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Patent application title: METHODS AND COMPOSITIONS FOR EVALUATING CHRONIC IMMUNE DISEASES
Marc Fremont (Zellik, BE)
Kenny Leo De Meirleir (Mechelen, BE)
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid
Publication date: 2010-09-02
Patent application number: 20100221732
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Patent application title: METHODS AND COMPOSITIONS FOR EVALUATING CHRONIC IMMUNE DISEASES
Marc Fremont
Kenny Leo De Meirleir
Origin: EAST PALO ALTO, CA US
IPC8 Class: AC12Q168FI
USPC Class:
Publication date: 09/02/2010
Patent application number: 20100221732
Methods and compositions are provided for evaluating a subject for chronic
immune disease, including predicting whether a subject is susceptible to
a chronic immune disease, diagnosing whether a subject has a chronic
immune disease and/or determining a treatment for a subject suffering
from a chronic immune disease. Aspects of the methods include genotyping
a subject to determine whether the subject has a least one polymorphism
in at least one gene chosen from TLR4, Hsp60, IL-23R, IL-6 and IL-17F. In
addition, reagents, devices and kits thereof that find use in practicing
the subject methods are provided.
1. A method of evaluating a subject for Chronic Fatigue Syndrome (CFS),
said method comprising:(a) genotyping said subject for at least a
polymorphism in at least one gene selected from the group consisting of
TLR4, Hsp60, IL-23R, IL-6 and IL-17F to obtain a result; and(b) employing
said result to provide an evaluation of a subject for CFS,
2. The method according to claim 1, wherein said TLR4 polymorphism is selected from rs4986790 and rs12344353 and combinations thereof.
3. The method according to claim 2, wherein identification of a G allele of rs4986790 or a C allele of rs12344353 is predictive of susceptibility to CFS or diagnostic of having CFS.
4. The method according to claim 1, wherein said Hsp60 polymorphism is rs2340690.
5. The method according to claim 4, wherein identification of two T alleles of rs2340690 is predictive of susceptibility to CFS or diagnostic of having CFS.
6. The method according to claim 1, wherein said IL-23R polymorphism is rs11209026.
7. The method according to claim 6, wherein identification of an A allele of rs11209026 is predictive of susceptibility to CFS or diagnostic of having CFS.
8. The method according to claim 1, wherein said IL-6 polymorphism is rs1800795.
9. The method according to claim 8, wherein identification of two G alleles of rs1800795 is predictive of susceptibility to CFS or diagnostic of having CFS.
10. The method according to claim 1, wherein said polymorphisms are rs2340890 in Hsp60 and rs1800795 in IL-6.
11. The method according to claim 10, wherein identification of two T alleles of rs2340890 and two G alleles of rs1800795 is predictive of susceptibility to CFS or diagnostic of having CFS.
12. The method according to claim 1, wherein said IL-17F polymorphism is rs763780.
13. The method according to claim 12, wherein identification of a C allele of rs763780 is predictive of resistance to CFS or diagnostic of not having CFS.
14. The method according to claim 1, wherein said evaluation is a prediction of whether a subject is susceptible to Chronic Fatigue Syndrome.
15. The method according to claim 1, wherein said evaluation is a diagnosis of CFS in a a subject suspected of having CFS.
16. The method according to claim 1, wherein said evaluation is a determination of a treatment for a subject with CFS.
17. An assay device for use in determining the CFS susceptibility phenotype of a source of a nucleic acid sample, said kit comprising:an element for genotyping said sample to identify a polymorphism in at least one gene selected from the group comprising TLR4, Hsp60, IL-23R, IL-6 and IL-17F.
18. The assay device according to claim 17, wherein said TLR4 polymorphism is selected from rs4986790 and rs12344353 and combinations thereof.
19. The assay device according to claim 17, wherein said Hsp60 polymorphism is rs2340690.
20. The assay device according to claim 17, wherein said IL-23R polymorphism is rs11209026.
21. The assay device according to claim 17, wherein said IL-6 polymorphism is rs1800795.
22. The assay device according to claim 17, wherein said IL-17F polymorphism is rs763780.
CROSS REFERENCE To RELATED APPLICATIONS
[0001]Pursuant to 35 U.S.C. §119 (e), this application claims priority to the filing date of the U.S. Provisional Patent Application Ser. No. 61/156,346 filed Feb. 27, 2009; the disclosure of which are herein incorporated by reference.
[0002]Chronic Fatigue Syndrome (CFS)/myalgic encephalomyelitis is defined by a severe and debilitating fatigue associated with a variety of symptoms including musculoskeletal pain, sore throat, tender lymph nodes, sleep abnormalities, and neurocognitive problems (Fukuda et al. (1994) Ann Intern Med 121:953-959). The pathogenesis of CFS is still poorly understood, but is likely to be multifactorial; viral infections, stress, neuroendocrine dysfunctions, exposure to toxins have all been proposed as contributing factors to the onset and maintenance of the disease (Devanur and Kerr (2006) J Clin Virol 37(3):139-50).
[0003]Methods and compositions are provided for evaluating a subject for a chronic immune disease, including predicting whether a subject is susceptible to a chronic immune disease, diagnosing whether a subject has a chronic immune disease and/or determining a treatment for a subject suffering from a chronic immune disease. Aspects of the methods include genotyping a subject to determine whether the subject has a least one polymorphism in at least one gene chosen from TLR4, Hsp60, IL-23R, IL-6 and IL-17F. In addition, reagents, devices and kits thereof that find use in practicing the subject methods are provided.
[0004]Methods and compositions are provided for evaluating a subject for a chronic immune disease, including predicting whether a subject is susceptible to a chronic immune disease, diagnosing whether a subject has a chronic immune disease and/or determining a treatment regimen for a subject suffering from a chronic immune disease. Aspects of the methods include genotyping a subject to determine whether the subject has a least one polymorphism in at least one gene chosen from TLR4, Hsp60, IL-23R, IL-6 and IL-17F. In addition, reagents, devices and kits thereof that find use in practicing the subject methods are provided.
[0005]Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0006]Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0007]Methods recited herein may be carried out in any order of the recited events which is logically possible, as well as the recited order of events.
[0008]Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
[0009]All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0010]It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.
[0011]The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
[0012]As summarized above, the subject invention is directed to methods of evaluating chronic immune disease including predicting whether a subject is susceptible to a chronic immune disease, diagnosing whether a subject has a chronic immune disease or determining a treatment for a subject suffering from a chronic immune disease, as well as reagents and kits thereof (and devices) for use in practicing the subject methods. In further describing the invention, the subject methods are described first, followed by a review of the reagents, kits and devices for use in practicing the subject methods.
Methods of Evaluating a Subject for Chronic Immune Disease
[0013]Embodiments of the subject invention are directed to methods for: (a) predicting whether a subject is susceptible to a chronic immune disease; (b) diagnosing a subject as having a chronic immune disease; and (c) determining a treatment in a subject suffering from a chronic immune disease. Examples of chronic immune diseases are Chronic Fatigue Syndrome and Multiple Sclerosis. In further describing aspects of the invention, the following description focuses on Chronic Fatigue Syndrome. However, the subject methods and the reagents, devices and kits thereof also find use in the prediction of susceptibility to, diagnosis of, and treatment of other chronic immune diseases as well, as described above.
[0014]Chronic Fatigue Syndrome (CFS), also known as myalgic encephalomyelitis (ME), is a condition in which the subject suffers from fatigue associated with symptoms which may include but are not limited to muscle and joint (musculoskeletal) pain, sore throat, tender lymph nodes, sleep abnormalities, and/or neurocognitive problems. Often, the symptoms last for six months or more. Often, the fatigue is debilitating. In certain embodiments, the methods are directed at CFS and particularly at whether a subject will or will not be susceptible to CFS, at diagnosing a subject as having CFS, or at determining what treatment to provide a subject with CFS.
[0015]Aspects of the invention include methods of predicting whether a subject is or is not susceptible to CFS, or is or is not resistant to CFS. A subject is susceptible to CFS if the subject is more likely to develop CFS than an individual of the general population, whereas a subject that is resistant to CFS is less likely to develop CFS than an individual of the general population.
[0016]Additional aspects of the invention include methods of diagnosing whether a subject suspected of having CFS has CFS. Subjects suspected of having CFS and thus amenable to these methods can be identified using any convenient protocol. One convenient protocol is diagnosis based on clinical symptoms. A number of different clinical symptoms may be used to identify subjects that may have or have CFS. For example, clinical symptoms of interest include: fatigue of six months or longer that causes a reduction in effort of greater than 50 percent of normal output, pain in multiple joints (arthralgia), muscle pain (myalgia), frequent or recurring sore throat, or tender cervical or axillary lymph nodes, headaches of a new kind or greater severity, cognitive dysfunction (e.g. memory loss, impaired concentration); and the like. The presence of one or more of the above symptoms may be used to identify subjects suspected of having CFS.
[0017]Aspects of the invention further include methods of determining a treatment protocol/regimen for a subject suffering from CFS. The subject methods may be used to predict the risk of a CFS complication, where that result may be used to determine a treatment protocol. In other words, the subject methods may be used to predict the risk of a CFS subject developing a complication, the results of such a prediction being useful in determining a prophylactic treatment to prevent the complication. The subject methods may also be used to diagnose the cause of an existing complication, wherein that result may be used to determine a treatment for that complication. A complication is an unfavorable evolution of a disease, a health condition or a medical treatment. Complications could be a natural evolution of CFS, or a result of a medical treatment that produces adverse effects and/or produces new health problem(s) by itself, or a new disease that develops as a result of CFS. Indications of complications include but are not limited to indications that the CFS has become worse in its severity or shows a increased number of signs, symptoms or new pathologies.
[0018]In practicing methods according to certain embodiments, a subject or patient sample, e.g., cells or collections thereof, e.g., tissues, is assayed to determine whether the host or subject from which the assayed sample was obtained is susceptible to CFS, or to diagnose a subject as having CFS, or determine a treatment for a person suffering from CFS. In practicing the subject diagnostic methods, the sample obtained from the host is assayed to determine the genotype of the host or subject from which the sample was obtained with respect to at least one, i.e., one or more, polymorphisms, where polymorphisms of interest are referred to herein as target polymorphisms. In certain embodiments, the at least one target polymorphism is in the TLR4, Hsp60, IL-23R, IL-6 or IL-17F gene.
[0019]A TLR4 polymorphism is a polymorphism present in the TLR4 (Toll-like receptor 4) protein. The amino acid sequence for TLR4 and the nucleic acid sequence that encodes it are deposited at Genbank NM--138554 (SEQ ID NO:1 and SEQ ID NO:2). TLR4 is a type I transmembrane protein that acts as a cell surface receptor, for example, as a pattern recognition receptor (PRR) on immune cells for pathogen associated molecular patterns (PAMPs), wherein recognition and binding of PAMPs by TLR4 often induces a pro-inflammatory cellular response. Often, recognition of PAMPs occurs when TLR4 heterodimerizes with another TLR. Polymorphisms of interest include but are not limited to those that modulate TLR4 function such that cellular responsiveness to, e.g. LPS, is diminished, including those that modulate TLR4 recognition of PAMPs, those that modulate dimerization of TLR4 with other TLRs, and those that modulate downstream signaling mechanism by which TLR4 stimulates a cell, for example, the rs4986790 polymorphism (Asp299Gly), wherein a Gly at residue 299 is correlated with a diminished cellular response to LPS and is predictive of susceptibility to CFS. Polymorphisms of particular interest include the rs4986790 A/G polymorphism, in which the A allele encodes a variant with an Asp at residue 299 and the G allele encodes a variant with a Gly at residue 299 ("Asp299Gly"), wherein the G allele is predictive of susceptibility to CFS; and the rs12344353 T/C polymorphism, in which there is a T/C genetic polymorphism in the first intron (i.e. the intron between exons 1 and 2) of the TL4R gene, where the C allele is predictive of susceptibility to CFS.
[0020]An Hsp60 polymorphism is a polymorphism present in the Hsp60 (Heat shock protein 60) protein. The amino acid sequence for Hsp60, also known as HSPD1 and chaperonin 60-kD (CPN60), and variants, and the nucleic acid sequences that encode them are deposited at Genbank NM--002156 (SEQ ID NO:3 and SEQ ID NO:4) and NM--199440 (SEQ ID NO:5 and SEQ ID NO:6). Hsp60 is a molecular chaperone, acting, for example, to assist in protein folding and in replication and transmission of mitochondrial DNA. Polymorphisms of interest include but are not limited to those that modulate Hsp60 function, for example, by modulating binding with proteins they are chaperoning, by modulating binding with ATP, by modulating binding with chaperonin proteins, or by modulating binding with single strand DNA. Polymorphisms of particular interest include the rs2340690 T/C polymorphism, in which there is a T/C genetic polymorphism in an intron of the Hsp60 gene, where two T alleles is predictive of susceptibility to CFS.
[0021]An IL-23R polymorphism is a polymorphism present in the IL-23R (interleukin 23 receptor) protein. The amino acid sequence for IL-23R and the nucleic acid sequence that encodes it are deposited at Genbank NM--144701 (SEQ ID NO:7 and SEQ ID NO:8). IL-23R is a type 1 transmembrane protein that acts as a receptor for cytokines including IL-23, wherein receptor ligand binding by IL-23R often induces a pro-inflammatory response. Polymorphisms of interest include but are not limited to those that modulate IL-23R function, for example, by modulating IL-23R binding to its ligands or by modulating downstream signaling. Polymorphisms of particular interest include the rs11209026 G/A polymorphism, wherein the G allele encodes a variant with an Arg at residue 381 and the A allele encodes a variant with a Gln at residue 381 ("Arg381 Gln", or "R381Q"), where the A allele is predictive of susceptibility to CFS.
[0022]An IL-6 polymorphism is a polymorphism present in the IL-6 (interleukin 6) protein. The amino acid amino acid sequence for IL-6, also known as interferon beta-2 (IFNβ2), B-cell stimulatory factor (BSF2), hepatocyte stimulatory factor (HSF), and hybridoma growth factor (HGF), and the nucleic acid sequence that encodes it are deposited at Genbank NM--000600 (SEQ ID NO:9 and SEQ ID NO:10). IL-6 is a cytokine that often produces a proinflammatory cellular response. Polymorphisms of interest include but are not limited to those which modulate IL-6 function, for example, by increasing IL-6 binding to its receptor or increasing IL-6 levels in the blood, as with, for example, the rs1800795 polymorphism, in which the G allele enhances promoter activity, resulting in increased levels of IL-6 in serum. Polymorphisms of particular interest include the rs1800795 C/G polymorphism, in which there is a C/G genetic polymorphism in the promoter region of the IL-6 gene, where the G allele is predictive of susceptibility to CFS.
[0023]An IL-17F polymorphism is a polymorphism present in the IL-17F (Interleukin 17F) protein. The amino acid sequence for IL-17F the nucleic acid sequence that encodes it are deposited at Genbank NM--052872 (SEQ ID NO:11 and SEQ ID NO:12). IL-17F is a cytokine that often produces a proinflammatory response. Polymorphisms of interest include but are not limited to polymorphisms that encode amino acid substitutions that modulate IL-17F function, for example, by antagonizing IL-17F function, e.g. proinflammatory effects, e.g. by diminishing IL-17F binding to its receptor or reducing IL-17F levels in the blood such that cellular response is diminished, as with, for example, the rs763780 polymorphism, in which an Arg at residue 161 is associated with diminished cellular response. Polymorphisms of particular interest include the rs763780 T/C polymorphism, wherein the T allele encodes a variant with a His at residue 161 and the C allele encodes a variant with an Arg at residue 161 ("His161Arg" or "H161R"), where the C allele confers resistance to CFS.
[0024]More information about the aforementioned polymorphisms as well as other polymorphisms in the aforementioned genes that may be of interest in the present invention may be found in the NCBI SNP database, searchable at the website having an address produced by placing "http://www." in front of "ncbi.nlm.nih.gov/PubMed/", using the polymorphism or the gene name as the search term and clicking on the link to the "gene view" in any entry.
[0025]In certain embodiments, the sample is assayed to determine the genotype of the host with respect to a single target polymorphism. In these embodiments, the single target polymorphism is a polymorphism in the TLR4, Hsp60, IL-23R, IL-6 or IL-17F gene. In certain embodiments, the sample is assayed to determine the genotype of the host with respect to two or more different target polymorphisms, where in these embodiments, the two or more different target polymorphisms are polymorphisms in the TLR4, Hsp60, IL-23R, IL-6 or IL-17F genes. In certain of these embodiments, at least two of the target polymorphisms are polymorphisms in different genes. In certain embodiments, the sample is assayed for both a first and a second polymorphism
[0026]Any convenient protocol for assaying a sample for the above one or more target polymorphisms may be employed in the subject methods. In certain embodiments, the target polymorphism is detected at the protein level, e.g., by assaying for a polymorphic protein. In yet other embodiments, the target polymorphism is detected at the nucleic acid level, e.g., by assaying for the presence of nucleic acid polymorphism, e.g., a single nucleotide polymorphism (SNP) that causes expression of the polymorphic protein.
[0027]For example, polynucleotide samples derived from (e.g., obtained from) an individual may be employed. Any biological sample that comprises a polynucleotide from the individual is suitable for use in the methods of the invention. The biological sample may be processed so as to isolate the polynucleotide. Alternatively, whole cells or other biological samples may be used without isolation of the polynucleotides contained therein. Detection of a target polymorphism in a polynucleotide sample derived from an individual can be accomplished by any means known in the art, including, but not limited to, amplification of a sequence with specific primers; determination of the nucleotide sequence of the polynucleotide sample; hybridization analysis; single strand conformational polymorphism analysis; denaturing gradient gel electrophoresis; mismatch cleavage detection; and the like. Detection of a target polymorphism can also be accomplished by detecting an alteration in the level of a mRNA transcript of the gene; aberrant modification of the corresponding gene, e.g., an aberrant methylation pattern; the presence of a non-wild-type splicing pattern of the corresponding mRNA; an alteration in the level of the corresponding polypeptide; and/or an alteration in corresponding polypeptide activity.
[0028]Detection of a target polymorphism by analyzing a polynucleotide sample can be conducted in a number of ways. A test nucleic acid sample can be amplified with primers which amplify a region known to comprise the target polymorphism(s). Genomic DNA or mRNA can be used directly. Alternatively, the region of interest can be cloned into a suitable vector and grown in sufficient quantity for analysis. The nucleic acid may be amplified by conventional techniques, such as a polymerase chain reaction (PCR), to provide sufficient amounts for analysis. The use of the polymerase chain reaction is described in a variety of publications, including, e.g., "PCR Protocols (Methods in Molecular Biology)" (2000) J. M. S. Bartlett and D. Stirling, eds, Humana Press; and "PCR Applications: Protocols for Functional Genomics" (1999) Innis, Gelfand, and Sninsky, eds., Academic Press. Once the region comprising a target polymorphism has been amplified, the target polymorphism can be detected in the PCR product by nucleotide sequencing, by Single Strand Conformation Polymorphism (SSCP) analysis, or any other method known in the art. In performing SSCP analysis, the PCR product may be digested with a restriction endonuclease that recognizes a sequence within the PCR product generated by using as a template a reference sequence, but does not recognize a corresponding PCR product generated by using as a template a variant sequence by virtue of the fact that the variant sequence no longer contains a recognition site for the restriction endonuclease. PCR may also be used to determine whether a polymorphism is present by using a primer that is specific for the polymorphism. Such methods may comprise the steps of collecting from an individual a biological sample comprising the individual's genetic material as template, optionally isolating template nucleic acid (genomic DNA, mRNA, or both) from the biological sample, contacting the template nucleic acid sample with one or more primers that specifically hybridize with a target polymorphic nucleic acid molecule under conditions such that hybridization and amplification of the template nucleic acid molecules in the sample occurs, and detecting the presence, absence, and/or relative amount of an amplification product and comparing the length to a control sample. Observation of an amplification product of the expected size is an indication that the target polymorphism contained within the target polymorphic primer is present in the test nucleic acid sample. Parameters such as hybridization conditions, polymorphic primer length, and position of the polymorphism within the polymorphic primer may be chosen such that hybridization will not occur unless a polymorphism present in the primer(s) is also present in the sample nucleic acid. Those of ordinary skill in the art are well aware of how to select and vary such parameters. See, e.g., Saiki et al. (1986) Nature 324:163; and Saiki et al (1989) Proc. Natl. Acad. Sci. USA 86:6230. As one non-limiting example, a PCR primer comprising the T78C variation described in Example 1 may be used.
[0029]Alternatively, various methods are known in the art that utilize oligonucleotide ligation as a means of detecting polymorphisms. See, e.g., Riley et al. (1990) Nucleic Acids Res. 18:2887-2890; and Delahunty et al. (1996) Am. J. Hum. Genet. 58:1239-1246.
[0030]A detectable label may be included in an amplification reaction. Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels, e.g. 32P, 35S, 3H; etc. The label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.
[0031]The sample nucleic acid may be sequenced by a dideoxy chain termination method or other well-known methods. Genomic DNA or mRNA may be used directly. If mRNA is used, a cDNA copy may first be made. If desired, the sample nucleic acid can be amplified using a PCR. A variety of sequencing reactions known in the art can be used to directly sequence the relevant gene, or a portion thereof in which a specific polymorphism is known to occur, and detect polymorphisms by comparing the sequence of the sample nucleic acid with a reference polynucleotide that contains a target polymorphism. Any of a variety of automated sequencing procedures can be used. See, e.g., WO 94/16101; Cohen et al. (1996) Adv. Chromatography 36:127-162.
[0032]Hybridization with the variant sequence may also be used to determine the presence of a target polymorphism. Hybridization analysis can be carried out in a number of different ways including, but not limited to Southern blots, Northern blots, dot blots, microarrays, etc. The hybridization pattern of a control and variant sequence to an array of oligonucleotide probes immobilized on a solid support, as described in U.S. Pat. No. 5,445,934, or in WO 95/35505, may also be used as a means of detecting the presence of variant sequences. Identification of a polymorphism in a nucleic acid sample can be performed by hybridizing a sample and control nucleic acids to high density arrays containing hundreds or thousands of oligonucleotide probes. Cronin et al. (1996) Human Mutation 7:244-255; and Kozal et al. (1996) Nature Med. 2:753-759.
[0033]Single strand conformational polymorphism (SSCP) analysis; denaturing gradient gel electrophoresis (DGGE); mismatch cleavage detection; and heteroduplex analysis in gel matrices can also be used to detect polymorphisms. Alternatively, where a polymorphism creates or destroys a recognition site for a restriction endonuclease (restriction fragment length polymorphism, RFLP), the sample is digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel or capillary electrophoresis, particularly acrylamide or agarose gels. The aforementioned techniques are well known in the art. Detailed description of these techniques can be found in a variety of publications, including, e.g., "Laboratory Methods for the Detection of Mutations and Polymorphisms in DNA" (1997) G. R. Taylor, ed., CRC Press, and references cited therein.
[0034]A number of methods are available for determining the expression level of a polymorphic nucleic acid molecule, e.g., a polymorphic polypeptide in a particular sample. Diagnosis may be performed by a number of methods to determine the absence or presence or altered amounts of normal or abnormal mRNA in a patient sample. For example, detection may utilize staining of cells or histological sections with labeled antibodies, performed in accordance with conventional methods. Cells are permeabilized to stain cytoplasmic molecules. The antibodies of interest are added to the cell sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes. The antibody may be labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or other labels for direct detection. Alternatively, a second stage antibody or reagent is used to amplify the signal. Such reagents are well known in the art. For example, the primary antibody may be conjugated to biotin, with horseradish peroxidase-conjugated avidin added as a second stage reagent. Alternatively, the secondary antibody conjugated to a fluorescent compound, e.g. fluorescein, rhodamine, Texas red, etc. Final detection uses a substrate that undergoes a color change in the presence of the peroxidase. The absence or presence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc. Detailed descriptions of how to make antibodies can be found in a variety of publications, including, e.g. "Making and using Antibodies: A Practical Handbook" (2006) G. C. Howard and M. R. Kaser, eds. CRC Press; "Antibody Engineering: Methods and Protocols" (2004) B. K. C. Lo, ed, Humana Press.
[0035]Alternatively, one may focus on the expression of mRNA. Biochemical studies may be performed to determine whether a sequence polymorphism in a coding region or control regions of interest is associated with disease. Disease associated polymorphisms may include deletion or truncation of the gene, mutations that alter expression level, that affect the activity of the protein, etc.
[0036]Alternatively, one may screen for polymorphisms at the protein level. Screening for mutations in a polymorphic polypeptide may be based on the functional or antigenic characteristics of the protein. Functional assays include cofactor binding assays, enzyme activity assays, substrate binding assays or surface expression assays. For example, protein truncation assays are useful in detecting deletions that may affect the biological activity of the protein. The activity of the encoded a polymorphic polypeptide may be determined by comparison with a reference polypeptide lacking a specific polymorphism. Alternatively, the three-dimensional structure of the protein may be assayed, for example by fluorescence polarization or circular dichroism spectroscopy, wherein the three-dimensional structure of the encoded a polymorphic polypeptide may be determined by comparison with purified protein carrying the opposing allele of the polymorphism. Alternatively, various immunoassays designed to detect polymorphisms in polymorphic polypeptides may be used. The absence or presence of antibody binding to a polymorphic polypeptide may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc. Detailed descriptions of how to make antibodies, including antibodies that are specific for epitopes, for example, single amino acid substitutions within epitopes, can be found in a variety of publications, including, e.g. "Making and using Antibodies: A Practical Handbook" (2006) G. C. Howard and M. R. Kaser, eds. CRC Press; "Antibody Engineering: Methods and Protocols" (2004) B. K. C. Lo, ed, Humana Press; and U.S. Pat. No. 6,054,632, the disclosure of which is herein incorporated by reference.
[0037]Diagnostic methods of the subject invention in which the level of polymorphic gene expression is of interest will typically involve comparison of the relevant nucleic acid abundance of a sample of interest with that of a control value to determine any relative differences, where the difference may be measured qualitatively and/or quantitatively, which differences are then related to the presence or absence of an abnormal gene expression pattern. A variety of different methods for determining the nucleic acid abundance in a sample are known to those of skill in the art, where particular methods of interest include those described in: Pietu et al., Genome Res. (June 1996) 6: 492-503; Zhao et al., Gene (Apr. 24, 1995) 156: 207-213; Soares, Curr. Opin. Biotechnol. (October 1997) 8: 542-546; Raval, J. Pharmacol Toxicol Methods (November 1994) 32: 125-127; Chalifour et al., Anal. Biochem (Feb. 1, 1994) 216: 299-304; Stolz & Tuan, Mol. Biotechnol. (December 19960 6: 225-230; Hong et al., Bioscience Reports (1982) 2: 907; and McGraw, Anal. Biochem. (1984) 143: 298. Also of interest are the methods disclosed in WO 97/27317, the disclosure of which is herein incorporated by reference.
[0038]Additional references describing various protocols for detecting the presence of a target polymorphism include, but are not limited to, those described in: U.S. Pat. Nos. 6,703,228; 6,692,909; 6,670,464; 6,660,476; 6,653,079; 6,632,606; 6,573,049; the disclosures of which are herein incorporated by reference.
[0039]Following obtainment of the genotype from the sample being assayed, the genotype is evaluated to determine if the subject is susceptible/resistant to CFS, or to determine a treatment in a subject that is suffering from CFS. In certain embodiments, the obtained genotype may be compared with a reference or control to make a diagnosis regarding the CFS phenotype of the cell or tissue, and therefore host, from which the sample was obtained/derived. The terms "reference" and "control" as used herein mean a standardized genotype to be used to interpret the genotype of a given patient and assign a prognostic class thereto. The reference or control may be a genotype that is obtained from a cell/tissue known to have a particular phenotype, e.g., a susceptibility phenotype, and therefore may be a positive reference or control genotype. Alternatively, the reference/control genotype may be from a cell/tissue known to not have the desired phenotype, and therefore be a negative reference/control genotype.
[0040]In certain embodiments, the obtained genotype is compared to a single reference/control genotype to obtain information regarding the phenotype of the cell/tissue being assayed. In yet other embodiments, the obtained genotype is compared to two or more different reference/control profiles to obtain more in depth information regarding the phenotype of the assayed cell/tissue. For example, the obtained genotype may be compared to a positive genotype and a negative genotype to obtain confirmed information regarding whether the cell/tissue has the phenotype of interest.
[0041]Representative examples of genotypes associated with susceptibility to or with having CFS include, but are not limited to: the G allele of TLR4 polymorphism rs4986790, the C allele of TLR4 polymorphism rs12344353, two T alleles of Hsp60 polymorphism rs2340690, the A allele of IL-23R polymorphism rs11209026, and two G alleles of the IL-6 polymorphism rs1800795. Representative examples of genotypes associated with resistance to CFS include, but are not limited to the C allele of the IL-17F polymorphism rs763780.
[0042]In certain embodiments, the above-obtained information about the cell/tissue being assayed is employed to determine if the subject is susceptible/resistant to CFS, or to diagnose a subject with CFS, or to predict if a subject suffering from CFS will develop a particular complication. In certain embodiments, the above-obtained information is employed to give a refined probability determination as to whether a subject is susceptible to CFS, or to diagnose a subject with CFS, or to give a refined determination as to whether a subject suffering from CFS will develop a particular complication. For example, an identification of at least one G allele of TLR4 polymorphism rs4986790, at least one C allele of TLR4 polymorphism rs12344353, two T alleles of Hsp60 polymorphism rs2340690, at least one A allele of IL-23R polymorphism rs11209026, or two G alleles of the IL-6 polymorphism rs1800795 may be employed to determine that the subject has at least a 70% chance, such as at least a 75% chance, including at least an 80% chance of developing CFS or a complication from CFS. Likewise, an identification of at least one C allele of the IL-17F polymorphism rs763780 may be employed to determine that the subject has less than 50% chance, such as a less than 45% chance, including a less than 40% chance of developing CFS or a complication from CFS.
[0043]In some embodiments, determining that the subject is susceptible/resistant to CFS, or diagnosing a subject with CFS, or predicting if a subject suffering from CFS will develop a particular complication may be made by employing the results of the aforementioned genotype analysis in conjunction with the identification of symptoms known in the art to be associated with CFS, e.g. a new onset (not lifelong) of unexplained, persistent fatigue unrelated to exertion and not substantially relieved by rest, impaired memory or concentration, post-exertional malaise, where physical or mental exertions bring on "extreme, prolonged exhaustion and sickness", unrefreshing sleep, muscle pain (myalgia), pain in multiple joints (arthralgia), headaches of a new kind or greater severity, frequent or recurring sore throat, or tender cervical or axillary lymph nodes. In addition, the artisan may consider the presence of symptoms that are attributable to other conditions, so as to exclude a determination of susceptibility to CFS or a CFS diagnosis, e.g. symptoms of mononucleosis, Lyme disease, lupus, multiple sclerosis, fibromyalgia, primary sleep disorders, severe obesity and major depressive disorders, as they are known in the art.
[0044]In some embodiments, the above-obtained information about the cell/tissue being assayed is employed to determine a treatment for a subject suffering from CFS. Treatments of interest include, but are not limited to, antihistamines, prescription sleep medications, analgesics, anti-depressants, dietary supplements, alternative medicine, cognitive behavior therapy (a form of psychological therapy used to treat chronically ill subjects), graded exercise therapy (a form of physical therapy), and pacing (an energy management strategy in which subjects are advised to set manageable daily activity/exercise goals and balance activity and rest to avoid over-exertion which may worsen symptoms). Classes of therapeutic treatments of interest include, but are not limited to, therapeutic antibodies, peptide mimetics, small molecule agonists or antagonists, siRNA-based therapeutics, and gene therapy.
[0045]In some instances, the treatment may be a pharmacogenomic treatment. In these applications, a subject/host/patient is first diagnosed for the presence of absence of a CFS phenotype using a protocol such as the diagnostic protocol described in the preceding section. The subject is then treated using a pharmacological protocol, where the suitability of the protocol for a particular subject/patient is determined using the results of the diagnosis step. In other words, the therapeutic protocol employed is a protocol that is tailored to the particular polymorphism identified in the patient sample. More specifically, where the identified phenotype is a polymorphism in one gene, a particular therapeutic treatment protocol is then employed to treat the subject. Alternatively, where a subject is identified as having a polymorphism in another gene, a different therapeutic treatment protocol is then employed. For example, a therapy for CFS or a CFS complication in a subject with a TLR4 polymorphism may include augmenting TLR4 signaling, for example with peptide mimetic or antibody therapy to promote signaling via the TLR4 Gly variant. As another example, a therapy for CFS or a CFS complication in a subject with an IL-6 rs18000795 polymorphism may include attenuating IL-6 signaling, for example with an IL-6 or IL-6 receptor blocking antibody, an IL-6-specific or IL-6 receptor-specific siRNA, or an IL-6 or IL-6 dominant negative peptide.
[0046]In some embodiments, providing an evaluation of a subject for CFS, e.g. a prediction of a subject's susceptibility to developing CFS, a diagnosis of CFS in a subject suspected of having CFS, and/or a determination of a treatment regimen for a CFS subject, includes generating a written report that includes the artisan's assessment of the subject's susceptibility to developing CFS, i.e. a "susceptibility assessment", the subject's current state of health i.e. a "CFS diagnosis assessment", and/or possible treatment regimens, i.e. a "treatment assessment". Thus, a subject method may further include a step of generating or outputting a report providing the results of a susceptibility assessment, a CFS diagnosis assessment, or treatment assessment, which report can be provided in the form of an electronic medium (e.g., an electronic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium).
[0047]A "report," as described herein, is an electronic or tangible document which includes report elements that provide information of interest relating to a susceptibility assessment, a CFS diagnosis assessment, or treatment assessment and its results. A subject report can be completely or partially electronically generated. A subject report includes at least a susceptibility prediction, i.e. a prediction as to the susceptibility of a subject to developing CFS; or a CFS diagnosis, i.e. whether a subject does or does not have CFS; or a suggested treatment regimen, i.e. a course of treatment to be followed. A subject report can further include one or more of: 1) information regarding the testing facility; 2) service provider information; 3) subject data; 4) sample data; 5) an assessment report, which can include various information including: a) test data, where test data can include genotypes for one or more polymorphism in at least one gene chosen from TLR4, Hsp60, IL-23R, IL-6 and IL-17F and b) reference values employed, if any; 6) other features.
[0048]The report may include information about the testing facility, which information is relevant to the hospital, clinic, or laboratory in which sample gathering and/or data generation was conducted. Sample gathering can include obtaining a fluid sample, e.g. blood, saliva, urine etc.; or a tissue sample, e.g. a tissue biopsy, a swab of the lining of the mouth or nose, a collect hair follicle, etc. from a subject. Data generation can include genotyping for one or more polymorphisms in TLR4, Hsp60, IL-23R, IL-6 and/or IL-17F. This information can include one or more details relating to, for example, the name and location of the testing facility, the identity of the lab technician who conducted the assay and/or who entered the input data, the date and time the assay was conducted and/or analyzed, the location where the sample and/or result data is stored, the lot number of the reagents (e.g., kit, etc.) used in the assay, and the like. Report fields with this information can generally be populated using information provided by the user.
[0049]The report may include information about the service provider, which may be located outside the healthcare facility at which the user is located, or within the healthcare facility. Examples of such information can include the name and location of the service provider, the name of the reviewer, and where necessary or desired the name of the individual who conducted sample gathering and/or data generation. Report fields with this information can generally be populated using data entered by the user, which can be selected from among pre-scripted selections (e.g., using a drop-down menu). Other service provider information in the report can include contact information for technical information about the result and/or about the interpretive report.
[0050]The report may include a subject data section, including subject medical history as well as administrative subject data (that is, data that are not essential to the susceptibility prediction, CFS diagnosis, or proposed treatment regimen) such as information to identify the subject (e.g., name, subject date of birth (DOB), gender, mailing and/or residence address, medical record number (MRN), room and/or bed number in a healthcare facility), insurance information, and the like), the name of the subject's physician or other health professional who ordered the susceptibility prediction and, if different from the ordering physician, the name of a staff physician who is responsible for the subject's care (e.g., primary care physician).
[0051]The report may include a sample data section, which may provide information about the biological sample analyzed in the susceptibility prediction, such as the source of biological sample obtained from the subject (e.g. blood, saliva, type of tissue, etc.), how the sample was handled (e.g. storage temperature, preparatory protocols) and the date and time collected. Report fields with this information can generally be populated using data entered by the user, some of which may be provided as pre-scripted selections (e.g., using a drop-down menu).
[0052]The report may include an assessment report section, which may include information generated after processing of the data as described herein. The interpretive report can include a prediction of the likelihood that the patient will develop CFS. The interpretive report can include, for example, results of genotyping for a TLR4 polymorphism; and/or results of genotyping for an Hsp60 polymorphism; and/or results of genotyping for a IL-23R polymorphism; and/or results of genotyping for a IL-6 polymorphism; and/or results of genotyping for a IL-17F polymorphism; and interpretation, i.e. prediction. The assessment portion of the report can optionally also include a Recommendation(s). For example, where the results indicate an increased susceptibility to developing CFS, the recommendation can include a recommendation that diet or lifestyle be altered or medical intervention be provided as recommended in the art.
[0053]It will also be readily appreciated that the reports can include additional elements or modified elements. For example, where electronic, the report can contain hyperlinks which point to internal or external databases which provide more detailed information about selected elements of the report. For example, the patient data element of the report can include a hyperlink to an electronic patient record, or a site for accessing such a patient record, which patient record is maintained in a confidential database. This latter embodiment may be of interest in an in-hospital system or in-clinic setting. When in electronic format, the report is recorded on a suitable physical medium, such as a computer readable medium, e.g., in a computer memory, zip drive, CD, DVD, etc.
[0054]It will be readily appreciated that the report can include all or some of the elements above, with the proviso that the report generally includes at least the elements sufficient to provide the analysis requested by the user (e.g., susceptibility prediction).
Reagents, Devices and Kits
[0055]Also provided are reagents, devices and kits thereof for practicing one or more of the above-described methods. The subject reagents, devices and kits thereof may vary greatly. Reagents and devices of interest include those mentioned above with respect to the methods of identifying the presence of the target polymorphisms, where such reagents may include nucleic acid primers, arrays of nucleic acid probes, antibodies to polymorphic polypeptides (e.g., immobilized on a substrate), signal producing system reagents, etc., depending on the particular detection protocol to be performed. For example, reagents may include PCR primers that are specific for one or more of the genes TLR4, Hsp60, IL-23R, IL-6 and IL-17F or one or more polymorphisms thereof, as described above. Other examples of reagents include arrays that comprise probes that are specific for one or more of the genes of interest or one or more polymorphisms thereof, and antibodies to epitopes of the proteins encoded by these genes of interest wherein the epitope comprises a polymorphism of interest.
[0056]In addition to the above components, the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
[0057]The following examples are offered by way of illustration and not by way of limitation.
Subjects and Methods
[0058]89 CFS patients (40±12 year old) were enrolled in the study. All were diagnosed for CFS according to the clinical criteria of Fukuda et al. ((1994) Ann. Intern. Med. 121:953-959). 56 aged-matched controls (37±12 year old) were also recruited. 79% of the patients, and 70% of the controls, were females. All subjects were of European origin.
[0059]Genomic DNA was extracted from whole blood using the Blood and Tissue DNA extraction kit, Qiagen (Venlo, Netherlands). Analysis of the rs763780 polymorphism in the IL-17F gene was performed with the SNPlex® genotyping technology, Applied Biosystems (Foster City, Calif., USA). Genotyping analyses were performed by DNAvision S. A. (Charleroi, Belgium), using ISO17025-accredited procedures.
[0060]A significantly lower prevalence of the His161Arg variant was observed in the CFS population as compared to the control population. 8 out of 89 patients have a CT genotype (8.9%), versus 14 out of 56 controls (25%). None of the CFS patients have the CC genotype, versus 2 out of 56 controls (3.6%). In total, 28.5% of the controls have a CC or CT genotype versus only 8.9% of the patients (OR=4.05, p=0.0018).
[0061]The observed lower frequency suggests an involvement of IL-17F, and more generally of Th17 cells, in the pathophysiology of CFS. The His161Arg variant antagonizes the pro-inflammatory effects of wild-type IL-17F, and thereby exerts a protective effect against asthma (Kawaguchi et al. (2006) J. Allergy Clin. Immunol. 117(4):795-801). Accordingly, the development and/or maintenance of CFS involves an increased production of IL-17F, and that expression of inactive variants such as His161Arg confer protection against the disease.
[0062]Increased IL-17F production has been observed in various inflammatory situations (Kolls and Linden (2004) Immunity 21(4):467-476; Kawaguchi et al. (2006) J. Allergy Clin. Immunol. 117(4):795-801). Interestingly, it also occurs in the context of intestinal disease, such as Crohn's disease or ulcerative colitis (Seiderer et al. (2008) Inflamm. Bowel Dis. 14(4):437-445). Intestinal dysfunction is a common symptom in CFS. Alterations of the intestinal microbial flora have been reported (Logan et al. (2003) Med. Hypotheses 60(6) 915-923); such alterations can lead to intestinal mucosal dysfunction, increased intestinal permeability (leaky gut), etc. that will cause an immune response to the LPS of gram-negative enterobacteria (Maes et al. (2007) J. Affect. Disord. 99(1-3):237-240; Maes et al. (2007) Neuro. Endocrinol. Lett. 28(6):739-44). Exposure of immune cells to LPS could therefore be the clue to the chronic immune activation observed in CFS patients. Considering this hypothesis, it is particularly noteworthy that in addition to IL-6 and TGB-β, optimal Th17 cell induction requires the action of Toll-like receptor (TLR)-activated peripheral blood mononuclear cells (PBMCs). TLR-activated monocytes may contribute to Th17 cell induction by cell-cell contact, through ligation of the T-cell receptor (Evans et al. (2007) Proc. Natl. Acad. Sci. USA 104(43):17034-9); alternatively, TLR-activated PBMCs may secrete a specific set of cytokines that will potentiate Th17 induction (Kattah et al. (2008) Arthritis Rheum. 58(6):1619-29). In this last report, it was shown that TLR-4 (receptor for LPS), as well as TLR-7/8, evoked the most robust induction of Th17, whereas stimulation of TLR-1, -2, -3 or -9 was not efficient.
[0063]Th17 cell induction could therefore be the link between exposure to enterobacterial LPS and the symptoms of chronic inflammation associated with CFS. Interestingly, Th17 and Th1 responses are mutually exclusive, since IFN-γ suppresses IL-17 and vice-versa (Kolls and Linden (2004) Immunity 21(4):467-476). Th17 induction is therefore consistent with a decreased production of Th1 cytokines, as seen in CFS. The pro-inflammatory effects of Th17-secreted cytokines are also consistent with other specific dysfunctions observed in CFS patients: IL-17 and IL-22 can disrupt the blood-brain barrier; Th17 lymphocytes transmigrate across the blood-brain barrier endothelial cells and promote inflammation of the central nervous system (Kebir et al. (2007) Nat. Med. 13(10):1173-1175). Blood-brain barrier permeability and CNS inflammation is thought to be a key aspect in the pathogenesis of CFS (Bested et al. (2001) Med. Hypotheses 57(2):231-237).
[0064]The implication of Th17 cell activation in the pathogenesis of CFS would be a significant progress in the understanding of the disease, opening new therapeutic perspectives. Other IL-17F-related genes, such as IL-23R, may also play a role in the pathogenesis. IL-17F is indeed specifically produced by IL-23R-expressing Th17 cells; IL-23R is also polymorphic and some variants of this receptor predispose to inflammatory bowel disease (Maloy (2008) J. Intern. Med. 263(6):584-590; Seiderer et al. (2008) Inflamm. Bowel Dis. 14(4):437-445). An association between CFS and polymorphisms of the IL-23R gene, or other functionally related genes, would provide additional support for the implication of the IL-17 axis in the pathogenesis of CFS.
Subjects and Methods
A. Study Subjects
[0065]Study subjects were 107 patients, 40±12 year old, previously diagnosed for CFS according to the clinical criteria of Fukuda et al. (Fukuda et al. 1994; Ann Intern Med 121:953-959), and 64 healthy controls (non-CFS subjects), 37±12 year old. 79% of patients, and 70% of controls, were females. All subjects were of European origin. Patients and controls were selected from medical practices in Oslo, Norway and Brussels, Belgium.
B. Blood Sampling and Genotyping Procedure
[0066]2 ml whole blood was collected in EDTA tubes, and stored at -80° C. until analysis. Genomic DNA was extracted from 200 μl whole blood using the Blood and Tissue DNA extraction kit, Qiagen (Venlo, Netherlands). Analysis of the rs4986790, rs4986791, rs12344353, rs11209026, rs1800795 and rs2340690 polymorphisms was performed with the SNPlex® genotyping technology, or with Taqman® SNP genotyping assays, both from Applied Biosystems (Foster City, Calif., USA).
[0067]A summary of all genotyping results is provided in Table 1.
TABLE-US-00001 TABLE 1 Frequency of specific alleles in controls and in CFS patients Hsp60 Gene TLR4 IL-23R (HSPD1) IL6 IL6 + Hsp60 SNP rs4986790 rs11209026 rs2340690 rs1800795 Rs1800795 in rs12344353 combination with rs2340690 Allele G allele of A allele T/T genotype G/G T/T genotype of rs4986790 genotype rs2340690 in C allele of combination rs12344353 with G/G genotype of rs1800795 Frequency 9% 0% 54% 13% 4% in controls Frequency 13% 10% 74% 29% 32% in patients
A. Toll-Like Receptor 4
[0068]Three different SNPs have been analyzed for this gene: rs4986790 (Asp299Gly), rs4986791 (Thr399lle) and rs12344353.
[0069]100% cosegregation level for the rs4986790 and rs12344353 SNPs was observed. rs4986791 cosegregrates with the two others, but less systematically (80% only). These high cosegregation levels were already described in the literature.
[0070]The G allele of rs4986790 is associated with a lower responsiveness to LPS stimulation. This is detrimental in that affected carriers are more susceptible to gram-negative infections (sepsis); on the other hand, these people tend to develop less inflammation, and seem to have less chance to develop cardiovascular disease or even Alzheimer's disease. TLR4 polymorphisms have been associated with intestinal diseases (Crohn's disease, inflammatory bowel disease), which may be explained by deficient host-bacteria interactions in the gut (Zacharowski et al. (2006) PNAS 103(16):6392-7; Franchimont et al. (2004) Gut 53(7):987-92).
[0071]The variant alleles were present in 6/64 (9%) of the controls, versus 14/107 (13%) of the patients (odds ratio 0.5). CFS patients therefore present a higher frequency of the TLR4 variant haplotype.
B. Heat-Shock Protein 60
[0072]Hsp60 is produced in stress situations (oxidative stress, toxic insult); it can be released by the cells and work extra-cellularly as a pro-inflammatory molecule (it is recognized by TLR4 and leads to the proinflammatory activation of macrophages) (Visser et al. (1998) J. Infect. Dis. 177(2):451-454). An intronic C/T substitution (rs2340690) has been described.
[0073]C/C or C/T genotypes of rs2340690 were observed in 26% of the patients versus in 46% of the controls (odds ratio 2.3). Therefore, carrying the T/T genotype of rs2340690 is a predisposing factor for CFS.
[0074]A SNP located on the IL-23R gene, rs11209026, has already been associated with Crohn's disease (Roberts et al. (2007) Am. J. Gastroenterol 102(12):2754-61). The A allele of this polymorphism was found in 10% of CFS patients, but in none of the controls.
D. IL-6, and IL-6 in Combination with Hsp60
[0075]IL-6 polymorphism rs1800795 is another SNP of interest. A higher prevalence of the G/G genotype of rs1800795 was observed in patients than in controls (29% vs. 13%, OR=0.35), indicating that IL-6 polymorphisms are predictive of CFS independent of other polymorphisms, e.g. polymorphisms in other cytokines, e.g. IFNgamma. The G/G genotype of rs1800795 is thought to confer susceptibility to inflammation (Tischendorf et al. (2007) Int J. Immunogenet 34(6):413-8).
[0076]A very discriminant genotype is formed by the combination of IL-6 and Hsp60: the frequency of subjects simultaneously carrying the G/G genotype of rs1800795 (IL-6) and the T/T genotype of rs2340690 (Hsp60) was found to be 32% in the patient population, versus only 4% in the control population.
[0077]All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
[0078]Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Sequence CWU
1215667DNAhomo sapiens 1ctcttgctgt ttctttagcc actggtctgc aggcgttttc ttcttctaac ttcctctcct 60gtgacaaaag agataactat tagagaaaca aaagtccaga atgctaaggt tgccgctttc 120acttcctctc accctttagc ccagaactgc tttgaataca ccaattgctg tggggcggct 180cgaggaagag aagacaccag tgcctcagaa actgctcggt cagacggtga tagcgagcca 240cgcattcaca gggccactgc tgctcacaga agcagtgagg atgatgccag gatgatgtct 300gcctcgcgcc tggctgggac tctgatccca gccatggcct tcctctcctg cgtgagacca 360gaaagctggg agccctgcgt ggaggtggtt cctaatatta cttatcaatg catggagctg 420aatttctaca aaatccccga caacctcccc ttctcaacca agaacctgga cctgagcttt 480aatcccctga ggcatttagg cagctatagc ttcttcagtt tcccagaact gcaggtgctg 540gatttatcca ggtgtgaaat ccagacaatt gaagatgggg catatcagag cctaagccac 600ctctctacct taatattgac aggaaacccc atccagagtt tagccctggg agccttttct 660ggactatcaa gtttacagaa gctggtggct gtggagacaa atctagcatc tctagagaac 720ttccccattg gacatctcaa aactttgaaa gaacttaatg tggctcacaa tcttatccaa 780tctttcaaat tacctgagta tttttctaat ctgaccaatc tagagcactt ggacctttcc 840agcaacaaga ttcaaagtat ttattgcaca gacttgcggg ttctacatca aatgccccta 900ctcaatctct ctttagacct gtccctgaac cctatgaact ttatccaacc aggtgcattt 960aaagaaatta ggcttcataa gctgacttta agaaataatt ttgatagttt aaatgtaatg 1020aaaacttgta ttcaaggtct ggctggttta gaagtccatc gtttggttct gggagaattt 1080agaaatgaag gaaacttgga aaagtttgac aaatctgctc tagagggcct gtgcaatttg 1140accattgaag aattccgatt agcatactta gactactacc tcgatgatat tattgactta 1200tttaattgtt tgacaaatgt ttcttcattt tccctggtga gtgtgactat tgaaagggta 1260aaagactttt cttataattt cggatggcaa catttagaat tagttaactg taaatttgga 1320cagtttccca cattgaaact caaatctctc aaaaggctta ctttcacttc caacaaaggt 1380gggaatgctt tttcagaagt tgatctacca agccttgagt ttctagatct cagtagaaat 1440ggcttgagtt tcaaaggttg ctgttctcaa agtgattttg ggacaaccag cctaaagtat 1500ttagatctga gcttcaatgg tgttattacc atgagttcaa acttcttggg cttagaacaa 1560ctagaacatc tggatttcca gcattccaat ttgaaacaaa tgagtgagtt ttcagtattc 1620ctatcactca gaaacctcat ttaccttgac atttctcata ctcacaccag agttgctttc 1680aatggcatct tcaatggctt gtccagtctc gaagtcttga aaatggctgg caattctttc 1740caggaaaact tccttccaga tatcttcaca gagctgagaa acttgacctt cctggacctc 1800tctcagtgtc aactggagca gttgtctcca acagcattta actcactctc cagtcttcag 1860gtactaaata tgagccacaa caacttcttt tcattggata cgtttcctta taagtgtctg 1920aactccctcc aggttcttga ttacagtctc aatcacataa tgacttccaa aaaacaggaa 1980ctacagcatt ttccaagtag tctagctttc ttaaatctta ctcagaatga ctttgcttgt 2040acttgtgaac accagagttt cctgcaatgg atcaaggacc agaggcagct cttggtggaa 2100gttgaacgaa tggaatgtgc aacaccttca gataagcagg gcatgcctgt gctgagtttg 2160aatatcacct gtcagatgaa taagaccatc attggtgtgt cggtcctcag tgtgcttgta 2220gtatctgttg tagcagttct ggtctataag ttctattttc acctgatgct tcttgctggc 2280tgcataaagt atggtagagg tgaaaacatc tatgatgcct ttgttatcta ctcaagccag 2340gatgaggact gggtaaggaa tgagctagta aagaatttag aagaaggggt gcctccattt 2400cagctctgcc ttcactacag agactttatt cccggtgtgg ccattgctgc caacatcatc 2460catgaaggtt tccataaaag ccgaaaggtg attgttgtgg tgtcccagca cttcatccag 2520agccgctggt gtatctttga atatgagatt gctcagacct ggcagtttct gagcagtcgt 2580gctggtatca tcttcattgt cctgcagaag gtggagaaga ccctgctcag gcagcaggtg 2640gagctgtacc gccttctcag caggaacact tacctggagt gggaggacag tgtcctgggg 2700cggcacatct tctggagacg actcagaaaa gccctgctgg atggtaaatc atggaatcca 2760gaaggaacag tgggtacagg atgcaattgg caggaagcaa catctatctg aagaggaaaa 2820ataaaaacct cctgaggcat ttcttgccca gctgggtcca acacttgttc agttaataag 2880tattaaatgc tgccacatgt caggccttat gctaagggtg agtaattcca tggtgcacta 2940gatatgcagg gctgctaatc tcaaggagct tccagtgcag agggaataaa tgctagacta 3000aaatacagag tcttccaggt gggcatttca accaactcag tcaaggaacc catgacaaag 3060aaagtcattt caactcttac ctcatcaagt tgaataaaga cagagaaaac agaaagagac 3120attgttcttt tcctgagtct tttgaatgga aattgtatta tgttatagcc atcataaaac 3180cattttggta gttttgactg aactgggtgt tcactttttc ctttttgatt gaatacaatt 3240taaattctac ttgatgactg cagtcgtcaa ggggctcctg atgcaagatg ccccttccat 3300tttaagtctg tctccttaca gaggttaaag tctagtggct aattcctaag gaaacctgat 3360taacacatgc tcacaaccat cctggtcatt ctcgagcatg ttctattttt taactaatca 3420cccctgatat atttttattt ttatatatcc agttttcatt tttttacgtc ttgcctataa 3480gctaatatca taaataaggt tgtttaagac gtgcttcaaa tatccatatt aaccactatt 3540tttcaaggaa gtatggaaaa gtacactctg tcactttgtc actcgatgtc attccaaagt 3600tattgcctac taagtaatga ctgtcatgaa agcagcattg aaataatttg tttaaagggg 3660gcactctttt aaacgggaag aaaatttccg cttcctggtc ttatcatgga caatttgggc 3720tagaggcagg aaggaagtgg gatgacctca ggaggtcacc ttttcttgat tccagaaaca 3780tatgggctga taaacccggg gtgacctcat gaaatgagtt gcagcagaag tttatttttt 3840tcagaacaag tgatgtttga tggacctctg aatctcttta gggagacaca gatggctggg 3900atccctcccc tgtacccttc tcactgccag gagaactacg tgtgaaggta ttcaaggcag 3960ggagtataca ttgctgtttc ctgttgggca atgctccttg accacatttt gggaagagtg 4020gatgttatca ttgagaaaac aatgtgtctg gaattaatgg ggttcttata aagaaggttc 4080ccagaaaaga atgttcatcc agcctcctca gaaacagaac attcaagaaa aggacaatca 4140ggatgtcatc agggaaatga aaataaaaac cacaatgaga tatcacctta taccaggtag 4200aatggctact ataaaaaaat gaagtgtcat caaggatata gagaaattgg aacccttctt 4260cactgctgga gggaatggaa aatggtgtag ccgttatgaa aaacagtacg gaggtttctc 4320aaaaattaaa aatagaactg ctatatgatc cagcaatctc acttctgtat atatacccaa 4380aataattgaa atcagaattt caagaaaata tttacactcc catgttcatt gtggcactct 4440tcacaatcac tgtttccaaa gttatggaaa caacccaaat ttccattgaa aaataaatgg 4500acaaagaaaa tgtgcatata cgtacaatgg gatattattc agcctaaaaa aagggggaat 4560cctgttattt atgacaacat gaataaaccc ggaggccatt atgctatgta aaatgagcaa 4620gtaacagaaa gacaaatact gcctgatttc atttatatga ggttctaaaa tagtcaaact 4680catagaagca gagaatagaa cagtggttcc tagggaaaag gaggaaggga gaaatgagga 4740aatagggagt tgtctaattg gtataaaatt atagtatgca agatgaatta gctctaaaga 4800tcagctgtat agcagagttc gtataatgaa caatactgta ttatgcactt aacattttgt 4860taagagggta cctctcatgt taagtgttct taccatatac atatacacaa ggaagctttt 4920ggaggtgatg gatatattta ttaccttgat tgtggtgatg gtttgacagg tatgtgacta 4980tgtctaaact catcaaattg tatacattaa atatatgcag ttttataata tcaattatgt 5040ctgaatgaag ctataaaaaa gaaaagacaa caaaattcag ttgtcaaaac tggaaatatg 5100accacagtca gaagtgtttg ttactgagtg tttcagagtg tgtttggttt gagcaggtct 5160agggtgattg aacatccctg ggtgtgtttc catgtctcat gtactagtga aagtagatgt 5220gtgcatttgt gcacatatcc ctatgtatcc ctatcagggc tgtgtgtatt tgaaagtgtg 5280tgtgtccgca tgatcatatc tgtatagaag agagtgtgat tatatttctt gaagaataca 5340tccatttgaa atggatgtct atggctgttt gagatgagtt ctctactctt gtgcttgtac 5400agtagtctcc ccttatccct tatgcttggt ggatacgttc ttagacccca agtggatctc 5460tgagaccgca gatggtacca aacctcatat atgcaatatt ttttcctata cataaatacc 5520taagataaag ttcatcttct gaattaggca cagtaagaga ttaacaataa ctaacaataa 5580aattgaatag ttataataat atattgtaat aaaagttatg tgaatgtgat ctctttcttt 5640ctctctctca aaaaaaaaaa aaaaaaa 56672839PRThomo sapiens 2Met Met Ser Ala Ser Arg Leu Ala Gly Thr Leu Ile Pro Ala Met Ala1 5 10 15Phe Leu Ser Cys Val Arg Pro Glu Ser Trp Glu Pro Cys Val Glu Val 20 25 30Val Pro Asn Ile Thr Tyr Gln Cys Met Glu Leu Asn Phe Tyr Lys Ile 35 40 45Pro Asp Asn Leu Pro Phe Ser Thr Lys Asn Leu Asp Leu Ser Phe Asn 50 55 60Pro Leu Arg His Leu Gly Ser Tyr Ser Phe Phe Ser Phe Pro Glu Leu65 70 75 80Gln Val Leu Asp Leu Ser Arg Cys Glu Ile Gln Thr Ile Glu Asp Gly 85 90 95Ala Tyr Gln Ser Leu Ser His Leu Ser Thr Leu Ile Leu Thr Gly Asn 100 105 110Pro Ile Gln Ser Leu Ala Leu Gly Ala Phe Ser Gly Leu Ser Ser Leu 115 120 125Gln Lys Leu Val Ala Val Glu Thr Asn Leu Ala Ser Leu Glu Asn Phe 130 135 140Pro Ile Gly His Leu Lys Thr Leu Lys Glu Leu Asn Val Ala His Asn145 150 155 160Leu Ile Gln Ser Phe Lys Leu Pro Glu Tyr Phe Ser Asn Leu Thr Asn 165 170 175Leu Glu His Leu Asp Leu Ser Ser Asn Lys Ile Gln Ser Ile Tyr Cys 180 185 190Thr Asp Leu Arg Val Leu His Gln Met Pro Leu Leu Asn Leu Ser Leu 195 200 205Asp Leu Ser Leu Asn Pro Met Asn Phe Ile Gln Pro Gly Ala Phe Lys 210 215 220Glu Ile Arg Leu His Lys Leu Thr Leu Arg Asn Asn Phe Asp Ser Leu225 230 235 240Asn Val Met Lys Thr Cys Ile Gln Gly Leu Ala Gly Leu Glu Val His 245 250 255Arg Leu Val Leu Gly Glu Phe Arg Asn Glu Gly Asn Leu Glu Lys Phe 260 265 270Asp Lys Ser Ala Leu Glu Gly Leu Cys Asn Leu Thr Ile Glu Glu Phe 275 280 285Arg Leu Ala Tyr Leu Asp Tyr Tyr Leu Asp Asp Ile Ile Asp Leu Phe 290 295 300Asn Cys Leu Thr Asn Val Ser Ser Phe Ser Leu Val Ser Val Thr Ile305 310 315 320Glu Arg Val Lys Asp Phe Ser Tyr Asn Phe Gly Trp Gln His Leu Glu 325 330 335Leu Val Asn Cys Lys Phe Gly Gln Phe Pro Thr Leu Lys Leu Lys Ser 340 345 350Leu Lys Arg Leu Thr Phe Thr Ser Asn Lys Gly Gly Asn Ala Phe Ser 355 360 365Glu Val Asp Leu Pro Ser Leu Glu Phe Leu Asp Leu Ser Arg Asn Gly 370 375 380Leu Ser Phe Lys Gly Cys Cys Ser Gln Ser Asp Phe Gly Thr Thr Ser385 390 395 400Leu Lys Tyr Leu Asp Leu Ser Phe Asn Gly Val Ile Thr Met Ser Ser 405 410 415Asn Phe Leu Gly Leu Glu Gln Leu Glu His Leu Asp Phe Gln His Ser 420 425 430Asn Leu Lys Gln Met Ser Glu Phe Ser Val Phe Leu Ser Leu Arg Asn 435 440 445Leu Ile Tyr Leu Asp Ile Ser His Thr His Thr Arg Val Ala Phe Asn 450 455 460Gly Ile Phe Asn Gly Leu Ser Ser Leu Glu Val Leu Lys Met Ala Gly465 470 475 480Asn Ser Phe Gln Glu Asn Phe Leu Pro Asp Ile Phe Thr Glu Leu Arg 485 490 495Asn Leu Thr Phe Leu Asp Leu Ser Gln Cys Gln Leu Glu Gln Leu Ser 500 505 510Pro Thr Ala Phe Asn Ser Leu Ser Ser Leu Gln Val Leu Asn Met Ser 515 520 525His Asn Asn Phe Phe Ser Leu Asp Thr Phe Pro Tyr Lys Cys Leu Asn 530 535 540Ser Leu Gln Val Leu Asp Tyr Ser Leu Asn His Ile Met Thr Ser Lys545 550 555 560Lys Gln Glu Leu Gln His Phe Pro Ser Ser Leu Ala Phe Leu Asn Leu 565 570 575Thr Gln Asn Asp Phe Ala Cys Thr Cys Glu His Gln Ser Phe Leu Gln 580 585 590Trp Ile Lys Asp Gln Arg Gln Leu Leu Val Glu Val Glu Arg Met Glu 595 600 605Cys Ala Thr Pro Ser Asp Lys Gln Gly Met Pro Val Leu Ser Leu Asn 610 615 620Ile Thr Cys Gln Met Asn Lys Thr Ile Ile Gly Val Ser Val Leu Ser625 630 635 640Val Leu Val Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe 645 650 655His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn 660 665 670Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val 675 680 685Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln 690 695 700Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala705 710 715 720Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val 725 730 735Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu 740 745 750Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe 755 760 765Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu 770 775 780Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser785 790 795 800Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu 805 810 815Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn 820 825 830Trp Gln Glu Ala Thr Ser Ile 83532339DNAhomo sapiens 3acgcggtgcc gcggggcggg agtagaggcg gagggagggg acacgggctc attgcggtgt 60gcgccctgca ctctgtccct cactcgccgc cgacgacctg tctcgccgag cgcacgcctt 120gccgccgccc cgcagaaatg cttcggttac ccacagtctt tcgccagatg agaccggtgt 180ccagggtact ggctcctcat ctcactcggg cttatgccaa agatgtaaaa tttggtgcag 240atgcccgagc cttaatgctt caaggtgtag accttttagc cgatgctgtg gccgttacaa 300tggggccaaa gggaagaaca gtgattattg agcagagttg gggaagtccc aaagtaacaa 360aagatggtgt gactgttgca aagtcaattg acttaaaaga taaatacaaa aacattggag 420ctaaacttgt tcaagatgtt gccaataaca caaatgaaga agctggggat ggcactacca 480ctgctactgt actggcacgc tctatagcca aggaaggctt cgagaagatt agcaaaggtg 540ctaatccagt ggaaatcagg agaggtgtga tgttagctgt tgatgctgta attgctgaac 600ttaaaaagca gtctaaacct gtgaccaccc ctgaagaaat tgcacaggtt gctacgattt 660ctgcaaacgg agacaaagaa attggcaata tcatctctga tgcaatgaaa aaagttggaa 720gaaagggtgt catcacagta aaggatggaa aaacactgaa tgatgaatta gaaattattg 780aaggcatgaa gtttgatcga ggctatattt ctccatactt tattaataca tcaaaaggtc 840agaaatgtga attccaggat gcctatgttc tgttgagtga aaagaaaatt tctagtatcc 900agtccattgt acctgctctt gaaattgcca atgctcaccg taagcctttg gtcataatcg 960ctgaagatgt tgatggagaa gctctaagta cactcgtctt gaataggcta aaggttggtc 1020ttcaggttgt ggcagtcaag gctccagggt ttggtgacaa tagaaagaac cagcttaaag 1080atatggctat tgctactggt ggtgcagtgt ttggagaaga gggattgacc ctgaatcttg 1140aagacgttca gcctcatgac ttaggaaaag ttggagaggt cattgtgacc aaagacgatg 1200ccatgctctt aaaaggaaaa ggtgacaagg ctcaaattga aaaacgtatt caagaaatca 1260ttgagcagtt agatgtcaca actagtgaat atgaaaagga aaaactgaat gaacggcttg 1320caaaactttc agatggagtg gctgtgctga aggttggtgg gacaagtgat gttgaagtga 1380atgaaaagaa agacagagtt acagatgccc ttaatgctac aagagctgct gttgaagaag 1440gcattgtttt gggagggggt tgtgccctcc ttcgatgcat tccagccttg gactcattga 1500ctccagctaa tgaagatcaa aaaattggta tagaaattat taaaagaaca ctcaaaattc 1560cagcaatgac cattgctaag aatgcaggtg ttgaaggatc tttgatagtt gagaaaatta 1620tgcaaagttc ctcagaagtt ggttatgatg ctatggctgg agattttgtg aatatggtgg 1680aaaaaggaat cattgaccca acaaaggttg tgagaactgc tttattggat gctgctggtg 1740tggcctctct gttaactaca gcagaagttg tagtcacaga aattcctaaa gaagagaagg 1800accctggaat gggtgcaatg ggtggaatgg gaggtggtat gggaggtggc atgttctaac 1860tcctagacta gtgctttacc tttattaatg aactgtgaca ggaagcccaa ggcagtgttc 1920ctcaccaata acttcagaga agtcagttgg agaaaatgaa gaaaaaggct ggctgaaaat 1980cactataacc atcagttact ggtttcagtt gacaaaatat ataatggttt actgctgtca 2040ttgtccatgc ctacagataa tttattttgt atttttgaat aaaaaacatt tgtacattcc 2100tgatactggg tacaagagcc atgtaccagt gtactgcttt caacttaaat cactgaggca 2160tttttactac tattctgtta aaatcaggat tttagtgctt gccaccacca gatgagaagt 2220taagcagcct ttctgtggag agtgagaata attgtgtaca aagtagagaa gtatccaatt 2280atgtgacaac ctttgtgtaa taaaaatttg tttaaagtta aaaaaaaaaa aaaaaaaaa 23394573PRThomo sapiens 4Met Leu Arg Leu Pro Thr Val Phe Arg Gln Met Arg Pro Val Ser Arg1 5 10 15Val Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe 20 25 30Gly Ala Asp Ala Arg Ala Leu Met Leu Gln Gly Val Asp Leu Leu Ala 35 40 45Asp Ala Val Ala Val Thr Met Gly Pro Lys Gly Arg Thr Val Ile Ile 50 55 60Glu Gln Ser Trp Gly Ser Pro Lys Val Thr Lys Asp Gly Val Thr Val65 70 75 80Ala Lys Ser Ile Asp Leu Lys Asp Lys Tyr Lys Asn Ile Gly Ala Lys 85 90 95Leu Val Gln Asp Val Ala Asn Asn Thr Asn Glu Glu Ala Gly Asp Gly 100 105 110Thr Thr Thr Ala Thr Val Leu Ala Arg Ser Ile Ala Lys Glu Gly Phe 115 120 125Glu Lys Ile Ser Lys Gly Ala Asn Pro Val Glu Ile Arg Arg Gly Val 130 135 140Met Leu Ala Val Asp Ala Val Ile Ala Glu Leu Lys Lys Gln Ser Lys145 150 155 160Pro Val Thr Thr Pro Glu Glu Ile Ala Gln Val Ala Thr Ile Ser Ala 165 170 175Asn Gly Asp Lys Glu Ile Gly Asn Ile Ile Ser Asp Ala Met Lys Lys 180 185 190Val Gly Arg Lys Gly Val Ile Thr Val Lys Asp Gly Lys Thr Leu Asn 195 200 205Asp Glu Leu Glu Ile Ile Glu Gly Met Lys Phe Asp Arg Gly Tyr Ile 210 215 220Ser Pro Tyr Phe Ile Asn Thr Ser Lys Gly Gln Lys Cys Glu Phe Gln225 230 235 240Asp Ala Tyr Val Leu Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln Ser 245 250 255Ile Val Pro Ala Leu Glu Ile Ala Asn Ala His Arg Lys Pro Leu Val 260 265 270Ile Ile Ala Glu Asp Val Asp Gly Glu Ala Leu Ser Thr Leu Val Leu 275 280 285Asn Arg Leu Lys Val Gly Leu Gln Val Val Ala Val Lys Ala Pro Gly 290 295
300Phe Gly Asp Asn Arg Lys Asn Gln Leu Lys Asp Met Ala Ile Ala Thr305 310 315 320Gly Gly Ala Val Phe Gly Glu Glu Gly Leu Thr Leu Asn Leu Glu Asp 325 330 335Val Gln Pro His Asp Leu Gly Lys Val Gly Glu Val Ile Val Thr Lys 340 345 350Asp Asp Ala Met Leu Leu Lys Gly Lys Gly Asp Lys Ala Gln Ile Glu 355 360 365Lys Arg Ile Gln Glu Ile Ile Glu Gln Leu Asp Val Thr Thr Ser Glu 370 375 380Tyr Glu Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser Asp Gly385 390 395 400Val Ala Val Leu Lys Val Gly Gly Thr Ser Asp Val Glu Val Asn Glu 405 410 415Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Val 420 425 430Glu Glu Gly Ile Val Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys Ile 435 440 445Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn Glu Asp Gln Lys Ile Gly 450 455 460Ile Glu Ile Ile Lys Arg Thr Leu Lys Ile Pro Ala Met Thr Ile Ala465 470 475 480Lys Asn Ala Gly Val Glu Gly Ser Leu Ile Val Glu Lys Ile Met Gln 485 490 495Ser Ser Ser Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe Val Asn 500 505 510Met Val Glu Lys Gly Ile Ile Asp Pro Thr Lys Val Val Arg Thr Ala 515 520 525Leu Leu Asp Ala Ala Gly Val Ala Ser Leu Leu Thr Thr Ala Glu Val 530 535 540Val Val Thr Glu Ile Pro Lys Glu Glu Lys Asp Pro Gly Met Gly Ala545 550 555 560Met Gly Gly Met Gly Gly Gly Met Gly Gly Gly Met Phe 565 57052319DNAhomo sapiens 5gccccgacgc gcaccgcgat tcgccccaag ggccctgcgc aggacgctga cgcgaagact 60cggaggcgga agaaaaaagg agctgtttct aggcttttct aggcgcccag ccgagaaatg 120cttcggttac ccacagtctt tcgccagatg agaccggtgt ccagggtact ggctcctcat 180ctcactcggg cttatgccaa agatgtaaaa tttggtgcag atgcccgagc cttaatgctt 240caaggtgtag accttttagc cgatgctgtg gccgttacaa tggggccaaa gggaagaaca 300gtgattattg agcagagttg gggaagtccc aaagtaacaa aagatggtgt gactgttgca 360aagtcaattg acttaaaaga taaatacaaa aacattggag ctaaacttgt tcaagatgtt 420gccaataaca caaatgaaga agctggggat ggcactacca ctgctactgt actggcacgc 480tctatagcca aggaaggctt cgagaagatt agcaaaggtg ctaatccagt ggaaatcagg 540agaggtgtga tgttagctgt tgatgctgta attgctgaac ttaaaaagca gtctaaacct 600gtgaccaccc ctgaagaaat tgcacaggtt gctacgattt ctgcaaacgg agacaaagaa 660attggcaata tcatctctga tgcaatgaaa aaagttggaa gaaagggtgt catcacagta 720aaggatggaa aaacactgaa tgatgaatta gaaattattg aaggcatgaa gtttgatcga 780ggctatattt ctccatactt tattaataca tcaaaaggtc agaaatgtga attccaggat 840gcctatgttc tgttgagtga aaagaaaatt tctagtatcc agtccattgt acctgctctt 900gaaattgcca atgctcaccg taagcctttg gtcataatcg ctgaagatgt tgatggagaa 960gctctaagta cactcgtctt gaataggcta aaggttggtc ttcaggttgt ggcagtcaag 1020gctccagggt ttggtgacaa tagaaagaac cagcttaaag atatggctat tgctactggt 1080ggtgcagtgt ttggagaaga gggattgacc ctgaatcttg aagacgttca gcctcatgac 1140ttaggaaaag ttggagaggt cattgtgacc aaagacgatg ccatgctctt aaaaggaaaa 1200ggtgacaagg ctcaaattga aaaacgtatt caagaaatca ttgagcagtt agatgtcaca 1260actagtgaat atgaaaagga aaaactgaat gaacggcttg caaaactttc agatggagtg 1320gctgtgctga aggttggtgg gacaagtgat gttgaagtga atgaaaagaa agacagagtt 1380acagatgccc ttaatgctac aagagctgct gttgaagaag gcattgtttt gggagggggt 1440tgtgccctcc ttcgatgcat tccagccttg gactcattga ctccagctaa tgaagatcaa 1500aaaattggta tagaaattat taaaagaaca ctcaaaattc cagcaatgac cattgctaag 1560aatgcaggtg ttgaaggatc tttgatagtt gagaaaatta tgcaaagttc ctcagaagtt 1620ggttatgatg ctatggctgg agattttgtg aatatggtgg aaaaaggaat cattgaccca 1680acaaaggttg tgagaactgc tttattggat gctgctggtg tggcctctct gttaactaca 1740gcagaagttg tagtcacaga aattcctaaa gaagagaagg accctggaat gggtgcaatg 1800ggtggaatgg gaggtggtat gggaggtggc atgttctaac tcctagacta gtgctttacc 1860tttattaatg aactgtgaca ggaagcccaa ggcagtgttc ctcaccaata acttcagaga 1920agtcagttgg agaaaatgaa gaaaaaggct ggctgaaaat cactataacc atcagttact 1980ggtttcagtt gacaaaatat ataatggttt actgctgtca ttgtccatgc ctacagataa 2040tttattttgt atttttgaat aaaaaacatt tgtacattcc tgatactggg tacaagagcc 2100atgtaccagt gtactgcttt caacttaaat cactgaggca tttttactac tattctgtta 2160aaatcaggat tttagtgctt gccaccacca gatgagaagt taagcagcct ttctgtggag 2220agtgagaata attgtgtaca aagtagagaa gtatccaatt atgtgacaac ctttgtgtaa 2280taaaaatttg tttaaagtta aaaaaaaaaa aaaaaaaaa 23196573PRThomo sapiens 6Met Leu Arg Leu Pro Thr Val Phe Arg Gln Met Arg Pro Val Ser Arg1 5 10 15Val Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe 20 25 30Gly Ala Asp Ala Arg Ala Leu Met Leu Gln Gly Val Asp Leu Leu Ala 35 40 45Asp Ala Val Ala Val Thr Met Gly Pro Lys Gly Arg Thr Val Ile Ile 50 55 60Glu Gln Ser Trp Gly Ser Pro Lys Val Thr Lys Asp Gly Val Thr Val65 70 75 80Ala Lys Ser Ile Asp Leu Lys Asp Lys Tyr Lys Asn Ile Gly Ala Lys 85 90 95Leu Val Gln Asp Val Ala Asn Asn Thr Asn Glu Glu Ala Gly Asp Gly 100 105 110Thr Thr Thr Ala Thr Val Leu Ala Arg Ser Ile Ala Lys Glu Gly Phe 115 120 125Glu Lys Ile Ser Lys Gly Ala Asn Pro Val Glu Ile Arg Arg Gly Val 130 135 140Met Leu Ala Val Asp Ala Val Ile Ala Glu Leu Lys Lys Gln Ser Lys145 150 155 160Pro Val Thr Thr Pro Glu Glu Ile Ala Gln Val Ala Thr Ile Ser Ala 165 170 175Asn Gly Asp Lys Glu Ile Gly Asn Ile Ile Ser Asp Ala Met Lys Lys 180 185 190Val Gly Arg Lys Gly Val Ile Thr Val Lys Asp Gly Lys Thr Leu Asn 195 200 205Asp Glu Leu Glu Ile Ile Glu Gly Met Lys Phe Asp Arg Gly Tyr Ile 210 215 220Ser Pro Tyr Phe Ile Asn Thr Ser Lys Gly Gln Lys Cys Glu Phe Gln225 230 235 240Asp Ala Tyr Val Leu Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln Ser 245 250 255Ile Val Pro Ala Leu Glu Ile Ala Asn Ala His Arg Lys Pro Leu Val 260 265 270Ile Ile Ala Glu Asp Val Asp Gly Glu Ala Leu Ser Thr Leu Val Leu 275 280 285Asn Arg Leu Lys Val Gly Leu Gln Val Val Ala Val Lys Ala Pro Gly 290 295 300Phe Gly Asp Asn Arg Lys Asn Gln Leu Lys Asp Met Ala Ile Ala Thr305 310 315 320Gly Gly Ala Val Phe Gly Glu Glu Gly Leu Thr Leu Asn Leu Glu Asp 325 330 335Val Gln Pro His Asp Leu Gly Lys Val Gly Glu Val Ile Val Thr Lys 340 345 350Asp Asp Ala Met Leu Leu Lys Gly Lys Gly Asp Lys Ala Gln Ile Glu 355 360 365Lys Arg Ile Gln Glu Ile Ile Glu Gln Leu Asp Val Thr Thr Ser Glu 370 375 380Tyr Glu Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser Asp Gly385 390 395 400Val Ala Val Leu Lys Val Gly Gly Thr Ser Asp Val Glu Val Asn Glu 405 410 415Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Val 420 425 430Glu Glu Gly Ile Val Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys Ile 435 440 445Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn Glu Asp Gln Lys Ile Gly 450 455 460Ile Glu Ile Ile Lys Arg Thr Leu Lys Ile Pro Ala Met Thr Ile Ala465 470 475 480Lys Asn Ala Gly Val Glu Gly Ser Leu Ile Val Glu Lys Ile Met Gln 485 490 495Ser Ser Ser Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe Val Asn 500 505 510Met Val Glu Lys Gly Ile Ile Asp Pro Thr Lys Val Val Arg Thr Ala 515 520 525Leu Leu Asp Ala Ala Gly Val Ala Ser Leu Leu Thr Thr Ala Glu Val 530 535 540Val Val Thr Glu Ile Pro Lys Glu Glu Lys Asp Pro Gly Met Gly Ala545 550 555 560Met Gly Gly Met Gly Gly Gly Met Gly Gly Gly Met Phe 565 57072826DNAhomo sapiens 7acaagggtgg cagcctggct ctgaagtgga attatgtgct tcaaacaggt tgaaagaggg 60aaacagtctt ttcctgcttc cagacatgaa tcaggtcact attcaatggg atgcagtaat 120agccctttac atactcttca gctggtgtca tggaggaatt acaaatataa actgctctgg 180ccacatctgg gtagaaccag ccacaatttt taagatgggt atgaatatct ctatatattg 240ccaagcagca attaagaact gccaaccaag gaaacttcat ttttataaaa atggcatcaa 300agaaagattt caaatcacaa ggattaataa aacaacagct cggctttggt ataaaaactt 360tctggaacca catgcttcta tgtactgcac tgctgaatgt cccaaacatt ttcaagagac 420actgatatgt ggaaaagaca tttcttctgg atatccgcca gatattcctg atgaagtaac 480ctgtgtcatt tatgaatatt caggcaacat gacttgcacc tggaatgctg ggaagctcac 540ctacatagac acaaaatacg tggtacatgt gaagagttta gagacagaag aagagcaaca 600gtatctcacc tcaagctata ttaacatctc cactgattca ttacaaggtg gcaagaagta 660cttggtttgg gtccaagcag caaacgcact aggcatggaa gagtcaaaac aactgcaaat 720tcacctggat gatatagtga taccttctgc agccgtcatt tccagggctg agactataaa 780tgctacagtg cccaagacca taatttattg ggatagtcaa acaacaattg aaaaggtttc 840ctgtgaaatg agatacaagg ctacaacaaa ccaaacttgg aatgttaaag aatttgacac 900caattttaca tatgtgcaac agtcagaatt ctacttggag ccaaacatta agtacgtatt 960tcaagtgaga tgtcaagaaa caggcaaaag gtactggcag ccttggagtt cactgttttt 1020tcataaaaca cctgaaacag ttccccaggt cacatcaaaa gcattccaac atgacacatg 1080gaattctggg ctaacagttg cttccatctc tacagggcac cttacttctg acaacagagg 1140agacattgga cttttattgg gaatgatcgt ctttgctgtt atgttgtcaa ttctttcttt 1200gattgggata tttaacagat cattccgaac tgggattaaa agaaggatct tattgttaat 1260accaaagtgg ctttatgaag atattcctaa tatgaaaaac agcaatgttg tgaaaatgct 1320acaggaaaat agtgaactta tgaataataa ttccagtgag caggtcctat atgttgatcc 1380catgattaca gagataaaag aaatcttcat cccagaacac aagcctacag actacaagaa 1440ggagaataca ggacccctgg agacaagaga ctacccgcaa aactcgctat tcgacaatac 1500tacagttgta tatattcctg atctcaacac tggatataaa ccccaaattt caaattttct 1560gcctgaggga agccatctca gcaataataa tgaaattact tccttaacac ttaaaccacc 1620agttgattcc ttagactcag gaaataatcc caggttacaa aagcatccta attttgcttt 1680ttctgtttca agtgtgaatt cactaagcaa cacaatattt cttggagaat taagcctcat 1740attaaatcaa ggagaatgca gttctcctga catacaaaac tcagtagagg aggaaaccac 1800catgcttttg gaaaatgatt cacccagtga aactattcca gaacagaccc tgcttcctga 1860tgaatttgtc tcctgtttgg ggatcgtgaa tgaggagttg ccatctatta atacttattt 1920tccacaaaat attttggaaa gccacttcaa taggatttca ctcttggaaa agtagagctg 1980tgtggtcaaa atcaatatga gaaagctgcc ttgcaatctg aacttgggtt ttccctgcaa 2040tagaaattga attctgcctc tttttgaaaa aaatgtattc acatacaaat cttcacatgg 2100acacatgttt tcatttccct tggataaata cctaggtagg ggattgctgg gccatatgat 2160aagcatatgt ttcagttcta ccaatcttgt ttccagagta gtgacatttc tgtgctccta 2220ccatcaccat gtaagaattc ccgggagctc catgcctttt taattttagc cattcttctg 2280cctcatttct taaaattaga gaattaaggt cccgaaggtg gaacatgctt catggtcaca 2340catacaggca caaaaacagc attatgtgga cgcctcatgt attttttata gagtcaacta 2400tttcctcttt attttccctc attgaaagat gcaaaacagc tctctattgt gtacagaaag 2460ggtaaataat gcaaaatacc tggtagtaaa ataaatgctg aaaattttcc tttaaaatag 2520aatcattagg ccaggcgtgg tggctcatgc ttgtaatccc agcactttgg taggctgagg 2580taggtggatc acctgaggtc aggagttcga gtccagcctg gccaatatgc tgaaaccctg 2640tctctactaa aattacaaaa attagccggc catggtggca ggtgcttgta atcccagcta 2700cttgggaggc tgaggcagga gaatcacttg aaccaggaag gcagaggttg cactgagctg 2760agattgtgcc actgcactcc agcctgggca acaagagcaa aactctgtct ggaaaaaaaa 2820aaaaaa 28268629PRThomo sapiens 8Met Asn Gln Val Thr Ile Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile1 5 10 15Leu Phe Ser Trp Cys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly 20 25 30His Ile Trp Val Glu Pro Ala Thr Ile Phe Lys Met Gly Met Asn Ile 35 40 45Ser Ile Tyr Cys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu 50 55 60His Phe Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile65 70 75 80Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys Asn Phe Leu Glu Pro His 85 90 95Ala Ser Met Tyr Cys Thr Ala Glu Cys Pro Lys His Phe Gln Glu Thr 100 105 110Leu Ile Cys Gly Lys Asp Ile Ser Ser Gly Tyr Pro Pro Asp Ile Pro 115 120 125Asp Glu Val Thr Cys Val Ile Tyr Glu Tyr Ser Gly Asn Met Thr Cys 130 135 140Thr Trp Asn Ala Gly Lys Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val145 150 155 160His Val Lys Ser Leu Glu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser 165 170 175Ser Tyr Ile Asn Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys Tyr 180 185 190Leu Val Trp Val Gln Ala Ala Asn Ala Leu Gly Met Glu Glu Ser Lys 195 200 205Gln Leu Gln Ile His Leu Asp Asp Ile Val Ile Pro Ser Ala Ala Val 210 215 220Ile Ser Arg Ala Glu Thr Ile Asn Ala Thr Val Pro Lys Thr Ile Ile225 230 235 240Tyr Trp Asp Ser Gln Thr Thr Ile Glu Lys Val Ser Cys Glu Met Arg 245 250 255Tyr Lys Ala Thr Thr Asn Gln Thr Trp Asn Val Lys Glu Phe Asp Thr 260 265 270Asn Phe Thr Tyr Val Gln Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile 275 280 285Lys Tyr Val Phe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp 290 295 300Gln Pro Trp Ser Ser Leu Phe Phe His Lys Thr Pro Glu Thr Val Pro305 310 315 320Gln Val Thr Ser Lys Ala Phe Gln His Asp Thr Trp Asn Ser Gly Leu 325 330 335Thr Val Ala Ser Ile Ser Thr Gly His Leu Thr Ser Asp Asn Arg Gly 340 345 350Asp Ile Gly Leu Leu Leu Gly Met Ile Val Phe Ala Val Met Leu Ser 355 360 365Ile Leu Ser Leu Ile Gly Ile Phe Asn Arg Ser Phe Arg Thr Gly Ile 370 375 380Lys Arg Arg Ile Leu Leu Leu Ile Pro Lys Trp Leu Tyr Glu Asp Ile385 390 395 400Pro Asn Met Lys Asn Ser Asn Val Val Lys Met Leu Gln Glu Asn Ser 405 410 415Glu Leu Met Asn Asn Asn Ser Ser Glu Gln Val Leu Tyr Val Asp Pro 420 425 430Met Ile Thr Glu Ile Lys Glu Ile Phe Ile Pro Glu His Lys Pro Thr 435 440 445Asp Tyr Lys Lys Glu Asn Thr Gly Pro Leu Glu Thr Arg Asp Tyr Pro 450 455 460Gln Asn Ser Leu Phe Asp Asn Thr Thr Val Val Tyr Ile Pro Asp Leu465 470 475 480Asn Thr Gly Tyr Lys Pro Gln Ile Ser Asn Phe Leu Pro Glu Gly Ser 485 490 495His Leu Ser Asn Asn Asn Glu Ile Thr Ser Leu Thr Leu Lys Pro Pro 500 505 510Val Asp Ser Leu Asp Ser Gly Asn Asn Pro Arg Leu Gln Lys His Pro 515 520 525Asn Phe Ala Phe Ser Val Ser Ser Val Asn Ser Leu Ser Asn Thr Ile 530 535 540Phe Leu Gly Glu Leu Ser Leu Ile Leu Asn Gln Gly Glu Cys Ser Ser545 550 555 560Pro Asp Ile Gln Asn Ser Val Glu Glu Glu Thr Thr Met Leu Leu Glu 565 570 575Asn Asp Ser Pro Ser Glu Thr Ile Pro Glu Gln Thr Leu Leu Pro Asp 580 585 590Glu Phe Val Ser Cys Leu Gly Ile Val Asn Glu Glu Leu Pro Ser Ile 595 600 605Asn Thr Tyr Phe Pro Gln Asn Ile Leu Glu Ser His Phe Asn Arg Ile 610 615 620Ser Leu Leu Glu Lys62591201DNAhomo sapiens 9aatattagag tctcaacccc caataaatat aggactggag atgtctgagg ctcattctgc 60cctcgagccc accgggaacg aaagagaagc tctatctccc ctccaggagc ccagctatga 120actccttctc cacaagcgcc ttcggtccag ttgccttctc cctggggctg ctcctggtgt 180tgcctgctgc cttccctgcc ccagtacccc caggagaaga ttccaaagat gtagccgccc 240cacacagaca gccactcacc tcttcagaac gaattgacaa acaaattcgg tacatcctcg 300acggcatctc agccctgaga aaggagacat gtaacaagag taacatgtgt gaaagcagca 360aagaggcact ggcagaaaac aacctgaacc ttccaaagat ggctgaaaaa gatggatgct 420tccaatctgg attcaatgag gagacttgcc tggtgaaaat catcactggt cttttggagt 480ttgaggtata cctagagtac ctccagaaca gatttgagag tagtgaggaa caagccagag 540ctgtgcagat gagtacaaaa gtcctgatcc agttcctgca gaaaaaggca aagaatctag 600atgcaataac cacccctgac ccaaccacaa atgccagcct gctgacgaag ctgcaggcac 660agaaccagtg gctgcaggac atgacaactc atctcattct gcgcagcttt aaggagttcc 720tgcagtccag cctgagggct cttcggcaaa tgtagcatgg gcacctcaga ttgttgttgt 780taatgggcat tccttcttct ggtcagaaac
ctgtccactg ggcacagaac ttatgttgtt 840ctctatggag aactaaaagt atgagcgtta ggacactatt ttaattattt ttaatttatt 900aatatttaaa tatgtgaagc tgagttaatt tatgtaagtc atatttatat ttttaagaag 960taccacttga aacattttat gtattagttt tgaaataata atggaaagtg gctatgcagt 1020ttgaatatcc tttgtttcag agccagatca tttcttggaa agtgtaggct tacctcaaat 1080aaatggctaa cttatacata tttttaaaga aatatttata ttgtatttat ataatgtata 1140aatggttttt ataccaataa atggcatttt aaaaaattca gcaaaaaaaa aaaaaaaaaa 1200a 120110212PRThomo sapiens 10Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala Phe Ser Leu1 5 10 15Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala Pro Val Pro Pro 20 25 30Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu Thr 35 40 45Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly Ile 50 55 60Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys Glu Ser65 70 75 80Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala 85 90 95Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu 100 105 110Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr 115 120 125Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala Val Gln 130 135 140Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys Asn145 150 155 160Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 165 170 175Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His 180 185 190Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala 195 200 205Leu Arg Gln Met 21011808DNAhomo sapiens 11gaacacaggc atacacagga agatacattc acagaaagag cttcctgcac aaagtaagcc 60accagcgcaa catgacagtg aagaccctgc atggcccagc catggtcaag tacttgctgc 120tgtcgatatt ggggcttgcc tttctgagtg aggcggcagc tcggaaaatc cccaaagtag 180gacatacttt tttccaaaag cctgagagtt gcccgcctgt gccaggaggt agtatgaagc 240ttgacattgg catcatcaat gaaaaccagc gcgtttccat gtcacgtaac atcgagagcc 300gctccacctc cccctggaat tacactgtca cttgggaccc caaccggtac ccctcggaag 360ttgtacaggc ccagtgtagg aacttgggct gcatcaatgc tcaaggaaag gaagacatct 420ccatgaattc cgttcccatc cagcaagaga ccctggtcgt ccggaggaag caccaaggct 480gctctgtttc tttccagttg gagaaggtgc tggtgactgt tggctgcacc tgcgtcaccc 540ctgtcatcca ccatgtgcag taagaggtgc atatccactc agctgaagaa gctgtagaaa 600tgccactcct tacccagtgc tctgcaacaa gtcctgtctg acccccaatt ccctccactt 660cacaggactc ttaataagac ctgcacggat ggaaacagaa aatattcaca atgtatgtgt 720gtatgtacta cactttatat ttgatatcta aaatgttagg agaaaaatta atatattcag 780tgctaatata ataaagtatt aataattt 80812163PRThomo sapiens 12Met Thr Val Lys Thr Leu His Gly Pro Ala Met Val Lys Tyr Leu Leu1 5 10 15Leu Ser Ile Leu Gly Leu Ala Phe Leu Ser Glu Ala Ala Ala Arg Lys 20 25 30Ile Pro Lys Val Gly His Thr Phe Phe Gln Lys Pro Glu Ser Cys Pro 35 40 45Pro Val Pro Gly Gly Ser Met Lys Leu Asp Ile Gly Ile Ile Asn Glu 50 55 60Asn Gln Arg Val Ser Met Ser Arg Asn Ile Glu Ser Arg Ser Thr Ser65 70 75 80Pro Trp Asn Tyr Thr Val Thr Trp Asp Pro Asn Arg Tyr Pro Ser Glu 85 90 95Val Val Gln Ala Gln Cys Arg Asn Leu Gly Cys Ile Asn Ala Gln Gly 100 105 110Lys Glu Asp Ile Ser Met Asn Ser Val Pro Ile Gln Gln Glu Thr Leu 115 120 125Val Val Arg Arg Lys His Gln Gly Cys Ser Val Ser Phe Gln Leu Glu 130 135 140Lys Val Leu Val Thr Val Gly Cys Thr Cys Val Thr Pro Val Ile His145 150 155 160His Val Gln
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text:
A new and bright way to buy and sell cranes and lifting equipment online is the website of machineryequipment .
Whether it is a new crane or a second hand crane that a company has out grown; the machineryequipment is the website to see. The website has all the top brands and the recent crane and lifting technology; the website does not sell the products direct; they advertise all good deals around Australia.
The beauty of the machineryequipment website is that one is in control of advertising one's product; the negation of purchasing is a simple as finding the product that one wants and contacting the supplier.

subset: commoncrawl


text:
Pre-Owned, Item code 11HB30
Free Packing and Shipping Cost Depending of the Zip Code or Pick Up in Countryside, IL 60525
Hobart 30 Quart Flat Beater "B"
Model# DS-30-B / 9875
Orginal Hobart attachment has been tested with Model D300.
Approx. size:
Approx. size of the Shaft Hole: 1 1/8" diameter.
Approx. weight: 2.5 Lbs.
NSF Approved.
Disk (W) W
Used Restaurant Equipment, Restaurant Equipment
9685 West 55th Street Countryside, Illinois 60525
Phone 708-354-1265 or Fax 708-354-1361
Monday-Friday 8:30 a.m. to 5:00 p.m. CSTSaturday 8:30 a.m. to 3:00 p.m. CST
Copyright © 2002-2050 Pierce Food Service Equipment Corporation Inc. All Rights Reserved.
Pierce Chef's Mart (TM) is a division of Pierce Food Service Equipment Corporation, Inc.
UPS, the UPS brandmark and the color
brown are trademarks that are used with permissionby the owner,
United Parcel Service of America, Inc. All rights reserved.


text:
Funny Commercial - 7-UpPosted by Danny Vice at 3:09 PM
This Funny 7up commercial ran a few years back. 7 up creates vending machines that come to you.
funny 7 up video, 7 up commercial, seven up commercial, funny seven up commercial, funny 7 up, 7 up commercials, funny commercials, funny beer commercials, beer commercials, soda commercial, 7 up, seven up, commercial, commercials, joke commercial, humorous commercials
Share Humor Vice with your friends, family or coworkers. Humor Vice sorts through literally thousands of funny pictures and videos and posts the very best of them. Enjoy the funniest videos, candid camera pranks, hidden camera pranks, practical jokes, funny commercials, the very best beer commercials, funny moments caught on camera, naughty humor, girl humor, silly humor, stand up comedy, funny mpegs, embarrassing videos, video clips, funny video collections, caught on camera, funny jokes, video outtakes, skits, sexy funny videos and more!
Funny Commercial - 7-Up
Danny Vice
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text:
Gauteng DestinationsFerndale, Randburg
One of Johannesburgs northern leafy burbs that falls just outside of the Randburg CBD, Ferndale lies close to some of the main business and entertainment areas that include Cresta Centre, Brightwater Commons (formerly known as the Randburg Waterfront), the Monte Casino and The Dome.
This is a particularly pretty part of Johannesburg. The elite, boomed suburbs of Sandton it is not, but Ferndale has an unassuming charm that gives it and other tree-lined northern suburbs their own appeal.
One of the main advantages of staying here is the central location - you can get anywhere relatively quickly, if you know where youre going and how best to get there. Sandton is a mere trip along Republic Road however it is best to venture along this route outside of peak hour. Ferndale is packed with shops, restaurants and business parks. Yet Ferndale and its neighbouring suburbs of Robindale, Fontainebleau and Blairgowrie are not short of open spaces and street trees as well as large, cultivated gardens that account for the feeling of space.
The Johannesburg Botanic Gardens at Emmarentia Dam in Emmarentia are not far from Ferndale and offer a wonderful sanctuary away from the rush of Johannesburg. The dam is regarded as the best dog walk in the city, and is also the site of a range of Summer Sunset Concerts.


text:
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A fifth grader from Laurel Elementary School created the art for an anti-tobacco campaign going up in areas of Southwest and Southside Virginia.
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