Diagnostic potential of cerebrospinal fluid biomarkers in Alzheimer’s disease combined with tau genotypes Mirjana Babić Leko1#, Nanet Willumsen2#, Matea Nikolac Perković3, Nataša Klepac4, Fran Borovečki4, Patrick R. Hof5, Nela Pivac3, Rohan de Silva2, Goran Šimić1* 1Department for Neuroscience, Croatian Institute for Brain Research, University of Zagreb Medical School, Zagreb, Croatia 2Reta Lila Weston Institute and Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK 3Ruđer Bošković Institute, Division of Molecular Medicine, Zagreb, Croatia 4Department for Functional Genomics, Center for Translational and Clinical Research, University of Zagreb Medical School, University Hospital Center Zagreb, Zagreb, Croatia 5Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA #equally contributing co-authors *correspondence to: gsimic@hiim.hr Croatian Science Foundation Materials and methods Introduction The study was conducted on 113 AD and 53 mild cognitive impairment (MCI) patients, 9 healthy controls and 54 patients with other causes of dementia (14 with vascular dementia (VaD), 22 with frontotemporal dementia (FTD), 7 with dementia with Lewy bodies, 3 with AD + VaD, 1 with corticobasal syndrome (CBS), 2 with Parkinson disease (PD), 1 with epilepsy and 4 with unspecified dementia). CSF was taken by lumbar puncture between intervertebral spaces L3/L4 or L4/L5. CSF samples were aliquoted, and stored at -80°C while levels of Aβ1-42, t-tau, p-tau181, VILIP-1, p-tau231 and p-tau199 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)), VILIP-1 (VILIP-1 Human ELISA), p-tau231 (Tau [pT231] Phospho-ELISA Kit) and p-tau199 (TAU [pS199] Phospho-ELISA Kit). Genomic DNA was extracted from peripheral blood using the salting-out method. Polymorphisms in MAPT gene (rs1467967, rs242557, rs3785883, rs2471738, del–In9, rs7521) were determined by restriction fragment analysis and using primers and probes purchased from Applied Biosystems as TaqMan® SNP Genotyping Assay by ABI Prism 7300 Real Time PCR System apparatus (Applied Biosystems, Foster City, CA, USA). Alzheimer's disease (AD) is the most common sporadic tauopathy. Although it is not caused by mutation in the microtubule-associated protein tau (MAPT) gene, previous studies showed that biomarkers of AD differ between patients with different MAPT genotypes. In this study, we tried to reveal 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 MAPT haplotype-tagging polymorphisms (rs1467967, rs242557, rs3785883, rs2471738, del–In9, rs7521). First, we compared if levels of Aβ1-42, t-tau, p-tau181, p-tau199, p-tau231, and VILIP-1 differed among patients with different MAPT genotypes (rs1467967, rs242557, rs3785883, rs2471738, del–In9, rs7521). Second, we analyzed the distribution of MAPT haplotypes (H1 and H2 haplotypes and their sub-haplotypes) in the Croatian population. Of the analyzed polymorphisms the del-In9 insertion-deletion in MAPT intron 9 defines the H1/H2 division caused by the inversion, while other single nucleotide polymorphisms (SNPs) define sub-haplotypes. Results Levels of t-tau were significantly higher in subjects with AG in comparison to GG rs1467967 genotype (when all patients were analyzed together (U=832, Z=-2.713, p=0.007*)) (Figure 1). This observation was also confirmed in the combined group of AD, MCI patients and healthy controls (U=500, Z=-2.671, p=0.008*), and in the combined group of AD and MCI patients (U=454, Z=-2.642, p=0.008*). Additionally, levels of p-tau181 were significantly higher in subjects with AG in comparison to GG rs1467967 tau genotype (in the combined group of AD and MCI patients) (U=456, Z=-2.434, p=0.015*) (Figure 2). Levels of t-tau were significantly higher in subjects with GG in comparison to the AG rs2471738 tau genotype (in the combined group of AD and MCI patients) (U=2190, Z=-2.477, p=0.013*) (Figure 3). Levels of p-tau231 were significantly higher in subjects with GG in comparison to AA+AG rs2471738 tau genotype (in AD patients - U=1112, Z=-2.119, p=0.034* and in the combined group of AD and MCI patients - U=2448.5, Z=-2.453, p=0.014* (Figure 4)). Levels of t-tau (U=2513, Z=-2.645, p=0.008*) (Figure 5) and p-tau181 (U=2506, Z=-2.350, p=0.019*) (Figure 6) were significantly higher in subjects with GG in comparison to AA + AG rs2471738 tau genotype (in the combined group of AD and MCI patients). These observations were also confirmed in the combined group of AD, MCI patients and healthy controls (for t-tau (U=2944.5, Z=-2.271, p=0.023*), p-tau181 (U=2888, Z=-2.126, p=0.034*) and p-tau231 (U=2739.5, Z=-2.499, p=0.012*)) and in the group of all patients (for p-tau231 (U=4895, Z=-2.387, p=0.017*)). Levels of t-tau (U=4517, Z=-2.089, p=0.037*) (Figure 7), p-tau181 (U=4308, Z=-2.112, p=0.035*) (Figure 8) and p-tau231 (U=4212, Z=-2.171, p=0.030*) (Figure 9) were significantly higher in subjects with H1H2 + H2H2 haplotype in comparison to H1H1 haplotype (when all patients were analyzed together). Additionally, we detected 23 H1 sub-haplotypes and 5 H2 sub-haplotypes in the Croatian population (Figures 10 and 11). Figure 1. Levels of t-tau in subjects with AG in comparison to GG rs1467967 tau genotype. Boxes represent the median, the 25th and 75th percentiles, and bars indicate the range of data distribution. Figure 2. Levels of p-tau181 in subjects with AG in comparison to GG rs1467967 tau genotype. Figure 3. Levels of t-tau in subjects with GG in comparison to AG rs2471738 tau genotype. Figure 4. Levels of p-tau231 in subjects with GG in comparison to AA + AG rs2471738 tau genotype. Figure 5. Levels of t-tau in subjects with GG in comparison to AA + AG rs2471738 tau genotype. Figure 6. Levels of p-tau181 in subjects with GG in comparison to AA + AG rs2471738 tau genotype. Figure 8. Levels of p-tau181 in subjects with H1H2 + H2H2 haplotype in comparison to H1H1 haplotype. Figure 9. Levels of p-tau231 in subjects with H1H2 + H2H2 haplotype in comparison to H1H1 haplotype. Figure 7. Levels of t-tau in subjects with H1H2 + H2H2 haplotype in comparison to H1H1 haplotype. Figure 11. Percentage (%) of 5 tau H2 haplotypes in Croatian population. Conclusion Figure 10. Percentage (%) of 23 tau H1 haplotypes in Croatian population. In this study, the difference in the levels of CSF biomarkers of AD (t-tau, p-tau181 and p-tau231) in patients with different rs1467967 and rs2471738 tau genotypes were detected. Levels of aforementioned CSF biomarkers also differed between patients with H1H1 in comparison to H1H2 + H2H2 haplotypes. Thus, potential of rs1467967 and rs2471738 tau genotypes and H1 and H2 tau haplotypes in early diagnosis of AD should be further tested. Acknowledgments This work was funded by the Croatian Science Foundation IP-2014-09-9730 “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.