RU2842506C1 - SET OF OLIGONUCLEOTIDE PRIMERS AND ALLELE-SPECIFIC FLUORESCENT PROBES FOR DETECTING POLYMORPHIC MARKER rs1990760 IN HUMAN IFIH1 GENE BY REAL-TIME POLYMERASE CHAIN REACTION - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, particularly to use of a set of oligonucleotide primers and allele-specific fluorescent probes for detecting polymorphic marker rs1990760 in human IFIH1 gene. Said set includes a forward primer with SEQ ID NO: 1; reverse primer with SEQ ID NO: 2; C-allele-specific fluorescent probe with SEQ ID NO: 3 and a T-allele-specific fluorescent probe with SEQ ID NO: 4.

EFFECT: present invention provides the determination of alleles C and T of the polymorphic marker rs1990760 of the IFIH1 gene in a human biological material by real-time polymerase chain reaction.

1 cl, 1 ex

Description

Index(es) of the heading of the current edition of the International Patent Classification adopted by the Strasbourg Agreement Concerning the International Patent Classification, concluded on March 24, 1971 in Strasbourg (Collection of current treaties, agreements and conventions concluded by the USSR with foreign states. Issue XXXI. - M., 1977. Pp. 106-115. The USSR acceded to this document on September 30, 1975. Date of entry into force for the Russian Federation - October 3, 1976) (IPC) C12Q 1/6876.

Field of technology to which the invention relates

The invention relates to the field of biotechnology, molecular genetic diagnostics, in particular to the determination of alleles of the polymorphic marker rs1990760 of the IFIH1 gene in samples of human biological material by the polymerase chain reaction method in real time and can be used in clinical practice and scientific research to determine the predisposition of carriers of a certain allele and genotype of the rs1990760 polymorphism to the development of various diseases and complications, in the pathogenesis of which RIG-like receptors are involved.

State of the art

RIG-like receptors (Retinoic acid-inducible gene I; Retinoic acid-inducible gene I) are among the receptors of innate immunity, which, after recognizing pathogen molecular patterns (PAMPs), trigger a cascade of innate immune reactions, accompanied by activation of phagocytosis and secretion of cytokines. These receptors are most effective in protecting against viral infections, since they recognize various types of viral RNA motifs. RIG-like receptors are also involved in the development of various autoimmune diseases: under certain pathological conditions, they can be activated by a person’s own RNA, leading to pronounced autoinflammatory processes.

Several single nucleotide polymorphisms in the genes of RIG-like receptors have been shown to be associated with the development of pathological conditions of the immune system associated with constitutive activation of endogenous RNA and leading to excessive expression of interferons. The polymorphic marker rs1990760 in the IFIH1 gene can affect the expression level of RIG-like receptors MDA5 and, consequently, type I IFN, thus contributing to the development of pathological conditions. It has been previously shown that the polymorphic marker rs1990760 in the IFIH1 gene is associated with the development of such autoimmune pathologies as type 1 diabetes mellitus [Looney BM, Xia C-Q, Concannon P, et al. Effects of type 1 diabetes-associated IFIH1 polymorphisms on MDA5 function and expression. Curr Diab Rep 2015; 15: 96. doi:10.1007/s11892-015-0656-8], Graves' disease [Sutherland A, Davies J, Owen CJ, et al. Genomic Polymorphism at the Interferon-Induced Helicase (IFIH1) Locus Contributes to Graves’ Disease Susceptibility. The Journal of clinical endocrinology and metabolism 2007; 92: 3338. doi:10.1210/jc.2007-0173], systemic lupus erythematosus [Enevold C, Kjær L, Nielsen CH, et al. Genetic polymorphisms of dsRNA ligating pattern recognition receptors TLR3, MDA5, and RIG-I. Association with systemic lupus erythematosus and clinical phenotypes. Rheumatol Int 2014; 34: 1401-1408. doi:10.1007/s00296-014-3012-4], multiple sclerosis [Wawrusiewicz-Kurylonek N, Gościk J, Chorąży M, et al. The interferon-induced helicase C domain-containing protein 1 gene variant (rs1990760) as an autoimmune-based pathology susceptibility factor. Immunobiology 2020; 225: 151864. doi:10.1016/j.imbio.2019.10.013] and many others. Also, the rs1990760 polymorphism is associated with the development of viral diseases, for example, COVID-19 [Maiti AK. The African-American population with a low allele frequency of SNP rs1990760 (T allele) in IFIH1 predicts less IFN-beta expression and potential vulnerability to COVID-19 infection. Immunogenetics 2020; 72: 387-391. doi:10.1007/s00251-020-01174-6]. This allows its use in genetic panels to assess a patient's predisposition to respiratory viral infections, as well as to type 1 diabetes mellitus and systemic lupus erythematosus.

A set of 10 polymorphic markers, including rs1990760, for diagnosing predisposition to severe viral respiratory diseases is known from the state of the art [Kamaeva Anna Stanislavovna (RU), Grigortsevich Natalia Yuryevna (RU), Minashkin Mikhail Mikhailovich (RU), et al. Method for predicting risks of severe viral respiratory diseases], however, this set does not offer a specific algorithm for detecting the proposed polymorphic markers in the human genome. A method for diagnosing lupus in humans is also known, including rs1990760 [Behrens Timothy W. (US), Graham Robert R. (US). Method for diagnosing lupus in humans], however, in this technique, a complex and limitedly accessible set of research methods was used to detect polymorphic markers. A method for determining the polymorphic marker rs1990760 in the human IFIH1 gene by real-time polymerase chain reaction using oligonucleotide primers and allele-specific fluorescent probes has not yet been developed.

Thus, in view of the high clinical and diagnostic significance of this polymorphic marker for the diagnosis of viral infections and autoimmune pathologies, there is a high need to develop an inexpensive and easy-to-implement method for determining the alleles and genotypes of the rs1990760 polymorphism in the IFIH1 gene.

Disclosure of the essence of the invention

The technical result of the claimed invention is aimed at determining the C and T alleles of the polymorphic marker rs1990760 (missense variant) in position chr2:162267541 (GRCh38.p14) of the IFIH1 gene in human biological material by the real-time polymerase chain reaction (RT-PCR) method using oligonucleotide primers and allele-specific fluorescent probes.

The technical result of the claimed invention is achieved by conducting a polymerase chain reaction in real time using a standard reaction mixture and a set of the following oligonucleotide primers and probes:

SEQ ID NO:1 (forward primer, 1990760_F): 5’-TCACCATTTATTTGATAGTCG-3’;

SEQ ID NO:2 (reverse primer, 1990760_R): 5’-CCATGAACTACACACTGGTA-3’;

SEQ ID NO:3 (C allele-specific fluorescent probe, 1990760_Z1_C):

5’-(FAM)ACACTTCTTTTGCAGTGCTTTG(RTQ-1)-3’;

SEQ ID NO:4 (T allele-specific fluorescent probe, 1990760_Z2_T):

5’-(ROX)CACACTTCTTTTGCAGTGTTTTG(BHQ-2)-3’

The following programs were used to develop the presented nucleotide sequences:

1. Ensembl database (https://www.ensembl.org/index.html) - to determine the target fragment of the genome sequence;

2. Multiple Primer Analyzer by Thermo Fisher Scientific Inc (https://www.thermofisher.com/ru/ru/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html) - to exclude the formation of "hairpins" and dimers in the selected oligonucleotide sequences, as well as to calculate the melting temperature.

3. BLAST database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) - to check the selected system for the probability of formation of “hairpins” and dimers, as well as the presence/absence of non-target regions of the gene with which the selected primer system may come into contact.

The selection of oligonucleotides was carried out so that the region with the detectable polymorphic marker was included in the probe sequence and was located between the direct and reverse primers. The length of the synthesized specific fragment was 134 pairs of oligonucleotides. The production of specific primers and probes was carried out to order in the company SINTOL (RF).

Brief description of drawings (if included in the application)

Implementation of the invention

Marker detection is carried out as follows:

1. Extraction of DNA from human biological material. For DNA extraction, it is recommended to use peripheral venous blood collected in EDTA tubes, but scrapings from the skin and mucous membranes and biopsy material can also be used. Extraction is proposed to be carried out by the sorption method using the commercial RIBO-sorb kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Russian Federation) according to the attached instructions or any other similar certified method.

2. Preparation of DNA samples for the amplification reaction. To assess the quality and quantity of the isolated DNA, it is recommended to use a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) or its direct analogues. The recommended permissible sample purity should be in the range of A260/280 from 1.5 to 2.0.

3. Carrying out RT-PCR using oligonucleotide primers and allele-specific fluorescent probes to detect the polymorphic marker rs1990760 in the human IFIH1 gene. To carry out the amplification reaction, it is proposed to prepare a reaction mixture using reagents from the "Reagent kit for RT-PCR" (Synthol, Russian Federation) and synthesized primers and probes. The reaction mixture should be prepared immediately before the amplification from the following components (volumes are given per 1 sample): deionized water (5 µl), buffer solution (1.5 µl), magnesium chloride - MgCl ₂ (1.5 µl), deoxynucleotide set - dNTP (1.5 µl), forward primer (0.5 µl), reverse primer (0.5 µl), C-allele-specific fluorescent probe (0.5 µl), T-allele-specific fluorescent probe (0.5 µl), Taq polymerase (0.5 µl). Then 12 µl of the prepared reaction mixture and 2 µl of the sample at a concentration of 10-12 ng/µl should be added to 0.2 ml microtubes. Another similar certified kit can also be used to prepare the reaction mixture. It is recommended to perform RT-PCR on the devices of the company DNA-Technology (RF): DT-96, DTprime 4 and DTprime 5; it is also acceptable to use any other similar amplifier with the detection channels FAM and ROX. The amplification program includes preliminary heating at 95°C for 3 minutes and 40 cycles with temperatures: 95°C (20 seconds) and 62°C (40 seconds). Fluorescence is read via the FAM and ROX channels: an increase in the fluorescence level via the FAM channel indicates the presence of the C allele in the sample, and via the ROX channel - the T allele.

The specificity of the forward and reverse primers was confirmed using the Sanger sequencing method, custom-made by Eurogen (RF), and was 100%. The calculated optimal conditions for conducting RT-PCR were optimized during practical experiments. The developed method was tested in the molecular immunology laboratory of the I.I. Mechnikov Research Institute of Vaccines and Sera on 286 biological samples.

Example. Genotyping of C and T alleles of the IFIH1 gene by rs1990760 polymorphism in human venous blood samples. A total of 286 samples were selected to determine the alleles and genotypes. The study was carried out according to the algorithm presented above, using unique synthesized oligonucleotide primers and probes SEQ ID NO: 1-4. Detection of the fluorescent signal was carried out via the FAM channel for the C allele and via the ROX channel for the T allele. For 97 samples, fluorescence was detected only via the ROX fluorophore channel, for 15 samples - only via the FAM fluorophore channel, for the remaining 174 samples - via both FAM and ROX channels, while the kinetics of fluorescent signal accumulation was exponential. Thus, the sample was found to contain 97 individuals with the TT genotype, 15 individuals with the CC genotype, and 174 individuals with the CT genotype for the rs1990760 marker in the IFIH1 gene.

Thus, the presented invention allows for an inexpensive and easy-to-implement method to detect the polymorphic marker rs1990760 in the human IFIH1 gene, which can be used both for prognostic purposes in relation to viral infections and autoimmune pathologies, and for further scientific research in the field of immunogenetics and personalized medicine.

List of nucleotide and/or amino acid sequences (if nucleotide and/or amino acid sequences are used to characterize the invention)

SEQ ID NO: 1: TCACCATTTATTTGATAGTCG;

SEQ ID NO: 2: CCATGAACTACACACTGGTA;

SEQ ID NO: 3: ACACTTCTTTTGCAGTGCTTTG;

SEQ ID NO: 4: CACACTTCTTTTGCAGTGTTTTG.

Information on the results of preclinical studies of medicinal products and clinical studies of medicinal products (provided at the initiative of the applicant on separate sheets for the purpose of removing information on the issuance of a patent when publishing).

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Claims (1)

Use of a set of oligonucleotide primers and allele-specific fluorescent probes for detecting the polymorphic marker rs1990760 in the human IFIH1 gene by real-time polymerase chain reaction, wherein said set, which is used to identify allelic variants of the gene, includes a forward primer 5'-TCACCATTTATTTGATAGTCG-3' (SEQ ID NO: 1); a reverse primer 5'-CCATGAACTACACACTGGTA-3' (SEQ ID NO: 2); C-allele-specific fluorescent probe 5'-(FAM)ACACTTCTTTTGCAGTGCTTTG(RTQ-1)-3' (SEQ ID NO: 3) and T-allele-specific fluorescent probe 5'-(ROX)CACACTTCTTTTGCAGTGTTTTG(BHQ-2)-3' (SEQ ID NO: 4).