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1.  Genetic associations of Nrf2-encoding NFE2L2 variants with Parkinson’s disease – a multicenter study 
BMC Medical Genetics  2014;15:131.
The 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.
Totally 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.
Meta-analyses of NFE2L2 haplotypes showed association of haplotype GAGCAAAA, including the fully functional promoter haplotype AGC, with decreased risk (OR = 0.8 per allele, p = 0.012) and delayed onset (+1.1 years per allele, p = 0.048) 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 G > A, −1.0 years per allele, p = 0.042; rs35652124 A > G, −1.1 years per allele, p = 0.045; rs2886161 A > G, −1.2 years per allele, p = 0.021; rs1806649 G > A, +1.2 years per allele, p = 0.029). One of these (rs35652124) is a functional SNP located in the NFE2L2 promoter. No individual SNP was associated with risk of Parkinson’s disease.
Our 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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12881-014-0131-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4335439  PMID: 25496089
Parkinson’s disease; PD; Nrf2; NFE2L2; Meta-analysis; Multicenter; SNP; Haplotype; Risk factor
2.  Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of therapeutic targets 
BMC Biotechnology  2014;14:57.
The dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels.
In order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells.
The unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation.
PMCID: PMC4074332  PMID: 24952598
3.  Assessment of a multimarker strategy for prediction of mortality in older heart failure patients: a cohort study 
BMJ Open  2013;3(3):e002254.
Primarily to develop a multimarker score for prediction of 3-year mortality in older patients with decompensated heart failure (HF).
Prospective cohort study.
Secondary care. Single centre.
Patients and biomarkers
131 patients, aged ≥65 years, with decompensated HF were included. Assessment of biomarkers was performed at discharge.
Primary outcome measure
3-year mortality.
Mean age was 73±11 years; mean left ventricular ejection fraction , 43±14%; 53% were male. The 3-year mortality was 53.4%. The following N-terminal brain natriuretic peptide (NTproBNP) levels could optimally stratify mortality: <2000 ng/l (n=39), 30.8% mortality; 2000–8000 ng/l (n=58), 51.7% mortality; and >8000 ng/l (n=34), 82.4% mortality. However, in the 2000–8000 ng/l range, NTproBNP levels had low-prognostic capacity, based on the area under the receiver operating characteristic curve (AUC=0.53; 95% CI 0.40 to 0.67). In this group, multivariate analysis identified age, cystatin C (CysC), and troponin T (TnT) levels as independent risk factors. A risk score based on these three risk factors separated a high-risk and low-risk groups within the NTproBNP range of 2000–8000 ng/l. The score exhibited a significantly higher AUC (0.75; 95% CI 0.62 to 0.86) than NTproBNP alone (p=0.03) in this NTproBNP group and had similar prognostic capacity as NTproBNP in patients below or above this NTproBNP range (p=0.57). Net reclassification improvement and integrated discriminatory improvement in the group with NTproBNP levels between 2000 and 8000 ng/l was 54% and 23%, respectively, and in the whole cohort 22% and 11%, respectively.
Our results suggested that, to assess risk in HF, older patients required significantly higher levels of NTproBNP than younger patients. Furthermore, a risk score that included TnT and CysC at discharge, and age could improve risk stratification for mortality in older patients with HF in particular when NTproBNP was moderately elevated.
PMCID: PMC3612770  PMID: 23474790
Biomarkers; Mortality; Heart Failure; Troponin T; Cystatin C; Ntprobnp Word Count; 2434 Words
4.  Cystatin C in a composite risk score for mortality in patients with infective endocarditis: a cohort study 
BMJ Open  2012;2(4):e000856.
To develop a multimarker prognostic score for infective endocarditis (IE).
Retrospective case–control.
Secondary care. Single centre.
125 patients with definite IE.
Primary outcome measures
90-day and 5-year mortality.
Mean age was 62.7±17 years. The 90-day and 5-year mortality was 10.4% and 33.6%, respectively. CysC levels at admission and over 20% increases in CysC levels during 2 weeks of treatment were prognostic for 90-day and 5-year mortality independent of creatinine estimated glomerular filtration rate. In multivariate analyses, CysC (OR 5.42, 95% CI 1.90 to 15.5, p=0.002) and age (OR 1.06, 95% CI 1.02 to 1.10, p=0.002) remained prognostic for 5-year mortality. NT-proBNP, TnT, C reactive protein and interleukin 6 were also linked to prognosis. A composite risk scoring system using levels of CysC, NT-proBNP, age and presence of mitral valve insufficiency was able to separate a high-risk and a low-risk group.
CysC levels at admission and increase in CysC after 2 weeks of treatment were independent prognostic markers for both 90-day and 5-year mortality in patients with IE. A multimarker composite risk scoring system including CysC identified a high-risk group.
Article summary
Article focus
Our aim was to develop a multimarker prognostic score for IE.
Key messages
CysC levels at admission and increase in CysC after 2 weeks of treatment were independent prognostic markers for both 90-day and 5-year mortality in patients with IE.
A prognostic score including CysC over 1.2 mg/l, NT-ProBNP over 2000 ng/l, presence of any grade of mitral valve insufficiency (MI) and aged 70 years or older could identify a high-risk and low-risk group in IE.
The prognostic score might be used to improve patient monitoring and assist treatment choices in IE.
Strengths and limitations of this study
We were able to monitor changes in levels of biomarkers during treatment in a large cohort of IE patients since blood samples were collected at admission and after 2 weeks of treatment.
One potential weakness was that 36.2% of patients treated for IE during the study period (71/196) were unavailable for biomarker studies since they lacked stored blood samples. The mortality was lower in the study group (125) compared with all IE patients treated for IE (196) during the study period.
PMCID: PMC3400063  PMID: 22798251
5.  Association of Nrf2-encoding NFE2L2 haplotypes with Parkinson's disease 
BMC Medical Genetics  2010;11:36.
Oxidative stress is heavily implicated in the pathogenic process of Parkinson's 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 Parkinson's disease.
The 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.
We 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.
These data suggest that variation in NFE2L2 modifies the Parkinson's disease process and provide another link between oxidative stress and neurodegeneration.
PMCID: PMC2843602  PMID: 20196834
6.  Correction: Numerical Analysis of Etoposide Induced DNA Breaks 
PLoS ONE  2009;4(6):10.1371/annotation/290cebfd-d5dc-4bd2-99b4-f4cf0be6c838.
PMCID: PMC2704093
7.  Numerical Analysis of Etoposide Induced DNA Breaks 
PLoS ONE  2009;4(6):e5859.
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.
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.
PMCID: PMC2689654  PMID: 19516899
8.  An optimized method for detecting gamma-H2AX in blood cells reveals a significant interindividual variation in the gamma-H2AX response among humans 
Nucleic Acids Research  2007;35(5):e36.
Phosphorylation of histone H2AX on serine 139 (gamma-H2AX, γH2AX) occurs at sites flanking DNA double-strand breaks (DSBs) and can provide a measure of the number of DSBs within a cell. Here we describe a rapid and simple flow-cytometry-based method, optimized to measure gamma-H2AX in non-fixed peripheral blood cells. No DSB induced signal was observed in H2AX−/− cells indicating that our FACS method specifically recognized gamma-H2AX accumulation. The gamma-H2AX assay was capable of detecting DNA damage at levels 100-fold below the detection limit of the alkaline comet assay. The gamma-H2AX signal was quantitative with a linear increase of the gamma-H2AX signal over two orders of magnitude. We found that all nucleated blood cell types examined, including the short-lived neutrophils induce gamma-H2AX in response to DSBs. Interindividual difference in the gamma-H2AX signal in response to ionizing radiation and the DSB-inducing drug calicheamicin was almost 2-fold in blood cells from patients, indicating that the amount of gamma-H2AX produced in response to a given dose of radiation varies significantly in the human population. This simple method could be used to monitor response to radiation or DNA-damaging drugs.
PMCID: PMC1865071  PMID: 17284459

Results 1-8 (8)