3.1 Sample Characteristics
Sample characteristics of the two groups are provided in . The proportion of females is increased in the HIBraak group, and the difference is significant (p=0.019). Subjects in the HIBraak category were significantly older at death than those in the LOBraak group (p<.0001); thus, we incorporated both gender and age-at-death as covariates. APOE allele frequencies(based on pre-determined APOE genotypes) did not differ significantly between groups (p=.556), nor did education level (p=.544).
The distribution of neuritic plaque density was different between groups (p<0.0001). Only 28% of subjects in the LOBraak category had “frequent” or “moderate” plaques, compared to 68% in the HI category. Hence, although Braak score was used to stratify groups by AD-associated neuropathological features, stratification by neuritic plaque burden also occurred.
Results of the test for population stratification indicated that all subjects were assigned to the same cluster, and there was no significant difference between pair-wise IBS distances in the LO and HI Braak groups. Thus, we detected no evidence for population stratification.
Allelic association for one SNP at a time resulted in one SNP, rs4298437 on chromosome 7, showing a Bonferroni-corrected significance level of p = 0.0341. Results for the ten SNPs with lowest p-values are shown in . The Q-Q plot in for all 279,911 SNPs demonstrates that most of the chi-square values perfectly follow theoretically expected values, thus confirming the conclusion of absence of population stratification. The plot also shows that at least ten SNPs have elevated test statistics.
Results of the allele association tests, showing the ten best SNPs, listed in order of significance
Q-Q plot based on nominal p-values resulting from the allelic association test
In the Set Association analysis, the sum of chi-squares for SNPs (; pSum) ranked 1-8 exhibited the smallest significance level, pmin = 0.0273. This analysis indicated that approximately eight SNPs jointly contribute to AD neuropathology although this conclusion is only marginally significant (the significance level associated with pmin, corrected for testing 15 sums, was p = 0.0577). Nonetheless, this result confirms an analogous conclusion based on the QQ plot. For the ten largest chi-square values, also shows p-values based on permutation tests (pRand) but these values are close to those obtained by Bonferroni correction for multiple testing, which indicates that test results for the 279,911 SNPs are largely independent.
For the logistic regression controlling for age of death and sex, the genome-wide distribution of all SNPs and their regression p-values are presented in . The ten SNPs with the lowest p-values for an association with the AD neuropathology phenotype are listed in .Two of the three most significant SNPs, rs4298437 and rs6943822, are in reelin (RELN); the p-value for rs4298437 reached genome-wide significance with the conservative Bonferroni correction (p = .0162). Both reelin SNPs are located in the 5’end of the gene.
Figure 2 Manhattan plot showing the distribution of analyzed SNPs and their corresponding genotypic association p-values from logistic regression. The SNPs are ordered by their positions on the chromosomes, and the chromosomes (number 1 to 22 from left to right) (more ...)
Results of the genotype association test using logistic regression analysis with covariates, showing the 10 SNPs with the lowest p-values, in order of significance
In the post-hoc analysis both reelin SNPs showed highly significant allelic association with the phenotype, as shown in . Alleles associated with the HIBraak group are the A allele of rs4298437 (OR=2.51), and the A allele of rs6943822 (OR=2.23).
Results of post-hoc allelic association analysis of SNPs in RELN
The overlap between the results of the allelic association test and the logistic regression analysis was minimal; three of the most significant SNPs in the former were included among the most significant SNPs in the latter. We attribute two reasons to this apparent discrepancy. First, we incorporated age-at-death and sex as covariates in the logistic regression analysis, whereas the allele test was carried out without inclusion of covariates. Also, the allele test may be less powerful than the genotype test. In spite of these differences, the prominent feature of this analysis is that the RELN SNP rs4298437 is the most significant SNP in both the allele and the genotype (with covariates) tests, with genome-wide significant Bonferroni-corrected p-values of .0341 and .0162, respectively; furthermore, in both tests, a second RELN SNP, rs6943822, is the third most significant SNP.
At the outset of this study, we excluded 101 subjects with mid-range (MED) Braak scores in order to maximize phenotypic differences between groups and avoid potential confounding effects of including subjects in this transitional category. Following the analysis, we examined the frequency of the RELN genotypes in rs4298437 and rs6943822 in this group of subjects. As indicated in , frequencies for both SNPs in the MED Braak subjects were intermediate between those of the LO and HI Braak subjects. This substantiates our approach of using extreme phenotypes rather than introducing the potential confounding effect of choosing an arbitrary midrange value for dichotomizing groups.
Frequencies(n, %) of RELNSNP genotypes in Braak score categories
Despite differences in AD neuropathology, there was no significant difference between HI and LO Braak subjects with respect to the frequency of the APOE
ε4 allele (). This lack of association between the ε4 allele and high levels of AD neuropathology may be related to the advanced age of these subjects (mean age at death in the HI Braak group = 87.8 years; ). This would be consistent with a number of studies that suggest that the positive association between APOE
ε4 and AD declines after the age of 85 years [Juva, et al. 2000
]. In order to further validate this finding, we examined the frequency of APOE
ε4 carriers (at least one ε4 allele) vs. non-carriers among the three genotypes for both RELN
SNPs. As shown in , there were no significant differences in the frequency of ε4 carriers and non-carriers across SNP genotypes for rs6943822
(p = .50) or rs4298437
(p = .13).
Relationship between APOE ε4 frequency and RELN genotypes
3.3 Reelin Immunohistochemistry
Reelin immunohistochemistry was performed on hippocampal sections from 19 (10 from the HI and 9 from the LO Braak group) subjects. Immunohistochemical staining for reelin was also performed on hippocampi from four subjects with clinical and neuropathological diagnoses of AD (Braak stage VI with moderate to frequent neuritic plaques). In general, reelin expression was confined to pyramidal neurons where it was present as variably intense cytoplasmic staining with highest expression in theCA2/3 sector of the hippocampus as previously described[Martinez-Cerdeno, et al. 2002
]. Reelin expression tended to be very pronounced in AD with strong staining encountered especially in regions with many neurons undergoing granulovacuolar degeneration (GVD), a cytoplasmic feature associated with AD (, top). Neurons with mild to moderate GVD in particular tended to have strong cytoplasmic reelin expression; granules and vacuoles themselves did not contain reelin and neurons with marked GVD tended to express less reelin due to extensive degeneration in general and to replacement of cytoplasmic with vacuolar lesions in particular. Reelin staining of neurofibrillary tangles and neuritic plaques was not conspicuous.
Figure 3 Reelin expression in hippocampal pyramidal neurons. Hippocampal immunohistochemistry was performed on paraffin sections of hippocampus and representative pyramidal neurons from the CA2 region are depicted in AD (upper panel) and in non-demented subjects (more ...)
In contrast, cognitively intact subjects in the LO Braak group tended to have lower reelin expression in the hippocampus , whereas reelin expression in the HI Braak group was similar to that observed in AD hippocampi. Hippocampal pyramidal neurons in both the LO and HI Braak groups displayed less GVD than in AD, but the tendency for increased reelin expression in the HI Braak group was still notable in neurons in which GVD was not present or was present to only a very limited extent (, bottom).
Total reelin expression was quantified as described in Methods. In both HI and LO Braak categories, subjects were stratified by two-SNP genotypes for rs4298437 and rs6943822. Total hippocampal reelin expression was significantly reduced in the LO Braak group (p = 0.025; ). In addition, when the LO and HI Braak groups were combined, there was a suggestion of an association between GG homozygotes (for either or both reelin SNPs) and reduced reelin expression, although this trend did not reach statistical significance (p = 0.096).
Figure 4 Analysis of reelin expression in hippocampus. Immunohistochemical (IHC) expression of reelin was scored in the hippocampus of OHSU cases with postmortem interval less than 24 hours. Scores are the sum of semiquantitative assessments of cytoplasmic reelin (more ...)