5hmC in human brain and its role in AD in human populations. Building on our prior work in human brain and animal models, we hypothesize that aberrant 5hmC modification is causally associated with AD pathology. Our goal is to identify causative 5hmC alterations associated with quantitative neuropathological measures for early features of AD pathology (e.g., amyloid plaques, neurofibrillary tangles). To achieve this, we propose four specific aims: (1) Identify differentially hydroxymethylated regions associated with AD pathology by genome-wide profiling of 5hmC in 740 postmortem brains collected by two large, community-based population studies of aging and dementia: the Religious Order Study (discovery sample) and the Rush Memory and Aging Project (replication sample). As traditional methods cannot discriminate between 5mC and 5hmC, we will perform 5hmC-capture sequencing, followed by TET-assisted bisulfite sequencing using novel techniques developed by our group and collaborators. (2) Conduct targeted methylation sequencing to identify additional AD-associated 5mC alterations that may have been missed by our previous EWAS as a result of the limited resolution and genome coverage of the Illumina platform. (3) Functionally validate the putative genes identified in Aims 1 and 2 using existing RNA-seq data from the same brain cortex and a fly model for AD. (4) Perform integrative `omics' analyses to test the joint and interactive effects of multi-layer `omic' markers on AD pathology. This innovative project leverages the wealth of deep clinical and neuropathological phenotypes and multi-level `omics' datasets generated in the same brain tissue, and provides unprecedented opportunities to uncover novel molecular mechanisms underlying AD pathology. Our proposal brings together an exceptionally strong and unique multi- disciplinary team with complementary expertise needed to achieve our goal. The work proposed represents the frontier in the interface between AD and `omics' research. Findings of this study will provide important mechanistic insights into disease etiology, and are highly likely to lead to the discovery of novel strategies for early detection, prevention and therapeutic intervention of AD.

Project Summary Alzheimer's disease (AD) is a devastating neurodegenerative disorder for which there is no cure or effective treatment. A thorough understanding of its molecular mechanisms is a prerequisite for discovering novel diagnostic and therapeutic strategies against AD. DNA methylation at the fifth position of cytosine (5mC) is a well-studied epigenetic mark that is implicated in AD. The newly discovered 5-hydroxymethylcytosine (5hmC) is an oxidative product of 5mC that is essential for DNA demethylation. 5hmC is particularly enriched in the brain, accumulates with aging process, and is dynamically regulated by life experiences. 5hmC exhibits distinctive genomic distribution as compared to 5mC, and altered 5hmC influences gene expression. These findings suggest that 5hmC represents a new dimension of epigenetic regulation for brain function and neurodegeneration. However, there is little research examining the genome-wide pattern of 5hmC in human brain and its role in AD in human populations. Building on our prior work in human brain and animal models, we hypothesize that aberrant 5hmC modification is causally associated with AD pathology. Our goal is to identify causative 5hmC alterations associated with quantitative neuropathological measures for early features of AD pathology (e.g., amyloid plaques, neurofibrillary tangles). To achieve this, we propose four specific aims: (1) Identify differentially hydroxymethylated regions associated with AD pathology by genome-wide profiling of 5hmC in 740 postmortem brains collected by two large, community-based population studies of aging and dementia: the Religious Order Study (discovery sample) and the Rush Memory and Aging Project (replication sample). As traditional methods cannot discriminate between 5mC and 5hmC, we will perform 5hmC-capture sequencing, followed by TET-assisted bisulfite sequencing using novel techniques developed by our group and collaborators. (2) Conduct targeted methylation sequencing to identify additional AD-associated 5mC alterations that may have been missed by our previous EWAS as a result of the limited resolution and genome coverage of the Illumina platform. (3) Functionally validate the putative genes identified in Aims 1 and 2 using existing RNA-seq data from the same brain cortex and a fly model for AD. (4) Perform integrative `omics' analyses to test the joint and interactive effects of multi-layer `omics' markers on AD pathology. This innovative project leverages the wealth of deep clinical and neuropathological phenotypes and multi-level `omics' datasets generated in the same brain tissue, and provides unprecedented opportunities to uncover novel molecular mechanisms underlying AD pathology. Our proposal brings together an exceptionally strong and unique multi- disciplinary team with complementary expertise needed to achieve our goal. The work proposed represents the frontier in the interface between AD and `omics' research. Findings of this study will provide important mechanistic insights into disease etiology, and are highly likely to lead to the discovery of novel strategies for early detection, prevention and therapeutic intervention of AD.

Project Summary American Indians (AIs) suffer disproportionately from type 2 diabetes (T2D). Discovery of novel mechanistic biomarkers is the key to identify at-risk individuals and to develop effective preventive strategies tailored to this high risk population. In response to PA-12-165, this project leverages the wealth of unique resources collected by the Strong Heart Study (SHS), the largest longitudinal cohort study of American Indians followed over 25 years, to identify sensitive and specific metabolic markers that are predictive of T2D risk at preclinical stages above and over standard clinical factors including obesity, fasting glucose and insulin resistance. Metabolomics is an emerging technology that can simultaneously identify and accurately quantify hundreds to thousands of metabolites in biofluids. Several metabolites, such as BCAAs, acylcarnitines, and lipids, have been associated with T2D, but these results were largely derived from cross-sectional studies in almost exclusively European Caucasians. However, given the genetic regulation of metabolism, metabolites identified in Caucasians may not be generalized to AIs who may have a different genetic make-up. In addition, cross- sectional analysis cannot capture the dynamic trajectory of metabolic changes over time. Moreover, most existing studies measured a list of pre-selected metabolites on a single platform, but given the complexity of the human metabolome and the substantial diversity of metabolites, no single analytical platform can detect all metabolites in a biological sample. We hypothesize that longitudinal changes in plasma metabolites predict T2D risk independent of fasting glucose, insulin resistance (IR) and obesity, and that metabolic profiles of T2D in AIs are similar to, but distinct from, those in Caucasians. Our goal here is to identify novel and sensitive T2D predictors that are specific to AIs beyond classical T2D indicators. To achieve this, we will repeatedly measure concentrations of over 500 metabolites, including BCAAs, carbohydrates, hydroxyl acids, lipids, as well as gut microbial-derived metabolites, in fasting plasma (~5 yr apart) from normoglycemic SHS participants followed >15 years. Putative metabolites will be replicated in an independent longitudinal sample of AIs followed for 10 years. To increase coverage, we will quantify metabolites concentrations on three complementary platforms. Each assay will be performed as a dual 'targeted' and 'untargeted' analyses to provide both hypothesis-driven quantitative data and discovery-driven semi-quantitative data of unidentified metabolites. Unknown compounds will be identified by well-established workflows. Multivariate analyses will be conducted to identify novel T2D predictors above and over standard clinical factors. Our multidisciplinary team consists of experts with complementary expertise in diabetes epidemiology, metabolomics, analytical chemistry, statistics and bioinformatics. Findings of this study will greatly advance our understanding of T2D pathology, and hold promise for reducing or eliminating T2D disparity in AIs, an ethnically important but traditionally understudied minority group suffering from alarmingly high rates of T2D and obesity.

Project Summary Alzheimer?s disease (AD) is the most common form of dementia among older people with no cure or effective treatment. A thorough understanding of its molecular mechanisms is required for discovering novel diagnostic and therapeutic strategies against AD. Chemical modifications of DNA such as methylation play critical roles in regulating gene expression and many other key biological processes, and altered DNA methylation pattern has been implicated in brain aging and AD. While much attention has focused on DNA methylation at the fifth position on cytosine (5mC), recent research identified a new form of DNA modification at the sixth position on adenine (6mA) in mammalian brains. However, little is known about its presence, genomic distribution, and possible functions in human brain and relevance to AD. Our preliminary data in mouse and human brain indicated that 6mA is dynamically responsive to environmental stress and accumulates in human AD brain. Our central hypothesis is that altered signature of 6mA modification is causally associated with AD neuropathology. The objectives of this project are to generate the first detailed map of brain 6mA methylome and identify causative genes harboring aberrant 6mA alterations associated with quantitative neuropathological measures for early features of AD pathology (e.g., amyloid plaques, neurofibrillary tangles). To achieve this, we propose three specific aims: (1) Genome-wide mapping of brain DNA 6mA methylome to identify differentially methylated genes/regions harboring altered 6mA sites (D6AMRs) associated with AD pathology in 1,200 postmortem brain tissue samples collected by two large, community-based population cohorts of aging and dementia. (2) Integrated multiomics analysis to elucidate the potential mechanistic role of 6mA alteration in AD pathology; and (3) Functionally validation of top-ranked candidate genes in 3D brain organoids derived from human iPSCs. This innovative project leverages the wealth of deep clinical and neuropathological phenotypes along with rich omics data including genetic (GWAS, WGS), epigenetic (5mC, 5hmC, 6mA, H3K9Ac), and transcriptome (RNA-seq) profiled on the same prefrontal cortex, and will provide unprecedented opportunities to uncover novel molecular mechanisms implicated in AD pathology. Our proposal brings together an exceptionally strong and unique multidisciplinary team with complementary expertise in genetic epidemiology, statistical genetics, bioinformatics, molecular and neuroepigenetics, and Alzheimer?s research. The work proposed represents the frontier in the interface between AD and omics research. Findings of this study will provide novel mechanistic insight into AD pathogenesis, and are likely to discover new molecular targets with important clinical and translational implications.

Project Summary Aging and age-related cardiometabolic diseases (CMDs) such as obesity, type 2 diabetes, hypertension, cardiovascular disease, and chronic kidney disease, along with their risk factors (e.g., insulin resistance, inflammation, dyslipidemia, etc.), result from the complex interplay between genetic, lifestyle, and environmental factors. American Indians (AIs) suffer disproportionately from these chronic cardiometabolic conditions. Gut microbiota (bacteria, viruses, fungi, multicellular parasites, and archaea in our intestine) has emerged as a novel, metabolically active ?organ? that regulates many key biological processes and physiological functions. Gut dysbiosis (imbalance in gut microbial community, e.g., loss of microbial diversity or beneficial microbes, expansion of pathogenic microbes) has been associated with chronic metabolic disorders. However, several fundamental knowledge gaps exist, e.g., what are the key microbial signatures associated with aging and CMDs? What host factors shape the gut flora and how? What are the specific microbes or microbial species in human gut, and how does their composition and function differ across different populations/ethnic groups? Is the variation in human gut microbiota influenced by host genome, and if so, to what extent? Despite these unknowns, it is well accepted that the gut microbiome varies significantly among individuals and its composition heavily depends on an individual?s age, gender, geography, dietary preference, lifestyle, health status, etc. Since AIs suffer from high rates of obesity and diabetes, live on reservations or other tribal lands, eat traditional food and medicine, and practice other unique lifestyles, it is possible that they harbor different sets of disease- and health-associated gut microbiomes compared to other populations/ethnic groups. The objectives of this study are to address these fundamental questions by generating the first complete map of the human gut microbiome and identifying key microbial features associated with aging and CMDs in American Indians. To achieve this, we will leverage the parent SHS Phase VII (funded by NHLBI as a contract, 2019-2026) that will re-exam all living participants (N~=3,000) in 2020-2024 to collect stool samples from 1,500 well-phenotyped AI participants. We will conduct whole-genome shotgun metagenomic sequencing and perform innovative statistical analyses to: (1) identify key age-related gut microbiome features associated with biological aging (assessed by leukocyte telomere length) and CMDs (Aim 1); (2) identify host factors that shape the human gut microbiota in AIs (Aim 2); (3) explore the mechanistic links between gut dysbiosis, aging, and CMDs (Aim 3). Our long-term goal is to understand the mechanisms through which gut microbes interact with host factors in leading to accelerated aging and CMDs, with an ultimate goal to develop novel, precision therapeutic interventions (e.g., diet, drugs, live organisms, fecal microbiota transplantation) to promote healthy aging and improve cardiometabolic health.