RU2729360C1 - Method of analyzing terminal mutations in brca1, brca2, atm and palb2 genes using multiplex pcr and subsequent hybridisation with an oligonucleotide biological microchip (biochip) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology and represents a method for determining terminal mutations in BRCA1, BRCA2, ATM and PALB2 genes, using multiplex PCR technology and subsequent hybridisation with an oligonucleotide biological microchip (biochip). Invention enables determination of 23 terminal mutations: in gene BRCA2 (NM_000059.3): c.752_753insAG (p.T251fs), c. 1010_1011insTG (p.Asn337fs), c.2808_2811delACAA (p.Ala938Profs), c.5286T>G (p.Tyr1762Ter), c.5586delG (p.Val1862fs), 5722_5723delCT (p.Leu1908Argfs), c.5946delT (p.Ser1982Argfs), C.6070C>T (p.Gln2024Ter), c.6298C>T (p.Gln2100Ter), c.7879A>T (p.Ile2627Phe), c.6997_6998insT (p.Pro2334Thrfs); in gene ATM(NM_000051.3): c.5932G>T (p.Glu1978Ter); in gene PALB2 (NM_024675.3): c.172_175delTTGT (p.Gln60Argfs), 509_510delGA (p.R170Ilefs); in gene BRCA1 (NM_007294.3): c.68_69delAG (p.Glu23Valfs), c.181T>G (p.Cys61Gly), c.5266dup (p.Gln1756Profs*74), c.3700_3704delGTAAA (p.Val1234Glnfs), c.3756_3759delGTCT (p.Ser1253Argfs), c.4035delA (p.Glu1346Lysfs), c.5161C>T (p.Gln1721Ter), c.5251C>T (p.Arg1751Ter), c.1961delA (p.Lys654Serfs). Diagnosis of mutations enables to establish with certain probability of effectiveness and feasibility of treating patients with platinum preparations and PARB-inhibitors.

EFFECT: invention allows performing analysis in a short time and with little material costs.

3 cl, 2 dwg, 2 ex

Description

A method for analyzing terminal mutations in genes BRCA1, BRCA2, ATM and PALB2 using multiplex PCR and subsequent hybridization with an oligonucleotide biological microchip (biochip).

The technical field to which the invention relates

The invention relates to the field of genetics, molecular biology and medicine and provides a method for identification using multiplex PCR and subsequent hybridization with an oligonucleotide biological microchip (biochip) terminal mutations in the genes BRCA1, BRCA2, ATM and PALB2, affecting the sensitivity of pancreatic cancer to therapy with platinum drugs and PARB inhibitors.

State of the art

A large number of methods for determining terminal mutations are known, which use various tools to analyze the unique nucleotide sequence of human DNA. Conventionally, there are six groups among them:

Ферментативные методы:

Enzymatic methods:

analysis of amplified fragment length polymorphism (AFLP);

restriction fragment length polymorphism (RFLP) analysis;

cleavage cleavage (CFLP);

cleavage by resolvase (EMD);

analysis based on ligase reaction (LDR, LCR);

Direct synthesis termination PCR (DT-PCR);

allele-specific PCR (AS-PCR, PCR-SSP);

Real-time PCR using TaqMan probes;

Sanger sequencing;

high throughput sequencing (NGS).

Химические методы:

Chemical methods:

chemical cleavage of heteroduplexes;

chemical ligation.

Методы, основанные на различной электрофоретической подвижности полиморфных участков ДНК:

Methods based on different electrophoretic mobility of polymorphic DNA regions:

analysis of the conformation of single-stranded fragments (SSCP);

heteroduplex analysis (HA);

conformation sensitive gel electrophoresis (CSGE);

separation of amplification products by capillary electrophoresis.

Детекция на твердой фазе:

Solid phase detection:

hybridization on oligonucleotide templates;

fiber-optic DNA hybridization analysis;

elongation of immobilized primers (minisequencing);

pyrosequencing.

Хроматографические методы:

Chromatographic methods:

high performance liquid chromatography (HPLC);

denaturing high performance liquid chromatography (DHPLC).

Физические методы:

Physical methods:

mass spectrometry;

resonant fluorescence quenching (FRET)

luminescence, depending on the local environment;

analysis of high resolution melting curves (HRM).

The most common methods presented are:

1. Sanger sequencing

Hereditary pancreatic cancer: a retrospective single-center study of 5143 Italian families with history of BRCA-related malignancies / A. Toss, M. Venturelli, E. Molinaro [et al.] // Cancers (Basel). - 2019. - Vol. 11 (2). - pii: E193. doi: 10.3390 / cancers11020193.

Screening for common mutations in BRCA1 and BRCA2 genes: interest in genetic testing of Tunisian families with breast and / or ovarian cancer / A. Fourati, M.M. Louchez, J. Fournier, [et al.] // Bulletin du Cancer. - 2014. - Vol. 101 (11). - E. 36-40. doi: 10.1684 / bdc.2014.2049.

Identification of BRCA1 / 2 founder mutations in Southern Chinese breast cancer patients using gene sequencing and high resolution DNA melting analysis / A. Kwong, E.K. Ng, C.L. Wong [et al.] // PLoS One. - 2012. - Vol. 7 (9). - e43994. doi: 10.1371 / journal.pone.0043994.

Germline mutations in the PALB2 gene are population specific and occur with low frequencies in familial breast cancer / H. Hellebrand, C. Sutter, E. Honisch [et al.] // Human Mutation. - 2011. -Vol. 32 (6). - E. 2176-88. doi: 10.1002 / humu.21478.

PALB2 mutations in European familial pancreatic cancer families / E.P. Slater, P. Langer, E. Niemczyk [et al.] // Clinical Genetics. - 2010. - Vol. 78 (5). - P. 490-494. doi: 10.1111 / j.1399-0004.2010.01425.x.

Contribution of inherited mutations in the BRCA2-interacting protein PALB2 to familial breast cancer / S. Casadei, B.M. Norquist, T. Walsh [et al.] // Cancer Research. - 2011. - Vol. 71 (6). - P. 2222-2229. doi: 10.1158 / 0008-5472.CAN-10-3958.

A novel germline PALB2 deletion in Polish breast and ovarian cancer patients / A. Dansonka-Mieszkowska, A. Kluska, J. Moes [et al.] // BMC Medical Genetics. - 2010. - Vol. 11. - 20.doi: 10.1186 / 1471-2350-11-20.

2. Analysis of the conformation of single-stranded DNA fragments (SSCP)

A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC / E. Gross, N. Arnold, J. Goette, [et al] // Human Genetics. - 1999. - Vol. 105 (1-2). - P. 72-78.

A novel germline PALB2 deletion in Polish breast and ovarian cancer patients / A. Dansonka-Mieszkowska, A. Kluska, J. Moes [et al.] // BMC Medical Genetics. - 2010. - Vol. 11. - 20.doi: 10.1186 / 1471-2350-11-20.

, S. Sheikhavandi, M. Telatar [et al.] / Human Mutation. - 1999. - Vol. 14(2). - P. 156-162.New mutations, polymorphisms, and rare variants in the ATM gene detected by a novel SSCP strategy / S.

, S. Sheikhavandi, M. Telatar [et al.] / Human Mutation. - 1999. - Vol. 14 (2). - P. 156-162.

3. Allele-specific PCR (AS-PCR)

Identification of a recurrent BRCA1 mutation in two breast / ovarian cancer predisposition families with distinct phenotypes, by using allele-specific multiplex-PCR / L. Negura, E. Carasevici, A. Negura [et al.] // Revista Romana de Medicina de Laborator. - 2010. - Vol. 18. - P. 53-61.

Germ-line BRCA1 mutations in Jewish and non-Jewish women with early-onset breast cancer / M.G. FitzGerald, D.J. MacDonald, M. Kramer [et al.] // The New England Journal of Medicine. - 1996. - Vol. 334 (3). - P. 143-149.

Simple and rapid detection of BRCA1 and BRCA2 mutations by multiplex mutagenically separated PCR / P.C. Chan, B.Y. Wong, H. Ozcelik, D.E. Cole // Clinical Chemistry. - 1999. - Vol. 45 (8 Pt 1). - P. 1285-1287.

4. Comparative genomic hybridization

Comparative genomic hybridization profiles in human BRCA1 and BRCA2 breast tumors highlight differential sets of genomic aberrations / E.H. van Beers, T. van Welsem, L.F. Wessels [et al.] // Cancer Research. - 2005. - Vol. 65 (3). - P. 822-827.

5. Analysis of restriction fragment length polymorphism (RFLP)

BRCA1 / 2 mutation screening in high-risk breast / ovarian cancer families and sporadic cancer patient surveilling for hidden high-risk families / D. Berzina, M. Nakazawa-Miklasevica, J. Zestkova [et al.] // BMC Medical Genetics. - 2013. - Vol. 14.-61.doi: 10.1186 / 1471-2350-14-61.

Allelic variants of breast cancer susceptibility genes PALB2 and RECQL in the Latvian population / P. Hilz, R. Heinrihsone, L.A. Patzold [et al.] // Hereditary Cancer in Clinical Practice.-2019. - Vol. 17. - 17.doi: 10.1186 / s13053-019-0116-6.

6. Conformation-sensitive gel electrophoresis (CSGE)

Detection of BRCA1 and BRCA2 mutations in breast cancer families by a comprehensive two-stage screening procedure / E. Spitzer, M.R. Abbaszadegan, F. Schmidt [et al.] // International Journal of Cancer. - 2000. - Vol. 85 (4). - P. 474-481.

7. Analysis of high-resolution melting curves

Identification of BRCA1 / 2 founder mutations in Southern Chinese breast cancer patients using gene sequencing and high resolution DNA melting analysis / A. Kwong, E.K. Ng, C.L. Wong [et al.] // PLoS One. - 2012. - Vol. 7 (9). - e43994. doi: 10.1371 / journal.pone.0043994.

PALB2 mutations in German and Russian patients with bilateral breast cancer / N. Bogdanova, A.P. Sokolenko, A.G. Iyevleva [et al.] // Breast Cancer Research and Treatment. - 2011. - Vol. 126 (2). - P. 545-550. doi: 10.1007 / s10549-010-1290-4.

Germline mutations in the PALB2 gene are population specific and occur with low frequencies in familial breast cancer / H. Hellebrand, C. Sutter, E. Honisch [et al.] // Human Mutation. - 2011. - Vol. 32 (6). - E2176-88. doi: 10.1002 / humu.21478.

8. Hybridization on oligonucleotide templates (biological microchips).

Analysis of point mutations in the BRCA1 gene by hybridization with hydrogel microchips / O.E. Fedorova, O. N. Sinitska, L.P. Tikhomirova [et al.] // Molecular Biology. - 2006. - Volume 40. - No. 1. - S. 31-36.

9. Real-time PCR using TaqMan probes

Detection of BRCA1 and BRCA2 mutations in breast cancer families by a comprehensive two-stage screening procedure / E. Spitzer, M.R. Abbaszadegan, F. Schmidt [et al.] // International Journal of Cancer. - 2000. - Vol. 85 (4). - P. 474-481.

Contribution of inherited mutations in the BRCA2-interacting protein PALB2 to familial breast cancer / S. Casadei, B.M. Norquist, T. Walsh [et al.] // Cancer Research. - 2011. - Vol. 71 (6). - P. 2222-2229. doi: 10.1158 / 0008-5472.CAN-10-3958.

10. Denaturing high performance liquid chromatography (DHPLC).

A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC / E. Gross, N. Arnold, J. Goette [et al.] // Human Genetics. - 1999. - Vol. 105 (1-2). - P. 72-78.

11. High-throughput sequencing (next generation sequencing, NGS)

Hereditary pancreatic cancer: a retrospective single-center study of 5143 Italian families with history of BRCA-related malignancies / A. Toss, M. Venturelli, E. Molinaro [et al.] // Cancers (Basel). - 2019. - Vol. 11 (2). - pii: E193. doi: 10.3390 / cancers11020193.

Multiple rare variants in high-risk pancreatic cancer-related genes may increase risk for pancreatic cancer in a subset of patients with and without germline CDKN2A mutations / X.R. Yang, M. Rotunno, Y. Xiao [et al.] // Human Genetics. - 2016. - Vol. 135 (11). - P. 1241-1249.

Breast cancer patients suggestive of Li-Fraumeni syndrome: mutational spectrum, candidate genes, and unexplained heredity / J. Penkert, G. Schmidt, W. Hofmann [et al.]. // Breast Cancer Research. -2018. - Vol. 20 (1). - 87. doi: 10.1186 / s13058-018-1011-1.

Mutation Analysis of BRCA1, BRCA2, PALB2 and BRD7 in a Hospital-Based Series of German Patients with Triple-Negative Breast Cancer / F. Pern, N. Bogdanova, P. Schurmann [et al.] // PLoS One. - 2012. - Vol. 7 (10). - e47993. doi: 10.1371 / journal.pone.0047993.

Deleterious germline mutations in patients with apparently sporadic pancreatic adenocarcinoma / K. Shindo, J. Yu, M. Suenaga [et al.] // Journal of Clinical Oncology. -2017. - Vol.35 (30). - P. 3382-3390. doi: 10.1200 / JCO.2017.72.3502.

Prevalence of germline mutations in cancer genes among pancreatic cancer patients with positive family history / K.G. Chaffee, A.L. Oberg, R.R. McWilliams [et al.] // Genetics in Medicine.-2018. - Vol. 20 (1). - P. 119-127. doi: 10.1038 / gim.2017.85.

Prospective Evaluation of Germline Alterations in Patients With Exocrine Pancreatic Neoplasms / M.A. Lowery, W. Wong, E.J. Jordan [et al.] // Journal of the National Cancer Institute.-2018. - Vol. 110 (10). - P. 1067-1074. doi: 10.1093 / jnci / djy024.

Method 1 is widespread and quite informative. It provides complete information about the DNA sequence at the locus of interest and allows de novo mutations to be identified. However, it has low performance. When analyzing a large number of loci, it requires a separate amplification / purification / sequencing reaction for each locus individually, which affects its laboriousness and cost of analysis.

Method 2 is difficult to execute and interpret the results, gives only indirect information about the nucleotide sequence and cannot be automated.

Method 3 is widespread enough, however, it requires setting up independent parallel PCR reactions corresponding to the number of analyzed single nucleotide substitutions, which reduces its value for large-scale genotyping.

Method 4 can be used for scientific purposes, but it requires highly qualified personnel and expensive equipment, which prevents its introduction into routine laboratory diagnostics.

Methods 5-6 in the classical form also require setting several independent reactions according to the number of analyzed loci. For method 5, after the amplification reaction, restriction is required, and then the electrophoretic separation of the restriction products is carried out. If for each patient it is necessary to study several loci, then the method becomes laborious, and the use of various restriction enzymes negatively affects its cost. It was one of the main methods for detecting terminal mutations and polymorphisms in the early 2000s, but is now inferior in prevalence to more convenient methods of analysis. Method 6 also requires the setting of parallel reactions when analyzing several loci in a patient and gives only an indirect idea of the nucleotide sequence of the analyzed loci. It allows to identify only the presence / absence of a mutation in a sample, but does not provide information about its nucleotide sequence. Methods 5 and 6 have little potential for multiplexing, however, it is limited to 2-4 loci.

Method 7 is widely used for the analysis of terminal mutations, including single nucleotide substitutions, but it has a number of significant limitations. This method requires the setting of parallel reactions according to the number of analyzed loci, each amplified locus should be rather short so that a change in one nucleotide in the sequence makes a significant contribution to the change in the high-resolution melting curve. This method gives an indirect idea of the nucleotide sequence, and to establish it requires additional analysis, for example, Sanger sequencing. When analyzing specific mutations, the picture of the melting curve is influenced by polymorphisms and other mutations localized in the analyzed locus, which can lead to difficulties in interpreting the analysis results.

Hybridization-based Method 8 is used in high-performance biological microarray platforms. GeneChip biological microarray technology (Affymetrix, USA) uses allele-specific probes 25 nucleotides in length, which are synthesized directly on a substrate by photolithography. Target gene loci carrying single nucleotide substitutions (polymorphisms and mutations) are amplified by PCR, cleaved into shorter fragments, labeled with biotin tags, and hybridized on a microchip. Hybridization results are visualized by fluorescent labeling with streptavidin dye. Several million different oligonucleotide probes can be located on each microchip, which makes it possible to determine 10 ⁴ -10 ⁵ single nucleotide substitutions in one analysis [Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays / H. Matsuzaki, S. Dong, H. Loi, [et al] // Nat Methods. - 2004. - Vol. 12). - P. 109-111]. The disadvantage of this method is the complexity of interpretation of the obtained fluorescent images and the high cost of consumables, which limits the application of this method in clinical practice.

An alternative approach using oligonucleotide microarrays is represented by the use of low density microarrays, which can differ in the method of applying oligonucleotides to the support, the type of support, and the procedure for detecting hybridization results. One of the options for methods belonging to this group is a two-stage multiplex PCR followed by hybridization of PCR products on low-density hydrogel microarrays. At the first stage, PCR is carried out with equal concentrations of forward and reverse primers to generate gene fragments carrying the analyzed mutations. In the second stage, the product of the first stage is used as a template for PCR. For the production of single-stranded DNA fragments required for hybridization, the reaction uses asymmetric PCR, performed using unequal concentrations of forward and reverse primers, and the reverse primer, which is added in excess, is labeled with a fluorescent dye. Hybridization of a single-stranded DNA fragment is carried out on hydrogel microchips. Hydrogel microchips are plastic substrates with immobilized hydrogel cells. In each cell, specific oligonucleotide probes are covalently attached, with which the products of the second stage of PCR hybridize. [Analysis of point mutations in the BRCA1 gene by hybridization with hydrogel microchips / O.E. Fedorova, O. N. Sinitska, L.P. Tikhomirova [et al.] // Molecular Biology. - 2006. - Volume 40. - No. 1. - S. 31-36]. The presented method is the closest analogue, inferior in diagnostic characteristics, and a prototype of the method proposed in this invention. The advantages of the above method include: low cost of consumables, relative ease of analysis and interpretation of results. The main disadvantage is a small (in comparison with the proposed method) number of simultaneously identified mutations in a smaller number of genes and a more laborious amplification process, consisting of two consecutive amplification reactions. The method proposed in the present invention provides multiplex analysis of 23 terminal mutations in the BRCA1, BRCA2, ATM and PALB2 genes in one amplification stage, which makes it more convenient and promising for use in clinical laboratory diagnostics.

Method 9 can be used for the analysis of terminal mutations; however, it has limitations on the multiplexity of the analysis: several loci can be identified in one reaction, but their number is very limited, primarily by the number of detection channels in the used real-time PCR device.

Method 10 can be used to analyze terminal mutations; however, it requires expensive equipment and highly qualified personnel.

Method 11 is the most promising for simultaneous genotyping of a large number of genetic loci with high analytical sensitivity. However, due to the high cost of equipment, consumables and the need for highly qualified personnel, its use in routine laboratory diagnostics is limited.

In addition to the above literature, there are currently a number of patents and patent applications describing various methods for detecting terminal mutations in one or more genes from the list (intended for analysis by the proposed method): in the BRCA2 gene (NM_000059.3): c.752_753insAG (p .T251fs), c.1010_1011insTG (p.Asn337fs), c.2808_2811delACAA (p.Ala938Profs), c.5286T> G (p.Tyr1762Ter), c.5586delG (p.Val1862fs), 5722_5723delC190 (p.ArgLefs) .5946delT (p.Ser1982Argfs), C.6070C> T (p.Gln2024Ter), c.6298C> T (p.Gln2100Ter), c.7879A> T (p.Ile2627Phe), c.6997_6998insT (p.Pro2334Thrfs); in the ATM gene (NM_000051.3): c.5932G> T (p.Glu1978Ter); in the PALB2 gene (NM_024675.3): c.172_175delTTGT (p.Gln60Argfs), 509_510delGA (p.R170Ilefs); in the BRCA1 gene (NM_007294.3): c.68_69delAG (p.Glu23Valfs), c.181T> G (p.Cys61Gly), c.5266dup (p.Gln1756Profs * 74), c.3700_3704delGTAAA (p.Val1234Glnfs) .3756_3759delGTCT (p.Ser1253Argfs), c.4035delA (p.Glu1346Lysfs), C.5161C> T (p.Gln1721Ter), c.5251C> T (p.Arg1751Ter), c.1961delA (p.Lys654Serfs).

Currently existing patents generally describe methods for analyzing mutations in one of the BRCA1, BRCA2, ATM, or PALB2 genes, rather than cover them all.

The Russian patent RU 2473700 C2 describes a method for detecting the 5382insC mutation in the BRCA1 gene, also known as c.5266dup (historically, the same mutation can have several alternative names, this is especially typical for the BRCA1 / 2 genes, they are indicated below in table 1) in genomic DNA using allele-specific polymerase chain reaction in real time on pooled DNA, characterized in that DNA pools are formed from individual DNA samples of various concentrations.

Russian patent RU 2445368 C2 describes a method for detecting only one 5382insC mutation in the BRCA1 gene based on the use of allele-specific primers with registration of real-time PCR results, characterized by the use of primers that differ for each allele, a common primer for two alleles and a common fluorescent probe.

Russian patent RU 2445369 C2 describes a method for determining the human genotype by the 185delAG mutation (also known as c.68_69delAG) of the BRCA1 gene, based on the use of allele-specific primers with registration of real-time PCR results, characterized by the use of primers that are different for each allele, a common primer for two alleles and a common fluorescent probe.

The invention described in patent RU 2445372 C1 can be used to determine the human genotype for the 6174delT mutation (also known as c.5946delT) of the BRCA2 gene. The method, like the two above-described patents RU 2445368 C2 and RU 2445369 C2, consists in using allele-specific primers with registration of the results of PCR in real time using fluorescently labeled samples. The reaction mixture contains primers that are different for each allele, and the primer and probe common to the two alleles, as well as a common fluorescent probe.

An obvious significant drawback of the above methods is the ability to detect only one mutation.

The invention described in Russian patent RU 2440415 C1 describes a set of synthetic oligonucleotides for performing genetic screening for BRCA1 gene mutations. Mutations are detected by determining the complete nucleotide sequence of the coding portion of the BRCA1 gene. Amplification of the coding part of the BRCA1 gene is carried out using 28 pairs of synthetic oligonucleotides, followed by sequencing of the obtained amplification products.

US 20030235819 A1 describes methods for identifying mutations in the BRCA1 gene resulting in stop codons. Analytical methods include oligonucleotides that specifically hybridize to codons in the analyzed sequence, which lead to the termination of protein synthesis.

The disadvantages of the above patents include the ability to detect mutations in only one gene.

There are several patents and patent applications describing methods for the simultaneous detection of multiple mutations in different genes:

Russian patent RU 2612894 C1 describes a method for determining the sequences of exons of the BRCA1 and BRCA2 genes using high throughput sequencing. The method includes two rounds of amplification of exons with splicing sites of the BRCA1 and BRCA2 genes, sequencing of the pooled DNA after amplification with MiSeq Illumina, and data analysis. The patent contains a set of primers for 86 monoplex amplification reactions of target fragments of these genes, which can be used in sample preparation for NGS. These primer sets fully cover the coding sequences of the BRCA1 and BRCA2 genes. however, the process is laborious and requires highly qualified personnel.

Invention CN 105779636 A describes a set of primers designed to amplify the coding regions of the BRCA1 and BRCA2 genes for subsequent analysis of the obtained fragments using high-throughput sequencing.

The invention CN 104694663 A describes a set of primers designed to amplify the exon and some intron regions of the BRCA1 and BRCA2 genes for subsequent analysis of the obtained fragments using high-throughput sequencing.

The disadvantages of patents RU 2612894 C1, CN 104694663 A, CN 105779636 A, include the need to use expensive equipment and highly qualified personnel.

US patent 9725759 B2 describes, among others, a method for analyzing mutations in the genes BRCA1 (185delAG, 5382insC) and BRCA2 (6174delT). The method is based on multiplex amplification of target gene regions, then a ligase reaction is carried out with allele-specific primers that carry fluorescent labels (one type of label for primers corresponding to the wild-type sequence, another for primers corresponding to the mutation). The presence of a mutation is assessed by hybridization with oligonucleotides immobilized on a support or by capillary electrophoresis.

Patent WO 2015089333 A1 describes a method for the analysis of mutations, including in the BRCA1 (185delAG, 5382insC) and BRCA2 (6174delT) genes. The method is based on obtaining cyclic polynucleotides, including a sequence of target regions, their subsequent analysis, for example, sequencing and interpretation of the results by comparison with a reference sequence.

The disadvantages of patents US 9725759 B2 and WO 2015089333 A1 can be attributed to the fact that they describe the analysis of only three mutations from the list of mutations, which offers to analyze the present invention.

EP 3447154 A1 describes an invention which describes a method for detecting mutations in three genes, including BRCA1 and BRCA2, using a matrix with DNA IMAGING technology.

WO 2016154584 A1 describes an algorithm for analyzing data obtained using high-throughput sequencing in order to search for mutations.

As seen above, there are currently patents and scientific articles describing the possibility of analyzing mutations in any of the BRCA1, BRCA2, ATM or PALB2 genes, but there are no sources describing a simple and reliable method for the simultaneous analysis of all 23 presented in the present invention clinical significant mutations in these genes. Since all currently available methods for detecting terminal mutations in the BRCA1, BRCA2, ATM, or PALB2 genes have one or another disadvantage, there is a real need to create a simplicity of analysis and low cost method for detecting terminal mutations associated with sensitivity. pancreatic cancer to platinum drugs or PARB inhibitors, with the aim of further introduction into any standard clinical laboratory.

Such a method is provided by the present invention.

Disclosure of invention

The essence of the invention is to provide a method for determining terminal mutations in the genes BRCA1, BRCA2, ATM and PALB2, which affect the sensitivity of pancreatic cancer to therapy with platinum drugs and PARB inhibitors. This method allows for the detection of terminal mutations, the list of which is presented in Table 1.

Pancreatic cancer ranks 12th in terms of frequency of occurrence among all oncological diseases, more than 400,000 new cases are registered annually [Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 32 cancer groups, 1990 to 2015: A Systematic Analysis for the Global Burden of Disease Study Global Burden / C. Fitzmaurice, C. Allen, RM Barber [et al] // JAMA Oncology. - 2017. - Vol. 3. - No. 4. - P. 524-548]. The number of new cases and deaths from pancreatic cancer is growing steadily every year [Cancer Facts & Figures 2013. Atlanta: American Cancer Society // American Cancer Society. - 2013]. Up to 95% of pancreatic neoplasms are ductal adenocarcinomas, which have a low sensitivity to conservative treatment. The median life expectancy of patients with metastatic pancreatic pouch is only 6-11 months.

According to scientific studies, tumors with a deficiency in homologous recombination have increased sensitivity to platinum derivatives or PARP inhibitors [Overall survival and clinical characteristics of pancreatic cancer in BRCA mutation carriers / T. Golan, Z.S. Kanji, R. Epelbaum [et al] // British Journal of Cancer. - 2014. - Vol. 111. - No. 6. - P. 1132-1138], [Randomized, double-blind phase II trial with prospective classification by ATM protein level to evaluate the efficacy and tolerability of olaparib plus paclitaxel in patients with recurrent or metastatic gastric cancer / Y.J. Bang, S.A. Im, K.W. Lee [et al] // Journal of Clinical Oncology. - 2015. - Vol 33. - No. 33. - P. 3858-3865], [FDA Approves Olaparib for Germline BRCA-mutated Metastatic: website. - URL: https://www.esmo.org/Oncology-News (Accessed: 01/21/2020). - [Electronic resource]], [Furuse, J. A PARP inhibitor in pancreatic cancer: Enhancement anti-tumor activity of chemoradiation therapy against pancreatic cancer? / J. Furuse // EBioMedicine. - 2019. - Vol. 40. - P. 9-10], [ATM dysfunction in pancreatic adenocarcinoma and associated therapeutic implications // S.A. Armstrong, C.W. Schultz, A. Azimi-Sadjadi [et al] // Molecular Cancer Therapeutics. - 2019. - Vol. 18. - No. 11. - P. 1899-1908], [Abstract CT234: A Phase II, single arm study of maintenance rucaparib in patients with platinum-sensitive advanced pancreatic cancer and a pathogenic germline or somatic mutation in BRCA1, BRCA2 or PALB2 / K.A.R. Binder, R. Mick, M. O'Hara [et al] // Clinical Trials. American Association for Cancer Research - 2019. - P. CT234-CT234], [Maintenance olaparib for germline BRCA-mutated metastatic pancreatic cancer / T. Golan, P. Hammel, M. Reni [et al] // The New England Journal of Medicine. - 2019. - Vol. 381 / - # 4. - P. 317-327].

Pathogenic mutations in the BRCA1, BRCA2, ATM, and PALB2 genes are one of the main causes of homologous recombination deficiency. In an unselected cohort of pancreatic cancer patients, the proportion of patients with terminal mutations in the BRCA1 / 2 genes is 2.4% - 4.6%, in the ATM and PALB2 gene - 1.4% and 0.7%, respectively [Germline BRCA mutations in a large clinic-based cohort of patients with pancreatic adenocarcinoma / S. Hotter, A. Borgida, A. Dodd [et al] // Journal of Clinical Oncology. - 2015. - Vol. 33. - No. 28. - P. 3124-3129], [Germline cancer susceptibility gene variants, somatic second hits, and survival outcomes in patients with resected pancreatic cancer / M.B. Yurgelun, A.B. Chittenden, V. Morales-Oyarvide [et al] // Genetics in Medicine. - 2019. - Vol. 21. - No. 1. - P. 213-223].

The integration into clinical practice of the analysis for the presence of a deficiency of homologous recombination in patients with pancreatic cancer opens up prospects for the individualization of anticancer therapy, an increase in the effectiveness of treatment and a decrease in mortality from this disease.

The main features of this invention are multiplex PCR and a biological microchip containing a set of immobilized differentiating oligonucleotides.

The first important feature of the invention is the primers for amplification of the target loci of the genes BRCA1, BRCA2, ATM and PALB2, the set of which is used to obtain the studied fluorescently labeled DNA fragments in the required amount. The primer sequences are shown in Sequence Listing 1 (SEQ ID NO: 1-44).

A second important feature of the invention is a biological microchip containing a set of immobilized differentiating oligonucleotides, the sequences of which are shown in Sequence Listing 2 (SEQ ID NO: 45-90). Differentiating oligonucleotides are immobilized in the cells of the hydrogel microchip, as described in the patent [A.D. Mirzabekov, A. Yu. Rubin, S.V. Pankov, A.N. Perov, V.V. Chupeeva. Composition for the immobilization of biological macromolecules in hydrogels, a method for preparing a composition, a biochip, a method for carrying out PCR on a biochip // Patent RU 2206575 C2, 2003] at a concentration of 200-400 μM. The layout of the cells of the biological microchip for the analysis of mutations in the BRCA1, BRCA2, ATM, and PALB2 genes is shown in FIG. 1.

A set of primers is used at the stage of amplification of DNA loci by means of multiplex PCR to prepare a target DNA for hybridization on a biochip. All reverse primers have an adapter that serves as a landing site for a short universal primer (SEQ ID NO: 44) bearing a fluorescent label (Cy-5) at the 5 'end. The conditions of the polymerase chain reaction are designed so that at the first stage of amplification, symmetric amplification of the target genetic loci occurs, and at the second stage, a single-stranded fluorescently labeled PCR product is obtained from a short universal primer. Then, the obtained fluorescently labeled single-stranded DNA fragments are hybridized with oligonucleotides immobilized in the gel cells and located on a plastic substrate. After hybridization and washing of the biological microchip, the obtained fluorescence pattern is analyzed, on the basis of which a conclusion is made about the genotype of the sample under study. An example of a hybridization pattern is shown in FIG. 2.

Sequence Lists

List of sequences 1. Sequences of primers for multiplex PCR followed by analysis by hybridization on a biochip for analysis of terminal mutations in the BRCAJ, BRCA2, ATM and PALB2 genes.

List of sequences 2. Sequences of oligonucleotide probes used for immobilization in the cells of a biological microchip.

A copy of the sequence listing provided on a computer-readable medium is identical to the sequence listing in printed form.

List of figures

Figure 1. Schematic of a biological microchip for the analysis of terminal mutations in the BRCA1, BRCA2, ATM and PALB2 genes. In the angular positions, cells (M) are marked, containing the fluorescent dye Cy 5, permanently glowing, for orientation and control of the glow intensity.

Figure 2. Hybridization pattern obtained using a biological microchip for the analysis of terminal mutations in the BRCA1, BRCA2, ATM and PALB2 genes in subject # XXX. The sample contains the 509_510delGA mutation in the PALB2 gene.

Implementation of the invention

The object of the present invention is to provide a rapid analysis method for detecting terminal mutations in the BRCA1, BRCA2, ATM and PALB2 genes.

To ensure the optimal composition and structure of the primers used in the multiplex PCR protocol, primers are needed that do not form high-energy structures (hairpins and duplexes) among themselves, provide specific amplification and, at the same time, are selected in such a way that their annealing on the target occurs at the same temperature (Sequence Listing 1 (SEQ ID NO: 1-43)). All reverse primers have an adapter that serves as a landing site for a short universal primer (Sequence Listing 1 (SEQ ID NO: 44)) bearing a fluorescent label (Cy-5) at the 5 'end. To ensure a balanced efficient amplification of the samples under study, parameters such as the concentration of MgCl ₂ and primers in the PCR mixture, the ratio of forward and reverse primers, the number of amplification cycles in both stages, and the elongation, denaturation and annealing times of primers at each stage must also be optimized. As a result of this work, primers were selected that allow efficient production of selected fragments of the BRCA1, BRCA2, ATM, and PALB2 genes.

Oligonucleotides for immobilization on a biological microchip are selected in such a way as to identify all terminal mutations selected for analysis in the BRCA1, BRCA2, ATM, and PALB2 genes.

Oligonucleotides must meet the following criteria:

1) The discriminating probe should have high specificity for the selected mutant locus for analysis, which is a region of the gene amplified by PCR with a fluorescent label included.

2) The variable nucleotide should be located in the middle region of the probe, since this position allows for greater discrimination between perfect and imperfect duplexes.

3) The selected oligonucleotides should not contain highly stable secondary structures, the presence of which can lead to a decrease in the efficiency of hybridization.

On the biochip for the determination of terminal mutations in the BRCA1, BRCA2, ATM and PALB2 genes, 46 highly specific differentiating oligonucleotide probes (Sequence Listing 2 (SEQ ID NO: 45-90)) are immobilized, the structure of which ensures binding only to completely complementary DNA targets, which causes bright fluorescent signal in the corresponding cells of the biological microchip and gives the clearest pictures of the distribution of hybridization signals. Nonspecific binding is minimized, which practically excludes false positive "triggering" of cells.

Here is the sequence of analysis using this method. The production of PCR products is carried out in one amplification reaction in one tube. Genomic DNA is used as a template.

PCR can be performed using any type of thermostable polymerase (for example, Hot Start Taq DNA polymerase (SibEnzyme-M LLC, Russia)). To build a new chain, a mixture of dNTPs (dATP, dGTP, dCTP, dTTP) is added to the buffer in the accepted concentrations, while dUTP can be used instead of dTTP. For PCR, ready-made commercially available kits containing all the necessary components, except for primers, can be used.

For multiplex PCR, primers SEQ ID NO: 1-44 are used as listed in Sequence Listing 1.

In the amplification program (temperature-time regime), two stages are conventionally distinguished: in the first half of the PCR program, symmetric amplification of the BRCA1, BRCA2, ATM and PALB2 gene loci takes place using specific primers (SEQ ID NO: 1-43). In the second part of the amplification program, a fluorescently-labeled single-stranded PCR product is obtained: by lowering the annealing temperature of the primers, it becomes possible to anneal and elongate the short universal fluorescently labeled primer (SEQ ID NO: 44) added in excess, which is annealed on the adapter portion of the reverse specific primers ... This allows one to obtain a single-stranded fluorescently labeled amplicon capable of hybridization with specific DNA probes on a biological microchip. Cy5 is used as a fluorescent label: a short universal primer carries a fluorescent label (Cy-5) at the 5'-end. Any fluorochrome without limitation (for example, FITC, Texas red, Su-3, etc.), as well as biotin, can also be used as a fluorescent label.

Primers and oligonucleotide probes are synthesized using various chemical approaches such as phosphodiester method, hydrophosphoryl method, etc., with the phosphoamidite synthesis method currently being the most common. Primer synthesis is carried out using automatic DNA / RNA synthesizers, for example, manufactured by Applied Biosystems (USA).

In the manufacture of a biochip, oligonucleotides can be used that carry an active group at the 5'- or 3'-end, providing immobilization. The modification of an oligonucleotide for the introduction of an active group can be carried out both automatically during synthesis using a wide range of commercially available modifiers, and postsynthetically in a manual mode. For example, when synthesizing oligonucleotide probes using the 3'-Amino-Modifier C7 CPG 500 (Glen Research), USA), a spacer with a free amino group is inserted into the 3'-end of oligonucleotides, which is used for subsequent immobilization of the oligonucleotide on the biochip.

Next, the fluorescently labeled DNA fragments obtained after PCR are hybridized with oligonucleotides immobilized in the gel cells, which are regions of the BRCA1, BRCA2, ATM and PALB2 genes and are complementary to the wild-type sequence or the sequence containing the mutation.

Hybridization can be carried out in any hybridization buffer known to the person skilled in the art, for example guanidine or SSPE buffer. Typical hybridization times are 18-24 hours at 37 ° C. The analysis of the genotype of the sample under study is carried out taking into account the location of the oligonucleotide probes on the biological microchip, the scheme of which makes it possible to determine which terminal mutations are present in a particular sample.

The analyzed DNA fragments form perfect hybridization duplexes only with completely complementary oligonucleotides. If the sequence of the analyzed DNA is completely complementary to the sequence of the probe, then a stable perfect duplex is formed (a fluorescence signal is detected). If the desired fragment is absent or there is a non-complementary base in it, then no stable duplex is formed (there is no fluorescence signal). Discrimination of perfect and imperfect duplexes is carried out after washing the biological microchip by comparing the intensities of the fluorescence signals of the corresponding cells of the biological microchip. Washing can be carried out in any salt-added buffer (SSC, SSPE, etc.) known in the art, or in deionized water, but in a shorter time [Sambrook, J.F. Molecular cloning: a laboratory manual / J.F. Sambrook, D. W. Russell - Cold Spring Harbor Laboratory Press, 2001].

Registration of the hybridization pattern can be performed using any detection system that recognizes a fluorescent signal (fluorescence microscope with a CCD camera, laser scanner, portable biochip analyzer, etc. commercially available fluorescent analyzers, for example, a portable biochip analyzer equipped with a CCD camera and special software, produced by OOO BIOCHIP-IMB (Russia)).

Biological microarrays can be manufactured by sequentially applying a matrix of acrylamide gel cells to the surface of a glass substrate, activating the cells and covalent immobilization in the cells of modified oligonucleotides carrying active groups [Kolchinsky, A. Analysis of SNPs and other genomic variations using gel-based chips / A Kolchinsky, A. Mirzabekov // Human Mutation. - 2002. Vol. 19 (4). - P. 343-360]. In addition to glass, other material can be used as a substrate, including metal, flexible membranes and plastic [S.V. Pankov, E. Ya. Kreindlin, O. G. Somova, O. V. Moiseeva, V.E. Barsky, A.S. Zasedatelev. The use of unmodified polymeric materials for the manufacture of a substrate for biochips, a biochip based on them and a method for its manufacture, a method for immobilizing hydrogels on unmodified polymeric materials // Patent RU 2309959, 2007]. Biochips can also be manufactured by any other methods known to the person skilled in the art [Seliger, H. Arrays of immobilized oligonucleotides-contributions to nucleic acids technology / H. Seliger, M. Hinz, E. Happ // CurrPharm Biotechnol. - 2003. - Vol. 4 (6). - P. 379-395].

For the manufacture of a biological microchip, the present invention uses a set of oligonucleotides (SEQ ID NO: 45-90) shown in Sequence Listing 2. A control DNA sample is used as a control for hybridization in the present invention. The location of specific oligonucleotide probes on a biological microchip can vary and is determined only by the convenience of interpreting the hybridization results.

The following are examples that show the application of the method for analyzing terminal mutations in the BRCA1, BRCA2, ATM and PALB2 genes. It should be understood that the examples given are for illustration only and are not intended to limit the scope of the claims. Based on the present description, a person skilled in the art will be able to easily propose his variations and modifications of the implementation of the invention without departing from the general concept of the present invention and without involving his own inventive activity, so it should be clear that such variations and modifications will also fall within the scope of the present invention. inventions.

Example 1. Amplification of fragments of genes BRCA1, BRCA2, ATM and PALB2 by multiplex PCR in order to obtain a fluorescently labeled PCR product in the required amount.

Genomic DNA was isolated using the DNeasy Blood & Tissue Kits (QIAGEN, Germany) from peripheral blood samples.

Amplification of the BRCA1, BRCA2, ATM and PALB2 gene loci was performed in one multiplex reaction containing the primers SEQ ID NO: 1-44. PCR was performed on a T100 device (Bio-Rad, United States). The PCR mixture of the first stage with a total volume of 25 μL included: 1 × PCR buffer (67 mM Tris-HCl, pH 8.6, 166 mM (NH ₄ ) ₂ SO ₄ , 0.01% Triton X-100), 4.4 mM MgCl ₂ , 0.2 mM of each dNTP (Sileks, Russia), 1.25 U Hot Start Taq DNA polymerase (SibEnzyme-M LLC, Russia), 0.04 μM specific primers (SEQ ID NO: 1-43), 0.4 μM universal fluorescently labeled primer (SEQ ID NO: 44) and 25 ng DNA. Amplification was performed according to the scheme indicated in Table 2.

Example 2. Oligonucleotide biological microchip for detecting terminal mutations in the BRCA1, BRCA2, ATM and PALB2 genes.

Oligonucleotides for immobilization on a microchip are synthesized on an automatic 394 DNA / RNA Synthesizer (Applied Biosystems, USA) using a standard phosphoamidite procedure. The 3'-end of the oligonucleotides contains a spacer with a free amino group, which is introduced into the oligonucleotide during synthesis using the 3'-Amino-Modifier C7 CPG 500 (Glen Research), USA). The attachment of a fluorescent label to oligonucleotides at the free amino group is carried out in accordance with the manufacturer's recommendations.

Biological microchips are made by copolymerizing an oligonucleotide in an acrylamide gel, as described earlier [C.V. Pankov, E. Ya. Kreindlin, O. G. Somova, et al. The use of unmodified polymeric materials for the manufacture of a substrate for biochips, a biochip based on them and a method for its manufacture, a method for immobilizing hydrogels on unmodified polymeric materials // Patent RU 2309959, 2007], [A.D. Mirzabekov, A. Yu. Rubin, S.V. Pankov, et al. A method for immobilizing oligonucleotides containing unsaturated groups in polymer hydrogels during microchip formation // Patent RU 2175972, 2001]. After manufacturing, biological microchips undergo application quality control, which includes visual control of the physical dimensions of the gel cells using a light microscope in reflected light and control of the concentration of immobilized oligonucleotides using the Su-3 dye (OOO BIOCHIP-IMB, Moscow, Russia). The biological microchip contains 46 immobilized oligonucleotide probes (SEQ ID NO: 45-90), a list of which is presented in Sequence Listing 2. The wells are applied according to the scheme in FIG. 1. Example 3. Hybridization of a labeled product on a biochip

The reaction mixture obtained after carrying out the multiplex PCR described in Example 1 is used for hybridization on a biochip. The hybridization mixture with a total volume of 40 μl contained 10 μl of formamide (Serva, USA), 10 μl of 20 × SSPE (Promega, USA), and 20 μl of PCR product. The finished hybridization mixture is thoroughly mixed and applied to a biological microchip in a 40 μl hybridization chamber (BIOCHIP-IMB, Moscow, Russia). Hybridization is carried out for 18-24 hours at 37 ° C. After the end of the incubation, the hybridization chamber is removed together with the unreacted hybridization mixture and washed in 1x SSPE buffer (Promega, USA) at room temperature for 15 min.

Example 4. Registration and interpretation of hybridization results

The registration of the hybridization pattern is carried out using a portable biochip analyzer equipped with a CCD camera manufactured by BIOCHIP-IMB LLC. A description of an automatic image analysis algorithm using Image Ware ™ is outside the scope of the present invention.

Determine the genotype according to the hybridization pattern shown in Fig. 2.

In the cells of the biological microchip corresponding to the c.509_510delGA (p.R170Ilefs) locus of the PALB2 gene, a positive signal is observed in two upper (corresponding to the wild-type sequence) and two lower cells (corresponding to the mutant sequence), respectively, the sample is heterozygous for the c.509_510delGA mutation (p.R170Ilefs) in the PALB2 gene.

Let us determine the genotype for the remaining loci of the BRCA2 gene (NM_000059.3): c.752_753insAG (p.T251fs), p. 1010_1011insTG (p.Asn337fs), c.2808_2811delACAA (p.Ala938Profs), c.5286T> G (p.Tyr1762Ter), c.5586delG (p.Val1862fs), 5722_5723delCT (p.Leu19.59Argfs) ), C.6070C> T (p.Gln2024Ter), c.6298C> T (p.Gln2100Ter), c.7879A> T (p.Ile2627Phe), c.6997_6998insT (p.Pro2334Thrfs); gene ATM (NM_000051.3): c.5932G> T (p.Glu1978Ter); gene PALB2 (NM_024675.3): c.172_175delTTGT (p.Gln60Argfs); gene BRCA1 (NM_007294.3): c.68_69delAG (p.Glu23Valfs), c.181T> G (p.Cys61Gly), c.5266dup (p.Gln1756Profs * 74), c.3700_3704delGTAAA (p.Val1234 .Glnfs), c.5266dup 3756_3759delGTCT (p.Ser1253Argfs), c.4035delA (p.Glu1346Lysfs), c.5161C> T (p.Gln1721Ter), c.5251C> T (p.Arg1751Ter), c.1961delA (p.Lys654Serfs). For each of the loci, an intense fluorescent signal is observed only in a pair of cells corresponding to the wild-type sequences.

Consequently, the genotype of the analyzed sample: heterozygote for the c.509_510delGA (p.R170Ilefs) mutation in the PALB2 gene

Claims (9)

1. A method for detecting terminal mutations in the BRCA1, BRCA2, ATM and PALB2 genes, which includes the following stages: (a) - amplification of BRCA1, BRCA2, ATM, and PALB2 gene fragments by multiplex PCR, in which a genomic DNA sample is used as a template for amplification: PCR uses primers SEQ ID NO: 1-44; the result is a fluorescently labeled PCR product; (b) - providing a biological microchip to identify terminal mutations in the BRCA1, BRCA2, ATM and PALB2 genes, containing a set of immobilized oligonucleotides with the sequences shown in SEQ ID NO: 45-90; (c) - hybridization of the labeled PCR product on a biological microchip; (d) - registration and interpretation of hybridization results; 2. The method according to p. 1, in which the registration of the results of hybridization is carried out using a device capable of registering and recording fluorescence signals, equipped with software that allows automatic processing of signal intensity with subsequent interpretation of the results; 3. The method according to claim 1, in which to identify mutations in the BRCA1, BRCA2, ATM and PALB2 genes amplified using a set of primers described in claim 1, a biological microchip is used, which is a support with a set of oligonucleotides immobilized on it with sequences shown in SEQ ID NO: 45-90.

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