vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RASvavilov-5234Research ArticleМЕДИЦИНСКАЯ ГЕНЕТИКАMEDICAL GENETICSАнализ влияния интронных вариантов c.940+3_940+6del, c.941-3C>G и c.2389+5G>A гена LDLR на сплайсинг пре-мРНК с использованием системы минигеновIn vitro analysis of the effects of intronic variants c.940+3_940+6del, c.941-3C>G, and c.2389+5G>A in the LDLR gene on pre-mRNA splicing using a minigene assayДанильченкоВ. Ю.DanilchenkoV. Yu.

Новосибирск

danilchenko_valeri@mail.ruИванощукД. Е.IvanoshchukD. E.

Новосибирск

Novosibirsk

ТимощенкоО. В.TimoshchenkoO. V.

Новосибирск

Novosibirsk

ПанинаЕ. А.PaninaE. A.

Новосибирск

ОрловП. С.OrlovP. S.

Новосибирск

Novosibirsk

ШахтшнейдерЕ. В.ShakhtshneiderE. V.

Новосибирск

Novosibirsk

Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesRussian FederationНаучно-исследовательский институт терапии и профилактической медицины – филиал Федерального исследовательского центра Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияInstitute of Internal and Preventive Medicine – Branch of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesRussian Federation202626082026305803813Copyright © Данильченко В.Ю., Иванощук Д.Е., Тимощенко О.В., Панина Е.А., Орлов П.С., Шахтшнейдер Е.В., 20262026Данильченко В.Ю., Иванощук Д.Е., Тимощенко О.В., Панина Е.А., Орлов П.С., Шахтшнейдер Е.В.Danilchenko V.Y., Ivanoshchuk D.E., Timoshchenko O.V., Panina E.A., Orlov P.S., Shakhtshneider E.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/5234

Семейная гиперхолестеринемия (СГХС) – аутосомно-кодоминантное заболевание, характеризующееся нарушением выведения липопротеинов низкой плотности из кровотока, выраженным повышением уровня общего холестерина и холестерина липопротеинов низкой плотности в крови и ранним развитием атеросклероза и сердечно-сосудистых заболеваний. СГХС является наиболее распространенным моногенным заболеванием у человека. Самые частые причины развития заболевания – мутации в трех генах: LDLR (OMIM: 143890), APOB (OMIM: 107730) и PCSK9 (OMIM: 607786). Более 80 % случаев СГХС обусловлено патогенными вариантами гена LDLR, локализованного на хромосоме 19. У 25–75 % пациентов с фенотипом СГХС патогенный вариант в указанных генах не обнаруживается. Полноэкзомное и таргетное секвенирование генов LDLR, APOB, PCSK9, LDLRAP1 было проведено у пациентов с фенотипом СГХС с последующим подтверждением выявленных вариантов секвенированием по методу Сэнгера. У трех неродственных пробандов обнаружены интронные варианты гена LDLR, для которых ранее не проводилась функциональная верификация. Целью работы стала функциональная оценка in vitro влияния трех интронных вариантов гена LDLR (NM_000527.5: c.940+3_940+6del, c.941-3C>G, c.2389+5G>A) на процесс сплайсинга с использованием генетических конструкций – минигенов. Для функциональной верификации in vitro были созданы минигены, включающие исследуемые экзоны с фланкирующими интронными областями. Анализ сплайсинга проводили в клеточных линиях HEK293 и HeLa. В результате анализа подтвержден патогенный эффект всех трех выявленных вариантов на сплайсинг пре-мРНК. Делеция четырех нуклеотидов c.940+3_940+6del приводила к образованию двух аберрантных транскриптов – включению шести нуклеотидов интрона 6, а также к полному удержанию интронной последовательности минигена. Вариант c.941-3C>G обусловливал потерю акцепторного сайта сплайсинга и активацию криптического, с включением нуклеотидов интрона 6. Замена c.2389+5G>A  приводила к пропуску экзона 16. Функциональный анализ in vitro – ключевой инструмент молекулярной верификации интронных вариантов с неопределенной клинической значимостью и в совокупности с клиническими данными помогает в установлении окончательного диагноза.

Familial hypercholesterolemia (FH) is an autosomal codominant disorder characterized by impaired clearance of low-density lipoproteins from the bloodstream, markedly elevated plasma total cholesterol and low density lipoprotein cholesterol levels, and early onset of atherosclerosis and cardiovascular disease. FH is one of the most common monogenic disorders in humans. The majority of FH cases are caused by pathogenic variants in three genes: LDLR (OMIM: 143890), APOB (OMIM: 107730), and PCSK9 (OMIM: 607786). More than 80 % of FH cases are associated with mutations in the LDLR gene, located on chromosome 19. In 25–75 % of patients with a clinical FH phenotype, no pathogenic variant is identified in these genes. Whole-exome sequencing and targeted gene panel sequencing of the LDLR, APOB, PCSK9, and LDLRAP1 genes were performed in patients with an FH phenotype, followed by confirmation of the identified variants by Sanger sequencing. Three unrelated probands were found to carry intronic LDLR variants for which functional evidence was previously unavailable. The aim of this study was to functionally assess in vitro the effects of three intronic LDLR variants (NM_000527.5: c.940+3_940+6del, c.941-3C>G, and c.2389+5G>A) on pre-mRNA splicing using a minigene assay. Minigene constructs encompassing target exons with flanking intronic sequences were generated and transfected into the HEK293 and HeLa cell lines. The deleterious effect of all three variants on pre-mRNA splicing was confirmed. The fournucleotide deletion c.940+3_940+6del resulted in two aberrant transcripts: inclusion of six nucleotides from intron 6 and complete retention of the minigene intronic sequence. The c.941-3C>G variant caused loss of the canonical acceptor splice site and activation of a cryptic site, with inclusion of intronic nucleotides from intron 6. The c.2389+5G>A variant resulted in exon 16 skipping. Functional in vitro analysis is a key tool for the molecular verification of intronic variants of uncertain clinical significance and, in conjunction with clinical data, supports the establishment of a definitive molecular diagnosis.

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