Generation of the ICGi019-B-1 and ICGi019-B-2 lines via correction of the p.Met659Ile (c.1977G>A) variant in MYH7 of patient-specific induced pluripotent stem cells using CRISPR/Cas9.

IF 0.9 Q3 AGRICULTURE, MULTIDISCIPLINARY
A E Shulgina, S V Pavlova, J M Minina, S M Zakian, E V Dementyeva
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引用次数: 0

Abstract

The problem of interpretation of the genetic data from patients with inherited cardiovascular diseases still remains relevant. To date, the clinical significance of approximately 40 % of variants in genes associated with inherited cardiovascular diseases is uncertain, which requires new approaches to the assessment of their pathogenetic contribution. A combination of the induced pluripotent stem cell (iPSC) technology and editing the iPSC genome with CRISPR/Cas9 is thought to be the most promising tool for clarifying variant pathogenicity. A variant of unknown significance in MYH7, p.Met659Ile (c.1977G>A), was previously identified in several genetic screenings of hypertrophic cardiomyopathy patients. In this study, the single nucleotide substitution was corrected with CRISPR/Cas9 in iPSCs generated from a carrier of the variant. As a result, two iPSC lines (ICGi019-B-1 and ICGi019-B-2) were generated and characterized using a standard set of methods. The iPSC lines with the corrected p.Met659Ile (c.1977G>A) variant in MYH7 possessed a morphology characteristic of human pluripotent cells, expressed markers of the pluripotent state (the OCT4, SOX2, NANOG transcription factors and SSEA-4 surface antigen), were able to give rise to derivatives of three germ layers during spontaneous differentiation, and retained a normal karyotype (46,XY). No CRISPR/Cas9 off-target activity was found in the ICGi019-B-1 and ICGi019-B-2 iPSC lines. The maintenance of the pluripotent state and normal karyotype and the absence of CRISPR/Cas9 off-target activity in the iPSC lines with the corrected p.Met659Ile (c.1977G>A) variant in MYH7 allow using the iPSC lines as an isogenic control for further studies of the variant pathogenicity and its impact on the hypertrophic cardiomyopathy development.

利用CRISPR/Cas9技术,通过纠正患者特异性诱导多能干细胞MYH7中的p.Met659Ile (c.1977G>A)变异,生成ICGi019-B-1和ICGi019-B-2细胞系。
解释来自遗传性心血管疾病患者的遗传数据的问题仍然具有相关性。迄今为止,大约40%与遗传性心血管疾病相关的基因变异的临床意义尚不确定,这需要新的方法来评估其致病作用。诱导多能干细胞(iPSC)技术与CRISPR/Cas9编辑iPSC基因组的结合被认为是澄清变异致病性的最有前途的工具。先前在肥厚性心肌病患者的几次遗传筛查中发现了MYH7中一种未知意义的变异p.Met659Ile (c.1977G . >A)。在这项研究中,用CRISPR/Cas9在由变体载体产生的iPSCs中纠正了单核苷酸替换。结果,生成了两个iPSC系(ICGi019-B-1和ICGi019-B-2),并使用一套标准方法对其进行了表征。经过MYH7 p.Met659Ile (c.1977G>A)修正的iPSC细胞系具有人类多能细胞的形态特征,表达多能状态的标志物(OCT4、SOX2、NANOG转录因子和SSEA-4表面抗原),在自发分化过程中能够产生三种胚层的衍生物,并保持正常的核型(46,XY)。在ICGi019-B-1和ICGi019-B-2 iPSC系中未发现CRISPR/Cas9脱靶活性。在MYH7 p.m at659ile (c.1977G>A)变异的iPSC系中,多能状态和正常核型的维持以及CRISPR/Cas9脱靶活性的缺失,允许将iPSC系作为等基因对照,进一步研究该变异的致病性及其对肥厚性心肌病发展的影响。
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来源期刊
Vavilovskii Zhurnal Genetiki i Selektsii
Vavilovskii Zhurnal Genetiki i Selektsii AGRICULTURE, MULTIDISCIPLINARY-
CiteScore
1.90
自引率
0.00%
发文量
119
审稿时长
8 weeks
期刊介绍: The "Vavilov Journal of genetics and breeding" publishes original research and review articles in all key areas of modern plant, animal and human genetics, genomics, bioinformatics and biotechnology. One of the main objectives of the journal is integration of theoretical and applied research in the field of genetics. Special attention is paid to the most topical areas in modern genetics dealing with global concerns such as food security and human health.
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