American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 136B:72–74 (2005)

Apolipoprotein E4 Is Probably Responsible for theChromosome 19 Linkage Peak for Parkinson’s DiseaseMaria Martinez,1 Alexis Brice,2 Jenny R. Vaughan,3 Alexander Zimprich,4 Monique M.B. Breteler,5

Giuseppe Meco,6 Alessandro Filla,7 Matthew J. Farrer,8 Christine Betard,9 Andrew Singleton,10

John Hardy,10* Giuseppe De Michele,7 Vincenzo Bonifati,6 Ben A. Oostra,11 Thomas Gasser,4

Nick W. Wood,12 Alexandra Durr,2 the French Parkinson’s Disease Genetics Study Group andthe European Consortium on Genetic Susceptibility in Parkinson’s Disease1INSERM EMI00-06, 523 Place des Terrasses de l’Agora, Evry, France2INSERM U289 and Departement de Genetique, Cytogenetique et Embryologie, Hopital La Pitite-Salpetriere, Paris, France3Department of Neurology, Charing Cross Hospital, London, United Kingdom4Department of Neurodegenerative Diseases, Hertie-Institute for Clinical Brain Research, University of Tubingen, Tubingen, Germany5Department of Epidemiology and Biostatistics, Erasmus MC, Rotterdam, The Netherlands6Department of Neurological Sciences, La Sapienza University, Rome, Italy7Department of Neurological Sciences, Federico II University, Naples, Italy8Neurogenetics Laboratory, Mayo Clinic Jacksonville, Florida9Centre National de Genotypage, Evry, France10Laboratory of Neurogenetics, National Institute on Aging, NIH, Bethesda, Maryland11Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands12Department of Molecular Neuroscience, Institute of Neurology, University College London, London, United Kingdom

Analysis of the apolipoprotein E genotype insibpairs with Parkinson’s disease showed the E4allele was over represented in those who sharedthe adjacent chromosome 19 markers. This sug-gests that apolipoprotein E4 is responsible for thelinkage peak in this region and that it is a modestrisk factor for Parkinson’s disease.� 2005 Wiley-Liss, Inc.

KEY WORDS: Parkinson’s disease; genetics; apo-lipoprotein E

INTRODUCTION

We undertook a genome-wide linkage study in a total of 227affected sib pairs from 199 pedigrees with Parkinson disease(PD) from Europe and North America [Martinez et al., in

press]. In that study, we identified 6 loci that reachedsuggestive linkage scores: chromosomes 2p (117 cM), 5q(130 cM), 6cen (85 cM), 7p (5 cM), 11q (91 cM), and 19q(73 cM). The chromosome 19 peak lies directly above theapolipoprotein E locus (69 cM). Apolipoprotein E4 has beenreported to be a risk factor locus for PD [Kruger et al., 1999]:but these results have been inconsistent [Maraganore et al.,2000; Li et al., 2004].With this background, the apolipoproteingene was a clear candidate gene for the chromosome 19 peak,and so we determined to genotype the sample series to test thehypothesis that the apolipoprotein allele frequencies would bedistorted in those sibpairs, which shared their chromosome 19markers. We have used this approach to indicate that theapolipoprotein E gene is responsible for the chromosome 19linkage peak in late onset Alzheimer’s disease [Myers et al.,2002].

CASES AND MOLECULAR WORK

We have previously described this series of cases (1): Allanalyses were performed using a narrow definition of disease,that is, subjects with definite or probable PD clinical type werecoded as affected. Subjects with possible PD clinical type werecoded as unknown and all remaining subjects were consideredas unaffected.

Two APOE markers genotyped (NCBI names are:APOE_429358 corresponding to SNP position codon 112, andAPOE_7412 corresponding to SNP position codon 158). http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId¼348. Geno-typing was carried out at the Centre National de Genotypage.APOE genotyping method used as previously described [Bollaet al., 1999].

ASSOCIATION METHODS

The null hypothesis of interest, here, is the absence ofassociation in presence of linkage, thus to detect LD withillness, we used the Pedigree Disequilibrium Test, as imple-mented in thePDTprogramv5.1 [Martin et al., 2000]. PDThas

The French Parkinson’s Disease Genetics Study Group: Y Agid,A-M Bonnet, M Borg, A Brice, E Broussolle, Ph Damier, A Destee,A Durr, F Durif, J Feingold, G Fenelon, E Lohmann, M Martinez,C Penet, P Pollak, O Rascol, F Tison, C Tranchant, M Verin, FViallet, M Vidailhet, and J-M Warter.

The European Consortium on Genetic Susceptibility in Parkin-son’s Disease: NW Wood and D Nicholl (UK); A Brice, A Durr, MMartinez, and Y Agid (France); T Gasser and B Muller-Myhsok(Germany); M Breteler, S Harhangi, and B Oostra (The Nether-lands); V Bonifati, E Fabrizio, N Vanacore, GMeco, G DeMichele,G Volpe, and A Filla (Italy).

*Correspondence to: John Hardy, Laboratory of Neurogenetics,National Institute on Aging, National Institutes of Health,Building 35, Room 1A1015, 35 Convent Drive, Room 1A1015MSC 3707, Bethesda, MD 20892-3707.E-mail: hardyj@mail.nih.gov

Received 14 December 2004; Accepted 21 April 2005

DOI 10.1002/ajmg.b.30196

� 2005 Wiley-Liss, Inc.

the ability to use pedigrees without having them broken downinto all individual nuclear families, though it only includesinformative pedigrees, that is, those contributing to the teststatistic. Informative pedigrees include at least one affectedoffspring genotype and can be of two types, depending onwhether or not both parents have DNA available. When bothparents are genotyped, PDT test is based, as the classical chi-square TDT test, on the contribution of marker allele parentaltransmissions to theaffected offspring (‘‘Triads’’). Otherwise, itusesDiscordant Sib Pairs (i.e., affected andunaffected siblingsgenotyped). The PDT test statistic uses a score function, Z, tomeasure allelic association, which is assumed to follow, underthe null hypothesis and asymptotically, a normal distributionwith a mean and a variance equal to 0 and 1, respectively. Inour PD family data, most parents do not have available DNA,thus Z is mainly based on the contribution of DSP data. Formarkers with more than two alleles, PDT provides globalP-values, which assess the significance of transmission distor-tion for all the tested alleles.

We performed additional analyses by generating morehomogenous subsets according to allele sharing in the 19qregion. The ‘‘Chr19q_Linked’’ subset included families with alod score value >0.20 at the D19S420–D19S902 region (i.e.,subset of families with excess (�>70%) sharing in the 19qregion). The remaining families were defined as subset‘‘Chr19q_Unlinked’’. Linkagewas testedusingnon-parametricaffected relative sharing statistics with Merlin [Holtzmanet al., 2000; Abecasis et al., 2002].

RESULTS AND DISCUSSION

In Table I, we show the lodscores generated on Chromosome19 adapted from reference 1. These data show that there is alocus that reaches significance at the position of the apolipo-protein E locus. Also shown is the lodscore and P-value forapolipoprotein E itself, which shows sub significant lodscores,presumably because it is a much less informative marker.Table II shows the genotype and allele frequencies in PD casesand controls showing that, overall, there were no significantdifferences in either allele or genotype frequencies betweenaffected and unaffected family members. These trends wereconfirmed under PDT analyses (Table III). However, in thosesibships in which the markers S420 and S902 cosegregatedwith disease there was a significantly increased frequency ofthe apolipoprotein E4 allele (nominal P-value¼ 0.02). Wefurther evaluated the significance of our PDT analyses using

simulations. Empirical P-values were estimated throughpermutation tests, that is under random permutations ofAPOE alleles in affected and unaffected siblings. One hundredthousands replicates of the (total and subsets) data weregenerated. These empirical P-values permit to estimate thesignificance of the best PDT result correcting for mul-tiple testing of tested alleles. We found that the empiricalP-value associated to our association result in the ‘‘Chr19-linked’’ subsetwas 0.032,which is slightly higher than our bestnominal P-value of 0.02.

These data are important for two reasons: first, theyillustrate the use of sibpair series to identify disease riskalleles in complex diseases and suggest that we and othersshould be able to test candidate genes for our linkage peaks forPD by this method: second they confirm, by a largelyindependent route, that apolipoprotein E4, or an allele inlinkage disequilibrium with apolipoprotein E 4, is a weak risklocus for PD. The mechanism by which apolipoprotein E4 maycontribute to the risk of PD is unclear: it appears to contributeto the risk of Alzheimer’s disease through a mechanisminvolving neuritic plaque formation [Holtzman et al., 2000]and for a similar reason shows an association with dementiawith Lewy bodies [Lippa et al., 1995]. One possibility may bethat E4 contributes to neuronal damage by this mechanismand this is additive to the damage caused by a synucleindependent mechanism. Such hypotheses must remain spec-

TABLE I. Pairwise Non-parametric Lodscore Values onChromosome 19

Marker cM LOD* (P-value)

D19S209 11 �0.13 (0.80)D19S216 20 0.21 (0.20)D19S884 26 0.07 (0.30)D19S221 36 �0.19 (0.80)D19S226 42 �0.06 (0.70)D19S414 54 �0.48 (0.90)D19S220 62 0.07 (0.30)D19S420 66 0.69 (0.04)APOE 72 0.35 (0.10)D19S902 73 0.88 (0.02)D19S418 93 0.00 (0.50)D19S210 100 �0.15 (0.80)

*Lod scores computed under the Kong and Cox affected-relatives sharinglikelihood ratio as implemented in Merlin program [Kong and Cox, 1997;Bolla et al., 1999].

TABLE II. Genotype and Allele Frequencies in Parkinson Casesand Their Unaffected Sibs

Parkinson cases(n¼387)

Unaffectedsibs (n¼ 257)

% (n) % (n)

Genotype22 1.0 (4) 0.8 (2)23 9.3 (36) 14.0 (36)24 2.6 (10) 2.3 (6)33 64.3 (249) 60.7 (156)34 21.2 (82) 21.4 (55)44 1.6 (6) 0.8 (2)

Allele2 7.0 (54) 9.0 (46)3 80.0 (616) 78.0 (403)4 14.0 (104) 13.0 (65)

TABLE III. PDT Results for Association Testing of PD andAPOE: Global and Allele-specific P-values

Allele-specifica

GlobalAPOE-2 APOE-3 APOE-4

Total dataset:103 informativefamilies (7 triads;352 DSPs)

0.21 (�) 0.88 (�) 0.33 (þ) 0.42

‘‘Chr19-linked’’ dataset:23 informativefamilies (0 triads;96 DSPs)

0.16 (�) 0.21 (�) 0.02 (þ) 0.054

Remaining dataset:80 informativefamilies (7 triads;256 DSPs)

0.55 (�) 0.64 (þ) 0.74 (�) 0.75

aP-values are two-sided. In parenthesis (þ or � sign) gives the associationtrend: þ means that allele is associated to a higher risk.Informative for PDT analyses.

Apolipoprotein E Is the Chromosome 19 Parkinson’s Gene 73

ulative until association studies can be carried out on largeseries with pathological confirmation.

REFERENCES

Abecasis GR, Cherny SS, Cookson WO, Cardon LR. 2002. Merlin—rapidanalysis of dense genetic maps using sparse gene flow trees. Nat Genet30:97–101.

Bolla MK, Wood N, Humphries SE. 1999. Rapid determination ofapolipoprotein E genotype using a heteroduplex generator. J Lipid Res40:2340–2345.

Holtzman DM, Bales KR, Tenkova T, Fagan AM, ParsadanianM, SartoriusLJ, Mackey B, Olney J, McKeel D, Wozniak D, Paul SM. 2000.Apolipoprotein E isoform-dependent amyloid deposition and neuriticdegeneration in a mouse model of Alzheimer’s disease. Proc Natl AcadSci USA 97(6):2892–2897.

Kong A, Cox NJ. 1997. Allele-sharing models: LOD scores and accuratelinkage tests. Am J Hum Genet 61:1179–1188.

Kruger R, Vieira-Saecker AM, Kuhn W, Berg D, Muller T, Kuhnl N, FuchsGA, Storch A, Hungs M, Woitalla D, Przuntek H, Epplen JT, Schols L,Riess O. 1999. Increased susceptibility to sporadic Parkinson’s diseaseby a certain combined alpha-synuclein/apolipoprotein E genotype. AnnNeurol 45(5):611–617.

Li YJ, Hauser MA, Scott WK, Martin ER, Booze MW, Qin XJ, Walter JW,Nance MA, Hubble JP, Koller WC, Pahwa R, Stern MB, Hiner BC,

Jankovic J, Goetz CG, Small GW, Mastaglia F, Haines JL, Pericak-VanceMA,Vance JM. 2004.ApolipoproteinE controls the riskandage atonset of Parkinson disease. Neurology 62(11):2005–2009.

Lippa CF, Smith TW, Saunders AM, Crook R, Pulaski-Salo D, Davies P,Hardy J, Roses AD, Dickson D. 1995. Apolipoprotein E genotype andLewy body disease. Neurology 45(1):97–103.

Maraganore DM, Farrer MJ, Hardy JA, McDonnell SK, Schaid DJ, RoccaWA. 2000. Case-control study of debrisoquine 4-hydroxylase, N-acetyltransferase 2, and apolipoprotein E gene polymorphisms inParkinson’s disease. Mov Disord 15(4):714–719.

MartinER,Monks SA,WarrenLL,KaplanNL. 2000.ATest for Linkage andAssociation in General Pedigrees: The Pedigree Disequilibrium Test.Am J Hum Genet 67:146–154.

Martinez M, Brice A, Vaughan J, Zimprich A, Breteler M, Meco G, Filla A,FarrerM,BetardC,Hardy J,DeMicheleG,Bonifati V,OostraB,GasserT, Wood N, Durr A. 2004. The French Parkinson’s Disease GeneticsStudyGroup&European ConsortiumGSPDGenome-wide scan linkageanalysis for Parkinson’s disease: The European Genetic Study of PD.J Med Genet 41:900–907.

MyersA,WavrantDe-VriezeF,HolmansP,HamshereM,CrookR,ComptonD,MarshallH,MeyerD, Shears S, Booth J, RamicD,KnowlesH,MorrisJC, Williams N, Norton N, Abraham R, Kehoe P, Williams H,Rudrasingham V, Rice F, Giles P, Tunstall N, Jones L, Lovestone S,Williams J, Owen MJ, Hardy J, Goate A. 2002. Full genome screen forAlzheimer disease: Stage II analysis. Am J Med Genet 114(2):235–244.

74 Martinez et al.