Distinctive features of single nucleotide alterations in induced pluripotent stem cells with different types of DNA repair deficiency disorders.

Kohji Okamura,Vaiyapuri Subbarayan Periasamy,Ken Nishimura,Ali Abdullah Alshatwi,Masashi Toyoda,Akihiro Umezawa,Kenichiro Hata,Kazuhiko Nakabayashi,Kazunori Hanaoka,Mayu Yamazaki-Inoue,Manami Ohtaka,Hironari Sakaguchi,Akon Higuchi,Mahito Nakanishi,Rie Sakamoto-Abutani,Shuji Takada

doi:10.1038/srep26342

Abstract

Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications.

Highlights

Shown to serve as an in vitro tool for understanding drug metabolism and toxicology[5, 6]
When the reprogramming factors OCT4/3, SOX2, KLF4 and c-MYC were introduced into 2.0 × 105 XP3OS and XPEMB-1 cells, induced pluripotent stem cells (iPSCs) from each Xeroderma pigmentosum (XP) cell were successfully generated and designated as XPAiPS-O1 and XPAiPS-E3, respectively
DNA is far more stable than RNA, DNA molecules are synthesized and decomposed repeatedly in cells. This reminds us that DNA is not chemically inert and that the DNA repair system is indispensable for maintaining life[18]

Summary

Introduction

Shown to serve as an in vitro tool for understanding drug metabolism and toxicology[5, 6]. Genotoxicity assays assess DNA damage such as single- or double-strand breaks, crosslinking, and point mutations. Human cells can be investigated for single nucleotide alterations and indels by genome-wide sequencing analysis. Unless the mutated cell attains proliferative predominance, the single nucleotide alteration/ mutation cannot reach a detectable level. Since iPSCs can be subcloned and assessed as a homogeneous population, they may be the key to development of next-generation genotoxicity tests. DNA repair is categorized into two categories, nucleotide excision repair and double-strand break repair, and the molecular mechanisms have been elucidated in detail. We generated iPSCs from patients with XP group A (XPA-iPSCs) and investigated numbers and types of detected SNAs in mutation-prone iPSCs, which may lead to a novel in vitro genotoxicity test using human iPSCs

Methods

Results

Conclusion