transplantologiyaТрансплантологияTransplantologiya. The Russian Journal of Transplantation2074-05062542-0909IPO Association of Transplantologists10.23873/2074-0506-2024-16-4-458-472transplantologiya-939Research ArticleПРОБЛЕМНЫЕ АСПЕКТЫPROBLEMATIC ASPECTSВлияние размера ДНК-мишени на эффективность измерения химеризма в циркулирующей свободной ДНК плазмыEffect of DNA target size on the efficiency of chimerism measurement in circulating free plasma DNAhttps://orcid.org/0000-0003-3914-8611НикулинаЕ. Е.NikulinaE. E.

Елена Евгеньевна Никулина - научный сотрудник лаборатории молекулярной гематологии.

125167, Москва, Новый Зыковский пр-д, д. 4

Elena E. Nikulina - Researcher, Laboratory of Molecular Hematology.

4 Noviy Zykovskiy Dr., Moscow 125167

lenysh2007@rambler.ruhttps://orcid.org/0000-0003-2957-1619РисинскаяН. В.RisinskayaN. V.

Наталья Владимировна Рисинская - канд. биол. наук, старший научный сотрудник лаборатории молекулярной гематологии.

125167, Москва, Новый Зыковский пр-д, д. 4

Natalia V. Risinskaya - Cand. Sci. (Biol.), Senior Researcher, Laboratory of Molecular Hematology.

4 Noviy Zykovskiy Dr., Moscow 125167

risinska@gmail.comhttps://orcid.org/0009-0005-1903-6200ДубоваО. Е.DubovaO. E.

Ольга Евгеньевна Дубова - стажер лаборатории молекулярной гематологии ФГБУ «НМИЦ гематологии» МЗ РФ; стажер-исследователь Института цифрового биодизайна и моделирования живых систем Первый МГМУ им. И.М. Сеченова МЗ РФ.

125167, Москва, Новый Зыковский пр-д, д. 4; 119991, Москва, ул. Трубецкая, д. 8, стр. 2

Olga E. Dubova - Researcher Assistant, Laboratory of Molecular Hematology, NMRCH; Research Assistant, Institute of Digital Biodesign and Simulation of Living Systems, I.M. Sechenov FMSMU (Sechenov University).

4 Noviy Zykovskiy Dr., Moscow 125167; 8 Bldg. 2 Trubetskaya St., Moscow 119991

dubovaolgaa@gmail.comhttps://orcid.org/0000-0003-3440-6685СумцоваО. В.SumtsovaO. V.

Ольга Васильевна Сумцова - врач-гепатолог, научный сотрудник отдела трансплантологии.

129110, Москва, ул. Щепкина, д. 61/2

Olga V. Sumtsova - Hepatologist, Researcher, Department of Transplantology.

61/2 Shchepkin St., Moscow 129110

olga.vasina.do4@gmail.comhttps://orcid.org/0000-0002-0002-9183МойсюкЯ. Г.MoysyukYa. G.

Ян Геннадьевич Мойсюк - проф., д-р мед. наук, руководитель отдела трансплантологии.

129110, Москва, ул. Щепкина, д. 61/2

Yan G. Moysyuk - Prof., Dr. Sci. (Med.), Head of the Department of Transplantology.

61/2 Shchepkin St., Moscow 129110

moysyuktrans@list.ruhttps://orcid.org/0000-0003-1739-1063ВасильеваВ. А.VasilievaV. A.

Вера Алексеевна Васильева - канд. мед. наук, заведующая отделением «Дневной стационар иммунохимиотерапии после трансплантации костного мозга и гемопоэтических стволовых клеток».

125167, Москва, Новый Зыковский пр-д, д. 4

Vera A. Vasilieva - Cand. Sci. (Med.), Head of the Department "Daytime Hospital for Immunochemotherapy after Transplantation of Bone Marrow and Hematopoietic Stem Cells".

4 Noviy Zykovskiy Dr., Moscow 125167

vasilievava4@mail.ruhttps://orcid.org/0000-0003-4142-171XСоловьеваМ. В.SolovevaM. V.

Майя Валерьевна Соловьева - канд. мед. наук, старший научный сотрудник отделения интенсивной высокодозной химиотерапии парапротеинемических гемобластозов.

125167, Москва, Новый Зыковский пр-д, д. 4

Maya V. Soloveva - Cand. Sci. (Med.), Hematologist, Department of Intensive High-Dose Chemotherapy for Paraproteinemic Hemoblastoses.

4 Noviy Zykovskiy Dr., Moscow 125167

firs-maia@yandex.ruhttps://orcid.org/0000-0002-0364-3242ЮшковаА. А.YushkovaA. A.

Анна Александровна Юшкова - ведущий специалист лаборатории молекулярной гематологии.

125167, Москва, Новый Зыковский пр-д, д. 4

Anna A. Yushkova - Leading Specialist, Laboratory of Molecular Hematology.

4 Noviy Zykovskiy Dr., Moscow 125167

ann.unikova@bk.ruhttps://orcid.org/0000-0002-8763-246XФевралеваИ. С.FevralevaI. S.

Ирина Серафимовна Февралева - канд. мед. наук, ведущий научный сотрудник лаборатории молекулярной гематологии.

125167, Москва, Новый Зыковский пр-д, д. 4

Irina S. Fevraleva - Senior Researcher, Laboratory of Molecular Hematology.

4 Noviy Zykovskiy Dr., Moscow 125167

irina_fevraleva@mail.ruhttps://orcid.org/0009-0009-1333-9437СкрипкинаА. С.SkripkinaA. S.

Анастасия Сергеевна Скрипкина - стажер лаборатории молекулярной гематологии ФГБУ «НМИЦ гематологии» МЗ РФ; стажер-исследователь Института цифрового биодизайна и моделирования живых систем Первый МГМУ им. И.М. Сеченова.

125167, Москва, Новый Зыковский пр-д, д. 4; 119991, Москва, ул. Трубецкая, д. 8, стр. 2

Anastasiya S. Skripkina - Trainee in Laboratory of Molecular Hematology, NMRCH; Research Assistant, Institute of Digital Biodesign and Simulation of Living Systems, I.M. Sechenov FMSMU (Sechenov University).

4 Noviy Zykovskiy Dr., Moscow 125167; 8 Bldg. 2 Trubetskaya St., Moscow 119991

stasia_a0@mail.ruhttps://orcid.org/0000-0002-4281-5051МакарикА. А.MakarikA. A.

Алина Артуровна Макарик - врач-ординатор отделения патологии беременности.

101000, Москва, ул. Покровка, д. 22А

Alina A. Makarik - Residency-training Physician, Department of Pregnancy Pathology.

22A Pokrovka St., Moscow 101000

alinamakarik@mail.ruhttps://orcid.org/0000-0001-9463-9187СудариковА. Б.SudarikovA. B.

Андрей Борисович Судариков - д-р биол. наук, заведующий отделом молекулярной генетики, заведующий лабораторией молекулярной гематологии.

125167, Москва, Новый Зыковский пр-д, д. 4

Andrey B. Sudarikov - Dr. Sci. (Biol.), Head of the Department of Molecular Genetics, Head of the Laboratory of Molecular Hematology.

4 Noviy Zykovskiy Dr., Moscow 125167

dusha@blood.ruФГБУ «НМИЦ гематологии» МЗ РФРоссияNational Medical Research Center for HematologyRussian FederationФГБУ «НМИЦ гематологии» МЗ РФ; ФГАОУ ВО Первый МГМУ им. И.М. Сеченова МЗ РФ (Сеченовский Университет)РоссияNational Medical Research Center for Hematology; I.M. Sechenov First Moscow State Medical University (Sechenov University)Russian FederationГБУЗ МО МОНИКИ им. М.Ф. ВладимирскогоРоссияMoscow Regional Research and Clinical Institute n.a. M.F. VladimirskiyRussian FederationГБУЗ МОНИАГ им. В.И. КраснопольскогоРоссияMoscow Regional Research Institute of Obstetrics and Gynecology n.a. Acad. V.I. KrasnopolskyRussian Federation202416122024164458472Copyright © Никулина Е.Е., Рисинская Н.В., Дубова О.Е., Сумцова О.В., Мойсюк Я.Г., Васильева В.А., Соловьева М.В., Юшкова А.А., Февралева И.С., Скрипкина А.С., Макарик А.А., Судариков А.Б., 20242024Никулина Е.Е., Рисинская Н.В., Дубова О.Е., Сумцова О.В., Мойсюк Я.Г., Васильева В.А., Соловьева М.В., Юшкова А.А., Февралева И.С., Скрипкина А.С., Макарик А.А., Судариков А.Б.Nikulina E.E., Risinskaya N.V., Dubova O.E., Sumtsova O.V., Moysyuk Y.G., Vasilieva V.A., Soloveva M.V., Yushkova A.A., Fevraleva I.S., Skripkina A.S., Makarik A.A., Sudarikov A.B.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.jtransplantologiya.ru/jour/article/view/939Актуальность

Актуальность. Анализ свободной циркулирующей ДНК открывает перспективы для молекулярной диагностики, однако ее фрагментация и низкая концентрация в крови могут осложнить проведение ПЦР.

Цель

Цель. Изучить влияние длины мишени на эффективность амплификации маркеров Y-хромосомы из свободной циркулирующей ДНК.

Материал и методы

Материал и методы. Пятьдесят образцов свободной циркулирующей ДНК полученных от 39 пациентов: после трансплантации печени (n=19), с острым лейкозом после аллогенной трансплантации гемопоэтических стволовых клеток (n=10) и беременных женщин (n=10). Кроме того, мы подготовили 16 образцов химер путем последовательного разведения мужской свободной циркулирующей ДНК в женской свободной циркулирующей ДНК от здоровых доноров. Мы определили долю мужской свободной циркулирующей ДНК с помощью маркера Y-хромосомы S02, длина которого составляет 211 пар нуклеотидов, как было предложено M. Alizadeh et al. Мы также модифицировали дизайн праймеров Alizadeh для получения ДНК-мишени длиной 138 пар нуклеотидов.

Доля мужской свободной циркулирующей ДНК также определялась с помощью фрагментного анализа с использованием маркера амелогенина Y (84 пар нуклеотидов) из набора COrDIS Plus (ООО «Гордиз», Россия).

Результаты

Результаты. В трех группах пациентов амплификация мужской свободной циркулирующей ДНК была более эффективной при использовании более коротких ДНК-мишеней (p<0,05). В искусственно созданных «химерах» с известным соотношением мужской и женской свободной циркулирующей ДНК анализ маркера длиной 84 пары нуклеотидов давал значения, наиболее близкие к реальным.

Выводы

Выводы. В количественных моделях, протестированных на данный момент, более короткие ПЦР-мишени предпочтительнее для анализа свободной циркулирующей ДНК.

Introduction

Introduction. The analysis of free circulating DNA (cfDNA) holds promise for molecular diagnostics, but its fragmentation and low concentration can complicate PCR analysis.

Objective

Objective. To investigate the effect of target length on the amplification efficiency of Y-chromosome markers from cfDNA.

Material and methods

Material and methods. Fifty cfDNA samples were obtained from 39 patients: patients after liver transplantation (n=19), patients with acute leukemia after allogeneic hematopoietic stem cell transplantation (n=10), and pregnant women (n=10). In addition, we prepared 16 chimeric samples by sequential dilution of male cfDNA into female cfDNA from healthy donors. We determined the proportion of male cfDNA using the Y-chromosome marker S02, which is 211 bp in length as suggested by M. Alizadeh et al. We also modified Alizadeh's primer design to obtain a DNA target with a length of 138 bp. The proportion of male cfDNA was also determined by fragment analysis using the amelogenin Y marker (84 bp) from the COrDIS Plus kit (Gordiz LLC, Russia).

Results

Results. In the three groups of patients, amplification of male cfDNA was more efficient when shorter DNA targets were used (p<0.05). In artificially created ‘chimeras’ with a known ratio of male to female cfDNA, analysis of a marker of 84 bp in length gave values closest to the real ones.

Conclusions

Conclusions. In the quantitative models tested so far, shorter PCR targets are preferred for the analysis of cfDNA.

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