A molecular biological investigation of the Kell blood group system

Murphy, Margo Taylor (1995) A molecular biological investigation of the Kell blood group system. PhD thesis, University of Glasgow.

Abstract

Molecular biology techniques are becoming increasingly widely used in the field of transfusion science. The investigation of the Kell blood group system, using a selection of these techniques, is described in this thesis. The first part of the investigation involved the synthesis of KEL cDNA from total RNA by reverse transcription and its subsequent amplification using the polymerase chain reaction (PCR). The resulting material was cloned into a plasmid and the cloned PCR products were then used for in situ hybridization studies to refine the location of the KEL gene on human chromosome 7 to 7q33- q35. This was a more precise location than had been previously published. Kell null is a rare phenotype within the Kell blood group system, where none of the Kell system antigens can be detected on the red cell surface. Southern blot investigation of total genomic DNA from one such individual revealed the presence of a KEL gene in a similar form to that present in normal Kell phenotype donors. These results indicate that this example of the null phenotype did not arise as a result of a major gene deletion. This suggests that the development of Kell system antigens, in this particular case, is blocked during transcription or translation of the protein. Direct sequence analysis of PCR products derived from individuals of predetermined Kell phenotype allowed the identification of a single C->T nucleotide substitution at position 701. This is responsible for the difference in expression of k (Cellano) and K (Kell) antigens on the red cell surface. This provided independent confirmation of the results published by another group of workers while this section of the work was in progress. The C->T nucleotide substitution creates a Bsm I restriction enzyme site when the sequence encoding the K antigen (the T nucleotide) is present. This enabled the development of a PCR-based genotyping assay for Kell (K1 or K) and Cellano (K2 or k). The assay involves the co-amplification from a total genomic DNA target of two PCR products, one which spans the K/k polymorphic site and a second which spans a conserved Bsm 1 site and which acts as an internal control for the assay. Several samples of KK, Kk and kk genomic DNA were analysed by this assay and definitive genotyping results were obtained for all samples. This should prove to be a useful tool for antenatal genotype determination in cases where haemolytic disease of the newborn is implicated, and could be incorporated into a battery of PCR tests to establish a panel of typed donors. The use of molecular biology techniques in the investigation of the Kell blood group system has allowed new information to be obtained regarding the molecular basis for the expression of antigens on the red cell surface. This should be a suitable foundation for the continuation of the work, allowing a more detailed investigation of the system as a whole, defining the basis for the other polymorphisms and enabling the development of diagnostic tests for the other allellic antigen sets.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from the Glasgow and West of Scotland Blood Transfusion Service.
• Keywords: Molecular biology.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Miles, Dr. John, Goddard, Dr. John and Fraser, Dr. Robin
• Date of Award: 1995
• Depositing User: Enlighten Team
• Unique ID: glathesis:1995-71525
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2019 14:24
• Last Modified: 12 Aug 2021 13:55
• Thesis DOI: 10.5525/gla.thesis.71525
• URI: https://theses.gla.ac.uk/id/eprint/71525

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