Molecular Hla Typing-the Brave New World

Soon after the first solid organ clinical transplants were performed, it became apparent that some degree of HLA typing was prudent, originally for matching and later as an aid to both cross-matching and antibody screening. Such typing was performed with a panel of antisera specific for different antigenic products of the system in an assay that became known as the complement-dependent cytotoxicity test (CDC*)(1), or serology. CDC is robust and can provide results within 3 hr, critical for reducing cold ischemia times in cadaveric transplantation. The close cooperation between HLA laboratories throughout the world in terms of serum exchange kept the cost of such tests to a minimum while ever-increasing the resolution. There are drawbacks to CDC; viable lymphocytes are required, the antibodies needed are generally nonrenewable, and the technique has limited powers of resolution, particularly at HLA class II. This problem of serological discrimination of class II antigens, arguably the most important antigens for solid organ transplantation, led to the first comprehensive molecular class II typing system using restriction fragment length polymorphism (RFLP)(2). Briefly, RFLP entails the restriction endonuclease digestion of genomic DNA followed by electrophoretic resolution of the endonucleolytic fragments which are denatured in situ and hybridized to a nylon membrane. The membrane is then probed with homologous labeled cDNA or genomic probes which yield hybridization signals characteristic of various HLA alleles. The relevance of RFLP was shown by Opelz et al. in the Collaborative Transplant Study (3) and by Mytilineos et al.(4, 5). These retrospective studies showed that RFLP-defined HLA-DR-matched renal grafts had improved survival compared with serology-defined HLA-DR-matched grafts, and that serology for class II was inadequate in many typing centers, with an overall discrepancy rate of up to 25% between serologically and RFLP-defined antigens. It soon became apparent that RFLP had too many drawbacks to be the method of choice for the 1990s. The main problem was the lengthy time from sampling to results (up to 10 days), but in addition, the resolution often depended on a strong association between neighboring sequences rather than on polymorphism within the DRβ1 domain. This restricted its use on mixed ethnic populations because of differences in linkage disequilibrium values. The development of the polymerase chain reaction (PCR)(6) allowed the evolution of improved molecular HLA-typing techniques. The PCR can generate specific amplified stretches of DNA sequences in vitro through repeated cycles of DNA denaturation, annealing of specific primer to a single strand, and nucleotide extension from primer pairs using a DNA polymerase. The PCR allowed extensive sequencing of HLA alleles from 1990 onward, as shown in Table 1. Transplantation laboratories were, for the first time, able to use PCR to amplify the polymorphic regions of HLA genes which could subsequently be analyzed for the polymorphism within the amplicon, thus establishing the tissue type. The new PCR tissue-typing techniques now allow histocompatibility scientists the choice of whether to use low-, medium-, or high-resolution methods: Low-resolution methods generally only identify broad specificities or groups of specificities. The term “medium resolution” can be used to describe a typing system that discriminates between all serological specificities but may also give some allele-specific results, whereas high-resolution typing is a term generally used to describe a typing system that discriminates between greater than 90% of the alleles in the loci analyzed. Thus, in solid organ transplantation, medium-resolution typing is generally the method of choice, whereas in unrelated bone marrow transplantation, low- or medium-resolution typing is always followed up with high-resolution typing. Obviously, high-resolution methods are not used routinely because they tend to be more time consuming and more costly compared with medium-resolution methods. Techniques for analyzing polymorphisms in amplified DNA can be divided into two basic groups: probe hybridization and direct amplicon analysis. Currently there are many different PCR-based tissue typing assays and it is up to individual laboratories as to which test or combination of tests they use and for which loci they choose to apply the tests. Most new molecular methods of tissue typing were initially developed for class II typing, particularly DQA1 and DQB1 (because of the relative simplicity of these loci compared with DR and class I). However, most molecular methods are now applicable to both class I and class II, the only difference being that in class I analysis, exons 2, 3, and sometimes exon 4 are required for discrimination of most alleles, whereas most class II alleles can be discriminated between by analyzing exon 2 only. A diagram illustrating PCR-sequence-specific oligonucleotide probing(SSOP), reverse PCR-SSOP, PCR-sequence-specific primers (SSP), and PCR-single-stranded conformational polymorphism (SSCP) is shown inFigure 1. PROBE HYBRIDIZATION TECHNIQUES Probe hybridization techniques rely on amplification of a target DNA sequence which is generally immobilized onto a support membrane (known as dot blotting). The initial amplification is normally generic but may be a mosaic of amplifications which when used together amplify all possible alleles of a given locus. The polymorphisms in the immobilized amplified DNA are subsequently detected by using specific single-stranded DNA probes in combination with highly stringent washes to remove non-specifically bound probe (7). In addition to the use of radioactive isotopes, hybridized probes can be detected by a variety of nonradioactive methods, such as horseradish peroxidase (8) and digoxigenin labeling(9). This technique became known as PCR-SSOP (PCR followed by sequence-specific oligonucleotide probing). PCR-SSOP was first applied to histocompatibility testing in HLA-DQA1 by Saiki et al.(7); subsequently, PCR-SSOP was applied to HLA-DRB1(10-14) and HLA-DQB1(15) and a combination of DR, DQB1, DQA1, DPA1, and DPB1(16). The assays were standardized and gained wide acceptance through the 1991 International Histocompatibility Workshop(13). In 1989, nonradioactive methods of PCR-SSOP analysis were introduced (15) which facilitated the spread of the technique to many laboratories. HLA class I PCR-SSOP techniques were slower to develop because complete sequence data for class I alleles was lacking. As more class I alleles were sequenced, it became clear that serology would not be able to discriminate between certain specificities, so researchers turned their attention to developing class I molecular techniques. The first PCR-SSOP class I dot blot techniques were described for differentiating HLA-B35 and HLA-B53 (17) and subtyping HLA-A2 and A28 (18). Complete single-locus typing systems were then developed for HLA-A (19, 20), HLA-B(19-24), and HLA-C(25, 26) such that a combination of these techniques could be used to completely type an individual for class I. PCR-SSOP is a generally accurate method of genotyping(27) and is especially suitable for large numbers of individual samples due to the fact that generic amplifications from many individuals can be hybridized to a single membrane. There is, however, a problem if you wish to obtain complete results on one individual within 24 hr, a problem that has led to the development of the reverse PCR-SSOP assay(28). In the reverse dot blot, the sequence-specific oligonucleotide probes are bound to a solid support membrane via an incorporated poly-(T) tail, which leaves the detection end of the probe free to interact with target DNA. When labeled DNA target is applied to the reverse dot blot membrane, it will only hybridize to those oligonucleotides that are complementary in sequence. Once hybridized, biotinylated products are detected by the addition of a reporter molecule: antibiotin antibody linked to streptavidin-horseradish peroxidase complex, which induces a color change in the substrate tetramethylbenzidine. Reverse dot or line blot methods have been published for class II typing (28, 29) and HLA-A(30). HLA-B and HLA-C reverse SSOP methods are currently being developed by various commercial companies. When combined, these reverse SSOP methods should allow complete results on one individual within 4-5 hr, which may be suitable for genotyping cadaver donors. However, the large number of probes needed for a parallel class I and II reverse SSOP system requires considerable quality assurance input, which means that complete reverse SSOP systems are most likely to be available only from commercial companies and may be too expensive for routine typing of all samples in a laboratory. DIRECT AMPLICON ANALYSIS TECHNIQUES Considerable impetus for rapid HLA typing comes from solid organ transplantation, where HLA matching is generally regarded as being beneficial and long cold ischemia time as damaging. Current PCR-SSOP approaches require lengthy post-PCR steps, which means that even the quickest reverse SSOP technique can not be completed within 5 hr, double that required for serology. There are molecular methods which, while not so efficient for large sample numbers, are more suitable for rapid limited sample number through-put. These include PCR-RFLP, PCR-SSP, nested PCR-SSP, heteroduplex analysis, and other conformational assays. PCR-RFLP As discussed earlier, restriction endonucleases have been used to detect polymorphism with significant success on genomic DNA, but the development of the PCR enabled the enzymes to be used in a much more precise way on much smaller blood samples without the necessity for detection probes. The principle of PCR-RFLP is that a precise region of target DNA is amplified and then cut. Since the sequences of the most common HLA alleles were already known, enzyme restriction sites could be defined within the PCR amplicon at sites of polymorphisms. Thus, a PCR amplicon of unknown HLA type would be digested with a variety of endonucleases, giving rise to PCR fragments of varying size depending on the alleles present. PCR-RFLP has been described for DQA1 (31), DQB1 (32-34), DPB1 (35), DRB1 (36-38), HLA-B44 subtyping (39), and HLA-C(40). PCR-RFLP can rival more established class II genotyping methods for sensitivity and accuracy (41), but complex methodology and interpretation have prevented its widespread use. PCR UTILIZING SSP In 1989, two separate groups (42, 43) described a system of PCR in which the specificity of the PCR reaction entailed matching the 3′ end of one primer with the target DNA sequence, thereby allowing the identification of any point mutation within one or two PCR reactions. Newton (43) described the detection of a single point mutation using one generic sense primer and two antisense primers: one antisense primer was specific for the “normal” form and was refractory to PCR on “mutant” DNA, and the other antisense primer for the “mutant” was refractory to PCR on “normal” DNA. This was termed the amplification refractory mutation system (ARMS). ARMS works because Taq polymerase lacks 3′ to 5′-exonucleolytic proof-reading activity (44, 45). Such an activity would correct the mismatched terminal base of an ARMS primer in a mismatched primer-template complex and subsequently permit efficient priming with the “repaired” primer. For efficient ARMS amplification without false priming, the conditions need to be highly stringent as it is possible for 3′-mismatch extension(42, 43, 46). Stringency is multifactorial, relying on the concentration of all the PCR constituents, such as target DNA,Taq, dNTPs, Tris, and free magnesium. PCR stringency kinetics also relies on individual primer factors, such as primer sequence and length and type of primer-template mismatches. An important feature of ARMS is that each individual reaction contains primers to amplify a so-called“housekeeping” gene which detects possible PCR inhibition and thus acts as a positive control. Without this positive control, it would be impossible to discriminate between a failed PCR reaction and a negative PCR reaction and hence all homozygous results would be questionable. ARMS was predicted to be applicable to HLA analysis in 1989(42) and was first applied in a limited fashion to HLA typing DPB in 1990 by Fugger et al. (47), while Lanchbury et al. (48) utilized the 3′-mismatch concept to obtain DR4 group-specific amplification prior to DR4 SSOP subtyping. The first comprehensive ARMS HLA typing system was described in 1992 by Olerup and Zetterquist (49) for low-resolution HLA-DRB1 typing, including group-specific detection of DRB3 and DRB4 by ARMS using 19 PCR reactions. Olerup and Zetterquist renamed the assay PCR-SSP (PCR using sequence-specific primers). Modern PCR-SSP features multiple PCR reactions where each reaction is specific for an allele, or more commonly a group of alleles that correspond to a serologically defined antigen. To type an individual completely at any given locus, multiple PCR-SSP reactions are set up and subjected to PCR under identical conditions. The presence or absence of PCR amplification is detected in a gel electrophoresis step with visualization by ethidium bromide incorporation. PCR-SSP was soon applied to detect polymorphisms in other class II loci, such as HLA-DQB1 (50, 51), HLA-DQA1(51), and DPB1 (52). Identification of class I alleles by PCR-SSP was first reported by Browning et al.(53), who described low-resolution typing of HLA-A. Thereafter, medium-resolution PCR-SSP systems were rapidly described for HLA-C(54, 55) and HLA-B (56), with low-resolution HLA-B typing systems also being described(57). Recently, a PCR-SSP method known as phototyping was developed that allows the simultaneous detection of HLA-A, -B, -C, -DRB1,-DRB3, -DRB4, -DRB5, and -DQB1 alleles (58). This method has a resolution and accuracy far greater than average serology, and only takes 3 hr to complete, making it suitable for genotyping cadaver donors. The development of automatic dispensing equipment and better electrophoresis equipment has facilitated the use of phototyping in many laboratories. In some laboratories, including our own (since September 1994), it has completely replaced serology. An advantage of PCR-SSP is that it detects polymorphisms linked on an individual chromosome (cis) whereas PCR-SSOP detects polymorphisms on both DNA chromosomes (cis or trans). Thus, PCR-SSP has greater power for discriminating between heterozygosity involving two closely related alleles, as illustrated in Figure In addition, the on can be by PCR In this a new is by using a sense primer that the from the initial molecular in combination with multiple antisense primers in multiple reactions. The positive and negative reactions with these primer can then be used to many of the polymorphisms. this is for it has been used to identify many new class and class II alleles alleles which have subsequently been correct by PCR-SSP is used as a medium-resolution but described allele-specific SSP for HLA-C while have applied allele-specific PCR-SSP to and to HLA-A PCR-SSP is by using a combination of group-specific amplifications with some allele-specific reactions. However, while this to allele-specific typing is applicable to all HLA antigens, it would only be possible by using a large of primer to obtain allele-specific typing, techniques such as nested PCR-SSP and conformational assays have been developed to be used as individual complete methods or methods used to medium-resolution typing by PCR-SSP or PCR-SSP PCR-SSP is a to HLA typing the region of is amplified in the first step and this amplicon is used of genomic DNA for the sequence-specific using primers which are to the first of amplification PCR-SSP was first described by et al. as a method for complete medium-resolution typing of In exon 2 of all HLA-DRB1 alleles was amplified and subjected to a amplification using PCR-SSP reactions which, PCR-SSP, used an amplification to the success of each individual The results were to PCR-SSP, but it was the technique that became PCR-SSP have PCR-SSP in that the of DNA required is In addition, allele-specific nested PCR-SSP is applicable to subtyping highly polymorphic alleles, such as For nested PCR-SSP, only the alleles of are so in the of only the alleles are amplified and these are then subjected to a with This has the advantage of allowing all alleles to be detected in all without the from closely related alleles as which would not be amplified in the initial ANALYSIS the end of any PCR the individual may with each other to form or they may with an unrelated DNA to form a or they may as single-stranded These different of PCR have conformational which may be by their electrophoretic in a or This PCR has been applied to HLA to tissue typing methods that can detect not only polymorphisms but new polymorphisms as PCR heteroduplex analysis has gained for HLA polymorphisms due to the of the gel analysis and the conditions. However, it has been used to individuals for and by and DNA are mixed the of the PCR to allow and to which may whether the are HLA The discrimination of heteroduplex analysis has been by the use of a heteroduplex The is a DNA that is to the polymorphic sequence of the target gene but contains at the polymorphic sites to the different to all known alleles. When the is incorporated into the the number of and hence the power of the is analysis has been used for class II typing, but PCR-SSOP, it of single alleles. of has been in class I DNA typing by et using a method of complementary analysis a biotinylated primer in the initial amplification that one DNA The DNA are then and using and then allowed to to a DNA The is from amplification of an unrelated sample of known HLA type. The hybridized alleles can then be using a gel system of and The different hybridized products to different in the gel to the conformational of the different The can then be of the gel and onto a membrane and subsequently analyzed by PCR-SSOP methods. The technique has two the alleles are and thus individuals can be more than with SSOP methods. the gel electrophoresis of the alleles can be used as a typing analysis on the fact that DNA of sequences conformational as a of complementary base The different single-stranded products different gel electrophoresis that can be used to the of an has been applied to HLA-A DQB1 and DQA1 and DPB1 and subtyping However, heteroduplex analysis, the of both the technique and the interpretation prevented the widespread of this PCR-SSP and PCR-SSOP methods are limited within the of sequence alleles may be or by both PCR methods. alleles are often by PCR-SSOP and by but these always require by sequencing a new is by the PCR-SSOP and can identify whereas sequencing The of typing are that the polymorphic regions of any given are amplified by PCR The PCR is by any one of a variety of different methods and analyzed by to the type. analysis is required because the from a individual will two sequences that need to be with all known sequences in to be and was initially described for HLA DQB1, and DQA1 by et al. and for by et is to class II typing because the of the polymorphism is in exon This means that a single sequencing reaction is to the class II type. of class I exons 2, 3, and 4 was first described by et al. but it is not used because of and approaches for class I have been developed that on exons 2 and 3, where the of class I polymorphisms are To the of of the highly polymorphic HLA-B locus, and used a combination of group-specific amplification followed by The main drawbacks of are the equipment and the time required to sequence one A with a single would to class and more than individuals a this is the advantage of high-resolution typing, but the of high-resolution typing is in and a which can not now be without some form of extensive high-resolution typing. is that all molecular methods, sequencing is not and many alleles have had to be due to most commonly In addition, even methods not always discriminate between all of alleles There are many for tissue matching for solid organ and bone marrow transplantation, as an important aid to association and of into The tissue typing method most to each is a of resolution, sample numbers, time, sample the used on and the of the individuals the typing. It is now possible to use molecular methods for all tissue typing without to serology. For the of a medium-resolution method is the It allows the to on loci, antigens, or alleles, which can then be defined by techniques. It allows the to antibody specificities or bone marrow and provide a of discrimination suitable for solid organ exchange In most it not which medium-resolution technique is so long as it is performed typing, whether by or the with a analysis In a association with serological antigens may clear when individual are In transplantation, the analysis of on multiple alleles can only be by high-resolution typing of all and The of high-resolution typing requires a different of analysis. of or with a of antigens, the of the will be required to or with and conformational It is also that will be required to with in class I or class II this may to the that certain of class I and class II antigens within an individual will that individual to or given to certain or antigens. molecular typing methods used without serological can not identify alleles or that may in certain Most alleles are the is common in most It is important that alleles are especially in unrelated bone marrow transplantation. The phototyping PCR-SSP a PCR-SSP reaction to identify the and it is possible to develop PCR-SSP reactions or probes to detect the alleles, such as the HLA-A2 as and when these alleles are In time, greater will be required so that not only will alleles be by molecular but will be as The method for transplantation, typing all HLA loci to a resolution, is arguably the PCR-SSP It has the advantage of resolution with of interpretation and of but has the that multiple reactions are needed that require dispensing of and of gel electrophoresis to detect PCR However, it is now possible to PCR and primer with automatic dispensing or dispensing and gel electrophoresis equipment has been to allow direct from This means that dispensing and detection of multiple reactions is now as as or than dispensing multiple serological reactions. it is now possible to completely PCR-SSP using the electrophoresis step will be completely as detection systems of commonly used typing methods: PCR-SSOP, reverse PCR-SSOP, PCR-SSP, and The amplification primers in each are on the and nucleotide The polymorphisms are between PCR-SSOP and PCR-SSP techniques in between PCR-SSOP methods two polymorphisms on an allele, whereas PCR-SSP This can in discriminating between some