Allelic variation of apolipoprotein E in Japanese sporadic Creutzfeldt-Jakob disease patients

ELSEVIER

Neuroscience Letters 187 (1995) 209-211

NEUROSCHC[ LETTERS

Allelic variation of apolipoprotein E in Japanese sporadic Creutzfeldt-Jakob disease patients Yasushi Nakagawa a,b,*, Tetsuyuki Kitamoto a, Hisako Furukawa a, Koji Ogomori b, Jun Tateishi a aDepartment of Neuropathology, Kyushu University, Fukuoka, Japan bDepartment of Neuropsychiatry, Facultyof Medicine, Kyushu University, Fukuoka, Japan Received 18 November1994; revised version received 30 January 1995; accepted 1 February 1995

Abstract

We analyzed apolipoprotein E (apo E) genotypes in 53 Japanese sporadic Creutzfeldt-Jakob disease (CJD) patients and 100 normal controls using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods. The apo E allelic frequencies in Japanese sporadic CJD patients and our control population were as follows: e2, 6.6% versus 7.5%; e3, 82.1% versus 81.5%; e4, 11.3% versus 11.0%. The mean ages at onset in Japanese sporadic CJD patients were as follows: e31e3, 63.8 years; e31e4, 66.3 years; e2/e3, 68.6 years. These results indicate that there is no association between apo E genotype and sporadic CJD in Japan. Keywords: Creutzfeldt-Jakob disease; Apolipoprotein E; Polymerase chain reaction; Restriction fragment length polymorphism; Genotype distribution; Age at onset

Apolipoprotein E (apo E) is a constituent of triglyceride-rich lipoproteins, which plays an important part in the brain regeneration process [2,7,8]. Apo E has three major isoforms (E2, E3, E4) and three alleles (e2, e3, e4) [8]. The observed correlation between apo E genotype and age at onset of Alzheimer's disease (AD) suggests that the apo E type e4 allele is one of the major susceptibility genes (risk factors) for AD [3,13,15]. Furthermore, the presence of apo E protein has recently been documented not only in senile plaques, neurofibrillary tangles, and vascular amyloid of AD [11, 14], but also in kuru plaques of Creutzfeldt-Jakob disease (CJD) [9,17]. In the present study, we therefore analyzed apo E genotypes in 53 Japanese sporadic CJD patients and 100 normal subjects using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) [6,16] and also using a slight modification method. As a source of control DNA, we collected peripheral blood lymphocytes from a randomly selected population * Corresponding author, Correspondence: Y. Nakagawa, Department of Neuropathology, Neurological Institute, Faculty of Medicine, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812, Japan. Fax: +81 92 632 3343.

of Japanese individuals without neurological diseases. The diagnoses for definite sporadic CJD were defined as autopsy-confirmed brains banked in the Department of Neuropathology at Kyushu University. Clinical diagnoses for probable sporadic CJD were made according to rapidly deteriorating dementia presentation with both myoclonus and periodic synchronous discharge on electroencephalogram, and without known family history of Gerstmann-Str/iussler syndrome (GSS), AD or other dementia such as Pick's disease. The genomic DNA from these patients has been banked over the past 5 years in the Department of Neuropathology at Kyushu University. We analyzed prion protein gene (PRNP) genotypes and excluded all patients with any mutations or codon 129 valine polymorphism [5]. The PCR protocol and the primers used were as described previously by Hixson et al. [6] and Wenham et al. [16]. Unfortunately this RFLP method may sometimes yield, on incomplete enzyme digestion with HhaI, a band of 81 bp which should be separated to two bands of 48 bp and 33 bp (Fig la). Therefore, we performed HaelI digestion to avoid incomplete digestion or misleading results. Fig. l b shows 2.5% Nuscieve gel-separated products of HaelI digestion samples after amplification by PCR using the primers reported by Hixson et al. [6].

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Y. Nakagawa et al. I Neuroscience Letters 187 (1995) 209-211

a)

Hha I cleavagemaps ~2

~ ~

~

~

~f

~f

~f 48bp ~¢33bp

~r ~

~

~ 48bp ~33bp

~3 E4

81bp

* sometimes generate incomplete digestion Hae

II cleavagemaps

Table 1 Prevalence (%) of apo E genotypes and alleles in the Japanese sporadic CJD cases and controls ApoE Japanese sporadic CJD genotype Definite Probable e2/e2 e2/e3 e3/e3 e21e4

244 bp

e3/e4

E2

~3

Hae II ~f 32bp

212bp

or

Control All

0 (0) 0(0) 0 (0) 15.4 (4/26) 11.1 (3/27) 13.2 (7153) 53.8 (14/26) 74.1 (20127) 64.2 (34/53) 0 (0)

0 (0)

0 (0)

1.0 (1/100) 13.0 (131100) 66.0 (66/100) 0 (0)

e4/e4

30.8 (8/26) 0(0)

14.8 (4/27) 0(0)

22.6(12/53) 0 (0)

18.0(18/100) 2.0 (21100)

e2 e3 e4

7.7 (4/52) 76.9(40/52) 15.4 (8/52)

5.6 (3/54) 87.0(47/54) 7.4 (4/54)

6.6 (7/106) 7.5 (15/200) 82.1(87/106)81.5(1631200) 11.3 (12/106) 11.0 (22/200)

E4

b) M1

2 3 4

489 267 80~

Fig. 1. Hhal cleavage maps, Haell cleavage maps and electrophoretic separation of HaelI fragments. (a) Haell should digest e3 or e4 PCR products amplified with the primers of Hixson et al. [6], but the absence of the 212 bp fragment suggests DNA from an e2 homozygote. (b) The fragment sizes in base pairs (bp) are shown to the left. Lanes M, 1, 2, 3 and 4 correspond to PHY marker (Takara) , e2 homozygote, e2 heterozygote, PCR product without e2 allele, and uncut sample, respectively. These samples represent an e2 homozygote, an e2 heterozygote, a PCR product without e2 allele, and an uncut sample. PCR products derived from bearers of e3 or e4 alleles, on H a e l I digestion, should yield a 212 bp fragment, while the absence o f this 212 bp fragment suggests D N A from an e2 homozygote. W e determined allelic frequencies for the control and sporadic CJD groups by counting alleles and calculating sample proportions, Group differences in allelic frequencies were analyzed by the g 2 test, and those in the age at onset o f Japanese sporadic CJD by the Kruskal-Wallis test. Table 1 shows the apo E allelic distribution patterns in Japanese sporadic CJD patients and our control population. The allelic frequencies o f the apo E gene in Japanese sporadic CJD patients were as follows: e2, 6.6%; e3, 82.1%; e4, 11.3%, while those in our control population were as follows: e2, 7.5%; e3, 81.5%; e4, 11.0%. Table 2 shows the correlation between mean age at onset and apo E genotypes in Japanese sporadic CJD. The mean age at onset o f the different apo E genotypes were as follows: e3/e3, 63.8 years; e3/e4, 66.3 years; e2/e3, 68.6 years.

A m o n g 53 CJD patients with codon 129 Met/Met polymorphism, the g 2 test showed no significant differences in the apo E allelic distribution patterns between probable, definite, or all sporadic CJD and the control population (e2 frequencies: definite, P = 1.000; probable, P=0.845; all, P = 0 . 9 5 5 . e3 frequencies: definite, P = 0 . 5 8 5 ; probable, P = 0 . 4 5 2 ; all, P = 1.000. e4 frequencies: definite, P = 0.529; probable, P = 0.603; all, P = 1.000). The K r u s k a l - W a l l i s test showed that there were no significant differences in mean age at onset among the three A p o E genotypes (definite, P = 0.051; probable, P = 0.702; all, P = 0.376). While it has been previously demonstrated that the frequency of e4 allele is not different from controls in familial CJD [12], A m o u y e l et al. [1] recently reported that the e4 allele of the apo E gene is a risk factor for CJD and e2 allele of the apo E gene delays occurrence o f death from a series of 61 patients with CJD. The CJD cases reported by A m o u y e l et al. [1] include 16 cases with known PRNP mutations and mix up polymorphisms at codon 129 (Met/Met, Met/Val, Val/Val). In our study, we excluded all patients with these mutations [5]. Moreover, because codon 129 polymorphism in PRNP influences disease duration as reported previously [4,10] and as A m o u y e l et al. mentioned [ 1], we restricted our analyses to CJD patients with both the wild type PRNP and codon 129 Met/Met polymorphism to avoid PRNP genetic influence on sporadic CJD. Our findings suggest no relation-

Table 2 Comparison of mean age at onset among Apo E genotypes CJD type

Probable Japanese CJD Definite Japanese CJD All CJD

Apo E geuotype e3/e3

e3/e4

e2/e3

65.0 62.0 63.8

66.8 66.0 66.3

63.0 72.8 68.6

Y. Nakagawa et al. I Neuroscience Letters 187 (1995) 209-211

ship b e t w e e n the apo E allele and sporadic C J D in Japan. Factors other than the apo E allele m a y function as promoters or p r o t e c t i v e factors in the d e v e l o p m e n t of sporadic C J D in addition to other specific ethnic backgrounds. W e thank all the doctors w h o p r o v i d e d materials for D N A analyses, Dr, H o q u e , M.Z. for clinical information and Mr. M. Y o n e d a for technical p h o t o g r a p h i c assistance. [1] Amouyel, P., Vidai, O. and Launay, J.M., The apolipoprotein E alleles as major susceptibility factors for Creutzfeldt-Jakob disease, Lancet, 344 (1994) 1315-1318. [2] Boyles, J.K., Pitas, R.E., Wilson, E., Mahley, R.W. and Taylor, J.M., Apolipoprotein E associated with Astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system, J. Clin. Invest., 76 (1985) 1501-1513. [3] Corder, E.H., Saunders, A.M., Strittmatter, W.J., Schmechel, D.E., Gaskell, P.C., Small, G.W., Roses, A.D., Haines, J.L. and Pericak-Vance, M.A., Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families, Science, 261 (1993) 921-923. [4] Doh-ura, K., Kitamoto, T., Sakaki, Y. and Tateishi, J., CJD discrepancy, Nature, 353 (1991) 801-802, [5] Kitamoto T. and Tateishi J., Human prion diseases with variant prion protein, Phil. Trans. R. Soc. London Ser. B, 343 (1994) 391-398. [6] Hixson, J.E. and Vernier, D.T., Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI, J. Lipid. Res., 31 (1990) 545-548. [7] LeBlanc, A.C. and Poduslo, J.F., Regulation of apolipoprotein E gene expression after injury of the rat sciatic nerve, J. Neurosci. Res., 25 (1990) 162-171. [8] Mahley, R.W., Apolipoprotein E: Cholesterol transport protein with expanding role in cell biology, Science, 240 (1988) 622630. [9] Namba, Y., Tomonaga, M., Kawasaki, H., Otomo, E. and Ikeda,

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