Nanopore Sequencing Indicates That Tandem Amplification of Chromosome 20q11.21 in Human Pluripotent Stem Cells Is Driven by Break-Induced Replication.

Jason A Halliwell,Peter W Andrews,Emma Betteridge,Duncan Baker,Michael A Quail,Karen Oliver,Ivana Barbaric,Jason Skelton,Kim Judge

doi:10.1089/scd.2021.0013

Abstract

Copy number variants (CNVs) are genomic rearrangements implicated in numerous congenital and acquired diseases, including cancer. The appearance of culture-acquired CNVs in human pluripotent stem cells (PSCs) has prompted concerns for their use in regenerative medicine. A particular problem in PSC is the frequent occurrence of CNVs in the q11.21 region of chromosome 20. However, the exact mechanism of origin of this amplicon remains elusive due to the difficulty in delineating its sequence and breakpoints. Here, we have addressed this problem using long-read Nanopore sequencing of two examples of this CNV, present as duplication and as triplication. In both cases, the CNVs were arranged in a head-to-tail orientation, with microhomology sequences flanking or overlapping the proximal and distal breakpoints. These breakpoint signatures point to a mechanism of microhomology-mediated break-induced replication in CNV formation, with surrounding Alu sequences likely contributing to the instability of this genomic region.

Highlights

Copy number variants (CNVs) are gains or losses of DNA segments ranging in size from *50 bp to several megabases [1]
These breakpoint signatures point to a mechanism of microhomology-mediated break-induced replication in CNV formation, with surrounding Alu sequences likely contributing to the instability of this genomic region
The profound effect of CNV acquisition on cellular phenotype has been described in human pluripotent stem cells (PSCs), which frequently gain a CNV located on chromosome 20 in the region q11.21 upon prolonged culture [2,3,4,5]

Summary

Introduction

Copy number variants (CNVs) are gains or losses of DNA segments ranging in size from *50 bp to several megabases [1]. The chromosome 20q11.21 CNV bestows on the variant PSC a growth advantage due to resistance to apoptosis [5,6]. The chromosome 20q11.21 CNV is typically gained as a tandem duplication, PSC lines with four or five copies of this CNV have been reported [2,8]. The shared region common to all of the reported variants contains a dosagesensitive antiapoptotic gene, BCL2L1, which has been identified as the driver gene, overexpression of which is responsible for the selective growth advantage of variant PSC carrying this CNV [5,6,8]. The nature of the mutational events that generate these chromosome 20q11.21 CNVs has not been elucidated in PSCs

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