RU2667006C1 - Method for determining posttransplantation chimerism in analysis of point mutations of base substitution in genes f2, f5, f7, f13, fgb, itga2, itgb3, pai-1 - Google Patents
Method for determining posttransplantation chimerism in analysis of point mutations of base substitution in genes f2, f5, f7, f13, fgb, itga2, itgb3, pai-1 Download PDFInfo
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- 238000004458 analytical method Methods 0.000 title claims abstract description 11
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Abstract
Description
Изобретение относится к области медицины, в частности к гематологии и может быть использовано для оценки донорского химеризма после трансплантации костного мозга и гемопоэтических стволовых клеток путем определения точечных мутаций замены оснований в генах F2, F5, F7, F13, FGB, ITGA2, ITGB3, PAI-1 методом полимеразной цепной реакции (ПЦР) в режиме реального времени.The invention relates to medicine, in particular to hematology, and can be used to evaluate donor chimerism after transplantation of bone marrow and hematopoietic stem cells by determining point mutations of base substitution in the F2, F5, F7, F13, FGB, ITGA2, ITGB3, PAI- 1 by real-time polymerase chain reaction (PCR).
Цель изобретения - разработка способа определения посттрансплантационного химеризма при анализе полиморфизма генов F2, F5, F7, F13, FGB, ITGA2, ITGB3, PAI-1.The purpose of the invention is the development of a method for determining post-transplant chimerism in the analysis of polymorphism of genes F2, F5, F7, F13, FGB, ITGA2, ITGB3, PAI-1.
Аллогенная трансплантация гемопоэтических стволовых клеток (аллоТГСК) является эффективным методом лечения тяжелых заболеваний системы крови и иммунной системы. Несмотря на достигнутые успехи в области трансплантологии, аллоТГСК сопряжена с развитием острой и хронической реакции «трансплантат против хозяина», возможностью отторжения трансплантата, возникновением рецидивов основного заболевания. Мониторинг приживления гемопоэтических стволовых клеток путем оценки посттрансплантационного химеризма необходим для оценки состояния кроветворной системы и планирования цитостатической и иммунносупрессивной терапии [1]. Генетический химеризм - это одновременное присутствие в организме двух и более маркеров генетически различных клеточных линий. Выделяют полный донорский химеризм, смешанный химеризм и отсутствие химеризма (отторжение трансплантата) [7].Allogeneic hematopoietic stem cell transplantation (alloTGSC) is an effective treatment for severe diseases of the blood system and the immune system. Despite the successes achieved in the field of transplantology, alloTGSC is associated with the development of an acute and chronic transplant versus host reaction, the possibility of transplant rejection, and the occurrence of relapses of the underlying disease. Monitoring the engraftment of hematopoietic stem cells by evaluating post-transplant chimerism is necessary to assess the condition of the hematopoietic system and plan cytostatic and immunosuppressive therapy [1]. Genetic chimerism is the simultaneous presence in the body of two or more markers of genetically different cell lines. Allocate complete donor chimerism, mixed chimerism and the absence of chimerism (transplant rejection) [7].
Известен способ диагностики химеризма цитогенетическим методом. Данный метод не обладает достаточной чувствительностью и используется при различиях донора и реципиента по полу или при наличии у пациента хромосомных особенностей. Недостатком является сложность получения метафазных пластинок хромосом при малой клеточности костного мозга. Метод флюоресцентной гибридизации in situ (FISH) обладает рядом преимуществ перед стандартной цитогенетикой, т.к. не предусматривает культивирования клеток, но, также как и метод количественного определения Y-хромосомы [5], данный способ может быть использован только при различиях донора и реципиента по полу [4].A known method for the diagnosis of chimerism by the cytogenetic method. This method does not have sufficient sensitivity and is used for differences in the donor and recipient by gender or in the presence of chromosomal features in the patient. The disadvantage is the difficulty of obtaining metaphase chromosome plates with low bone marrow cellularity. In situ fluorescence hybridization (FISH) has several advantages over standard cytogenetics, as it does not provide for the cultivation of cells, but, like the method for quantitative determination of the Y chromosome [5], this method can only be used for differences in the donor and recipient sex [4].
Известен способ мониторинга химеризма по результатам исследования антигенов эритроцитов. Необходимым условием для проведения данного метода являются различия между донором и реципиентом по каким-либо антигенам систем АВО, Резус, Келл, MNS Даффи, Левис, Кидд. Преимуществами способа являются простота и высокая скорость выполнения. Недостатками данного способа являются относительно низкая чувствительность (1-5%) и возможность использования только через 4 недели и позже после аллоТГСК [3].A known method of monitoring chimerism according to the results of a study of red blood cell antigens. A necessary condition for carrying out this method is the difference between the donor and the recipient for any antigens of the ABO, Rhesus, Kell, MNS Duffy, Levis, Kidd systems. The advantages of the method are simplicity and high speed of execution. The disadvantages of this method are the relatively low sensitivity (1-5%) and the possibility of use only after 4 weeks and later after alloTGSK [3].
Известен способ выявления сателлитных последовательностей, характеризующихся высокой степенью полиморфизма. Исследование заключается в определении минисателлитов (10-70 пар оснований) (variable number tandem repeats, VNTR) или микросателлитов (2-5 пар оснований) (short tandem repeats, STR) [6]. Методы исследования, основанные на анализе STR, отличаются высокой чувствительностью (от 1 до 0,001%), быстротой и простотой в исполнении, возможностью количественной оценки химеризма.A known method for detecting satellite sequences characterized by a high degree of polymorphism. The study consists in determining minisatellites (10-70 base pairs) (variable number tandem repeats, VNTR) or microsatellites (2-5 base pairs) (short tandem repeats, STR) [6]. Research methods based on the STR analysis are characterized by high sensitivity (from 1 to 0.001%), speed and simplicity in execution, and the ability to quantify chimerism.
Наиболее близким к предполагаемому изобретению является исследование полиморфизма InDel, типа генетической вариации, при котором специфическая нуклеотидная последовательность присутствует (инсерция) или отсутствует (делеция) [8]. Главным преимуществом InDel ПЦР по сравнению с мультиплексной STR ПЦР является отсутствие конкуренции и влияния эффекта «плато» на результат реакции ПЦР [1], что делает данный способ наиболее чувствительным из всех перечисленных.Closest to the proposed invention is the study of InDel polymorphism, a type of genetic variation in which a specific nucleotide sequence is present (insertion) or absent (deletion) [8]. The main advantage of InDel PCR compared to multiplex STR PCR is the absence of competition and the influence of the “plateau” effect on the result of the PCR reaction [1], which makes this method the most sensitive of all listed.
Техническим результатом заявляемого изобретения является создание способа оценки гемопоэтического химеризма на основании анализа информативных аллелей в генах F2, F5, F7, F13, FGB, ITGA2, ITGB3, PAI-1. Технология анализа однонуклеотидных полиморфизмов (single nucleotide polymorphisms - SNPs) позволяет выявить замену в нуклеотидной последовательности ДНК и три варианта генотипа: гомозиготность с исходной последовательностью нуклеотидов, гетерозиготность и гомозиготность с заменой в последовательности нуклеотидов. Предлагаемый способ позволяет определить маркерные локусы, необходимые для оценки состояния больного после трансплантации ГСК.The technical result of the claimed invention is the creation of a method for evaluating hematopoietic chimerism based on the analysis of informative alleles in the F2, F5, F7, F13, FGB, ITGA2, ITGB3, PAI-1 genes. The technology of analysis of single nucleotide polymorphisms (single nucleotide polymorphisms - SNPs) makes it possible to identify a replacement in the nucleotide sequence of DNA and three variants of the genotype: homozygosity with the original nucleotide sequence, heterozygosity and homozygosity with replacement in the nucleotide sequence. The proposed method allows to determine marker loci necessary for assessing the patient's condition after HSC transplantation.
Для достижения указанного результата в соответствии с аналогом (Alizadeh М., 2002) проводят типирование генов донора и реципиента и выявляют информативные аллели, позволяющие дифференцировать ДНК донора и реципиента. В отличие от прототипа, в котором проводится ГТЦР коротких тандемных повторов (STR-PCR) биаллельных генетических систем S01-S11, в предлагаемом способе осуществляется анализ однонуклеотидных полиморфизмов генов F2, F5, F7, F13, FGB, ITGA2, ITGB3, PAI-1. Анализ химеризма проводится методом ПЦР в режиме реального времени.To achieve this result, in accordance with the analogue (Alizadeh M., 2002), the donor and recipient genes are typed and informative alleles are identified that allow differentiating the donor and recipient DNA. In contrast to the prototype, in which GTCR of short tandem repeats (STR-PCR) of biallelic genetic systems S01-S11 is performed, the proposed method analyzes the single nucleotide polymorphisms of the F2, F5, F7, F13, FGB, ITGA2, ITGB3, PAI-1 genes. Chimerism analysis is carried out by real-time PCR method.
В процессе проведения патентно-информационного поиска не выявлено источников, порочащих новизну предполагаемого изобретения.In the process of conducting a patent information search, no sources were found discrediting the novelty of the alleged invention.
Заявляемое изобретение разработано в лаборатории иммуногематологии ФГБУН КНИИГиПК ФМБА России в соответствии с планом научно-исследовательской работы.The claimed invention was developed in the laboratory of immunohematology FGBUN KNIIIGiPK FMBA of Russia in accordance with the plan of research work.
Способ осуществляется следующим образом: Этап 1. Исследуют полиморфизм генов F2, F5, F7, F13, ITGA, ITGB, FGB, PAI-1 у донора и реципиента. С этой целью проводят выделение ДНК из периферической крови донора и реципиента согласно инструкции, прилагающейся к наборам реагентов. Маркируют пробирки для каждого исследуемого образца и отрицательного контроля в расчете на восемь полиморфизмов. В пробирки вносят соответствующие смеси для амплификации, ПЦР-буфер, Taq-полимеразу, исследуемый образец ДНК (100 нг) и отрицательный контрольный образец. Пробирки устанавливают в блок детектирующего амплификатора. ПЦР проводят при следующих параметрах: 2 мин при 80°С, 5 мин при 94°С с последующей амплификацией продуктов в течение 5 циклов (94°С - 30 сек, 67°С - 10 сек), 45 циклов (94°С - 5 сек, 67°С - 5 сек),1 цикл (25°С - 30 сек), 50 циклов (25°С - 15 сек). Смесь для амплификации содержит флуоресцентные метки Fam и Hex для соответствующего варианта полиморфизма. В конце амплификации осуществляется температурное плавление дуплексов (ампликон + сигнальный зонд), изменяющее уровень флуоресценции. Анализ кривых плавления позволяет определить специфичность фрагментов ДНК. Оба варианта искомой последовательности определяются в одной пробирке при одновременной гибридизации с двумя альтернативными типирующими зондами с различными флуорофорами. Описание SNP-генов, используемых в исследовании, представлено в таблице 1.The method is as follows: Step 1. Investigate the polymorphism of genes F2, F5, F7, F13, ITGA, ITGB, FGB, PAI-1 in the donor and recipient. To this end, DNA is extracted from the peripheral blood of the donor and recipient according to the instructions attached to the reagent kits. Label tubes for each test sample and negative control per eight polymorphisms. Appropriate amplification mixtures, PCR buffer, Taq polymerase, a test DNA sample (100 ng) and a negative control sample were added to the tubes. The tubes are installed in a detecting amplifier unit. PCR is carried out at the following parameters: 2 min at 80 ° C, 5 min at 94 ° C followed by amplification of the products for 5 cycles (94 ° C - 30 sec, 67 ° C - 10 sec), 45 cycles (94 ° C - 5 sec, 67 ° С - 5 sec), 1 cycle (25 ° С - 30 sec), 50 cycles (25 ° С - 15 sec). The amplification mixture contains Fam and Hex fluorescent labels for the corresponding polymorphism variant. At the end of amplification, the temperature melting of duplexes (amplicon + signal probe) is carried out, which changes the level of fluorescence. Analysis of the melting curves allows us to determine the specificity of DNA fragments. Both variants of the desired sequence are determined in one tube while hybridizing with two alternative typing probes with different fluorophores. Description of SNP genes used in the study are presented in table 1.
Этап 2. Определение информативных генов, позволяющих выявить аллельные различия донора и реципиента. Аллель считается информативным, если он позитивен для реципиента и негативен для донора или наоборот. Для определения процента донорского химеризма строят калибровочную кривую для выбранных информативных аллелей в серии разведений ДНК реципиента в ДНК донора (100, 10-1, 10-2, 10-4, 10-5), содержащих следующее отношения ДНК:Stage 2. Determination of informative genes, allowing to identify allelic differences between the donor and the recipient. An allele is considered informative if it is positive for the recipient and negative for the donor or vice versa. To determine the percentage of donor chimerism, a calibration curve is constructed for the selected informative alleles in a series of dilutions of the recipient’s DNA in the donor’s DNA (10 0 , 10 -1 , 10 -2 , 10 -4 , 10 -5 ) containing the following DNA ratios:
- 100% ДНК донора;- 100% donor DNA;
- 10% ДНК реципиента и 90% ДНК донора;- 10% of the recipient’s DNA and 90% of the donor’s DNA;
- 1% ДНК реципиента и 99% ДНК донора;- 1% recipient DNA and 99% donor DNA;
- 0,1% ДНК реципиента и 99,9% ДНК донора;- 0.1% of the recipient’s DNA and 99.9% of the donor’s DNA;
- 0,01% ДНК реципиента и 99,99% ДНК донора.- 0.01% of the recipient’s DNA and 99.99% of the donor’s DNA.
Этап 3. Исследование гемопоэтического химеризма проводят на 28, 42, 56, 70, 100, 120 дни после трансплантации, при этом анализируют информативные аллели. Протокол амплификации должен соответствовать описанному в «Этап 1». Оценку количественных значений химеризма осуществляют с использованием формулы:Stage 3. The study of hematopoietic chimerism is carried out on 28, 42, 56, 70, 100, 120 days after transplantation, while informative alleles are analyzed. The amplification protocol should be as described in “Step 1”. Quantification of chimerism is carried out using the formula:
Химеризм, %=2-DDCt × 100%,Chimerism,% = 2 -DDCt × 100%,
где DDCt - разница между уровнями пороговой флуоресценции исследуемого маркера и референсного гена в исследуемом образце разведения и в образце ДНК донора.where DDCt is the difference between the threshold fluorescence levels of the studied marker and the reference gene in the studied dilution sample and in the donor DNA sample.
Представленные ниже клинические примеры подтверждают возможность использования заявляемого способа при оценке гемопоэтического химеризма после аллоТГСК.The following clinical examples confirm the possibility of using the proposed method in assessing hematopoietic chimerism after alloTGSC.
Пример 1.Example 1
Пациент С, 44 года, с диагнозом: Миелодиспластический синдром: рефрактерная анемия с избытком бластов (RAEB-1) IPSS-1.5. АллоТКМ после немиелоаблативного режима кондиционирования выполнена 21.01.2016 от HLA-идентичного родственного донора (сестра, возраст 47 лет). Результаты типирования генов тромбофилии методом ПЦР в режиме реального времени представлены в таблице 2. Перед трансплантацией были выявлены информативные маркеры - гены F7 и F13, в которых у реципиента имелись аллельные специфичности, отсутствующие у донора.Patient C, 44 years old, with a diagnosis of Myelodysplastic syndrome: refractory anemia with excess blasts (RAEB-1) IPSS-1.5. AlloTKM after a non-myelo-ablative conditioning regimen was performed on January 21, 2016 from an HLA-identical related donor (sister, age 47). The results of typing of thrombophilia genes by real-time PCR are presented in Table 2. Before transplantation, informative markers were identified - genes F7 and F13, in which the recipient had allelic specificities that were missing from the donor.
Выстроена калибровочная кривая в серии разведений ДНК донора в ДНК реципиента (100, 10-1, 10-2, 10-4, 10-5). Мониторинг химеризма проведен через 28, 42, 60, 70, 90, 100, 120, 300, 330 дней после аллоТГСК. Донорский химеризм >95% зарегистрирован в 28 и 42 дни. Донорский химеризм >99% определен с 60 дня и далее. Клинической картины отторжения или недостаточности трансплантата не наблюдалось. Больной в настоящее время не получает иммуносупрессивную терапию, ведет полноценную жизнь хорошего качества.A calibration curve is constructed in a series of dilutions of donor DNA in recipient DNA (10 0 , 10 -1 , 10 -2 , 10 -4 , 10 -5 ). Chimerism was monitored after 28, 42, 60, 70, 90, 100, 120, 300, 330 days after alloTGSK. Donor chimerism> 95% registered on days 28 and 42. Donor chimerism> 99% determined from day 60 onwards. The clinical picture of rejection or transplant failure was not observed. The patient currently does not receive immunosuppressive therapy, leads a full life of good quality.
Пример 2.Example 2
Пациент С., 24 года с диагнозом Острый миелобластный лейкоз высокий риск. АллоТГСК с немиелоаблативным режимом кондиционирования выполнена 05.09.2016 для консолидации ремиссии 1. Донором явилась HLA-идентичная сестра больного (27 лет). С целью выявления маркеров посттрансплантационного химеризма проведено исследование генов тромбофилии. Результаты типирования представлены в таблице 3. Информативным маркером явился ген F13, в котором у донора был определен аллель G, отсутствующий у реципиента. Построена калибровочная кривая в серии разведений ДНК донора и реципиента. Исследование гемопоэтического химеризма выполнено на 28, 42, 60 дни после аллоТГСК. Донорский химеризм >95% регистрировался в 28 и 45 дни. При исследовании на 60 день донорский химеризм не превышал 5%. Наблюдались признаки отторжения трансплантата. Больной умер на +65 сутки после трансплантации от тяжелого сепсиса, септического шока.Patient S., 24 years old, with a diagnosis of Acute myeloid leukemia high risk. AlloTGSK with non-myelo-ablative conditioning was performed on September 5, 2016 to consolidate remission 1. The donor was the HLA-identical patient's sister (27 years old). In order to identify markers of post-transplant chimerism, a study of thrombophilia genes was carried out. The typing results are presented in table 3. An informative marker was the F13 gene, in which the G allele was absent in the donor, which was absent in the recipient. A calibration curve was constructed in a series of dilutions of the donor and recipient DNA. The study of hematopoietic chimerism was performed on 28, 42, 60 days after alloTGSC. Donor chimerism> 95% was recorded on days 28 and 45. In the study on day 60, donor chimerism did not exceed 5%. There were signs of transplant rejection. The patient died at +65 days after transplantation from severe sepsis, septic shock.
Список литературы:Bibliography:
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