DETECTION OF AN ACQUIRED MUTATION IN AN HLA–A*03 ALLELE IMPAIRING GENE EXPRESSION IN LEUKEMIA CELLS OF A PATIENT WITH ALL

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TITLE: DETECTION OF AN ACQUIRED MUTATION IN AN HLA–A*03 ALLELE IMPAIRING GENE EXPRESSION IN LEUKEMIA CELLS OF A PATIENT WITH ALL

A. G. Smith,¹ M. Sprague,¹ P. Peterson,¹ L. Worley,¹ S. McKinney,¹ S. Warnock,¹ J. A. Hansen.²

¹Clinical Immunogenetics, Seattle Cancer Care Alliance, Seattle, WA; ²Human Immunogenetics, Fred Hutchinson Cancer Research Center, Seattle, WA

Extensive publications document clonal gene mutations resulting from chromosome rearrangements among patients with malignant hematopoietic diseases. We report here the detection of an acquired, de novo single nucleotide polymorphism (SNP) in an HLA–A*03 allele in a patient with Acute Lymphocytic Leukemia (ALL) during clinical HLA typing prior to hematopoietic cell transplantation. DNA isolated from 2 separate peripheral blood draws for the proband was DNA sequenced for HLA–A, B and DRB1 genes. Both samples exhibited the sequence A*0101, 0301, B*0702, 5501, DRB1*0407, 1501, except for a single nucleotide difference in either the HLA–A*01 or A*03 allele at nucleotide 513 exon 3 (codon 147), where the consensus sequence, TGG, was found, but also a novel sequence, TGA. To confirm the results and determine which allele was novel, parents were sequenced, but neither parent (A*0101, 1101 and A*0301, 0301) exhibited the novel sequence. HLA–A*03 specific amplification of patient DNA found the novel G to A SNP in the HLA–A*03 gene (GenBank accession numbers AY065963, AY065964, exons 2, 3). DNA from a patient skin biopsy revealed the normal A*0301 allele. Cryopreserved peripheral blood mononuclear cells, from a blood draw with the novel A*03 sequence, were immunophenotyped and flow cytometry cell sorting provided CD45/19+ leukemia cells versus normal CD3+ T–cells. Sequencing found the novel TGA sequence at codon 147 in leukemia cells, but not in normal T–cells. Flow cytometry analysis with an anti–HLA–A3 monoclonal showed HLA–A3 expression on normal cells, but not on leukemia cells. These results document a de novo SNP producing a stop codon and resulting in non–expression of in an HLA–A*03 allele in a malignant cell population in a patient with ALL. This interesting case strongly suggests that new HLA alleles, especially those detected in patients with malignant diseases, should be confirmed to be of germline origin using a skin biopsy or in a relation sharing the same haplotype.