Karl J. Lackner

Genetics and Beyond – The Transcriptome of Human Monocytes and Disease Susceptibility

Background Variability of gene expression in human may link gene sequence variability and phenotypes; however, non-genetic variations, alone or in combination with genetics, may also influence expression traits and have a critical role in physiological and disease processes. Methodology/Principal Findings To get better insight into the overall variability of gene expression, we assessed the transcriptome of circulating monocytes, a key cell involved in immunity-related diseases and atherosclerosis, in 1,490 unrelated individuals and investigated its association with >675,000 SNPs and 10 common cardiovascular risk factors. Out of 12,808 expressed genes, 2,745 expression quantitative trait loci were detected (P<5.78×10−12), most of them (90%) being cis-modulated. Extensive analyses showed that associations identified by genome-wide association studies of lipids, body mass index or blood pressure were rarely compatible with a mediation by monocyte expression level at the locus. At a study-wide level (P<3.9×10−7), 1,662 expression traits (13.0%) were significantly associated with at least one risk factor. Genome-wide interaction analyses suggested that genetic variability and risk factors mostly acted additively on gene expression. Because of the structure of correlation among expression traits, the variability of risk factors could be characterized by a limited set of independent gene expressions which may have biological and clinical relevance. For example expression traits associated with cigarette smoking were more strongly associated with carotid atherosclerosis than smoking itself. Conclusions/Significance This study demonstrates that the monocyte transcriptome is a potent integrator of genetic and non-genetic influences of relevance for disease pathophysiology and risk assessment.

Serial Changes in Highly Sensitive Troponin I Assay and Early Diagnosis of Myocardial Infarction

CONTEXT Introduction of highly sensitive troponin assays into clinical practice has substantially improved the evaluation of patients with chest pain. OBJECTIVE To evaluate the diagnostic performance of a highly sensitive troponin I (hsTnI) assay compared with a contemporary troponin I (cTnI) assay and their serial changes in the diagnosis of acute myocardial infarction (AMI). DESIGN, SETTING, AND PATIENTS A total of 1818 patients with suspected acute coronary syndrome were consecutively enrolled at the chest pain units of the University Heart Center Hamburg, the University Medical Center Mainz, and the Federal Armed Forces Hospital Koblenz, all in Germany, from 2007 to 2008. Twelve biomarkers including hsTnI (level of detection, 3.4 pg/mL) and cTnI (level of detection, 10 pg/mL) were measured on admission and after 3 and 6 hours. MAIN OUTCOME MEASURES Diagnostic performance for AMI of baseline and serial changes in hsTnI and cTnI results at 3 hours after admission to the emergency department. RESULTS Of the 1818 patients, 413 (22.7%) were diagnosed as having AMI. For discrimination of AMI, the area under the receiver operating characteristic (ROC) curve was 0.96 (95% CI, 0.95-0.97) for hsTnI on admission and 0.92 (95% CI, 0.90-0.94) for cTnI on admission. Both were superior to the other evaluated diagnostic biomarkers. The use of hsTnI at admission (with the diagnostic cutoff value at the 99th percentile of 30 pg/mL) had a sensitivity of 82.3% and a negative predictive value (for ruling out AMI) of 94.7%. The use of cTnI (with the diagnostic cutoff value at the 99th percentile of 32 pg/mL) at admission had a sensitivity of 79.4% and a negative predictive value of 94.0%. Using levels obtained at 3 hours after admission, the sensitivity was 98.2% and the negative predictive value was 99.4% for both hsTnI and cTnI assays. Combining the 99th percentile cutoff at admission with the serial change in troponin concentration within 3 hours, the positive predictive value (for ruling in AMI) for hsTnI increased from 75.1% at admission to 95.8% after 3 hours, and for cTnI increased from 80.9% at admission to 96.1% after 3 hours. CONCLUSIONS Among patients with suspected acute coronary syndrome, hsTnI or cTnI determination 3 hours after admission may facilitate early rule-out of AMI. A serial change in hsTnI or cTnI levels from admission (using the 99th percentile diagnostic cutoff value) to 3 hours after admission may facilitate an early diagnosis of AMI.

Copeptin Improves Early Diagnosis of Acute Myocardial Infarction

OBJECTIVES Early identification of myocardial infarction in chest pain patients is crucial to identify patients at risk and to maintain a fast treatment initiation. BACKGROUND The aim of the current investigation is to test whether determination of copeptin, an indirect marker for arginin-vasopressin, adds diagnostic information to cardiac troponin in early evaluation of patients with suspected myocardial infarction. METHODS Between January 2007 and July 2008, patients with suspected acute coronary syndrome were consecutively enrolled in this multicenter study. Copeptin, troponin T (TnT), myoglobin, and creatine kinase-myocardial band were determined at admission and after 3 and 6 h. RESULTS Of 1,386 (66.4% male) enrolled patients, 299 (21.6%) had the discharge diagnosis of acute myocardial infarction, 184 (13.3%) presented with unstable angina, and in 903 (65.2%) an acute coronary syndrome could be excluded. Combined measurement of copeptin and TnT on admission improved the c-statistic from 0.84 for TnT alone to 0.93 in the overall population and from 0.77 to 0.9 in patients presenting within 3 h after chest pain onset (CPO) (p < 0.001). In this group the combination of copeptin with a conventional TnT provided a negative predictive value of 92.4%. CONCLUSIONS In triage of chest pain patients, determination of copeptin in addition to troponin improves diagnostic performance, especially early after CPO. Combined determination of troponin and copeptin provides a remarkable negative predictive value virtually independent of CPO time and therefore aids in early and safe rule-out of myocardial infarction.

Genetic Analysis of the Interleukin-18 System Highlights the Role of the Interleukin-18 Gene in Cardiovascular Disease

Background—Interleukin (IL)-18 plays a key role in atherosclerosis and its complications. The present study investigated the genetic variability of 4 genes of the IL-18 system—IL18, IL18R1, IL18RAP, and IL18BP—in relation to circulating IL-18 levels and cardiovascular mortality. Methods and Results—Twenty-two polymorphisms were genotyped in 1288 patients with coronary artery disease prospectively followed up during a median period of 5.9 years. The end point was death from cardiovascular causes (n=142). Baseline IL-18 levels were predictive of cardiovascular deaths occurring during ≤4 years of follow-up (HR=2.96, 95% CI 1.54 to 5.70, P=0.001 for the top compared with the bottom quartile) but not of later deaths. Haplotypes of the IL18 gene were associated with IL-18 levels (P=0.002) and cardiovascular mortality (P=0.006) after adjustment for cardiovascular risk factors. The same haplotype was associated with both a 9% lowering effect on IL-18 levels and a protective effect on risk (HR=0.57, 95% CI 0.36 to 0.92). IL18 haplotypes explained only 2% of IL-18 variability. Adjustment for baseline IL-18 levels abolished the association of haplotypes with cardiovascular risk. The haplotype associated with phenotypes was the only one carrying the minor allele of the IL18/A+183G polymorphism located in the 3′untranslated region and potentially affecting mRNA stability. The other genes of the system were not related to IL-18 levels or cardiovascular outcome. Conclusion—Variations of the IL18 gene consistently influence circulating levels of IL-18 and clinical outcome in patients with coronary artery disease, which supports the hypothesis of a causal role of IL-18 in atherosclerosis and its complications.

Genetic variants associated with cardiac structure and function: A meta-analysis and replication of genome-wide association data

CONTEXT Echocardiographic measures of left ventricular (LV) structure and function are heritable phenotypes of cardiovascular disease. OBJECTIVE To identify common genetic variants associated with cardiac structure and function by conducting a meta-analysis of genome-wide association data in 5 population-based cohort studies (stage 1) with replication (stage 2) in 2 other community-based samples. DESIGN, SETTING, AND PARTICIPANTS Within each of 5 community-based cohorts comprising the EchoGen consortium (stage 1; n = 12 612 individuals of European ancestry; 55% women, aged 26-95 years; examinations between 1978-2008), we estimated the association between approximately 2.5 million single-nucleotide polymorphisms (SNPs; imputed to the HapMap CEU panel) and echocardiographic traits. In stage 2, SNPs significantly associated with traits in stage 1 were tested for association in 2 other cohorts (n = 4094 people of European ancestry). Using a prespecified P value threshold of 5 x 10(-7) to indicate genome-wide significance, we performed an inverse variance-weighted fixed-effects meta-analysis of genome-wide association data from each cohort. MAIN OUTCOME MEASURES Echocardiographic traits: LV mass, internal dimensions, wall thickness, systolic dysfunction, aortic root, and left atrial size. RESULTS In stage 1, 16 genetic loci were associated with 5 echocardiographic traits: 1 each with LV internal dimensions and systolic dysfunction, 3 each with LV mass and wall thickness, and 8 with aortic root size. In stage 2, 5 loci replicated (6q22 locus associated with LV diastolic dimensions, explaining <1% of trait variance; 5q23, 12p12, 12q14, and 17p13 associated with aortic root size, explaining 1%-3% of trait variance). CONCLUSIONS We identified 5 genetic loci harboring common variants that were associated with variation in LV diastolic dimensions and aortic root size, but such findings explained a very small proportion of variance. Further studies are required to replicate these findings, identify the causal variants at or near these loci, characterize their functional significance, and determine whether they are related to overt cardiovascular disease.

Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus

Central corneal thickness (CCT) is associated with eye conditions including keratoconus and glaucoma. We performed a meta-analysis on >20,000 individuals in European and Asian populations that identified 16 new loci associated with CCT at genome-wide significance (P < 5 × 10−8). We further showed that 2 CCT-associated loci, FOXO1 and FNDC3B, conferred relatively large risks for keratoconus in 2 cohorts with 874 cases and 6,085 controls (rs2721051 near FOXO1 had odds ratio (OR) = 1.62, 95% confidence interval (CI) = 1.4–1.88, P = 2.7 × 10−10, and rs4894535 in FNDC3B had OR = 1.47, 95% CI = 1.29–1.68, P = 4.9 × 10−9). FNDC3B was also associated with primary open-angle glaucoma (P = 5.6 × 10−4; tested in 3 cohorts with 2,979 cases and 7,399 controls). Further analyses implicate the collagen and extracellular matrix pathways in the regulation of CCT.

Different Calculations of Ankle-Brachial Index and Their Impact on Cardiovascular Risk Prediction

Background— An ankle-brachial index (ABI; ratio of ankle and brachial systolic blood pressure) <0.9 indicates peripheral arterial disease (PAD) and is a strong predictor of cardiovascular events. The aim of the present study was to address the prognostic value of different methods of ABI calculation. Methods and Results— In 831 patients admitted with chest pain for diagnostic heart catheterization, blood pressure of both anterior and posterior tibial arteries was measured. ABI was calculated for each leg with the higher of the 2 ankle pressures (current definition of the American Heart Association) or with the lower of the 2 ankle pressures (modified definition) in relation to the higher of the left or right brachial systolic blood pressure. For each patient, the lower ABI from both legs was used for further evaluation. Fifteen patients (1.8%) with ABI >1.5 were excluded. We compared patients with ABI <0.9 according to the current definition (with PAD, n=204 [25.0%]), those with ABI ≥0.9 according to the modified definition (without PAD, n=524 [64.2%]), and those with ABI <0.9 according to the modified definition and ≥0.9 according to the current definition (suspected PAD, n=88 [10.8%]). Follow-up data (median 6.6 years) were available for 812 patients (99.5%); 157 patients (19.3%) experienced cardiovascular events (cardiovascular death, myocardial infarction, or stroke). Patients without PAD had the lowest cardiovascular event rate, whereas event rates were comparable for patients with PAD and those with suspected PAD (14.8% versus 28.4% versus 25.0%, respectively). In a fully adjusted Cox regression analysis that included patients without PAD as the reference group, the hazard ratio (95% CI) was 1.56 (0.97 to 2.53) for patients with suspected PAD and 1.67 (1.16 to 2.40) for patients with PAD. Conclusions— When the higher ankle pressure is used for ABI calculation, a group of patients at high risk for cardiovascular events is overlooked. With a simple modification of ABI (use of the lower instead of the higher ankle pressure), more patients at risk could be identified.

Possible Protective Role for C-Reactive Protein in Atherogenesis Complement Activation by Modified Lipoproteins Halts Before Detrimental Terminal Sequence

Background—Previous work indicated that enzymatically remodeled LDL (E-LDL) might activate complement in atherosclerotic lesions via a C-reactive protein (CRP)–dependent and CRP-independent pathway. We sought to substantiate this contention and determine whether both pathways drive the sequence to completion. Methods and Results—E-LDL was prepared by sequential treatment of LDL with a protease and cholesteryl esterase. Trypsin, proteinase K, cathepsin H, or plasmin was used with similar results. Functional tests were used to assess total complement hemolytic activity, and immunoassays were used to demonstrate C3 cleavage and to quantify C3a, C4a, C5a, and C5b-9. E-LDL preparations activated complement to completion, independent of CRP, when present above a threshold concentration (100 to 200 μg/mL in 5% serum). Below the threshold, all E-LDL preparations activated complement in dependence of CRP, but the pathway then halted before the terminal sequence. Native LDL and oxidized LDL did not activate complement under any circumstances tested. Immunohistological analyses corroborated the concept that CRP-dependent complement activation inefficiently generates C5b-9. Conclusions—Binding of CRP to E-LDL is the first trigger for complement activation in the atherosclerotic lesion, but the terminal sequence is thereby spared. This putatively protective function of CRP is overrun at higher E-LDL concentrations, so that potentially harmful C5b-9 complexes are generated.

A Genome-wide Association Study Identifies LIPA as a Susceptibility Gene for Coronary Artery Disease

Background— eQTL analyses are important to improve the understanding of genetic association results. We performed a genome-wide association and global gene expression study to identify functionally relevant variants affecting the risk of coronary artery disease (CAD). Methods and Results— In a genome-wide association analysis of 2078 CAD cases and 2953 control subjects, we identified 950 single-nucleotide polymorphisms (SNPs) that were associated with CAD at P<10−3. Subsequent in silico and wet-laboratory replication stages and a final meta-analysis of 21 428 CAD cases and 38 361 control subjects revealed a novel association signal at chromosome 10q23.31 within the LIPA (lysosomal acid lipase A) gene (P=3.7×10−8; odds ratio, 1.1; 95% confidence interval, 1.07 to 1.14). The association of this locus with global gene expression was assessed by genome-wide expression analyses in the monocyte transcriptome of 1494 individuals. The results showed a strong association of this locus with expression of the LIPA transcript (P=1.3×10−96). An assessment of LIPA SNPs and transcript with cardiovascular phenotypes revealed an association of LIPA transcript levels with impaired endothelial function (P=4.4×10−3). Conclusions— The use of data on genetic variants and the addition of data on global monocytic gene expression led to the identification of the novel functional CAD susceptibility locus LIPA, located on chromosome 10q23.31. The respective eSNPs associated with CAD strongly affect LIPA gene expression level, which was related to endothelial dysfunction, a precursor of CAD.

6-18F-fluoro-L-dihydroxyphenylalanine positron emission tomography is superior to 123I-metaiodobenzyl-guanidine scintigraphy in the detection of extraadrenal and hereditary pheochromocytomas and paragangliomas: correlation with vesicular monoamine transporter expression.

CONTEXT Pheochromocytomas (PHEOs) and paragangliomas (PGLs) may be better detected by (18)F-fluorodihydroxyphenylalanine-positron emission tomography (FDOPA-PET) than (123)I-metaiodobenzyl-guanidine (123-I-MIBG) scintigraphy. OBJECTIVE The objective of the study was to correlate functional imaging results with immunohistochemical, molecular-genetic, and biochemical findings. DESIGN AND SETTING Thirty consecutive patients with suspected PHEO/PGL presenting at a tertiary referral centre were investigated in a prospective study. PATIENTS Twenty-five patients had confirmed PHEO/PGL. Thirteen of 25 patients had a hereditary PHEO/PGL syndrome (two multiple endocrine neoplasia II, six succinate dehydrogenase complex, subunit D, two succinate dehydrogenase complex, subunit B, one von Hippel Lindau tumor suppressor protein, two Neurofibromatosis-1), and 12 of 25 were classified as sporadic. Five patients had hormonally inactive adrenal incidentalomas. MAIN OUTCOME MEASURES In all patients computed tomography scan and/or magnetic resonance imaging as well as both 123-I-MIBG scintigraphy and FDOPA-PET were performed. Resected tumors were examined by immunohistochemistry for expression of the vesicular monoamine transporter (VMAT)-1 and -2 and other markers. RESULTS A total of 64 lesions were found with both functional imaging modalities. FDOPA-PET detected 62 lesions, whereas only 34 lesions were detected by 123-I-MIBG scintigraphy. This resulted in an overall sensitivity and specificity for FDOPA-PET of 98 and 100% and for MIBG of 53 and 91%, respectively. Comparable sensitivities were found for adrenal and extraadrenal abdominal lesions (94 vs. 97%), whereas in thoracic/cervical lesions, the sensitivity for 123-I-MIBG scintigraphy (15%) was inferior to that of FDOPA-PET imaging (100%). Immunohistochemistry demonstrated a lack of VMAT-1 expression in all MIBG-negative tumors. Clinical predictors for MIBG negativity were a predominant norepinephrine/normetanephrine secretion, an age less than 45 yr, and a hereditary cause. CONCLUSION FDOPA-PET is superior to 123-I-MIBG scintigraphy in patients with extraadrenal, predominantly noradrenaline-secreting, and hereditary types of PHEO/PGL. The lack of VMAT-1 expression predicts negativity for MIBG-scintigraphy.