Use of UITma DNA polymerase improves the PCR detection of rearranged immunoglobulin heavy chain CDR3 junctions

Abstract

The development of rapid PCR protocols for amplification of rearranged IgH gene sequences has greatly facilitated the identification of clonal IGH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias. However, the 15-35% incidence of false negative results with this approach has been a constant and unresolved problem. To assess the reliability of a previously published framework region 3 (FR3A) IgH-CDR3-PCR for detection of monoclonal IgH gene rearrangements we compared the PCR and Southern results in a series of 44 NHL and leukemias of B cell lineage showing a JH-rearrangement in Southern analysis with genomic DNA and hybridization with a IgH joining region (JH) probe. IgH-CDR3 regions were amplified using DNA extracted from clinical specimens by PCR using fluorescent dye-labeled consensus primers homologous to conserved regions within the variable (VH) and the joining (JH) gene segments. The PCR products were size separated on a high resolution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an automated DNA sequencer. With commonly used DNA polymerases monoclonal IgH-CDR3 junctions were identified in 36/44 samples (82%). However, in the remaining eight cases (18%) with pathohistologically clearly demonstrated B cell malignancies which were also monoclonal on JH-Southern analysis, monoclonality could be demonstrated by FR3A-IgH-CDR3-PCR only with the proofreading UITma DNA polymerase. In four of these monoclonal VH--N--DH--N--JH junctions sequence analysis was performed which showed a point mutation in one and a single nucleotide deletion at the 3' terminus of the primer target site in the other case. In the remaining two cases no primer mismatches could be identified. Thus we conclude that the marked improvement of the PCR-detection rate of monoclonal IgH-CDR3 junctions was achieved at least in part due to the ability of UITma DNA polymerase to remove mismatched bases at the 3' terminus of the primers with respect to the target during the first amplification cycles. Our results suggest, that UITma is the DNA polymerase of choice for amplification of IgH-CDR3 junctions with consensus FR3A-VH- and JH-primers.