#34
SYNONYMOUS, BUT ALTERNATIVELY SPLICED IN HLA-A*0111N: HLA-A SPLICING INFLUENCES ALLELE EXPRESSION.
Judith Reinders 1, Erik H. Rozemuller 1, Henny G. Otten 1, Anna J.S. Houben 1, Anne Dormoy 2, Arend Mulder 3, Jan G. van den Tweel 1, Eefke J. Petersen 1 and Marcel G.J. Tilanus 1. 1 Department of Pathology, Immunology and Haematology, University Medical Centre Utrecht (UMC-U), Utrecht, Netherlands ; 2 Histocompatibility Laboratory, EFS Alsace, Strasbourg, France and 3 Department of Immunohaematology and Bloodtransfusion, Leiden University Medical Center (LUMC), Leiden, Netherlands .
Alternative splicing affects the expression of a new variant of the HLA-A*010101 allele, designated as HLA-A*0111N; found in a patient requiring a stem cell transplantation. The new variant was identified due to discrepant results between serological typing (A2,-) and routine DNA typing (A*010101,0206). DNA typing revealed the presence of a substitution from G to T at position 597, codon 175, of exon 3 of this new HLA-A*0111N allele. This variation results in a synonymous amino acid substitution. Additional FACS analysis with monoclonal antibodies specific for the A1 and A2 molecule confirmed cell membrane expression loss of the A*0111N allele. RNA sequenced-based-typing (SBT) revealed that 24 nucleotides, corresponding to codons 175 through 182 in exon 3, were alternatively spliced in the A*010101var allele. One-dimensional isoelectric focusing (1D-IEF) on total cell lysate from a cell line established from patient
s cells, showed no cell surface or cytoplasmic A*010101var expression, confirming serology and FACS results. The silent mutation resulted in the introduction of a new splice site, with a higher affinity for splicing than the conserved exon 3 / intron 3 splice site giving rise to this new HLA-A null allele. The alternative splicing of the A*0111N allele prevented the formation of a stable class I molecule on the cell surface, giving rise to this new HLA-A null allele.