Patent application title: GENETIC MARKERS ASSOCIATED WITH AGE-RELATED MACULAR DEGENERATION, METHODS OF DETECTION AND USES THEREOF
Inventors:
John R. W. Yates (Cambridge, GB)
Assignees:
Cambridge Enterprise Limited
IPC8 Class: AC40B3004FI
USPC Class:
506 9
Class name: Combinatorial chemistry technology: method, library, apparatus method of screening a library by measuring the ability to specifically bind a target molecule (e.g., antibody-antigen binding, receptor-ligand binding, etc.)
Publication date: 2012-07-26
Patent application number: 20120190568
Abstract:
Disclosed is a method for identifying an individual who has an altered
risk for developing age related macular degeneration comprising detecting
a single nucleotide polymorphism (SNP)Claims:
1-29. (canceled)
30. A method for assessing the probability of a therapeutic; or toxic response to a chemical, protein, nucleic acid-based therapeutic or other biological, naturally or synthetic entity in a human subject who has or who may have age-related macular degeneration or any pathological process substantially similar, the method comprising the steps of: i) obtaining a biological sample from a human subject; ii) analyzing the sample to determine whether the subject carries one or more of: a) C or G at rs2230199 of the complement 3 (C3) gene, or b) the amino acid arginine or glycine at position 102 of the C3 protein.
31. A method according to claim 30 wherein the biological sample is analyzed by a method comprising assaying DNA in said sample for the presence of at least one allele of the complement component 3 (C3) gene comprising a G nucleotide at the single nucleotide polymorphism (SNP) rs2230199 at the position corresponding to position 366 of SEQ ID NO:1, wherein the presence of at least one allele of the C3 gene comprising a G nucleotide at the single nucleotide polymorphism (SNP) rs2230199 at the position corresponding to position 366 of SEQ ID NO:1 is indicative of an increased probability of a therapeutic or toxic response to a chemical, protein, nucleic acid-based therapeutic or other biological, naturally or synthetic entity in the human subject.
32. A method of claim 31 wherein said assaying comprises; amplifying the DNA in the presence of a pair of primers wherein a first of the primers comprises at least 10 consecutive nucleotides selected from the sequence identified as SEQ ID NO 1 and located upstream of the base located at position 366 of the sequence and a second primer comprising at least 10 consecutive nucleotides selected from within the same sequence and located downstream of the base located at position 366; and determining the identity of the base in the amplified genetic material that corresponds to position 366.
33. A method according to claim 32 wherein the amplified genetic material is between about 16 and about 2000 nucleotides in length,
34. A method according to claim 31 wherein said assaying comprises; contacting the biological sample with a reagent which specifically hybridizes under stringent hybridization conditions to an allele of the complement component 3 (C3) gene comprising a G nucleotide at the single nucleotide polymorphism (SNP) rs2230199 at the position corresponding to position 366 of SEQ ID NO:1, or a reagent which specifically hybridizes under stringent hybridization conditions to an allele of the complement component 3 (C3) gene comprising a C nucleotide at the single nucleotide polymorphism (SNP) rs2230199 at the position corresponding to position 366 of SEQ ID NO:1, and detecting the formation of a hybridized duplex.
35. A method according to claim 34 in which detection is carried out by a process which may be selected from the group consisting of: allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, sequencing, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, and single-stranded conformation polymorphism.
36. A method according to claim 34 wherein the reagent is a polynucleotide probe.
37. A method according to claim 36 wherein the polynucleotide probe is contained in a rnicroarray.
38. A method according to claim 30 wherein the biological sample is analyzed by a method comprising assaying said sample for the presence of a C3 protein comprising a glycine amino acid at the position corresponding to position 102 of SEQ ID NO:2; wherein the presence of a C3 protein comprising a glycine amino acid at the position corresponding to position 102 of SEQ ID N0:2 is indicative of an increased probability of a therapeutic; or toxic response to a chemical, protein, nucleic acid-based therapeutic; or other biological, naturally or synthetic; entity in the human subject.
39. A method according to claim 38 wherein said assaying comprises; contacting a test sample with a specific binding member which binds to a variant polypeptide encoded by a nucleotide sequence which comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO: 3 with a polymorphic variant sequence at a site of single nucleotide polymorphism (SNP) therein, and detecting the binding of the specific binding member to polypeptide in the sample.
40. A method according to claim 30, wherein the subject is asymptomatic of macular degeneration.
41. A method according to claim 30, wherein the subject has been diagnosed as having symptoms of macular degeneration.
42. A method according to claim 30, wherein the sample is from blood, saliva, sputum, urine, cell scrapings or biopsy tissue.
43. The method of claim 31 further comprising determining whether the subject is homozygous or heterozygous for said polymorphism.
44. A method comprising the steps of: assaying a biological sample from a human and detecting the presence of (i) at least one allele of the complement component 3 (C3) gene comprising a G nucleotide at the single nucleotide polymorphism (SNP) rs2230199 at the position corresponding to position 366 of SEQ ID NO: 1; or (ii) a C3 protein comprising a glycine amino acid at the position corresponding to position 102 of SEQ ID NO: 2; wherein the presence of at least one allele of the C3 gene comprising a nucleotide at the single nucleotide polymorphism (SNP) rs2230199 at the position corresponding to position 366 of SEQ ID NO: 1 or a C3 protein comprising a glycine amino add at the position corresponding to position 102 of SEQ ID NO:2 is indicative of an increased risk of development of macular degeneration in said human.
45. The method of claim 44, wherein said biological sample is DNA and said assaying comprises: amplifying the DNA in the presence of a pair of primers wherein a first primer comprises at least 10 consecutive nucleotides of one of SEQ ID NO: 1 or the complement of SEQ ID NO:1 and is located upstream of the base located at position 366 of SEQ ID NO:1, and a second primer comprises at least 10 consecutive nucleotides of the other of SEQ ID NO:1 or the complement of SEQ ID NO:1 and is located downstream of the base located at position 366 of SEQ ID NO:1; and determining the identity of the base in the amplified genetic material that corresponds to position 366 of SEQ ID NO: 1.
46. A method comprising the steps of: assaying a biological sample from a human and detecting the presence of (i) at least one allele of the complement component 3 (C3) gene comprising a G nucleotide at the single nucleotide polymorphism (SNP) rs2230199 at the position corresponding to position 366 of SEQ ID NO: 1; or (ii) a C3 protein comprising a glycine amino acid at the position corresponding to position 102 of SEQ ID NO: 2; wherein the presence of at least one allele of the C3 gene comprising a nucleotide at the single nucleotide polymorphism (SNP) rs2230199 at the position corresponding to position 366 of SEQ ID NO: 1 or a C3 protein comprising a glycine amino add at the position corresponding to position 102 of SEQ ID NO:2 is indicative of an increased risk of development of macular degeneration characterized by geographic atrophy or exudative disease in said human.
47. The method of claim 44, wherein said biological sample is DNA and said assaying comprises: amplifying the DNA in the presence of a pair of primers wherein a first primer comprises at least 10 consecutive nucleotides of one of SEQ ID NO: 1 or the complement of SEG ID Nal and is located upstream of the base located at position 366 of SEQ ID NO:1, and a second primer comprises at least 10 consecutive nucleotides of the other of SEQ ID NO:1 or the complement of SEC) ID NO:1 and is located downstream of the base located at position 366 of SEQ ID NO:1; and determining the identity of the base in the amplified genetic material that corresponds to position 366 of SEQ ID NO: 1.
Description:
[0001] This application is a divisional of Ser. No. 12/382,569 (allowed),
filed Mar. 18, 2009 (published as US 2009-0269761-A1), which claims the
benefit of U.S. Provisional Application No. 61/037,411 filed on Mar. 18,
2008, the entire contents of each of which is hereby incorporated by
reference.
TECHNICAL FIELD OF THE INVENTION
[0002] This invention is related to the area of genetic testing, drug discovery, and Age-Related Macular Degeneration. In particular, it relates to genetic variants found within the complement cascade C3 gene which increase the risk of Age-Related Macular Degeneration.
BACKGROUND OF THE INVENTION
[0003] Age-related macular degeneration (AMD) causes progressive impairment of central vision and is the leading cause of irreversible vision loss in older Americans(1). The most severe form of AMD involves neovascular/exudative (wet) and/or atrophic (dry) changes to the macula. Although the etiology of AMD remains largely unknown, implicated risk factors include age, ethnicity, smoking, hypertension, obesity and diet (2). Familial aggregation (3), twin studies (4), and segregation analysis(5) suggest that there is also a significant genetic contribution to the disease. The candidate gene approach and genome-wide association studies have consistently implicated the CFH, ARMS2 and C2/BF genes, all members of the complement-mediated inflammatory cascade.
[0004] Age-related macular degeneration (AMD) is a common complex disorder that affects the central region of the retina (macula) and is the leading cause of legal blindness in older American adults. The prevalence of AMD and its significant morbidity will rise sharply as the population ages. AMD is a clinically heterogeneous disorder with a poorly understood etiology. Population-based longitudinal studies(6-8) have established that the presence of extracellular protein/lipid deposits (drusen) between the basal lamina of the retinal pigment epithelium (RPE) and the inner layer of Bruchs' membrane is associated with an increased risk of progressing to an advanced form of AMD, either geographic atrophy or exudative disease. The presence of large and indistinct (soft) drusen coupled with RPE abnormalities is considered an early form of the disorder and is often referred to as age-related maculopathy (ARM).
[0005] Epidemiology: AMD is a complex disorder with contributions of environmental factors as well as genetic susceptibility(2). Many environmental and lifestyle factors have been postulated, but by far the most consistently implicated non-genetic risk factor for AMD is cigarette smoking (6). Much progress has been made in identifying and characterizing the genetic basis of AMD. In a remarkable example of the convergence of methods for disease gene discovery, multiple independent research efforts identified the Y402H variant in the complement factor H (CFH [(MIM 134370]) gene on chromosome 1q32 as the first major AMD susceptibility allele (9-14). While one of the studies was able to pinpoint CFH on the basis of a whole-genome association study (11), most studies focused on the 1q32 region because it had consistently been implicated by several whole-genome linkage scans. Disease associated haplotypes within the CFH gene are also associated with AMD (15). A second genomic region with similarly consistent linkage evidence is chromosome 10q26, which was identified as the single most promising region by a recent meta-analysis of published linkage screens (16).
[0006] Two studies have suggested specific AMD susceptibility genes located on chromosome 10q26. One used a combination of family-based and case-control analyses to implicate the PLEKHA1 gene (pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 1 [MIM 607772]) and the predicted ARMS2 gene (14;17;18). ARMS2 appears to be a mitochondrial membrane protein involved in inflammation (19) A second study using two independent case-control datasets concluded that the T allele of SNP rs10490924 in ARMS2, a coding change (Ala69Ser) in exon 1 of this gene, was the most likely AMD susceptibility allele (16). Both studies reported that the chromosome 10q26 variant confers an AMD risk similar in magnitude to that of the Y402H variant in CFH. A locus with less strong association, but reproducible association with AMD is the complement component 2 (C2) and Factor B (C2/BF) locus within the major histocompatability complex III locus found on chromosome 6 The L9H variant of BF and the E318D variant of C2 , as well as a variant in intron 10 of C2 and the R32Q variant of BF, confer a significantly reduced risk of AMD (20).
SUMMARY OF THE INVENTION
[0007] Here, we describe highly significant association of SNPs within the C3 gene (NCBI GeneID: 718), specifically rs2230199 (Arg102Gly) found on chromosome 19 with age related macular degeneration and its use, alone or in combination, in predicting predisposition to this disease (21). We have thus established that identification of the nucleotide residue at rs2230199 can predict the predisposition of an individual to AMD. Related findings have since been published by Maller et al. (22).
[0008] According to some embodiments of the invention, a method is provided for assessing increased risk of Age Related Macular Degeneration. The identity is determined of at least one nucleotide residue of the genomic germ-line C3 coding sequence of an individual The nucleotide residue is identified as normal or variant by comparing it to a normal genomic germ-line sequence of C3 coding sequence as shown in SEQ ID NO:1 (coding sequence) or SEQ ID NO: 3 (genomic sequence). A normal nucleotide residue is identical to the corresponding nucleotide residue in the normal genomic germ-line sequence of C3. A variant nucleotide residue is not identical to the corresponding nucleotide residue in the normal genomic germ-line sequence of C3. A variant C3 coding sequence may contain at least one variant nucleotide residue relative to the normal C3 coding sequence. An individual with a variant sequence has a higher risk of Age Related Macular Degeneration than an individual with a normal sequence.
[0009] According to some embodiments, a method is provided for assessing increased risk of Age Related Macular Degeneration. The identity is determined of at least one amino acid residue of the C3 protein of an individual. The at least one amino acid residue is identified as normal or variant by comparing it to a normal sequence of the C3 protein as shown in SEQ ID NO: 2. A person with a variant sequence has a higher risk of Age Related Macular Degeneration than a person with a normal sequence.
[0010] Further embodiments of the invention provide a method to assess risk of AMD in an individual. The presence of a G or C allele at the single nucleotide polymorphism (SNP)rs 2230199 within the genomic sequence is determined in an individual. The person is identified as being at high risk of AMD if the patient has one or two copies of the G allele on the negative genomic strand at this SNP (or conversely one or two copies of the C allele on the positive genomic strand) in relation to the March 2006 human reference sequence (NCBI Build 36.1). . The SNP rs2230199 is found in the first position of codon 102 (corresponding to position 366 in the C3 coding sequence of SEQ ID NO: 1 or 304 nucleotides downstream of the start of the initiation codon). SNP rs2230199 is located at position 6669387 on human chromosome 19 ((NCBI Build 36.1). The G allele changes the amino acid specified from arginine to glycine. The patient is identified as being at lower risk of AMD if the patient does not have one or two copies of the G allele at rs2230199.
[0011] Further embodiments provide a method for assessing increased risk of Age Related Macular Degeneration. The identity of the residue at position 102 of the pro-C3 protein sequence or position 80 of the mature C3 protein sequence is determined in an individual. The residue is identified as normal or variant by comparing it to a normal sequence of the pro-C3 protein or C3 protein as shown in SEQ ID NO: 2. An individual with a variant sequence has a higher risk of Age Related Macular Degeneration than an individual with a normal sequence. For example, an individual with Gly at position 102 has a higher risk of Age Related Macular Degeneration than an individual with Arg at position 102.
[0012] While not being bound by any theory, this marker, or one in linkage disequilibrium, may change the composition, function or abundance of the elements of cellular constituents resulting in a predisposition to age related macular degeneration. Measuring this marker in individuals who do not ostensibly have age related macular degeneration may identify those at heightened risk for the subsequent development of age related macular degeneration, providing benefit for, but not limited to, individuals, insurers, care givers and employers. Information obtained from the detection of SNPs associated with age related macular degeneration is of great value in the treatment and prevention of this condition.
[0013] In the context of this invention, a marker is said to be in "linkage disequilibrium" with the residue at rs2230199 when the correlation coefficient (r2) between the marker and rs2230199 is >0.5 (23).
[0014] Further scope of the applicability of the present invention will become apparent from the detailed description provided below. It should be understood however, that the following detailed description and examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modification within the spirit and scope of the invention will become apparent to those skilled in the art from the following
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present inventors have discovered that polymorphic variants in the C3 gene, which is shown in sequences, SEQ ID NOs: 1-3 are associated with an altered risk of developing age related macular degeneration in subjects. The present invention thus provides a SNP associated with age related macular degeneration, nucleic acid molecules containing the SNP, methods and reagents for the detection of the SNP disclosed herein, uses of this SNP for the development of detection reagents, and assays or kits that utilize such reagents. The age related macular degeneration-associated SNP disclosed herein may be useful for diagnosing, screening for, and evaluating predisposition to age related macular degeneration and related pathologies in humans.
[0016] The age related macular degeneration-associated SNP has been identified by genotyping DNA from 1548 individuals, 847 of these individuals having been previously diagnosed with age related macular degeneration and 701 being "control" or individuals thought to be free of age related macular degeneration.
[0017] Aspects of the present invention thus provides an individual SNP associated with age related macular degeneration, genomic sequences (SEQ ID NO: 3) containing SNPs, transcript sequences (SEQ ID NO: 1) and amino acid sequences (SEQ ID NO: 2). Aspects of the invention include methods of detecting these polymorphisms in a test sample, methods of determining the risk of an individual of having or developing age related macular degeneration, methods of using the disclosed SNPs to select a treatment strategy, and methods of using the SNPs of the present invention for human identification.
[0018] When the presence in the genome of an individual of a particular base, e.g., guanine, at a particular location in the genome (e.g. the SNP rs2230199) correlates with an increased probability of that individual contracting age related macular degeneration vis-a-vis a population not having that base at that location in the genome, that individual is said to be at "increased risk" of contracting age related macular degeneration , i.e., to have an increased susceptibility. In the present case, such increased probability exists when the base is present in one or the other or both alleles of the individual. Furthemore, the probability is increased when the base is present in both alleles of the individual rather than one allele of the individual.
[0019] When the presence in the genome of an individual of a particular base, e.g., cytosine, at a particular location in the genome (e.g. the SNP rs2230199) decreases the probability of that individual contracting age related macular degeneration vis-a-vis a population not having that base at that location in the genome, that individual is said to be at "decreased risk" of contracting age related macular degeneration, i.e., to have a decreased susceptibility. Such an allele is sometimes referred to in the art as being "protective". As with increased risk, it is also possible for a decreased risk to be characterized as dominant or recessive.
[0020] An "altered risk" means either an increased or a decreased risk.
[0021] The genetic analysis detailed below-linked age related macular degeneration with a SNP in the human genome. A SNP is a particular type of polymorphic site, a polymorphic site being a region in a nucleic acid sequence at which two or more alternative nucleotides are observed in a significant number of individuals from a population. A polymorphic site may be a nucleotide sequence of two or more nucleotides, an inserted nucleotide or nucleotide sequence, a deleted nucleotide or nucleotide sequence, or a microsatellite, for example. A polymorphic site that is two or more nucleotides in length may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 30 or more, 50 or more, 75 or more, 100 or more, 500 or more, or about 1000 nucleotides in length, where all or some of the nucleotide sequences differ within the region. The specific polymorphic site found in the genomic sequences identified as SEQ ID NOs: 1 and 3 is a "single nucleotide polymorphism" or a "SNP" i.e. a polymorphic site which is one nucleotide in length.
[0022] Where there are two, three, or four alternative nucleotide sequences at a polymorphic site, each nucleotide sequence is referred to as a "polymorphic variant" or "nucleic acid variant." Where two polymorphic variants exist, for example, the polymorphic variant represented in a majority of samples from a population is sometimes referred to as a "prevalent allele" and the polymorphic variant that is less prevalently represented is sometimes referred to as an "uncommon allele." An individual who possesses two prevalent alleles or two uncommon alleles is "homozygous" with respect to the polymorphism, and an individual who possesses one prevalent allele and one uncommon allele is "heterozygous" with respect to the polymorphism. Individuals who are homozygous with respect to one allele are sometimes predisposed to a different phenotype as compared to individuals who are heterozygous or homozygous with respect to another allele.
[0023] A genotype or polymorphic variant may also be expressed in terms of a "haplotype," which refers to the identity of two or more polymorphic variants occurring within genomic DNA on the same strand of DNA. For example, two SNPs may exist within a gene where each SNP position may include a cytosine variation or an adenine variation. Certain individuals in a population may carry an allele (heterozygous) or two alleles (homozygous) having the gene with a cytosine at each SNP position. As the two cytosines corresponding to each SNP in the gene travel together on one or both alleles in these individuals, the individuals can be characterized as having a cytosine/cytosine haplotype with respect to the two SNPs in the gene.
[0024] A "phenotype" is a trait which can be compared between individuals, such as presence or absence of a condition, for example, occurrence of age related macular degeneration.
[0025] Polymorphic variants are often reported without any determination of whether the variant is represented in a significant fraction of a population. Some reported variants are sequencing errors and/or not biologically relevant. Thus, it is often not known whether a reported polymorphic variant is statistically significant or biologically relevant until the presence of the variant is detected in a population of individuals and the frequency of the variant is determined.
[0026] A polymorphic variant may be detected on either or both strands of a double-stranded nucleic acid. Also, a polymorphic variant may be located within an intron or exon of a gene or within a portion of a regulatory region such as a promoter, a 5' untranslated region (UTR), a 3' UTR, and in DNA (e.g., genomic DNA (gDNA) and complementary DNA (cDNA)), RNA (e.g., mRNA, tRNA, and rRNA), or a polypeptide. Polymorphic variations may or may not result in detectable differences in gene expression, polypeptide structure, or polypeptide function.
[0027] In our genetic analysis associating age related macular degeneration with the polymorphic variants set forth in Table 1, samples from individuals diagnosed with age related macular degeneration and individuals not having age related macular degeneration were allelotyped and genotyped. The allele frequency for each polymorphic variant among cases and controls was determined. These allele frequencies were compared in cases and controls, or combinations. Particular SNPs were thus found to be associated with age related macular degeneration when genotype and haplotype frequency differences calculated between case and control pools were established to be statistically significant.
[0028] As mentioned above, polymorphic variants can travel together. Such variants are said to be in "linkage disequilibrium" so that heritable elements e.g., alleles that have a tendency to be inherited together instead of being inherited independently by random assortment are in linkage disequilibrium. Alleles are randomly assorted or inherited independently of each other if the frequency of the two alleles together is the product of the frequencies of the two alleles individually. For example, if two alleles at different polymorphic sites are present in 50% of the chromosomes in a population, then they would be said to assort randomly if the two alleles are present together on 25% of the chromosomes in the population. A higher percentage would mean that the two alleles are linked. For example, a first polymorphic site P1 having two alleles, e.g. A and C--each appearing in 50% of the individuals in a given population, is said to be in linkage disequilibrium with a second polymorphic site P2 having two alleles e.g. G and T--each appearing in 50% of the individuals in a given population, if particular combinations of alleles are observed in individuals at a frequency greater than 25% (if the polymorphic sites are not linked, then one would expect a 50% chance of an individual having A at P1 and a 50% chance of having G at P2 thus leading to a 25% chance of having the combination of A at P1 and G at P2 together). Heritable elements that are in linkage disequilibrium are said to be "linked" or "genetically linked" to each other.
[0029] One can see that in the case of a group of SNPs that are in linkage disequilibrium with each other, knowledge of the existence of all such SNPs in a particular individual generally provides redundant information. Thus, when identifying an individual who has an altered risk for developing age related macular degeneration according to this invention, it is necessary to detect only one SNP of such a group of SNPs associated with an altered risk of developing age related macular degeneration.
[0030] The data set out below shows that one or more SNPs in the C3 genomic sequences identified herein as SEQ ID NOs: 1 and 3 are associated with the occurrence of age related macular degeneration. Thus, featured herein are methods for identifying a risk of age related macular degeneration in a subject, which includes detecting the presence or absence of a polymorphic variant at one or more of the SNPs described herein in a human nucleic acid sample. For example, the presence or absence of a polymorphic variant at rs2230199 (e.g. the G allele) may be detected in a human nucleic acid sample.
[0031] Three different analyses were performed for each marker and significant results reported below as follows: (a) a test of trend across the 3 genotypes(24) , (b) a dominant model where the homozygous genotype for allele "B" is combined with the prevalent heterozygote genotype; and (c) a recessive model where the homozygous genotype for allele "A" is combined with the heterozygous genotype. An empirical p-value for the largest of these three test statistics was calculated by permutations. In addition, a Mantel-Haenszel odds ratio measuring the change in risk associated with each additional copy of allele B is also calculated and reported.
[0032] Pertinent results for the SNP are summarized in Table 1: Chromosomal number and position-using the International Human Genome Sequencing Consortium build 35 (http://www.ncbi.nlm.nih.gov/genome/seq/) as made available by the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Bethesda, Md. 20894 U.S.A., gene marker name-using the nomenclature of the NCBI dbSNP (URL[colon][slash][slash] www[dot]ncbi[dot]nlm[dot]nih[dot]gov[slash]SNP[slash]) and gene name-using the unigene naming convention. Under the "Case Flag" the number 1 designates Cases and the number 0 designates Controls. The identity of the base designated "A" in the analysis is indicated where 1=A (adenine), 2=C (cytosine), 3=G (guanine) and 4=T (thymidine). "B" indicates the polymorphic allele. AA, AB, BB are the counts of the number of individuals with the given genotype, by cases/controls. The odds ratio is the Mantel-Haenszel odds ratio across the three genotypes.
[0033] It has been discovered that polymorphic variation at SNPs in the C3 genomic sequences which are identified herein as SEQ ID NOs: 1 or 3 is associated with the occurrence of age related macular degeneration. Thus, featured herein are methods for identifying a risk of age related macular degeneration in a subject, which comprises detecting the presence or absence of one or more of the polymorphic variations described herein in a human nucleic acid sample. The polymorphic variations and SNPs are detailed in the table. In some embodiments, the presence of a polymorphic variant at rs2230199 is indicative of an altered risk of age related macular degeneration. For example, the presence of the uncommon G allele at rs2230199 may be indicative of an increased risk of age related macular degeneration, relative to individuals with the prevalent C allele at rs2230199.
[0034] Methods for determining whether a subject is at risk of age related macular degeneration are provided herein. These methods include detecting the presence or absence of one or more polymorphic variations at SNPs which are associated with age related macular degeneration, in a sample from a subject.
[0035] SNPs may be associated with a disease state such as AMD, in humans or in animals. The association can be direct, as in conditions where the substitution of a base results in alteration of the protein coding sequence of a gene which contributes directly to the pathophysiology of the condition. Common examples of this include diseases such as sickle cell anemia and cystic fibrosis. The association can be indirect when the SNP plays no role in the disease, but is located close to the defective gene such that there is a strong association between the presence of the SNP and the disease state. Because of the high frequency of SNPs within the genome, there is a greater probability that a SNP will be linked to a genetic locus of interest than other types of genetic markers.
[0036] Disease-associated SNPs may occur in coding and non-coding regions of the genome. When located in the coding region altered function of the ensuing protein sequence may occur. For example, polymorphic variation at SNP rs2230199 may alter the amino acid residue at position 102 of the C3 pro-protein. If it occurs in the regulatory region of a gene it may affect expression of the protein. Ifthe protein is involved in protecting the body against pathological conditions this can result in disease susceptibility.
[0037] Numerous methods exist for the measurement of specific SNP genotypes. Individuals carrying mutations in one or more SNPs of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
[0038] The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis (25). RNA or cDNA may also be used in the same ways. As an example, PCR primers complementary to the nucleic acid of one or more SNPs of the present invention can be used to identify and analyze the presence or absence of the SNP. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled SNP RNA of the present invention or alternatively, radiolabeled SNP antisense DNA sequences of the present invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
[0039] Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method. For example, a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
[0040] Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures(26).
[0041] Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method(27).
[0042] Thus, the detection of a specific DNA sequence may be achieved by methods which include, but are not limited to, hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., restriction fragment length polymorphisms ("RFLP") and Southern blotting of genomic DNA).
[0043] Hybridisation may be carried out under stringent hybridization conditions, for example for detection of sequences that are about 80-90% identical suitable conditions include hybridization overnight at 42° C. in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55° C. in 0.1×SSC, 0.1% SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65° C. in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60° C. in 0.1×SSC, 0.1% SDS.
[0044] In addition to more conventional gel-electrophoresis and DNA sequencing, mutations also can be detected by in situ analysis.
[0045] Genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes(28;29). For example, genetic mutations can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
[0046] Specific mutations can also be determined through direct sequencing of one or both strands of DNA using dideoxy nucleotide chain termination chemistry, electrophoresis through a semi-solid matrix and fluorescent or radioactive chain length detection techniques. Further mutation detection techniques may involve differential susceptibility of the polymorphic double strand to restriction endonuclease digestion, or altered electrophoretic gel mobility of single or double stranded gene fragments containing one polymorphic form. Other techniques to detect specific DNA polymorphisms or mutation may involve evaluation of the structural characteristics at the site of polymorphism using nuclear magnetic resonance or x-ray diffraction techniques.
[0047] These genetic tests are useful for prognosing and/or diagnosing age related macular degeneration and often are useful for determining whether an individual is at an increased or decreased risk of developing or having age related macular degeneration .
[0048] Thus, the invention includes a method for identifying a subject at risk of age related macular degeneration , which includes detecting in a nucleic acid sample from the subject the presence or absence of a polymorphic variant at a SNP associated with age related macular degeneration in a nucleotide sequence identified as SEQ ID NOs:1 and 3.
[0049] For example, the presence of one or two copies of the G allele at SNP rs2230199 may be indicative of the subject being at risk of age related macular degeneration i.e. an individual at risk of AMD may be heterozygous (genotype GC) or homozygous (genotype GG) at SNP rs2230199 in the C3 gene,
[0050] Results from prognostic tests may be combined with other test results to diagnose age related macular degeneration. For example, prognostic results may be gathered, a patient sample may be ordered based on a determined predisposition to age related macular degeneration , the patient sample analyzed, and the results of the analysis may be utilized to diagnose age related macular degeneration . Also age related macular degeneration diagnostic methods can be developed from studies used to generate prognostic/diagnostic methods in which populations are stratified into subpopulations having different progressions of age related macular degeneration . In some embodiments, prognostic results may be gathered; a patient's risk factors for developing age related macular degeneration analyzed (e.g., age, family history, smoking); and a patient sample may be ordered based on a determined predisposition to age related macular degeneration. In some embodiments, the results from predisposition analyses may be combined with other test results, epidemiologic or genetic in nature, indicative of age related macular degeneration, which were previously, concurrently, or subsequently gathered with respect to the predisposition testing. In these embodiments, the combination of the prognostic test results with other test results can be probative of age related macular degeneration, and the combination can be utilized as a age related macular degeneration diagnostic.
[0051] Risk of age related macular degeneration sometimes is expressed as a probability, such as an odds ratio, percentage, or risk factor. The risk is based upon the presence or absence of the SNP variant described herein, and also may be based in part upon phenotypic traits of the individual being tested. Methods for calculating risk based upon patient data are well known (30). Allelotyping and genotyping analyses may be carried out in populations other than those exemplified herein to enhance the predictive power of the prognostic method. These further analyses are executed in view of the exemplified procedures described herein, and may be based upon the same polymorphic variations or additional polymorphic variations. Risk determinations for age related macular degeneration are useful in a variety of applications. In some embodiments, age related macular degeneration risk determinations may be used by clinicians to direct appropriate detection, preventative and treatment procedures to subjects who most require these. In other embodiments, age related macular degeneration risk determinations may be used by health insurers for preparing actuarial tables and for calculating insurance premiums.
[0052] The nucleic acid sample typically is isolated from a biological sample obtained from a subject. For example, nucleic acid can be isolated from blood, saliva, sputum, urine, cell scrapings, and biopsy tissue. The nucleic acid sample can be isolated from a biological sample using standard techniques. The nucleic acid sample may be isolated from the subject and then directly utilized in a method for determining the presence of a polymorphic variant, or alternatively, the sample may be isolated and then stored (e.g., frozen) for a period of time before being subjected to analysis.
[0053] The presence or absence of a polymorphic variant may be determined using one or both chromosomal complements represented in the nucleic acid sample. Determining the presence or absence of a polymorphic variant in both chromosomal complements represented in a nucleic acid sample is useful for determining the zygosity of an individual for the polymorphic variant (i.e., whether the individual is homozygous or heterozygous for the polymorphic variant). For example, a homozygous individual having the GG genotype at SNP rs2230199 (i.e. the G allele in both copies of the C3 gene) may have an increased risk of AMD relative to a heterozygous individual having the GC genotype at SNP rs2230199 (i.e. the G allele in one copies of the C3 gene and the C allele in the other)
[0054] Any oligonucleotide-based diagnostic may be utilized to determine whether a sample includes the presence or absence of a polymorphic variant in a sample. For example, primer extension methods, ligase sequence determination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, and WO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat. Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), microarray sequence determination methods, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism detection (SSCP) (e.g., U.S. Pat. Nos. 5,891,625 and 6,013,499), PCR-based assays (e.g., TAQMAN® PCR System (Applied Biosystems)), and nucleotide sequencing methods may be used.
[0055] Oligonucleotide extension methods typically involve providing a pair of oligonucleotide primers in a polymerase chain reaction (PCR) or in other nucleic acid amplification methods for the purpose of amplifying a region from the nucleic acid sample that comprises the polymorphic variation. One oligonucleotide primer is complementary to a region 3' of the polymorphism and the other is complementary to a region 5' of the polymorphism. A PCR primer pair may be used in methods disclosed in U.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01/27327; and WO 01/27329 for example. PCR primer pairs may also be used in any commercially available machines that perform PCR, such as any of the GENEAMPTM, systems available from Applied Biosystems. Also, those of ordinary skill in the art will be able to design oligonucleotide primers based upon the nucleotide sequences set forth in SEQ ID NOs: 1 and 3.
[0056] Also provided is an extension oligonucleotide that hybridizes to the amplified fragment adjacent to the polymorphic variation. An adjacent fragment refers to the 3' end of the extension oligonucleotide being often 1 nucleotide from the 5' end of the polymorphic site, and sometimes 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polymorphic site, in the nucleic acid when the extension oligonucleotide is hybridized to the nucleic acid. The extension oligonucleotide then is extended by one or more nucleotides, and the number and/or type of nucleotides that are added to the extension oligonucleotide determine whether the polymorphic variant is present. Oligonucleotide extension methods are disclosed, for example, in U.S. Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702; 6,046,005; 6,087,095; 6,210,891; and WO 01/20039. Oligonucleotide extension methods using mass spectrometry are described, for example, in U.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031; and 6,194,144. Multiple extension oligonucleotides may be utilized in one reaction, which is referred to as multiplexing.
[0057] A microarray can be utilized for determining whether a SNP is present or absent in a nucleic acid sample. A microarray may include any oligonucleotides described herein, and methods for making and using oligonucleotide microarrays suitable for diagnostic use are disclosed in U.S. Pat. Nos. 5,492,806; 5,525,464; 5,589,330; 5,695,940; 5,849,483; 6,018,041; 6,045,996; 6,136,541; 6,142,681; 6,156;501; 6,197,506; 6,223,127; 6,225,625; 6,229,911; 6,239,273; WO 00/52625; WO 01/25485; and WO 01/29259. The microarray typically comprises a solid support and the oligonucleotides may be linked to this solid support by covalent bonds or by non-covalent interactions. The oligonucleotides may also be linked to the solid support directly or by a spacer molecule. A microarray may comprise one or more oligonucleotides complementary to a nucleotide sequence which includes a SNP set forth in Table 1. The one or more oligonucleotides may for example, hybridise specifically to a nucleotide sequence which comprises a particular polymorphic variant at the SNP, but not to nucleotide sequences which comprise other polymorphic variants at the SNP.A kit also may be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A kit may include one or more pairs of oligonucleotide primers useful for amplifying a fragment of a nucleotide sequence of interest, where the fragment includes a polymorphic site. The kit may comprise a polymerizing agent, for example, a thermostable nucleic acid polymerase such as one disclosed in U.S. Pat. Nos. 4,889,818 or 6,077,664. Also, the kit may comprise an elongation oligonucleotide that hybridizes to the nucleotide sequence in a nucleic acid sample adjacent to the polymorphic site. Where the kit includes an elongation oligonucleotide, it may also comprise chain elongating nucleotides, such as dATP, dTTP, dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTP and dITP, provided that such analogs are substrates for a thermostable nucleic acid polymerase and can be incorporated into a nucleic acid chain elongated from the extension oligonucleotide. Along with chain elongating nucleotides may be one or more chain terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP. The kit may comprise one or more oligonucleotide primer pairs, a polymerizing agent, chain elongating nucleotides, at least one elongation oligonucleotide, and one or more chain terminating nucleotides. Kits optionally include buffers, vials, microtiter plates, and instructions for use.
[0058] An individual identified as being susceptible to age related macular degeneration may be heterozygous or homozygous with respect to the allele associated with an increased risk of age related macular degeneration, as indicated in the table. For example, the individual may be heterozygous or homozygous with respect to the G allele of rs2230199 which is shown herein to be associated with an increased risk of age related macular degeneration. A subject homozygous for an allele associated with an increased risk of age related macular degeneration is at a comparatively high risk of age related macular degeneration as far as that SNP is concerned whether or not the allelic effect has been determined to be dominant or recessive. A subject who is heterozygous for an allele associated with an increased risk of age related macular degeneration , in which the allelic effect is recessive would likely be at a comparatively reduced risk of age related macular degeneration predicted by that SNP. The allelic effect of the G allele of rs2230199 is shown herein to be dominant and an individual who is heterozygous for the G allele may be at an increased risk of age related macular degeneration relative to individuals who lack the G allele.
[0059] Individuals carrying mutations in one or more SNP of the present invention may be detected at the protein level by a variety of techniques. Cells suitable for diagnosis may be obtained from a patient's blood, urine, saliva, tissue biopsy and autopsy material.
[0060] Also featured are methods for determining risk of age related macular degeneration and/or identifying a subject at risk of age related macular degeneration by contacting a polypeptide or protein encoded by a nucleotide sequence from a subject with an antibody that specifically binds to an epitope associated with an altered, usually increased risk of age related macular degeneration in the polypeptide.
[0061] Another aspect of the invention provides an isolated nucleic acid molecule comprising at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25, or at least 26, or at least 27, or at least 28, or at least 29, or at least 30, or at least 31, or at least 32, or at least 33, or at least 34, or at least 35, or at least 36, or at least 37, or at least 38, or at least 39, or at least 40, or at least 41, or at least 42, or at least 43, or at least 44, or at least 45, or at least 46, or at least 47, or at least 48, or at least 49, or at least 50, or at least 51, or at least 52, or at least 53, or at least 54, or at least 55, or at least 56, or at least 57, or at least 58, or at least 59, or at least 60, or at least 61, or at least 62, or at least 63, or at least 64, or at least 65, or at least 66, or at least 67, or at least 68, or at least 69, or at least 70, or at least 71, or at least 72, or at least 73, or at least 74, or at least 75, or at least 76, or at least 77, or at least 78, or at least 79, or at least 80, or at least 81, or at least 82, or at least 83, or at least 84, or at least 85, or at least 86, or at least 87, or at least 88, or at least 89, or at least 90, or at least 91, or at least 92, or at least 93, or at least 94, or at least 95, or at least 96, or at least 97, or at least 98, or at least 99, or at least 100 contiguous nucleotides from any one of SEQ NOS: 1 or 3 wherein one of the nucleotides is located at the site of single nucleotide polymorphism (SNP) corresponding to single nucleotide polymorphism (SNP) at rs2230199 on human chromosome 19 as set out herein or the complement thereof, and optionally; wherein the isolated nucleic acid molecule has a maximum length of 100 said contiguous nucleotides, or a maximum length of 90 said contiguous nucleotides, or a maximum length of 80 said contiguous nucleotides, or a maximum length of 70 said contiguous nucleotides, or a maximum length of 60 said contiguous nucleotides, or a maximum length of 50 said contiguous nucleotides, or a maximum length of 40 said contiguous nucleotides, or a maximum length of 30 said contiguous nucleotides, or a maximum length of 20 said contiguous nucleotides.
[0062] Oligonucleotides can be linked to a second moiety, which can be another nucleic acid molecule to provide, for example, a tail sequence (e.g., a polyadenosine tail), an adapter sequence (e.g., phage M13 universal tail sequence), etc. Alternatively, the moiety might be one that facilitates linkage to a solid support or a detectable label, e.g., a radioactive label, a fluorescent label, a chemiluminescent label, a paramagnetic label, etc.
[0063] Nucleic acid sequences shown in SEQ ID NO: 1, 3 or 4, or fragments thereof, may be used for diagnostic purposes for detection of polypeptide expression.
[0064] DNA encoding a polypeptide can also be used in the diagnosis of age related macular degeneration. For example, the nucleic acid sequence can be used in hybridization assays of biopsies or autopsies to polymorphic variants associated with increased risk of AMD (e.g., Southern or Northern blot analysis, in situ hybridization assays).
[0065] Expression of a polypeptide during embryonic development can also be determined using nucleic acid encoding the polypeptide, particularly production of a functionally impaired polypeptide that is the cause of age related macular degeneration. In situ hybridizations using a polypeptide as a probe can be employed to predict problems related to age related macular degeneration.
[0066] Included as part of this invention are nucleic acid vectors, often expression vectors, which contain a nucleotide sequence set forth in the SEQ ID NO:1 or 3, or a fragment thereof. A vector is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid, or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors may include replication defective retroviruses, adenoviruses and adeno-associated viruses for example.
[0067] A vector can include a nucleotide sequence from SEQ ID NO: 1 or 3 or a fragment thereof, in a form suitable for expression of an encoded protein or nucleic acid in a host cell. The recombinant expression vector generally includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. A regulatory sequence includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. Expression vectors can be introduced into host cells to produce the desired polypeptides, including fusion polypeptides.
[0068] Recombinant expression vectors can be designed for expression of polypeptides in prokaryotic or eukaryotic cells. For example, the polypeptides can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further by Goeddel (31).A recombinant expression vector can also be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
[0069] Expression of polypeptides in prokaryotes can be carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide. Such fusion vectors typically serve to increase expression of recombinant polypeptide, to increase the solubility of the recombinant polypeptide and/or to aid in the purification of the recombinant polypeptide by acting as a ligand during purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;), pMAL (New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.
[0070] Purified fusion polypeptides can be used in screening assays and to generate antibodies specific for polypeptides.
[0071] Expressing a polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide can be used to maximize recombinant polypeptide expression (32). The nucleotide sequence of the nucleic acid to be inserted into an expression vector can be changed so that the individual codons for each amino acid are those preferentially utilized in E. coli (33).
[0072] When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Recombinant mammalian expression vectors can be capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Examples of suitable tissue-specific promoters include an albumin promoter(34), lymphoid-specific promoters (35) (36), promoters of immunoglobulins(37;38), neuron-specific promoters (39), pancreas-specific promoters (40), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are sometimes utilized, for example, the murine hox promoters(41) and the α-fetopolypeptide promoter(42).
[0073] Vectors can be introduced into host cells via conventional transformation or transfection techniques. The terms transformation and transfection refer to a variety of techniques known for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, transduction/infection, DEAE-dextran-mediated transfection, lipofection, or electroporation.
[0074] A host cell can be used to produce a polypeptide. Accordingly, methods for producing a polypeptide using the host cells are included as part of this invention. Such a method can include culturing host cells into which a recombinant expression vector encoding a polypeptide has been introduced in a suitable medium such that the polypeptide is produced. The method can further include isolating the polypeptide from the medium or the host cell.
[0075] Polypeptides can be expressed in transgenic animals or plants by introducing a nucleic acid encoding the polypeptide into the genome of an animal. In certain embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Also included is a population of cells from a transgenic animal.
[0076] Isolated polypeptides encoded by a nucleotide sequence from SEQ ID NO: 1 or 3, or a fragment thereof, can be synthesized. Isolated polypeptides include both the full-length polypeptide and the mature polypeptide (i.e., the polypeptide minus the signal sequence or propeptide domain). An isolated, or purified, polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or is substantially free from chemical precursors or other chemicals when chemically synthesized. Substantially free means a preparation of a polypeptide having less than about 5% (by dry weight) of contaminating protein, or of chemical precursors or non-target chemicals. When the desired polypeptide is recombinantly produced, it is typically substantially free of culture medium, specifically, where culture medium represents less than about 10% of the polypeptide preparation.
[0077] Also, polypeptides may exist as chimeric or fusion polypeptides. As used herein, a "target chimeric polypeptide" or "target fusion polypeptide" includes a target polypeptide linked to a different polypeptide. The target polypeptide in the fusion polypeptide can correspond to an entire or nearly entire polypeptide as it exists in nature or a fragment thereof. The other polypeptide can be fused to the N-terminus or C-terminus of the target polypeptide.
[0078] Fusion polypeptides can include a moiety having high affinity for a ligand. For example, the fusion polypeptide can be a GST-target fusion polypeptide in which the target sequences are fused to the C-terminus of the GST sequences, or a polyhistidine-target fusion polypeptide in which the target polypeptide is fused at the N- or C-terminus to a string of histidine residues. Such fusion polypeptides can facilitate purification of recombinant target polypeptide. Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide), and a nucleotide sequence from SEQ ID NO: 1, 3 or 4, or a fragment thereof, or a substantially identical nucleotide sequence thereof, can be cloned into an expression vector such that the fusion moiety is linked in-frame to the target polypeptide. Further, the fusion polypeptide can be a target polypeptide containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression, secretion, cellular internalization, and cellular localization of a target polypeptide can be increased through use of a heterologous signal sequence. Fusion polypeptides can also include all or a part of a serum polypeptide (e.g., an IgG constant region or human serum albumin).
[0079] Target polypeptides can be used as immunogens to produce anti-target antibodies in a subject, to purify the polypeptide ligands or binding partners.
[0080] Polypeptides can be differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any known modification including specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc. may be used. Additional post-translational modifications include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.
[0081] Pharmacogenomics is a discipline that involves tailoring a treatment for a subject according to the subject's genotype. For example, based upon the outcome of a prognostic test, a clinician or physician may target pertinent information and preventative or therapeutic treatments to a subject who would be benefited by the information or treatment and avoid directing such information and treatments to a subject who would not be benefited (e.g., the treatment has no therapeutic effect and/or the subject experiences adverse side effects). As therapeutic approaches for age related macular degeneration continue to evolve and improve, the goal of treatments for age related macular degeneration related disorders is to intervene even before clinical signs manifest themselves. Thus, genetic markers associated with susceptibility to age related macular degeneration prove useful for early diagnosis, prevention and treatment of age related macular degeneration.
[0082] The following is an example of a pharmacogenomic embodiment. A particular treatment regimen can exert a differential effect depending upon the subject's genotype. Where a candidate therapeutic exhibits a significant beneficial interaction with a prevalent allele and a comparatively weak interaction with an uncommon allele (e.g., an order of magnitude or greater difference in the interaction), such a therapeutic typically would not be administered to a subject genotyped as being homozygous for the uncommon allele, and sometimes not administered to a subject genotyped as being heterozygous for the uncommon allele. In another example, where a candidate therapeutic is not significantly toxic when administered to subjects who are homozygous for a prevalent allele but is comparatively toxic when administered to subjects heterozygous or homozygous for an uncommon allele, the candidate therapeutic is not typically administered to subjects who are genotyped as being heterozygous or homozygous with respect to the uncommon allele.
[0083] Methods of the invention are applicable to pharmacogenomic methods for detecting, preventing, alleviating and/or treating age related macular degeneration. For example, a nucleic acid sample from an individual may be subjected to a genetic test. Where one or more polymorphic variants associated with increased risk of age related macular degeneration are identified at SNPs in the individual, information for detecting, preventing or treating age related macular degeneration and/or one or more age related macular degeneration detection, prevention and/or treatment regimens then may be directed to and/or prescribed to that individual.
[0084] In certain embodiments, a detection, preventative and/or treatment regimen is specifically prescribed and/or administered to individuals who will most benefit from it based upon their risk of developing age related macular degeneration assessed by the methods described herein. Methods are thus provided for identifying a subject at risk of age related macular degeneration and then prescribing a detection, therapeutic or preventative regimen to individuals identified as being at increased risk of age related macular degeneration. Thus, certain embodiments are directed to methods for treating age related macular degeneration in a subject, reducing risk of age related macular degeneration in a subject, or early detection of age related macular degeneration in a subject, which comprise: detecting the presence or absence of a polymorphic variant associated with age related macular degeneration at a SNP in a nucleotide sequence set forth in SEQ ID NOs:1 and 3, and prescribing or administering an age related macular degeneration treatment regimen, preventative regimen and/or detection regimen to a subject from whom the sample originated where the presence of polymorphic variants associated with age related macular degeneration are detected at one or more SNPs in the nucleotide sequence. In these methods, genetic results may be utilized in combination with other test results to diagnose age related macular degeneration as described above.
[0085] The use of certain age related macular degeneration treatments are known in the art, and include surgery, chemotherapy and/or radiation therapy. Any of the treatments may be used in combination to treat or prevent age related macular degeneration (e.g., surgery followed by radiation therapy or chemotherapy).
[0086] Pharmacogenomics methods also may be used to analyze and predict a response to a age related macular degeneration treatment or a drug. For example, if pharmacogenomics analysis indicates a likelihood that an individual will respond positively to a age related macular degeneration treatment with a particular drug, the drug may be administered to the individual.
[0087] Conversely, if the analysis indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects. The response to a therapeutic treatment can be predicted in a background study in which subjects in any of the following populations are genotyped: a population that responds favorably to a treatment regimen, a population that does not respond significantly to a treatment regimen, and a population that responds adversely to a treatment regiment (e.g., exhibits one or more side effects). These populations are provided as examples and other populations and subpopulations may be analyzed. Based upon the results of these analyses, a subject is genotyped to predict whether he or she will respond favorably to a treatment regimen, not respond significantly to a treatment regimen, or respond adversely to a treatment regimen.
[0088] The methods described herein also are applicable to clinical drug trials. Polymorphic variants indicative of response to an agent for treating age related macular degeneration or to side effects to an agent for treating age related macular degeneration may be identified at one or more SNPs. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems.
[0089] Thus, another embodiment is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: (a) obtaining a nucleic acid sample from an individual; (b) determining the identity of a polymorphic variant which is associated with a positive response to the treatment or the drug, or a polymorphic variant which is associated with a negative response to the treatment or the drug at at least one SNP in the nucleic acid sample, and (c) including the individual in the clinical trial if the nucleic acid sample contains the polymorphic variant associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said polymorphic variant associated with a negative response to the treatment or the drug. The SNP may be in a sequence selected individually or in any combination from the C3 genomic sequence disclosed in the table. Step (c) can also include administering the drug or the treatment to the individual if the nucleic acid sample contains the polymorphic variant associated with a positive response to the treatment or the drug and the nucleic acid sample lacks the polymorphic variant associated with a negative response to the treatment or the drug.
[0090] A peptide nucleic acid, or PNA, refers to a nucleic acid mimic such as a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. Synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described, for example, in Hyrup et al. (71), and Perry-O'Keefe et al. (70).
[0091] PNA nucleic acids can be used in prognostic and diagnostic applications. For example, PNAs can be used in the analysis of SNPs in a gene, (e.g., by PNA-directed PCR clamping); as artificial restriction enzymes when used in combination with other enzymes, (e.g., S1 nucleases(71) or as probes or primers for DNA sequencing or hybridization (71;72).
[0092] A further aspect of the invention provides an antibody molecule that binds specifically to a vairiant C3 polypeptide i.e. a polypeptide encoded by a nucleotide sequence comprising polymorphic variants at one or more SNPs described herein. For example, an antibody molecule may bind specifically to the C3F polypeptide which comprises an R102G substitution which is encoded by the coding sequence comprising the G allele of SNP rs2230199. Such an antibody binds preferentially to the C3F polypeptide relative to C3S polypeptide which lacks the R102G substitution.
[0093] A method of identifying and/or obtaining an antibody specific for C3F polypeptide may comprise; [0094] providing a population of antibody molecules specific for C3F polypeptide, [0095] contacting said population with a C3S polypeptide, [0096] identifying one or more members of said population which bind preferentially to C3F relative to C3S polypeptide.
[0097] Antibody molecules may be useful both in the diagnosis of AMD, in accordance with the invention.
[0098] Antibodies that are specific for a C3 polypeptide may be obtained using techniques that are standard in the art. An immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal). An appropriate immunogenic preparation can contain, for example, recombinantly expressed chemically synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.
[0099] Amino acid polymorphisms can be detected using antibodies specific for the altered epitope by western analysis after the electrophoresis of denatured proteins. Protein polymorphism can also be detected using fluorescently identified antibodies which bind to specific polymorphic epitopes and detected in whole cells using fluorescence activated cell sorting techniques (FACS). Polymorphic protein sequence may also be determined by NMR spectroscopy or by x-ray diffraction studies. Further, determination of polymorphic sites in proteins may be accomplished by observing differential cleavage by specific or non specific proteases.
[0100] An antibody is an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. An antibody can be polyclonal, monoclonal, or recombinant (e.g., a chimeric or humanized), fully human, non-human (e.g., murine), or a single chain antibody.
[0101] A full-length polypeptide or antigenic peptide fragment encoded by a target nucleotide sequence can be used as an immunogen or can be used to identify antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. An antigenic peptide often includes at least 8 amino acid residues of the amino acid sequences encoded by a nucleotide sequence of one of SEQ ID NOs:1 and 3, and encompasses an epitope. Antigenic peptides sometimes include 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, or 30 or more amino acids. Hydrophilic and hydrophobic fragments of polypeptides sometimes are used as immunogens.
[0102] Epitopes encompassed by the antigenic peptide are regions located on the surface of the polypeptide (e.g., hydrophilic regions) as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human polypeptide sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the polypeptide and are thus likely to constitute surface residues useful for targeting antibody production. The antibody may bind an epitope on any domain or region on polypeptides for use in the invention.
[0103] An antibody (e.g., monoclonal antibody) can be used to isolate target polypeptides by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an antibody can be used to detect a target polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide. Antibodies can be used diagnostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H. Also, an antibody can be utilized as a test molecule for determining whether it can treat age related macular degeneration , and as a therapeutic for administration to a subject for treating age related macular degeneration.
[0104] An antibody can be made by immunizing with a purified antigen, or a fragment thereof, a membrane associated antigen, tissues, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions.
[0105] Included as part of this invention are antibodies which bind only a native polypeptide, only denatured or otherwise non-native polypeptide, or which bind both, as well as those having linear or conformational epitopes. Conformational epitopes sometimes can be identified by selecting antibodies that bind to native but not denatured polypeptide. Also featured are antibodies that specifically bind to a polypeptide variant associated with age related macular degeneration.
[0106] Preferably, an antibody displays increased binding to the C3F polypeptide relative to the C3S polypeptide.
[0107] The examples set forth below are intended to illustrate but not limit the invention.
[0108] Age-related macular degeneration is the most common cause of blindness in Western populations. Susceptibility is influenced by age and by genetic and environmental factors.
[0109] Complement activation is implicated in the pathogenesis. We tested for an association between age-related macular degeneration and 13 single nucleotide polymorphisms (SNPs) spanning the complement genes C3 and C5 in case subjects and control subjects from the southeastern region of England. All subjects were examined by an ophthalmologist and had independent grading of fundus photographs to confirm their disease status. To test for replication of the most significant findings, we genotyped a set of Scottish cases and controls. The common functional polymorphism rs2230199 (Arg102Gly) in the C3 gene, corresponding to the electrophoretic variants C3S (slow) and C3F (fast), was strongly associated with age-related macular degeneration in both the English group (603 cases and 350 controls, P=5.9×10-5) and the Scottish group (244 cases and 351 controls, P=5.0×10-5). The odds ratio for age-related macular degeneration in C3 S/F heterozygotes as compared with S/S homozygotes was 1.7 (95% confidence interval [CI], 1.3 to 2.1); for F/F homozygotes, the odds ratio was 2.6 (95% CI, 1.6 to 4.1). The estimated population attributable risk for C3F was 22%. Complement C3 is important in the pathogenesis of age-related macular degeneration. This finding further underscores the influence of the complement pathway in the pathogenesis of this disease.
[0110] The inventors of the present invention have discovered a single base pair polymorphism that is present in a highly significant percentage of the genetic DNA of individuals affected with age related macular degeneration while only present in a smaller percentage of individuals who are not known to be affected by the disease.
[0111] For individuals with age-related macular degeneration, the distribution of polymorphic alleles at position 6669387 of chromosome 19, found within the C3 gene, was different from those without age-related macular degeneration (Table 1). The trend test for risk associated with carrying the C allele (on the positive reference strand of the human genome) had an empirical p-value of 0.000059225, and the corresponding Mantel-Haenszel odds ratio for trend is 1.600 (Table 1). These data further suggest that this marker, located within the C3 gene, is associated with age-related macular degeneration risk and that the C allele at position 6669387 of chromosome 19 is associated with an increased risk of developing age-related macular degeneration. The C allele at position 6669387 of the positive strand corresponds to the G allele within the negative strand, in which is found the coding sequence for C3.
TABLE-US-00001 TABLE 1 rs no. 2230199 Chromosome; Position 19; 6669387 Gene Name C3 SEQ ID NO; Position 3; 2274 Genotype; Phenotype n = C; increased risk (positive strand relative to the human reference sequence version 36.1) Hardy-Weinberg 0.86594 Case Allele Odds Flag B AA AB BB Model p-Value Ratio 0 C 223 109 14 Trend 0.000059 1.600 1 C 303 242 45
[0112] The present invention has been described in detail by way of illustration and example in order to acquaint others skilled in the art with the invention, its principles and its practical application. Particular formulations and processes of the present invention are not limited to the descriptions of the specific embodiments presented, but rather the descriptions and examples should be viewed in terms of the claims that follow and their equivalents. While some of the examples and descriptions above include some conclusions about the way the invention may function, the inventors do not intend to be bound by those conclusions and functions, but put them forth only as possible explanations.
[0113] It is to be further understood that the specific embodiments of the present invention as set forth are not intended as being exhaustive or limiting of the invention, and that many alternatives, modifications and variations will be apparent to those of ordinary skill in the art in light of the foregoing examples and detailed description. Accordingly, this invention is intended to embrace all such alternatives, modifications and variations that fall within the spirit and scope of the following claims.
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Sequence CWU
1
315101DNAHomo sapiens 1cactcctccc catcctctcc ctctgtccct ctgtccctct
gaccctgcac tgtcccagca 60ccatgggacc cacctcaggt cccagcctgc tgctcctgct
actaacccac ctccccctgg 120ctctggggag tcccatgtac tctatcatca cccccaacat
cttgcggctg gagagcgagg 180agaccatggt gctggaggcc cacgacgcgc aaggggatgt
tccagtcact gttactgtcc 240acgacttccc aggcaaaaaa ctagtgctgt ccagtgagaa
gactgtgctg acccctgcca 300ccaaccacat gggcaacgtc accttcacga tcccagccaa
cagggagttc aagtcagaaa 360aggggcgcaa caagttcgtg accgtgcagg ccaccttcgg
gacccaagtg gtggagaagg 420tggtgctggt cagcctgcag agcgggtacc tcttcatcca
gacagacaag accatctaca 480cccctggctc cacagttctc tatcggatct tcaccgtcaa
ccacaagctg ctacccgtgg 540gccggacggt catggtcaac attgagaacc cggaaggcat
cccggtcaag caggactcct 600tgtcttctca gaaccagctt ggcgtcttgc ccttgtcttg
ggacattccg gaactcgtca 660acatgggcca gtggaagatc cgagcctact atgaaaactc
accacagcag gtcttctcca 720ctgagtttga ggtgaaggag tacgtgctgc ccagtttcga
ggtcatagtg gagcctacag 780agaaattcta ctacatctat aacgagaagg gcctggaggt
caccatcacc gccaggttcc 840tctacgggaa gaaagtggag ggaactgcct ttgtcatctt
cgggatccag gatggcgaac 900agaggatttc cctgcctgaa tccctcaagc gcattccgat
tgaggatggc tcgggggagg 960ttgtgctgag ccggaaggta ctgctggacg gggtgcagaa
cccccgagca gaagacctgg 1020tggggaagtc tttgtacgtg tctgccaccg tcatcttgca
ctcaggcagt gacatggtgc 1080aggcagagcg cagcgggatc cccatcgtga cctctcccta
ccagatccac ttcaccaaga 1140cacccaagta cttcaaacca ggaatgccct ttgacctcat
ggtgttcgtg acgaaccctg 1200atggctctcc agcctaccga gtccccgtgg cagtccaggg
cgaggacact gtgcagtctc 1260taacccaggg agatggcgtg gccaaactca gcatcaacac
acaccccagc cagaagccct 1320tgagcatcac ggtgcgcacg aagaagcagg agctctcgga
ggcagagcag gctaccagga 1380ccatgcaggc tctgccctac agcaccgtgg gcaactccaa
caattacctg catctctcag 1440tgctacgtac agagctcaga cccggggaga ccctcaacgt
caacttcctc ctgcgaatgg 1500accgcgccca cgaggccaag atccgctact acacctacct
gatcatgaac aagggcaggc 1560tgttgaaggc gggacgccag gtgcgagagc ccggccagga
cctggtggtg ctgcccctgt 1620ccatcaccac cgacttcatc ccttccttcc gcctggtggc
gtactacacg ctgatcggtg 1680ccagcggcca gagggaggtg gtggccgact ccgtgtgggt
ggacgtcaag gactcctgcg 1740tgggctcgct ggtggtaaaa agcggccagt cagaagaccg
gcagcctgta cctgggcagc 1800agatgaccct gaagatagag ggtgaccacg gggcccgggt
ggtactggtg gccgtggaca 1860agggcgtgtt cgtgctgaat aagaagaaca aactgacgca
gagtaagatc tgggacgtgg 1920tggagaaggc agacatcggc tgcaccccgg gcagtgggaa
ggattacgcc ggtgtcttct 1980ccgacgcagg gctgaccttc acgagcagca gtggccagca
gaccgcccag agggcagaac 2040ttcagtgccc gcagccagcc gcccgccgac gccgttccgt
gcagctcacg gagaagcgaa 2100tggacaaagt cggcaagtac cccaaggagc tgcgcaagtg
ctgcgaggac ggcatgcggg 2160agaaccccat gaggttctcg tgccagcgcc ggacccgttt
catctccctg ggcgaggcgt 2220gcaagaaggt cttcctggac tgctgcaact acatcacaga
gctgcggcgg cagcacgcgc 2280gggccagcca cctgggcctg gccaggagta acctggatga
ggacatcatt gcagaagaga 2340acatcgtttc ccgaagtgag ttcccagaga gctggctgtg
gaacgttgag gacttgaaag 2400agccaccgaa aaatggaatc tctacgaagc tcatgaatat
atttttgaaa gactccatca 2460ccacgtggga gattctggct gtgagcatgt cggacaagaa
agggatctgt gtggcagacc 2520ccttcgaggt cacagtaatg caggacttct tcatcgacct
gcggctaccc tactctgttg 2580ttcgaaacga gcaggtggaa atccgagccg ttctctacaa
ttaccggcag aaccaagagc 2640tcaaggtgag ggtggaacta ctccacaatc cagccttctg
cagcctggcc accaccaaga 2700ggcgtcacca gcagaccgta accatccccc ccaagtcctc
gttgtccgtt ccatatgtca 2760tcgtgccgct aaagaccggc ctgcaggaag tggaagtcaa
ggctgctgtc taccatcatt 2820tcatcagtga cggtgtcagg aagtccctga aggtcgtgcc
ggaaggaatc agaatgaaca 2880aaactgtggc tgttcgcacc ctggatccag aacgcctggg
ccgtgaagga gtgcagaaag 2940aggacatccc acctgcagac ctcagtgacc aagtcccgga
caccgagtct gagaccagaa 3000ttctcctgca agggacccca gtggcccaga tgacagagga
tgccgtcgac gcggaacggc 3060tgaagcacct cattgtgacc ccctcgggct gcggggaaca
gaacatgatc ggcatgacgc 3120ccacggtcat cgctgtgcat tacctggatg aaacggagca
gtgggagaag ttcggcctag 3180agaagcggca gggggccttg gagctcatca agaaggggta
cacccagcag ctggccttca 3240gacaacccag ctctgccttt gcggccttcg tgaaacgggc
acccagcacc tggctgaccg 3300cctacgtggt caaggtcttc tctctggctg tcaacctcat
cgccatcgac tcccaagtcc 3360tctgcggggc tgttaaatgg ctgatcctgg agaagcagaa
gcccgacggg gtcttccagg 3420aggatgcgcc cgtgatacac caagaaatga ttggtggatt
acggaacaac aacgagaaag 3480acatggccct cacggccttt gttctcatct cgctgcagga
ggctaaagat atttgcgagg 3540agcaggtcaa cagcctgcca ggcagcatca ctaaagcagg
agacttcctt gaagccaact 3600acatgaacct acagagatcc tacactgtgg ccattgctgg
ctatgctctg gcccagatgg 3660gcaggctgaa ggggcctctt cttaacaaat ttctgaccac
agccaaagat aagaaccgct 3720gggaggaccc tggtaagcag ctctacaacg tggaggccac
atcctatgcc ctcttggccc 3780tactgcagct aaaagacttt gactttgtgc ctcccgtcgt
gcgttggctc aatgaacaga 3840gatactacgg tggtggctat ggctctaccc aggccacctt
catggtgttc caagccttgg 3900ctcaatacca aaaggacgcc cctgaccacc aggaactgaa
ccttgatgtg tccctccaac 3960tgcccagccg cagctccaag atcacccacc gtatccactg
ggaatctgcc agcctcctgc 4020gatcagaaga gaccaaggaa aatgagggtt tcacagtcac
agctgaagga aaaggccaag 4080gcaccttgtc ggtggtgaca atgtaccatg ctaaggccaa
agatcaactc acctgtaata 4140aattcgacct caaggtcacc ataaaaccag caccggaaac
agaaaagagg cctcaggatg 4200ccaagaacac tatgatcctt gagatctgta ccaggtaccg
gggagaccag gatgccacta 4260tgtctatatt ggacatatcc atgatgactg gctttgctcc
agacacagat gacctgaagc 4320agctggccaa tggtgttgac agatacatct ccaagtatga
gctggacaaa gccttctccg 4380ataggaacac cctcatcatc tacctggaca aggtctcaca
ctctgaggat gactgtctag 4440ctttcaaagt tcaccaatac tttaatgtag agcttatcca
gcctggagca gtcaaggtct 4500acgcctatta caacctggag gaaagctgta cccggttcta
ccatccggaa aaggaggatg 4560gaaagctgaa caagctctgc cgtgatgaac tgtgccgctg
tgctgaggag aattgcttca 4620tacaaaagtc ggatgacaag gtcaccctgg aagaacggct
ggacaaggcc tgtgagccag 4680gagtggacta tgtgtacaag acccgactgg tcaaggttca
gctgtccaat gactttgacg 4740agtacatcat ggccattgag cagaccatca agtcaggctc
ggatgaggtg caggttggac 4800agcagcgcac gttcatcagc cccatcaagt gcagagaagc
cctgaagctg gaggagaaga 4860aacactacct catgtggggt ctctcctccg atttctgggg
agagaagccc aacctcagct 4920acatcatcgg gaaggacact tgggtggagc actggcccga
ggaggacgaa tgccaagacg 4980aagagaacca gaaacaatgc caggacctcg gcgccttcac
cgagagcatg gttgtctttg 5040ggtgccccaa ctgaccacac ccccattccc ccactccaga
taaagcttca gttatatctc 5100a
510121663PRTHomo sapiens 2Met Gly Pro Thr Ser Gly
Pro Ser Leu Leu Leu Leu Leu Leu Thr His1 5
10 15Leu Pro Leu Ala Leu Gly Ser Pro Met Tyr Ser Ile
Ile Thr Pro Asn 20 25 30Ile
Leu Arg Leu Glu Ser Glu Glu Thr Met Val Leu Glu Ala His Asp 35
40 45Ala Gln Gly Asp Val Pro Val Thr Val
Thr Val His Asp Phe Pro Gly 50 55
60Lys Lys Leu Val Leu Ser Ser Glu Lys Thr Val Leu Thr Pro Ala Thr65
70 75 80Asn His Met Gly Asn
Val Thr Phe Thr Ile Pro Ala Asn Arg Glu Phe 85
90 95Lys Ser Glu Lys Gly Arg Asn Lys Phe Val Thr
Val Gln Ala Thr Phe 100 105
110Gly Thr Gln Val Val Glu Lys Val Val Leu Val Ser Leu Gln Ser Gly
115 120 125Tyr Leu Phe Ile Gln Thr Asp
Lys Thr Ile Tyr Thr Pro Gly Ser Thr 130 135
140Val Leu Tyr Arg Ile Phe Thr Val Asn His Lys Leu Leu Pro Val
Gly145 150 155 160Arg Thr
Val Met Val Asn Ile Glu Asn Pro Glu Gly Ile Pro Val Lys
165 170 175Gln Asp Ser Leu Ser Ser Gln
Asn Gln Leu Gly Val Leu Pro Leu Ser 180 185
190Trp Asp Ile Pro Glu Leu Val Asn Met Gly Gln Trp Lys Ile
Arg Ala 195 200 205Tyr Tyr Glu Asn
Ser Pro Gln Gln Val Phe Ser Thr Glu Phe Glu Val 210
215 220Lys Glu Tyr Val Leu Pro Ser Phe Glu Val Ile Val
Glu Pro Thr Glu225 230 235
240Lys Phe Tyr Tyr Ile Tyr Asn Glu Lys Gly Leu Glu Val Thr Ile Thr
245 250 255Ala Arg Phe Leu Tyr
Gly Lys Lys Val Glu Gly Thr Ala Phe Val Ile 260
265 270Phe Gly Ile Gln Asp Gly Glu Gln Arg Ile Ser Leu
Pro Glu Ser Leu 275 280 285Lys Arg
Ile Pro Ile Glu Asp Gly Ser Gly Glu Val Val Leu Ser Arg 290
295 300Lys Val Leu Leu Asp Gly Val Gln Asn Pro Arg
Ala Glu Asp Leu Val305 310 315
320Gly Lys Ser Leu Tyr Val Ser Ala Thr Val Ile Leu His Ser Gly Ser
325 330 335Asp Met Val Gln
Ala Glu Arg Ser Gly Ile Pro Ile Val Thr Ser Pro 340
345 350Tyr Gln Ile His Phe Thr Lys Thr Pro Lys Tyr
Phe Lys Pro Gly Met 355 360 365Pro
Phe Asp Leu Met Val Phe Val Thr Asn Pro Asp Gly Ser Pro Ala 370
375 380Tyr Arg Val Pro Val Ala Val Gln Gly Glu
Asp Thr Val Gln Ser Leu385 390 395
400Thr Gln Gly Asp Gly Val Ala Lys Leu Ser Ile Asn Thr His Pro
Ser 405 410 415Gln Lys Pro
Leu Ser Ile Thr Val Arg Thr Lys Lys Gln Glu Leu Ser 420
425 430Glu Ala Glu Gln Ala Thr Arg Thr Met Gln
Ala Leu Pro Tyr Ser Thr 435 440
445Val Gly Asn Ser Asn Asn Tyr Leu His Leu Ser Val Leu Arg Thr Glu 450
455 460Leu Arg Pro Gly Glu Thr Leu Asn
Val Asn Phe Leu Leu Arg Met Asp465 470
475 480Arg Ala His Glu Ala Lys Ile Arg Tyr Tyr Thr Tyr
Leu Ile Met Asn 485 490
495Lys Gly Arg Leu Leu Lys Ala Gly Arg Gln Val Arg Glu Pro Gly Gln
500 505 510Asp Leu Val Val Leu Pro
Leu Ser Ile Thr Thr Asp Phe Ile Pro Ser 515 520
525Phe Arg Leu Val Ala Tyr Tyr Thr Leu Ile Gly Ala Ser Gly
Gln Arg 530 535 540Glu Val Val Ala Asp
Ser Val Trp Val Asp Val Lys Asp Ser Cys Val545 550
555 560Gly Ser Leu Val Val Lys Ser Gly Gln Ser
Glu Asp Arg Gln Pro Val 565 570
575Pro Gly Gln Gln Met Thr Leu Lys Ile Glu Gly Asp His Gly Ala Arg
580 585 590Val Val Leu Val Ala
Val Asp Lys Gly Val Phe Val Leu Asn Lys Lys 595
600 605Asn Lys Leu Thr Gln Ser Lys Ile Trp Asp Val Val
Glu Lys Ala Asp 610 615 620Ile Gly Cys
Thr Pro Gly Ser Gly Lys Asp Tyr Ala Gly Val Phe Ser625
630 635 640Asp Ala Gly Leu Thr Phe Thr
Ser Ser Ser Gly Gln Gln Thr Ala Gln 645
650 655Arg Ala Glu Leu Gln Cys Pro Gln Pro Ala Ala Arg
Arg Arg Arg Ser 660 665 670Val
Gln Leu Thr Glu Lys Arg Met Asp Lys Val Gly Lys Tyr Pro Lys 675
680 685Glu Leu Arg Lys Cys Cys Glu Asp Gly
Met Arg Glu Asn Pro Met Arg 690 695
700Phe Ser Cys Gln Arg Arg Thr Arg Phe Ile Ser Leu Gly Glu Ala Cys705
710 715 720Lys Lys Val Phe
Leu Asp Cys Cys Asn Tyr Ile Thr Glu Leu Arg Arg 725
730 735Gln His Ala Arg Ala Ser His Leu Gly Leu
Ala Arg Ser Asn Leu Asp 740 745
750Glu Asp Ile Ile Ala Glu Glu Asn Ile Val Ser Arg Ser Glu Phe Pro
755 760 765Glu Ser Trp Leu Trp Asn Val
Glu Asp Leu Lys Glu Pro Pro Lys Asn 770 775
780Gly Ile Ser Thr Lys Leu Met Asn Ile Phe Leu Lys Asp Ser Ile
Thr785 790 795 800Thr Trp
Glu Ile Leu Ala Val Ser Met Ser Asp Lys Lys Gly Ile Cys
805 810 815Val Ala Asp Pro Phe Glu Val
Thr Val Met Gln Asp Phe Phe Ile Asp 820 825
830Leu Arg Leu Pro Tyr Ser Val Val Arg Asn Glu Gln Val Glu
Ile Arg 835 840 845Ala Val Leu Tyr
Asn Tyr Arg Gln Asn Gln Glu Leu Lys Val Arg Val 850
855 860Glu Leu Leu His Asn Pro Ala Phe Cys Ser Leu Ala
Thr Thr Lys Arg865 870 875
880Arg His Gln Gln Thr Val Thr Ile Pro Pro Lys Ser Ser Leu Ser Val
885 890 895Pro Tyr Val Ile Val
Pro Leu Lys Thr Gly Leu Gln Glu Val Glu Val 900
905 910Lys Ala Ala Val Tyr His His Phe Ile Ser Asp Gly
Val Arg Lys Ser 915 920 925Leu Lys
Val Val Pro Glu Gly Ile Arg Met Asn Lys Thr Val Ala Val 930
935 940Arg Thr Leu Asp Pro Glu Arg Leu Gly Arg Glu
Gly Val Gln Lys Glu945 950 955
960Asp Ile Pro Pro Ala Asp Leu Ser Asp Gln Val Pro Asp Thr Glu Ser
965 970 975Glu Thr Arg Ile
Leu Leu Gln Gly Thr Pro Val Ala Gln Met Thr Glu 980
985 990Asp Ala Val Asp Ala Glu Arg Leu Lys His Leu
Ile Val Thr Pro Ser 995 1000
1005Gly Cys Gly Glu Gln Asn Met Ile Gly Met Thr Pro Thr Val Ile
1010 1015 1020Ala Val His Tyr Leu Asp
Glu Thr Glu Gln Trp Glu Lys Phe Gly 1025 1030
1035Leu Glu Lys Arg Gln Gly Ala Leu Glu Leu Ile Lys Lys Gly
Tyr 1040 1045 1050Thr Gln Gln Leu Ala
Phe Arg Gln Pro Ser Ser Ala Phe Ala Ala 1055 1060
1065Phe Val Lys Arg Ala Pro Ser Thr Trp Leu Thr Ala Tyr
Val Val 1070 1075 1080Lys Val Phe Ser
Leu Ala Val Asn Leu Ile Ala Ile Asp Ser Gln 1085
1090 1095Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu
Glu Lys Gln Lys 1100 1105 1110Pro Asp
Gly Val Phe Gln Glu Asp Ala Pro Val Ile His Gln Glu 1115
1120 1125Met Ile Gly Gly Leu Arg Asn Asn Asn Glu
Lys Asp Met Ala Leu 1130 1135 1140Thr
Ala Phe Val Leu Ile Ser Leu Gln Glu Ala Lys Asp Ile Cys 1145
1150 1155Glu Glu Gln Val Asn Ser Leu Pro Gly
Ser Ile Thr Lys Ala Gly 1160 1165
1170Asp Phe Leu Glu Ala Asn Tyr Met Asn Leu Gln Arg Ser Tyr Thr
1175 1180 1185Val Ala Ile Ala Gly Tyr
Ala Leu Ala Gln Met Gly Arg Leu Lys 1190 1195
1200Gly Pro Leu Leu Asn Lys Phe Leu Thr Thr Ala Lys Asp Lys
Asn 1205 1210 1215Arg Trp Glu Asp Pro
Gly Lys Gln Leu Tyr Asn Val Glu Ala Thr 1220 1225
1230Ser Tyr Ala Leu Leu Ala Leu Leu Gln Leu Lys Asp Phe
Asp Phe 1235 1240 1245Val Pro Pro Val
Val Arg Trp Leu Asn Glu Gln Arg Tyr Tyr Gly 1250
1255 1260Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe Met
Val Phe Gln Ala 1265 1270 1275Leu Ala
Gln Tyr Gln Lys Asp Ala Pro Asp His Gln Glu Leu Asn 1280
1285 1290Leu Asp Val Ser Leu Gln Leu Pro Ser Arg
Ser Ser Lys Ile Thr 1295 1300 1305His
Arg Ile His Trp Glu Ser Ala Ser Leu Leu Arg Ser Glu Glu 1310
1315 1320Thr Lys Glu Asn Glu Gly Phe Thr Val
Thr Ala Glu Gly Lys Gly 1325 1330
1335Gln Gly Thr Leu Ser Val Val Thr Met Tyr His Ala Lys Ala Lys
1340 1345 1350Asp Gln Leu Thr Cys Asn
Lys Phe Asp Leu Lys Val Thr Ile Lys 1355 1360
1365Pro Ala Pro Glu Thr Glu Lys Arg Pro Gln Asp Ala Lys Asn
Thr 1370 1375 1380Met Ile Leu Glu Ile
Cys Thr Arg Tyr Arg Gly Asp Gln Asp Ala 1385 1390
1395Thr Met Ser Ile Leu Asp Ile Ser Met Met Thr Gly Phe
Ala Pro 1400 1405 1410Asp Thr Asp Asp
Leu Lys Gln Leu Ala Asn Gly Val Asp Arg Tyr 1415
1420 1425Ile Ser Lys Tyr Glu Leu Asp Lys Ala Phe Ser
Asp Arg Asn Thr 1430 1435 1440Leu Ile
Ile Tyr Leu Asp Lys Val Ser His Ser Glu Asp Asp Cys 1445
1450 1455Leu Ala Phe Lys Val His Gln Tyr Phe Asn
Val Glu Leu Ile Gln 1460 1465 1470Pro
Gly Ala Val Lys Val Tyr Ala Tyr Tyr Asn Leu Glu Glu Ser 1475
1480 1485Cys Thr Arg Phe Tyr His Pro Glu Lys
Glu Asp Gly Lys Leu Asn 1490 1495
1500Lys Leu Cys Arg Asp Glu Leu Cys Arg Cys Ala Glu Glu Asn Cys
1505 1510 1515Phe Ile Gln Lys Ser Asp
Asp Lys Val Thr Leu Glu Glu Arg Leu 1520 1525
1530Asp Lys Ala Cys Glu Pro Gly Val Asp Tyr Val Tyr Lys Thr
Arg 1535 1540 1545Leu Val Lys Val Gln
Leu Ser Asn Asp Phe Asp Glu Tyr Ile Met 1550 1555
1560Ala Ile Glu Gln Thr Ile Lys Ser Gly Ser Asp Glu Val
Gln Val 1565 1570 1575Gly Gln Gln Arg
Thr Phe Ile Ser Pro Ile Lys Cys Arg Glu Ala 1580
1585 1590Leu Lys Leu Glu Glu Lys Lys His Tyr Leu Met
Trp Gly Leu Ser 1595 1600 1605Ser Asp
Phe Trp Gly Glu Lys Pro Asn Leu Ser Tyr Ile Ile Gly 1610
1615 1620Lys Asp Thr Trp Val Glu His Trp Pro Glu
Glu Asp Glu Cys Gln 1625 1630 1635Asp
Glu Glu Asn Gln Lys Gln Cys Gln Asp Leu Gly Ala Phe Thr 1640
1645 1650Glu Ser Met Val Val Phe Gly Cys Pro
Asn 1655 1660342783DNAHomo sapiens 3ctcctcccca
tcctctccct ctgtccctct gtccctctga ccctgcactg tcccagcacc 60atgggaccca
cctcaggtcc cagcctgctg ctcctgctac taacccacct ccccctggct 120ctggggagtc
ccatgtgagt ggttatgact ctacccacaa acagggctgg ttctggggtg 180gaagcagaca
tttgggggtc caggtccctg tagaattcag ggtgcatttg ggtgtttgtg 240gattcagggg
ttagcaggtt gggaatgatt atatatattt gggctgcctg tgagtttggg 300tgtttgtggt
tgggtgtttg tggaatccag gtatcatgga attggagttt atatacattt 360gggctgcctg
tgagtttggg tgtttgtggt tgggtgtttg tggaatccag gtatcgtgga 420attggagttt
atatacattt gggctgcctg agagtttggg tgtttatggg ttgggtgttt 480gtggaatcca
ggtatggtgg aattggagtt tgggatgttt ctagaattga ggtcatctgt 540tggtttaggg
tgtatgtggt gttcattgat ggtgcggttg ggggtgtttg gagactcgga 600ggtttggact
ttacaagatt tgggagtttg cagcttgggg acttgcaatt ttcagtgtgg 660gtttaaagat
tggctacttc gggttcatgt atagttgggg catttggaat tgattgtatt 720tattaggact
ggggtgttgg aggtttaggc tgggtttggg gtgctctaag atttgaggtt 780tagaggtttt
ggcgtatgtg ggtttgggta ggtagagttg agggtgtccg ggagtttgag 840tgtttacata
tttggagtgt ttagagaggt agaggtttag ggtttggggc atgtgtgggt 900ttaggcgatt
gtgggtctgg aagtccagag acttggagga gttgctgacg ctggttggaa 960ggttcagggt
ttggtgggat gtgtggcccc ctcgttgccc aggctttcaa aggccaggcc 1020cagctggctg
agagtgggag tcatggtggc tgctgtcctg cccatgtggt tgagacggtg 1080gcagtgccca
gagaagataa tggcattggc aagtgcgccg gcagtcactg gatcctctcc 1140aggaccagag
gctggggcac acagcctgcc aggcgctgac tccagtgagg actggcgtct 1200cacatccgtg
gaatgacaag cccactcccg tgccccactc cgacaggtac tctatcatca 1260cccccaacat
cttgcggctg gagagcgagg agaccatggt gctggaggcc cacgacgcgc 1320aaggggatgt
tccagtcact gttactgtcc acgacttccc aggcaaaaaa ctagtgctgt 1380ccagtgagaa
gactgtgctg acccctgcca ccaaccacat gggcaacgtc accttcacgg 1440tgagtgcaga
ctggcgcagg acccggctga cacccacagc cacgcccact ccccccctcc 1500tcctgagccc
ctccccttct gtcttctccc tttctaagcc ctgcccttcc ctgagactcc 1560accccttcgg
agtcgcctct ccttctaagc ccctcccttc tctgagactc caccccttct 1620gagtctcctc
cccttataag cccctccctt ttctgagacc cccccccacc ccttctgaat 1680ctcctcccct
tctaagccct gaccttccct gagaccccac cccttctgag actcctcccc 1740ttctgagtcc
ctcccttccc tgagacccca ccccttctga ggttcctccc cttctctgag 1800actccacccc
ttctgagtct cctccccctc taagtccctc ccactgaatt ccttttccaa 1860gcccctcccc
ctcgaagtct cctcttctga actcctcccc tcttagtctc catcactttc 1920taagttccct
cacctgagtc cctccccctt tctgagcccc tcccatgtca gccccttccc 1980tttctgagtc
cccgcccctt ctgagcccct cctcctataa gctctctcct ccttgtgagc 2040tcttcttttt
gagttccctc cctggtcccc cctctcccct cgcacctcct tcacatgccc 2100ctccctcccc
aaaacggcca cctcggaaga ccaagaataa tgggcaggca aggagggacc 2160cagcccaaga
tccggaagct ggaccgtggg catggggcct tggaacagac ccctgacaat 2220gccctgccca
cgcctagatc ccagccaaca gggagttcaa gtcagaaaag gggcgcaaca 2280agttcgtgac
cgtgcaggcc accttcggga cccaagtggt ggagaaggtg gtgctggtca 2340gcctgcagag
cgggtacctc ttcatccaga cagacaagac catctacacc cctggctcca 2400caggtgaggc
tgggggcggc tggagagggc ggggcaccgg cgtgggcggg ctagggtctc 2460acgaggcctc
tttgtctctc cccagttctc tatcggatct tcaccgtcaa ccacaagctg 2520ctacccgtgg
gccggacggt catggtcaac attgaggtgc cagccagagg gggccccagg 2580ggaagcaggg
gcacaggctt aggagaggca aagagatcga gagagacaga gaaagacaca 2640ccggaagggg
tgcagtggca gagacacaga ggcaaagaga tatgcagaca cacacccaca 2700caacacacac
acatacagca cacaacatgc acacacacag cacacaatac acacacagag 2760gcaaagagat
atgcagacac atgtgcacac acaatgcaca cacacaatgc aacacacaca 2820aacacacaac
atacacgacc acacaacaca cacaacacaa cacacaacac aatacacaca 2880gcacaacgtg
catgaccaca cacacaacac acaacacaca caacacaata cacaacatac 2940acaaccacgc
aatacacaca aaacacacac aacacaacac aacatacata accacaccac 3000acacaacaca
caaccacaca acactatcac acaacacaca caaacacaca caacacacaa 3060cacacacaac
acacacaaaa cacaacacac acacaacata cacaaccaca caacacacaa 3120ccacacaaca
tacacgacca cacaacacag tgcacacaaa catagcacac acaacacaca 3180acccaacaca
caaccacaca atacaccata tggcgcgcac acacacacac acacacacac 3240aggctgagag
acaaggtgga gatccaggga gaccccaggg agcagtgcag gtgtccgtgg 3300attctgcttt
cagttaaacc cctgatcact tcacctccct gagcctcagt taccttatct 3360gaatatcggg
atcatgacgg ataattgtat gtcatctatt ctaccgacgg cagccagagg 3420acgcctgtga
gcacctgagt cagggcccat ccctgctctg cctacagccc tccatggctc 3480ccaccttcct
atgcgtcaaa gcccaagtcc tccctgcagt ccacaaggcc ctgcacacct 3540tgccctgtcc
cttccctgcc ctcccctcct ccctctctcc ccctcgttca ctcttctgga 3600gccacacggg
ccatcctccc tgttcctcca acacccaggt gcagtcctgc cttggcgcct 3660tggcacgggc
tgtgccctct tctcaagaaa accctcttct tccaaatatc cacacagctt 3720gttctctctc
ctcctttaag tctttgctca aatgtcacca atgtctcaat tttacaatga 3780ggtctctctg
agtaacctat aaagtcgcaa atacccaccc tgagcgtccc ccctccccgc 3840tacacacact
cctccttcct gccatgtcct gcaaatgaga tttattcatt tgataattgc 3900ttctcccatc
gcctcgccct ctattgaacc taaatccctc caggaaggaa ttgttatgtt 3960tgttgagggt
tttgtcacct gaactcagca caatgctggt atatagttgg gtttaataaa 4020aaacttactg
gaagaagcga gaaggatggg aggagagaag gggaaggagg gtgttctcat 4080agaattatca
tgaggatgtg ttgaaatcat acaaggctag gtgcagtggc tcacacttgt 4140aatcccagct
gtttgggagg ccaaggcggg aggatcgctt gagcccaaga gtccaagacc 4200agcctgggca
acacagccag accctgtctc tacaaaaaag aaaagttaaa aacaaacaaa 4260aaaacagctg
tgtgtggtgg tgcttgcttg tggttgcagc taccccagga ggctgaggca 4320ggaggatcac
ttgagcccag gaattccagg ctgcagtgag ccgtgatcgc accactgcac 4380tccagcctgg
gtggcagagt gagaccctgt ctcaaaaaat aattggggca aatgcaatgg 4440ctcaagcctg
taattccaac atttcgggag gcagaggtgg gaagactgct cgaggccaag 4500agttcaagac
cagcctggga aagctaggga gactacatct ctacaaaaaa aatgtaaaaa 4560ttatctagat
ttagggattg atgtggtctg tggggaacag agaccacaca tctcttgtaa 4620aggcacaaca
gttgcccagc tccaattaga tgtctcctgc taaccagagt acactatcca 4680cagaaatttc
cttgtttcca acagaagcta gaaaaacaga tttttggcca ggtgcagtgg 4740ctcactccta
taatcccagc actttgggag gtggaggcgg gcagatcacg aggtcaggag 4800atcgagacca
tcctggctaa cacggtgaaa ccccgtcttt attaaaagta caaaaaaaaa 4860attagctggg
cgtggtggcg ggcacctgta gtcccagcta ctcgagaggc tgaggcagga 4920gaatggtgtg
aacccgggag gcggagcctg cagtgagccg agatctcgcc attacactcc 4980agcctgggcg
acagagcaag actccgtctc aaaaaaaaaa acaaaaaaaa caaaaaaaaa 5040acagattttt
atatgtttta attcctaaag ccagctcacg gccttcagat atgccacttg 5100cctgatccct
gttacctctg tacaatttct tttaaactta tttattcatt cattcattca 5160ttattattat
ttttgagaca gggtctcatt ctgttgccca ggctagagtg cagtggcaca 5220atcacagctc
actgcagcat tgacctcctg ggcccaagct gtcctcctgt ctcagcctcc 5280tgggtagctg
ggaccacaga cgtgcgccac cacatccagc taattttaaa aaatttttgt 5340agagatggag
tctccctaca tttcccaggc tggtcttgaa cccttgacct tgagcaatct 5400tcccacttct
gcctctcaaa gtgctgggat tacaggcttg agccattgcg ctcgccctaa 5460tacattattt
tttgagatgg ggtctcgctc tttcacccag actggagtgc agtggtgcaa 5520tgatgtctca
tgatgtttaa atgttggcag caaatgaaat gacactacta gttattagta 5580ttcagagaga
cactgaaaaa atgagcccct actcatatga actatgtccc aagccaacac 5640agtaggtgcc
attataatct cctgtttcaa gatttgcaca ttgagcacag agaggttagg 5700taacttgccc
agggtcacac agcttgtaag tggcacagta gagattgaaa cctaaggttg 5760actgactccg
gtccttgttc tttttttcga gacagactct cactctgtct cccaggctgg 5820agtgcagtgg
agtgatcttg gctctctgca atctccgcct cccgggttca agcgattctc 5880ccgcctcagc
ctcctgagta gctgggatta cgggtgccta ccaccatgcc tggctaattt 5940ttgtattttt
agtagagaca gggtttcatc acgttggcca ggctggtctt gaactcctga 6000cctcaggtga
tctgcccgcc tcagcctccc agagtgctgg gatgacaggc gtgagccgct 6060gcgcccacct
gggtccctgt tcttaaccac agtagacact gtgcacagag aatgtccaga 6120cacaggtcgg
ggagagctga gaggctaagc ccagcctccg aagagccact ttatcctcta 6180tccttccctc
ctgcctccca cagaacccgg aaggcatccc ggtcaagcag gactccttgt 6240cttctcagaa
ccagcttggc gtcttgccct tgtcttggga cattccggaa ctcgtcaagt 6300atgtcaggtt
cttgaggagg gggctcaggg ctcccctatc cccggagagg gagcaggggg 6360gctccgaggc
ctgagagacc actcatccgc cctcctcaca gcatgggcca gtggaagatc 6420cgagcctact
atgaaaactc accacagcag gtcttctcca ctgagtttga ggtgaaggag 6480tacggtaaga
ggaggagggg ctggggggag tcagtgccca gaacgcctgg cccagcgccg 6540gccccaccaa
cgccatctct cccccagtgc tgcccagttt cgaggtcata gtggagccta 6600cagagaaatt
ctactacatc tataacgaga agggcctgga ggtcaccatc accgccaggt 6660gagggactgg
gggtggggcc aggtaagagc caggtgaggg accaggtgaa gaccaggtgg 6720gggactgggg
gtggagtcag gtggggggct ggagatggga ccaggtgggg ggctgggggt 6780ggagtcaggt
ggggggctgg gggtggggaa ggtggggggc tgggggtggg gcaaggtgag 6840gggctggggg
tgggaccagg tggggggctg gggggtggag tcaggtgggg gctgggagtg 6900gggaaggtgg
ggggctgggg gtggggccag gtgaggggct ggaggtggga ccatgtgggg 6960ggtgggagtg
gggcaaggtg gggggctggg ggtggggcca ggtgaggggc tggaggtggg 7020gccaggtgag
aggccagcag tgggttgggg gctccagtct tcagcacagg caggagaagc 7080tgggggagat
cccattctcc aggagggatg gacctgaagc cctccttgtc tgtcccgtag 7140gttcctctac
gggaagaaag tggagggaac tgcctttgtc atcttcggga tccaggatgg 7200cgaacagagg
atttccctgc ctgaatccct caagcgcatt ccggtaccat agacggaggc 7260cgctttgatc
cctgccccag tccccgccac ctctgagccc gctcccctct ctgagccctc 7320ctctcccttc
tcagattgag gatggctcgg gggaggttgt gctgagccgg aaggtactgc 7380tggacggggt
gcagaacccc cgagcagaag acctggtggg gaagtctttg tacgtgtctg 7440ccaccgtcat
cttgcactca ggtgaggccc agtctgaagg ccaggctcag gaccaccaag 7500tgggccggtc
tgagagggga gaccaggtca gaagagaaag cctagtctaa ggagggaggc 7560tcagagtgaa
agtggggttc agtctgatgg ggtaggccca gtctgagagg ggaggccgag 7620tatgaagatg
gattccagcc tgatgggggg aggcagggcc agtataaagg tggggtccgg 7680gctgatgggg
gcacaggccc agtatgaagt ctgtgtccag tctgatgagg gaggcagggc 7740cagtataaag
atgggtccag tctgatgggg gaggcagggc cagtataaag gtggggtccg 7800gtctgatggg
ggtcacaggc ccagtatgaa gtctgtgcca gtctgatgga ggaggcaagg 7860ccagtataaa
ggtggagtcc agtctgatgg ggggcacagg cccagtatga aagtggactc 7920tactctgagg
gaggaggtct agtctgaagt tggggtccat tctgagggag gaggtctaat 7980cctgaggggt
ggcccagaag cctacactca cagctggtcc cctcaggcag tgacatggtg 8040caggcagagc
gcagcgggat ccccatcgtg acctctccct accagatcca cttcaccaag 8100acacccaagt
acttcaaacc aggaatgccc tttgacctca tggtgagacc cggggcggga 8160aggggtccca
ctcctccctt cggggacacc ggccacagcc ctgagcctgc ctgaacttcc 8220cccacctgca
ccccacatca caggtgttcg tgacgaaccc tgatggctct ccagcctacc 8280gagtccccgt
ggcagtccag ggcgaggaca ctgtgcagtc tctaacccag ggagatggcg 8340tggccaaact
cagcatcaac acacacccca gccagaagcc cttgagcatc acggtgcgtc 8400tgggcccagc
ctcggaaccc catcactggg aagacggtac aggggttctg gtgtttgcac 8460agtggggtcc
tgtcatttgc atacagatat tctcatctgc atagagaggt tctctcctgc 8520gcagaggggt
cctgccattt gcatagagat actctcatct gcatagaggg gttctgtcct 8580gcacagtggg
gtcctgccat ttgcatagac attctcattt gcctagaggg gttctgtcct 8640gcacagtggg
gtcctgccgt ctgcatggag gggtccgcag tttgaggaaa caggaatctt 8700cctcttgcat
gccctgctcc ttccacttac acggagaggc gctccatcca cgcacagtct 8760ttccactccc
atgggggaag gagcctgaat ctcacaagga gggttgtgta gtgtttggga 8820caggcccatt
gttgtgaggt ggtctcagtt ctcctggctt ctgtgcacgt ggctctgttg 8880cccctcactg
ggagggaagc aagtctcatg acagctgcgg aggttgcaga tggcctccca 8940gtccctctgc
agctcccagg ctgcgcaccc cacttacccc tccctgtgct cagcatgtgc 9000gtgaatttcc
ggtggctacc atgagaaatg gccacagcct agtgatctaa agcaacacac 9060atttatgggt
ctatagtttg agaggtcaga agtcctggct ctgggggaaa gttcgctccc 9120ttgctttttc
cagtgtcgcc agggcaccct aaaggcctgg ctcatggccc cttcctccac 9180ctttaaaggc
agcagcatag catcttccag tgtctctctt tctctctgtc tctgtctctc 9240ctttctcccc
tgcccctgct taataaagac ccttatgatt acattagctc cacctacata 9300atccaggata
atgattccat ctccagatcc ctaacttaat cccatctgca aagccccttt 9360tgttaagaaa
ggccaccaat tcccaggtct cagggattcg ggtgtgggta tcctcgggcg 9420gcgaccagca
ggcatccctc tttccccacc caggtgcgca cgaagaagca ggagctctcg 9480gaggcagagc
aggctaccag gaccatgcag gctctgccct acagcaccgt gggcaactcc 9540aacaattacc
tgcatctctc agtgctacgt acagagctca gacccgggga gaccctcaac 9600gtcaacttcc
tcctgcgaat ggaccgcgcc cacgaggcca agatccgcta ctacacctac 9660ctggtccgtg
gccacctgga aacctcagcc cccgcctcct ccttgtttct tccgcacccc 9720tgggactcct
tcccccatcc cggatccctc cctgcgttcc ctgccactca ccctccccag 9780cctgatgcca
gcctgtcccc ccagatcatg aacaagggca ggctgttgaa ggcgggacgc 9840caggtgcgag
agcccggcca ggacctggtg gtgctgcccc tgtccatcac caccgacttc 9900atcccttcct
tccgcctggt ggcgtactac acgctgatcg gtgccagcgg ccagagggag 9960gtggtggccg
actccgtgtg ggtggacgtc aaggactcct gcgtgggctc ggtaagtgtg 10020ccctgggctc
gctcgccccc tctccctctc cctactcctc tctctctctc tctctccctg 10080tctcctctct
ctctctctct ccctttctcc ttttctctct cctttctctc tcttctcttc 10140ctctcccttt
ctctcctccc tctctgtctc tcaactgtct ctctttttat ctctctttcc 10200ctctctctac
atctctcttt ccctctctct ttatttctct ttccttctct ctctccctct 10260ctcgatctct
ctttctctcc atctctctcc ttttctctct ccctctctct ctccttttct 10320ctctccctgt
ctctttccct ttccctctct ctcccctctc tttctctccc tctctctttc 10380cctctccctc
tctctctccc tttctctctc tccctctctc tccttctctc tccctctttc 10440tctccttctc
tctttccctc tctctctccc tctctctttc cctctctctc cctctccctt 10500tctctccctc
tttccctttc cctctctccc ccctcactct ccctctctct gtctctccgt 10560ctctctccct
ctctccctgt ctctccgtct ctctccctgt ctctcccttt ctctctctct 10620cccgccctct
ctccctctct ctccctccct ctctcccttt ctctctctct ccctctctct 10680ccccctcccc
agccccacgg ctccccccaa cctttctgtc tttccactct agcccagcac 10740ccactccatc
ccaggcactc ctctctccca gggctgactt ctttcggcgt ctccaccctc 10800cccacagctg
gtggtaaaaa gcggccagtc agaagaccgg cagcctgtac ctgggcagca 10860gatgaccctg
aagatagagg gtgaccacgg ggcccgggtg gtactggtgg ccgtggacaa 10920gggcgtgttc
gtgctgaata agaagaacaa actgacgcag agtaaggtaa gggccagtga 10980cccaaggctg
ctgagaagag gcggaggcac ggagctgggg ctgggggagg tgggtgggac 11040tggagagggc
agtgcagtgg ggggcatgcg ctgaaagcag agatcggagc agaccagaca 11100cagggatggt
tgaagctgaa gatgggaatg aggttggaca tgggttccaa ttggggatgg 11160tcctgagaat
tggacttttt tttctgtttg tttgtttgtt tttgagacag agtctctctc 11220tgtcaccagg
ctggagtgca gtggcacaat ctcggctcac tgcaacctct gcctcccagg 11280ttcaagcgat
tctcctgcct cagcttccct agtagctggg actacaggtg cccatcacca 11340cgcccagcta
atttttgtat ttttagtgaa gacgggggtt tcaccatgtt ggccaggatg 11400gtctcgatct
cttggccttg tgatccaccc gcctcgacct cccaaagtgt tgggattaca 11460ggcgtgagcc
actgcgcccg gctgagaatt ggacactttc aactggggcc ctgagaggct 11520ggtggcagca
cacccagggt cattcagtgg ggaaggtttc cggagtaggg acgaagatgg 11580agatggggtt
ggcttgggat caggagtgag gatgggaatg cagatggaat cagaggggaa 11640atggagataa
gatttggaat ggaggccagg tgcggtggct cacgtctgga atcccagcac 11700tttgggaggt
caaggtggga ggatcacttg aggccaggag ttcagaccag cttgggcaac 11760atggcaagac
cccatctcta cagaaaaaat tttaaaatag ctgggcatga tggcgcatgc 11820ctgtagtccc
atctgctcag gaggcagagg tgcgaggatt gcttgagccc aggaatttga 11880ggctgcagtg
agctatgcct gcaccactgc actccagcct gggagacagt ggaaaatccc 11940aacttaaaaa
aaaaaaaaaa gaatggaaag aaaggaggaa aaaaaaagaa gagagagaga 12000aacagagaga
aagaaaaaga aaggagataa agaggaaggg agggagggag tgaagaatga 12060aggaaggaaa
gaaggaagga aggaaggagg gaaggaggga aggaaagggg gagcaaagga 12120aggaggaaag
gaggaatgga gggaggaagg gagggagagg aaggaaggga aagaaagaag 12180acagaaagaa
aagaaaaaga aggccgggca tggtggctca ctcctgtaat ccctttggga 12240ggccaagcac
tttgggaggc caagacaggc gaatcatttc aggtcaggag ttcgagacca 12300gcctggccaa
catggtgaaa tcccgtctct actaaatata taaaaattag ctgggcatgg 12360tggcatgcac
ctgtagtccc agatactcgg gaggctgagg caggaaaatt gcctgaacct 12420gggagttgga
ggttacagtg agcggagatc acaccactgc actccagcct gggtgacaga 12480gcaagactcc
atctcgaaag aaagaaagag agagagtgag aaagagaaag aaaaagagaa 12540ggaaggagag
agaaggaagg aaggaaagag aaagagaaag gaagggcaga agcaggaatg 12600ggggagatga
gagtgggaca gggtggggtc atttgggaag agatacacag gtgcatatgt 12660gggggatccc
aattgtcagc ctggcctccc tgcgtcccgc cacccctatg ccccccgcag 12720atctgggacg
tggtggagaa ggcagacatc ggctgcaccc cgggcagtgg gaaggattac 12780gccggtgtct
tctccgacgc agggctgacc ttcacgagca gcagtggcca gcagaccgcc 12840cagagggcag
gtgaggtcgc caccaggggc cggtgcaggg acagacagca cctccacctc 12900ccagatgctg
ggagcagagc tctggaaacc gggggcctgg gttcaagccc cgcctccacc 12960accacctagt
aaatccctcc cctctgagcc tcagtttgct cttccatcaa atgggagcag 13020gaacaccccc
acctcacacg atcgtgaggg gtgaaccgag gacacctagt aggtgcctca 13080tccatcttct
tctcggtccg cctgccctgc agaacttcag tgcccgcagc cagccgcccg 13140ccgacgccgt
tccgtgcagc tcacggagaa gcgaatggac aaaggtggga gcctttccta 13200cccactcctg
cccccgagcc ccaccccagg agaccccagc ccggccgtgc aggagccaga 13260gagggaggag
gggaggccct ggcggcgggg aagtcctccc tggggtccgt cccgcgtccc 13320tcctgctgcc
ggcccccggc tgagggtgtg gcctggggga acacgtgctc ccgcagtcgg 13380caagtacccc
aaggagctgc gcaagtgctg cgaggacggc atgcgggaga accccatgag 13440gttctcgtgc
cagcgccgga cccgtttcat ctccctgggc gaggcgtgca agaaggtctt 13500cctggactgc
tgcaactaca tcacagagct gcggcggcag cacgcgcggg ccagccacct 13560gggcctggcc
aggagtaggt cccacggggt ggggacaggg ggagggggcc gtctgatggg 13620ggaggagact
cctgtctgag gagggaggat gccctgtctg gtgggggtgg ggctggagga 13680ggccgctgtc
tgagggggga ggaggcccct gtctgagggg gcaggaggtc cctgtctcag 13740gggggaggag
gcccctgtct gaggagggag gaaacctccg tctgaggagg gaggaggtcc 13800ctgtctgagg
agggaggagg ccttgagggg ggaggaggtc cccgtctgag gagggaggag 13860gcctctgtct
gaggagagag gaggtacctg tctgaggggg gaggaggcct ctgtctgagg 13920ggggaggatg
cccctgtctg agggggtagg aggaggcctc tgtctcgggg ggaggagtcc 13980cctgtctgag
gagggaggag gcctctgtct gaggggggag gatgccgctg tctgagaggg 14040taggaggagg
cctctgtctg ttgggagagg aggcccctgt ctgagggtga tgccgatgag 14100gtgatgccct
gccagcgtga ggtagagaag acccaggtct gaagagggga ggatcaagtc 14160agagaagcgt
agatgcccat ctgagatgga ggaggctccc gtccgagggg aggggacact 14220cctgtctgga
agggacagag gccttcagat gaggagccag gaggcccagg cctgagggag 14280gagaagggcc
tagtctgatg gggagaaggg cccttgcctg aaggcagagc agtttcctgc 14340ctgggaaggt
catcccagcc ccacccatca gtctgaattg gacatcacca gtgcccagga 14400cattggaggt
ctgagggaaa agtctagaaa gatgatgggg ctggtcacac actaattacc 14460aatgggaaag
ctaaggtgag ttccaagttt ggcttcacca gagaaaacta atttgtgtgg 14520cattccagaa
agacctgcca aactcgatga gtgaacaggc agcccttctt cattcatgca 14580tgcattcagt
ttttgaatca ggtgagactt tagatctcac gtgaaataag tcttaagtga 14640aacaaagaga
aatttatctt ataataagag aaaattggcc gggcatggtg gctcacaccg 14700gcaatcgcag
cactttggga ggccgaggtg gatggatcac ttgaggtcag gagttcaaga 14760ctagtctggc
caacatggtg aaaccccgtc tctactaaaa atgcaaaaat agcctggcga 14820gctggcaggc
gcctgtaatc ccagctactc aggaggctga ggtgggagaa tcgcttgaac 14880ctggtaggtt
taggttgcag tgagctgaga ttgtgccact gcactccagc ctgggcaaca 14940gagcaagact
ccgtctcaaa aacaaaacaa aacaaaacaa aaaaagaaag gaaaaagaaa 15000attggccggg
cacggtggct cacacctgta atgcccacac tttgcgaggc cgagaagggt 15060ggattgcttg
agtccagaaa tttgagacca gcctgggcaa catggcagaa ccccatatct 15120acaaaaataa
aataaaataa ttagccgggt gtgggggtgc acacctgtag tcccagctac 15180tcaggaggct
gaggtgggag gatcgtttga acccaggaga tggaggcgtc aatgagccaa 15240aatcacacca
ccgcactcca gcctgggcaa cagagcaaga ccctgtctca aaaaagaaaa 15300aaaaaaaaag
agagagaaaa gaaaagaaaa tgaaaagaaa aaattcaagc aaatttagaa 15360tgatctcctt
cacaaagagg cgatagtgtg agggtcactg ggaaaattag acaaaaagtc 15420tggtctactg
aaatatggtt tacatccaca tggatggtgg gctgtacttt tctccagaat 15480tgtgtaattc
ctttggccca ttgggggtca gaaaaagaat ggctaaatgt tactatccca 15540agacacttgg
attgattatt ccagagtgtg agtaaattca ggtatctctt ttaggaattc 15600catctacttt
gggctgggct tagtggctca cacctgtgat cccagcactt tgggaggctg 15660aggcagcggg
atcgcttgag ctctggagtt tgagagcagt ctgggcagcg tagtgagact 15720ttgtacggac
gaaaactttt tttttttttt ttgagatgga atcttgctct gtcacccagg 15780ctgaagtaca
gtggcacaac ctcggctcac cgcaacctcc acctcatggg ttcaagcgat 15840tctcctgcct
cagcctcctg agtagctgag attattatta tttgtttttt tgagatggag 15900tctcgctctg
tcacacaggc tgcagtacag tggtgcaatc ttggctcact acaacctccg 15960cctcccgtgt
tcaagtgatt ctcctgcctc agcctcccaa gtagctggga ttacaggcac 16020ctgccaccac
acccagctaa tttttgtatt tttagtagaa aagaggtttc accgtgttgg 16080ccaggctggt
gtcgaactcc caaccttcgg ggatctgccc gcctccgcct cccaaagtat 16140tgggattaca
ggcatgagcc actgtgcctg gctgaaaaat attaaaatat atatattttt 16200taagggattc
cagctacttt gttgttatgg agatccagaa cccaattaaa gcctgtctat 16260catgtttgag
gaaagtgcag tttgagtcaa agcctagtcc agtccaattt catttacttg 16320ctggtagtgt
caagctgttt ttgtttattt atatatttat ttagaggcag gatcttgctc 16380tttcgcccag
gctggagtgc agtggtgcga tcacagctca ctgcagcgtc aacctcttgg 16440gctcaaggag
tccttctgtc tcatcctcag ccttctgagt agctaggact acaggtgcat 16500gccagcatgc
ccagctaatt tttaaattat tatttgtaga gagagggtct cagtgtgttg 16560cccaggctgg
tctcaaactc ctgggctcaa gccatcctcc caccttggcc tctcagagcg 16620ctgggatgat
agcaccacat ccagcctatc gagatttttt ttgtgttttt ttctttgttt 16680tttgtttgtt
tgtttgtttg tttgagaggg agtctcgctc tgtcgccagg ctggagtgca 16740gttgcgcagt
ctcggctcac tgtaacctcc gcctcctgga ttcaagagat tctcatccct 16800cagcctcccg
agtagctggg attacaggcg catgccatca cacccagcta atttttgtat 16860taggtggttt
ttaaaggcca ccgcttcttc agtgttctgc accaggtctg ggaatgttct 16920cagctcacct
agtcatgttc agaatggaca aatccctcag aggaagcaga cacggtttct 16980cgggacggtg
atcctttaga gccacatgca catgcttgct ttcttttatt attatctttt 17040tttgagatgg
agtctcactc cgtcaccgag gctggagtgc agtggcataa tcttggctca 17100ctacaacctc
tgcctcccgg gttcaagcga ttctcctgcc tcagcctccc gagtatctgg 17160gactacaggt
gcccgctgcc aagcctggct aattttcata tttttagtag aggcgcggtt 17220ttgccacatt
ggccaggctg tctcgaactc ctgacctcaa gtgatccacc cgcctcggcc 17280tcccaaagtg
ctggaattac agatgtgagc cactgtgcct ggccaaatgc tttcgtttct 17340ttaaaaatca
aagggaaagg aatgactata atccagtctg cattgtatat gtccttatac 17400cagtacattt
gtgggatata atttttagtt ctttttatgg agaagaagtt cccaaggcag 17460atgtgtctgg
ggctcgtgaa aattcatcct gaagtcctcc atgtccggga tgtatttcac 17520tgctaggaat
ccctcctggg cagaggtagg atctaaaggt gtgaccgctg aggaagtagg 17580tcggctctct
ttttgtttgt tttttgtttt tgttttcaga tggagtctgt ctctgtcgcc 17640tgggctggag
tgtagtcgtg tgatctcagc tcactgcaac ctccacctcc tgggttcaag 17700tgattctgct
gcctcagcct ccacagtagc tgggatcaca ggcacgcgcc accacaccca 17760gctaattttt
gtgtttttag tagagatggg gtttcaccat gttgtccagg ctggtctcaa 17820agtcctgacc
tcaagcgatc cacccacctc agcctcccaa agtgctggga ttacaggggt 17880gagccaccgt
gcccagcctt aatttttgta tttttagtag agatgggttt caccatgtta 17940gctaggctgg
tctccaactc ctggcctcaa gtgatccacc tgccttggcc tccctaagtg 18000ctgggatttc
aggcatgagc catggcaact ggcctgctct gttctaaatg cagatctaaa 18060ccccctgcag
gtaacctgga tgaggacatc attgcagaag agaacatcgt ttcccgaagt 18120gagttcccag
agagctggct gtggaacgtt gaggacttga aagagccacc gaaaaatggg 18180taaggccggg
gtacccccgg tacaacccac cccagagtca gaccgtttaa tttgcatgca 18240cctgctatct
ctggtcttct ctggaatcac agtgcaaccc cacagcccaa cctagaaaaa 18300tcaggaattg
ggtgacctac atggaggcac ccccagaccc ttccagcctg tcccttgggg 18360tccctctgca
ccagttcttc ccctctacca ccctgctaga tgacatctcc taatacccca 18420acctcttctc
catccagaat ctctacgaag ctcatgaata tatttttgaa agactccatc 18480accacgtggg
agattctggc tgtgagcatg tcggacaaga aaggtgagag aggatgctgg 18540ctggtccccg
ggaggcaggg accccagggt gtctgagtgt catctcattt tatccaaact 18600caatcaaccc
tatgtttctt ggcactttat tctctgccct ggttaccaca gaggtgttgt 18660taccaggaac
tgtgggaatc cttagttcct gtctaacttg gaagaaagaa ttcagccaag 18720agtcacatag
caagggttaa gtagcagagt ttattgaagg aagaaacagc tctgggctgg 18780tccccctgga
aaaatagtag tagcaatgct tatttaaaga gacagggcca gcctcgatgg 18840ctcacaccta
taatcccagc actttgggag gctgaggcag gggaatcact tcaggtcagg 18900agttcaagac
cagcctggtc aacgtggtga aaccccgtct ctactgaaag tacaaaacaa 18960ttagccaggc
agggggtggc gggcgcctat aatcccagct actcgggagg ctgaggcagg 19020agatttggtt
gaacccggga ggtggaggtt gcggtgagct gagattgtgc cactgcactc 19080cagcctgggc
aacaagagca aaactccttc tctaaataaa taaaaagtga ccgtatgctc 19140tgaaagacga
cacagacatg gctgctcaac agaacgagcc agcagcagat actgctggta 19200gactcttttt
atgagactct tacatgattt ttcgtgaagg ggcgtgagtg ggtgtcactt 19260gtaagcatgt
tttgggaggt ctctttgggc gagcaggctc tgtggctgta ggtactagca 19320tgcacgtggc
atgtctcatt agcatcgaaa atctccaccc agaggtgtgt tttttactat 19380gataatgagc
aaaacacaac tctagggtgt tttcggagca gtgcacatgc tcatcatcgg 19440ggaaaatccc
tagcaaagtt atttccagct aggacctgat aagtcccctt cagggccaga 19500ggaccccaac
cacaaggcca tgtgtagcta aagtagccat cgtccttttc gctgactgcc 19560agtgagcagc
gctgtcagta ggcagcctgt ctgggacttc ttttcccaga aagctcccct 19620gcctgctcat
ttccgcctat ctgcctactc taacagtgtc aaaagctaga cagggtgggg 19680gtacagtctc
taaaattgat gcttttcttt ctttcttttg tttttgagaa ggagtctcac 19740tcggtcatcc
agccataatt tatatggttt attataattt ataataaatt taattataat 19800atttatttat
atatttatta attgtaatgt ttataattat aatatataat tatatattac 19860ataatatatt
tcatatctac atatcacata ttacatatgc aatatattat ataccacata 19920ttacatatat
aacataccac atattacata tataatatat catatattat atattacata 19980tataatatat
catatattat atattacata tataatatat catatattat atattacata 20040tataatatat
catatattat atattacata tataatatat catatattac atatattata 20100tattacatat
ataatatatc atattacata tattatatat tacatatata atatatcata 20160ttacatatat
tatatattac atatataaca tatatattac atatatcata ttacatatat 20220catatattac
atatataata tatcatatta catatatatc atatattaca tatataatat 20280atcatattac
atatatatca tatattacat attacatgta atatgttata ttacatataa 20340tatatattgc
atatcacata tataatatgt tatatgttgc atattacata tataatatat 20400tatatattgt
atattacata tataatatat atgtaatata tacatattac acatgtaata 20460tattatgtaa
acatataata tgtattataa tttataagaa atttaattat aatataattt 20520aatgaattat
aataaaccat aattcattat aatttaatac attataataa accataattt 20580attataattt
aattttgttg taatgtataa ttataattta ctactaatat gtcatttgtt 20640attgttgaca
tgttaacata tataatgtat attttattag atatataata taaatgatgt 20700atcatttatt
attgattaca tatctataat tataccatat cataacttat tacaaaacat 20760tctatttaat
ttaaatatac ccaaaatagt atcatttcaa cattttgtaa aaagttgcaa 20820aaccacaacc
cactaataat gtgactataa ccttttaata tttgataata atctactagt 20880atatcaaaat
tactgatgat atattttact tctgtttgca ctaagtcttc aaaatccagc 20940atgtgtttta
caattcagtg catctcattt aggatactag attttctttc tttttttttt 21000ttgatacagg
agcttgctct gtcacctagg atggagtgca gtggtgtaaa caggatgcta 21060agttttcttt
ttttagtaga gacagggtgt caccatgttg gccaggctgg tctcaaactc 21120ctggcctcaa
gcaatctgcc ttcctcagcc tcccagagtg ctggaattac aggcgtgagc 21180caccgcgccc
agcgcaggat gctaggtttt cactggaaat actttgatct gtattttagg 21240tttcataaaa
tttacagttg aaaaggtaga ttctcaggcc gggtgcaaag gctcaagcct 21300gtaatcccat
tactttcaga ggctgaggcc ggcaaatcat ttgaggtcgg agtttgagac 21360cagcctgggc
aacatggcaa agccccgtct ctacaaaaaa aaaaaagaaa agaaaagaaa 21420agagaaagaa
aaggtagatc ctcatactca agtagttgca aaaatactta aacgttttcc 21480actcaatcat
catttttaaa aaattaagat ttaattcact tactatatgt caccctttta 21540aaatgtacaa
ctcaggtcgg gcacggtggc tcacacctgt aatcccagca ctttgggagg 21600cccaggcagg
cagatcacct gaggtcagga ggtggagaac agcctggcca acatggtgaa 21660accctgtctc
tactaaaaat acaaaaaatt agcaggacat gcgggtgggt gcctgtaatc 21720ccagctactc
aggaggctga ggcaggagaa ttgcttgaac ccaggatata gaggttgtag 21780tgagccaaga
tcacgccact gcactccagc ctgggtgaca gagcgagacc ccatctcaaa 21840aaataaataa
ataaaaaata ataaaatata taattcagtg gtgtttcata tatttaaaat 21900gagcatcagt
tgtttgtttt gtttcattgg gtttggtttt acagacagga tctcactctg 21960ttgcccaggc
tggagcacag tggtgcgatc atagctcact gcagccttga actcctgggc 22020tcaagcaatc
ctcctgcctc agcctcccaa agtgctgtga ttacaggcat gagccaccgc 22080acctagctag
atcatcaggt ttaaagttta agtctgaatt aaattaaata catttaaata 22140caagtacatc
aaataaaagt acaaatccag tttctcactc aggcaaaccc catttcaagt 22200gctcagcgct
cccccacagc ttggggctac catatcagac aagcagatat attttggaga 22260tttctcttcc
tccctacacg tagatctctg agtcaaacta caaacagaat gtaaatcatt 22320aaatagtggt
aactccggcc aggcgcagtg gctcacgcct gtaatctcag cacttgggag 22380gctgaggcgg
gtggatcgtg aggtcaagag atcgagacca tcctggccaa catggtgaaa 22440ccccatctct
actaaatata caaaaattag ctggacatgg tggtgcgtgc ctgcagtccc 22500agctactcga
gaggctgagg caggagaatt gcttgaaccc aggaggcgga ggttgcgttg 22560agccgagatg
gcgccactgc actccagcct ggcgacagag tcttgctctg tctcaaataa 22620ttaataataa
taataataat aataataata ataataataa ataatggtaa ctcccagcca 22680ccaccatcat
catctgtcat ttgtcgccat tgacagcgtt tagttcacag gcttcagcaa 22740agacaggctg
agttagggag agctcctgcg gagtggacta agagctgaga cccaggagcc 22800tggccttgtc
cactccccga ccttgacact ccgtgttctg tctctgcccg agcagggatc 22860tgtgtggcag
accccttcga ggtcacagta atgcaggact tcttcatcga cctgcggcta 22920ccctactctg
ttgttcgaaa cgagcaggtg gaaatccgag ccgttctcta caattaccgg 22980cagaaccaag
agctcaaggt gggtcccggg gtggcagagg cttcttggag gctgccaggg 23040ggtaggtagc
ctgttgcaca cacacttgcc cggatccttt ctctccctgg caggtgaggg 23100tggaactact
ccacaatcca gccttctgca gcctggccac caccaagagg cgtcaccagc 23160agaccgtaac
catccccccc aagtcctcgt tgtccgttcc atatgtcatc gtgccgctaa 23220agaccggcct
gcaggaagtg gaagtcaagg ctgctgtcta ccatcatttc atcagtgacg 23280gtgtcaggaa
gtccctgaag gtcgtggtga gtgcttgggg cacccacaaa cccttgtcct 23340tcagagaggg
ctcctggtct tcgtactatt gactcaggtt ggagatccag gctctgagac 23400actaagaatc
atagtgtcca gcttaggaaa tttggaagtc ccagaatttc agaagcagag 23460ccaggattgg
ggtaaagtga gtgagatgac cccaggctta gaattttagg tggtgccaaa 23520aacctcgtcg
accatcacca atcaataatt tttttatact cgatttgaaa ttttttattt 23580atttatttat
ttgtttgttt atttttttga gacagagtct cactctgttc cccaggctgg 23640agtgcagtgg
cgcgatctca gctcactgca atatccgcct cccgggttca cgccatcctc 23700ctgcctcagc
ctcccgagta gctgggacta caggcgccag ccaccacgcc cggctaattt 23760ttttgtattt
ttagtagaga cagggtttca ctgtgttagc caggatggtc tcgatcttct 23820gacctcgtga
tccacccacc tcggcctccc aaagtgctag gatcacaggc acgagccacc 23880gcgcccggca
atgctagggt gatcctaagg acagtgccct gctgaccatc tgtgtgtctg 23940tctgttcttt
tattcatcca acgactcccc ccacctctaa cactgcgtag ccggaaggaa 24000tcagaatgaa
caaaactgtg gctgttcgca ccctggatcc agaacgcctg ggccgtggtg 24060agtcggctgc
agggggaggg gctgaggggc tggcagggta aggggggtaa atgacctggg 24120tttagtgagg
taggataggg cgggagggag ctagagccat cggtatctct cactcaccct 24180gcagaaggag
tgcagaaaga ggacatccca cctgcagacc tcagtgacca agtcccggac 24240accgagtctg
agaccagaat tctcctgcaa ggtgagacac ccttgacccc gaccccatgg 24300gtcccaggag
ggcatggatg gagccaaatt ccatctcatt ctggaggtgt ttaacccgca 24360cctttctctt
ccccttcagc tagaacagcc catctgtgat ctgttttccc tcttttacat 24420tttttttttt
tttttttttt gagacagagt ctggctctgt cacccaggct ggagtgcagt 24480ggcgcgacct
cagctcgctg caagctccgc ctcccgggtt cacgccattc tcctgcctca 24540gcctcccgag
tagctgggac tacagccacc cgccaccacg cccggctaat ttttttgtat 24600ttttagtaga
gacagggttt caccgtgtta gccaggatgg tctcgatctc ctgacctcgt 24660gatccacccg
cctcagcctc ccaaagtgct gggattacag gcatgagcca ttatgcccgg 24720cctaaaaatt
tttttaacca tacagatatt atttgctatg atcggtttta tagaagcctc 24780cagatagcat
ttagttcagc aaagagcttt cgctgataca tcagtttatt ttaatttttc 24840tagaccttct
gtgcttctta gatgggaaac cagcttaaat gagactcaat agcctgtaat 24900cccagcactt
tgggaggccg aggcaggcag accacctgag gtaggagttt gagaccagcc 24960tggccaacat
ggtgaaaccc tgtctctact aaaaatacaa aagttagctg ggcgtggtgg 25020cacatgcctg
taatcccagc cactcgggag gctgaagcag gataatcgat tgaacgtggg 25080aggcgtaggt
tgcagtaagc cgagatcagg ccactgcact ccagcctggg cggcagagca 25140agactttgtc
tcaaacaaaa acaaacaaac aaacaaacaa aaagacaagc aacatagtac 25200aagagcagaa
attctggagg tcatttcttg ccccaggagg gaagactgga gaaagaaagg 25260gacttgcaac
ctgtaagcta taaggctttg gggcaagagc cttggttttt tcacctttgg 25320taggggtaga
ataatagtat ctacctccaa gggttggtgt gatgattttt tttttttttt 25380tgaggcggag
tctcactctg tcgccaggct agagtgcagt ggcgtgatct cggctcactg 25440caaccccagc
ctcccgggtt caagtgattc ttgtgcctca gcctcccaag tagctgggac 25500tacaggcgcc
cgccaccatg cccactaatt tttgtatttt tagtagagac ggtgtttcac 25560catattggtc
aggctggtct tgaactcctg acctcaggtg atccacccac cccagcctcc 25620caaagtgctg
agattacagg cttcagccac ggcgcccagc ctcgttgact attaagtgag 25680acactctatg
gtattctctt agaacagtct ggaaagtaac attaagcgtg atataagtat 25740tcctgaatat
tgttactgga attattttac tgctggtgaa atgagaccca aggaccaggg 25800tgcccctgtg
aagcacctcc cactcctaac agtgcagacc cccgaacagc cactcagcca 25860tgcagcctcc
cctccccgca gtcacatcct ccccagtcct cgcctgtccc taaccccttg 25920gccctggctg
gttgggaggc tggaaccctt ttcacgccac cccaaggtgg gtcacccacc 25980tggcttgagc
aacgtcctct tcccacctgc tgcagggacc ccagtggccc agatgacaga 26040ggatgccgtc
gacgcggaac ggctgaagca cctcattgtg accccctcgg gctgcgggga 26100acagaacatg
atcggcatga cgcccacggt catcgctgtg cattacctgg atgaaacgga 26160gcagtgggag
aagttcggcc tagagaagcg gcagggggcc ttggagctca tcaagaaggg 26220tgggctccct
gcccctcttg gagaccccag ggaccccttt ccgagcgcat ccctccccta 26280agatcccacc
tcatctcaag accacgccct cccctgaggc tccaccttct ctcctagcca 26340ctcccctcat
ttgaggcccc acctcttctc aaggctacgc cctctgaggc cctgactcct 26400cccaggccag
gcttttcatg agaccccgcc tctcctcaag gccatgccca tcccctgagg 26460gccccccacc
tcttctcaag gccacgccct ctgaggccct gactcctccc aggccaggct 26520cttcatgaga
ccccgcctct cctcaaggcc atgcccatcc cctgagggcc ccccacctct 26580tctcaaggcc
acgccctctg aggccctgac tcctcccagg ccaggctctt catgagaccc 26640cgcctctcct
caaggccatg cccatcccct gagggcctcc cacctcttct caaggccacg 26700ccctctgagg
ccctgactcc tcccaggcca gaatctcgag accctgcctc ttttcaaggc 26760cacgcccatc
ccctgggtcc ccacatcttc tcaaggccac acccttctgt gaggcgccac 26820ctcctgtccc
agccactctc atctgaggcc ccacgtcctc tccaggccat gcctcttccc 26880tgagactcca
ccccctctct gagagccctc ccctccctga aagcccccca ccctcaatat 26940ccttctcctc
tctgaatccc ttgtcctctt gagaactttt ccacctcctc gttctgatcc 27000cccaccctct
ttgagtcctt ccctttttaa ggtcccctcc tcccagaacc cctccgccac 27060cctgagcccc
tgtcccctct ctgcaccccg cccctgccct ttctggcgtg ccccctctgc 27120tcagccccgg
ctcttttggg ggttcctctc tcttctctgc agggtacacc cagcagctgg 27180ccttcagaca
acccagctct gcctttgcgg ccttcgtgaa acgggcaccc agcacctggt 27240gagtcccaac
agccagctca ggccatgcat actccccacc ctcaaccccc agcagggccc 27300ggaccctggc
caggggtggt cccttaggcc agccttgccc aaacagccct ggacctgcag 27360agtccaggca
agcgctggct gagtggccgg cggtcattaa gcatccttaa gcacggaccg 27420catacaacag
ctgggtcctg gggcctggga aggcaaacca ggcaaactgg gccaggccct 27480ggtccctccc
ccacgctcat tggctggttg acatggcagt ctctggatct cagagccgat 27540tggctcatgc
tctgtgccca ctccaggctg accgcctacg tggtcaaggt cttctctctg 27600gctgtcaacc
tcatcgccat cgactcccaa gtcctctgcg gggctgttaa atggctgatc 27660ctggagaagc
agaagcccga cggggtcttc caggaggatg cgcccgtgat acaccaagaa 27720atgattgtaa
gaggctggga tttagggcaa aatggaagag aggggctcct gagtctcgca 27780ggatgaacac
gagagagagc cccacctcca tgtgcccact gcccaattcc ctttgcaaag 27840attgggctgg
ggggtggggg caggcagata tatgagccag aggcgtcact ccagcattgc 27900aaaaaccaga
gacctgcgaa gcccagcgca aaatgaagag acacggcccc tcgctcagaa 27960attattaaga
atttcattaa accaagtgca ggggtcctgc ctgggaatcc ctttctcaca 28020ttcaatccat
caacacctgc attctcccat gatgttataa gaatcacctc cttctctcca 28080tccttatggc
cagcccctgg tccaagcaac actctccccg cccctcctta tttggagacc 28140ttgtagaaac
cacctcctgg tcatcatcct ggtggcctcc cacttttgtt ggctctcaga 28200cactcaccac
atagcagttg gggtgatttt ttcaaatcca gctggatcag ttcttagaaa 28260gtcccgtggc
tccccctgtg gcacttaaac acaaaactcc ttcgagcact ggttctcgaa 28320gtgtgatcct
cagaccagcg gcagcaacag cacccatgac ttactaaaaa tgtgcattct 28380gtggctgggc
tcgacggccc atgcctgtaa tcccagcgct ttgggaggcc gaggcaggag 28440gatggcttga
gcccaggagg tcgaggctgc agtgagccat gatcatgaca ctgcactcca 28500ggctgataac
agagtgagac cctgtctcaa aaacaaaaca tattctgaga ccggacccca 28560gactcactga
atcagaaatt ctaggggcag gacccaggaa tctgaggggt gtgagtgtgt 28620gtgtgtgtgt
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt ttgagatgga gttttgctct 28680tgtcacccag
gctggagtgc aatggcccga tcttggctca ctgcaacctc cacctcccag 28740gttcaagcaa
ttctcctacc tcaacctcct cagtagctgg gattacaggt gcccgctcca 28800ccatgcccag
ctgatttttg tatttttagt agagacgggg tttcaccatg ttggccaggc 28860tggtcttgaa
ctcctgacct caggtgatcc gcccaccttg gcctcccaaa gtgctgggat 28920tacaggcatg
agccaccgcg cccggcctag gaatctgagt ttttaaaagt gcccgcattc 28980ctccaggtga
tgctaatgtg tgcttgagat ggagaatcac tgcctcagtc tcacctttca 29040ggcttccaga
cttccagcct ttcttttctt tccaggctcc atccattgat aggagccttg 29100ctctattgtt
ctacagggcc tttgcacatg ctgtttctgc cacctagtat gctaatccct 29160gccgtctgtg
agagttgact ccctcaggga cactttttct gacctcccca actgggtcac 29220actcccacag
ttcattatcg ctgcgatgtc ctctttccct tgcacagaac tcatccactt 29280ataagtatat
atctcttggc tgggcgcagt ggctcatgcc tgtaatccca gcactgtggg 29340aggccgaggc
aggtggatca cctgaggtca ggagttcggg accagcctga ccaacagggg 29400aaaccccatc
tctactaaat acaaaaaaat tagcttggtg tggtggtgca tgcttgtaat 29460cccagctact
tcggaggctg aggcaggaga attgcttgaa tccaggaggc ggaggttgca 29520gggagtcgag
attgcgccat tgcactccag cctgggcaac aagagcaaaa ctgtcccaaa 29580aaaaaaaaaa
aaaagtgtat atctcttgag gagctggatg gaccatgtcc atcttcccta 29640ctagacaaaa
gctctgtgag ggctagagcc tgtgtctggt tttacaatgg atcagaccgt 29700tgtacccatt
gtacattgca cattgtacat tgacatttgc agaaggaaca aattgttgca 29760tgaattaata
ctaagaagtt tgaccttcct agggtagcgg ggtaacacct agaagagact 29820cagccctgcc
cagaccccct gattctgaat ctgcaagggg ggatgactgc catgtgtgga 29880cacaccggtg
accccatcct tgctttctgc tctctatctc agggtggatt acggaacaac 29940aacgagaaag
acatggccct cacggccttt gttctcatct cgctgcagga ggctaaagat 30000atttgcgagg
agcaggtcaa cgtaagtgcc ctccatcttc ccaccctacc ctaccttacc 30060cgatgcagag
cacagccacc ttggagagtg agaggttgcc ttcagggaat ttgcagctct 30120cccagtgcaa
taacagacat cactgcagtc atgttaatag ctaacatctt ttgagcactt 30180aactcatcta
atacagaccc gccctctaat agtttcacat gttaagtctc ataatccttt 30240tagcagcctg
aaaggtaagt cactcttatt atccccagtt tgcagatgag aaaactgagg 30300cacaaagaga
tcaaaggtgg ggattctttc tgtctgcctt acaattttca gagggttttc 30360agcccatttc
caaaagtgct ttctacatca gtgctacatg atcagtacag ttgcgtactt 30420gctacttcct
taaagaaaac ttgggataca gagctaagac tatttcctta gtccagagga 30480tctttcaggt
gattttcaaa gggatccgtg actccaaaca ggaaacggtg aacactgttg 30540gctcatcact
gtctcttttt cctctggttt tgattctgaa gcagggaagc ttggaaagat 30600gggccgctga
gagtctggaa tgcctttgtc tgctttattg tggttgtttg tttgtttgtt 30660tattttttgt
gatggagtct cactctgtcg cccaggctgc aatgcagtgg catgatctca 30720gctcactgca
ccctttgcct cccaggttca agggacttta ctgtttcagc ctccagagca 30780tctgggatta
caggcacccg ccaccatacc cggctaattt ttgtcttttt agtagacatg 30840aggtttcacc
atattggcca ggctggtctc gaactcctga cctcaggtga tctgcctggc 30900gtggcctccc
aaagtgctgg gattacaggc atgagccact gcacccagcc taattgttgt 30960atttttagta
gagatggggt ttcaccatgt tggccaggct ggtttcgaac tcctgacctc 31020aagtgatcca
cccaccttag cctcccaaag tgctcggatt acaggcgtga gtcactgcac 31080ctagctgatc
gtggggtttt gagtgggttg tttaacgttt agctttccaa gtgggaagcc 31140caggattcca
ccctcagcta gtggcttctc cccccttagg aaaagagatg gaggggaggg 31200gccagtgaag
agaaaaacaa acacagggct gttgcctcta acacccaaga gggaccaagg 31260cagagagaga
gagagagaga gagagagagg gagggaggga gggagggagg gagggaggga 31320ggtaggtaga
gagagagaga gagagagaga ggagaggtgg ggtcagacaa atctgacttc 31380aaatcctgac
tcatgggcac ttccaccctt gagcctcact caggatgtgc atctgtaaat 31440tggggataat
aaataacgat ctctgtattt ttaggcctct gagttgtccc agatataaca 31500cacatgtgac
ccagattata caaaaattga tggggaattt atgtgcaggc accaaggcat 31560caaatagaga
tgaaggtggc ctcagggact ctgccaggat gctttgctcc tctctcccgt 31620gatcttcatt
ccgttcttgg ccaataattc agttcaggca gaatatggct gccttcctta 31680gagaaaatat
cagatcaagg ttagggccgc catattccca ggaaaggact ctgattggct 31740cagcctgggt
cagatgacta tatctggacc aatcagctaa ggacaggaag taggtctcag 31800ggggcagaca
tggctgtttc cactgtggcc acgtgaatgg aagggagaag aagttcttac 31860aaaaggagtg
gatgtcagag aggcaaatgg gcaggaataa aagagatttg tttctgctac 31920aacatagcaa
cattgtagca gagtatagca caggctgtga aaccagactc ctggggtcaa 31980gagtgtgctg
taatcccaac tactcaagat gctgaggcag gagaatcact tgaaccaggg 32040aggtggaggt
tgcagtgagc cgagattgcg ccactgcact ccagcctggg caacacagca 32100agactccttt
tcaaaaaaaa aaaaagtgtg ctataactag cttgctggag cccagtgtta 32160aatttccagg
aatttttcaa gctggtcatt aaatacaatt attattaaaa actaaatatt 32220aggccaggca
cagtgagcct gtaatcccgg cactttggga agccaaggcc ggcagatcac 32280ctgaggtcag
gagttcaaaa ccaccctggc caacatggca aaaccccgtc tctactaaaa 32340atacaaaaat
tagccgggca tggtggaggg gggcgcctgt aatcccagct acgcaggagg 32400ctaaggcaca
agaatcgctt gaacccggga ggcggaggtt gcagtgagcc gagattgcgc 32460catgcactcc
agcctgggcc agagcgagac tccgtctcaa aaaaaaggcc aggcgcggtg 32520gctcacgcct
gtaatcccag cactttggga ggccgaggtg ggcggatcac gaggtcagga 32580gatcgagacc
acggtgaaac cccgtctcta ctaaaaatac aaaaaattag ccgggcgcgg 32640tggcggacgc
ctgtagtccc agctactggg aaggctgagg caggagaatc acttgaaccc 32700gagaggcgga
gcttgcagtg agccgagatc gcgccactgc actccagcgt gggcgacaga 32760gcaagactcc
gtctcaaaaa aaaaaaaaag caacaacaaa aaacccaacc aaccaaccaa 32820acaaacaaag
ttataaaagt tacagttaaa taaattatat taaacacaaa ggttagaaac 32880actcaaactc
atcgcttcct aaacgcctta ctcccataat ctatactctt ggggttactt 32940atgtctgttg
gatctgtata gtgaaaatac tatataatac tgtggtactg caaagctctt 33000cccaactcta
cattcaacga caccatattg gtaggttgaa atcagtgatg gaagtattta 33060catcatggaa
atgagaaaac agtacaaatc atgtcttccc ccatccccag aaggctgtgt 33120ttggatccta
actctgccac ttatttccta ggtggtcttt gcaaaattac tgcatctctc 33180agggctcagt
atgctcatca ggttttatga gattaaatgt gtgggtatct gaatgacaca 33240aagtaagtgt
gagctatgat gatgaagaag ataaagatga tgatgacgat gatgatgatg 33300actggatgag
gtgttcacag tggtatactg aatctggcgc atactagttt atgagtaaca 33360atttggagaa
tgtctcccca ggactttgtt cagtgatgtc gcattgacac cgtgaaattg 33420gcccctggtg
ggagtattta caccacagaa attgtaaatc attataaacc aaggatccct 33480caaccctccc
actggagagc tggctgttaa acttttacca gcacaccacg gggtacgtgg 33540atttctccag
atacataata gatatgcagc aacaaggcag ctcatggtgg ctaaaatatc 33600tgggaaattc
tcaaaaatgg acaaatctaa gacaggtgtg tcccaaggac agaaatccct 33660gatgctcagg
aagtgctgct cgaatgatcc ttactaacgt gacagcaatg cccacatgac 33720cggagaatct
gatcctcttt ctcatagagc ctgccaggca gcatcactaa agcaggagac 33780ttccttgaag
ccaactacat gaacctacag agatcctaca ctgtggccat tgctggctat 33840gctctggccc
agatgggcag gctgaagggg cctcttctta acaaatttct gaccacagcc 33900aaaggtgagg
gttggcctgg aggggtgaag ggagatgcat ggctgaagtt cagggcggga 33960gatactgagc
tgggatgcat ggcttttagc tgagctggga cagatgaccc taagccaagc 34020tgagatggat
agtcctaagg tatcaagctg ggatgcataa ccctgagctg agctgggatg 34080cacggctcta
agttttcgca ggtcctcatt gtaaaccaca cgagaaagtt tgttgcgtca 34140tttattcaac
aaatgcgtat taagcattca tttcaaaggg agaagtgaga gttgatgaaa 34200caagagaggt
aaggcaggag ccaagtaatt gagagcctcg aatgtcagcc aggacaccca 34260aacaccagga
agtctagcat gcatctcttt ctgagctttc tctgagccat ccccaggctg 34320gacagagcag
tgagcactgg ggatggggta tcttctttgc agataagaac cgctgggagg 34380accctggtaa
gcagctctac aacgtggagg ccacatccta tgccctcttg gccctactgc 34440agctaaaaga
ctttgacttt gtgcctcccg tcgtgcgttg gctcaatgaa cagagatact 34500acggtggtgg
ctatggctct acccaggcaa gtgggcccac agcccctagg cacatgcatc 34560cctgtctcct
gcggcttccc actggcctcc tagagaagac actgaggccc agcgaggcag 34620ttcttcattc
ccacgagcca gtgtgattgc agtggagttg agaatcagtt tttattactt 34680gcaaacccat
ctataggttc tagaatacaa tctgggtact ccaagctgtg tgttgagcct 34740tcttcttgcc
ccaggtgtct agatcatgtt ctcagggccc aggttcaggt ctaagcctct 34800ctctccacct
ggtgggctct agaccaggtt cccagttcta tctcacaatc ttaccctgtc 34860ttgctggtgg
gttctagacc atgttcccag ttctaccagg ctcccaatgt cacattgcct 34920cactggcggg
ctctatagta tgttcccagt taccctgggg cattacgcaa accctcttct 34980aggccatggt
ttcagtaact tcaggcttca gcaacttcag gctccagttg gcctcctttc 35040tttctggtgg
tctgtcactc acgttctcag tgttacagtg tcactcttgg gttgtagatt 35100atatgctcag
tatcctctgg ctacggtttc attctgttct tcatgagtgg gttctagaca 35160tattctcagt
gtctccaagc cctggtctaa gactctctcc tcttgatggg tctagactgc 35220atcctcaggg
tcgctagaca ttcagtctta catttggact ttctgatgga ttctagacat 35280gttctcagca
tctccaagtc ctggtgtaag tttctgtctc tcggagagtt ctgaacatgt 35340cctcagagtc
cagtgacctc cagttatcac ccctgcactc tctagtaggt tctaggccac 35400attttgatgt
cccagctctg atttgaacct ctttatcccc cactggattc tagccacttt 35460cccaggctcc
cagatcacca tctttctctc ttgtgggttc taggccacct tcatggtgtt 35520ccaagccttg
gctcaatacc aaaaggacgc ccctgaccac caggaactga accttgatgt 35580gtccctccaa
ctgcccagcc gcagctccaa gatcacccac cgtatccact gggaatctgc 35640cagcctcctg
cgatcagaag aggtacagtc acccagccaa gccctcctca ctctggctgt 35700ctccccctac
actagccagg gtttactggg aagcaagagg gagggccagg tgaccatcac 35760aggcagcaga
aggcttaatt cccaacatgc tctcttctct cttttcactc tgcagaccaa 35820ggaaaatgag
ggtttcacag tcacagctga aggaaaaggc caaggcacct tgtcggtaag 35880gaacagaaac
ccacacctgc ctggcccatg cccctctgcc ccagagggac catctcctct 35940tgtccccagc
agtcctagtc ctgtgggctg acattgtgtc tcctctccca tcttaccagg 36000tggtgacaat
gtaccatgct aaggccaaag atcaactcac ctgtaataaa ttcgacctca 36060aggtcaccat
aaaaccagca ccggaaacag gtaaaaggaa tcaaggcctt atctgtcacc 36120ttcctcctac
ccctcttcta atgtcttccc cgctcctgaa tcaacacaca ggtataccct 36180ctcccatctt
tctctcttct gtgtttctag aaaagaggcc tcaggatgcc aagaacacta 36240tgatccttga
gatctgtacc aggtaagaag ctaggtcacc ggggttcatc ttggccatcc 36300ctctatctct
agcaagaatt cttgcaaata atatccatga tattcagtac tttccaagta 36360cactgtgtat
ctgatactgt tctaagtatc caccatgagg tagacaacac agacagtcct 36420tgctttgcat
gttaatgtga gaccacagca atgaccacgt aagctgagac tgtcaaagca 36480tcttagtaat
caatggagga aagtacacaa tcattccatg acctttaaag ttttcttttt 36540ttctttttag
agagataggg tcttgctctg tcagccaggc tggagtgcag tggcacaatc 36600atagctcact
gtaacctcaa actccctggc tcaagcgatc ctcctgcctc agccactcaa 36660gtagctggga
ctacaggcgt gtgccatgac acctggctga tttttatttt ttattctttc 36720tagaggcagg
gcctcactgt gttgcccagg ctggtctcga actcctagcc ttgagcattc 36780ctctgccttg
ggctgccaaa gttttgggat cacaagcatg agccactatg cccagcctaa 36840atgtttctat
tacaacattt aaaattatca tactgccagt tataaagata cagggaaatg 36900gccgggtgtg
gcggctcgcg cctgtaatcc cagcactttg ggaggctgag gcgggcagat 36960cacgaggtca
ggagatcgag accatcctgg ctaacacggt gaaacaccgt ctctactaaa 37020aatacaaaaa
aattagccgg gcatggtggc gggtgcctgt agtcccagct acttgggagg 37080ctgaggcaga
agaatggcgt gaacccagga ggcggagctt gcagtgagct gagatcacgc 37140cactgcactc
cagcctgggc gaaagagcaa gactctgtct caaaaaaaaa aaaaaaaaaa 37200aaatagaata
aaacaaaata aagatacagg gaaatgaaat tcatagtaag atgagtattt 37260gactacaccg
taatttaaaa cattagaaca ttgagatgca aggtgtattt gttgtttttt 37320ttttcctttg
tatgacactt acggagagta ctttagttca aaaaaatgct tgccttcttc 37380tctttgtata
atttacaaca tggagtaaac atcttttcta tgccttagta ccttgtcttg 37440ctcctttcta
agtttggatc agcttccaat attttatcct ttgagctttc catgacacaa 37500aattcctcca
agagttcctt taaagtgact ttgtattcta taatgtccct tcctctggga 37560catcttcatc
ctttttgtcc ccatgacctt ccttatttat gctaatacat ttgccttccc 37620tgagttcctc
tacactacct atctctcaaa tggcagcagg gtcaacatca ccatagtctg 37680ctattctttg
ataactccat ttatgctgtc tttgaagttc acttctggca ttatcacttt 37740tcatttcttt
gctgcatttt tatctttgtt ggccagttcc ctcttttcgt gatacattgt 37800tgtaaaatct
catgggagtt agccacctgg agacagggag gcaacagaac tacacacttt 37860gctgtctgtg
cataaattga agagcagaag ctcagtgacc aatcactgat ggactttgaa 37920aggagtgaca
gtaattggcc ctcaattatg atgcttatct tttatttatg tcgtgatttc 37980tagactgaag
agttagcaac aaagtttata ccatatgcaa ctactcgtga tcaatatacc 38040aaggtactga
aaaagaacca tgtcactggg ctactagtgt tatttaactg aatcatgcag 38100agtgagggct
gcctgtattc ttgccttgtt ttctagaact gaagcatgga gggtcaaata 38160atgcatccaa
tgttatttag agctggaatt tgaatccatg cagttgggtg cagagtctga 38220gctcttaatc
accttgacca ttacattacc ttgcttttta tttcctttgg ggaaatgttt 38280cctaaaaaat
gtaacgcccc tctgtgctgc tatgtgggaa tcagaagtct cagtgcctga 38340tcagacctcc
ttgtccagga acagaccctt ggggctgacc cctccttggg acccaatgcc 38400cttctttctg
cactatccag gtaccgggga gaccaggatg ccactatgtc tatattggac 38460atatccatga
tgactggctt tgctccagac acagatgacc tgaagcaggt atgaagggct 38520caggagctgg
gataagtgga aaggagcctg ggttctggaa gaggctgcag ggagagaggg 38580gtccaggagg
gatttttcac aggctccacc tttccccagc tggccaatgg tgttgacaga 38640tacatctcca
agtatgagct ggacaaagcc ttctccgata ggaacaccct catcatctac 38700ctggacaagg
taaggctgca tcatcctccc ctgggaggct tccaggggca ccctgacctc 38760tatctggctg
gtctttcttt tcctttcagc ttttgtctct gggtcagact aaccctgggc 38820cagaggagac
agggtctgtg ctgctgagtt gtaggggaag gagcttgtaa aataaggggg 38880tcaacccagc
atcttctata aacatctcat cttctgacca tttgcctcct ccaacttgtt 38940atcagagtct
taaacaacca ttgaaaaaaa gcccttttgg tttttttggt ttttttttta 39000agtgctttgt
agagagcaag gtcttgcctc gttccctaac ccaatcctgg gctttgtttc 39060tttctttgat
ctatttctct cttctgttgt tttctttctt tcaggagaca gggtcttgct 39120ctgtcaccca
gactggagta cagtgtcttg atactagctc actgcaaagt caaattcctg 39180ggctcaaggg
atcctcctgc ctcagccacc tgaggagctg gaactgcagg cctgcgacac 39240tgcacccagc
taattttttt ttcataaata ttatgctttt gtacccagct tttttttttt 39300ttttttttta
actgcagcct tgacctccca ggcttacatg atcctcccac ctctgcttcc 39360tgagtagctg
tgattacagg tgcatgccac catgcccagt gaattaaaaa aaaaaaaagt 39420ttgtagatat
ggggtcccac tgtactgcct aggctggtct taaactcctg agctcaagtg 39480attctcccac
ctcagcctcc taaagtgctg agattacagg cataagcccc tggtgcctgg 39540ccccagctga
atttttgttc ttgtttcttc ataaatattc tgtgtaagta cccagctgat 39600tgttttattt
tttgtagaga tgggggtctt gatatgttgc tcaagttggt ctcaaactac 39660tggcctcaag
cgatcttcct gcctcagcct cccaaagggc tgggattcca agcatgagcc 39720accacacctg
ccacctcttc tgttattttc tctccatctg gcattctctg actctttcat 39780ctctaccatg
atttgggctt tctcctctcc cttctcttat ttcttcccat tctcctatcc 39840ccatatcctc
cctgctaact cctgataccc acagggcccc tcaatcccat tttagtcagc 39900ttaagtaaca
atagctacta aaacaaaacc cctaagaata tggggtctta acacaacaga 39960cttgtatttc
tcactcatgt aaagtccagt tggcatgggg ggtaaggaag ggtccctctg 40020ctccatgtag
tctctcaggg atccaagcac cttccatcct gtggctctgc aatccttagg 40080atcttctgta
gttctctgca ggattcattc attctagatg gaaataagat tgtgcatggg 40140ttgtttttat
gggcatagat agcaatctgt tcagccacct ggccacacct aattgaaaga 40200ggagctgaga
aaggtagtct cactgtgagt ctaggaagaa aagtaaatgg atttgctgaa 40260ttgctcattc
atctttgcca cttcctcctt gatccttcag tttctccacc actgcctcag 40320ctcccaagac
aatgctggac tccctcccac atcaccccac tgaccaagct cctccttccc 40380cctcaggtct
cacactctga ggatgactgt ctagctttca aagttcacca atactttaat 40440gtagagctta
tccagcctgg agcagtcaag gtctacgcct attacaacct gggtgagcag 40500ccaacctagg
gcctggggtc tgatggttcc aggggcctga gagtcccagg tatatatgaa 40560ttgtggggat
ctgagaatga aggtctaagg agtccaggga tttgagcatt cgtagtatga 40620aggtcccacg
ggtctgaggg tcccaaggat ctatgagttg aggttctgag gttctgaggg 40680gatctgagaa
tgatggtcta agcaggccag ggatttcagg attagtaatc tgaaggtccc 40740agggtctgag
agtcccaagg atctatgagt tggttctagg gatctgagac ttgggggtct 40800gatgggttca
ggggtctcag ggtcttagga atatgtgagt tgcagggggt tctgaaaata 40860agggtctaag
gattctagat atatgagggt tggaggcctg cgtgtcccag gaatctatga 40920atttggggtc
tgagggtccc aggcttctgt gagttgagag tctaagagac tcaagggtct 40980gagaatccca
aagatcagaa agtagagggg gtcttggggt ctgagggatc tgaggggttg 41040aagacctagc
atctccaggt ctgaagactg agaactgggg atctgggcct cccaggcatg 41100gtctttggag
ggaggccctt atcctctcat cttcacatca catctgcccg cagaggaaag 41160ctgtacccgg
ttctaccatc cggaaaagga ggatggaaag ctgaacaagc tctgccgtga 41220tgaactgtgc
cgctgtgctg agggtgagtt ccctggagcc gggaacaggt gggtctgagc 41280aagccacact
tacccaggtc atctatccca tggtcaggga cccccagacc catacccagg 41340ggataccaag
gggggtaggc tcccagggct ggccacaccc atgggcagta ggccccagat 41400aaggagtggg
acttagaccc tgtctccacc ccaccctgca gagaattgct tcatacaaaa 41460gtcggatgac
aaggtcaccc tggaagaacg gctggacaag gcctgtgagc caggagtgga 41520ctatggtgag
tgggtgatgg gtgggggtca cgcatgttta gctgtgtgtg tccaattgtg 41580tggtgggtgg
taggtgtggt tgtcatggtg tggcttcagg ctgtgggtgt gggtgactgt 41640ggtgtgtgtg
agagcatgta ttgtgagggg ccatgattgt gtggggaacc atgactgtga 41700gtggcctagg
tatgctcatg tgagaaaagg tagatgtggt tgtatgcatc attgcgtggg 41760tggctgtgag
gttgtagttg tgtgtggctg tggttgtgtg aggctgtgtg gttgtagatg 41820gcagtgagtg
tgaggtcctg aagttacgta tatgactgta gttttccgtg gctatggttg 41880tgtgcatggc
catgaggcta cagtattttg tgcatatgag tcactctcat tgcatagtat 41940gaatagtatg
ttactagaca ttgtgggtgg ctgtgacctc tgtgcatgcc tatgagcacg 42000actgtgtgtg
gatggtgaca tgggaccctc tatggttgtg tgtgtaatga ggggtgggcc 42060atagtgtgac
tggctgtgat tctgcaactt tctgcttggg agagagagcc acatgcccgg 42120gtgcacttgc
aaaccagggt gcccctcatg gtcaacctag cccaccaccc aaactgtctg 42180cctctccccc
acagtgtaca agacccgact ggtcaaggtt cagctgtcca atgactttga 42240cgagtacatc
atggccattg agcagaccat caagtcaggt caggctcagc acgctgcctc 42300ccgtggctct
tccctggctt cctccccacg actcagcttc ttccctctcc cctccactcc 42360aggctcggat
gaggtgcagg ttggacagca gcgcacgttc atcagcccca tcaagtgcag 42420agaagccctg
aagctggagg agaagaaaca ctacctcatg tggggtctct cctccgattt 42480ctggggagag
aagcccaagt gagtgctttc cctgcgcgtg cgcgcgaccg cccgactgcc 42540ccgcccatgc
cacgcccaca ccattgtcac gcccctgcgc cacgcccaca ccacgcccct 42600tcctgacctg
ccattcttcc ctccagcctc agctacatca tcgggaagga cacttgggtg 42660gagcactggc
ccgaggagga cgaatgccaa gacgaagaga accagaaaca atgccaggac 42720ctcggcgcct
tcaccgagag catggttgtc tttgggtgcc ccaactgacc acacccccat 42780tcc
42783
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