Combining Polygenic and Proteomic Risk Scores With Clinical Risk Factors to Improve Performance for Diagnosing Absence of Coronary Artery Disease in Patients With de novo Chest Pain.

BACKGROUND: Patients with de novo chest pain, referred for evaluation of possible coronary artery disease (CAD), frequently have an absence of CAD resulting in millions of tests not having any clinical impact. The objective of this study was to investigate whether polygenic risk scores and targeted proteomics improve the prediction of absence of CAD in patients with suspected CAD, when added to the PROMISE (Prospective Multicenter Imaging Study for Evaluation of Chest Pain) minimal risk score (PMRS). METHODS: Genotyping and targeted plasma proteomics (N=368 proteins) were performed in 1440 patients with symptoms suspected to be caused by CAD undergoing coronary computed tomography angiography. Based on individual genotypes, a polygenic risk score for CAD (PRSCAD) was calculated. The prediction was performed using combinations of PRSCAD, proteins, and PMRS as features in models using stability selection and machine learning. RESULTS: Prediction of absence of CAD yielded an area under the curve of PRSCAD-model, 0.64±0.03; proteomic-model, 0.58±0.03; and PMRS model, 0.76±0.02. No significant correlation was found between the genetic and proteomic risk scores (Pearson correlation coefficient, -0.04; P=0.13). Optimal predictive ability was achieved by the full model (PRSCAD+protein+PMRS) yielding an area under the curve of 0.80±0.02 for absence of CAD, significantly better than the PMRS model alone (P<0.001). For reclassification purpose, the full model enabled down-classification of 49% (324 of 661) of the 5% to 15% pretest probability patients and 18% (113 of 611) of >15% pretest probability patients. CONCLUSIONS: For patients with chest pain and low-intermediate CAD risk, incorporating targeted proteomics and polygenic risk scores into the risk assessment substantially improved the ability to predict the absence of CAD. Genetics and proteomics seem to add complementary information to the clinical risk factors and improve risk stratification in this large patient group. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT02264717.

Association of gene polymorphisms in MYH11 and TGF-ß signaling with the susceptibility and clinical outcomes of DeBakey type III aortic dissection.

To investigate the association of myosin heavy chain protein 11 (MYH11) and transforming growth factor ß signaling-related gene polymorphisms with the susceptibility of DeBakey type III aortic dissection (AD) and its clinical outcomes. Four single-nucleotide polymorphism (SNPs) (MYH11 rs115364997, rs117593370, TGFB1 rs1800469, and TGFBR1 rs1626340) were analyzed in patients with DeBakey III AD (173) and healthy participants (335). Gene-gene and gene-environment interactions were evaluated using generalized multifactor dimensionality reduction. The patients were followed up for a median of 55.7 months. MYH11 rs115364997 G or TGFBR1 rs1626340 A carriers had an increased risk of DeBakey type III AD. MYH11, TGFB1, TGFBR1, and environment interactions contributed to the risk of DeBakey type III AD (cross-validation consistency = 10/10, P = 0.001). Dominant models of MYH11 rs115364997 AG + GG genotype (HR = 2.443; 95%CI: 1.096-5.445, P = 0.029), TGFB1 rs1800469 AG + GG (HR = 2.303; 95%CI: 1.069-4.96, P = 0.033) were associated with an increased risk of mortality in DeBakey type III AD. The dominant model of TGFB1 rs1800469 AG + GG genotype was associated with an increased risk of recurrence of chest pain in DeBakey type III AD (HR = 1.566; 95%CI: 1.018-2.378, P = 0.041). In conclusions, G carriers of MYH11 rs115364997 or TGFB1 rs1800469 may be the poor prognostic indicators of mortality and recurrent chest pain in DeBakey type III AD. The interactions of gene-gene and gene-environment are associated with the risk of DeBakey type III AD.

Polygenic Risk Score-Enhanced Risk Stratification of Coronary Artery Disease in Patients With Stable Chest Pain.

BACKGROUND: Polygenic risk scores (PRSs) are associated with coronary artery disease (CAD), but the clinical potential of using PRSs at the single-patient level for risk stratification has yet to be established. We investigated whether adding a PRS to clinical risk factors (CRFs) improves risk stratification in patients referred to coronary computed tomography angiography on a suspicion of obstructive CAD. METHODS: In this prespecified diagnostic substudy of the Dan-NICAD trial (Danish study of Non-Invasive testing in Coronary Artery Disease), we included 1617 consecutive patients with stable chest symptoms and no history of CAD referred for coronary computed tomography angiography. CRFs used for risk stratification were age, sex, symptoms, prior or active smoking, antihypertensive treatment, lipid-lowering treatment, and diabetes. In addition, patients were genotyped, and their PRSs were calculated. All patients underwent coronary computed tomography angiography. Patients with a suspected ≥50% stenosis also underwent invasive coronary angiography with fractional flow reserve. A combined end point of obstructive CAD was defined as a visual invasive coronary angiography stenosis >90%, fractional flow reserve <0.80, or a quantitative coronary analysis stenosis >50% if fractional flow reserve measurements were not feasible. RESULTS: The PRS was associated with obstructive CAD independent of CRFs (adjusted odds ratio, 1.8 [95% CI, 1.5-2.2] per SD). The PRS had an area under the curve of 0.63 (0.59-0.68), which was similar to that for age and sex. Combining the PRS with CRFs led to a CRF+PRS model with area under the curve of 0.75 (0.71-0.79), which was 0.04 more than the CRF model (P=0.0029). By using pretest probability (pretest probability) cutoffs at 5% and 15%, a net reclassification improvement of 15.8% (P=3.1×10-4) was obtained, with a down-classification of risk in 24% of patients (211 of 862) in whom the pretest probability was 5% to 15% based on CRFs alone. CONCLUSIONS: Adding a PRS improved risk stratification of obstructive CAD beyond CRFs, suggesting a modest clinical potential of using PRSs to guide diagnostic testing in the contemporary clinical setting. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02264717.

Association of 25-hydroxyvitamin D with HDL-cholesterol and other cardiovascular risk biomarkers in subjects with non-cardiac chest pain.

BACKGROUND: Chest pain is a serious symptom that is routinely investigated as a sign of coronary artery disease. Non-cardiac chest pain (NCCP) is indistinguishable from ischemic chest pain and both are considered serious and receive similar medical investigations. Although NCCP is not associated with cardiovascular diseases (CVDs), patients with NCCP may become anxious and frightened from developing coronary events. So, it will be valuable to improve modifiable cardiovascular risk factors in such subjects to reduce fear from CVDs. Because vitamin D deficiency was considered as a possible modifiable cardiovascular risk factor, our aim was to investigate association between serum vitamin D and cardiovascular risk variables in subjects with NCCP. METHODS: A cross-sectional study involved 104 subjects who underwent cardiac catheterization that did not reveal any cardiac origin for their chest pain. 25-hydroxyvitamin D was measured by electrochemiluminescence immunoassay, glucose was measured by hexokinase method, hemoglobin A1c (HbA1c) was measured by turbidimetric inhibition immunoassay and lipid profile was measured by enzymatic colorimetric assays. RESULTS: High density lipoprotein cholesterol (HDL-C) was significantly higher in subjects with sufficient vitamin D compared to those with insufficient or deficient vitamin D (p-value< 0.01). 25-hydroxyvitamin D was positively associated with HDL-C (p-value< 0.01) and inversely associated with HbA1c (p-value = 0.02). 25-hydroxyvitamin D was not significantly correlated with other cardiovascular biomarkers including blood pressure, glucose, and other components of lipid profile (p-values> 0.05). CONCLUSIONS: low serum vitamin D could be involved in reducing HDL-C and increasing HbA1c and thus it may increase cardiovascular risk in subjects with NCCP.

Birt-Hogg-Dubé syndrome: spontaneous pneumothorax as a first symptom.

Birt-Hogg-Dubé syndrome (BHDS) is a rare hereditary autosomal dominant condition characterised by benign cutaneous lesions, lung cysts, increased risk of spontaneous pneumothorax and renal cancer. It shows great heterogenous presentation within and between affected families. We report a case of a Danish female patient with recurrent pneumothoraces as the first symptom of BHDS. Over the years, she developed skin changes, and a family history of skin changes, pneumothoraces and renal cancer was discovered. BHDS was suspected, a genetic analysis was performed and a pathogenic variation c.1285delC in FLCN gene was detected in the patient. As we stated the diagnosis BHDS, we discovered several undiagnosed family members all of them now entering a lifelong follow-up programme with abdominal imaging because of the increased risk of developing renal cancer. BHDS should be known to oncologists, dermatologists and pulmonologists as the patients most often present to these medical disciplines.

Evaluating Diagnostic and Prognostic Value of Plasma miRNA133a in Acute Chest Pain Patients Undergoing Coronary Angiography.

Circulating microRNA has recently emerged as a promising biomarker for cardiovascular disease. This study sought to evaluate the diagnostic and prognostic value of circulating miR-133a as a marker of acute myocardial infarction in acute chest pain patients undergoing coronary angiography.Plasma was collected from 312 patients with chest pain on admission in the emergency department and 67 healthy controls. MiR-133a was detected using real-time quantitative PCR and enhanced accu-TnI, creatinine kinase-MB mass, and myoglobin were measured by immunoassay. End-point events (serious adverse cardiovascular events which require hospitalization or cardiovascular death) were examined in the AMI (acute myocardical infarction) group within 1, 6, 12, and 24 months.The miR-133a level was higher in AMI patients than in non-AMI patients (P < 0.001). In the ROC analysis, the sensitivity of miR-133a in diagnosis of AMI is 0.61 and the specificity is 0.68. In the prognostic analysis, only 1 endpoint event was observed in the non-AMI group; the amount of cases with end-point events in the AMI group at 1,6,12, and 24 months were 8, 19, 28, and 35, respectively. The cutoff value of miR-133a was determined using the median value of the AMI group and separated the patients into a positive group and a negative group. The Kaplan-Meier survival curve showed no significant difference in survival was detected in AMI patients between the miR-133a positive group and negative group after follow-up (12-month: x2 = 1.353, P = 0.245; 24-month: x2 = 3.722, P = 0.054). After adjusting for age, gender, Killip classes, prior myocardiac infarction history, myoglobin, LVEF (left ventricular ejection fraction), diabetes, hypertension, smoking and systolic blood pressure, miR133a had a significant association with the risk of events at 12 months (HR = 2.869, P = 0.024) and 24 months (HR = 3.936, P = 0.001).In patients undergoing coronary angiography, circulating miR-133a is upregulated in AMI patients, but it does not provide enough accuracy for clinical AMI diagnosis because it also rises in unstable angina patients. Its prognostic value in AMI is uncertain mainly for the number of cases with end-point event was small and may be further validated in a larger, better designed study.

A patient with Factor V Leiden and a related pulmonary embolism.

Venous thromboembolism (VTE) is a complication associated with the hypercoaguable state produced in patients with Factor V Leiden deficiency. Clinically, patients can present with pleuritic chest pain, dyspnea, tachycardia, and unilateral leg swelling, warmth, erythema, and pain. Treatment with heparin is urgent, and patients should continue an anticoagulation drug regimen at home.

Targeted next-generation sequencing (NGS) of nine candidate genes with custom AmpliSeq in patients and a cardiomyopathy risk group.

BACKGROUND: Hypertrophic cardiomyopathy is a common genetic cardiac disease. Prevention and early diagnosis of this disease are very important. Because of the large number of causative genes and the high rate of mutations involved in the pathogenesis of this disease, traditional methods of early diagnosis are ineffective. METHODS: We developed a custom AmpliSeq panel for NGS sequencing of the coding sequences of ACTC1, MYBPC3, MYH7, MYL2, MYL3, TNNI3, TNNT2, TPM1, and CASQ2. A genetic analysis of student cohorts (with and without cardiomyopathy risk in their medical histories) and patients with cardiomyopathies was performed. For the statistical and bioinformatics analysis, Polyphen2, SIFT, SnpSift and PLINK software were used. To select genetic markers in the patients with cardiomyopathy and in the students of the high risk group, four additive models were applied. RESULTS: Our AmpliSeq custom panel allowed us to efficiently explore targeted sequences. Based on the score analysis, we detected three substitutions in the MYBPC3 and CASQ2 genes and six combinations between loci in the MYBPC3, MYH7 and CASQ2 genes that were responsible for cardiomyopathy risk in our cohorts. We also detected substitutions in the TNNT2 gene that can be considered as protective against cardiomyopathy. CONCLUSION: We used NGS with AmpliSeq libraries and Ion PGM sequencing to develop improved predictive information for patients at risk of cardiomyopathy.

Enhanced assessment of chest pain and related symptoms in the primary care setting through the use of a novel personalized medicine genomic test: results from a prospective registry study.

Novel approaches for assessing patients with chest pain and related symptoms may improve outpatient care. The REGISTRY I study measured the impact of a personalized gene expression score (GES) on subsequent cardiac referral decisions by primary care providers. Of the 342 stable, nonacute patients evaluated, the mean age was 55 years, 53% were female, and mean (SD) GES was 16 (±10) (range = 1-40). Low GES (≤15), indicating a low current likelihood of obstructive coronary artery disease (CAD), was observed in 49% of patients. After clinical covariate adjustment, each 10-point GES decrease was associated with a 14-fold decreased odds of cardiac referral (P < .0001). Low GES patients had 94% reduced odds of referral relative to elevated GES patients (P < .0001), with follow-up supporting a favorable safety profile. This genomic-based test demonstrated clinical utility by guiding decision making during assessment of symptomatic patients with suspected obstructive CAD.

Distal myosin heavy chain-7 myopathy due to the novel transition c.5566G>A (p.E1856K) with high interfamilial cardiac variability and putative anticipation.

Myosin-heavy-chain 7 (MYH7)-myopathy manifests clinically with a distal, scapuloperoneal, limb-girdle (proximal), or axial distribution and may involve the respiratory muscles. Cardiac involvement is frequent, ranging from relaxation impairment to severe dilative cardiomyopathy. Progression and earlier onset of cardiac disease in successive generations with MYH7-myopathy is unreported. In a five-generation family MYH7-myopathy due to the novel c.5566G > A (p.E1856K) mutation manifested with late-onset, distal > proximal myopathy and variable degree of cardiac involvement. The index patient developed distal myopathy since age 49 y and anginal chest pain. Her mother had distal myopathy and impaired myocardial relaxation. The daughter of the index patient had discrete myopathy but left ventricular hypertrabeculation/noncompaction and ventricular arrhythmias requiring an implantable cardioverter defibrillator. The granddaughter of the index patient had infantile dilated cardiomyopathy without overt myopathy. Cardiac involvement may be present in MYH7-myopathy and may be progressive between the generations, ranging from relaxation abnormality to noncompaction, ventricular arrhythmias, and dilated cardiomyopathy.

The HDL anti-inflammatory function is impaired in myocardial infarction and may predict new cardiac events independent of HDL cholesterol.

BACKGROUND: Intrinsic functional properties of high density lipoproteins (HDL) are considered to be physiologically important for atheroprotection. We compared the HDL anti-inflammatory capacity between patients with acute myocardial infarction (MI) and patients with non-cardiac chest pain, and prospectively determined the association of new major adverse cardiovascular events (MACE) with this metric of HDL function. METHODS: A prospective study was carried out in 93 patients referred for acute chest pain (65 patients with acute MI). The HDL anti-inflammatory capacity was determined as the ability to suppress tumor necrosis factor-α-induced vascular cell adhesion molecule-1 mRNA expression in endothelial cells in vitro. RESULTS: Acute MI at admission was associated with impaired HDL anti-inflammatory capacity (p=0.001), even after adjustment for HDL cholesterol and apolipoprotein A-I (p=0.003). Twenty nine MACE were ascertained during a median follow-up of 1210 (910-1679) days. New MACE was associated with impaired HDL anti-inflammatory capacity (hazard ratio: 1.80 (1.17-2.77) per SD change, p=0.007) in age, sex, HDL cholesterol and apolipoprotein-AI adjusted analysis. CONCLUSIONS: The ability of HDL to attenuate endothelial inflammation is impaired in acute MI, and this metric of HDL function may serve as a predictor of new MACE, even independent of HDL cholesterol and apolipoprotein A-I.

Magnetic resonance imaging can detect thoracic inflammation due to familial Mediterranean fever.

A 32-year-old man presented to our hospital complaining of periodic fever and unilateral chest pain. We suspected that he had familial Mediterranean fever because of his symptoms. Magnetic resonance imaging (MRI) showed an increased intensity within the anterior chest wall, which was consistent with the site of his pain. Genomic analysis showed the patient to be heterozygous for the E148Q/M694I mutation in the MEFV gene, and we diagnosed familial Mediterranean fever. The ability of MRI to detect inflammatory changes could provide useful additional information for evaluating thoracic symptoms in FMF patients, and the detection of inflammatory changes using MRI may aid in early diagnosis, thus contributing to early and adequate treatment.

Unexpectedly low mutation rates in beta-myosin heavy chain and cardiac myosin binding protein genes in Italian patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Fourteen sarcomeric and sarcomere-related genes have been implicated in HCM etiology, those encoding ß-myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) reported as the most frequently mutated: in fact, these account for around 50% of all cases related to sarcomeric gene mutations, which are collectively responsible for approximately 70% of all HCM cases. Here, we used denaturing high-performance liquid chromatography followed by bidirectional sequencing to screen the coding regions of MYH7 and MYBPC3 in a cohort (n = 125) of Italian patients presenting with HCM. We found 6 MHY7 mutations in 9/125 patients and 18 MYBPC3 mutations in 19/125 patients. Of the three novel MYH7 mutations found, two were missense, and one was a silent mutation; of the eight novel MYBPC3 mutations, one was a substitution, three were stop codons, and four were missense mutations. Thus, our cohort of Italian HCM patients did not harbor the high frequency of mutations usually found in MYH7 and MYBPC3. This finding, coupled to the clinical diversity of our cohort, emphasizes the complexity of HCM and the need for more inclusive investigative approaches in order to fully understand the pathogenesis of this disease.

PPARgamma gene C161T substitution is associated with reduced risk of coronary artery disease and decreased proinflammatory cytokine expression.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARgamma) is a transcription factor implicated in the expression of proinflammatory cytokines in atheroma-associated cells, and the expression of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha) and matrix metalloproteinases (MMPs), represents a critical step in atherogenesis and atherosclerosis. In this study, we test the hypothesis of whether a polymorphism corresponding to C161T substitution in exon 6 of the PPARgamma gene may affect the expression of proinflammatory cytokines and the onset of coronary artery diseases (CADs) in a Chinese population. METHODS: We have studied distribution of the PPARgamma C161T substitution at exon 6 in 247 patients with CAD and 214 patients with chest pain syndrome. The plasma concentrations of MMP-9 and TNF-alpha were measured by enzyme-linked immunosorbent assay. RESULTS: The results showed that the frequencies of the CC, CT, and TT genotypes were 61.9%, 34.0%, and 4.1% in CAD, and 51.4%, 45.3%, and 3.3% in chest pain syndrome, respectively. There was a significant association between the PPARgamma C161T polymorphism and CAD. The T allele carriers (CT + TT) had significantly reduced CAD risk compared with the CC homozygotes (odds ratio 0.547, 95% CI 0.327-0.831, P = .012) in a logistic regression model after controlling known independent factors for CAD. The CC homozygotes also had increased MMP-9 and TNF-alpha levels compared with T allele carriers. Moreover, the CC homozygotes were more susceptible to acute coronary syndrome than T allele carriers. CONCLUSIONS: PPARgamma C161T polymorphism was associated with angiographically documented CAD in a Chinese population. The T allele of the PPARgamma gene might have a protective effect on the progression of CAD and reduce the onset of acute coronary syndrome, which might be associated with the decreased expression of MMP-9 and TNF-alpha in patients with CAD.

ET-1 and ecNOS gene polymorphisms andsusceptibility to acute chest syndrome and painful vaso-occlusive crises in children with sickle cell anemia.

The association of endothelin 1 (ET-1) and endothelial constitutive nitric oxide synthase (ecNOS) gene polymorphisms (G5665T and T8002C, VNTR and T-786C respectively) with the occurrence of acute chest syndrome and painful vaso-occlusive crises was evaluated in homozygous SS children. This retrospective study reveals that ET-1 T8002 and ecNOS C-786 alleles are associated with, respectively, an increased and a decreased risk of acute chest syndrome.

The T(-786)C endothelial nitric oxide synthase genotype predicts cardiovascular mortality in high-risk patients.

OBJECTIVES: This study sought to investigate the impact of a common T(-786)C single-nucleotide polymorphism (SNP) in the promoter of the endothelial nitric oxide synthase (eNOS, NOS3) gene on cardiovascular (CV) death in a prospective cohort study. BACKGROUND: The T(-786)C SNP eNOS gene implies a blunted endothelium-dependent vasodilation in hypertensive patients and was associated with multivessel coronary artery disease in cross-sectional studies, but it remained unsettled whether it carried prognostic information. METHODS: In consecutive white patients of the GENICA (Genetic and Environmental Factors in Coronary Atherosclerosis) study, who underwent coronary angiography between 1999 and 2001, we determined the incidence of CV death at follow-up. The eNOS T(-786)C and the exon 7 G(894)T SNPs were determined by melting curve analysis of amplicons from allele-specific fluorescence resonance energy transfer probes. Plasma levels of nitrate/nitrite, nitrotyrosine, and myeloperoxidase were also measured. The Kaplan-Meier and Cox regression analyses were used to assess the impact of SNPs on event-free survival. RESULTS: Complete follow-up data were obtained in 1,086 (98%) patients. After a median follow-up of 1,296 days (range 4 to 2,057 days), we observed 85 (8.2%) CV deaths. There was a significant impact of the T(-786)C eNOS genotype on CV death-free (p = 0.0102) survival, but no differences in CV death rates across G(894)T genotypes. The TT individuals, who showed a lower survival, exhibited higher plasma myeloperoxidase (p < 0.0001) and lower levels of nitrotyrosine (p < 0.0001) than CC patients. CONCLUSIONS: The T(-786)C SNP in the promoter of eNOS bears independent prognostic information and is associated with changes in markers of oxidant stress in high-risk white patients referred for coronary angiography.