Development of Portable Electronic Health Record Based Algorithms to Identify Individuals with Diabetic Retinopathy.

Objectives: To develop, validate and implement algorithms to identify diabetic retinopathy (DR) cases and controls from electronic health care records (EHR)s. Methods : We developed and validated EHR-based algorithms to identify DR cases and individuals with type I or II diabetes without DR (controls) in three independent EHR systems: Vanderbilt University Medical Center Synthetic Derivative (VUMC), the VA Northeast Ohio Healthcare System (VANEOHS), and Massachusetts General Brigham (MGB). Cases were required to meet one of three criteria: 1) two or more dates with any DR ICD-9/10 code documented in the EHR, or 2) at least one affirmative health-factor or EPIC code for DR along with an ICD9/10 code for DR on a different day, or 3) at least one ICD-9/10 code for any DR occurring within 24 hours of an ophthalmology exam. Criteria for controls included affirmative evidence for diabetes as well as an ophthalmology exam. Results: The algorithms, developed and evaluated in VUMC through manual chart review, resulted in a positive predictive value (PPV) of 0.93 for cases and negative predictive value (NPV) of 0.97 for controls. Implementation of algorithms yielded similar metrics in VANEOHS (PPV=0.94; NPV=0.86) and lower in MGB (PPV=0.84; NPV=0.76). In comparison, use of DR definition as implemented in Phenome-wide association study (PheWAS) in VUMC, yielded similar PPV (0.92) but substantially reduced NPV (0.48). Implementation of the algorithms to the Million Veteran Program identified over 62,000 DR cases with genetic data including 14,549 African Americans and 6,209 Hispanics with DR. Conclusions/Discussion: We demonstrate the robustness of the algorithms at three separate health-care centers, with a minimum PPV of 0.84 and substantially improved NPV than existing high-throughput methods. We strongly encourage independent validation and incorporation of features unique to each EHR to enhance algorithm performance for DR cases and controls.

Plasma Arginine and Citrulline are Elevated in Diabetic Retinopathy.

PURPOSE: To determine if plasma levels of six arginine-related and citrulline-related metabolites (arginine, citrulline, asymmetric dimethylarginine [ADMA], ornithine, proline, and argininosuccinate) differ between patients with type 2 diabetes and diabetic retinopathy (DR) and type 2 diabetic controls or between patients with proliferative DR (PDR) and non-proliferative DR (NPDR). DESIGN: Cross-sectional study. METHODS: Adults with type 2 diabetes were recruited from the Vanderbilt Eye Institute. Exclusion criteria included non-diabetic retinal disease. Plasma metabolite levels were quantified in 159 diabetic controls and 156 DR patients (92 NPDR, 64 PDR) using isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Metabolite levels were compared using Wilcoxon Rank Sum test and logistic regressions adjusting for age, sex, hemoglobin A1c, diabetes duration, statin use, and angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker use. A secondary analysis that included creatinine in the regression model was performed for the subset of patients with available creatinine values (135 diabetic controls, 100 DR patients [58 NPDR, 42 PDR]). RESULTS: Multivariable logistic regression analyses determined that arginine (OR = 1.20, [1.06-1.38], P = .0067) and citrulline (OR = 1.53, [1.20-1.98], P = .0025) were significantly elevated in DR patients compared to diabetic controls. While ADMA differed between NPDR and PDR patients in the primary analysis (OR = 1.56, [1.15-2.16], P = .0051), it was not significantly different when adjusting for creatinine (OR = 1.30, [0.90-1.91], P = .15). CONCLUSIONS: Plasma arginine and citrulline were significantly elevated in type 2 diabetic patients with DR compared to diabetic controls. None of the tested metabolites significantly differed between NPDR and PDR patients in the adjusted analysis.

Plasma Metabolomics of Intermediate and Neovascular Age-Related Macular Degeneration Patients.

To characterize metabolites and metabolic pathways altered in intermediate and neovascular age-related macular degeneration (IAMD and NVAMD), high resolution untargeted metabolomics was performed via liquid chromatography-mass spectrometry on plasma samples obtained from 91 IAMD patients, 100 NVAMD patients, and 195 controls. Plasma metabolite levels were compared between: AMD patients and controls, IAMD patients and controls, and NVAMD and IAMD patients. Partial least-squares discriminant analysis and linear regression were used to identify discriminatory metabolites. Pathway analysis was performed to determine metabolic pathways altered in AMD. Among the comparisons, we identified 435 unique discriminatory metabolic features. Using computational methods and tandem mass spectrometry, we identified 11 metabolic features whose molecular identities had been previously verified and confirmed the molecular identities of three additional discriminatory features. Included among the discriminatory metabolites were acylcarnitines, phospholipids, amino acids, and steroid metabolites. Pathway analysis revealed that lipid, amino acid, and vitamin metabolism pathways were altered in NVAMD, IAMD, or AMD in general, including the carnitine shuttle pathway which was significantly altered in all comparisons. Finally, few discriminatory features were identified between IAMD patients and controls, suggesting that plasma metabolic profiles of IAMD patients are more similar to controls than to NVAMD patients.

Pharmacogenetics of hypoglycemia associated with sulfonylurea therapy in usual clinical care.

Hypoglycemia is a common complication among type 2 diabetes mellitus (T2DM) patients receiving sulfonylurea therapy. The aim of this study was to determine if genetic contributions to sulfonylurea pharmacokinetics or pharmacodynamics substantially affect the risk of hypoglycemia in these patients. In a retrospective case-control study in European American patients with T2DM, we examined the potential association between CYP2C9 reduced-function variants and sulfonylurea-related hypoglycemia. We also explored the relationship between sulfonylurea-related hypoglycemia and several candidate genetic variants previously reported to alter the response to sulfonylureas. We detected no evidence of association between CYP2C9 reduced-function alleles or any of the candidate genetic variants and sulfonylurea-related hypoglycemia. In conclusion, we identified no clinically significant predictors of hypoglycemia among genes associated with sulfonylurea pharmacokinetics or pharmacodynamics.

Arginine and Carnitine Metabolites Are Altered in Diabetic Retinopathy.

Purpose: To determine plasma metabolite and metabolic pathway differences between patients with type 2 diabetes with diabetic retinopathy (DR) and without retinopathy (diabetic controls), and between patients with proliferative DR (PDR) and nonproliferative DR (NPDR). Methods: Using high-resolution mass spectrometry with liquid chromatography, untargeted metabolomics was performed on plasma samples from 83 DR patients and 90 diabetic controls. Discriminatory metabolic features were identified through partial least squares discriminant analysis, and linear regression was used to adjust for age, sex, diabetes duration, and hemoglobin A1c. Pathway analysis was performed using Mummichog 2.0. Results: In the adjusted analysis, 126 metabolic features differed significantly between DR patients and diabetic controls. Pathway analysis revealed alterations in the metabolism of amino acids, leukotrienes, niacin, pyrimidine, and purine. Arginine, citrulline, glutamic γ-semialdehyde, and dehydroxycarnitine were key contributors to these pathway differences. A total of 151 features distinguished PDR patients from NPDR patients, and pathway analysis revealed alterations in the ß-oxidation of saturated fatty acids, fatty acid metabolism, and vitamin D3 metabolism. Carnitine was a major contributor to the pathway differences. Conclusions: This study demonstrates that arginine and citrulline-related pathways are dysregulated in DR, and fatty acid metabolism is altered in PDR patients compared with NPDR patients.

The Carnitine Shuttle Pathway is Altered in Patients With Neovascular Age-Related Macular Degeneration.

Purpose: To identify metabolites and metabolic pathways altered in neovascular age-related macular degeneration (NVAMD). Methods: We performed metabolomics analysis using high-resolution C18 liquid chromatography-mass spectrometry on plasma samples from 100 NVAMD patients and 192 controls. Data for mass/charge ratio ranging from 85 to 850 were captured, and metabolic features were extracted using xMSanalyzer. Nested feature selection was used to identify metabolites that discriminated between NVAMD patients and controls. Pathway analysis was performed with Mummichog 2.0. Hierarchical clustering was used to examine the relationship between the discriminating metabolites and NVAMD patients and controls. Results: Of the 10,917 metabolic features analyzed, a set of 159 was identified that distinguished NVAMD patients from controls (area under the curve of 0.83). Of these features, 39 were annotated with confidence and included multiple carnitine metabolites. Pathway analysis revealed that the carnitine shuttle pathway was significantly altered in NVAMD patients (P = 0.0001). Tandem mass spectrometry confirmed the molecular identity of five carnitine shuttle pathway acylcarnitine intermediates that were increased in NVAMD patients. Hierarchical cluster analysis revealed that 51% of the NVAMD patients had similar metabolic profiles, whereas the remaining 49% displayed greater variability in their metabolic profiles. Conclusions: Multiple long-chain acylcarnitines that are part of the carnitine shuttle pathway were significantly increased in NVAMD patients compared to controls, suggesting that fatty acid metabolism may be involved in NVAMD pathophysiology. Cluster analysis suggested that clinically indistinguishable NVAMD patients can be separated into distinct subgroups based on metabolic profiles.

Hi-MC: a novel method for high-throughput mitochondrial haplogroup classification.

Effective approaches for assessing mitochondrial DNA (mtDNA) variation are important to multiple scientific disciplines. Mitochondrial haplogroups characterize branch points in the phylogeny of mtDNA. Several tools exist for mitochondrial haplogroup classification. However, most require full or partial mtDNA sequence which is often cost prohibitive for studies with large sample sizes. The purpose of this study was to develop Hi-MC, a high-throughput method for mitochondrial haplogroup classification that is cost effective and applicable to large sample sizes making mitochondrial analysis more accessible in genetic studies. Using rigorous selection criteria, we defined and validated a custom panel of mtDNA single nucleotide polymorphisms that allows for accurate classification of European, African, and Native American mitochondrial haplogroups at broad resolution with minimal genotyping and cost. We demonstrate that Hi-MC performs well in samples of European, African, and Native American ancestries, and that Hi-MC performs comparably to a commonly used classifier. Implementation as a software package in R enables users to download and run the program locally, grants greater flexibility in the number of samples that can be run, and allows for easy expansion in future revisions. Hi-MC is available in the CRAN repository and the source code is freely available at https://github.com/vserch/himc.

Mitochondrial Haplogroups Modify the Effect of Diabetes Duration and HbA1c on Proliferative Diabetic Retinopathy Risk in Patients With Type 2 Diabetes.

Purpose: We previously demonstrated an association between European mitochondrial haplogroups and proliferative diabetic retinopathy (PDR). The purpose of this study was to determine how the relationship between these haplogroups and both diabetes duration and hyperglycemia, two major risk factors for diabetic retinopathy (DR), affect PDR prevalence. Methods: Our population consisted of patients with type 2 diabetes with (n = 377) and without (n = 480) DR. A Kruskal-Wallis test was used to compare diabetes duration and hemoglobin A1c (HbA1c) among mitochondrial haplogroups. Logistic regressions were performed to investigate diabetes duration and HbA1c as risk factors for PDR in the context of European mitochondrial haplogroups. Results: Neither diabetes duration nor HbA1c differed among mitochondrial haplogroups. Among DR patients from haplogroup H, longer diabetes duration and increasing HbA1c were significant risk factors for PDR (P = 0.0001 and P = 0.011, respectively). Neither diabetes duration nor HbA1c was a significant risk factor for PDR in DR patients from haplogroup UK. Conclusions: European mitochondrial haplogroups modify the effects of diabetes duration and HbA1c on PDR risk in patients with type 2 diabetes. In our patient population, longer diabetes duration and higher HbA1c increased PDR risk in patients from haplogroup H, but did not affect PDR risk in patients from haplogroup UK. This relationship has not been previously demonstrated and may explain, in part, why some patients with nonproliferative DR develop PDR and others do not, despite similar diabetes duration and glycemic control.

Altering the Mitochondrial Fatty Acid Synthesis (mtFASII) Pathway Modulates Cellular Metabolic States and Bioactive Lipid Profiles as Revealed by Metabolomic Profiling.

Despite the presence of a cytosolic fatty acid synthesis pathway, mitochondria have retained their own means of creating fatty acids via the mitochondrial fatty acid synthesis (mtFASII) pathway. The reason for its conservation has not yet been elucidated. Therefore, to better understand the role of mtFASII in the cell, we used thin layer chromatography to characterize the contribution of the mtFASII pathway to the fatty acid composition of selected mitochondrial lipids. Next, we performed metabolomic analysis on HeLa cells in which the mtFASII pathway was either hypofunctional (through knockdown of mitochondrial acyl carrier protein, ACP) or hyperfunctional (through overexpression of mitochondrial enoyl-CoA reductase, MECR). Our results indicate that the mtFASII pathway contributes little to the fatty acid composition of mitochondrial lipid species examined. Additionally, loss of mtFASII function results in changes in biochemical pathways suggesting alterations in glucose utilization and redox state. Interestingly, levels of bioactive lipids, including lysophospholipids and sphingolipids, directly correlate with mtFASII function, indicating that mtFASII may be involved in the regulation of bioactive lipid levels. Regulation of bioactive lipid levels by mtFASII implicates the pathway as a mediator of intracellular signaling.

Mitochondrial variation and the risk of age-related macular degeneration across diverse populations.

Substantial progress has been made in identifying susceptibility variants for age-related macular degeneration (AMD). The majority of research to identify genetic variants associated with AMD has focused on nuclear genetic variation. While there is some evidence that mitochondrial genetic variation contributes to AMD susceptibility, to date, these studies have been limited to populations of European descent resulting in a lack of data in diverse populations. A major goal of the Epidemiologic Architecture for Genes Linked to Environment (EAGLE) study is to describe the underlying genetic architecture of common, complex diseases across diverse populations. This present study sought to determine if mitochondrial genetic variation influences risk of AMD across diverse populations. We performed a genetic association study to investigate the contribution of mitochondrial DNA variation to AMD risk. We accessed samples from the National Health and Nutrition Examination Surveys, a U.S population-based, cross-sectional survey collected without regard to health status. AMD cases and controls were selected from the Third NHANES and NHANES 2007-2008 datasets which include non-Hispanic whites, non-Hispanic blacks, and Mexican Americans. AMD cases were defined as those > 60 years of age with early/late AMD, as determined by fundus photography. Targeted genotyping was performed for 63 mitochondrial SNPs and participants were then classified into mitochondrial haplogroups. We used logistic regression assuming a dominant genetic model adjusting for age, sex, body mass index, and smoking status (ever vs. never). Regressions and meta-analyses were performed for individual SNPs and mitochondrial haplogroups J, T, and U. We identified five SNPs associated with AMD in Mexican Americans at p < 0.05, including three located in the control region (mt16111, mt16362, and mt16319), one in MT-RNR2 (mt1736), and one in MT-ND4 (mt12007). No mitochondrial variant or haplogroup was significantly associated in non-Hispanic blacks or non- Hispanic whites in the final meta-analysis. This study provides further evidence that mitochondrial variation plays a role in susceptibility to AMD and contributes to the knowledge of the genetic architecture of AMD in Mexican Americans.

Investigating the relationship between mitochondrial genetic variation and cardiovascular-related traits to develop a framework for mitochondrial phenome-wide association studies.

BACKGROUND: Mitochondria play a critical role in the cell and have DNA independent of the nuclear genome. There is much evidence that mitochondrial DNA (mtDNA) variation plays a role in human health and disease, however, this area of investigation has lagged behind research into the role of nuclear genetic variation on complex traits and phenotypic outcomes. Phenome-wide association studies (PheWAS) investigate the association between a wide range of traits and genetic variation. To date, this approach has not been used to investigate the relationship between mtDNA variants and phenotypic variation. Herein, we describe the development of a PheWAS framework for mtDNA variants (mt-PheWAS). Using the Metabochip custom genotyping array, nuclear and mitochondrial DNA variants were genotyped in 11,519 African Americans from the Vanderbilt University biorepository, BioVU. We employed both polygenic modeling and association testing with mitochondrial single nucleotide polymorphisms (mtSNPs) to explore the relationship between mtDNA variants and a group of eight cardiovascular-related traits obtained from de-identified electronic medical records within BioVU. RESULTS: Using polygenic modeling we found evidence for an effect of mtDNA variation on total cholesterol and type 2 diabetes (T2D). After performing comprehensive mitochondrial single SNP associations, we identified an increased number of single mtSNP associations with total cholesterol and T2D compared to the other phenotypes examined, which did not have more significantly associated SNPs than would be expected by chance. Among the mtSNPs significantly associated with T2D we identified variant mt16189, an association previously reported only in Asian and European-descent populations. CONCLUSIONS: Our replication of previous findings and identification of novel associations from this initial study suggest that our mt-PheWAS approach is robust for investigating the relationship between mitochondrial genetic variation and a range of phenotypes, providing a framework for future mt-PheWAS.

Characterization of mitochondrial haplogroups in a large population-based sample from the United States.

Mitochondrial DNA (mtDNA) haplogroups are valuable for investigations in forensic science, molecular anthropology, and human genetics. In this study, we developed a custom panel of 61 mtDNA markers for high-throughput classification of European, African, and Native American/Asian mitochondrial haplogroup lineages. Using these mtDNA markers, we constructed a mitochondrial haplogroup classification tree and classified 18,832 participants from the National Health and Nutrition Examination Surveys (NHANES). To our knowledge, this is the largest study to date characterizing mitochondrial haplogroups in a population-based sample from the United States, and the first study characterizing mitochondrial haplogroup distributions in self-identified Mexican Americans separately from Hispanic Americans of other descent. We observed clear differences in the distribution of maternal genetic ancestry consistent with proposed admixture models for these subpopulations, underscoring the genetic heterogeneity of the United States Hispanic population. The mitochondrial haplogroup distributions in the other self-identified racial/ethnic groups within NHANES were largely comparable to previous studies. Mitochondrial haplogroup classification was highly concordant with self-identified race/ethnicity (SIRE) in non-Hispanic whites (94.8 %), but was considerably lower in admixed populations including non-Hispanic blacks (88.3 %), Mexican Americans (81.8 %), and other Hispanics (61.6 %), suggesting SIRE does not accurately reflect maternal genetic ancestry, particularly in populations with greater proportions of admixture. Thus, it is important to consider inconsistencies between SIRE and genetic ancestry when performing genetic association studies. The mitochondrial haplogroup data that we have generated, coupled with the epidemiologic variables in NHANES, is a valuable resource for future studies investigating the contribution of mtDNA variation to human health and disease.

KIAA1462, a coronary artery disease associated gene, is a candidate gene for late onset Alzheimer disease in APOE carriers.

Alzheimer disease (AD) is a devastating neurodegenerative disease affecting more than five million Americans. In this study, we have used updated genetic linkage data from chromosome 10 in combination with expression data from serial analysis of gene expression to choose a new set of thirteen candidate genes for genetic analysis in late onset Alzheimer disease (LOAD). Results in this study identify the KIAA1462 locus as a candidate locus for LOAD in APOE4 carriers. Two genes exist at this locus, KIAA1462, a gene associated with coronary artery disease, and "rokimi", encoding an untranslated spliced RNA The genetic architecture at this locus suggests that the gene product important in this association is either "rokimi", or a different isoform of KIAA1462 than the isoform that is important in cardiovascular disease. Expression data suggests that isoform f of KIAA1462 is a more attractive candidate for association with LOAD in APOE4 carriers than "rokimi" which had no detectable expression in brain.

The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation.

Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

Trans-ethnic fine-mapping of lipid loci identifies population-specific signals and allelic heterogeneity that increases the trait variance explained.

Genome-wide association studies (GWAS) have identified ~100 loci associated with blood lipid levels, but much of the trait heritability remains unexplained, and at most loci the identities of the trait-influencing variants remain unknown. We conducted a trans-ethnic fine-mapping study at 18, 22, and 18 GWAS loci on the Metabochip for their association with triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), respectively, in individuals of African American (n = 6,832), East Asian (n = 9,449), and European (n = 10,829) ancestry. We aimed to identify the variants with strongest association at each locus, identify additional and population-specific signals, refine association signals, and assess the relative significance of previously described functional variants. Among the 58 loci, 33 exhibited evidence of association at P<1 × 10(-4) in at least one ancestry group. Sequential conditional analyses revealed that ten, nine, and four loci in African Americans, Europeans, and East Asians, respectively, exhibited two or more signals. At these loci, accounting for all signals led to a 1.3- to 1.8-fold increase in the explained phenotypic variance compared to the strongest signals. Distinct signals across ancestry groups were identified at PCSK9 and APOA5. Trans-ethnic analyses narrowed the signals to smaller sets of variants at GCKR, PPP1R3B, ABO, LCAT, and ABCA1. Of 27 variants reported previously to have functional effects, 74% exhibited the strongest association at the respective signal. In conclusion, trans-ethnic high-density genotyping and analysis confirm the presence of allelic heterogeneity, allow the identification of population-specific variants, and limit the number of candidate SNPs for functional studies.

Effects of meals high in carbohydrate, protein, and fat on ghrelin and peptide YY secretion in prepubertal children.

CONTEXT: Ghrelin and peptide YY (PYY) are two hormones produced by the gastrointestinal tract that have effects on appetite. However, little is known about their secretion in response to meals high in individual macronutrients in prepubertal children. OBJECTIVE: We sought to understand how meals high in carbohydrate, protein, and fat affect serum concentrations of total ghrelin and total PYY, hypothesizing that these macronutrients would exert differential effects on their secretion. DESIGN AND SETTING: This was a cross-sectional study at one tertiary care center. SUBJECTS: Subjects were 7- to 11-yr-old healthy normal-weight (NW) and obese (OB) volunteers recruited from local advertisements. INTERVENTIONS: After an overnight fast, the subjects were given a breakfast high in carbohydrate, protein, or fat at 0800 h. Blood samples for total ghrelin and total PYY were taken at baseline, 30 min, and hourly from 0900 to 1200 h. MAIN OUTCOME MEASURE: We assessed postprandial ghrelin suppression and PYY elevation, as well as changes in reported hunger and satiety, after the three test meals. RESULTS: After the high-protein meal, ghrelin declined gradually in both groups over the study period without subsequent increase, whereas ghrelin suppressed more rapidly to a nadir at 60 min after the high-carbohydrate meal in both NW and OB children, followed by rebound in ghrelin levels. Similarly, after the high-protein meal, PYY concentrations increased steadily over the course of the morning in both groups without decline, whereas PYY levels peaked 30 min after the high-carbohydrate meal in both NW and OB subjects with significant decline thereafter. Ghrelin and PYY responses to the high-fat meal were somewhat intermediate between that observed with high carbohydrate and high protein. The OB children reported higher hunger and lower satiety after the high-carbohydrate meal compared to the NW subjects, whereas appetite ratings were similar between the groups after the high-protein and high-fat meals. Additionally, within the OB group, area under the curve (AUC) analysis revealed significantly greater PYY response, as well as lower AUC hunger and higher AUC satiety, to the high-protein meal than the high-carbohydrate and high-fat meals. CONCLUSIONS: The patterns of secretion of ghrelin and PYY in our study of prepubertal children suggest that they may play a role in the effectiveness of high-protein/low-carbohydrate diets in promoting weight loss.