KR101626648B1 - Method and kit for determining in vitro the probability for an individual to suffer from colorectal cancer - Google Patents

Abstract

The present invention provides a method for quantifying the amount of the expression product of the nucleic acid of the gene of the individual to be tested by comparing the amount of the expression product of the nucleic acid of the same gene obtained from the CRC patient group constituting the positive control and Comparing the amount of expression product of the nucleic acid of the gene to determine the probability that the individual will suffer from colorectal cancer in a peripheral blood sample in vitro; And a specific binding partner for the expression product.

Description

METHOD AND KIT FOR DETERMINING IN VITRO THE PROBABILITY FOR INDIVIDUAL TO SUFFER FROM COLORECTAL CANCER BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

The present invention relates to the detection of colorectal cancer, and in particular to a method and kit for measuring the probability of having such cancer.

Colorectal cancer (CRC), also referred to as colon cancer or colon cancer, is the fifth most common form of cancer in the United States, the fourth most common cancer in China, and the third leading cause of cancer-related deaths in Europe. The initial detection of CRC is a key to successful treatment and patient survival and represents a major public health challenge. In fact, CRC is often healable, especially when diagnosed at an early stage. Several screening techniques have already been implemented in various countries. Typical CRC screening tests include fecal occult blood test (FOBT), sigmoidoscopy, colonoscopy, dual contrast barium enema or digital rectal examinations. While all of these have their advantages and limitations, adherence is often lower than expected due to the patient's performance plan or discomfort.

Retrieval of peripheral blood biomarkers for the initial detection of CRC has been noted for several years because of its convenience. On the other hand, the feasibility of a blood-based test was supported by only a few studies showing that genomic biomarkers in blood can distinguish CRC patients from controls. These studies were based on flow cytometry, a technique for suspending microscopic particles, such as cells, in a fluid stream and counting and examining them by analyzing them using an electronic detector.

The inventors have found that differentially expressed genes represent important biomarkers in peripheral blood samples. They did not use classical flow cytometry techniques and used confirmation of different expression of genes from whole blood. It is not uncommon to measure the level of expression of a gene through analysis of transcripts in whole blood because it is very difficult to retrieve the information without specific purification steps if certain information is diluted in a complex mixture of RNA (total RNA) Since they are commonly recognized by those skilled in the art. The advantage of this method is also to avoid this RNA purification step.

Accordingly, the present invention relates to a method for measuring the probability of an individual having a colorectal cancer in a peripheral blood sample in vitro, comprising the steps of:

a) measuring in the peripheral blood sample the amount of at least one expression product from at least one nucleic acid sequence selected from the sequences identified in SEQ ID NOS: 1-11 and up to 7 nucleic acid sequences;

b) comparing the amount of the expression product measured in step a) with a reference amount of expression product for a group of individuals previously diagnosed as a colorectal cancer patient and a reference amount of expression product for a group of individuals previously verified as non- Comparing with a reference quantity; And

c) performing an analysis of the result of step b)

- if the result on the tested individual is similar or equivalent to the results obtained from a group of individuals previously diagnosed as a colorectal cancer patient, the tested individual is classified as a colorectal cancer patient,

- the tested individual is classified as a non-colorectal cancer entity if the results on the tested individual are similar or equivalent to those obtained from a group of individuals previously validated as non-colon carcinoma individuals.

The amount of expression product is directly related to the expression level of the gene defined by its nucleic acid sequence.

The level of expression of one or more of the nucleic acids is sufficient information to determine whether the individual is a CRC patient or not. However, in a preferred embodiment of the present invention, in step a), SEQ ID NO: 1; SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; SEQ ID NO: 5 or SEQ ID NO: 6; SEQ ID NO: 7 or SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; And the amount of the expression product from the nucleic acid sequence selected from the group consisting of SEQ ID NO: 11 is measured.

The amount of expression product (s) from the nucleic acid (s) is determined by contacting the expression product (s) with one or more binding partners specific for each expression product.

The expression product (s) means RNA transcript (s) or polypeptide (s). Thus, in the method of the present invention, the amount of one or more RNA transcripts or one or more polypeptides is measured.

The term RNA transcript is intended to mean total RNA, either coding or non-coding RNA obtained directly from a peripheral blood sample or obtained indirectly from a blood sample after cell lysis. In particular, total RNA also includes mRNA and ribosomal RNA transcribed from transcribed RNA (tRNA), messenger RNA (mRNA), such as mRNA transcribed from a target gene, as well as from any other gene.

For example, if the RNA is intracellular RNA, the RNA may be extracted from cells present in the blood sample by a lysis step of the cell to release nucleic acids contained in the cells of the individual to be tested. For example, the dissolution methods described in the following patent application publications may be used: International Patent Application Publication No. WO 00/05338 for mixed magnetic and mechanical dissolution, International Patent Application Publication No. WO 99 / 53304, and International Patent Application Publication No. WO 99/15321 on mechanical dissolution. Those skilled in the art can use other well-known dissolution methods, such as chemical dissolution using heat or osmotic shock or chaotropic agents, such as guanidinium salts (US Pat. No. 5,234,809). It is also possible to provide an additional step of separating nucleic acids from other cellular components released in the lysis step. This generally makes it possible to concentrate the nucleic acid.

In the method of the present invention, the RNA transcript can be detected and quantified by hybridization, amplification or sequencing. In particular, the RNA transcript to be detected and quantified is contacted with said one or more probes or said one or more primers under predetermined conditions to enable hybridization of said RNA transcript with one or more probes and / or one or more probes . However, in another embodiment of the present invention, there is provided a method for preparing a DNA transcript of an RNA transcript, which comprises contacting the DNA transcript with one or more probes and / or one or more primers under predetermined conditions, The DNA copy is measured by contacting with at least one probe or at least one primer.

More precisely, in the above-described method, an RNA transcript or a DNA copy is contacted with at least one hybridization probe and at least one primer, more particularly at least one hybridization probe and two primers.

The term "hybridization" is intended to mean a process in which two nucleotide fragments under appropriate conditions are combined with a stable specific hydrogen bond to form a double-stranded complex. These hydrogen bonds are formed between the complementary adenine (A) and the thymine (T) (or uracil (U)) bases (this is referred to as AT binding) or complementary guanine (G) (This is referred to as GC coupling). The hybridization of the two nucleotide fragments can be complete (in this case, referred to as complementary nucleotide fragment or sequence) (i.e., the double stranded conjugate obtained during this hybridization contains only the A-T and C-G bonds). This hybridization can be partial (in this case, referred to as a sufficiently complementary nucleotide fragment or sequence) (i. E., AT binding and CG binding, which allows the resulting double stranded conjugate to form a double stranded conjugate, Including bases which are not bonded to an atomic base. The hybridization between the two nucleotide fragments depends on the working conditions used and in particular on the stringency. Strictness is defined not only as a function of the base composition of the two nucleotide fragments, but also by the degree of discrepancy between the two nucleotide fragments. The degree of stringency may also depend on the reaction parameters, such as the concentration and type of ionic species present in the hybridization solution, the nature and concentration of the denaturant, and / or the hybridization temperature. All of these data are well known, and suitable conditions can be determined by one of ordinary skill in the art. Generally, depending on the length of the nucleotide fragments to be hybridized, the hybridization temperature is about 20 ° C to 70 ° C, especially 35 ° C to 65 ° C in a saline solution at a concentration of about 0.5 M to 1 M. A sequence, nucleotide fragment, oligonucleotide or polynucleotide is a series of nucleotide motifs assembled together by a phosphoester linkage, characterized by the information sequence of the native nucleic acid and capable of hybridizing to the nucleotide fragment, May contain monomers and may be obtained from natural nucleic acid molecules and / or may be obtained by genetic recombination and / or chemical synthesis. The motif is a derivative of a monomer in which the constituent is a natural nucleotide of a nucleic acid which is a sugar, a phosphate group and a nitrogenous base. In the DNA, the sugar is deoxy-2-ribose. In the RNA, the sugar is ribose. , The nitrogenous base is selected from adenine, guanine, uracil, cytosine and thymine, alternatively the monomer is a nucleotide modified in at least one of the three components, for example, Can be prepared by reacting a modified base such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine, Can occur at the base level to have any other modified base capable of hybridizing, or can occur at the sugar level (e. G., By substitution of one or more deoxyriboses with polyamides (PE Nielsen et al., Science, 254, 1497-1500 (1991)), or at phosphate group levels (e. G., Especially in the form of diphosphates, alkylphosphonates, arylphosphonates, Substitution of phosphate group by ester selected from porothioate).

For purposes of the present invention, the term "amplification primer" refers to a nucleotide fragment comprising 5 to 100 nucleotides, preferably 15 to 30 nucleotides, which enables the initiation of enzymatic polymerization, . The term "enzyme amplification reaction " is intended to mean the process of generating multiple copies of a nucleotide fragment through the action of one or more enzymes. Such amplification reactions are well known to those skilled in the art, and in particular the following techniques may be mentioned: PCR (polymerase chain reaction) as described in U.S. Patent No. 4,683,195, U.S. Patent No. 4,683,202 and U.S. Patent No. 4,800,159; For example, the LCR (ligase chain reaction) disclosed in European Patent Application No. 0 201 184; The RCR (repair chain reaction) described in International Patent Application Publication No. WO 90/01069; 3SR (self-sustained sequence replication) as described in International Patent Application Publication No. WO 90/06995; NASBA (nucleic acid sequence-based amplification) described in International Patent Application Publication No. WO 91/02818; TMA (transcription-mediated amplification) described in U.S. Patent No. 5,399,491; And RT-PCR.

When the enzyme amplification is PCR, two or more kinds of amplification primers specific to the target gene are used, which enable an amplification substance specific for the target gene. The substance specific to the target gene is preferably a complementary DNA (referred to as target gene-specific cDNA) obtained by reverse transcription of messenger RNA derived from the target gene, or a cDNA specific for the target gene (Referred to as target gene-specific cRNA). If the enzyme amplification is PCR performed after the reverse transcription reaction, this amplification is referred to as RT-PCR.

The term "hybridization probe" refers to a nucleic acid molecule having 5 or more nucleotides, such as 5 to 100 nucleotides, in particular 10 to 75 nucleotides, having a hybridization specificity under predetermined conditions to form a hybridization complex with a substance specific for the target gene. For example, a nucleotide fragment comprising 15 to 35 nucleotides and 60 to 70 nucleotides. In the present invention, a substance specific to the target gene includes a nucleotide sequence (referred to as target gene-specific mRNA) contained in messenger RNA derived from the target gene, a complementary DNA obtained by complementary DNA obtained by reverse transcription of the messenger RNA (Referred to as the target gene specific cDNA) contained in the complementary RNA obtained by the nucleotide sequence (referred to as the target gene specific cDNA) or the above-described transcription of the cDNA. The hybridization probe may include a label for its detection. The term "detection" is intended to mean a direct detection, such as a counting method, or an indirect detection by a detection method using a label. Many nucleic acid detection methods exist (see, for example, Kricka et al., Clinical Chemistry, 1999, no 45 (4), p. 453-458) or Keller GH et al., DNA Probes, 2nd Ed ., Stockton Press, 1993, sections 5 and 6, p. 173-249). The term "cover" is intended to mean a tracer capable of generating a signal that can be detected. A non-limiting list of these tracers includes colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase or glucose-6-phosphate dehydrogenase An enzyme that generates a signal that can be detected by dehydrogenase; Chromophores, such as fluorescence, luminescent or dye compounds; An electron tightener which can be detected by electron microscopic observation, by their electrical properties, for example, by conductivity, by current measurement or voltage measurement, or by impedance measurement; A group which can be detected by optical methods such as diffraction, surface plasmon resonance or contact angle changes, or by physical methods such as nuclear spectroscopy, tunneling effects, and the like; And radioactive molecules, such as ³² P, ³⁵ S or ¹²⁵ I.

For purposes of the present invention, the hybridization probe may be a "detection" probe. In this case, the "detection" probe is labeled by the label. The detection probe may be a "molecular beacon" detection probe, particularly as described in Tyagi & Kramer, Nature biotech, 1996, 14: 303-308. These "molecular beacons" will fluoresce during hybridization. They have a stem-loop type structure and contain fluorescent moieties and "quencher" groups. The combination of a particular loop sequence with its complementary target nucleic acid sequence causes the stem to loosen and cause emission of the fluorescent signal during excitation at the appropriate wavelength. The detection probe may be a " reporter probe ", especially comprising a "color-coded barcode" according to the NanoString (TM) technology.

For detection of the hybridization reaction, the target sequence labeled directly (particularly by introduction of the label into the target sequence) or indirectly (particularly by use of the detection probe defined above) may be used. In particular, for example, a step consisting of labeling and / or cleavage of a target sequence using a deoxyribonucleotide triphosphate labeled during an enzyme amplification reaction may be performed prior to the hybridization step. The cleavage can be carried out in particular by the action of imidazole or manganese chloride. The target sequence may be labeled after the amplification step, for example, by hybridizing the detection probe according to the sandwich hybridization technique described in International Patent Application Publication No. WO 91/19812. Another particular preferred method of labeling nucleic acids is described in French patent application no. 2780059.

According to a preferred embodiment of the present invention, the detection probe comprises a fluorescent dye and a quencher. According to a still more preferred embodiment of the present invention, the hybridization probe comprises a FAM (6-carboxy-fluororesin) or ROX (6-carboxy-X-rhodamine) fluorophore at its 5 ' (Dabsyl). ≪ / RTI >

The hybridization probe may be a "capture" probe. In this case, the "capture" probe may be immobilized on a solid substrate or fixed by any suitable means, i.e., directly or indirectly, e.g. by covalent or adsorption. Optionally chemically modified synthetic or natural materials, in particular polysaccharides such as cellulose-based materials such as paper, cellulose derivatives such as cellulose acetate and nitrocellulose or dextran, polymers, copolymers, especially styrene Monomer-based copolymers of the type, natural fibers such as cotton, and synthetic fibers such as nylon; Inorganic materials such as silica, quartz, glass or ceramics; Latex; Magnetic particles; Metal derivatives; Gel or the like can be used as a solid substrate. The solid substrate may be in the form of a microtiter plate, a film as described in International Patent Application Publication No. WO 94/12670, or in the form of particles. It is also possible to immobilize various different capture probes, each specific for the target gene, on the substrate. In particular, a biochip where a large number of probes can be fixed can be used as a substrate. The term "biochip" is intended to mean a small size solid substrate having a large number of capture probes attached at predetermined locations. The concept of biochip or DNA chip has appeared since the early 1990s. The chip is based on a variety of disciplines that integrate microelectronics, nucleic acid chemistry, image analysis and information technology. The working principle is based on the hybridization phenomenon that is the basis of molecular biology, that is, the pairing by the complementarity of the bases of two DNA and / or RNA sequences. The biochip method is based on the use of a capture probe attached to a solid substrate, wherein the probe is made to act on a sample of a target nucleotide fragment labeled directly or indirectly with a fluorescent dye. The capture probe is specifically located on a substrate or chip, and each hybridization provides specific information related to the target nucleotide fragment. The information obtained is cumulative and makes it possible, for example, to quantify the expression level of one or more target genes. In order to analyze the expression of the target gene, a substrate containing a plurality of probes corresponding to all or a part of the target gene to be transcribed into mRNA can be produced. For purposes of the present invention, the term "low density substrate" is intended to mean a substrate comprising less than 50 probes. For purposes of the present invention, the term "medium density substrate" is intended to mean a substrate comprising 50 to 10,000 probes. For the purposes of the present invention, the term "high density substrate" is intended to mean a substrate comprising more than 10,000 probes.

CRNA or cDNA specific for the nucleic acid of the target gene to be analyzed is hybridized to, for example, a specific capture probe. After hybridization, the substrate or chip is washed and the labeled cDNA or cRNA / capture probe conjugate is labeled, for example, by a high affinity ligand bound to the label of the fluorescent dye type. Fluorescence is read by a scanner, for example, and the analysis of fluorescence is processed by information technology. For example, DNA chips developed by Affymetrix for molecular diagnostics (see "Accessing Genetic Information with High-Density DNA arrays ", M. Chee et al., Science, 1996, 274, 610-614 ], ["Light-generated oligonucleotide arrays for rapid DNA sequence analysis", A. Caviani Pease et al., Proc Natl Acad Sci USA, 1994, 91, 5022-5026). In this technique, capture probes are generally small in size (about 25 nucleotides). Other examples of biochips are described in G. Ramsay, Nature Biotechnology, 1998, No. 16, p. 40-44]; F. Ginot, Human Mutation, 1997, No. 10, p. 1-10]; J. Cheng et al., Molecular diagnosis, 1996, no. 1 (3), p. 183-200]; T. Livache et al., Nucleic Acids Research, 1994, No. 22 (15), p. 2915-2921; J. Cheng et al., Nature Biotechnology, 1998, no. 16, p. 541-546, US 4,981,783, US 5,700,637, US 5,445,934, US 5,744,305, and US 5,807,522. The main feature of the solid substrate should be to generate minimal background noise for the detection method while at the same time preserving the hybridization characteristics of the capture probe to the target nucleotide fragment. Three major fabrication methods for immobilizing probes on a substrate can be distinguished.

Above all, there is a first technique consisting of depositing a pre-synthesized probe. Attachment of the probe is performed by direct delivery, micropipette or microdot, or inkjet device. This technique allows the attachment of probes of sizes ranging from a few bases (5 to 10) to a relatively large size of 60 bases (printing) or hundreds of bases (micro-deposition).

Printing is a modification of the method used by inkjet printers. Printing is based on propulsion of very small fluid sumps (volume < 1 nl) at a rate that can reach 4000 droplets / s. Printing does not involve any contact between the system that emits the fluid and the surface on which it is deposited.

Micro-deposition consists of attaching long probes consisting of tens to hundreds of bases to the surface of the glass slide. These probes are generally extracted from the database, and are present in the form of amplified and purified products. This technique makes it possible to produce chips called microarrays having about 10,000 DNA spots (referred to as recognition bands) on a small surface area of less than 4 cm.sup.2. However, the use of nylon membranes, commonly referred to as "macroarrays, " which have products amplified by PCR and have diameters of 0.5 mm to 1 mm and a maximum density of 25 spots / cm.sup.2 . This highly flexible technique is used by many laboratories. In the present invention, the latter technique is considered to be included in the biochip. However, a certain volume of sample may be deposited on the bottom of each well of the microtiter plate, as in the case of WO 00/71750 and in French Patent Application No. 00/14896, Water droplets can be deposited on the bottom of one and the same petri dish in accordance with another French patent application No. 00/14691.

The second technique of attaching probes to a substrate or chip is referred to as in situ synthesis. This technique allows short probes to be generated directly on the surface of the chip. This technique is based on in situ oligonucleotide synthesis (in particular, International Patent Application Publication No. WO 89/10977 and International Patent Application Publication No. WO 90/03382) and oligonucleotide synthesizer processes. The technique consists of moving the reaction chamber along which the oligonucleotide extension reaction takes place along the glass surface.

Finally, the third technique is referred to as photolithography, which is the basis of the biochip developed by Apimetrix. This technique is also in situ synthesis. Photolithography is derived from microprocessor techniques. The surface of the chip is altered by the attachment of photo-labile chemical groups that can be optically activated. Once illuminated, these groups can react with the 3 ' end of the oligonucleotide. By protecting the surface with a mask of defined type, the area of the chip to which one of the four kinds of nucleotides is to be attached can be selectively illuminated and activated. Successful use of different masks allows for the generation of oligonucleotide probes on spots of approximately several tens of cubic microns (탆 ² ) by alternating cycles of protection / reaction. This method makes it possible to generate up to several hundred thousand spots on a surface area of several cubic centimeters (cm &lt; ² &gt;). Photolithography has the advantage that it makes it possible to produce large quantities of N-MER chips simultaneously in only four N cycles. All of these techniques can be used with the present invention. According to a preferred embodiment of the invention, the at least one specific reagent of step b), defined above, preferably comprises at least one hybridization probe immobilized on a substrate. The substrate is preferably a low density substrate, a high density substrate or a medium density substrate defined above.

After these hybridization steps on a substrate comprising a plurality of probes to increase the amount of target dielectric material, an enzyme amplification reaction step as defined above may be performed.

Measurement of the expression level of the target gene can be performed by any protocol known to those skilled in the art. In general, the expression of a target gene can be analyzed by detecting mRNA (messenger RNA) transcribed from the target gene at a given moment.

The present invention is preferably directed to determining the level of expression of this target gene by detecting mRNA derived from the target gene according to any protocol well known to those skilled in the art. According to certain embodiments of the present invention, the expression levels of different target genes are simultaneously measured by detection of several different mRNAs (each mRNA is derived from the target gene).

Amplification can be used to determine the expression level of the target gene as follows: 1) Total RNA (including transfer RNA (tRNA), ribosomal RNA (rRNA) and messenger RNA (mRNA)) is extracted from whole blood, A reverse transcription step is performed to obtain the complementary DNA (or cDNA) of the mRNA. For example, the reverse transcription reaction can be performed by using a reverse transcriptase that makes it possible to obtain a complementary DNA fragment from the RNA fragment. In particular, reverse transcriptase from AMV (Myoblastosis virus) or MMLV (Moloney Murine leukemia virus) can be used. More specifically, when it is desired to obtain only cDNA of mRNA, this reverse transcription step is carried out in the presence of a nucleotide fragment (poly T) containing only a thymine base complementarily hybridizing to the poly A sequence of the mRNA, To form a poly T-poly A conjugate that serves as a starting point for the reverse transcription reaction. CDNA (target gene specific cDNA) complementary to the mRNA derived from the target gene and cDNA complementary to the mRNA derived from the gene other than the target gene (cDNA not specific to the target gene) are obtained. 2) The amplification primer (s) specific for the target gene is brought into contact with the target gene-specific cDNA and the cDNA that is not specific for the target gene. The amplification primer (s) specific for the target gene hybridizes with the target gene specific cDNA and the predetermined region of the known length of the cDNA derived from the mRNA derived from the target gene is specifically amplified. CDNA that is not specific to the target gene is not amplified while a large amount of target gene-specific cDNA is obtained. For the purpose of the present invention, "target gene specific cDNA" or "cDNA derived from mRNA derived from a target gene" is mentioned without any distinction. This step may be carried out, in particular, by an amplification reaction of the PCR type or by any other amplification technique as defined above. It is also possible to simultaneously amplify several different cDNAs (each of which is specific for a different target gene) by PCR using multiple pairs of different amplification primers (each of which is specific for the target gene) (referred to as multiple amplifications) ). 3) Expression of the target gene is measured by detecting and quantifying the target gene-specific cDNA obtained in the step 2). This detection can be performed after electrophoretic migration of target gene specific cDNAs according to their size. The gel and medium for the movement can contain ethidium bromide to allow direct detection of target gene specific cDNAs through the emission of a light signal when the gel is placed on a UV-ray (ultraviolet) light table after a given migration time have. The greater the amount of target gene specific cDNA, the brighter the optical signal. These electrophoresis techniques are well known to those skilled in the art. The target gene-specific cDNA may be detected and quantified by using the quantification range obtained by the amplification reaction performed until saturation. To simultaneously account for variability in enzyme efficiency that can be observed during various steps (reverse transcription, PCR, etc.), the expression of the "howls skipping" gene, which has similar expression in various patient groups, Expression can be standardized. By obtaining the ratio of the expression of the target gene to the expression of the Haas Skipping gene, that is, by obtaining the ratio of the amount of the target gene specific cDNA to the amount of the Haas Skipping gene specific cDNA, an arbitrary variability . Those skilled in the art can in particular refer to the following references: Bustin SA, J Mol Endocrinol, 2002, 29: 23-39 and Giulietti A Methods, 2001, 25: 386-401.

The expression of the target gene can be measured by hybridization as follows: 1) Total RNA is extracted from whole blood, and then the reverse transcription step is performed as described above to amplify the cDNA complementary to the mRNA derived from the target gene cDNA) and cDNA complementary to the mRNA derived from the gene other than the target gene (cDNA not specific to the target gene). 2) The hybridization reaction between the target gene-specific cDNA and the capture probe is carried out by contacting all the cDNAs with a substrate on which capture probes specific for the target gene (to analyze its expression) are immobilized, Lt; / RTI &gt; does not hybridize to the capture probe. A hybridization reaction may be performed on a solid substrate including all the above-indicated materials. According to a preferred embodiment, the hybridization probe is immobilized on a substrate. Preferably, the substrate is a low density, high density or medium density substrate as defined above. After the hybridization reaction, a step composed of enzyme amplification of the target gene-specific cDNA as described above is performed to obtain a large amount of target gene-specific cDNAs, and the target gene-specific cDNA is introduced into a capture probe specific for the target gene The probability of hybridization can be increased. In addition, after the hybridization reaction, for example, a labeled deoxyribonucleotide triphosphate may be used in an amplification reaction to perform a step consisting of labeling and / or cleavage of the target gene specific cDNA as described above. The cleavage can be carried out in particular by the action of imidazole and manganese chloride. For example, a labeled probe may be hybridized according to the sandwich hybridization technique described in WO 91/19812 to label the target gene-specific cDNA after the amplification step. Other preferred nucleic acid labeling and / or cleavage methods are disclosed in International Patent Application Publication No. WO 99/65926, International Patent Application Publication No. WO 01/44507, International Patent Application Publication No. WO 01/44506, International Patent Application Publication WO 02 / 090584 and International Patent Application Publication No. WO 02/090319. 3) The subsequent step is performed by detecting the hybridization reaction. Detection can be performed by contacting a substrate with a capture probe specific for the target gene hybridized with the target gene specific cDNA with a labeled "detection" probe and detecting the signal emitted by the label. When the target gene-specific cDNA is pre-labeled with a label, the signal emitted by the label is directly detected.

Expression of the target gene may be measured in the following manner: 1) Total RNA is extracted from whole blood, and then the reverse transcription step is performed as described above to obtain cDNA of the mRNA of the biological material. The polymerisation of the complementary RNA of the cDNA is then carried out using a T7 polymerase which is capable of acting under the control of the promoter and obtaining complementary RNA from the DNA template. Next, cRNA (referred to as target gene-specific cRNA) of cDNA of mRNA specific to the target gene and cRNA of cDNA of mRNA not specific to the target gene are obtained. 2) All cRNAs are contacted with a substrate onto which capture probes specific for the target gene (to analyze its expression) are immobilized to perform hybridization reactions between the target gene-specific cRNAs and the capture probes, The cRNA that is not specific for the gene is not hybridized to the capture probe. When simultaneous expression of multiple target genes is desired, multiple different capture probes (each of which is specific for the target gene) can be immobilized on the substrate. In addition, after the hybridization reaction, a step consisting of labeling and / or cleavage of the target gene-specific cRNA may be performed as described above. 3) The subsequent step is performed by detecting the hybridization reaction. Detection may be performed by contacting a substrate with a capture probe specific for the target gene hybridized with the target gene-specific cRNA, with a labeled "detection" probe and detecting the signal emitted by the label. When the target gene-specific cRNA is pre-labeled with a label, the signal emitted by the label is directly detected. The use of cRNA is particularly advantageous when a substrate of the biochip type is used in which a large number of probes are hybridized.

If the expression product is a polypeptide, the polypeptide may be detected by contacting it with one or more specific ligands, such as a ligand as defined below. In a preferred embodiment, the expressed polypeptide is contacted with two or more specific ligands, such as the ligands defined below. Certain ligands refer to, for example, antibodies or affinity proteins named "Nanofitin ".

Nanophytes are affinity proteins with competitive properties. Nanophytes exhibit competitive affinities similar to antibodies.

The term "antibody or antibodies" encompasses a polyclonal antibody, a monoclonal antibody, a humanized antibody, and a recombinant antibody. Methods for their preparation are well known to those skilled in the art.

The present invention relates to a method for screening for the presence or absence of one or more binding partners specific for one or more nucleic acid sequences, Wherein the one or more binding partners are specific for one or more expression products of one or more nucleic acid sequences selected from the group consisting of the sequences set forth in SEQ ID NOS: 1-11.

In particular, the kit comprises SEQ ID NO: 1; SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; SEQ ID NO: 5 or SEQ ID NO: 6; SEQ ID NO: 7 or SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; And a combination of seven binding partners specific for the expression products of the seven nucleic acid sequences having the sequences set forth in SEQ ID NO: 11.

In the kit, the specific binding partner may comprise at least one hybridization probe, at least one hybridization probe and at least one primer, at least one hybridization probe and at least two primers, or at least one specific ligand or at least two specific Such as antibodies and / or affinity proteins.

Finally, the present invention relates to the use of one or more specific binding partners for at least one expression product of one or more nucleic acid sequences to seven nucleic acid sequences for the production of a composition for in vitro measurement of the probability that an individual will suffer from colorectal cancer Wherein the one or more nucleic acid sequences have a sequence selected from the group consisting of the nucleic acid sequences set forth in SEQ ID NOS: 1 to 11, and the use of no more than seven specific binding partners for the seven expression products of the sequence.

In particular, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; SEQ ID NO: 5 or SEQ ID NO: 6; SEQ ID NO: 7 or SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; And the use of a combination of seven specific binding partners specific for the seven expression products of the seven nucleic acid sequences having the sequences set forth in SEQ ID NO: 11.

The specific binding partner may comprise one or more hybridization probes, one or more hybridization probes and one or more primers, one or more hybridization probes and two primers, or one or more specific ligands or two or more specific ligands, Antibodies and / or affinity proteins.

Example

I) Materials and Methods

1. Patient and sample collection

Peripheral blood samples from 161 colorectal cancer patients (CRC) and 148 colonoscopy negative control patients (CNC) were collected between 2006 and 2010. CRC patients were mobilized in the colon rectus operation room of FDUSCC in China. Tumors were grouped according to the International Cancer Federation (UICC) and the recommended tumor-node metastasis (TNM) system. No patients received preoperative radiation or chemotherapy. Patients with hereditary colorectal cancer or inflammatory bowel disease (Crohn's disease or ulcerative colitis) were excluded from this study. CNCs without any symptoms of colon or colorectal cancer confirmed by colonoscopy were registered from local hospitals and FDUSCC in the Shanghai area. For each patient, 2.5 ml of peripheral blood was collected into a PAXgene (TM) blood RNA tube (PreAnalytiX GmbH, Hombruck, Switzerland) and processed according to the manufacturer's instructions Respectively.

This study includes two separate groups of participants. Group 1 consisted of 100 CRC patients and 100 CNC patients. For CRC patients, blood samples were collected from FDUSCC prior to surgery after 1 week or more from preoperative colonoscopy. For CNC patients, blood samples were collected at local hospitals in Shanghai one week prior to colonoscopy. The gene expression profiles from these samples were analyzed as a training set to search for significant genes associated with CRC and to confirm the molecular signature. Group 2 included 61 CRC patients and 48 CNC patients. Samples were collected in the same manner as group 1. Group 2 was used as an independent set of tests to verify the signature performance observed in Group 1.

2. RNA  Extraction and Microarray  Experiment

Total RNA was extracted using the Fax Jean (TM) blood RNA system (free antigenic) according to the manufacturer's instructions. The amount of total RNA was measured spectrophotometrically at an optical density of 260 nm and analyzed on an RNA 6000 LabChip ™ instrument on a BioAnalyzer Agilent 2100 (Agilent Technologies, Palo Alto, CA) Lt; RTI ID = 0.0 &gt; RNA &lt; / RTI &gt; name) kit. Only samples with an RNA Integrity Index of 7 to 10 were analyzed. Then, a WT-Ovation RNA amplification system (NuGEN Technologies Inc., San Carlos, Calif., USA) was used to construct a ribo- 50 ng of total RNA was reverse transcribed and amplified proportionally using single-stranded cDNA using SPIA: trademark technology, and the product was amplified using QIAquick (TM) PCR purification kit (QIAGEN GmbH, Material). Then 2 μg of amplified and purified cDNA was fragmented with RQ1 RNase-free DNase (Promega Corp., Pittsburgh, Wis., USA) and ligated with end-transferase (Roche Diagnostics Corp.). ) And Biotinylated Deoxyne (GeneChip®) DNA Marking Reagent (Affymetrix Inc., Santa Clara, Calif., USA) and GeneChip ™ DNA Marking Reagent (Affymetrix Inc., Indianapolis, Ind. Lt; / RTI &gt; triphosphate. The labeled cDNA was hybridized for 18 hours on a GeneChip HG U133 plus 2.0 array (ApiMetrix) at 60 ° C in a hybridization oven 640 (Agilent Technologies) rotating at 60 ° C per minute. The HG U133 plus 2.0 array contains 54,675 probe sets representing approximately 39,000 of the best characterized human genes. After hybridization, the arrays were washed and stained according to the Affymetrix protocol EukGE-WS2v4 using Jinchip (R) Fluidics Station 450 (Affymetrix). The array was scanned with a GinChip (registered trademark) scanner 3000 (ApiMetrix).

3. Statistical analysis

Microarray data quality control was performed according to the recommendations of the standard apical quality control parameters. (Irizarry RA et al., Biostatistics 20 3; 4: 249-64) with background correction, side-position normalization, and median polish summarization Lt; RTI ID = 0.0 &gt; apiMetrix &lt; / RTI &gt; expression array.

For group 1 data, a set of probes having excessive signal intensity (lower than 2E14 log2 (50) lower) was filtered out. Then, biological knowledge-based filtering was performed using information from the Entrez Gene Database (Maglot D et al., Nucleic Acids Research 2007; 35: D26-31). A set of probes without an Entreze gene ID annotation was removed. For a plurality of probe sets mapped to the same entrease gene ID, only the probe set with the largest value of the mutual position range was retained and the rest was removed. After two-step filtering, 9,859 probe sets were stored for downstream analysis. To reduce the possibility of a batch effect, the Combat method was applied to the filtered expression data (Johnson WE et al., Biostatistics 2007; 8: 118-27). Analysis of differentially expressed genes (DEG) was performed by the significance analysis (SAM) method of microarray (false detection rate = 0.05; type = "two unpaired classes"; test statistic = "t- statistic" Number = 1,000) (Tusher VG et al., PNAS USA 2001,98: 5116-21). Significant gene selection and prediction models were constructed using the 5-fold cross-validation procedure with the RFE-SVM method. Of the 200 samples in the training set, 160 samples were randomly selected to form a learning set and a prediction model with different sizes ranging from 1 to 100 genes scored by RFE-SVM was generated, Forty samples were used to evaluate the model performance. This process was repeated 1,000 times. Our results suggest that up to 97% accuracy can be achieved using 100 gene-based SVM prediction models. Signature size optimization takes into account prediction performance, signature complexity and economy. Finally, the present inventors confirmed seven core genes that meet the target performance of the present inventors with a total accuracy of 90%. The seven genes were selected by t-test P- value, multiples of variation, biological function and non-relevance to age or gender factors.

II) Results

1. Characterization of colorectal cancer patients and control patients

Of the 309 participants in the two groups, 161 had CRC and 148 had CNC. The demographic and clinical characteristics of the patients are summarized in Table 1.

2. 7 genetic CRC biomarker panels: identification and validation

Training set: We performed significant gene selection and prediction model construction based on a 5x cross validation procedure. The above procedure was repeated 1000 times. Within each iteration, we recorded a unique set of seven top genes and their corresponding predictive model performance evaluated by internal test folds. Finally, the overall performance was estimated by taking the average performance of the 1,000 prediction models in the internal test run. The results show that a full accuracy performance of 90.0% can be achieved using the predictive model. We chose the seven best gene prediction models (90.0% accuracy, 89.0% sensitivity, and 91.0% specificity for the training set).

Test set: We verified the performance of the signature of the predictive model identified in a training set of independent groups (test sets) containing 109 samples (61 CRC samples and 48 CNC samples). The overall performance of this signature is 83.0% (CI%: 73.9, 88.9) accuracy, 84.0% (CI%: 71.5, 91.4) sensitivity and 81.0% (CI%: 66.9, 86.6) specificity.

3. Analysis of differential performance of individual genes from signatures observed from training set

Table 2 summarizes the individual performance of the seven genes. For each gene, individual characteristics such as probe set_id (identifying the aprimetric probe set), T_check P value observed between 100 CNC samples and 100 CRC samples, and 100 CNC samples Multiple observed variations between 100 CRC samples are provided.

Upward means that the average signal for the CRC group is higher than for the CNC group.

Downward means that the average signal for the CRC group is lower than for the CNC group.

* Means the probe set_id according to the 2010 apimetrix annotation version (https://www.affymetrix.com/analysis/netaffx/xmlquery.affx?netaffx=netaffx4_annot&_requested=403680).

** means the same gene and its variable or related sequence for the gene or variable.

*** means the average signal observed in 100 CRC and 100 CNC array experiments.

                         SEQUENCE LISTING <110> Biomerieux   <120> Method and kit for determining in vitro the probability for an        individual to suffer from colorectal cancer <130> P2012813C <140> PCT / CN2012 / 072931 <141> 2012-03-23 <150> PCT / CN2011 / 072155 <151> 2011-3-25 <160> 11 <170> PatentIn version 3.3 <210> 1 <211> 22743 <212> DNA <213> Homo sapiens <400> 1 ggagttagcg acagggaggg atgcgcgcct gggtgtagtt gtgggggagg aagtggctag 60 ctcagggctt caggggacag acagggagag atgactgagt tagatgagac gagggggcgg 120 gctgggggtg cgagaaggaa gcttggcaag gagactaggt ctagggggac cacagtgggg 180 caggctgcat ggaaaatatc cgcagggtcc cccaggcaga acagccacgc tccaggccag 240 gctgtcccta ctgcctggtg gagggggaac ttgacctctg ggagggcgcc gctcttgcat 300 agctgagcga gcccgggtgc gctggtctgt gtggaaggag gaaggcaggg agaggtagaa 360 ggggtggagg agtcaggagg aataggccgc agcagccctg gaaatgatca ggaaggcagg 420 cagtgggtgc agggctgcag gagggccggg agggctaatc ttcaacttgt ccatgccagc 480 agcccctttt tttccagacc aagggctgtg aacccgcctg gggatgaggc ctggtcttgt 540 ggaactgaac ttagctcgac ggggctgacc gctctggccc agggtggtat gtaattttcg 600 ctcggcctgg gacggggccc aggccgggcc cagcctggtg gagcgtccag gtctgggtgc 660 gaagccaggc ccctgggcgg aggtgagggg tggtctgagg agtgatgtgg agttaaggcg 720 ccatcctcac cggtgactgg tgcggcacct agcatgtttg acaggcgggg actgcgaggc 780 acgctgctcg ggtgttgggg acaacattga ccaacgcttt attttccagg tggcagtgct 840 ccttttggac ttttctctag gtttggcgct aaactcttct tgtgagctca ctccacccct 900 tcttcctccc tttaacttat ccattcactt aaaacattac ctggtcatct ggtaagcccg 960 ggacagtaag ccgagtggct gttggagtcg gtattgttgg taatggtgga ggaagagagg 1020 ccttcccgct gaggctgggg tggggcggat cggtgttgct tgcctgcaga gagggtgggg 1080 agtgaatgtg cacccttggg tgggcctgca gccatccagc tgaaagttac aaaaatgctt 1140 catggaccgt ggtttgttac tatagtgttc ctcatggcga gcagatggaa ccgggagaca 1200 tggagtccct ggccagtgtg agtcctagca ttgcaggagg ggagaccctg gaggagag 1260 cccgcctcaa ttgatgcctg cagattgaat ttccagaggc ttaggaggag gaagttctcc 1320 aatgttctgt ttccaggcct tgctcaggaa gccctgtatt caggaggcta ccatttaaag 1380 tttgcagatg agcttatggg gggcaatctt aaaaagtcca cagcagatgc atccggctcg 1440 aggggccatc agctttgaat aaatgcttgt tccagagccc atgaatgcca gcaggcaccc 1500 ctcctttcct ggggtaaagg ttttcagatg ctgcatcttc taaattgagc ctccggtcat 1560 actagttttg tgcttggaac cttgcttcaa gaagatccct aagctgtaga acattttaac 1620 gttgatgcca caacgcagat tgatgccttg tagatggagc ttgcagatgg agccccgtga 1680 cctctcacct acccacctgt ttgcctgcct tcttgtgcgt ttctcggaga agttcttagc 1740 ctgatgaaat aacttggggc gttgaagagc tgtttaattt taaatgcctt agactgggga 1800 tatattagag gaagcagatt gtcaaattaa gggtgtcatt gtgttgtgct aaacgctggg 1860 agggtacaag ttggtcattc ctaaatctgt gtgtgagaaa tggcaggtct agtttgggca 1920 ttgtgattgc attgcagatt actaggagaa gggaatggtg ggtacaccgg tagtgctctt 1980 ttgttcttgc ttcgtttttt taaacttgaa ctttacttcg ttagatttca taatactttc 2040 ttggcattct agtaagagga ccctgaggtg ggagttgtgg gggacgggga gaaggggaca 2100 gcttggcacc ggtcccgtgg gcgttgcagt gtgggggatg ggggtatgca gcttggcact 2160 ggtactggga gggatgaggg tgaagaaggg gagagggttg gttagagata cagtgtgggt 2220 ggtgggggtg gtaggaaatg caggttgaag ggaattctct ggggctttgg ggaatttagt 2280 gcgtgggtga gccaagaaaa tactaattaa taatagtaag ttgttagtgt tggttaagtt 2340 gttgcttgga agtgagaagt tgcttagaaa ctttccaaag tgcttagaac tttaagtgca 2400 aacagacaaa ctaacaaaca aaaattgttt tgctttgcta caaggtgggg aagactgaag 2460 aagtgttaac tgaaaacagg tgacacagag tcaccagttt tccgagaacc aaagggaggg 2520 gtgtgtgatg ccatctcaca ggcaggggaa atgtctttac cagcttcctc ctggtggcca 2580 agacagcctg tttcagaggg ttgttttgtt tggggtgtgg gtgttatcaa gtgaattagt 2640 cacttgaaag atgggcgtca gacttgcata cgcagcagat cagcatcctt cgctgcccct 2700 tagcaactta ggtggttgat ttgaaactgt gaaggtgtga ttttttcagg agctggaagt 2760 cttagaaaag ccttgtaaat gcctatattg tgggctttta acgtatttaa gggaccactt 2820 aagacgagat tagatgggct cttctggatt tgttcctcat ttgtcacagg tgtcttgtga 2880 ttgaaaatca tgagcgaagt gaaattgcat tgaatttcaa gggaatttag tatgtaaatc 2940 gtgccttaga aacacatctg ttgtcttttc tgtgtttggt cgatattaat aatggcaaaa 3000 tttttgccta tctagtatct tcaaattgta gtctttgtaa caaccaaata accttttgtg 3060 gtcactgtaa aattaatatt tggtagacag aatccatgta cctttgctaa ggttagaatg 3120 aataatttat tgtattttta atttgaatgt ttgtgctttt taaatgagcc aagactagag 3180 gggaaactat cacctaaaat cagtttggaa aacaagacct aaaaagggaa ggggatgggg 3240 attgtgggga gagagtgggc gaggtgcctt tactacatgt gtgatctgaa aaccctgctt 3300 ggttctgagc tgcgtctatt gaattggtaa agtaatacca atggcttttt atcatttcct 3360 tcttcccttt aagtttcact tgaaatttta aaaatcatgg ttatttttat cgttgggatc 3420 tttctgtctt ctgggttcca ttttttaaat gtttaaaaat atgttgacat ggtagttcag 3480 ttcttaacca atgacttggg gatgatgcaa acaattactg tcgttgggat ttagagtgta 3540 ttagtcacgc atgtatgggg aagtagtctc gggtatgctg ttgtgaaatt gaaactgtaa 3600 aagtagatgg ttgaaagtac tggtatgttg ctctgtatgg taagaactaa ttctgttacg 3660 tcatgtacat aattactaat cacttttctt cccctttaca gcacaaataa agtttgagtt 3720 ctaaactcat tagaattgtt gtattgctat gttacatttc tcgaccccta tcacattgcc 3780 ttcataacga ctttggatgt atcttcatat tgtagattta ggtctagatt tgctagctcc 3840 aagtaattaa ggccatgtag gagagcatgg taaccacaga tagaactggt attatcccaa 3900 gtggtctgca gactgctgag tggggatggg atctgctctc tgttgagagt tggtaatcat 3960 tggtttgaaa tgtgatgaaa ccactcaagc caatgaaggt gggtgtgtag gtggggagta 4020 ctttgccata atattttaaa acattacctg gttagagttc taagtggtac ttatttttgt 4080 ttggttaggg gaaagcctga ataaaaacag aaatggacac ataatatgca tattccatag 4140 tctttgggag gctggaatgt gcctgggatt tgggtctaag tgtatgcgta attcttacct 4200 cactaaagaa tttgccttgt ttttttcctt ttggtgagtg actaaaacgt ctgggcttcc 4260 ctgtgtgcgt gctacagtaa gcaagcagag gctgtgcaaa ggtgtgagca ggatcacgtg 4320 gaatctggag gatacatctt ggcttgcaaa ctgcctctgt ctcctgggtg ggactgttct 4380 gtccttgcac tgctgttctg tgttacctct tggggtgtaa ggttttgctt acaggagaca 4440 aactttgggc gtagaatgga agccactgcc agcctctgtg ctgagaagga aggtgcttgt 4500 ttcaaaggga gcagcaaggg aggcttgttc tactcacctg ggcctgtttg cctgagaagg 4560 ggagataagg gctgaactgg gactagccag ggggaccaac acaaatggtg ggggatcatg 4620 acctgaagga ttctttcctt cccatgagct gcagggctgg ttgccgtcct tgcaactgtg 4680 tcttatttgc ctgtgccgtt atatcttggt gacccctcca cgtgtacact actgacaaac 4740 gggtggagtg ctggggagaa gtcactgtgc cgcccaccta gtaaaccttc tgtctgtgct 4800 catggcatct ccaagatggg gcactgctgt gtgcagaatc cagggtcctc tttctgcttg 4860 caactccttt ccctggatgc cccagaaaca atccaggcct cctttcctat cttacccctt 4920 tgctttgctt tttaccccag cacctctata accgccttct cttcttttca gaactccttg 4980 tttctcgtcc tgttttttat gattacaaaa ctcttgcttc caccctggaa gataactgct 5040 atagatgcct gtatgtaaat ggtgctgtct ccagcaactg gcatgctgaa gaagaattga 5100 ttcacggggt ataaatgttg gggattggaa gtggggatga aatggcactt gttgatacag 5160 gagcagagag gtgaggccga ctgctgaaga cagctcgcca ccctccttgc ctccactcca 5220 atccaggggc tggggccaca ttctttgcct tcatttatcc tcagatcagg tgagatcgac 5280 aggaggtgtt gatggcagtg ccagcaatta ttgctaatcc gtttgcatcc ttatgcatag 5340 atctgaattc agactttgtg aatttccaga ggtgtgggta atataataga attcagtgag 5400 tgggcatggc tgatcttgtg caaattaaaa gttatggggc ataagaatag caaaagttga 5460 acttctttta aaaaggaaag taccctgaga gccagtattg gttgaggctc ttcagtatgc 5520 ccaggttggc agcactgaga accgcaggaa cggcctgttg ttacaaaaag gagattgact 5580 cagctgccct tggtgcatct gactgactat gactgctgag agattccaag gacccttaat 5640 gccagggcta acctctccat gtgcagtgag acctctggag gaagtgtcat cctctggctt 5700 tgtgtggtac tcattatggt gcagtgcggg catgaaatga agacacccaa ataggcttac 5760 agatacgata tgttttaaat gttcgtattt aacaaaaaca tactgacact gtttggaaat 5820 ggcaacagga agatagcaaa atgaatacta acattacgaa aagatgaaca ggtacatgtt 5880 ccaaggcagg tggctgtgaa cttcctctga gtgaaggcat cccctccagc acctttcagc 5940 ctgctagtta ggacgacccg ccgccaccct ccaggacctc cagccctgca ctgcctttcc 6000 tctcttttaa ataattcttc attgagttct aatatgtaaa aaaaaaaagt ttactgtaaa 6060 gtttgcaaat aaggaaattt tttttaaaag tcctcagtaa tcttaccagt aacaattgtt 6120 atgggcacat ttgcttttgg aagatttctt ttgtatgcat gggataagta catttttaaa 6180 caaaaatggg attatgccat aaattctatt ttgtgacttt aatatatagt gaacaccttt 6240 tttaatgatg acaggatgtt cccttgcatg gctgtatcaa tttaaacaat cttgtttcaa 6300 tgggcataca gggtattttc tagttttttt ttcctcttag aaaataatac ttgcgatgac 6360 tttccttgta gctcagactt tttcacgtct gttgttatct ctttgggaat gctgaataca 6420 tacatttcga gaaggaaatg actgttaaac tcttaagact tcaggttcat attgctaaac 6480 tgcccagcag ggagggattt tttcaattag tgttctcact ggtgaggcaa acctgatgcc 6540 ttcccctctt cctcagaacc ggctttatca cattgaaaac ctttgctcct ccgacggatc 6600 gagtctgctt tccctctgga tgtgagcatt gctttgtctg ctggtgactg aacatctcta 6660 ccttgtgtca attggccatt tgtggtgtgt gtgtgtgtgc gtgtgtgtgt gtgtgtgtgt 6720 gtatgatttt ctaattccta gtcatttttc tattgattgt tttgcaaaag ccatttacat 6780 cttaaggata ttgataatct tttgttatat ttgatgcaaa tatttttttc cagtttatag 6840 gttgcctttt aattttgtgt ttcaggtaga taaaagttaa acgattttct taggttagtt 6900 tatcactgtg gtttctgaac ttgttatgtg tagatctttt ccaccccaag agtacataaa 6960 tattaatcca tactttctta tggaacttgt atggtttcgt tttttacatt taaaccttct 7020 tccccgtggt gtgtgttgtg gaatctgtgt ttgtgtgagg aggggcatgg tgctctcaga 7080 acccacctcc tgtggccaga gagccctgtc ctgtgagggt ggttgtcaca gtggcagggt 7140 tcaattcaga agaccttgag ggcaggctga tgtttcctga atgggcccct ggttgttgct 7200 tgtccctgac tctccatttc cccatctgag tggatttgga cctaataggg cactggagct 7260 ggttcgaatc ctgactggac tacttggcaa ctttatgtct gggagcaagt tacttaacct 7320 ccccaagcct gtgtctgtga aatgcgggta aatgaatgta gatgtttggc agcagctact 7380 ccttgttgag ctctcacagt gaactctcct gcctctgccc tccttccccg cctcccctgg 7440 tgcctagcgt caggtctagc cacttcctcc tgggcccctc tcccttttct gtggctggct 7500 gcctgcccgc ctggcgctgg acctttcatg taacgggaat cagcatgtat attctggtct 7560 ggtctgtttc tacacttaat tttgtttcca gtagtatttc cctgtaccgg cagagttcac 7620 aaacacattt gaagaggctt tttctcagga ttcttaacct tcccaaagga agtcccatgg 7680 atgggtttct agaagtctat aaatgctctg aaattgtatt tttctgtgga aagcataact 7740 ttcatctgct tgttcgtgct caaaaaagat catgaatgaa tgattgcatg attttatgcc 7800 attgtgctta tactaaagga tatgtagccc atctcttgag ctgttaaact gttttgacta 7860 ctttaaatcg tgcagctgtg agcatctctg taaatttagt gtacacatgt atcccctgga 7920 gtggcattgc ctcggcagtg agcacttatg gttttataac tctcttcaca gactcaaatg 7980 actccagaaa gctacacttc ctgttgtgag tatatgatat ccatttccct acatagccac 8040 taacatcagg tttttacaat tttatttatt tcttgctact ttaagaaatt tttgtggtga 8100 aatacatata atagaagttg actatctgaa tcatttttaa gtatacattc agtagtgtta 8160 agtatgtcgc cattgttgta caaccaatct ccagaacttt ttcatcttgc aaaacaaact 8220 ctgtacccat taaataacat taaacattcc attccctcca gcctcagcaa ccccattcta 8280 ctttctgttt ctgtgagttt gactattcca agcacttcat atcagttaaa tcatgaagta 8340 tttgtctgtc tgtgactggc ttatttctct gagcacagtg tcctcgagat gcgtctatgt 8400 tgtagcatat gtcagaattt ccttcctttt taaaagatcc aaataatatt cttattttat 8460 atcttttttt tatccattca tccattagtg gacacttggg ttgcttttgg ctattgtaaa 8520 taatggtgct atgtacaaat atctatatta ttgtatttac aagtataatg ctgtaatgta 8580 cacacatctt tttgagatcc taccttcagt tcttttgagt atatagccag aagtggtatt 8640 actaaatctt acgatatttc tatttttaat ttattgagga accactgtag tttttcatag 8700 caactgcacc attttacgtt ctcaccaaga gtgcacaagg gttccgaggt tcccacatcc 8760 tccccaacac ttgttatttt ctgctttttt tagattgcag ccatcatagt gggtgtgagg 8820 tgacatttca ttgtggtttt gatttgcatt tccctaatga ggagtgatgc tgagcatctt 8880 ttcatatgct tactggtcat ttgtatgttg tctttggaaa aatgtctatt caagtccttt 8940 gactatttta aaaattgggt tattagagtt atcgttgttg ttgacttgta ggagtttctt 9000 tctatattct ggatattaat cccctatcag atatatgatt tgcaaatatc ttctcttatt 9060 ccataaggtt actttttcac tttgttgatt gtgttctttg atgtatagaa gtttttagtt 9120 ttgaaatagt ctaatttatc tgtttttact tttgtggtct gtgcttttgg tgtcatatcc 9180 aagaaatcct tgccaaatcc aacgttataa ggtactttta aggtatttta gttgtcttag 9240 tctatatttc tgtactcacc tttctttatc cactcatcag ttgatgggca tgtaggttgg 9300 ttccatatct ttgcaattct gaattgtgct atgatcaggt gtctttttag tataatgatt 9360 tactctcctt tgggtagata cccagtagtg ggattgctgg atcgaatggt ttttataatt 9420 ttctatttta ccacagtttc tctctgcatt tttcctcttt gaccactaac catgtgaaat 9480 tctcatattg acctttataa tgatcatgaa ctcttagtat cattgggaag gccacatttg 9540 ccacttatga ttgtaaacct tatcctccat ttttcctgtt attgttggtg caaaaagcac 9600 ctattatacc aggactttaa aaatcagtct gataagtctt tgataagtct aataataata 9660 actgataagt ccattgaatt tgcttctgat tactttttct ttagtagcta aacatgtatg 9720 tactcctatg attacaatga acactcctct ccatttaaat taattattta cattgatgaa 9780 atagcaaaat gttaatgact aaatactgtc ttggtttttt cgttccaggt cagtcaatat 9840 taacttctta taattttctt ttttttcttt atgtgtgtgt gtgtgtgtat tttttttttt 9900 ttaatttcaa tggcttttgg ggtacaaatg gcttttggtc atatagatga attctacagt 9960 agtgaagtct gagattttac tgcaccggtc acctgagtag tgtacattgt acccaatatg 10020 tggtttttta taccttgccc ccctcttacc ctccccactt tgagtctcta gtgtccatta 10080 tgtcactctg tatacctttt tgtacccata agttagctct cacttataag tgagaacaca 10140 cagtatttgg ttttccattc ctgagttgct tcacttagaa taatatcctc cagctccatc 10200 caaaattgct gcaaaaaaaa aaaaaaccac aaacattatt ttgttctttt ttattgctaa 10260 gtcatattcc atggtgtaga gataccacat tttatttatc cactcactgg ttgatgggtt 10320 ggttccacat ctttgcaatt gtgacttgta ctgccatcaa gtgtctttct ggtataatga 10380 cttcttttcc tttgggtaga tacccaggag tgggattgct agatcaaatg gttcttaaca 10440 ttttctctct ggatctattt ctggaaattt taggctccag tttttgttgt tgttgttaat 10500 aaaatgcaat ggaatgtaat gatcatcact tttcattatg ctttaaaatc tggtaaatgg 10560 aggctagaac actcctgtaa ggcaagaata ttctctctgt tggaactcaa atacacagaa 10620 ctgggtaaat ctcaatctta atctttgatt caggacacaa catggctctc ttttacttgc 10680 tttctttaat tgttttttaa taatgtggta agcatttctg aatctcctat ccaatacaaa 10740 aactaggaca atacaca taactcctat ggttacaatg aacactcctc tccacttaaa 10800 ttaattattt acactgatga aattgaaata gcaaaatttt aatgactaaa tactgtcttt 10860 gattttttgt tccaggtctg tcaatattaa cttcttataa ttttcttttt ttttctttat 10920 gtgtgtgtgt gtgtgtgtat atatatatat ttaatttcaa tggcttttgg ggtacaaatg 10980 gcttttggtc atatatatga gttctacagt agtgaagtct gagattttac tacaccttcc 11040 acttatgtgg tcccacacca cccgcctccc ctgccgcctc ctgccacccc ctaggccaag 11100 gtaataatca tcctgaatcc tgggtttatc tctcacttgc tttcttttca tataattttg 11160 caaaagaatc tgatctaaat gtgtttttca gagtatatat ttatatttta gctgttctta 11220 gagaaaattt attattttgc atgtaatctt atggaacatt ctcatttaat accatggtaa 11280 gattcagccc ttgcccaggg gatagttcat ttagtttgtt tactggatag agctcatcat 11340 gtgactatac ctcagttagt ttatcagttc tcccatccat ggtgactagg ttgcctctca 11400 gcctctcaac aacactgttt ctcagtgtcc ttgtagaagt gatatgtggg tgttttctcc 11460 ttacacagag ttgaaaggtg acgacaacaa cgttggcact accaatcccc caccctccag 11520 aggggtaacc agtgttacca gtttgctgtg tttcctgcta cacctcgcct tattcacttc 11580 catttgtatc tgaaaaacgt gttgcatggt ttcttttcta tagaagtggt aaaatgctat 11640 tgtgtcctgt acattattga ttactttttt tcatttaaca gtagggagat gcctgggagt 11700 acacagagaa ctgccctcat tgttttcaac ttctgcactg tatgtctgtg agtttagcca 11760 ttctgctgtt aatggaaatt tacagtattc taatcttttg atattacaaa cagttctgtg 11820 cgatcatcgt catacacaac cccttgtgca caatgcatga gtgtttctca gggtaggtac 11880 caagaagtga aattcctggg tcatagggcg tgagtccgac atttttctcc attctgccct 11940 gttgccctcc agagtgggtg tccagctttg catacctaag tatgagagta tctgttgttc 12000 atatcctcta cgacgctcca tatatgaaac ttaagtttct gctagttgcc atctttgatc 12060 tatcatgtat gcagtgacct actaagactg taattggtac agtagattct tgtcatctgt 12120 gtgtgaattt agcattcatg ggcttaatgc tgacaaggcc cccagggtcc aagacatata 12180 atcatgtata attttgtcaa ggtataattt tttaaattgc ttttgtcatg tgtctgctgg 12240 tgatgcccaa cccagtgctc tgcacccagg tcacactgtg gctttgtcct ctgcttatgc 12300 ctgcattgca gcaactgtcc tgaagagacc aaaattatgc agatttaggt aagtccatgg 12360 ctaatgttat tatattatgt gctattgtaa tggatggggc tgtggagtgt atgaatttat 12420 aaatcactgg tcttgtaatt aaaattcaaa cactatagaa aaaggccatg tagaagataa 12480 aagttcctct ataatcccgg acccctaaga taactactaa tgacaacttc atttatattc 12540 cttcagacat tttctggctg tggatgtact aaaatgtatc ctattattct ctgccctaaa 12600 atggaatcat acaaggtgta ctgttatttt tatggctcta taacatgtca tattgtacgt 12660 gttggtatgg tcattttaac catttttcta gtgatggctt tgaggttatt tgcagtttcc 12720 tagccatctc aaagtgtgct gcggggatct cttttgcatc cctctgggtg cagagctgag 12780 gcacccagag gcagtgtcca gaggaggcag catctgtagg tgtcttcacc tgctctggct 12840 cttggcacat ctggttggtg acactgtttt gtgagatggg ttgaaagcac gtgctgccaa 12900 aatagaataa tgttggtcct ctcctcatgt gccgtggaac tggggtaaaa ctgcgtagtg 12960 gctgcagctg cctgtccata ccggaatcga gtataacacg gtgcctggct tagcacaaaa 13020 cagtagtggg tcctgcaggc cccagagtct aattcctggt attctttccc ctacacagat 13080 taaataaacc aaaaacaaac tattctagga aagcgtctgt gacatttgta aaaagtggta 13140 tttaatgatc ttttattcac ttgtctgttt agtttgttga aatcttaagt ggcatcctgg 13200 tctgggaagg agtgctgtct gcgcctgccc tccgctgggc acagcgtggc tgcttcaggg 13260 gctaagcaca cactttctgt cttctaaagg gccgccacat gccaggagct caggtgtgag 13320 cccggctctg gctcttacct catagggtca ctcatagggg cacagggagc agaacattgt 13380 acacagcgag gcaccacccg gcttggcatc tgcctcggtg gacttactac ctctagaagg 13440 aaatacctga gttcctctgg cctcagctcc tagagtgact ggtgtgctgt ccctgttact 13500 cttctgtcaa ggtgacaact gtgtgaccca tcatctgtgt gtcaaagcaa ggccctgcct 13560 gggcctctgc tcctgtgctg accccaaagg caaatgcttt gctagtttcc ttccagttaa 13620 tttcacctat gaatagatgt gtgaaaactg ttcaaagcca tacctgcaca tgtttgaact 13680 tcaaaccctg tgggtgattc agtggcatct ttctctaacc cccagcctcc cttcccacag 13740 aggccaccgt catggccagt tgctgcagtt tctttccaga gaacctgtgt atgtgtaaag 13800 ctgtacaggc gtgggtacac cacacagcct gtcttgcact gtggactgtt gagttactag 13860 tacatctagg taagcaccgc atatctgtat tcatgtctgc cttggtcttt tcaacatctg 13920 tgtggtagcc gtgtttgaat tacccattcc ctttttgggg aaccattaag ttgtttcagc 13980 aatttttact gtagataagg ctataccgca tatctgtgta catgggtttt tatgtacatg 14040 ggcaagtata tctgtgagag aaaagtttcc tcaggaggaa ttctgggcac agcatgtgta 14100 aatttctaaa tatgatggac acccccagct tccacctcaa ggaggttggt cccattgaca 14160 tttccccaca ccttcaccca ggctgtgccc ttaaacttgg ttatttgtca atgtgagaag 14220 tggaaaatag tatttaattg tagtttggat ttgtatttct attgggttgt atacttactg 14280 attaataata agagctcttt acatattaag gaaattaacc cttttcaaat acattcctat 14340 ttctcactaa tctttaagtt ttattgtaat attttgctct ttagtttata tatatatgta 14400 tatatatata tatgatatata tatatatata catatatata tacatatata tatactaatt 14460 ttcttttatg gttcctggat tttgtgagta gtttgaaaag gctaatccag ctgaagattt 14520 tgttgttgtt gttaaacccc atgttttctc ctaactcttt ttatttttat tttggaggac 14580 tctatctaga cttaatttta gcataacaag tgacagggtt agttagcctg ttgtccttac 14640 accattttct ggctaataca gctattaact attgatctgt ctattcacgt gccagttcct 14700 aatggtttta catagtgtaa tctgcacttc aaaatagcga agggaagccc tacctcatta 14760 ttctactttt ccagaattct cctggctatt ccaggctgca tgtttacctt aaccttccct 14820 gtgatgtctt catgccgttg tcttcttatg caagaataag gtacgtcttt ccatccactc 14880 acgtctattt aatttgactt tgcattacac agaaagctgg tcttggtctg tctacctcgg 14940 catctagttg tcctcactgc cccctagccg accccacccc atctgactga ctaccccatc 15000 acagagtact tttatttacg ttttgctctg cctaatggtt acttgatact gtcacgccga 15060 cagtgtccag ttcagtggtc tttgcagttg aaatgctccc gtacacactg tcttgttaaa 15120 aatgccagta agttcataca aacccagctt gcacccaagg tcacattcag agagcgtagg 15180 gctgggatgg gttgttttcc aagcttctgc cactgtgtgg ctagctcttc ccactgggaa 15240 gttctgtgta cccggaatgt cggagtggag tcctgttcta gtgtccagca cctgaccctg 15300 tgcccaaccc ctcaacagcc tattcctgct gtccacagcc tgctggaact ttttacaaaa 15360 tatgttgcca tgctggaccc tgggcactgg acataagccc cctggcagcc tttttcatgt 15420 cacccaaagg ggtaattgtc ctactggtgg tctgtaagat gagttagggt gacttgctaa 15480 tagacattgt aaatcttaat atttatgtat gtattttatt attaccggtt ttccatttat 15540 gatggtaata ttgtttcttc taagaatatt tatttttcct tctaaatatt gagataaaat 15600 tcatgctttt gaaatgttct attcagtggc ttttagtata tttgctatgt tgtgcaacca 15660 tcgacactat ccatttctag aactttttcg tcatcccaaa cagacgctct gtattcataa 15720 aaaaataact tcctacctgt ctctccccct agtctttggt aacctttgtt atactggtaa 15780 actttgttgt gctctctgtc tgtgtgaatt tgcctattct aggggcctca tataagtgta 15840 atcatacagt atttgtcttt ttgggtctgt ctgatttcac ttagcgggtt ttcagggttc 15900 attcatgttg cagcatataa cagtactgcg ttcctttttc tggctgaata atattccact 15960 gtatggatag accccatttt gtttattcac acatcatttg gacatttgga ttatttctgg 16020 tttttggcta ttatgaacaa tggtgctatg aacagttgcg tacaagtttt tgtgtgaaca 16080 tatgttttca attctctcat tatataccta ggagtagaat tactgggtca tatggtaact 16140 gtatattttt gaggaactgc caaactattt tcccacgtcc atgcaccatt tcacattccc 16200 accagtaagt aagagggttc caatttctgc gcattcttgc caacactagt tattatctga 16260 ctttctggtt ataatcattc taatgagtgt gaagtagcct ctggtgtcat ttggatttgc 16320 atttctctga tgagtgatgc tatcaagcac ctttgctggt gctgttggcc atatgtgtat 16380 gttccctgga gaagtgtctg tgctgagcct tggcccactt tttaattagg cgtttgtctt 16440 tttattactg agttgtaaga gttctttata tattctggat tctagaccct tatcagatac 16500 atggtttgca aatattttct cccattctgt gggttgtgtt ttcactttat cgataatgtc 16560 cttagacata taataaattt gtattttaaa agtgacttga tttggctgtg caaggtggct 16620 cacgcttgta atcccagcac tttgggagac tgaggtgggt ggatcatatg aggaggctag 16680 gagttcgagg tcagcctggc cagcatagcg aaaacttgtc tctactaaaa atacaaaaat 16740 tagtcaggca tggtggtgca cgtctgtaat accagcttct caggaggctg aggcacgagg 16800 atcacttgaa cccaggagga ggaggttgca gtgagctgag atcatgccag ggcaacagaa 16860 tgagactttg tttaaaaaaa aaaaaaagtg acttgattta agggaaaaaa tgactggcta 16920 tattcagtca gatatggcaa aaagtctcaa ggtgttaatg tgaatgatta aggtcttggg 16980 gggggtgtcc cctatcagac tacaggtgtt tagaggcaca gaaaaaggtg cagttgggtt 17040 cttaatgtga aatgatgaga agcacaactc cagtgtgtct ctttgtgtag aatgtcagca 17100 gacaccccct gctagatgtg ctggatcatg ggaaagcatt tccatttgtt actagattgt 17160 tcagaagttt taatttatga tgggtgtggt ggctcatgcc tgtagtccca gcactgtggg 17220 aggctgaggc aggaggatca tctgaggcca agagttcaag atcagcctgg gcaacatagt 17280 gataccctat ctcttaaaaa agaagaagtt tttaaatttg aaataataat aggtactgga 17340 tttatgcaaa tgtcttttct gcgtcttttg agatgagtat caggtttttt tttttccttt 17400 tatcatctga tgatgaactt aatgtttcca tttgtattaa tggaatacta agtccctctg 17460 tgatttctga accaagctat tcctaggcct gagttttatt ttgttgacac agaaataaat 17520 tagaaggcca agcgtggtgg catgtgcctg tagtcctagt tgctgaggta agaggattgc 17580 ttgagcccag gagttcaagg ctgcagcaag ctttgattgc gccactgcac tccagccttg 17640 gcgacagact aagacgctgt ctcaaaaaaa aacaaaaacg acaaaaaaaa aacaaaacag 17700 aaaaaataaa ctaaggcaat gacagtccct ggcaaatgct gggagggagg cagcagtggt 17760 cagggaaggt aaccctgaag caggacttgt aaagcaaata agattgggag gccaaggtgg 17820 gtggatcacg aggtcaggag ttcgagacca gcctggccaa catagtgaaa ccccgtcttt 17880 actaaaaata caaaaaaatt agccaggtgt ggtggtgggt gcctgtagtc ccagctactt 17940 gggaggctga ggcaggagaa tctcgaaccc aggaggcgga ggttacagtc agctgagacc 18000 gcaccattgc actccagcct gggtgacaga gcaagattcc gtctcaaaaa aaaaaaaaaa 18060 aaaaaaacca agaagaaaag gaatgaatta gaacttcttc tgcttggact taagggcatc 18120 atcaggcagg ttttgggtag gatagcaggg gaggcagaga catagtcggg gtcagtggtc 18180 atgagtgtgg ctttgagccc aaaaacttgg tttctgttcc ctactttgcc actcagtagt 18240 gcatgacttt ggccaaattt cttaaattca tgaagcaagt ttccgggtga atgaaatggg 18300 gataaaaata gtgttcaaac ctatccgttg gtttgtgtga aactgaaatg aatagtatcg 18360 tgcaggtact tgtgagcaag gggagctgct gtttcctgtc cctttatgat gggaaatatc 18420 tagacaagtt cccaaccctc tgcactgcag gctgcatggc acggagggtc ttgtaacacc 18480 agctggggct ggccttcttt taggagcttc agtggttctg aaaactttta tttgtttgtt 18540 tgttttagta gatgtggggt ctttctgtgt tgcccggact ggtctcaaac ttctggactc 18600 aagtgatcct cccccgctca acctcccaaa gtgttgggat tacaggtgtg agccactgtg 18660 cccagccttg aaaacttttt caggttcttc cagggttact gggctattaa atatttctat 18720 ttcattataa gtcagttttt caaagttata ttatcttaat tacctttttt atatgtatta 18780 gtgtagagta gcattttata ttttgatatc ctccttatgc atagtttttc actttttatt 18840 cctagttttt cgtttttaat aagactttca agaaatttat tttattggcc ttttgaaaaa 18900 agcagcttta gataaagtaa gcagttctgc tttcatttta taatttattt ctacttttgt 18960 ttcattaatc ttttcctccg gcatgccttg gattttgttg tgttactctt tttctagagg 19020 ctcgcattgt gtgtctggtt cacttatgat cacgcttgcc tacttttaag aatggaagag 19080 gggaggtgga gggtggctgc acagtcgagg gtgtgaggca gtcttgctct agccccacca 19140 tgccctcagc ccgctgtggc cacgctggtt cctcaattgc tggggcgtgc agtgtctgta 19200 agggaggcta ctgatgccat ccgaggaaga tgtaaggttt cgtgtgggca gcgagagcct 19260 agcaggcatg tggggtgccc agcaaagggt aacagtggac agttgttgcc tcattccaca 19320 gagttttgat tttttttttt tttttaatgg tcactccatc aacatccccc atggccagag 19380 cctgagctgg tccccagaga cacaggcatt cagctgacag cctcgccttc acgctgctgc 19440 tgttctcatg ggggacaggc ctcaggtggc aatgcacaaa tcattagtta agggcagttg 19500 tgacagttac caaggagtgt agtcccccgc cccccgccca gtgaaaacag ccctaaccag 19560 gggtggggac ctttgggctc tgacccgaag ggtaggagaa gctggaagga cagcattcct 19620 gtctgcgaag gcaggagcaa agctgccagg ctatgaagga aatggctgga gcctgaagtc 19680 atgcaagctg gggctggcag ggacagggcc aacttccagg cctgggggcc accatgagga 19740 ttcaggacgt gacccccagg gcacatgaag gccttccatc tgtatttaag aaaagacttt 19800 atcagacgag tatggtggct cacgcctgaa tcttagcact ttgggaggct gaggcaggtg 19860 gatcacgagg tcaggagttc aataccagcc tggccaatat ggtaaaaccc catctctact 19920 aaaactacaa aaattagcca ggcatggtgg cgcacgcctg tagtcccagc tactcgggag 19980 gctgaggcag aagaatcact tgaacccggg aggtggaggt tacagtgagc caagatcgcg 20040 ccactacact ccagcctggg tgacagagtg agactccgtc tcaaaaaaac caaaagactt 20100 tatcttattt cctatatgtt tgtggtttca gtcctgatgt ataatttgac cctagttaga 20160 atggttatct gaggaagtgg cctgtacgat ttctgctttt ttaaatgtgt ggctcccttt 20220 cttcattgat taacgtatga ttatttttat aaatgttcca tggcagtggg aagggattct 20280 ctgtcacatt ccacatctgg atcagttcct ccccattttg ttggtcaaat ccgatctgcc 20340 atatcctgtg taatgacaag tgagttgcat tctcaccgtc actcctgggg tctctccgct 20400 tcccctgagc tggctcagca gtctgctcca tgtgttttga tgcagggtga cccattggta 20460 ttcccgacac taacgccccc gtctgtggac tgcttgctgc ttgggcttca ctgtgtctgg 20520 tgttgacagt gcagacctaa aggtgtgcac acatgtgcac acacactccg ctgtcttctt 20580 gtttgcactg gacttaaata tctatgaggg ttattttcaa ctgctgaatt tggaatgatt 20640 tttatatctt ttctgctttc tgcccatgta catgtgttta ttttacactg ttgtgattgg 20700 tagttactat gtggggacac aattacttgg gctgaaataa tccacctgtt gtggttgggg 20760 tcctctgggg cattccaggg tgagaggttg tcactgccac ctgggccatg tgggccggca 20820 ccagcatttt gtggttacga attctacagt cacaaatatc tttgggcaaa tccccttcta 20880 tacctcaagg cagcttttgg tttgcaaccc cactggccag agggaagggc cagtcacttg 20940 gctctctcac tgccctgcgc cccagatggt tctagggctg ctgttttccc ttggccctgc 21000 caacaccact gtttttactt ctgctcattg gctgagtgca gtggttcctg gaagccagtg 21060 gcacgtttcc ccgcgtagct cgcttatccc acagcacaca cccaagggtt ctgttgctaa 21120 cacgctgaat taattctttg ctcatcttac agagtgtgtt ttgactgccc ccatttctga 21180 ggccttgtaa ggccagagct ttgttgcttc atcggcaggt tgggacttag atggccgtga 21240 atgtttcctc tctgctgctg cagtaagtaa gtgcccgcac catagtgtgt ttggaggctg 21300 aagttgaagc gaggctgtga ggggagatgg acgtgtgagg agggatgatg gggcttgagc 21360 aaagtggggg agggggcaaa ggcagttggc ccaacacatt ccccacccct ttgagaggtc 21420 tgaggcctgc agacctggct cggagcccac ctggtagtcc tcagactgtg tgtgtgtgtg 21480 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtaaaag agagaagttg 21540 tggagaaatg gggggctgat tctgctcaga ttcatcagga tgagtagaag gcacccagct 21600 ctcaccctgg cctgacatgt gtgtccctga gcaggttaca gtcctctctg agcctctgct 21660 tcccatctgg accctgctgg gcagggcttc tgagctcctt agcactagca ggaggggctc 21720 caggggccct ccctccatgg cagccaggac aggactctca aatgaggaca gcagagctcg 21780 tggggggctc ccacggaccc gccgtgggcc caggggaggc agagcctgag ccaacagcag 21840 tggtgctgtg gaccgtggat cctgagggtg gcctggggca agtaccggct gagggtccag 21900 gtgggctttg tgtacctttg ggtcctgggg ccctggtgac ttggactcca ggttagagtc 21960 aagtgacagg agaaaggctg gtggggccct gtgcttccga cttcatttcg agtgatggca 22020 gttcccagga aggaatccac agctgacggt ggctgacaga tcagagaatg gaaggcgagg 22080 caggcgggcg tctgcgtgac ctcaggtgct tggggcccag cagacccaga gaaccatttc 22140 cactaggcca gggtgccgga agtgtccaca ggtcttagat tccctgttca gatgaaaaga 22200 tttgtgcctt taatgataaa agtgatctgc atagagtcaa aaattcaagc catgggtata 22260 aaatgcaagt aaaatccctg ccctcaccta tcccacccta ctacacagag atgtcctctc 22320 gagtttccta gactcactct ggaaatttct gtatacacac agaagcttgt gcctctgctc 22380 gtgaaggcag agggagggag agctgaaggg ccagcacctt ctcacctgtg ggccccctca 22440 gtgctcggtc ccagagcatg caggactgtg cctcgtgttc agtttgctgg tctgacttca 22500 tgctccttgg gcaggatatg catgtgccat gctaggagac atgtggatgt gaagctgggg 22560 gacaatgtcc cctggctatg cctttacaag ggaagtaagg aaggtaggag gtgagcctgg 22620 gagggaggga gggaggcgcg gagccgccgc aggtgtttct tttactgagt gcagcccatg 22680 gccgcactca ggttttgctt ttcaccttcc catctgtgaa agagtgagca ggaaaaagca 22740 aaa 22743 <210> 2 <211> 4833 <212> DNA <213> Homo sapiens <400> 2 gtgaactgtt gcaccgtgca attgcacact ataaatgtct ttccttatct gtgtgtactc 60 ttatctcact gttctatttt ttctcctcat ttatattaac tctttcttac ctttttttct 120 gaacttctag gccttctctt tccagaactg gtggaagaca aatgaaacgg ccaagatggt 180 aagaaacaag ccgcatttct ccttggggag actgataatt taaaaggttt gttgtgtcag 240 aaacattccc agcttcatca ccaacccttt ccttccacct ctgcccactg gagaccactt 300 atatcccgaa gcggacgcgg cagctgaagt caggaaacca tgcatcacat tagcaggagc 360 caactgcaga ctttaaactc cgttcaacat gtggatgcgg cagagaaatg acctgtccag 420 acaagccggg gcagctcata aactggttca tctgctccct gtgcgtcccg cgggtgcgta 480 agctctggag cagccggcgt ccaaggaccc ggagaaacct tctgctgggc actgcgtgtg 540 ccatctactt gggcttcctg gtgagccagg tggggagggc ctctctccag catggacagg 600 cggctgagaa ggggccacat cgcagccgcg acaccgccga gccatccttc cctgagatac 660 ccctggatgg taccctggcc cctccagagt cccagggcaa tgggtccact ctgcagccca 720 atgtggtgta cattacccta cgctccaagc gcagcaagcc ggccaatatc cgtggcaccg 780 tgaagcccaa gcgcaggaaa aagcatgcag tggcatcggc tgccccaggg caggaggctt 840 tggtcggacc atcccttcag ccgcaggaag cggcaaggga agctgatgct gtagcacctg 900 ggtacgctca gggagcaaac ctggttaaga ttggagagcg accctggagg ttggtgcggg 960 gtccgggagt gcgagccggg ggcccagact tcctgcagcc cagctccagg gagagcaaca 1020 ttaggatcta cagcgagagc gccccctcct ggctgagcaa agatgacatc cgaagaatgc 1080 gactcttggc ggacagcgca gtggcagggc tccggcctgt gtcctctagg agcggagccc 1140 gtttgctggt gctggagggg ggcgcacctg gcgctgtgct ccgctgtggc cctagcccct 1200 gtgggcttct caagcagccc ttggacatga gtgaggtgtt tgccttccac ctagacagga 1260 tcctggggct caacaggacc ctgccgtctg tgagcaggaa agcagagttc atccaagcag 1320 cagcagcagc gtgtctttcc atgcgcttgg cattctttat tttcccagcc tgggaggata 1380 tgagagttcc agggaaatgc tgtattggac atgcaagact cacctgggga acttatcagc 1440 agttgctgaa acagaaatgc tggcagaatg gccgagtacc caagcctgaa tcgggttgta 1500 ctgaaataca tcatcatgag tggtccaaga tggcactctt tgattttttg ttacagattt 1560 ataatcgctt agatacaaat tgctgtggat tcagacctcg caaggaagat gcctgtgtac 1620 agaatggatt gaggccaaaa tgtgatgacc aaggttctgc ggctctagca cacattatcc 1680 agcgaaagca tgacccaagg catttggttt ttatagacaa caagggtttc tttgacagga 1740 gtgaagataa cttaaacttc aaattgttag aaggcatcaa agagtttcca gcttctgcag 1800 tttctgtttt gaagagccag cacttacggc agaaacttct tcagtctctg tttcttgata 1860 aagtgtattg ggaaagtcaa ggaggtagac aaggaattga aaagcttatc gatgtaatag 1920 aacacagiqc caaaattctt atcacctata tcaatgcaca cggggtcaaa gtattaccta 1980 tgaatgaatg acaaaagaat cttctggcta gggtgttaga tatatttatg catttttggt 2040 tttgttttta aatcaagcac atcaacctca agcccgttta gcaatgaggc agtgtagatg 2100 aatacgtaaa ataaatgact ttaaccaagt agctataatg ggacttagca ctgtatgcat 2160 acttaaaaag gttttgaaaa acaaactact tgagaaatat ttgtttatat ttttctctaa 2220 catcatgcta tgtgtcagtc tgaacatctg acaacagaaa tttcagttat tattctagct 2280 aagttttgaa aacatttgtc atgctgttta atagaaaact gcaaaccaga gacactgact 2340 ccattaataa accatatttt gtgccgtttt gactgttctg accaaatact aatgggaaca 2400 attcttgacg tttttctgtt gctgattgtt aacatagagc agtctctaca ctaccctgag 2460 gcaactctac attggaacac tgaggcttac agcctgcaag agcatcagag ctgaccatac 2520 atttaaacag aaatgctggt ttatttgcaa aatcaccagt atattttcta ttgtgtctat 2580 aaaaaatcag tcatttaagt acaagaatca tattttccat tcctttttag aaatttattt 2640 tgttgtccct atggaaatca ttcacatctg acaatttata tgttaaagag ttttactctc 2700 tctattttgg tccaatttgt atctagtggc tgagaaatta aataattcta aagtatgaag 2760 ttacctatct gaaaatgtac ttacagagta tcattttaaa atggatgtct ctttaaaaat 2820 tttgttactt ttaccaacaa tgtaatataa tttatgtata ttttattaat aatagtgaat 2880 tccttaaaat ttgttctatg tacttatatt taatttgatt taatggttac tgcccagata 2940 ttgagaattg gttcaaatat tgagtgtgtt tcaatatatt atctggctta tttcaacatg 3000 agtaatatga gcaaaataag ttaaaacctg cgtctgatca attttcctca tgactagaac 3060 taaaacagta aatttggaca atattaagcc tcaaataatc atctccaaac tccttctaac 3120 actttttaaa tcagattgga agacatggac aaatcaggtt catgtgttgc atctttatgt 3180 cctttgccaa tatccaagat catcacatat ggtagatatt cacatggagt ttcaaattca 3240 gaatagatta ccattacctt cctgccctta cacatcctac tccttattta aaagttctat 3300 ttgtgacttt tcatttcctg aaagtttaaa aatacaattt gagaatgttt ataatacatt 3360 ctctcctgtc ttttcacggt tacgtctgtt attgctgaaa tacaccacat tttctttgtt 3420 ctggtcaagg ttaactcaat atctgtgtga aagagaacta ctaacaacgt tacaatagag 3480 gctagatttg aaaaaaaaaa tctatagatc taattgatac aattgtagaa caaaatgtca 3540 aaataatgtt ttaagtataa gagaagatgg accaaggaga gagagatcat ttgaaaatct 3600 aattgtagct tttctaggct cacattcatg tactactttt agcaccctta tgggctgtgc 3660 tcgccccctg gacagttgag ctttggatta tcttcctctt caattttccc tctattgacc 3720 cgagtgtctc cctctgcttc tacagattta tagtactcct tggctctttt gagtctccac 3780 ttttactcac tgtctctggg atttttaaga tccttttctt ctcttataaa tcatcctctt 3840 aatgaaaatt agcctaacaa aagtttggag actggaatcc tactttgagc cactgacttg 3900 aaataactct tttggcaagt tgcctgacat cctgtcttac caaggtggca tatttgcatt 3960 tttactgctt aaaacatttt ttttttttta ccatctttat ccaaatttat catattgatg 4020 gtaggactaa caggcttttt agaagctggc tttaactttg agtctcaagc tacaatgctg 4080 ttgggcagcc tggtcttccc acgtgagggt ttaactttgt ttatttgcct ccagttattc 4140 caaaatgctt attaaatgaa agtcccagga acatgtttat tttagtcacc tttgcttttt 4200 aacaattttg ttttgtaatc aatgagtaat tcatgatgaa ttatttttga ctaatggata 4260 gccgaaggcc aggcttttaa ttctaatagg taatgttctt cttttgtctt attgaaacaa 4320 tgagaatact ctgtgcattt caaatgcact ccgattatgc tgtggtttta ttcacataag 4380 cacaatatgt gttttattta taacttcata acaaacttat aatataataa tttaccttag 4440 cagacatgca aaagcttatt cttgtgtgac ttactttctt taagctaata atataaaaat 4500 aaatatgtat cttaaaaatc tataataaaa cattagaaat taaagatatg tgctttttat 4560 tttgcagatg agttcatttg cttttgtaga tgtgttttca gagctaggta cagaggaatg 4620 tttgctacct ttagcggtga aaaaagaaag agagtcaaga attttgttgg attgtgtttg 4680 tgtgtgcata tatttgatat catcattata tttgtaatct ttggacttgt aatcatagcc 4740 tgtttattct actgtgccat taaatatact ttaccttata cataacgaat aaaataccta 4800 gaagtagatt tatttacaaa aaaaaaaaaa aaa 4833 <210> 3 <211> 4854 <212> DNA <213> Homo sapiens <400> 3 gtttttaaaa gctttgtatc tcttaaaacc atgcagcagt cagtttccaa gttttgcttt 60 gcaatcagta gttttcaagg gagcttttaa agctgaactg aaatgtttga aatgtggaac 120 actcttgacc atgaaatatg ttctacttac atgcctcagc ctttaaaagt tctttgcatt 180 agagtcaagg attacattct tcctggagcc aagcatgggg ccagctgtaa acaagccgca 240 tttctccttg gggagactga taatttaaaa ggtttgttgt gtcagaaaca ttcccagctt 300 catcaccaac cctttccttc cacctctgcc cactggagac cacttatatc ccgaagcgga 360 cgcggcagct gaagtcagga aaccatgcat cacattagca ggagccaact gcagacttta 420 aactccgttc aacatgtgga tgcggcagag aaatgacctg tccagacaag ccggggcagc 480 tcataaactg gttcatctgc tccctgtgcg tcccgcgggt gcgtaagctc tggagcagcc 540 ggcgtccaag gacccggaga aaccttctgc tgggcactgc gtgtgccatc tacttgggct 600 tcctggtgag ccaggtgggg agggcctctc tccagcatgg acaggcggct gagaaggggc 660 cacatcgcag ccgcgacacc gccgagccat ccttccctga gatacccctg gatggtaccc 720 tggcccctcc agagtcccag ggcaatgggt ccactctgca gcccaatgtg gtgtacatta 780 ccctacgctc caagcgcagc aagccggcca atatccgtgg caccgtgaag cccaagcgca 840 ggaaaaagca tgcagtggca tcggctgccc cagggcagga ggctttggtc ggaccatccc 900 ttcagccgca ggaagcggca agggaagctg atgctgtagc acctgggtac gctcagggag 960 caaacctggt taagattgga gagcgaccct ggaggttggt gcggggtccg ggagtgcgag 1020 ccgggggccc agacttcctg cagcccagct ccagggagag caacattagg atctacagcg 1080 agagcgcccc ctcctggctg agcaaagatg acatccgaag aatgcgactc ttggcggaca 1140 gcgcagtggc agggctccgg cctgtgtcct ctaggagcgg agcccgtttg ctggtgctgg 1200 aggggggcgc acctggcgct gtgctccgct gtggccctag cccctgtggg cttctcaagc 1260 agcccttgga catgagtgag gtgtttgcct tccacctaga caggatcctg gggctcaaca 1320 ggaccctgcc gtctgtgagc aggaaagcag agttcatcca agatggccgc ccatgcccca 1380 tcattctttg ggatgcatct ttatcttcag caagtaatga cacccattct tctgttaagc 1440 tcacctgggg aacttatcag cagttgctga aacagaaatg ctggcagaat ggccgagtac 1500 ccaagcctga atcgggttgt actgaaatac atcatcatga gtggtccaag atggcactct 1560 ttgatttttt gttacagatt tataatcgct tagatacaaa ttgctgtgga ttcagacctc 1620 gcaaggaaga tgcctgtgta cagaatggat tgaggccaaa atgtgatgac caaggttctg 1680 cggctctagc acacattatc cagcgaaagc atgacccaag gcatttggtt tttatagaca 1740 acaagggttt ctttgacagg agtgaagata acttaaactt caaattgtta gaaggcatca 1800 aagagtttcc agcttctgca gtttctgttt tgaagagcca gcacttacgg cagaaacttc 1860 ttcagtctct gtttcttgat aaagtgtatt gggaaagtca aggaggtaga caaggaattg 1920 aaaagcttat cgatgtaata gaacacagag ccaaaattct tatcacctat atcaatgcac 1980 acggggtcaa agtattacct atgaatgaat gacaaaagaa tcttctggct agggtgttag 2040 atatatttat gcatttttgg ttttgttttt aaatcaagca catcaacctc aagcccgttt 2100 agcaatgagg cagtgtagat gaatacgtaa aataaatgac tttaaccaag tagctataat 2160 gggacttagc actgtatgca tacttaaaaa ggttttgaaa aacaaactac ttgagaaata 2220 tttgtttata tttttctcta acatcatgct atgtgtcagt ctgaacatct gacaacagaa 2280 atttcagtta ttattctagc taagttttga aaacatttgt catgctgttt aatagaaaac 2340 tgcaaaccag agacactgac tccattaata aaccatattt tgtgccgttt tgactgttct 2400 gaccaaatac taatgggaac aattcttgac gtttttctgt tgctgattgt taacatagag 2460 cagtctctac actaccctga ggcaactcta cattggaaca ctgaggctta cagcctgcaa 2520 gagcatcaga gctgaccata catttaaaca gaaatgctgg tttatttgca aaatcaccag 2580 tatattttct attgtgtcta taaaaaatca gtcatttaag tacaagaatc atattttcca 2640 ttccttttta gaaatttatt ttgttgtccc tatggaaatc attcacatct gacaatttat 2700 atgttaaaga gttttactct ctctattttg gtccaatttg tatctagtgg ctgagaaatt 2760 aaataattct aaagtatgaa gttacctatc tgaaaatgta cttacagagt atcattttaa 2820 aatggatgtc tctttaaaaa ttttgttact tttaccaaca atgtaatata atttatgtat 2880 attttattaa taatagtgaa ttccttaaaa tttgttctat gtacttatat ttaatttgat 2940 ttaatggtta ctgcccagat attgagaatt ggttcaaata ttgagtgtgt ttcaatatat 3000 tatctggctt atttcaacat gagtaatatg agcaaaataa gttaaaacct gcgtctgatc 3060 aattttcctc atgactagaa ctaaaacagt aaatttggac aatattaagc ctcaaataat 3120 catctccaaa ctccttctaa cactttttaa atcagattgg aagacatgga caaatcaggt 3180 tcatgtgttg catctttatg tcctttgcca atatccaaga tcatcacata tggtagatat 3240 tcacatggag tttcaaattc agaatagatt accattacct tcctgccctt acacatccta 3300 ctccttattt aaaagttcta tttgtgactt ttcatttcct gaaagtttaa aaatacaatt 3360 tgagaatgtt tataatacat tctctcctgt cttttcacgg ttacgtctgt tattgctgaa 3420 atacaccaca ttttctttgt tctggtcaag gttaactcaa tatctgtgtg aaagagaact 3480 actaacaacg ttacaataga ggctagattt gaaaaaaaaa atctatagat ctaattgata 3540 caattgtaga acaaaatgtc aaaataatgt tttaagtata agagaagatg gaccaaggag 3600 agagagatca tttgaaaatc taattgtagc ttttctaggc tcacattcat gtactacttt 3660 tagcaccctt atgggctgtg ctcgccccct ggacagttga gctttggatt atcttcctct 3720 tcaattttcc ctctattgac ccgagtgtct ccctctgctt ctacagattt atagtactcc 3780 ttggctcttt tgagtctcca cttttactca ctgtctctgg gatttttaag atccttttct 3840 tctcttataa atcatcctct taatgaaaat tagcctaaca aaagtttgga gactggaatc 3900 ctactttgag ccactgactt gaaataactc ttttggcaag ttgcctgaca tcctgtctta 3960 ccaaggtggc atatttgcat ttttactgct taaaacattt tttttttttt accatcttta 4020 tccaaattta tcatattgat ggtaggacta acaggctttt tagaagctgg ctttaacttt 4080 gagtctcaag ctacaatgct gttgggcagc ctggtcttcc cacgtgaggg tttaactttg 4140 tttatttgcc tccagttatt ccaaaatgct tattaaatga aagtcccagg aacatgttta 4200 ttttagtcac ctttgctttt taacaatttt gttttgtaat caatgagtaa ttcatgatga 4260 attatttttg actaatggat agccgaaggc caggctttta attctaatag gtaatgttct 4320 tcttttgtct tattgaaaca atgagaatac tctgtgcatt tcaaatgcac tccgattatg 4380 ctgtggtttt attcacataa gcacaatatg tgttttattt ataacttcat aacaaactta 4440 taatataata atttacctta gcagacatgc aaaagcttat tcttgtgtga cttactttct 4500 ttaagctaat aatataaaaa taaatatgta tcttaaaaat ctataataaa acattagaaa 4560 ttaaagatat gtgcttttta ttttgcagat gagttcattt gcttttgtag atgtgttttc 4620 agagctaggt acagaggaat gtttgctacc tttagcggtg aaaaaagaaa gagagtcaag 4680 aattttgttg gattgtgttt gtgtgtgcat atatttgata tcatcattat atttgtaatc 4740 tttggacttg taatcatagc ctgtttattc tactgtgcca ttaaatatac tttaccttat 4800 acataacgaa taaaatacct agaagtagat ttatttacaa aaaaaaaaaa aaaa 4854 <210> 4 <211> 4809 <212> DNA <213> Homo sapiens <400> 4 gtgaactgtt gcaccgtgca attgcacact ataaatgtct ttccttatct gtgtgtactc 60 ttatctcact gttctatttt ttctcctcat ttatattaac tctttcttac ctttttttct 120 gaacttctag gccttctctt tccagaactg gtggaagaca aatgaaacgg ccaagatggt 180 aagaaacaag ccgcatttct ccttggggag actgataatt taaaaggttt gttgtgtcag 240 aaacattccc agcttcatca ccaacccttt ccttccacct ctgcccactg gagaccactt 300 atatcccgaa gcggacgcgg cagctgaagt caggaaacca tgcatcacat tagcaggagc 360 caactgcaga ctttaaactc cgttcaacat gtggatgcgg cagagaaatg acctgtccag 420 acaagccggg gcagctcata aactggttca tctgctccct gtgcgtcccg cgggtgcgta 480 agctctggag cagccggcgt ccaaggaccc ggagaaacct tctgctgggc actgcgtgtg 540 ccatctactt gggcttcctg gtgagccagg tggggagggc ctctctccag catggacagg 600 cggctgagaa ggggccacat cgcagccgcg acaccgccga gccatccttc cctgagatac 660 ccctggatgg taccctggcc cctccagagt cccagggcaa tgggtccact ctgcagccca 720 atgtggtgta cattacccta cgctccaagc gcagcaagcc ggccaatatc cgtggcaccg 780 tgaagcccaa gcgcaggaaa aagcatgcag tggcatcggc tgccccaggg caggaggctt 840 tggtcggacc atcccttcag ccgcaggaag cggcaaggga agctgatgct gtagcacctg 900 ggtacgctca gggagcaaac ctggttaaga ttggagagcg accctggagg ttggtgcggg 960 gtccgggagt gcgagccggg ggcccagact tcctgcagcc cagctccagg gagagcaaca 1020 ttaggatcta cagcgagagc gccccctcct ggctgagcaa agatgacatc cgaagaatgc 1080 gactcttggc ggacagcgca gtggcagggc tccggcctgt gtcctctagg agcggagccc 1140 gtttgctggt gctggagggg ggcgcacctg gcgctgtgct ccgctgtggc cctagcccct 1200 gtgggcttct caagcagccc ttggacatga gtgaggtgtt tgccttccac ctagacagga 1260 tcctggggct caacaggacc ctgccgtctg tgagcaggaa agcagagttc atccaagatg 1320 gccgcccatg ccccatcatt ctttgggatg catctttatc ttcagcaagt aatgacaccc 1380 attcttctgt taagctcacc tggggaactt atcagcagtt gctgaaacag aaatgctggc 1440 agaatggccg agtacccaag cctgaatcgg gttgtactga aatacatcat catgagtggt 1500 ccaagatggc actctttgat tttttgttac agatttataa tcgcttagat acaaattgct 1560 gtggattcag acctcgcaag gaagatgcct gtgtacagaa tggattgagg ccaaaatgtg 1620 atgaccaagg ttctgcggct ctagcacaca ttatccagcg aaagcatgac ccaaggcatt 1680 tggtttttat agacaacaag ggtttctttg acaggagtga agataactta aacttcaaat 1740 tgttagaagg catcaaagag tttccagctt ctgcagtttc tgttttgaag agccagcact 1800 tacggcagaa acttcttcag tctctgtttc ttgataaagt gtattgggaa agtcaaggag 1860 gtagacaagg aattgaaaag cttatcgatg taatagaaca cagagccaaa attcttatca 1920 cctatatcaa tgcacacggg gtcaaagtat tacctatgaa tgaatgacaa aagaatcttc 1980 tggctagggt gttagatata tttatgcatt tttggttttg tttttaaatc aagcacatca 2040 acctcaagcc cgtttagcaa tgaggcagtg tagatgaata cgtaaaataa atgactttaa 2100 ccaagtagct ataatgggac ttagcactgt atgcatactt aaaaaggttt tgaaaaacaa 2160 actacttgag aaatatttgt ttatattttt ctctaacatc atgctatgtg tcagtctgaa 2220 catctgacaa cagaaatttc agttattatt ctagctaagt tttgaaaaca tttgtcatgc 2280 tgtttaatag aaaactgcaa accagagaca ctgactccat taataaacca tattttgtgc 2340 cgttttgact gttctgacca aatactaatg ggaacaattc ttgacgtttt tctgttgctg 2400 attgttaaca tagagcagtc tctacactac cctgaggcaa ctctacattg gaacactgag 2460 gcttacagcc tgcaagagca tcagagctga ccatacattt aaacagaaat gctggtttat 2520 ttgcaaaatc accagtatat tttctattgt gtctataaaa aatcagtcat ttaagtacaa 2580 gaatcatatt ttccattcct ttttagaaat ttattttgtt gtccctatgg aaatcattca 2640 catctgacaa tttatatgtt aaagagtttt actctctcta ttttggtcca atttgtatct 2700 agtggctgag aaattaaata attctaaagt atgaagttac ctatctgaaa atgtacttac 2760 agagtatcat tttaaaatgg atgtctcttt aaaaattttg ttacttttac caacaatgta 2820 atataattta tgtatatttt attaataata gtgaattcct taaaatttgt tctatgtact 2880 tatatttaat ttgatttaat ggttactgcc cagatattga gaattggttc aaatattgag 2940 tgtgtttcaa tatattatct ggcttatttc aacatgagta atatgagcaa aataagttaa 3000 aacctgcgtc tgatcaattt tcctcatgac tagaactaaa acagtaaatt tggacaatat 3060 taagcctcaa ataatcatct ccaaactcct tctaacactt tttaaatcag attggaagac 3120 atggacaaat caggttcatg tgttgcatct ttatgtcctt tgccaatatc caagatcatc 3180 acatatggta gatattcaca tggagtttca aattcagaat agattaccat taccttcctg 3240 cccttacaca tcctactcct tatttaaaag ttctatttgt gacttttcat ttcctgaaag 3300 tttaaaaata caatttgaga atgtttataa tacattctct cctgtctttt cacggttacg 3360 tctgttattg ctgaaataca ccacattttc tttgttctgg tcaaggttaa ctcaatatct 3420 gtgtgaaaga gaactactaa caacgttaca atagaggcta gatttgaaaa aaaaaatcta 3480 tagatctaat tgatacaatt gtagaacaaa atgtcaaaat aatgttttaa gtataagaga 3540 agatggacca aggagagaga gatcatttga aaatctaatt gtagcttttc taggctcaca 3600 ttcatgtact acttttagca cccttatggg ctgtgctcgc cccctggaca gttgagcttt 3660 ggattatctt cctcttcaat tttccctcta ttgacccgag tgtctccctc tgcttctaca 3720 gatttatagt actccttggc tcttttgagt ctccactttt actcactgtc tctgggattt 3780 ttaagatcct tttcttctct tataaatcat cctcttaatg aaaattagcc taacaaaagt 3840 ttggagactg gaatcctact ttgagccact gacttgaaat aactcttttg gcaagttgcc 3900 tgacatcctg tcttaccaag gtggcatatt tgcattttta ctgcttaaaa catttttttt 3960 tttttaccat ctttatccaa atttatcata ttgatggtag gactaacagg ctttttagaa 4020 gctggcttta actttgagtc tcaagctaca atgctgttgg gcagcctggt cttcccacgt 4080 gagggtttaa ctttgtttat ttgcctccag ttattccaaa atgcttatta aatgaaagtc 4140 ccaggaacat gtttatttta gtcacctttg ctttttaaca attttgtttt gtaatcaatg 4200 agtaattcat gatgaattat ttttgactaa tggatagccg aaggccaggc ttttaattct 4260 aataggtaat gttcttcttt tgtcttattg aaacaatgag aatactctgt gcatttcaaa 4320 tgcactccga ttatgctgtg gttttattca cataagcaca atatgtgttt tatttataac 4380 ttcataacaa acttataata taataattta ccttagcaga catgcaaaag cttattcttg 4440 tgtgacttac tttctttaag ctaataatat aaaaataaat atgtatctta aaaatctata 4500 ataaaacatt agaaattaaa gatatgtgct ttttattttg cagatgagtt catttgcttt 4560 tgtagatgtg ttttcagagc taggtacaga ggaatgtttg ctacctttag cggtgaaaaa 4620 agaaagagag tcaagaattt tgttggattg tgtttgtgtg tgcatatatt tgatatcatc 4680 attatatttg taatctttgg acttgtaatc atagcctgtt tattctactg tgccattaaa 4740 tatactttac cttatacata acgaataaaa tacctagaag tagatttatt tacaaaaaaa 4800 aaaaaaaaa 4809 <210> 5 <211> 4745 <212> DNA <213> Homo sapiens <400> 5 ttttctgccc ttctttgctt tggtggcttc cttgtggttc ctcagtggtg cctgcaaccc 60 ctggttcacc tccttccagg ttctggctcc ttccagccat ggctctcaga gtccttctgt 120 taacagcctt gaccttatgt catgggttca acttggacac tgaaaacgca atgaccttcc 180 aagagaacgc aaggggcttc gggcagagcg tggtccagct tcagggatcc agggtggtgg 240 ttggagcccc ccaggagata gtggctgcca accaaagggg cagcctctac cagtgcgact 300 acagcacagg ctcatgcgag cccatccgcc tgcaggtccc cgtggaggcc gtgaacatgt 360 ccctgggcct gtccctggca gccaccacca gcccccctca gctgctggcc tgtggtccca 420 ccgtgcacca gacttgcagt gagaacacgt atgtgaaagg gctctgcttc ctgtttggat 480 ccaacctacg gcagcagccc cagaagttcc cagaggccct ccgagggtgt cctcaagagg 540 atagtgacat tgccttcttg attgatggct ctggtagcat catcccacat gactttcggc 600 ggatgaagga gtttgtctca actgtgatgg agcaattaaa aaagtccaaa accttgttct 660 ctttgatgca gtactctgaa gaattccgga ttcactttac cttcaaagag ttccagaaca 720 accctaaccc aagatcactg gtgaagccaa taacgcagct gcttgggcgg acacacacgg 780 ccacgggcat ccgcaaagtg gtacgagagc tgtttaacat caccaacgga gcccgaaaga 840 atgcctttaa gatcctagtt gtcatcacgg atggagaaaa gtttggcgat cccttgggat 900 atgaggatgt catccctgag gcagacagag agggagtcat tcgctacgtc attggggtgg 960 gagatgcctt ccgcagtgag aaatcccgcc aagagcttaa taccatcgca tccaagccgc 1020 ctcgtgatca cgtgttccag gtgaataact ttgaggctct gaagaccatt cagaaccagc 1080 ttcgggagaa gatctttgcg atcgagggta ctcagacagg aagtagcagc tcctttgagc 1140 atgagatgtc tcaggaaggc ttcagcgctg ccatcacctc taatggcccc ttgctgagca 1200 ctgtggggag ctatgactgg gctggtggag tctttctata tacatcaaag gagaaaagca 1260 ccttcatcaa catgaccaga gtggattcag acatgaatga tgcttacttg ggttatgctg 1320 ccgccatcat cttacggaac cgggtgcaaa gcctggttct gggggcacct cgatatcagc 1380 acatcggcct ggtagcgatg ttcaggcaga acactggcat gtgggagtcc aacgctaatg 1440 tcaagggcac ccagatcggc gcctacttcg gggcctccct ctgctccgtg gacgtggaca 1500 gcaacggcag caccgacctg gtcctcatcg gggcccccca ttactacgag cagacccgag 1560 ggggccaggt gtccgtgtgc cccttgccca gggggcagag ggctcggtgg cagtgtgatg 1620 ctgttctcta cggggagcag ggccaaccct ggggccgctt tggggcagcc ctaacagtgc 1680 tgggggacgt aaatggggac aagctgacgg acgtggccat tggggcccca ggagaggagg 1740 acaaccgggg tgctgtttac ctgtttcacg gaacctcagg atctggcatc agcccctccc 1800 atagccagcg gatagcaggc tccaagctct ctcccaggct ccagtatttt ggtcagtcac 1860 tgagtggggg ccaggacctc acaatggatg gactggtaga cctgactgta ggagcccagg 1920 ggcacgtgct gctgctcagg tcccagccag tactgagagt caaggcaatc atggagttca 1980 atcccaggga agtggcaagg aatgtatttg agtgtaatga tcaggtggtg aaaggcaagg 2040 aagccggaga ggtcagagtc tgcctccatg tccagaagag cacacgggat cggctaagag 2100 aaggacagat ccagagtgtt gtgacttatg acctggctct ggactccggc cgcccacatt 2160 cccgcgccgt cttcaatgag acaaagaaca gcacacgcag acagacacag gtcttggggc 2220 tgacccagac ttgtgagacc ctgaaactac agttgccgaa ttgcatcgag gacccagtga 2280 gccccattgt gctgcgcctg aacttctctc tggtgggaac gccattgtct gctttcggga 2340 acctccggcc agtgctggcg gaggatgctc agagactctt cacagccttg tttccctttg 2400 agaagaattg tggcaatgac aacatctgcc aggatgacct cagcatcacc ttcagtttca 2460 tgagcctgga ctgcctcgtg gtgggtgggc cccgggagtt caacgtgaca gtgactgtga 2520 gaaatgatgg tgaggactcc tacaggacac aggtcacctt cttcttcccg cttgacctgt 2580 cctaccggaa ggtgtccacg ctccagaacc agcgctcaca gcgatcctgg cgcctggcct 2640 gtgagtctgc ctcctccacc gaagtgtctg gggccttgaa gagcaccagc tgcagcataa 2700 accaccccat cttcccggaa aactcagagg tcacctttaa tatcacgttt gatgtagact 2760 ctaaggcttc ccttggaaac aaactgctcc tcaaggccaa tgtgaccagt gagaacaaca 2820 tgcccagaac caacaaaacc gaattccaac tggagctgcc ggtgaaatat gctgtctaca 2880 tggtggtcac cagccatggg gtctccacta aatatctcaa cttcacggcc tcagagaata 2940 ccagtcgggt catgcagcat caatatcagg tcagcaacct ggggcagagg agcctcccca 3000 tcagcctggt gttcttggtg cccgtccggc tgaaccagac tgtcatatgg gaccgccccc 3060 aggtcacctt ctccgagaac ctctcgagta cgtgccacac caaggagcgc ttgccctctc 3120 actccgactt tctggctgag cttcggaagg cccccgtggt gaactgctcc atcgctgtct 3180 gccagagaat ccagtgtgac atcccgttct ttggcatcca ggaagaattc aatgctaccc 3240 tcaaaggcaa cctctcgttt gactggtaca tcaagacctc gcataaccac ctcctgatcg 3300 tgagcacagc tgagatcttg tttaacgatt ccgtgttcac cctgctgccg ggacaggggg 3360 cgtttgtgag gtcccagacg gagaccaaag tggagccgtt cgaggtcccc aaccccctgc 3420 cgctcatcgt gggcagctct gtcgggggac tgctgctcct ggccctcatc accgccgcgc 3480 tgtacaagct cggcttcttc aagcggcaat acaaggacat gatgagtgaa gggggtcccc 3540 cgggggccga accccagtag cggctccttc ccgacagagc tgcctctcgg tggccagcag 3600 gactctgccc agaccacacg tagcccccag gctgctggac acgtcggaca gcgaagtatc 3660 cccgacagga cgggcttggg cttccatttg tgtgtgtgca agtgtgtatg tgcgtgtgtg 3720 caagtgtctg tgtgcaagtg tgtgcacatg tgtgcgtgtg cgtgcatgtg cacttgcacg 3780 cccatgtgtg agtgtgtgca agtatgtgag tgtgtccaag tgtgtgtgcg tgtgtccatg 3840 tgtgtgcaag tgtgtgcatg tgtgcgagtg tgtgcatgtg tgtgctcagg ggcgtgtggc 3900 tcacgtgtgt gactcagatg tctctggcgt gtgggtaggt gacggcagcg tagcctctcc 3960 ggcagaaggg aactgcctgg gctcccttgt gcgtgggtga agccgctgct gggttttcct 4020 ccgggagagg ggacggtcaa tcctgtgggt gaagacagag ggaaacacag cagcttctct 4080 ccactgaaag aagtgggact tcccgtcgcc tgcgagcctg cggcctgctg gagcctgcgc 4140 agcttggatg gagactccat gagaagccgt gggtggaacc aggaacctcc tccacaccag 4200 cgctgatgcc caataaagat gcccactgag gaatgatgaa gcttcctttc tggattcatt 4260 tattatttca atgtgacttt aattttttgg atggataagc ttgtctatgg tacaaaaatc 4320 acaaggcatt caagtgtaca gtgaaaagtc tccctttcca gatattcaag tcacctcctt 4380 aaaggtagtc aagattgtgt tttgaggttt ccttcagaca gattccaggc gatgtgcaag 4440 tgtatgcacg tgtgcacaca caccacacat acacacacac aagctttttt acacaaatgg 4500 tagcatactt tatattggtc tgtatcttgc tttttttcac caatatttct cagacatcgg 4560 ttcatattaa gacataaatt actttttcat tcttttatac cgctgcatag tattccattg 4620 tgtgagtgta ccataatgta tttaaccagt cttcttttga tatactattt tcattctctt 4680 gttattgcat caatgctgag ttaataaatc aaatatatgt catttttgca tatatgtaag 4740 gataa 4745 <210> 6 <211> 4742 <212> DNA <213> Homo sapiens <400> 6 ttttctgccc ttctttgctt tggtggcttc cttgtggttc ctcagtggtg cctgcaaccc 60 ctggttcacc tccttccagg ttctggctcc ttccagccat ggctctcaga gtccttctgt 120 taacagcctt gaccttatgt catgggttca acttggacac tgaaaacgca atgaccttcc 180 aagagaacgc aaggggcttc gggcagagcg tggtccagct tcagggatcc agggtggtgg 240 ttggagcccc ccaggagata gtggctgcca accaaagggg cagcctctac cagtgcgact 300 acagcacagg ctcatgcgag cccatccgcc tgcaggtccc cgtggaggcc gtgaacatgt 360 ccctgggcct gtccctggca gccaccacca gcccccctca gctgctggcc tgtggtccca 420 ccgtgcacca gacttgcagt gagaacacgt atgtgaaagg gctctgcttc ctgtttggat 480 ccaacctacg gcagcagccc cagaagttcc cagaggccct ccgagggtgt cctcaagagg 540 atagtgacat tgccttcttg attgatggct ctggtagcat catcccacat gactttcggc 600 ggatgaagga gtttgtctca actgtgatgg agcaattaaa aaagtccaaa accttgttct 660 ctttgatgca gtactctgaa gaattccgga ttcactttac cttcaaagag ttccagaaca 720 accctaaccc aagatcactg gtgaagccaa taacgcagct gcttgggcgg acacacacgg 780 ccacgggcat ccgcaaagtg gtacgagagc tgtttaacat caccaacgga gcccgaaaga 840 atgcctttaa gatcctagtt gtcatcacgg atggagaaaa gtttggcgat cccttgggat 900 atgaggatgt catccctgag gcagacagag agggagtcat tcgctacgtc attggggtgg 960 gagatgcctt ccgcagtgag aaatcccgcc aagagcttaa taccatcgca tccaagccgc 1020 ctcgtgatca cgtgttccag gtgaataact ttgaggctct gaagaccatt cagaaccagc 1080 ttcgggagaa gatctttgcg atcgagggta ctcagacagg aagtagcagc tcctttgagc 1140 atgagatgtc tcaggaaggc ttcagcgctg ccatcacctc taatggcccc ttgctgagca 1200 ctgtggggag ctatgactgg gctggtggag tctttctata tacatcaaag gagaaaagca 1260 ccttcatcaa catgaccaga gtggattcag acatgaatga tgcttacttg ggttatgctg 1320 ccgccatcat cttacggaac cgggtgcaaa gcctggttct gggggcacct cgatatcagc 1380 acatcggcct ggtagcgatg ttcaggcaga acactggcat gtgggagtcc aacgctaatg 1440 tcaagggcac ccagatcggc gcctacttcg gggcctccct ctgctccgtg gacgtggaca 1500 gcaacggcag caccgacctg gtcctcatcg gggcccccca ttactacgag cagacccgag 1560 ggggccaggt gtccgtgtgc cccttgccca gggggagggc tcggtggcag tgtgatgctg 1620 ttctctacgg ggagcagggc caaccctggg gccgctttgg ggcagcccta acagtgctgg 1680 gggacgtaaa tggggacaag ctgacggacg tggccattgg ggccccagga gaggaggaca 1740 accggggtgc tgtttacctg tttcacggaa cctcaggatc tggcatcagc ccctcccata 1800 gccagcggat agcaggctcc aagctctctc ccaggctcca gtattttggt cagtcactga 1860 gtgggggcca ggacctcaca atggatggac tggtagacct gactgtagga gcccaggggc 1920 acgtgctgct gctcaggtcc cagccagtac tgagagtcaa ggcaatcatg gagttcaatc 1980 ccagggaagt ggcaaggaat gtatttgagt gtaatgatca ggtggtgaaa ggcaaggaag 2040 ccggagaggt cagagtctgc ctccatgtcc agaagagcac acgggatcgg ctaagagaag 2100 gacagatcca gagtgttgtg acttatgacc tggctctgga ctccggccgc ccacattccc 2160 gcgccgtctt caatgagaca aagaacagca cacgcagaca gacacaggtc ttggggctga 2220 cccagacttg tgagaccctg aaactacagt tgccgaattg catcgaggac ccagtgagcc 2280 ccattgtgct gcgcctgaac ttctctctgg tgggaacgcc attgtctgct ttcgggaacc 2340 tccggccagt gctggcggag gatgctcaga gactcttcac agccttgttt ccctttgaga 2400 agaattgtgg caatgacaac atctgccagg atgacctcag catcaccttc agtttcatga 2460 gcctggactg cctcgtggtg ggtgggcccc gggagttcaa cgtgacagtg actgtgagaa 2520 atgatggtga ggactcctac aggacacagg tcaccttctt cttcccgctt gacctgtcct 2580 accggaaggt gtccacgctc cagaaccagc gctcacagcg atcctggcgc ctggcctgtg 2640 agtctgcctc ctccaccgaa gtgtctgggg ccttgaagag caccagctgc agcataaacc 2700 accccatctt cccggaaaac tcagaggtca cctttaatat cacgtttgat gtagactcta 2760 aggcttccct tggaaacaaa ctgctcctca aggccaatgt gaccagtgag aacaacatgc 2820 ccagaaccaa caaaaccgaa ttccaactgg agctgccggt gaaatatgct gtctacatgg 2880 tggtcaccag ccatggggtc tccactaaat atctcaactt cacggcctca gagaatacca 2940 gtcgggtcat gcagcatcaa tatcaggtca gcaacctggg gcagaggagc ctccccatca 3000 gcctggtgtt cttggtgccc gtccggctga accagactgt catatgggac cgcccccagg 3060 tcaccttctc cgagaacctc tcgagtacgt gccacaccaa ggagcgcttg ccctctcact 3120 ccgactttct ggctgagctt cggaaggccc ccgtggtgaa ctgctccatc gctgtctgcc 3180 agagaatcca gtgtgacatc ccgttctttg gcatccagga agaattcaat gctaccctca 3240 aaggcaacct ctcgtttgac tggtacatca agacctcgca taaccacctc ctgatcgtga 3300 gcacagctga gatcttgttt aacgattccg tgttcaccct gctgccggga cagggggcgt 3360 ttgtgaggtc ccagacggag accaaagtgg agccgttcga ggtccccaac cccctgccgc 3420 tcatcgtggg cagctctgtc gggggactgc tgctcctggc cctcatcacc gccgcgctgt 3480 acaagctcgg cttcttcaag cggcaataca aggacatgat gagtgaaggg ggtcccccgg 3540 gggccgaacc ccagtagcgg ctccttcccg acagagctgc ctctcggtgg ccagcaggac 3600 tctgcccaga ccacacgtag cccccaggct gctggacacg tcggacagcg aagtatcccc 3660 gacaggacgg gcttgggctt ccatttgtgt gtgtgcaagt gtgtatgtgc gtgtgtgcaa 3720 gtgtctgtgt gcaagtgtgt gcacatgtgt gcgtgtgcgt gcatgtgcac ttgcacgccc 3780 atgtgtgagt gtgtgcaagt atgtgagtgt gtccaagtgt gtgtgcgtgt gtccatgtgt 3840 gtgcaagtgt gtgcatgtgt gcgagtgtgt gcatgtgtgt gctcaggggc gtgtggctca 3900 cgtgtgtgac tcagatgtct ctggcgtgtg ggtaggtgac ggcagcgtag cctctccggc 3960 agaagggaac tgcctgggct cccttgtgcg tgggtgaagc cgctgctggg ttttcctccg 4020 ggagagggga cggtcaatcc tgtgggtgaa gacagaggga aacacagcag cttctctcca 4080 ctgaaagaag tgggacttcc cgtcgcctgc gagcctgcgg cctgctggag cctgcgcagc 4140 ttggatggag actccatgag aagccgtggg tggaaccagg aacctcctcc acaccagcgc 4200 tgatgcccaa taaagatgcc cactgaggaa tgatgaagct tcctttctgg attcatttat 4260 tatttcaatg tgactttaat tttttggatg gataagcttg tctatggtac aaaaatcaca 4320 aggcattcaa gtgtacagtg aaaagtctcc ctttccagat attcaagtca cctccttaaa 4380 ggtagtcaag attgtgtttt gaggtttcct tcagacagat tccaggcgat gtgcaagtgt 4440 atgcacgtgt gcacacacac cacacataca cacacacaag cttttttaca caaatggtag 4500 catactttat attggtctgt atcttgcttt ttttcaccaa tatttctcag acatcggttc 4560 atattaagac ataaattact ttttcattct tttataccgc tgcatagtat tccattgtgt 4620 gagtgtacca taatgtattt aaccagtctt cttttgatat actattttca ttctcttgtt 4680 attgcatcaa tgctgagtta ataaatcaaa tatatgtcat ttttgcatat atgtaaggat 4740 aa 4742 <210> 7 <211> 5188 <212> DNA <213> Homo sapiens <400> 7 aaaaccctgc aggagactgc gagccctgca gaactgctag ctgcggggga gagggcaggg 60 gtcgggcgcc tgtggcggag ccgggctggg gccagggcag ggaggctgac aagcggcggg 120 agaagccggc ggagggcggg atcgcgcctc ctgacatgtt gggggtatcc ctggccgggc 180 cgggccgggg ctaagagcgg cgctgcgggc cggggtcggg gtcgggtcgc ggtccgcccc 240 cgctgtccct ccgtcctgcc ctgtcgagga cgtgcgttcc gcactcggcc gcctccagag 300 ggagcgaggg aagcggctag aggatcgggg agaaggagca ttcgccggag gctggaggag 360 gctgacccgc gtccccgccc agcctgctcc tatgcggtac ttgaaggatg gcgaagaggt 420 cgcgcagtga ggatgaggat gatgaccttc agtatgccga tcatgattat gaagtaccac 480 aacaaaaagg actgaagaaa ctctggaaca gagtaaaatg gacaagggac gaggatgata 540 aattaaagaa gttggttgaa caacatggaa ctgatgattg gactctaatt gctagtcatc 600 ttcaaaatcg ctctgatttt cagtgccagc atcgatggca gaaagtttta aatcctgaat 660 tgataaaggg tccttggact aaagaagaag atcagagggt tattgaatta gttcagaaat 720 atgggccaaa aagatggtct ttaattgcaa aacatttaaa aggaagaata ggcaagcagt 780 gtagagaaag atggcataat catctgaatc ctgaggtaaa gaaatcttcc tggacagaag 840 aggaggacag gatcatctat gaagcacata agcggttggg aaatcgttgg gcagaaattg 900 ccaaactact tccaggaagg actgataatt ctatcaaaaa tcattggaat tctactatgc 960 gaagaaaagt ggaacaggag ggctatttac aagatggaat aaaatcagaa cgatcttcat 1020 ctaaacttca acacaaacct tgtgcagcta tggatcatat gcaaacccag aatcagtttt 1080 acatacctgt tcagatccct gggtatcagt atgtgtcacc tgaaggcaat tgtatagaac 1140 atgttcagcc tacttctgcc tttattcagc aacccttcat tgatgaagat cctgataagg 1200 aaaagaaaat aaaggaactt gagatgcttc ttatgtcagc tgagaatgaa gttagaagaa 1260 agcgaattcc atcacagcct ggaagttttt ctagctggtc tggtagtttc ctcatggatg 1320 ataacatgtc taatactcta aatagccttg acgagcacac tagtgagttt tacagtatgg 1380 atgaaaatca gcctgtgtct gctcagcaga attcacccac aaagttcctg gccgtggagg 1440 caaacgctgt gttatcctct ttgcagacca tcccagaatt tgcagagact ctagaactta 1500 ttgaatctga tcctgtagca tggagtgacg ttaccagttt tgatatttct gatgctgctg 1560 cttctcctat caaatccacc ccagttaaat taatgagaat tcagcacaat gaaggagcca 1620 tggaatgcca atttaacgtc agtcttgtac ttgaagggaa aaaaaacact tgtaatggtg 1680 gcaacagtga agctgttcct ttaacatccc caaatatagc caagtttagc actccaccag 1740 ccatcctcag aaagaagaga aaaatgcgag tgggtcattc cccaggcagc gaacttaggg 1800 atggctcatt gaacgatggt ggtaatatgg cgctaaaaca tacaccactg aaaacactac 1860 cattttctcc ttcacagttt ttcaacacat gtcctggtaa tgaacaactt aatatagaaa 1920 atccttcatt tacatcaacc cctatttgtg ggcagaaagc tctcattaca actcctcttc 1980 ataaggaaac aactcccaaa gatcaaaagg aaaatgtagg gtttagaaca cctactatta 2040 gaagatctat actgggtacc acaccaagaa ctcctactcc ttttaagaat gcgcttgctg 2100 ctcaggagaa aaaatatgga cctcttaaaa ttgtgtccca gccacttgct ttcttggaag 2160 aagatattcg ggaagtttta aaagaagaaa ctggaacaga cctattcctc aaagaggaag 2220 atgaacctgc ttacaaaagc tgcaaacaag agaataccgc ttctgggaag aaagtcagaa 2280 aatcactagt cttagataat tgggaaaaag aagaatcagg cactcaactg ttgactgaag 2340 acatttcaga catgcagtca gaaaatagat ttactacatc cttattaatg ataccattat 2400 tggaaataca tgacaatagg tgcaacttga ttcctgaaaa acaagatata aattcaacca 2460 acaaaacata tacacttact aaaaagaaac caaaccctaa cacttccaaa gttgtcaaat 2520 tggaaaagaa tgtcagtca aattgtgaat gggaaacagt ggtttatggg aagacagaag 2580 accaacttat tatgactgaa caagcaagaa gatatctgag tacttacaca gctaccagta 2640 gtacttcaag agctctcata ctgtaattgt tattaaaatt gatgaaatgc cccactccct 2700 tactgcagtc tctactaaat taggttgcag tgaaattttt ctcaattagt tgtttttaaa 2760 gttgtaagat agccctttta atacagcatc ttttttctat tctatatagt aggcagaaag 2820 ctagtaagtc acttaagggg tagatagttt catagtttat tttttaagag atgagatttt 2880 taaaaattgt ttttaaagaa caagatggga aaataataga atgttcatgg atttctaaaa 2940 gtaaattctc atatattttc ttcacaagat atatgttgct actctcttga tgctgcagtt 3000 ttgttataga taggtgtatg agtatatatg atttctgaaa ttagtctatg tatggaaagc 3060 acacatgatt ttatgaagta cttttgccca tgtgctgatt tacttaggct accatttaca 3120 aagaaacaca ttgaaaagga atttaaagga aggatagaaa gttgcactac taattttttg 3180 tttttttttt cagaagcagt aaaattaact acagtgttaa atgtatttat ttgagcatag 3240 tactgaaaac aaaaagcatt caaaaaagag ttttttcttt attagtaaat agtattttct 3300 taatctcaga ggagctgaga gttttgttga atgtattgta cagtatgtag gagcaggaga 3360 actttgtaaa ttggaaagaa gtctgttttt ataatttatt tttattttta aagcttaaat 3420 gtagatattt atacgtatac agggtgccta gaagccaatg ttgtttcctg ttattacagc 3480 taacacagta aagaataatt ttgactttaa gtatgaaaca gtagtaagtt atagctgcaa 3540 agaatacaat atctatactg tatgtcacat ctacctaaat gttgcactat gccctttaaa 3600 tcatgctggt tataaagtag ttctaaaaat gtactaaata ataatttaat attttctttt 3660 taaattatat cgggggtggt catatacatt aatctggtga tttgtatatg tgtttgaaat 3720 ttttgcattt tgtttaaaaa ataatatggt accttggtcc ctaaaaacag tctgcactta 3780 gt; attttgtcct tttttaaatg ttttgtgttt ctctacaggt tacaacagtt gcttcagttg 3900 cctgttttag gtgtttgcac ttattttatt tcttcttgaa agaattttta tttgcttttg 3960 tggtagagat tatatgtaat tttttttcag tcatataatg gtgtgctgtc aacttaaaca 4020 ctgacaggta aatagaattg tacactgtag tttgaattat ttataattga cacactctct 4080 ccctctccac tcctgaagta tgctgctata gaaaatagca gaatcggctt gctgctacga 4140 gagaaggaaa gagcgaccac cacttgcact gtgtgaaaag ataaaaaaca aatgatggca 4200 agttctcaag ttaactaaat ggaatcaacc attaccaggc aaattcttgc aaataccaaa 4260 atactactat gccttataaa acaaaatgaa agcaggttaa gattttctgc tctgtttgta 4320 tgttaataga aatggaaata ctaagtattt taatgcttag ctcttgaaca gtagacctaa 4380 aagggtttta agctatttaa atctacttgc tagtttttgc atattttata tatatatata 4440 tttatatata tatatagtga gaagtgaaga aaatgtatgg tactaagatt atgccttatt 4500 gataaataga taaaccaatt tgaatcctct tagcatgttt aagtatgttg attgctttct 4560 aattaatgaa cttctcacag aaatttcact tagtgaaacc aatgattgta gcaaactcat 4620 actggatcat ttcagttacc ttgaactaat agcacataat ggttttttgt tgttgttgtt 4680 tttaatgtag cccttacctg gatatacata gtctgcaatc accaaagtat aatatcttgt 4740 aaggctatat tttttaaagc atattttttc ttgagcatta aattatccta aatggtaata 4800 tattgtggat aagtctgggc ttattggaca taatacatat ttgggttggt actggttgaa 4860 tccttcagtt aactgctttg ttgctttttg caagattttt tatcttaaac atgtcaggca 4920 tcttaagtca cctttatact gttttgttcc tctgagtttc tttcagtatg ttatacaaat 4980 gccagacata acatgtagca gccatacttg catggaaact gactacacat acataatact 5040 gcattttatt gtaaggtttt cacattaata cagcaattac cctgactaaa ttgagttttg 5100 tgatatatgg aaaacttcat tgtaagagaa tcttgcatac aatgttgaca tattaacatc 5160 caaaataaag catctgtgta caagctga 5188 <210> 8 <211> 5008 <212> DNA <213> Homo sapiens <400> 8 aaaaccctgc aggagactgc gagccctgca gaactgctag ctgcggggga gagggcaggg 60 gtcgggcgcc tgtggcggag ccgggctggg gccagggcag ggaggctgac aagcggcggg 120 agaagccggc ggagggcggg atcgcgcctc ctgacatgtt gggggtatcc ctggccgggc 180 cgggccgggg ctaagagcgg cgctgcgggc cggggtcggg gtcgggtcgc ggtccgcccc 240 cgctgtccct ccgtcctgcc ctgtcgagga cgtgcgttcc gcactcggcc gcctccagag 300 ggagcgaggg aagcggctag aggatcgggg agaaggagca ttcgccggag gctggaggag 360 gctgacccgc gtccccgccc agcctgctcc tatgcggtac ttgaaggatg gcgaagaggt 420 cgcgcagtga ggatgaggat gatgaccttc agtatgccga tcatgattat gaagtaccac 480 aacaaaaagg actgaagaaa ctctggaaca gagtaaaatg gacaagggac gaggatgata 540 aattaaagaa gttggttgaa caacatggaa ctgatgattg gactctaatt gctagtcatc 600 ttcaaaatcg ctctgatttt cagtgccagc atcgatggca gaaagtttta aatcctgaat 660 tgataaaggg tccttggact aaagaagaag atcagagggt tattgaatta gttcagaaat 720 atgggccaaa aagatggtct ttaattgcaa aacatttaaa aggaagaata ggcaagcagt 780 gtagagaaag atggcataat catctgaatc ctgaggtaaa gaaatcttcc tggacagaag 840 aggaggacag gatcatctat gaagcacata agcggttggg aaatcgttgg gcagaaattg 900 ccaaactact tccaggaagg actgataatt ctatcaaaaa tcattggaat tctactatgc 960 gaagaaaagt ggaacaggag ggctatttac aagatggaat aaaatcagaa cgatcttcat 1020 ctaaacttca acacaaacct tgtgcagcta tggatcatat gcaaacccag aatcagtttt 1080 acatacctgt tcagatccct gggtatcagt atgtgtcacc tgaaggcaat tgtatagaac 1140 atgttcagcc tacttctgcc tttattcagc aacccttcat tgatgaagat cctgataagg 1200 aaaagaaaat aaaggaactt gagatgcttc ttatgtcagc tgagaatgaa gttagaagaa 1260 agcgaattcc atcacagcct ggaagttttt ctagctggtc tggtagtttc ctcatggatg 1320 ataacatgtc taatactcta aatagccttg acgagcacac tagtgagttt tacagtatgg 1380 atgaaaatca gcctgtgtct gctcagcaga attcacccac aaagttcctg gccgtggagg 1440 caaacgctgt gttatcctct ttgcagacca tcccagaatt tgcagagact ctagaactta 1500 ttgaatctga tcctgtagca tggagtgacg ttaccagttt tgatatttct gatgctgctg 1560 cttctcctat caaatccacc ccagttaaat taatgagaat tcagcacaat gaaggagcca 1620 tggaatgcca atttaacgtc agtcttgtac ttgaagggaa aaaaaacact tgtaatggtg 1680 gcaacagtga agctgttcct ttaacatccc caaatatagc caagtttagc actccaccag 1740 ccatcctcag aaagaagaga aaaatgcgag tgggtcattc cccaggcagc gaacttaggg 1800 atggctcatt gaacgatggt ggtaatatgg cgctaaaaca tacaccactg aaaacactac 1860 cattttctcc ttcacagttt ttcaacacat gtcctggtaa tgaacaactt aatatagaaa 1920 atccttcatt tacatcaacc cctatttgtg ggcagaaagc tctcattaca actcctcttc 1980 ataaggaaac aactcccaaa gatcaaaagg aaaatgtagg gtttagaaca cctactatta 2040 gaagatctat actgggtacc acaccaagaa ctcctactcc ttttaagaat gcgcttgctg 2100 ctcaggagaa aaaatatgga cctcttaaaa ttgtgtccca gccacttgct ttcttggaag 2160 aagatattcg ggaagtttta aaagaagaaa ctggaacaga cctattcctc aaagaggaag 2220 atgaacctgc ttacaaaagc tgcaaacaag agaataccgc ttctgggaag aaagtcagaa 2280 aatcactagt cttagataat tgggaaaaag aagaatcagg cactcaactg ttgactgaag 2340 acatttcaga catgcagtca aattgtgaat gggaaacagt ggtttatggg aagacagaag 2400 accaacttat tatgactgaa caagcaagaa gatatctgag tacttacaca gctaccagta 2460 gtacttcaag agctctcata ctgtaattgt tattaaaatt gatgaaatgc cccactccct 2520 tactgcagtc tctactaaat taggttgcag tgaaattttt ctcaattagt tgtttttaaa 2580 gttgtaagat agccctttta atacagcatc ttttttctat tctatatagt aggcagaaag 2640 ctagtaagtc acttaagggg tagatagttt catagtttat tttttaagag atgagatttt 2700 taaaaattgt ttttaaagaa caagatggga aaataataga atgttcatgg atttctaaaa 2760 gtaaattctc atatattttc ttcacaagat atatgttgct actctcttga tgctgcagtt 2820 ttgttataga taggtgtatg agtatatatg atttctgaaa ttagtctatg tatggaaagc 2880 acacatgatt ttatgaagta cttttgccca tgtgctgatt tacttaggct accatttaca 2940 aagaaacaca ttgaaaagga atttaaagga aggatagaaa gttgcactac taattttttg 3000 tttttttttt cagaagcagt aaaattaact acagtgttaa atgtatttat ttgagcatag 3060 tactgaaaac aaaaagcatt caaaaaagag ttttttcttt attagtaaat agtattttct 3120 taatctcaga ggagctgaga gttttgttga atgtattgta cagtatgtag gagcaggaga 3180 actttgtaaa ttggaaagaa gtctgttttt ataatttatt tttattttta aagcttaaat 3240 gtagatattt atacgtatac agggtgccta gaagccaatg ttgtttcctg ttattacagc 3300 taacacagta aagaataatt ttgactttaa gtatgaaaca gtagtaagtt atagctgcaa 3360 agaatacaat atctatactg tatgtcacat ctacctaaat gttgcactat gccctttaaa 3420 tcatgctggt tataaagtag ttctaaaaat gtactaaata ataatttaat attttctttt 3480 taaattatat cgggggtggt catatacatt aatctggtga tttgtatatg tgtttgaaat 3540 ttttgcattt tgtttaaaaa ataatatggt accttggtcc ctaaaaacag tctgcactta 3600 gaagtttata tttactcagt gtttcagaag tggagaacat tatcttttat ttataaaaat 3660 attttgtcct tttttaaatg ttttgtgttt ctctacaggt tacaacagtt gcttcagttg 3720 cctgttttag gtgtttgcac ttattttatt tcttcttgaa agaattttta tttgcttttg 3780 tggtagagat tatatgtaat tttttttcag tcatataatg gtgtgctgtc aacttaaaca 3840 ctgacaggta aatagaattg tacactgtag tttgaattat ttataattga cacactctct 3900 ccctctccac tcctgaagta tgctgctata gaaaatagca gaatcggctt gctgctacga 3960 gagaaggaaa gagcgaccac cacttgcact gtgtgaaaag ataaaaaaca aatgatggca 4020 agttctcaag ttaactaaat ggaatcaacc attaccaggc aaattcttgc aaataccaaa 4080 atactactat gccttataaa acaaaatgaa agcaggttaa gattttctgc tctgtttgta 4140 tgttaataga aatggaaata ctaagtattt taatgcttag ctcttgaaca gtagacctaa 4200 aagggtttta agctatttaa atctacttgc tagtttttgc atattttata tatatatata 4260 tttatatata tatatagtga gaagtgaaga aaatgtatgg tactaagatt atgccttatt 4320 gataaataga taaaccaatt tgaatcctct tagcatgttt aagtatgttg attgctttct 4380 aattaatgaa cttctcacag aaatttcact tagtgaaacc aatgattgta gcaaactcat 4440 actggatcat ttcagttacc ttgaactaat agcacataat ggttttttgt tgttgttgtt 4500 tttaatgtag cccttacctg gatatacata gtctgcaatc accaaagtat aatatcttgt 4560 aaggctatat tttttaaagc atattttttc ttgagcatta aattatccta aatggtaata 4620 tattgtggat aagtctgggc ttattggaca taatacatat ttgggttggt actggttgaa 4680 tccttcagtt aactgctttg ttgctttttg caagattttt tatcttaaac atgtcaggca 4740 tcttaagtca cctttatact gttttgttcc tctgagtttc tttcagtatg ttatacaaat 4800 gccagacata acatgtagca gccatacttg catggaaact gactacacat acataatact 4860 gcattttatt gtaaggtttt cacattaata cagcaattac cctgactaaa ttgagttttg 4920 tgatatatgg aaaacttcat tgtaagagaa tcttgcatac aatgttgaca tattaacatc 4980 caaaataaag catctgtgta caagctga 5008 <210> 9 <211> 2632 <212> DNA <213> Homo sapiens <400> 9 agctgaggga cgcgtcagcc aggcaccccg gggtgtggcc agaggacttc ggcgacgctt 60 ccccgagagt agcccccctc ctcaacccag aaaagacaac cccgcggggc tgcagcgagc 120 caggcatgct cactggcgca ggcccggccc gcagcccgag caggaagcgc cggcgctagg 180 cggccccctg cgctgccagc tggagccggg cggagccagc gccccggcgc agggtggctc 240 tgccagtccc cgcgcgcctg ggcggccgca cacgtgtcca ggcgtcacgt ccgcgcgcgc 300 ccccggggct tgcgtcagcg gctgttccag aagcgggtgg gccagggctc tgcgcaccgc 360 tggggttcgg ggcccgggac gccgccggga ggagggcacc gcgcggggtc cgacgcggag 420 gcgtgctcgg aacgccgggg gctgcggagt gcatcagcgc ggtccagccc tccgcctgcc 480 gggcgccgag cgtctccgcc gcccggacct gggctgggcg ccgtggcgtt gcctcggagc 540 tcgctgcccg cggggcgcgc accgccttga cccgggcggc cccgcggcag gcaggcgccc 600 gcagttccat ggttggttcg gagcgcgatg agccgcccgt cctccaccgg ccccagcgct 660 aataaaccct gcagcaagca gccgccgccg cagccccagc acactccgtc cccggctgcg 720 cccccggccg ccgccaccat ctcggctgcg ggccccggct cgtccgcggt gcccgccgcg 780 gcggcggtga tctcgggccc cggcggcggc ggcggggccg gcccggtgtc cccgcagcac 840 cacgagctga cctcgctctt cgagtgtccg gtctgctttg actatgtcct gcctcctatt 900 ctgcagtgcc aggccgggca cctggtgtgt aaccaatgcc gccagaagtt gagctgctgc 960 ccgacgtgca ggggcgccct gacgcccagc atcaggaacc tggctatgga gaaggtggcc 1020 tcggcagtcc tgtttccctg taagtatgcc accacgggct gttccctgac cctgcaccat 1080 acggagaaac cagaacatga agacatatgt gaataccgtc cctactcctg cccatgtcct 1140 ggtgcttcct gcaagtggca ggggtccctg gaagctgtga tgtcccatct catgcacgcc 1200 cacaagagca ttaccaccct tcagggagaa gacatcgtct ttctagctac agacattaac 1260 ttgccagggg ctgtcgactg ggtgatgatg cagtcatgtt ttggccatca cttcatgctg 1320 gtgctggaga aacaagagaa gtacgaaggc caccagcagt tttttgccat cgtcctgctc 1380 attggcaccc gcaagcaagc cgagaacttt gcctacagac tggagttgaa tgggaaccgg 1440 cggagattga cctgggaggc cacgccccgt tcgattcatg acggtgtggc tgcggccatc 1500 atgaacagcg actgccttgt tttcgacaca gccatagcac atctttttgc agataatggg 1560 aaccttggaa tcaatgttac tatttctaca tgttgtccat gatgtgactt tcgtaaacct 1620 tcaaaattat ttgggcatag tgctctatgt ttaataaagg tttttataga tgttttattc 1680 catatgtctt cacaagtcag gacccacaat tacccgtgtt ttgtttgaac agcagtgtcc 1740 catctggctt cgacccaaca aagttcatta acctgggatg aatggggttg gcctgttggt 1800 gatttggatg ctgttctgtg atctaaaaca actcttattg aattgtattt actccctaaa 1860 caacacttga caggctgttg cacagggctt ctatagatca gtgtgttagg aatgggaggc 1920 cccttcctgc ctgccttccc atattggtcc cttgacattg acaaaagcac agtgactgtc 1980 agcagattcc tttacttttg tttgtgggag gtaggaattg ttttaatgca ttttaaacag 2040 tgtttctgaa attggatggc tggctaatag acactgaatc acccggagtg cttatcttaa 2100 aattgcagat ttagggagcc tgccaattta acagtctcat caggtgattc ttttcaacag 2160 taatgtttga gaattactgg gttaaattgt gggaaagggt ccagatttta aaggtgcttt 2220 aaggttgccc tctgccgata ctgtttgtct ttctactgtt tcatccccta acttccccca 2280 accctcaaat taaaactaga actatagatc cacatgaacg cacgcctgag atttggccac 2340 tcacctatgt tttgggtgga ttgcctagga aagcaagtca tatggccatt gatagttctc 2400 atgtaattag ttttgctcac cactagtaca gatgacccgt ttacacgtgg cttccctcgg 2460 aagccctcct caacagtagc tggtgtgaaa gactaaatca gtagagttgg aaaagcttta 2520 gt; attcactagt tttttaataa atattttcca aaaatggata aaaaaaaaaa aa 2632 <210> 10 <211> 894 <212> DNA <213> Homo sapiens <400> 10 gcccgtcttc gtgtctcctc cctccctcgc cttcctcctt cctagctcct ctcctccagg 60 gccagactga gcccaggttg atttcaggcg gacaccaata gactccacag cagctccagg 120 agcccagaca ccggcggcca gaagcaaggc taggagctgc tgcagccatg tcggccctca 180 gcctcctcat tctgggcctg ctcacggcag tgccacctgc cagctgtcag caaggcctgg 240 ggaaccttca gccctggatg cagggcctta tcgcggtggc cgtgttcctg gtcctcgttg 300 caatcgcctt tgcagtcaac cacttctggt gccaggagga gccggagcct gcacacatga 360 tcctgaccgt cggaaacaag gcagatggag tcctggtggg aacagatgga aggtactctt 420 cgatggcggc cagtttcagg tccagtgagc atgagaatgc ctatgagaat gtgcccgagg 480 aggaaggcaa ggtccgcagc accccgatgt aaccttctct gtggctccaa ccccaagact 540 cccaggcaca tgggatggat gtccagtgct accacccaag ccccctcctt ctttgtgtgg 600 aatctgcaat agtgggctga ctccctccag ccccatgccg gccctacccg cccttgaagt 660 atagccagcc aaggttggag ctcagaccgt gtctaggttg gggctcggct gtggccctgg 720 ggtctcctgc tcagctcaga agagccttct ggagaggaca gtcagctgag cacctcccat 780 cctgctcaca cgtccttccc cataactatg gaaatggccc taatttctgt gaaataaaga 840 ctttttgtat ttctggggct gaggctcagc aacagcccct caggcttcca gtga 894 <210> 11 <211> 5003 <212> DNA <213> Homo sapiens <400> 11 agttgggctc ccgcctggct gggaggcggg agggatcccg ctcctgttgt tttccgccgg 60 caggagtagg ctggcgggcg cagggggcgg ggtgcgccct ccctccccgg ccagggcgct 120 cgggagcggg gacccgagcc tgcagccgag ctccgctgcc ggccctggac actcggctca 180 gccaagcatc cttcctgggg gccgaggaag tggggccact ctgccgttcc gaggacctgg 240 gaggagccct cggtaccccg ggccccgggg ccctggggca cacacgtcca gcccagcccg 300 agcctgcgtt tcctgagccg ggatctgggg cgagatggcc gcaggcggca gtgcgcccga 360 gccccgcgtc ctcgtctgcc tcggggcgct cctggccggc tgggtcgccg taggattgga 420 ggctgttgtc attggagaag ttcatgagaa tgttactctg cactgtggca acatctcggg 480 actgaggggc caggtgacct ggtaccggaa caactcggag cctgtcttcc ttctctcgtc 540 caactctagc ctccggccag ctgagcctcg cttctctcta gtggatgcca cctccctgca 600 cattgaatcg ctgagcctgg gagatgaggg aatctacacc tgccaggaga tcctgaatgt 660 gactcagtgg ttccaagtgt ggctgcaggt ggccagcggc ccctatcaga ttgaggtcca 720 catcgtggcc accggcacac tccccaacgg caccctctac gcagccaggg gctcccaggt 780 ggacttcagc tgcaacagca gctccaggcc accacccgtg gttgaatggt ggttccaggc 840 cctgaattcc agcagcgagt cctttggcca caacctgaca gtcaactttt tctcactgtt 900 actgatatcg ccaaacctcc aagggaacta cacctgttta gccttgaatc agctcagcaa 960 gagacatcga aaggtgacca ccgagctcct ggtctactat ccccctccat cagctcccca 1020 gtgctgggca cagatggcat caggatcgtt catgttgcag cttacctgtc gctgggatgg 1080 gggataccct gaccctgact tcctgtggat agaagagcca ggaggtgtaa tcgtggggaa 1140 gtcaaagctg ggggtggaaa tgctgagcga gtcccagctg tcggatggca agaagttcaa 1200 gtgtgttaca agccacatag ttgggccaga gtcgggcgcc agctgcatgg tgcagatcag 1260 gggtccctcc cttctctctg agcccatgaa gacttgcttc actgggggca atgtgacgct 1320 tacatgccag gtgtctgggg cctacccccc tgccaagatc ctgtggctga ggaaccttac 1380 ccagcccgag gtgatcatcc agcctagcag ccgccatctc attacccagg atggccagaa 1440 ctccaccctc actatccaca actgctccca ggacctggat gagggctact acatctgccg 1500 agctgacagc cctgtagggg tgagggagat ggaaatctgg ctgagtgtga aagaaccttt 1560 aaatatcggg gggattgtgg gaaccattgt gagcctcctt ctgctgggac tggccattat 1620 ctcagggctt ctgttgcatt atagccctgt gttctgctgg aaagtaggaa acacttccag 1680 gggacaaaac atggatgatg tcatggtttt ggtggattca gaagaggaag aggaggagga 1740 ggaggaggag gaggaagatg ctgcagtagg ggaacaggag ggagcacgtg agagagagga 1800 gttgccaaaa gaaataccta agcaggacca cattcacaga gtgaccgcct tggtgaatgg 1860 gaacatagaa cagatgggaa atggattcca ggatcttcaa gatgacagca gtgaggagca 1920 aagtgacatt gttcaagaag aagacaggcc agtctgaaga agaggatggt ccatggttgt 1980 cttgctctga aagcttggag agctacattg aagacgagct cttcattcag ctttgactcc 2040 acctgcaccc ctggcggggg cttgcactaa caatgtttgg gtctcagcaa aaaacaaaac 2100 caagcacaca catctttcct tccatgtatt gaaaaacatt ggtttgattt gctctaagtt 2160 ttcccaatga tgtttaaaag ctttgagaag gaaagctgct ttggtgtctg aggtgccact 2220 tctgctgtga atcctggctt tatccaggtt gatctactgt gatagatgct gatttagagg 2280 gaacagaggt cagggaagca ctgggtcttg gtgccttttg ccgctttttt tttttttttt 2340 tttttttttt gagacggagt ctccctctat tgcccaggct ggagtgcaat ggcacgatct 2400 tggctcacca caacttctgc ctcccaggtt caagcgattc tcctgcctca gccacggcac 2460 cttgcaaata tcagctcctt ggaacaggtg aagttccagg taccaatgcc aatcagagga 2520 aggcagtttg gttcaggctt tggagttaga aacacctgaa gttgaatctg ggctctgttg 2580 cttccttctt tcatgggcta gagcacgact ctttacctct ctcttggcct caatttcctc 2640 acctgtaaaa tagatgagga agctgctcac ttattattgt ctcgttctga aagcttggaa 2700 agctacatcg aagatgagct cttcattcag ctttgatttg acctgtaccc ctggtggggg 2760 attgcactgg caacatttgg gtctcagcaa aaaaaccaag cacacacatc tttccttcca 2820 tttattgaaa aacatctttg taagatccat tcattgaaaa acataatcca tttattgaaa 2880 aatatctttg taagatcacc tgctaaatat gaaaatctga cttgaatttg tactctttaa 2940 agttgcgtat ctgctctagt gggcaggacc tagggcttaa aggggaactt cctttctcca 3000 tttctaagaa ctgggactct aaaatgagaa gctggttgtc tgaagtaacc ctgcaggtgt 3060 ggttggggaa ggtctgtttt cttggatgaa ggaactaaac taagcatatc agagcactgt 3120 cttaaccagt tttatttccc tggagataga attcttttaa aaagagttag ggagctggta 3180 ataggaagtg cctttcatta taactacatt ttgcagagct tcatatttat atacaagcct 3240 cctaggtgat acactgttag cttgcagact ttcctatgct tcatttctcc tgttgctttc 3300 aaagaaggca ggagacacgt ttaataacgg agtatctggt gataagaatt gcttgggcaa 3360 accagctcat ctggactctt tctcagtctt ggaagtggga agaggaaaac ttgtttcctt 3420 cctgcttctt aaggatattc tgagggtaca ctgatcaata acactaaatt tggaatgaaa 3480 ataccatgtg atgagtttag cctgctgatg cttccagtag atccttgtat agtttcaaga 3540 tttaagtttt ccgatttcat ataaatttct taaagtcgag gaccttataa gggtgcaatg 3600 gatgtttgct aaatatgaaa aactgacttg aatttgcact ctttaatgtt gcgtatctgc 3660 tctagtgggc aggacctagg gcttaaaggg gaacttcctt tctccaattc taggaactgg 3720 gactctaaaa tgagaagctg gttgtctgaa gtaaccctgc aggtgtggtt ggggaaggtc 3780 tgttttcttg gatgaaggga ttaaactgag caaatcacta gaagtatgcc ctgtcccctg 3840 ctcagaacac tggggagctc aagagtgggc tgcaatgtgc acccctcagg aatagctgtg 3900 aattgcaggt ctactggctt tttgcttttt gtcttttgct gcaaggtacc ccacgtactt 3960 aaccattctc aacagtgtaa atcagtgtca ttttagaatg agatactcag cttgcttcta 4020 aagtcactga attactgagt gagtctctcc tttagagtct tcggcaacca aattccagaa 4080 ttgaagagtc tactactcag aggcaacaag attaaaaaaa gaaaacacaa aaactgttga 4140 ggtgaaaaaa aaaaaaaacc ctagctagga acacagagaa tgttttgtag gatcactggg 4200 atattttcca caacttcctc ttctctagca cacacatctg ttgataggaa atatttgagg 4260 gtttttccac taccaaatgg gagcttcatg gtcctggtgt caaacactat aaacctttga 4320 ccagctgagc tgtgactgct gtcacatatc tgagtcctgt gtgcacagta atatcctggg 4380 tcaggtaaaa tccaggtctt caagttttaa ggattttttg aagaattcgg gcttctttaa 4440 gacgatccat gcccaaatcc acaagcttgt tgacagtggga ttacagtttg tgtggcaaag 4500 tccaagttgt tacactgtgc tttaaaaaaa atcttatctg catgtattgt taacttagag 4560 accatgagat ctatttatca ggaccaggaa gatacacact tcaggtccat tgcaactgac 4620 ttttttcttg tttttctcaa accctggtgg agcctgggaa gggggcctcc acaattctgt 4680 ggctttgata ttagccccaa ttctcacaag cacatacaag ccccataatt gccgcaggaa 4740 aacacaagat ggaaaattgc aataacccat gcactgagac ttagaaaatc atccttacta 4800 ggcaaaatgt attatgatgc aataagtgcc aactgatatt tctcacgttg ggactggcca 4860 ggaactgctg caaagaaaaa taagcagctc cttctccatt atttacattt taagatgtgg 4920 tggggggagg ttgggagaaa ttagttctga ggttatcata tgcctttttt aaaagataat 4980 ggaataaagc tatttttaag taa 5003

Claims (25)

CLAIMS What is claimed is: 1. A method for in vitro measurement of the probability of an individual having a colorectal cancer in a peripheral blood sample, comprising the steps of:
a) measuring the amount of the expression product of the ITGAM gene shown in SEQ ID NO: 5 or 6 and the VSIG10 gene shown in SEQ ID NO: 11 in the peripheral blood sample;
b) comparing the amount of the expression product measured in step a) with a reference amount of expression product for a group of individuals previously diagnosed as a colorectal cancer patient and a reference amount of expression product for a group of individuals previously verified as non- Comparing with a reference quantity; And
c) performing an analysis of the result of step b)
- if the result on the tested individual is similar or equivalent to the results obtained from a group of individuals previously diagnosed as a colorectal cancer patient, the tested individual is classified as a colorectal cancer patient,
- the tested individual is classified as a non-colorectal cancer entity if the results on the tested individual are similar or equivalent to those obtained from a group of individuals previously validated as non-colon carcinoma individuals. The method according to claim 1,
The step a) comprises, in the peripheral blood sample, the NEAT1 gene shown in SEQ ID NO: 1, the FAM198B gene shown in SEQ ID NO: 2, 3 or 4, the MYBL1 gene shown in SEQ ID NO: 7 or 8, The SIAH2 gene, and the PDZK1IP1 gene shown in SEQ ID NO: 10. 3. The method according to claim 1 or 2,
Wherein the amount of expression product in step a) is determined by contacting said expression product with one or more binding partners specific for this expression product. 3. The method of claim 2,
In step a), from the peripheral blood sample, the amount of the expression product of the ITGAM gene shown in SEQ ID NO: 5 or 6, the VSIG10 gene shown in SEQ ID NO: 11, and the FAM198B gene shown in SEQ ID NO: 2, How. 3. The method of claim 2,
In step a), from the peripheral blood sample, the ITGAM gene shown in SEQ ID NO: 5 or 6, the VSIG10 gene shown in SEQ ID NO: 11, the FAM198B gene shown in SEQ ID NO: 2, 3 or 4, Wherein the amount of the expression product of the NEAT1 gene is measured. 3. The method of claim 2,
In step a), from the peripheral blood sample, the ITGAM gene shown in SEQ ID NO: 5 or 6, the VSIG10 gene shown in SEQ ID NO: 11, the FAM198B gene shown in SEQ ID NO: 2, 3 or 4, NEAT1 shown in SEQ ID NO: And the amount of the expression product of the MYBL1 gene shown in SEQ ID NO: 7 or 8 is measured. 3. The method of claim 2,
In step a), from the peripheral blood sample, the ITGAM gene shown in SEQ ID NO: 5 or 6, the VSIG10 gene shown in SEQ ID NO: 11, the FAM198B gene shown in SEQ ID NO: 2, 3 or 4, NEAT1 shown in SEQ ID NO: The amount of the expression product of the gene, the MYBL1 gene shown in SEQ ID NO: 7 or 8, and the SIAH2 gene shown in SEQ ID NO: 9 is measured. 3. The method of claim 2,
In step a), from the peripheral blood sample, the ITGAM gene shown in SEQ ID NO: 5 or 6, the VSIG10 gene shown in SEQ ID NO: 11, the FAM198B gene shown in SEQ ID NO: 2, 3 or 4, NEAT1 shown in SEQ ID NO: The amount of the expression product of the gene, the MYBL1 gene shown in SEQ ID NO: 7 or 8, the SIAH2 gene shown in SEQ ID NO: 9, and the PDZK1IP1 gene shown in SEQ ID NO: 10 is measured. 3. The method according to claim 1 or 2,
Wherein the expression product is at least one RNA transcript or at least one polypeptide. 10. The method of claim 9,
Wherein the expression product is at least one mRNA. 11. The method of claim 10,
Wherein the RNA transcript is detected and quantified by hybridization, amplification or sequencing. 10. The method of claim 9,
Wherein the RNA transcript is contacted with said one or more probes or one or more primers under predetermined conditions to enable hybridization of said RNA transcript with at least one probe or at least one primer. 10. The method of claim 9,
Wherein the RNA transcript is contacted with said one or more probes and one or more primers under predetermined conditions to enable hybridization of said RNA transcript with at least one probe and at least one primer. 12. The method of claim 11,
Wherein a DNA copy of an RNA transcript is produced and said DNA copy is contacted with said one or more probes or one or more primers under predetermined conditions to enable hybridization of said DNA copy with one or more probes or one or more primers, Way. 12. The method of claim 11,
Wherein a DNA copy of an RNA transcript is produced and said DNA copy is contacted with said one or more probes and one or more primers under predetermined conditions to enable hybridization of said DNA copy with one or more probes and one or more primers, Way. 10. The method of claim 9,
Wherein the expressed polypeptide is detected by contact with at least one specific ligand selected from the group consisting of an antibody and an affinity protein. 17. The method of claim 16,
Wherein the expressed polypeptide is contacted with two or more specific ligands selected from the group consisting of two antibodies, two affinity proteins, or one antibody and one affinity protein. A kit for in vitro measurement of the probability of an individual having colorectal cancer, comprising an ITGAM gene shown in SEQ ID NO: 5 or 6 and a binding partner specific for the expression product of the VSIG10 gene shown in SEQ ID NO: 11. 19. The method of claim 18,
The NEAT1 gene shown in SEQ ID NO: 1, the FAM198B gene shown in SEQ ID NO: 2, 3 or 4, the MYBL1 gene shown in SEQ ID NO: 7 or 8, the SIAH2 gene shown in SEQ ID NO: 9, and PDZK1IP1 shown in SEQ ID NO: Further comprising a binding partner specific for an expression product of one or more genes selected from the genes. 20. The method of claim 19,
The NEAT1 gene shown in SEQ ID NO: 1, the FAM198B gene shown in SEQ ID NO: 2, 3 or 4, the MYBL1 gene shown in SEQ ID NO: 7 or 8, the SIAH2 gene shown in SEQ ID NO: 9, the PDZK1IP1 gene shown in SEQ ID NO: , And a combination of seven binding partners specific for the expression product of the VSIG10 gene shown in SEQ ID NO: 11. 20. The method according to claim 18 or 19,
Wherein the at least one specific binding partner comprises at least one hybridization probe. 22. The method of claim 21,
Wherein the specific binding partner comprises at least one hybridization probe and at least one primer. 22. The method of claim 21,
Wherein the specific binding partner comprises at least one hybridization probe and two primers. 20. The method according to claim 18 or 19,
Wherein the at least one specific binding partner comprises at least one specific ligand selected from the group consisting of one antibody and one affinity protein. 25. The method of claim 24,
Wherein the specific binding partner comprises two or more specific ligands selected from the group consisting of two antibodies or two affinity proteins, or one kind of antibody and one kind of affinity protein.