medgenМедицинская генетикаMedical Genetics2073-7998Publishing House «Genius Media» LLC10.25557/2073-7998.2023.08.20-29medgen-2331Research ArticleОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯORIGINAL RESEARCHМетод анализа метилирования CpG-островка, принадлежащего промоторной области гена MGMT, с использованием таргетного клонального бисульфитного секвенирования ДНК нового поколенияMethod for the analysis of methylation of the CpG island belonging to the promoter region of the MGMT gene using targeted new generation clonal bisulfite DNA sequencingМусатоваВ. В.MusatovaV. V.

115522, г. Москва, ул. Москворечье, д. 1

1, Moskvorechye st., Moscow, 115522

Shkarupo@mail.ruНиколаеваА. Ф.NikolaevaA. F.

115522, г. Москва, ул. Москворечье, д. 1

1, Moskvorechye st., Moscow, 115522

ПетроваК. О.PetrovaK. O.

115522, г. Москва, ул. Москворечье, д. 1

1, Moskvorechye st., Moscow, 115522

АлексееваЕ. А.AlekseevaE. A.

115522, г. Москва, ул. Москворечье, д. 1

1, Moskvorechye st., Moscow, 115522

ФГБНУ «Медико-генетический научный центр имени академика Н.П. Бочкова»РоссияResearch Centre for Medical GeneticsRussian Federation2023161020232282029Copyright © Мусатова В.В., Николаева А.Ф., Петрова К.О., Алексеева Е.А., 20232023Мусатова В.В., Николаева А.Ф., Петрова К.О., Алексеева Е.А.Musatova V.V., Nikolaeva A.F., Petrova K.O., Alekseeva E.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.medgen-journal.ru/jour/article/view/2331

Проведено сравнение методов секвенирования по Сэнгеру, секвенирования нового поколения и метилчувствительной ПЦР (МЧПЦР) с целью определения метилированного состояния целевого локуса. Обсуждается клиническая значимость получаемых расхождений в приложении к определению статуса метилирования промоторной области гена MGMT при глиобластоме. Показано, что для МЧ-ПЦР характерны ложноотрицательные результаты, а также сложна оценка низких уровней метилирования, не имеющих клинической значимости. Секвенирование по Сэнгеру не даёт возможности профилирования отдельных клеточных клонов, требует отдельного программного обеспечения для количественной оценки метилирования. Для рутинной лабораторной практики предложена технология таргетного клонального бисульфитного секвенирования ДНК с использованием метода секвенирования нового поколения для определения статуса метилирования CpG-островка, включающего в свой состав промоторную область гена MGMT. Рекомендовано использование прибора Ion Torrent PGM для избегания расчёта поправочных коэффициентов на определение смещения уровня метилирования.

Sanger sequencing, next generation sequencing, and methyl-sensitive PCR were compared to determine the methylated state of the target locus. The clinical significance of the resulting discrepancies in application to determining the methylation status of the promoter region of the MGMT gene in glioblastoma is discussed. It has been shown that MP-PCR is characterized by false-negative results, and it is also difficult to assess low levels of methylation that do not have clinical significance. Sanger sequencing does not allow profiling individual cell clones and requires separate software to quantify methylation. For routine laboratory practice, a technology of targeted clonal bisulfite DNA sequencing was proposed using a new generation sequencing method to determine the methylation status of a CpG island that includes the MGMT gene promoter region. The use of the Ion Torrent PGM is recommended to avoid calculating correction factors for methylation bias.

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