An increased level of serum C‐reactive protein (CRP) is a known prognostic factor for acute coronary events and sudden cardiac death, and it is associated with coronary calcification. CRP is expressed in coronary arteries, but its role in the development of coronary plaques is unclear.
To investigate CRP immunoreactivity in relation to the severity of coronary artery disease and plaque morphology in human left anterior descending coronary arteries (LAD).
A prospective, consecutive autopsy series of 66 patients (mean age 63.4 years) in Tampere University Hospital, Tampere, Finland.
CRP immunoreactivity was seen in 59% of the cases. In logistic regression analysis with age, sex and body mass index as confounders, CRP immunoreactivity in LAD was associated with >50% stenosis and plaque calcification. All three cases with acute coronary thrombosis due to rupture or erosion of the plaque showed a clear immunopositive reaction. CRP‐positive cells were never detected in normal arteries, but were often found in early fibrous plaques (75%) and almost invariably present in the shoulder area of plaques with necrotic core (96%). CRP immunoreactivity adjacent to calcified areas in more stable plaques (71%) was less consistent with one‐third of these plaques showing no immunoreactivity.
CRP immunoreactivity is associated with the progression of atherosclerosis, and especially with unstable coronary plaques. The immunoreactivity could cease at the stable calcified stages of atherosclerosis.
Although sudden cardiac death (SCD) is heritable, its genetic underpinnings are poorly characterized. The QT interval appears to have a graded relationship to SCD and 35–45% of its variation is heritable. We examined the relationship among recently reported common genetic variants, QT interval and SCD.
Methods and Results
We genotyped 15 common (minor allele frequency >1%) candidate SNPs, based on association to the QT interval in prior studies, in individuals in 2 cohort studies (Health 2000, n=6,597; Mini-Finland, n=801). After exclusions, we identified 116 incident SCDs from the remaining sample (n=6,808). We constructed a QT genotype score (QTscore) using the allele copy number and previously reported effect estimates for each SNP. Cox proportional hazards models adjusting for age, sex, and geographical area were used time to SCD analyses. The QTscore was a continuous independent predictor of the heart rate-corrected QT interval (P<10−107). Comparing the top to the bottom quintile of QTscore, there was a 15.6 msec higher group mean QT interval (P<10−84). A 10 msec increase in the observed QT was associated with an increased risk of SCD (HR 1.19, 95% CI 1.07–1.32, P=0.002). There was no linear relationship between QTscore and SCD risk; although, in post-hoc secondary analysis there was increased risk in the top compared with the middle QTscore quintile (HR of 1.92, 95% CI 1.05–3.58, P=0.04).
Our study strongly replicates the relationship between common genetic variants and the QT interval, confirms the relationship between the QT interval and SCD, but does not show evidence for a linear relationship between QTscore and SCD risk.
death; sudden; genetics; QT interval; electrocardiography; mortality; electrophysiology
The frequency of adult-type hypolactasia (lactase non-persistence) varies widely among different ethnic groups. The cultural historical hypothesis assumes a link between the occurrence of hypolactasia and the distribution of dairy farming. The nomadic Nenets have been reindeer herders for generations and have therefore not consumed any dairy products. The hypotheses here was that the prevalence of lactase non-persistence (−13910 C/C genotype) among Nenets people having four Nenets grandparents is high, while the prevalence among Nenets originating from ethnically mixed families is lower.
The material was collected in four typical Nenets settlements in the Nenets Autonomous Okrug in Russia. One-third of the adult Nenets population were invited to answer a questionnaire and to donate buccal samples for genotyping by a doctor from the team of medical professionals who make rounds in this area. The total number of available participants was 177.
Genotyping was performed with the AbiPrism system. We used the method of concordance of grandparents’ national origin to ascribe ethnicity.
The prevalence of adult-type hypolactasia (−13910 C/C) among Nenets who had four Nenets grandparents was found to be 90%. The figures among others reporting three, two and one grandparent of Nenets origin were 72, 60 and 28%, respectively.
The findings are in accord with the cultural historical hypothesis.
adult-type hypolactasia; lactase non-persistence; cultural historical hypothesis; nomadic Nenets; genotyping
USF1 is a ubiquitous transcription factor governing the expression of numerous genes of lipid and glucose metabolism. APOA5 is a well-established candidate gene regulating triglyceride (TG) levels and has been identified as a downstream target of upstream stimulatory factor. No detailed studies about the effect of APOA5 on atherosclerotic lesion formation have been conducted, nor has its potential interaction with USF1 been examined.
Methods and Results
We analyzed allelic variants of USF1 and APOA5 in families (n=516) ascertained for atherogenic dyslipidemia and in an autopsy series of middle-aged men (n=300) with precise quantitative measurements of atherosclerotic lesions. The impact of previously associated APOA5 variants on TGs was observed in the dyslipidemic families, and variant rs3135506 was associated with size of fibrotic aortic lesions in the autopsy series. The USF1 variant rs2516839, associated previously with atherosclerotic lesions, showed an effect on TGs in members of the dyslipidemic families with documented coronary artery disease. We provide preliminary evidence of gene-gene interaction between these variants in an autopsy series with a fibrotic lesion area in the abdominal aorta (P=0.0028), with TGs in dyslipidemic coronary artery disease subjects (P=0.03), and with high-density lipoprotein cholesterol (P=0.008) in a large population cohort of coronary artery disease patients (n=1065) in which the interaction for TGs was not replicated.
Our findings in these unique samples reinforce the roles of APOA5 and USF1 variants on cardiovascular phenotypes and suggest that both genes contribute to lipid levels and aortic atherosclerosis individually and possibly through epistatic effects.
genes; USF1; APOA5; lipids; atherosclerosis; epistasis
APOE is the strongest risk gene for sporadic Alzheimer's disease (AD) so far. Recent genome wide association studies found links for sporadic AD with CLU and CR1 involved in Aβ clearance, and PICALM affecting intracellular trafficking.
We investigated the associations of senile plaques (SP) and neurofibrillary tangles (NFT) with the proposed risk genes and APOE, in the Tampere Autopsy Study (TASTY) series (603 cases), a sample of the general population (0 to 97 yrs), who died out-of-hospital.
Age and the APOEε4 allele associated strongly with all phenotypes of SP, as expected. In age and APOEε4 adjusted analyses, compared to the most common homozygous genotype, burnt out SP were more common among carriers of the C-allele of CLU, whereas the T-allele of PICALM and C-allele of CR1 were linked with lower SP coverage. We found no significant associations between any of the genetic variants and NFT.
Marginal effects from CLU, CR1 and PICALM suggest that these genes have minimal effects on the development of AD lesions.
Sudden unexpected postoperative hemodynamic collapse with a high mortality develops in 1–3% of patients undergoing coronary artery bypass surgery (CABG). The contribution of surgical graft complications to this serious condition is poorly known and their demonstration at autopsy is a challenging task. Isolated CABG was performed in 8,807 patients during 1988–1999. Of the patients, 76 (0.9%) developed sudden postoperative hemodynamic collapse resulting in subsequent emergency reopening of the median sternotomy and open cardiac massage. Further emergency reoperation could be performed in 62 (82%) whereas 14 patients died prior to reoperation and a further 21 did not survive the reoperation or died a few days later. All 35 (46%) patients who did not survive were subjected to medico-legal autopsy combined with postmortem cast angiography. By combining clinical data with autopsy and angiography data, various types of graft complications were observed in 27 (36%, 1.3 per patient) of the 76 patients with hemodynamic collapse. There were no significant differences in the frequency (33 vs. 40%) or number of complicated grafts per patient (1.2 vs. 1.4) between those who survived reoperation and who did not. Autopsy detected 25 major and minor findings not diagnosed clinically. Postmortem cast angiography visualized 2 graft twists not possible to detect by autopsy dissection only. Surgical graft complications were the most frequent single cause for sudden postoperative hemodynamic collapse in CABG patients leading to a fatal outcome in almost half of the cases. Postmortem angiography improved the accuracy of autopsy diagnostics of graft complications.
Malpractice; CABG; Postmortem angiography; Operative complication; Mortality; Autopsy
Adult human heart mitochondrial DNA (mtDNA) has recently been shown to have a complex organization with abundant dimeric molecules, branched structures and four-way junctions. In order to understand the physiological significance of the heart-specific mtDNA maintenance mode and to find conditions that modify human heart mtDNA structure and replication, we analyzed healthy human heart of various ages as well as several different heart diseases, including ischemic heart disease, dilated as well as hypertrophic cardiomyopathies, and several mitochondrial disorders. By using one- and two-dimensional agarose gel electrophoresis, various enzymatic treatments and quantitative PCR we found that in human newborns heart mtDNA has a simple organization, lacking junctional forms and dimers. The adult-type branched forms are acquired in the early childhood, correlating with an increase in mtDNA copy number. Mitochondrial disorders involving either mutations in the mtDNA polymerase γ (PolGα) or mtDNA helicase Twinkle, while having no obvious cardiac manifestation, show distinct mtDNA maintenance phenotypes, which are not seen in various types of diseased heart or in mitochondrial disorders caused by point mutations or large-scale deletions of mtDNA. The findings suggest a link between cardiac muscle development, mtDNA copy number, replication mode and topological organization. Additionally, we show that Twinkle might have a direct role in the maintenance of four-way junctions in human heart mtDNA.
Analysis of human heart mitochondrial DNA (mtDNA) by electron microscopy and agarose gel electrophoresis revealed a complete absence of the θ-type replication intermediates seen abundantly in mtDNA from all other tissues. Instead only Y- and X-junctional forms were detected after restriction digestion. Uncut heart mtDNA was organized in tangled complexes of up to 20 or more genome equivalents, which could be resolved to genomic monomers, dimers, and linear fragments by treatment with the decatenating enzyme topoisomerase IV plus the cruciform-cutting T7 endonuclease I. Human and mouse brain also contained a population of such mtDNA forms, which were absent, however, from mouse, rabbit, or pig heart. Overexpression in transgenic mice of two proteins involved in mtDNA replication, namely human mitochondrial transcription factor A or the mouse Twinkle DNA helicase, generated abundant four-way junctions in mtDNA of heart, brain, and skeletal muscle. The organization of mtDNA of human heart as well as of mouse and human brain in complex junctional networks replicating via a presumed non-θ mechanism is unprecedented in mammals.
USF1 regulates the transcription of more than 40 cardiovascular related genes and is well established as a gene associated with familial combined hyperlipidemia, a condition increasing the risk for coronary heart disease. No detailed data, however, exists on the impact of this gene to the critical outcome at the tissue level: different types of atherosclerotic lesions.
Methods and Results
We analyzed the USF1 in 2 autopsy series of altogether 700 middle-aged men (the Helsinki Sudden Death Study) with quantitative morphometric measurements of coronary atherosclerosis. SNP rs2516839, tagging common USF1 haplotypes, associated with the presence of several types of atherosclerotic lesions, particularly with the proportion of advanced atherosclerotic plaques (P=0.02) and area of calcified lesions (P<0.001) of the coronary arteries. Importantly, carriers of risk alleles of rs2516839 also showed a 2-fold risk for sudden cardiac death (genotype TT versus CC; OR 2.10, 95% CI 1.17 to 3.75, P=0.04). The risk effect of rs2516839 was present also in aorta samples of the men.
Our findings in this unique study sample suggest that USF1 contributes to atherosclerosis, the pathological arterial wall phenotype resulting in coronary heart disease and in its most dramatic consequence—sudden cardiac death.
atherosclerosis; coronary; genes; genetics; death; sudden
The local immunoreactivity of C-reactive protein (CRP) was studied in a heterotopic porcine model of posttranplant obliterative bronchiolitis (OB). Bronchial allografts and control autografts were examined serially 2–28 days after subcutaneous transplantation. The autografts stayed patent. In the allografts, proliferation of inflammatory cells (P < .0001) and fibroblasts (P = .02) resulted in occlusion of the bronchial lumens (P < .01). Influx of CD4+ (P < .001) and CD8+ (P < .0001) cells demonstrated allograft immune response. CRP positivity simultaneously increased in the bronchial walls (P < .01), in macrophages, myofibroblasts, and endothelial cells. Local CRP was predictive of features characteristic of OB (R = 0.456–0.879, P < .05−P < .0001). Early obliterative lesions also showed CRP positivity, but not mature, collagen-rich obliterative plugs (P < .05). During OB development, CRP is localized in inflammatory cells, myofibroblasts and endothelial cells probably as a part of the local inflammatory response.
Disturbed cellular cholesterol homeostasis may lead to accumulation of cholesterol in human atheroma plaques. Cellular cholesterol homeostasis is controlled by the sterol regulatory element-binding transcription factor 2 (SREBF-2) and the SREBF cleavage-activating protein (SCAP). We investigated whole genome expression in a series of human atherosclerotic samples from different vascular territories and studied whether the non-synonymous coding variants in the interacting domains of two genes, SREBF-2 1784G>C (rs2228314) and SCAP 2386A>G, are related to the progression of coronary atherosclerosis and the risk of pre-hospital sudden cardiac death (SCD).
Whole genome expression profiling was completed in twenty vascular samples from carotid, aortic and femoral atherosclerotic plaques and six control samples from internal mammary arteries. Three hundred sudden pre-hospital deaths of middle-aged (33–69 years) Caucasian Finnish men were subjected to detailed autopsy in the Helsinki Sudden Death Study. Coronary narrowing and areas of coronary wall covered with fatty streaks or fibrotic, calcified or complicated lesions were measured and related to the SREBF-2 and SCAP genotypes.
Whole genome expression profiling showed a significant (p = 0.02) down-regulation of SREBF-2 in atherosclerotic carotid plaques (types IV-V), but not in the aorta or femoral arteries (p = NS for both), as compared with the histologically confirmed non-atherosclerotic tissues. In logistic regression analysis, a significant interaction between the SREBF-2 1784G>C and the SCAP 2386A>G genotype was observed on the risk of SCD (p = 0.046). Men with the SREBF-2 C allele and the SCAP G allele had a significantly increased risk of SCD (OR 2.68, 95% CI 1.07–6.71), compared to SCAP AA homologous subjects carrying the SREBF-2 C allele. Furthermore, similar trends for having complicated lesions and for the occurrence of thrombosis were found, although the results were not statistically significant.
The results suggest that the allelic variants (SREBF-2 1784G>C and SCAP 2386A>G) in the cholesterol homeostasis regulating SREBF-SCAP pathway may contribute to SCD in early middle-aged men.
Cyclooxygenase (COX) catalyzes formation of prostaglandins that contribute to the inflammation in atherosclerosis. Our objective was to study whether the functional C variant of the −765G→C polymorphism in the human COX-2 gene associates with
the severity of coronary atherosclerosis measured at the coronary
artery level. The Helsinki sudden death study autopsy material
(n = 300) comprised of Finnish men who died suddenly.
The area of atherosclerotic lesions in the coronary arteries was
quantitated, and coronary narrowing was measured. The occurrence
of myocardial infarction (MI) was assessed. Genotyping was by
restriction endonuclease analysis. Men carrying the minor C allele
had larger areas of complicated lesions (P = .024)
and a higher number of coronary arteries that had over 50%
stenosis (P = .036) compared to men representing the
common GG genotype. The COX-2 polymorphism was not associated with
MI. Our data suggest that COX-2 may be involved in plaque
Genetic variation in proprotein convertase subtilisin/kexin type 9 (PCSK9) gene has been recently identified as an important determinant of plasma LDL-cholesterol and severity of coronary heart disease. We studied whether the PCSK9 gene is linked to the risk of ischemic stroke (IS) and with the development of intracranial atherosclerosis.
The pivotal E670G polymorphism, tagging an important haplotype of the PCSK9 gene, was genotyped in two independent studies. The Belgium Stroke Study included 237 middle aged (45–60) Belgian patients, with small-vessel occlusion (SVO) and large-vessel atherosclerosis stroke (LVA), and 326 gender and ethnicity matched controls (>60 yrs) without a history of stroke. In multivariate analysis the minor allele (G) carriers appeared as a significant predictor of LVA (OR = 3.52, 95% CI 1.25–9.85; p = 0.017). In a Finnish crossectional population based consecutive autopsy series of 604 males and females (mean age 62.5 years), G-allele carriers tended to have more severe allele copy number-dependent (p = 0.095) atherosclerosis in the circle of Willis and in its branches.
Our findings in this unique combination of clinical and autopsy data, provide evidence that PCSK9 gene associates with the risk of LVA stroke subtype, and suggest that the risk is mediated by the severity of intracranial atherosclerosis.
The mammalian mitochondrial transcription termination factor mTERF binds with high affinity to a site within the tRNALeu(UUR) gene and regulates the amount of read through transcription from the ribosomal DNA into the remaining genes of the major coding strand of mitochondrial DNA (mtDNA). Electrophoretic mobility shift assays (EMSA) and SELEX, using mitochondrial protein extracts from cells induced to overexpress mTERF, revealed novel, weaker mTERF-binding sites, clustered in several regions of mtDNA, notably in the major non-coding region (NCR). Such binding in vivo was supported by mtDNA immunoprecipitation. Two-dimensional neutral agarose gel electrophoresis (2DNAGE) and 5′ end mapping by ligation-mediated PCR (LM-PCR) identified the region of the canonical mTERF-binding site as a replication pause site. The strength of pausing was modulated by the expression level of mTERF. mTERF overexpression also affected replication pausing in other regions of the genome in which mTERF binding was found. These results indicate a role for TERF in mtDNA replication, in addition to its role in transcription. We suggest that mTERF could provide a system for coordinating the passage of replication and transcription complexes, analogous with replication pause-region binding proteins in other systems, whose main role is to safeguard the integrity of the genome whilst facilitating its efficient expression.
We used the Tampere Autopsy Study (TASTY) series (n = 603, age 0-97 yrs), representing an unselected population outside institutions, to investigate the pathogenic involvement of inflammation in Alzheimer's disease-related lesions.
We studied senile plaque (SP), neurofibrillary tangles (NFT) and SP phenotype associations with 6 reported haplotype tagging single nucleotide polymorphisms (SNPs) in the CRP gene. CRP and Aβ immunohistochemistry was assessed using brain tissue microarrays.
In multivariate analyses (age- and APOE-adjusted), non-neuritic SP were associated with the high-CRP TA-genotype (3.0% prevalence) of rs3091244 and CA-genotype (10.8%) of rs3093075 compared to common genotypes. Conversely, the low-CRP C allele (39.3%) of rs2794521 reduced the risk of harbouring early non-neuritic SP, compared to the TT genotype. CRP haplotype TAGCC (high) associated with non-neuritic SP, whereas haplotype CCGCC offered protection. TT genotypes (high) of rs3091244 and rs1130864 were associated with CRP staining. There were no associations between SNPs or haplotypes and NFT. CRP staining of the hippocampal CA1/2 region correlated with Aβ staining.
CRP gene variation affects early SP development in prodromal Alzheimer's disease, independent of APOE genotype.