The invention relates to cancer diagnostics and in particular, the detection of an increased risk of kidney cancer in humans. More specifically, the present invention relates to methods for the identification of persons with an increased risk of renal cell carcinoma based on single nucleotide polymorphisms occurring in the genes Grainyhead-like 1 (GRHL1 ), Grainyhead-like 2 (GRHL2) and Grainyhead-like 3 (GRHL3). The present invention may be used in prognostic genetic studies designed to identify patients at an increased risk for acquiring renal cancer, and in medical diagnostics.

Cancer is a disease causing the death more than 8 million people annually (data from the World Health Organization). According to the American Cancer Society, cancer of the kidney is one of the 10 most common types of cancer. The risk of developing kidney cancer is estimated to be 1/63, meaning that 1 .6% of people (16 per 1000) in their lifetime will develop kidney cancer. The most common variant of kidney cancer is a clear cell carcinoma (ccRCC). It represents about 70% of kidney cancer cases.

Chemotherapy and radiotherapy currently used in the treatment of renal cell carcinoma are inadequate and approved compounds with an immunotherapeutic effect often have serious side effects. If, after surgical removal of the tumor, it is followed by a relapse, patient outcomes are not favorable. In the case of metatastases of the cancer to lymph nodes, the chance of 5-year survival rate ranges from 5 to 15%. If the metastases are to other organs, survival is less than 5%. A complete remission can only be achieved in 12-20% of cases (source: News- Medical. net). There is therefore a strong need for new methods of combating this type of cancer. So far, we have identified a number of potential molecular markers of RCC, but none have so far been validated and implemented into clinical practice (Audenet et al, 2012).

It should be emphasized that kidney cancer at an early stage of development may not cause any symptoms, and therefore the early diagnosis of kidney cancer is very difficult. In its later stages, the symptoms are similar to the symptoms of other diseases such as kidney stones or urinary tract infections. These symptoms include: • fatigue

• loss of appetite

• weight loss

• increased body temperature not not caused by an infection

• anemia

• presence of blood in the urine (hematuria)

• lower back pain not caused by an injury

• a palpable tumor in the lower back or side

Awareness of the increased risk of developing kidney cancer will be very helpful in the early diagnosis of the disease and can help to save the lives of many patients. Identification of single nucleotide polymorphisms (SNP) in the regulatory sequences of the genes GRHL1 and / or GRHL2 and / or GRHL3 may be useful for determining increased risk of cancer of the kidney.

Risk factors for kidney cancer include: smoking obesity

• Occupational exposure on contact with certain substances (cadmium, some herbicides and organic solvents such as trichloroethene)

• genetic factors

The occurrence of atypical variants of genes responsible for the proper functioning of the kidney may lead to an increased risk of developing renal cancer. An example of this is the VHL gene. Mutations in this gene cause von Hippel-Lindau disease , also called cerebello-retinal angiomatosis. The VHL gene is a tumor suppressor that controls the rate of cell proliferation. Mutations in this gene can be inherited from parents. A mutant VHL gene is no longer able to suppress the abnormal growth of cells, which increases the risk of kidney cancer. Mutations in other genes are also associated with the risk of developing kidney cancer, such as MET, FH, FLCN, SDHB and SDHC (Clague et al, 2009). These mutations have a high penetration, meaning that a high percentage of persons with these mutations get cancer. However, such mutations are rare proabably a majority of kidney cancer cases are not related to them. Other mutations of lower penetration may occur with greater frequency in the human population, and they may be responsible for most cases of kidney cancer (Hemminki & Li, 2004). This category may include mutations in the GRHL1 and / or GRHL2 and / or GRHL3.

Transcription factors of the GRHL (Grainyhead-like) family are involved in many biological processes, such as:

• the development and functioning of the epidermis;

• the development and functioning of various epithelia;

• the development of the central nervous system;

• the development of the ectoderm;

• positive regulation of transcription by RNA polymerase type II.

Changes in expression of genes from the GRHL family are associated with the development of a variety of human cancers such as breast cancer (Werner et al, 2013), skin (Darido et al, 201 1 ), liver (Tanaka et al, 2008), stomach (Xiang et al, 2013), colon (Quan et al, 2014) and in fetal neuroma (Fabian et al, 2014). In most cases, the tumour growth is accompanied by a decrease in the expression of GRHL genes. Studies using tissue cultures have shown that in many tumour cell lines, GRHL gene expression levels are lower than in non-cancer cell lines obtained from corresponding tissues. Muted GRHL gene expression in non-tumour cell lines often results in acquiring the characteristics of tumour cells, and increased expression of GRHL genes in tumour cell lines results in the loss of the characteristics of tumour cells. Research has shown that the role of the GRHL1 gene in fetal neuroma (Fabian et al, 2014), GRHL2 gene in gastric cancer (Xiang et al, 2013) and GRHL2 gene in colorectal cancer (Quan et al, 2014). Moreover, by using animal models, it has demonstrated that genes of the GRHL family provide protection against the development of cancer. In mouse models of gene expression, silencing GrhM and Grhl3 increases the risk of skin cancer (Darido et al, 201 1 ; Mlacki et al, 2014). It is therefore a reasonable hypothesis that people with low levels of gene expression of the GRHL family may have an increased risk of developing cancer.

In the case of renal cell carcinoma, the following examples show the relationship of the genes GRHL1 , GRHL2 and GRHL3 with the formation and development of this type of cancer in humans.

The publication by Peyrard-Janvid et al., "Mutations in GRHL3 Dominant Cause of Van der Woude Syndrome and Oral Periderm Disrupt Development of" The American Journal of Human Genetics 2014; 94 (1 ): 23-32, relates to mutations in the gene GRHL3 in patients with Van der Woude (cleft lip and palate), and refers to renal cancer.

The publication by Petros et al., "Mutations in GRHL2 Result in an autosomal- recessive Ectodermal Dysplasia Syndrome" AJGH 2014; 95 (3): 308-314 refers to a mutation in GRHL2 in patients with ectodermal dysplasia and refers to renal cancer.

Publication of Lin et al., "The grainyhead-like 2 gene (GRHL2) single nucleotide polymorphism is not associated with age-related hearing impairment in the Han Chinese," The Laryngoscope 201 1 ; 121 (6): 1303-1307 refers to an SNP in the gene GRHL2 in terms of hearing loss and refers to renal cancer.

The publication by Van Laer et al., "The grainyhead like 2 gene (GRHL2), aka TFCP2L3, is associated with age-related hearing impairment" Human Molecular Genetics 2008; 17 (2): 159-169, relates to an SNP in the GRHL2 gene, but only in the context of deafness and does not apply to cancer.

The publication by Peters et al., "Mutation of a transcription factor, TFCP2L3, Causes autosomal dominant progressive hearing loss, DFNA28" Human Molecular Genetics 2002; 1 1 (23): 2877-2885 refers to SNP in a gene GRHL2, but only with respect to deafness and does not apply to cancer.

The publication by Kamiyama et al., "Polymorphisms in the 3'UTR in the neurocalcin δ: affect gene imRNA stability, and Confer Susceptibility is diabetic nephropathy" Human Genetics 2007; 122 (3-4): 397-407 refers to an SNP, but only in the context of diabetes and does not apply to cancer.

The publication by Bhandari et al., "The Grainyhead transcription factor Grhl3 / Get1 suppresses imiR-21 expression and tumourigenesis in skin: modulation of the miR-21 target MSH2 by RNA-binding protein DND1 " Oncogene 32: 1497-1507 relates to a functional role of GRHL3 in cancer of the skin, but without reference to SNPs. The publication by Darido et al., "Targeting Tumour Suppressor of the GRHL3 to a imiR-21 - Dependent Proto-Oncogenic Network Results in Loss of PTEN and Tumourigenesis" Cancer Cell 201 1 ; 20 (5): 635-648 refers to the functional role of GRHL3 in skin cancer, but without reference to SNPs.

The publication by Panis et al. "Putative circulating markers of the early and advanced stages of breast cancer IDENTIFIED BY high-resolution label-free proteomics" Cancer Letters 2013 330 (1 ): 57-66 mentions GRHL3 in tumours, but applies only to breast cancer, and she does not refer to SNPs.

International Patent Application WO20130291 16A1 relates, inter alia, a functional role of GRHL3 in skin cancer but does not refer to an SNP or renal cancer.

The present invention provides a method of identifying persons with increased risk for renal cell carcinoma based on single nucleotide polymorphisms that occur in the genes GRHL1 , GRHL2 and GRHL3. Methods for the detection of an increased risk of cancer of the kidney are characterized in that they comprise:

A) The identification of at least one single nucleotide polymorphism (SNP) in the area of regulatory region of the GRHL1 and / or GRHL2 and / or GRHL3 genes, present homozygously or heterozygously a position mentioned in Table 1 or Table 2 or Table 3, in a biological sample taken from a patient.

Table 1 Listing of SNPs in the promotor and regulatory sequences of GRHL genes in patients with renal cell carcinoma

8 GRHL2 102506074 rs688324 25 46 0,92 0,793 - 0,00188

8 GRHL2 102508080 17 17 0,001 0,293 + 1 ,90x10 ^"3 '

8 GRHL2 10251 1670 rs554509 22 41 0,92 0,707 - 1 ,81 x10 ^"

8 GRHL2 102512007 5 5 0,001 0,0862 + 4,38x10 ^"9

8 GRHL2 102648952 6 6 0,001 0,103 + 3,87x10 "

8 GRHL2 102649525 rs7017592 1 2 0,24 0,0345 - 3,84x10 ^"b

Polymorphisms labeled "+" are present in the patient population in excess of the European population and "-" less frequently. The numbering of the items in the column "position" is consistent with the GRCh37/hg19 annotation.

B) The determination of the genotype which leads to an increased risk of cancer of the kidney, in the identification of at least one of the single nucleotide polymorphisms listed in Table 1 or Table 2 or Table 3.

In one embodiment, the cancer is renal clear cell carcinoma.

In the second embodiment, the cancer is a renal cell cancer of another type.

In the method specified in the invention, the presence of the variant genotypic is detected based on the analysis of DNA, RNA or proteins.

Preferably, the DNA, RNA or protein test is carried out using any methods which allow the identification of homozygous and heterozygous SNPs in the genomic, gene or protein, sequence including Western blot using specific antibodies, microarrays of SNPs, SNP-RFLP (restriction fragment length polymorphism ), the dynamic hybridization of specific alleles (dynamic allele-specific hybridization - DASH), molecular beacons, probes of the TaqMan type, primer extension, MALDI-TOF (matrix-assisted laser desorption/ionization - time of flight), ELISA (enzyme-linked immunosorbent assay) and the like, an oligonucleotide single-strand conformation polymorphism, gel electrophoresis, temperature gradient gel electrophoresis (TGGE), denaturing high performance liquid chromatography (HPLC), high resolution melting polymerase chain reaction (HRM PCR) SNPlex and/or new generation sequencing (NGS) performed on material isolated from samples of tissue or blood collected from a patient, wherein at least one of these SNPs present at a statistically significant level indicates the functional disruption of GRHL1 and/or GRHL2 and/or GRHL3 dependent processes.

Another subject of the present invention is the use of single nucleotide polymorphisms in GRHL genesl and/or GRHL2 and/or GRHL3 to predict the in vitro or ex vivo increased risk of kidney cancer in humans.

Using the information about the single nucleotide polymorphisms in GRHL genesl and/or GRHL2 and/or GRHL3 in kidney cancer (homozygous or heterozygous), it is preferred that variants of genotypes are detected through DNA, RNA or protein analysis.

Preferably, the methods are applicable to samples of tissue or blood collected from a human donor, in which at least one of these SNPs is present at a statistically significant level, indicating the functional disruption of GRHL1 and/or GRHL2 and/or GRHL3 dependent processes.

Preferably, during the use of the present invention, the DNA, RNA or protein test is carried out using any methods which allow the identification of homozygous and heterozygous SNPs in the genomic, gene or protein, sequence including Western blot using specific antibodies, microarrays of SNPs, SNP-RFLP (restriction fragment length polymorphism ), the dynamic hybridization of specific alleles (dynamic allele- specific hybridization - DASH), molecular beacons, probes of the TaqMan type, primer extension, MALDI-TOF (matrix-assisted laser desorption/ionization - time of flight), ELISA (enzyme-linked immunosorbent assay) and the like, an oligonucleotide single-strand conformation polymorphism, gel electrophoresis, temperature gradient gel electrophoresis (TGGE), denaturing high performance liquid chromatography (HPLC), high resolution melting polymerase chain reaction (HRM PCR) SNPlex and/or new generation sequencing (NGS) performed on material isolated from samples of tissue or blood collected from a patient, wherein at least one of these SNPs present at a statistically significant level indicates the functional disruption of GRHL1 and/or GRHL2 and/or GRHL3 dependent processes.

In one embodiment, the cancer is renal clear cell carcinoma.

In the second embodiment, the cancer is a renal cell cancer of another type.

Detailed description

The first subject of the present application are the single nucleotide polymorphisms identified in the regulatory sequences and the coding sequences of The GRHL1 , GRHL2 and GRHL3 genes and specific for patients with renal cell carcinoma. Their presence in the DNA sequence can lead to the impaired gene expression of GRHL1 and/or GRHL2 and/or GRHL3 and/or the functioning of the encoded proteins GRHL1 and/or GRHL2 and/or GRHL3 and, therefore, may cause disturbances in the functioning of pathways and pathway signals involving GRHL1 and/or GRHL2 and/or GRHL3 gene products.

The second subject of the present application is a method of determining a patient's increased risk of developing kidney cancer, especially cancer of the clear cell, based on the identification of at least one of said single nucleotide polymorphisms in The GRHL1 , GRHL2 and GRHL3 genes. The third subject of the present application is the use of methods of determining patients at increased risk of developing kidney cancer, in particular of the clear cell type, based on the identification of at least one of said single nucleotide polymorphisms in the GRHL1 , GRHL2 and GRHL3 genes in human population screening for the detection of an increased risk of kidney cancer.

The fourth subject of the present application is the use of methods of identifying determining an increased risk of developing kidney cancer in patients, in particular the clear cell type, based on the identification of at least one of the said single nucleotide polymorphisms in the GRHL1 , GRHL2 and GRHL3 genes in population prevention programs and early detection of increased risk of cancer of the kidney.

The essence of the present invention is illustrated by the following examples which do not exhaust of all the possibilities of the invention. During routine laboratory efforts, a considerable number of procedural changes and variations may be found which, however, fall within the scope of the present invention.

Example 1

Identification of single nucleotide polymorphisms characteristic of patients with renal cell cancer.

Cancerous changes in the kidney with a margin of healthy tissue are surgically removed and undergo an initial pathological evaluation. Fragments of tumour tissue and normal tissue from the margin of excised changes were collected from 29 patients with clear cell renal carcinoma. The tissues were stored at -80 ° C until analysis. Then, using a DNA isolation kit (DNeasy Blood & Tissue Kit, Qiagen) we isolated genomic DNA. The enrichment of samples in the selected target sequences was performed using an individually designed HaloPlex kit (Agilent), and the prepared DNA library containing the fragments of GRHL1 , GRHL2 and GRHL3 genes was targeted deep Next Generation Sequencing using a MiSeq (lllumina) system.

Other known methods which may be used to detect the presence of polymorphisms in the gene sequences GRHL1 and/or GRHL2 and/or GRHL3 tissue or body fluids from patients include, but are not limited to: Western blot using specific antibodies, SNP microarrays, SNP-RFLP (restriction fragment length polymorphism), dynamic allele- specific hybridization (DASH), molecular beacons, TaqMan probes, primer extension, MALDI-TOF (matrix-assisted laser desorption/ionization - time of flight), ELISA (enzyme-linked immunosorbent assay), and the like, oligonucleotide single- strand conformation polymorphism, temperature gradient gel electrophoresis (TGGE), denaturing high performance liquid chromatography (high performance liquid chromatography - HPLC), high resolution melting polymerase chain reaction (HRM PCR), SNPlex and/or New Generation sequencing - NGS) and restriction digestion preceded by a PCR.

Example 2

Analysis of the frequency and correlation of alleles in the population

We determined the incidence of single nucleotide polymorphisms in patients with kidney cancer compared to their prevalence in the European population based on data from the database l OOOGenomes. To determine the statistical significance of variations (at p <0.05) for the SNP frequency in the population, we used a binomial test. The SNP distribution with a different frequency in the group of patients is shown in a Manhattan plot (Fig. 1 ).

Based on the above-described analysis we defined single nucleotide polymorphisms in the regulatory sequences of the genes GRHL1 , GRHL2 and GRHL3, which occur in the renal cell carcinoma patient population with a significantly altered frequency in comparison to the European population. The described single nucleotide polymorphisms are shown in Table 1 , Table 2 and Table 3.

Example 3

Changes in the level of GRHL gene expression in clear cell carcinomas of the kidneys The GRHL family of transcription factors is an evolutionarily conserved, tissue- specific group of proteins. Their role is to regulate transcription (through activation or repression) of genes essential for maintaining the proper structure and function of the epithelium. They control the expression of, among others, genes such as DSG1 , CDH1 , RAB25, CLDN3 and CLDN4, the genes of the EDC complex, hTERT and PCNA. Disturbances in the expression level of GRHL genes may be indirectly associated with multiple kidney diseases and may contribute to tumourigenesis.

RNA was isolated from tumour tissue and normal tissue taken from the margin of the excised change, a mortar-and-pestle and a the GeneMATRIX Universal RNA Purification Kit (Euryx). Reverse transcription was carried out for 250 ng of imRNA using the First Strand RevertAid cDNA Synthesis Kit (Thermo Scientific). Detection of changes in the level of expression in tumour tissue with respect to the healthy tissue of the patient were performed using Thermo Scientific Maxima™ SYBR Green/ROX qPCR Master Mix:

The Quantitative Real-Time PCR - (Q-RT-PCR) was carried out in the Eco lllumina system. Samples of clear cell renal cancer showed a decrease in the level of gene expression in GRHL1 and GRHL2 compared with surrounding normal tissue (Fig. 2).

Use: The decrease in GRHL gene expression is a molecular marker of renal cancer.

Effect of single-nucleotide polymorphisms on the level of GRHL gene expression in kidney cancers

The regulation of gene expression is a complex process and depends on many factors. Furthermore, each of the stages of gene expression can also be regulated by different mechanisms. GRHL expression in kidney cells depends on the cell type and on the state of metabolic and physiological activities. a) Single nucleotide polymorphisms in the promoter and regulatory sequences

Next generation sequencing of GRHL genes enabled us to determine single nucleotide polymorphisms in the promoter and regulatory regions of GRHL genes in patients with renal cell cancer (Table 1 ). On the basis of the analysis, we made a Manhattan Plot (described earlier) indicating single nucleotide polymorphisms that may be associated with an increased risk of cancer of the kidney. The frequency changes in their occurrence in the patient population with clear cell renal carcinoma compared with the European population was statistically significant.

Biological significance

The presence of single nucleotide polymorphisms in the sequence recognized by transcription factors can lead to the loss, weakness, gain, or the formation of a protein/DNA binding site, which leads to changes in gene expression level.

b) Single nucleotide polymorphisms in the 3'UTR regions of GRHL genes

In patients with clear cell renal carcinoma, we showed a decrease in the level of GRHL1 and GRHL2 gene expression. The level of gene expression may be dependent on regulation by microRNAs (imiRNA). imiRNAs regulate gene expression levels by binding to a specific 7-nucleotide sequence in the 3'UTR region of imRNA molecules. The reduction of the efficiency of the translation of genetic information into protein, the gene expression is silenced. The dysfunction of gene expression can be crucial in the process of carcinogenesis. One of the causes of the abnormal imiRNA/mRNA interaction may be the presence of single nucleotide polymorphisms in the 3'UTR sequence. In effect, this may abolish existing or create new specific 6-8- nucleotide sequences recognized by imiRNAs, resulting in an undesirable increase or decrease in the level of expression of a given gene. Next generation sequencing of the GRHL genen made it possible to identify single nucleotide polymorphisms in patients with renal cell carcinoma in the 3'UTR regions of the GRHL genes. On the basis of the prepared Manhattan Plot analysis, we indicated those single nucleotide polymorphisms that may be associated with an increased risk of kidney cancer. The changed frequency of their occurrence in the patient population with clear cell renal carcinomas was statistically significant compared with the European population.

Table 2 Listing of variants in the 3 'UTR sequences of GRHL genes in patients with renal cell carcinoma

Polymorphisms labeled "+" are present in the patient population in excess of the European population and "-" if less frequently. The numbering of the items in the column "position" is consistent with the GRCh37/hg19 annotation.

Biological significance

The presence of single nucleotide polymorphisms in the recognition sequence of the miRNA may lead to loss, weakness, enhancement or to the creation of a miRNA/mRNA binding site.

c) Single nucleotide polymorphisms in the coding regions of GRHL genes

The GRHL3 transcription factor directly positively regulates the expression of the PTEN gene whose protein product is an inhibitor of the PI3K/AKT/mTOR pathway (Darido et al, 201 1 ). One of the subjects of the present application is a single nucleotide polymorphism that was identified in the coding sequence of the GRHL3 gene and is specific for patients with renal cell carcinoma. Its presence in the DNA sequence can lead to the impaired function of the GRHL3 protein and thus can affect the reduction of protein activity of PTEN and the activation of the PI3K/AKT/mTOR pathway that promotes cell proliferation and tumour growth. According to the UCSC database, this polymorphism is found in all alternative splice variants of GRHL3 that encode the protein (http://genome.ucsc.edu). Thus, the presence of this polymorphism leads to a change in the amino acid sequence and can potentially affect the proper functioning of all isoforms GRHL3. Moreover, the amino acid residue at the codon which is located in this polymorphism is located in the motif potentially recognized by protein kinases such as GSK-3P, ERK1 , ERK2, and CDK5 (http://kinasephos.mbc.nctu.edu.tw/) . It is very likely that the phosphorylation of GRHL3 by these kinases is essential for its proper functioning.

Tabela 3 Listing of SNPs in the coding sequences of GRHL genes in patients with renal cell carcinoma

Polymorphisms labeled "+" are present in the patient population in excess of the European population and "-" if less frequently. The numbering of the items in the column "position" is consistent with the GRCh37/hg19 annotation.

GRCh37/hg19.

Biological significance

The occurrence of polymorphism rs41268753 causes the loss of a phosphorylation site in the GRHL3 protein. Its molecular consequences in post-translational modifications and functioning of the GRHL3 protein are the subject of further studies by the authors.

Sequence listing: Table 1 Table 2 Table 3

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