Repertoire Sequencing of B Cells Elucidates the Role of UNG and Mismatch Repair Proteins in Somatic Hypermutation in Humans.

Hanna Ijspeert,Claudia Blattmann,Pauline A Van Schouwenburg,Gertjan J Driessen,Laurence Brugieres,Ingrid Pico-Knijnenburg,Heinz Jacobs,Jan Loeffen,Marcus G Seidel,Mirjam Van Der Burg,Manon Suerink,Amedeo A Azizi,Danuta Januszkiewicz-Lewandowska

doi:10.3389/fimmu.2019.01913

Abstract

The generation of high-affinity antibodies depends on somatic hypermutation (SHM). SHM is initiated by the activation-induced cytidine deaminase (AID), which generates uracil (U) lesions in the B-cell receptor (BCR) encoding genes. Error-prone processing of U lesions creates a typical spectrum of point mutations during SHM. The aim of this study was to determine the molecular mechanism of SHM in humans; currently available knowledge is limited by the number of mutations analyzed per patient. We collected a unique cohort of 10 well-defined patients with bi-allelic mutations in genes involved in base excision repair (BER) (UNG) or mismatch repair (MMR) (MSH2, MSH6, or PMS2) and are the first to present next-generation sequencing (NGS) data of the BCR, allowing us to study SHM extensively in humans. Analysis using ARGalaxy revealed selective skewing of SHM mutation patterns specific for each genetic defect, which are in line with the five-pathway model of SHM that was recently proposed based on mice data. However, trans-species comparison revealed differences in the role of PMS2 and MSH2 in strand targeting between mice and man. In conclusion, our results indicate a role for UNG, MSH2, MSH6, and PMS2 in the generation of SHM in humans comparable to their function in mice. However, we observed differences in strand targeting between humans and mice, emphasizing the importance of studying molecular mechanisms in a human setting. The here developed method combining NGS and ARGalaxy analysis of BCR mutation data forms the basis for efficient SHM analyses of other immune deficiencies.

Highlights

The formation of high-affinity antibodies is based on the efficient introduction of somatic point mutations in the variable region of the B-cell receptor (BCR) encoding genes
To study the molecular mechanism of somatic hypermutations (SHM) in human, we included a unique cohort of 10 patients with UNG, MSH2, MSH6, or PMS2 deficiency, and 15 previously published agematched HCs (Table 1) [32]
Since the amount of blood that was available for most of the patients was very limited, we decided to analyze the IGHA and IGHG rearrangements from total PBMCs. These rearrangements are derived from antigen-experienced B cells that have undergone class switch recombination (CSR) and SHM

Summary

Introduction

The formation of high-affinity antibodies is based on the efficient introduction of somatic point mutations in the variable region of the B-cell receptor (BCR) encoding genes. Initiation of these somatic hypermutations (SHM) requires the activity of activation-induced cytidine deaminase (AID) that deaminates cytosines (C) to uracils (U), thereby generating highly mutagenic U lesions [1, 2]. Bi-allelic mutations in MMR result in constitutional mismatch repair deficiency (CMMRD) (OMIM #276300). This is a rare childhood cancer predisposition syndrome without overt clinical signs of an immunodeficiency [4]

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Conclusion