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The Advantages of Haplotype Analysis of the Promoter Region of the Human Apolipoprotein E Gene
Analytical Biochemistry 299, 183–187 (2001) doi:10.1006/abio.2001.5425, available online at http://www.idealibrary.com on
The Advantages of Haplotype Analysis of the Promoter Region of the Human Apolipoprotein E Gene Daniel Peter McLaughlin, Anita Sharma, Ann McGinley, and Gurdip Singh Samra Academic Department of Anaesthesia and Intensive Care, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, The Royal London Hospital, Whitechapel, London E1 1BB, United Kingdom
Received May 3, 2001; published online November 10, 2001
Polymorphisms in the regulatory region of the human apolipoprotein E gene (gene, APOE; protein, apoE) have been implicated in Alzheimer’s disease. Here we describe in detail the advantages of a simple method for haplotype analysis of this region (at ⴚ491 and ⴚ427 bases relative to the transcription start site of the gene). The promoter region of the APOE gene was amplified by polymerase chain reaction (PCR) and this fragment was then used as a template for PCR with “nested” primers to generate a 228-bp product incorporating both the ⴚ491 and the ⴚ427 loci. PCR products were then digested with DraI and AluI together and subjected to polyacrylamide gel electrophoresis. The distinct pattern of bands appearing on the gel was then used to ascribe [ⴚ491,ⴚ427] haplotypes to each subject, from which ⴚ491 and ⴚ427 genotypes were inferred. ⴚ491 and ⴚ427 genotypes were also confirmed by digestion with DraI alone or AluI alone. Haplotype analysis was successful in all 20 samples analyzed and was 100% consistent with genotyping. We suggest that this is a reliable, time-saving method that the will be useful in large-scale APOE promoter genotyping studies. © 2001 Elsevier Science Key Words: apolipoprotein E; human; genotyping; haplotyping; polymerase chain reaction.
Together with polymorphisms in the coding region of the apolipoprotein E gene (1, 2) (gene, APOE; protein, apoE), 1 polymorphisms in the regulatory region (promoter) have been implicated in the development of Alzheimer’s disease (3–7). Two of these polymorphisms (⫺491A/T and ⫺427T/C) play a major role in determining the level of expression of the protein, with certain 1
Abbreviations used: APOE, apolipoprotein E gene; apoE, apolipoprotein E protein; PCR, polymerase chain reaction; PSF, purified salt-free; BSA, bovine serum albumin. 0003-2697/01 $35.00 © 2001 Elsevier Science All rights reserved.
genotypes being associated with AD (⫺427C (3) and ⫺491A (4, 5)). Given the ongoing intense research effort into the pathophysiology of APOE genotype, it is important that researchers have reliable methods for assigning patient genotypes. A problem with genotyping in the APOE promoter is that it is time-consuming. Two rounds of polymerase chain reaction (PCR) and subsequent digestion with restriction enzymes are usually required (3–5). One research group has utilized a onestage PCR analysis (6), but this has not proved reliable even in their laboratory (3). In this paper, we highlight a simple and reliable method for APOE promoter genotyping that reduces the number of manipulations and restriction digests and the number of lanes of analytical polyacrylamide gel required for the assignment of APOE ⫺491 and ⫺427 genotypes. This may help reduce the amount of time taken to conduct large-scale studies. Restriction digestion of PCR products with two enzymes together has been used extensively in molecular biology in the past. In the interest of brevity, we have cited only one here (8). However, this is the first time that such a method has been systematically applied to the study of APOE promoter polymorphisms and compared to separate genotyping at the individual loci. MATERIALS AND METHODS
Subjects and DNA Extraction All procedures described in this study were approved by the research ethics committee of East London and the City Health Authority. The first 20 subjects (16 male and 4 female) recruited in a larger group of former severely head-injured patients who had previously been treated at the Royal London Hospital attended a follow-up clinic and had a small (8 ml) blood sample taken. The mean age of subjects at the time of injury (⫾SD) was 38.6 ⫾ 18 years and they were re183
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cruited to the study on average 5 years after their injury. These statistics are representative of a group of head injury patients in that they are predominantly young and cohorts generally have a preponderance of males (around 80% (9)). Genomic DNA was extracted from the blood samples using a commercial kit (Qiagen, Germany). After spectrophotometric quantification of the amount of DNA extracted, samples were stored at ⫺20°C until use. DNA Amplification First-round PCR. Genomic DNA was diluted down to 20 ng/l in sterile deionized water and 5 l of this DNA was amplified by PCR in a total volume of 12.5 l using primers spanning the promoter region of the human APOE gene. The primers (purified salt-free (PSF) quality, MWG Biotech, Milton Keynes, UK) had the following sequences: upstream 5⬘-CAAGGTCACACAGCTGGCAAC-3⬘; downstream 5⬘-TCCAATCGACGGCTAGCTACC-3⬘ (2). In first- and second-round PCR, a single frozen aliquot of primers (10 M) was used on each day. The final concentration of reagents in the PCR tube was DNA 8 ng/l; 15 mM Tris–HCl, pH 8; 50 mM KCl; primers 1 M each; MgCl 2 2 mM; dNTPs 0.4 mM each; 0.1 Units/l Amplitaq Gold DNA polymerase (Applied Biosystems). First-round PCR was carried out using the following conditions: 12 min at 94°C (1 cycle, hot start); 30 s at 94°C, 30 s at 54°C, 1 min at 72°C (40 cycles); 10 min at 72°C (1 cycle, extension). After the program was complete, tubes were held at 4°C until products were analyzed. To check the fidelity of the first round of PCR, 2.5 l of the PCR reaction was electrophoresed on a 1% agarose/1⫻ TAE gel. The expected product for this PCR spans bases ⫺1017 to ⫹406 relative to the transcription start site of the APOE gene and is 1423 bp in size (EMBL Accession No. AF055343). Commonly, two major products of around 500 and 1400 bp were amplified. Second-round PCR. PCR products from first-round amplification were diluted 100-fold in sterile deionized water and 5 l of this sample underwent PCR in a final volume of 12.5 l with a pair of “nested” primers. The primers (PSF quality, MWG Biotech) had the following sequences: upstream (mismatches underlined) 5⬘-TGTTGGCCAGGCTGGTTTTAA-3⬘; downstream 5⬘-CCTCCTTTCCTGACCCTGTCC-3⬘ (2). The mismatched upstream primer, corresponding to bases ⫺512 to ⫺492 of the APOE gene, introduces DraI sites (5⬘-TTTAAA-3⬘) in the PCR product amplified from individuals with one or more ⫺491A alleles. The final concentration of reagents (except DNA) in the reaction tube was identical to that described above for first-round PCR. Second-round PCR was carried out using the following conditions: 12 min at 94°C (1 cycle, hot start); 30 s
at 94°C, 30 s at 52°C, 1 min at 72°C (35 cycles, amplification); 10 min at 72°C (1 cycle, extension). After the program was complete, tubes were held at 4°C until products were analyzed. To check the fidelity of the second round of PCR, 2.5 l of this PCR reaction was electrophoresed on a 1.8% agarose/1⫻ TAE gel. The expected product was 228 bp in size. Haplotyping To haplotype the regulatory region of the APOE gene, 2.5 l of the second-round PCR product was digested in a final volume of 10 l. The restriction digests contained 10 units of DraI (Roche Molecular Biochemicals), 4 units of AluI (Promega Corporation), and 100 ng/l BSA in 1⫻ Promega restriction enzyme buffer B and were incubated for 3 h at 37°C. Following digestion, enzymes were denatured by heating at 65°C for 15 min before the samples were held at 4°C. Two microliters of a gel loading buffer (0.25% bromphenol blue, 40% sucrose) was added to each sample and the mixture was electrophoresed at 100 V (5– 8 mA) on a 16% nondenaturing polyacrylamide gel (1⫻ TBE) in a minigel system (Bio-Rad Mini-Protean II) for 230 min. After electrophoresis, the gel was stained with 0.5 g/ml ethidium bromide (Sigma) in 1⫻ TBE for 30 min before being viewed by ultraviolet transillumination. The APOE [⫺491, ⫺427] haplotypes of subjects were determined from the pattern of restriction fragments on the gel: [A-C], 209 bp; [A-T], 144 and 65 bp; [T-T], 144 and 84 bp; and [T-C], 228 bp. The 10 possible combinations of these four haplotypes of the APOE promoter each produce a distinct pattern of bands (Fig. 2). ⫺491 and ⫺427 Genotyping APOE ⫺491 and ⫺427 genotypes were confirmed by digestion of the 228-bp second-round PCR products with DraI alone or AluI alone, respectively. A total of 2.5 l of PCR product was digested with either 10 Units of DraI (Roche) or 4 Units of AluI (Promega) in a final volume of 10 l for 3 h at 37°C. Following digestion, the enzymes were denatured and the digested fragments were analyzed as for haplotyping. The pattern of bands on the gel indicated a subject’s genotype: ⫺491 A/A 209 bp, T/T 228 bp, and A/T 228 and 209 bp; ⫺427, T/T 144 and 84 bp, C/C 228 bp, and T/C 228, 144, and 84 bp. These genotypes were compared to those that had been inferred from haplotype analysis. RESULTS
First-round amplifications of the promoter region of the APOE gene produced several products of varying size (Fig. 1a). The first-round of PCR was more reliable
HAPLOTYPE ANALYSIS OF THE APOLIPOPROTEIN E GENE
FIG. 1. Agarose gel electrophoresis of (a) first- and (b) secondround PCR of the polymorphic region of the human APOE promoter in five representative subjects (Nos. 1, 5, 18, 3, and 4). Note the multiple bands in first-round PCR (including the target at 1423 bp, indicated by the arrow) and the single band at 228 bp in secondround PCR. B, PCR reactions with no starting DNA; , DNA marker cut with EcoRI and HindIII; , X174 DNA marker cut with HinfI. Sizes of molecular weight markers (bp) are indicated along the side of the gel.
if, on a regular basis, primers were freshly diluted from 100 M stock solutions and frozen in small aliquots at ⫺20°C until use. A single aliquot was only ever used once. In each case the 1423-bp product was apparent (Fig. 1a) and thus the 228-bp fragment was always amplified in the second round with the nested primers (Fig. 1b). Despite varying annealing temperature, primer concentration, and magnesium ion concentration, we have
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been unable to eliminate the major 500-bp band in first-round PCR. In initial experiments, when firstround PCR failed to produce a band at 1423 bp due to problems with the primers (rectified by using fresh aliquots), the major 500-bp band was amplified, but second-round PCR failed to produce a band at 228 bp (data not shown). Therefore, it is unlikely that the 500-bp product of first-round PCR is an internal fragment of the APOE promoter that could interfere with subsequent analysis of genotype. Also, we have purified, cloned, and sequenced this fragment, which is actually 479 bp. The only matches in the nucleotide database (using a BLAST search) in the human APOE gene relate to the upstream primer sequence. No significant matches to any other sequences were found. Digestion of the 228-bp PCR product with DraI and AluI together produced a range of different fragment lengths on the polyacrylamide gel (Figs. 2 and 3a). The haplotypes assigned allowed us to infer ⫺491 and ⫺427 genotypes for each of the subjects (Fig. 3a). ⫺491 and ⫺427 genotyping using DraI or AluI alone confirmed that our inferences were correct in all cases (Figs. 3b and 3c). The distribution of haplotype combinations and relative haplotype and allele frequencies for the ⫺491 and ⫺427 loci are shown in Tables 1 and 2. Even given the small number of subjects studied here, these frequencies are reasonably close to those described for control subjects in previous studies at these loci (2– 6). DISCUSSION
This study confirms the utility of double-digestion of PCR fragments to ascribe APOE [⫺491, ⫺427] haplotype. From each of the haplotypes, it is always possible to infer both the ⫺491 and the ⫺427 genotypes of a
FIG. 2. Schematic gel image showing the pattern of bands on a polyacrylamide gel from subjects with the 10 possible [⫺491, ⫺427] haplotype combinations. Fragment sizes are indicated by the arrows. , X174 DNA marker cut with HinfI (marker sizes (bp) indicated).
186
MCLAUGHLIN ET AL. TABLE 1
APOE [⫺491, ⫺427]/[⫺491, ⫺472] Haplotype Combinations and ⫺491A/T and ⫺427T/C Genotypes from 20 Subjects Subject
[⫺491,⫺427] haplotype combination
⫺491 genotype
⫺427 genotype
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
[T-T]/[T-T] [A-C]/[T-T] [A-T]/[T-T] [A-T]/[A-C] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-C] [A-T]/[T-T] [A-T]/[T-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-C]/[A-C] [A-T]/[A-T] [A-T]/[A-T]
T/T A/T A/T A/A A/A A/A A/A A/A A/A A/A A/T A/T A/A A/A A/A A/A A/A A/A A/A A/A
T/T T/C T/T T/C T/T T/T T/T T/T T/T T/C T/T T/T T/T T/T T/T T/T T/T C/C T/T T/T
Note. Haplotypes and genotypes were determined independently.
Furthermore, haplotype analysis is more reliable than single digestion with DraI at locus ⫺491 in the APOE gene. Figure 3b illustrates that complete digestion with DraI of DNA amplified from ⫺491A homozygous (subjects 4, 5, and 18) or ⫺491 heterozygous individuals (subject 3) was often not possible. In order to assign genotypes, a degree if inference (“guesswork”) FIG. 3. Polyacrylamide gel electrophoresis of 228-bp products from PCR of the polymorphic region of the APOE promoter in five subjects (Nos. 1, 5, 18, 3, and 4). (a) Products cut with DraI and AluI together (the 65-bp band in 03 is clearer on the original gel); (b) products cut with DraI alone; (c) products cut with AluI alone. , X174 DNA marker cut with HinfI. Sizes of molecular weight markers are indicated along the side of the gel.
subject, whereas it is not always possible to infer [⫺491, ⫺427]/[⫺491, ⫺427] haplotype combination from the individual genotypes. For example, a subject who has a genotype of ⫺491A/T and ⫺427T/C (like subject 2 in this study) could have either of the two following haplotype combinations: [A-T]/[T-C] or [A-C]/ [T-T]. If the ⫺491A and ⫺427C alleles are associated with higher levels of expression of apoE (3), it is evident that the latter of these combinations could have implications for the likelihood of the subject developing Alzheimer’s disease or other diseases related to apoE (especially if the subject’s apoE phenotype contains the deleterious form apoE4). In the specific case of subject 2, we know that his apoE phenotype is purely apoE3.
TABLE 2
Frequencies for ⫺491, ⫺427 Haplotypes and the Possible Combinations Thereof, Together with Relative Allele Frequencies at APOE ⫺491 and ⫺427 Loci [⫺491, ⫺427] haplotype combination A-T/A-T A-T/T-T A-T/A-C T-T/T-T A-C/T-T A-C/A-C
Haplotype combination frequency 0.60 (12/20) 0.15 (3/20) 0.10 (2/20) 0.05 (1/20) 0.05 (1/20) 0.05 (1/20)
[⫺491, ⫺427] haplotype A-T T-T A-C
Relative haplotype frequency 0.725 0.15 0.125
allele ⫺491A ⫺491T ⫺427T ⫺427C
Relative allele frequency 0.850 0.150 0.875 0.125
HAPLOTYPE ANALYSIS OF THE APOLIPOPROTEIN E GENE
must be employed based upon the relative intensity of band staining on the gel. In this study, we have used the enzyme from the supplier that has given least problems. Stocks of DraI purchased from other suppliers were even less reliable. Whether this problem arises because of difficulty in fully digesting a nested PCR product or a problem with all supplies of DraI is unclear. What is clear is that haplotype analysis with double digestions poses no such problems (Fig. 3a). Another potentially interesting point is that in our study, the haplotype combinations [T-T]/[T-T], [A-C]/ [T-T], and [A-C]/[A-C] are represented slightly more often (each 5% of subjects) than in a previous study where less than 5% of control subjects had these genotypes (3). Given the small number of patients studied here, no real conclusions can be drawn and whether these relatively rare haplotype combinations are associated with a more favorable outcome following traumatic brain injury remains to be seen. We should add that the technique of haplotyping by restriction digestion of PCR products with two enzymes together has been used over the past 20 years or more. One example is a study of prenatal diagnosis of ␣-thalassemia by Lebo et al. (8). In this study, the authors extracted and concentrated genomic DNA from fetuses and expectant parents before digesting the samples with BglII and Asp718. This then allowed the detection of the most common deletions in the gene that are responsible for the disease in the South East Asian population by subsequent Southern blotting of the digested DNA and analysis of fragment sizes. Although the two methods have a similar digestion with two restriction enzymes together, their method differed somewhat to that reported here. First, they used Southern blotting of digested genomic DNA rather than PAGE of digested PCR products for accurate sizing and also relied upon existing restriction enzyme sites in the ␣-globin gene, rather than introducing one of the sites by using mismatched primers. In conclusion, this method cuts down substantially on manipulations of samples and tubes in an already
187
laborious procedure and is likely to be very useful in large-scale studies of APOE promoter polymorphisms. A further advantage is that the more cryptic combinations of APOE [⫺491, ⫺427] haplotypes are readily distinguishable and need not be investigated “after the fact.” REFERENCES 1. Wenham, P. R., Price, W. H., and Blundell, G. (1991) Apolipoprotein E genotyping by one-stage PCR. Lancet 337, 1158 –1159. 2. Strittmatter, W. J., Saunders, A. M., Schmechel, D., PericakVance, M., Enghild, J., Salvesen, G. S., and Roses, A. D. (1993) Apolipoprotein E: High-avidity binding to -amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc. Natl. Acad. Sci. USA 90, 1977–1981. 3. Artiga, M. J., Bullido, M. J., Frank, A., Sastre, I., Recuero, M., Garcia, M. A., Lendon, C. L., Han, S. W., Morris, J. C., Vazquez, J., Goate, A., and Valdivieso, F. (1998) Risk for Alzheimer’s disease correlates with transcriptional activity of the APOE gene. Hum. Mol. Genet. 7, 1887–1892. 4. Laws, S. M., Taddei, K., Martins, G., Paton, A., Fisher, C., Clarnette, R., Hallmayer, J., Brooks, W. S., Gandy, S. E., and Martins, R. N. (1999) The -491AA polymorphism in the APOE gene is associated with increased plasma apoE levels in Alzheimer’s disease. NeuroReport 10, 879 – 882. 5. Ahmed, A. R. H., MacGowan, S. H., Culpan, D., Jones, R. W., and Wilcock, G. K. (1999) The ⫺491A/T polymorphism of the apolipoprotein E gene is associated with the apoE⑀4 allele and Alzheimer’s disease. Neurosci. Lett. 263, 217–219. 6. Artiga, M. J., Bullido, M. J., Sastre, I., Recuero, M., Garcia, M. A., Aldudo, J., Vazquez, J., and Valdivieso, F. (1998) Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E gene. FEBS Lett. 421, 105–108. 7. Thome, J., Gewirtz, J. C., Sakai, N., Zachariou, V., Retz-Junginger, P., Retz, W., Duman, R. S., and Ro¨sler, M. (1999) Polymorphisms of the human apolipoprotein E promoter and bleomycin hydrolase gene: Risk factors for Alzheimer’s dementia? Neurosci. Lett. 273, 37– 40. 8. Lebo, R. V., Saiki, R. K., Swanson, K., Montano, M. A., Erlich, H. A., and Golbus, M. S. (1990) Prenatal diagnosis of alphathalassemia by polymerase chain reaction and dual restriction enzyme analysis. Hum. Genet. 85, 293–299. 9. Wilson, J. T. L., Pettigrew, L. E. L., and Teasdale, G. M. (2000) Emotional and cognitive consequences of head injury in relation to the Glasgow outcome scale. J. Neurol. Neurosurg. Psychiatr. 69, 204 –209.
The Advantages of Haplotype Analysis of the Promoter Region of the Human Apolipoprotein E Gene Daniel Peter McLaughlin, Anita Sharma, Ann McGinley, and Gurdip Singh Samra Academic Department of Anaesthesia and Intensive Care, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, The Royal London Hospital, Whitechapel, London E1 1BB, United Kingdom
Received May 3, 2001; published online November 10, 2001
Polymorphisms in the regulatory region of the human apolipoprotein E gene (gene, APOE; protein, apoE) have been implicated in Alzheimer’s disease. Here we describe in detail the advantages of a simple method for haplotype analysis of this region (at ⴚ491 and ⴚ427 bases relative to the transcription start site of the gene). The promoter region of the APOE gene was amplified by polymerase chain reaction (PCR) and this fragment was then used as a template for PCR with “nested” primers to generate a 228-bp product incorporating both the ⴚ491 and the ⴚ427 loci. PCR products were then digested with DraI and AluI together and subjected to polyacrylamide gel electrophoresis. The distinct pattern of bands appearing on the gel was then used to ascribe [ⴚ491,ⴚ427] haplotypes to each subject, from which ⴚ491 and ⴚ427 genotypes were inferred. ⴚ491 and ⴚ427 genotypes were also confirmed by digestion with DraI alone or AluI alone. Haplotype analysis was successful in all 20 samples analyzed and was 100% consistent with genotyping. We suggest that this is a reliable, time-saving method that the will be useful in large-scale APOE promoter genotyping studies. © 2001 Elsevier Science Key Words: apolipoprotein E; human; genotyping; haplotyping; polymerase chain reaction.
Together with polymorphisms in the coding region of the apolipoprotein E gene (1, 2) (gene, APOE; protein, apoE), 1 polymorphisms in the regulatory region (promoter) have been implicated in the development of Alzheimer’s disease (3–7). Two of these polymorphisms (⫺491A/T and ⫺427T/C) play a major role in determining the level of expression of the protein, with certain 1
Abbreviations used: APOE, apolipoprotein E gene; apoE, apolipoprotein E protein; PCR, polymerase chain reaction; PSF, purified salt-free; BSA, bovine serum albumin. 0003-2697/01 $35.00 © 2001 Elsevier Science All rights reserved.
genotypes being associated with AD (⫺427C (3) and ⫺491A (4, 5)). Given the ongoing intense research effort into the pathophysiology of APOE genotype, it is important that researchers have reliable methods for assigning patient genotypes. A problem with genotyping in the APOE promoter is that it is time-consuming. Two rounds of polymerase chain reaction (PCR) and subsequent digestion with restriction enzymes are usually required (3–5). One research group has utilized a onestage PCR analysis (6), but this has not proved reliable even in their laboratory (3). In this paper, we highlight a simple and reliable method for APOE promoter genotyping that reduces the number of manipulations and restriction digests and the number of lanes of analytical polyacrylamide gel required for the assignment of APOE ⫺491 and ⫺427 genotypes. This may help reduce the amount of time taken to conduct large-scale studies. Restriction digestion of PCR products with two enzymes together has been used extensively in molecular biology in the past. In the interest of brevity, we have cited only one here (8). However, this is the first time that such a method has been systematically applied to the study of APOE promoter polymorphisms and compared to separate genotyping at the individual loci. MATERIALS AND METHODS
Subjects and DNA Extraction All procedures described in this study were approved by the research ethics committee of East London and the City Health Authority. The first 20 subjects (16 male and 4 female) recruited in a larger group of former severely head-injured patients who had previously been treated at the Royal London Hospital attended a follow-up clinic and had a small (8 ml) blood sample taken. The mean age of subjects at the time of injury (⫾SD) was 38.6 ⫾ 18 years and they were re183
184
MCLAUGHLIN ET AL.
cruited to the study on average 5 years after their injury. These statistics are representative of a group of head injury patients in that they are predominantly young and cohorts generally have a preponderance of males (around 80% (9)). Genomic DNA was extracted from the blood samples using a commercial kit (Qiagen, Germany). After spectrophotometric quantification of the amount of DNA extracted, samples were stored at ⫺20°C until use. DNA Amplification First-round PCR. Genomic DNA was diluted down to 20 ng/l in sterile deionized water and 5 l of this DNA was amplified by PCR in a total volume of 12.5 l using primers spanning the promoter region of the human APOE gene. The primers (purified salt-free (PSF) quality, MWG Biotech, Milton Keynes, UK) had the following sequences: upstream 5⬘-CAAGGTCACACAGCTGGCAAC-3⬘; downstream 5⬘-TCCAATCGACGGCTAGCTACC-3⬘ (2). In first- and second-round PCR, a single frozen aliquot of primers (10 M) was used on each day. The final concentration of reagents in the PCR tube was DNA 8 ng/l; 15 mM Tris–HCl, pH 8; 50 mM KCl; primers 1 M each; MgCl 2 2 mM; dNTPs 0.4 mM each; 0.1 Units/l Amplitaq Gold DNA polymerase (Applied Biosystems). First-round PCR was carried out using the following conditions: 12 min at 94°C (1 cycle, hot start); 30 s at 94°C, 30 s at 54°C, 1 min at 72°C (40 cycles); 10 min at 72°C (1 cycle, extension). After the program was complete, tubes were held at 4°C until products were analyzed. To check the fidelity of the first round of PCR, 2.5 l of the PCR reaction was electrophoresed on a 1% agarose/1⫻ TAE gel. The expected product for this PCR spans bases ⫺1017 to ⫹406 relative to the transcription start site of the APOE gene and is 1423 bp in size (EMBL Accession No. AF055343). Commonly, two major products of around 500 and 1400 bp were amplified. Second-round PCR. PCR products from first-round amplification were diluted 100-fold in sterile deionized water and 5 l of this sample underwent PCR in a final volume of 12.5 l with a pair of “nested” primers. The primers (PSF quality, MWG Biotech) had the following sequences: upstream (mismatches underlined) 5⬘-TGTTGGCCAGGCTGGTTTTAA-3⬘; downstream 5⬘-CCTCCTTTCCTGACCCTGTCC-3⬘ (2). The mismatched upstream primer, corresponding to bases ⫺512 to ⫺492 of the APOE gene, introduces DraI sites (5⬘-TTTAAA-3⬘) in the PCR product amplified from individuals with one or more ⫺491A alleles. The final concentration of reagents (except DNA) in the reaction tube was identical to that described above for first-round PCR. Second-round PCR was carried out using the following conditions: 12 min at 94°C (1 cycle, hot start); 30 s
at 94°C, 30 s at 52°C, 1 min at 72°C (35 cycles, amplification); 10 min at 72°C (1 cycle, extension). After the program was complete, tubes were held at 4°C until products were analyzed. To check the fidelity of the second round of PCR, 2.5 l of this PCR reaction was electrophoresed on a 1.8% agarose/1⫻ TAE gel. The expected product was 228 bp in size. Haplotyping To haplotype the regulatory region of the APOE gene, 2.5 l of the second-round PCR product was digested in a final volume of 10 l. The restriction digests contained 10 units of DraI (Roche Molecular Biochemicals), 4 units of AluI (Promega Corporation), and 100 ng/l BSA in 1⫻ Promega restriction enzyme buffer B and were incubated for 3 h at 37°C. Following digestion, enzymes were denatured by heating at 65°C for 15 min before the samples were held at 4°C. Two microliters of a gel loading buffer (0.25% bromphenol blue, 40% sucrose) was added to each sample and the mixture was electrophoresed at 100 V (5– 8 mA) on a 16% nondenaturing polyacrylamide gel (1⫻ TBE) in a minigel system (Bio-Rad Mini-Protean II) for 230 min. After electrophoresis, the gel was stained with 0.5 g/ml ethidium bromide (Sigma) in 1⫻ TBE for 30 min before being viewed by ultraviolet transillumination. The APOE [⫺491, ⫺427] haplotypes of subjects were determined from the pattern of restriction fragments on the gel: [A-C], 209 bp; [A-T], 144 and 65 bp; [T-T], 144 and 84 bp; and [T-C], 228 bp. The 10 possible combinations of these four haplotypes of the APOE promoter each produce a distinct pattern of bands (Fig. 2). ⫺491 and ⫺427 Genotyping APOE ⫺491 and ⫺427 genotypes were confirmed by digestion of the 228-bp second-round PCR products with DraI alone or AluI alone, respectively. A total of 2.5 l of PCR product was digested with either 10 Units of DraI (Roche) or 4 Units of AluI (Promega) in a final volume of 10 l for 3 h at 37°C. Following digestion, the enzymes were denatured and the digested fragments were analyzed as for haplotyping. The pattern of bands on the gel indicated a subject’s genotype: ⫺491 A/A 209 bp, T/T 228 bp, and A/T 228 and 209 bp; ⫺427, T/T 144 and 84 bp, C/C 228 bp, and T/C 228, 144, and 84 bp. These genotypes were compared to those that had been inferred from haplotype analysis. RESULTS
First-round amplifications of the promoter region of the APOE gene produced several products of varying size (Fig. 1a). The first-round of PCR was more reliable
HAPLOTYPE ANALYSIS OF THE APOLIPOPROTEIN E GENE
FIG. 1. Agarose gel electrophoresis of (a) first- and (b) secondround PCR of the polymorphic region of the human APOE promoter in five representative subjects (Nos. 1, 5, 18, 3, and 4). Note the multiple bands in first-round PCR (including the target at 1423 bp, indicated by the arrow) and the single band at 228 bp in secondround PCR. B, PCR reactions with no starting DNA; , DNA marker cut with EcoRI and HindIII; , X174 DNA marker cut with HinfI. Sizes of molecular weight markers (bp) are indicated along the side of the gel.
if, on a regular basis, primers were freshly diluted from 100 M stock solutions and frozen in small aliquots at ⫺20°C until use. A single aliquot was only ever used once. In each case the 1423-bp product was apparent (Fig. 1a) and thus the 228-bp fragment was always amplified in the second round with the nested primers (Fig. 1b). Despite varying annealing temperature, primer concentration, and magnesium ion concentration, we have
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been unable to eliminate the major 500-bp band in first-round PCR. In initial experiments, when firstround PCR failed to produce a band at 1423 bp due to problems with the primers (rectified by using fresh aliquots), the major 500-bp band was amplified, but second-round PCR failed to produce a band at 228 bp (data not shown). Therefore, it is unlikely that the 500-bp product of first-round PCR is an internal fragment of the APOE promoter that could interfere with subsequent analysis of genotype. Also, we have purified, cloned, and sequenced this fragment, which is actually 479 bp. The only matches in the nucleotide database (using a BLAST search) in the human APOE gene relate to the upstream primer sequence. No significant matches to any other sequences were found. Digestion of the 228-bp PCR product with DraI and AluI together produced a range of different fragment lengths on the polyacrylamide gel (Figs. 2 and 3a). The haplotypes assigned allowed us to infer ⫺491 and ⫺427 genotypes for each of the subjects (Fig. 3a). ⫺491 and ⫺427 genotyping using DraI or AluI alone confirmed that our inferences were correct in all cases (Figs. 3b and 3c). The distribution of haplotype combinations and relative haplotype and allele frequencies for the ⫺491 and ⫺427 loci are shown in Tables 1 and 2. Even given the small number of subjects studied here, these frequencies are reasonably close to those described for control subjects in previous studies at these loci (2– 6). DISCUSSION
This study confirms the utility of double-digestion of PCR fragments to ascribe APOE [⫺491, ⫺427] haplotype. From each of the haplotypes, it is always possible to infer both the ⫺491 and the ⫺427 genotypes of a
FIG. 2. Schematic gel image showing the pattern of bands on a polyacrylamide gel from subjects with the 10 possible [⫺491, ⫺427] haplotype combinations. Fragment sizes are indicated by the arrows. , X174 DNA marker cut with HinfI (marker sizes (bp) indicated).
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MCLAUGHLIN ET AL. TABLE 1
APOE [⫺491, ⫺427]/[⫺491, ⫺472] Haplotype Combinations and ⫺491A/T and ⫺427T/C Genotypes from 20 Subjects Subject
[⫺491,⫺427] haplotype combination
⫺491 genotype
⫺427 genotype
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
[T-T]/[T-T] [A-C]/[T-T] [A-T]/[T-T] [A-T]/[A-C] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-C] [A-T]/[T-T] [A-T]/[T-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-T]/[A-T] [A-C]/[A-C] [A-T]/[A-T] [A-T]/[A-T]
T/T A/T A/T A/A A/A A/A A/A A/A A/A A/A A/T A/T A/A A/A A/A A/A A/A A/A A/A A/A
T/T T/C T/T T/C T/T T/T T/T T/T T/T T/C T/T T/T T/T T/T T/T T/T T/T C/C T/T T/T
Note. Haplotypes and genotypes were determined independently.
Furthermore, haplotype analysis is more reliable than single digestion with DraI at locus ⫺491 in the APOE gene. Figure 3b illustrates that complete digestion with DraI of DNA amplified from ⫺491A homozygous (subjects 4, 5, and 18) or ⫺491 heterozygous individuals (subject 3) was often not possible. In order to assign genotypes, a degree if inference (“guesswork”) FIG. 3. Polyacrylamide gel electrophoresis of 228-bp products from PCR of the polymorphic region of the APOE promoter in five subjects (Nos. 1, 5, 18, 3, and 4). (a) Products cut with DraI and AluI together (the 65-bp band in 03 is clearer on the original gel); (b) products cut with DraI alone; (c) products cut with AluI alone. , X174 DNA marker cut with HinfI. Sizes of molecular weight markers are indicated along the side of the gel.
subject, whereas it is not always possible to infer [⫺491, ⫺427]/[⫺491, ⫺427] haplotype combination from the individual genotypes. For example, a subject who has a genotype of ⫺491A/T and ⫺427T/C (like subject 2 in this study) could have either of the two following haplotype combinations: [A-T]/[T-C] or [A-C]/ [T-T]. If the ⫺491A and ⫺427C alleles are associated with higher levels of expression of apoE (3), it is evident that the latter of these combinations could have implications for the likelihood of the subject developing Alzheimer’s disease or other diseases related to apoE (especially if the subject’s apoE phenotype contains the deleterious form apoE4). In the specific case of subject 2, we know that his apoE phenotype is purely apoE3.
TABLE 2
Frequencies for ⫺491, ⫺427 Haplotypes and the Possible Combinations Thereof, Together with Relative Allele Frequencies at APOE ⫺491 and ⫺427 Loci [⫺491, ⫺427] haplotype combination A-T/A-T A-T/T-T A-T/A-C T-T/T-T A-C/T-T A-C/A-C
Haplotype combination frequency 0.60 (12/20) 0.15 (3/20) 0.10 (2/20) 0.05 (1/20) 0.05 (1/20) 0.05 (1/20)
[⫺491, ⫺427] haplotype A-T T-T A-C
Relative haplotype frequency 0.725 0.15 0.125
allele ⫺491A ⫺491T ⫺427T ⫺427C
Relative allele frequency 0.850 0.150 0.875 0.125
HAPLOTYPE ANALYSIS OF THE APOLIPOPROTEIN E GENE
must be employed based upon the relative intensity of band staining on the gel. In this study, we have used the enzyme from the supplier that has given least problems. Stocks of DraI purchased from other suppliers were even less reliable. Whether this problem arises because of difficulty in fully digesting a nested PCR product or a problem with all supplies of DraI is unclear. What is clear is that haplotype analysis with double digestions poses no such problems (Fig. 3a). Another potentially interesting point is that in our study, the haplotype combinations [T-T]/[T-T], [A-C]/ [T-T], and [A-C]/[A-C] are represented slightly more often (each 5% of subjects) than in a previous study where less than 5% of control subjects had these genotypes (3). Given the small number of patients studied here, no real conclusions can be drawn and whether these relatively rare haplotype combinations are associated with a more favorable outcome following traumatic brain injury remains to be seen. We should add that the technique of haplotyping by restriction digestion of PCR products with two enzymes together has been used over the past 20 years or more. One example is a study of prenatal diagnosis of ␣-thalassemia by Lebo et al. (8). In this study, the authors extracted and concentrated genomic DNA from fetuses and expectant parents before digesting the samples with BglII and Asp718. This then allowed the detection of the most common deletions in the gene that are responsible for the disease in the South East Asian population by subsequent Southern blotting of the digested DNA and analysis of fragment sizes. Although the two methods have a similar digestion with two restriction enzymes together, their method differed somewhat to that reported here. First, they used Southern blotting of digested genomic DNA rather than PAGE of digested PCR products for accurate sizing and also relied upon existing restriction enzyme sites in the ␣-globin gene, rather than introducing one of the sites by using mismatched primers. In conclusion, this method cuts down substantially on manipulations of samples and tubes in an already
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laborious procedure and is likely to be very useful in large-scale studies of APOE promoter polymorphisms. A further advantage is that the more cryptic combinations of APOE [⫺491, ⫺427] haplotypes are readily distinguishable and need not be investigated “after the fact.” REFERENCES 1. Wenham, P. R., Price, W. H., and Blundell, G. (1991) Apolipoprotein E genotyping by one-stage PCR. Lancet 337, 1158 –1159. 2. Strittmatter, W. J., Saunders, A. M., Schmechel, D., PericakVance, M., Enghild, J., Salvesen, G. S., and Roses, A. D. (1993) Apolipoprotein E: High-avidity binding to -amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc. Natl. Acad. Sci. USA 90, 1977–1981. 3. Artiga, M. J., Bullido, M. J., Frank, A., Sastre, I., Recuero, M., Garcia, M. A., Lendon, C. L., Han, S. W., Morris, J. C., Vazquez, J., Goate, A., and Valdivieso, F. (1998) Risk for Alzheimer’s disease correlates with transcriptional activity of the APOE gene. Hum. Mol. Genet. 7, 1887–1892. 4. Laws, S. M., Taddei, K., Martins, G., Paton, A., Fisher, C., Clarnette, R., Hallmayer, J., Brooks, W. S., Gandy, S. E., and Martins, R. N. (1999) The -491AA polymorphism in the APOE gene is associated with increased plasma apoE levels in Alzheimer’s disease. NeuroReport 10, 879 – 882. 5. Ahmed, A. R. H., MacGowan, S. H., Culpan, D., Jones, R. W., and Wilcock, G. K. (1999) The ⫺491A/T polymorphism of the apolipoprotein E gene is associated with the apoE⑀4 allele and Alzheimer’s disease. Neurosci. Lett. 263, 217–219. 6. Artiga, M. J., Bullido, M. J., Sastre, I., Recuero, M., Garcia, M. A., Aldudo, J., Vazquez, J., and Valdivieso, F. (1998) Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E gene. FEBS Lett. 421, 105–108. 7. Thome, J., Gewirtz, J. C., Sakai, N., Zachariou, V., Retz-Junginger, P., Retz, W., Duman, R. S., and Ro¨sler, M. (1999) Polymorphisms of the human apolipoprotein E promoter and bleomycin hydrolase gene: Risk factors for Alzheimer’s dementia? Neurosci. Lett. 273, 37– 40. 8. Lebo, R. V., Saiki, R. K., Swanson, K., Montano, M. A., Erlich, H. A., and Golbus, M. S. (1990) Prenatal diagnosis of alphathalassemia by polymerase chain reaction and dual restriction enzyme analysis. Hum. Genet. 85, 293–299. 9. Wilson, J. T. L., Pettigrew, L. E. L., and Teasdale, G. M. (2000) Emotional and cognitive consequences of head injury in relation to the Glasgow outcome scale. J. Neurol. Neurosurg. Psychiatr. 69, 204 –209.