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Novel germline mutation KMT2A G3131S confers genetic susceptibility to familial myeloproliferative neoplasms

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Abstract

The current study analyzed the clinical and genetic characteristics of a family with familial myeloproliferative neoplasms (MPNs). Whole-exome sequencing was conducted, and a germline heterozygous mutation in lysine methyltransferase 2A (KMT2A, also known as MLL1), G3131S (c.9391G > A, p.Gly3131Ser, rs150804738), was identified. Somatic DNA and germline DNA were collected from 8 family members, 120 healthy donors (somatic DNA), and 30 healthy donors (germline DNA). Using Sanger sequencing, the KMT2A G3131S mutation was analyzed. Four individuals, the proband (II-1), his sister (patient II-2), and family members II-3 and III-1 (somatic DNA and germline DNA), tested positive for the KMT2A G3131S mutation. We did not observe the KMT2A G3131S mutation in healthy donors (somatic DNA and germline DNA), indicating that this is not a SNP. Bioinformatics analysis of KMT2A G3131S suggested that protein structure changes could be caused by this mutation. To further elucidate the function of KMT2A G3131S, the CRISPR-Cas9 technique was applied to generate a KMT2A G3131S heterozygous K562 cell line. The colony formation potency, apoptosis, and cell cycle of KMT2A G3131S mutant K562 cells were analyzed. The results demonstrated that KMT2A G3131S mutant K562 cells showed increased proliferation and colony formation ability. Immunophenotyping was performed using flow cytometry to analyze the surface marker expression of gene-edited KMT2A G3131S mutant K562 cells. A significant increase in CD11b and mild increases in CD61 and CD235a were observed in KMT2A G3131S mutant K562 cells, suggesting that the KMT2A G3131S mutant could cause an increase in myeloproliferation. May-Giemsa staining showed that the morphological changes in KMT2A G3131S mutant K562 cells were consistent with the flow cytometry analysis. To verify which downstream genes were affected by the KMT2A G3131S mutant, we performed real-time PCR to evaluate the expression of previously reported KMT2A-related genes and found that C-MYB expression was significantly decreased. Western blotting was applied to investigate the expression of Kmt2a and C-myb proteins, and the results showed that in KMT2A G3131S mutant K562 cells, the expression of C-myb was decreased. Our findings suggested that KMT2A G3131S could affect the myeloproliferation of K562 cells and decrease C-myb expression. In conclusion, KMT2A G3131S could be considered a novel genetic susceptibility gene in familial MPN.

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Funding

This work was supported in part by the National Natural Science Foundation of China (Nos. 82070175, 81800198, 81700168) and the Natural Science Foundation of Hunan Province (No. 2019JJ50863).

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Author notes

  1. Le Yin and Sisi Xie are contributed equally to this work.

Authors and Affiliations

  1. Department of Hematology, The Second Xiangya Hospital, Central South University, Changsha, People’s Republic of China

    Le Yin, Sisi Xie, Yi Chen, Wang Li, Xian Jiang, Heng Li, Ji Li, Zefang Wu, Xiang Xiao, Guangsen Zhang, Zhao Cheng & Hongling Peng

  2. Institute of Molecular Hematology, Central South University, Changsha, People’s Republic of China

    Zhao Cheng & Hongling Peng

  3. Hunan Key Laboratory of Tumor Models and Individualized Medicine, Changsha, Hunan, 410011, People’s Republic of China

    Hongling Peng

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  1. Le Yin
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Contributions

Professor Hongling Peng diagnosed patient and designed and composed this study. Dr. Zhao Cheng designed and composed this study and wrote the manuscript. Miss Le Yin and Sisi Xie collected data and conducted the study. Mr. Yi Chen helped with performing experiments. Mr. Wang Li and Xian Jiang helped with data collection and experiments. Dr. Heng Li and Dr. Ji Li helped with clinic sample collection. Mr. Zefang Wu helped with clinic sample collection. Dr. Xiang Xiao helped with flow cytometry and RNA reverse-transcription and transcriptome sequencing. Professor Guangsen Zhang partially participate the study and helped with clinic sample collection.

Corresponding authors

Correspondence to Zhao Cheng or Hongling Peng.

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The study was approved by the Ethics Committee of the Second Xiangya Hospital, Central South University. Informed consent was obtained in writing in accordance with the Declaration of Helsinki.

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Yin, L., Xie, S., Chen, Y. et al. Novel germline mutation KMT2A G3131S confers genetic susceptibility to familial myeloproliferative neoplasms. Ann Hematol 100, 2229–2240 (2021). https://doi.org/10.1007/s00277-021-04562-4

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