Semiquantitative and qualitative assessment of B-lymphocyte V H repertoire by a fluorescent multiplex PCR.
We established a new tool to perform semiquantitative and qualitative screening for V(H) gene usage frequency during IgH rearrangements in human B-lymphocytes. In two separate multiplex PCRs, the rearranged VDJ regions were amplified with V(H) family-specific primers labeled with different fluorescent dyes (FAM, HEX, NED, or ROX). The relative amount of each of the particular V(H) family products and their ratios were determined by fragment analysis on a ABI PRISM 377 sequencer. We verified that the fluorescent multiplex PCR (FMPCR) shows high specificity and sensitivity, acceptable reproducibility and reliability. Data obtained were well in agreement with results revealed by sequencing following single-cell PCR. Ten healthy volunteers showed a comparable semiquantitative V(H) family distribution. The FMPCR also correctly detected a monoclonal peak in a CLL patient. Thus, labeling primers with various fluorescent dyes allows for an assessment of V(H) family usage and an immediate determination of the involved V(H) gene family if any clonal peaks are present. This method provides a quick, easy, and reliable tool for V(H) repertoire screening of larger populations of patients suffering from diseases with changes in the V(H) repertoire allowing for selection of cases worth a more detailed and cumbersome sequence analysis later on.
Medizinische Poliklinik, University of Würzburg, Würzburg, Germany.
Martin
M
Feuchtenberger
Hans-Peter
HP
Tony
Anne-Sophie
AS
Rouzière
Anette
A
Jacobi
Thomas
T
Dörner
Christian
C
Kneitz
Petr
P
Starostik
D000328:Adult; D000368:Aged; D001327:Autoimmune Diseases / Q000175:diagnosis*; D001402:B-Lymphocytes / Q000276:immunology; D005192:Family Health; D005456:Fluorescent Dyes; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D005456:Fluorescent Dyes; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region