Apolipoprotein-E genotype in normal aging, age-associated memory impairment, Alzheimer's disease and vascular dementia patients

Neuroscience Letters 231 (1997) 59–61

Apolipoprotein-E genotype in normal aging, age-associated memory impairment, Alzheimer’s disease and vascular dementia patients B. Palumbo a ,*, L. Parnetti a, G. Nocentini b, L. Cardinali c, S. Brancorsini a, C. Riccardi b, U. Senin a a

Section of Gerontology and Geriatrics, Department of Clinical Medicine, Pathology and Pharmacology, University of Perugia, Via Eugubina 42, 06122 Perugia, Italy b Section of Pharmacology, Department of Clinical Medicine, Pathology and Pharmacology, University of Perugia, Perugia, Italy c Chair of Nuclear Medicine, University of Perugia, Perugia, Italy Received 31 January 1997; received in revised form 10 July 1997; accepted 17 July 1997

Abstract Apolipoprotein-E (Apo-E) genotype and allele frequencies were evaluated in patients with late-onset probable Alzheimer’s disease (LOAD; n = 64), early-onset probable Alzheimer’s disease (EOAD; n = 32), possible Alzheimer’s disease (pAD; n = 44), vascular dementia (VD; n = 12), age-associated memory impairment (AAMI; n = 15) and 40 healthy age-matched controls. APO-E was performed by polymerase chain reaction products digested by the restriction enzyme HhaI. A statistically significant increase of e4 frequency was found in LOAD as compared to the other groups, and in pAD with respect to controls, while VD and AAMI groups did not disclose any difference as regards to control subjects. Multivariate analysis showed a significant association of e4 with female gender. Our results confirm the increased frequency of e4 in both probable and possible LOAD, failing to show a similar trend in VD and AAMI.  1997 Elsevier Science Ireland Ltd. Keywords: Apolipoprotein-E (Apo-E) genotype; e4 Allele; Probable Alzheimer’s disease; Possible Alzheimer’s disease; Vascular dementia; Age-associated memory impairment; Aging brain

Apolipoprotein-E (APO-E, gene; Apo-E, protein) is defined by three alleles, e2, e3 and e4, resulting in six genotypes (homozygous e2/2, e3/3, e4/4, heterozygous e4/3, e3/2, e4/2) [11]. APO-Ee4 is considered the first identified genetic susceptibility factor for late-onset probable Alzheimer’s disease (LOAD) [2,11], while the e2 allele seems to have a protective effect [17]. Neuropathological findings show that plaques containing b-amyloid protein are more common in patients with e4/4 and e4/3 genotype. e4 isoform shows affinity for b-amyloid protein, acting probably as a ‘pathological chaperone’ to promote the tertiary structure typical of non-soluble amyloid [15], whilst e2 allele is associated with a reduced deposition of amyloid, probably due to a lower affinity for b-amyloid protein [8]. The e4 allele may also interact with intraneuronal microtubule-associated t protein, favoring the hyper-phosphorylation responsible for the easier formation of neurofibrillary tangles [15]. Almost 50% of sporadic LOAD and more than 65% of * Corresponding author. Tel.: +39 75 5855822; fax: +39 75 5730259.

familial forms carry the e4 allele in comparison with a prevalence of 20% of this allele in normal population [2]. e4 allele frequency has been found to be increased also in vascular dementia, Parkinson’s disease, Parkinson plus, Lewy body disease and frontal dementia, although figures observed in these entities did not reach the statistical significance with respect to controls [6]. Other observations [4] showed a similar e4 allele frequency in Alzheimer’s disease (AD; 45%) and vascular dementia (VD; 46%). According to the NINCDS-ADRDA criteria [10] in clinical practice one can formulate the diagnosis of probable, possible or definite AD. No data are available on the prevalence of APO-E in possible AD (pAD), a clinical subtype where concomitant diseases contribute to the pathogenesis of cognitive impairment. At present, scarce is also the knowledge about the e4 prevalence in ‘age-associated memory impairment’ (AAMI). With the aim of further contributing to the knowledge of APO-E frequency in different conditions related to aging brain often encountered in geriatric practice, we investi-

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0538-7

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B. Palumbo et al. / Neuroscience Letters 231 (1997) 59–61

Table 1 Distribution of APO-E and alleles in the groups studied n

EOAD LOAD pAD VD AAMI Controls

APO-E

32 64 44 12 15 40

Allele frequency

e3/2 (%)

e3/3 (%)

e4/3 (%)

e4/4 (%)

e2

e3

e4

0 0 1 (2) 2 (17) 0 11 (27.5)

26 25 30 8 12 25

6 34 13 2 3 4

0 5 (8) 0 0 0 0

0.00 0.00 0.01 0.08 0.00 0.14

0.91 0.66 0.84 0.83 0.90 0.81

0.09 0.34 0.15 0.08 0.10 0.05

(81) (39) (68) (66) (80) (62.5)

(19) (53) (30) (17) (20) (10)

Both genotypes (x2 = 39.78, P , 0.0001) and allele frequencies (x2 = 38.28, P , 0.0001) differed significantly across study groups. As regards to genotypes: LOAD/EOAD, P , 0.0001; LOAD/pAD, P , 0.002; LOAD/AAMI, P , 0.008; LOAD/VD, P , 0.009; LOAD/C, P , 0.0001; pAD/C, P , 0.05; allele frequencies: LOAD/EOAD, P , 0.0001; LOAD/pAD, P , 0.002; LOAD/AAMI, P , 0.01; LOAD/VD, P , 0.02; LOAD/C, P , 0.0001; pAD/C, P , 0.05.

15 ± 6). According to the NINDS-AIREN criteria [16] 12 were diagnosed as suffering from probable VD (5 males and 7 females, mean age 71 ± 14 years, age at onset 74 ± 9 years, duration of disease 3 ± 1 years, MMSE score 19 ± 4, Hachinski Ischemic Score (HIS) [5] 8 ± 1); 15 suffered from AAMI defined according to the Crook at al.’s criteria [1] (7 males and 8 females, mean age 73 ± 6 years, age at onset 70 ± 6 years, MMSE score ≥25). Genomic DNA was extracted from whole blood and amplified by polymerase chain reaction (PCR) as described by Hixson and Vernier [7]. The PCR products were digested by the enzyme HhaI. Table 1 shows the distribution of the different genotypes and allele frequencies in the categories studied. In EOAD, LOAD and AAMI no heterozygous e3/2 was observed, while carriers of the homozygosity e4/4 were recorded only in LOAD group. Two out of the four e4 carrier controls were relatives of LOAD patients. Frequencies of APO-E and alleles were significantly higher in LOAD with respect

gated the APO-E genotype and alleles frequency in probable and possible AD, AAMI, VD, and healthy elderly subjects. APO-E genotyping was performed in 167 subjects (64 males and 103 females) consecutively referred to our Aging Brain Centre for diagnostic purposes and 40 healthy agematched controls (12 males and 28 females, mean age 71 ± 4 years) who consecutively referred to our out-patient section for routine check-up. All patients had no history of neuropsychiatric disorders and a Mini Mental State Examination (MMSE) score ≥26. According to NINCDSADRDA criteria [10], 64 suffered from LOAD (19 males and 45 females, mean age 77 ± 5 years, age at onset 73 ± 5 years, duration of disease 4 ± 2 years, MMSE score [3] 15 ± 4); 32 were affected by early onset probable AD (EOAD) (13 males and 19 females, mean age 64 ± 6 years, age at onset 59 ± 5 years, duration of disease 5 ± 2 years, MMSE score 15 ± 5); 44 were defined as pAD (20 males and 24 females, mean age 78 ± 8 years, age at onset 75 ± 8 years, duration of disease 4 ± 2 years, MMSE score Table 2

Clinical characteristics of each group according to absence (e4−) or presence (e4 + ) of e4 allele Probable AD EOAD n = 32

Number M/F Mean-age (Range) Age at onset (Range) Duration of disease (Range) HIS (Range) MMSE (Range)

LOAD n = 64

Possible AD

VD

AAMI

Controls

n = 44

n = 12

n = 15

n = 40

e4−

e4+

e4−

e4+

e4−

e4+

e4−

e4+

e4−

e4+

e4−

e4+

26 13/13 66 ± 6 (51–73) 60 ± 5 (50–65) 4±2

6 0/6 60 ± 5 (50–68) 55 ± 4 (47–59) 5±2

25 10/15 78 ± 5 (71–90) 73 ± 5 (67–86) 5±1

39 9/30 76 ± 5 (67–86) 72 ± 5 (65–84) 4±1

31 14/17 78 ± 9 (68–92) 75 ± 9 (66–87) 3±1

13 6/7 78 ± 6 (67–87) 75 ± 6 (66–85) 3±2

10 4/6 76 ± 10 (50–85) 73 ± 10 (49–84) 3±1

2 1/1 77 ± 4 (74–81) 74 ± 6 (70–79) 3±1

12 6/6 74 ± 7 (62–90) 70 ± 7 (59–88)

3 0/3 71 ± 1 (70–72) 68 ± 2 (66–71)

36 12/24 72 ± 13 (40–100)

4 0/4 60 ± 14 (50–82)

(2–11) 1±1 (0–4) 15 ± 6 (3–25)

(3–9) 1 ± 0.7 (0–2) 15 ± 5 (8–21)

(2–9) 1±1 (0–4) 15 ± 5 (5–26)

(1–8) 1±1 (0–4) 14 ± 4 (3–24)

(1–7) 2±2 (0–6) 15 ± 6 (5–27)

(2–10) 2±2 (0–5) 17 ± 5 (5–22)

(1–6) 8±1 (7–11) 20 ± 4 (11–24)

(2–4) 7 ± 0.7 (7–8) 16 ± 0.7 (16–17)

1±2 (0–4) 27 ± 1 (25–28)

0±1 (0–2) 27 ± 0.5 (26–28)

0±1 (0–6) 27 ± 2 (26–30)

0±0 (0–0) 29 ± 0.5 (28–30)

HIS, Hachinski Ischemic Score; MMSE, Mini Mental State Examination.

B. Palumbo et al. / Neuroscience Letters 231 (1997) 59–61

to the other groups. Also pAD showed higher frequencies as compared to controls. In Table 2 clinical details of each subgroup according to presence or absence of e4 allele are reported. It can be noted that prevalence of APO-Ee4 is constantly higher in females. The multivariate analysis (linear logistic regression analysis) carried out in order to evaluate the actual relationship between APO-Ee4/4 or e4/3 and main clinical characteristics (gender, age, age at onset, duration of disease, severity of disease) showed the association of these genotypes with the female gender (Odds ratio 2.66, P = 0.0088), while the other variables failed to show any significant relationship. The significant association with female gender is in agreement with some recent data showing that the risk of developing AD in men carriers of at least one e4 is lower than in women carriers [14]. A great neurobiological overlap exists among dementia categories, where clinical diagnosis is based on exclusion rather than inclusion criteria. Heterogeneity of patients defined as possible AD is higher, due to the actual difficulty in defining the pathogenetic contribution of associated diseases. This aspect could explain the result we obtained in pAD where figures of APO-E e4/3-4/4 (30%) and e4 allele (0.15) frequency were in between those observed in LOAD (61%, 0.34) and controls (10%, 0.05, respectively). AAMI is either considered as a preclinical phase of AD [13] or a paraphysiological condition [9]; in our series APO-E prevalence in AAMI did not show any difference with respect to controls; similarly, APO-E frequency in VD group was not different to controls, in contrast to some studies showing a prevalence of e4 in VD as high as in AD [4,12]. Since the small number of subjects examined does not allow definite conclusions, it will be important to definitely ascertain e4 prevalence in these entities. In conclusion, APO-E determination does not represent a diagnostic test for dementia disorders, being rather an element for better defining the level of probability in the diagnostic process of LOAD; in the case of possible AD, APO-E genotype could actually be a factor capable of supporting the main role played by degeneration in this kind of dementia. The authors are grateful to Dr. Antonella Santucci, for the statistical analysis and to Dr. Fausto Chionne and Dr. Carlo Raymondi for their technical advice. [1] Crook, T., Bartus, R.T., Ferris, S.H., Whitehouse, P., Cohen, G.D. and Gershon, S., Age-associated memory impairment. Proposed diagnostic criteria and measures of clinical change, Dev. Neuropsychol., 24 (1986) 261–271. [2] Dal Forno, G., Rasmusson, R., Brandt, J., Carson, K.A., Brookmeyer, R., Troncoso, J. and Kawas, C.H., Apolipoprotein E genotype and rate of decline in probable Alzheimer’s disease, Arch. Neurol., 53 (1996) 345–350.

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