Sonja Zehetmayer

Biological Variation of the Platelet Proteome in the Elderly Population and Its Implication for Biomarker Research

Knowledge about the extent of total variation experienced between samples from different individuals is of great importance for the design of not only proteomics but every clinical study. This variation defines the smallest statistically significant detectable signal difference when comparing two groups of individuals. We isolated platelets from 20 healthy human volunteers aged 56–100 years because this age group is most commonly encountered in the clinics. We determined the technical and total variation experienced in a proteome analysis using two-dimensional DIGE with IPGs in the pI ranges 4–7 and 6–9. Only spots that were reproducibly detectable in at least 90% of all gels (n = 908) were included in the study. All spots had a similar technical variation with a median coefficient of variation (cv) of about 7%. In contrast, spots showed a more diverse total variation between individuals with a surprisingly low median cv of only 18%. Because most known biomarkers show an effect size in a 1–2-fold range of their cv, any future clinical proteomics study with platelets will require an analytical method that is able to detect such small quantitative differences. In addition, we calculated the minimal number of samples (sample size) needed to detect given protein expression differences with statistical significance.

Gene Expression as Peripheral Biomarkers for Sporadic Alzheimer's Disease

Alzheimers disease (AD), the most common cause of dementia, is a progressive neurodegenerative disease. At present, diagnosis of AD is rather late in the disease. Therefore, we attempted to find peripheral biomarkers for the early diagnosis of AD. We investigated the profiles of 33 genes, previously found by our group to have altered expression in postmortem brains of AD. The gene profiles were studied via quantitative-real-time-reverse-transcription-polymerase-chain-reaction, in whole blood samples (collected with the PAXgene blood RNA system) isolated from a population clinically diagnosed with AD and healthy controls (1-year period/ up to 4 samples). Five genes showed significant correlation to the dementia score, Mini-Mental State Examination (MMSE). Focusing on the two genes with the smallest p-value, H3-histone and cannabinoid-receptor-2, notable increases in these genes were found in peripheral blood mRNA in subjects with lower MMSE scores. Seasonal variations in gene expression were not significant due to sample size, but did seem to vary due to time of sample withdrawal. In conclusion, gene expression profiling might be a promising method to investigating a large population with the aim of developing an early diagnosis of AD.

Genetic risk factors and markers for Alzheimer's disease and/or depression in the VITA study.

OBJECTIVES In ageing population, both Alzheimers disease (AD) and depression are common. Significant depressive symptoms are often co-morbid with cognitive impairment and dementia. In this study, we attempted to find various factors and markers for both AD and depression in a longitudinal cohort, the Vienna-Transdanube-Aging (VITA)-study. METHODS The VITA-Study consisted of 305 healthy subjects, 174 subjects with depression only, 55 subjects diagnosed with AD only and 72 subjects with depression as well as AD. Associations between AD and/or depression to gene polymorphisms APO E (epsilon4), choline acetyltransferase (ChAT) 4G to A, serotonin-transporter gene promoter-length, dopamine-D4-receptor, ciliary-neurotrophic-factor-null mutation and brain-derived neurotrophic factor (C270T) and to various known factors were analyzed. RESULTS AD and depression were significant associated. Significant risk factors found for AD were low education, low folic acid and depressive-symptoms, while for depression were low education and higher nonsteroidal anti-inflammatory drugs (NSAID) consume. Moreover, the ChAT polymorphism associated significant to depression. Gender, education, and ChAT significantly associated with the combination AD and/or depression. CONCLUSION Such studies must be conducted cautiously, as co-morbidities and gene-environmental-social influences may sway the results dramatically. We found in the VITA-study significant association between depression and AD and between ChAT polymorphism and depression.

Genome-wide CpG island methylation analyses in non-small cell lung cancer patients

DNA methylation is part of the epigenetic gene regulation complex, which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stages I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis. Overall, we identified 2414 genomic positions differentially methylated between tumor and non-malignant lung tissue samples. Ninety-seven percent of them were found to be tumor-specifically methylated. Annotation of these genomic positions resulted in the identification of 477 tumor-specifically methylated genes of which many are involved in regulation of gene transcription and cell adhesion. Tumor-specific methylation was confirmed by a gene-specific approach. In the majority of tumors, methylation of certain genes was associated with loss of their protein expression determined by immunohistochemistry. Treatment of NSCLC cells with epigenetically active drugs resulted in upregulated expression of many tumor-specifically methylated genes analyzed by gene expression microarrays suggesting that about one-third of these genes are transcriptionally regulated by methylation. Moreover, comparison of methylation results with certain clinicopathological characteristics of the patients suggests that methylation of HOXA2 and HOXA10 may be of prognostic relevance in squamous cell carcinoma (SCC) patients. In conclusion, we identified a large number of tumor-specifically methylated genes in NSCLC patients. Expression of many of them is regulated by methylation. Moreover, HOXA2 and HOXA10 methylation may serve as prognostic parameters in SCC patients. Overall, our findings emphasize the impact of methylation on the pathogenesis of NSCLCs.

Data From the VITA Study Do Not Support the Concept of Vascular Depression

OBJECTIVE Cerebrovascular lesions that are apparent in magnetic resonance scans and regioselective atrophy of the brain have been proposed as a causative or exacerbating factor in depression with late-life onset. The objective of this study was to investigate whether deep white matter or periventricular hyperintensities, small ischemic lesions, and brain atrophy contribute to late-onset depression in the nondemented elderly. METHOD Based on a group of 606 individuals of identical age (75.8 years, standard deviation: 0.45 years) residing in two districts of Vienna, the authors built a case-control cohort (ratio: 1:4) consisting of 51 individuals with late-onset major or minor depression matched with 204 subjects of identical gender and education status without depression, resulting in two groups that were homogenous with respect to age, place of residence, gender, and education. Scores for focal brain lesions, mediotemporal lobe atrophy, and ventricular enlargement as well as risk factors for vascular disease were compared with cognition and depression status. RESULTS Depressed individuals had significantly lower scores than nondepressed subjects in all measures of cognitive and executive function. No significant relation was found between a diagnosis of depression and any type of discrete brain lesions, but measures of brain atrophy (Cella Media indices, mediotemporal atrophy) showed a clear statistical relation to depression. No relationship was found between depression and lipid parameters. CONCLUSION The authors found no indication that white matter hyperintensities or minor ischemic lesions played a role in our depressed cohort, casting doubt on the vascular hypothesis of late-onset depression.

Evaluation of the 2010 McDonald multiple sclerosis criteria in children with a clinically isolated syndrome

Background: Magnetic resonance imaging diagnostic criteria for paediatric multiple sclerosis have been established on the basis of brain imaging findings alone. The 2010 McDonald criteria for the diagnosis of multiple sclerosis, however, include spinal cord imaging for detection of lesion dissemination in space. The new criteria have been recommended in paediatric multiple sclerosis. Objective: (1) To evaluate the 2010 McDonald multiple sclerosis criteria in children with a clinically isolated syndrome and to compare them with recently proposed magnetic resonance criteria for children; (2) to assess whether the inclusion of spinal cord imaging provided additional value to the 2010 McDonald criteria. Methods: We performed a retrospective analysis of brain and spinal cord magnetic resonance imaging scans from 52 children with a clinically isolated syndrome. Sensitivity, specificity and accuracy of the magnetic resonance criteria were assessed. Results and conclusion: The 2010 McDonald dissemination in space criteria were more sensitive (85% versus 74%) but less specific (80% versus 100%) compared to the 2005 McDonald criteria. The Callen criteria were more accurate (89%) compared to the 2010 McDonald (85%), the 2005 McDonald criteria for dissemination in space (81%), the KIDMUS criteria (46%) and the Canadian Pediatric Demyelinating Disease Network criteria (76%). The 2010 McDonald criteria for dissemination in time were more accurate (93%) than the dissemination in space criteria (85%). Inclusion of the spinal cord did not increase the accuracy of the McDonald criteria.

DNA repair polymorphisms associated with cytogenetic subgroups in B-cell chronic lymphocytic leukemia†

Genetic polymorphisms in DNA repair genes can affect the risk of developing different forms of cancer. Therefore, we have studied the putative association of seven single nucleotide polymorphisms (SNPs) in five DNA repair genes with the incidence of chronic lymphocytic leukemia (CLL). We included 461 CLL patients and the same number of age‐ and sex‐matched controls. As chromosomal aberrations are important prognostic markers in CLL, we additionally correlated the SNPs with the occurrence of favorable and unfavorable cytogenetic aberrations in CLL patients. Patients with del(13q) as a sole aberration were allocated to the favorable cytogenetic risk group, and patients with del(17p) and/or del(11q) to the unfavorable cytogenetic risk group. All investigated SNPs were equally distributed between patients with the favorable cytogenetic aberration and controls. However, differences were observed in the distribution of rs13181 in ERCC2 between all CLL patients and controls. Moreover, the clearest differences were found for rs13181 in ERCC2 and rs25487 in XRCC1 between CLL patients with unfavorable cytogenetic aberrations and controls. These data suggest that inborn genetic polymorphisms may predict the outcome of CLL.

Optimized multi‐stage designs controlling the false discovery or the family‐wise error rate

When a large number of hypotheses are investigated, we propose multi-stage designs where in each interim analysis promising hypotheses are screened, which are investigated in further stages. Given a fixed overall number of observations, this allows one to spend more observations for promising hypotheses than with single-stage designs, where the observations are equally distributed among all considered hypotheses. We propose multi-stage procedures controlling either the family-wise error rate (FWER) or the false discovery rate (FDR) and derive asymptotically optimal stopping boundaries and sample size allocations (across stages) to maximize the power of the procedure. Optimized two-stage designs lead to a considerable increase in power compared with the classical single-stage design. Going from two to three stages additionally leads to a distinctive increase in power. Adding a fourth stage leads to a further improvement, which is, however, less pronounced. Surprisingly, we found only small differences in power between optimized integrated designs, where the data of all stages are used in the final test statistics, and optimized pilot designs where only the data from the final stage are used for testing. However, the integrated design controlling the FDR appeared to be more robust against misspecifications in the planning phase. Additionally, we found that with increasing number of stages the drop in power when controlling the FWER instead of the FDR becomes negligible. Our investigations show that the crucial point is not the choice of the error rate or the type of design, but the sequential nature of the trial where non-promising hypotheses are dropped in the early phases of the experiment.

Risk Factors for Alzheimer Dementia in a Community-Based Birth Cohort at the Age of 75 Years

Background: Few prospective community-based cohort studies have so far concentrated specifically on the risk factors for Alzheimer dementia (AD) with onset after the age of 75 years. Methods: We prospectively investigated a birth cohort of 585 nondemented inhabitants in the area on the East bank of the river Danube who were born between 1925 and 1926. They were investigated at the age of 75 years and followed up after 30 months. The follow-up was possible with 488 probands; 36 died, and 61 refused to participate. Results: In multivariate analysis an elevated risk for late-onset AD could be found for (1) history of depressive episodes (OR = 2.09; 95% CI = 1.25–3.48); (2) the Ε4 allele of the APOE gene (OR = 1.86; 95% CI = 1.08–3.23); (3) lower serum level of folate (OR = 0.92; 95% CI = 0.87–0.98); (4) no chronic use of nonsteroidal anti-inflammatory drugs (OR = 0.40; 95% CI = 0.20–0.81), and (5) lower education (OR = 1.43; 95% CI = 1.03–2.00). Conclusions: Five risk factors for late-onset AD could be confirmed, which might be targets for preventive strategies.

Relation between vascular risk factors and cognition at age 75

Objective –  Recent trends in dementia research emphasize that not only cerebrovascular events but also vascular risk factors induce, favour or cause cognitive impairment and Alzheimers disease.