Genome-wide screen identifies rs646776 near sortilin as a regulator of progranulin levels in human plasma.

Recent studies suggest progranulin (GRN) is a neurotrophic factor. Loss-of-function mutations in the progranulin gene (GRN) cause frontotemporal lobar degeneration (FTLD), a progressive neurodegenerative disease affecting ∼10% of early-onset dementia patients. Using an enzyme-linked immunosorbent assay, we previously showed that GRN is detectable in human plasma and can be used to predict GRN mutation status. This study also showed a wide range in plasma GRN levels in non-GRN mutation carriers, including controls. We have now performed a genome-wide association study of 313,504 single-nucleotide polymorphisms (SNPs) in 533 control samples and identified on chromosome 1p13.3 two SNPs with genome-wide significant association with plasma GRN levels (top SNP rs646776; p = 1.7 × 10⁻³°). The association of rs646776 with plasma GRN levels was replicated in two independent series of 508 controls (p = 1.9 × 10⁻¹9) and 197 FTLD patients (p = 6.4 × 10⁻¹²). Overall, each copy of the minor C allele decreased GRN levels by ∼15%. SNP rs646776 is located near sortilin (SORT1), and the minor C allele of rs646776 was previously associated with increased SORT1 mRNA levels. Supporting these findings, overexpression of SORT1 in cultured HeLa cells dramatically reduced GRN levels in the conditioned media, whereas knockdown of SORT1 increased extracellular GRN levels. In summary, we identified significant association of a locus on chromosome 1p13.3 with plasma GRN levels through an unbiased genome-wide screening approach and implicated SORT1 as an important regulator of GRN levels. This finding opens avenues for future research into GRN biology and the pathophysiology of neurodegenerative diseases.

Investigation of 15 of the top candidate genes for late-onset Alzheimer's disease.

The 12 genome-wide association studies (GWAS) published to-date for late-onset Alzheimer's disease (LOAD) have identified over 40 candidate LOAD risk modifiers, in addition to apolipoprotein (APOE) ε4. A few of these novel LOAD candidate genes, namely BIN1, CLU, CR1, EXOC3L2 and PICALM, have shown consistent replication, and are thus credible LOAD susceptibility genes. To evaluate other promising LOAD candidate genes, we have added data from our large, case-control series (n=5,043) to meta-analyses of all published follow-up case-control association studies for six LOAD candidate genes that have shown significant association across multiple studies (TNK1, GAB2, LOC651924, GWA_14q32.13, PGBD1 and GALP) and for an additional nine previously suggested candidate genes. Meta-analyses remained significant at three loci after addition of our data: GAB2 (OR=0.78, p=0.007), LOC651924 (OR=0.91, p=0.01) and TNK1 (OR=0.92, p=0.02). Breslow-Day tests revealed significant heterogeneity between studies for GAB2 (p<0.0001) and GWA_14q32.13 (p=0.006). We have also provided suggestive evidence that PGBD1 (p=0.04) and EBF3 (p=0.03) are associated with age-at-onset of LOAD. Finally, we tested for interactions between these 15 genes, APOE ε4 and the five novel LOAD genes BIN1, CLU, CR1, EXOC3L2 and PICALM but none were significant after correction for multiple testing. Overall, this large, independent follow-up study for 15 of the top LOAD candidate genes provides support for GAB2 and LOC651924 (6q24.1) as risk modifiers of LOAD and novel associations between PGBD1 and EBF3 with age-at-onset.

Erratum to: Genetically-controlled Vesicle-Associated Membrane Protein 1 expression may contribute to Alzheimer's pathophysiology and susceptibility.

Following publication of this work, we noticed that we inadvertently failed to include Dr Ferenc Deák in the author list. The author list has now been corrected and the amended authors' contributions section has been modified accordingly below.

Genetically-controlled Vesicle-Associated Membrane Protein 1 expression may contribute to Alzheimer's pathophysiology and susceptibility.

BACKGROUND: Alzheimer's disease is a neurodegenerative disorder in which extracellular deposition of ß-amyloid (Aß) oligomers causes synaptic injury resulting in early memory loss, altered homeostasis, accumulation of hyperphosphorylated tau and cell death. Since proteins in the SNAP (Soluble N-ethylmaleimide-sensitive factor Attachment Protein) REceptors (SNARE) complex are essential for neuronal Aß release at pre-synaptic terminals, we hypothesized that genetically controlled SNARE expression could alter neuronal Aß release at the synapse and hence play an early role in Alzheimer's pathophysiology. RESULTS: Here we report 5 polymorphisms in Vesicle-Associated Membrane Protein 1 (VAMP1), a gene encoding a member of the SNARE complex, associated with bidirectionally altered cerebellar VAMP1 transcript levels (all p<0.05). At the functional level, we demonstrated that control of VAMP1 expression by heterogeneous knockdown in mice resulted in up to 74% reduction in neuronal Aß exocytosis (p<0.001). We performed a case-control association study of the 5 VAMP1 expression regulating polymorphisms in 4,667 Alzheimer's disease patients and 6,175 controls to determine their contribution to Alzheimer's disease risk. We found that polymorphisms associated with increased brain VAMP1 transcript levels conferred higher risk for Alzheimer's disease than those associated with lower VAMP1 transcript levels (p=0.03). Moreover, we also report a modest protective association for a common VAMP1 polymorphism with Alzheimer's disease risk (OR=0.88, p=0.03). This polymorphism was associated with decreased VAMP1 transcript levels (p=0.02) and was functionally active in a dual luciferase reporter gene assay (p<0.01). CONCLUSIONS: Genetically regulated VAMP1 expression in the brain may modify both Alzheimer's disease risk and may contribute to Alzheimer's pathophysiology.

Linking protective GAB2 variants, increased cortical GAB2 expression and decreased Alzheimer's disease pathology.

GRB-associated binding protein 2 (GAB2) represents a compelling genome-wide association signal for late-onset Alzheimer's disease (LOAD) with reported odds ratios (ORs) ranging from 0.75-0.85. We tested eight GAB2 variants in four North American Caucasian case-control series (2,316 LOAD, 2,538 controls) for association with LOAD. Meta-analyses revealed ORs ranging from (0.61-1.20) with no significant association (all p>0.32). Four variants were hetergeneous across the populations (all p<0.02) due to a potentially inflated effect size (OR = 0.61-0.66) only observed in the smallest series (702 LOAD, 209 controls). Despite the lack of association in our series, the previously reported protective association for GAB2 remained after meta-analyses of our data with all available previously published series (11,952-22,253 samples; OR = 0.82-0.88; all p<0.04). Using a freely available database of lymphoblastoid cell lines we found that protective GAB2 variants were associated with increased GAB2 expression (p = 9.5×10(-7)-9.3×10(-6)). We next measured GAB2 mRNA levels in 249 brains and found that decreased neurofibrillary tangle (r = -0.34, p = 0.0006) and senile plaque counts (r = -0.32, p = 0.001) were both good predictors of increased GAB2 mRNA levels albeit that sex (r = -0.28, p = 0.005) may have been a contributing factor. In summary, we hypothesise that GAB2 variants that are protective against LOAD in some populations may act functionally to increase GAB2 mRNA levels (in lymphoblastoid cells) and that increased GAB2 mRNA levels are associated with significantly decreased LOAD pathology. These findings support the hypothesis that Gab2 may protect neurons against LOAD but due to significant population heterogeneity, it is still unclear whether this protection is detectable at the genetic level.

Association and heterogeneity at the GAPDH locus in Alzheimer's disease.

Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and its paralogs were implicated in late-onset Alzheimer's disease (LOAD), although the strength and direction of association have not been consistent. We genotyped 3 previously reported single nucleotide polymorphisms (SNPs; rs3741916-GAPDH 5' UTR, rs2029721-pGAPD, and rs4806173-GAPDHS) in 3 case-control series (2112 cases and 3808 controls). Rs3741916 showed the strongest LOAD association (p = 0.003). The minor allele of rs3741916 showed a protective effect in our combined series (odds ratio [OR] = 0.87%, 95% confidence interval [CI] = 0.79-0.96). This is consistent with results from the 2 published follow-up studies and in opposite direction of the original report. Meta-analysis of the published series with ours suggests presence of heterogeneity (Breslow-Day p < 0.0001). Meta-analysis of only the follow-up series including ours revealed a significant protective effect for the minor allele of rs3741916 (OR = 0.85%, 95% CI = 0.76-0.96, p = 0.009). Our results support the presence of LOAD variants and heterogeneity at the GAPDH locus. The most promising rs3741916 variant is unlikely to be functional given opposing effects in different series. Identification of functional variant(s) in this region likely awaits deep sequencing.

Replication of BIN1 association with Alzheimer's disease and evaluation of genetic interactions.

The most recent late-onset Alzheimer's disease (LOAD) genome-wide association study revealed genome-wide significant association of two new loci: rs744373 near BIN1 (p = 1.6 × 10-11) and rs597668 near EXOC3L2/BLOC1S3/MARK4 (p = 6.5 × 10-9). We have genotyped these variants in a large (3,287 LOAD, 4,396 controls), independent dataset comprising eleven case-control series from the USA and Europe. We performed meta-analyses of the association of these variants with LOAD and also tested for association using logistic regression adjusted by age-at-diagnosis, gender, and APOE ε4 status. Meta-analysis results showed no evidence of series heterogeneity and logistic regression analysis successfully replicated the association of BIN1 (rs744373) with LOAD with an odds ratio (OR = 1.17, p = 1.1 × 10-4) comparable to that previously reported (OR = 1.15). The variant near EXOC3L2 (rs597668) showed only suggestive association with LOAD (p = 0.09) after correcting for the presence of the APOE ε4 allele. Addition of our follow-up data to the results previously reported increased the strength of evidence for association with BIN1 (11,825 LOAD, 32,570 controls, rs744373 Fisher combined p = 3.8 × 10-20). We also tested for epistatic interaction between these variants and APOE ε4 as well as with the previously replicated LOAD GWAS genes (CLU: rs11136000, CR1: rs3818361, and PICALM: rs3851179). No significant interactions between these genes were detected. In summary, we provide additional evidence for the variant near BIN1 (rs744373) as a LOAD risk modifier, but our results indicate that the effect of EXOC3L2 independent of APOE ε4 should be studied further.

Replication of EPHA1 and CD33 associations with late-onset Alzheimer's disease: a multi-centre case-control study.

BACKGROUND: A recently published genome-wide association study (GWAS) of late-onset Alzheimer's disease (LOAD) revealed genome-wide significant association of variants in or near MS4A4A, CD2AP, EPHA1 and CD33. Meta-analyses of this and a previously published GWAS revealed significant association at ABCA7 and MS4A, independent evidence for association of CD2AP, CD33 and EPHA1 and an opposing yet significant association of a variant near ARID5B. In this study, we genotyped five variants (in or near CD2AP, EPHA1, ARID5B, and CD33) in a large (2,634 LOAD, 4,201 controls), independent dataset comprising six case-control series from the USA and Europe. We performed meta-analyses of the association of these variants with LOAD and tested for association using logistic regression adjusted by age-at-diagnosis, gender, and APOE ε4 dosage. RESULTS: We found no significant evidence of series heterogeneity. Associations with LOAD were successfully replicated for EPHA1 (rs11767557; OR = 0.87, p = 5 × 10-4) and CD33 (rs3865444; OR = 0.92, p = 0.049), with odds ratios comparable to those previously reported. Although the two ARID5B variants (rs2588969 and rs494288) showed significant association with LOAD in meta-analysis of our dataset (p = 0.046 and 0.008, respectively), the associations did not survive adjustment for covariates (p = 0.30 and 0.11, respectively). We had insufficient evidence in our data to support the association of the CD2AP variant (rs9349407, p = 0.56). CONCLUSIONS: Our data overwhelmingly support the association of EPHA1 and CD33 variants with LOAD risk: addition of our data to the results previously reported (total n > 42,000) increased the strength of evidence for these variants, providing impressive p-values of 2.1 × 10-15 (EPHA1) and 1.8 × 10-13 (CD33).

Replication of CLU, CR1, and PICALM associations with alzheimer disease.

OBJECTIVE: To test for replication of the association between variants in the CLU, CR1, and PICALM genes with Alzheimer disease. DESIGN: Follow-up case-control association study. SETTING: The Mayo Clinics at Jacksonville, Florida, and Rochester, Minnesota. PARTICIPANTS: Community-based patients of European descent with late-onset Alzheimer disease (LOAD) and controls without dementia who were seen at the Mayo clinics, and autopsy-confirmed cases and controls whose pathology was evaluated at the Mayo Clinic in Jacksonville. Additional samples were obtained from the National Cell Repository for Alzheimer Disease (NCRAD). A total of 1829 LOAD cases and 2576 controls were analyzed. INTERVENTIONS: The most significant single-nucleotide polymorphisms in CLU (rs11136000), CR1 (rs3818361), and PICALM (rs3851179) were tested for allelic association with LOAD. Main Outcome Measure Clinical or pathology-confirmed diagnosis of LOAD. RESULTS: Odds ratios for CLU, CR1, and PICALM were 0.82, 1.15, and 0.80, respectively, comparable in direction and magnitude with those originally reported. P values were 8.6 x 10(-5), .014, and 1.3 x 10(-5), respectively; they remain significant even after Bonferroni correction for the 3 single-nucleotide polymorphisms tested. CONCLUSION: These results show near-perfect replication and provide the first additional evidence that CLU, CR1, and PICALM are associated with the risk of LOAD.