抗体基因点突变、插入和缺失体外检测方法的建立及应用

doi:10.3969/j.issn.1674-8115.2022.04.015

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (4): 518-527.doi: 10.3969/j.issn.1674-8115.2022.04.015

• 论著 · 技术与方法 • 上一篇下一篇

抗体基因点突变、插入和缺失体外检测方法的建立及应用

罗思敏(), 叶菱秀(), 郝茜()

上海交通大学基础医学院免疫学与微生物学系，上海市免疫学研究所，上海 200025

收稿日期:2021-12-31 接受日期:2022-03-25 出版日期:2022-04-28 发布日期:2022-04-28
通讯作者: 叶菱秀,郝茜 E-mail:siminluo@126.com;yeaplengsiew@shsmu.edu.cn;haoqian_2019@shsmu.edu.cn
作者简介:罗思敏（1997—），女，硕士生；电子信箱：siminluo@126.com。
基金资助:
国家重点研发计划(2020YFA0112900);上海交通大学医学院“新进青年教师启动”计划

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

LUO Simin(), YEAP Lengsiew(), HAO Qian()

Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China

Received:2021-12-31 Accepted:2022-03-25 Online:2022-04-28 Published:2022-04-28
Contact: YEAP Lengsiew,HAO Qian E-mail:siminluo@126.com;yeaplengsiew@shsmu.edu.cn;haoqian_2019@shsmu.edu.cn
Supported by:
National Key R&D Program of China(2020YFA0112900);Foundation of “New Young Teachers Initiative” Program of Shanghai Jiao Tong University School of Medicine

摘要/Abstract

摘要：

目的·建立由激活诱导胞苷脱氨酶（activation-induced cytidine deaminase，AID）引起的体细胞高频突变过程中，快速检测抗体基因可变（variable，V）、多样（diversity，D）、连接（joining，J）区发生突变（包括点突变、插入和缺失事件）的体外方法。应用该方法检测经2种DNA聚合酶β（polymerase β，Polβ）抑制剂处理的细胞中抗体基因VDJ片段的突变情况。方法·用慢病毒感染法处理CH12F3细胞（即处理组），感染前的CH12F3细胞为对照组。采用蛋白质印迹法（Western blotting）检测2组细胞中AID的表达水平。构建高通量测序文库，应用生物信息学的方法分析处理组和对照组细胞中抗体基因VDJ片段的点突变、插入及缺失频率。采用不同浓度的Polβ抑制剂［扎西他滨（2'，3'-dideoxycytidine，DDC）、5-甲氧基黄铜（5-methoxyflavone，5-MF）］处理CH12F3细胞，并采用台盼蓝拒染法测定该2种抑制剂对细胞增殖的影响。分别以筛选得到的DDC浓度、5-MF浓度处理CH12F3细胞（即实验组），以0.9% NaCl处理的细胞及DMSO处理的细胞依次记为上述实验组的对照组，使用上述已建立的方法进行处理，最终行高通量测序，分析该2种抑制剂对抗体基因VDJ片段中点突变、插入及缺失频率的影响。结果·Western blotting结果显示，和CH12F3细胞对照组相比，CH12F3细胞处理组中AID表达较高；且高通量测序结果显示，该组在抗体基因VDJ片段上存在大量的点突变，且插入和缺失的频率均较高（均P=0.000）。台盼蓝拒染法检测显示，处理CH12F3细胞最适宜的DDC浓度为100 μmol/L、5-MF浓度为25 μmol/L。最终的高通量测序结果显示，和0.9 %NaCl对照组相比，经100 μmol/L DDC处理的细胞在抗体基因VDJ片段的点突变频率较低（P=0.000），长度为1 bp的缺失频率亦较低（P=0.009）；和DMSO对照组相比，经25 μmol/L 5-MF处理的细胞在抗体基因VDJ片段的点突变频率较低（P=0.000），长度大于1 bp的插入和缺失频率亦有所下降（均P=0.000）。结论·该研究建立的体外检测方法可用于分析AID引起的抗体基因VDJ片段的突变事件。Polβ抑制剂DDC和5-MF对由AID引起的抗体基因VDJ片段的突变事件具有抑制作用。

关键词: 激活诱导胞苷脱氨酶, 点突变, 插入, 缺失

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

中图分类号:

R392.33

罗思敏, 叶菱秀, 郝茜. 抗体基因点突变、插入和缺失体外检测方法的建立及应用[J]. 上海交通大学学报（医学版）, 2022, 42(4): 518-527.

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.

图/表 4

图1 高通量测序样品准备流程图及PCR策略Note： A. Schematic of the study design. B. Schematic of PCR strategies to amplify VDJ regions in the productive allele of CH12F3 cells using allele-specific primers. The first PCR primers are labelled with yellow and parallel red solid arrows. The second PCR primers are labelled with blue and parallel red solid arrows.

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

图 2 CH12F3细胞处理组和对照组中，AID的表达及抗体基因VDJ片段的点突变、插入和缺失频率的分析Note： A. Expression of AID in CH12F3 cells with and without AID overexpression by Western blotting. B. Map of point mutations in the VDJ regions of antibody genes. Positions of AGCT and RGYW motifs are marked with orange and yellow bars, respectively. C/D. Insertion (C) and deletion (D) frequencies over total reads. F/G. Insertion (F) and deletion (G) frequencies of the indicated sizes in the VDJ regions of antibody genes. E/H. Mean frequencies of insertion (E) and deletion (H) on the start and end points of sequencing. CDRs are labelled with black solid lines. ①P=0.000,②P=0.001, compared with CH12F3 cells without AID overexpression.

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

图3 DDC对CH12F3细胞增殖及对抗体基因VDJ片段的点突变、插入和缺失频率的影响Note： A. Effects of different concentrations of DDC on the proliferation of CH12F3 cells. ①P=0.003, ②P=0.038, ③P=0.000, ④P=0.001, compared with 0.9% NaCl control group. B. Detection of the expression of AID and Polβ in the control group and experimental group by Western blotting. C. Mean frequencies over total reads that contain a point mutation. ③P=0.000, compared with the control group. D. Map of point mutations in the VDJ regions of antibody genes. E. Insertion frequencies over total reads. F. Mean frequencies of insertion on the start and end points of sequencing. G. Deletion frequencies over total reads. ⑤P=0.009, compared with the control group. H. Mean frequencies of deletion on the start and end points of sequencing.

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

图4 5-MF对CH12F3细胞增殖及对抗体基因VDJ片段的点突变、插入和缺失频率的影响Note： A. Effects of different concentrations of 5-MF on the proliferation of CH12F3 cells. ①P=0.000, ②P=0.002, compared with DMSO control group. B. Detection of expression of AID and Polβ in the control group and experimental group by Western blotting. C. Mean frequencies of sequences over total reads that contain a point mutation. ①P=0.000, compared with the control group. D. Map of point mutations in the VDJ regions of antibody genes. E. Insertion frequencies over total reads. ①P=0.000, compared with the control group. F. Mean frequencies of insertion on the start and end points of sequencing. G. Deletion frequencies over total reads. ①P=0.000, compared with the control group. H. Mean frequencies of deletion on the start and end points of sequencing.

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

参考文献 26

1	DI NOIA J M, NEUBERGER M S. Molecular mechanisms of antibody somatic hypermutation[J]. Annu Rev Biochem, 2007, 76: 1-22.
2	MURAMATSU M, KINOSHITA K, FAGARASAN S, et al. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme[J]. Cell, 2000, 102(5): 553-563.
3	YEAP L S, HWANG J K, DU Z, et al. Sequence-intrinsic mechanisms that target AID mutational outcomes on antibody genes[J]. Cell, 2015, 163(5): 1124-1137.
4	HAYNES B F, MOODY M A, LIAO H X, et al. B cell responses to HIV-1 infection and vaccination: pathways to preventing infection[J]. Trends Mol Med, 2011, 17(2): 108-116.
5	KWONG P D, MASCOLA J R. Human antibodies that neutralize HIV-1: identification, structures, and B cell ontogenies[J]. Immunity, 2012, 37(3): 412-425.
6	WALKER L M, PHOGAT S K, CHAN-HUI P Y, et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target[J]. Science, 2009, 326(5950): 285-289.
7	KEPLER T B, LIAO H X, ALAM S M, et al. Immunoglobulin gene insertions and deletions in the affinity maturation of HIV-1 broadly reactive neutralizing antibodies[J]. Cell Host Microbe, 2014, 16(3): 304-313.
8	YU L, GUAN Y J. Immunologic basis for long HCDR3s in broadly neutralizing antibodies against HIV-1[J]. Front Immunol, 2014, 5: 250.
9	METHOT S P, DI NOIA J M. Molecular mechanisms of somatic hypermutation and class switch recombination[M]//Advances in Immunology. Amsterdam: Elsevier, 2017: 37-87.
10	ZANOTTI K J, GEARHART P J. Antibody diversification caused by disrupted mismatch repair and promiscuous DNA polymerases[J]. DNA Repair (Amst), 2016, 38: 110-116.
11	BENNETT S E, SUNG J S, MOSBAUGH D W. Fidelity of uracil-initiated base excision DNA repair in DNA polymerase β-proficient and -deficient mouse embryonic fibroblast cell extracts[J]. J Biol Chem, 2001, 276(45): 42588-42600.
12	GU H, MARTH J D, ORBAN P C, et al. Deletion of a DNA polymerase β gene segment in T cells using cell type-specific gene targeting[J]. Science, 1994, 265(5168): 103-106.
13	NIU J Y, XIONG J, HU D, et al. 2', 3'-dideoxycytidine protects dopaminergic neurons in a mouse model of Parkinson's disease[J]. Neurochem Res, 2017, 42(10): 2996-3004.
14	MERLO S, BASILE L, GIUFFRIDA M L, et al. Identification of 5-methoxyflavone as a novel DNA polymerase-beta inhibitor and neuroprotective agent against β-amyloid toxicity[J]. J Nat Prod, 2015, 78(11): 2704-2711.
15	KIM A, HAN L, YU K F. Immunoglobulin class switch recombination is initiated by rare cytosine deamination events at switch regions[J]. Mol Cell Biol, 2020, 40(16): e00125-e00120.
16	STOCKDALE A M, DUL J L, WIEST D L, et al. The expression of membrane and secreted immunoglobulin during the in vitro differentiation of the murine B cell lymphoma CH12[J]. J Immunol, 1987, 139(10): 3527-3535.
17	YANG D P, SUN Y, CHEN J J, et al. REV7 is required for processing AID initiated DNA lesions in activated B cells[J]. Nat Commun, 2020, 11(1): 2812.
18	LANGMEAD B, SALZBERG S L. Fast gapped-read alignment with Bowtie 2[J]. Nat Methods, 2012, 9(4): 357-359.
19	ROBERT P A, MARSCHALL A L, MEYER-HERMANN M. Induction of broadly neutralizing antibodies in Germinal Centre simulations[J]. Curr Opin Biotechnol, 2018, 51: 137-145.
20	SCHEID J F, MOUQUET H, UEBERHEIDE B, et al. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding[J]. Science, 2011, 333(6049): 1633-1637.
21	WU X L, YANG Z Y, LI Y X, et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1[J]. Science, 2010, 329(5993): 856-861.
22	CORTI D, CAMERONI E, GUARINO B, et al. Tackling influenza with broadly neutralizing antibodies[J]. Curr Opin Virol, 2017, 24: 60-69.
23	MURIRA A, LAPIERRE P, LAMARRE A. Evolution of the humoral response during HCV infection: theories on the origin of broadly neutralizing antibodies and implications for vaccine design[J]. Adv Immunol, 2016, 129: 55-107.
24	GILCHUK P, KUZMINA N, ILINYKH P A, et al. Multifunctional pan-Ebolavirus antibody recognizes a site of broad vulnerability on the Ebolavirus glycoprotein[J]. Immunity, 2018, 49(2): 363-374.e10.
25	ROBBIANI D F, BOZZACCO L, KEEFFE J R, et al. Recurrent potent human neutralizing antibodies to Zika virus in Brazil and Mexico[J]. Cell, 2017, 169(4): 597-609.e11.
26	TAN J, PIEPER K, PICCOLI L, et al. A LAIR1 insertion generates broadly reactive antibodies against malaria variant antigens[J]. Nature, 2016, 529(7584): 105-109.

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抗体基因点突变、插入和缺失体外检测方法的建立及应用

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

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