Matching for HLA alleles between patients and their potential donors is critical for the success of haematopoietic stem cell transplantation. Current genotyping methods identify HLA alleles but not the haplotypes in which they reside, although both are important for transplantation outcome. The aim of this thesis was to identify HLA specific polymorphisms and tagSNPs that could identify 3 common HLA haplotypes present in population of Northern European (NE) Caucasian ethnicity, HLA-A*01-B*08-DRB1*03, HLA-A*02-B*44-DRB1*04, and HLA-A*03-B*07-DRB1*15. Phylogenetic and bioinformatics methods were used to select polymorphisms for each of the nine HLA alleles that were unique, or only found at low frequency in other HLA alleles. PCR -sequence specific priming (SSP) was used to validate the nine selected polymorphisms, which were then incorporated into a multiplex SSP-PCR assay. However, this method does not differentiate between homozygous and heterozygous samples positive for the HLA alleles and therefore an allele specific primer extension (ASPE)-Luminex assay was developed to allow the simultaneous detection of the selected HLA polymorphisms. In order to determine the HLA haplotypes, sets of tagSNPs exhibiting high specificity for the common haplotypes were then identified. This was achieved by using British Birth Control Cohort (~2,000 samples) SNP genotyping data generated from Illumina and Affymetrix gene chips. Eight tagSNPs were identified; rs2187668 and rs2734986 for HLA-A*01-B*08-DRB1*03; rs2844821, rs2596477 and rs660895 for HLA-A*02-B*44-DRB1*04 and rs3094170, rs3129860 and rs3130933 for HLA-A*03-B*07-DRB1*15 haplotypes. These tagSNPs capture the three haplotypes with a specificity ranging from 96 to 100 %. Luminex-ASPE multiplex assays were developed to validate the eight tagSNPs using 59 homozygous and 70 heterozygous samples previously typed at high resolution level by sequence-based typing. A further validation was then performed using a blind cohort of 93 previously HLA typed samples. This Luminex-ASPE tagSNP multiplex method correctly identified the three most common HLA haplotypes present in the Northern European population. In summary, this is the first time when a novel tagSNPs HLA genotyping assay has been developed and successfully applied to detect three common HLA haplotypes in a blinded retrospective study.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:625784 |
| Date | January 2012 |
| Creators | Lemnrau, A. G. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1344057/ |
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