Apolipoprotein E (ApoE)


LASAB870, LAS2B870

Contact: Natasja van Schoor

Background
The ε4 allele of the APOE gene has been established to be a major genetic risk factor for late-onset Alzheimer's Disease (Strittmatter et al, 1993; Henderson et al, 1995). APOE plays a central role in lipid metabolism, and has as such been associated with atherosclerosis (Davignon et al, 1988; Mahley, 1988).

Measurements in LASA (first cohort)

Blood collection
Blood samples were taken from the LASA respondents. At the LASA B-cycle of the first cohort, this was done in the regions Amsterdam and Zwolle, but not in the region Oss. At the LASA C-cycle of the first cohort, this was done in all three regions. Therefore, for subjects living in the Oss region ApoE phenotypes were determined from blood samples on C. Also, additional phenotypes were determined for subjects whose blood samples were available from C, but not from B (see Table 1). As ApoE is based on a genetic expression, it is a stable factor and could therefore be determined in blood either from B or from C. The blood samples were frozen at -80°C until determination of ApoE phenotypes in 1997/1998.

Measurement procedure & variable information

ApoE phenotyping

The ApoE phenotyping was done at the Immunochemisch Laboratorium of the VU University Medical Center, Amsterdam (supervision Gerard J. van Kamp, PhD, Dept. of Clinical Chemistry). In short, the ApoE phenotypes were determined by isoelectric focusing of delipidated serum samples, followed by immunoblotting. This method has been developed at the Gaubius Laboratory TNO-Leiden and is described by Havekes et al. (1987).

ApoE isoforms

The APOE gene is located on the long arm of chromosome 19. The APOE gene has three common alleles, ε2, ε3 and ε4, which determine the protein isoforms e2, e3 and e4, respectively. Please mind the notation: APOE [all capitals] gene with alleles ε2, ε3, ε4; apoE protein with isoforms e2, e3, e4. (Sometimes written as: APOE*2 allele with apoE2 isoform.) As the isoforms are encoded on two chromosomes, three homozygous (e2/2, e3/3 and e4/4) and three heterozygous (e3/2, e4/2, and e4/3) phenotypes are found in the general population. The ApoE phenotype frequencies in the LASA sample are given in Table 2. Gerdes et al. (1992) describes the phenotype frequencies found in 46 study populations around the world. Although mild discrepancies occurred for ApoE4 and substantial discrepancies for ApoE2 between phenotypes and genotypes, as reported in the HAAS study, the ApoE genotypes and phenotypes showed similar associations with Alzheimer's Disease (Kardaun et al, 2000). The robustness of these relationships supports the use of phenotypes, especially when DNA specimens are not available.

ApoE classification

ApoE e3 is the most commonly occurring isoform. ApoE4 is a risk factor for cognitive decline and Alzheimer's Disease, and works in a dose-response relationship. ApoE2 is thought to be protective, but the results are inconsistent. There are several possible ways to classify the phenotypes. To increase the number of ApoE e4, we classified ApoE status as e4 for subjects with the ApoE e4 isoform (phenotypes e2/4, e3/4, e4/4) and as non-e4 for subjects without the ApoE e4 isoform (phenotypes e2/2, e2/3, e3/3) (Dik et al, 2000). One could also choose to study e3/4 and e4/4 separately, in order to take advantage of the dose-response relationship. Some investigators leave e2/4 out of the analyses, because of their antagonistic effect (e2 being protective, and e4 being a risk factor).

Measurements in LASA (second cohort)

Blood collection
At the LASA 2B-cycle and G-cycle of the second LASA cohort, blood samples were collected in all three regions (n=807) (Table 3). At the 2B-cycle, DNA was extracted from buffy coats; at the G-cycle, DNA was extracted from full blood. If present, DNA from full blood was used; otherwise DNA from buffy coats was used. The DNA samples were frozen at -20°C until APOE genotyping in 2013. In 46 persons the DNA concentration was too low, and in 8 persons APOE genotype could not be determined, resulting in a total n of 753.

Measurement procedure & variable information

ApoE genotyping

APOE genotyping was performed by Sanger sequencing of a 428 bp PCR fragment of the APOE gene containing codons 112 and 158. Both alleles were scored for homo-and heterozygosity and the presence versus absence of the wildtype or variant alleles using Sequencher (version 5). The  haplotypes were defined accordingly; ε2/ε2 (TT/TT), ε2/ε3 (TT/CT), ε2/ε4 (TC/CT), ε3/ε3 (TT/CC), ε3/ε4 (TC/CC), ε4/ε4 (CC/CC) (Table 4).

Availability of data per wave

Numbers per wave

Table 1. Serum samples available for ApoE phenotyping in first cohort (n=2233)

 

B

C

Amsterdam

1007

117

Zwolle

729

66

Oss

XXX

314

All regions

1736

497

  

Table 2. ApoE phenotype frequencies in first cohort (n=2233)

Phenotype

N

%

E2/2

15

0.7

E2/3

262

11.7

E3/3

1350

60.5

E2/4

67

3.0

E3/4

475

21.3

E4/4

64

2.9

 

Table 3. DNA samples available for genotyping in second cohort (n=753)

 

2B 

G

All regions

235

518

 

Table 4. ApoE genotype frequencies in second cohort (n=753)

Genotype

N

%

ε2/ε2 (TT/TT)

9

1.2

ε2/ε3 (TT/CT)

98

13.0

ε2/ε4 (TC/CT)

33

4.4

ε3/ε3 (TT/CC)

413

54.8

ε3/ε4 (TC/CC)

180

23.9

ε4/ε4 (CC/CC).

20

2.7

 

Previous use in LASA

 

References

  1. Davignon J, Gregg RE, Sing CF. Apolipoprotein E polymorphism and atherosclerosis. Arteriosclerosis 1988; 8: 1-21.
  2. Gerdes LU, Klausen IC, Sihm I, Faergeman O. Apolipoprotein E polymorphism in a Danish population compared to findings in 45 other study populations around the world. GenEpidemiol 1992;9:155-67.
  3. Havekes LM, De Knijff P, Beisiegel U, Havinga J, Smit M, Klasen E. A rapid micromethod for apolipoprotein E phenotyping directly in serum. J Lipid Res 1987;28:455-463.
  4. Henderson AS, Easteal S, Jorm AF, Mackinnon AJ, Korten A, Christensen H, Croft L, Jacomb PA. Apolipoprotein E allele epsilon 4, dementia, and cognitive decline in a population sample. Lancet 1995;346:1387-1390.
  5. Kardaun JWPF, White L, Resnick HE, Petrovitch H, Marcovina SM, Saunders AM, Foley DJ, Havlik RJ. Genotypes and phenotypes for Apolipoprotein E and Alzheimer Disease in the Honolulu-Asia Aging Study. Clinical Chemistry 2000;46:1548-1554.
  6. Mahley RW. Apolipoprotein E: Cholesterol transport protein with expanding role in cell biology. Science 1988; 240: 622-630.
  7. Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, Enghild J, Salvesen GS, Roses AD. Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci USA 1993;90:1977-1981.