masterThesis
HLA-A Typing in formalin-fixed paraffin embedded tissue samples : towards potential retrospective analysis
Fecha
2011Registro en:
TMM 0005 2011
Autor
Leon Rodriguez, Daniel Arturo
Institución
Resumen
Human Leukocyte Antigen (HLA) has been described in many cases as prognostic factor for cancer. The main feature of HLA genes, located on chromosome 6 (6p21.3), is their extensive polymorphism. Nucleotide sequence analysis of alleles shows that the variation is restricted predominantly to exons that encode the peptide-binding domain of the protein. Thus, HLA polymorphism defines the repertoire of peptides that bind to HLA allotypes and this fact defines an individual s ability to respond to exposure to many infections agents throughout his life. HLA typing has become an important analysis in the clinic. Formalin-fixed paraffin embedded (FFPE) tissue samples are routinely collected in the oncological clinic. This could be used as a good DNA source, since in past studies DNA collecting assays were not normally performed from almost any kind of tissue or sample on regular clinic procedures. Considering that the most important problem with FFPE DNA is fragmentation, we proposed a new method for HLA-A allele typing from FFPE samples based on the sequences of exons 2, 3 and 4. We designed a set of twelve primers: four for HLA-A exon 2, three for HLA-A exon 3 and five for HLA-A exon 4, each one according to the flanking sequences for their respective exon and the sequence variation between different alleles. 17 FFPE samples collected in Karolinska University Hospital, Stockholm Sweden, and 1 control blood sample underwent amplification reactions and the products were submitted to sequencing. Finally all the obtained sequences were analyzed and compared with IMGT-HLA database. The FFPE samples were previous to this HLA PCR-SSP typed in a certified laboratory and then compared to the results from our novel method. According to this, the samples could be correctly sequenced compared to the previous typing. With this procedure, we conclude that our study is the first Sequence Based Typing method which allows the analysis of damaged DNA samples. It opens the possibility to develop retrospective analysis in order to establish new relationships between HLA and different diseases as well cancer.