RU2723090C1 - Diagnostic technique for predisposition to renal cell carcinoma based on pcr-rflp - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention represents a diagnostic technique for predisposition to renal cell carcinoma based on PCR-RFLP, consisting in isolation of DNA from pre-selected biological material of patients with PCR reaction using primers GCAAGATACACAGTAAAGG (SEQ ID NO: 1) and ACTTGCCTTCATCATGCC (SEQ ID NO: 2), as well as further restriction analysis of the obtained amplicon. A restriction enzyme ApaLI is used for amplicon restriction. Restriction enzyme forms unique restriction fragments in the presence of mutations and is normal (55 bp and 164 bp normal; 219 bp in the presence of a mutation). If observing a mutation in the course of the conducted reaction, a person from the European population has predisposition to renal cell cancer.

EFFECT: invention enables detecting predispositions to renal cell carcinoma in a human from the European population for 4½–6 hours (depending on the selected DNA extraction method) without the need to sequencing DNA samples.

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Description

The present invention relates to medicine, in particular oncology, and can be used to diagnose a predisposition to human renal cell carcinoma from the European human population (EUR code in accordance with the nomenclature of the 1000 Genomes project).

Renal cell carcinoma (RCC) accounts for approximately 4.6% of all new malignancies, with a mean age of 62.1 years [Tsimafeyeu et al. 2017. Five-year Survival of Patients With Metastatic Renal Cell Carcinoma in the Russian Federation: Results From the RENSUR5 Registry. Clin Genitourin Cancer. 15 (6), e1069-e1072.]. In Russia, as of 2017, the incidence of kidney cancer was 3.4% among women and 4.8% among men of the total number of cancer incidence. RCC is the most common form of kidney cancer. RCC occurs in both single and hereditary forms. Studies of environmental factors and lifestyles contributing to the risk of RCC focus almost exclusively on sporadic RCC. The development of RCC is a complex process in which changes are found in more than 20 genes located on different chromosomes [Velickovic M., Delahunt B., Grebe S.K. 1999. Loss of heterozygosity at 3p14.2 in clear cell renal cell carcinoma is an early event and is highly localized to the FHITgene locus. Cancer Res. 59, 1323-1326; Lam JS, Leppert JT, Figlin RA, Belldegrun AS. 2005. Role of molecular markers in the diagnosis and therapy of renal cell carcinoma. Urology. 66, 1-9]. Non-invasive methods are used to diagnose kidney cancer: ultrasound and x-ray computed tomography, magnetic resonance imaging. Less commonly used is a puncture biopsy of a kidney tumor followed by histological examination. Often, kidney cancer is detected in the early stages only due to random detection by ultrasound. About a quarter of patients already have metastases at the time of diagnosis.

Identifying a predisposition to this type of human cancer would allow us to detect the onset of RCC in time and treat this disease in the early stages of the formation of malignant neoplasms.

A known method for predicting the risk of malignant neoplasms of the female genital organs and mammary glands (RU 2578459 C1, publ. 03/27/2016, Bull. No. 9). To check women of reproductive age, signs of hereditary disorders (NNST) of connective tissue in different systems and organs of at least one genetic and at least one anamnestic factors of thrombogenic risk (PTF) and genital tract infection are revealed. In accordance with the identified signs, a low, medium or high risk of malignant neoplasms of the female genital organs and mammary glands is established. An important disadvantage of this method is the need to evaluate several factors at once, including genetic, which is long in time and in terms of labor. The method does not allow to identify a predisposition to renal cell carcinoma.

A known method for predicting a hereditary predisposition to breast cancer based on the identification of a mutation in the BLM gene (RU 2522501 C1, publ. 07.20.2014, Bull. No. 20). The method is characterized in that amplification of short fragments of the BLM gene with a length of up to 200 bp is carried out, followed by high-resolution melting, including the stage of heteroduplex formation optimized for the BLM gene: rapid heating to 95 ° C and slow temperature decrease to 50 ° C; one fragment with an aberrant melting profile is selected for sequencing, the selected fragment is sequenced and a hereditary predisposition to breast cancer is predicted when a BLM gene mutation is detected. The disadvantage of this method is the need for sequencing a DNA fragment, which increases the complexity, time and cost of analysis. The method does not allow to identify a predisposition to renal cell carcinoma.

A known method for the diagnosis of susceptibility to breast cancer (RU 2470998 C2, publ. 12/27/2012, Bull. No. 36). This is a method for detecting an increased risk of a human subject having breast cancer, based on the analysis of a DNA sample for the presence of SNP at positions 142, 355 and 4326 of the CYP1B1 gene; deletion variants of 1100 delC, del5395, as well as IVS2 + 1G> A of the CHEK2 gene and C5972T substitution in the BRCA2 gene. Moreover, the method determines combined genotypes for all the indicated positions of the three named genes, in which the risk of developing breast cancer exceeds the sum of the effects determined by the corresponding variants of each of the three genes separately, which are considered as a sign of a particular susceptibility of the subject to the disease (disproportionately high risk of the disease ) breast cancer. This method of determining a predisposition to cancer by identifying genotypic combinations of specific variants of the genes cyp1b1, brca2 and CHEC2 is taken as a prototype. The disadvantage of this method is the lack of laboratory methods for the detection of these mutations associated with a predisposition to kidney cancer. In addition, the method does not specify for which kidney cancer a predisposition is determined.

In the course of studies at the Voronezh State University, in which the genotypes of patients with renal cell carcinoma were analyzed using high-throughput sequencing, the mutation chr11: 108200993_G / C (region COSM6196808), which is associated with renal cell carcinoma, was analyzed.

Identification of mutations in the described method is carried out on the basis of restriction analysis by PCR-RFLP (polymerase chain reaction - polymorphism of the lengths of restriction fragments).

The technical result is the identification of a predisposition to renal cell carcinoma in humans from the European population within 4.5-6 hours (depending on the chosen method of DNA extraction) without the need for sequencing of DNA samples.

The technical result is achieved by the fact that in the method for diagnosing a predisposition to renal cell carcinoma based on PCR-RFLP, including the extraction of DNA from pre-selected biological material of patients with the NDP reaction, the direct primer GCAAGATACACAGTAAAGG (SEQ ID NO: 1) and the reverse are used according to the invention primer ACTTGCCTTCATCATGCC (SEQ ID NO: 2), restriction analysis of the resulting amplicon is carried out, while for restriction of the amplicon, the restriction enzyme ApaLI is used, which forms unique restriction fragments in the presence of mutations and normally - 55 bp and 164 bp normal; 219 bp in the presence of a mutation, when a mutation is detected during the reaction, a person from the European population is predisposed to renal cell carcinoma in relation to the general European population of people.

A method for detecting a predisposition to RCC based on the amplification of the COSM6196808 region with subsequent restriction analysis of the amplified region is implemented as follows:

1. Is the extraction of DNA from the blood of the patient. For this, both organic extraction methods and commercially available kits for DNA and blood isolation can be used.

2. The reaction of the NDP is carried out with the following primers and reaction conditions: forward primer GCAAGATACACAGTAAAGG (SEQ ID NO: 1), reverse primer ACTTGCCTTCATCATGCC (SEQ ID NO: 2); reaction conditions: 94 ° C 4 min, 35 cycles: 94 ° C 30 sec, 55 ° C 30 sec, 72 ° C 30 sec., final elongation 72 ° C 5 min. During the PNR reaction, a 219 bp PCR product is formed, which is used for the restriction reaction according to the following scheme: 10 μl of the PCR product are taken, 1.5 μl of 10X reaction buffer are added, and 5 units restriction enzymes ApaLI. The volume was adjusted to 15 μl with deionized water. The mixture is incubated for 1.5 hours at 37 ° C, the enzyme is inactivated by heating to 75 ° C for 15 minutes. The restriction products are visualized by electrophoresis on a 3% agarose gel. In the absence of mutation (normal) there will be two fragments of restriction length of 55 bp and 164 bp If there is a mutation, one product with a length of 219 bp will be visible on the electrophoregram.

If a mutation is detected during the reaction, a person from the European population has a predisposition to renal cell carcinoma.

The proposed method for detecting a predisposition to RCC based on the amplification of the COSM6196808 region with subsequent restriction analysis of the amplified region is highly sensitive, well reproducible and economical. Depending on the method of DNA extraction from biological samples, the analysis takes from 4.5 to 6 hours. The analysis can be carried out in laboratories having a thermal cycler, a centrifuge, an electrophoresis chamber and related reagents, and consumables.

Primer sequence listing

Claims (1)

A method for diagnosing a predisposition to renal cell carcinoma based on PCR-RFLP, comprising isolating DNA from previously selected biological material of patients using a PCR reaction, characterized in that the primers are SEQ ID NO: 1 and SEQ ID NO: 2 shown in the List of sequences and also carry out a restriction analysis of the resulting amplicon, while for restriction of the amplicon, the restriction enzyme ApaLI is used, which forms unique restriction fragments in the presence of mutations and normally - 55 bp and 164 bp normal; 219 bp in the presence of a mutation; when a mutation is detected during the reaction, a person from the European population is predisposed to renal cell carcinoma in relation to the general European population of people.