High throughput methods of HLA typing

Details High throughput methods of HLA typing High throughput methods of HLA typing

2000-12-20

2003-12-30

Fredman, Jeffrey (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S024300, C536S023100, C536S024310

Reexamination Certificate

active

06670124

ABSTRACT:

FIELD OF THE INVENTION
In general, this invention relates to typing and matching human leukocyte antigens or alleles of human leukocyte antigens and in particular, to high throughput screening methods of human leukocyte antigen matching or alleles of human leukocyte antigens.
BACKGROUND OF THE INVENTION
The human leukocyte antigen complex (also known as the major histocompatibility complex) spans approximately 3.5 million base pairs on the short arm of chromosome 6. It is divisible into 3 separate regions which contain the class I, the class II and the class III genes. In humans, the class I HLA complex is about 2000 kb long and contains about 20 genes. Within the class I region exist genes encoding the well characterized class I MHC molecules designated HLA-A, HLA-B and HLA-C. In addition, there are nonclassical class I genes that include HLA-E, HLA-F, HLA-G, HLA-H, HLA-J and HLA-X as well as a new family known as MIC. The class II region contains three genes known as the HLA-DP, HLA-DQ and HLA-DR loci. These genes encode the &agr; and &bgr; chains of the classical class II MHC molecules designated HLA-DR, DP and DQ. In humans, nonclassical genes designated DM, DN and DO have also been identified within class II. The class III region contains a heterogeneous collection of more than 36 genes. Several complete components are encoded by three genes including TNF-&agr; and TNF-&bgr;.
Any given copy of chromosome 6 can contain many different alternative versions of each of the preceding genes and thus can yield proteins with distinctly different sequences. The loci constituting the MHC are highly polymorphic, that is, many forms of the gene or alleles exist at each locus. Several hundred different allelic variants of class I and class II MHC molecules have been identified in humans. However, any one individual only expresses up to 6 different class I molecules and up to 12 different class II molecules.
The foregoing regions play a major role in determining whether transplanted tissue will be accepted as self (histocompatible) or rejected as foreign (histoincompatible). For instance, within the class II region, three loci i.e., HLA-DR, DQ and DP are known to express functional products. Pairs of A and B genes within these three loci encode heterodimeric protein products which are multi-allelic and alloreactive. In addition, combinations of epitopes on DR and/or DQ molecules are recognized by alloreactive T cells. This reactivity has been used to define “Dw” types by cellular assays based upon the mixed lymphocyte reaction (MLR). It has been demonstrated that matching of donor and recipient HLA-DR and DQ alleles prior to allogeneic transplantation has an important influence on allograft survival. Therefore, HLA-DR and DQ matching is now generally undertaken as a clinical prerequisite for renal and bone marrow transplantation as well as cord blood applications.
Until recently, matching has been confined to serological and cellular typing. For instance, in the microcytotoxicity test, white blood cells from the potential donor and recipient are distributed in a microtiter plate and monoclonal antibodies specific for class I and class II MHC alleles are added to different wells. Thereafter, complement is added to the wells and cytotoxicity is assessed by uptake or exclusion to various dyes by the cells. If the white blood cells express the MHC allele for a particular monoclonal antibody, then the cells will be lysed on addition of complement and these dead cells will take up the dye. (see, Terasaki and McClelland, (1964)
Nature,
204:998). However, serological typing is frequently problematic, due to the availability and crossreactivity of alloantisera and because live cells are required. A high degree of error and variability is also inherent in serological typing, which ultimately affects transplant outcome and survival (Sasazuki et al., (1998)
New England J. of Medicine
339: 1177-1185). Therefore, DNA typing is becoming more widely used as an adjunct, or alternative, to serological tests.
Initially, the most extensively employed DNA typing method for the identification of these alleles has been restriction fragment length polymorphism (RFLP) analysis. This well established method for HLA class II DNA typing suffers from a number of inherent drawbacks. RFLP typing is too time-consuming for clinical use prior to cadaveric renal transplantation for example, and for this reason it is best suited to live donor transplantation or retrospective studies. Furthermore, RFLP does not generally detect polymorphism within the exons which encode functionally significant HLA class II epitopes, but relies upon the strong linkage between alleles-specific nucleotide sequences within these exons and restriction endonuclease recognition site distribution within surrounding, generally noncoding, DNA.
In addition to restriction fragment length polymorphism (PCR-RFLP), an even more popular approach has been the hybridization of PCR amplified products with sequence-specific oligonucleotide probes (PCR-SSO) to distinguish between HLA alleles (see, Tiercy et al., (1990)
Blood Review
4: 9-15). This method requires a PCR product of the HLA locus of interest be produced and then dotted onto nitrocellulose membranes or strips. Then each membrane is hybridized with a sequence specific probe, washed, and then analyzed by exposure to x-ray film or by colorimetric assay depending on the method of detection. Similar to the PCR-SSP methodology, probes are made to the allelic polymorphic area responsible for the different HLA alleles. Each sample must be hybridized and probed at least 100-200 different times for a complete Class I and II typing. Hybridization and detection methods for PCR-SSO typing include the use of non-radioactive labeled probes, microplate formats, etc. (see e.g., Saiki et al. (1989)
Proc. Natl. Acad. Sci., U.S.A.
86: 6230-6234; Erlich et al. (1991)
Eur. J. Immunogenet.
18(1-2): 33-55; Kawasaki et al. (1993)
Methods Enzymol.
218:369-381), and automated large scale HLA class II typing. A common drawback to these methods, however, is the relatively long assay times needed—generally one to two days—and their relatively high complexity and resulting high cost. In addition, the necessity for sample transfers and washing steps increases the chances that small amounts of amplified DNA might be carried over between samples, creating the risk of false positives.
More recently, a molecular typing method using sequence specific primer amplification (PCR-SSP) has been described (see, Olerup and Zetterquist (1992)
Tissue Antigens
39: 225-235). This PCR-SSP method is simple, useful and fast relative to PCR-SSO, since the detection step is much simpler. In PCR-SSP, allelic sequence specific primers amplify only the complementary template allele, allowing genetic variability to be detected with a high degree of resolution. This method allows determination of HLA type simply by whether or not amplification products (collectively called an “amplicon”) are present or absent following PCR. In PCR-SSP, detection of the amplification products is usually done by agarose gel electrophoresis followed by ethidium bromide (EtBr) staining of the gel. Unfortunately, the electrophoresis process takes a long time and is not very suitable for large number of samples, which is a problem since each clinical sample requires testing for many potential alleles. Gel electrophoresis also is not easily adapted for automatic HLA-DNA typing.
Another HLA typing method is SSCP—Single-Stranded Conformational Polymorphism. Briefly, single stranded PCR products of the different HLA loci are run on non-denaturing Polyacrylamide Gel Electrophoresis (PAGE). The single strands will migrate to a unique location based on their base pair composition. By comparison with known standards, a typing can be deduced. It is the only method that can determine true homozygosity. However, many PAGE have to be run and many controls have to be run to make it a viable typing method. This method is very time consuming, labor intensive, and not really su

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