IT201900000130A1 - METHOD FOR IDENTIFYING SUBJECTS RESISTANT TO TREATMENT WITH PLATINUM-BASED DRUGS AND RELATIVE KIT - Google Patents

Description

Patent application for industrial invention entitled:

"METHOD FOR IDENTIFYING SUBJECTS RESISTANT TO TREATMENT WITH PLATINUM-BASED DRUGS AND RELATIVE KIT"

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to a method for identifying oncological subjects resistant to treatment with drugs of the category of platinum complexes. In particular, the invention relates to a method that allows the physician to be provided with a useful tool to assess whether a specific cancer subject will be able to respond positively to treatment with complex platinum-based anticancer drugs or whether he will develop resistance, as well as to predict the effectiveness of such treatment. Furthermore, the invention relates to a kit for the identification of said subjects.

PRIOR STATE OF THE ART

[0002] Platinum complexes are a category of drugs that include cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, lipoplatin and constitute first-line clinical therapies in the treatment of many types of solid tumors (Kang et al., 2015). Although most patients initially respond very well to treatment with these compounds, the development of platinum-resistant (PT) tumor cells is a very frequent occurrence (Kang et al., 2015; Siddik, 2003).

Furthermore, it is known that platinum treatment causes rather severe side effects for the subject subjected to it. In other words, these treatments cause substantial toxicity with the appearance of more or less severe skin rash accompanied by itching, temperature rise, chills, dizziness, headache, asthenia, nausea and vomiting, alteration of renal function, hair loss, in association with less frequent phenomena such as pain and ulcers of the oral cavity, diarrhea, cardio toxicity, neuro toxicity.

[0004] Therefore, there is a strong need on the part of clinicians to have a valid tool available to carefully evaluate the opportunity to treat oncologists with potentially very toxic drugs. At the same time, this tool should provide a diagnosis and prognosis on the treatment with platinum complexes so that the physician can have further important information for this evaluation and for a targeted planning of dosages and administration times.

[0005] In this regard, some methods of diagnosis, prognosis and treatment of primary and metastatic tumors have been proposed through the use of biomarkers. Among these biomarkers various molecules of an oligonucleotide nature and hundreds of genes have been specifically proposed, whose activities influence the behavior and the state of cellular differentiation of cancer cells as well as their sensitivity or resistance to therapeutic agents such as the aforementioned platinum complexes. In any case, the proposed methods have proved to be of little use in providing clinicians with useful tools for assessing the status of cancer subjects to be treated.

[0006] In fact, it has been found that these methods focus on the use of single analytes without taking into account at least the known dozens of interactions between different tumor markers. Furthermore, the known diagnostic methods that are based on immunohistochemistry are not standardized.

SUMMARY OF THE INVENTION

[0007] The technical problem faced by the present invention is therefore that of providing a method for identifying oncological subjects sensitive to the treatment of platinum-based drugs in an effective, simple and reliable way.

[0008] This problem is solved by a method comprising the detection of a group of ten specific and selected genes as molecular signature identifying the presence of drug resistance to platinum complexes by oncological subjects.

[0009] Therefore, a first object of the invention is a method for identifying oncological subjects resistant to treatment with a therapeutic agent based on or including platinum, comprising a phase of simultaneous detection of the expression of ten specific genes.

[0010] A second object of the invention is a method for the prognosis of oncological subjects to be subjected to treatment with a platinum-based therapeutic agent.

[0011] A third object is a method as previously in which the identification of genes is carried out by means of probes targeted against said genes.

[0012] A further object is a method as above in which a "weighing" step of the single genes is carried out.

[0013] A further object is a kit for the identification of an oncological subject resistant to platinum-based therapeutic agents including probes targeted against the mRNA of said ten genes.

BRIEF DESCRIPTION OF THE FIGURES

[0014] The further characteristics and advantages of the method and of the kit according to the present invention will become more evident in the following description relating to a preferred embodiment given purely by way of non-limiting example, with reference to the following figures in which:

- Fig. 1 shows a table showing the ten most over-expressed (upregulated) genes in cancer cells that respond to the combined treatment with platinum complexes and USP1 inhibitors by modulating the expression of the Snail protein;

- Fig. 2 is a graph showing a Kaplan-Mayer curve for the evaluation of progression-free survival for patients with EOC using the ten over-expressed genes listed in figure 1 to discriminate the patients with better and worse prognosis;

- Fig. 3 is a graph showing a Kaplan-Mayer curve for the evaluation of progression-free survival for patients with TNBC using the ten over-expressed genes listed in figure 1 to discriminate the patients with better and worse prognosis;

- Fig. 4 is a graph showing a Kaplan-Mayer curve for the evaluation of progression-free survival for patients with LC using the ten over-expressed genes listed in figure 1 to discriminate the patients with the best and worst prognosis;

- Fig. 5 is a graph showing a Kaplan-Mayer curve for the evaluation of progression-free survival for ER + BC patients using the ten over-expressed genes listed in figure 1 to discriminate the patients with better and worse prognosis.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Ovarian epithelial cancer (EOC, Epithelial Ovarian Cancer) is a prototype of a pathology resistant to PT treatment, associated with a very poor prognosis (Jayson et al., 2014). Resistant recurrent EOC grows as a metastatic disease that invades the peritoneum, omentum and organs located in the peritoneal cavity (Thibault et al., 2014; Weidle et al., 2016).

[0016] EOC is unfortunately a high mortality disease that afflicts thousands of women every year. Although the survival rate in the first five years is very high for localized tumors, this rate plummets after distal metastases. Furthermore, unfortunately only a small percentage (15%) of cases are diagnosed when the cancer is still in the primary stage, while the vast majority of EOC diagnoses are made following distal metastases that have already occurred. Therefore, EOC is considered a deadly pathology.

The need, therefore, for a better understanding of the procedures and pathways leading to the development of tumors as well as resistance to the most common treatments available to clinicians is of primary importance.

[0018] To achieve this goal, the identification of biomarkers is undoubtedly an important avenue of medical research especially for the evaluation of treatment with certain therapeutic agents such as platinum complexes.

Furthermore, both drug resistance and tumor spread have been correlated with Epithelial-to-Mesenchymal Transition (EMT), a complex molecular and cellular program by which cancer cells acquire aggressive characteristics , including cellular plasticity, invasiveness and resistance to apoptosis (Polyak and Weinberg, 2009). Whether and how this phenotype is connected with an alteration of the DNA Damage Response (DDR, DNA Damage Response) activated by chemotherapy is still unclear.

[0020] The idea behind the present invention was therefore to start from the investigation of mediating and regulatory proteins connected with EMT and DDR. In particular, among the EMT-inducing proteins, it is known that the transcription factor Snail can contribute to PT-resistance in EOC by mediating cell survival and the acquisition of stem-like characteristics (Haslehurst et al., 2012; Kurrey et al. al., 2009). Snail is a short-lived protein as it is rapidly polyubiquitinated and degraded by the proteasome complex (Zhou et al., 2004). Several ubiquitin ligases have been identified for the ubiquitination and degradation of Snail, but no other factor correlates with Snail's stability in order to allow it to come into play in PT-resistance.

Following numerous investigations, it was found that the USP1 protein (Ubiquitin-Specific Protease 1) is capable of de-ubiquitinating and stabilizing Snail, thereby causing PT-resistance and metastatic dissemination in EOC cells.

[0022] Consequently, methods have been developed to verify the response to the inhibition of USP1 in EOC cell lines treated with PT-complexes in the presence or absence of a USP1 inhibitor. Subsequently, the expression of the Snail protein was verified. The result of these investigations showed that in eight out of ten cell lines tested, called "responsive" cells compared to "non-responsive" cells, the USP1 inhibitor prevents the increase (upregulation) of Snail after PT-treatment.

[0023] Once the activity of USP1 was demonstrated, it was decided to also verify whether the molecular signature of the mRNA associated with said activity could somehow predict the prognosis of EOC patients.

In this regard, it has been observed that about five hundred genes are upregulated in "responsive" cells. These genes were selected using gene expression profiles available in the literature (Kreuzinger et al., 2015). In particular, during the tumor mass reduction surgery, the tumor tissues were taken and directly analyzed for confirmation of the histological type. Subsequently, seven different cell lines were obtained from this material from patients with HGSOC.

Of these seven cell lines, the following have been considered in the present invention: 8587, 13699 and 13914 all responsive after six months of treatment, 14433 non-responsive after six months of treatment. Line 8587 derives from tumor tissue, while the others derive from ascites. The ascites were centrifuged and then the erythrocytes eliminated thanks to a centrifugation step with Histopaque 1077 (Sigma-Aldrich, St. Louis, USA). Tumor tissues were cut into small pieces and digested with collagenase (1 mg / mL, 1453 CDU / mg, Sigma-Aldrich) at 37 ° C for 1 h. Cells were cultured in DMEM medium, with 10% fetal bovine serum (FBS), 100 units / mL penicillin and 100 µg / mL streptomycin (PS; all from Gibco by Life Technologies, CA, USA) at 37 ° C and 5 % CO 2. VenorGeM Classic Mycoplasma Detection Kit for conventional PCR (Minerva Biolabs, Berlin, Germany) was used to control mycoplasma contamination.

[0026] A Short Tandem Repeats analysis of 7 markers (TPOX, vWA, CSF180, D16S539, D7S820, D13S317, D5S818, Applied Biosystems Life Technologies) was also carried out. The analysis was carried out using ABI Prism 310 Genetic Analyzer (Applied Biosystems, Life Technologies) in order to authenticate the cell lines.

[0027] Subsequently, the DNA and RNA were isolated from fresh frozen tumor tissue cell lysate (Mikro-Dismembrator U; B.Braun Biotech International, Melsungen, Germany) using the AllPrep DNA / RNA Mini Kit (Qiagen, Hilden, Germany). Nucleic acid concentration was measured with BioPhotometer (Eppendorf, Hamburg, Germany).

At this point, complete Shot-gun genomic libraries were prepared using the KAPA Library Preparation Kit (KAPA Biosystems). Following the instructions given by the manufacturer, they were quantified and sequenced thanks to a HiSeq2000 (Illumina) with low coverage generating reads of 50bp. The raw sequences were then mapped to the reference human genome (NCBI37 / hg19) with Burrows – Wheeler Aligner (BWA v0.5.8a).

[0029] The RNA libraries, useful for quantifying the gene mappings, were created using Illumina TruSeq RNA sample preparation kit V2 following the manufacturer's instructions and were subsequently sequenced with HiSeq2000 (Illumina) using a V3 flowcell generating 1 × 50bp reads.

[0030] The raw sequences were then mapped to the transcriptome and the human reference genome (NCBI37 / hg19) using the tools TopHat 2.0 and Bowtie 2.0.32 859 670 readings were then associated to the respective genes thanks to the HTSeq software package and have been normalized with EDASeq.

[0031] From the analysis just described it was therefore possible to construct the molecular signature of the mRNA associated with the activity of USP1. Therefore, to verify the possibility of predicting the prognosis, we proceeded to select the overexpressed genes in an arbitrary manner by placing a fold change cut off greater than 100. The first 10 genes, shown below, were then chosen, which are at least 100 times more expressed in "responsive" cells, that is, responding to treatment with platinum complexes, compared to "non-responsive" cells, that is, not responding to treatment with platinum complexes:

CLDN6 (SEQ ID NO: 1), COL11A1 (SEQ ID NO: 2), SIM1 (SEQ ID NO: 3), DKK1 (SEQ ID NO: 4), GAL3ST3 (SEQ IDE NO: 5), IGFBP5 (SEQ ID NO : 6), DKK2 (SEQ ID NO: 7), COL1A2 (SEQ ID NO: 8), COL3A1 (SEQ ID NO: 9), MAP3K7CL (SEQ ID NO: 10).

[0032] On the basis of the aforementioned investigations, it was thought to employ this molecular signature of the aforementioned ten genes for the realization of a method for identifying oncological subjects resistant to treatment with therapeutic agents based on or including platinum complexes, comprising the steps of :

- make available a biological sample isolated from the human body of a cancer patient;

- extract the RNA from said sample;

- identify the contemporary up-regulation of the above ten genes.

The term "biological sample isolated from the human body" means a fresh or preserved tissue fragment obtained from a tumor, either by biopsy or by needle aspiration, or any biological fluid containing tumor cells taken from the human body. Biological fluids include, for example, whole blood, plasma, serum, peritoneal fluid, lymphatic fluid, urine, seminal fluid.

[0034] The "tumor cells" or cancerous cells can be of origin from tumor or primary cancer of various kinds or from metastases, such as cancer of the ovarian epithelium (EOC), cancer of the pancreas, stomach, kidney, colon -rectal, liver, lung, prostate or breast. In particular, for ovarian cancer, the most frequent tumor is the serous one, considered the most aggressive and lethal form of EOC.

The term "nucleic acids" means both natural and artificial nucleotides including polynucleotides or oligonucleotides, generally ribonucleotides (RNA) or deoxyribonucleotides (DNA), both modified and unmodified. Therefore, the above term includes single or double stranded RNA or DNA, single or double stranded RNA or DNA, RNA-DNA hybrid molecules, cDNA, mRNA, tRNA, mi-RNA, sh-RNA. Purified nucleic acids consist of molecules substantially free from other cellular material or culture media when synthetically produced; essentially free means 60%, 70%, 80%, 90% and even over 99% pure. The degree of purity is determined through conventional purification methods such as column chromatography, polyacrylamide gel electrophoresis or HPLC.

[0036] The term "probes" refers to sense or anti-sense nucleic acids used in conventional hybridization techniques and provided with, ie labeled, detectable molecules, ie a signal molecule, ie a molecule capable of being detected through conventional analytical methods, such as for example a fluorophore, a radioisotope, an enzyme, a heavy metal.

By the term "molecular signature" is meant any group of biological molecular markers, in particular a nucleic acid sequence, specifically an mRNA sequence. The sequences are characterized by having at least 60%, 70%, 80%, 90% identity or complementarity with the SEQ ID NO: 1-10 gene sequences according to the present invention, taking into account variants, non-functional mutations and the degeneration of the code.

The term "gene expression profiles" indicates a specific condition of a cell. In practice, on the basis of the evaluation of several genes simultaneously, a unique gene profile of the state of a cell can be produced. In other words, a normal tissue can be distinguished from a diseased (cancerous) tissue, and within the same cancerous tissue different prognoses (good or poor long-term survival prospects) can be distinguished. For example, the comparison between sequences that are expressed differently between normal or cancerous cells and also between cancer cells and cancer cells can help to identify different chemotherapy treatment regimens.

A "poor prognosis" indicates a decrease in the progression-free survival rate. Progression-free survival is the period of time in which the disease does not progress and that elapses between the date of randomization in the clinical study and the date of progression assessed for example by CT or MRI.

"Gene amplification" means a process by which multiple copies of a gene or gene fragments are formed in a particular cell or cell line or by PCR. The duplicated region is generally called the "amplicon".

The term "responsive" referring to a tumor condition with respect to treatment with chemotherapy means that the degree of growth of the tumor is inhibited compared to the degree of growth without treatment with chemotherapy. "Unresponsive", on the other hand, indicates a tumor growth rate that is not inhibited or with a poorly reduced growth rate or with a statistically insignificant rate of growth.

[0042] In the method according to the present invention, the biological sample is taken according to conventional systems during, for example, a surgical procedure for the removal of the tumor mass, removal of a small amount of tissue, needle aspiration. Alternatively, the withdrawal takes place through a syringe, spatula or other known device capable of obtaining a quantity of a body fluid to be treated in order to extract the nucleic acids.

[0043] The extraction of nucleic acids from said sample takes place through as many conventional methods. For example, mRNA is extracted from a tissue sample that has been fixed by embedding in paraffin and fixed with formalin. However, general mRNA extraction methods are well known in the field and described in molecular biology books and manuals such as Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997), Rupp and Locker, Lab Invest .56: A67 (1987), De Andrés et al., BioTechniques 18: 42044 (1995), Molecular Cloning - a Laboratory Manual, Sambrook and Russel. Many RNA extraction methods employ strong denaturants such as guanidine hydrochloride or guanidine thiocyanate to break cells, solubilize its components and denature endogenous RNase. In particular, the isolation of RNA is carried out using commercially available purification kits, buffer sets and proteases. For example, total RNA from cultured cells can be extracted using Qiagen RNeasy mini-columns, MasterPure <TM> Complete DNA and RNA purification kit (EPICENTRE <®>, Madison, WI), Paraffin Block RNA isolation kit ( Ambion, inc.), Stat-60 RNA (Tel-Test) used for total RNA from tissue samples, density gradient centrifugation in cesium chloride.

Preferably, the TRI (Molecular Reserach Center), TriZol (Ambion), Isogen (Nippon Gene) reagents are used to extract the total RNA. The protocols are widely known and generally provide for the preparation of reagents in which to homogenize the samples, separate the protein and DNA phases from that of the RNA, precipitate the RNA and wash it from the reagent residues with appropriate standardized solutions. Preferred is the separation of mRNA by means of affinity chromatography on an oligo (dT) -cellulose column or, in the case of many samples to be processed or small quantities, bach chromatography on an identical column.

[0044] For the determination of the molecular signature of mRNA of interest from the extracted and isolated RNA as previously, it is possible to use two general known methods which are based on the hybridization analysis of polynucleotides and on the sequencing of polynucleotides. Among these, the most used include northern blotting; in situ hybridization; RNase protection assays; reverse transcription polymerase chain reaction (RT-PCR). Alternatively, monoclonal or polyclonal antibodies that recognize specific duplexes of RNA, DNA-RNA, RNA-protein can be used. In particular, the methods of quantitative real-time RT-PCR (qRT-PCR) are very useful.

[0045] Among the aforementioned different techniques, the most sensitive and flexible is qRT-PCR which in fact can be used to compare the mRNA levels in different populations of samples, both normal and tumor, with or without chemotherapy treatment. In this way, it is possible to discriminate between structurally very close mRNAs.

[0046] In determining a gene expression profile by RT-PCR, the first step usually consists in retro-transcribing the RNA into cDNA followed by its amplification in the PCR reaction. For this purpose, known reverse transcriptases are used which catalyze this reaction: among the most used we can mention the reverse transcriptase of the avian myeloblastosis virus and the reverse transcriptase of the Moloney murine leukemia virus. Transcriptases typically require specific primers, random hexamers or nonamers, or oligo-dT primers. Examples of such kits are the GeneAmp RNA PCR kit (Perkin Elmer, CA, USA) and the AMW reverse transcriptase kit (Promega).

Numerous varieties of DNA-dependent thermostable DNA polymerases can be used for the next step of the PCR. Typically, Taq DNA polymerase can be employed; for example, TaqMan® PCR exploits the 5-nuclease activity of the Taq or Tth polymerase to hydrolyze a hybridization bond of a probe to its target amplicon, but any equivalent enzyme with 5'-nuclease activity can be used. Two primer oligonucleotides are used to generate the typical amplicon of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequences located between the two primers. The probe is labeled with a fluorescent reporter dye and a fluorescent quencher dye. During the amplification reaction, the Taq polymerase cleaves the probe, releasing the reporter dye from the masking effect of the quencher dye. Therefore, a reporter dye molecule is released, and therefore identifiable, for each synthesized molecule, thus allowing the quantitative evaluation of the data.

[0048] Devices usually used for this type of PCR are for example ABI PRISM 7700TM Sequence Detection System TM, GeneAmp 5700 Sequence Detection System (Perkin-Elmer Biosystems), LightCycler (Roche), MyiQ2 Two Color Real-time PCR Detection System (Biorad ).

[0049] A different approach for the evaluation of a gene expression profile is represented by the microarray technique, ie DNA chip. A series of sequences of interest (oligonucleotides) are attached to the surface of a substrate such as a slide, a plastic sheet, a silicon chip, in order to give rise to an array or matrix of sequences. These sequences are then hybridized with specific DNA probes. In particular, the amplified PCR inserts of cDNA clones are fixed on a substrate. Specific cDNA probes with fluorescent labels hybridize at precise points on the matrix with said inserts. After removing the probes that have not hybridized, the support is analyzed with an appropriate fluorescence detection equipment.

[0050] Further mRNA detection systems include second and third generation sequencing technologies such as sequencing according to Illumina, Ion Torrent, SMRT, nanopore, SeqLL.

[0051] The use of immunohistochemical methods are also suitable for detecting the expression levels of markers. As is known, with these methods, monoclonal or polyclonal antibodies are produced targeted against the markers of interest (Antibodies, "A laboratory Manual", Cold Spring Harbor Laboratory Press, Second Edition, 2014; "Critical Steps in the Production of Polyclonal and Monoclonal Antibodies: Evaluation and Recommendations ", Marlies Leenaars, Coenraad F. M. Hendriksen (ILAR Journal, Volume 46, Issue 3, 1 January 2005, pages 269-279; M. Schreier et al." Hybridoma Techniques ", 1980; Hammerling et al., "Monoclonal Antibodies and T-cell Hybridomas", 1981; Kennet et al., Monoclonal Antibodies, 1980; Harlow et al.). Antibodies can be detected by labels attached to them such as radioactive elements, fluorescent compounds, haptens such as biotin or enzymes such as horseradish peroxidase or alkaline phosphatase. Alternatively, an unlabeled primary antibody and a labeled secondary antibody can be used. By means of suitable detectors such as radioactivity counters, scin tillers, spectrophotometers, colorimeters, fluorimeters, luminometers and densitometers.

[0052] The probes used to detect in the samples of interest the quantity of mRNA of the molecular signature according to the invention are produced in accordance with conventional methods among which the most widespread is PCR, but priming with random, nick oligonucleotides can also be used -translation. In any case, the probes are small fragments of oligonicleotides synthesized in such a way as to complement, and thus hybridize, with specific short sequences of a target such as a portion of a gene or an mRNA. The probes are built to carry radioactive elements such as <32> P so as to be detectable by autoradiography, or elements or chemical groups conjugated with nucleic acids by enzymatic, photochemical or chemical reaction. An example of this widely used method is constituted by biotin which allows detection by interaction with streptavidin conjugated to a reporter enzyme. Another non-radioactive detection system is represented by DIG.

[0053] The detection systems of the probes are generally the colorimetric assays, the chemoluminescence and the fluorescent assays, widely known to the skilled in the art.

[0054] In particular, it has been seen that the over-expression index of the ten genes of the invention is determined when, based on the aforementioned analyzes conducted on at least ten samples, there is a corresponding amount of mRNA higher than the median value of the ten samples considered. This threshold value for establishing the presence of overexpression was found for example using qRT-PCR analysis. However, it should be kept in mind that by varying the analytical method, the quantities found could also vary. In any case, using the procedure described below it is still possible to correlate quantities of mRNA with a situation of over-expression.

[0055] In the method according to the present invention, the step of determining the overexpression of the aforementioned ten selected genes comprises a list of genes differentially expressed in the lines responsive to the combined platinum + USP1 inhibitor treatment and were selected according to the methodology described in Kreuzinger et al., Cancer letters July 1, 2015: 362 (2), 218–228. For each gene the value corresponds to the average of three technical replicates for each cell line and the fold change (FC) is calculated according to the formula FC = mean (C1 + 0.5, C2 + 0.5, C3 + 0.5) / mean (C4 + 0.5, C5 + 0.5), where C1-C3 are cell lines # 8587, 13914_1 and 13699, while C4-5: 14333 and 12370 (0.5 is added to all values to avoid case N / 0). The C1-C5 values correspond to the number of reads for each gene for each cell line obtained from the RNA seq assay. Genes with FC <4 (in both directions) were discarded.

[0056] In accordance with a preferred embodiment of the invention, the ten genes are weighed. The term "weight" refers to the absolute size of an element in a mathematical calculation. Therefore, the weight of each of the ten genes of the invention in the molecular signature can be determined on a data set of patient samples using learning methods known in the field.

[0057] In particular, for each type of tumor the weights for each gene were chosen in an "arbitrary" manner after having conducted an analysis on the survival curve considering each gene individually. The genes that separated the two curves better (genes whose low expression correlates with greater survival) had a higher value (max 3.5), the genes that did not separate the two curves very much had the lowest weight ( min 0). The genes that went the other way around (their high expression was indicative of a higher survival) had an "inverted" weight.

[0058] Table 1 below shows the HR (Hazard Ratio) values, as produced by the KM Plotter program available online, of the ten genes according to the invention.

Table 1

[0059] Once the above-mentioned values have been obtained, the above-mentioned "arbitrary" weights have been defined as reported in the following Table 2.

Table 2

[0060] Therefore, using the values of the aforementioned weights of table 2 and correlating them with each of the ten genes, it is possible to obtain the weights necessary to obtain the curves in accordance with the KM Plotter program, as explained in more detail with reference to the specific examples of realization. These weights are shown in the following Table 3.

Table 3

[0061] It has therefore been established that for weight values between 0.1 and 0.5 there is a low single gene weight; values between 0.6 and 1.5 have an average weight; values between 1.6 and 2 have a high weight and between 2.1 and 3.5 there is a very high weight. It follows that the corresponding gene expression becomes variously important, depending on the type of cancer, in the evaluation of the administration of the therapy.

[0062] According to a further embodiment, the method comprises a step of evaluating the prediction of a treatment with therapeutic agents based on or including platinum complexes, in which said step involves evaluating the expression of each gene using the "Best cut off" as a threshold to distinguish a gene with high or low expression.

[0063] A further object of the invention is a kit for the identification of oncological subjects resistant to the treatment of complex platinum-based therapeutic agents, preferably comprising a microplate whose wells are coated with an antibody or by probes constructed as previously , a container for a control sample comprising the mRNA sequences of the aforementioned ten genes, a

container for a diluent substance for the control sample and one for the sample to be tested, a container for a buffer, a container for a substrate solution, a container for a reaction termination solution, container for a wash buffer. In addition, the kit includes a package insert with explanations of how to prepare the samples, incubation and washing times, reading conditions of the signal detected with a photometer or alternative equipment depending on the signal to be detected.

[0064] In accordance with a preferred form of the invention, the kit can comprise a reference sample or a table of predetermined data for comparing the values and / or types of molecules detected and corresponding quantities referred to the threshold values indicative of the presence of resistance to complex platinum treatment.

[0065] Some embodiments of the invention will now be described, given purely by way of non-limiting example.

[0066] The mRNA was extracted from a biopsy in accordance with the procedure described in Kreuzinger et al. 2015, previously reported.

[0067] To confirm the role of prognostic and predictive biomarkers, an online database containing microarray expression data for ovarian cancer is used. This database contains the clinical data of these patients and it is also possible to extract data on the patients' Overall Survival and disease free survival. This tool, the KmPlotter is capable of drawing survival curves of a cohort of patients for individual genes. Through the KM Plotter online tool (http://kmplot.com) the names of the various signature genes can be entered in the Gene symbol box (choosing the one with the green indication among possible probes as they are considered the most reliable by the program) and selecting the option “Use multigene classifier”. The same steps were carried out for triple negative breast cancer, estrogen receptor positive breast cancer and for lung cancer.

MODEL OF OVARIAN EPITHELIUM CANCER (EOC)

[0068] In the main screen of the KM Plotter program, enter the names of the genes relating to the molecular signature according to the invention in the "Affy id / Gene symbol" space using the "Use multi gene classifier" option, select "Auto select best cutoff "in the" Split patients by "box, in the" Restrict analysis to subtypes "boxes where the options relating to the stage, grade, histotype and p53 status of the patients to be considered are chosen, the option is chosen for all items Finally, in the boxes "Restrict analysis to treatment groups" where the options relating to the type of surgery and chemotherapy are chosen, also in this case the option "all" is chosen for all items. analysis by clicking on “Draw Kaplan-Meier plot.” The result is the diagram of figure 2 in which the HR (Hazard Ratio) value calculated by the program is the average of the individual HRs of each of the ten genes according to the present invention.

TRIPLE NEGATIVE BREAST CANCER

[0069] In the main screen of the KM Plotter program, enter the names of the genes relating to the molecular signature in the "Affy id / Gene symbol" space using the "Use multi gene classifier" option, select "Auto select best cutoff" in the "Split patients by" box, in the "Restrict analysis to subtypes" boxes only patients with negative ER status, PR status and HER2 status are selected, while for the other items the "all" option is selected for all. "Restrict analysis to selected cohorts" where no option is selected. At this point the analysis is launched by clicking on "Draw Kaplan-Meier plot". The result is the diagram of figure 3 in which the HR (Hazard Ratio) value calculated by the program is the average of the individual HRs of each of the ten genes according to the present invention.

LUNG CANCER

[0070] In the main screen of the KM Plotter program, enter the names of the genes relating to the molecular signature in the "Affy id / Gene symbol" space using the "Use multi gene classifier" option, select "Auto select best cutoff" in the "Split patients by" box, in the "Restrict analysis to subtypes" boxes where the options relating to the stage, grade, histotype of the tumor, and the sex of the patients to be taken into consideration are chosen, the option "all Finally, in the boxes "Restrict analysis to treatment groups" where the options relating to the type of surgery, chemotherapy and radiotherapy are chosen, also in this case the option "all" is chosen for all items. analysis by clicking on “Draw Kaplan-Meier plot.” The result is the diagram of figure 4 in which the HR (Hazard Ratio) value calculated by the program is the average of the individual HRs of each of the ten genes according to the present invention.

BREAST CANCER POSITIVE TO ESTROGEN RECEPTOR

[0071] In the main screen of the KM Plotter program, enter the names of the genes relating to the molecular signature in the "Affy id / Gene symbol" space using the "Use multi gene classifier" option, select "Auto select best cutoff" in the "Split patients by" box, in the "Restrict analysis to subtypes" boxes only patients with positive ER status are selected, while for the other items the "all" option is selected for all. Finally, in the "Restrict analysis to selected cohorts" boxes "Where no option is selected. At this point the analysis is launched by clicking on" Draw Kaplan-Meier plot ". The result is the diagram in figure 2 in which the HR (Hazard Ratio) value calculated by the program is the average of the individual HRs of each of the ten genes according to the present invention.

LIST OF SEQUENCES

<110> Oncology Reference Center

<120> Method to identify subjects resistant to treatment with platinum-based drugs and related kit

<130> B18-143

<170> PatentIn version 3.5

<210> 1

<211> 1389

<212> DNA

<213> Homo sapiens

<400> 1

cgacactcgg cctaggaatt tcccttatct ccttcgcagt gcagctcctt caacctcgcc 60 atggcctctg ccggaatgca gatcctggga gtcgtcctga cactgctggg ctgggtgaat 120 ggcctggtct cctgtgccct gcccatgtgg aaggtgaccg ctttcatcgg caacagcatc 180 gtggtggccc aggtggtgtg ggagggcctg tggatgtcct gcgtggtgca gagcaccggc 240 cagatgcagt gcaaggtgta cgactcactg ctggcgctgc cacaggacct gcaggctgca 300 cgtgccctct gtgtcatcgc cctccttgtg gccctgttcg gcttgctggt ctaccttgct 360 ggggccaagt gtaccacctg tgtggaggag aaggattcca aggcccgcct ggtgctcacc 420 tctgggattg tctttgtcat ctcaggggtc ctgacgctaa tccccgtgtg ctggacggcg 480 catgccatca tccgggactt ctataacccc ctggtggctg aggcccaaaa gcgggagctg 540 ggggcctccc tctacttggg ctgggcggcc tcaggccttt tgttgctggg tggggggttg 600 ctgtgctgca cttgcccctc gggggggtcc cagggcccca gccattacat ggcccgctac 660 tcaacatctg cccctgccat ctctcggggg ccctctgagt accctaccaa gaattacgtc 720 tgacgtggag gggaatgggg gctccgctgg cgctagagcc atccagaagt ggcagtgccc 780 aacagctttg ggatgggttc gtaccttttg tttctgcctc ctgctatttt tcttttgact 840 gaggatattt aaaattcatt tgaaaactga gccaaggtgt tgactcagac tctcacttag 900 gctctgctgt ttctcaccct tggatgatgg agccaaagag gggatgcttt gagattctgg 960 atcttgacat gcccatctta gaagccagtc aagctatgga actaatgcgg aggctgcttg 1020 ctgtgctggc tttgcaacaa gacagactgt ccccaagagt tcctgctgct gctgggggct 1080 gggcttccct agatgtcact ggacagctgc cccccatcct actcaggtct ctggagctcc 1140 tctcttcacc cctggaaaaa caaatgatct gttaacaaag gactgcccac ctccggaact 1200 tctgacctct gtttcctccg tcctgataag acgtccaccc cccagggcca ggtcccagct 1260 atgtagaccc ccgcccccac ctccaacact gcacccttct gccctgcccc cctcgtctca 1320 ccccctttac actcacattt ttatcaaata aagcatgttt tgttagtgca aaaaaaaaaa 1380 aaaaaaaaa 1389

<210> 2

<211> 7291

<212> DNA

<213> Homo sapiens

<400> 2

acacagtact ctcagcttgt tggtggaagc ccctcatctg ccttcattct gaaggcaggg 60 cccggcagag gaaggatcag agggtcgcgg ccggagggtc ccggccggtg gggccaactc 120 agagggagag gaaagggcta gagacacgaa gaacgcaaac catcaaattt agaagaaaaa 180 gccctttgac tttttccccc tctccctccc caatggctgt gtagcaaaca tccctggcga 240 taccttggaa aggacgaagt tggtctgcag tcgcaatttc gtgggttgag ttcacagttg 300 tgagtgcggg gctcggagat ggagccgtgg tcctctaggt ggaaaacgaa acggtggctc 360 tgggatttca ccgtaacaac cctcgcattg accttcctct tccaagctag agaggtcaga 420 ggagctgctc cagttgatgt actaaaagca ctagattttc acaattctcc agagggaata 480 tcaaaaacaa cgggattttg cacaaacaga aagaattcta aaggctcaga tactgcttac 540 agagtttcaa agcaagcaca actcagtgcc ccaacaaaac agttatttcc aggtggaact 600 ttcccagaag acttttcaat actatttaca gtaaaaccaa aaaaaggaat tcagtctttc 660 cttttatcta tatataatga gcatggtatt cagcaaattg gtgttgaggt tgggagatca 720 cctgtttttc tgtttgaaga ccacactgga aaacctgccc cagaagacta tcccctcttc 780 agaactgtta acatcgctga cgggaagtgg catcgggtag caatcagcgt ggagaagaaa 840 actgtgacaa tgattgttga ttgtaagaag aaaaccacga aaccacttga tagaagtgag 900 agagcaattg ttgataccaa tggaatcacg gtttttggaa caaggatttt ggatgaagaa 960 gtttttgagg gggacattca gcagtttttg atcacaggtg atcccaaggc agcatatgac 1020 tactgtgagc attatagtcc agactgtgac tcttcagcac ccaaggctgc tcaagctcag 1080 gaacctcaga tagatgagta tgcaccagag gatataatcg aatatgacta tgagtatggg 1140 gaagcagagt ataaagaggc tgaaagtgta acagagggac ccactgtaac tgaggagaca 1200 atagcacaga cggaggcaaa catcgttgat gattttcaag aatacaacta tggaacaatg 1260 gaaagttacc agacagaagc tcctaggcat gtttctggga caaatgagcc aaatccagtt 1320 gaagaaatat ttactgaaga atatctaacg ggagaggatt atgattccca gaggaaaaat 1380 tctgaggata cactatatga aaacaaagaa atagacggca gggattctga tcttctggta 1440 gatggagatt taggcgaata tgatttttat gaatataaag aatatgaaga taaaccaaca 1500 agccccccta atgaagaatt tggtccaggt gtaccagcag aaactgatat tacagaaaca 1560 agcataaatg gccatggtgc atatggagag aaaggacaga aaggagaacc agcagtggtt 1620 gagcctggta tgcttgtcga aggaccacca ggaccagcag gacctgcagg tattatgggt 1680 cctccaggtc tacaaggccc cactgga ccc cctggtgacc ctggcgatag gggcccccca 1740 ggacgtcctg gcttaccagg ggctgatggt ctacctggtc ctcctggtac tatgttgatg 1800 ttaccgttcc gttatggtgg tgatggttcc aaaggaccaa ccatctctgc tcaggaagct 1860 caggctcaag ctattcttca gcaggctcgg attgctctga gaggcccacc tggcccaatg 1920 ggtctaactg gaagaccagg tcctgtgggg gggcctggtt catctggggc caaaggtgag 1980 agtggtgatc caggtcctca gggccctcga ggcgtccagg gtccccctgg tccaacggga 2040 aaacctggaa aaaggggtcg tccaggtgca gatggaggaa gaggaatgcc aggagaacct 2100 ggggcaaagg gagatcgagg gtttgatgga cttccgggtc tgccaggtga caaaggtcac 2160 aggggtgaac gaggtcctca aggtcctcca ggtcctcctg gtgatgatgg aatgagggga 2220 gaagatggag aaattggacc aagaggtctt ccaggtgaag ctggcccacg aggtttgctg 2280 ggtccaaggg gaactccagg agctccaggg cagcctggta tggcaggtgt agatggcccc 2340 ccaggaccaa aagggaacat gggtccccaa ggggagcctg ggcctccagg tcaacaaggg 2400 aatccaggac ctcagggtct tcctggtcca caaggtccaa ttggtcctcc tggtgaaaaa 2460 ggaccacaag gaaaaccagg acttgctgga cttcctggtg ctgatgggcc tcctggtcat 2520 cctgggaaag aaggccagtc tggagaaaag gg ggctctgg gtccccctgg tccacaaggt 2580 cctattggat acccgggccc ccggggagta aagggagcag atggtgtcag aggtctcaag 2640 ggatctaaag gtgaaaaggg tgaagatggt tttccaggat tcaaaggtga catgggtcta 2700 aaaggtgaca gaggagaagt tggtcaaatt ggcccaagag gggaagatgg ccctgaagga 2760 cccaaaggtc gagcaggccc aactggagac ccaggtcctt caggtcaagc aggagaaaag 2820 ggaaaacttg gagttccagg attaccagga tatccaggaa gacaaggtcc aaagggttcc 2880 actggattcc ctgggtttcc aggtgccaat ggagagaaag gtgcacgggg agtagctggc 2940 aaaccaggcc ctcggggtca gcgtggtcca acgggtcctc gaggttcaag aggtgcaaga 3000 ggtcccactg ggaaacctgg gccaaagggc acttcaggtg gcgatggccc tcctggccct 3060 ccaggtgaaa gaggtcctca aggacctcag ggtccagttg gattccctgg accaaaaggc 3120 cctcctggac cacctgggaa ggatgggctg ccaggacacc ctgggcaacg tggggagact 3180 ggatttcaag gcaagaccgg ccctcctggg ccagggggag tggttggacc acagggacca 3240 accggtgaga ctggtccaat aggggaacgt gggcatcctg gccctcctgg ccctcctggt 3300 gagcaaggtc ttcctggtgc tgcaggaaaa gaaggtgcaa agggtgatcc aggtcctcaa 3360 ggtatctcag ggaaagatgg accagcagga ttacgtgg tt tcccagggga aagaggtctt 3420 cctggagctc agggtgcacc tggactgaaa ggaggggaag gtccccaggg cccaccaggt 3480 ccagttggct caccaggaga acgtgggtca gcaggtacag ctggcccaat tggtttacca 3540 gggcgcccgg gacctcaggg tcctcctggt ccagctggag agaaaggtgc tcctggagaa 3600 aaaggtcccc aagggcctgc agggagagat ggagttcaag gtcctgttgg tctcccaggg 3660 ccagctggtc ctgccggctc ccctggggaa gacggagaca agggtgaaat tggtgagccg 3720 ggacaaaaag gcagcaaggg tgacaaggga gaaaatggcc ctcccggtcc cccaggtctt 3780 caaggaccag ttggtgcccc tggaattgct ggaggtgatg gtgaaccagg tcctagagga 3840 cagcagggga tgtttgggca aaaaggtgat gagggtgcca gaggcttccc tggacctcct 3900 ggtccaatag gtcttcaggg tctgccaggc ccacctggtg aaaaaggtga aaatggggat 3960 gttggtccca tggggccacc tggtcctcca ggcccaagag gccctcaagg tcccaatgga 4020 gctgatggac cacaaggacc cccagggtct gttggttcag ttggtggtgt tggagaaaag 4080 ggtgaacctg gagaagcagg gaacccaggg cctcctgggg aagcaggtgt aggcggtccc 4140 aaaggagaaa gaggagagaa aggggaagct ggtccacctg gagctgctgg acctccaggt 4200 gccaaggggc caccaggtga tgatggccct aagggtaacc cgg gtcctgt tggttttcct 4260 ggagatcctg gtcctcctgg ggaacctggc cctgcaggtc aagatggtgt tggtggtgac 4320 aagggtgaag atggagatcc tggtcaaccg ggtcctcctg gcccatctgg tgaggctggc 4380 ccaccaggtc ctcctggaaa acgaggtcct cctggagctg caggtgcaga gggaagacaa 4440 ggtgaaaaag gtgctaaggg ggaagcaggt gcagaaggtc ctcctggaaa aaccggccca 4500 gtcggtcctc agggacctgc aggaaagcct ggtccagaag gtcttcgggg catccctggt 4560 cctgtgggag aacaaggtct ccctggagct gcaggccaag atggaccacc tggtcctatg 4620 ggacctcctg gcttacctgg tctcaaaggt gaccctggct ccaagggtga aaagggacat 4680 cctggtttaa ttggcctgat tggtcctcca ggagaacaag gggaaaaagg tgaccgaggg 4740 ctccctggaa ctcaaggatc tccaggagca aaaggggatg ggggaattcc tggtcctgct 4800 ggtcccttag gtccacctgg tcctccaggt ttaccaggtc ctcaaggccc aaagggtaac 4860 aaaggctcta ctggacccgc tggccagaaa ggtgacagtg gtcttccagg gcctcctggg 4920 tctccaggtc cacctggtga agtcattcag cctttaccaa tcttgtcctc caaaaaaacg 4980 agaagacata ctgaaggcat gcaagcagat gcagatgata atattcttga ttactcggat 5040 ggaatggaag aaatatttgg ttccctcaat tccctgaaac aagacattg a gcatatgaaa 5100 tttccaatgg gtactcagac caatccagcc cgaacttgta aagacctgca actcagccat 5160 cctgacttcc cagatggtga atattggatt gatcctaacc aaggttgctc aggagattcc 5220 ttcaaagttt actgtaattt cacatctggt ggtgagactt gcatttatcc agacaaaaaa 5280 tctgagggag taagaatttc atcatggcca aaggagaaac caggaagttg gtttagtgaa 5340 tttaagaggg gaaaactgct ttcatactta gatgttgaag gaaattccat caatatggtg 5400 caaatgacat tcctgaaact tctgactgcc tctgctcggc aaaatttcac ctaccactgt 5460 catcagtcag cagcctggta tgatgtgtca tcaggaagtt atgacaaagc acttcgcttc 5520 ctgggatcaa atgatgagga gatgtcctat gacaataatc cttttatcaa aacactgtat 5580 gatggttgtg cgtccagaaa aggctatgaa aagactgtca ttgaaatcaa tacaccaaaa 5640 attgatcaag tacctattgt tgatgtcatg atcaatgact ttggtgatca gaatcagaag 5700 ttcggatttg aagttggtcc tgtttgtttt cttggctaag attaagacaa agaacatatc 5760 aaatcaacag aaaatatacc ttggtgccac caacccattt tgtgccacat gcaagttttg 5820 aataaggatg gtatagaaaa caacgctgca tatacaggta ccatttagga aataccgatg 5880 cctttgtggg ggcagaatca catggcaaaa gctttgaaaa tcataaagat ataa gttggt 5940 gtggctaaga tggaaacagg gctgattctt gattcccaat tctcaactct ccttttccta 6000 tttgaatttc tttggtgctg tagaaaacaa aaaaagaaaa atatatattc ataaaaaata 6060 tggtgctcat tctcatccat ccaggatgta ctaaaacagt gtgtttaata aattgtaatt 6120 attttgtgta cagttctata ctgttatctg tgtccatttc caaaacttgc acgtgtccct 6180 gaattccatc tgactctaat tttatgagaa ttgcagaact ctgatggcaa taaatatatg 6240 tattatgaaa aaataaagtt gtaatttctg atgactctaa gtccctttct ttggttaata 6300 ataaaatgcc tttgtatata ttgatgttga agagttcaat tatttgatgt cgccaacaaa 6360 attctcagag ggcaaaaatc tggaagactt ttggaagcac actctgatca actcttctct 6420 gccgacagtc attttgctga atttcagcca aaaatattat gcattttgat gctttattca 6480 aggctatacc tcaaactttt tcttctcaga atccaggatt tcacaggata cttgtatata 6540 tggaaaacaa gcaagtttat atttttggac agggaaatgt gtgtaagaaa gtatattaac 6600 aaatcaatgc ctccgtcaag caaacaatca tatgtatact ttttttctac gttatctcat 6660 ctccttgttt tcagtgtgct tcaataatgc aggttaatat taaagatgga aattaagcaa 6720 ttatttatga atttgtgcaa tgttagattt tcttatcaat caagttcttg aatttgattc 6780 taagttgcat attataacag tctcgaaaat tattttactt gcccaacaaa tattactttt 6840 ttcctttcaa gataatttta taaatcattt gacctaccta attgctaaat gaataacata 6900 tggtggactg ttattaagag tatttgtttt aagtcattca ggaaaatcta aacttttttt 6960 tccactaagg tatttacttt aaggtagctt gaaatagcaa tacaatttaa aaattaaaaa 7020 ctgaattttg tatctatttt aagtaatata tgtaagactt gaaaataaat gttttatttc 7080 ttatataaag tgttaaatta attgatacca gatttcactg gaacagtttc aactgataat 7140 ttatgacaaa agaacatacc tgtaatattg aaattaaaaa gtgaaatttg tcataaagaa 7200 tttcttttat ttttgaaatc gagtttgtaa atgtcctttt aagaagggag atatgaatcc 7260 aataaataaa ctcaagtctt ggctacctgg a 7291

<210> 3

<211> 3995

<212> DNA

<213> Homo sapiens

<400> 3

cgaattttca aagaacatat tttccgttca cccccgctgg tcttttactg ccatcaatac 60 actgttcttg gtgcaaatac ttcagcctct ttattcaaag tatgttttat gtttttgcca 120 aatatgatct ctaattgaaa gtttattttt ggttttggat gaatctgcgg agcttaagtt 180 gtgagaagaa agggggaaca agacacaatg aaagaaaagt ccaaaaatgc tgcgcggact 240 aggagggaga aggaaaacag tgaattttat gaactggcta aattactgcc tttgccctcg 300 gctatcacct cgcagctgga caaagcatcc ataatcagac tcacgaccag ctatctcaaa 360 atgagagtgg tgttcccaga agggctcggc gaggcgtggg gccactcaag tcggaccagc 420 cccctggaca acgttggccg agaactgggc tcccatctgc tccagaccct ggatggcttc 480 atcttcgtgg tagccccaga tgggaagatc atgtacatct cagagacagc ctcagtccac 540 ttgggtcttt ctcaggtaga gctgaccgga aacagcattt atgaatacat tcacccggca 600 gaccacgacg agatgacggc ggtgctcacc gcccatcaac cctaccactc tcacttcgtg 660 caggagtatg agatcgagcg ctccttcttc ctgaggatga agtgcgtctt ggccaagcgt 720 aacgccggcc tcacctgtgg cggctacaag gtcatccact gcagcggcta cttgaagatc 780 cgccagtaca gcctggacat gtcccccttc gacggctgct accaaaacgt gggcctggtg 840 gccgtgggcc actcgctgcc tcccagcgcc gtcacggaga tcaagctaca cagcaatatg 900 tttatgttcc gcgccagcct ggacatgaag ctcatctttc tggactccag ggtggcggag 960 ctgacggggt acgaacctca ggacctgatt gagaagactc tgtaccacca tgtgcacggc 1020 tgcgacacct tccacctgcg ctgcgcgcac catttgctgc tggtgaaggg acaggtgacc 1080 accaagtact acaggttcct ggcgaaacac ggcggctggg tatgggtgca gagctacgcg 1140 accatcgtgc acaacagtcg ctcctccagg ccacactgta tcgtcagcgt caactatgtc 1200 ctcacagaca cagaatacaa agggctgcag ctctccctgg atcagatctc agcctccaaa 1260 ccagccttct cctataccag cagctccacc cccaccatga ctgacaacag aaagggggcc 1320 aaatcccggc tctccagctc aaagtcaaaa tccaggactt ccccataccc tcagtattcg 1380 ggatttcaca cagaaagatc ggaatctgat catgacagcc agtggggcgg aagtcccttg 1440 accgacacgg cctctccgca gcttctggac cccgccgata ggcctggctc ccagcacgac 1500 gcatcgtgcg cctacagaca gttttcggac cgcagctctc tctgctatgg ctttgcgctt 1560 gaccactcga ggctggtgga agagaggcat ttccataccc aggcctgtga aggaggccga 1620 tgtgaggcag gcaggtactt cctgggaacg ccgcaggccg ggagggagcc ctggtggggc 1680 tctcgcgcag ccttgcccct gacaaag gcc tccccagaaa gcagagaagc ctatgaaaac 1740 agcatgcctc acatcgcttc agtccacagg atccatgggc gaggtcattg ggatgaagat 1800 agtgtggtca gttctccaga ccctgggtcg gccagtgaat caggtgaccg atatcgtact 1860 gagcagtatc aaagtagccc acatgaaccc agcaaaattg aaactcttat aagagccact 1920 cagcaaatga ttaaagaaga agagaacaga ttacagctaa ggaaagcccc ctcagaccaa 1980 ctggcttcca ttaatggggc tgggaaaaaa cactccctgt gttttgcaaa ctaccaacag 2040 cccccaccaa caggtgaagt ctgccatggc tctgctcttg ccaacacttc accatgtgac 2100 catatccagc agagagaggg aaaaatgttg agcccccatg aaaatgacta tgacaacagt 2160 cccaccgcac tatctcggat aagtagtccc aattcggatc gcatttcaaa atccagtttg 2220 atcctagcta aagactatct gcattcggat atatctcctc atcagacagc aggagaccac 2280 cctactgtct ctccaaactg ctttggctct caccggcagt attttgacaa gcatgcttac 2340 acattaactg gatatgccct ggagcactta tatgacagcg aaaccattag aaactattcc 2400 ttgggctgta atggctcaca ctttgatgta acttcccatc tgaggatgca accagaccca 2460 gcacaaggac acaagggaac atctgttata ataaccaacg gaagctgatg ttttgctgaa 2520 atattttgtt ctttaaggat ctctgaaaca ta tttatagt ttaatacccc attaccagca 2580 tttactatgc cacagattgt tagagagtat aacttaagtt actgggtatt tgatacgtgt 2640 tcctataaaa tcaaagaaaa catagcacta gcattcaggg ttatacacag aaaagggagc 2700 taaattgaat acacaaattt cccctctaat tatatgggaa ccagaataga taaattttga 2760 cttgaaaaat attcatgtag atcaagtgtg catatatact acatgagagg actgatgaat 2820 gacaacattg cattgtgact atccagtgat cctcaaacac acaaactatt acttacaaac 2880 tgcggtatac attttacata tggaaatata ggctatgtaa tgtaaataca tcaaaaatgg 2940 gtaattttct ttgactctgt cacactaaac ttcttaacga aatttccatt cccaaaataa 3000 ctgagaaaga gagagataca tcttataaac tgacttcttt gtggtttcaa atcagccagc 3060 tcatttggtt caggcataaa ttagagaaat ggttctggat atggtgcaaa aatgagtttt 3120 cacctggtat ccattataaa caatcaggaa gaggtaattt ttcaccttgc ttttcagtta 3180 gacaaggacc aggattgcac tgacatggcg ctgagggttt ttctaagtaa gaacactgag 3240 atattgggac acacatcaaa aacctggagt gctcaattgg aagtagttct atgaatatgg 3300 aaaggccaga ggcagagtga aataaaatgc tatctcaaag tttaacacaa tttaagggct 3360 cagcataagt aaacaacata tttggggttt gcttgtaa aa ccaactaaat aaaaaattca 3420 aaccaattca cccagaaaaa agaccaatag gtgcaaaaat aaaaggaaaa ccagtgaagt 3480 gccacatgac agcagtgtta agtgtttgaa aacgtttcaa agcacatatg tgccaatgtg 3540 acaacatgtg gaaagcctca ggagagagtc taagataaaa gcttaggctg atagacaagt 3600 agttaagagc taagagcagt actctgaagg aataggcaaa atgtttattt tccttattgt 3660 ttgtaaacaa caaacttggt cttacatctg tgtggtatag tagaaaggcc agctgactag 3720 atctctggat tctaattttg gccctacctg taacttaatt ttgtgaccac agttgtacca 3780 ttcaccgtgc ctgggctcta gtttcctggt ttgtaaggca gccccagcgt tcatgttctg 3840 tgatagagca gaactgaact tattacctaa ttaactctct gctatgagtt gtcaagactg 3900 atcattctgt tttttctgta cacagaagtt tagatgcttt gtgacttaag caggtgtgtgttg 3960 ggctccttacta ggtagataggc 3960 ggctccttacta ggtaggc

<210> 4

<212> DNA

<213> Homo sapiens

<400> 4

gcgctttgaa atcccatccc ggctttgttg tctccctccc aaggggccgg aatgctccgg 60 gcccgcggta taaaggcagc cgcggtggcg gtggcggcgc agagctctgt gctccctgca 120 gtcaggactc tgggaccgca gggggctccc ggaccctgac tctgcagccg aaccggcacg 180 gtttcgtggg gacccaggct tgcaaagtga cggtcatttt ctctttcttt ctccctcttg 240 agtccttctg agatgatggc tctgggcgca gcgggagcta cccgggtctt tgtcgcgatg 300 gtagcggcgg ctctcggcgg ccaccctctg ctgggagtga gcgccacctt gaactcggtt 360 ctcaattcca acgctatcaa gaacctgccc ccaccgctgg gcggcgctgc ggggcaccca 420 ggctctgcag tcagcgccgc gccgggaatc ctgtacccgg gcgggaataa gtaccagacc 480 attgacaact accagccgta cccgtgcgca gaggacgagg agtgcggcac tgatgagtac 540 tgcgctagtc ccacccgcgg aggggacgca ggcgtgcaaa tctgtctcgc ctgcaggaag 600 cgccgaaaac gctgcatgcg tcacgctatg tgctgccccg ggaattactg caaaaatgga 660 atatgtgtgt cttctgatca aaatcatttc cgaggagaaa ttgaggaaac catcactgaa 720 agctttggta atgatcatag caccttggat gggtattcca gaagaaccac cttgtcttca 780 aaaatgtatc acaccaaagg acaagaaggt tctgtttgtc tccggtcatc agactgtgcc 840 tcaggattgt gttgtgctag acacttctgg tccaagatct gtaaacctgt cctgaaagaa 900 ggtcaagtgt gtaccaagca taggagaaaa ggctctcatg gactagaaat attccagcgt 960 tgttactgtg gagaaggtct gtcttgccgg atacagaaag atcaccatca agccagtaat 1020 tcttctaggc ttcacacttg tcagagacac taaaccagct atccaaatgc agtgaactcc 1080 ttttatataa tagatgctat gaaaaccttt tatgaccttc atcaactcaa tcctaaggat 1140 atacaagttc tgtggtttca gttaagcatt ccaataacac cttccaaaaa cctggagtgt 1200 aagagctttg tttctttatg gaactcccct gtgattgcag taaattactg tattgtaaat 1260 tctcagtgtg gcacttacct gtaaatgcaa tgaaactttt aattattttt ctaaaggtgc 1320 tgcactgcct atttttcctc ttgttatgta aatttttgta cacattgatt gttatcttga 1380 ctgacaaata ttctatattg aactgaagta aatcatttca gcttatagtt cttaaaagca 1440 taacccttta ccccatttaa ttctagagtc tagaacgcaa ggatctcttg gaatgacaaa 1500 tgataggtac ctaaaatgta acatgaaaat actagcttat tttctgaaat gtactatctt 1560 aatgcttaaa ttatatttcc ctttaggctg tgatagtttt tgaaataaaa tttaacattt 1620 aatatcatga aatgttataa gtagacatac attttgggat tgtgatctta gaggtttgtg 1680 tgtgtgtacg tatgtgtgtg ttctaca aga acggaagtgt gatatgttta aagatgatca 1740 gagaaaagac agtgtctaaa tataagacaa tattgatcag ctctagaata actttaaaga 1800 aagacgtgtt ctgcattgat aaactcaaat gatcatggca gaatgagttagt aaatcttataa ctctacataa tataaacta 1860

<210> 5

<211> 3335

<212> DNA

<213> Homo sapiens

<400> 5

ctagcggacg ctgccacctg atcccatcca ggcgctgaga ggcaagccac cggcactcgg 60 cgcgcagggc cccgggcacc ctcggacccc gtgcggattt cgagacaagg gtccagagat 120 agcggcggcc gcccccaacc cgggccagag gcagagccca ggcgtgcggt gcttctcggg 180 aacactggcc ctgctaccag agccgcagga tcccccatga gtgcctgtga agggagtcct 240 ggatagtgag gccctggctg gtccaggggt gggtactccg ccatgccacc catcctccag 300 cgcctgcagc aggccaccaa gatgatgagc cgccggaaaa tcctgctgct ggtgctaggg 360 tgcagcaccg taagccttct catccaccag ggggcgcagc tcagctggta ccccaagctg 420 ttccccttga gctgccctcc tctgcggaac tcgccgccgc gccccaagca catgactgtg 480 gccttcctga agactcacaa gacggcaggc acgacggtgc agaacatcct gtttcgcttt 540 gccgagcgcc acaacctgac ggtggccctg ccgcacccga gctgcgagca ccagttctgc 600 tacccccgca acttctcggc gcacttcgtg cacccggcca cgcggccgcc gcacgtgctg 660 gccagccacc tgcgcttcga ccgtgcggag ctggagcgcc tcatgccgcc cagcaccgtc 720 tatgtcacca tcctgcgcga gccggccgcc atgttcgagt cgctcttcag ctactacaac 780 cagtactgcc cggccttccg gcgcgtaccc aacgcctcgc tcgaggcctt cctgcgcgcg 840 cccgaggcat actaccgcgc tggcgagcac ttcgccatgt tcgcacacaa cacgctggcc 900 tacgacctgg gcggcgacaa tgagcgcagc ccgcgcgacg acgccgccta cctggcgggc 960 ctcatccgcc aggtggagga ggttttctcg ctcgtcatga tcgccgagta cttcgacgag 1020 tcgctagtgc tgctgcggcg cctactggcc tgggacctgg acgacgtgct ctacgccaag 1080 ctcaacgcgc gcgccgccag ctcgcgcctg gccgccatcc ccgcggcgct ggcgcgggcg 1140 gcgcgcacct ggaacgccct ggacgccggc ctctacgacc acttcaacgc caccttctgg 1200 cgccacgtgg cgcgcgcggg ccgcgcgtgc gtggagcgcg aggcgcgcga gctgcgcgag 1260 gcccgccagc gcctactgcg gcgctgcttc ggggacgagc cactgctgcg gcctgccgcg 1320 cagatccgca ccaagcagct gcagccgtgg cagcccagcc gcaaggtgga cattatgggc 1380 tatgacctgc ccggcggcgg cgccggcccg gccaccgagg cctgcctcaa gctggccatg 1440 cccgaggtcc agtactcgaa ctacctgttg cgcaagcaga agcgccgggg cggtgcgcgg 1500 gctcggcccg agcccgtcct ggacaatccc ccgcctcggc ccatccgagt gctgcctcgc 1560 ggccctcaag gtccctgagc tccgcaggcc tccaggagta tgggtcctgc cctgccctcc 1620 caggagtcca gccaggggct gcctccatgt cctgccccca tgagagccct ttcttttcaa 1680 ggggcttgag tccctcgctg aactttg ggg tacgcctccc tatctgagcc cccgctcccg 1740 atcgtgggct gaaccttgcc ttcgggcacg gcgctgatag gctggatcca gcgctgggcc 1800 accctctcct tcccagctgg ttggtgattt tgacaagagg cctgagggct gtccagaact 1860 gctcaccctg cctttttctg ggggctgagc ccccacaacc tgtcttttcc aagtggataa 1920 gcccaggccc catggagaga ggctcagccc tagggaaagg ctgctcttgc tctccctccc 1980 agggcttgag gctccctata cttttctgtc ttccaggttc taccctcccc acagacctaa 2040 gctgctccca gtctcccaga accaagagtt cttgggttgg gatttttgtt tggttggttt 2100 ttcttttttt taataaaaca tctttaaaat gtgcgagttg tatgtcagtc atagaagccg 2160 gattccccac atttgaggat gaaacggatg cccaggagga gctgtggctg gacccatgag 2220 gatgggggcc tggactcaag gtcccccgcc acctggcata ggcctacaac cctcctcaac 2280 cgcttgaccg actgggcccc caacacagac tggccacctt agccccactc actttccaca 2340 tcgttctttt tcacaaaatt ctgcgacatt tactgagcat catctctgta acttggggat 2400 acaggtgaac cagatagata gggccttatt ttcatggagc ttatggggaa acattggaac 2460 aggagcaaga caggtaagca aagaggaagg actggagggg ttagagcaca acacctcttt 2520 gggtggaagg aggatcattt tcagataagg ag ctgcctaa ctcagaattt gacctcctcc 2580 tgtgaatcac tgacttcact aatggcctgc tttcctcaca agaagacaac ttggactcag 2640 atgaagacca gtccctttct gcagtttcct ttaacacagc aagatagcca gcacttccag 2700 gtgaaatctc ccttaagact agcagagaat tggaactttt tctgttactt tcttcagagc 2760 tatatcgacg tatctggaca agtgataaaa aacaatagct attatggtct acggtgtgcc 2820 agccactgtt taagggcttt atgtatgtta actttggaga tgggtgccgt tttccaggtg 2880 agggaactga aacttggggg aaattaagtc agggtcacac agccagaaag tggccactag 2940 gatctcaacc aaggcagtgt ggcttcagag tttgtgctca caagtgctgt ctgtagtgcc 3000 attcaaataa aaaaggtcac atgccacaag ctaggctgat tcccagcacc ttcccccaaa 3060 agaatgtaaa gggaagaacc aggacactgc tatcagcaac tctttatcca gtttttcatg 3120 gagacaacgg ttgacctgtt tggacaatgg acattacagt gattgattgg caggatgcat 3180 gacacgctgg gcttgaaaag ggactgtttg tatggcaccc cactttttac cagcagagct 3240 ctgaaaaagg caataattag aatcatgcaa aagcataatt aagtttctga cattaagcag 3300 cagtaaagaa ttgtaccaga aaaaaaaaaa aaaaa 3335

<210> 6

<211> 6333

<212> DNA

<213> Homo sapiens

<400> 6

gtagtctctt tggaaacttc tgcaggggaa aagagctagg aaagagctgc aaagcagtgt 60 gggctttttc ccttttttgc tccttttcat tacccctcct ccgttttcac ccttctccgg 120 acttcgcgta gaacctgttttttga atttcga taga tcaggtaggt 180

ctcccaccct ctcggctgca gccaacgcct cttacctgtt ctgcggcgcc gcgcaccgct 300 ggcagctgag ggttagaaag cggggtgtat tttagatttt aagcaaaaat tttaaagata 360 aatccatttt tctctcccac ccccaacgcc atctccactg catccgatct cattatttcg 420 gtggttgctt gggggtgaac aattttgtgg ctttttttcc cctataattc tgacccgctc 480 aggcttgagg gtttctccgg cctccgctca ctgcgtgcac ctggcgctgc cctgcttccc 540 ccaacctgtt gcaaggcttt aattcttgca actgggacct gctcgcaggc accccagccc 600 tccacctctc tctacatttt tgcaagtgtc tgggggaggg cacctgctct acctgccaga 660 aattttaaaa caaaaacaaa aacaaaaaaa tctccggggg ccctcttggc ccctttatcc 720 ctgcactctc gctctcctgc cccaccccga ggtaaagggg gcgactaaga gaagatggtg 780 ttgctcaccg cggtcctcct gctgctggcc gcctatgcgg ggccggccca gagcctgggc 840 tccttcgtgc actgcgagcc ctgcgacgag aaagccctct ccatgtgccc ccccagcccc 900 ctgggctgcg agctggtcaa ggagccgggc tgcggctgct gcatgacctg cgccctggcc 960 gaggggcagt cgtgcggcgt ctacaccgag cgctgcgccc aggggctgcg ctgcctcccc 1020 cggcaggacg aggagaagcc gctgcacgcc ctgctgcacg gccgcggggt ttgcctcaac 1080 gaaaagagct accgcga gca agtcaagatc gagagagact cccgtgagca cgaggagccc 1140 accacctctg agatggccga ggagacctac tcccccaaga tcttccggcc caaacacacc 1200 cgcatctccg agctgaaggc tgaagcagtg aagaaggacc gcagaaagaa gctgacccag 1260 tccaagtttg tcgggggagc cgagaacact gcccaccccc ggatcatctc tgcacctgag 1320 atgagacagg agtctgagca gggcccctgc cgcagacaca tggaggcttc cctgcaggag 1380 ctcaaagcca gcccacgcat ggtgccccgt gctgtgtacc tgcccaattg tgaccgcaaa 1440 ggattctaca agagaaagca gtgcaaacct tcccgtggcc gcaagcgtgg catctgctgg 1500 tgcgtggaca agtacgggat gaagctgcca ggcatggagt acgttgacgg ggactttcag 1560 tgccacacct tcgacagcag caacgttgag tgatgcgtcc ccccccaacc tttccctcac 1620 cccctcccac ccccagcccc gactccagcc agcgcctccc tccaccccag gacgccactc 1680 atttcatctc atttaaggga aaaatatata tctatctatt tgaggaaact gaggacctcg 1740 gaatctctag caagggctca acttcgaaaa tggcaacaac agagatgcaa aaagctaaaa 1800 agacaccccc cccctttaaa tggttttctt tttgaggcaa gttggatgaa cagagaaggg 1860 aagagaggaa gaacgagagg aagagaaggg aaggaagtgt ttgtgtagaa gagagagaaa 1920 gacgaataga gttaggaaaa gg aagacaag caggtgggca ggaaggacat gcaccgagac 1980 caggcagggg cccaactttc acgtccagcc ctggcctggg gtcgggagag gtgggcgcta 2040 gaagatgcag cccaggatgt ggcaatcaat gacactattg gggtttccca ggatggattg 2100 gtcaggggga gaaaggaaaa ggcaaaacac tccaggacct ctcccggatc tgtctcctcc 2160 tctagccagc agtatggaca gctggacccc tgaacttcct ctcctcttac ctgggcagag 2220 tgttgtctct ccccaaattt ataaaaacta aaatgcattc cattcctctg aaagcaaaac 2280 aaattcataa ttgagtgata ttaaatagag aggttttcgg aagcagatct gtgaatatga 2340 aatacatgtg catatttcat tccccaggca gacatttttt agaaatcaat acatgcccca 2400 atattggaaa gacttgttct tccacggtga ctacagtaca tgctgaagcg tgccgtttca 2460 gccctcattt aattcaattt gtaagtagcg cagcagcctc tgtgggggag gataggctga 2520 aaaaaaaaag tgggctcgta tttatctaca ggactccata tagtcatata taggcatata 2580 aatctattct ttttctttgt ttttttcttt cttcctttct ttcaaaggtt tgcattaact 2640

tttcaaagta gttcctatag gggcattgag gagcttcctc attctgggaa aactgagaaa 2700 acccatattc tcctaataca acccgtaata gcatttttgc ctgcctcgag gcagagtttc 2760 ccgtgagcaa taaactcagc ttttttgtgg ggcacagtac tggatttgac agtgattccc 2820 cacgtgtgtt catctgcacc caccgagcca ggcagaggcc agccctccgt ggtgcacaca 2880 gcacgcgcct cagtccatcc cattttagtc tttaaaccct caggaagtca cagtctccgg 2940 acaccacacc acatgagccc aacaggtcca cgatggatcc accagtccca ccccagcctt 3000 ttcctttcat ctgaacagaa tgtgcatttt tggaagcctc cctcactctc catgctggca 3060 gagcaggagg gagactgaag taagagatgg cagagggaga tggtggcaaa aaggtttaga 3120 tgcaggagaa cagtaagatg gatggttccg gccagagtcg atgtggggag gaacagaggg 3180 ctgaagggag agggggctga ctgttccatt ctagctttgg cacaaagcag cagaaagggg 3240 gaaaagccaa tagaaatttc cttagcttcc ccaccatatg tattttctag gatttgagag 3300 gaaagagagg aaaatggggg aatgggttgc aaaatagaaa tgagcttaat ccaggccgca 3360 gagccaggga aggtgagtaa ctttaggagg gtgctagact ttagaagcca gataggaaga 3420 atcagtctaa actggccatg ctttggaagg gacaagacta tgtgctccgc tgcccacctt 3480 cagcct gcaa tgagggactg aggcccacga gtctttccag ctcttcctcc attctggcca 3540 gtccctgcat cctccctggg gtggaggatg gaaggaaagc tgggacaagc agggaacgca 3600 tgattcaggg atgctgtcac tcggcagcca gattccgaaa ctcccattct ccaatgactt 3660 cctcaaccaa tgggtggcct tgtgactgtt ctttaaggct gaagatatcc aggaaagggg 3720 gcttggacac tggccaagga gaccccttcg tgctgtggac acagctctct tcactctttg 3780 ctcatggcat gacacagcgg agaccgcctc caacaacgaa tttggggcta cgaagaggaa 3840 tagcgaaaaa gcaaatctgt ttcaactgat gggaacccta tagctataga acttgggggc 3900 tatctcctat gcccctggac aggacagttg gctggggaca ggagaagtgc tcaatcttca 3960 tgagacaaag gggcccgata gggccagcag ccacaaggcc ttgacctgcc gagtcagcat 4020 gccccatctc tctgcacagc tgtcccctaa acccaactca cgtttctgta tgtcttaggc 4080 cagtatccca aacctcttcc acgtcactgt tctttccacc cattctccct ttgcatcttg 4140 agcagttatc caactaggat ctgccaagtg gatactgggg tgccactccc ctgagaaaag 4200 actgagccag gaactacaag ctccccccac attcctccca gcctggacct aattcttgag 4260 aggggctctc tcttcacgga ctgtgtctgg actttgagca ggcttctgcc ccttgcgttg 4320 gctctttgct g ccagccatc aggtggggga ttagagcctg gtgtaagtgc gccagactct 4380 tccggtttcc aaagttcgtg cctgcgaacc caaacctgtg agtctcttct gcatgcagga 4440 gtttctcctg ggcagctggt cactccccag agaagctggg ccttcatgga cacatggaac 4500 taagcctccc aaatgggagt tctggctgag cccagggtgg ggagatcctg ggaagggagg 4560 cactggagga agacggcacc tcttccccca tggcagggtg tgagggaggc aggtttggaa 4620 tggtgcgagt atggcaatct aagcaggggt ctggtctctt tgactccagg ctggcctttg 4680 gccgactgtc tgctcaccca gagaccttgg actccggact atccatggct ccgaatctaa 4740 gtgctgccca ctcccatgct cacacccaca gaaggtcttc ccatcccctt tagattcgtg 4800 cctcactcca ccagtgagga agatgcctct gtctttccca cgactgccag gagataggga 4860 agcccagcca ggactgaccc tccttcctcc agcctgccct gacccacctg gcaaagcagg 4920 gcacatgggg aggaagagac tggaaccttt ctttgacagc caggcctaga cagacaggcc 4980 tggggacact ggccccatga ggggaggaag gcaggcgcac gaggtccagg gaggcccttt 5040 tctgatcatg ccccttctct cccaccccat ctccccacca ccacctctgt ggcctccatg 5100 gtacccccac agggctggcc tcccctagag ggtgggcctc aaccacctgc tcccgccacg 5160 caccggttag tgagaca ggg ctgccacggc aaccgccaag cccccctcaa ggtgggacag 5220 taccccggac ccatccactc actcctgaga gggctccggc ccagaatggg aacctcagag 5280 aagagctcta aggagaagaa accccatagc gtcagagagg atatgtctgg cttccaagag 5340 aaaggaggct ccgttttgca aagtggagga gggacgaggg acaggggttt caccagccag 5400 caacctgggc cttgtactgt ctgtgttttt aaaaccacta aagtgcaaga attacattgc 5460 actgtttctc cactttttat tttctcttag gcttttgttt ctatttcaaa catactttct 5520

tggttttcta atggagtata tagtttagtc atttcacaga ctctggcctc ctctcctgaa 5580 atccttttgg atggggaaag ggaaggtggg gagggtccga ggggaagggg accccagctt 5640 ccctgtgccc gctcacccca ctccaccagt ccccggtcgc cagccggagt ctcctctcta 5700 ccgccactgt cacaccgtag cccacatgga tagcacagtt gtcagacaag attccttcag 5760 attccgagtt gcctaccggt tgttttcgtt gttgttgttg ttgtttttct ttttcttttt 5820

ttttttgaag acagcaataa ccacagtaca tattactgta gttctctata gttttacata 5880 cattcatacc ataactctgt tctctcctct tttttgtttt caactttaaa aacaaaaata 5940 aacgatgata atctttactg gtgaaaagga tggaaaaata aatcaacaaa tgcaaccagt 6000 ttgtgagaaa aaaaaaaaaa agccgaaaaa aaaaaaaaaa acacctgaat gcggaagagc 6060 tcggctcccg tttagcattt tgtacttaag gaaataaaaa accaacaaag gatctcacat 6120 tttcttaaaa agtgaagatt gctgtatact atttattcaa cttataattt atgttactcc 6180

ttgatctttg tcttttgtca tgacaaagca tttatttaat aaagttatgc attcagttaa 6240 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa aaaaaaaaaa aaaaaaaaaa 6300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 6333

<210> 7

<211> 3659

<212> DNA

<213> Homo sapiens

<400> 7

cgggagcccg cggcgagcgt agcgcaagtc cgctccctag gcatcgctgc gctggcagcg 60 attcgctgtc tcttgtgagt caggggacaa cgcttcgggg caactgtgag tgcgcgtgtg 120 ggggacctcg attctcttca gatctcgagg attcggtccg gggacgtctc ctgatcccct 180 actaaagcgc ctgctaactt tgaaaaggag cactgtgtcc tgcaaagttt gacacataaa 240 ggataggaaa agagaggaga gaaaagcaac tgagttgaag gagaaggagc tgatgcgggc 300 ctcctgatca attaagagga gagttaaacc gccgagatcc cggcgggacc aaggaggtgc 360 ggggcaagaa ggaacggaag cggtgcgatc cacagggctg ggttttcttg caccttgggt 420 cacgcctcct tggcgagaaa gcgcctcgca tttgattgct tccagttatt gcagaacttc 480 ctgtcctggt ggagaagcgg gtctcgcttg ggttccgcta atttctgtcc tgaggcgtga 540 gactgagttc atagggtcct gggtccccga accaggaagg gttgagggaa cacaatctgc 600 aagcccccgc gacccaagtg aggggccccg tgttggggtc ctccctccct ttgcattccc 660 acccctccgg gctttgcgtc ttcctgggga ccccctcgcc gggagatggc cgcgttgatg 720 cggagcaagg attcgtcctg ctgcctgctc ctactggccg cggtgctgat ggtggagagc 780 tcacagatcg gcagttcgcg ggccaaactc aactccatca agtcctctct gggcggggag 840 acgcctggtc aggccgccaa tcgatctgcg ggcatgtacc aaggactggc attcggcggc 900 agtaagaagg gcaaaaacct ggggcaggcc tacccttgta gcagtgataa ggagtgtgaa 960 gttgggaggt attgccacag tccccaccaa ggatcatcgg cctgcatggt gtgtcggaga 1020 aaaaagaagc gctgccaccg agatggcatg tgctgcccca gtacccgctg caataatggc 1080 atctgtatcc cagttactga aagcatctta acccctcaca tcccggctct ggatggtact 1140 cggcacagag atcgaaacca cggtcattac tcaaaccatg acttgggatg gcagaatcta 1200 ggaagaccac acactaagat gtcacatata aaagggcatg aaggagaccc ctgcctacga 1260 tcatcagact gcattgaagg gttttgctgt gctcgtcatt tctggaccaa aatctgcaaa 1320 ccagtgctcc atcaggggga agtctgtacc aaacaacgca agaagggttc tcatgggctg 1380 gaaattttcc agcgttgcga ctgtgcgaag ggcctgtctt gcaaagtatg gaaagatgcc 1440 acctactcct ccaaagccag actccatgtg tgtcagaaaa tttgatcacc attgaggaac 1500 atcatcaatt gcagactgtg aagttgtgta tttaatgcat tatagcatgg tggaaaataa 1560 ggttcagatg cagaagaatg gctaaaataa gaaacgtgat aagaatatag atgatcacaa 1620 aaagggagaa agaaaacatg aactgaatag attagaatgg gtgacaaatg cagtgcagcc 1680 agtgtttcca ttatgcaact tgtctat gta aataatgtac acatttgtgg aaaatgctat 1740 tattaagaga acaagcacac agtggaaatt actgatgagt agcatgtgac tttccaagag 1800 tttaggttgt gctggaggag aggtttcctt cagattgctg attgcttata caaataacct 1860 acatgccaga tttctattca acgttagagt ttaacaaaat actcctagaa taacttgtta 1920 tacaataggt tctaaaaata aaattgctaa acaagaaatg aaaacatgga gcattgttaa 1980 tttacaacag aaaattacct tttgatttgt aacactactt ctgctgttca atcaagagtc 2040 ttggtagata agaaaaaaat cagtcaatat ttccaaataa ttgcaaaata atggccagtt 2100 gtttaggaag gcctttagga agacaaataa ataacaaaca aacagccaca aatacttttt 2160 tttcaaaatt ttagttttac ctgtaattaa taagaactga tacaagacaa aaacagttcc 2220 ttcagattct acggaatgac agtatatctc tctttatcct atgtgattcc tgctctgaat 2280 gcattatatt ttccaaacta tacccataaa ttgtgactag taaaatactt acacagagca 2340 gaattttcac agatggcaaa aaaatttaaa gatgtccaat atatgtggga aaagagctaa 2400 cagagagatc attatttctt aaagattggc cataacctgt attttgatag aattagattg 2460 gtaaatacat gtattcatac atactctgtg gtaatagaga cttgagctgg atctgtactg 2520 cactggagta agcaagaaaa ttgggaaaac tt tttcgttt gttcaggttt tggcaacaca 2580 tagatcatat gtctgaggca caagttggct gttcatcttt gaaaccaggg gatgcacagt 2640 ctaaatgaat atctgcatgg gatttgctat cataatattt actatgcaga tgaattcagt 2700 gtgaggtcct gtgtccgtac tatcctcaaa ttatttattt tatagtgctg agatcctcaa 2760 ataatctcaa tttcaggagg tttcacaaaa tggactcctg aagtagacag agtagtgagg 2820 tttcattgcc ctctataagc ttctgactag ccaatggcat catccaattt tcttcccaaa 2880 cctctgcagc atctgcttta ttgccaaagg gctagtttcg gttttctgca gccattgcgg 2940 ttaaaaaata taagtaggat aacttgtaaa acctgcatat tgctaatcta tagacaccac 3000 agtttctaaa ttctttgaaa ccactttact acttttttta aacttaactc agttctaaat 3060 actttgtctg gagcacaaaa caataaaagg ttatcttata gtcgtgactt taaacttttg 3120 tagaccacaa ttcacttttt agttttcttt tacttaaatc ccatctgcag tctcaaattt 3180 aagttctccc agtagagatt gagtttgagc ctgtatatct attaaaaatt tcaacttccc 3240 acatatattt actaagatga ttaagactta cattttctgc acaggtctgc aaaaacaaaa 3300 attataaact agtccatcca agaaccaaag tttgtataaa caggttgcta taagcttggt 3360 gaaatgaaaa tggaacattt caatcaaaca tttcctat at aacaattatt atatttacaa 3420 tttggtttct gcaatatttt tcttatgtcc acccttttaa aaattattat ttgaagtaat 3480 ttatttacag gaaatgttaa tgagatgtat tttcttatag agatatttct tacagaaagc 3540 tttgtagcag aatatatttg cagctattga ctttgtaatt taggaaaaat gtataataag 3600 ataaaatcta ttaaattttt ctcctctaaa aactgaaaaa aaaaaaaaaa aaaaaaaaa 3659

<210> 8

<211> 5411

<212> DNA

<213> Homo sapiens

<400> 8

gtgtcccata gtgtttccaa acttggaaag ggcgggggag ggcgggagga tgcggagggc 60 ggaggtatgc agacaacgag tcagagtttc cccttgaaag cctcaaaagt gtccacgtcc 120 tcaaaaagaa tggaaccaat ttaagaagcc agccccgtgg ccacgtccct tcccccattc 180 gctccctcct ctgcgccccc gcaggctcct cccagctgtg gctgcccggg cccccagccc 240 cagccctccc attggtggag gcccttttgg aggcacccta gggccaggga aacttttgcc 300 gtataaatag ggcagatccg ggctttatta ttttagcacc acggcagcag gaggtttcgg 360 ctaagttgga ggtactggcc acgactgcat gcccgcgccc gccaggtgat acctccgccg 420 gtgacccagg ggctctgcga cacaaggagt ctgcatgtct aagtgctaga catgctcagc 480 tttgtggata cgcggacttt gttgctgctt gcagtaacct tatgcctagc aacatgccaa 540 tctttacaag aggaaactgt aagaaagggc ccagccggag atagaggacc acgtggagaa 600 aggggtccac caggcccccc aggcagagat ggtgaagatg gtcccacagg ccctcctggt 660 ccacctggtc ctcctggccc ccctggtctc ggtgggaact ttgctgctca gtatgatgga 720 aaaggagttg gacttggccc tggaccaatg ggcttaatgg gacctagagg cccacctggt 780 gcagctggag ccccaggccc tcaaggtttc caaggacctg ctggtgagcc tggtgaacct 840 ggtcaaactg gtcctgcagg tgctcgtggt ccagctggcc ctcctggcaa ggctggtgaa 900 gatggtcacc ctggaaaacc cggacgacct ggtgagagag gagttgttgg accacagggt 960 gctcgtggtt tccctggaac tcctggactt cctggcttca aaggcattag gggacacaat 1020 ggtctggatg gattgaaggg acagcccggt gctcctggtg tgaagggtga acctggtgcc 1080 cctggtgaaa atggaactcc aggtcaaaca ggagcccgtg ggcttcctgg tgagagagga 1140 cgtgttggtg cccctggccc agctggtgcc cgtggcagtg atggaagtgt gggtcccgtg 1200 ggtcctgctg gtcccattgg gtctgctggc cctccaggct tcccaggtgc ccctggcccc 1260 aagggtgaaa ttggagctgt tggtaacgct ggtcctgctg gtcccgccgg tccccgtggt 1320 gaagtgggtc ttccaggcct ctccggcccc gttggacctc ctggtaatcc tggagcaaac 1380 ggccttactg gtgccaaggg tgctgctggc cttcccggcg ttgctggggc tcccggcctc 1440 cctggacccc gcggtattcc tggccctgtt ggtgctgccg gtgctactgg tgccagagga 1500 cttgttggtg agcctggtcc agctggctcc aaaggagaga gcggtaacaa gggtgagccc 1560 ggctctgctg ggccccaagg tcctcctggt cccagtggtg aagaaggaaa gagaggccct 1620 aatggggaag ctggatctgc cggccctcca ggacctcctg ggctgagagg tagtcctggt 1680 tctcgtggtc ttcctggagc tgatggc aga gctggcgtca tgggccctcc tggtagtcgt 1740 ggtgcaagtg gccctgctgg agtccgagga cctaatggag atgctggtcg ccctggggag 1800 cctggtctca tgggacccag aggtcttcct ggttcccctg gaaatatcgg ccccgctgga 1860 aaagaaggtc ctgtcggcct ccctggcatc gacggcaggc ctggcccaat tggcccagct 1920 ggagcaagag gagagcctgg caacattgga ttccctggac ccaaaggccc cactggtgat 1980 cctggcaaaa acggtgataa aggtcatgct ggtcttgctg gtgctcgggg tgctccaggt 2040 cctgatggaa acaatggtgc tcagggacct cctggaccac agggtgttca aggtggaaaa 2100 ggtgaacagg gtccccctgg tcctccaggc ttccagggtc tgcctggccc ctcaggtccc 2160 gctggtgaag ttggcaaacc aggagaaagg ggtctccatg gtgagtttgg tctccctggt 2220 cctgctggtc caagagggga acgcggtccc ccaggtgaga gtggtgctgc cggtcctact 2280 ggtcctattg gaagccgagg tccttctgga cccccagggc ctgatggaaa caagggtgaa 2340 cctggtgtgg ttggtgctgt gggcactgct ggtccatctg gtcctagtgg actcccagga 2400 gagaggggtg ctgctggcat acctggaggc aagggagaaa agggtgaacc tggtctcaga 2460 ggtgaaattg gtaaccctgg cagagatggt gctcgtggtg ctcctggtgc tgtaggtgcc 2520 cctggtcctg ctggagccac aggtgaccgg gg cgaagctg gggctgctgg tcctgctggt 2580 cctgctggtc ctcggggaag ccctggtgaa cgtggtgagg tcggtcctgc tggccccaat 2640 ggatttgctg gtcctgctgg tgctgctggt caacctggtg ctaaaggaga aagaggagcc 2700 aaagggccta agggtgaaaa cggtgttgtt ggtcccacag gccccgttgg agctgctggc 2760 ccagctggtc caaatggtcc ccccggtcct gctggaagtc gtggtgatgg aggcccccct 2820 ggtatgactg gtttccctgg tgctgctgga cggactggtc ccccaggacc ctctggtatt 2880 tctggccctc ctggtccccc tggtcctgct gggaaagaag ggcttcgtgg tcctcgtggt 2940 gaccaaggtc cagttggccg aactggagaa gtaggtgcag ttggtccccc tggcttcgct 3000 ggtgagaagg gtccctctgg agaggctggt actgctggac ctcctggcac tccaggtcct 3060 cagggtcttc ttggtgctcc tggtattctg ggtctccctg gctcgagagg tgaacgtggt 3120 ctaccaggtg ttgctggtgc tgtgggtgaa cctggtcctc ttggcattgc cggccctcct 3180 ggggcccgtg gtcctcctgg tgctgtgggt agtcctggag tcaacggtgc tcctggtgaa 3240 gctggtcgtg atggcaaccc tgggaacgat ggtcccccag gtcgcgatgg tcaacccgga 3300 cacaagggag agcgcggtta ccctggcaat attggtcccg ttggtgctgc aggtgcacct 3360 ggtcctcatg gccccgtggg tcctgctggc aaacatgg aa accgtggtga aactggtcct 3420 tctggtcctg ttggtcctgc tggtgctgtt ggcccaagag gtcctagtgg cccacaaggc 3480 attcgtggcg ataagggaga gcccggtgaa aaggggccca gaggtcttcc tggcttaaag 3540 ggacacaatg gattgcaagg tctgcctggt atcgctggtc accatggtga tcaaggtgct 3600 cctggctccg tgggtcctgc tggtcctagg ggccctgctg gtccttctgg ccctgctgga 3660 aaagatggtc gcactggaca tcctggtaca gttggacctg ctggcattcg aggccctcag 3720 ggtcaccaag gccctgctgg cccccctggt ccccctggcc ctcctggacc tccaggtgta 3780 agcggtggtg gttatgactt tggttacgat ggagacttct acagggctga ccagcctcgc 3840 tcagcacctt ctctcagacc caaggactat gaagttgatg ctactctgaa gtctctcaac 3900 aaccagattg agacccttct tactcctgaa ggctctagaa agaacccagc tcgcacatgc 3960 cgtgacttga gactcagcca cccagagtgg agcagtggtt actactggat tgaccctaac 4020 caaggatgca ctatggatgc tatcaaagta tactgtgatt tctctactgg cgaaacctgt 4080 atccgggccc aacctgaaaa catcccagcc aagaactggt ataggagctc caaggacaag 4140 aaacacgtct ggctaggaga aactatcaat gctggcagcc agtttgaata taatgtagaa 4200 ggagtgactt ccaaggaaat ggctacccaa cttgccttca tgc gcctgct ggccaactat 4260 gcctctcaga acatcaccta ccactgcaag aacagcattg catacatgga tgaggagact 4320 ggcaacctga aaaaggctgt cattctacag ggctctaatg atgttgaact tgttgctgag 4380 ggcaacagca ggttcactta cactgttctt gtagatggct gctctaaaaa gacaaatgaa 4440 tggggaaaga caatcattga atacaaaaca aataagccat cacgcctgcc cttccttgat 4500 attgcacctt tggacatcgg tggtgctgac caggaattct ttgtggacat tggcccagtc 4560 tgtttcaaat aaatgaactc aatctaaatt aaaaaagaaa gaaatttgaa aaaactttct 4620 ctttgccatt tcttcttctt cttttttaac tgaaagctga atccttccat ttcttctgca 4680 catctacttg cttaaattgt gggcaaaaga gaaaaagaag gattgatcag agcattgtgc 4740 aatacagttt cattaactcc ttcccccgct cccccaaaaa tttgaatttt tttttcaaca 4800 ctcttacacc tgttatggaa aatgtcaacc tttgtaagaa aaccaaaata aaaattgaaa 4860 aataaaaacc ataaacattt gcaccacttg tggcttttga atatcttcca cagagggaag 4920 tttaaaaccc aaacttccaa aggtttaaac tacctcaaaa cactttccca tgagtgtgat 4980 ccacattgtt aggtgctgac ctagacagag atgaactgag gtccttgttt tgttttgttc 5040 ataatacaaa ggtgctaatt aatagtattt cagatacttg aagaatgtt g atggtgctag 5100 aagaatttga gaagaaatac tcctgtattg agttgtatcg tgtggtgtat tttttaaaaa 5160 atttgattta gcattcatat tttccatctt attcccaatt aaaagtatgc agattatttg 5220 cccaaatctt cttcagattc agcatttgtt ctttgccagt ctcattttca tcttcttcca 5280 tggttccaca gaagctttgt ttcttgggca agcagaaaaa ttaaattgta cctattttgt 5340 atatgtgaga tgtttaaata aattgtgaaa aaaatgaaat aaagcatgtt tggttttcca 5400 aaagaacata t 5411

<210> 9

<211> 5490

<212> DNA

<213> Homo sapiens

<400> 9

ggctgagttt tatgacgggc ccggtgctga agggcaggga acaacttgat ggtgctactt 60 tgaactgctt ttcttttctc ctttttgcac aaagagtctc atgtctgata tttagacatg 120 atgagctttg tgcaaaaggg gagctggcta cttctcgctc tgcttcatcc cactattatt 180 ttggcacaac aggaagctgt tgaaggagga tgttcccatc ttggtcagtc ctatgcggat 240 agagatgtct ggaagccaga accatgccaa atatgtgtct gtgactcagg atccgttctc 300 tgcgatgaca taatatgtga cgatcaagaa ttagactgcc ccaacccaga aattccattt 360 ggagaatgtt gtgcagtttg cccacagcct ccaactgctc ctactcgccc tcctaatggt 420 caaggacctc aaggccccaa gggagatcca ggccctcctg gtattcctgg gagaaatggt 480 gaccctggta ttccaggaca accagggtcc cctggttctc ctggcccccc tggaatctgt 540 gaatcatgcc ctactggtcc tcagaactat tctccccagt atgattcata tgatgtcaag 600 tctggagtag cagtaggagg actcgcaggc tatcctggac cagctggccc cccaggccct 660 cccggtcccc ctggtacatc tggtcatcct ggttcccctg gatctccagg ataccaagga 720 ccccctggtg aacctgggca agctggtcct tcaggccctc caggacctcc tggtgctata 780 ggtccatctg gtcctgctgg aaaagatgga gaatcaggta gacccggacg acctggagag 840 cgaggattgc ctggacctcc aggtatcaaa ggtccagctg ggatacctgg attccctggt 900 atgaaaggac acagaggctt cgatggacga aatggagaaa agggtgaaac aggtgctcct 960 ggattaaagg gtgaaaatgg tcttccaggc gaaaatggag ctcctggacc catgggtcca 1020 agaggggctc ctggtgagcg aggacggcca ggacttcctg gggctgcagg tgctcggggt 1080 aatgacggtg ctcgaggcag tgatggtcaa ccaggccctc ctggtcctcc tggaactgcc 1140 ggattccctg gatcccctgg tgctaagggt gaagttggac ctgcagggtc tcctggttca 1200 aatggtgccc ctggacaaag aggagaacct ggacctcagg gacacgctgg tgctcaaggt 1260 cctcctggcc ctcctgggat taatggtagt cctggtggta aaggcgaaat gggtcccgct 1320 ggcattcctg gagctcctgg actgatggga gcccggggtc ctccaggacc agccggtgct 1380 aatggtgctc ctggactgcg aggtggtgca ggtgagcctg gtaagaatgg tgccaaagga 1440 gagcccggac cacgtggtga acgcggtgag gctggtattc caggtgttcc aggagctaaa 1500 ggcgaagatg gcaaggatgg atcacctgga gaacctggtg caaatgggct tccaggagct 1560 gcaggagaaa ggggtgcccc tgggttccga ggacctgctg gaccaaatgg catcccagga 1620 gaaaagggtc ctgctggaga gcgtggtgct ccaggccctg cagggcccag aggagctgct 1680 ggagaacctg gcagagatgg cgtccct gga ggtccaggaa tgaggggcat gcccggaagt 1740 ccaggaggac caggaagtga tgggaaacca gggcctcccg gaagtcaagg agaaagtggt 1800 cgaccaggtc ctcctgggcc atctggtccc cgaggtcagc ctggtgtcat gggcttcccc 1860 ggtcctaaag gaaatgatgg tgctcctggt aagaatggag aacgaggtgg ccctggagga 1920 cctggccctc agggtcctcc tggaaagaat ggtgaaactg gacctcaggg acccccaggg 1980 cctactgggc ctggtggtga caaaggagac acaggacccc ctggtccaca aggattacaa 2040 ggcttgcctg gtacaggtgg tcctccagga gaaaatggaa aacctgggga accaggtcca 2100 aagggtgatg ccggtgcacc tggagctcca ggaggcaagg gtgatgctgg tgcccctggt 2160 gaacgtggac ctcctggatt ggcaggggcc ccaggactta gaggtggagc tggtccccct 2220 ggtcccgaag gaggaaaggg tgctgctggt cctcctgggc cacctggtgc tgctggtact 2280 cctggtctgc aaggaatgcc tggagaaaga ggaggtcttg gaagtcctgg tccaaagggt 2340 gacaagggtg aaccaggcgg tccaggtgct gatggtgtcc cagggaaaga tggcccaagg 2400 ggtcctactg gtcctattgg tcctcctggc ccagctggcc agcctggaga taagggtgaa 2460 ggtggtgccc ccggacttcc aggtatagct ggacctcgtg gtagccctgg tgagagaggt 2520 gaaactggcc ctccaggacc tgctggtttc cc tggtgctc ctggacagaa tggtgaacct 2580 ggtggtaaag gagaaagagg ggctccgggt gagaaaggtg aaggaggccc tcctggagtt 2640 gcaggacccc ctggaggttc tggacctgct ggtcctcctg gtccccaagg tgtcaaaggt 2700 gaacgtggca gtcctggtgg acctggtgct gctggcttcc ctggtgctcg tggtcttcct 2760 ggtcctcctg gtagtaatgg taacccagga cccccaggtc ccagcggttc tccaggcaag 2820 gatgggcccc caggtcctgc gggtaacact ggtgctcctg gcagccctgg agtgtctgga 2880 ccaaaaggtg atgctggcca accaggagag aagggatcgc ctggtgccca gggcccacca 2940 ggagctccag gcccacttgg gattgctggg atcactggag cacggggtct tgcaggacca 3000 ccaggcatgc caggtcctag gggaagccct ggccctcagg gtgtcaaggg tgaaagtggg 3060 aaaccaggag ctaacggtct cagtggagaa cgtggtcccc ctggacccca gggtcttcct 3120 ggtctggctg gtacagctgg tgaacctgga agagatggaa accctggatc agatggtctt 3180 ccaggccgag atggatctcc tggtggcaag ggtgatcgtg gtgaaaatgg ctctcctggt 3240 gcccctggcg ctcctggtca tccaggccca cctggtcctg tcggtccagc tggaaagagt 3300 ggtgacagag gagaaagtgg ccctgctggc cctgctggtg ctcccggtcc tgctggttcc 3360 cgaggtgctc ctggtcctca aggcccacgt ggtgacaa ag gtgaaacagg tgaacgtgga 3420 gctgctggca tcaaaggaca tcgaggattc cctggtaatc caggtgcccc aggttctcca 3480 ggccctgctg gtcagcaggg tgcaatcggc agtccaggac ctgcaggccc cagaggacct 3540 gttggaccca gtggacctcc tggcaaagat ggaaccagtg gacatccagg tcccattgga 3600 ccaccagggc ctcgaggtaa cagaggtgaa agaggatctg agggctcccc aggccaccca 3660 gggcaaccag gccctcctgg acctcctggt gcccctggtc cttgctgtgg tggtgttgga 3720 gccgctgcca ttgctgggat tggaggtgaa aaagctggcg gttttgcccc gtattatgga 3780 gatgaaccaa tggatttcaa aatcaacacc gatgagatta tgacttcact caagtctgtt 3840 aatggacaaa tagaaagcct cattagtcct gatggttctc gtaaaaaccc cgctagaaac 3900 tgcagagacc tgaaattctg ccatcctgaa ctcaagagtg gagaatactg ggttgaccct 3960 aaccaaggat gcaaattgga tgctatcaag gtattctgta atatggaaac tggggaaaca 4020 tgcataagtg ccaatccttt gaatgttcca cggaaacact ggtggacaga ttctagtgct 4080 gagaagaaac acgtttggtt tggagagtcc atggatggtg gttttcagtt tagctacggc 4140 aatcctgaac ttcctgaaga tgtccttgat gtgcagctgg cattccttcg acttctctcc 4200 agccgagctt cccagaacat cacatatcac tgcaaaaata gca ttgcata catggatcag 4260 gccagtggaa atgtaaagaa ggccctgaag ctgatggggt caaatgaagg tgaattcaag 4320 gctgaaggaa atagcaaatt cacctacaca gttctggagg atggttgcac gaaacacact 4380 ggggaatgga gcaaaacagt ctttgaatat cgaacacgca aggctgtgag actacctatt 4440 gtagatattg caccctatga cattggtggt cctgatcaag aatttggtgt ggacgttggc 4500 cctgtttgct ttttataaac caaactctat ctgaaatccc aacaaaaaaa atttaactcc 4560 atatgtgttc ctcttgttct aatcttgtca accagtgcaa gtgaccgaca aaattccagt 4620 tatttatttc caaaatgttt ggaaacagta taatttgaca aagaaaaatg atacttctct 4680 ttttttgctg ttccaccaaa tacaattcaa atgctttttg ttttattttt ttaccaattc 4740 caatttcaaa atgtctcaat ggtgctataa taaataaact tcaacactct ttatgataac 4800 aacactgtgt tatattcttt gaatcctagc ccatctgcag agcaatgact gtgctcacca 4860 gtaaaagata acctttcttt ctgaaatagt caaatacgaa attagaaaag ccctccctat 4920 tttaactacc tcaactggtc agaaacacag attgtattct atgagtccca gaagatgaaa 4980 aaaattttat acgttgataa aacttataaa tttcattgat taatctcctg gaagattggt 5040 ttaaaaagaa aagtgtaatg caagaattta aagaaatatt tttaaagcc a caattatttt 5100 aatattggat atcaactgct tgtaaaggtg ctcctctttt ttcttgtcat tgctggtcaa 5160 gattactaat atttgggaag gctttaaaga cgcatgttat ggtgctaatg tactttcact 5220 tttaaactct agatcagaat tgttgacttg cattcagaac ataaatgcac aaaatctgta 5280 catgtctccc atcagaaaga ttcattggca tgccacaggg gattctcctc cttcatcctg 5340 taaaggtcaa caataaaaac caaattatgg ggctgctttt gtcacactag catagagaat 5400 gtgttgaaat ttaactttgt aagcttgtat gtggttgttg atcttttttt tccttacaga 5460 cacccataat aaaatatcat attaaaattc 5490

<210> 10

<211> 2269

<212> DNA

<213> Homo sapiens

<400> 10

cgcagccccg gttcctgccc gcacctctcc ctccacacct ccccgcaagc tgagggagcc 60 ggctccggcc tcggccagcc caggaaggcg ctcccacagc gcagtggtgg gctgaagggc 120 tcctcaagtg ccgccaaagt gggagcccag gcagaggagg cgccgagagc gagggagggc 180 tgtgaggact gccagcacgc tgtcacctct caatagcagc ccaaacagat taagacatgg 240 gaggtgaaag acaacttgag tggttaaatt actgtcatgc aaagcgacta gatggttcag 300 ctgattgcac ctttagaagt tatgtggaac gaggcagcag atcttaagcc ccttgctctg 360 tcacgcaggc tggaatgcag tggtggaatc atggctcact acagccctga cctcctgggc 420 ccagagatgg agtctcgcta ttttgcccag gttggtcttg aacacctggc ttcaagcagt 480 cctcctgctt ttggcttctt gaagtgcttg gattacagta tttcagtttt atgctctgca 540 acaagtttgg ccatgttgga ggacaatcca aaggtcagca agttggctac tggcgattgg 600 atgctcactc tgaagccaaa gtctattact gtgcccgtgg aaatccccag ctcccctctg 660 gattgtcagt ggctgctatg cagcaggtgc agcctggtct ctcactgagt ctctactcca 720 caaaggcaac gactggccaa ggcagtggct ggctctgggt tacacaagtg cagacactca 780 actaagtgag ctggaagacc caggagaagg cggaggctca ggtgcccaca tgatcagcac 840 agccagggta cctgctgaca agcctgtacg catcgccttt agcctcaatg acgcctcaga 900 tgatacaccc cctgaagact ccattccttt ggtctttcca gaattagacc agcagctaca 960 gcccctgccg ccttgtcatg actccgagga atccatggag gtgttcaaac agcactgcca 1020 aatagcagaa gaataccatg aggtcaaaaa ggaaatcacc ctgcttgagc aaaggaagaa 1080 ggagctcatt gccaagttag atcaggcaga aaaggagaag gtggatgctg ctgagctggt 1140 tcgggaattc gaggctctga cggaggagaa tcggacgttg aggttggccc agtctcaatg 1200 tgtggaacaa ctggagaaac ttcgaataca gtatcagaag aggcagggct cgtcctaact 1260 ttaaattttt cagtgtgagc atacgaggct gatgactgcc ctgtgctggc caaaagattt 1320 ttattttaaa tgaatagtga gtcagatcta ttgcttctct gtattaccca catgacaact 1380 gtctataatg agtttactgc ttgccagctt ctagcttgag agaagggata ttttaaatga 1440 gatcattaac gtgaaactat tactagtata tgtttttgga gatcagaatt cttttccaaa 1500 gatatatgtt tttttctttt ttaggaagat atgatcatgc tgtacaacag ggtagaaaat 1560 gataaaaata gactattgac tgacccagct aagaatcgtg ggctgagcag agttaaacca 1620 tgggacaaac ccataacatg ttcaccatag tttcacgtat gtgtattttt aaatttcatg 1680 cctttaatat ttcaaatatg ctcaaat tta aactgtcaga aacttctgtg catgtattta 1740 tatttgccag agtataaact tttatactct gatttttatc cttcaatgat tgattatact 1800 aagaataaat ggtcacatat cctaaaagct tcttcatgaa attattagca gaaaccatgt 1860 ttgtaaccaa agcacatttg ccaatgctaa ctggctgttg taataataaa cagataaggc 1920 tgcatttgct tcatgccatg tgacctcaca gtaaacatct ctgcctttgc ctgtgtgtgt 1980 tctgggggag gggggacatg gaaaaatatt gtttggacat tacttgggtg agtgcccatg 2040 aaaacatcag tgaacttgta actattgttt tgttttggat ttaaggagat gttttagatc 2100 agtaacagct aataggaata tgcgagtaaa ttcagaattg aaacaatttc tccttgttct 2160 acctatcacc acattttctc aaattgaact ctttgttata tgtccatttc tattcatgta 2220 acttcttttt cattaaacat ggatcaaaac tgacaaaaaa aaaaaaaaa 2269

Claims (6)

Patent claims for industrial invention entitled: "METHOD FOR IDENTIFYING SUBJECTS RESISTANT TO TREATMENT WITH PLATINUM-BASED DRUGS AND RELATIVE KIT" CLAIMS 1. A method for identifying cancer subjects resistant to treatment with therapeutic agents based on or including platinum complexes, comprising the steps of: - making available a biological sample isolated from the human body of a cancer patient; - separating the tumor cells from said biological sample; - extract the RNA from said cells; - identify the contemporary up-regulation of the following ten genes: CLDN6 (SEQ ID NO: 1), COL11A1 (SEQ ID NO: 2), SIM1 (SEQ ID NO: 3), DKK1 (SEQ ID NO: 4), GAL3ST3 (SEQ IDE NO: 5), IGFBP5 (SEQ ID NO: 6), DKK2 (SEQ ID NO: 7), COL1A2 (SEQ ID NO: 8), COL3A1 (SEQ ID NO: 9), MAP3K7CL ( SEQ ID NO: 10). Method according to claim 1, wherein the overexpression value is determined when the expression value of said ten genes is at least 100 times higher in the cells responding to the treatment than in the cells not responding to the treatment. Method according to claim 2, wherein said expression value of the ten genes is found by Northern Blotting, in situ hybridization, RNase protection assays, RT-PCR, monoclonal or polyclonal antibodies, microarrays, second and third sequencing generation, immunohistochemistry. Method according to any one of claims 1 to 3, further comprising a weighing step of the ten genes in which each of said ten genes is subjected to analysis on the survival curve consisting of calculating the HR values by means of the KM Plotter program, giving a score for each of said HR values in an arbitrary manner, correlating said score with said ten genes so as to obtain survival curves by means of said KM Plotter program. Method according to any one of claims 1 to 4, further comprising a step of evaluating the prediction of a treatment with therapeutic agents based on or including platinum complexes, wherein said step consists in evaluating the expression of each gene using the value of "Best cut off" as a threshold to distinguish a gene with high expression or low expression. 6. Kit for the identification of cancer subjects resistant to the treatment of therapeutic agents based on or including platinum complexes, comprising a microplate whose wells are coated with an antibody or probes capable of binding to the mRNA of the ten genes according to the claims 1, a container for a control sample comprising the mRNA sequences of the above ten genes, a container for a diluent substance for the control sample and one for the sample to be tested, a container for a swab, a container for a substrate solution, a container for a reaction termination solution, a container for a wash buffer, a package insert with explanations of how to prepare the samples, incubation times and washings, conditions for reading the signal detected with a photometer or alternative equipment to depending on the signal to be detected.