Multiple sclerosis and cognitive decline: isApoE-4 a surrogate marker?

Introduction

Multiple sclerosis (MS) is a chronic autoimmunedisease of unknown aetiology, which mostly affectswhite matter. About 43–65% of patients willdevelop subcortical cognitive decline during thedisease (1, 2), thus affecting their daily life.Although several studies have associated atrophy,T1-weighted lesion and new T2 lesion load withearly cognitive impairment, the main aetiologyremains unclear (3, 4).Attention, information processing speed,

memory and executive functions seem to be themost affected areas. Disturbances in the first twodomains are early markers of cognitive decline inMS and are affected even in patients with nomemory complaints (5).Depression, which affects almost 50% of

patients with MS (6), can be a confoundingfactor in cognitive performance, and it is usuallyassessed using the Beck Depression Inventory(BDI) (7).

The apolipoprotein E (ApoE)-4 polymorphismhas been associated with an increased risk ofdeveloping Alzheimer�s disease and �age-relatedmemory decline� (8, 9). Several studies havefocused on the relationship between this alleleand cognitive disturbances in MS patients but theresults have been inconsistent (10–12).Recent genome-wide association studies have

identified a number of single-nucleotide polymor-phisms (SNPs) associated with susceptibility toMS (13–16); among the heritable factors, MHCclass II genes are strongly involved not only insusceptibility but also in disease severity andcognitive decline (17). Although the researchabout genetics and MS has been multiplied inthe last years, we have to take in account thecomplex molecular background of multiple sclero-sis.The aims of the present study were as follows: (i)

to assess the association between the ApoE-4 alleleand cognitive decline in 50 patients with relapsing-remitting MS (RRMS) and mild disability; (ii) to

Acta Neurol Scand 2011: 124: 258–263 DOI: 10.1111/j.1600-0404.2010.01473.x � 2011 John Wiley & Sons A ⁄ SACTA NEUROLOGICA

SCANDINAVICA

Carmona O, Masuet C, Santiago O, Alıa P, Moral E, Alonso-Magdalena L, Casado V, Arbizu T. Multiple sclerosis and cognitivedecline: is ApoE-4 a surrogate marker?Acta Neurol Scand: 2011: 124: 258–263.� 2011 John Wiley & Sons A ⁄S.

Background – The role of the apolipoprotein E (ApoE) polymorphismhas been well demonstrated in neurodegenerative disorders such asAlzheimer. However, its role in multiple sclerosis (MS) remainsunclear. Aims – The aims of our study were as follows: (i) to assesswhether ApoE-4 might be a surrogate marker of cognitive decline inMS; (ii) to confirm the presence of cognitive impairment in mildlydisabled patients treated with interferon-beta; and (iii) to analyse thecorrelation between cognitive disturbances and clinical variables.Material and methods – Fifty relapsing-remitting MS patientsunderwent a battery of neuropsychological tests and were genotypedfor ApoE. Their scores were compared with those of 35 controls.Results – No association was found between ApoE-4 and cognitiveimpairment. Significant differences in most domains were observedbetween MS and the control group. Cognitive decline was not relatedto disability progression. Conclusion – No association betweencognitive impairment and ApoE-4 or clinical markers was detected inour MS patients.

O. Carmona1, C. Masuet2*,O. Santiago1*, P. Al�a3*,E. Moral1*, L. Alonso-Magdalena1*, V. Casado1*,T. Arbizu1*1Multiple Sclerosis Unit, Neurology Department,Hospital Universitari de Bellvitge, L�Hospitalet deLlobregat, Barcelona, Spain; 2Preventive MedicineDepartment, Hospital Universitari de Bellvitge,Barcelona, Spain; 3Biochemistry Department, HospitalUniversitari de Bellvitge, Barcelona, Spain

Key words: apolipoprotein E-4; clinical markers;cognitive decline; interferon-beta; multiple sclerosis

Olga Carmona i Codina, Unitat d�Esclerosi Mfflltiple,Hospital de Bellvitge, Edificio, ICO Consultas externas(Dur�n i Reynals) 2 planta, Gran V�a s ⁄ n. Km. 2,7,08907 Hospitalet de Llobergat, Barcelona, SpainTel.: 93 ⁄ 2607813 – 932607436Fax: 93 2607778e-mail: occodina@gmail.com

*These authors contributed equally to this work.

Accepted for publication November 16, 2010

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assess cognitive impairment in patients with mild-disability MS when compared with healthy controlsubjects; and (iii) to analyse the correlationbetween cognitive disturbances and clinical diseasevariables in our patients.

Material and methods

Patients

The sample comprised 50 patients diagnosed withRRMS according to the McDonald criteria (18,19) and followed up prospectively in the MS Unitof the Hospital de Bellvitge (Barcelona) between1995 and 2005. Patients were recruited consecu-tively from the clinical site if they had RRMSand were treated with interferon-beta-1b. Allparticipants gave written informed consent toparticipate in this study (clinical data and molec-ular genetic studies), which was previouslyapproved by the hospital�s ethics committee(IRB).Patients were excluded if they showed any of the

following: motor and ⁄or sensory disability thatcould interfere with their test performance; ahistory of drug or alcohol abuse; clinical historyof other neurological or psychiatric diseases; arelapse in the previous month, which neededsteroid treatment.The clinical variables analysed were age, sex,

mean duration of the disease and interferontreatment, total number of relapses and degree ofdisability [measured by the Expanded DisabilityStatus Scale, (EDSS)] (20), age at onset of MS andlevel of education.

ApoE genotyping

Blood samples of all patients were obtained duringusual laboratory controls. DNA was extracted withstandard commercial kits and preserved at )20�C.DNA samples were amplified by polymerase chainreaction (PCR), followed by enzymatic digestionand restriction fragment length polymorphism anal-ysis (21). The PCR was made using the followingprimers: forward 5¢ TAA GCT TGG CAC GGCTGT CCA AGG A 3¢ and reverse 5¢ ACA GAATTC GCC CCG GCC TGG TAC AC 3¢.The PCR program was 96�C for 4 min, followed

by 35 cycles of 95�C for 10 s, 61�C for 25 s and72�C for 25 s and a final extension at 72�C for5 min. The 244-bp product was digested with theenzyme Cfo I (Roche). DNA fragments wereseparated by electrophoresis, and band patternswere used to distinguish E2, E3 and E4 genevariants.

Neuropsychological assessment

The neuropsychological assessment consisted of astandardized battery of tests, specifically designedfor cognitive decline in MS. The battery wasadministered in a single, 45-min session by thesame neuropsychologist.The following functions and tests were selected:

attention: direct and reverse digits (Barcelona Test)(22); speed of information processing: Symbol DigitModalities Test (SDMT) (23); Paced AuditorySerial Attention Test (PASAT 3s) (24), and StroopTest (25); verbal fluency: animals for 1 min andwords beginning with �P� for 1 min; memory: visualreproductive memory, immediate and deferred textmemory and word series recalling (Barcelona Test)(22); problem solving: Wisconsin card sorting test(WCST) (26) and the arithmetic subtest of theWechsler Adult Intelligence Scale (WAIS) (27).Mood was assessed with the Spanish version of theBDI (28).All 50 patients and 35 healthy controls under-

went the battery of tests; controls were selectedfrom among patients� relatives and friends whoagreed to participate in the study and we usedgroup matching to obtain comparable distributionsof sex, age and educational level (see Table 1).Tests were performed during routine visits

except when patients were in relapse or had beentreated with steroids in the last month, in whichcase the examination was postponed.Patients were divided into three different groups

(29) based on their test scores: no impairment (0–2failed subtests), mild impairment (3–5 failed sub-tests) and moderate impairment (more than fivefailed subtests).

Statistical analysis

Descriptive analysis was performed and weobtained central (mean and median) and disper-sion (standard deviation, interquartile range) mea-sures depending on whether there was a normal

Table 1 Demographic data for patients and controls

MS patients(n = 50)

Controls(n = 35) P-value

Age (mean � SD) 37.08 � 8.6 38.34 � 14.2 0.64Sex (women) 66% 65.7% 0.97Education in years (mean � SD) 10.62 � 3.6 11.66 � 3.7 0.20EDSS (mean � SD) 2.25 � 1.6 n-aDuration of MS (mean � SD) 9.44 � 7.1 n-aDuration of treatment (mean � SD) 3.24 � 1.34 n-a

n-a, non-attributable.

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distribution (Kolmogorov–Smirnov test). Forquantitative variables, the T–test or Mann–Whit-ney U test was used as appropriate. The chi-square test was used for qualitative comparisonswith normal distribution, or Fisher�s whenrequired.We used linear regression to correlate the risk of

cognitive dysfunction with clinical variables ofdisease. Statistically significant variables withoutinteractions were included in the final model.Significance was set at the 0.05 level (spss 12.0,Chicago, IL, USA).

Results

The mean age of patients at the time of neuropsy-chological assessment was 37.08 (SD 8.6) and 66%(33 ⁄50) were women. The mean time of educationwas 10.62 years (SD 3.6). Median EDSS was 2.25(SD 1.6), and the mean duration of MS was9.44 years (SD 7.1). Patients had been treated withinterferon-beta-1b for a mean time of 3.24 years(SD 1.34).Significant differences were found when com-

paring neuropsychological performance of MSpatients with that of healthy controls on visualand verbal memory, learning subtests and infor-mation processing speed. Unexpectedly, depressionscores (Beck inventory) did not differ between thegroups: 5.63 (SD 6.84) for controls vs 7.98 (SD5.85) for the MS group (P = 0.094).When the two groups were adjusted for sex, no

differences were found in test performance ordepression scores. The neuropsychological testresults are described in Table 2.Most patients (41 ⁄50) presented no signs of

cognitive impairment after nearly 10 years of MS(0–2 failed tests), and only 18% of patients (9 ⁄50)failed 3–5 tests (mild cognitive decline). No patientfulfilled the criteria for moderate cognitive impair-ment (more than five failed tests).Moreover, no correlation was found between the

cognitive decline and disability (EDSS), the pro-gression index, the number of relapses, the meanduration of MS and the exposure to immunomod-ulatory treatment.The ApoE genotype distribution in our sample

was as follows: E2–E2 0%, E2–E3 16%, E3–E368%, E3–E4 10%, E2–E4 4%, E4–E4 2%; becauseof the reduced number of ApoE-4 in our popula-tion we used the subgroup E4 carriers (16%) andE4 non-carriers (84%).No differences between E4 carriers and non-

carriers were found in neuropsychological tests,scores on the BDI or clinical variables. Results areshown in Table 3.

Table 2 Neuropsychological test scores in patients and controls

MS patients Controls

P-value(n = 50) (n = 35)

Verbal fluencyAnimals 60 s 18.56 (5.8) 20.65 (5.1) 0.92*Words beginning with �P � 60 s 12.60 (4.7) 13.54 (4.3) 0.35*

Processing speedStroop words 94 (20) 100 (9.50) 0.032�Stroop colour 59.46 (14.7) 65.37 (14.1) 0.08*Stroop word-colour 36.19 (9.7) 40.78 (13.8) 0.087*PASAT 3 s 35.65 (16.4) 47.76 (10) 0.001*Digit symbol test 41.68 (12.1) 52.14 (18.2) 0.005*

Mental controlInterference )82.74 (24.3) )89.96 (18.7) 0.160*

AttentionDirect digit test 6.00 (1) 5.00 (1) 0.204�Reverse digit test 4.00 (1) 4.00 (2) 0.065�

Abstract reasoning and problem solvingArithmetic WAIS 8.24 (2.7) 9.42 (2.3) 0.046*WSCT number of categories 3.00 (2.25) 4.00 (2.0) 0.113�WSCT correct responses 45.30 (9.1) 47.82 (9.1) 0.221*WSCT trials first category 3.00 (12.0) 2.00 (2.0) 0.057�WSCT perseverations 5.39 (4.5) 3.54 (3.7) 0.052*

Immediate and recall memoryLearning trial 1 4.90 (1.3) 4.42 (1.6) 0.164*Learning trial 2 6.54 (1.3) 6.68 (1.5) 0.641*Learning trial 3 7.00 (2.0) 8.00 (2.0) 0.030�Global learning 18.62 (3.1) 18.88 (3.9) 0.730*Recall learning 4.90 (2.1) 5.85 (2.1) 0.060*Immediate verbal memory 6.76 (2.5) 8.02 (2.8) 0.036*Recall verbal memory 7.24 (2.7) 8.80 (2.8) 0.014*Immediate verbal memory questions 9.00 (4.0) 10.00 (4.0) 0.091�Recall verbal memory questions 8.60 (2.7) 9.62 (2.6) 0.085*

Visual memoryReproduction visual memory 11.10 (2.4) 12.82 (2.4) 0.002*

*T-test (mean and standard deviation).�Mann–Whitney U test (median and interquartile range).

Table 3 Demographic characteristics, clinical characteristics and neuropsycho-logical scores of patients with MS according to ApoE-4 status

E4+(n = 8)

E4)(n = 42) P-value

Sex (women) 66.7% 62.5% 0.558*Age, mean (SD) 33.25 (2.69) 37.81 (1.34) 0.062Educational level, mean (SD) 11.50 (1.39) 10.68 (0.51) 0.550Duration (in years), mean (SD) 10.73 (2.69) 12.05 (1.08) 0.157EDSS, mean (SD) 2.12 (1.38) 2.73 (1.70) 0.292BDI, mean (SD) 5.13 (4.58) 8.54 (5.95) 0.092Verbal fluency, mean (SD) 3.49 (0.22) 3.37 (0.31) 0.312Processing speed, mean (SD) 255.00 (31.92) 260.70 (57.94) 0.716Mental control, mean (SD) 108.50 (16.75) 100.84 (21.32) 0.345Attention, mean (SD) 37.50 (14.94) 47.06 (17.56) 0.165Immediate verbal memory, mean (SD) 39.37 (5.37) 39.97 (8.15) 0.265Recall verbal memory, mean (SD) 23.87 (6.60) 20.02 (7.39) 0.184Mild cognitive impairment ⁄ no impaired 25.0% 23.8% 0.627*

*Chi-square test or Student t-test as required.ApoE, apolipoprotein E; BDI, Beck Depression Inventory; EDSS, Expanded DisabilityStatus Scale.

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We used linear regression to study the risk ofcognitive decline (three or more failed tests),considering the following variables as possibledeterminants: sex (P = 0.776), mean duration ofdisease (P = 0.251), EDSS (P = 0.603), BDIscores (P = 0.622), education level (P = 0.032)and ApoE4 allele (P = 0.757). The education levelwas the only independent variable that predictedcognitive impairment in our patients with anOR = 0.73.

Discussion

In agreement with the literature (10, 11, 29, 30),our results show significant differences in neuro-psychological test performance between healthysubjects and MS patients, including visual andverbal memory, learning subtests and informationprocessing speed, confirming the presence of cog-nitive decline in mildly disabled patients withRRMS.Cognitive deficits may develop in all MS sub-

types (31) and at any disease stage, includingpatients with clinically isolated syndrome (32, 33);moreover, even in this very early stage of disease,cognitive dysfunction may predict disease activity(32) or early conversion to clinical definite MS (34).However, in the present study, cognitive impair-

ment was not related to clinical markers ofprogression (measured by EDSS or progressionindex) as reported in previous series. One possibleexplanation for this could be the low degree ofdisability of our MS patients; in this regard, Amatoet al. (29) failed to find a significant correlationbetween EDSS and cognitive decline in their firstpublished series in the early stages of disease,whereas when they reanalysed the same patientsafter 10 years (30) EDSS was found to be apredictor of poor performance on neuropsycho-logical tests. At all events, this would only partiallyexplain our results, taking into account that ourpatients had been suffering from MS for nearly10 years; differences in disability between the twocohorts (median EDSS 2.25 in our cohort vs 3.5 inthe Italian one) or the effect of immunomodulatorytreatment on cognitive decline (only 24% of Italianpatients were under active treatment) could bealternative explanations. In line with our hypoth-esis, Zivadinov et al. (35) and Koutsis et al. (36)failed to find a correlation between cognitivedecline and EDSS in mildly disabled patients; inthis regard, a possible effect of immunomodulatorytreatment on our patients might explain the lowpercentage of cognitive decline after nearly10 years of disease (18%), which was indeed only�mild� impairment.

In relation to the ApoE polymorphism and inagreement with the findings of other groups (37–41), we found no significant differences in cognitivedecline between ApoE 4 carriers and the remainingpatients. Savettieri (11) reported an inconsistentcorrelation between this polymorphism and cogni-tive impairment: differences were only significantfor the male subgroup, whereas the relationshipwas lost when they analysed all patients; theauthors suggested a possible oestrogenic protectoreffect to explain their results. According to thispoint of view, Beatty et al. (41) previously per-formed a within-gender analysis of neuropsycho-logical performance in 64 patients with MS andonly found differences on verbal and non-verbalmemory tests. The authors adjusted their results bysex following the meta-analysis of Herlitz et al.(42), suggesting a gender difference in verballearning and memory in the healthy population.In contrast, our study found no significant sex

differences among patients or controls, althoughwe are aware that our sample is too small to drawfirm conclusions in this respect.In agreement with the present results, Oliveri

et al. (10), and Parmenter et al. (43) failed tocorrelate cognitive decline with gender or theApoE4 allele (the proportion of allele carriers inboth series was low, below 20%).Unfortunately, none of these studies (including

our own) obtained lesion burden magnetic reso-nance image (MRI) data, which has shown astronger correlation with the ApoE4 allele (44).Recently, Portaccio et al. (45) published their

work on ApoE4 allele and cognitive impairment;although no relation between the allele and cogni-tive decline was found they demonstrated anassociation with cortical damage and verbal learn-ing tasks and subcortical damage with worseperformance on attention and concentration tasksin MS patients. A similar conclusion was found inCOGIMUS study (46), which showed thatincreased T2 lesion volume predicted for anincreased risk of cognitive impairment in non-treated RRMS patients.No correlation between time of exposure to

immunomodulatory treatment and cognitiveimpairment was found in our sample, in contrastto the findings of Flechter et al. (47) and Kapposet al. in BENEFIT trial (48). Longer longitudinalwell-controlled studies would be needed to clarifythe effect of interferon-beta on cognitive decline.

Conclusion

To conclude, our findings confirm the presence ofcognitive disturbances (around 18%) in the mild-

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disability stages of MS with respect to healthycontrols. However, neither clinical markers, sex orthe ApoE4 allele was found to be predictors ofcognitive decline. Large longitudinal studies withMRI data are necessary to clarify the main causesof early cognitive decline in MS, since thesedisturbances can affect their daily life even in theabsence of physical disability.

Acknowledgements

The authors thank Susanna Pobla for her technical supportand to Ana Gonzalez and Mireia Bures for their help incollecting samples.

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