Pediatric Hematology/Oncology and ImmunopathologyPediatric Hematology/Oncology and ImmunopathologyВопросы гематологии/онкологии и иммунопатологии в педиатрии1726-17082414-9314Fund Doctors, Innovations, Science for Children69910.24287/1726-1708-2023-22-3-48-57ORIGINAL ARTICLESОРИГИНАЛЬНЫЕ СТАТЬИMolecular genetic diagnosis in the group of hemophilia A patients in Belarus: 12 new allelic variants in the F8 geneМолекулярно-генетическая диагностика в группе пациентов с гемофилией А в Республике Беларусь: 12 новых аллельных вариантов в гене F8https://orcid.org/0000-0002-6127-7404LiubushkinA. V.ЛюбушкинА. В.
Belarus

Aliaksandr V. Liubushkin - junior researcher of Laboratory of Molecular Genetic Research, Scientific Department, Belarusian Research Center for Pediatric Oncology, Hematology and Immunology, Belarus

43 Frunzenskaya St., Borovlyany 223053, Minsk District

Любушкин Александр Владимирович - младший научный сотрудник лаборатории молекулярно-генетических исследований научного отдела.

223053, Минский район, д. Боровляны, ул. Фрунзенская, 43

aleksandr.liubushkin@gmail.comhttps://orcid.org/0000-0002-9696-3949GuryanovaI. E.ГурьяноваИ. Е.
Belarus

Minsk Region, Borovlyany; Minsk

Минский район, д. Боровляны; Минск

https://orcid.org/0000-0003-0233-7718DmitrievE. V.ДмитриевЕ. В.

Minsk Region, Borovlyany

Минский район, д. Боровляны

https://orcid.org/0000-0003-0274-2107VertelkoV. R.ВертелкоВ. Р.
Belarus

Minsk Region, Borovlyany

Минский район, д. Боровляны

https://orcid.org/0000-0002-0706-6622PolyakovaE. A.ПоляковаЕ. А.
Belarus

Minsk Region, Borovlyany; Minsk

Минский район, д. Боровляны

https://orcid.org/0000-0003-1054-0054VolkovaL. I.ВолковаЛ. И.
Belarus

Minsk

Минск

https://orcid.org/0000-0003-0143-1921AleinikovaO. V.АлейниковаО. В.
Russian Federation

Moscow

Москва

The Belarusian Research Center for Pediatric Oncology, Hematology and Immunology, the Republic of BelarusГУ «Республиканский научно-практический центр детской онкологии, гематологии и иммунологии»Belarusian State Medical University, the Republic of BelarusУО «Белорусский государственный медицинский университет»The Belarusian Medical Academy of Postgraduate Education of Ministry of Health of the Republic of Belarus, the Republic of BelarusГУО «Белорусская медицинская академия последипломного образования» Минздрава РеспубликиThe Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of Ministry of Healthcare of the Russian FederationФГБУ «Национальный медицинский исследовательский центр детской гематологии, онкологии и иммунологии им. Дмитрия Рогачева» Минздрава России0310202322348572802202320062023Copyright ©; 2023, «D. Rogachev NMRCPHOI»Copyright ©; 2023, ФГБУ «НМИЦ ДГОИ им. Дмитрия Рогачева» Минздрава России2023«D. Rogachev NMRCPHOI»ФГБУ «НМИЦ ДГОИ им. Дмитрия Рогачева» Минздрава Россииhttps://creativecommons.org/licenses/by/4.0https://hemoncim.com/jour/article/view/699

Hemophilia A is the most common severe bleeding disorder caused by various genetic changes in the F8 gene, leading to coagulation factor VIII deficiency. Hemophilia A is characterized by high heterogeneity of genetic defects. The severity of hemophilia A varies depending on the type of genetic defects in the F8 gene. More than 3000 unique variants of the F8 gene are associated with the hemophilia A. Approximately 30% of genetic defects occur de novo. The aim of this study is to determine the spectrum of genetic defects in the F8 gene in children with hemophilia A in Belarus. The study was approved by the Independent Ethics Committee and the Scientific Council of the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (the Republic of Belarus). The study included 98 patients with hemophilia A, who had been treated or followed up at the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (the Republic of Belarus). Patients were categorized into 3 groups based on the severity of their disease: severe (n = 82), moderate (n = 3), and mild (n = 13). Twenty (20.4%) patients had a history of inhibitors to factor VIII. For our study, we used venous blood samples. Genomic DNA was isolated from leukocyte suspension (obtained from the whole blood samples) using phenol-chloroform extraction. All severe hemophilia A patients were prescreened for intron 22 and 1 inversions in the F8 gene using inverse and multiplex polymerase chain reaction assays, respectively. Sequencing of F8 coding regions was carried out by next generation sequencing. All clinically relevant variants were confirmed by Sanger sequencing. Genetic testing revealed that 99% of the patients with hemophilia A (n = 97) had pathogenic variants in the F8 gene. Intron 22 and intron 1 inversion mutations within the F8 gene were detected in 45.1% (n = 37) and 1.2% (n = 1) patients with severe hemophilia A, respectively. Two patients had an abnormal pattern of intron 1 inversion, not previously described in the literature. A total of 48 different variants in the F8 gene were detected in 57 patients using next generation sequencing. Eleven of the 48 genetic variants identified have not been previously reported.

Гемофилия А является наиболее распространенным тяжелым нарушением свертываемости крови, обусловленным различными генетическими нарушениями в гене F8, приводящими к дефициту VIII фактора свертывания крови. Гемофилия А характеризуется крайне высокой гетерогенностью в отношении генетических нарушений. Степень тяжести гемофилии А варьирует в зависимости от типа нарушения в гене F8. Идентифицировано и описано более 3000 уникальных вариантов гена F8, ассоциированных с фенотипом гемофилии А. При этом примерно 30% генетических нарушений возникают de novo. Цель настоящего исследования – определить спектр генетических нарушений в гене F8 у детей и подростков с гемофилией А в Республике Беларусь. Настоящее исследование одобрено независимым этическим комитетом и утверждено решением ученого совета ГУ «Республиканский научно-практический центр детской онкологии, гематологии и иммунологии» (Республика Беларусь). В исследование были включены 98 пациентов с клиническим диагнозом гемофилии А, находившихся на обследовании и лечении в ГУ «Республиканский научнопрактический центр детской онкологии, гематологии и иммунологии» (Республика Беларусь). В зависимости от тяжести заболевания распределение пациентов было следующим: 82 пациента – с тяжелой, 3 – со среднетяжелой и 13 – с легкой формой гемофилии А. У 20 (20,4%) пациентов в анамнезе имелись данные о развитии ингибиторов к VIII фактору свертывания крови. Материалом исследования послужила венозная кровь. Геномную ДНК выделяли стандартным методом фенолхлороформной экстракции из полученной суспензии лейкоцитов. Всем пациентам с тяжелой формой гемофилии А выполняли предскрининг на наличие инверсии 22-го и 1-го интронов гена F8 методами инвертированной и мультиплексной полимеразной цепной реакции соответственно. Секвенирование кодирующих областей гена F8 проводили методом высокопроизводительного секвенирования. Все клинически значимые изменения в нуклеотидной последовательности подтверждали с помощью секвенирования по Сэнгеру. В результате проведенного исследования генетические нарушения в гене F8 были обнаружены у 99% (n = 97) пациентов с гемофилией А. Инверсии 22-го и 1-го интронов гена F8 были детектированы у 45,1% (n = 37) и 1,2% (n = 1) пациентов с тяжелой формой гемофилии А соответственно. У 2 пациентов выявлен аномальный паттерн инверсии 1-го интрона, ранее не описанный в литературе. Высокопроизводительное секвенирование выявило 48 различных генетических нарушений в гене F8 у 57 пациентов. Из 48 обнаруженных генетических нарушений 11 ранее не были описаны в литературных источниках.

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