AU2016224709B2 - Method for assisting in prognostic diagnosis of colorectal cancer, recording medium and determining device - Google Patents

Method for assisting in prognostic diagnosis of colorectal cancer, recording medium and determining device Download PDF

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AU2016224709B2
AU2016224709B2 AU2016224709A AU2016224709A AU2016224709B2 AU 2016224709 B2 AU2016224709 B2 AU 2016224709B2 AU 2016224709 A AU2016224709 A AU 2016224709A AU 2016224709 A AU2016224709 A AU 2016224709A AU 2016224709 B2 AU2016224709 B2 AU 2016224709B2
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Yuichiro Yoshida
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Sysmex Corp
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

The present invention measures an SCEL gene expression amount in a biological specimen taken from a colorectal cancer patient and assists in the prognostic diagnosis of colorectal cancer on the basis of the measured SCEL gene expression amount.

Description

The present invention measures an SCEL gene expression amount in a biological specimen taken from a colorectal cancer patient and assists in the prognostic diagnosis of colorectal cancer on the basis of the measured SCEL gene expression amount.
2016224709 10 Oct 2018
DESCRIPTION
TITLE OF INVENTION: METHOD FOR ASSISTING IN PROGNOSTIC DIAGNOSIS
OF COLORECTAL CANCER, RECORDING MEDIUM AND DETERMINING DEVICE
TECHNICAL FIELD [0001]
The present invention relates to a method for assisting in the prognostic diagnosis of colorectal cancer. More specifically, the present invention relates to a method, a recording medium, and a determining device for assisting in the prognostic diagnosis of colorectal cancer in a patient based on expression level data of SCEL gene that are obtained for a nucleic acid obtained from a tissue of a colorectal cancer patient.
BACKGROUND ART [0001 A]
Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.
[0002]
Colorectal cancer is a generic term for carcinomas occurring in the cecum, colon, and rectum. As with many cancers, early detection is important for the treatment of colorectal cancer. In the treatment of cancer, an anticancer drug having a strong side effect is sometimes used. In such a case, the patient is forced to bear a heavy burden. In order to reduce such burden on the patient, it is important for a doctor to select an optimal treatment method for the patient. For this purpose, the doctor has to accurately grasp the stage of cancer progression, degree of malignancy, symptoms, and the like of the patient. [0003]
In addition, accurate prediction of a patient’s prognosis is important for improving
1002317435
2016224709 10 Oct 2018 the quality of life (QOL) of the patient in the prognosis. In recent years, as a pathological prognostic factor of colorectal cancer, presence or absence of budding has been drawing attention. Non Patent Literature 1 reports that budding can be useful as a prognostic factor of mucinous cancers in rectum and colon.
CITATIONS LIST
Patent Literature [0004]
Non Patent Literature 1: Okuyama T. et al., Budding (sprouting) as a useful prognostic marker in colorectal mucinous carcinoma, Jpn J Clin Oncol, 2002; 32 (10)
412-416
SUMMARY
TECHNICAL PROBLEMS [0005]
In the above-mentioned conventional method, the diagnosis result is subjective since a pathologist makes the diagnosis through microscopic observation. The present inventor has found a problem that there is a risk of overlook of the budding through microscopic observation since the budding is a nest containing 1 to 4 cancer cells. The inventor has also found a problem that it is difficult to make a prognostic determination because highly specialized knowledge is required for the prognostic determination by the conventional method.
It is an aspect of the present invention to provide a method, a recording medium, and a determining device for assisting in a more objective prognostic diagnosis of colorectal cancer.
SOLUTIONS TO PROBLEMS [0006]
As a result of extensive research to solve the above-mentioned problems, the
1002317435 present inventor has found that the expression of SCEL gene correlates with the presence or absence of budding, and completed the present invention.
[0007]
According to the present invention, there is provided a method for assisting in prognostic diagnosis of colorectal cancer, the method including the steps of measuring the expression level of SCEL gene in a biological sample collected from a colorectal cancer patient, and determining the prognosis of colorectal cancer based on the measured expression level.
ADVANTAGEOUS EFFECTS OF INVENTION [0008]
According to the present invention, it is possible to acquire intermediate information for assisting in the prognostic diagnosis of colorectal cancer by a doctor or the like.
BRIEF DESCRIPTION OF DRAWINGS [0009]
Fig. 1 is a schematic diagram showing an example of a diagnosis assisting apparatus.
Fig. 2 is a block diagram showing a functional configuration of software of a diagnosis assisting apparatus.
Fig. 3 is a block diagram showing a configuration of hardware of a diagnosis assisting apparatus.
Fig. 4 is a flowchart showing an example of an operation of a diagnosis assisting apparatus.
Fig. 5 is a flowchart showing an example of the operation of the diagnosis assisting apparatus.
Fig. 6 is a box-and-whisker plot showing a correlation between a BSS and a budding grade.
Fig. 7 is an ROC curve in a case of Example 1 (training set).
Fig. 8 is a Kaplan-Meier curve showing the results of comparing the survival periods between a high-risk group (poor prognosis) and a low-risk group (good prognosis) in the case of Example 1 (training set).
Fig. 9 is an ROC curve in a case of Example 2 (validation set).
Fig. 10 is a Kaplan-Meier curve showing the results of comparing the disease free survival periods between a high-risk group (poor prognosis) and a low-risk group (good prognosis) in the case of Example 2 (validation set).
Fig. 11 is an ROC curve in a case of Example 3.
Fig. 12 is a Kaplan-Meier curve showing the results of comparing the relapse free survival periods between a high-risk group (poor prognosis) and a low-risk group (good prognosis) in the case of Example 3.
Fig. 13 is an ROC curve in a case of Example 4.
Fig. 14 is a Kaplan-Meier curve showing the results of comparing the relapse free survivals between a high-risk group (poor prognosis) and a low-risk group (good prognosis) in the case of Example 4.
DESCRIPTION OF EMBODIMENTS [0010]
In the prognostic determination method for colorectal cancer in the present embodiment (hereinafter sometimes referred to as “determination method”), first, a step of measuring the expression level of SCEL gene in a biological sample collected from a colorectal cancer patient is performed.
[0011]
The “biological sample” is not particularly limited as long as it contains a nucleic acid (e.g., mRNA) derived from a tumor cell of a colorectal cancer patient.
For example, a clinical specimen can be used. Specific examples of the clinical specimen include tissues, blood, and serum collected by surgery or biopsy. Preferably, the clinical specimen can be a tumor tissue collected by surgery or biopsy, particularly a tissue surrounding the invasive front of cancer.
[0012]
The nucleotide sequence of cDNA of SCEL (sciellin) gene is represented in SEQ ID NO: 1. This nucleotide sequence is publicly known under Accession No.
NM 001160706 in the human genome database GenBank.
[0013]
In another embodiment, in the measurement step, the expression level of at least one gene selected from MGAT3 gene, SLC4A11 gene, MSLN gene, FOXC1 gene, RUNX2 gene, and WNT11 gene may be further measured in addition to the expression level of SCEL gene.
[0014]
The nucleotide sequence of cDNA of MGAT3 (mannosyl (beta-l,4-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase) gene is represented in SEQ ID NO: 2. This nucleotide sequence is publicly known under Accession No. ΝΜ 001098270 in the human genome database GenBank.
[0015]
The nucleotide sequence of cDNA of SLC4A11 (solute carrier family 4, sodium borate transporter, member 11) gene is represented in SEQ ID NO: 3. This nucleotide sequence is publicly known under Accession No. NM 001174089 in the human genome database GenBank.
[0016]
The nucleotide sequence of cDNA of MSLN (mesothelin) gene is represented in SEQ ID NO: 4. This nucleotide sequence is publicly known under Accession No. NM 001177355 in the human genome database GenBank.
[0017]
The nucleotide sequence of cDNA of FOXC1 (forkhead box Cl) gene is represented in SEQ ID NO: 5. This nucleotide sequence is publicly known under Accession No. NM 001453 in the human genome database GenBank.
[0018]
The nucleotide sequence of cDNA of RUNX2 (runt-related transcription factor 2) gene is represented in SEQ ID NO: 6. This nucleotide sequence is publicly known under Accession No. NM 001015051 in the human genome database GenBank.
[0019]
The nucleotide sequence of cDNA of WNT11 (wingless-type MMTV integration site family, member 11) gene is represented in SEQ ID NO: 7. This nucleotide sequence is publicly known under Accession No. NM 004626 in the human genome database GenBank.
[0020]
Budding is a nest that contains 1 to 4 cancer cells and is present in the interstitium near the invasive front of cancer. According to the “Clinical Practice Guideline” of the Japan Society of Clinical Oncology, when referring to colorectal cancer, budding is cancer cells that are liberated from a cancer tissue and proliferated, and is known as a risk factor for lymph node metastasis of colorectal cancer. The grade of the budding is evaluated by selecting an area with the most advanced budding, observing the invasive front of cancer development in a field of view of 20 x 10 times, and counting the number of budding foci. An individual with 5 or more counted budding foci (Grades 2 and 3) has a significantly increased lymph node metastasis rate compared to an individual with less than 5 budding foci (Grade 1).
[0021]
The present inventor found for the first time that the expression levels of the above-mentioned seven genes are significantly increased in the invasive front of cancer having the budding. Therefore, the seven genes can serve as useful genetic markers in the prognostic determination of colorectal cancer. Since the gene expression level can be measured and quantified according to a method known to those skilled in the art, measuring the expression levels of these genes enables the objective prognostic determination of colorectal cancer.
[0022]
A “gene transcription product” is a product obtained by transcription of a gene. Specific examples of the transcription product include messenger RNA (mRNA) and a precursor of mRNA.
The “gene expression level” refers to the abundance of a gene transcription product or the amount of a substance reflecting the abundance in the biological sample. Therefore, in the present embodiment, the amount of mRNA, or the amount of complementary deoxyribonucleic acid (cDNA) or complementary RNA (cRNA) obtained from mRNA is measured as the gene expression level. Since mRNA is present in a biological sample usually in a trace amount, it is preferred to measure the amount of cDNA or cRNA obtained from the mRNA by reverse transcription and in vitro transcription (IVT).
[0023]
As a method for extracting a gene transcription product from a biological sample, an RNA extraction method known in the art can be mentioned. For example, it is possible to obtain an RNA extract by centrifuging a biological sample to precipitate RNA-containing cells, and disrupting the cells by a physical technique, a chemical technique, or an enzymatic technique to remove the cell debris. The extraction of RNA can also be performed using a commercially available RNA extraction kit or the like.
[0024]
It is also possible to perform a treatment of removing, from the extract of the gene transcription product obtained as described above, a contaminant component derived from a biological sample (for example, globin mRNA when the biological sample is blood) which is preferably not incorporated in the extract at the time of measuring the gene expression level.
[0025]
The method for measuring the gene expression level is not particularly limited as long as it is a quantitative method. For example, the expression level can be measured using a microarray containing DNA, RNA, artificial nucleic acid or the like as a probe (hereinafter sometimes simply referred to as “microarray”) or quantitative PCR (for example, quantitative RT-PCR). In a preferred embodiment, a method using a microarray can be used.
In the case of measuring the gene expression level using a microarray, the expression level of the target gene can be measured, for example, by bringing an extract of a gene transcription product, or cDNA or cRNA prepared from a gene transcription product into contact with a nucleic acid probe of about 20 to 25 mer fixed on a substrate, and confirming the presence or absence of hybridization by measuring the change in indices such as fluorescence, color, and current.
The number of required nucleic acid probes used for one gene transcription product is at least one, and a plurality of probes may be used depending on the length and the like of the gene transcription product. A person skilled in the art can appropriately determine the sequence of the probe in accordance with the sequence of the gene transcription product to be measured. For example, in the present embodiment, polynucleotides represented by SEQ ID NOS: 8 to 84 can be used as the probe.
As a method for measuring the gene expression level using a microarray, for example, a GeneChip (registered trademark) system provided by Affymetrix, Inc. can be used.
[0026]
When a microarray is used, the gene transcription product, or cDNA or cRNA thereof may be fragmented to facilitate hybridization with the nucleic acid probe. The fragmentation can be carried out by a method known in the art. For example, the fragmentation can be carried out using a nucleolytic enzyme such as ribonuclease or deoxyribonuclease.
[0027]
The amount of the gene transcription product, or cDNA or cRNA thereof to be brought into contact with a nucleic acid probe in a microarray may be usually about 5 to 20 pg. The contact conditions are usually about 16 hours at 45°C.
[0028]
The gene transcription product, or cDNA or cRNA thereof that has hybridized by contact with a nucleic acid probe can be detected for the presence or absence of hybridization and the amount of hybridization based on the fluorescent substance, dye, or a change in the amount of current flowing on the microarray due to hybridization.
When the hybridization is measured by detection of a fluorescent substance or a dye, it is preferred that the gene transcription product, or cDNA or cRNA thereof is labeled with a labeling substance for detection of a fluorescent substance or a dye. A labeling substance commonly used in the art can be used as the labeling substance. Usually, biotinylated nucleotide or biotinylated ribonucleotide can be mixed as a nucleotide or ribonucleotide substrate when synthesizing cDNA or cRNA to label the obtained cDNA or cRNA with biotin. When cDNA or cRNA is biotin-labeled, avidin or streptavidin, which is a binding partner for biotin, can be bound on the microarray. Hybridization can be detected by binding of avidin or streptavidin to an appropriate fluorescent substance or dye. Examples of the fluorescent substance include fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), luciferin, and phycoerythrin. It is usually convenient to use a commercially available conjugate of phycoerythrin-streptavidin.
In addition, it is also possible to detect a fluorescent substance or a dye of a labeled antibody by bringing an antibody capable of being labeled with avidin or streptavidin into contact with avidin or streptavidin.
[0029]
The gene expression level obtained in this step is not particularly limited as long as it is a value that relatively expresses the abundance of each gene transcription product in the biological sample. In the case of performing the measurement with the above-mentioned microarray, the expression level can be a signal obtained from the microarray based on the fluorescence intensity, color intensity, current amount, and the like.
These signals can be measured using a measuring device for a microarray.
[0030]
Next, in the determination step, the prognosis of colorectal cancer is determined based on the gene expression level obtained in the measurement step. Preferably, the determination step includes a step of comparing the gene expression level or a logarithm thereof with a predetermined reference value. As the gene expression level, a value obtainable by the above-mentioned measurement method can be used as it is. For example, when the expression level is measured with a microarray, the value of the fluorescence intensity can be used as the gene expression level. When the expression level is measured by quantitative RT-PCR, values such as a PCR cycle number, and an mRNA copy number calculated from the PCR cycle number can be used.
The base of the logarithm of the gene expression level is not particularly limited, and can be 2 or 10.
In the case where the determination step is performed based on the expression levels of a plurality of genes, an average value of the gene expression levels, a median value of the gene expression levels, an average value of logarithms of the gene expression levels, a median value of the logarithms, an average value of values obtained by standardizing the gene expression levels, a median value of the standardized values, and the like can be used. In the comparison step, such value is compared with a predetermined reference value.
When the value is not less than the predetermined reference value, a patient may be determined to have a poor prognosis. When the value is less than the reference value, a patient may be determined to have a good prognosis.
[0031]
The “reference value” can be appropriately set based on accumulated data of gene expression levels. More specifically, the reference value may be a threshold that allows accurate classification into patients with a poor prognosis and patients with a good prognosis. For example, the reference value is a value that is obtained by measuring gene expression levels of a plurality of patients whose prognosis is known, and that enables most accurate classification into a patient group with a poor prognosis and a patient group with a good prognosis. Measuring the gene expression level of a patient whose prognosis is unknown and comparing the gene expression level with the reference value by the method of the present embodiment makes it possible to determine whether the patient has a good prognosis or not.
[0032]
In the present embodiment, the reference value may be a threshold that is obtained based on an ROC curve through an ROC analysis using an average value of logarithms (base = 2) of gene expression levels measured by a microarray for a plurality of specimens. In the present embodiment, when the average value of logarithms (base = 2) of gene expression levels measured by a microarray for specimens derived from an individual as a target of prognostic determination is equal to or more than the above-mentioned threshold, the individual may be determined to have a poor prognosis of colorectal cancer. On the other hand, when the average value is less than the above-mentioned threshold, the individual may be determined to have a good prognosis of colorectal cancer.
[0033]
The present invention also includes a program product for making a computer execute the prognostic determination of colorectal cancer in a patient. Examples of such a program product include a program that can be downloaded via the Internet or the like, and a computer-readable recording medium recording the program therein. [0034]
For example, a program for making a computer execute the following steps can be mentioned:
acquiring, from a measuring device, information relating to a gene expression level in a biological sample collected from a colorectal cancer patient; and determining the prognosis of colorectal cancer in the patient based on the acquired information. [0035]
Hereinafter, one mode of an apparatus suitable for implementing the method of the present embodiment will be described with reference to the drawings. However, the present invention is not limited to this embodiment. Fig. 1 is a schematic diagram showing an example of a diagnosis assisting apparatus used for prognostic determination of colorectal cancer in a patient. A diagnosis assisting apparatus 10 shown in Fig. 1 includes a measuring device 20 and a determining device 30 connected to the measuring device 20.
[0036]
In the present embodiment, the measuring device 20 may be a measuring device for a microarray. The measuring device 20 can acquire the gene expression level itself, and information relating to the gene expression level such as the fluorescence hue and fluorescence intensity of the microarray. When a biological sample collected from a colorectal cancer patient is set in the measuring device 20, the measuring device 20 can acquire information relating to the gene expression level in the biological sample, and provide the acquired information to the determining device 30. [0037]
The determining device 30 includes a computer main body 300, an input unit 301 including a keyboard and a mouse, and a display unit 302 that includes an LCD or a CRT and displays specimen information, a determination result, and the like. The determining device 30 acquires information relating to the expression level of each gene from the measuring device 20. Based on the information, the determining device 30 executes a program for determining the prognosis of colorectal cancer in a subject.
Via the input unit 301, it is possible to enter “perform single-gene determination”, “perform three-gene determination” and the like described later.
Note that the determining device 30 may be a device separate from the measuring device 20 as shown in Fig. 1 or may be a device including the measuring device 20. In the latter case, the determining device 30 may itself be the diagnosis assisting apparatus 10.
[0038]
Fig. 2 is a block diagram showing software of the computer main body 300 of the determining device 30 in functional blocks. As shown in Fig. 2, the computer includes an acquisition unit 321, a storage unit 322, a calculation unit 323, a determination unit 324, and an output unit 325. The acquisition unit 321 is communicably connected to the measuring device 20 via a network. Into the determination unit 324, information necessary for the prognostic determination of colorectal cancer, specifically, information on whether or not to perform single-gene determination, and/or whether or not to perform three-gene determination can be entered via the input unit 301.
[0039]
The acquisition unit 321 acquires information provided from the measuring device 20. The storage unit 322 stores a reference value necessary for the determination, and formulae and processing programs for calculating the gene expression level. The calculation unit 323 calculates the gene expression level according to the stored formulae using the information acquired by the acquisition unit
321. The determination unit 324 determines whether or not the gene expression level acquired by the acquisition unit 321 or calculated by the calculation unit 323 is not less than the reference value stored in the storage unit 322. The output unit 325 outputs the determination result by the determination unit 324 to the display unit 302 as a prognostic determination result of colorectal cancer in a subject.
[0040]
Fig. 3 is a block diagram showing a hardware configuration of the computer main body 300 shown in Fig. 2. As shown in Fig. 3, the computer main body 300 includes a CPU (Central Processing Unit) 310, a ROM (Read Only Memory) 311, a RAM (Random Access Memory) 312, a hard disk 313, an input/output interface 314, a reading device 315, a communication interface 316, and an image output interface 317. The CPU 310, the ROM 311, the RAM 312, the hard disk 313, the input/output interface 314, the reading device 315, the communication interface 316, and the image output interface 317 are connected by a bus 318 so that they are capable of data communication.
[0041]
The CPU 310 can execute a program stored in the ROM 311 and a program loaded in the RAM 312. When the CPU 310 executes the programs, the functions shown in Fig. 2 are executed. In this manner, the determining device 30 functions as a determining device for determining the prognosis of colorectal cancer in a subject. [0042]
The ROM 311 may be a mask ROM, a PROM, an EPROM, an EEPROM, or the like. In the ROM 311, the program executed by the CPU 310 and data used for the program are recorded as described above.
[0043]
The RAM 312 may be an SRAM, a DRAM, or the like. The RAM 312 is used for reading out the programs recorded in the ROM 311 and the hard disk 313.
The RAM 312 is also used as a work area of the CPU 310 when these programs are executed.
[0044]
In the hard disk 313, an operating system and programs such as an application program (a program for prognostic determination of colorectal cancer in a subject) to be executed by the CPU 310, and data used for executing the programs are installed.
[0045]
The reading device 315 may be a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like. The reading device 315 can read out a program or data recorded in a portable recording medium 40.
[0046]
The input/output interface 314 includes, for example, a serial interface such as USB, IEEE 1394, or RS-232C, a parallel interface such as SCSI, IDE, or IEEE 1284, and an analog interface including a D/A converter, an A/D converter, and the like. To the input/output interface 314, the input unit 301 such as a keyboard and a mouse is connected. An operator can enter various commands into the computer main body 300 via the input unit 301.
[0047]
The communication interface 316 is, for example, an Ethernet (registered trademark) interface. The computer main body 300 can also transmit print data to a printer or the like through the communication interface 316.
[0048]
The image output interface 317 is connected to the display unit 302 including an LCD, a CRT, or the like. As a result, the display unit 302 can output a video signal corresponding to the image data provided by the CPU 310. The display unit 302 displays an image (screen) according to the input video signal.
[0049]
Next, the processing procedure for prognostic determination of colorectal cancer in a subject by the diagnosis assisting apparatus 10 will be described.
Fig. 4 is an example of a flowchart of prognostic determination of colorectal cancer. Herein, an example will be described about a case where the fluorescence intensity is calculated from the fluorescence information obtained using a biological sample derived from a subject, the gene expression level is calculated from the obtained fluorescence intensity, and a determination is made as to whether or not the obtained expression level is not less than a reference value. However, the present invention is not limited to this embodiment alone.
[0050]
First, in step S1-1, the acquisition unit 321 of the diagnosis assisting apparatus 10 acquires fluorescence information relating to the expression level of the SCEL gene, MGAT3 gene, SLC4A11 gene, MSLN gene, FOXC1 gene, RUNX2 gene, and/or WNT11 gene from the measuring device 20.
[0051]
Next, in step SI-2, the calculation unit 323 calculates the fluorescence intensity from the acquired fluorescence information and transmits the fluorescence intensity information to the storage unit 322. In step Sl-3, the calculation unit 323 calculates the gene expression level according to the stored formula based on the stored fluorescence intensity.
[0052]
Then, in step SI-4, the determination unit 324 determines whether or not the expression level calculated in step SI-3 is not less than the reference value stored in the storage unit 322. When the expression level is not less than the reference value, the routine proceeds to step SI-5. Then, the determination unit 324 transmits a determination result indicating that the subject has a poor prognosis of colorectal cancer to the output unit 325. On the other hand, when the expression level is less than the reference value, the routine proceeds to step SI-6. Then, the determination unit 324 transmits a determination result indicating that the subject has a good prognosis of colorectal cancer to the output unit 325.
[0053]
Finally, in step SI-7, the output unit 325 outputs the prognostic determination result of colorectal cancer in the subject and causes the display unit 302 to display the result. In this manner, the diagnosis assisting apparatus 10 can provide a doctor or the like with information that assists in diagnosing whether the subject has a good prognosis of colorectal cancer or not.
[0054]
In the present embodiment, the gene used for the prognostic determination may be only the SCEL gene, or two or more genes including the SCEL gene and at least one additional gene selected from the MGAT3 gene, SLC4A11 gene, MSLN gene, FOXC1 gene, RUNX2 gene, and WNT11 gene.
[0055]
Further, in another embodiment, it is also possible to enable a user to select a gene to be used for prognostic determination. Such a processing procedure will be described with reference to Fig. 5 as an example. In this embodiment, a user can select whether to use only the SCEL gene (single-gene determination), use the SCEL gene,
MGAT3 gene, and SLC4A11 gene (three-gene determination), or use the SCEL gene, MGAT3 gene, SLC4A11 gene, MSLN gene, FOXC1 gene, RUNX2 gene, and WNT11 gene (seven-gene determination).
[0056]
First, in step S2-1, when an operator enters “perform single-gene determination” via the input unit 301, the routine proceeds to S2-3. Then, the acquisition unit 321 of the determining device 30 acquires fluorescence information relating to the expression level of the SCEL gene from the measuring device 20 (single-gene determination).
[0057]
On the other hand, when an operator does not enter “perform single-gene determination” via the input unit, the routine proceeds to S2-2. Then, in step S2-2, when the operator enters “perform three-gene determination” via the input unit 301, the routine proceeds to S2-4. Then, the acquisition unit 321 of the diagnosis assisting apparatus 10 acquires fluorescence information relating to the expression levels of the SCEL gene, MGAT3 gene, and SLC4A11 gene from the measuring device 20 (three-gene determination).
[0058]
When the operator does not enter “perform three-gene determination” in step S2-2, the routine proceeds to S2-5. Then, the acquisition unit 321 of the diagnosis assisting apparatus 10 acquires fluorescence information relating to the expression levels of the SCEL gene, MGAT3 gene, SLC4A11 gene, MSLN gene, FOXC1 gene, RUNX2 gene, and WNT11 gene from the measuring device 20 (seven-gene determination).
[0059]
Next, in step S2-6, the calculation unit 323 calculates the fluorescence intensity from the acquired fluorescence information and transmits the fluorescence intensity information to the storage unit 322. In step S2-7, the calculation unit 323 calculates the gene expression level according to the stored formula based on the stored fluorescence intensity.
[0060]
Then, in step S2-8, the determination unit 324 determines whether or not the expression level calculated in step S2-7 is not less than the reference value stored in the storage unit 322. When the expression level is not less than the reference value, the routine proceeds to step S2-9. Then, the determination unit 324 transmits a determination result indicating that the subject has a poor prognosis of colorectal cancer to the output unit 325. On the other hand, when the expression level is less than the reference value, the routine proceeds to step S2-10. Then, the determination unit 324 transmits a determination result indicating that the subject has a good prognosis of colorectal cancer to the output unit 325.
[0061]
Finally, in step S2-11, the output unit 325 outputs the prognostic determination result of colorectal cancer in the subject and causes the display unit 302 to display the result. In this manner, the diagnosis assisting apparatus 10 can provide a doctor or the like with information that assists in diagnosing whether the subject has a good prognosis of colorectal cancer or not.
[0062]
The present invention also includes a determining device suitable for prognostic determination of colorectal cancer in a subject.
[0063]
It should be noted that the storage unit 322 records therein a program for making the determining device 30 execute the following steps: acquiring, from a measuring device, information relating to a gene expression level in a biological sample collected from a colorectal cancer patient; and determining the prognosis of colorectal cancer in the patient based on the acquired information.
[0064]
In the present embodiment, it is possible to acquire information on the gene expression level measured by the microarray, and determine the prognosis of colorectal cancer in a subject based on the acquired information. For example, it is possible to provide a determination result that the subject has a good/poor prognosis of colorectal cancer. By providing the above-mentioned determination result to a doctor or the like, it is possible to assist in diagnosis by a doctor or the like about the prognosis of colorectal cancer.
EXAMPLES [0065]
Example 1: Prognostic determination of colorectal cancer using seven genes (training set) (1) Search for markers
Budding markers were searched according to the following procedure. Specifically, first, with respect to each two positions in total in the invasive front and the base part of three colorectal cancer tissue specimens in which budding is observed, (1) 23,509 genes were selected, the genes had an average expression value as measured by a microarray (manufactured by Affymetrix, Inc.) of 200 or more. Then, (2) 73 genes were selected, the genes had a minimal expression ratio between the invasive front and the base part in the three specimens of 2 or more (genes whose expression level in the invasive front with budding was about 2-fold that in the base part). Then, (3) 34 genes were selected, the genes had an expression ratio between the invasive front and the whole tissues of 1 or more (genes whose expression level in the invasive front with budding was higher than that in the whole tissue).
[0066]
Then, (4) using 85 specimens of colorectal cancer tissues including the above-mentioned three specimens, through a T test between 26 specimens positive for budding (Grade 3) and 44 negative specimens (Grade 1), seven genes with significant differences (p < 0.05) and whose expression was elevated in the budding-positive specimens were selected from the above-mentioned 34 genes. The selected seven genes and the IDs of the probe sets used for measuring the expression levels of the genes are shown in Table 1 below. In addition, the nucleotide sequences of the probes (all antisense strands) are represented by SEQ ID NOS: 8 to 84. [0067] [Table 1]
Gene symbol Gene name cDNA SEQ ID NO: Probe set ID Sequence ID No. of probe included in probe set
SCEL sciellin 1 206884 s at 8-18
MGAT3 mannosyl (beta-1,4-)glycoprotein beta-1,4-Nacetylglucosaminyltransferase 2 209764_at 19-29
SLC4A11 solute carrier family 4, sodium borate transporter, member 11 3 223748_at 30-40
MSLN mesothelin 4 204885 s at 41-51
FOXC1 forkhead box C1 5 1553613_s_at 52-62
RUNX2 runt-related transcription factor 2 6 23223 l_at 63-73
WNT11 wingless-type MMTV integration site family, member 11 7 206737_at 74 - 84
A correlation between the average value of logarithms (base = 2) of the [0068] expression level calculated above (hereinafter referred to as Budding Signature Score (BSS); the specific calculation formula is shown below) and the budding grade by a pathological diagnosis is shown in Fig. 6. Herein, the budding grade was determined as defined in [Grade of budding] in the Clinical Practice Guideline of the Japan Society of Clinical Oncology. That is, an area with the most advanced budding in the specimen was selected, the invasive front of cancer development was observed in a field of view of 20 x 10 times, and the number of budding foci were counted. Based on the result, when the number of budding foci was 0 to 4, the area was classified as Grade 1, when the number was 5 to 9, the area was classified as Grade 2, and when the number was 10 or more, the area was classified as Grade 3. From Fig. 6, it was found that the BSS increases as the budding grade increases. That is, the higher the budding grade is, the more genes are expressed.
[0069] [Mathematical Expression 1]
Budding Signature Score = [Log2 (signal value of DNA chip of MSLN gene) + Log2 (signal value of DNA chip of SCEL gene) +
Log2 (signal value of DNA chip of RUNX2 gene) + Log2 (signal value of DNA chip of FOXC1 gene) +
Log2 (signal value of DNA chip of MGAT3 gene) + Log2 (signal value of DNA chip of SLC4A11 gene) + Log2 (signal value of DNA chip of WNT11 gene)]/7 [0070] (2) Prognostic determination
An ROC analysis was performed on the 85 specimens using the BSS value calculated as described above, and a threshold was set. The results are shown in Fig. 7. In the ROC curve of Fig. 7, the threshold (8.436) was set to the value at which the sensitivity and specificity were the highest (the value corresponds to the point closest to (sensitivity, specificity) = (1, 1) on the ROC curve). The area under the curve (AUC) was 0.602.
[0071]
Subsequently, the survival periods were compared between the specimens having a BSS not less than the threshold and the specimens having a BSS less than the threshold. The results are shown in Fig. 8. As shown in Fig. 8, a significant difference (p = 0.0479) was observed from the viewpoint of survival probability (probability) between the specimens having a BSS not less than the threshold and the specimens having a BSS less than the threshold. As described above, it was suggested that it is possible to determine whether a colorectal cancer patient has a high-risk prognosis or a low-risk prognosis based on the expression levels of seven genes, that is, the SCEL gene, MGAT3 gene, SLC4A11 gene, MSLN gene, FOXC1 gene, RUNX2 gene, and WNT11 gene.
[0072]
Example 2: Prognostic determination of colorectal cancer using seven genes (validation set)
The usefulness of the seven genes as colorectal cancer prognostic markers was further validated using published gene expression level data of colorectal cancer. The used data were GSE39582 (461 specimens) of Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE39582), a public database.
The ROC analysis was performed in the same manner as in Example 1, and a threshold (7.686, AUC = 0.5752) was set. The survival periods were compared between the specimens having a BSS not less than the threshold and the specimens having a BSS less than the threshold. The results are shown in Figs. 9 and 10. As shown in Fig. 10, a significant difference (p = 0.000473) was observed from the viewpoint of disease free survival between the specimens having a BSS not less than the threshold and the specimens having a BSS less than the threshold. This result reproduces the result of Example 1. Therefore, it was confirmed that the above-mentioned seven genes are useful as prognostic markers for colorectal cancer. [0073]
Example 3: Prognostic determination of colorectal cancer using three genes The present inventor conducted the same experiment as in Example 2 using the same database as in Example 2 on the three genes of the SCEL gene, MGAT3 gene, and SLC4A11 gene. Specifically, the ROC analysis was performed in the same manner as in Example 2, and a threshold (6.112, AUC = 0.5828) was set. The relapse free survival periods (r.f.s. delay, number of days) were compared between the specimens having a BSS not less than the threshold (the specific calculation formula of the BSS in the case of three-gene measurement is shown below) and the specimens having a BSS less than the threshold. The results are shown in Figs. 11 and 12. As shown in Fig. 12, a significant difference (p = 0.000684) was observed from the viewpoint of survival probability between the specimens having a BSS not less than the threshold and the specimens having a BSS less than the threshold. Therefore, it was shown that the prognosis of colorectal cancer can be determined based on the expression levels of the above-mentioned three genes.
[0074] [Mathematical Expression 2]
Budding Signature Score = [Log2 (signal value of DNA chip of SCEL gene) + Log2 (signal value of DNA chip of MGAT3 gene) +
Log2 (signal value of DNA chip of SLC4A11 gene)]/3 [0075]
Example 4: Prognostic determination of colorectal cancer using SCEL gene Whether or not the prognostic determination of colorectal cancer can be made based on the expression level of the SCEL gene was validated. That is, the same experiment as in Example 2 was conducted on the SCEL gene using the same database as in Example 2. Specifically, the ROC analysis was performed in the same manner as in Example 2, and a threshold (5.300, AUC = 0.6003) was set. The relapse free survival periods (number of days) were compared between the specimens having a BSS not less than the threshold (the specific calculation formula of the BSS in the case of one-gene measurement is shown below) and the specimens having a BSS less than the threshold. The results are shown in Figs. 13 and 14. As shown in Fig. 14, a significant difference (p = 0.000702) was observed from the viewpoint of survival probability between the specimens having a BSS not less than the threshold and the specimens having a BSS less than the threshold. Therefore, it was shown that the prognosis of colorectal cancer can be determined based on the expression level of the SCEL gene.
[0076] [Mathematical Expression 3]
Budding Signature Score = Log2 (signal value of DNA chip of SCEL gene)
REFERENCE SIGNS LIST
Diagnosis assisting apparatus
Measuring device
Determining device
Recording medium [0077]
Computer main body Input unit Display unit
CPU
ROM
RAM
Hard disk
Input/output interface Reading device
Communication interface
Image output interface
Bus
Acquisition unit Storage unit
Calculation unit
Determination unit
Output unit
2016224709 10 Oct 2018

Claims (10)

1. A method for assisting in prognostic diagnosis of colorectal cancer, the method comprising the steps of:
measuring an expression level of SCEL gene in a biological sample collected from a colorectal cancer patient, and determining a prognosis of colorectal cancer based on the expression level, wherein the expression level or a logarithm of the gene expression level which is not less than a reference value indicates poor prognosis, and the expression level or a logarithm of the gene expression level which is less than the reference value indicates good prognosis.
2. The method according to claim 1, wherein in the determination step, the expression level or a logarithm thereof is compared with a predetermined reference value, and when the expression level or a logarithm thereof is not less than the reference value, the patient is determined to have a poor prognosis, whereas when the expression level or a logarithm thereof is less than the reference value, the patient is determined to have a good prognosis.
3. A method for assisting in prognostic diagnosis of colorectal cancer, the method comprising the steps of:
measuring an expression level of SCEL gene in a biological sample collected from a colorectal cancer patient, and determining a prognosis of colorectal cancer based on the expression level, in the measurement step, an expression level of at least one gene selected from the
1002317435
2016224709 10 Oct 2018 group consisting of MGAT3 gene, SLC4A11 gene, MSLN gene, FOXC1 gene, RUNX2 gene, and WNT11 gene is further measured, and in the determination step, the prognosis of colorectal cancer is determined based on the gene expression levels measured in the measurement step.
4. The method according to claim 3, wherein in the measurement step, expression levels of MGAT3 gene and SLC4A11 gene are further measured, and in the determination step, the prognosis of colorectal cancer is determined based on the gene expression levels measured in the measurement step.
5. The method according to claim 4, wherein in the measurement step, an expression level of MSLN gene, an expression level of FOXC1 gene, an expression level of RUNX2 gene, and an expression level of WNT11 gene are further measured, and in the determination step, the prognosis of colorectal cancer is determined based on the gene expression levels measured in the measurement step.
6. The method according to claim 1, wherein in the determination step, a logarithm of the gene expression level measured in the measurement step is calculated, the logarithm is compared with a predetermined reference value, and when the logarithm is not less than the reference value, the patient is determined to have a poor prognosis, whereas when the logarithm is less than the reference value, the patient is determined to have a good prognosis.
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2016224709 10 Oct 2018
7. The method according to any one of claims 3 to 5, wherein in the determination step, an average value of the gene expression levels measured in the measurement step or a logarithm of the average value is calculated, the average value or the logarithm is compared with a predetermined reference value, and when the average value or the logarithm is not less than the reference value, the patient is determined to have a poor prognosis, whereas when the average value or the logarithm is less than the reference value, the patient is determined to have a good prognosis.
8. A computer program for making a computer execute the steps of:
acquiring, from a measuring device, information relating to an expression level of SCEL gene in a biological sample collected from a colorectal cancer patient, and determining a prognosis of colorectal cancer in the patient based on the acquired information, wherein the expression level or a logarithm of the gene expression level which is not less than a reference value indicates poor prognosis, and the expression level or a logarithm of the gene expression level which is less than the reference value indicates good prognosis.
9. A determining device for determining a prognosis of colorectal cancer, comprising at least a computer including a processor and a memory, wherein the memory records therein a program for making the computer execute the steps of:
acquiring, from a measuring device, information relating to an expression level of SCEL gene in a biological sample collected from a colorectal cancer patient, and
1002317435
2016224709 10 Oct 2018 determining a prognosis of colorectal cancer in the patient based on the acquired information, wherein the expression level or a logarithm of the gene expression level which is not less than a reference value indicates poor prognosis, and the expression level or a logarithm of the gene expression level which is less than the reference value indicates good prognosis.
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31J
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1 2 3
Budding grade [Fig. 7] [Fig. 8]
Low (60) i-ή High ¢25) ^4—44+--4*-W4-44-~44- ¢4 60 G Si«) -iOOO 1560 2®» 2500
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2016224709 10 Oct 2018
Specifietiy [Fig. 10]
100 g
>80
Έ □
φ © tow (337) b 40 r; ο B
5 · '-4 f i+
High (124) p= 0.000473
2000
4000 (Day)
10 OS 06 ¢4 0,2 0 0
Specificity
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2016224709 10 Oct 2018 [Fig. 12] [Fig. 13]
Spcetoty [Fig. 14]
Low (221)
High (240) p = 0.000702 rfs delay
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aagaaacct c t gaact gt t c act aat acag t caggt agag gt t gagact c cact gaat aa 60 act ct aggt t cccat 11 ct t t cagccagat cct cccaggg aat cact aca ggct ggt t ag 120 ccaaaaagt c ct gat t t t ct gct caat aga ggt cct t act ggaaggcagc at gt ccaat g 180 t t acct t gag aaaaat gt ct cccacaggaa at gagat gaa gagcaccact cagggaacca 240 cacggaagca gcaggat 111 cacgaggt ga acaaaagaag aact t t ct t a caggat aaca 300 gt t ggat aaa gaaacgccct gaagaagaaa aagat gaaaa tt acggt agg gt ggt gct ca 360 accgacat aa 11 cccat gat gcat t ggaca ggaaagt aaa t gagagagat gt gccaaaag 420 ct acaat t ag t cggt acagt t ct gat gaca ct t t ggacag gat ct cagac agaaat gat g 480 ct gct aaaac at at aaggcc aat acct t gg at aaccaact aaccaat agg agcat gt cca 540 t gt t t agat c act ggaagt a acaaagt t gc aacct ggcgg t t cat t gaat gccaacacct 600 ccaacaccat agcat ccact t ct gct act a ct cct gt aaa gaagaagagg cagt cct ggt 660 t t ccaccgcc ccct ccaggt t acaat gcct cct cgagcac aggaaccagg agacgggaac 720 caggt gt t ca ccct ccaat a cct ccaaagc ccagt t ct cc t gt t t ct t ct cct aaccagc 780 t gagacagga t aat aggcag at acat ccac ct aaaccagg t gt at at aca gaaaccaaca 840 gat ct gct ga aagaaat at a agt gaagaat t ggat aat ct cat caaaat g aacaaaagct 900 t gaat aggaa t caaggt ct t gat agt ct ct t cagagcaaa t ccaaaggt a gaagaaagag 960 agaaaagagc caaaagcct t gaaagt ct ca t ct at at gag t acccggaca gat aaagat g 1020 gcaaaggaat ccaaagcct t ggaagt ccga t t aaagt t aa t caaaggact gacaaaaat g 1080 agaaaggaag acaaaat ct c gaat ct gt t g ct aaagt gaa t gccaggat g aat aaaacga 1140 gcagaagaag t gaagacct t gat aat gct a ct gaagt aaa t cccaaagga cat gaaaat a 1200 ccact ggaaa aaaagacct t gat gggct t a t t aaagt gga t cct gaaaca aat aaaaat a 1260 t t acgagggg ccagagcct t gat aat ct ca t caaagt gac ccct gaagt a aagagaagt a 1320 accaaggt t c caaagacct t aat aact t ca t caaagt gt a t ccaggaaca gaaaaaagt a 1380 ct gaaggggg ccaaagt ct c gacagcct ca t t aaagt gac t cct gaaaga aacagaact a 1440 accaagggaa ccaagact t g gaaaat ct t a t caaagt gat Page 1 ccct t cagca aacaaaagca 1500
1- 2014- 041PCT_Sequence l i st i ng. TXT
gt gaacaagg t ct t gat gaa cat at t aat g t cagccccaa agct gt caaa aacact gat g 1560 gaaaacaaga t ct t gat aaa ct cat caagg t gaat cct ga aat t t t caca aacaaccaaa 1620 gaaaccaaga t ct t gct aac ct cat caaag t aaat cct gc agt aat caga aacaat caga 1680 gccaagact t ggacaat ct t at t aaagt ga aacct t cagc t ct t agaaac act aat cgag 1740 accagaacct ggaaaat t t a at t gaagt aa at t ct cat gt gt ct gaaaac aagaat ggaa 1800 gct ct aacac t ggagccaag caggcaggac cacaggat ac tgttgt gtac acaaggacat 1860 at gt ggagaa t agt aaat ca cccaaggat g gat at cagga gaat at ct ct ggaaaat aca 1920 t acaaact gt 11 at t caact t ct gat aggt ct gt cat t ga aagagat at g t gcact t act 1980 gccgaaaacc ct t gggt gt a gaaact aaaa t gat 111 aga t gaat t acaa at 11 gct gcc 2040 at t ct act t g ct t t aagt gt gaaat at gca agcagcct t t ggaaaat ct a caagcgggt g 2100 at agt at t t g gat t t at aga cagacaat ac act gt gaacc t t gct act ct aaaat t at gg 2160 caaagt ggat t ccat aact c t ggcacaagg aaat caagat gaaaagcact cat t aaggaa 2220 t t aaagt t ac aagt t t t at c t t aat aat at gt aat ct aga aaagct t t ca cat t gaagat 2280 caact ct t gt acaaaat t aa caat t ct gt t at t gcat aag t aat ct aat t gt ct t caat a 2340 aggt cacaca cat aaaaaga gccat ct ggt ct ct ggct ag agt t agcaat aaaaagt t ca 2400 aat ggt t cca gat t ccagt g t caaaggagt gat gcat t ac act ccagcca ggt ccat ccc 2460 t gct ccgt at gttggct gt g agt ggt ggt t t ccat 11 aaa ccaagt t t ct cat t t ct t ca 2520 cct t t t t t t c t ct aagaat t t ggat t cgt a gacat t gaca t cccgaagaa ct gt caagga 2580 agcaagat at gct 11 ct t ca t ct gcaaaag aaat act aac aacaat t t t c 11 at acagt t 2640 t ggcagaaag at gt t aacat aaaaagt t t a t at acct caa aaat cact aa act 11 ccaga 2700 t ct ct gt cct at t at t t gt a acacaagggg cat t ggat aa aat gat t t ct agggt t cct t 2760 t t gct t ccca aat t ct ct ga 11 ct aaagca gt t t t t agaa t cat t agct c 111 ggaaaca 2820 t at at gcat a cat gt t t gt t aagcct at t g aact aggt ag gacat at aaa caat t t aat t 2880 t t agt gt cat t gt 11 aat ca cagact t agt gt t t gaaaac t gt gt t t t aa aaacagaaac 2940 agat t gat gg gt aacaggt a aaat at gaca t gt at agct t acat gt t at t at t t gt t aaa 3000 t t t t ct t t gt at acat 11 ca aaat ct gggt at act t at aa t ccat t agaa gt aat ggt t a 3060 t ggact aaaa agat at gt t c 111 agt at gt t at at at act cat at t acat agcagt at gt 3120 t t acaaaagg ct t at aaaaa t aaaat gaac t at cagt t ac at agaaaaaa aaaaaaaaaa 3180
<210> 2 <211> 4987 <212> DNA <213> Homo sapi ens <400> 2 ggggcagcag gtgctggcca ccacat t gt c cagcaaggt g gcagcagagg cct cct aggt cccct t cct a ggaaaggagc ct gggct gcc ct gat gagt c t cct gt ct ct ct ct ct cccg
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caggat gaag at gagacgct acaagct ct t t ct cat gt t c t gt at ggccg gcct gt gcct 180 cat ct cct t c ct gcact t ct t caagaccct gt cct at gt c acct t ccccc gagaact ggc 240 ct ccct cagc cct aacct gg t gt ccagct t 111 ct ggaac aat gccccgg t cacgcccca 300 ggccagcccc gagccaggag gccct gacct gct gcgt acc ccact ct act cccact cgcc 360 cct gct gcag ccgct gccgc ccagcaaggc ggccgaggag ct ccaccggg tggacttggt 420 gct gcccgag gacaccaccg agt at t t cgt gcgcaccaag gccggcggcg t ct gct t caa 480 acccggcacc aagat gct gg agaggccgcc cccgggacgg ccggaggaga agcct gaggg 540 ggccaacggc t cct cggccc ggcggccacc ccggt acct c ct gagcgccc gggagcgcac 600 ggggggccga ggcgcccggc gcaagt gggt ggagt gcgt g t gcct gcccg gct ggcacgg 660 acccagct gc ggcgt gccca ct gt ggt gca gt act ccaac ct gcccacca aggagcggct 720 ggt gcccagg gaggt gccgc gccgcgt cat caacgccat c aacgt caacc acgagt t cga 780 cct gct ggac gt gcgct t cc acgagct ggg cgacgt ggt g gacgcct t t g t ggt gt gcga 840 gt ccaact t c acggct t at g gggagccgcg gccgct caag t t ccgggaga t gct gaccaa 900 t ggcacct t c gagt acat cc gccacaaggt gct ct at gt c t t cct ggacc act t cccgcc 960 cggcggccgg caggacggct ggat cgccga cgact acct g cgcacct t cc t cacccagga 1020 cggcgt ct cg cggct gcgca acct gcggcc cgacgacgt c t t cat cat t g acgat gcgga 1080 cgagat cccg gcccgt gacg gcgt cct 111 cct caagct c t acgat ggct ggaccgagcc 1140 ct t cgcct t c cacat gcgca agt cgct ct a cggct t ct t c t ggaagcagc cgggcaccct 1200 ggaggt ggt g t caggct gca cggt ggacat gct gcaggca gt gt at gggc t ggacggcat 1260 ccgcct gcgc cgccgccagt act acaccat gcccaact t c agacagt at g agaaccgcac 1320 cggccacat c ct ggt gcagt ggt cgct ggg cagccccct g cact t cgccg gct ggcact g 1380 ct cct ggt gc 11 cacgcccg agggcat ct a ct t caagct c gt gt ccgccc agaat ggcga 1440 ct t cccacgc t ggggt gact acgaggacaa gcgggacct g aact acat cc gcggcct gat 1500 ccgcaccggg ggctggttcg acggcacgca gcaggagt ac ccgcct gcag accccagcga 1560 gcacat gt at gcgcccaagt acct gct gaa gaact acgac cggt t ccact acct gct gga 1620 caacccct ac caggagccca ggagcacggc ggcgggcggg t ggcgccaca ggggt cccga 1680 gggaaggccg cccgcccggg gcaaact gga cgaggcggaa gt ct agagct gcat gat ct g 1740 at agggt t t g t gacagggcg ggggt ggcgg cggcccct ag cgct at ct cc ct gcct cct g 1800 ccggct cct t ggt t ct t gag gggaccagga gt gggt gggg agt gggggt g ggggt agggt 1860 t t ccct act g aagccct t gt gaat caaggg t caggcct 11 gagct cagaa aat at ccct c 1920 ct gt t gggag agggcgcagg ccgt gacgt c t gggt ggccc t t at gact gc caagact gct 1980 gt ggccagga ggt gccact g gagt gt gcgt ggt ggt ccct gggt agcggg ggagggt agg 2040 caggat t ggg gaagagagcc t gcaggat ct caccaggcag cct ct ggggg gt ggccaggc 2100 cgggaaaaag cccaccat 11 ggcat ccct g ggcct t gggc t ccgt gt ggg agaccggcct 2160
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1- 2014- 041PCT_Sequence l i st i ng. TXT
gccaggagga cccagggct c t gt aagt aga t gcat t t ggg t ccaggagga agcgt ggaca 2220 cct cgt aggg aagagat gaa aaagccacat cct accaaga ggaggt gct g agggatgctt 2280 t gcagt gt ag t cagaagt gc t gggccagat ggagacagaa ct ccaccccc t gccgcaaag 2340 gacaggacct ggct gccct g ggat gct ggt gcct gagt ct gt ct ct gt gc acccct cagg 2400 ct gt cgt gag ccaacacagg ggcct ggaga accct gagga gct t t cct t t t ggt t ct aaa 2460 cccggcgt t g acgt t cct t c t ccct t t cac at t gct gt ct t gt ggact gt gcact cagt c 2520 ct t gcaaggc caagagt cca gt t gt aggt g t ggcct t gag ggggaagt gg ggaggagaag 2580 act gacat ga gt cct ct gca cggat ccgt c t ct ccct ccc cat cacccct t cct t ct gac 2640 acccagt ccc agct gt ccac t gt cccaggt gcagt cact g t t gt gccct t ccttggggca 2700 ggct ggct gg gggccagaaa ggggccat ga ggct gt ct t g ggcccaaaaa gggacaat aa 2760 ggccagt t gt at gct t cct g t t cct cat ag ct t gcct t gg t ggggat gt c t t t gt t ggag 2820 t t gat t ct ga gct gct gt ga t t aggagacc ct gaaat aca gt ggt t t aag caagat ggaa 2880 gct t gt t t ct aat t agt ct a gat t gagat g gcccagagct ggt agggcag ct ct gcgt t t 2940 ct t cat acgc acct t ccaat t ct gggt aca cagcggct gc t ccagcgccc accct cct gt 3000 gt gcat ccaa gcct ggggga agcagaaat a gacaagaggg cacacccact 1111 gct aaa 3060 ggcat gagcc agaat t ggca ggct cacct c t gct ggcct c t cat t ggct g ggact cagt c 3120 acat ggccac aagcagct gc t agggaacct gggaagt gt a gt ct t cagcg gggccgccat 3180 gt gcct ggcc t cacct t ggg agt t at ct t a t t gat ggagg agaagagaat ggat at gggg 3240 gaccagt agc at ct ct ggga gagggggagg gagcagcaat aact cagt cg t cggat ccag 3300 ct ct cat t gt cagagt 11 cc ggaacagct t gct cct gt t t ccct cact gt gcagcccagg 3360 gct gggggca gt gaggagct t gcagct ct g t gggaagggg aaacaccccc t cccct cggc 3420 ccct cagacg ct acccaat g at gccggt t t gcagagt t gg cct gt ggaat ggct cat gt t 3480 t gt gcgt gt g t gt gt gt at a t t t at gggca t gggt gcat g ct t ggt gt gt at 11 gt acat 3540 gt ct gt at t g ct gt gt ccct gt aaat acat gct t gt gt at ggat ggaaga ggccaggccc 3600 aggcct ggcc t ct t cct cgg gcct gt ggcc acacct cct g cagct cccca aaat gact ga 3660 ggcagaaagc cct t ggggag cct agaaagc aaagct aaag gggat gcagg gt ct gt ct gt 3720 ct gt ct gt ct 11 cagt ct ga ggaat gagaa t cct gacct g agggct gt gc agct gagagc 3780 ccact acct c cccagcccct ct cggcccca gccgcat cat cccacct gt c ccct cccccc 3840 cacct ccagt ggggcttt ct ccagat gt ct t at ggt t ggg ggt t t cct ga t gggccagga 3900 gaggagggca t ct t ct t gcg acagcact gt ct gggt t aag t gcccagt ga gggcat ggt g 3960 t ggggagct g gcct cagagg agccgct ggt gggcaagcgt gaagt gggct gaggggct ct 4020 gagccact t t gct cccat ct aggggact gc cccccat gga act cct t t ga agt cacagca 4080 gcct t cct t t ct gt 11 gct c ttggggctga gaggt ggct c aaacact cgg ggt ccct at g 4140 gct ct gggt c aat ct aggcc aggct gcacc ccat ggacag ggagt ct cag ggct cct gat 4200
Page 4
cat gcccagg ccct ggcct g 1-2014-041PCT Sequence listing.TXT gggcct ccct cct t ggcagc t t t cccaccc ccacgcccct 4260 ggcat cct ca gttgct atgg gat gcccct c cagggcacca gct cagggct aagcgaagga 4320 agat aggagc agct cagagc t gccaggct c t gcct t cct c acagacct gg t ggggcaggt 4380 cct gt t caca gcagcaggag t gaaggcct g gccat cggt g gagagggcag ct gt cagagg 4440 gct gggggcc agggcacagg at t gaagagt 11 cacat at c at cacagcat acact gggaa 4500 tttggtgggg gcagaagaac ccagggccac t ccct caat a t gaagggaaa ccaagct gaa 4560 t gt gaccacc ggcacact gc t gccat gt cc cat gt ccacc t t t ct ccccg ggaat aact g 4620 gccct gagac ccct agaccc aaggaggcct gt ccat gcca agcat ccggg aagcat ggct 4680 ggcct t at cc acccat gggt cacgt cggt t cccaggggca gcat gggaga tctttggggg 4740 caacagggag agt ct gggt g gggagacggg act t gt ccaa gcagaaggca ggaccct ggg 4800 aaat gcat aa t gt aaggaca t caat aat ag t at t at 1111 t t t gt aaggg aaaat caat a 4860 t gt acat t ct gaaat cat t t t ct ct gt aaa tggttggatt t cat t t cacc ct t aaaggga 4920 t gct t aaagg agaagat aat at t aat aat a aaaacagct a caaagt ct ga aaaaaaaaaa 4980
aaaaaaa 4987 <210> 3 <211> 3268
<212> DNA <213> Homo sapi ens <400> 3 ccct t t t gga ccaacggct c t ggct t ccag gcggccggga cgcggt ccca ggact ggaga 60 ccgt t gcct g t cggcccccg t gt gacccgg ggcgcgt gac gggggt cggg ggaact gcgc 120 ct gcaat ggg cgt t t at ggc ccccaggacc ggt ct gagag t gagaagagg gat gt gcaga 180 gagat ccccc gcct t ggcat ccgaggagag agggggagag gcccgct cgg gcccggt ccc 240 t t cct ct cgc t gcagcgggg cagggt 111 c t caggaaaac ct ggat t agc gaacat gaaa 300 act ct cccac cat gt cgcag aat ggat act t cgaggat t c aagct act ac aagt gt gaca 360 cagat gacac ct t cgaagcc cgagaggaga t cct ggggga t gaggcct t c gacact gcca 420 act cct ccat cgt gt ct ggc gagagt at cc gt t t t t t t gt caat gt caac ct t gagat gc 480 aggccaccaa cact gagaat gaagcgact t ccggt ggct g t gt gct cct g cacacct ccc 540 gaaagt acct gaagt t aaag aact t caagg aagagat ccg t gcgcaccgc gacct agat g 600 gct t cct ggc gcaggccagc at cgt cct ga acgagacggc cacct ccct g gat aacgt gc 660 t gcggaccat gct t cgccgc tt cgccaggg accct gacaa caat gagccc aact gcaacc 720 t ggacct gct cat ggccat g ct ct t caccg at gccggggc acccat gcgg ggt aaagt cc 780 acct gct gt c agat accat c caaggggt ca ccgccacagt gacaggggt g cggt accagc 840 agt cgt ggct ct gcat cat c t gt accat ga aggccct aca gaagcggcac gt gt gcat ca 900 gccgcct ggt t cgcccacag aact gggggg agaat t cct g t gaggt t cgg 11 cgt cat cc 960 t ggt gct ggc cccacccaag at gaaaagca ct aagact gc gat ggaggt g gcgcgcacgt 1020
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1- 2014- 041PCT_Sequence l i st i ng. TXT
11 gccaccat gt t ct cggat at cgcct t cc gccagaagct cct ggagacc cgcacagagg 1080 aggaat t caa ggaggccttg gt gcat caga gacagct gct caccat ggt g agccacggt c 1140 cagt ggcgcc gagaacgaag gaacgcagca cagt ct ccct ccct gcccac agacacccag 1200 agcccccaaa gt gcaaggac t t t gt ccct t ttgggaaggg cat ccgggag gacat cgcac 1260 gcaggt t ccc ct t gt acccc t t ggact t ca ct gat ggcat t at t gggaaa aacaaggct g 1320 t gggcaaat a cat caccacc accct gt t cc t ct act t cgc ct gcct cct g cccaccat cg 1380 ct t t cgggt c t ct caat gac gagaacacag acggggccat cgacgt gcag aagaccat ag 1440 ccgggcagag cat cgggggc ct gct ct acg cgct ct t ct c t gggcagcca ttggtgattc 1500 t gct gaccac cgcgcccct g gcgct ct aca t ccaggt gat t cgt gt cat c t gt gat gact 1560 at gacct gga ct t caact cc t t ct acgcat ggacgggcct gt ggaat agt t t ct t cct t g 1620 cgct t t at gc ct 1111 caac ct cagcct gg t cat gagt ct ct t caagagg t cgacggagg 1680 agat cat cgc cct ct t cat t t ccat cacgt t t gt gct gga t gccgt caag ggcacggt t a 1740 aaat ct t ct g gaagt act ac t at gggcat t act t ggacga ct at cacaca aaaaggact t 1800 cat ccct t gt cagcct gt ca ggcct cggcg ccagcct caa cgccagcct c cacact gccc 1860 t caacgccag ct t cct cgcc agccccacgg agct gccct c ggccacacac t caggccagg 1920 cgaccgccgt gct cagcct c ct cat cat gc t gggcacgct ct ggct gggc t acaccct ct 1980 accaat t caa gaagagcccc t acct gcacc cct gcgt gcg agagat cct g t ccgact gcg 2040 ccct gcccat cgcggt gct c gcct t ct ccc t cat cagct c ccat ggct t c cgggaaat cg 2100 agat gagcaa gt t ccgct ac aaccccagcg agagcccct t t gcgat ggcg cagat ccagt 2160 cgct gt ccct gagggccgt c agcggt gcca t gggcct cgg ct t cct gct g t ccat gct ct 2220 t ct t cat cga gcagaact t g gt ggccgcct t ggt gaat gc accggagaac aggct ggt ga 2280 agggcact gc ct accact gg gacct cct gc t cct cgccat cat caacaca gggct gt ct c 2340 tgtttgggct gcct t ggat c cat gccgcct acccccact c cccgct gcac gt gcgagccc 2400 t ggcct t agt ggaggagcgt gt ggagaacg gacacat ct a t gacacgat t gt gaacgt ga 2460 aggagacgcg gct gacct cg ct gggcgcca gcgt cct ggt gggcct gt cc ct gt t gct gc 2520 t gccggt ccc gcttcagtgg at ccccaagc ccgt gct ct a t ggcct ct t c ct ct acat cg 2580 cgct cacct c cct cgat ggc aaccagct cg t ccagcgcgt ggccct gct g ct caaggagc 2640 agact gcgt a ccccccgaca cact acat cc ggagggt gcc ccagaggaag at ccact act 2700 t cacgggcct gcaggtgctt cagct gct gc t gct gt gt gc ct t cggcat g agct ccct gc 2760 cct acat gaa gat gat ct t t cccct cat ca t gat cgccat gat ccccat c cgct at at cc 2820 t gct gccccg aat cat t gaa gccaagt act t ggat gt cat ggacgct gag cacaggcct t 2880 gact ggcaga ccct gcccac gccccat t cg ccagccct cc acgt cct ccc aggct ggct c 2940 t ggagct gt g aggggaggt g t aggt gt gt g ggt gact gct ct gt gct gcg cct t ct cat g 3000 gct gact cag gcct ggggca t ct gggcat t gt aggggt gc Page 6 agt ggt at gt gcccacccct 3060
1- 2014- 041PCT_Sequence l i st i ng. TXT
ct cccat t at cct 11 agct t t aggccaaga gcgt t gct ca gggcagct t c t gcccagggt 3120 gggt gggact gagcaggat g gat t t t ct t t t gat aaaaga gt cgat gcct gaaagagaaa 3180 ccat t t cct t gat t gt gt aa ggaact t gct ggacgcacat t agagaat aa agct cct gt t 3240 t ct aggct cc t aaaaaaaaa aaaaaaaa 3268
<210> 4 <211> 2187 <212> DNA <213> Homo sapi ens <400> 4
t gccaggct c t ccaccccca ct t cccaat t gaggaaaccg aggcagagga ggct cagcgc 60 cacgcact cc t ct 11 ct gcc t ggccggcca ct cccgt ct g ct gt gacgcg cggacagaga 120 gct accggt g gacccacggt gcct ccct cc ct gggat ct a cacagaccat ggcct t gcca 180 acggct cgac ccct gt t ggg gt cct gt ggg acccccgccc t cggcagcct cct gt t cct g 240 ct ct t cagcc t cggat gggt gcagccct cg aggaccct gg ct ggagagac agggcaggag 300 gct gcgcccc t ggacggagt cct ggccaac ccacct aaca t t t ccagcct ct cccct cgc 360 caact cct t g gct t cccgt g t gcggaggt g t ccggcct ga gcacggagcg t gt ccgggag 420 ct ggct gt gg cct t ggcaca gaagaat gt c aagct ct caa cagagcagct gcgct gt ct g 480 gct caccggc t ct ct gagcc ccccgaggac ct ggacgccc t cccat t gga cct gct gct a 540 t t cct caacc cagat gcgt t ct cggggccc caggcct gca cccgt t t ct t ct cccgcat c 600 acgaaggcca at gt ggacct gct cccgagg ggggct cccg agcgacagcg gct gct gcct 660 gcggct ct gg cct gct gggg t gt gcggggg t ct ct gct ga gcgaggct ga t gt gcgggct 720 ct gggaggcc tggcttgcga cct gcct ggg cgct t t gt gg ccgagt cggc cgaagt gct g 780 ct accccggc t ggt gagct g cccgggaccc ct ggaccagg accagcagga ggcagccagg 840 gcggct ct gc agggcggggg acccccct ac ggccccccgt cgacat ggt c t gt ct ccacg 900 at ggacgct c t gcggggcct gct gcccgt g ct gggccagc ccat cat ccg cagcat cccg 960 cagggcat cg t ggccgcgt g gcggcaacgc t cct ct cggg acccat cct g gcggcagcct 1020 gaacggacca t cct ccggcc gcggtt ccgg cgggaagt gg agaagacagc ct gt cct t ca 1080 ggcaagaagg cccgcgagat agacgagagc ct cat ct t ct acaagaagt g ggagct ggaa 1140 gcct gcgt gg at gcggccct gct ggccacc cagat ggacc gcgt gaacgc cat cccct t c 1200 acct acgagc agct ggacgt cct aaagcat aaact ggat g agct ct accc acaaggt t ac 1260 cccgagt ct g t gat ccagca cct gggct ac ct ct t cct ca agat gagccc t gaggacat t 1320 cgcaagt gga at gt gacgt c cct ggagacc ct gaaggct t t gct t gaagt caacaaaggg 1380 cacgaaat ga gt cct caggt ggccaccct g at cgaccgct ttgtgaaggg aaggggccag 1440 ct agacaaag acaccct aga caccct gacc gcct t ct acc ct gggt acct gt gct ccct c 1500 agccccgagg agct gagct c cgt gcccccc agcagcat ct gggcggt cag gccccaggac 1560
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ct ggacacgt 1-2014-041PCT Sequence listing.TXT gt gacccaag gcagct ggac gt cct ct at c ccaaggcccg cct t gct 11 c 1620 cagaacat ga acgggt ccga at act t cgt g aagat ccagt cct t cct ggg t ggggccccc 1680 acggaggat t t gaaggcgct cagt cagcag aat gt gagca tggacttggc cacgt t cat g 1740 aagct gcgga cggat gcggt gctgccgttg act gt ggct g aggt gcagaa act t ct ggga 1800 ccccacgt gg agggcct gaa ggcggaggag cggcaccgcc cggt gcggga ct ggat cct a 1860 cggcagcggc aggacgacct ggacacgct g gggct ggggc t acagggcgg cat ccccaac 1920 ggct acct gg t cct agacct cagcat gcaa gaggccct ct cggggacgcc ct gcct cct a 1980 ggacct ggac ct gt t ct cac cgt cct ggca ct gct cct ag cct ccaccct ggcct gaggg 2040 ccccact ccc 11 gct ggccc cagccct gct ggggat cccc gcct ggccag gagcaggcac 2100 gggt ggt ccc cgt t ccaccc caagagaact cgcgct cagt aaacgggaac at gccccct g 2160 cagacacgt a aaaaaaaaaa aaaaaaa 2187 <210> 5 <211> 3452 <212> DNA <213> Homo sapi ens <400> 5 at gcaggcgc gct act ccgt gt ccagcccc aact ccct gg gagt ggt gcc ct acct cggc 60 ggcgagcaga gct act accg cgcggcggcc gcggcggccg ggggcggct a caccgccat g 120 ccggccccca t gagcgt gt a ct cgcaccct gcgcacgccg agcagt accc gggcggcat g 180 gcccgcgcct acgggccct a cacgccgcag ccgcagccca aggacat ggt gaagccgccc 240 t at agct aca t cgcgct cat caccat ggcc at ccagaacg ccccggacaa gaagat cacc 300 ct gaacggca t ct accagt t cat cat ggac cgct t cccct t ct accggga caacaagcag 360 ggct ggcaga acagcat ccg ccacaacct c t cgct caacg agt gct t cgt caaggt gccg 420 cgcgacgaca agaagccggg caagggcagc t act ggacgc t ggacccgga ct cct acaac 480 at gt t cgaga acggcagct t cct gcggcgg cggcggcgct t caagaagaa ggacgcggt g 540 aaggacaagg aggagaagga caggct gcac ct caaggagc cgcccccgcc cggccgccag 600 cccccgcccg cgccgccgga gcaggccgac ggcaacgcgc ccggt ccgca gccgccgccc 660 gt gcgcat cc aggacat caa gaccgagaac ggt acgt gcc cct cgccgcc ccagcccct g 720 t ccccggccg ccgccct ggg cagcggcagc gccgccgcgg t gcccaagat cgagagcccc 780 gacagcagca gcagcagcct gt ccagcggg agcagccccc cgggcagcct gccgt cggcg 840 cggccgct ca gcct ggacgg t gcggat t cc gcgccgccgc cgcccgcgcc ct ccgccccg 900 ccgccgcacc at agccaggg ct t cagcgt g gacaacat ca t gacgt cgct gcgggggt cg 960 ccgcagagcg cggccgcgga gct cagct cc ggcct t ct gg cct cggcggc cgcgt cct cg 1020 cgcgcgggga t cgcaccccc gct ggcgct c ggcgcct act cgcccggcca gagct ccct c 1080 t acagct ccc cct gcagcca gacct ccagc gcgggcagct cgggcggcgg cggcggcggc 1140 gcgggggccg cggggggcgc gggcggcgcc gggacct acc act gcaacct gcaagccat g 1200
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1- 2014- 041PCT_Sequence l i st i ng. TXT
agcct gt acg cggccggcga gcgcgggggc cact t gcagg gcgcgcccgg gggcgcgggc 1260 ggct cggccg t ggacgaccc cct gcccgac t act ct ct gc ct ccggt cac cagcagcagc 1320 t cgt cgt ccc t gagt cacgg cggcggcggc ggcggcggcg ggggaggcca ggaggccggc 1380 caccaccct g cggcccacca aggccgcct c acct cgt ggt acct gaacca ggcgggcgga 1440 gacct gggcc acttggcgag cgcggcggcg gcggcggcgg ccgcaggct a cccgggccag 1500 cagcagaact t ccact cggt gcgggagat g t t cgagt cac agaggat cgg ct t gaacaac 1560 t ct ccagt ga acgggaat ag t agct gt caa at ggcct t cc ct t ccagcca gt ct ct gt ac 1620 cgcacgt ccg gagct 11 cgt ct acgact gt agcaagt t t t gacacaccct caaagccgaa 1680 ct aaat cgaa ccccaaagca ggaaaagct a aaggaaccca t caaggcaaa at cgaaact a 1740 aaaaaaaaaa at ccaat t aa aaaaaacccc t gagaat at t caccacacca gcgaacagaa 1800 t at ccct cca aaaat t cagc t caccagcac cagcacgaag aaaact ct at 111 ct t aacc 1860 gat t aat t ca gagccacct c cact t t gcct t gt ct aaat a aacaaacccg t aaact gt 11 1920 t at acagaga cagcaaaat c t t ggt t t at t aaaggacagt gt t act ccag at aacacgt a 1980 agt t t ct t ct t gct 111 cag agacct gct t t cccct cct c ccgt ct cccc t ct ct t gcct 2040 t ct t cct t gc ct ct cacct g t aagat at t a t t t t at cct a t gt t gaaggg agggggaaag 2100 t ccccgt t t a t gaaagt cgc 111 ct 1111 a t t cat ggact t gt t t t aaaa t gt aaat t gc 2160 aacat agt aa 111 at 1111 a at t t gt agt t ggat gt cgt g gaccaaacgc cagaaagt gt 2220 t cccaaaacc t gacgt t aaa 11 gcct gaaa ct t t aaat t g t gct t t t t t t ct cat t at aa 2280 aaagggaaac t gt at t aat c 11 at t ct at c ct ct t t t ct t t ct t t t t gt t gaacat at t c 2340 at t gt t t gt t t at t aat aaa 11 accat t ca gt t t gaat ga gacct at at g t ct ggat act 2400 t t aat agagc 111 aat t at t acgaaaaaag at t t cagaga t aaaacact a gaagt t acct 2460 at t ct ccacc t aaat ct ct g aaaaat ggag aaaccct ct g act agt ccat gt caaat 111 2520 act aaaagt c tttttgttta gat t t at t t t cct gcagcat ct t ct gcaaa at gt act at a 2580 t agt cagct t gctttgaggc t agt aaaaag at at t t t t ct aaacagat t g gagttggcat 2640 at aaacaaat acgt 111 ct c act aat gaca gt ccat gat t cggaaat t t t aagcccat ga 2700 at cagccgcg gt ct t accac ggt gat gcct gt gt gccgag agat gggact gt gcggccag 2760 at at gcacag at aaat at t t ggct t gt gt a t t ccat at aa aat t gcagt g cat at t at ac 2820 at ccct gt ga gccagat gct gaat agat at t t t cct at t a t t t cagt cct t t at aaaagg 2880 aaaaat aaac cagt 1111 aa at gt at gt at at aat t ct cc cccat t t aca at cct t cat g 2940 t at t acat ag aaggat t gct 11111 aaaaa t at act gcgg gt t ggaaagg gat at t t aat 3000 ct t t gagaaa ct at t t t aga aaat at gt t t gt agaacaat t at t t t t gaa aaagat t t aa 3060 agcaat aaca agaaggaagg cgagaggagc agaacat t t t ggt ct agggt ggt t t ct t t t 3120 t aaaccat t t 111 ct t gt t a at t t acagt t aaacct aggg gacaat ccgg at t ggccct c 3180 cccct t t t gt aaat aaccca ggaaat gt aa t aaat t cat t Page 9 at ct t agggt gat ct gccct 3240
1- 2014- 041PCT_Sequence l i st i ng. TXT
gccaat caga ct t t ggggag at ggcgat t t gat t acagac gt t cgggggg gtggggggct 3300 t gcagt t t gt t t t ggagat a at acagt t t c ct gct at ct g ccgct cct at ct agaggcaa 3360 cact t aagca gt aat t gct g t t gct t gt t g t caaaat t t g at cat t gt t a aaggat t gct 3420 gcaaat aaat acact 11 aat t t cagt caaa aa 3452
<210> 6 <211> 5553
<212> DNA <213> Homo sapi ens <400> 6 gt gt gaat gc 11 cat t cgcc t cacaaacaa ccacagaacc acaagt gcgg t gcaaact 11 60 ct ccaggagg acagcaagaa gt ct ct ggt t t t t aaat ggt t aat ct ccgc aggt cact ac 120 cagccaccga gaccaacaga gt cat t t aag gct gcaagca gt at t t acaa cagagggt ac 180 aagt t ct at c t gaaaaaaaa aggagggact at ggcat caa acagcct ct t cagcacagt g 240 acaccat gt c agcaaaact t ct t t t gggat ccgagcacca gccggcgct t cagccccccc 300 t ccagcagcc t gcagcccgg caaaat gagc gacgt gagcc cggt ggt ggc t gcgcaacag 360 cagcagcaac agcagcagca gcaacagcag cagcagcagc agcaacagca gcagcagcag 420 caggaggcgg cggcggcggc t gcggcggcg gcggcggct g cggcggcggc agct gcagt g 480 ccccggt t gc ggccgcccca cgacaaccgc accat ggt gg agat cat cgc cgaccacccg 540 gccgaact cg t ccgcaccga cagccccaac t t cct gt gct cggt gct gcc ct cgcact gg 600 cgct gcaaca agaccct gcc cgt ggcct t c aaggt ggt ag ccct cggaga ggt accagat 660 gggact gt gg 11 act gt cat ggcgggt aac gat gaaaat t at t ct gct ga gct ccggaat 720 gcct ct gct g 11 at gaaaaa ccaagt agca aggt t caacg at ct gagat t t gt gggccgg 780 agt ggacgag gcaagagt 11 cacct t gacc at aaccgt ct t cacaaat cc t ccccaagt a 840 gct acct at c acagagcaat t aaagt t aca gt agat ggac ct cgggaacc cagaaggcac 900 agacagaagc 11 gat gact c t aaacct agt t t gt t ct ct g accgcct cag tgatttaggg 960 cgcat t cct c at cccagt at gagagt aggt gt cccgcct c agaacccacg gccct ccct g 1020 aact ct gcac caagt cct 11 t aat ccacaa ggacagagt c agat t acaga ccccaggcag 1080 gcacagt ct t ccccgccgt g gt cct at gac cagt ct t acc cct cct acct gagccagat g 1140 acgt ccccgt ccat ccact c t accaccccg ct gt ct t cca cacggggcac t gggct t cct 1200 gccat caccg at gt gcct ag gcgcat 11 ca gat gat gaca ct gccacct c t gact t ct gc 1260 ct ct ggcct t ccact ct cag t aagaagagc caggcaggt g ct t cagaact gggccct t t t 1320 t cagacccca ggcagt t ccc aagcat t t ca t ccct cact g agagccgct t ct ccaaccca 1380 cgaat gcact at ccagccac ct t t act t ac accccgccag t cacct cagg cat gt ccct c 1440 ggt at gt ccg ccaccact ca ct accacacc t acct gccac caccct accc cggct ct t cc 1500 caaagccaga gt ggaccct t ccagaccagc agcact ccat at ct ct act a t ggcact t cg 1560
Page 10
1- 2014- 041PCT_Sequence l i st i ng. TXT
t caggat cct at cagt 11 cc cat ggt gccg gggggagacc ggt ct cct t c cagaat gct t 1620 ccgccat gca ccaccacct c gaat ggcagc acgct at t aa at ccaaat t t gcct aaccag 1680 aat gat ggt g 11 gacgct ga t ggaagccac agcagt t ccc caact gt t t t gaat t ct agt 1740 ggcagaat gg at gaat ct gt t t ggcgacca t at t gaaat t cct cagcagt ggcccagt gg 1800 t at ct ggggg ccacat ccca cacgt at caa t at at acat a t at agagaga gt gcat at at 1860 at gt at at cg at t agct at c t acaaagt gc ct at t t t t t a gaagat t t t t cat t cact ca 1920 ct cagt cat g at ct t gcagc cat aagaggg t agat at t ga gaagcagaag gct caagaga 1980 gacaat t gca at cgagct t c agat t gt t t a ct at t t aaga t gt act t t t a caaaggaaca 2040 aagaagggaa aaggt at t t t tgtttttgtt gt t t ggt ct g t t at cat caa t aacct gt t c 2100 at at gccaat t cagagaggt ggact ccagg t t caggaggg agaagagcaa agccgct t cc 2160 t ct ct gt gct 11 gaaact t c acaccct cac ggt ggcagct gt gt at ggac cagt gccct c 2220 cgcagacagc t cacaaaacc agt t gaggt g cact aaaggg acat gaggt a gaat ggat gc 2280 t t ccat caca gt accat cat t cagaat aac t ct t ccaat t t ct gct t t ca gacat gct gc 2340 aggt cct cat ct gaact gt t gggt t cgt t t tttttttttt t t t t cct gct ccaagaaagt 2400 gact t caaaa at aact gat c aggat agat t at t t t at t t t act t t t t aac act cct t ct c 2460 ccct t t t ccc act gaaccaa aaagaaat cc cat ccct aaa acct gcct t c t cct 111 at g 2520 caaaact gaa aat ggcaat a cat t at t at a gccat aat gg t at agat agt gat t gcgt t t 2580 ggct at gt gt t gt 111 ct 11 t t t t t t aaat t at gaat at g t gt aaaat ct gaggt aact t 2640 gct aacgt ga at ggt cat at aact t t aaag at at at t t at aat t at t t aa t gacat 11 gg 2700 accct t gaaa cat t t ct t ag t gt at t gat a t gt t gact t c ggt ct ct aaa agt gct ct 11 2760 at t aaat aac aaat t t ct t c agt ggt ct ag agccat at ct gaaat at t gc t aagcaat 11 2820 cagt t cat cc aggcacaat g t gat t t t aaa aaat act t cc at ct ccaaat at t t t agat a 2880 t agat t gt t t 11 gt gat gt a t gaaggaaat gt t at gt t t a gt t ct t t cag at ct t t gaat 2940 gcct ct aaca cagct 11 gcc t t ct aaagcg gt aat t aggg at t t aaaaaa caacct t t ag 3000 ccct t t at ca gcat gaaat g ct ggagt gat gt ggt t t t ct aat t t ct t t g gggt aat t at 3060 gact ct t gt c at at t aaaaa gacaagcaca agt aaat cat t gaact acag aaaaat gt t c 3120 tgtggtttca t agt t aagca aaact ct aaa t cgccaggct t cat agcaaa gacat agt ca 3180 gct aaaagcc gcacat gt gg at agagggt t caat t at gag acacct agt a caggagagca 3240 aaat t gcacc agagat t ct t aaccaaccag cct t accaaa caacacaaca ggggaacccc 3300 aat ct gcct t acccaaggcc ccact ggcag ct t t ccacag aat t t gcat t t agaggagca 3360 gaat gacat c act gt cct t t gggagt aggt cct ct gaaaa ggcagcaggt t ccagcaggt 3420 agct gagct g agaggacat a t ggcccacgg ggacct acag acagcct t t g acat t t gt at 3480 t t ct t acaat ggagggccaa ggagggcaag gggct gt gga gt t t ggt gt c t act agt gt g 3540 t at gaat t t g agct agagt c ct t ct gt ggc at gcact t t g accact cct g gcagt cacat 3600
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1- 2014- 041PCT_Sequence l i st i ng. TXT
ggcagat t t c caagt gcaaa t cct t aat cc aaacaaggat cat ct aat ga caccaccagg 3660 ccaat ccct g ct ct cct ccc cgaaaagt ca gggt ccct t c at t ggaat cc t ccacccacc 3720 caagcagaat 11 agcagaga 111 gcct t ca aaccct aacg gccccct t gt t ct ct ggt cc 3780 t t ct caaacc cacct 11 gt a ggccacccag cat t gcagga cagcgt gt gg ggcagct gga 3840 cct gt gct t c ct gcct ggga gt ct ccct t g gaat t cat cc t gact cct t c t aat aaaaat 3900 ggat gggaaa gcaaaacact 11 gcct t ct a aaggccgt at accaagt at g ct t agat aaa 3960 t aagccact t t t ct at t act t aagt aagaa ggaagt agt a at t gat act a tttattgttt 4020 gt gt gt ggt a gct t gaagca caccact gt c cat t t at t t g t aagt gt aaa at at gt gt gt 4080 t t gt t t cagc agcact t aaa aaagccagt g t ct ggt t aca cat t t caat t t t aat t aat t 4140 gacat aaaaa t gct accgcc agt gccagct gcat cct at t t aat t aaaaa ggt act at at 4200 t t gt acat t a t t t t t t aat g 11 aaaagggc t t t t t t aagt t t acagt aca cat accgagt 4260 gact t t aggg at gct 111 gt gt t gaaat gt t act at agt g gct gcaggca gcaacccaga 4320 aacact t t ag aagct 11111 t cct t gggaa aaat t caagc act t ct t ccc t ccaccct ca 4380 ct ccaaccac cccaat gggg gt aat t caca t t t ct t agaa caaat t ct gc cct t t t t t gg 4440 t ct agggat t aaaat t t t gt 1111 ct 11 ct t t ct t t t t t t t t t t t t t t ca ct gaaccct t 4500 aat t t gcact gggt cat gt g 111 gat 11 gt gat t t caaga ccaaagcaaa gt ct t act ac 4560 t act gt ggaa ccat gt act a gt t cct ggga at t aaaat ag cgt ggt t ct c 111 gt agcac 4620 aaacat t gct ggaat t t gca gt ct t t t caa t gcagccaca t t t t t at cca 111 cagt t gt 4680 ct cacaaat t 11 aacccat a t cagagt t cc agaacaggt a ccacagct t t ggt t t t agat 4740 t agt ggaat a acat t cagcc cagaact gag aaact caaca gat t aact at cgt 11 gct ct 4800 t t agacggt c t cact gcct c t cact t gcca gagccct t t c aaaat gagca gagaagt cca 4860 caccat t agg gaccat ct gt gat aaat t ca gaagggagga gat gt gt gt a cagct t t aag 4920 gat t ccct ca at t ccgagga aagggact gg cccagaat cc aggt t aat ac at ggaaacac 4980 gaagcat t ag caaaagt aat aat t at acct at ggt at t t g aaagaacaat aat aaaagac 5040 act t ct t cca aacct t gaat ttgttgtttt t agaaaacga at gcat t t aa aaat at t t t c 5100 t at gt gagaa 111111 agat gt gt gt t t ac t t cat gt t t a caaat aact g tttgcttttt 5160 aat gcagt ac 111 gaaat at at cagccaaa accat aact t acaat aat t t ct t aggt at t 5220 ct gaat aaaa 11 ccat 11 ct t t t ggat at g ct t t accat t ct t aggt t t c t gt ggaacaa 5280 aaat at t t gt agcat 111 gt gt aaat acaa gct t t cat t t t t at t t t t t c caat t gct at 5340 t gcccaagaa 11 gct 11 cca t gcacat at t gt aaaaat t c cgct t t gt gc cacaggt cat 5400 gat t gt ggat gagt t t act c t t aact t caa agggact at t t gt at t gt at gt t gcaact g 5460 t aaat t gaat t at 11 ggcat t t t t ct cat g at t gt aat at t aat t t gaag 111 gaat 11 a 5520 at t t t caat a aaat ggct 11 t t t ggt t t t g tta 5553
<210> 7
Page 12
1- 2014- 041PCT_Sequence l i st i ng. TXT <211> 1927 <212> DNA <213> Homo sapi ens <400> 7
t aacccgccg cct ccgct ct ccccggct gc aggcggcgt g caggaccagc ggcggccgt g 60 caggcggagg act t cggcgc ggct cct cct gggt gt gacc ccgggcgcgc ccgccgcgcg 120 acgat gaggg cgcggccgca ggt ct gcgag gcgct gct ct t cgccct ggc gct ccagacc 180 ggcgt gt gct at ggcat caa gt ggct ggcg ct gt ccaaga caccat cggc cct ggcact g 240 aaccagacgc aacact gcaa gcagct ggag ggt ct ggt gt ct gcacaggt gcagct gt gc 300 cgcagcaacc t ggagct cat gcacacggt g gt gcacgccg cccgcgaggt cat gaaggcc 360 t gt cgccggg cct 11 gccga cat gcgct gg aact gct cct ccat t gagct cgcccccaac 420 t at t t gct t g acct ggagag agggacccgg gagt cggcct t cgt gt at gc gct gt cggcc 480 gccgccat ca gccacgccat cgcccgggcc t gcacct ccg gcgacct gcc cggct gct cc 540 t gcggccccg t cccaggt ga gccacccggg cccgggaacc gct ggggagg at gt gcggac 600 aacct cagct acgggct cct cat gggggcc aagt t t t ccg at gct cct at gaaggt gaaa 660 aaaacaggat cccaagccaa t aaact gat g cgt ct acaca acagt gaagt ggggagacag 720 gct ct gcgcg cct ct ct gga aat gaagt gt aagt gccat g gggt gt ct gg ct cct gct cc 780 at ccgcacct gct ggaaggg gct gcaggag ct gcaggat g t ggct gct ga cct caagacc 840 cgat acct gt cggccaccaa ggt agt gcac cgacccat gg gcacccgcaa gcacct ggt g 900 cccaaggacc t ggat at ccg gcct gt gaag gact cggaac t cgt ct at ct gcagagct ca 960 cct gact t ct gcat gaagaa t gagaaggt g ggct cccacg ggacacaaga caggcagt gc 1020 aacaagacat ccaacggaag cgacagct gc gacct t at gt gct gcgggcg t ggct acaac 1080 ccct acacag accgcgt ggt cgagcggt gc cact gt aagt accact ggt g ct gct acgt c 1140 acct gccgca ggt gt gagcg t accgt ggag cgct at gt ct gcaagt gagg ccct gccct c 1200 cgccccacgc aggagcgagg act ct gct ca aggaccct ca gcaact gggg ccaggggcct 1260 ggagacact c cat ggagct c t gct t gt gaa 11 ccagat gc caggcat ggg aggcggcttg 1320 t gct t t gcct t cact t ggaa gccaccagga acagaaggt c t ggccaccct ggaaggaggg 1380 caggacat ca aaggaaaccg acaagat t aa aaat aact t g gcagcct gag gct ct ggagt 1440 gcccacaggc t ggt gt aagg agcggggct t gggat cggt g agact gat ac agact t gacc 1500 t t t cagggcc acagagacca gcct ccggga aggggt ct gc ccgcct t ct t cagaat gt t c 1560 t gcgggaccc cct ggcccac cct ggggt ct gagcct gct g ggcccaccac at ggaat cac 1620 t agct t gggt t gt aaat gt t 11 ct 111 gt t ttttgctttt t ct t cct t t g ggat gt ggaa 1680 gct acagaaa t at 11 at aaa acat agct 11 t t ct t t gggg t ggcact t ct caat t cct ct 1740 t t at at at t t t at at at at a aat at at at g t at at at at a at gat ct ct a 1111 aaaact 1800 agct t t t t aa gcagct gt at gaaat aaat g ct gagt gagc cccagcccgc ccct gcagt t 1860 cccggcct cg t caagt gaac t cggcagacc ct ggggct gg cagagggagc t ct ccagt 11 1920
Page 13
1- 2014- 041PCT_Sequence l i st i ng. TXT ccaggca 1927 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220>
<223> A probe i ncl uded in the pr obe set I D206884. s at <400> 8 aat cacccaa ggat ggat at cagga 25 <210> 9 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206884. s at <400> 9 at t caact t c t gat aggt ct gt cat 25 <210> 10 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206884. s at <400> 10 atgtgcactt act gccgaaa accct 25 <210> 11 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206884. s at <400> 11 t act gccgaa aacccttggg t gt ag 25 <210> 12 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206884. s at <400> 12 gatgaattac aaatttgctg ccat t 25 <210> 13 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e
Page 14
1- 2014- 041PCT_Sequence l i st i ng. TXT <220>
<223> <400> t t t gct A probe i ncl uded in the 13 gcca t t ct act t gc t t t aa pr obe set I D206884 s at 25 <210> 14 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206884 s at <400> 14 gt gaaat at g caagcagcct ttgga 25 <210> 15 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206884 s at <400> 15 agacaat aca ct gt gaacct t gct a 25 <210> 16 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206884 s at <400> 16 gtggattcca t aact ct ggc acaag 25 <210> 17 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206884 s at <400> 17 gctttcacat t gaagat caa ct ct t 25 <210> 18 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206884 s at <400> 18 at ct ggt ct c t ggct agagt t agca 25
Page 15
1- 2014- 041PCT_Sequence l i st i ng. TXT <210> 19 <211> 25 <212> DNA <213> Ar t i f i ci al Sequence <220>
<223> A probe i ncl uded in the pr obe set I D209764_ at <400> 19 gcaggagtga aggcct ggcc at cgg 25 <210> 20 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D209764_ at <400> 20 gcctggccat cggtggagag ggcag 25 <210> 21 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D209764_ at <400> 21 agagtttcac at at cat cac agcat 25 <210> 22 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D209764_ at <400> 22 cagggccact ccct caat at gaagg 25 <210> 23 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D209764_ at <400> 23 aaccaagct g aat gt gacca ccggc 25 <210> 24 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D209764_ at Page 16
1- 2014- 041PCT_Sequence l i st i ng. TXT <400> 24 t ccccgggaa t aact ggccc t gaga 25 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220>
<223> <400> aagcat A probe i ncl uded in the 25 ccgg gaagcatggc tggcc pr obe set I D209764_ at 25 <210> 26 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D209764_ at <400> 26 tcggttccca ggggcagcat gggag 25 <210> 27 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D209764_ at <400> 27 gcatgggaga tctttggggg caaca 25 <210> 28 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D209764_ at <400> 28 ggggagacgg gacttgtcca agcag 25 <210> 29 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D209764_ at <400> 29 ggaccct ggg aaat gcat aa t gt aa 25 <210> 30 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e
Page 17
1- 2014- 041PCT_Sequence l i st i ng. TXT <220>
<223> <400> gaggt g A probe i ncl uded in the 30 t agg t gt gt gggt g act gc pr obe set I D223748_ at 25 <210> 31 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D223748_ at <400> 31 tgtaggggtg cagt ggt at g t gccc 25 <210> 32 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D223748_ at <400> 32 cccat t at cc tttagcttta ggcca 25 <210> 33 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D223748_ at <400> 33 gctttaggcc aagagcgttg ct cag 25 <210> 34 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D223748_ at <400> 34 caagagcgtt gct cagggca gcttc 25 <210> 35 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D223748_ at <400> 35 gggact gagc aggat ggat t t t ct t 25
Page 18
1- 2014- 041PCT_Sequence l i st i ng. TXT <210> 36 <211> 25 <212> DNA <213> Ar t i f i ci al Sequence <220>
<223> A probe i ncl uded in the pr obe set I D223748_ at <400> 36 t aaaagagt c gatgcctgaa agaga 25 <210> 37 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D223748_ at <400> 37 gt gt aaggaa cttgctggac gcaca 25 <210> 38 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D223748_ at <400> 38 ggaacttgct ggacgcacat t aaag 25 <210> 39 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D223748_ at <400> 39 agaat aaagc t cct gt t t ct aggct 25 <210> 40 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D223748_ at <400> 40 gctcctgttt ct aggct cct aaaaa 25 <210> 41 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D204885_ s_at Page 19
1- 2014- 041PCT_Sequence l i st i ng. TXT <400> 41 tttccagaac atgaacgggt ccgaa 25 <210> 42 <211> 25 <212> DNA <213> Ar t i f i ci al Sequence <220>
<223> A probe i ncl uded in the pr obe set I D204885 s at <400> 42 gaacgggt cc gaat act t cg t gaag 25 <210> 43 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D204885 s at <400> 43 cttcgtgaag at ccagt cct t cct g 25 <210> 44 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D204885 s at <400> 44 ggcccccacg gaggat t t ga aggcg 25 <210> 45 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D204885 s at <400> 45 ggatttgaag gcgct cagt c agcag 25 <210> 46 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D204885 s at <400> 46 ggcgct cagt cagcagaat g t gagc 25 <210> 47 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e
Page 20
1- 2014- 041PCT_Sequence l i st i ng. TXT <220>
<223> <400> agcaga A probe i ncl uded in the 47 at gt gagcatggac ttggc pr obe set I D204885_s_at 25 <210> 48 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D204885_s_at <400> 48 gccacgttca tgaagctgcg gacgg 25 <210> 49 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D204885_s_at <400> 49 gttcatgaag ct gcggacgg at gcg 25 <210> 50 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D204885_s_at <400> 50 t gaagct gcg gacggatgcg gt gct 25 <210> 51 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D204885_s_at <400> 51 tgctgccgtt gactgtggct gaggt 25 <210> 52 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D1553613_s_at <400> 52 aacct gcaag ccat gagcct gt acg 25
Page 21
1- 2014- 041PCT_Sequence l i st i ng. TXT <210> 53 <211> 25 <212> DNA <213> Ar t i f i ci al Sequence <220>
<223> <400> acct cg A probe i ncl uded in the 53 t ggt acct gaacca ggcgg pr obe set I D1553613_s at 25 <210> 54 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D1553613 s at <400> 54 agaacttcca ct cggt gcgg gagat 25 <210> 55 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D1553613 s at <400> 55 tgttcgagtc acagaggat c ggctt 25 <210> 56 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D1553613 s at <400> 56 ggatcggctt gaacaact ct ccagt 25 <210> 57 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D1553613 s at <400> 57 tgtaccgcac gtccggagct ttcgt 25 <210> 58 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D1553613 s at Page 22
1- 2014- 041PCT_Sequence l i st i ng. TXT <400> 58 ggagctttcg t ct acgact g t agca 25 <210> 59 <211> 25 <212> DNA <213> Ar t i f i ci al Sequence <220>
<223> A probe i ncl uded in the probe set <400> 59 tgtagcaagt tttgacacac cctca I D1553613_s_at 25 <210> 60 <211> 25 <212> DNA <213> Ar t i f i ci al Sequence <220> <223> A probe i ncl uded in the probe set I D1553613_s_at <400> 60 acacaccct c aaagccgaac t aaat <210> 61 <211> 25 <212> DNA <213> Ar t i f i ci al Sequence <220> <223> A probe i ncl uded in the probe set I D1553613_s_at 25 <400> 61 gaact aaat c gaaccccaaa gcagg <210> 62 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> A probe i ncl uded in the probe set I D1553613_s_at 25 <400> 62 ggaacccat c aaggcaaaat cgaaa <210> 63 <211> 25 <212> DNA <213> Ar t i f i ci al Sequence <220> <223> A probe i ncl uded in the probe set I D232231_at 25 <400> 63 aagacacttc 11 ccaaacct t gaat <210> 64 <211> 25 <212> DNA <213> Ar t i f i ci al Sequence 25
Page 23
1- 2014- 041PCT_Sequence l i st i ng. TXT <220>
<223> <400> gat gt g A probe i ncl uded in the 64 t gt t t act t cat gt t t aca pr obe set I D232231 _at 25 <210> 65 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D232231 _at <400> 65 at cagccaaa accataactt acaat 25 <210> 66 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D232231 _at <400> 66 ttggatatgc 111 accat t c 11 agg 25 <210> 67 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D232231 _at <400> 67 accat t ct t a ggt t t ct gt g gaaca 25 <210> 68 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D232231 _at <400> 68 11111 ccaat t gct at t gcc caaga 25 <210> 69 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D232231 _at <400> 69 gctattgccc aagaattgct 11 cca 25
Page 24
1- 2014- 041PCT_Sequence l i st i ng. TXT <210> 70 <211> 25 <212> DNA <213> Ar t i f i ci al Sequence <220>
<223> A probe i ncl uded in the pr obe set I D232231 at <400> 70 gaattgcttt ccat gcacat at t gt 25 <210> 71 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D232231 at <400> 71 11 gt aaaaat tccgctttgt gccac 25 <210> 72 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D232231 at <400> 72 gctttgtgcc acaggt cat g at t gt 25 <210> 73 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D232231 at <400> 73 agggact at t t gt at t gt at gt t gc 25 <210> 74 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206737 at <400> 74 gccttcactt ggaagccacc aggaa 25 <210> 75 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206737 at Page 25
1- 2014- 041PCT Sequence l i st i ng. TXT <400> 75 gggcttggga t cggt gagac t gat a 25 <210> 76 <211> 25 <212> DNA <213> Art i f i ci al Sequence <220> <223> A probe i ncl uded in the pr obe set I D206737_at <400> 76 gagact gat a cagacttgac ct 11 c 25 <210> 77 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206737_at <400> 77 cttgaccttt cagggccaca gagac 25 <210> 78 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206737_at <400> 78 gagaccagcc t ccgggaagg ggt ct 25 <210> 79 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206737_at <400> 79 cccgccttct tcagaatgtt ct gcg 25 <210> 80 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206737_at <400> 80 cttcagaatg 11 ct gcggga ccccc 25 <210> 81 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e
Page 26
1- 2014- 041PCT_Sequence l i st i ng. TXT <220>
<223> A probe i ncl uded in the pr obe set I D206737_ at <400> 81 gggccaccac at ggaat cac t agct 25 <210> 82 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206737_ at <400> 82 at cact agct t cgggt t gt a aat gt 25 <210> 83 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206737_ at <400> 83 ct 11 ggggt g gcacttctca at t cc 25 <210> 84 <211> 25 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> A probe i ncl uded in the pr obe set I D206737_ at <400> 84 ccggcct cgt caagt gaact cggca 25
Page 27
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