Establishment and application of an in vitro detection method for antibody gene point mutation, insertion and deletion

doi:10.3969/j.issn.1674-8115.2022.04.015

Abstract

Abstract: Objective

·To establish an in vitro method for rapidly detecting mutations, including point mutation, insertion and deletion events in the variable (V), diversity (D) and joining (J) regions of antibody genes in the process of somatic hypermutations caused by activation-induced cytidine deaminase (AID). The above method was used to detect the effects of two DNA polymerase β (Polβ) inhibitors on the mutations in the VDJ regions of antibody genes.

Methods

·CH12F3 cells were treated with lentivirus infection (i.e. treatment group), and the CH12F3 cells before infection were used as control group. Western blotting was used to detect the expression level of AID in the two groups of cells. The high-throughput sequencing libraries were constructed, and the frequencies of point mutation, insertion and deletion in the VDJ regions of antibody genes in the two groups of cells were analyzed by bioinformatics. CH12F3 cells were treated with different concentrations of Polβ inhibitors [2', 3'-dioxycytidine (DDC) and 5- methoxyflavone (5-MF)], and the effects of the two inhibitors on cell proliferation were measured by Trypan blue exclusion method. CH12F3 cells (i.e. experimental group) were treated with the selected DDC concentration or 5-MF concentration, respectively; the cells treated with 0.9% NaCl or DMSO were recorded as the control group of the above experimental group, respectively. The four groups of cells before and after lentivirus infection were detected by the above established methods, and finally high-throughput sequencing was carried out to analyze the effects of the two inhibitors on the frequencies of point mutation, insertion and deletion in the VDJ regions of antibody genes.

Results

·Western blotting results showed that compared with CH12F3 cells in the control group, the expression of AID in CH12F3 cells in the treatment group was higher; and the high-throughput sequencing results showed that there were a large number of point mutations in the VDJ regions of antibody genes of CH12F3 cells in the treatment group, and its frequencies of insertion and deletion were also higher than those of the control group (both P=0.000). Trypan blue exclusion method showed that the optimal concentration of DDC and 5-MF for CH12F3 cells was 100 μmol/L and 25 μmol/L, respectively. The high-throughput sequencing results showed that compared with the 0.9% NaCl control group, the point mutation frequencies in the VDJ regions of antibody genes were decreased after cells were treated by 100 μmol/L DDC (P=0.000), and its 1 bp deletion frequencies decreased slightly (P=0.009); compared with DMSO control group, the frequencies of point mutation (P=0.000), longer than 1 bp insertion and deletion (both P=0.000) in the VDJ regions of antibody genes were decreased after cells were treated by 25 μmol/L 5-MF.

Conclusion

·The in vitro detection method established in this study can be used to analyze the AID-induced mutation events of VDJ regions of antibody genes. The Polβ inhibitors DDC and 5-MF can inhibit the mutations in the VDJ regions of antibody genes induced by AID.

Key words: activation-induced cytidine deaminase (AID), point mutation, insertion, deletion

CLC Number:

R392.33

LUO Simin, YEAP Lengsiew, HAO Qian. Establishment and application of an in vitro detection method for antibody gene point mutation, insertion and deletion[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(4): 518-527.

Figures/Tables 4

Fig 1 Flowchart of preparation for high-throughput sequencing and PCR strategies

Fig 2 Analysis of point mutation, insertion and deletion frequencies in the VDJ regions of antibody genes and AID expression in CH12F3 cells with or without AID overexpression

Fig 3 Effects of DDC on the proliferation of CH12F3 cells and the frequencies of point mutation, insertion and deletion in the VDJ regions of antibody genes

Fig 4 Effects of 5-MF on the proliferation of CH12F3 cells and the frequencies of point mutation, insertion and deletion in the VDJ regions of antibody genes

TrendMD

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