Simultaneous Genotyping of DRB1/3/4/5 Loci by Oligonucleotide Microarray

Abstract

Matching of the HLA antigens for donor-recipient in transplantation, disease predisposition or protection, population studies, and forensic testing requires accurate but simple typing methods. Here, we describe a DNA-based tissue-typing assay that determines the haplotype of the DRB1/3/4/5 loci in hybridization of oligonucleotide array after sample amplification. Using this multianalyte DNA hybridization system, we analyzed seven regions of exon 2 of DRB loci that have single-base discrimination. Thirtysix oligonucleotide probes complementary to the alleles of interest were immobilized on each microslide. The efficiency and specificity of identifying DRB genotypes using the oligonucleotide arrays was evaluated by blinded analysis of 147 samples from reference standards and subjects. The established method provides a rapid and inexpensive DRB "low-resolution" typing tool for prescreening a large number of samples. (J Mol Diagn 2005, 7:592-599)The human leukocyte antigen (HLA) class II molecules of the human major histocompatibility complex are encoded on the short arm of human chromosome 6p21.3 in the HLA-D region. These glycoproteins consist of an ␣-and a ␤-chain associated as heterodimers on the cell surface of antigen-presenting cells such as B cells and macrophages. They play a central role in the regulation of the immune system 1 in transplantation biology, 2,3 as well as in susceptibility to a number of diseases, including autoimmune disorders 4,5 and certain cancers. 6,7 The HLA-D region contains several class II genes and has three subregions: HLA-DR, -DQ and -DP according to the position of gene. For HLA-DR, one gene coding for the DR ␣-chain, DRA, and one gene coding for ␤-chain, DRB1, is always present. Depending on the DRB1 type, a DRB3, DRB4, or DRB5 may also be present and may be accompanied by pseudogenes. With the exception of the DRA molecule, the genes encoding the functional class II molecules are highly polymorphic with virtually all of the variability localized to the second exon. This exon encodes the amino-terminal extracellular domain, which functions as the antigen binding site for processed peptides.With respect to the multiple functions of the HLA antigens, adequate typing analyses are extremely important. Matching for donor-recipient in transplantation and typing for disease predisposition or protection, for population studies, as well as for paternity or other forensic testing are all applications demanding an accurate but simple typing method. Traditionally, HLA antigens were tested by serological typing. Recent advances in DNA technology have vastly improved the detail to which HLA antigens can be characterized. Several different DNAbased techniques have been developed to improve HLA-class II typing such as polymerase chain reaction (PCR)-restriction fragment length polymorphism, 8 PCR sequence-specific oligonucleotide probe (PCR-SSOP), 9 reverse dot blot hybridization, 10 PCR with sequence specific primers (PCR-SSP), 11,12 oligonucleotide array, 13,14 multiplex primer extension, 15...

Development and clinical evaluation of a microarray for HLA‐A and ‐DRB1 genotyping

“…Consequently, almost all alleles of the HLA complex can be arrayed on a single slide. These microarray characteristics exploiting both PCR‐SSP and PCR‐SSOP (9, 10) allow the simultaneous typing of several HLA loci.…”

“…Thus, these genes are suitable targets for microarray technology. Several recent studies using oligonucleotide microarrays for HLA‐A, ‐B (8), and HLA‐DRB1 genotyping (9–11) show that microarrays are an alternative reliable method for HLA genotyping. We have developed a system whereby exons 2 and 3 of HLA‐A are amplified in a single reaction; this differs from previously described methods (8, 9) using a dual amplification system.…”

Molecular Typing of Blood Cell Antigens

“…MALDI-TOF MS analysis in comparison to PCR using Sequence Specifi c Priming (PCR-SSP) and a novel real-time PCR high-resolution melt curve (HRM) analysis, delivered 100 % concordance rates for all investigated HPA and blood group Indian genotypes, respectively [ 33 , 35 ]. A third group of researchers reported about polymorphisms linked to 22 different blood groups, that both the error rate of the MALDI-TOF MS assay, as measured by the strand concordance rate, and the no-call rate were very low (0.1 %) [ 32 ]. With respect to donor genotyping, all authors positively commented on the ability of MALDI-TOF MS based methodology for automation, high throughput, and cost effi ciency.…”

Human leucocyte antigen typing: techniques and technology, a critical appraisal

“…Although there have been some reports of the use of oligonucleotide arrays specific for HLA typing (Guo et al. , 1999, 2002; Bang‐Ce et al. , 2005; Palmisano et al.…”