Common genetic variation within the Low-Density Lipoprotein Receptor-Related Protein 6 and late-onset Alzheimer’s disease Giancarlo V. De Ferrari*†‡§, Andreas Papassotiropoulos¶ʈ, Travis Biechele*†, Fabienne Wavrant De-Vrieze**, Miguel E. Avila‡, Michael B. Major*†, Amanda Myers**, Katia Sa´ez††, Juan P. Henrı´quez‡‡, Alice Zhao**, M. Axel Wollmerʈ, Roger M. Nitschʈ, Christoph Hockʈ, Chris M. Morris§§, John Hardy**, and Randall T. Moon*†§ *Howard Hughes Medical Institute and †Department of Pharmacology and Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, WA 98195; Departamentos de ‡Bioquı´micay Biologı´aMolecular, ††Estadı´stica,y de ‡‡Biologı´aCelular, Universidad de Concepcio´n, P.O. Box 160-C Concepcio´n 4089100, Chile; ¶Division of Molecular Psychology and Life Sciences Training Facility, Biozentrum, University of Basel, 4055 Basel, Switzerland; ʈDivision of Psychiatry Research, University of Zurich, Lenggstrasse 31, 8029 Zurich, Switzerland; **Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892; and §§Institute for Aging and Health, MRC Building, Newcastle General Hospital, Newcastle-upon-Tyne NE4 6BE, United Kingdom Edited by Elaine Fuchs, The Rockefeller University, New York, NY, and approved April 20, 2007 (received for review May 1, 2006) Genome-wide linkage studies have defined a broad susceptibility of Wnt signaling components may be involved in AD (15–19), region for late-onset Alzheimer’s disease on chromosome 12, leading us to examine whether the LRP6 gene, which encodes a Wnt which contains the Low-Density Lipoprotein Receptor-Related Pro- coreceptor (20–22), is associated with this disease. tein 6 (LRP6) gene, a coreceptor for Wnt signaling. Here, we report the association between common LRP6 variants and late-onset Results Alzheimer’s disease in a multicenter case-control series as well as Genetic Variation in LRP6 Is Associated with Late-Onset AD. We in a large family-based series ascertained by the National Institute sought to confirm the existence of single-nucleotide polymorphisms of Mental Health–National Institute on Aging Genetics Initiative. (SNPs) in exons 7, 11, 14, 16, and 18 of LRP6, which were described As shown in the genome-wide linkage studies, our association in the dbSNP database (National Center for Biotechnology Infor- depends mainly on apolipoprotein E-␧4(APOE-␧4) carrier status. mation) (Fig. 1B). We observed that a nonsynonymous coding Haplotype tagging single-nucleotide polymorphisms (SNPs) with a sequence SNP Ile-1062 3 Val in exon 14 (14e, rs2302685; T 3 C) set of seven allelic variants of LRP6 identified a putative risk and a synonymous SNP in exon 18 (18e, rs1012672; C 3 T) were haplotype, which includes a highly conserved coding sequence polymorphic and therefore we continued our analysis with LRP6 SNP: Ile-1062 3 Val. Functional analyses revealed that the associ- SNPs 14e and 18e. ated allele Val-1062, an allele previously linked to low bone mass, Upon genotyping, 398 Caucasian cases and 339 age- and ethnicity- has decreased ␤-catenin signaling in HEK293T cells. Our study matched controls coming from the Zurich series (23), the Newcastle unveils a genetic relationship between LRP6 and APOE and sup- Brain Bank (U.K. series) (24), and various brain banks throughout ports the hypothesis that altered Wnt/␤-catenin signaling may be the United States (U.S. series) (24) (see Methods), we noted a trend involved in this neurodegenerative disease. toward association in single-locus tests for 14e (P ϭ 0.075, Table 1) and significant association for 18e with AD (P ϭ 0.037, Table 1) in neurodegenerative ͉ LRP-6 ͉ single-nucleotide polymorphism ͉ the combined multicenter sample (Zurich/U.K./U.S. series). Al- APOE ͉ Wnt though 14e was not associated with AD in any of the case-control series analyzed separately, we found that 18e was highly associated ϭ ϭ lzheimer’s disease (AD) [Online Mendelian Inheritance in in the Zurich series (P 0.0048, P 0.011, allelic and genotypic Man (OMIM) 104300], the most common form of age- frequencies, respectively), accounting for most of the effect in the A combined sample (Table 1). LRP6 18e was also associated in the associated dementia, is a progressive neurodegenerative disorder U.K. series (P ϭ 0.039, genotypic frequency, data not shown). characterized by a deficit in cognitive processes that manifest as Given that the evidence for linkage to chromosome 12 is signif- alterations in memory, judgment, and reasoning (1). Although the icant mainly when the analysis has been limited to individuals etiology of AD remains to be fully understood, it is well accepted lacking an APOE-␧4 allele (Fig. 1A), we then stratified the multi- that, along with age, family history is the most prominent risk factor center sample on the basis of whether individuals possessed at least for the development of the disease. one APOE-␧4 allele (7, 8, 10). The combined sample thus stratified Inheritance of the apolipoprotein E-␧4(APOE-␧4) allele is a risk gave 361 individuals corresponding to the ␧4-positive stratum (at factor for AD, including both sporadic and late-onset familial forms of the disease (2, 3). Nevertheless, epidemiological studies estimate ␧ that 42–68% of AD patients do not present the APOE- 4 allele, Author contributions: G.V.D.F., A.P. and T.B. contributed equally to this work; G.V.D.F., suggesting that additional genetic or environmental factors could A.P., T.B., F.W.D.-V., A.M., J.H., and R.T.M. designed research; G.V.D.F., A.P., T.B., M.E.A., play essential roles in the disease (4). A fact consistent with this M.B.M., A.M., J.P.H., A.Z., M.A.W., R.M.N., and C.H. performed research; C.M.M. contrib- uted new reagents/analytic tools; G.V.D.F., A.P., T.B., F.W.D.-V., A.M., K.S., J.H., and R.T.M. observation is that genome-wide screens have identified several analyzed data; and G.V.D.F. wrote the paper. regions that show significant linkage to AD, of which the most likely The authors declare no conflict of interest. to harbor new risk factors are chromosomes 9, 10, and 12 (5–13). The reported linkage peaks for chromosome 12 show significant This article is a PNAS Direct Submission. association with AD, mainly when samples have been stratified Abbreviations: AD, Alzheimer’s disease; SNP, single-nucleotide polymorphism; htSNP, haplotype-tagging SNP; LDLR, Low-Density Lipoprotein Receptor; LRP6, LDLR-Related according to APOE carrier status and cluster into two distinct Protein 6; APOE, apolipoprotein E. Ϸ regions (Fig. 1A). One region is located at the p-ter, from 6–30 §To whom correspondence may be addressed. E-mail: [email protected] or cM, in the vicinity of the Low Density Lipoprotein Receptor [email protected]. (LDLR)-Related Protein 6 (LRP6) (14) gene (at Ϸ26 cM), whereas This article contains supporting information online at www.pnas.org/cgi/content/full/ the other is pericentromeric, from Ϸ48–68 cM close to the LRP1 0603523104/DC1. gene (Ϸ68 cM). We and others have proposed that altered function © 2007 by The National Academy of Sciences of the USA 9434–9439 ͉ PNAS ͉ May 29, 2007 ͉ vol. 104 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603523104 Downloaded by guest on September 27, 2021 A within LRP6 were responsible for the association signal seen for 18e in the nonstratified Zurich case-control series, we assessed the 0 50 100 150 Mayeux et al. Scott et al. Chromosome 12, distance from pter (cM) contribution of the whole gene with a panel of seven haplotype- APOE ε4- APOE ε4- tagging SNPs (htSNPs): rs7316466, rs2284396, rs1012672 (18e), Myers et al. Rogaeva et al. rs2302685 (14e), rs7294695, rs2417086, and rs7308022 (Fig. 1 and APOE ε4- APOE ε4+ 2). Consistent with what has been determined for the LRP6 gene Wu et al. Pericak-Vance et al. in the CEPH population (i.e., Caucasians with ancestry from ε ε APOE 4- APOE 4- northern and western Europe) by the International HAPMAP GAPD Project (25), the region described by our htSNPs had a high degree OLR1 LRP6/BCL-G LRP1 of linkage disequilibrium (Fig. 2B and SI Fig. 4) and did not extend α2M LBP-1c to other chromosome 12 candidate genes previously associated with AD (SI Fig. 4 and A.P., personal communication on OLR1 and B LRP1 in this sample, rs1050286 and rs1799986, respectively). Single LRP6 locus tests for each marker revealed that 18e was the only LRP6 rs2302685 (14e) rs7316466 rs2284396 rs1012672 (18e) rs7294695 rs2417086 rs7308022 htSNP showing association with AD (Fig. 2A and Table 2). Nonetheless, haplotype analysis revealed that a 5-htSNP haplotype could be drawn (Block 1: TTTTC; rs7316466, rs2284396, rs1012672 Chromosome 12 pter ATG (18e), rs2302685 (14e),and rs7294695, respectively; Fig. 2B) and ϭ Fig. 1. Late onset AD and chromosome 12. (A) Summary of reported that this haplotype is strongly associated with AD (P 0.0069, genome-wide linkage regions according to APOE-␧4 carrier status and loca- Table 2). Finally, in agreement with our findings regarding tion of the LRP6 locus relative to candidate genes showing association to AD APOE-␧4 stratification, haplotype analysis of the combined multi- (see also www.alzforum.org/res/com/gen/alzgene/default.asp). (B) Diagram center sample revealed that 14eT/18eT was associated in the of exon–intron boundaries of LRP6 gene and chromosomal position of SNPs combined (P ϭ 0.04) and highly significant in the ␧4-negative (arrows) analyzed in this study. stratum (P ϭ 0.0086) of the multicenter sample (SI Table 5). LRP6 Variants Are Associated with AD in the Original Linkage Sample least one APOE-␧4 allele: 48.9%) and 377 individuals within the to Chromosome 12.