A Preliminary Study On Translocation Of The Immunoglobulin Heavy Chain Gene In KM3 Myeloma Cell Line

Backgreunds: Multiple myeloma is a kind of plasma cell clonal disease, Which ischaracterized by producing monoclonal immunoglobulin. Its tumorigenesis is not clearRecellt molecular analysis demonstraed IgH translocation in 93% in human myelomacell lines (HMCLs) and in 73.9 % of multiple myeloma (MM) patients. The consequenceof the IgH translocation is dysregulation, increased expression of the paner gene as aresult of its juxtaposition to intronic Eμ and/or 3'enhancers of IgH gene, which would beresponsible of MM genesis. To date, several involved oncogenes have been cloned. Therest remain to be identified.Compared with that in U.S.-European, multiple myeloma of Chinse patients has itsown specific clinical characteristics as well as different genetic backgrounds. However,no study about the IgH translocation of multiple myeloma has been reported thus far inChina. Here we aimed at making a preliminary study of IgH translocation in ChinseMM and trying to evaluate the chromosomal differences between Chinese and WesternpatientsMethods: According to the published reference, we designed 5 pairs of DNAprobes specific for the upsttean and downstueam sequences of IgH switch regions: σμ/ σδ, S μ,Sγ, S α,S ε. The probes were synthesized and DIG-labeled usingPCR assap The sizes of the synthesized probes were calculated after gel electrophoresis.After purification, the estimation of the yield of the DIG-labeled probes was condutedin a spot test with a DIG-labeled control. Dot blotting was made for the qualitativescreening of genome DNA. Southern blot assny was performed to identify anddistinguish IgH translocation in KM3 myeloma cell line established from Chinesepatients. Relevant restriction enzymes, Hind III. Bgl II and Sph I, were used to digestgenomic DNA at sites outside the pairs of switch probes. Placental genome was used ascontrol. Different recombination events are reflected in different patterns ofcohybridizaion of the probes. A restriction fragment to which only 1 of the 10 switchprobes hybridized is recognized as an illegitimate fragment.ReuIts: 1. The POsition of each probe in ngarose gel is identical to the rePOrted one.2. The DIG-labeling efficiency of 3' o p probe is 0. lpg/ P l; all of the others were 0.0lpg/ P l. 3. Dot blOt assay showed that all the probes could hybridize to placental genomicDNA. 4. Southem blot indicated that an illegitimate 5.6Kb Hind III fragrnent onlyhybridized tO 3'S P probe, while the l0Kb Hind III fragment hybridized to both 3'S uand 5'S ll Probes as in placental genomic DNA. The other Probes shown no restrictionfragInens in KM3 genome except 5'S P and 5'S E probes with the sazne fragment as inplacenta.Conclu8ion: l.The 5 Pairs of DIG-labeled probes proved to be just wha weexPected. 2.The IgH translocation occurred in the KM3 myeloma cell line and thebreakPoint located in the IgH S ll region .The partner chromosome and its breakPoint areleft to be detected.