Association of MAPT haplotype‐tagging polymorphisms with cerebrospinal fluid biomarkers of Alzheimer's disease: A preliminary study in a Croatian cohort

Abstract Introduction Alzheimer's disease (AD) is the world leading cause of dementia. Early detection of AD is essential for faster and more efficacious usage of therapeutics and preventive measures. Even though it is well known that one ε4 allele of apolipoprotein E gene increases the risk for sporadic AD five times, and that two ε4 alleles increase the risk 20 times, reliable genetic markers for AD are not yet available. Previous studies have shown that microtubule‐associated protein tau (MAPT) gene polymorphisms could be associated with increased risk for AD. Methods The present study included 113 AD patients and 53 patients with mild cognitive impairment (MCI), as well as nine healthy controls (HC) and 53 patients with other primary causes of dementia. The study assessed whether six MAPT haplotype‐tagging polymorphisms (rs1467967, rs242557, rs3785883, rs2471738, del–In9, and rs7521) and MAPT haplotypes are associated with AD pathology, as measured by cerebrospinal fluid (CSF) AD biomarkers amyloid β1–42 (Aβ1–42), total tau (t‐tau), tau phosphorylated at epitopes 181 (p‐tau181), 199 (p‐tau199), and 231 (p‐tau231), and visinin‐like protein 1 (VILIP‐1). Results Significant increases in t‐tau and p‐tau CSF levels were found in patients with AG and AA MAPT rs1467967 genotype, CC MAPT rs2471738 genotype and in patients with H1H2 or H2H2 MAPT haplotype. Conclusions These results indicate that MAPT haplotype‐tagging polymorphisms and MAPT haplotypes should be further tested as potential genetic biomarkers of AD.


| INTRODUC TI ON
Alzheimer's disease (AD), the most common primary cause of dementia, is a complex disease with poorly understood etiology. The characteristic amyloid plaques and neurofibrillary changes seen in AD brains are frequently observed in other neurodegenerative diseases. As a consequence, many AD cases are misdiagnosed. One such autopsy-confirmed series showed sensitivity of AD diagnosis to range from 70.9% to 87.3%, while specificity ranged from 44.3% to 70.8% (with controls as reference groups; Beach, Monsell, Phillips, & Kukull, 2012;Gay, Taylor, Hohl, Tolnay, & Staehelin, 2008;Joachim, Morris, & Selkoe, 1988). This creates substantial difficulties in interpretations of results obtained by different studies that include patients with probable AD. One possibility to avoid this problem would be to use intermediate quantitative traits (endophenotypes) rather than clinical diagnoses (case-control studies) as indices of AD pathology. Endophenotypes are any biomarkers that signal the presence of AD pathology. For example, for this study, we used core cerebrospinal fluid (CSF) biomarkers of AD, such as amyloid β 1-42 (Aβ 1-42 ), total tau (t-tau), and tau phosphorylated at epitope 181 (p-tau 181 ), and potential CSF biomarkers, such as tau phosphorylated at epitopes 199 (p-tau 199 ), and 231 (p-tau 231 ), and visinin-like protein 1 (VILIP-1) as endophenotypes. Aβ 1-42 indicates the presence of senile plaques in the brain (Grimmer et al., 2009), t-tau and VILIP-1 are markers of neurodegeneration (Babić et al., 2014;Babić Leko, Borovečki, Dejanović, Hof, & Šimić, 2016), while p-tau 181 , p-tau 199 , and p-tau 231 reflect the presence of neurofibrillary tangles in the brain (Bürger et al., 2006). CSF core biomarkers (Aβ 1-42 , t-tau, and p-tau 181 ) were previously used in genome-wide association studies (GWAS) as endophenotypes for detection of AD risk genes (Cruchaga et al., 2013;Kim et al., 2011). In this study, we used these biomarkers to assess whether certain variants of microtubule-associated protein tau (MAPT) gene were associated to their pathological levels in CSF. Although AD is not caused by mutations in the MAPT gene, previous studies demonstrated that CSF biomarkers of AD differ among patients with different MAPT genotypes (Compta et al., 2011;Kauwe et al., 2008). Besides comparing the levels of CSF biomarkers Aβ 1-42 , t-tau, p-tau 181 , p-tau 199 , p-tau 231 , and VILIP-1 among patients with six different MAPT genotypes, we also analyzed the distribution of MAPT H1 and H2 haplotypes and their subhaplotypes in a Croatian patient cohort.
The majority of AD patients are late-onset sporadic cases, whose heritability for the disease has been estimated to be as high as 58%-79% (Gatz et al., 2006). In addition to apolipoprotein E gene (APOE), more than 20 common loci have been associated with risk for sporadic AD, age at onset, and progression of cognitive decline, but reported genome-wide significant loci do not account for all the estimated heritability and provide little information about underlying biological mechanisms (Šimić et al., 2016a, 2016b test, and had a negative serology for syphilis or Lyme's disease. criteria (Román et al., 1993), as well as the Hachinski Ischemic Score (HIS) (Hachinski et al., 1975). The most important clinical discriminative factors for VaD vs. AD are stepwise progression, prominent impairment of the executive functions, higher probability of VaD when HIS is >4, and focal neurological signs implying cortical or subcortical lesions (Desmond et al., 1999). Clinical criteria for FTD were based on consensus published by Neary and collaborators (Neary et al., 1998). Conditions overlapping AD are very difficult to study, but biomarkers that we have chosen are again superior in this respect in comparison to other approaches, such as correlation with clinical diagnosis.

| DNA analysis of MAPT polymorphisms
Venous blood samples (4 ml) were collected into plastic syringes with 1 ml of acid citrate dextrose as an anticoagulant. Genomic DNA was extracted from peripheral blood using the salting-out method (Miller, Dykes, & Polesky, 1988

| Ethical approval
The present study was conducted according to the 6th revised Declaration of Helsinki (Edinburgh, 2000) and Good Clinical Practice

| Comparison of protein CSF biomarkers between subjects with different MAPT genotypes
The concentrations of CSF protein biomarkers (Aβ 1-42 , t-tau, ptau 181 , p-tau 199 , p-tau 231 , and VILIP-1) in the analyzed subjects are presented in Table 1. The measured inter assay coefficient of variability (CV) was <10%. The intra assay CV was <15% for all biomarkers used. No significant differences were found between males and females in any of the groups. There was no significant difference in the levels of CSF protein biomarkers among subjects with different MAPT rs242557, MAPT rs3785883, and MAPT rs7521 genotypes.

| MAPT rs1467967 genotype
The difference in t-tau levels was detected between patients with MAPT rs1467967 genotype (H = 11.655, df = 2, p = 0.003). T-tau levels were significantly higher in patients with AG compared to GG MAPT rs1467967 genotype (p = 0.002) and AA compared to GG MAPT rs1467967 genotype (p = 0.022) when all patients were analyzed together (Figure 1a). The observation of increased ttau levels in patients with AG compared to GG MAPT rs1467967 genotype was confirmed when combining AD and MCI patients, and healthy controls (p = 0.004; Figure 1b) and in AD and MCI patients (p = 0.005; Figure 1c). The difference in p-tau 181 levels was detected between patients with MAPT rs1467967 genotype (H = 6.955, df = 2, p = 0.031). P-tau 181 levels were significantly higher in patients with AG in comparison to GG MAPT rs1467967 genotype when combining AD and MCI patients (p = 0.025; Figure 2). There was no significant difference in levels of Aβ 1-42 , p-tau 199 , p-tau 231 , and VILIP-1 among subjects with different MAPT rs1467967 genotype.

| MAPT rs2471738 genotype
The difference in t-tau levels was detected between patients with

| D ISCUSS I ON
This preliminary study of a Croatian cohort investigated whether certain variants of MAPT gene were associated with AD pathology as it was shown that polymorphisms in the MAPT gene increase the risk of tauopathies (Di Maria et al., 2010;Myers et al., 2005;Pittman et al., 2005). While a limitation of this study is a low number of HC (n = 9), our analysis of MAPT polymorphisms was conducted in all patients including the HC group (228 subjects in total), as such, the small number of HC is unlikely to have influenced the outcome of the analysis. The levels of t-tau and p-tau 181 were significantly higher in patients with AG compared to GG MAPT rs1467967 genotype.
Levels of t-tau were significantly higher in patients with AA compared to GG MAPT rs1467967 genotype. Levels of t-tau were significantly higher in patients with CC compared to TC MAPT rs2471738 genotype. Additionally, levels of t-tau, p-tau 181 and p-tau 231 were significantly higher in patients with CC compared to patients with TT or TC MAPT rs2471738 genotypes. Also, levels of t-tau and p-tau 231 were significantly higher in patients with H1H2 or H2H2 haplotypes compared to patients with the H1H1 haplotype.

Compta and collaborators found that carriers of the A-allele in
MAPT rs242557 polymorphism had increases in CSF t-tau and ptau 181 levels (Compta et al., 2011). This was observed in patients with PD, but only in those with dementia and pathological Aβ 1-42 levels (lower than 500 pg/ml). Although Compta et al. compared F I G U R E 5 Levels of t-tau (a, b) and p-tau 181 (c, d) in AD, MCI patients and HC and in AD and MCI patients with the MAPT rs2471738 genotype. Boxes represent the median, the 25th and 75th percentiles, and bars indicate the range of data distribution. Circles represent outliers, and asterisks represent extreme data points. *p < 0.05 F I G U R E 6 Levels of (a) t-tau and (b) p-tau 231 in all patients with H1H1 and H1H2 + H2H2 MAPT haplotypes. Boxes represent the median, the 25th and 75th percentiles, and bars indicate the range of data distribution. Circles represent outliers, and asterisks represent extreme data points. *p < 0.05 the levels of t-tau and p-tau 181 in PD patients with different genotype with t-tau and p-tau 181 that was also tested in that study was not observed (Kauwe et al., 2008). Although Laws et al. (2007) analyzed the association of CSF t-tau with all polymorphisms included in our study, only an association of MAPT rs242557 genotype with t-tau levels was observed. Laws et al. proposed that association between CSF tau levels and MAPT polymorphisms (or haplotypes) could occur through MAPT expression. In other words, individuals carrying risk MAPT alleles have a higher MAPT brain expression and consequently an increased neurodegeneration and leakage of tau protein in CSF (Laws et al., 2007).
The study of Ning et al. (2011) showed that MAPT rs1467967 polymorphism could serve as a genetic biomarker for VaD, since the rs1467967 genotypes differed between VaD patients and HC. Also, the MAPT rs2471738 polymorphism was associated with an increased risk for AD (Vázquez-Higuera et al., 2009). While the metaanalysis of Yuan, Du, Ge, Wang, & Xia (2018) showed that none of the polymorphisms analyzed in the present study (rs1467967, rs3785883, rs2471738, and rs7521), except for rs242557 that showed an association with AD, the meta-analysis of Zhou and Wang (2017) demonstrated an association of rs242557 and rs2471738 polymorphisms (but not rs3785883 or rs1467967 polymorphisms) with AD.
The association of the H1U and H1H haplotypes and H1C haplotype with t-tau levels was previously demonstrated (Kauwe et al., 2008;Laws et al., 2007). The H1C haplotype was shown to be a risk factor for progressive supranuclear palsy and CBS , late-onset AD (LOAD; Myers et al., 2005), and MCI (Di Maria et al., 2010), while the H2 haplotype was associated with a reduced risk for LOAD (Allen et al., 2014;Zhang et al., 2017). Our results do not support observations that individuals with the H1 haplotype have increased CSF t-tau levels (Kauwe et al., 2008;Laws et al., 2007) and increased risk for AD or other tauopathies (Di Maria et al., 2010;Myers et al., 2005;Pittman et al., 2005), as our patients with the H2 haplotype had pathological CSF t-tau and p-tau levels. However, several studies failed to detect the association of the H1 haplotype with an increased risk for AD (Abraham et al., 2009;Mukherjee, Kauwe, Mayo, Morris, & Goate, 2007;Russ et al., 2001).
Additionally, in the study of Min et al. (2014), patients with FTD and MAPT H1 haplotype had an increase in CSF p-tau 181 levels, while there was no difference in the levels of t-tau. Another study found no association between MAPT haplotypes and CSF t-tau, p-tau 181 or Aβ 1-42 levels (Johansson, Zetterberg, Håkansson, Nissbrandt, & Blennow, 2005).
In conclusion, the present study resulted in several notable findings. The association of MAPT rs1467967 polymorphism with AD pathology measured by levels of CSF biomarkers was demonstrated, with CSF t-tau and p-tau 181 levels being significantly higher in patients with AG compared to GG MAPT rs1467967 genotype and t-tau levels being significantly higher in patients with AA compared to GG MAPT rs1467967 genotype. Additionally, we detected an association of the MAPT rs2471738 polymorphism with AD pathology that was also observed in previous studies (Kauwe et al., 2008;Vázquez-Higuera et al., 2009). However, the MAPT rs2471738 risk allele detected in our study (C-allele) differs from the risk allele detected in the study of Myers et al. (2005) and Vázquez-Higuera et al. (2009) (T-allele), warranting further investigation. We also observed an increase in CSF t-tau and ptau levels in patients with H1H2 or H2H2 haplotypes. This finding differs from studies in which the H1 haplotype was detected as a risk haplotype for AD and other tauopathies (Di Maria et al., 2010;Myers et al., 2005), and this issue also will require additional research. We used potentially novel CSF biomarkers of AD as endophenotypes (p-tau 199 , p-tau 231 , and VILIP-1), while previous studies testing the association of MAPT polymorphisms with AD used only core CSF biomarkers as endophenotypes (Aβ 1-42 , t-tau, p-tau 181 ). The p-tau 231 endophenotype showed significant difference between groups of patients with different MAPT genotypes.
Finally, we detected 23 H1 and 5 H2 MAPT subhaplotypes in this Croatian cohort, revealing that MAPT haplotype-tagging polymorphisms and MAPT haplotypes should be further tested as potential genetic biomarkers of AD.

DATA AVA I L A B I L I T Y
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

CO N FLI C T O F I NTE R E S T
None declared.