Apolipoprotein E in sporadic and familial Creutzfeldt-Jakob disease

Neuroscience Letters 199 (1995) 95-98

Apolipoprotein E in sporadic and familial Creutzfeldt-Jakob disease Mirella Salvatoreav*, Angela Claudia Seebera, Benedetta Nacmiasb, Rosella Petrarolia, Marco D’Alessandroa, Sandro Sorbib, Maurizio Pocchiaria aLnboratory of Virology, Istituto Superiore di Sanitci. Viale Regina Elena, 299, 00161 Rome, Italy bDepartment of Neurological and Psychiatric Sciences, University of Florence, Florence, Italy

Received 7 July 1995; revised version received 5 September 1995; accepted 11 September 1995

Abstract

We assessed the apolipoprotein E (ApoE) genotype in 49 sporadic and ten familial Creutzfeldt-Jakob disease (CJD) patients, in seven healthy siblings with a PRNP mutation and in 84 controls. In sporadic CJD, ApoE genotypes and allelic frequencies do not significantly differ from that of controls. No influence of ApoE genotypes on age at onset was found. In familial cases, the disease appeared in mutated subjects showing the same ApoE genotype as members who have not yet developed CJD. Our results provide further evidence that ApoE is not a risk factor for CJD. Keywords:

Apolipoprotein E; Creutzfeldt-Jakob disease, PRNP; Allele frequency; Genotype distribution

Apolipoprotein E (ApoE) is a lipoprotein present in the amyloid plaques of Alzheimer’s disease (AD), Creutzfeldt-Jakob disease (CJD) and other amyloid forming diseases [l] that may act as a chaperon protein in facilitating the accumulation of amyloid [2]. The ApoE gene is located on chromosome 19 and has three alleles (~2, ~3 and ~4) coding for three common isoforms. ApoE ~4 allele has been identified as a susceptibility factor for AD [3] but its role in other amyloid-forming diseases is still controversial [4-71. CJD is a fatal transmissible neurological disease characterised by accumulation, in the brain of affected individuals, of a partially protease-resistant amyloid protein known as PrP-res that ‘most likely derives from a conformational modification of a cellular ‘normal’ protein (PrPsen). What is responsible for the formation of the pathological protein is still unknown. The methionine/valine homozygosity at the polymorphic codon 129 of the PrP gene (PRNP) causes a predisposition to sporadic [8] and iatrogenic CJD [9], while a number of PRNP point and insert mutations have been linked to familial CJD [IO]. However, the existence of other genetic and environmental factors is claimed to explain the incomplete penetrance and the clinical variability of these mutations. * Corresponding author. Tel.: +39 6 49903312; fax: +39 6 4457582.

Amouyel et al. [5] found a higher frequency of the ~4 allele in sporadic CJD patients than controls and suggested that the ApoE ~4 allele is a susceptibility factor for CJD and that in affected patients the ApoE e2 allele delays the occurrence of death. However, this seems not to be the case for Japanese CJD patients [6], whose different genetic background might be thought to explain this difference, for German sporadic CJD patients [7] and for ten familial CJD patients [4]. Thus, the definitive role of ApoE alleles in affecting CJD predisposition still has to be ascertained. Our study of sporadic and familial CJD patients and of healthy relatives carrying incompletely penetrant PRNP mutations gave us the opportunity to provide evidence that ApoE ~4 is not associated with CJD development. Forty-nine sporadic Italian CJD patients without PRNP mutations, ten familial CJD patients carrying the partially penetrant PRNP mutations at codon 200 [l l] or codon 210 [ 121, seven mutated siblings who have not yet developed the disease (age ranging from 65 to 82 years) and 84 randomly selected Italian individuals without neurological diseases [ 131 were studied. Patients from all over Italy were referred to the Italian National survey for CJD. Thirty-three sporadic patients were definite CJD whose diagnosis was confirmed by neuropathological examination of the brain and/or by Western blot detection of PrP-

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M. Salvatore et al. /Neuroscience Letters 199 (1995) 95-98

Table 1 ApoE genotypes (%) and allele frequencies in Italian CJD patients and controls Sporadic CJD

Controls

Definite

Probable

All

Met/Met

ApoE genotype &4/&4 &4/&3 ~4182 &3/&3 ~31~2 &V&2

(n = 33) 1 (3.0) 4 (12.1) 1 (3.0) 24 (72.7) 3 (9.1) 0 (0.0)

(n = 16) 0 (0.0) 2 (12.5) 0 (0.0) 10 (62.5) 4 (25.0) 0 (0.0)

(n = 49) 1 (2.0) 6 (12.2) 1 (2.0) 34 (69.4) 7 (14.3) 0 (0.0)

(n = 40) 0 (0.0) 6 (15.0) 1 (2.5) 26 (65.0) 7 (17.5) 0 (0.0)

(n = 84) 0 (0.0) 14 (16.7) 0 (0.0) 62 (73.8) 8 (9.5) 0 (0.0)

Allele frequency* E2 E3 E4

(n = 66) 0.061 0.833 0.106

(n = 32) 0.125 0.813 0.063

(n = 98) 0.082 0.827 0.092

(n = 80) 0.100 0.813 0.088

(n = 168) 0.048 0.869 0.083

Age (years; mean f SD)

62.2 + 10.3

61.3 + 7.4

63.9 + 9.7

64.6 f 10.1

59.3 f 6.5

*&2:definite P = 0.94, probable P = 0.20, all P = 0.26, Met/Met P = 0.12; ~3: definite P = 0.48, probable P = 0.57, all P = 0.34, Met/Met P = 0.24; ~4: definite P = 0.77, probable P = 0.97, all P = 0.81, Met/Met P = 0.91; chi-square test.

res from cortical samples of frozen brains [12]. Clinical diagnosis for probable CJD was made in 16 sporadic patients based on the presence of dementia, myoclonus, pseudoperiodic EEG, and the exclusion of other neurological diseases [ 141. To determine the ApoE genotype, genomic DNA samples were prepared from either whole blood or postmortem brain tissues, amplified in a Thermal Cycler Perkin Elmer-Cetus 9600, and digested with CfoI restriction endonuclease. The restriction fragment length polymorphism was detected as described [15]. Insertion and

deletion of PRNP were tested by direct electrophoresis of amplified DNA on agarose gels. PRNP point mutations (at codon 102, 117, 178, 198, 200, 210 and 217) and codon 129 polymorphism were tested as described [ 8,121. Allele frequencies for patients and control subjects were estimated by counting the alleles and calculating sample proportions. Frequencies of ApoE genotypes and alleles were compared using the chi-square test with the Yates correction for continuity when necessary. The odd ratio (OR) and 95% CI were computed between subjects with or without at least one ApoE ~4 allele. Age at onset ac-

Table 2 ApoE genotypes, PRNP mutation

Family NST Family IT-91

Family Family Family Family Family Family

PZZ GFF DNG RPN CTL TSC

Family FRN

CJD Unaffected CJD Unaffected Unaffected CJD CJD CJD CJD CJD CJD CJD Unaffected Unaffected Unaffected Unaffected CJD

and codon 129 genotype

in familial CJD patients and healthy relatives

Sex

ApoE genotype

PRNP mutation

Codon 129

Age at onset

Age

M F F F F F F F F M F M F F F F F

313 313 2l3 2J3 2f3 313 313 2l3 2l3 2l3 313 313 313 313 313 2i3 313

210 210 210 210 210 210 210 210 210 210 200 200 200 200 200 200 200

M/M M/M

49

_

*24 bp deletion in the octapeptide repeats of the non-mutated allele.

65 68

65 70 52 55 71 61 58

81 82

-

66 69 82 80

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M. Salvatore et al. /Neuroscience Letters 199 (1995) 95-98

cording to ApoE genotype was compared using nonparametric procedures (Kruskal-Wallis test). Statistical analyses were done by using SAS software. In sporadic CJD, no significant departures from Hardy-Weinberg equilibrium were observed. ApoE allele frequencies and genotype distribution did not significantly differ from that of controls (Table 1). The relative risk of developing CJD for subjects with or without ApoE ~4 was 0.98 (95% CI: 0.38-2.52) for all CJD patients and 1.06 (95% CI: 0.39-2.88) when only methionine homozygous patients are considered. Mean age at onset (and SD) was: ~41~4 73 years; ~41~3 58.5 (15.2); &3/E3 63.9 (8.8); ~31~2 67 (9.0); ~41~2 66; and did not significantly vary according to ApoE genotypes (P = 0.60, Kruskal-Wallis test). To exclude any effect of codon 129 polymorphism, the analysis was performed only on methionine homozygous (M/M) patients (n = 40). Also in this case, no significant effect on age at onset was found (Table 1). In familial CJD (Table 2), none of the patients had the ~4 allele while four of them had the allele ~2 that is supposed to be protective:. The remaining six patients were genotype did not ~31~3. Patients with the ApoE E2/E3 have an age at onset different from that of patients with ~3/&3 (Table 2). Our results indicate that the ApoE ~4 allele is not overrepresented in sporadic CJD patients and that it is not a risk factor for CJD. These data support the studies carried out in German [6] and in Japanese [7] patients but differs from the study carried out in French patients in which ApoE ~4 allele bearers had a two-fold increased risk of developing CJD (OR 1.8; 95% CI 1 .O-3.2) or a three-fold increased risk (OR 2.9; 95% CI 1.0-8.1) when only methionine homozygous patients were analysed [5,16]. The discrepancy found between the French and other studies could be due to an incorrect selection of controls (either 15 years older or 10 years younger than CJD patients) who show a lower frequency of the ApoE ~4 allele. The frequency of the ~4 allele in our control group did not differ from that previously reported in large Italian populations [ 17,181 and was not elevated as in the German neurological controls, where it was similar to that recorded in AD patients [7,16]. Moreover, we found a low ApoE ~4 allele frequency in patients with a definite CJD diagnosis (positive neuropathology or PrP-res detection by Western blot, or both), and in patients homozygous for methionine at codon 129. We also tested whether the ApoE genotype could act synergistically with the partially penetrant PRNP mutations at codon 200 or 210 determining or influencing their penetrance in members of the same CJD family. Although none of the mutated subjects carried the ApoE ~4 allele, patients with ~21~3 genotype had an age at onset similar to that of patients with, ~3/&3 (Table 2). Moreover, family members who died from CJD and members of the same family who have not yet developed the disease (age

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ranging 65-82) not only had the same PRNP mutation and codon 129 polymorphism but also the same ApoE genotype. These data provide further evidence that factors different from ApoE genotype determine the appearance of sporadic or familial CJD in subjects carrying incompletely penetrant PRNP mutations. We thank Prof. Giorgio Macchi and Dr. Maurizio Genuardi for helpful discussion and Ms. Deborah Wool for editorial assistance. This work was partially supported by the National Registry of Creutzfeldt-Jakob disease, financed by the Italian Ministry of Health-Istituto Superiore di Sanid and by Italian National Research Council (CNR) Target Project on Aging (Grant no. 952608). BN was also supported by Telethon Italia (no. E.304).

VI Namba, Y., Tomoraga,

VI

[31

[41

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