Quantitative determination of immunoglobulin heavy chain gene rearrangements in childhood acute lymphoblastic leukemia

Objective To establish the methodology for quantitative detection of immunoglabulin heavy chain(IgH)gene rearrangements in childhood acute lymphoblastic leukemia(ALL)by real-time quantitative polymerase chain reaction(RQ-PCR).Methods Clonal IgH gene rearrangements in 109 B cell acute lymphoblastie leukemia(B-ALL)patients at diagnosis were identified by multiplex PCR assay followed by sequencing.Allele specific oligonucleotide(ASO)primers were designed complementary to the junctional region.Germline JH TaqMan probe and primer was used to establish RQ-PCR for determination of IgH gene rearrangements in 41 childhood B-ALL The sensitivity and specificity were assessed as well.Results IgH rearrangements was identified in 48 cases out of 109 patients with B-ALL.The sequence analysis showed that the most frequently used V,D,J segments were from V3 family,D3 family and J4 family,respectively.41 cases were repeatable among 48 cases.Both sensitivity and specificity was less than 10~(-4).The Ct values are more than 40 in all nonspeeific amplification.The difference is more than 3 compared with that in specific amplification.The mean slope of the standard curves was-3.31±0.19 and the mean intercept was 37.66 ±1.23.The correlation coefficients of all standard curves were above 0.97.Conclusions RQ-PCR analysis of IgH gene rearrangements is highly sensitive(≤10~(-4))and specific.It is applicable to detect MRD in childhood ALL.