abstract

• Haematopoietic stem cell transplantation (HSCT) is a widely used therapy for malignant and genetic disorders. However the main complication following HSCT is Graft versus Host Disease (GVHD). GVHD is mainly the result of HLA mismatches between donor and patient, it is therefore of paramount importance that key HLA alleles are matched, including HLA-DRB. Molecular methods such as Sequence Specific Oligonucleotide Probing (SSOP), Sequence Specific Priming (SSP) and Sequencing Based Typing (SBT) are all currently used methods for HLA-DRB typing. However, using SSOP and SBT it is often impossible to distinguish the cis or trans orientation of certain sequence motifs which leading to ambiguous typing results. We are now able to show the application of a generic HLA-DRB1 PCR amplification coupled to the technique Reference Strand Conformational Analysis (RSCA) to accurately type a total of over 70 different HLA-DRB1 alleles. A set of generic primers residing within the introns surrounding exon 2 for the DRB1 gene were used to amplify from DNA samples which had been previously typed by conventional methods by the Histocompatibility Typing laboratory. These amplicons were hybridised to three separate Fluorescently Labelled References (FLRs) after which they were electrophoresed through native Polyacrylamide gels on an ALFexpress automated sequencer. Each allele was run a minimum of five times before a statistical analysis was earned out on the mobility values. These mobility values form a database which is being used to type samples to high resolution. Over 200 routine donor samples were typed for HLA-DRB1 using RSCA and we were able to successfully confirm the results obtained by conventional method in each case and in some instances increase the level of resolution previously achieved. For high throughput histocompatibility laboratories the application of a single generic amplification for HLA-DRB1 followed by RSCA which subsequently gives a high resolution DRB1 type despite the complexity of HLA-DRB1 is extremely desirable. Furthermore, this approach can potentially generate a high resolution DRB1 type for the whole of exon 2. This significantly improves on the conventional techniques such as SSP where the primers reside within exon 2 and as a result may not target existing or new polymorphisms. © 2001 Blackwell Science Ltd.

authors

• Corell
• García Sepúlveda Christian Alberto
• Little A.-M.
• Madrigal J.A.
• Marsh S.G.E.
• McWhinnie A.
• Ngui S.L.
• Soteriou B.A.
• Tavarozzi F.

publication date

• 2001-01-01

published in

• European Journal of Immunogenetics  Journal

start page

• 344

end page

volume

• 28

issue

• 2