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1 Deutsches Herzzentrum München and 1. Medizinische Klinik rechts der Isar, Technische Universität München, D-80636 München, Germany.
2 Technion—Israel Institute of Technology, The Bruce Rappaport Faculty of Medicine, Haifa, Israel.
aAddress correspondence to this author at: Deutsches Herzzentrum München, Experimentelle Kardiologie, Lazarettstrasse 36, D-80636 Munich, Germany. Fax 49-89-1218-3053; e-mail wkoch{at}dhm.mhn.de.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Taking advantage of the selectivity of PCR, we amplified DNA segments specifically representing haptoglobin alleles Hp 1 and Hp 2 from genomic DNA. The products were analyzed by agarose gel electrophoresis. Haptoglobin phenotyping of plasma samples was performed by polyacrylamide gel electrophoresis and peroxidase staining.
Results: Exploiting the known size difference between Hp 1 and Hp 2, we amplified allele-specific DNA molecules with one pair of oligonucleotide primers. As an alternative, we used separate primer pairs to generate amplification products indicative of alleles Hp 1 and Hp 2. Because of the primer design, genotype determination was not compromised by sequence variations specifying haptoglobin allele subtypes S and F. For the same reason, the sequence similarity between the haptoglobin gene and the haptoglobin-related gene did not interfere with the accuracy of genotyping. Analysis with restriction enzymes demonstrated the authenticity of the allele-specific DNA products. Haptoglobin DNA genotyping and protein phenotyping, performed in parallel, yielded fully corresponding results. In a group of 249 individuals, the haptoglobin genotype distribution was as follows: 14.5% Hp 1-1, 48.2% Hp 2-1, and 37.3% Hp 2-2.
Conclusion: The new method can be used for genotyping of a common haptoglobin polymorphism.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1700 bp in Hp 2 but not Hp 1 (2). Most likely, formation of the Hp 2 allele is the result of a breakage and reunion event at nonhomologous positions within the fourth and second introns of two Hp 1 genes. As a consequence of this illegitimate crossing-over event, exons 5 and 6 of allele Hp 2 originate from exons 3 and 4, respectively, of one of these Hp 1 genes. The haptoglobin Hp 1/2 polymorphism has been associated with the prevalence of infections, autoimmune diseases, cardiovascular diseases, and other disorders, thus suggesting a broad clinical significance (1). For example, the Hp 2-2 phenotype was overrepresented in patients with more severe forms of myocardial infarction (3). More recently, the Hp 1-1 phenotype was reported to give protection from two critical vascular complications of diabetes mellitus: diabetic nephropathy and restenosis after percutaneous transluminal coronary angioplasty (4). Conventionally, assay systems established for phenotypic distinction between protein variants Hp 1-1, Hp 2-1, and Hp 2-2 are used for haptoglobin typing (5). To allow the use of genomic DNA, we have developed a haptoglobin genotyping method based on PCR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dna sequences specifying haptoglobin alleles Hp 1 and Hp 2 and the haptoglobin-related gene
The Hp 1- and Hp 2-specific sequences are contained in the EMBL/GenBank Data Libraries under accession numbers AC004682 and M69197, respectively (6)(7). In these sequences, the haptoglobin gene is represented by the allele subtypes Hp 1S and Hp 2FS (2). According to the sequence present in AC004682, the Hp 1-specific DNA region has a length of 1711 bp (Fig. 1
); it extends, in an inverse orientation, from nucleotide position 188616 to nucleotide position 186906. In the sequence present in M69197, the 3435-bp Hp 2-specific DNA segment starts at position 2804 and ends at position 6238; it contains two units of similar sequences, consisting of 1724 and 1711 bp (Fig. 1
). The sequences of the 1711-bp segments present in AC004682 and M69197 are complementary with the exception of one divergence located at position 188141 in AC004682, which corresponds to position 5003 in M69197. The sequence of the haptoglobin-related gene is also contained in M69197.
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The sizes of the PCR products and DNA fragments generated by restriction enzymes were calculated on the basis of the sequences present in AC004682 or M69197, as appropriate.
determination of haptoglobin genotypes
Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA Blood Kit as suggested by the supplier (Qiagen). Oligonucleotide primers A (5'-GAGGGGAGCTTGCCTTTCCATTG-3') and B (5'-GAGATTTTTGAGCCCTGGCTGGT-3') were used for amplification of a 1757-bp Hp 1 allele-specific sequence and a 3481-bp Hp 2 allele-specific sequence (Fig. 1
). Primers C (5'-CCTGCCTCGTATTAACTGCACCAT-3') and D (5'-CCGAGTGCTCCACATAGCCATGT-3') were used to amplify a 349-bp Hp 2 allele-specific sequence (Fig. 1
). Primers were synthesized by Applied Biosystems.
In Hp 1 and Hp 2, the annealing sites for primer A are located immediately upstream of the 1711-bp unit and the 1724-bp unit, respectively (Fig. 1
). The nucleotide at the 5' end of primer A corresponds to position 188639 in AC004682 (Hp 1) and position 2781 in M69197 (Hp 2). Primer B has binding sites just downstream of the 1711-bp elements of Hp 1 and Hp 2 (Fig. 1
). The nucleotide at the 5' end of primer B corresponds to position 186883 in AC004682 (Hp 1) and position 6261 in M69197 (Hp 2). Depending on the genotype represented by the template DNA, a Hp 1-specific product of 1757 bp and/or a Hp 2-specific product of 3481 bp is generated in PCRs with primers A and B (Fig. 1
). Primers C and D have one binding site in allele Hp 1 and two binding sites in allele Hp 2 (Fig. 1
). In reactions with primers C and D, a PCR product, 349 bp in length, is generated only in the presence of the Hp 2 template, whereas no product is formed in the presence of the Hp 1 template (Fig. 1
). This kind of allelic specificity is attributable to the relative positions of the binding sites and the 5'
3' orientation of primers C and D (Fig. 1
). The template for the 349-bp Hp 2-specific amplification product, including the annealing sites for primers C and D, extends from nucleotide position 4352 to 4700 in M69197. There is also one binding site each for primers C and D at the corresponding positions in the haptoglobin-related gene, although the sequences of primers and annealing sites are not 100% complementary. Importantly, with the haptoglobin-related gene as a template, amplification reactions with primers C and D proceed in opposite directions, which does not allow a PCR product to be generated.
The 20-µL reactions contained 2 U of Taq polymerase (Qiagen), 1–100 ng of DNA, and 200 µM each of dATP, dCTP, dGTP, and dTTP (Invitrogen); PCR buffer was used as suggested by the supplier (Qiagen) with no supplements added. After initial denaturation at 95 °C for 2 min, the two-step thermocycling procedure consisted of denaturation at 95 °C for 1 min and annealing and extension at 69 °C for 2 min (in the presence of primers A and B or primers A, B, C, and D) or 1 min (in the presence of primers C and D only), repeated for 35 cycles, and followed by a final extension at 72 °C for 7 min. The thermocyclers used were GeneAmp PCR systems 9600 and 9700 (Applied Biosystems). For genotype assignments, the PCR products were separated in 0.7% agarose gels. Eight percent polyacrylamide gels (Invitrogen) were also suitable in cases where primers C and D were used for the determination of the Hp 2 allele instead of primers A and B. Genotype determinations were done without knowledge of the phenotyping results.
restriction enzyme analysis
Restriction enzyme analysis was done to verify the identity of Hp 1- and Hp 2-specific PCR products. The 1757- and 3481-bp products were digested with restriction enzyme MlsI, and the 349-bp product was digested with DraI, as recommended by the supplier (MBI Fermentas). DNA fragments were separated by gel electrophoresis.
determination of haptoglobin phenotypes
Haptoglobin phenotype was determined from 10 µL of hemoglobin-enriched plasma by polyacrylamide gel electrophoresis and peroxidase staining (8). Briefly, serum (10 µL) was mixed with 2 µL of a 10% hemoglobin solution, and the samples were incubated for 5 min at room temperature to permit the haptoglobin-hemoglobin complexes to form. An equal volume of sample buffer containing 125 mmol/L Tris base (pH 6.8), 200 g/L glycerol, and 0.01 g/L bromphenol blue was added to each sample before electrophoresis. The haptoglobin-hemoglobin complex was resolved by polyacrylamide electrophoresis using a buffer containing 25 mmol/L Tris base and 192 mmol/L glycine. The stacking gel was 4% polyacrylamide (29:1 acrylamide–bis-acrylamide) in 125 mmol/L Tris base, pH 6.8, and the separating gel was 4.7% polyacrylamide (29:1 acrylamide–bis-acrylamide) in 360 mmol/L Tris base, pH 8.8. Electrophoresis was performed at a constant voltage of 250 V for 3 h. After the electrophoresis was completed, the haptoglobin-hemoglobin complexes were visualized by soaking the gel in freshly prepared staining solution. The staining solution contained 5 mL of 2 g/L 3,3',5,5'-tetramethylbenzidine in methanol, 0.5 mL of dimethyl sulfoxide, 10 mL of a 50 mL/L solution of glacial acetic acid, 1 mL of a 10 g/L potassium ferricyanide solution, and 150 µL of 300 g/L solution of hydrogen peroxide. The bands corresponding to the haptoglobin-hemoglobin complex were readily visible within 15 min and were stable for more than 48 h. All gels were documented with photographs. Phenotypes Hp 1-1, Hp 2-2, and Hp 2-1 were distinguished by a characteristic pattern of bands representing the haptoglobin-hemoglobin complexes. Phenotype determinations were done without knowledge of the genotyping results.
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Results |
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). After electrophoresis of the reaction products in 0.7% agarose gels, Hp genotype-specific banding patterns were obtained: genotypes Hp 1-1 and Hp 2-2 were characterized by single bands representing the 1757- and 3481-bp products, respectively, and genotype Hp 2-1 was characterized by the presence of both the 1757- and 3481-bp products (Fig. 2A
).
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With the heterozygous genotype Hp 2-1, the Hp 1-specific 1757-bp band was considerably more intense than the Hp 2-specific 3481-bp band (Fig. 2A
, lane 3). Occasionally, in the presence of the 1757-bp product, it was not possible to conclusively determine whether the 3481-bp Hp 2-specific PCR product was also present. In these cases, an alternative protocol (protocol 2), consisting of two separate reactions, was chosen: one reaction, using primers A and B, was aimed at detecting the 1757-bp Hp 1-specific product, and the other reaction, using primers C and D, was aimed at detecting the 349-bp Hp 2-specific product (Fig. 1
). Using 0.7% agarose gels, we either electrophoresed the products of the two reactions in separate lanes (Fig. 2B
, lanes 2–7) or, after combination of aliquots of the products, in the same lane (Fig. 2B
, lanes 8–10).
DNA amplifications using the four primers, A, B, C, and D, in one reaction offered another modification of the PCR protocol (protocol 3). In this case, the product pattern was more complex than that obtained with the combined samples of the simplex reactions (Fig. 2C
). As predicted from the genomic sequences, four PCR products were generated in addition to the 1757-bp Hp 1-specific product and the 349-bp Hp 2-specific product: two Hp 2-specific products of 1923 bp (with primers A and D) and 1910 bp (with primers B and C) and two haptoglobin allele-nonspecific products of 196 bp (with primers A and D) and 195 bp (with primers B and C). In reactions with four primers, a PCR product of 450 bp was generated, which appeared to be Hp 2-specific, because it occurred only together with the 349-bp product (Fig. 2C
, lanes 3 and 4). We do not know which primer pair gave rise to this PCR product. Importantly, the appearance of the additional products, characteristic of the PCR system using four primers, did not interfere with genotype determination. The 3481-bp Hp 2-specific product was not synthesized in detectable amounts in the presence of the four primers combined. Comparative testing with DNA samples from 50 individuals showed that the three protocols for haptoglobin genotyping yielded identical results (data not shown). With protocol 1, 10 ng of genomic DNA was required for genotyping, whereas with protocols 2 and 3, 1 ng of DNA was sufficient.
The PCR products critical for genotype determination were identified by analysis with restriction enzymes MlsI and DraI. In accordance to the Hp 1- and Hp 2-specific genomic DNA sequences, MlsI cleaved the 1757-bp Hp 1-specific product into two fragments of 1206 and 551 bp and the 3481-bp Hp 2-specific product into three fragments of 1715, 1215, and 551 bp (Fig. 3A
). DraI digested the 349-bp Hp 2-specific product into two fragments of 193 and 156 bp (Fig. 3B
).
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The haptoglobin genotypes of 249 consecutive patients were determined with genomic DNA prepared from blood samples. With PCR protocol 1, we successfully genotyped 244 of the 249 samples, whereas we could not establish the genotype of 5 samples. In these five cases, the 1757-bp Hp 1-specific product was present as a relatively weak band, probably because of low concentrations of genomic DNA. In the same lanes, the 3481-bp Hp 2-specific band was not visible, and it was unclear whether individual samples did not contain the Hp 2 allele or whether the 3481-bp product was present at concentrations too low to be detected in the gel. Thus, in these cases, it was not possible to decide whether the genotype was Hp 1-1 or Hp 2-1. Subsequent genotyping of the five samples with PCR protocols 2 and 3 independently demonstrated that two samples were Hp 1-1 and three samples were Hp 2-1.
The accuracy of haptoglobin genotyping was tested by comparison with the results of haptoglobin protein phenotyping performed with plasma samples prepared from blood of the same 249 individuals (data not shown). There was no discrepancy between the genotyping and phenotyping results. The genotype/phenotype distribution obtained from the 249 probands was as follows: Hp 1-1, 14.5%; Hp 2-1, 48.2%; and Hp 2-2, 37.3%. Accordingly, the Hp 1 allele frequency was 0.39.
The haptoglobin genotyping protocols described here were suitable for use with DNA samples that had been stored at -20 °C or lower for up to several years, and they worked well with DNA samples that were repeatedly frozen and thawed during this period of time. We also found that DNA prepared from blood that had been stored for years at -20 °C or lower and that had gone through several freezing and thawing cycles was still in a condition that allowed accurate genotype determination. We have not examined the usefulness of the genotyping method with DNA isolated from sources other than blood, e.g., from paraffin sections, or obtained from samples stored under unfavorable conditions for extended periods of time.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1700-bp-long sequence, each copy including two of the exons encoding the haptoglobin 
-chain (Fig. 1
) (2). The two haptoglobin alleles are defined by this distinction of their structures rather than by a typical difference between the nucleotide sequences of the 1700-bp elements. A comparison of the 1711-bp subtype Hp 1S-specific sequence in EMBL/GenBank accession number AC004682 (7) and the 1711-bp subtype Hp 2S-specific sequence in EMBL/GenBank accession number M69197 (6) revealed, as the only difference, an exchange of a single base pair. However, we do not know whether this particular difference is consistently present and, therefore, might be useful for discrimination among genotypes Hp 1-1, Hp 2-1, and Hp 2-2. For this reason, we sought to design a PCR system suitable for haptoglobin genotyping that exploited the structural difference of alleles Hp 1 and Hp 2. Oligonucleotide primers A and B, which bind to allele-nonspecific sites immediately outside of the upstream and downstream borders of the allele-specific regions, respectively, gave rise to PCR products of 1757 bp (genotype Hp 1-1), 3481 bp (genotype Hp 2-2), or both (genotype Hp 2-1). The PCR system also took into account the sequence similarities between the haptoglobin gene and the haptoglobin-related gene. Although the haptoglobin-related gene contains a cryptic binding site for primer A, with 18 of 23 nucleotide positions matching, we did not detect, by sequence comparison, a potential binding site for primer B in this gene. We intentionally did not design the PCR system to discriminate between the different haptoglobin genotypes specified by allele subtypes S and F (for example, between Hp 1S/Hp 2FS and Hp 1S/Hp 2SS). Variants S and F are defined by known sequence differences within the 1700-bp element (2) that are located at positions that do not overlap the annealing sites of primers C and D. Thus, they had no influence on our genotyping results.
Selection of PCR primers A, B, C, and D allowed the use of three different haptoglobin genotyping protocols: (1) use of primers A and B to identify the haptoglobin genotype in one assay; (2) use of primer pairs A/B and C/D to determine the haptoglobin genotype after combination of the results of two separate assays; and (3) use of primers A, B, C, and D to identify the haptoglobin genotype in one multiplex assay. Protocol 2 or 3 was especially useful in situations in which the presence or absence of the 3481-bp product could not be determined conclusively. We observed that such a problem occurred in cases in which the PCR was run under suboptimal conditions, with extensively degraded DNA, or with limited quantities of DNA. Requiring two amplification reactions for each DNA sample, protocol 2 was more laborious than protocol 3. However, protocol 2 gave rise to relatively simple patterns of DNA bands. In contrast, although protocol 3 involved only one amplification reaction, it produced a relatively complex pattern of DNA bands.
Analyzing blood samples from a group of Japanese (n = 148) and a small cohort of Germans (n = 20), Yano et al. (9) introduced a PCR method for haptoglobin genotyping. Being potentially able to discriminate among alleles Hp 1S, Hp 1F, and Hp 2, their system relied on the presence of the Hp 2 subtype Hp 2FS, which is almost exclusively the case in Japanese populations (5). However, in Caucasian populations carrying relevant proportions of Hp 2 subtypes other than Hp 2FS, e.g., Hp 2SS and Hp 2FF (5), the system of Yano et al. (9) may yield incorrect genotype assignments; for example, the heterozygous genotype Hp 1S/Hp 2SS would be erroneously typed as the homozygous genotype Hp 1S/Hp 1S.
In conclusion, differentiation among haptoglobin genotypes Hp 1-1, Hp 2-1, and Hp 2-2 may be useful in a broad spectrum of basic studies and clinical examinations. In some cases, e.g., in forensic examinations, haptoglobin typing that discriminates between subtypes S and F may be required. To our knowledge, a globally applicable subtype-specific system for haptoglobin genotyping is not available at present. However, once the principal genotype has been determined, e.g., by use of one of the protocols presented here, subtype-related approaches, based on PCR, might subsequently be used.
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