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Improved method facilitates reliable APOE genotyping of genomic DNA extracted from formaldehyde-fixed pathology specimens
Journal of Neuroscience Methods 79 (1998) 229 – 231
Improved method facilitates reliable APOE genotyping of genomic DNA extracted from formaldehyde-fixed pathology specimens Estifanos Ghebremedhin *, Heiko Braak, Eva Braak, Ju¨rgen Sahm Department of Anatomy, J.W. Goethe-Uni6ersity, Theodor Stern Kai 7, D-60590 Frankfurt/Main, Germany Received 18 July 1997; received in revised form 14 November 1997; accepted 15 November 1997
Abstract Apolipoprotein E (APOE) genotyping of genomic DNA extracted from formaldehyde-fixed specimens is cumbersome: there is not only a low yield or failure of PCR amplification (presumably due to degradation of DNA in the formaldehyde-fixed and paraffin-embedded tissue), but the standard method also involves the separation of DNA fragments as small as 48, 72, 81 and 91 bp requiring high-yield PCR products. Here we report about a semi-nested PCR method suitable for providing specific high-yield PCR products from DNA that has been extracted from formaldehyde-fixed specimens which initially generate low-quality templates. This method facilitates reliable APOE genotyping of DNA from difficult templates. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Formaldehyde-fixation; Apolipoprotein E; Genotyping; Semi-nested PCR
1. Introduction Apolipoprotein E (APOE) is one of the constituents of plasma lipoproteins that participates in the transport of cholesterol and specific lipids (Mahley, 1988). APOE provides three common isoforms E2, E3, and E4, encoded by the e2, e3, and e4 alleles which determine six genotypes. These three isoforms differ from each other by means of cysteine-arginine interchanges at positions 112 and 158. Various studies have documented the association of APOE e4 allele with an increased risk factor for both Alzheimer’s disease (Saunders et al., 1993; Strittmatter et al., 1993) and cardiovascular disease (Davignon et al., 1988). There is currently much interest in employing well-documented collections of pathology specimens for retrospective genotyping of APOE alleles. However, the use of formaldehyde-fixed specimens for this procedure is cumbersome owing to the failure of the polymerase chain reaction (PCR) or to the generation of low-yield products in PCR and the * Corresponding author. Tel.: +49 69 63016912; fax: + 49 69 63016425. 0165-0270/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 0 2 7 0 ( 9 7 ) 0 0 1 9 7 - 0
separation of small fragments (48, 72, 81 and 91 bp DNA) in the subsequent restriction isotyping which require high yield PCR products. The introduction of a semi-nested PCR assay, which increases the yield of PCR product, overcomes this difficulty and is described in detail in the present study.
2. Materials and methods Material for PCR included archival formaldehydefixed and paraffin-embedded brain, liver, spleen or stored post-mortem blood samples of subjects autopsied between 1980 and 1996. The tissue was fixed in an aqueous solution of formaldehyde for a period ranging from a few days (liver, spleen) up to 1 month (brain) prior to paraffin-embedding. Genomic DNA was extracted from paraffin blocks using previously described protocols (Ko¨sel and Graeber, 1994). Initial studies were conducted in order to establish the feasibility of amplifying DNA obtained from 150 archival samples for APOE genotyping. The samples were subjected to PCR for APOE genotyping using the method previ-
230
E. Ghebremedhin et al. / Journal of Neuroscience Methods 79 (1998) 229–231
Fig. 1. APOE genotype analysis of PCR products generated using DNA prepared from formaldehyde-fixed and paraffin-embedded specimens. (A) Evaluation of DNA amplification by agarose gel electrophoresis. Lanes 1, 2 and 3 = PCR products (227 bp) from first round PCR; Lanes 5, 6 and 7 = PCR products (197 bp) from second round PCR (semi-nested PCR). Twelve microliters of PCR products were loaded in each lane. (B) 15% polyacrylamide gel showing HhaI restriction fragment length polymorphism of the APOE gene PCR product obtained using the semi-nested PCR. Lanes 1 =MspI-digested plasmid pBR 322 size marker; 2 and 3 =APOE genotypes E3/E3 and E4/E4, respectively.
ously described (Wenham et al., 1991). Fifty out of 150 samples yielded low PCR product which impeded subsequent DNA processing. These 50 samples were selected for the present study. For each of the samples a two-stage PCR protocol was employed to amplify the target DNA encompassing codons 112 and 158 of the APOE gene. The first PCR was carried out with the outer primer sequences (Ape-1: 5%-TCCAAGGAGCTGCAGGCGGCGCA-3% and Ape-3: 5%-ACAGAATTCGCCCCGGCCTGGTACACTGCCA-3%) according to the protocol previously described (Wenham et al., 1991), amplifying a 227 bp DNA product. One microliter of 10 – 100-fold diluted aliquot of the initial PCR was used as a template for the second PCR (semi-nested PCR) with the nested primer R-1: 5%-CTGGGCGCGGACATGGAG-3% reported elsewhere (Egensperger et al., 1995), and the outer primer Ape-3, and gave rise to a 197 bp DNA product. The semi-nested PCR was performed in a 50 ml reaction mixture containing 25 pmol of each primer, 1.5 mmol/l MgCl2, 200 mmol/l each dNTP, 50 mmol/l KCl, 10 mmol/l Tris – HCl (pH 9), 0,1% Triton X-100, and 1.4 units Thermus aquaticus polymerase (MWGBiotech, Ebersberg). One cycle of denaturation at 95°C for 3 min, was followed by 35 cycles at 95°C for 30 s, 68°C for 30 s, and 72°C for 40 s. The reaction was terminated with a 10-min extension step at 72°C in an Omn-E Thermal Cycler (Hybaid, Middlesex, UK). An aliquot (25 ml) of the PCR-amplified product was digested with restriction enzyme HhaI. For genotype analysis, the small fragment-sized cleavage products of HhaI (48, 72, 81 and 91 bp) were electrophoresed
through gels consisting of 5% MetaPhor agarose gel (FMC BioProducts, Crook et al., 1994) or polyacrylamide gels stained with ethidium bromide and visualized with ultraviolet illumination.
3. Results and discussion First-round PCR amplification was performed on samples containing templates with primers Ape-1 and Ape-3 (see Section 2). A 15 ml portion from each of the completed PCR reaction was subjected to 2% agarose gels and DNA bands were visualized under UV light by ethidium bromide fluorescence. In most instances no bands were detected (Fig. 1A). In some other cases the bands were within the detection limits. Therefore, we attempted to devise a procedure that would enhance the PCR analysis. The use of a semi-nested PCR with PCR products from first round PCR as a starting template (see Section 2) produced excellent results with the expected 197 bp bands (Fig. 1A). Fig. 1B shows gel-separated, HhaI-digested products of semi-nested PCR. Many factors influence the quality and quantity of DNA prepared from archival tissue samples, beginning from preservation to DNA extraction steps (Ko¨sel and Graeber, 1994). DNA obtained from the majority of cases could be used successfully for APOE genotyping by employing the standard methods. Nonetheless, in some samples the yield of PCR product is insufficient for further processing. Introduction of a new semi-nested PCR assay enabled us to enhance the amplification products. The technique has been applied
E. Ghebremedhin et al. / Journal of Neuroscience Methods 79 (1998) 229–231
successfully to all of the samples included in this study, it is convenient, and it permits wide-ranging applicability in APOE genotyping of archival tissue specimens.
Acknowledgements This study was kindly supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie. The authors express their thanks to Prof Bratzke (Department of Forensic Medicine of the University of Frankfurt) as well as Prof Goebel and Dr Bohl (Department of Neuropathology of the University of Mainz) for providing us with autopsy tissue specimens.
References Crook R, Hardy J, Duff K. Single-day apolipoprotein E genotyping. J Neurosci Methods 1994;53:125–7.
.
231
Davignon J, Gregg RE, Sing CF. Apolipoprotein E polymorphism and atherosclerosis. Arteriosclerosis 1988;8:1 – 21. Egensperger R, Ko¨sel S, Schnabel R, Mehraein P, Graeber MB. Apolipoprotein E genotype and neuropathological phenotype in two members of a German family with chromosome 14-linked early onset Alzheimer’s disease. Acta Neuropathol 1995;90:257– 65. Ko¨sel S, Graeber MB. Use of neuropathological tissue for molecular genetic studies: parameters affecting DNA extraction and polymerase chain reaction. Acta Neuropathol 1994;88:19 – 25. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 1988;240:622 – 30. Saunders AM, Strittmatter WJ, Schmechel D, George-Hyslop PH, Pericak-Vance MA, Joo SH, Rosi BL, Gusella JF, CrapperMacLachlan DR, Alberts MJ, Hulette C, Crain B, Goldgaber D, Roses AD. Association of apolipoprotein E, allele E4 with lateonset familial and sporadic Alzheimer’s disease. Neurology 1993;43:1467 – 72. Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, Enghild J, Salvesen GS, Roses AD. Apolipoprotein E: high-avidity binding to b-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer’s disease. Proc Natl Acad Sci USA 1993;90:1977 – 81. Wenham PR, Price WH, Blundell G. Apolipoprotein E genotyping by one-stage PCR. Lancet 1991;337:1158 – 9.
Improved method facilitates reliable APOE genotyping of genomic DNA extracted from formaldehyde-fixed pathology specimens Estifanos Ghebremedhin *, Heiko Braak, Eva Braak, Ju¨rgen Sahm Department of Anatomy, J.W. Goethe-Uni6ersity, Theodor Stern Kai 7, D-60590 Frankfurt/Main, Germany Received 18 July 1997; received in revised form 14 November 1997; accepted 15 November 1997
Abstract Apolipoprotein E (APOE) genotyping of genomic DNA extracted from formaldehyde-fixed specimens is cumbersome: there is not only a low yield or failure of PCR amplification (presumably due to degradation of DNA in the formaldehyde-fixed and paraffin-embedded tissue), but the standard method also involves the separation of DNA fragments as small as 48, 72, 81 and 91 bp requiring high-yield PCR products. Here we report about a semi-nested PCR method suitable for providing specific high-yield PCR products from DNA that has been extracted from formaldehyde-fixed specimens which initially generate low-quality templates. This method facilitates reliable APOE genotyping of DNA from difficult templates. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Formaldehyde-fixation; Apolipoprotein E; Genotyping; Semi-nested PCR
1. Introduction Apolipoprotein E (APOE) is one of the constituents of plasma lipoproteins that participates in the transport of cholesterol and specific lipids (Mahley, 1988). APOE provides three common isoforms E2, E3, and E4, encoded by the e2, e3, and e4 alleles which determine six genotypes. These three isoforms differ from each other by means of cysteine-arginine interchanges at positions 112 and 158. Various studies have documented the association of APOE e4 allele with an increased risk factor for both Alzheimer’s disease (Saunders et al., 1993; Strittmatter et al., 1993) and cardiovascular disease (Davignon et al., 1988). There is currently much interest in employing well-documented collections of pathology specimens for retrospective genotyping of APOE alleles. However, the use of formaldehyde-fixed specimens for this procedure is cumbersome owing to the failure of the polymerase chain reaction (PCR) or to the generation of low-yield products in PCR and the * Corresponding author. Tel.: +49 69 63016912; fax: + 49 69 63016425. 0165-0270/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 0 2 7 0 ( 9 7 ) 0 0 1 9 7 - 0
separation of small fragments (48, 72, 81 and 91 bp DNA) in the subsequent restriction isotyping which require high yield PCR products. The introduction of a semi-nested PCR assay, which increases the yield of PCR product, overcomes this difficulty and is described in detail in the present study.
2. Materials and methods Material for PCR included archival formaldehydefixed and paraffin-embedded brain, liver, spleen or stored post-mortem blood samples of subjects autopsied between 1980 and 1996. The tissue was fixed in an aqueous solution of formaldehyde for a period ranging from a few days (liver, spleen) up to 1 month (brain) prior to paraffin-embedding. Genomic DNA was extracted from paraffin blocks using previously described protocols (Ko¨sel and Graeber, 1994). Initial studies were conducted in order to establish the feasibility of amplifying DNA obtained from 150 archival samples for APOE genotyping. The samples were subjected to PCR for APOE genotyping using the method previ-
230
E. Ghebremedhin et al. / Journal of Neuroscience Methods 79 (1998) 229–231
Fig. 1. APOE genotype analysis of PCR products generated using DNA prepared from formaldehyde-fixed and paraffin-embedded specimens. (A) Evaluation of DNA amplification by agarose gel electrophoresis. Lanes 1, 2 and 3 = PCR products (227 bp) from first round PCR; Lanes 5, 6 and 7 = PCR products (197 bp) from second round PCR (semi-nested PCR). Twelve microliters of PCR products were loaded in each lane. (B) 15% polyacrylamide gel showing HhaI restriction fragment length polymorphism of the APOE gene PCR product obtained using the semi-nested PCR. Lanes 1 =MspI-digested plasmid pBR 322 size marker; 2 and 3 =APOE genotypes E3/E3 and E4/E4, respectively.
ously described (Wenham et al., 1991). Fifty out of 150 samples yielded low PCR product which impeded subsequent DNA processing. These 50 samples were selected for the present study. For each of the samples a two-stage PCR protocol was employed to amplify the target DNA encompassing codons 112 and 158 of the APOE gene. The first PCR was carried out with the outer primer sequences (Ape-1: 5%-TCCAAGGAGCTGCAGGCGGCGCA-3% and Ape-3: 5%-ACAGAATTCGCCCCGGCCTGGTACACTGCCA-3%) according to the protocol previously described (Wenham et al., 1991), amplifying a 227 bp DNA product. One microliter of 10 – 100-fold diluted aliquot of the initial PCR was used as a template for the second PCR (semi-nested PCR) with the nested primer R-1: 5%-CTGGGCGCGGACATGGAG-3% reported elsewhere (Egensperger et al., 1995), and the outer primer Ape-3, and gave rise to a 197 bp DNA product. The semi-nested PCR was performed in a 50 ml reaction mixture containing 25 pmol of each primer, 1.5 mmol/l MgCl2, 200 mmol/l each dNTP, 50 mmol/l KCl, 10 mmol/l Tris – HCl (pH 9), 0,1% Triton X-100, and 1.4 units Thermus aquaticus polymerase (MWGBiotech, Ebersberg). One cycle of denaturation at 95°C for 3 min, was followed by 35 cycles at 95°C for 30 s, 68°C for 30 s, and 72°C for 40 s. The reaction was terminated with a 10-min extension step at 72°C in an Omn-E Thermal Cycler (Hybaid, Middlesex, UK). An aliquot (25 ml) of the PCR-amplified product was digested with restriction enzyme HhaI. For genotype analysis, the small fragment-sized cleavage products of HhaI (48, 72, 81 and 91 bp) were electrophoresed
through gels consisting of 5% MetaPhor agarose gel (FMC BioProducts, Crook et al., 1994) or polyacrylamide gels stained with ethidium bromide and visualized with ultraviolet illumination.
3. Results and discussion First-round PCR amplification was performed on samples containing templates with primers Ape-1 and Ape-3 (see Section 2). A 15 ml portion from each of the completed PCR reaction was subjected to 2% agarose gels and DNA bands were visualized under UV light by ethidium bromide fluorescence. In most instances no bands were detected (Fig. 1A). In some other cases the bands were within the detection limits. Therefore, we attempted to devise a procedure that would enhance the PCR analysis. The use of a semi-nested PCR with PCR products from first round PCR as a starting template (see Section 2) produced excellent results with the expected 197 bp bands (Fig. 1A). Fig. 1B shows gel-separated, HhaI-digested products of semi-nested PCR. Many factors influence the quality and quantity of DNA prepared from archival tissue samples, beginning from preservation to DNA extraction steps (Ko¨sel and Graeber, 1994). DNA obtained from the majority of cases could be used successfully for APOE genotyping by employing the standard methods. Nonetheless, in some samples the yield of PCR product is insufficient for further processing. Introduction of a new semi-nested PCR assay enabled us to enhance the amplification products. The technique has been applied
E. Ghebremedhin et al. / Journal of Neuroscience Methods 79 (1998) 229–231
successfully to all of the samples included in this study, it is convenient, and it permits wide-ranging applicability in APOE genotyping of archival tissue specimens.
Acknowledgements This study was kindly supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie. The authors express their thanks to Prof Bratzke (Department of Forensic Medicine of the University of Frankfurt) as well as Prof Goebel and Dr Bohl (Department of Neuropathology of the University of Mainz) for providing us with autopsy tissue specimens.
References Crook R, Hardy J, Duff K. Single-day apolipoprotein E genotyping. J Neurosci Methods 1994;53:125–7.
.
231
Davignon J, Gregg RE, Sing CF. Apolipoprotein E polymorphism and atherosclerosis. Arteriosclerosis 1988;8:1 – 21. Egensperger R, Ko¨sel S, Schnabel R, Mehraein P, Graeber MB. Apolipoprotein E genotype and neuropathological phenotype in two members of a German family with chromosome 14-linked early onset Alzheimer’s disease. Acta Neuropathol 1995;90:257– 65. Ko¨sel S, Graeber MB. Use of neuropathological tissue for molecular genetic studies: parameters affecting DNA extraction and polymerase chain reaction. Acta Neuropathol 1994;88:19 – 25. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 1988;240:622 – 30. Saunders AM, Strittmatter WJ, Schmechel D, George-Hyslop PH, Pericak-Vance MA, Joo SH, Rosi BL, Gusella JF, CrapperMacLachlan DR, Alberts MJ, Hulette C, Crain B, Goldgaber D, Roses AD. Association of apolipoprotein E, allele E4 with lateonset familial and sporadic Alzheimer’s disease. Neurology 1993;43:1467 – 72. Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, Enghild J, Salvesen GS, Roses AD. Apolipoprotein E: high-avidity binding to b-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer’s disease. Proc Natl Acad Sci USA 1993;90:1977 – 81. Wenham PR, Price WH, Blundell G. Apolipoprotein E genotyping by one-stage PCR. Lancet 1991;337:1158 – 9.