CRP Gene Variation Affects Early Development of Alzheimer's Disease-related Plaques

Eloise Helena Kok; Mervi Alanne-Kinnunen; Karita Isotalo; Teemu Luoto; Satu Haikonen; Sirkka Goebeler; Markus Perola; Mikko A Hurme; Hannu Haapasalo; Pekka J Karhunen

Disclosures

J Neuroinflammation. 2011;8(96) 

In This Article

Methods

Cohort

The Tampere Autopsy Study (TASTY) cohort comprises 603 men and women aged 0–97 years who were subjected to medico-legal autopsy and generally died out-of-hospital in Finland during the years 2002–2004, representing around 4% of deaths in the Tampere region. None died of AD causes, although 6 (< 1%) were clinically diagnosed with AD during life, 22 (3.7%) were demented and 10 (1.7%) had memory problems. Recorded causes of death are given in Table 1; more detailed causes of death are not available. Further data on illnesses and/or medication use during life are not accessible to the researchers. Autopsies were performed by the department of Forensic Medicine at the University of Tampere and data pertaining to the cases were obtained from doctors and family members where possible. The study was approved by the Board of Medicolegal Affairs of Finland.

Senile Plaques and Neurofibrillary Tangles

SP and NFT assessments were made as previously described.[28] A large number (70%) of cases had 'no SP' and using this skewed data as a continuous variable would make analyses invalid; therefore we categorised the SP into the following categorisations: ≥1 SP (yes/no), and SP typing (no SP, non-neuritic SP (diffuse/primitive), neuritic SP (classic/burnt out)). Analyses also investigated SP density in a semi-quantitative manner, dividing SP counts into 'no SP', 'sparse SP', 'moderate SP' and 'frequent SP', comprising a scoring system based on the CERAD protocol (but without age adjustment). We categorised NFT as: ≥1 NFT (yes/no). NFT and SP were defined by a neuropathologist assessing grid regions of complete brain samples on Bielschowsky-stained slides of frontal cortex (SP) and hippocampus (NFT) in each case. In our cohort, females were older on average by 10 years, causing the category of gender to represent age, however analyses showed similar results when split by gender. Therefore gender was excluded as a covariate in our analyses.

Tissue Microarrays

Tissue microarrays (TMAs) were also constructed (as described in [28]), to allow easier and simultaneous analysis of multiple cases, and held approximately 10–14 cases per block. TMAs were utilised for immunohistochemistry for CRP and Aβ staining. Brain regions that were incorporated into the TMAs were the hippocampal regions CA1, CA2, CA3, and CA4; cerebellum, neocortex (frontal lobe), gyrus cinguli and cerebrum (white matter). Technical difficulties and sample damage precluded inclusion of all TASTY cases, but 92.5% were incorporated.

Genotyping

CRP genotyping was performed at Biomedicum, Helsinki (MA) on the Sequenom MassArray system with the homogeneous Mass Extension (hME) reaction (Sequenom, San Diego, USA) for 6 reported haplotype tagging single nucleotide polymorphisms (SNPs), including rs2794521 (T > C), rs3091244 (C > T > A), rs1800947 (G > C), rs1130864 (C > T), rs1205 (C > T) and rs3093075 (C > A). Haplotyping was calculated with 5 SNPs (SNP order: rs2794521, rs3091244, rs1800947, rs1130864 and rs1205; rs3093075 was excluded as it produced too many low prevalence haplotypes) using the PHASE program[31,32] (version 2.1.1) and indicated five haplotypes with prevalence above 5%.

Immunohistochemistry

Fluorescent immunohistochemical (F-IHC) staining was performed on the TASTY-TMAs in the hippocampal CA1/2 area and utilised DAPI (Sigma-Aldrich, Germany), rabbit anti-CRP (BioLegend, USA), mouse anti-Aβ (Acris Antibodies, Germany), anti-mouse IgG FITC conjugated (Novus Biologicals, USA), anti-rabbit IgG rhodamine conjugated (Antibodies-online, Germany), all according to manufacturer's instructions. For analyses, cases were assessed as positive or negative for staining.

Statistics

Statistical analyses were performed with an SPSS program (version 14.0). Analyses for CRP SNPs and haplotypes used the most common genotype or previously reported 'risk' allele as the reference group and included APOE4 carriership and age as covariates where possible. Their associations were analysed using logistic regression. Chi square analysis was used to determine association with IHC staining. False discovery rate (FDR) multiple correction calculations were performed assuming there were 11 independent tests (6 SNPs and 5 haplotypes), using the calculation below and assuming an FDR value of < 0.05 was acceptable.

Comments

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