vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/vjgb-25-38vavilov-4601Research ArticleМОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯMOLECULAR AND CELL BIOLOGYПолучение линий ICGi019-B-1 и ICGi019-B-2 посредством исправления с помощью системы CRISPR/Cas9 варианта p.Met659Ile (c.1977G>A) в гене MYH7 в пациент-специфичных индуцированных плюрипотентных стволовых клеткахGeneration 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/Cas9ШульгинаА. Е.ShulginaA. E.

Новосибирск

Novosibirsk

ПавловаС. В.PavlovaS. V.

Новосибирск

Novosibirsk

МининаЮ. М.MininaJ. M.

Новосибирск

Novosibirsk

ЗакиянС. М.ZakianS. M.

Новосибирск

Novosibirsk

ДементьеваЕ. В.DementyevaE. V.

Новосибирск

Novosibirsk

dementyeva@bionet.nsc.ruФедеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesRussian Federation202503062025293349357Copyright © Шульгина А.Е., Павлова С.В., Минина Ю.М., Закиян С.М., Дементьева Е.В., 20252025Шульгина А.Е., Павлова С.В., Минина Ю.М., Закиян С.М., Дементьева Е.В.Shulgina A.E., Pavlova S.V., Minina J.M., Zakian S.M., Dementyeva E.V.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/4601

Проблема интерпретации результатов генетического анализа пациентов, страдающих наследственными сердечно-сосудистыми заболеваниями, по-прежнему сохраняет свою актуальность. На сегодняшний день клиническое значение около 40 % вариантов в генах, ассоциированных с наследственными сердечно-сосудистыми заболеваниями, остается неясным, что приводит к необходимости использования новых подходов для оценки патогенетического вклада этих вариантов. Совместное применение технологии индуцированных плюрипотентных стволовых клеток и редактирования их генома с помощью системы CRISPR/ Cas9 считается наиболее перспективным способом выяснения патогенности генетических вариантов.

Ранее в нескольких генетических скринингах пациентов с гипертрофической кардиомиопатией был выявлен вариант с неясным клиническим значением в гене MYH7, p.Met659Ile (c.1977G>A). В настоящем исследовании данная однонуклеотидная замена с помощью системы CRISPR/Cas9 была исправлена в индуцированных плюрипотентных стволовых клетках, полученных от носителя этого генетического варианта. В результате получены и охарактеризованы с использованием стандартного набора методов две линии индуцированных плюрипотентных стволовых клеток (ICGi019-B-1 и ICGi019-B-2). Линии индуцированных плюрипотентных стволовых клеток с исправленным вариантом p.Met659Ile (c.1977G>A) в гене MYH7 имели характерную для плюрипотентных клеток человека морфологию, экспрессировали маркеры плюрипотентного состояния (транскрипционные факторы OCT4, SOX2, NANOG и поверхностный антиген SSEA-4), были способны давать производные трех зародышевых листков при спонтанной дифференцировке и сохраняли нормальный кариотип (46,XY). В линиях индуцированных плюрипотентных стволовых клеток ICGi019-B-1 и ICGi019-B-2 не обнаружено нецелевой активности системы CRISPR/Cas9. Поддержание плюрипотентного состояния и нормального кариотипа, а также отсутствие нецелевой активности системы CRISPR/Cas9 в линиях индуцированных плюрипотентных стволовых клеток с исправленным вариантом p.Met659Ile (c.1977G>A) в гене MYH7 позволят использовать полученные линии в качестве изогенного контроля для дальнейших исследований патогенности данного генетического варианта и его влияния на развитие гипертрофической кардиомиопатии.

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 in herited 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.

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The authors declare that there are no conflicts of interest present.