Gut microbiome and metabolome profiling in Framingham heart study reveals cholesterol-metabolizing bacteria.

Accumulating evidence suggests that cardiovascular disease (CVD) is associated with an altered gut microbiome. Our understanding of the underlying mechanisms has been hindered by lack of matched multi-omic data with diagnostic biomarkers. To comprehensively profile gut microbiome contributions to CVD, we generated stool metagenomics and metabolomics from 1,429 Framingham Heart Study participants. We identified blood lipids and cardiovascular health measurements associated with microbiome and metabolome composition. Integrated analysis revealed microbial pathways implicated in CVD, including flavonoid, γ-butyrobetaine, and cholesterol metabolism. Species from the Oscillibacter genus were associated with decreased fecal and plasma cholesterol levels. Using functional prediction and in vitro characterization of multiple representative human gut Oscillibacter isolates, we uncovered conserved cholesterol-metabolizing capabilities, including glycosylation and dehydrogenation. These findings suggest that cholesterol metabolism is a broad property of phylogenetically diverse Oscillibacter spp., with potential benefits for lipid homeostasis and cardiovascular health.

Use of Narrative Concepts in Electronic Health Records to Validate Associations Between Genetic Factors and Response to Treatment of Inflammatory Bowel Diseases.

Crohn's disease (CD) and ulcerative colitis (UC) are heterogeneous. With availability of therapeutic classes with distinct immunologic mechanisms of action, it has become imperative to identify markers that predict likelihood of response to each drug class. However, robust development of such tools has been challenging because of need for large prospective cohorts with systematic and careful assessment of treatment response using validated indices. Most hospitals in the United States use electronic health records (EHRs) that warehouse a large amount of narrative (free-text) and codified (administrative) data generated during routine clinical care. These data have been used to construct virtual disease cohorts for epidemiologic research as well as for defining genetic basis of disease states or discrete laboratory values.1-3 Whether EHR-based data can be used to validate genetic associations for more nuanced outcomes such as treatment response has not been examined previously.

Statin Use Is Associated With Reduced Risk of Colorectal Cancer in Patients With Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Inflammatory bowel diseases (IBDs) such as Crohn's disease and ulcerative colitis are associated with an increased risk of colorectal cancer (CRC). Chemopreventive strategies have produced weak or inconsistent results. Statins have been associated inversely with sporadic CRC. We examined their role as chemopreventive agents in patients with IBD. METHODS: We collected data from 11,001 patients with IBD receiving care at hospitals in the Greater Boston metropolitan area from 1998 through 2010. Diagnoses of CRC were determined using validated International Classification of Diseases, 9th revision, Clinical Modification codes. Statin use before diagnosis was assessed through analysis of electronic prescriptions. We performed multivariate logistic regression analyses, adjusting for potential confounders including primary sclerosing cholangitis, smoking, increased levels of inflammation markers, and CRC screening practices to identify an independent association between statin use and CRC. We performed sensitivity analyses using propensity score adjustment and variation in the definition of statin use. RESULTS: In our cohort, 1376 of the patients (12.5%) received 1 or more prescriptions for a statin. Patients using statins were more likely to be older, male, white, smokers, and have greater comorbidity than nonusers. Over a follow-up period of 9 years, 2% of statin users developed CRC compared with 3% of nonusers (age-adjusted odds ratio, 0.35; 95% confidence interval, 0.24-0.53). On multivariate analysis, statin use remained independently and inversely associated with CRC (odds ratio, 0.42; 95% confidence interval, 0.28-0.62). Our findings were robust on a variety of sensitivity and subgroup analyses. CONCLUSIONS: Statin use was associated inversely with the risk of CRC in a large IBD cohort. Prospective studies on the role of statins as chemopreventive agents are warranted.

Using an Electronic Medical Records Database to Identify Non-Traditional Cardiovascular Risk Factors in Nonalcoholic Fatty Liver Disease.

OBJECTIVES: Among adults with nonalcoholic fatty liver disease (NAFLD), 25% of deaths are attributable to cardiovascular disease (CVD). CVD risk reduction in NAFLD requires not only modification of traditional CVD risk factors but identification of risk factors unique to NAFLD. METHODS: In a NAFLD cohort, we sought to identify non-traditional risk factors associated with CVD. NAFLD was determined by a previously described algorithm and a multivariable logistic regression model determined predictors of CVD. RESULTS: Of the 8,409 individuals with NAFLD, 3,243 had CVD and 5,166 did not. On multivariable analysis, CVD among NAFLD patients was associated with traditional CVD risk factors including family history of CVD (OR 4.25, P=0.0007), hypertension (OR 2.54, P=0.0017), renal failure (OR 1.59, P=0.04), and age (OR 1.05, P<0.0001). Several non-traditional CVD risk factors including albumin, sodium, and Model for End-Stage Liver Disease (MELD) score were associated with CVD. On multivariable analysis, an increased MELD score (OR 1.10, P<0.0001) was associated with an increased risk of CVD. Albumin (OR 0.52, P<0.0001) and sodium (OR 0.96, P=0.037) were inversely associated with CVD. In addition, CVD was more common among those with a NAFLD fibrosis score >0.676 than those with a score ≤0.676 (39 vs. 20%, P<0.0001). CONCLUSIONS: CVD in NAFLD is associated with traditional CVD risk factors, as well as higher MELD scores and lower albumin and sodium levels. Individuals with evidence of advanced fibrosis were more likely to have CVD. These findings suggest that the drivers of NAFLD may also promote CVD development and progression.

Development and Validation of an Algorithm to Identify Nonalcoholic Fatty Liver Disease in the Electronic Medical Record.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. Risk factors for NAFLD disease progression and liver-related outcomes remain incompletely understood due to the lack of computational identification methods. The present study sought to design a classification algorithm for NAFLD within the electronic medical record (EMR) for the development of large-scale longitudinal cohorts. METHODS: We implemented feature selection using logistic regression with adaptive LASSO. A training set of 620 patients was randomly selected from the Research Patient Data Registry at Partners Healthcare. To assess a true diagnosis for NAFLD we performed chart reviews and considered either a documentation of a biopsy or a clinical diagnosis of NAFLD. We included in our model variables laboratory measurements, diagnosis codes, and concepts extracted from medical notes. Variables with P < 0.05 were included in the multivariable analysis. RESULTS: The NAFLD classification algorithm included number of natural language mentions of NAFLD in the EMR, lifetime number of ICD-9 codes for NAFLD, and triglyceride level. This classification algorithm was superior to an algorithm using ICD-9 data alone with AUC of 0.85 versus 0.75 (P < 0.0001) and leads to the creation of a new independent cohort of 8458 individuals with a high probability for NAFLD. CONCLUSIONS: The NAFLD classification algorithm is superior to ICD-9 billing data alone. This approach is simple to develop, deploy, and can be applied across different institutions to create EMR-based cohorts of individuals with NAFLD.

Autosis is a Na+,K+-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia.

A long-standing controversy is whether autophagy is a bona fide cause of mammalian cell death. We used a cell-penetrating autophagy-inducing peptide, Tat-Beclin 1, derived from the autophagy protein Beclin 1, to investigate whether high levels of autophagy result in cell death by autophagy. Here we show that Tat-Beclin 1 induces dose-dependent death that is blocked by pharmacological or genetic inhibition of autophagy, but not of apoptosis or necroptosis. This death, termed "autosis," has unique morphological features, including increased autophagosomes/autolysosomes and nuclear convolution at early stages, and focal swelling of the perinuclear space at late stages. We also observed autotic death in cells during stress conditions, including in a subpopulation of nutrient-starved cells in vitro and in hippocampal neurons of neonatal rats subjected to cerebral hypoxia-ischemia in vivo. A chemical screen of ~5,000 known bioactive compounds revealed that cardiac glycosides, antagonists of Na(+),K(+)-ATPase, inhibit autotic cell death in vitro and in vivo. Furthermore, genetic knockdown of the Na(+),K(+)-ATPase α1 subunit blocks peptide and starvation-induced autosis in vitro. Thus, we have identified a unique form of autophagy-dependent cell death, a Food and Drug Administration-approved class of compounds that inhibit such death, and a crucial role for Na(+),K(+)-ATPase in its regulation. These findings have implications for understanding how cells die during certain stress conditions and how such cell death might be prevented.

The glycosylation-dependent interaction of perlecan core protein with LDL: implications for atherosclerosis.

Perlecan is a major heparan sulfate (HS) proteoglycan in the arterial wall. Previous studies have linked it to atherosclerosis. Perlecan contains a core protein and three HS side chains. Its core protein has five domains (DI-DV) with disparate structures and DII is highly homologous to the ligand-binding portion of LDL receptor (LDLR). The functional significance of this domain has been unknown. Here, we show that perlecan DII interacts with LDL. Importantly, the interaction largely relies on O-linked glycans that are only present in the secreted DII. Among the five repeat units of DII, most of the glycosylation sites are from the second unit, which is highly divergent and rich in serine and threonine, but has no cysteine residues. Interestingly, most of the glycans are capped by the negatively charged sialic acids, which are critical for LDL binding. We further demonstrate an additive effect of HS and DII on LDL binding. Unlike LDLR, which directs LDL uptake through endocytosis, this study uncovers a novel feature of the perlecan LDLR-like DII in receptor-mediated lipoprotein retention, which depends on its glycosylation. Thus, perlecan glycosylation may play a role in the early LDL retention during the development of atherosclerosis.

Colonoscopy is associated with a reduced risk for colon cancer and mortality in patients with inflammatory bowel diseases.

BACKGROUND & AIMS: Crohn's disease and ulcerative colitis are associated with an increased risk of colorectal cancer (CRC). Surveillance colonoscopy is recommended at 2- to 3-year intervals beginning 8 years after diagnosis of inflammatory bowel disease (IBD). However, there have been no reports of whether colonoscopy examination reduces the risk for CRC in patients with IBD. METHODS: In a retrospective study, we analyzed data from 6823 patients with IBD (2764 with a recent colonoscopy, 4059 without a recent colonoscopy) seen and followed up for at least 3 years at 2 tertiary referral hospitals in Boston, Massachusetts. The primary outcome was diagnosis of CRC. We examined the proportion of patients undergoing a colonoscopy within 36 months before a diagnosis of CRC or at the end of the follow-up period, excluding colonoscopies performed within 6 months before a diagnosis of CRC, to avoid inclusion of prevalent cancers. Multivariate logistic regression was performed, adjusting for plausible confounders. RESULTS: A total of 154 patients developed CRC. The incidence of CRC among patients without a recent colonoscopy (2.7%) was significantly higher than among patients with a recent colonoscopy (1.6%) (odds ratio [OR], 0.56; 95% confidence interval [CI], 0.39-0.80). This difference persisted in multivariate analysis (OR, 0.65; 95% CI, 0.45-0.93) and was robust when adjusted for a range of assumptions in sensitivity analyses. Among patients with CRC, a colonoscopy within 6 to 36 months before diagnosis was associated with a reduced mortality rate (OR, 0.34; 95% CI, 0.12-0.95). CONCLUSIONS: Recent colonoscopy (within 36 months) is associated with a reduced incidence of CRC in patients with IBD, and lower mortality rates in those diagnosed with CRC.

Circulating CD34(+) progenitor cell frequency is associated with clinical and genetic factors.

Circulating blood CD34(+) cells consist of hematopoietic stem/progenitor cells, angiogenic cells, and endothelial cells. In addition to their clinical use in hematopoietic stem cell transplantation, CD34(+) cells may also promote therapeutic neovascularization. Therefore, understanding the factors that influence circulating CD34(+) cell frequency has wide implications for vascular biology in addition to stem cell transplantation. In the present study, we examined the clinical and genetic characteristics associated with circulating CD34(+) cell frequency in a large, community-based sample of 1786 Framingham Heart Study participants.Among subjects without cardiovascular disease (n = 1595), CD34(+) frequency was inversely related to older age, female sex, and smoking. CD34(+) frequency was positively related to weight, serum total cholesterol, and statin therapy. Clinical covariates accounted for 6.3% of CD34(+) variability. CD34(+) frequency was highly heritable (h(2) = 54%; P < .0001). Genome-wide association analysis of CD34(+) frequency identified suggestive associations at several loci, including OR4C12 (chromosome 11; P = 6.7 × 10(-7)) and ENO1 and RERE (chromosome 1; P = 8.8 × 10(-7)). CD34(+) cell frequency is reduced in older subjects and is influenced by environmental factors including smoking and statin use. CD34(+) frequency is highly heritable. The results of the present study have implications for therapies that use CD34(+) cell populations and support efforts to better understand the genetic mechanisms that underlie CD34(+) frequency.

Mode of childbirth and long-term outcomes in women with inflammatory bowel diseases.

INTRODUCTION: Inflammatory bowel diseases [IBD; Crohn's disease (CD), ulcerative colitis] often affect women in their reproductive years. Few studies have analyzed the impact of mode of childbirth on long-term IBD outcomes. METHODS: We used a multi-institutional IBD cohort to identify all women in the reproductive age-group with a diagnosis of IBD prior to pregnancy. We identified the occurrence of a new diagnosis code for perianal complications, IBD-related hospitalization and surgery, and initiation of medical therapy after either a vaginal delivery or caesarean section (CS). Cox proportional hazards models adjusting for potential confounders were used to estimate independent effect of mode of childbirth on IBD outcomes. RESULTS: Our cohort included 360 women with IBD (161 CS). Women in the CS group were likely to be older and more likely to have complicated disease behavior prior to pregnancy. During follow-up, there was no difference in the likelihood of IBD-related surgery (multivariate hazard ratio 1.75, 95 % confidence interval (CI) 0.40-7.75), IBD-related hospitalization (HR 1.39), initiation of immunomodulator therapy (HR 1.45), or anti-TNF therapy (HR 1.11). Among the 133 CD pregnancies with no prior perianal disease, we found no excess risk of subsequent new diagnosis perianal fistulae with vaginal delivery compared to CS (HR 0.19, 95 % CI 0.04-1.05). CONCLUSIONS: Mode of delivery did not influence natural history of IBD. In our cohort, vaginal delivery was not associated with increased risk of subsequent perianal disease in women with CD.

Association of circulating endothelial microparticles with cardiometabolic risk factors in the Framingham Heart Study.

OBJECTIVE: To examine the relation of endothelial microparticles (EMPs) with cardiometabolic risk in the community. BACKGROUND: Circulating EMPs are small membrane vesicles released after endothelial cell injury. Endothelial microparticles are reportedly increased among individuals with a high burden of cardiovascular risk factors. However, prior investigations have been limited to small, highly selected samples. METHODS: We studied 844 individuals without a history of cardiovascular disease in the Framingham Offspring cohort (mean age 66 ± 9 years, 57% women). We used standardized flow cytometry methods to identify and quantify circulating CD144+ and CD31+/CD41- EMPs. We then used multivariable regression analyses to investigate the relations of EMP phenotypes with cardiovascular and metabolic risk factors. RESULTS: In multivariable analyses, the following cardiovascular risk factors were associated with one or more of the circulating EMP populations: hypertension (P = 0.025 for CD144+,), elevated triglycerides (P = 0.002 for CD144+, P < 0.0001 for CD31+/CD41-), and metabolic syndrome (P < 0.0001 for CD144+,). Overall, each tertile increase in the Framingham risk score corresponded to a 9% increase in log-CD31+/CD41- EMPs (P = 0.022). Furthermore, the presence of hypertriglyceridaemic waist status was associated with 38% higher levels of CD144+ EMPs (P < 0.0001) and 46% higher levels of CD31+/CD41- EMPs (P < 0.0001). CONCLUSION: In a large community-based sample, circulating EMP levels were associated with the presence of cardiometabolic risk factors, particularly dyslipidaemia. These data underscore the potential influence of high-risk metabolic profiles on endothelial integrity.

Improving the power of genetic association tests with imperfect phenotype derived from electronic medical records.

To reduce costs and improve clinical relevance of genetic studies, there has been increasing interest in performing such studies in hospital-based cohorts by linking phenotypes extracted from electronic medical records (EMRs) to genotypes assessed in routinely collected medical samples. A fundamental difficulty in implementing such studies is extracting accurate information about disease outcomes and important clinical covariates from large numbers of EMRs. Recently, numerous algorithms have been developed to infer phenotypes by combining information from multiple structured and unstructured variables extracted from EMRs. Although these algorithms are quite accurate, they typically do not provide perfect classification due to the difficulty in inferring meaning from the text. Some algorithms can produce for each patient a probability that the patient is a disease case. This probability can be thresholded to define case-control status, and this estimated case-control status has been used to replicate known genetic associations in EMR-based studies. However, using the estimated disease status in place of true disease status results in outcome misclassification, which can diminish test power and bias odds ratio estimates. We propose to instead directly model the algorithm-derived probability of being a case. We demonstrate how our approach improves test power and effect estimation in simulation studies, and we describe its performance in a study of rheumatoid arthritis. Our work provides an easily implemented solution to a major practical challenge that arises in the use of EMR data, which can facilitate the use of EMR infrastructure for more powerful, cost-effective, and diverse genetic studies.

Serum inflammatory markers and risk of colorectal cancer in patients with inflammatory bowel diseases.

BACKGROUND & AIMS: Patients with inflammatory bowel diseases (IBDs) (Crohn's disease, ulcerative colitis) are at increased risk of colorectal cancer (CRC). Persistent inflammation is hypothesized to increase risk of CRC in patients with IBD; however, the few studies in this area have been restricted to cross-sectional assessments of histologic severity. No prior studies have examined association between C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR) elevation and risk of CRC in an IBD cohort. METHODS: From a multi-institutional validated IBD cohort, we identified all patients with at least one measured CRP or ESR value. Patients were stratified into quartiles of severity of inflammation on the basis of their median CRP or ESR value, and subsequent diagnosis of CRC was ascertained. Logistic regression adjusting for potential confounders was used to identify the independent association between CRP or ESR elevation and risk of CRC. RESULTS: Our study included 3145 patients with at least 1 CRP value (CRP cohort) and 4008 with at least 1 ESR value (ESR cohort). Thirty-three patients in the CRP cohort and 102 patients in the ESR cohort developed CRC during a median follow-up of 5 years at a median age of 55 years. On multivariate analysis, there was a significant increase in risk of CRC across quartiles of CRP elevation (P(trend) = .017; odds ratio for quartile 4 vs quartile 1, 2.72; 95% confidence interval, 0.95-7.76). Similarly higher median ESR was also independently associated with risk of CRC across the quartiles (odds ratio, 2.06; 95% confidence interval, 1.14-3.74) (P(trend) = .007). CONCLUSIONS: An elevated CRP or ESR is associated with increased risk of CRC in patients with IBD.

Association between reduced plasma 25-hydroxy vitamin D and increased risk of cancer in patients with inflammatory bowel diseases.

BACKGROUND & AIMS: Vitamin D deficiency is common among patients with inflammatory bowel diseases (IBD) (Crohn's disease or ulcerative colitis). The effects of low plasma 25-hydroxy vitamin D (25[OH]D) on outcomes other than bone health are understudied in patients with IBD. We examined the association between plasma level of 25(OH)D and risk of cancers in patients with IBD. METHODS: From a multi-institutional cohort of patients with IBD, we identified those with at least 1 measurement of plasma 25(OH)D. The primary outcome was development of any cancer. We examined the association between plasma 25(OH)D and risk of specific subtypes of cancer, adjusting for potential confounders in a multivariate regression model. RESULTS: We analyzed data from 2809 patients with IBD and a median plasma level of 25(OH)D of 26 ng/mL. Nearly one-third had deficient levels of vitamin D (<20 ng/mL). During a median follow-up period of 11 years, 196 patients (7%) developed cancer, excluding nonmelanoma skin cancer (41 cases of colorectal cancer). Patients with vitamin D deficiency had an increased risk of cancer (adjusted odds ratio, 1.82; 95% confidence interval, 1.25-2.65) compared with those with sufficient levels. Each 1-ng/mL increase in plasma 25(OH)D was associated with an 8% reduction in risk of colorectal cancer (odds ratio, 0.92; 95% confidence interval, 0.88-0.96). A weaker inverse association was also identified for lung cancer. CONCLUSIONS: In a large multi-institutional IBD cohort, a low plasma level of 25(OH)D was associated with an increased risk of cancer, especially colorectal cancer.

Thromboprophylaxis is associated with reduced post-hospitalization venous thromboembolic events in patients with inflammatory bowel diseases.

BACKGROUND & AIMS: Patients with inflammatory bowel diseases (IBDs) have increased risk for venous thromboembolism (VTE); those who require hospitalization have particularly high risk. Few hospitalized patients with IBD receive thromboprophylaxis. We analyzed the frequency of VTE after IBD-related hospitalization, risk factors for post-hospitalization VTE, and the efficacy of prophylaxis in preventing post-hospitalization VTE. METHODS: In a retrospective study, we analyzed data from a multi-institutional cohort of patients with Crohn's disease or ulcerative colitis and at least 1 IBD-related hospitalization. Our primary outcome was a VTE event. All patients contributed person-time from the date of the index hospitalization to development of VTE, subsequent hospitalization, or end of follow-up. Our main predictor variable was pharmacologic thromboprophylaxis. Cox proportional hazard models adjusting for potential confounders were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: From a cohort of 2788 patients with at least 1 IBD-related hospitalization, 62 patients developed VTE after discharge (2%). Incidences of VTE at 30, 60, 90, and 180 days after the index hospitalization were 3.7/1000, 4.1/1000, 5.4/1000, and 9.4/1000 person-days, respectively. Pharmacologic thromboprophylaxis during the index hospital stay was associated with a significantly lower risk of post-hospitalization VTE (HR, 0.46; 95% CI, 0.22-0.97). Increased numbers of comorbidities (HR, 1.30; 95% CI, 1.16-1.47) and need for corticosteroids before hospitalization (HR, 1.71; 95% CI, 1.02-2.87) were also independently associated with risk of VTE. Length of hospitalization or surgery during index hospitalization was not associated with post-hospitalization VTE. CONCLUSIONS: Pharmacologic thromboprophylaxis during IBD-related hospitalization is associated with reduced risk of post-hospitalization VTE.

Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection.

BACKGROUND & AIMS: Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. METHODS: We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) × Villin-cre) or CD11c(+) cells (Atg16l1(f/f) × CD11c-cre); these mice were used to assess cell type-specific antibacterial autophagy. All responses were compared with Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells. RESULTS: Autophagy was induced in small intestine and cecum after infection with S typhimurium, and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3ß (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) × Villin-cre mice. Atg16l1(f/f) × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1(f/f) × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. CONCLUSIONS: Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria.

Association between low density lipoprotein and rheumatoid arthritis genetic factors with low density lipoprotein levels in rheumatoid arthritis and non-rheumatoid arthritis controls.

OBJECTIVES: While genetic determinants of low density lipoprotein (LDL) cholesterol levels are well characterised in the general population, they are understudied in rheumatoid arthritis (RA). Our objective was to determine the association of established LDL and RA genetic alleles with LDL levels in RA cases compared with non-RA controls. METHODS: Using data from electronic medical records, we linked validated RA cases and non-RA controls to discarded blood samples. For each individual, we extracted data on: first LDL measurement, age, gender and year of LDL measurement. We genotyped subjects for 11 LDL and 44 non-HLA RA alleles, and calculated RA and LDL genetic risk scores (GRS). We tested the association between each GRS and LDL level using multivariate linear regression models adjusted for age, gender, year of LDL measurement and RA status. RESULTS: Among 567 RA cases and 979 controls, 80% were female and mean age at the first LDL measurement was 55 years. RA cases had significantly lower mean LDL levels than controls (117.2 vs 125.6 mg/dl, respectively, p<0.0001). Each unit increase in LDL GRS was associated with 0.8 mg/dl higher LDL levels in both RA cases and controls (p=3.0×10(-7)). Each unit increase in RA GRS was associated with 4.3 mg/dl lower LDL levels in both groups (p=0.01). CONCLUSIONS: LDL alleles were associated with higher LDL levels in RA. RA alleles were associated with lower LDL levels in both RA cases and controls. As RA cases carry more RA alleles, these findings suggest a genetic basis for epidemiological observations of lower LDL levels in RA.

Disease allele-dependent small-molecule sensitivities in blood cells from monogenic diabetes.

Even as genetic studies identify alleles that influence human disease susceptibility, it remains challenging to understand their functional significance and how they contribute to disease phenotypes. Here, we describe an approach to translate discoveries from human genetics into functional and therapeutic hypotheses by relating human genetic variation to small-molecule sensitivities. We use small-molecule probes modulating a breadth of targets and processes to reveal disease allele-dependent sensitivities, using cells from multiple individuals with an extreme form of diabetes (maturity onset diabetes of the young type 1, caused by mutation in the orphan nuclear receptor HNF4α). This approach enabled the discovery of small molecules that show mechanistically revealing and therapeutically relevant interactions with HNF4α in both lymphoblasts and pancreatic ß-cells, including compounds that physically interact with HNF4α. Compounds including US Food and Drug Administration-approved drugs were identified that favorably modulate a critical disease phenotype, insulin secretion from ß-cells. This method may suggest therapeutic hypotheses for other nonblood disorders.

Striatal neurons expressing full-length mutant huntingtin exhibit decreased N-cadherin and altered neuritogenesis.

The expanded CAG repeat that causes striatal cell vulnerability in Huntington's disease (HD) encodes a polyglutamine tract in full-length huntingtin that is correlated with cellular [ATP] and [ATP/ADP]. Since striatal neurons are vulnerable to energy deficit, we have investigated, in Hdh CAG knock-in mice and striatal cells, the hypothesis that decreased energetics may affect neuronal (N)-cadherin, a candidate energy-sensitive adhesion protein that may contribute to HD striatal cell sensitivity. In vivo, N-cadherin was sensitive to ischemia and to the effects of full-length mutant huntingtin, progressively decreasing in Hdh(Q111) striatum with age. In cultured striatal cells, N-cadherin was decreased by ATP depletion and STHdh(Q111) striatal cells exhibited dramatically decreased N-cadherin, due to decreased Cdh2 mRNA and enhanced N-cadherin turnover, which was partially normalized by adenine supplementation to increase [ATP] and [ATP/ADP]. Consistent with decreased N-cadherin function, STHdh(Q111) striatal cells displayed profound deficits in calcium-dependent N-cadherin-mediated cell clustering and cell-substratum adhesion, and primary Hdh(Q111) striatal neuronal cells exhibited decreased N-cadherin and an abundance of immature neurites, featuring diffuse, rather than clustered, staining for N-cadherin and synaptic vesicle markers, which was partially rescued by adenine treatment. Thus, mutant full-length huntingtin, via energetic deficit, contributes to decreased N-cadherin levels in striatal neurons, with detrimental effects on neurite maturation, strongly suggesting that N-cadherin-mediated signaling merits investigation early in the HD pathogenic disease process.

Dextran-coated iron oxide nanoparticles: a versatile platform for targeted molecular imaging, molecular diagnostics, and therapy.

Advances in our understanding of the genetic basis of disease susceptibility coupled with prominent successes for molecular targeted therapies have resulted in an emerging strategy of personalized medicine. This approach envisions risk stratification and therapeutic selection based on an individual's genetic makeup and physiologic state (the latter assessed through cellular or molecular phenotypes). Molecularly targeted nanoparticles can play a key role in this vision through noninvasive assessments of molecular processes and specific cell populations in vivo, sensitive molecular diagnostics, and targeted delivery of therapeutics. A superparamagnetic iron oxide nanoparticle with a cross-linked dextran coating, or CLIO, is a powerful and illustrative nanoparticle platform for these applications. These structures and their derivatives support diagnostic imaging by magnetic resonance (MRI), optical, and positron emission tomography (PET) modalities and constitute a versatile platform for conjugation to targeting ligands. A variety of conjugation methods exist to couple the dextran surface to different functional groups; in addition, a robust bioorthogonal [4 + 2] cycloaddition reaction between 1,2,4,5-tetrazene (Tz) and trans-cyclooctene (TCO) can conjugate nanoparticles to targeting ligands or label pretargeted cells. The ready availability of conjugation methods has given rise to the synthesis of libraries of small molecule modified nanoparticles, which can then be screened for nanoparticles with specificity for a specific cell type. Since most nanoparticles display their targeting ligands in a multivalent manner, a detailed understanding of the kinetics and affinity of a nanoparticle's interaction with its target (as determined by surface plasmon resonance) can yield functionally important insights into nanoparticle design. In this Account, we review applications of the CLIO platform in several areas relevant to the mission of personalized medicine. We demonstrate rapid and highly sensitive molecular profiling of cancer markers ex vivo, as part of detailed, individualized molecular phenotyping. The CLIO platform also facilitates targeted magnetic resonance and combined modality imaging (such as MR/PET/fluorescence/CT) to enable multiplexed measurement of molecular phenotypes in vivo for early diagnosis and disease classification. Finally, the targeted delivery of a photodynamic therapy agent as part of a theranostic nanoparticle successfully increased local cell toxicity and minimized systemic side effects.