1. COMBINATION OF CSF PROTEIN BIOMARKERS AND BDNF, IL10 AND IL6 GENOTYPES IN EARLY DIAGNOSIS OF ALZHEIMER’S DISEASE  
Mirjana Babić Leko1, Matea Nikolac Perković2, Nataša Klepac3,
Fran Borovečki3, Nela Pivac2, Goran Šimić1*
1Department of Neuroscience, Croatian Institute for Brain Research, University of Zagreb School of Medicine, Zagreb, Croatia
2Ruđer Bošković Institute, Division of Molecular Medicine, Zagreb, Croatia
3Department for Functional Genomics, Center for Translational and Clinical Research, University of Zagreb Medical School, University Hospital Center Zagreb, Zagreb, Croatia.
*correspondence to:
Abstract
Cerebrospinal fluid (CSF) biomarkers are mostly used for early detection of Alzheimer’s disease (AD) in the group of patients with mild cognitive impairment (MCI). However, detection of AD in asymptomatic individuals (preclinical AD) is critical. In this study we tested whether the diagnostic potential of core CSF biomarkers (amyloid β42 (Aβ42), total tau (t-tau) and tau phosphorylated at epitope 181 (p-tau181)) could be improved by genetic biomarkers related to inflammatory pathways (IL1α, IL1β, IL10 and IL6) and survival of neurons (BDNF). We compared if levels of Aβ42, t-tau and p-tau181 differed between patients with different BDNF (rs6265), IL1α (rs ), IL1β (rs ), IL10 (rs ) and IL6 (rs ) genotypes. Levels of p-tau181 were significantly higher in the mixed group (AD, MCI and control subjects) with GA in comparison to GG BDNF genotype (t = , df = 221, p = 0.039). Levels of Aβ42 were significantly higher in the group of control subjects with TT in comparison to TC IL10 genotype (t = 2.500, df = 7, p = 0.041), while p-tau181 levels were significantly higher in the group of MCI patients with GC in comparison to CC IL6 genotype (t = , df = 34, p = 0.028). Thus, the potential of selected BDNF (rs6265), IL10 (rs ) and IL6 (rs ) polymorphisms in early diagnosis of AD should be further tested and validated on larger cohorts of patients.
Introduction
Materials and methods
This study included 233 patients recruited at the University Hospital Centre „Zagreb”, of whom 118 fulfilled NINCDS-ADRDA criteria for AD, 53 fulfilled criteria for MCI, 10 were healthy controls (HC) and 52 had other primary causes of dementia. Due to the limited number of patients the analysis was carried out across all the groups as well as separately for each group. Genomic DNA was extracted from peripheral blood using the salting-out method. SNPs in the genes for BDNF (rs6265), IL1α (rs ), IL1β (rs ), IL10 (rs ) and IL6 (rs ) were determined in all subjects by ABI Prism 7300 Real Time PCR System apparatus (Applied Biosystems, Foster city, CA, USA) using primers and probes purchased from Applied Biosystems as TaqMan® SNP Genotyping Assay (C_904973_10 ND C_ _20). CSF was obtained by lumbar puncture between intervertebral spaces L3/L4 or L4/L5, always between 9 a.m. and 11 a.m. Samples were centrifuged for 10 min at 2,000 g, aliquoted, and stored at -80°C. Aβ42, t-tau and p-tau181 were measured using enzyme-linked immunosorbent assay (ELISA); Aβ42 (Innotest β-amyloid42), t-tau (Innotest hTau Ag), p-tau181 (Innotest Phospho-Tau(181P)).
The final goal of the studies on Alzheimer's disease (AD) biomarkers is detection of AD in asymptomatic individuals (preclinical AD). In comparison to other biomarkers, genetic biomarkers give us the opportunity for the earliest detection of AD. In this study we tested if the diagnostic potential of the core CSF biomarkers (amyloid β42 (Aβ42), total tau (t-tau) and tau phosphorylated at epitope 181 (p-tau181)) could be improved by potential genetic biomarkers related to inflammatory pathways (genes for IL1α, IL1β, IL10 and IL6) and survival of neurons (BDNF gene). We compared if levels of Aβ42, t-tau and p-tau181 differed between patients with different BDNF (rs6265), IL1α (rs ), IL1β (rs ), IL10 (rs ) and IL6 (rs ) genotypes.
Results
Levels of p-tau181 were significantly higher in the mixed group of patients and healthy controls (AD, MCI and cognitively normal subjects) with GA in comparison to GG BDNF genotype (t = , df = 221, p = 0.039). Levels of Aβ42 were significantly higher in the group of control subjects with TT in comparison to TC IL10 genotype (t = 2.500, df = 7, p = 0.041), while p-tau181 levels were significantly higher in the group of MCI patients with GC in comparison to CC IL6 genotype (t = , df = 34, p = 0.028). There was no difference in Aβ42, t-tau and p-tau181 levels between patients with different genotypes of IL1α (rs ) and IL1β (rs ) in either group. The potential of selected BDNF (rs6265), IL10 (rs ) and IL6 (rs ) polymorphisms in early diagnosis of AD should be further tested and validated on larger cohorts of patients.
Figure 3. Levels of p-tau181 in the group of MCI patients with different IL6 genotypes (GG, CC, GC). Boxes represent the median with the 25th and the 75th percentiles, and bars indicate the range of data distribution. Circles represent outliers. *Statistically significant at p < 0.05.
Figure 2. Levels of Aβ42 in the group of cognitively healthy controls with different IL10 genotypes (TT, CC, TC). Boxes represent the median with the 25th and the 75th percentiles, and bars indicate the range of data distribution. Circles represent outliers. *Statistically significant at p < 0.05.
Figure 1. Levels of p-tau181 in the mixed group of patients and controls with different BDNF genotype (GG, AA, GA). Boxes represent the median with the 25th and the 75th percentiles, and bars indicate the range of data distribution. Circles represent outliers. *Statistically significant at p < 0.05.
Conclusion
Acknowledgments
The results of this study indicate that patients with GA genotype of BDNF, TC genotype of IL10 and GC genotype of IL6 could have higher susceptibility for AD since levels of known protein biomarkers in CSF were pathological in these subjects. However, these findings should be further validated in future prospective multicentre studies in larger cohorts of MCI and AD patients, and cognitively healthy controls.
This work was funded by the Croatian Science Foundation IP “Tau protein hyperphosphorylation, aggregation and trans-synaptic transfer in Alzheimer's disease: cerebrospinal fluid analysis and assessment of potential neuroprotective compounds“ to G.Š. The authors declare no conflict of interest.