Using heterogeneous, longitudinal EHR data for risk assessment and early detection of cardiovascular disease

Cardiovascular disease (CVD) affects millions of people and is a leading cause of death worldwide. CVD consists of a broad set of conditions including structural heart disease, coronary artery disease and stroke. Risk for each of these conditions accumulates over long periods of time depending on several risk factors. In order to reduce morbidity and mortality due to CVD, preventative treatments administered prior to first CVD event are critical. According to clinical guidelines, such treatments should be guided by an individual’s total risk within a window of time. A related objective is secondary prevention, or early detection, wherein the aim is to identify and mitigate the impact of a disease that has already taken effect. With the widespread adoption of electronic health records (EHRs), there is tremendous opportunity to build better methods for risk assessment and early detection. However, existing methods which use EHRs are limited in several ways: (1) they do not leverage the full longitudinal history of patients, (2) they use a limited feature set or specific data modalities, and (3) they are rarely validated in broader populations and across different institutions. In this dissertation, I address each of these limitations. In Aim 1, I explore the challenge of handling longitudinal, irregularly sampled clinical data, proposing discriminative and generative approaches to model this data. In Aim 2, I develop a multimodal approach for the early detection of structural heart disease. Finally, in Aim 3, I study how different feature inclusion choices affect the transportability of deep risk assessment models of coronary artery disease across institutions. Collectively, this dissertation contributes important insights towards building better approaches for risk assessment and early detection of CVD using EHR data and systematically assessing their transportability across institutions and populations

Mechanisms by Which 17β-Estradiol (E2) Suppress Neuronal cox-2 Gene Expression.

E2 attenuates inflammatory responses by suppressing expression of pro-inflammatory genes. Given that inflammation is increasingly being associated with neurodegenerative and psychiatric processes, we sought to elucidate mechanisms by which E2 down-regulates a component of an inflammatory response, cyclooxygenase- 2 (COX-2) expression. Although inflammatory processes in the brain are usually associated with microglia and astrocytes, we found that the COX-2 gene (cox-2) was expressed in a neuronal context, specifically in an amygdalar cell line (AR-5). Given that COX-2 has been reported to be in neurons in the brain, and that the amygdala is a site involved in neurodegenerative and neuropsychiatric processes, we investigated mechanisms by which E2 could down-regulate cox-2 expression in the AR-5 line. These cells express estrogen receptors alpha (ERα) and beta (ERβ), and as shown here cox-2. At the level of RNA, E2 and the ERβ selective ligand diarylpropionitrile (DPN) both attenuated gene expression, whereas the ERα selective ligand propyl pyrazole triol (PPT) had no effect. Neither ligand increased ERβ at the cox-2 promoter. Rather, DPN decreased promoter occupancy of NF-κB p65 and histone 4 (H4) acetylation. Treatment with the non-specific HDAC inhibitor Trichostatin A (TSA) counteracted DPN's repressive effects on cox-2 expression. In keeping with the TSA effect, E2 and DPN increased histone deacetylase one (HDAC1) and switch-independent 3A (Sin3A) promoter occupancy. Lastly, even though E2 increased CpG methylation, DPN did not. Taken together, the pharmacological data indicate that ERβ contributes to neuronal cox-2 expression, as measured by RNA levels. Furthermore, ER ligands lead to increased recruitment of HDAC1, Sin3A and a concomitant reduction of p65 occupancy and Ac-H4 levels. None of the events, however, are associated with a significant recruitment of ERβ at the promoter. Thus, ERβ directs recruitment to the cox-2 promoter, but does so in the absence of being recruited itself

PHTPP reverses E2 and DPN repression of COX-2 hnRNA.

<p>A, Cells were treated for 24 hrs with PHTPP (100nm), E2 + PHTPP or DPN + PHTPP. (n = 5). B, Western blot showing relative protein levels. Polyclonal anti-COX-2 (Abcam) and anti-β-actin (Cell signaling) were used at a dilution of 1:1000. Data represents mean ± SEM. A, (*) p = 0.001 E2 and DPN compared to VEH, (#) p = 0.023 E2+PHTPP compared to E2, (^) p = 0.026 DPN+PHTPP compared to DPN.</p

E2 and DPN fail to induce ERβ occupancy at the <i>cox-2</i> proximal promoter in the region of the NF-κB element.

<p>A, Schematic of <i>cox-2</i> proximal promoter region. The numbers indicate the bases from the transcription start site (arrow). (P1) forward primer, (P2) reverse primer. B and C, Cells were treated for 24 hrs here and in subsequent experiments. Fold occupancy at the <i>cox-2</i> NF-<b>κ</b>B and <i>c-fos</i> ERE regions, respectively. A polyclonal anti-ERβ antibody (Abcam) was used. (n = 5). Data represent mean ± SEM C, (*) p = 0.043, E2 compared to VEH, and p = 0.008, DPN compared to VEH.</p

E2 increases the overall methylation of the <i>cox-2</i> proximal promoter.

<p>A, Raw methylation data; each row is an independent clone and each column is one of eight CpG sites in the CpG island. B, Percent CpG island methylation (n = 3). Data represents mean ± SEM. (*) p = 0.035 E2 compared to VEH, (#) p = 0.049 E2 compared to DPN.</p

Characterization of COX-2 expression in AR-5 cells.

<p>A, left, duplicate samples revealed a band with a molecular mass of approximately 69 kDa, the molecular weight of COX-2. A, right, Immunocytochemistry (ICC) for COX-2 merged with Hoechst fluorescence. Polyclonal anti-COX-2 (Abcam) and anti-β-actin (Cell signaling) were used at a dilution of 1:250 for ICC and 1:1000 for western blot (WB) analysis. Scale bar = 50 μm. B and C, E2 and DPN suppress COX-2 mRNA and hnRNA at 24 hrs. Cells were treated with E2 (10^-7M), DPN (10^-7M) and PPT (10^-7M) for the indicated times. Expression of COX-2 mRNA and hnRNA was measured by real time RT-qPCR. (n ≥ 3), data represent the mean ± SEM. B, (*) p = 0.03 E2 and DPN compared to VEH. C, (*) p = 0.01 E2 compared to VEH, (*) p = 0.002 DPN compared to VEH. VEH (Vehicle) represented by the gray line here and in subsequent graphs.</p

E2 and DPN decrease p65 occupancy and Ac-H4 levels.

<p>ChIP analyses were performed and all antibodies were polyclonal. A, p65 (Santa Cruz), B, pan acetylated H4 (Active Motif), and C, pan acetylated H3 (Millipore). (n = 3 for all experiments). Data represents mean ± SEM and are expressed as fold of VEH. A, (*) p <i>=</i> 0.042 E2 compared to VEH; (*) p <i>=</i> 0.003 DPN compared to VEH. B, (*) p <i>=</i> 0.013 E2 compared to VEH, (*) p <i>=</i> 0.001 DPN compared to VEH. (#) p = 0.05 E2 compared to DPN.</p

Deacetylation plays a role in E2 and DPN repression of basal.

<p>A, TSA increases COX-2 hnRNA. Cells were treated for 24 hrs with E2, DPN, TSA (100nm) or E2+TSA and DPN+TSA. ChIP analyses were performed for B, HDAC1 (Abcam), C, HDAC3 (Cell Signaling), and D, Sin3A (Sigma-aldrich). (n≥3). Data represents mean ± SEM and is expressed as fold of VEH. A, (*) p = 0.007 for E2, p = 0.050 for DPN, p = 0.025 for TSA p = 0.013 for E2+TSA, p = 0.005 for DPN+TSA. (#) p = 0.004 E2+TSA compared to E2, (^) p = 0.003 DPN+TSA compared to DPN. B, (*) p = 0.008 E2, p = 0.002 DPN compared to VEH. (#) p = 0.04 E2 compared to DPN. D, (*) p = 0.002 E2, p = 0.048 DPN compared to VEH.</p

Human SMAD4 Genomic Variants Identified in Individuals with Heritable and Early-Onset Thoracic Aortic Disease

Thoracic aortic aneurysms (TAAs) that progress to acute thoracic aortic dissections (TADs) are life-threatening vascular events that have been associated with altered transforming growth factor (TGF) β signaling. In addition to TAA, multiple genetic vascular disorders, including hereditary hemorrhagic telangiectasia (HHT), involve altered TGFβ signaling and vascular malformations. Due to the importance of TGFβ, genomic variant databases have been curated for activin receptor-like kinase 1 (ALK1) and endoglin (ENG). This case report details seven variants in SMAD4 that are associated with either heritable or early-onset aortic dissections and compares them to pathogenic exon variants in gnomAD v2.1.1. The TAA and TAD variants were identified through whole exome sequencing of 346 families with unrelated heritable thoracic aortic disease (HTAD) and 355 individuals with early-onset (age ≤ 56 years old) thoracic aortic dissection (ESTAD). An allele frequency filter of less than 0.05% was applied in the Genome Aggregation Database (gnomAD exome v2.1.1) with a combined annotation-dependent depletion score (CADD) greater than 20. These seven variants also have a higher REVEL score (&gt;0.2), indicating pathogenic potential. Further in vivo and in vitro analysis is needed to evaluate how these variants affect SMAD4 mRNA stability and protein activity in association with thoracic aortic disease