Robert Roberts

Genetic Variants Influencing Circulating Lipid Levels and Risk of Coronary Artery Disease

Objective—Genetic studies might provide new insights into the biological mechanisms underlying lipid metabolism and risk of CAD. We therefore conducted a genome-wide association study to identify novel genetic determinants of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides. Methods and Results—We combined genome-wide association data from 8 studies, comprising up to 17 723 participants with information on circulating lipid concentrations. We did independent replication studies in up to 37 774 participants from 8 populations and also in a population of Indian Asian descent. We also assessed the association between single-nucleotide polymorphisms (SNPs) at lipid loci and risk of CAD in up to 9 633 cases and 38 684 controls. We identified 4 novel genetic loci that showed reproducible associations with lipids (probability values, 1.6×10−8 to 3.1×10−10). These include a potentially functional SNP in the SLC39A8 gene for HDL-C, an SNP near the MYLIP/GMPR and PPP1R3B genes for LDL-C, and at the AFF1 gene for triglycerides. SNPs showing strong statistical association with 1 or more lipid traits at the CELSR2, APOB, APOE-C1-C4-C2 cluster, LPL, ZNF259-APOA5-A4-C3-A1 cluster and TRIB1 loci were also associated with CAD risk (probability values, 1.1×10−3 to 1.2×10−9). Conclusion—We have identified 4 novel loci associated with circulating lipids. We also show that in addition to those that are largely associated with LDL-C, genetic loci mainly associated with circulating triglycerides and HDL-C are also associated with risk of CAD. These findings potentially provide new insights into the biological mechanisms underlying lipid metabolism and CAD risk.

Specificity of elevated serum MB creatine phosphokinase activity in the diagnosis of acute myocardial infarction

Creatine phosphokinase (CPK) isoenzyme determinations are useful in the diagnosis of myocardial infarction. However, until suitably sensitive and precise quantitative procedures became available, the diagnostic specificity of serum CPK isoenzyme elevations could not be thoroughly examined. In this study an assay procedure capable of accurately determining activity of individual CPK isoenzymes even in serum samples with normal total CPK activity was employed to obtain two types of information. First, CPK isoenzyme profiles were examined in extracts of a spectrum of human tissues obtained at operation to determine whether the isoenzyme associated with myocardium is presented in other human tissues in quantities sufficient to produce increased activity in serum. In addition, CPK isoenzymes were analyzed quantitatively in serial serum samples from 50 hospitalized control subjects, 100 patients with acute myocardial infarction, 100 patients undergoing non-cardiac surgery and 50 patients undergoing cardiac catheterization to determine whether insult to tissues other than the heart is associated with increased myocardial CPK isoenzyme activity in serum. Results from analyses of tissue extracts indicated that myocardium is the only tissue surveyed containing sufficient MB CPK to account for substantial increases in serum MB activity. Results from analyses of serial serum samples indicated that MB CPK activity levels are consistently elevated after myocardial infarction, averaging 0.089 IU/ml. However, after cardiac cathetrization or noncardiac surgery peak serum MB activity remains low, averaging only 0.004 IU/ml despite marked elevations in total serum CPK activity. Thus, elevated serum MB CPK activity is a highly specific as well as sensitive criterion of myocardial injury.

Increased congestive heart failure after myocardial infarction of modest extent in patients with diabetes mellitus

To elucidate the factors involved in the reduced survival rate of diabetic patients after acute myocardial infarction (AMI), we prospectively evaluated 100 patients with well-documented diabetes and 426 control patients. We characterized infarct size and analyzed the incidence and severity of congestive heart failure (CHF) and subsequent death with respect to infarct size. The extent of the index infarct was less in diabetic compared to nondiabetic patients, 16.2 +/- 2.2 CK-gm-eq/m2 compared with 19.2 +/- 0.9 (p less than 0.02). However, CHF was more prevalent in diabetic patients (31.2% of the diabetic patients compared to 15.7%). The difference was most prominent in diabetic patients who had sustained prior infarction (50% compared to 16%), but was evident also in diabetic patients with initial infarction (26% compared to 16%). The mortality rate was greater in diabetic patients (p less than 0.04). When diabetic and nondiabetic patients were stratified with respect to the presence or absence of CHF, survival curves were comparable. The increased incidence of CHF despite a smaller infarct size suggests that additional factors must contribute to myocardial dysfunction and the resultant excess in mortality.

Serial plasma catecholamine response early in the course of clinical acute myocardial infarction: Relationship to infarct extent and mortality

Clinical and experimental evidence suggest that sympathoadrenal activation contributes to mortality in patients with ischemic heart disease. To determine the level of sympathoadrenal activation in the very early phase of acute myocardial infarction (AMI) and to determine if location of infarction (anterior versus inferior) was related to sympathoadrenal activation, we studied norepinephrine (NE) and epinephrine (E) within 4 hours after the onset of symptoms and prior to any rise in plasma creatine kinase (CK). Mean (+/- SE) initial (NE = 591 +/- 111 pg/ml and E = 73 +/- 19 pg/ml), peak (NE = 1356 +/- 178 and E +/- 1098 +/- 608) and average (NE = 815 +/- 142 and E = 252 +/- 68) plasma catecholamine concentrations were considerably above normal (NE = 228 +/- 10 and E = 34 +/- 2 pg/ml, n 60) and values were similar for inferior and anterior infarctions. During an 18-month follow-up, three patients died in whom the AMI mean NE and E and peak CK were higher than in the eight late survivors. Thus the three AMI patients with peak EP values greater than 1000 died, whereas the eight AMI patients with peak EP values less than 1000 survived (p less than 0.01). The magnitude of sympathoadrenal activation early in the course of clinical AMI appeared related to the extent of myocardial damage and late mortality.

Deleterious effects due to hemorrhage after myocardial reperfusion

Abstract The effect of coronary reperfusion on evolving myocardial infarction was examined in 16 conscious dogs. Coronary arterial occlusion was produced by inflating an exteriorized balloon cuff, and reperfusion was initiated 5 hours later by deflating the cuff. Infarct size calculated from 24 hour serial serum creatine phosphokinase (CPK) values was compared to infarct size predicted from log normal curves best fit to initial 5 hour serial CPK changes. With sustained occlusion, predicted and observed infarct size correlated closely ( r = 0.94, no. = 11). Reperfusion produced two disparate effects. In nine dogs, observed infarct size was less than predicted, reflecting 42 ± 6 percent (standard error) salvage of myocardium. In seven dogs, observed infarct size exceeded predicted size by more than 100 percent. Thus, reperfusion resulted in extension of myocardial infarction in 44 percent of the animals. Extension of infarction was associated with myocardial hemorrhage. Hemoglobin content in biopsy specimens from the center of the infarct was 10 times greater in the animals with extension of infarction than in those with myocardial salvage. Thus, reperfusion is not without hazard during evolving infarction in the conscious dog; although it frequently diminishes ischemic injury, it may precipitate myocardial hemorrhage and extension of infarction.

Localization of a Gene Responsible for Arrhythmogenic Right Ventricular Dysplasia to Chromosome 3p23

BACKGROUNDnArrhythmogenic right ventricular dysplasia (ARVD), a familial cardiomyopathy occurring with a prevalence of 1 in 5000, is characterized by replacement of myocytes with fatty and fibrous tissue. Clinical manifestations include structural and functional abnormalities of the right ventricle and arrhythmias, leading to a sudden death rate of 2.5% per year. Four loci have been mapped, but no gene has been identified as yet.nnnMETHODS AND RESULTSnWe identified a large family of >200 members with ARVD segregating as an autosomal dominant trait affecting 10 living individuals. The diagnosis of ARVD was based on international diagnostic criteria including history, physical examination, ECG, echocardiogram, right ventricular angiogram, endomyocardial biopsy, and 24-hour ambulatory ECG. Blood was collected for DNA from 149 family members. Analysis of 257 polymorphic microsatellite markers by genetic linkage excluded previously known loci for ARVD and identified a novel locus at 3p23. Analysis of an additional 20 markers further defined the region. A peak logarithm of the odds score of 6.91 was obtained with marker D3S3613 at theta=0% recombination. Haplotype analysis identified a shared region between markers D3S3610 and D3S3659 of 9. 3 cM.nnnCONCLUSIONSnA novel locus for ARVD has been mapped to 3p23 and the region narrowed to 9.3 cM. Identification of the gene will allow genetic screening and a specific diagnosis for a disease with protean nonspecific findings. It should also provide insight fundamental to understanding cardiac chamber-specific gene expression and/or the mechanism of myocyte apoptosis observed in this disease.

Factors presaging early recurrent myocardial infarction (“Extension”)

A prospective study of 200 consecutive patients with acute myocardial infarction was undertaken to characterize the frequency and severity of early recurrent infarction (extension), manifested by secondary plasma MB creatine kinase (CK) and myoglobin peaks, and to identify patients at particularly high risk. Serial MB CK and myoglobin determinations and continuous electrocardiographic recordings were obtained in all patients for 14 days, and serial radioventriculograms were obtained in selected patients. Chest pain and S-T segment changes occurred often, in 57 and 43 percent, respectively, of the entire group of patients. However, a secondary rise in plasma MB CK levels indicative of recurrent infarction, occurring an average of 10 +/- 4 days after the initial infarct, was evident in only 17 percent of patients. Forty-three percent (25 of 58) of patients with initial subendocardial infarction exhibited recurrent infarction compared with only 8 percent of those with initial transmural infarction. The mortality rate was 7 percent in patients with subendocardial infarction without early recurrence compared with 16 percent among those with recurrent. Logistic regression analysis indicated that obese women with initial subendocardial infarction and repeated episodes of prolonged chest pain had a high probability rate (60 percent) of recurrence in contrast to the low probability (2 percent) in patients without these features. Thus, early recurrent infarction is frequent after subendocardial infarction and is associated with a marked increase in mortality. These results suggest that patients with subendocardial infarction are at particularly high risk for recurrent infarction and that patients with this type of infarction require vigorous monitoring and prolonged surveillance.

Effects of dobutamine in patients with acute myocardial infarction

This study was designed to evaluate the effects of dobutamine, a new cardioselective beta adrenergic agonist, on cardiac performance and myocardial injury in patients with evolving myocardial infarction. Results in 16 patients given dobutamine (1 to 40 microng/kg per min for 24 hours) were compared with those in two groups of control patients: one of 16 patients matched for predicted infarct size, and the other of 16 patients matched for early ventricular dysrhythmia, analyzed by computer. Infarct size was predicted from plasma creatine kinase (CK) values during the first 7 hours after the initial elevation, before infusion of dobutamine. Overall observed infarct size was estimated from hourly CK values for 48 hours (including those before and after administration of dobutamine). In all patients technetium-99m (stannous) pyrophosphate scans were positive for myocardial infarction. Dobutamine increased cardiac output (assessed by thermodilution) from 4.9 +/- 0.37 (mean +/- standard error) to 6.0 +/- 0.38 liters/min (P less than 0.05) and decreased pulmonary arterial occlusive pressure from 21.5 +/- 2.7 to 16.7 +/- 1.6 mmHg (P less than 0.01) without significantly altering heart rate or systemic arterial blood pressure. The ratio of observed to predicted infarct size, the frequency of independently detected reinfarction or extension of infarction and the frequency of premature ventricular complexes were similar in control and treated patients. Thus administration of dobutamine in doses sufficient to improve ventricular performance after myocardial infarction does not exacerbate myocardial injury or ventricular dysrhythmia.

Quantification of serum creatine phosphokinase isoenzyme activity

Abstract Three serum creatine phosphokinase (CPK) isoenzymes (MM, MB and BB) have been recognized. Myocardium is richly endowed with MB CPK. Accordingly, increased serum MB CPK activity reflects myocardial injury. Although recent advances have facilitated detection of activity of CPK isoenzymes, available techniques have unavoidable quantitative limitations. Accordingly, we used a new procedure to quantify CPK isoenzymes in serum samples with normal or increased total CPK activity. Samples were diluted or concentrated so that total CPK activity was 0.100 to 1.200 IU/ml and then subjected to electrophoresis on cellulose acetate. Regions of the strips encompassing each isoenzyme were cut out, immersed in 0.5 to 3 ml of assay medium in which the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) was generated as a function of isoenzyme activity. NADPH was detected by serial determinations of fluorescence in the medium. The assay was linear with respect to time and isoenzyme activity (0.010 to 1.2 ID/ml, no . = 100) and recovery averaged 83 ± 1.8 percent (standard deviation). Reproducibility was within 2 percent. Accuracy of the method was validated with selected mixtures of BB and MM isoenzymes. Serum MB CPK activity averaged only 0.002 ± 0.001 lU/ml in normal persons ( no . = 27), compared to 0.064 ± 0.043 lU/ml in 30 patients with myocardial infarction. Serum MB CPK activity was not increased in 23 patients after intramuscular injections or abdominal surgery. The procedure described permits quantitative determination of activity of individual CPK isoenzymes in serum samples with or without increased total CPK activity and thus is of potential value in the quantitative assessment of myocardial infarction by serum enzyme analysis.

The Locus of a Novel Gene Responsible for Arrhythmogenic Right-Ventricular Dysplasia Characterized by Early Onset and High Penetrance Maps to Chromosome 10p12-p14

Arrhythmogenic right-ventricular dysplasia (ARVD), a cardiomyopathy inherited as an autosomal-dominant disease, is characterized by fibro-fatty infiltration of the right-ventricular myocardium. Four loci for ARVD have been mapped in the Italian population, and recently the first locus was mapped in inhabitants of North America. None of the genes have been identified. We have now identified another North American family with early onset of ARVD and high penetrance. All of the children with the disease haplotype had pathological or clinical evidence of the disease at age <10 years. The family spans five generations, having 10 living and 2 dead affected individuals, with ARVD segregating as an autosomal-dominant disorder. Genetic linkage analysis excluded known loci, and a novel locus was identified on chromosome 10p12-p14. A peak two-point LOD score of 3.92 was obtained with marker D10S1664, at a recombination fraction of 0. Additional genotyping and haplotype analysis identified a shared region of 10.6 cM between marker D10S547 and D10S1653. Thus, a novel gene responsible for ARVD resides on the short arm of chromosome 10. This disease is intriguing, since it initiates exclusively in the right ventricle and exhibits pathological features of apoptosis. Chromosomal localization of the ARVD gene is the first step in identification of the genetic defect and the unraveling of the molecular basis responsible for the pathogenesis of the disease.