1. Fran Borovečki3, Patrick R. Hof4, Nela Pivac2, Goran Šimić1*
Cerebrospinal fluid and genetic biomarkers in early detection of Alzheimer’s disease
Mirjana Babić Leko1#, Matea Nikolac Perković2#, Lea Langer Horvat1, Nataša Klepac3,
Fran Borovečki3, Patrick R. Hof4, Nela Pivac2, Goran Šimić1*
1Department for Neuroscience, Croatian Institute for Brain Research, University of Zagreb Medical School, 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
4Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA
#Equally contributing co-authors
*correspondence to:
Subjects and methods
Background
The study was conducted on 115 AD and 53 mild cognitive impairment (MCI) patients, 10 healthy controls (HC) and 56 patients with other causes of dementia (14 with vascular dementia (VaD), 23 with frontotemporal dementia, 7 with dementia with Lewy bodies, 3 with AD + VaD, 1 with corticobasal degeneration, 1 with hydrocephalus, 2 with Parkinson disease, 1 with epilepsy and 4 with unspecified dementia). Genomic DNA was extracted from peripheral blood using the salting-out method. DBH (rs ), IL1α (rs ), IL1β (rs ), IL6 (rs ), IL10 (rs ), TNFα (rs ), 5-HT2A (rs6313), 5-HT1B (re ), 5-HT2C (rs ), COMT (rs4680), BDNF (rs6265) and MAOB (rs ) polymorphisms 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. CSF was obtained by lumbar puncture between intervertebral spaces L3/L4 or L4/L5. Samples were aliquoted, and stored at -80°C. Aβ1-42, t-tau, p-tau181, p-tau199, p-tau231 and VILIP-1 were measured using enzyme-linked immunosorbent assay (ELISA): Aβ1-42 (Innotest β-amyloid1-42), t-tau (Innotest hTau Ag), p-tau181 (Innotest Phospho-Tau(181P)), p-tau199 (TAU [pS199] Phospho-ELISA Kit), p-tau231 (Tau [pT231] Phospho-ELISA Kit) and VILIP-1 (VILIP-1 Human ELISA).
Early diagnosis of Alzheimer’s disease (AD) in asymptomatic individuals is crucial because potential therapeutics should be administrated as early as possible, when neurodegeneration is not yet advanced. In this study we assessed whether the diagnostic potential of cerebrospinal fluid (CSF) biomarkers amyloid β1-42 (Aβ1-42), total tau (t-tau), tau phosphorylated at epitope 181 (p-tau181), epitope 199 (p-tau199), epitope 231 (p-tau231) and visinin-like protein 1 (VILIP-1) could be improved by genetic biomarkers related to serotonin metabolism (5-HT2A, 5-HT1B, 5-HT2C, and MAOB), inflammatory pathways (IL1α, IL1β, IL10, IL6, and TNFα), catecholamine metabolism (COMT, DBH), and survival of neurons (BDNF). We compared whether levels of Aβ1-42, t-tau, p-tau181, p-tau199, p-tau231, and VILIP-1 differ between patients with different DBH, IL1α, IL1β, IL6, IL10, TNFα, 5-HT2A, 5-HT1B, 5-HT2C, COMT, BDNF and MAOB genotypes.
Results
Figure 3. Levels of p-tau181 were significantly higher in AD patients with GA in comparison to AA and GG BDNF genotype and in MCI patients with CT in comparison to CC DBH genotype. Levels of Aβ1-42 were significantly lower in MCI patients with AA in comparison to GG MAOB genotype.
Figure 4. Levels of VILIP-1 were significantly higher in MCI patients with GC in comparison to GG IL6 genotype, while levels of p-tau199 were significantly higher in MCI patients and levels of t-tau were significantly higher in mixed group of patients with GC in comparison to CC IL6 genotype.
Figure 1. Levels of t-tau, p-tau181, p-tau199, p-tau231 and VILIP-1 were significantly higher in subjects with AA in comparison to GG and AG TNFα genotype in the mixed group of patients and in the group of AD patients. Boxes represent the median, the 25th and 75th percentiles, and bars indicate the range of data distribution.
Figure 5. Levels of p-tau181 were significantly higher in MCI patients with TT in comparison to CC and TC IL10 genotype, while levels of p-tau199 and p-tau231 were significantly higher in AD patients with CC in comparison to TC IL10 genotype.
Figure 2. Levels of t-tau, p-tau181 and p-tau231 were significantly higher in mixed group of patients with AA in comparison to AG COMT genotype, while levels of Aβ1-42 were significantly lower in patients with GG in comparison to AG COMT genotype. Boxes represent the median, the 25th and 75th percentiles, and bars indicate the range of data distribution.
Figure 6. Levels of p-tau199 and p-tau231 were significantly higher in in mixed group of patients with CG in comparison to CC IL1β genotype. Boxes represent the median, the 25th and 75th percentiles, and bars indicate the range of data distribution.
Conclusion
Acknowledgments
This study showed the difference in the levels of cerebrospinal fluid biomarkers of Alzheimer’s disease in patients with different TNFα, IL1β, COMT, IL10, IL6, BDNF, DBH and MAOB genotype. Thus, potential of TNFα (rs ), IL1β (rs ), COMT (rs4680), IL10 (rs ), IL6 (rs ), BDNF (rs6265), DBH (rs ) and MAOB (rs ) polymorphisms in early diagnosis of Alzheimer’s disease should be further tested and validated on larger cohorts of patients.
All procedures involving human subjects were done in accord with the approval of the Central Ethical Committee of the University of Zagreb Medical School case no / /90, class /11-02, signed on 19th May This work was funded by the Croatian Science Foundation grant no. IP to G.Š. The authors declare no conflict of interest.