Carol Haynes

Association of a peripheral blood metabolic profile with coronary artery disease and risk of subsequent cardiovascular events

Background—Molecular tools may provide insight into cardiovascular risk. We assessed whether metabolites discriminate coronary artery disease (CAD) and predict risk of cardiovascular events. Methods and Results—We performed mass–spectrometry–based profiling of 69 metabolites in subjects from the CATHGEN biorepository. To evaluate discriminative capabilities of metabolites for CAD, 2 groups were profiled: 174 CAD cases and 174 sex/race-matched controls (“initial”), and 140 CAD cases and 140 controls (“replication”). To evaluate the capability of metabolites to predict cardiovascular events, cases were combined (“event” group); of these, 74 experienced death/myocardial infarction during follow-up. A third independent group was profiled (“event-replication” group; n=63 cases with cardiovascular events, 66 controls). Analysis included principal-components analysis, linear regression, and Cox proportional hazards. Two principal components analysis–derived factors were associated with CAD: 1 comprising branched-chain amino acid metabolites (factor 4, initial P=0.002, replication P=0.01), and 1 comprising urea cycle metabolites (factor 9, initial P=0.0004, replication P=0.01). In multivariable regression, these factors were independently associated with CAD in initial (factor 4, odds ratio [OR], 1.36; 95% CI, 1.06 to 1.74; P=0.02; factor 9, OR, 0.67; 95% CI, 0.52 to 0.87; P=0.003) and replication (factor 4, OR, 1.43; 95% CI, 1.07 to 1.91; P=0.02; factor 9, OR, 0.66; 95% CI, 0.48 to 0.91; P=0.01) groups. A factor composed of dicarboxylacylcarnitines predicted death/myocardial infarction (event group hazard ratio 2.17; 95% CI, 1.23 to 3.84; P=0.007) and was associated with cardiovascular events in the event-replication group (OR, 1.52; 95% CI, 1.08 to 2.14; P=0.01). Conclusions—Metabolite profiles are associated with CAD and subsequent cardiovascular events.

Early‐onset Alzheimer's disease: Clinical predictors of institutionalization and death

Follow-up observations were made of 92 white patients with early-onset Alzheimers disease to determine the demographic, clinical, and neuropsychological factors predictive of institutionalization or death. The cumulative mortality rate 5 years after entry into the study was 23.9%, compared with an expected rate of 9.5%. The 5-year cumulative rate of admission to nursing homes was 62.8%. The language ability of the patients on entry to the study, their scores on a brief screening test of cognitive function, and their overall ratings of clinical dementia were found to be predictors of subsequent institutional care and death. The age of the patients had a significant modifying effect on these predictive factors, resulting in a greater risk of institutionalization and death in younger patients with severe cognitive impairment as compared with older individuals with the same degree of dysfunction.

A Genomewide Scan for Early-Onset Coronary Artery Disease in 438 Families: The GENECARD Study

A family history of coronary artery disease (CAD), especially when the disease occurs at a young age, is a potent risk factor for CAD. DNA collection in families in which two or more siblings are affected at an early age allows identification of genetic factors for CAD by linkage analysis. We performed a genomewide scan in 1,168 individuals from 438 families, including 493 affected sibling pairs with documented onset of CAD before 51 years of age in men and before 56 years of age in women. We prospectively defined three phenotypic subsets of families: (1) acute coronary syndrome in two or more siblings; (2) absence of type 2 diabetes in all affected siblings; and (3) atherogenic dyslipidemia in any one sibling. Genotypes were analyzed for 395 microsatellite markers. Regions were defined as providing evidence for linkage if they provided parametric two-point LOD scores >1.5, together with nonparametric multipoint LOD scores >1.0. Regions on chromosomes 3q13 (multipoint LOD = 3.3; empirical P value <.001) and 5q31 (multipoint LOD = 1.4; empirical P value <.081) met these criteria in the entire data set, and regions on chromosomes 1q25, 3q13, 7p14, and 19p13 met these criteria in one or more of the subsets. Two regions, 3q13 and 1q25, met the criteria for genomewide significance. We have identified a region on chromosome 3q13 that is linked to early-onset CAD, as well as additional regions of interest that will require further analysis. These data provide initial areas of the human genome where further investigation may reveal susceptibility genes for early-onset CAD.

Genetic linkage studies in Alzheimer's disease families

Alzheimers disease is a devastating neurological disorder and the leading cause of dementia among the elderly. Recent studies have localized the gene for familial Alzheimers disease to chromosome 21 in a series of early onset AD families (mean age of onset less than 60). Familial late onset AD (mean age of onset greater than 60) is a more common clinical form of the disorder. Thirteen families with multiply affected Alzheimers disease family members were identified and sampled. Ten of these families were of the late onset Alzheimers disease type. Simulation studies were used to evaluate the usefulness of these pedigrees in linkage studies in familial Alzheimers disease. Linkage studies undertaken to test the localization of both early onset and late onset Alzheimers disease families to chromosome 21 failed to establish linkage and excluded linkage from a large portion of the region where the early onset Alzheimers disease gene was localized. These findings suggest that more than one etiology may exist for familial Alzheimers disease and indicate the need for continued screening of the genome in familial Alzheimers disease families.

Baseline metabolomic profiles predict cardiovascular events in patients at risk for coronary artery disease

BACKGROUND Cardiovascular risk models remain incomplete. Small-molecule metabolites may reflect underlying disease and, as such, serve as novel biomarkers of cardiovascular risk. METHODS We studied 2,023 consecutive patients undergoing cardiac catheterization. Mass spectrometry profiling of 69 metabolites and lipid assessments were performed in fasting plasma. Principal component analysis reduced metabolites to a smaller number of uncorrelated factors. Independent relationships between factors and time-to-clinical events were assessed using Cox modeling. Clinical and metabolomic models were compared using log-likelihood and reclassification analyses. RESULTS At median follow-up of 3.1 years, there were 232 deaths and 294 death/myocardial infarction (MI) events. Five of 13 metabolite factors were independently associated with mortality: factor 1 (medium-chain acylcarnitines: hazard ratio [HR] 1.12 [95% CI, 1.04-1.21], P = .005), factor 2 (short-chain dicarboxylacylcarnitines: HR 1.17 [1.05-1.31], P = .005), factor 3 (long-chain dicarboxylacylcarnitines: HR 1.14 [1.05-1.25], P = .002); factor 6 (branched-chain amino acids: HR 0.86 [0.75-0.99], P = .03), and factor 12 (fatty acids: HR 1.19 [1.06-1.35], P = .004). Three factors independently predicted death/MI: factor 2 (HR 1.11 [1.01-1.23], P = .04), factor 3 (HR 1.13 [1.04-1.22], P = .005), and factor 12 (HR 1.18 [1.05-1.32], P = .004). For mortality, 27% of intermediate-risk patients were correctly reclassified (net reclassification improvement 8.8%, integrated discrimination index 0.017); for death/MI model, 11% were correctly reclassified (net reclassification improvement 3.9%, integrated discrimination index 0.012). CONCLUSIONS Metabolic profiles predict cardiovascular events independently of standard predictors.

High heritability of metabolomic profiles in families burdened with premature cardiovascular disease

Integration of genetic and metabolic profiling holds promise for providing insight into human disease. Coronary artery disease (CAD) is strongly heritable, but the heritability of metabolomic profiles has not been evaluated in humans. We performed quantitative mass spectrometry‐based metabolic profiling in 117 individuals within eight multiplex families from the GENECARD study of premature CAD. Heritabilities were calculated using variance components. We found high heritabilities for amino acids (arginine, ornithine, alanine, proline, leucine/isoleucine, valine, glutamate/glutamine, phenylalanine and glycine; h2=0.33–0.80, P=0.005–1.9 × 10−16), free fatty acids (arachidonic, palmitic, linoleic; h2=0.48–0.59, P=0.002–0.00005) and acylcarnitines (h2=0.23–0.79, P=0.05–0.0000002). Principal components analysis was used to identify metabolite clusters. Reflecting individual metabolites, several components were heritable, including components comprised of ketones, β‐hydroxybutyrate and C2‐acylcarnitine (h2=0.61); short‐ and medium‐chain acylcarnitines (h2=0.39); amino acids (h2=0.44); long‐chain acylcarnitines (h2=0.39) and branched‐chain amino acids (h2=0.27). We report a novel finding of high heritabilities of metabolites in premature CAD, establishing a possible genetic basis for these profiles. These results have implications for understanding CAD pathophysiology and genetics.

Peakwide Mapping on Chromosome 3q13 Identifies the Kalirin Gene as a Novel Candidate Gene for Coronary Artery Disease

A susceptibility locus for coronary artery disease (CAD) has been mapped to chromosome 3q13-21 in a linkage study of early-onset CAD. We completed an association-mapping study across the 1-LOD-unit-down supporting interval, using two independent white case-control data sets (CATHGEN, initial and validation) to evaluate association under the peak. Single-nucleotide polymorphisms (SNPs) evenly spaced at 100-kb intervals were screened in the initial data set (N=468). Promising SNPs (P<.1) were then examined in the validation data set (N=514). Significant findings (P<.05) in the combined initial and validation data sets were further evaluated in multiple independent data sets, including a family-based data set (N=2,954), an African American case-control data set (N=190), and an additional white control data set (N=255). The association between genotype and aortic atherosclerosis was examined in 145 human aortas. The peakwide survey found evidence of association in SNPs from multiple genes. The strongest associations were found in three SNPs from the kalirin (KALRN) gene, especially in patients with early-onset CAD (P=.00001-00028 in the combined CATHGEN data sets). In-depth investigation of the gene found that an intronic SNP, rs9289231, was associated with early-onset CAD in all white data sets examined (P<.05). In the joint analysis of all white early-onset CAD cases (N=332) and controls (N=546), rs9289231 was highly significant (P=.00008), with an odds-ratio estimate of 2.1. Furthermore, the risk allele of this SNP was associated with atherosclerosis burden (P=.03) in 145 human aortas. KALRN is a protein with many functions, including the inhibition of inducible nitric oxide synthase and guanine-exchange-factor activity. KALRN and two other associated genes identified in this study (CDGAP and MYLK) belong to the Rho GTPase-signaling pathway. Our data suggest the importance of the KALRN gene and the Rho GTPase-signaling pathway in the pathogenesis of CAD.

Branched chain amino acids are novel biomarkers for discrimination of metabolic wellness

OBJECTIVE To identify novel biomarkers through metabolomic profiles that distinguish metabolically well (MW) from metabolically unwell (MUW) individuals, independent of body mass index (BMI). MATERIALS/METHODS This study was conducted as part of the Measurement to Understand the Reclassification of Disease of Cabarrus/Kannapolis (MURDOCK) project. Individuals from 3 cohorts were classified as lean (BMI<25kg/m²), overweight (BMI≥25kg/m², BMI<30kg/m²) or obese (BMI≥30kg/m²). Cardiometabolic abnormalities were defined as: (1) impaired fasting glucose (≥100mg/dL and ≤126mg/dL); (2) hypertension; (3) triglycerides ≥150mg/dL; (4) HDL-C <40mg/dL in men, <50mg/dL in women; and (5) insulin resistance (calculated Homeostatic Model Assessment (HOMA-IR) index of >5.13). MW individuals were defined as having <2 cardiometabolic abnormalities and MUW individuals had≥two cardiometabolic abnormalities. Targeted profiling of 55 metabolites used mass-spectroscopy-based methods. Principal components analysis (PCA) was used to reduce the large number of correlated metabolites into clusters of fewer uncorrelated factors. RESULTS Of 1872 individuals, 410 were lean, 610 were overweight, and 852 were obese. Of lean individuals, 67% were categorized as MUW, whereas 80% of overweight and 87% of obese individuals were MUW. PCA-derived factors with levels that differed the most between MW and MUW groups were factors 4 (branched chain amino acids [BCAA]) [p<.0001], 8 (various metabolites) [p<.0001], 9 (C4/Ci4, C3, C5 acylcarnitines) [p<.0001] and 10 (amino acids) [p<.0002]. Further, Factor 4, distinguishes MW from MUW individuals independent of BMI. CONCLUSION BCAA and related metabolites are promising biomarkers that may aid in understanding cardiometabolic health independent of BMI category.

Risk of ischemic heart disease in patients with TIA

A prospective study was made of the morbidity and mortality from ischemic heart disease in 390 patients with focal TIA caused by atherosclerotic vascular disease. The 5-year cumulative rate of myocardial infarction or sudden death in these patients was 21.0%, a rate only slightly less than that of fatal or nonfatal cerebral infarction (22.7%). Risk factors including diabetes, angina, and ECG abnormalities were associated with an increase in morbidity and mortality from ischemic heart disease. A major factor associated with these cardiac events was the presence of atherosclerotic obstructive or ulcerative lesions in the carotid arteries. These observations indicate that focal TIA caused by carotid atherosclerosis is a predictor not only of cerebral infarction, but also of serious cardiac disease and death.

Neuropeptide Y Gene Polymorphisms Confer Risk of Early-Onset Atherosclerosis

Neuropeptide Y (NPY) is a strong candidate gene for coronary artery disease (CAD). We have previously identified genetic linkage to familial CAD in the genomic region of NPY. We performed follow-up genetic, biostatistical, and functional analysis of NPY in early-onset CAD. In familial CAD (GENECARD, N = 420 families), we found increased microsatellite linkage to chromosome 7p14 (OSA LOD = 4.2, p = 0.004) in 97 earliest age-of-onset families. Tagged NPY SNPs demonstrated linkage to CAD of a 6-SNP block (LOD = 1.58–2.72), family-based association of this block with CAD (p = 0.02), and stronger linkage to CAD in the earliest age-of-onset families. Association of this 6-SNP block with CAD was validated in: (a) 556 non-familial early-onset CAD cases and 256 controls (OR 1.46–1.65, p = 0.01–0.05), showing stronger association in youngest cases (OR 1.84–2.20, p = 0.0004–0.09); and (b) GENECARD probands versus non-familial controls (OR 1.79–2.06, p = 0.003–0.02). A promoter SNP (rs16147) within this 6-SNP block was associated with higher plasma NPY levels (p = 0.04). To assess a causal role of NPY in atherosclerosis, we applied the NPY1-receptor–antagonist BIBP-3226 adventitially to endothelium-denuded carotid arteries of apolipoprotein E-deficient mice; treatment reduced atherosclerotic neointimal area by 50% (p = 0.03). Thus, NPY variants associate with atherosclerosis in two independent datasets (with strong age-of-onset effects) and show allele-specific expression with NPY levels, while NPY receptor antagonism reduces atherosclerosis in mice. We conclude that NPY contributes to atherosclerosis pathogenesis.