J7ournal ofNeurology, Neurosurgery, and Psychiatry 1996;61:347-351

Frequency of the apolipoprotein E 84 allele in a

case-control study of early onset Parkinson'sdisease

Alexander S Whitehead, Solange Bertrandy, Finola Finnan, Aileen Butler,George Davey Smith, Yoav Ben-Shlomo

Department ofGenetics andBiotechnologyInstituteA S WhiteheadS BertrandyA ButlerDepartment ofCommunity Healthand General Practice,Trinity College,Dublin, IrelandF FinnanDepartment of SocialMedicine, BristolUniversity, Bristol, UKG Davey SmithY Ben-ShlomoDepartment ofEpidemiology andPublic Health,University College,London, UKY Ben-ShlomoCorrespondence to:Dr Y Ben-Shlomo,Department of SocialMedicine, Canynge Hall,Whiteladies Road, Bristol,England.Received 6 February 1996and in final revised form18 June 1996Accepted 25 June 1996

AbstractObjectives-It has been suggested thatParkinson's disease and Alzheimer's dis-ease may share a common or at least over-

lapping aetiology. The prevalence ofdementia among cases of Parkinson's dis-ease is known to be greater than expectedin the general population. The frequencyof the apolipoprotein E4 allele in a largecase-control study of early onsetParkinson's disease has been examined.Methods-215 patients and 212 populationbased controls were recruited from theRepublic of Ireland between 1992 and1994. Cases had to have disease onset at 55years or younger and be born after 1925.Results-The frequency of the E4 allelewas almost identical between cases ofParkinson's disease (14.6%) and healthycontrols (13.3%). There was no relationbetween E4 status and disease onset, dis-ease duration, Hoehn and Yahr score, anddisease progression. The frequency of theE4 allele was not increased among 10patients with Parkinson's disease withdementia (10.0%) compared with theother patients without dementia (14.8%).There was no association between e4 allelestatus and either a history of smoking,family history of dementia, or Parkinson'sdisease, or being born in a rural area. Theodds ratio for the ApoE e4 allele associ-ated with Parkinson's disease was 1 10(95% confidence interval (95% CI)0.68-179), adjusting for age group, sex,and residential status. The pooled oddsratio from a meta-analysis of six studies ofApoE e4 status and Parkinson's diseasewas 0X94 (95% CI 0X69-1X27).Conclusions-The results from our studyas well as the pooled meta-analysisexclude any important role for ApoE E4status in the development of Parkinson'sdisease. Our results similarly do not sup-

port its role either in dementia associatedwith Parkinson's disease or disease prog-nosis.

(J Neurol Neurosurg Psychiatry 1996;61:347-35 1)

Keywords: Parkinson's disease; apolipoprotein E4;case-control study; meta-analysis

Parkinson's disease is a progressive neurode-generative disorder that affects a significant

number of the elderly population.1 Althoughclinically and pathologically well defined, theunderlying aetiology remains poorly under-stood.2 It is now generally accepted, however,that the pathogenesis of Parkinson's diseasemay well involve both genetic and environ-mental factors.3 Apolipoprotein E (ApoE) is apolymorphic protein with three major iso-forms, E2, E3, and E44 that is present in highconcentrations in brain5 where it is synthe-sised by astrocytes6 7 and can be internalisedby nerve cells utilising a receptor mediatedpathway.8 It may therefore play a part in theaetiology of neurodegenerative diseases.Several studies have established a significantassociation between the E4 allele and bothfamilial late onset Alzheimer's disease andsporadic late onset senile dementia of theAlzheimer type,9-'2 and subsequent studieshave noted similar associations with seniledementia of the Lewy body type. 13-18

There are several reasons for examiningwhether the E4 allele is also a risk factor in thedevelopment of Parkinson's disease: firstly,some investigators have postulated thatParkinson's disease and Alzheimer's diseasemay have a common, or at least overlapping,aetiology.'9 20 Some studies have noted familialaggregation of dementia, amyotrophic lateralsclerosis, and Parkinson's disease2' 22; sec-ondly, the Lewy body is the pathognomonicpathological lesion in Parkinson's disease23 24;thirdly, dementia is more prevalent thanexpected in patients with Parkinson's dis-ease25; fourthly, one report has noted anincreased risk of coronary heart diseaseamong a cohort of patients with Parkinson'sdisease compared with a control population.26As the E4 allele is also associated with anincreased risk of coronary heart disease,27 itcould provide a common link between thesetwo diseases.We report the results of a large genetic

and epidemiological case-control study ofearly onset Parkinson's disease using popula-tion based controls. The aims of which were(a) to establish more precise estimates ofthe risk of Parkinson's disease associated withthe ApoE e4 allele, (b) to examine the inter-actions of £4 with features of clinical diseaseseverity, and (c) to test for interactionsbetween the e4 allele and other knownrisk factors for Parkinson's disease such assmoking habit,28 family history of disease,29and being born and residing in an urban orrural environment.303'

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Subjects and methodsSAMPLE SELECTIONBetween 1992 and 1994 a large populationbased case-control study of early onsetParkinson's disease was conducted in theRepublic of Ireland to examine both environ-mental and genetic risk factors. Cases wereascertained from all neurologists in theRepublic of Ireland, national inpatient data,Parkinson's disease self help groups, and drugadministration records. Only patients fulfillingthe Parkinson's Disease Brain Bank criteriawere included-2 that is, patients wereenrolled if they had bradykinesia and at leastone of the following criteria: (a) resting tremor(4-6 Hz); (b) muscular rigidity; (c) posturalinstability unrelated to primary visual, cerebel-lar, vestibular, or proprioceptive dysfunction.All patients had a physicians' diagnosis ofParkinson's disease and were personally exam-ined by one of the study investigators (YB-S)for the above criteria. Additional inclusion cri-teria for cases were that the initial symptomsof disease had started at the age of 55 oryounger and that date of birth was after 1925.All cases were administered a modified minimental state examination33 excluding the firstfive preliminary questions. A cut off of 17 orless out of 25 was taken to indicate possibledementia, instead of the normal cut off of 20or less out of 30.

Population based controls were ascertainedfrom the electoral register, which is comput-erised and covers 98% of the total popula-tion.34 Controls were randomly selected fromdistrict electoral divisions stratified by anurban-rural indicator from the proportion ofthe population living in towns or cities. Onecontrol was found for every case and was fre-quency matched by sex and age in five yearage groups.35Those who were unable to provide a history

because of dementia, aphasia, or serious psy-chiatric illness were excluded from the studyboth as cases and controls.

EPIDEMIOLOGICAL DATASubjects were all interviewed and details con-cerning clinical history and disability wereobtained. The disability was classified accord-ing to Hoehn and Yahr criteria.'6 Age at onset ofdisease and diagnosis were determined anddisease progression was dichotomised intogood and normal groups. The good groupcomprised patients who were in the top tertileof disease duration for their Hoehn and Yahrscore-that is, a long duration of illness forthat degree of severity of disease. Self reportdata about each residence were collected andthese were classified into "urban" (city/town)or "rural" (village/country). A complete ciga-rette, cigar, and pipe smoking history wasobtained and used to classify subjects intonever smokers, ex-smokers, and current smok-ers. A positive family history for dementia orParkinson's disease was recorded if subjectsstated that a parent, sibling, uncle, aunt, orcousin had had the disease. The positive identi-fication or recollection of tremor only was notaccepted as evidence of Parkinson's disease.

DNA PREPARATIONBlood samples were drawn into EDTA tubes.All samples were blind coded before transferfor the laboratory analysis. Blood wasprocessed to provide crude cell lysates suitablefor amplification by polymerase chain reaction(PCR). Briefly, white blood cell pellets werelysed in 5 ml lysis buffer (50 mM KCI,2-5 mM MgCl2, 20 mM Tris (pH 8 3),0 45% nonidet P-40, 0 45% Tween-20)with 0-2 mg/ml proteinase K (BoehringerMannheim). After incubation for at least 30minutes to dissolve the cell pellet completely,lysates were boiled for 10 minutes to inactivatethe enzyme.

APOLIPOPROTEIN GENOTYPE ANALYSISDNA samples were genotyped for the com-mon ApoE polymorphisms e2, e3, and e4essentially by the method of Hixson andVernier.'7 In brief, the oligonucleotide pri-mers 5'-ACAGAATTCGCCCCGGCCTG-GTACAC-3' and 5'-TAAGCTTGGC-ACGGCTGTCCAAGGA-3' were synthe-sised using an Applied Biosystems PCR-Matemodel; PCR amplification conditions were95°C, five minutes, one cycle; 60°C, oneminute, 70°C, two minutes, 95°C, oneminute, 30 cycles. Products were digested tocompletion with Hha I and resolved by elec-trophoresis through 6% polyacrylamide gels.The ApoE alleles represented in each of thecoded samples were determined by two investi-gators independently, without knowledge ofcase-control status, and before assigning theresulting ApoE genotypes to the correspond-ing cases and controls.

STATISTICAL ANALYSISThe frequency of the ApoE alleles were com-pared by X2 test and Fisher's exact test if theexpected cell count was less than five.Differences in proportions were calculatedusing the Z statistic.'8 For continuous vari-ables, the Kruskal-Wallis one way analysis ofvariance was used as the variables were notnormally distributed.39 Logistic regressionanalysis was used to calculate odds ratios and95% confidence intervals (95% CIs) andadjusted for potential confounders. As con-trols were matched by sex, five year age bands,and stratified by an urban-rural indicator,these variables were included in the multivari-ate model as indicator terms.'5

All papers that have reported the frequencyof the E4 allele in Parkinson's disease wereidentified by using both a Medline search andBIDS, including a citation search. The calcu-lation of the weighted odds ratio in the meta-analysis method was developed by Yusuf andcolleagues.40

Results215 cases and 212 controls were recruited intothe study and blood samples were obtainedfrom 195 (91%) and 166 (78%) respectively.Five cases that did not fulfil the inclusion crite-ria were not included in the analysis. FiveDNA samples (from one case and four con-

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Frequency of the apolipoprotein E e4 allele in a case-control study of early onset Parkinson's disease

Table 1 Description ofsample characteristics

Parkinson's disease Control subjects

Characteristic All Women Men All Women Men

No (%) 189 (100) 66 (34 9) 123 (65-1) 162 (100) 61 (37 7) 101 (62 4)Mean age at entry in study (SD) (y) 56-9 (6 6) 56-8 (7 2) 57 0 (6-3) 58-0 (7-1) 58-1 (8-1) 57-9 (6 4)Mean age at disease onset (SD) (y) (y) 45-6 (7 6) 44-6 (8 7) 46 1 (6 9) N/A N/A N/AFamilyhistoryofdementia 18 (9-5) 6 (9-1) 12 (9 8) 9 (5 5) 2 (3-3) 7 (6 9)Family history of

Parkinson's disease 36 (19-1) 15 (22-7) 21 (17-1) 18 (11 1) 8 (13-1) 10 (9 9)

Table 2 ApoE genotypes and allele frequencies

Parkinson's disease Control subjects

All Women Men All Women Men

Genotype(%) (n = 189) (n = 66) (n = 123) (n = 162) (n = 61) (n = 101)t4/4 1-6 0.0 2-4 1-2 1-6 1.0t3/3 58-7 66-7 54-5 63-0 70 5 58-4t2/2 0-0 0-0 0-0 0-0 0 0 0.0t4/3 23-3 24-2 22-8 23-5 21-3 24-8t3/2 13 8 9.1 16-3 11-7 6-6 14-9A4/2 2-7 0.0 4-1 0-6 0.0 1 0

Allele frequency (n = 378) (n = 132) (n = 246) (n = 324) (n = 122) (n = 202)&2 8-2 4-6 10-2 6-2 3-3 7 9t3 77-3 83-3 74-0 80-6 84-4 78-2t4 14-6 12-1 15-9 13-3 12-3 13 9

trols) could not be amplified by PCR leaving351 subjects (189 cases and 162 controls) forwhom ApoE genotypes could be determined.

Table 1 shows the basic demographicdetails of the subjects. The only significant dif-ference between the cases and controls was in apositive family history of Parkinson's disease(x = 4-2 on 1 df, P = 0-04). The odds ratioof reporting a family history of Parkinson'sdisease was 1*88 (95% CI 101-3*50) havingadjusted for sex, age groups and residentialstatus. Table 2 shows the frequencies of thecommon ApoE alleles; no significant differ-ences in ApoE allele frequencies were appar-ent between cases and controls.

Table 3 shows the relation between theApoE alleles and clinical disease. The meanage of onset for the total sample was 45-6 (SD7-6) years). Cases with the e2 allele were morelikely to have an older age of onset (P =0 046) but did not differ significantly in anyother respect. The prognosis of disease wasnot associated with the £4 allele. There were10 cases with a diagnosis of dementia based onthe mini mental state score. There was littledifference in the frequency of the £4 alleleamong cases of Parkinson's disease either withor without dementia (10% v 14-8%).No significant differences or interactions

were noted in the frequency of the E4 allelewith a positive history of smoking (differencein E4 status for cases compared with controls2-3% v 0-8%), a family history of eitherParkinson's disease (6- 1% v 6-9%) or dementia

Table 3 Relation between clinicalfeatures and allele status

Clinicalfeature £2 allele (31) t3 allele (292) t4 allele (55)

Age at onset of disease (y)* 48-5 45-2 45-8Disease duration (y) 9 0 11-6 11-4Mean Hoehn and Yahr score 2-2 2-5 2-5Proportion with good prognosis 29 0 33-7 29-1Dementia (based on MMSE)

Present 5 0 85-0 10 0Absent 8-4 76-8 14-8

*Kruskal-Wallis X2 = 6-2 on 2 df, P = 0-046. MMSE = mini mental state examination.

(5 4% v 3 6%) , or with being born in a ruralrather than urban environment (1.5% v6-7%). The unadjusted odds ratio for theApoE £4 allele associated with Parkinson's dis-ease was 1 12 (95% CI 070-1-81). The multi-variate odds ratio, adjusting for age group, sex,and residential status, was 1v10 (95% CI0-68-1 79). There were no significant interac-tion terms. A repeat analysis of cases and con-trols that were aged 50 years or younger, with amean age of onset of 35-5 (range 19-45) yearsshowed the e4 allele to be less common incases with Parkinson's disease but this was notstatistically significant (odds ratio 0 35, 95%CI 0d12-1-08).From the seven published reports13 16 41-45

only four papers published the genotype datato enable the calculation of an odds ratio asso-ciated with e4 allele status. We were able toobtain additional data from the author of oneof the other three reports. Only non-dementedcases of Parkinson's disease were includedfrom the report by Arai et al.42 The figureshows the results of the meta-analysis . Four ofthe studies showed a reduced risk associated

Study

Harrington etal13K0

Marder etal44

Rubinsztein et al43

Arai et aI42 K

Koller etal45

Whithead et al

Total

0.1

F- 0-H

hiH1

Odds ratios

III, , , ,10

Meta-analysis ofodds ratios for the association betweenApolipoprotein E E4 status and Parkinson's disease. x2 forhomogeneity = 3 21, Sdf, P = 0-67.

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with the £4 allele status whereas the other twoshowed an increased risk. However, all the95% CIs included unity, reflecting randomvariation. The pooled odds ratio was 094(95% CI 0-69-1-27). This confirms the lack ofany significant association between ApoE £4status and Parkinson's disease, and excludesany clinically important raised risk as the 95%CIs for the pooled estimate are reasonably nar-row. The homogeneity test showed no signifi-cant difference between studies (P = 0 67).

DiscussionRISK OF PARKINSON'S DISEASESeveral studies to date have attempted toassess the possible involvement of the £4 allelein Parkinson's disease.'3 1641 45 All have deter-mined the ApoE genotypes of small samples;although no significant increase in £4 allele fre-quency was found, none of these studies havehad sufficient statistical power to exclude anodds ratio less than three. These studies didnot always have representative samples ofpatients and may have been biased towardsmore atypical cases. No study has specificallyexamined possible interactions between the £4allele and various environmental factors andits association with early onset patients, inwhom the risk conferred by a given geneticcomponent might be more pronounced.Our results provide the most precise esti-

mates of the risk associated between ApoE £4status and Parkinson's disease. We confirmprevious reports that there is no associationbetween ApoE polymorphism and the risk ofParkinson's disease regardless of age at onset.When our results are pooled with other pub-lished reports in the meta-analysis, we canconfidently conclude that if there is any "true"5risk associated with £4 status, it is small and ofvery limited clinical relevance.

PARKINSON'S DISEASE PROGNOSISApoE and the low density lipoprotein receptorhave been postulated to play a part in repairingand maintaining the nervous system.6 Thisfunction, when compromised, may have a rolein the progression of nerve cell degenerationand thus be related to clinical disease progres-sion. Furthermore, there is compelling evi-dence that ApoE is massively expressed afterexperimentally induced nerve damage.58 It ispossible that although the £4 allele is not asso-ciated with risk of developing Parkinson's dis-ease, it could be related to accelerated diseaseprogression and hence worse prognosis. Ourresults, however, do not show any relationbetween £4 status or clinical features, progres-sion, and disability.

PARKINSON'S DISEASE AND DEMENTIAWe failed to find an association between £4status and patients with Parkinson's diseasewith dementia, a finding similar to that of twoother studies4449 but unlike another.42 A recentreport of 50 patients with Alzheimer's diseasewith concomitant pathological damage associ-ated with Parkinson's disease, did show anassociation with £4 status. These patients were

selected on the basis of Alzheimer's diseaseand the parkinsonian pathology "might repre-sent merely the coincidental occurrence ofanother common disorder of the elderly."46The number of cases of dementia in our samplewas small and the diagnosis was based on themini mental state examination. It is possiblethat we failed to show an association becauseof misclassification and an insufficient numberof cases. Alternatively, dementia inParkinson's disease may be of mixed aetiology;some patients may have concurrentAlzheimer's pathology and hence an associa-tion with £4 status whereas other patients donot.47 For example, one study which comparedfive non-demented patients with Parkinson'sdisease with seven cognitively impairedpatients failed to detect any difference in theaccumulation of paired helical filaments in thecingulate and occipital cortices48 suggesting anon-Alzheimer's cause of dementia

APOE AND OTHER ENVIRONMENTAL FACTORSRiggs49 has postulated that the inverse relationfound between smoking and both Parkinson'sdisease and Alzheimer's disease may be linkedto a genetic predisposition to accelerated aging(and therefore premature death) that interactswith smoking. His hypothesis suggests thatstudies of aged (older average age of onset)patients may be depleted of those carrying the£4 allele, because of premature death fromother causes, thereby, at least partly, maskingany inherent risk of Parkinson's disease in car-riers of the £4 allele due to selective loss ofsuch people. It is therefore important to exam-ine the genetic and environmental interactionsbetween smoking, ApoE genotype, andParkinson's disease in a cohort of youngpatients in whom premature mortality from £4associated disease(s) will be less problematic.The results of van Duijn and colleagues failedto show any relation between £4 status, smok-ing, and Alzheimer's disease.50 We too failedto show any difference in £4 status betweensmokers and never smokers either in cases orcontrols. These results do not provide anysupport for Riggs' hypothesis, but cannotrefute it as it is still possible that this hypothesismay be valid for another "genetic candidate".

MISDIAGNOSIS OF PARKINSON'S DISEASEOur study was based on a clinical diagnosis ofParkinson's disease. We know that evenamong patients diagnosed by clinicians with aspecial interest in movement disorders, around25% of patients are misdiagnosed.32 This isreduced to 18% if the Parkinson's DiseaseSociety Brain Bank criteria are applied,5' ashas been done in this study. If it is assumedthat the "other" conditions wrongly labelled asParkinson"s disease have no relation withApoE status, then our study will have underes-timated the potential true relation. This isunlikely to be a serious problem as our nega-tive results are very similar to smaller studieswhich in some cases have been validated atpostmortem'6 and the small degree of misclas-sification would be insufficient to totallyremove a twofold to threefold association.

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ConclusionsThe results of this and other research clearlyindicate no relation between ApoE status andeither the risk of developing Parkinson's dis-ease or its natural history. Furthermore, thelack of an association in this young samplereduces the possibility that selective mortalitymight have depleted the ApoE e4 gene pooland thus produced a spurious negative result.Similarly, no associations were seen with vari-ous environmental risk factors. The differentia-tion between Parkinson's disease, and bothAlzheimer's disease and the "Lewy body vari-ant of Alzheimer's disease" suggests that ApoEcannot provide a unifying explanation forthese conditions.

This work was supported by The Parkinson's Disease Society ofThe United Kingdom. The environmental data came from anepidemiological study funded by the Wellcome Trust. YB-S wassupported by a Wellcome Trust clinical epidemiology fellow-ship. AB was supported by the Irish Health Research Board.Professor Brendan Whelan (Economic and Social ResearchInstitute) provided help with ascertaining control subjects. Inaddition, we thank all the consultant neurologists, theParkinson's Disease Society of Ireland, anonymous referees,and finally all the subjects who made the study possible.

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