Whole-genome sequencing suggests a chemokine gene cluster that modifies age at onset in familial Alzheimer’s disease

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We have sequenced the complete genomes of 72 individuals affected with early-onset familial Alzheimer’s disease caused by an autosomal dominant, highly penetrant mutation in the presenilin-1 (PSEN1) gene, and performed genome-wide association testing to identify variants that modify age at onset (AAO) of Alzheimer’s disease.

Our analysis identified a haplotype of single-nucleotide polymorphisms (SNPs) on chromosome 17 within a chemokine gene cluster associated with delayed onset of mild-cognitive impairment and dementia.

Individuals carrying this haplotype had a mean AAO of mild-cognitive impairment at 51.0±5.2 years compared with 41.1±7.4 years for those without these SNPs.

This haplotype thus appears to modify Alzheimer’s AAO, conferring a large (~10 years) protective effect.

The associated locus harbors several chemokines including eotaxin-1 encoded by CCL11, and the haplotype includes a missense polymorphism in this gene.

Validating this association, we found plasma eotaxin-1 levels were correlated with disease AAO in an independent cohort from the University of California San Francisco Memory and Aging Center.

In this second cohort, the associated haplotype disrupted the typical age-associated increase of eotaxin-1 levels, suggesting a complex regulatory role for this haplotype in the general population.

Altogether, these results suggest eotaxin-1 as a novel modifier of Alzheimer’s disease AAO and open potential avenues for therapy.

About Eotaxin

The eotaxins are a CC chemokine subfamily of eosinophil chemotactic proteins.

Results

Association of a novel locus on chromosome 17 modifying Alzheimer’s AAO

Association analysis revealed a cluster of single-nucleotide variants comprising a haplotype on chromosome 17 approaching genome-wide significance for association with AAO of MCI (best P-value 6.43E−08). This P-value is genome-wide significant at the empirical threshold for European populations29 and was well supported by association of multiple variants in the region (Figure 1 and Supplementary Table 1). Adjusted for APOE status, the regression model identified the same peak at chromosome 17 and improved the best P to a genome-wide significant 4.85E−08. APOE ε4 was not significant in this model. Permutation testing empirically supported the chromosome 17 peak as the top associated locus in our study (P=1E−06, the lowest achievable P-value; Supplementary Table 2). The regression model using AAO of dementia as the trait of interest also indicated this same region as the top hit, although the overall P-value was increased as a result of the diminished sample size for this variable (Supplementary Table 3). The Mendel software program, accounting for relatedness among the samples, identified the same locus as the top associated region in this study and achieved genome-wide significance (P=2.86E−08; Supplementary Table 4). Remarkably, the effect size of this haplotype was almost 10 years of delayed onset in carriers. Average AAO of MCI in carriers of the protective haplotype was 51.0±5.2 years compared with non-carriers age at MCI 41.1±7 years (mean±s.d.) (Figure 2).

Examination of the regional association plot on chromosome 17 revealed that the associated haplotype spans several chemokines, including some proinflammatory factors reported to be elevated in AD (Figure 3).37 One chemokine subfamily, the monocyte chemoattractant proteins (MCPs), is implicated in neuronal death during sustained inflammation.38 Four major MCPs reside within the genomic neighborhood associated in this study—MCP1 encoded by the CCL2 gene, MCP2 encoded by CCL7 gene, MCP3 encoded by CCL8 gene and MCP4 encoded by CCL13gene. Two additional genes in this locus include CCL1, encoding a well-characterized proinflammatory chemokine,39 and CCL11, which encodes eotaxin-1, a chemokine whose serum and cerebrospinal fluid levels increase with age and correlate with reduced neurogenesis,40 making it a promising candidate for modifying Alzheimer’s AAO.

To identify the genetic variants potentially causal for the large-effect size of the association peak, we functionally annotated all single-nucleotide variants in linkage disequilibrium with our association peak. The haplotype associated with AAO of MCI comprises 22 single-nucleotide variants spanning almost 80kb (Figure 3 and Supplementary Table 5). Of these, the only single-nucleotide variant located within the coding portion of a gene, rs1129844 (NG_012212.1:g.5208G>A, NP_002977.1:p.Ala23Thr), encodes an alanine-to-threonine missense polymorphism at codon 23 in eotaxin-1 (CCL11 A23T). This missense polymorphism lies directly at the signal peptide cleavage site of the eotaxin-1 precursor protein and is predicted to alter its cleavage in silico (Supplementary Figure 3).41Differential secretion of mutant and wild-type eotaxin-1 in HEK-293T cells suggests that this mutation may enhance the secretion of eotaxin-1 (Supplementary Figure 3).

Association of eotaxin-1 levels with Alzheimer’s AAO and effect of the associated haplotype on eotaxin-1 levels in sporadic AD

A prerequisite for generalizability of our results is the presence of this allele in the general population. Within the Colombian data set, rs1129844 (G>A) was observed with a minor allele frequency of 0.14. In the general population, the minor allele frequency for rs1129844 is identical (0.14), making this variant a fairly common polymorphism with a prevalence in the general population matching that of APOE ε4 (minor allele frequency=0.15). Given the presence of this haplotype in the general population, we tested whether plasma levels of eotaxin-1 correlate with AAO in an independent Alzheimer’s cohort at the UCSF Memory and Aging Center (n=152). After controlling for CDR, APOE ε4 genotype and the age gap variable, we found that higher plasma eotaxin-1 levels were significantly correlated with higher AAO (F(6, 145)=2.81; P=0.012; β=0.0252, s.e.=0.0122; t-value=2.07, F-test for linear regression). APOE ε4 genotype was not significant in this model.

Finally, we asked whether the haplotype identified in the Antioquian early-onset Alzheimer’s cohort affects eotaxin-1 levels in the UCSF Memory and Aging Center Alzheimer’s cohort. In agreement with previous studies,40 eotaxin-1 levels increased with age in the total cohort. However, when we stratified these samples by the presence of the onset-associated haplotype, we found that this haplotype decoupled the relationship between age and plasma levels of eotaxin-1 (Figure 4). In other words, we observed a linear increase in eotaxin-1 levels for non-haplotype carriers but not for haplotype carriers. This decoupling suggests that this haplotype exerts a complex regulatory effect on eotaxin-1 levels. Although the age-associated increase in eotaxin-1 levels has been correlated with reduced neurogenesis and memory impairment,40 eotaxin-1 may elicit a hormetic response curve with normal or even neuroprotective effects within a certain interval, and deleterious effects at higher levels. By abrogating the linear increase between eotaxin-1 levels and age, the haplotype identified in this study may effectively constrain eotaxin-1 levels within this normal/protective realm. We observed a trend toward a protective effect of the associated haplotype on AAO in the UCSF cohort, although this effect was not significant (Supplementary Figure 5).

Figure 4.

Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorThe associated haplotype decouples plasma eotaxin-1 levels from age in haplotype carriers. Shaded regions show 95% confidence limits for the mean.

Full figure and legend (70K)Download PowerPoint slide (200 KB)


In silico identification of functional variants in extreme onset individuals

A complementary approach to association testing is bioinformatically screening the catalog of genetic variation we generated through whole-genome sequencing to identify putative functional variants. This bioinformatic screen for functional variants present in individuals at one extreme for AAO predicted eight variants to be deleterious to protein function (SIFT score less than or equal to0.1) (Supplementary Table 6). Among these was rs150955128 (NM_000418.3:c.554G>A, NP_000409.1:p.Arg185His), an arginine-to-histidine amino-acid substitution at codon 185 of the interleukin 4 receptor (IL4R). The variant is present in seven of the late-onset genomes and absent from the early-onset genomes. An additional 8 of the 72 PSEN1 mutation carriers also have this variant. Of the predicted deleterious variants, carriers of rs150955128 displayed the largest difference in AAO versus non-carriers (50.5 versus 43.2 years), suggesting a protective effect in carriers of this variant. This variant is present with a minor allele frequency around 1% (G>A) but the Exome Aggregation Consortium browser (http://exac.broadinstitute.org/) revealed that the variant is largely restricted to Latino populations and virtually non-existent in Asian, African or European populations. This finding also underscores the importance of including racially and ethnically diverse populations in GWAS.42

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Discussion

Through whole-genome sequencing of 72 individuals affected with early-onset familial AD caused by an E280A mutation in PSEN1, we have identified a haplotype on chromosome 17 associated with delayed AAO of MCI and dementia. The identified haplotype spans several chemokines, including CCL2, a proinflammatory chemokine implicated in the chronic neuroinflammation accompanying AD.18 In our study, the associated haplotype confers ~10 years of protection against AD onset among carriers in the Antioquian early-onset AD cohort. In the general population, this haplotype is relatively common, with an expected prevalence of around one in four people. Within this haplotype, we identified a missense polymorphism in eotaxin-1 (CCL11 A23T) lying directly at the signal peptide cleavage site and therefore we predict this variant may alter the cleavage and secretion of this chemokine. Indeed, we found enhanced secretion of the variant protein in an in vitro expression study. Extensive pathological commonalities between the Antioquian familial AD and the general form of the disease, and the relatively common prevalence of the identified haplotype, lend hope that this haplotype functions similarly (i.e., protectively) in the general population. In the UCSF cohort, we observed a protective trend for this haplotype on AAO, which requires a larger sample to validate.

Eotaxin-1 levels increase throughout life and this increase is correlated with reduced neurogenesis.40 This raises the intriguing possibility that eotaxin-1 is a molecular effector of aging, the largest risk factor for developing AD. Here we show that plasma eotaxin-1 levels are correlated with AAO in an independent cohort of Alzheimer’s patients, implicating this chemokine as a novel modulator of Alzheimer’s AAO. Furthermore, carriers of the chromosome 17 haplotype in the UCSF cohort did not exhibit the typical increase of chemokine levels with age. As chemokines mediate both neuroprotection and injury,43 we postulate a hormesis response model for eotaxin-1 levels whereby low to moderate levels of this chemokine elicit a normal or protective response, but higher levels ultimately lead to neurodegeneration and memory impairment. By decoupling eotaxin-1 levels from age, the haplotype identified in this study may protect against the deleterious effects accompanying high levels of this chemokine.

The protein product of CCL11 resulting from differential signal peptide cleavage is predicted to retain additional amino-acid residues at its N terminus, a key region specifying binding and activity of chemokines.44 Recent structural studies have implicated a critical role for this region of eotaxin-1 in binding and activation of its receptor CCR3.45 Similar to other AD-associated immune-response genes, this receptor is expressed highly in microglia.14 Therefore, it is reasonable to hypothesize altered eotaxin-1 signaling modulates neuroinflammation among carriers of the CCL11 A23T mutation.

Individuals at the extremes of a trait distribution are likely to be enriched for functional variants underlying that trait.

Through bioinformatic prediction of variants likely to alter gene function, we identified a Latino-specific variant in IL4R enriched in the latest-onset individuals in this study.

Similar to the eotaxin-1 receptor, IL4R is also highly expressed in microglia14 and its ligand, the anti-inflammatory cytokine interleukin-4, attenuates AD symptoms in transgenic mice.46

Another ligand for IL4R is interleukin-13.

Taken together, interleukin-4 and interleukin-13 induce clearance of β-amyloid and improve memory in transgenic mice.47

As these cytokines are known to cause the release of eotaxin-1 in certain human cell types upon binding IL4R,48 we suggest that eotaxin-1 release may mediate β-amyloid clearance.

This finding thus dovetails with our implication of eotaxin-1 in modulating AAO, and highlights cytokine-mediated neuroinflammation as a promising pathway for therapeutic intervention in AD.

Clarifying the precise relationship between the identified haplotype and eotaxin-1 levels, and identifying the primary cellular sources and regulators of this chemokine in both healthy and diseased states, warrant further study.

As the incidence of AD doubles approximately every 5 years,49 delaying the onset by this amount would consequently halve the disease incidence.

In this study, we identified a protective haplotype conferring a ~10-year protective effect in one monogenic early-onset Alzheimer’s population.

Therapies based on this protective haplotype offer the potential to reduce markedly the incidence of AD while enhancing the quality of life of millions of individuals.

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