Detection Of IgV_H Gene Segments Usage And Mutation Status In Patients With Chronic Lymphocytic Leukemia By Multiplex PCR

Objective Mutational status of the variable region of theimmunoglobulin heavy chain gene (IgV_H) is an important prognosticmarker in B-cell chronic lymphocytic leukaemia (B-CLL), with mutatedpatients having improved outcome. However, routine PCR technologiestook very long time and were expensive. The objective of present study isto examine the mutational status and V_H gene segment location inChinese patients with B-CLL using new multiplex PCR method.Methods IgV_H gene segments were amplified by multiplex PCR in 52B-CLL patients and purified PCR amplification products were directlysequenced. IgV_H gene segments usage and somatic hypermutation statuswere analysed by IMGT/V-QUEST. Three-color flow cytometry wasused to explore the ZAP-70 and CD38 expression in CLL.Results Using multiplex PCR, somatic hypermutation status of IgV_Hgenes were easily and rapidly analyzed. The results showed that 38patients had mutated IgV_H, whereas 14 others had unmutated IgV_H. Themost frequently expressed V_H gene family was found to be V_H3 (46%)followed by V_H4 (40.4%), V_H1 (5.8%), V_H2 (5.8%) and V_H7 (2%), withno expression of V_H5 and V_H6 gene families. It was reported that V_H1-69and V_H3-21 were commonly overused in Western B-CLL with worstoutcome, whereas, both of them were absent in our study.Conclusions Multiplex PCR is a rapid and easy method which solvedthe limitations and pitfalls of routine PCR in detecting IgV_H mutation status; it is worth extensively used both in clinic and scientific researches.The frequency of IgV_H gene families' usage indicates significantdifference between Chinese B-CLL and Western patients, suggestinginvolvement of ethnic and/or environmental factors in B-CLL diseaseinitiation, which maybe one of the most important reasons of significantlydifferent incidence.