Genetic Markers Enhance Coronary Risk Prediction in Men: The MORGAM Prospective Cohorts

Background

More accurate coronary heart disease (CHD) prediction, specifically in middle-aged men, is needed to reduce the burden of disease more effectively. We hypothesised that a multilocus genetic risk score could refine CHD prediction beyond classic risk scores and obtain more precise risk estimates using a prospective cohort design.

Methods

Using data from nine prospective European cohorts, including 26,221 men, we selected in a case-cohort setting 4,818 healthy men at baseline, and used Cox proportional hazards models to examine associations between CHD and risk scores based on genetic variants representing 13 genomic regions. Over follow-up (range: 5–18 years), 1,736 incident CHD events occurred. Genetic risk scores were validated in men with at least 10 years of follow-up (632 cases, 1361 non-cases). Genetic risk score 1 (GRS1) combined 11 SNPs and two haplotypes, with effect estimates from previous genome-wide association studies. GRS2 combined 11 SNPs plus 4 SNPs from the haplotypes with coefficients estimated from these prospective cohorts using 10-fold cross-validation. Scores were added to a model adjusted for classic risk factors comprising the Framingham risk score and 10-year risks were derived.

Results

Both scores improved net reclassification (NRI) over the Framingham score (7.5%, p = 0.017 for GRS1, 6.5%, p = 0.044 for GRS2) but GRS2 also improved discrimination (c-index improvement 1.11%, p = 0.048). Subgroup analysis on men aged 50–59 (436 cases, 603 non-cases) improved net reclassification for GRS1 (13.8%) and GRS2 (12.5%). Net reclassification improvement remained significant for both scores when family history of CHD was added to the baseline model for this male subgroup improving prediction of early onset CHD events.

Conclusions

Genetic risk scores add precision to risk estimates for CHD and improve prediction beyond classic risk factors, particularly for middle aged men.     

Intracranial aneurysm risk locus 5q23.2 is associated with elevated systolic blood pressure

Although genome-wide association studies (GWAS) have identified hundreds of complex trait loci, the pathomechanisms of most remain elusive. Studying the genetics of risk factors predisposing to disease is an attractive approach to identify targets for functional studies. Intracranial aneurysms (IA) are rupture-prone pouches at cerebral artery branching sites. IA is a complex disease for which GWAS have identified five loci with strong association and a further 14 loci with suggestive association. To decipher potential underlying disease mechanisms, we tested whether there are IA loci that convey their effect through elevating blood pressure (BP), a strong risk factor of IA. We performed a meta-analysis of four population-based Finnish cohorts (n(FIN) = 11 266) not selected for IA, to assess the association of previously identified IA candidate loci (n = 19) with BP. We defined systolic BP (SBP), diastolic BP, mean arterial pressure, and pulse pressure as quantitative outcome variables. The most significant result was further tested for association in the ICBP-GWAS cohort of 200 000 individuals. We found that the suggestive IA locus at 5q23.2 in PRDM6 was significantly associated with SBP in individuals of European descent (p(FIN) = 3.01E-05, p(ICBP-GWAS) = 0.0007, p(ALL) = 8.13E-07). The risk allele of IA was associated with higher SBP. PRDM6 encodes a protein predominantly expressed in vascular smooth muscle cells. Our study connects a complex disease (IA) locus with a common risk factor for the disease (SBP). We hypothesize that common variants in PRDM6 can contribute to altered vascular wall structure, hence increasing SBP and predisposing to IA. True positive associations often fail to reach genome-wide significance in GWAS. Our findings show that analysis of traditional risk factors as intermediate phenotypes is an effective tool for deciphering hidden heritability. Further, we demonstrate that common disease loci identified in a population isolate may bear wider significance.     

Biomechanical testing in experimental bone interventions--May the power be with you

Total variation in any measured variable, in conjunction with expected treatment effect, defines the minimum sample size (minSS) required to detect the expected effect with statistical confidence should the effect truly exist. A comprehensive literature survey of 3472 original studies was carried out to identify studies with biomechanical testing of whole bones. Total variation in common biomechanical traits and expected treatment effects in typical interventions were statistically determined. According to this survey, total variation in biomechanical traits between different species of experimental animals was similar, justifying the use of rat femur as a model in further analyses. Due to poorer precision, stiffness and energy absorption assessment require substantially larger sample size than breaking load. Due to same reason, minSS for femoral neck compression test is considerably larger than for femoral shaft three-point bending test. For the bending test, minSS to show a 10% treatment effect in the breaking load with 80% statistical power is 11rats/group, while corresponding minSS is 23 for the stiffness, and 53 for the energy absorption. For the femoral neck compression test, minSSs are 16, 51, and 134rats/group, respectively. Among the reviewed studies, the mean sample size was 11animals/group. This underscores the need for considerably larger sample sizes in experimental bone interventions which employ mechanical traits as primary outcome variables. In particular, poor precision and generally small expected treatment effects compromise the utility of stiffness and energy absorption assessments in experimental bone interventions.     

Ring-cleaving cyanuric acid amidohydrolase activity in the atrazine-mineralizing Ralstonia basilensis M91-3

The purpose of this study was to characterize the cyanuric acid amidohydrolase reaction in Ralstonia basilensis M91-3, an atrazine-mineralizing soil bacterium. This ring fission reaction is the last aromatic step in the degradative pathway of atrazine and other s-triazines. The products and molar stoichiometry of the cyanuric acid amidohydrolase reaction were one mol biuret (H₂N·CO·NH·CO·NH₂) and one mol CO₂ per mol cyanuric acid hydrolyzed, as confirmed by ¹³C-NMR and gas chromatography. The optimum pH and temperature, substrate specificity, and kinetic parameters were also characterized for the purified enzyme. The native enzyme had two forms of different sizes, 204 kDa and 160 kDa. Each was a tetramer or pentamer of 44 kDa and 33 kDa, respectively.     

Lipid-free NisI: interaction with nisin and contribution to nisin immunity via secretion

Nisin-producing Lactococcus lactis cells protect their own cytoplasmic membrane by specific immunity proteins, NisF/E/G and NisI, a transporter complex and a lipoprotein, respectively. A portion of NisI is secreted to the medium in a lipid-free form (LF-NisI). Here, kinetics of the interaction between nisin and LF-NisI was examined by surface plasmon resonance analysis. The affinity constant K_D for the interaction was calculated to be in the micromolar range. Contribution of the secreted LF-NisI to nisin immunity was studied by replacing the lipoprotein specific nisI signal sequence with a secretion signal of non-lipoprotein origin. Secretion of LF-NisI in NisF/E/G-expressing L. lactis strain NZ9840 increased significantly its nisin tolerance suggesting that the lipid-free form of NisI could have a supportive role in nisin immunity.     

The Genome-wide Patterns of Variation Expose Significant Substructure in a Founder Population

Although high-density SNP genotyping platforms generate a momentum for detailed genome-wide association (GWA) studies, an offshoot is a new insight into population genetics. Here, we present an example in one of the best-known founder populations by scrutinizing ten distinct Finnish early- and late-settlement subpopulations. By determining genetic distances, homozygosity, and patterns of linkage disequilibrium, we demonstrate that population substructure, and even individual ancestry, is detectable at a very high resolution and supports the concept of multiple historical bottlenecks resulting from consecutive founder effects. Given that genetic studies are currently aiming at identifying smaller and smaller genetic effects, recognizing and controlling for population substructure even at this fine level becomes imperative to avoid confounding and spurious associations. This study provides an example of the power of GWA data sets to demonstrate stratification caused by population history even within a seemingly homogeneous population, like the Finns. Further, the results provide interesting lessons concerning the impact of population history on the genome landscape of humans, as well as approaches to identify rare variants enriched in these subpopulations.     

Novel method for cell debris removal in the flow cytometric cell cycle analysis using carboxy-fluorescein diacetate succinimidyl ester.

BACKGROUND: Cell cycle analysis with flow cytometry using propidium iodide (PI) can be difficult in some cases because of the cell debris. Here, we introduce debris removal using intranuclear protein staining (DRIPS), a novel method for separating intact nuclei and cell debris to different populations using carboxy-fluorescein diacetate succinimidyl ester (CFSE). METHODS: To study the apoptosis-sensitivity, chicken DT40 B cell lymphoma cell line was gamma irradiated. After the irradiation, the cells were incubated up to 8 h and the stages of the cell cycle were followed with flow cytometry. RESULTS: CFSE staining, done simultaneously with PI, stained the cell debris brighter than intact nuclei and could be excluded from the histogram with a simple gating procedure. The method is reliable and reproducible and can be executed within 15 min. CONCLUSIONS: DRIPS-method greatly enhances the analysis of difficult cell cycle samples.     

Novel Nano-Sized MR Contrast Agent Mediates Strong Tumor Contrast Enhancement in an Oncogene-Driven Breast Cancer Model

The current study was carried out to test the potential of a new nanomaterial (Spago Pix) as a macromolecular magnetic MR contrast agent for tumor detection and to verify the presence of nanomaterial in tumor tissue. Spago Pix, synthesized by Spago Nanomedical AB, is a nanomaterial with a globular shape, an average hydrodynamic diameter of 5 nm, and a relaxivity (r₁) of approximately 30 (mM Mn)⁻¹ s⁻¹ (60 MHz). The material consists of an organophosphosilane hydrogel with strongly chelated manganese (II) ions and a covalently attached PEG surface layer. In vivo MRI of the MMTV-PyMT breast cancer model was performed on a 3 T clinical scanner. Tissues were thereafter analyzed for manganese and silicon content using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The presence of nanomaterial in tumor and muscle tissue was assessed using an anti-PEG monoclonal antibody. MR imaging of tumor-bearing mice (n = 7) showed a contrast enhancement factor of 1.8 (tumor versus muscle) at 30 minutes post-administration. Contrast was retained and further increased 2–4 hours after administration. ICP-AES and immunohistochemistry confirmed selective accumulation of nanomaterial in tumor tissue. A blood pharmacokinetics analysis showed that the concentration of Spago Pix gradually decreased over the first hour, which was in good agreement with the time frame in which the accumulation in tumor occurred. In summary, we demonstrate that Spago Pix selectively enhances MR tumor contrast in a clinically relevant animal model. Based on the generally higher vascular leakiness in malignant compared to benign tissue lesions, Spago Pix has the potential to significantly improve cancer diagnosis and characterization by MRI.