Ola Hammarsten

Association of Nrf2-encoding NFE2L2 haplotypes with Parkinson's disease

BackgroundOxidative stress is heavily implicated in the pathogenic process of Parkinsons disease. Varying capacity to detoxify radical oxygen species through induction of phase II antioxidant enzymes in substantia nigra may influence disease risk. Here, we hypothesize that variation in NFE2L2 and KEAP1, the genes encoding the two major regulators of the phase II response, may affect the risk of Parkinsons disease.MethodsThe study included a Swedish discovery case-control material (165 cases and 190 controls) and a Polish replication case-control material (192 cases and 192 controls). Eight tag single nucleotide polymorphisms representing the variation in NFE2L2 and three representing the variation in KEAP1 were chosen using HapMap data and were genotyped using TaqMan Allelic Discrimination.ResultsWe identified a protective NFE2L2 haplotype in both of our European case-control materials. Each haplotype allele was associated with five years later age at onset of the disease (p = 0.001) in the Swedish material, and decreased risk of PD (p = 2 × 10-6), with an odds ratio of 0.4 (95% CI 0.3-0.6) for heterozygous and 0.2 (95% CI 0.1-0.4) for homozygous carriers, in the Polish material. The identified haplotype includes a functional promoter haplotype previously associated with high transcriptional activity. Genetic variation in KEAP1 did not show any associations.ConclusionThese data suggest that variation in NFE2L2 modifies the Parkinsons disease process and provide another link between oxidative stress and neurodegeneration.

Numerical analysis of etoposide induced DNA breaks.

Background Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs. Methodology/Principal Findings To examine etoposide-induced DNA damage in more detail we compared the relative amount of SSBs and DSBs, survival and H2AX phosphorylation in cells treated with etoposide or calicheamicin, a drug that produces free DSBs and SSBs. With this combination of methods we found that only 3% of the DNA strand breaks induced by etoposide were DSBs. By comparing the level of DSBs, H2AX phosphorylation and toxicity induced by etoposide and calicheamicin, we found that only 10% of etoposide-induced DSBs resulted in histone H2AX phosphorylation and toxicity. There was a close match between toxicity and histone H2AX phosphorylation for calicheamicin and etoposide suggesting that the few etoposide-induced DSBs that activated H2AX phosphorylation were responsible for toxicity. Conclusions/Significance These results show that only 0.3% of all strand breaks produced by etoposide activate H2AX phosphorylation and suggests that over 99% of the etoposide induced DNA damage does not contribute to its toxicity.

Inflammation increases NT-proBNP and the NT-proBNP/BNP ratio

Plasma BNP and NT-proBNP are often regarded as interchangeable parameters in assessing heart failure (HF) severity and prognosis. Renal failure results in disproportionate increases of NT-proBNP and an increased NT-proBNP/BNP ratio. Low kidney function is therefore considered particularly when NT-proBNP is used to assess HF. The purpose of this study was to identify other conditions affecting the NT-proBNP/BNP ratio. We examined the NT-proBNP/BNP ratio, 26 other lab parameters, and clinical factors in 218 patients admitted to the HF ward. In addition to renal function, we also found significant correlations between the NT-proBNP/BNP ratio and inflammation as measured by orosomucoid (rxa0=xa00.525, pxa0<xa00.0001), CRP (rxa0=xa00.333, pxa0<xa00.0001), haptoglobulin (rxa0=xa00.201, pxa0=xa00.02), and alpha1-antitrypsin (rxa0=xa00.223, pxa0=xa00.01). Reverse correlation was found with transferrin (rxa0=xa0−0.323, pxa0<xa00.0001), albumin (rxa0=xa0−0.251, pxa0=xa00.003), and S–Fe (rxa0=xa0−0.205, pxa0=xa00.02), parameters known to decrease during inflammation. Inflammation increased levels of NT-proBNP more than BNP, resulting in an increased NT-proBNP/BNP ratio. Our findings indicate that NT-proBNP should be evaluated concomitantly with inflammatory status to avoid overestimation of HF severity.

Nrf2-encoding NFE2L2 haplotypes influence disease progression but not risk in Alzheimer's disease and age-related cataract.

Alzheimers disease (AD) and age-related cataract, disorders characterized by protein aggregation causing late-onset disease, both involve oxidative stress. We hypothesize that common variants of NFE2L2 and KEAP1, the genes encoding the main regulators of the Nrf2 system, an important defence system against oxidative stress, may influence risk of AD and/or age-related cataract. This case-control study combines an AD material (725 cases and 845 controls), and a cataract material (489 cases and 182 controls). Genetic variation in NFE2L2 and KEAP1 was tagged by eight and three tag single nucleotide polymorphisms (SNPs), respectively. Single SNPs and haplotypes were analyzed for associations with disease risk, age parameters, MMSE and AD cerebrospinal fluid biomarkers. NFE2L2 and KEAP1 were not associated with risk of AD or cataract. However, one haplotype allele of NFE2L2 was associated with 2 years earlier age at AD onset (p(c)=0.013) and 4 years earlier age at surgery for posterior subcapsular cataract (p(c)=0.019). Another haplotype of NFE2L2 was associated with 4 years later age at surgery for cortical cataract (p(c)=0.009). Our findings do not support NFE2L2 or KEAP1 as susceptibility genes for AD or cataract. However, common variants of the NFE2L2 gene may affect disease progression, potentially altering clinically recognized disease onset.

Seasonal variations in serum 25-hydroxy vitamin D levels in a Swedish cohort

To study seasonal inter-individual and intra-individual variations in serum 25-hydroxy vitamin D (25(OH)D) and to explore parameters associated with 25(OH)D in a healthy Swedish adult population. 540 blood donors (60xa0% men; mean age 41xa0±xa013xa0years) and 75 thrombocyte donors (92xa0% men, aged 46xa0±xa011xa0years) were included. Serum was collected during 12xa0months and analyzed for 25(OH)D and parathyroid hormone (S-iPTH). The blood donors answered questionnaires concerning vitamin D supplements, smoking, physical activity, sunbed use and sun holidays. Repeated serum samples were collected from the thrombocyte donors to study the intra-individual variations in S-25(OH)D. S-25(OH)D varied greatly over the year correlating with the intensity of the UV-B irradiation (rSxa0=xa00.326; pxa0<xa00.001). During January–March, a S-25(OH)D level below the thresholds of 50 and 75xa0nmol/L was observed in 58 and 88xa0%, respectively, and during July–September in 11 and 50xa0% (pxa0<xa00.001). S-25(OH)D was negatively correlated with body mass index and S-iPTH, but was significantly higher in holiday makers in sunny destinations, sunbed users, non-smokers, and in the physically active. The intra-individual analyses showed a mean increase in S-25(OH)D by 8xa0nmol/L/month between April and August. Approximately 75xa0% had serum 25(OH)D values <75xa0nmol/L during 75xa0% of the year and 50xa0% had serum 25(OH)D <50xa0nmol/L during 50xa0% of the year. Serum 25(OH)D was strongly associated with parameters related to sun exposure, but only weakly with intake of vitamin D supplements.

High-sensitive cardiac troponin, NT-proBNP, hFABP and copeptin levels in relation to glomerular filtration rates and a medical record of cardiovascular disease

BACKGROUNDnElevation of cardiac markers in patients with renal dysfunction has not been fully assessed reducing the diagnostic usefulness of these biomarkers.nnnOBJECTIVEnTo examine the effects of renal function and a medical record of cardiovascular disease on levels of cardiac biomarkers.nnnMETHODSnSerum samples were collected from 489 patients referred for GFR measurement using Cr51-EDTA or iohexol plasma clearance (measured GFR). The cardiac biomarkers Troponin T (hs-cTnT), Troponin I (hsTnI), N-Terminal pro-Brain Natriuretic Peptide (NTproBNP), Copeptin, Human Fatty Acid-Binding Protein (hFABP), as well as the kidney function biomarkers creatinine and cystatin C, were measured. Regression was used to analyse the relationship between biomarker levels and the glomerular filtration rate (GFR) between 15 and 90mL/min/1.73m(2).nnnRESULTSnCompared with normal kidney function, the estimated increases in the studied cardiac biomarkers at a GFR of 15mL/min/1.73m(2) varied from 2-fold to 15-fold but were not very different between patients with or without a medical record of cardiovascular disease and were most prominent for cardiac biomarkers with low molecular weight. hs-cTnT levels correlated more strongly to measured GFR and increased more at low GFR compared to hs-cTnI. For hFABP and NTproBNP increases at low kidney function were more correctly predicted by a local Cystatin C-based eGFR formula compared with creatinine-based eGFR (using the MDRD or CKD-EPI equations).nnnCONCLUSIONnThe extent of the elevation of cardiac markers at low renal function is highly variable. For hFABP and NTproBNP Cystatin C-based eGFR provides better predictions of the extent of elevation compared to the MDRD or CKD-EPI equations.

Genetic associations of Nrf2-encoding NFE2L2 variants with Parkinson’s disease – a multicenter study

BackgroundThe transcription factor Nrf2, encoded by the NFE2L2 gene, is an important regulator of the cellular protection against oxidative stress. Parkinson’s disease is a neurodegenerative disease highly associated with oxidative stress. In a previously published study, we reported associations of NFE2L2 haplotypes with risk and age at onset of idiopathic Parkinson’s disease in a Swedish discovery material and a Polish replication material. Here, we have extended the replication study and performed meta-analyses including the Polish material and four new independent European patient-control materials. Furthermore, all SNPs included in the haplotype windows were investigated individually for associations with Parkinson’s disease in meta-analyses including all six materials.MethodsTotally 1038 patients and 1600 control subjects were studied. Based on previous NFE2L2 haplotype associations with Parkinson’s disease, five NFE2L2 tag SNPs were genotyped by allelic discrimination and three functional NFE2L2 promoter SNPs were genotyped by sequencing. The impact of individual SNPs and haplotypes on risk and age at onset of Parkinson’s disease were investigated in each material individually and in meta-analyses of the obtained results.ResultsMeta-analyses of NFE2L2 haplotypes showed association of haplotype GAGCAAAA, including the fully functional promoter haplotype AGC, with decreased risk (ORu2009=u20090.8 per allele, pu2009=u20090.012) and delayed onset (+1.1xa0years per allele, pu2009=u20090.048) of Parkinson’s disease. These results support the previously observed protective effect of this haplotype in the first study. Further, meta-analyses of the SNPs included in the haplotypes revealed four NFE2L2 SNPs associated with age at onset of Parkinson’s disease (rs7557529 Gu2009>u2009A, −1.0xa0years per allele, pu2009=u20090.042; rs35652124 Au2009>u2009G, −1.1xa0years per allele, pu2009=u20090.045; rs2886161 Au2009>u2009G, −1.2xa0years per allele, pu2009=u20090.021; rs1806649 Gu2009>u2009A, +1.2xa0years per allele, pu2009=u20090.029). One of these (rs35652124) is a functional SNP located in the NFE2L2 promoter. No individual SNP was associated with risk of Parkinson’s disease.ConclusionOur results support the hypothesis that variation in the NFE2L2 gene, encoding a central protein in the cellular protection against oxidative stress, may contribute to the pathogenesis of Parkinson’s disease. Functional studies are now needed to explore these results further.

Stable High-Sensitivity Cardiac Troponin T Levels and Outcomes in Patients With Chest Pain

BACKGROUNDnThere is a paucity of data on the association between high-sensitivity cardiac troponin (hs-cTn) levels and outcomes in patients with chest pain but no myocardial infarction (MI), or any other condition that may lead to acutely elevated troponin levels.nnnOBJECTIVESnThe authors hypothesized that any detectable high-sensitivity cardiac troponin T (hs-cTnT) level is associated with adverse outcomes.nnnMETHODSnAll patients (Nxa0=xa022,589) >25 years of age with chest pain and hs-cTnT analyzed concurrently in thexa0emergency department of Karolinska University Hospital, Stockholm, Sweden from 2011 to 2014 were eligible for inclusion. After excluding all patients with acute conditions that may have affected hs-cTnT, or MI associated with thexa0visit, or insufficient information to determine whether troponin levels were stable, Cox regression was used to estimate risks for all-cause, cardiovascular, and noncardiovascular mortality, MI, and heart failure at different levels of troponins.nnnRESULTSnA total of 19,460 patients with a mean age of 54 ± 17 years were included. During a mean follow-up of 3.3xa0±xa01.2 years, 1,349 (6.9%) patients died. Adjusted hazard ratios (with 95% confidence intervals) for all-cause mortality were 2.00 (1.66 to 2.42), 2.92 (2.38 to 3.59), 4.07 (3.28 to 5.05), 6.77 (5.22 to 8.78), and 9.68 (7.18 to 13.00) in patientsxa0with hs-cTnT levels of 5 to 9, 10 to 14, 15 to 29, 30 to 49, andxa0≥50xa0ng/l, respectively, compared with patients with hs-cTnT levelsxa0<5xa0ng/l. There was a strong and graded association between all detectable levels of hs-cTnT and riskxa0for MI, heart failure, and cardiovascular and noncardiovascular mortality.nnnCONCLUSIONSnAmong patients with chest pain and stable troponin levels, any detectable level of hs-cTnT is associated with an increased risk of death and cardiovascular outcomes and should merit further attention.

Red blood cell distribution width and its relation to cardiac function and biomarkers in a prospective hospital cohort referred for echocardiography.

BACKGROUNDnRed blood cell distribution width (RDW), a measure of anisocytosis, is a prognostic biomarker for heart failure (HF). However it is still unclear how RDW is associated with heart function and established cardiac biomarkers.nnnMETHODS AND RESULTSnIn a prospective hospital cohort of 296 patients referred for echocardiography because of suspected HF, blood sampling and clinical examination were performed within 24h after echocardiography. The patients were divided into four HF groups, including one group where the HF diagnosis was uncertain (gray zone). In the patients the mean age was 70 ± 11 years, 44% with systolic HF (SHF), 18% with heart failure with normal ejection fraction (HFNEF), 17% with gray zone and 21% without HF (non-HF). RDW was higher among patients with SHF and HFNEF, compared with gray zone and non-HF patients. The distribution of different variables over the RDW quartiles showed an inverse correlation between RDW levels and LVEF and a positive correlation between RDW and NT-proBNP levels. Further analysis with stepwise multiple linear regression demonstrated that NT-proBNP levels, but not LVEF, were independently correlated with RDW.nnnCONCLUSIONnIn patients referred for echocardiography because of suspected HF, RDW levels were higher in patients with SHF and HFNEF. Moreover, NT-proBNP levels were independently linked with elevated RDW.

Clearance of cardiac troponin T with and without kidney function

OBJECTIVEnThe extent of kidney-dependent clearance of the cardiac damage biomarker cardiac troponin T (cTnT) is not known.nnnMETHODS AND RESULTSnWe examined clearance of cTnT after injection of heart extracts in rats with or without clamped kidney vessels. The extent of degradation of cTnT to fragments able to pass the glomerular membrane and the kidney extraction index of cTnT was examined in human subjects. After a bolus injection of rat cardiac extract, simulating a large myocardial infarction, there was no significant difference in clearance of cTnT with or without kidney function. However, a slower clearance was observed late in the clearance process, when cTnT levels were low. When low levels of rat cardiac extract were infused at a constant rate to steady state, clamping of the renal vessels resulted in significant 2-fold reduction in clearance of cTnT. Over 60% of the measured cTnT in human subjects had a molecular weight below 17kDa, expected to have a relatively free passage over the glomerular membrane. The extraction index of cTnT in three heart failure patients undergoing renal vein catheterization was 8-19%. Kidney function adjusted cTnT levels increased the area under the ROC curve for diagnosis of myocardial infarction of the cTnT analysis in an emergency room cohort.nnnCONCLUSIONSnAt high concentrations, often found after a large myocardial infarction, extrarenal clearance of cTnT dominates. At low levels of cTnT, often found in patients with stable cTnT elevations, renal clearance also contribute to the clearance of cTnT. This potentially explains why stable cTnT levels tend to be higher in patients with low kidney function.