Association of IL-1β, IL-1α and IL-10 single nucleotide polymorphisms with Mini-Mental State Examination and event-related potentials CURRENT

Background: Neuroinflammation is enhanced in Alzheimer’s disease (AD) brain. Its association with both amyloid and tau pathology is well documented. Activated microglia in the AD brain release pro-inflammatory cytokines that can damage neurons, while anti-inflammatory cytokines are also released to oppose this process. Association of IL-1β -1473C/G, IL-1α -889C/T and IL-10 -1082G/A polymorphisms with AD has been amply documented previously. In this study we assessed whether people carrying certain genotypes in these polymorphisms were more prone to disease progression as tested by the Mini‐Mental State Examination (MMSE) scores and event-related potentials (ERP). Methods: After blood collection, isolation of DNA and determination of polymorphisms, 226 subjects were tested neuropsychologically using MMSE (including AD patients, mild cognitive impairment patients, patients with other causes of dementia, and healthy controls). ERP were measured by electroencephalography (EEG) in this cohort. Results: MMSE scores were significantly lower in patients carrying the G allele in the IL-1β -1473, T allele in the IL-1α -889, and A allele in the IL-10 -1082 polymorphism. The P300 latency was significantly prolonged in patients carrying the G allele in the IL-1β -1473 polymorphism. Conclusions: Patients carrying risk genotypes in IL-1β -1473, IL-1α -889 and IL-10 -1082 polymorphisms may be susceptible to faster disease progression. Additionally, IL-1β -1473 polymorphism may represent a strong genetic biomarker of AD.

polymorphisms may present with increased amount of pro-inflammatory cytokines (IL-1β and IL-1α) and decreased amount of anti-inflammatory cytokines (IL-10) and consequently be more vulnerable to inflammatory mechanisms that could lead to AD [3,31]. Because event-related potentials (ERP) measured by electroencephalography (EEG) and Mini-Mental State Examination (MMSE) scores show potential in early and differential diagnosis of AD [32,33], we assessed whether MMSE scores and evoked potentials differed among patients carrying particular IL-1β -1473C/G, IL-1α -889C/T and IL-10 -1082G/A genotypes, which would in turn indicate a genetic predisposition to develop AD or for being prone to faster disease progression.

Determination of polymorphisms
Venous blood was collected in plastic syringes with 1 ml of acid citrate dextrose as an anticoagulant.

Measurement of event-related potentials
ERP (P300 and N200) were measured using EEG in the Laboratory for Cognitive and Experimental Neurophysiology at the University Hospital Centre, Zagreb. For ERP measurement, 32 electrodes were placed on the head of patients according to the international 10/20 system. Overall 54 subjects (31 AD, 19 MCI patients and 4 HC) were tested by an auditory oddball paradigm. During the testing, participants were sitting in an auditory sound-proof chamber with headphones on. They had to count all target auditory tones among non-target and interfering tones. Some subjects participated in the auditory oddball paradigm with two frequencies, while the others participated in the auditory oddball paradigm with three frequencies. In the first paradigm, ERP were measured while subjects tried to differentiate target from non-target tone, whereas in the second paradigm, participants tried to differentiate target tone from non-target and interfering tones, as described previously [32].

Statistical analysis
Statistical analysis was performed with SPSS 19.0.1 (SPSS, Chicago, IL, USA), with the level of statistical significance set at α = 0.05. We tested data normality using the Kolmogorov-Smirnov test.
However, non-parametric statistics were mostly used due to the small number of subjects in some groups. MMSE scores and ERP latencies were compared among groups using the non-parametric Kruskal-Wallis test. A post-hoc non-parametric test to correct p values was used for pairwise comparisons.

Discussion
The goal of this study was to test whether polymorphisms in genes for IL-1β, IL-1α and IL-10 are associated with MMSE scores and ERP. Polymorphisms in IL-1β, IL-1α and IL-10 genes could lead to different transcription products and consequently influence the amount of the produced proteins [26][27][28][29][30]. Increase in production of pro-inflammatory cytokines (IL-1β and IL-1α) and decrease in production of anti-inflammatory cytokines (IL-10) would result in increased inflammation that favours development of AD [1,2,31]. We show that MMSE scores are significantly lower in carriers of a G allele in IL-1β -1473, T allele in IL-1α -889, and A allele in IL-10 -1082 polymorphisms, while P300 latency is significantly prolonged in carriers of a G allele in IL-1β -1473 polymorphism.
Association of IL-1β polymorphisms with AD was reported before by numerous studies [43][44][45][46]. IL-1β +3953, IL-1β -31 and IL-1β -511 polymorphisms were mainly tested [43][44][45][46], although the results of our previous study [18] showed that levels of various cerebrospinal fluid (CSF) biomarkers (amyloid The association of SNPs in genes for other pro-inflammatory cytokines and vulnerability of AD was also tested. Pro-inflammatory IL-1α cytokine is overexpressed in AD brain [49]. As presence of a T allele in the IL-1α -889 polymorphism leads to increase in transcriptional activity of the IL-1α gene [29,30], this could explain why carriers of a T allele in the IL-1α -889 polymorphism have higher risk for AD [8][9][10][11][12][13][14][15][16][17]. Our recent study showed that there is no association between CSF AD biomarkers and IL-1α -889 genotypes [18]. However, the present study indicates that although the IL-1α -889 polymorphism is not suitable as an early genetic biomarker of AD, it could be an index of disease severity as patients carrying the T allele in IL-1α -889 polymorphism have significantly lower MMSE scores. According to these results, T allele carriers could be more vulnerable to disease progression.
Our results agree with previous studies showing increased risk of AD in carriers of a T allele in the IL-1α -889 polymorphism [8][9][10][11][12][13][14][15][16][17]46,[50][51][52]. However, due to the facts that many studies did not reveal an association of the IL-1α -889 polymorphism and AD  and that our recent study failed to detect an association of this polymorphism with CSF AD biomarkers [18], these results should be validated in larger cohorts.

Conclusion
In conclusion, our study shows that patients with the IL-1β -1473 G, IL-1α -889 T and IL-10 -1082 A genotypes have significantly lower MMSE scores. Additionally, patients carrying a G allele in theIL-1β -1473 polymorphism have prolonged P300 latencies. These results indicate that these risk genotypes could represent genetic biomarkers of disease progression [87] and as such should be further correlated with neuroimaging and genetic biomarkers of AD. The most important finding of this study is the association of the IL-1β -1473 polymorphism with MMSE scores and P300 ERP. This finding together with our previous results [18] indicates that the IL-1β -1473 polymorphism could be strong genetic biomarker of AD.