Apolipoprotein E-Ε4 Is Associated with Lesser Cognitive Dysfunction in HCV-Afflicted Patients

Wozniak 1

Apolipoprotein E-ε4 deficiency and cognitive function in hepatitis C virus -infected patients

M. A. Wozniak, PhD,1 L. M. Lugo Iparraguirre,2 M. Dirks, MD,2 M. Deb-Chatterji,MD,2 H. Pflugrad,MD,2 A. Goldbecker,MD,2 A. B.Tryc, MD,2 H.Worthmann,MD,2 M. Gess, MD, 3 M. M.E. Crossey,4 D. M. Forton,MD,3 S. D. Taylor-Robinson, Professor,4 R. Itzhaki, Professor,1* K. Weissenborn,Professor.2

1Faculty of Life Sciences, University of Manchester, Manchester M60 1QD, United Kingdom

2Dept. of Neurology, Hannover Medical School, 30623 Hannover, Germany

3Dept. of Gastroenterology and Hepatology, St George’s Hospital and Medical School, London SW17 0QT, United Kingdom

4 Dept. of Medicine, St Mary´s Hospital Campus, Imperial College London, London W2 1NY, United Kingdom

*Present address: Nuffield Dept. Clinical Neurosciences, University of Oxford, Level 6, West Wing, John Radcliffe Hospital, Oxford, OX3 9DU

Corresponding Author:

Professor Karin Weissenborn

Department of Neurology

Hannover Medical School,

30623 Hannover, Germany

Phone: 0049-511-532-2339; Fax: 0049-511-532-3115;

e-mail:

running title: ApoE and cognition in HCV patients
e-mail addresses of all authors:

Matthew A Wozniak:

Lea Margrit Lugo Iparraguirre:

Meike Dirks:

Milani Deb-Chatterji:

Henning Pflugrad:

Annemarie Goldbecker:

Anita Blanka Tryc:

Hans Worthmann:

Markus Gess:

Mary ME Crossey:

Daniel M Forton:

Simon D Taylor-Robinson:

Ruth Itzhaki:

Karin Weissenborn:

The statistical analysis of the data was conducted by M.A. Wozniak; Faculty of Life Sciences, University of Manchester, Manchester M60 1QD, United Kingdom

Supplemental references and tables are available electronically (SR-1 to SR-10, table e-1, table e-2)

Study funding: The genotyping was partially financed by a grant from Pfizer Inc. to SDT-R. SDT-R and MMEC are grateful to the United Kingdom National Institute for Health Research (NIHR) Biomedical Facility at Imperial College London for infrastructure support. MMEC is supported by a Fellowship from the Sir Halley Stewart Trust (Cambridge, UK).

Search terms: All Clinical Neurology [14], All Cognitive Disorders [25], Viral Infections [142

Word count:

Abstract: 227 words

Main Text: 2878 words

Number of tables: 2

Number of figures: 0

Authors’ Contributions

Matthew A Wozniak: study concept and design, APO E genotyping, analysis and interpretation of data, statistical analysis, drafting of the manuscript; critical revision of the manuscript for important intellectual content

Lea Margret Lugo Iparraguirre: recruitment of participants, psychometric testing, analysis and interpretation of the data, critical revision of the manuscript for important intellectual content

Meike Dirks: recruitment of participants, psychometric testing, analysis and interpretation of the data, critical revision of the manuscript for important intellectual content

Milani Deb-Chatterji: recruitment of participants, critical revision of the manuscript for important intellectual content

Henning Pflugrad: recruitment of participants, psychometric testing, analysis and interpretation of the data, critical revision of the manuscript for important intellectual content

Annemarie Goldbecker: recruitment of participants, analysis and interpretation of the data, critical revision of the manuscript for important intellectual content

Anita Blanka Tryc: recruitment of participants, critical revision of the manuscript for important intellectual content

Hans Worthmann: recruitment of participants, critical revision of the manuscript for important intellectual content

Markus Gess: critical revision of the manuscript for important intellectual content

Mary ME Crossey: critical revision of the manuscript for important intellectual content

Daniel M Forton: drafting of the manuscript, critical revision of the manuscript for important intellectual content

Simon D Taylor-Robinson: drafting of the manuscript, critical revision of the manuscript for important intellectual content, obtained funding

Ruth Itzhaki: study concept, interpretation of data; drafting of the manuscript; critical revision of the manuscript for important intellectual content

Karin Weissenborn: study concept and design, recruitment of participants, analysis and interpretation of data, drafting of the manuscript; critical revision of the manuscript for important intellectual content

Disclosures

Matthew A Wozniak reports no disclosures.

Lea Margrit Lugo Iparraguirre reports no disclosures

Meike Dirks reports no disclosures

Milani Deb-Chatterji reports no disclosures

Henning Pflugrad reports no disclosures

Annemarie Goldbecker reports no disclosures

Anita Blanka Tryc reports no disclosures

Hans Worthmann reports no disclosures

Markus Gess reports no disclosures

Mary ME Crossey reports no disclosures

Daniel M Forton reports no disclosures

Simon D Taylor-Robinson reports a grant from Pfizer Inc. that helped financing the genotyping

Ruth Itzhaki reports no disclosures

Karin Weissenborn reports no disclosures

List of abbreviations

HCV – hepatitis C virus

APOE - apolipoprotein E gene

apo E – apolipoprotein E protein

HIV – human immunodeficiency virus

DNA - deoxyribonucleic acid

PCR - Polymerase-Chain-Reaction

TAP- test battery for the assessment of attention

HADS - Hospital Anxiety and Depression Scale

BDI - Beck Depression Inventory

FIS - Fatigue Impact Scale

WFMT - word-figure memory test

RFT - Recurring Figures Test

BBB – blood brain barrier

MRS magnetic resonance spectroscopy

PET - positron emission tomography

Abstract

Objective: Hepatitis C virus causes not only liver damage in certain patients but can also lead to neuropsychiatric symptoms. Previous studies have shown that the type 4 allele of the gene for apolipoprotein E is strongly protective against hepatitis C virus-induced damage in liver. In the present study, we have investigated the possibility that APOE genotype is involved in the action of hepatitis C virus in brain.

Methods: 100 hepatitis C virus-infected patients with mild liver disease underwent a neurological examination and a comprehensive psychometric testing of attention and memory function. In addition, patients completed questionnaires for the assessment of fatigue, health-related quality of life and mood disturbances. APOE-genotyping was done on saliva using buccal swabs.

Results: The APOE-ε4 allele frequency was significantly lower in patients with an impairment of working memory, compared to those with a normal working memory test result (p=0.003). A lower APOE-ε4 allele frequency was also observed in patients with definitely altered attention ability (p=0.008), but here the p-value missed the level of significance after application of the Bonferroni correction.

Conclusions: Our data suggest that the APOE-ε4 allele is protective against attention deficit and especially against poor working memory in HCV-infected subjects with mild liver disease. Considering the role of apolipoprotein E in the life cycle of the virus the findings shed interesting new light upon possible pathomechanisms behind the development of neuropsychiatric symptoms in hepatitis C infection.

Keywords: virus replication, neurodegeneration, cerebral lipoprotein metabolism, attention, HCV encephalopathy

Introduction

Current World Health Organisation estimates suggest that, globally, 170 million people are infected with hepatitis C virus (HCV), with 3-4 million new infections each year. Like many infectious agents, HCV causes a variety of outcomes: patients can experience no symptoms, but liver involvement may encompass a spectrum from mild liver disease, cirrhosis to liver cancer. A considerable amount number of patients also exhibit neuropsychiatric symptoms, such as depression, fatigue, and deficits in attention, concentration and memory.1 The reason why there are different clinical syndromes probably reflects both underlying host and viral factors. In the case of liver disease caused by HCV, one factor that is involved is apolipoprotein E (gene: APOE; protein: apoE). This polymorphic gene has three common alleles (APOE-ε2, -ε3 and -ε4) that give rise to three common protein isoforms (apoE2, apoE3 and apoE4).2 The main functions of the apoE protein are the transport of lipids around the body and the repair of damaged tissue.2 APOE is also a key determinant of the outcome of infection, with possession of certain alleles conferring risk of, or protection against, damage caused by several infectious agents, including HIV3, Herpes simplex virus types 14 and 25 ,Mycobacterium tuberculosis6 and Plasmodium falciparum7 For HCV, the APOE-ε4 allele confers protection against severe liver disease.8 Subsequent studies have confirmed the beneficial role of APOE-ε4 in HCV disease: Richardson and colleagues9 demonstrated that HCV-induced liver damage progression was more rapid in those who lacked an APOE-ε4 allele; Price and colleagues10 found that the APOE-ε2 and APOE-ε4 alleles were both associated with increased clearance of the virus and that the APOE-ε3 allele was a risk for HCV persistence; and Toniutto and colleagues11,12 showed that HCV-infected individuals who received a liver transplant and had an APOE-ε4 allele had a slower liver disease progression rate than recipients carrying the other alleles.

We hypothesised that HCV-induced development of neuropsychiatric symptoms might also depend on possession of a particular APOE allele.. We therefore investigated the role of APOE in the occurrence of neuropsychiatric complications in HCV-infected patients.

Materials and Methods

Patients

100 HCV-infected patients with mild liver disease (32 male) aged 25-75 years were recruited at Hannover Medical School, Hannover, Germany. 84 patients were infected with HCV genotype 1. Eighty-one patients were HCV-RNA positive. Forty-six patients had undergone interferon treatment several years previously. The time period since infection was more than 25 years in most of those in whom the route of transmission was clear. The majority of patients had been infected via blood transfusion (n=37), or by treatment with immunoglobulins (n=34). Seven patients had a history of intravenous drug abuse more than 25 years ago. In four patients, occupational transmission was considered likely, and in one, maternal transmission. In 17 patients, the route of transmission remained unclear. Clinical findings, liver biopsy results and/or APRI scores showed that none of the patients had cirrhosis. Patients with concomitant diseases, or with medication which might impair brain function, were excluded from the study, as were patients with ongoing alcohol or drug abuse.

Clinical assessment

Besides neurological examination the patients underwent comprehensive psychometric testing. In addition, they completed questionnaires for the assessment of health-related quality of life and mood disturbances. The psychometric test battery applied comprised the PSE-Syndrome-TestSR-1 the cancelling “d”-testSR-2 and several subtests of the TAP-battery, a battery of attention testsSR-3. Furthermore, the Hospital Anxiety and Depression Scale (HADS)SR-4, Beck Depression Inventory (BDI)SR-5, the Fatigue Impact Scale (FIS)SR-6, and the SF-36 questionnaireSR-7 were performed. Seventy-four patients performed Luria’s list of wordsSR-8, the word-figure memory test (WFMT)SR-9 and the Recurring Figures Test (RFT)SR-10 for the assessment of learning ability and memory.

The individual results of the cancelling “d”-test, the TAP-battery subtests and the RFT were compared to the available norm data and expressed as percentiles. Since in practice percentiles less than 10 are considered clinically relevant, the 10th percentile (accordingly z-scores ≤ -1.3) was used as cut-off between normal and abnormal results. To achieve a sum score for each patient’s individual attention ability, we calculated the ratio between of the maximal achievable number of abnormal results and to the patient’s number of abnormal results (attention test sum score). Attention tests results were summarized in a score ranging from 0-1. Scores above 0.35 are were considered abnormal.

Test results of the WFMT are were expressed as z-scores after comparison with normal data, adjusted for age and education. In accordance with the evaluation of the attention tests, z-scores ≤ -1.3 were scored as abnormal. PSE-test results of less than -4 points were regarded as abnormal, as recommended in the test manual.

APOE genotyping

Buccal swabs were used for APOE genotyping. DNA was prepared from the swabs using the Nucleospin tissue preparation kit (ABgene, Epsom, UK) according to manufacturer’s instructions. Swabs were left in PBS for 1 hour and then proteinase K and lysis buffer were added. After a 10-min incubation at 70ºC, the DNA was ethanol-precipitated, bound to a column, washed and then eluted. The extracted DNA was checked for quality and quantity using the Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, USA) and stored at -20C. The APOE genotype was then determined using the method of Wenham et al.13, but using Hotstartaq® (Qiagen, Crawley, UK) and agarose gel electrophoresis.

Standard Protocol Approvals, Registrations, and Patient Consents

The study was approved by the institutional ethics committee and complied with the precepts set out in the 1975 Helsinki Declaration on human rights. Patients were included after giving their written, informed consent.

Statistics

To determine the influence of APOE on the psychometric tests conducted, subjects were divided into those with and those without a specific allele. For example, subjects were divided into APOE-ε2 allele possessors (i.e., those subjects with genotypes APOE-ε2ε2, ε2ε3 and ε2ε4) and those who did not possess an APOE-ε2 allele (i.e., APOE-ε3ε3, ε3ε4 and ε4ε4 subjects). The means of the different tests were compared by ANOVA using SPSS version 15.0 for Windows. Similarly, to determine the influence of the APOE-ε4 allele, subjects were divided into APOE-ε4 allele possessors and non-possessors. The subjects were not divided into those with and those without an APOE-ε3 as there were far too few subjects without an APOE-ε3 allele. For some categories, subjects were divided into abnormal and normal scores and the APOE allele frequencies were determined for each group. Statistical significance in these cases was determined using c2 analysis. A Bonferroni correction was applied and results were considered significant if p≤0.003.

Results

There were no differences in any of the psychometric test results between APOE-ε2 allele possessors and non-possessors, and the same was true for APOE-ε4 (Tables e-1 and e-2). There were also no differences in terms of allele frequency between the groups when divided into those individuals with abnormal and normal fatigue, depression and anxiety scores (data not shown). However, there were statistically significant differences in the working memory test of the TAP battery (table 1). In this test, the APOE- ε4 allele frequency was higher (25.9 %) in those patients who performed normally on this task compared to patients who performed expressing an abnormally high number of errors (4.5%, p=0.003).

The results were also analysed for males and females separately. In women, the APOE-ε4 allele frequency was higher in those with correct responses (25%) compared to those with a high number of errors (2.8%, p=0.004). A difference was also seen in the men (27.5 % v 12.5%) but this did not reach statistical significance.

There were further trends for a beneficial effect of the APOE-ε4 allele in performance in attention tasks (Intermodal comparison test p=0.01, Incompatibility test p=0.05, Alertness test p=0.008) but these did not meet our criterion for statistical significance (table 1). Finally, when we analysed the attention tests sum score, the APOE-ε4 allele frequency was significantly higher in those with preserved cognitive function compared to those with a high number of pathological scores (24.6 % v 9.5 %, p=0.008). There were no significant results for the APOE-ε2 and APOE-ε3 alleles. Memory function was not found to be related to the frequency of any of the APOE alleles (table 2).