| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1 Deutsches Herzzentrum München and 1. Medizinische Klinik rechts der Isar, Technische Universität München, D-80636 München, Germany;
2
Institute of Forensic Medicine, University of Oslo, Rikshospitalet, N-0027 Oslo, Norway
aaddress correspondence to this author at: Deutsches Herzzentrum München, Experimentelle Kardiologie, Lazarettstrasse 36, D-80636 München, Germany; fax 49-89-1218-3053, e-mail wkoch{at}dhm.mhn.de
Haptoglobin is an acute-phase protein synthesized by the liver in response to inflammatory cytokines (1)(2). Its major function is to form a stable complex with free hemoglobin released from old red blood cells or during episodes of hemolysis, thus preventing iron loss and renal damage (1)(2). Haptoglobin consists of two chains, the 
-chain and the ß-chain, which are derived from a single polypeptide after proteolytic cleavage (3). The -chain exists in two major forms, the 1-chain and the 2-chain, which are characterized by the presence (2-chain) or absence (1-chain) of a direct repeat of 63 amino acids (1)(2)(3). The structural heterogeneity of the -chain is the result of an intragenic duplication of 
1700 bp (3). The 1700-bp sequence of the Hp 1 allele and the upstream 1700-bp sequence of the Hp 2 allele contain exons 3 and 4, and the downstream 1700-bp sequence of the Hp 2 allele carries exons 5 and 6 (3). Three principal genotypes, Hp 1-1, Hp 2-1, and Hp 2-2, result from the haptoglobin gene polymorphism, and we have previously established a technique, based on PCR, for genotype determination (4).
Two common sequence versions of the 1700-bp unit exist, which are correlated with the presence of amino acids Asp-Lys (version F) or Asn-Glu (version S) at amino acid positions 52 and 53 in the 1- and 2-chains of haptoglobin and amino acid positions 111 and 112 in the 2-chain (3)(5). Additional differences between the nucleotide sequences of the F-specific and S-specific 1700-bp units occur, including point mutations and small insertions or deletions; however, these differences have no effect on the amino acid sequence (3)(5). Conventional assays for haptoglobin subtyping exploited the specific migration velocities during electrophoresis or characteristic isoelectric points of the different polypeptide species in gel matrices (1)(6)(7)(8). Because of the F/S-related heterogeneity of the Hp 1 and Hp 2 alleles, 15 different subtypes of haptoglobin have been identified in population studies (9)(10)(11)(12)(13)(14).
As an advancement of the genotyping method, we now describe an assay for haptoglobin subtyping that is based on PCR and analysis of the PCR products with a restriction enzyme. In essence, the assay relies on the presence of a particular recognition site for the restriction enzyme DraI in the S-specific 1700-bp unit, a site that is absent from the corresponding position of the F-specific 1700-bp unit [accession nos. AC004682 (15) and M69197(16) deposited with the EMBL/GenBank data libraries]. The S-specific DraI cleavage site is located 72 bp upstream of exon 4 (Hp 1 and Hp 2) and exon 6 (Hp 2). Our own sequence analyses provided evidence for a firm association of this DraI site with the S-specific 1700-bp unit. A subtype-nonspecific DraI site relevant for the subtyping assay is present 648 bp downstream of exon 4 in Hp 1F and Hp 2F, a position corresponding to 641 bp downstream of exon 4 in Hp 1S and Hp 2S.
Genomic DNA was extracted from peripheral blood leukocytes with use of the QIAamp DNA Blood Kit, as suggested by the supplier (Qiagen). Six different PCR primers (synthesized by Applied Biosystems) were used for haptoglobin genotyping and subtyping:
After genotype determination, which was done with primer pairs A/B and C/D as outlined in our previous report (4), the haptoglobin subtype was determined. Depending on the genotype, we used one of three different subtyping protocols: (a) subtyping of samples genotyped as Hp 1-1 involved digestion, with DraI, of an aliquot of the PCR product that was generated with primer pair A/B in the genotyping step; (b) samples genotyped as Hp 2-2 underwent separate amplification reactions with primer pairs E/D and C/F, followed by digestion with DraI; (c) samples genotyped as Hp 2-1 were subtyped using a combination of the assays used for subtyping of Hp 1-1 and Hp 2-2. Fig. 1
shows the DNA fragments that resulted from digestion of the PCR products with DraI. Regarding genotypes Hp 2-1 and Hp 2-2, all types of Hp 2-specific DNA fragments resulting from digestion with DraI were separated from each other during gel electrophoresis. For this reason, it was possible to combine the PCR products generated with primer pairs D/E and C/F before restriction enzyme analysis.
|
To test the accuracy of the new method, we determined the haptoglobin subtypes in DNA samples obtained from 20 individuals who had previously been protein subtyped. The protein subtypes had been firmly established in an experienced routine haptoglobin-subtyping laboratory by a combination of isoelectric focusing and immunoblotting (12). Operators who conducted the DNA analysis were not aware of the protein-based subtyping data. The results of DNA subtyping and protein subtyping fully corresponded, which demonstrated the accuracy of the new haptoglobin gene subtyping method. Nine different subtypes were present in the test series.
We determined the major haptoglobin genotypes and, using the new method, the subtypes in 805 individuals of Caucasian origin. These individuals were recruited to examine a possible association of the haptoglobin polymorphism with adverse events commonly occurring after interventions in coronary arteries. Written informed consent was obtained from all participating patients. The study protocol was in accordance with the current revision of the Helsinki Declaration and approved by the institutional ethics committee. In this series of patients, the distribution of the three major haptoglobin genotypes was as follows: 113 (14.0%) were homozygous Hp 1-1, 366 (45.5%) were heterozygous Hp 2-1, and 326 (40.5%) were homozygous Hp 2-2. The observed genotype distribution was in Hardy–Weinberg equilibrium (P = 0.53). Subtype analysis revealed that among the 113 individuals with genotype Hp 1-1, 56 (49.6%) had subtype Hp 1F-1S, 33 (29.2%) subtype Hp 1S-1S, and 24 (21.2%) subtype Hp 1F-1F. In the group of the 366 individuals with genotype Hp 2-1, 207 (56.6%) carried subtype Hp 2FS-1S, 143 (39.1%) subtype Hp 2FS-1F, 9 (2.5%) subtype Hp 2SS-1F, and 7 (1.9%) subtype Hp 2SS-1S. Of the 326 individuals with genotype Hp 2-2, 290 (89.0%) had subtype Hp 2FS-2FS, 35 (10.7%) subtype Hp 2FS-2SS, and 1 (0.3%) subtype Hp 2FF-2FS. Typical results from subtyping of samples representing genotypes Hp 1-1, Hp 2-1, and Hp 2-2 are shown in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol49/issue11/. In total, we observed 10 different subtype-specific haptoglobin genotypes and 5 distinct subtype-specific haptoglobin alleles (Table 1
).
|
On the basis of subtype-specific DraI banding patterns, unequivocal subtype assignment was achieved with the DNA samples of the 805 study participants. Regarding genotype Hp 2-2, unique DraI fragment patterns can be predicted for 8 of the 10 theoretically possible Hp 2-2 subtypes, including the 3 subtypes we have observed (Hp 2FS-2FS, Hp 2FS-2SS, and Hp 2FF-2FS), whereas identical patterns are expected for Hp 2FF-2SS and Hp 2FS-2SF (Fig. 1B
). None of the DNA samples that we have subtyped to date has given rise to the banding pattern that would indicate subtypes Hp 2FF-2SS and Hp 2FS-2SF. In theory, PCR with primers designed for amplification of F- and S-specific sequences in the upstream and downstream 1700-bp units would allow a distinction between Hp 2FF-2SS and Hp 2FS-2SF. Subtype Hp 2FF-2SS is extremely rare in the populations examined to date. For example, subtyping of 6668 individuals from Norway, which was done by isoelectric focusing of reduced serum proteins and subsequent immunoblotting, revealed 3 cases (0.04%) with this subtype (12). To our knowledge, the presence of subtype Hp 2FS-2SF has not been reported.
We observed a high degree of similarity between the subtype distribution in our cohort and the distributions determined previously, by protein subtyping, in other European populations (9)(10)(11)(12)(13)(14). In addition to the result of the test experiment, this finding strongly suggests that our method allows correct haptoglobin subtype assignments.
The new technique for DNA-based haptoglobin subtyping may be useful in population-related and clinical studies. Of particular interest is the question whether the repeatedly demonstrated clinical relevance of the haptoglobin polymorphism (1)(2)(17)(18)(19)(20)(21) is associated with a specific subtype.
References
2FSß and 1Sß variants. FEBS Lett 1984;168:103-107.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
chain antibodies. Electrophoresis 1990;11:483-488.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
2-variant. Electrophoresis 1990;11:61-65.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
3' orientation of the primers relative to the coding strand of DNA. Hp 1 contains one binding site each for primers C, D, E, and F, and Hp 2 contains a second binding site for each of these primers (shown in italics). Because of their relative locations, these sites were not relevant for PCR with primer pairs E/D and C/F. Arrows indicate the recognition sites of DraI present within the PCR products obtained with DNA specific for HP 1F, Hp 1S, Hp 2SF, Hp 2FF, Hp 2SS, or Hp 2FS. Also shown are the sizes of the fragments obtained by digestion of the PCR products with DraI. The DraI fragments of 39 bp (Hp 1), 156 bp (Hp 2), and 193 bp (Hp 2) are subtype-nonspecific, and their presence indicates the technical success of individual restriction enzyme digestions. The presence or absence of the other DraI fragments is subtype-specific. Note that (a) with primer pair A/B, a Hp 1F-specific PCR product of 1766 bp or a Hp 1S-specific PCR product of 1757 bp is generated, (b) with primer pair E/D, a Hp 2F-specific PCR product of 1465 bp or a Hp 2S-specific PCR product of 1456 bp is obtained, and (c) primer pair C/F gives rise to a Hp 2F-specific PCR product of 1224 bp or a Hp 2S-specific PCR product of 1222 bp. Calculations of the sizes of the PCR products and DNA fragments were done on the basis of the nucleotide sequences deposited under accession numbers AC004682 (Hp 1S) and M69197 (Hp 2FS) in the EMBL/GenBank sequence libraries and our own sequence analyses.