Гипометилирование LINE-1 и гиперметилирование HIST1H4F как онкомаркеры в жидкостной биопсии колоректального рака

   Введение. Локальное гиперметилирование промоторов уникальных генов и глобальное гипометилирование генома – хорошо известные проявления ассоциированного с канцерогенезом аберрантного метилирования. Мы исследовали этот феномен в качестве возможного диагностического маркера для жидкостной биопсии колоректального
рака, используя оригинальный метод количественного анализа плавления гибридов ДНК-зонд (quantitative DNA melting analysis with hybridization probes, qDMA-HP).

   Цель исследования – количественная оценка метилирования гена HIST1H4F и транспозона LINE-1 в циркулирующей ДНК плазмы крови больных колоректальным раком.

   Материалы и методы. Проанализированы обработанные бисульфитом образцы ДНК, выделенные из плазмы крови здоровых доноров и онкологических больных. Метилирование HIST1H4F оценивали с помощью асимметричной полимеразной цепной реакции с гибридизуемым зондом и плавлением гибридов ампликон / зонд после завершения амплификации. Для тестирования многократно повторяющихся и характеризующихся выраженным полиморфизмом
последовательностей LINE-1 использовали асимметричную полимеразную цепную реакцию с гибридизуемым зондом и интеркалирующим красителем SYBR Green, после чего плавили гибриды и анализировали многокомпонентные профили плавления.

   Результаты. Показана высокая диагностическая эффективность панели маркеров LINE-1 и HIST1H4F в жидкостной биопсии колоректального рака: площадь под ROC-кривой 0,92; чувствительность 100 %; специфичность 84 %. Перекрестная валидизация подтверждает этот результат. Гиперметилирование HIST1H4F и гипометилирование LINE-1
статистически значимо коррелируют (коэффициент корреляции Спирмена r = 0,4; p = 0,01).

   Заключение. Метод qDMA-HP можно использовать для количественной оценки аберрантного метилирования клинически значимых генов.

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