Patent application title: GENES INVOLVED IN INFLAMMATORY BOWEL DISEASES AND USE THEREOF
Inventors:
Jean Pierre Hugot (Paris, FR)
Gilles Thomas (Paris, FR)
Mohamed Zouali (Bagneux, FR)
Suzanne Lesage (Sainte-Honorine, FR)
Mathias Chamaillard (Joue-Les-Tours, FR)
Assignees:
FONDATION JEAN DAUSSET-CEPH
IPC8 Class: AC07H2104FI
USPC Class:
506 16
Class name: Library, per se (e.g., array, mixture, in silico, etc.) library containing only organic compounds nucleotides or polynucleotides, or derivatives thereof
Publication date: 2013-02-07
Patent application number: 20130035260
Abstract:
The invention concerns genes involved in inflammatory and/or immune
diseases and some cancers, in particular intestinal cryptogenic
inflammatory diseases, and proteins coded by said genes. The invention
also concerns methods for diagnosing inflammatory diseases.Claims:
1-25. (canceled)
26. A purified or isolated nucleic acid probe, wherein said nucleic acid probe is specific for a variant nucleic acid sequence selected from the group consisting of SEQ ID NO:3 having a C to T mutation at nucleotide 16467, SEQ ID NO:3 having a G to C mutation at nucleotide 27059, and SEQ ID NO:3 having a C insertion at nucleotide 34296.
27. The nucleic acid probe of claim 26, wherein said nucleic acid probe is specific for SEQ ID NO:3 having a C to T mutation at nucleotide 16467.
28. The nucleic acid probe of claim 26, wherein said nucleic acid probe is specific for SEQ ID NO:3 having a G to C mutation at nucleotide 27059.
29. The nucleic acid probe of claim 26, wherein said nucleic acid probe is specific for SEQ ID NO:3 having a C insertion at nucleotide 34296.
30. The nucleic acid probe of claim 26, wherein said nucleic acid probe is about 15 to 30 nucleotides in length.
31. The nucleic acid probe of claim 26, wherein said nucleic acid probe comprises a detectable label.
32. The nucleic acid probe of claim 31, wherein said detectable label is selected from the group consisting of a radioactive isotope, a ligand, and a luminescent agent.
33. The nucleic acid probe of claim 32, wherein said ligand is selected from the group consisting of biotin, avidin, streptavidin, dioxygenin, a hapten, and a dye.
34. The nucleic acid probe of claim 32, wherein said luminescent agent is selected from the group consisting of a radioluminescent agent, a chemiluminescent agent, a bioluminescent agent, a fluorescent agent, and a phosphorescent agent.
35. The nucleic acid probe of claim 26, wherein said nucleic acid probe is immobilized on a support.
36. A kit comprising the nucleic acid probe of claim 26.
37. The kit of claim 36, wherein said kit comprises nucleic acid probes specific for at least two of said variant nucleic acid sequences.
38. The kit of claim 36, wherein said kit comprises nucleic acid probes specific for three of said variant nucleic acid sequences.
39. A DNA chip comprising the nucleic acid probe of claim 26.
40. The DNA chip of claim 39, wherein said DNA chip comprises nucleic acid probes specific for at least two of said variant nucleic acid sequences.
41. The DNA chip of claim 39, wherein said DNA chip comprises nucleic acid probes specific for three of said variant nucleic acid sequences.
Description:
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser. No. 12/370,543, filed Feb. 12, 2009, which application is a continuation of U.S. application Ser. No. 10/240,046, filed Jan. 15, 2003, now U.S. Pat. No. 7,592,437 which issued on Sep. 22, 2009, which application was a National Stage application of PCT/FR01/00935, filed Mar. 27, 2001, which application claims priority to FR 0003832, filed Mar. 27, 2000, all of which are incorporated by reference in their entirety.
[0002] The present invention relates to genes involved in inflammatory and/or immune diseases and certain cancers, in particularly cryptogenetic inflammatory bowel diseases, and also to the proteins encoded by these genes. The present invention also relates to methods for diagnosing inflammatory diseases.
[0003] Cryptogenetic inflammatory bowel diseases (IBDs) are diseases characterized by an inflammation of the digestive tract, the cause of which is unknown. Depending on the location and the characteristics of the inflammation, two different nosological entities are distinguished: ulcerative colitis (UC) and Crohn's disease (CD). UC was described by S Wilkes in 1865, whereas the first case of regional ileitis was reported by Crohn in 1932. In reality, it is possible that these two diseases go back much further.
[0004] IBDs are chronic diseases which evolve throughout life and which affect approximately 1 to 2 individuals per 1000 inhabitants in western countries, which represents between 60000 and 100000 individuals suffering from these diseases in France. They are diseases which appear in young individuals (peak instance is in the third decade), progressing via attacks interspersed with remissions, with frequent complications such as undernutrition, retarded growth in children, bone demineralization and, in the end, malignant degeneration to colon cancer. No specific treatment exists. Conventional therapeutics make use of anti-inflammatories, of immunosuppressors and of surgery. All these therapeutic means are, themselves, a source of considerable iatrogenic morbidity. For all these reasons, IBDs appear to be a considerable public health problem.
[0005] The etiology of IBDs is currently unknown. Environmental factors are involved in the occurrence of the disease, as witnessed by the secular increase in incidence of the disease and the incomplete concordance in monozygous twins. The only environmental risk factors currently known are 1) tobacco, the role of which is harmful in CD and beneficial in UC, and 2) appendectomy which has a protective role for UC.
[0006] Genetic predisposition has been suspected for a long time due to the existence of ethnic and familial aggregation of these diseases. In fact, IBDs are more common in the Caucasian population, and in particular in the Jewish population of central Europe. Familial forms represent from 6 to 20% of IBD cases. They are particularly common when the disease begins early. However, it is studies in twins which have made it possible to confirm the genetic nature of these diseases. In fact, the concordance rate between twins for these diseases is greater in monozygous twins than in dizygous twins, which pleads strongly in favor of a hereditary component to IBDs, in particular to CD. In all probability, IBDs are complex genetic diseases involving several different genes, interacting with one another and with environmental factors. IBDs can therefore be classified within the context of multifactor diseases.
[0007] Two major strategies have been developed in order to demonstrate the IBD-susceptibility genes. The first is based on the analysis of genes which are candidates for physiopathological reasons. Thus, many genes have been proposed as potentially important for IBDs. They are often genes which have a role in inflammation and the immune response. Mention may be made of the HLA, TAP, TNF and MICA genes, lymphocyte T receptor, ICAM1, interleukin 1, CCR5, etc. Other genes participate in diverse functions, such as GAI2, motilin, MRAMP, HMLH1, etc. In reality, none of the various candidate genes studied has currently definitively proved itself to have a role in the occurrence of IBDs.
[0008] The recent development of human genome maps using highly polymorphic genetic markers has enabled geneticists to develop a nontargeted approach over the entire genome. This approach, also called reverse genetics or positional cloning, makes no hypothesis regarding the genes involved in the disease and attempts to discover them through systematic screening of the genome. The method most used for complex genetic diseases is based on studying identity by decendance of the affected individuals of the same family. This value is calculated for a large number (300-400) of polymorphism markers distributed evenly (every 10cM) over the genome). In the case of excess identity between affected individuals, the marker(s) tested indicate(s) a region supposed to contain a gene for susceptibility to the disease. In the case of complex genetic diseases, since the model underlying the genetic predisposition (number of genes and relative importance of each of them) is unknown, the statistical methods to be used will have to be adjusted.
[0009] The present invention relates to the demonstration of the nucleic acid sequence of genes involved in IBDs, and other inflammatory diseases, and also the use of these nucleic acid sequences.
[0010] In the context of the present invention, preliminary studies by the inventors have already made it possible to locate a CD-susceptibility gene. Specifically, the inventors (Hugot et al., 1996) have shown that a CD-susceptibility gene is located in the pericentromeric region of chromosome 16 (FIG. 1). It was the first gene for susceptibility to a complex genetic disease located by positional cloning and satisfying the strict criteria proposed in the literature (Lander and Kruglyak, 1995). This gene was named IBD1 (for inflammatory bowel disease 1). Since then, other locations have been proposed by other authors, in particular on chromosomes 12, 1, 3, 6 and 7 (Satsangi et al., 1996; Cho et al., 1998). Although they have been located, it has currently not been possible to identify any of these IBD-susceptibility genes.
[0011] Some authors have not been able to replicate this location (Rioux et al., 1998). This is not, however, surprising in the case of complex genetic diseases in which genetic heterogeneity is probable.
[0012] It is interesting to note that, according to the same approach of positional cloning, locations have also been proposed on chromosome 16 for several immune and inflammatory diseases, such as ankylosing spondylarthritis, Blau's syndrome, psoriasis, etc. (Becker et al., 1998; Tromp et al., 1996). All these diseases may then share the same gene (or the same group of genes) located on chromosome 16.
[0013] A maximum of genetic linkage tests is virtually always located at the same position, in the region of D16S409 or D16S411, separated only by 2cM. This result contradicts the considerable size (usually greater than 20cM) of the confidence interval which can be attributed to the genetic location according to an approach using nonparametric linkage analyses.
[0014] Comparison of the statistical tests used in the studies by the inventors shows that the tests based on complete identity by decendance (Tz2) are better than the tests based on the mean of identity by decendance (Tz) (FIG. 1). Such a difference can be explained by a recessive effect of IBD1.
[0015] Several genes known to be in the pericentromeric region of chromosome 16, such as the interleukin 4 receptor, CD19, CD43 or CD11, appear to be good potential candidates for CD. Preliminary results do not however plead in favor of these genes being involved in CD.
[0016] In particular, the present invention provides not only the sequence of IBD1 gene, but also the partial sequence of another gene, called IBD1prox due to it being located in proximity to IBD, and demonstrated as reported in the examples below. These genes, the cDNA sequence of which corresponds, respectively, to SEQ ID No. 1 and SEQ ID No. 4, are therefore potentially involved in many inflammatory and/or immune diseases and also in cancers.
[0017] The peptide sequence expressed by the IBD1 and IBD1prox genes is represented by SEQ ID No. 2 and SEQ ID No. 5, respectively; the genomic sequence of these genes is represented by SEQ ID No. 3 and SEQ ID No. 6, respectively.
[0018] Thus, a subject of the present invention is a purified or isolated nucleic acid, characterized in that it Comprises a nucleic acid sequence chosen from the following group of sequences: [0019] a) SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 6; [0020] b) the sequence of a fragment of at least 15 consecutive nucleotides of a sequence chosen from SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 6; [0021] c) a nucleic acid sequence having a percentage identity of at least 80%, after optimal alignment, with a sequence defined in a) or b); [0022] d) a nucleic acid sequence which hybridizes, under high stringency conditions, with a nucleic acid sequence defined in a) or b); [0023] e) the complementary sequence or the RNA sequence corresponding to a sequence as defined in a), b), c) or d).
[0024] The nucleic acid sequence according to the invention defined in c) has a percentage identity of at least 80%, after optimal alignment, with a sequence as defined in a) or b) above, preferably 90%, most preferably 98%.
[0025] The terms "nucleic acid", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", terms which will be employed indifferently in the present description, are intended to denote a precise series of nucleotides, which may or may not be modified, making it possible to define a fragment or a region of a nucleic acid, which may or may not comprise unnatural nucleotides, and which may correspond equally to a double-stranded DNA, a single-stranded DNA and transcription products of said DNAs. Thus, the nucleic acid sequences according to the invention also encompass PNAs (Peptide Nucleic Acids), or the like.
[0026] It should be understood that the present invention does not relate to the nucleotide sequences in their natural chromosomal environment, that is to say in the natural state. They are sequences which have been isolated and/or purified, that is to say they have been taken directly or indirectly, for example by copying, their environment having been at least partially modified. Thus, nucleic acids obtained by chemical synthesis are also intended to be denoted.
[0027] For the purpose of the present invention, the term "percentage identity" between two nucleic acid or amino acid sequences is intended to denote a percentage of nucleotides or of amino acid residues which are identical between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. The term "best alignment" or "optimal alignment" is intended to denote the alignment for which the percentage identity determined as below is highest. Sequence comparisons between two nucleic acid or amino acid sequences are conventionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out by segment or by "window of comparison" so as to identify and compare local regions of sequence similarity. The optimal alignment of the sequences for the comparison may be carried out, besides manually, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970), by means of the similarity search method of Pearson and Lipman (1988), by means of computer programs using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.). In order to obtain the optimal alignment, the BLAST program is preferably used, with the BLOSUM 62 matrix. The PAM or PAM250 matrices may also be used.
[0028] The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences aligned optimally, the nucleic acid or amino acid sequence to be compared possibly comprising additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The percentage identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, dividing this number of identical positions by the total number of positions compared and multiplying the resultant number by 100 so as to obtain the percentage identity between these two sequences.
[0029] The expression "nucleic acid sequences having a percentage identity of at least 80%, preferably 90%, more preferably 98%, after optimal alignment with a reference sequence" is intended to denote the nucleic acid sequences which, compared to the reference nucleic acid sequence, have certain modifications, such as in particular a deletion, a truncation, an extension, a chimeric fusion and/or a substitution, in particular of the point type, and the nucleic acid sequence of which exhibits at least 80%, preferably 90%, more preferably 98%, identity, after optimal alignment, with the reference nucleic acid sequence. They are preferably sequences whose complementary sequences are capable of hybridizing specifically with the sequence SEQ ID No. 1 or SEQ ID No. 4 of the invention. Preferably, the specific or high stringency hybridization conditions will be such that they ensure at least 80%, preferably 90%, more preferably 98%, identity, after optimal alignment, between one of the two sequences and the sequence complementary to the other.
[0030] Hybridization under high stringency conditions means that the conditions of temperature and of ionic strength are chosen such that they allow the hybridization between two complementary DNA fragments to be maintained. By way of illustration, high stringency conditions for the hybridization step for the purposes of defining the polynucleotide fragments described above are advantageously as follows.
[0031] The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5×SSC (1×SSC corresponds to a solution of 0.15 M NaCl+0.015 M sodium citrate), 50% of formamide, 7% of sodium dodecyl sulfate (SDS), 10×Denhardt's, 5% of dextran sulfate and 1% of salmon sperm DNA; (2) hybridization per se for 20 hours at a temperature which depends on the length of the probe (i.e.: 42° C. for a probe >100 nucleotides in length), followed by 2 washes of 20 minutes at 20° C. in 2×SSC+2% SDS and 1 wash of 20 minutes at 20° C. in 0.1×SSC+0.1% SDS. The final wash is carried out in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for a probe >100 nucleotides in length. The high stringency hybridization conditions described above for a polynucleotide of defined length may be adjusted by those skilled in the art for longer or shorter oligonucleotides, according to the teaching of Sambrook et al., 1989.
[0032] Among the nucleic acid sequences having a percentage identity of at least 80%, preferably 90%, more preferably 98%, after optimal alignment, with the sequence according to the invention, preference is also given to the variant nucleic acid sequences of SEQ ID No. 1 or of SEQ ID No. 4, or of fragments thereof, that is to say all the nucleic acid sequences corresponding to allelic variants, that is to say individual variations of the sequence SEQ ID No. 1 or SEQ ID No. 4. These natural mutated sequences correspond to polymorphisms present in mammals, in particular in humans and, in particular, to polymorphisms which may lead to the occurrence of a pathological condition. Preferably, the present invention relates to the variant nucleic acid sequences in which the mutations lead to a modification of the amino acid sequence of the polypeptide, or of fragments thereof, encoded by the normal sequence of SEQ ID No. 1 or SEQ ID No. 4.
[0033] The expression "variant nucleic acid sequence" is also intended to denote any RNA or cDNA resulting from a mutation and/or variation of a splice site of the genomic nucleic acid sequence the cDNA of which has the sequence SEQ ID No. 1 or SEQ ID No. 4.
[0034] The invention preferably relates to a purified or isolated nucleic acid according to the present invention, characterized in that it comprises or consists of one of the sequences SEQ ID No. 1 or SEQ ID No. 4, of the sequences complementary thereto, or of the RNA sequences corresponding to SEQ ID No. 1 or SEQ ID No. 4.
[0035] The probes or primers, characterized in that they comprise a sequence of a nucleic acid according to the invention, are also part of the invention.
[0036] Thus, the present invention also relates to the primers or the probes according to the invention which may make it possible in particular to demonstrate or to distinguish the variant nucleic acid sequences, or to identify the genomic sequence of the genes the cDNA of which is represented by SEQ ID No. 1 or SEQ ID No. 4, in particular using an amplification method such as the PCR method or a related method.
[0037] The invention also relates to the use of a nucleic acid sequence according to the invention, as a probe or primer, for detecting, identifying, assaying or amplifying a nucleic acid sequence.
[0038] According to the invention, the polynucleotides which can be used as a probe or as a primer in methods for detecting, identifying, assaying or amplifying a nucleic acid sequence are a minimum of 15 bases, preferably 20 bases, or better still 25 to 30 bases in length.
[0039] The probes and primers according to the invention may be labeled directly or indirectly with a radioactive or nonradioactive compound using methods well known to those skilled in the art, in order to obtain a detectable and/or quantifiable signal.
[0040] The polynucleotide sequences according to the invention which are unlabeled can be used directly as a probe or primer.
[0041] The sequences are generally labeled so as to obtain sequences which can be used in many applications. The primers or the probes according to the invention are labeled with radioactive elements or with nonradio-active molecules.
[0042] Among the radioactive isotopes used, mention may be made of 32P, 33P, 35S, 3H or 125I. The nonradioactive entities are selected from ligands such as biotin, avidin, streptavidin or dioxygenin, haptens, dyes and luminescent agents, such as radioluminescent, chemiluminescent, bioluminescent, fluorescent or phosphorescent agents.
[0043] The polynucleotides according to the invention may thus be used as a primer and/or probe in methods using in particular the PCR (polymerase chain reaction) technique (Rolfs et al., 1991). This technique requires choosing pairs of oligonucleotide primers bordering the fragment which must be amplified. Reference may, for example, be made to the technique described in U.S. Pat. No. 4,683,202. The amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatographic technique such as gel filtration or ion exchange chromatography, and then sequenced. The specificity of the amplification can be controlled using, as primers, the nucleotide sequences of polynucleotides of the invention and, as matrices, plasmids containing these sequences or else the derived amplification products. The amplified nucleotide fragments may be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments.
[0044] The invention is also directed toward the nucleic acids which can be obtained by amplification using primers according to the invention.
[0045] Other techniques for amplifying the target nucleic acid may advantageously be employed as an alternative to PCR (PCR-like) using a pair of primers of nucleotide sequences according to the invention. The term "PCR-like" is intended to denote all the methods using direct or indirect reproductions of nucleic acid sequences, or else in which the labeling systems have been amplified; these techniques are of course, known. In general, they involve amplifying the DNA with a polymerase; when the sample of origin is an RNA a reverse transcription should be carried out beforehand. A large number of methods currently exist for this amplification, such as, for example, the SDA (strand displacement amplification) technique (Walker et al., 1992), the TAS (transcription-based amplification system) technique described by Kwoh et al. (1989), the 3SR (self-sustained sequence replication) technique described by Guatelli et al. (1990), the NASBA (nucleic acid sequence based amplification) technique described by Kievitis et al. (1991), the TMA (transcription mediated amplification) technique, the LCR (ligase chain reaction) technique described by Landegren et al. (1988), the RCR (repair chain reaction) technique described by Segev (1992), the CPR (cycling probe reaction) technique described by Duck et al. (1990), and the Q-beta-replicase amplification technique described by Miele et al. (1983). Some of these techniques have since been improved.
[0046] When the target polynucleotide to be detected is an mRNA, an enzyme of the reverse transcriptase type is advantageously used, prior to carrying out an amplification reaction using the primers according to the invention or to carrying out a method of detection using the probes of the invention, in order to obtain a cDNA from the mRNA contained in the biological sample. The cDNA obtained will then serve as a target for the primers or the probes used in the amplification or detection method according to the invention.
[0047] The probe hybridization technique may be carried out in many ways (Matthews et al., 1988). The most general method consists in immobilizing the nucleic acid extracted from the cells of various tissues or from cells in culture, on a support (such as nitrocellulose, nylon or polystyrene), and in incubating the immobilized target nucleic acid with the probe, under well-defined conditions. After hybridization, the excess probe is removed and the hybrid molecules formed are detected using the appropriate method (measuring the radioactivity, the fluorescence or the enzymatic activity linked to the probe).
[0048] According to another embodiment of the nucleic acid probes according to the invention, the latter may be used as capture probes. In this case, a probe, termed "capture probe", is immobilized on a support and is used to capture, by specific hybridization, the target nucleic acid obtained from the biological sample to be tested, and the target nucleic acid is then detected using a second probe, termed "detection probe", labeled with a readily detectable element.
[0049] Among the advantageous nucleic acid fragments, mention should thus be made in particular of antisense oligonucleotides, i.e. oligonucleotides, the structure of which ensures, by hybridization with the target sequence, inhibition of expression of the corresponding product. Mention should also be made of sense oligonucleotides, which, by interacting with proteins involved in regulating the expression of the corresponding product, will induce either inhibition or activation of this expression.
[0050] In both cases (sense and antisense), the oligo-nucleotides of the invention may be used in vitro and in vivo.
[0051] The present invention also relates to an isolated polypeptide, characterized in that it comprises a polypeptide chosen from: [0052] a) a polypeptide of sequence SEQ ID No. 2 or SEQ ID No. 5; [0053] b) a variant polypeptide of a polypeptide of sequence defined in a); [0054] c) a polypeptide homologous to a polypeptide defined in a) or b), comprising at least 80% identity with said polypeptide of a); [0055] d) a fragment of at least 15 consecutive amino acids of a polypeptide defined in a), b) or c); [0056] e) a biologically active fragment of a polypeptide defined in a), b) or c).
[0057] For the purpose of the present invention, the term "polypeptide" is intended to denote proteins or peptides.
[0058] The expression "biologically active fragment" is intended to mean a fragment having the same biological activity as the peptide fragment from which it is deduced, preferably within the same order of magnitude (to within a factor of 10). Thus, the examples show that the IBD1 protein (SEQ ID No. 2) has a potential role in apoptosis phenomena. A biologically active fragment of the IBD1 protein therefore consists of a polypeptide derived from SEQ ID No. 2, also having a role in apoptosis. The examples below propose biological functions for the IBD1 and IBD1prox proteins, as a function of the peptide domains of these proteins, and thus allow those skilled in the art to identify the biologically active fragments.
[0059] Preferably, a polypeptide according to the invention is a polypeptide consisting of the sequence SEQ ID No. 2 (corresponding to the protein encoded by the IBD1 gene) or of the sequence SEQ ID No. 5 (corresponding to the protein encoded by IBD1prox) or of a sequence having at least 80% identity with SEQ ID No. 2 or SEQ ID No. 5 after optimal alignment.
[0060] The sequence of the polypeptide has a percentage identity of at least 80%, after optimal alignment, with the sequence SEQ ID No. 2 or SEQ ID No. 5, preferably 90%, more preferably 98%.
[0061] The expression "polypeptide, the amino acid sequence of which has a percentage identity of at least 80%, preferably 90%, more preferably 98%, after optimal alignment, with a reference sequence" is intended to denote the polypeptides having certain modifications compared to the reference polypeptide, such as in particular one or more deletions and/or truncations, an extension, a chimeric fusion and/or one or more substitutions.
[0062] Among the polypeptides, the amino acid sequence of which has a percentage identity of at least 80%, preferably 90%, more preferably 98%, after optimal alignment, with the sequence SEQ ID No. 2 or SEQ ID No. 5 or with a fragment thereof according to the invention, preference is given to the variant polypeptides encoded by the variant nucleic acid sequences as defined previously, in particular the polypeptides, the amino acid sequence of which has at least one mutation corresponding in particular to a truncation, deletion, substitution and/or addition of at least one amino acid residue compared with the sequence SEQ ID No. 2 or SEQ ID No. 5 or with a fragment thereof, more preferably the variant polypeptides having a mutation associated with the pathological condition.
[0063] The present invention also relates to the cloning and/or expression vectors comprising a nucleic acid or encoding a polypeptide according to the invention. Such a vector may also contain the elements required for the expression and, optionally, the secretion of the polypeptide in a host cell. Such a host cell is also a subject of the invention.
[0064] The vectors characterized in that they comprise a promoter and/or regulator sequence according to the invention are also part of the invention.
[0065] Said vectors preferably comprise a promoter, translation initiation and termination signals, and also regions suitable for regulating transcription. It must be possible for them to be maintained stably in the cell and they may optionally contain particular signals specifying secretion of the translated protein.
[0066] These various control signals are chosen as a function of the cellular host used. To this effect, the nucleic acid sequences according to the invention may be inserted into vectors which replicate autonomously in the chosen host, or vectors which integrate in the chosen host.
[0067] Among the systems which replicate autonomously, use is preferably made, depending on the host cell, of systems of the plasmid or viral type, the viral vectors possibly being in particular adenoviruses (Perricaudet et al., 1992), retroviruses, lentiviruses, poxviruses or herpesviruses (Epstein et al., 1992). Those skilled in the art are aware of the technology which can be used for each of these systems.
[0068] When integration of the sequence into the chromosomes of the host cell is desired, use may be made, for example, of systems of the plasmid or viral type; such viruses are, for example, retroviruses (Temin, 1986), or AAVs (Carter, 1993).
[0069] Among the nonviral vectors, preference is given to naked polynucleotides such as naked DNA or naked RNA according to the technology developed by the company VICAL, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) for expression in yeast, mouse artificial chromosomes (MACs) for expression in murine cells and, preferably, human artificial chromosomes (HACs) for expression in human cells.
[0070] Such vectors are prepared according to the methods commonly used by those skilled in the art, and the clones resulting therefrom can be introduced into a suitable host using standard methods, such as, for example, lipofection, electroporation, heat shock, transformation after chemical permeabilization of the membrane, or cell fusion.
[0071] The invention also comprises host cells, in particular the eukaryotic and prokaryotic cells, transformed with the vectors according to the invention, and also the transgenic animals, preferably the mammals, except humans, comprising one of said transformed cells according to the invention. These animals may be used as models, for studying the etiology of inflammatory and/or immune diseases, in particular of the inflammatory diseases of the digestive tract, or for studying cancers.
[0072] Among the cells which can be used for the purpose of the present invention, mention may be made of bacterial cells (Olins and Lee, 1993), but also yeast cells (Buckholz, 1993) as well as animal cells, in particular mammalian cell cultures (Edwards and Aruffo, 1993), and especially Chinese hamster ovary (CHO) cells. Mention may also be made of insect cells in which it is possible to use methods employing, for example, baculo viruses (Luckow, 1993). A preferred cellular host for expressing the proteins of the invention consists of COS cells.
[0073] Among the mammals according to the invention, animals such as rodents, in particular mice, rats or rabbits, expressing a polypeptide according to the invention are preferred.
[0074] Among the mammals according to the invention, preference is also given to animals such as mice, rats or rabbits, characterized in that the gene encoding the protein of sequence SEQ ID No. 2 or SEQ ID No. 5, or the sequence of which is encoded by the homologous gene in these animals, is not functional, has been knocked out or has at least one mutation.
[0075] These transgenic animals are obtained, for example, by homologous recombination on embryonic stem cells, transfer of these stem cells to embryos, selection of the chimeras affected in the reproductive lines, and growth of said chimeras.
[0076] The transgenic animals according to the invention may thus overexpress the gene encoding the protein according to the invention, or their homologous gene, or express said gene into which a mutation is introduced. These transgenic animals, in particular mice, are obtained, for example, by transfection of a copy of this gene under the control of a promoter which is strong and ubiquitous, or selective for a tissue type, or after viral transcription.
[0077] Alternatively, the transgenic animals according to the invention may be made deficient for the gene encoding one of the polypeptides of sequence SEQ ID No. 2 or SEQ ID No. 5, or their homologous genes, by inactivation using the LOXP/CRE recombinase system (Rohlmann et al., 1996) or any other system for inactivating the expression of this gene.
[0078] The cells and mammals according to the invention can be used in a method for producing a polypeptide according to the invention, as described below, and may also be used as a model for analysis.
[0079] The cells or mammals transformed as described above can also be used as models in order to study the interactions between the polypeptides according to the invention, and the chemical or protein compounds involved directly or indirectly in the activities of the polypeptides according to the invention, this being in order to study the various mechanisms and interactions involved.
[0080] They may in particular be used for selecting products which interact with the polypeptides according to the invention, in particular the protein of sequence SEQ ID No. 2 or SEQ ID No. 5 or variants thereof according to the invention, as a cofactor or as an inhibitor, in particular a competitive inhibitor, or which have an agonist or antagonist activity with respect to the activity of the polypeptides according to the invention. Preferably, said transformed cells or transgenic animals are used as a model in particular for selecting products for combating pathological conditions associated with abnormal expression of this gene.
[0081] The invention also relates to the use of a cell, of a mammal or of a polypeptide according to the invention, for screening chemical or biochemical compounds which may interact directly or indirectly with the polypeptides according to the invention, and/or which are capable of modulating the expression or the activity of these polypeptides.
[0082] Similarly, the invention also relates to a method for screening compounds capable of interacting, in vitro or in vivo, with a nucleic acid according to the invention, using a nucleic acid, a cell or a mammal according to the invention, and detecting the formation of a complex between the candidate compounds and the nucleic acid according to the invention.
[0083] The compounds thus selected are also subjects of the invention.
[0084] The invention also relates to the use of a nucleic acid sequence according to the invention, for synthesizing recombinant polypeptides.
[0085] The method for producing a polypeptide of the invention in recombinant form, which is itself included in the present invention, is characterized in that the transformed cells, in particular the cells or mammals of the present invention, are cultured under conditions which allow the expression of a recombinant polypeptide encoded by a nucleic acid sequence according to the invention, and in that said recombinant polypeptide is recovered.
[0086] The recombinant polypeptides, characterized in that they can be obtained using said method of production, are also part of the invention.
[0087] The recombinant polypeptides obtained as indicated above can be in both glycosylated and nonglycosylated form, and may or may not have the natural tertiary structure.
[0088] The sequences of the recombinant polypeptides may also be modified in order to improve their solubility, in particular in aqueous solvents.
[0089] Such modifications are known to those skilled in the art, such as, for example, deletion of hydrophobic domains or substitution of hydrophobic amino acids with hydrophilic amino acids.
[0090] These polypeptides may be produced using the nucleic acid sequences defined above, according to the techniques for producing recombinant polypeptides known to those skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals which allow its expression in a cellular host.
[0091] An effective system for producing a recombinant polypeptide requires having a vector and a host cell according to the invention.
[0092] These cells can be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, and then culturing said cells under conditions which allow the replication and/or expression of the transfected nucleotide sequence.
[0093] The methods used for purifying a recombinant polypeptide are known to those skilled in the art. The recombinant polypeptide may be purified from cell lysates and extracts or from the culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using specific monoclonal or polyclonal antibodies, etc.
[0094] The polypeptides according to the present invention can also be obtained by chemical synthesis using one of the many known forms of peptide synthesis, for example techniques using solid phases (see in particular Stewart et al., 1984) or techniques using partial solid phases, by fragment condensation or by conventional synthesis in solution.
[0095] The polypeptides obtained by chemical synthesis and which may comprise corresponding unnatural amino acids are also included in the invention.
[0096] The mono- or polyclonal antibodies, or fragments thereof, chimeric antibodies or immunoconjugates, characterized in that they are capable of specifically recognizing a polypeptide according to the invention, are part of the invention.
[0097] Specific polyclonal antibodies may be obtained from a serum of an animal immunized against the polypeptides according to the invention, in particular produced by genetic recombination or by peptide synthesis, according to the usual procedures.
[0098] The advantage of antibodies which specifically recognize certain polypeptides, variants or immunogenic fragments thereof according to the invention is in particular noted.
[0099] The mono- or polyclonal antibodies, or fragments thereof, chimeric antibodies or immunoconjugates characterized in that they are capable of specifically recognizing the polypeptides of sequence SEQ ID No. 2 or SEQ ID No. 5 are particularly preferred.
[0100] The specific monoclonal antibodies may be obtained according to the conventional method of hybridoma culture described by Kohler and Milstein (1975).
[0101] The antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, or Fab or F(ab')2 fragments. They may also be in the form of immunoconjugates or of labeled antibodies, in order to obtain a detectable and/or quantifiable signal.
[0102] The invention also relates to methods for detecting and/or purifying a polypeptide according to the invention, characterized in that they use an antibody according to the invention.
[0103] The invention also comprises purified polypeptides, characterized in that they are obtained using a method according to the invention.
[0104] Moreover, besides their use for purifying the polypeptides, the antibodies of the invention, in particular the monoclonal antibodies, may also be used for detecting these polypeptides in a biological sample.
[0105] They thus constitute a means for the immunocytochemical or immunohistochemical analysis of the expression of the polypeptides according to the invention, in particular the polypeptides of sequence SEQ ID No. 2 or SEQ ID No. 5, or a variant thereof, on specific tissue sections, for example using immunofluorescence, gold labeling and/or enzymatic immunoconjugates.
[0106] They may in particular make it possible to demonstrate abnormal expression of these polypeptides in the biological specimens or tissues.
[0107] More generally, the antibodies of the invention may advantageously be used in any situation where the expression of a polypeptide according to the invention, normal or mutated, must be observed.
[0108] Thus, a method for detecting a polypeptide according to the invention, in a biological sample, comprising the Steps of bringing the biological sample into contact with an antibody according to the invention and demonstrating the antigen-antibody complex formed, is also a subject of the invention, as is a kit for carrying out such a method. Such a kit in particular contains: [0109] a) a monoclonal or polyclonal antibody according to the invention; [0110] b) optionally, reagents for constituting a medium suitable for the immunoreaction; [0111] c) the reagents for detecting the antigen-antibody complex produced during the immunoreaction.
[0112] The antibodies according to the invention may also be used in the treatment of an inflammatory and/or immune disease, or of a cancer, in humans, when abnormal expression of the IBD1 gene or of the IBD1prox gene is observed. Abnormal expression means overexpression or the expression of a mutated protein.
[0113] These antibodies may be obtained directly from human serum, or may be obtained from animals immunized with polypeptides according to the invention, and then "humanized", and may be used as such or in the preparation of a medicinal product intended for the treatment of the abovementioned diseases.
[0114] The methods for determining an allelic variability, a mutation, a deletion, a loss of heterozygocity or any genetic abnormability of the gene according to the invention, characterized in that they use a nucleic acid sequence, a polypeptide or an antibody according to the invention, are also part of the invention.
[0115] The invention in fact provides the sequence of the IBD1 and IBD1prox genes involved in inflammatory and/or immune diseases, and in particular IBDs. One of the teachings of the invention is to specify the mutations, in these nucleic acid or polypeptide sequences, which are associated with a phenotype corresponding to one of these inflammatory and/or immune diseases.
[0116] These mutations can be detected directly by analysis of the nucleic acid and of the sequences according to the invention (genomic DNA, RNA or cDNA), but also via the polypeptides according to the invention. In particular, the use of an antibody according to the invention which recognizes an epitope bearing a mutation makes it possible to distinguish between a "healthy" protein and a protein "associated with a pathological condition".
[0117] Thus, the study of the IBD1 gene in various inflammatory and/or immune human diseases thus shows that sequence variants of this gene exist in Crohn's disease, ulcerative colitis and Blau's syndrome, as demonstrated by the examples. These sequence variations result in considerable variations in the deduced protein sequence. In fact, they are either located on very conserved sites of the protein in important functional domains, or they result in the synthesis of a truncated protein. It is therefore extremely probable that these deleterious modifications lead to a modification of the function of the protein and therefore have a causal effect in the occurrence of these diseases.
[0118] The variety of diseases in which these mutations are observed suggests that the IBD1 gene is potentially important in many inflammatory and/or immune diseases. This result should be compared with the fact that the pericentromeric region of chromosome 16 has been described as containing genes for susceptibility to various human diseases, such as ankylosing spondylarthritis or psoriatic arthropathy. It may therefore be considered that IBD1 has an important role in a large number of inflammatory and/or immune diseases.
[0119] In particular, IBD1 can be associated with granulomatous inflammatory diseases. Blau's syndrome and CD are in fact diseases which are part of this family. It is therefore hoped that variations in the IBD1 gene will be found for the other diseases of the same family (sarcoidosis, Behcet's disease, etc.).
[0120] In addition, the involvement of IBD1 in the cellular pathways leading to apoptosis raises the question of its possible carcinogenic role. In fact, it is expected that a dysregulation of IBD1 may result in a predisposition to cancer. This hypothesis is supported by the fact that a predisposition to colon cancer exists in inflammatory bowel diseases. IBD1 may in part explain this susceptibility to cancer and define new carcinogenic pathways.
[0121] The precise description of the mutations which can be observed in the IBD1 gene thus makes it possible to lay down the foundations of a molecular diagnosis for the inflammatory or immune diseases in which this role is demonstrated. Such an approach, based on searching for mutations in the gene, will make it possible to contribute to the diagnosis of these diseases and possibly to reduce the extent of certain additional examinations which are invasive or expensive. The invention lays down the foundations of such a molecular diagnosis based on searching for mutations in IBD1.
[0122] The molecular diagnosis of inflammatory diseases should also make it possible to improve the nosological classification of these diseases and to more clearly define subgroups of particular diseases by their clinical characteristics, the progressive nature of the disease or the response to certain treatments. By way of example, the dismantling of the existing mutations may thus make it possible to classify the currently undetermined forms of colitis which represent more than 10% of inflammatory bowel diseases. Such an approach will make it possible to propose an early treatment suitable for each patient. In general, such an approach makes it possible to hope that it will eventually be possible to define an individualized treatment for the disease, depending on the genetic area of each disease, including curative and preventive measures.
[0123] In particular, preference is given to a method of diagnosis and/or of prognostic assessment of an inflammatory disease or of a cancer, characterized in that the presence of at least one mutation and/or a deleterious modification of expression of the gene corresponding to SEQ ID No. 1 or SEQ ID No. 4 is determined, using a biological specimen from a patient, by analyzing all or part of a nucleic acid sequence corresponding to said gene. The genes SEQ ID No. 3 or SEQ ID No. 6 may also be studied.
[0124] This method of diagnosis and/or of prognostic assessment may be used preventively (a study of predisposition to inflammatory diseases or to cancer), or in order to serve in establishing and/or confirming a clinical condition in a patient.
[0125] Preferably, the inflammatory disease is an inflammatory disease of the digestive tract, and the cancer is a cancer of the digestive tract (small intestine or colon).
[0126] The teaching of the invention in fact makes it possible to determine the mutations which exhibit a linkage disequilibrium with inflammatory diseases of the digestive tract, and which are therefore associated with such diseases.
[0127] The analysis may be carried out by sequencing all or part of the gene, or by other methods known to those skilled in the art. Methods based on PCR, for example PCR-SSCP, which makes it possible to detect point mutations, may in particular be used.
[0128] The analysis may also be carried out by attaching a probe according to the invention, corresponding to one of the sequences SEQ ID No. 1, 3, 4 or 6, to a DNA chip, and hybridization on these microplates. A DNA chip containing a sequence according to the invention is also one of the subjects of the invention.
[0129] Similarly, a protein chip containing an amino acid sequence according to the invention is also a subject of the invention. Such a protein chip makes it possible to study the interactions between the polypeptides according to the invention and other proteins or chemical compounds, and may thus be useful for screening compounds which interact with the polypeptides according to the invention. The protein chips according to the invention may also be used to detect the presence of antibodies directed against the polypeptides according to the invention in the serum of patients. A protein chip containing an antibody according to the invention may also be used.
[0130] Those skilled in the art are also able to carry out techniques for studying the deleterious modification of the expression of a gene, for example by studying the mRNA (in particular by Northern blotting or with RT-PCR experiments, with probes or primers according to the invention), or the protein expressed, in particular by Western blotting, using antibodies according to the invention.
[0131] The gene tested is preferably the gene of sequence SEQ ID No. 1, the inflammatory disease for which the intention is to predict susceptibility being a disease of the digestive tract, in particular Crohn's disease or ulcerative colitis. If the intention is to detect a cancer, it is preferably colon cancer.
[0132] The invention also relates to methods for obtaining an allele of the IBD1 gene, associated with a detectable phenotype, comprising the following steps: [0133] a) obtaining a nucleic acid sample from an individual expressing said detectable phenotype; [0134] b) bringing said nucleic acid sample into contact with an agent capable of specifically detecting a nucleic acid encoding the IBD1 protein; [0135] c) isolating said nucleic acid encoding the IBD1 protein.
[0136] Such a method may be followed by a step of sequencing all or part of the nucleic acid encoding the IBD1 protein, which makes it possible to predict susceptibility to inflammatory disease or of a cancer.
[0137] The agent capable of specifically detecting a nucleic acid encoding the IBD1 protein is advantageously an oligonucleotide probe according to the invention, which may be made up of DNA, RNA or PNA, which may or may not be modified. The modifications may include radioactive or fluorescent labeling, or may be due to modifications in the bonds between the bases (phosphorothioates or methyl phosphonates, for example). Those skilled in the art are aware of the protocols for isolating a specific DNA sequence. Step b) of the method described above may also be an amplification step as described above.
[0138] The invention also relates to a method for detecting and/or assaying a nucleic acid according to the invention, in a biological sample, comprising the following steps of bringing a probe according to the invention into contact with a biological sample, and detecting and/or assaying the hybrid formed between said polynucleotide and the nucleic acid of the biological sample.
[0139] Those skilled in the art are capable of carrying out such a method, and may in particular use a kit of reagents, comprising: [0140] a) a polynucleotide according to the invention, used as a probe; [0141] b) the reagents required for carrying out a hybridization reaction between said probe and the nucleic acid of the biological sample; [0142] c) the reagents required for detecting and/or assaying the hybrid formed between said probe and the nucleic acid of the biological sample; which is also a subject of the invention.
[0143] Such a kit may also contain positive or negative controls in order to ensure the quality of the results obtained.
[0144] However, in order to detect and/or assay a nucleic acid according to the invention, those skilled in the art may also perform an amplification step using primers chosen from the sequences according to the invention.
[0145] Finally, the invention also relates to the compounds chosen from a nucleic acid, a polypeptide, a vector, a cell or an antibody according to the invention, or the compounds obtained using the screening methods according to the invention, as a medicinal product, in particular for preventing and/or treating an inflammatory and/or immune disease, or a cancer, associated with the presence of at least one mutation of the gene corresponding to SEQ ID No. 1 or SEQ ID No. 4, preferably an inflammatory disease of the digestive tract, in particular Crohn's disease or ulcerative colitis.
[0146] The following examples make it possible to understand more clearly the advantages of the invention, and should not be considered to limit the scope of the invention.
DESCRIPTION OF THE FIGURES
[0147] FIG. 1: Nonparametric genetic linkage tests for Crohn's disease in the pericentromeric region of chromosome 16 (according to Hugot et al., 1996). Multipoint linkage analysis based on identity by decendance for the markers of the pericentromeric region of chromosome 16. The genetic distances between markers were estimated using the CRIMAP program. The lod score (MAPMAKER/SIBS) is indicated on the left-hand figure. Two pseudoprobability tests were developed and reported on the right-hand figure. The first (Tz) is analogous to the test of the means. The second (Tz2) is analogous to the test of the proportion of affected pairs sharing two alleles.
[0148] FIG. 2: Multipoint nonparametric genetic linkage analysis. 78 families with several relatives suffering from Crohn's disease were genotyped for 26 polymorphism markers in the pericentromeric region of chromosome 16. The location of each marker is symbolized by an arrow. The order of the markers and the distance separating them derive from the analysis of the experimental data with the Crimap software. The arrows under the curve indicate the markers SPN, D16S409 and D16S411 used in the first study published (Hugot et al., 1996). The arrows located at the top of the figure correspond to the markers D16S3136, D16S541, D16S3117, D16S416 and D16S770 located at the maximum of the genetic linkage test. The typing data were analyzed using the multipoint nonparametric analysis program of the Genehunter software version 1.3. The maximum NPL score is 3.33 (p=0.0004).
[0149] FIG. 3: Diagrammatic representation of the protein encoded by IBD1. The protein encoded by IBD1 is represented horizontally. The various domains of which it is composed are indicated on the figure with the amino acid reference number corresponding to the start and to the end of each domain. The protein consists of a CARD domain, a nucleotide-binding domain (NBD) and leucine-rich motifs (LRR).
[0150] FIG. 4: Diagrammatic representation of the IBD1/NOD2 protein in three variants associated with CD.
[0151] A: The translation produced deduced from the cDNA sequence of the IBD1 candidate gene is identical to that of NOD2 (Ogura et al., 2000). The polypeptide contains 2 CARD domains (CAspase Recruitment Domains), a nucleotide-binding domain (NBD) and 10 repeats of 27 amino acids, leucine-rich motifs (LRR). The consensus sequence of the ATP/GTP-binding site of the motif A (P loop) of the NBD is indicated with a black circle. The sequence changes encoded by the three main variants associated with CD are SNP 8 (R675W), SNP 12 (G881R) and SNP 13 (frame shift 980). The frame shift changes a leucine codon to a proline codon at position 980, which is immediately followed by a stop codon.
[0152] B: Rare missense variants of NOD2 in 457 CD patients, 159 UC patients and 103 unaffected, unrelated individuals. The positions of the rare missense variants are indicated for the three groups. The scale on the left indicates the number of each variant identified in the groups under investigation and that on the right measures the frequency of the mutation. The allelic frequencies of the polymorphism V928I was not significantly different (0.92:0.08) in the three groups and the corresponding genotypes were in Hardy-Weinberg equilibrium.
EXAMPLES
Example 1
Fine Location of IBD1
[0153] The first step toward identifying the IBD1 gene was to reduce the size of the genetic region of interest, initially centered on the marker D16S411 located between D16S409 and D16S419 (Hugot et al., 1996 and FIG. 1). A group of close markers (high resolution genetic map) was used in order to more clearly specify the genetic region, and made it possible to complete the genetic linkage analyses and to search for a genetic linkage disequilibrium with the disease.
[0154] The study related to 78 families comprising at least 2 relatives suffering from CD, which corresponded to 119 affected pairs. The families comprising sick individuals suffering from UC were excluded from the study.
[0155] Twenty-six genetic polymorphism markers of the micro-satellite type were studied. These markers together made up a high resolution map with an average distance between markers of the order of 1cM in the genetic region of interest. The characteristics of the markers studied are given in table 1.
TABLE-US-00001 TABLE 1 Polymorphic markers of the microsatellite type used for the fine location of IBD1 Name of polymorphism Cumulative marker distance (cM) PCR primers D16S3120 0 SEQ ID No. 7 (AFM326vc5) SEQ ID No. 8 D16S298 2.9 SEQ ID No. 9 (AFMa189wg5) SEQ ID No. 10 D16S299 3.4 SEQ ID No. 11 SEQ ID No. 12 SPN 3.9 SEQ ID No. 13 SEQ ID No. 14 D16S383 4.3 SEQ ID No. 15 SEQ ID No. 16 D16S753 4.9 SEQ ID No. 17 (GGAA3G05) SEQ ID No. 18 D16S3044 5.8 SEQ ID No. 19 (AFMa222za9) SEQ ID No. 20 D16S409 5.8 SEQ ID No. 21 (AFM161xa1) SEQ ID No. 22 D16S3105 6.1 SEQ ID No. 23 (AFMb341zc5) SEQ ID No. 24 D16S261 6.8 SEQ ID No. 25 (MFD24) SEQ ID No. 26 D16S540 6.9 SEQ ID No. 27 (GATA7B02) SEQ ID No. 28 D16S3080 7 SEQ ID No. 29 (AFMb068zb9) SEQ ID No. 30 D16S517 7 SEQ ID No. 31 (AFMa132we9) SEQ ID No. 32 D16S411 8 SEQ ID No. 33 (AFM186xa3) SEQ ID No. 34 D16S3035 10.4 SEQ ID No. 35 (AFMa189wg5) SEQ ID No. 36 D16S3136 10.4 SEQ ID No. 37 (AFMa061xe5) SEQ ID No. 38 D16S541 11.4 SEQ ID No. 39 (GATA7E02) SEQ ID No. 40 D16S3117 11.5 SEQ ID No. 41 (AFM288wb1) SEQ ID No. 42 D16S416 12.4 SEQ ID No. 43 (AFM210yg3) SEQ ID No. 44 D16S770 13.2 SEQ ID No. 45 (GGAA20G02) SEQ ID No. 46 D16S2623 15 SEQ ID No. 47 (GATA81B12) SEQ ID No. 48 D16S390 16.5 SEQ ID No. 49 SEQ ID No. 50 D16S419 20.4 SEQ ID No. 51 (AFM225zf2) SEQ ID No. 52 D16S771 21.8 SEQ ID No. 53 (GGAA23C09) SEQ ID No. 54 D16S408 25.6 SEQ ID No. 55 (AFM137xf8) SEQ ID No. 56 D16S508 38.4 SEQ ID No. 57 (AFM304xf1) SEQ ID No. 58
[0156] Each marker is listed according to international nomenclature and mostly by the name proposed by the laboratory of origin. The markers appear according to their order on the chromosome (from 16p to 16q). The genetic distance between the markers (in Kosambi centiMorgans, calculated from the experimental data using the Crimap program) is indicated in the second column. The first polymorphic marker is taken randomly as a reference point. The oligonucleotides which were used for the polymerase chain reaction (PCR) are indicated in the third column.
[0157] The genotyping of these microsatellite markers was based on automatic sequencer technology using fluorescent primers. Briefly, after amplification, the fluorescent polymerase chain reaction (PCR) products were loaded onto a polyacrylamide gel on an automatic sequencer according to the manufacturer's recommendations (Perkin Elmer). The size of the alleles for each individual was deduced using the Genescan® and Genotyper® software. The data were then kept on an integrated computer base containing the genealogical, phenotypic and genetic data. They were then used for the genetic linkage analyses.
[0158] Several quality controls were carried out throughout the genotyping procedure: [0159] independent double reading of the genotyping data, [0160] use of a standard DNA as an internal control for each electrophoretic migration, [0161] control of the size range for each allele observed, [0162] search for mendelian transmission errors, [0163] calculation of the genetic distance between markers (CRIMAP program) and comparison of this distance with the data from the literature, [0164] further typing of the markers for which recombination between close markers was observed.
[0165] The genotyping data were analyzed by multipoint non-parametric genetic linkage methods (GENEHUNTER program version 1.3). The informativeness of the marker system was greater than 80% for the region studied. The test maximum (NPL=3.33; P=0.0004) was obtained for the markers D16S541, D16S3117, D16S770 and D16S416 (FIG. 2).
[0166] The typing data for these 26 polymorphism markers were also analyzed so as to search for a transmission disequilibrium. Two groups of 108 and 76 families with one or more sick individuals suffering from CD were studied. The statistical test for transmission disequilibrium has been described by Spielman et al. (1993). In this study, only one sick individual per family was taken into account, and the value of p was corrected by the number of alleles tested for each marker studied.
[0167] A transmission disequilibrium was observed for alleles 4 and 5 (size 205 and 207 base pairs, respectively) of the marker D16S3136 (p=0.05 and p=0.01, respectively).
[0168] These results, which suggest an association between the marker D16S3136 and CD, led to the construction of a physical map of the genetic region centered on D16S3136 and to establishment of the sequence of a large genomic DNA segment (BAC) containing this polymorphic site. It was then possible to identify and analyze a larger number of polymorphism markers in the region of D16S3136, and also to define and study the transcribed sequences present in the region.
Example 2
Physical Mapping of the IBD1 Region
[0169] A contig of genomic DNA fragments, centered on the markers D16S3136, D16S3117, D16S770 and D16S416, was generated from the human genomic DNA libraries of the Jean Dausset foundation/CEPH. The chromosomal DNA segments were identified based on certain polymorphism markers used in fine genetic mapping (D16S411, D16S416, D16S541, D16S770, D16S2623, D16S3035, D16S3117 and D16S3136). For each marker, a bacterial artificial chromosome (BAC) library was screened by PCR so as to search for clones containing the marker sequence. Depending on whether or not the sequences tested were present on the BAC clones, it was then possible to organize the clones among one another using the Segmap software version 3.35.
[0170] It was possible to establish, for the BACs, a continuous organization (contig) covering the genetic region of interest, according to a method known to those skilled in the art (Rouquier et al., 1994; Kim et al., 1996; Asakawa et at., 1997). To do this, the ends of the BACs identified were sequenced and these new sequence data were then used to repeatedly screen the BAC libraries. At each screening, the BAC contig then progressed by a step until a continuum of overlapping clones was obtained. The size of each BAC contributing to the contig was deduced from its migration profile on a pulsed field agarose gel.
[0171] A BAC contig containing 101 BACs and extending over an overall distance of more than 2.5 Mb, with an average redundancy of 5.5 BACs at each point of the contig, was thus constructed. The average size of the BACs is 136 kb.
Example 3
Sequencing of BAC hb87b10
[0172] The BAC of this contig containing the polymorphism marker D16S3136 (called hb87b10), the size of which was 163761 bp, was sequenced according to the "shotgun" method. Briefly, the BAC DNA was fragmented by sonication. The DNA fragments thus generated were subjected to agarose gel electrophoresis and those with a size greater than 1.5 kb were eluted in order to be analyzed. These fragments were then cloned into the m13 phage, which was itself introduced into bacteria made competent, by electroporation. After culturing, the DNA of the clones was recovered and sequenced by automatic sequencing methods using fluorescent primers of the m13 vector on an automatic sequencer.
[0173] 1526 different sequences with an average size of 600 bp were generated, which were organized with respect to one another using the Polyphredphrap® software, resulting in a sequence contig covering the entire BAC. The sequence thus generated had an average redundancy of 5.5 genomic equivalents. The rare (n=5) sequence gaps not represented in the m13 clone library were filled by generating specific PCR primers, on either side of these gaps, and analyzing the PCR product derived from the genomic DNA of a healthy individual.
[0174] Sequence homologies with sequences available in public genetic databases (Genbank) were sought. No known gene could be identified in this region of 163 kb. Several ESTs were positioned, suggesting that unknown genes were contained in this sequence. These ESTs derived from the public genetic databases (Genbank, GDB, Unigene, dbEST) bore the following references: AI167910, A1011720, Rn24957, Mm30219, hs132289, AA236306, hs87296, AA055131, hs151708, AA417809, AA417810, hs61309, hs116424, HUMGS01037, AA835524, hs105242, SHGC17274, hs146128, hs122983, hs87280 and hs135201. The search for putative exons using the GRAIL computer program made it possible to identify several potential exons, polyadenylation sites and promoter sequences.
Example 4
Transmission Disequilibrium Studies
[0175] 12 biallelic polymorphism markers (SNPs) were identified in a region extending over approximately 250 kb and centered on the BAC hb87b10. These polymorphisms were generated by analyzing the sequence of ten or so independent sick individuals suffering from CD. The sequencing was mostly carried out at known ESTs positioned on the BAC or in the region thereof. Putative exons, predicted by the GRAIL computer program, were also analyzed. The characteristics of the polymorphic markers thus identified are given in table 2.
TABLE-US-00002 TABLE 2 Characteristics of biallelic polymorphism markers studied in the region of IBD1 I II III IV V VI 1 KIAA0849ex9 AS-PCR SEQ ID No. 88 to 90 116 2 hb27G11F PCR- BsrI SEQ ID No. 86, 87 185 RFLP 116 69 3 Ctg22Ex1 PCR- RsaI SEQ ID No. 84, 85 381 RFLP 313 69 4 SNP1 AS-PCR SEQ ID No. 81 to 83 410 5 ctg2931- LO SEQ ID No. 78 to 80 51 3ac/ola 49 6 ctg2931- LO SEQ ID No. 75 to 77 44 5ag/ola 42 7 SNP3-2931 AS-PCR SEQ ID No. 72 to 74 245 8 Ctg25Ex1 PCR- BsteII SEQ ID No. 70, 71 207 RFLP 122 85 9 CTG35ExA AS-PCR SEQ ID No. 67 to 69 333 10 ctg35ExC AS-PCR SEQ ID No. 64 to 66 198 11 D16S3136 SEQ ID No. 37, 38 12 hb133D1f PCR- TaqI SEQ ID No. 62, 63 369 RFLP 295 74 13 D16S3035 SEQ ID No. 35, 36 14 ADCY7int7 AS-PCR SEQ ID No. 59 to 61 140 AS-PCR: allele-specific PCR; LO: ligation of oligonucleotides
[0176] The 12 biallelic polymorphism markers newly described in this study are listed in this table. For each one of them, the following are indicated: [0177] the locus (column I) [0178] the name (column II) [0179] the genotyping technique used (column III) [0180] the restriction enzyme possibly used (column IV) [0181] the oligonucleotide primers used for the polymerase chain reaction or for the ligation (column V) [0182] the size of the products expected during typing (column VI)
[0183] 199 families comprising 1 or more sick individuals suffering from CD were typed for these 12 polymorphism markers and also for the markers D16S3035 and D16S3136 located on the BAC hb87b10. The families comprising sick individuals suffering from UC were not taken into account. The methods for typing the polymorphisms studied were variable depending on the type of polymorphism, using: [0184] the PCR-RFLP technique (amplification followed by enzymatic digestion of the PCR product) when the polymorphism was located on an enzymatic restriction site. [0185] PCR with primers specific for the polymorphic site: differential amplification of two alleles using primers specific for each allele. [0186] Oligoligation test: differential ligation using oligonucleotides specific for each allele, followed by polyacrylamide gel electrophoresis.
[0187] The typing data were then analyzed using a transmission disequilibrium test (TDT computer program of the GENEHUNTER software version 2). For the families comprising several affected relatives, a single sufferer was taken into account for the analysis. In fact, if several related sufferers are taken into account, this poses the problem of nonindependence of the data in the statistical calculations and can induce an inflation of the value of the test. The sufferer used for the analysis was drawn by lots, within each family, using an automatic randomization procedure. Given this randomization, the value of the statistical test obtained represented only one possible sample derived from the group of families studied. So as not to limit the analysis to this one possible sample, and in order to understand more clearly the soundness of the results obtained, for each test, about one hundred random samples were thus generated and analyzed.
[0188] The markers were studied separately and then grouped according to their order on the chromosomal segment (KIAA0849ex9 (locus 1), hb27G11F (locus 2), Ctg22Ex1 (locus 3), SNP1 (locus 4), ctg2931-3ac/ola (locus 5), ctg2931-5ag/ola (locus 6), SNP3-2931 (locus 7), Ctg25Ex1 (locus 8), CTG35ExA (locus 9), ctg35ExC (locus 10), d16s3136 (locus 11), hb133D1f (locus 12), D16S3035 (locus 13), ADCY7int7 (locus 14)) (table 2). The haplotypes comprising 2, 3 and 4 consecutive markers were thus analyzed still using the same strategy (100 random samples, taking a single affected individual for each family).
[0189] For each sample tested, only the genotypes (or haplotypes) carried by at least 10 parental chromosomes were taken into account. On average, 250 different tests were thus carried out for each sample. It was then possible to deduce the number of tests expected to be positive for each significance threshold and to compare this distribution to the distribution observed. For the healthy individuals, the distribution of the tests is not different from that expected on a random basis (χ2=2.85, ddl=4, p=0.58). For the sick individuals, on the other hand, there is an excess of positive tests, reflecting the existence of a transmission disequilibrium in the region studied.
[0190] The results of the transmission disequilibrium test for each polymorphism marker taken separately or for the haplotypes showing the strongest transmission disequilibriums showed that the following markers and the disease are in linkage disequilibrium: Ctg22Ex1 (locus 3), SNP1 (locus 4), ctg2931-5ag/ola (locus 6), SNP3-2931 (locus 7), Ctg25Ex1 (locus 8) and ctg35ExC (locus 10). These markers extend over a region of approximately 50 kb (positions 74736 to 124285 on the sequence of hb87b10).
[0191] The haplotypes the most strongly associated with Crohn's disease themselves also extend over this region. Thus, for the majority of the random samples, the transmission test was positive (p<0.01) for haplotypes combining the following markers: [0192] locus 5-6, locus 6-7, locus 7-8, locus 8-9, locus 9-10, locus 10-11 [0193] locus 5-6-7, locus 6-7-8, locus 7-8-9, locus 8-9-10, locus 9-10-11 [0194] locus 5-6-7-8, locus 6-7-8-9, locus 7-8-9-10.
[0195] The susceptibility haplotype most at risk is defined by the loci 7 to 10. This is the haplotype 1-2-1-2 (table 2).
[0196] The markers tested are, as expected, in linkage disequilibrium with respect to one another.
[0197] More recently, a new test, the Pedigree Disequilibrium Test (PDT), published in July 2000 (Martin et al., 2000), was used to understand more clearly the meaning of the results obtained with the TDT computer program. This new statistic in fact makes it possible to use all of the information available in a family, both from the sick individuals and from the healthy individuals, and to counterbalance the importance of each relative in an overall statistic for each family. The values of p corresponding to the PDT tests and obtained for an enlarged group of 235 families with one or more relatives suffering from Crohn's disease are given in table 3. This new analysis confirms that the region of the BAC hb87b10 is indeed associated with Crohn's disease.
TABLE-US-00003 TABLE 3 Results of the PDT tests carried out on 235 families suffering from Crohn's disease LOCUS VALUE p OF THE PDT TEST KIAA0849ex9 NS hb27g11f 0.05 ctg22ex1 0.01 SNP1 0.001 ctg2931-3ac/ola NS ctg2931-5ag/ola 0.0001 SNP3-2931 0.0001 ctg25ex1 0.0006 ctg35exA NS ctg35exC 0.00002 D16S3136 NS hb133d1f NS D16S3035 NS (NS: not significant)
Example 5
Identification of the IBD1 Gene
[0198] The published EST groups (Unigene references: Hs135201, Hs87280, Hs122983, Hs146128, H5105242, Hs116424, Hs61309, Hs151708, Hs87296 and Hs132289) present on the BAC hb87b10 were studied in the search for a more complete complementary DNA (cDNA) sequence. For IBD1prox, the clones available in public libraries were sequenced and the sequences were organized with respect to one another. For IBD1, a peripheral blood complementary DNA library (Stratagene human blood cDNA lambda zapexpress ref 938202) was screened with the PCR products generated from known ESTs according to the methods proposed by the manufacturer. The sequence of the cDNAs thus identified was then used for further screening of the cDNA library, and so on, until the presented cDNA was obtained.
[0199] The EST hs135201 (UniGene) made it possible to identify a cDNA not appearing on the available genetic databases (Genbank). It therefore corresponds to a new human gene. Comparison of the sequence of the cDNA and of the genomic DNA showed that this gene consists of 11 exons and 10 introns. An additional exon, positioned 5' to the cDNA identified, is predicted by analysis of the sequence with the Grail program. These exons are very homologous to the first exons of the CARD4/NOD1 gene. Taking into consideration all of the exons identified and the putative additional exon, this new gene appears to have a genomic structure very close to that of CARD4/NOD1. Moreover, a transcription initiation site appears upstream of the first putative exon. For all of these reasons, the putative exon was considered to contribute to this new gene. The cDNA reproduced in the annex (SEQ ID No. 1) therefore comprises all of the identified sequence plus the sequence predicted by the computer modeling, the complementary DNA beginning randomly at the first ATG codon of the predicted coding sequence. On this basis, the gene would therefore comprise 12 exons and 11 introns. The intron-exon structure of the gene is reported on SEQ ID No. 3.
[0200] The protein sequence deduced from the nucleotide sequence comprises 1041 amino acids (SEQ ID No. 2). This sequence has not been found on the biological databases either (Genpept, pir, swissprot).
[0201] Now, more recently, it has not been possible to confirm the putative exon described above. The IBD1 gene therefore effectively comprises only 11 exons and 10 introns and encodes a protein of 1013 amino acids (i.e. 28 amino acids less than initially determined).
[0202] The study of the deduced protein sequence shows that this gene contains three different functional domains (FIG. 3): [0203] A CARD domain (Caspase Recruitment Domain) known to be involved in the interaction between proteins regulating apoptosis and activation of the NFkappa B pathway. The CARD domain makes it possible to classify this new protein in the CARD protein family, the most longstanding members of which are CED4, APAF1 and RICK. [0204] An NBD domain (Nucleotide-Binding Domain) comprising an ATP-recognition site and a magnesium-binding site. The protein should therefore very probably have kinase activity. [0205] An LRR domain (Leucine-Rich Domain) presumed to participate in the interaction between proteins, by analogy with other described protein domains.
[0206] Moreover, the LRR domain of the protein makes it possible to affiliate the protein to a family of proteins involved in intracellular signaling and present both in plants and in animals.
[0207] Comparison of this new gene with previously identified genes available in the public databases shows that this gene is very homologous to CARD4/NOD1 (Bertin et al., 1999; Inohara et al., 1999). This homology relates to the sequence of the complementary DNA, the intron-exon structure of the gene and the protein sequence. The sequence identity of the two complementary DNAs is 58%. A similarity is also observed at the level of the intron-exon structure. The sequence homology at the protein level is of the order of 40%.
[0208] The similarity between this new gene and CARD4/NOD1 suggests that, like CARD4/NOD1, the IBD1 protein is involved in the regulation of apoptosis and of the activation of NF-kappa B (Bertin et al., 1999; Inohara et al., 1999). The regulation of cellular apoptosis and activation of NF-kappa B are intracellular signaling pathways which are essential in immune reactions. Specifically, these signal translation pathways are the effector pathways of the proteins of the TNF (Tumor Necrosis Factor) receptor family involved in cell-cell interactions and the cellular response to the various mediators of inflammation (cytokines). The new gene therefore appears to be potentially important in the inflammatory reaction in general.
[0209] Several bodies of proof support bacteria-induced deregulation of NF-kB in Crohn's disease. First of all, spontaneous susceptibility to IBD in mice has been associated with mutations in Tlr4, a molecule known to bind to LPS via its LRR domain (Poltorak et al., 1998 and Sundberg et al., 1994) and to be a member of the activators of the NF-kB family. Second, treatment with antibiotics causes a provisional improvement in patients suffering from CD, giving credit to the hypothesis that enteric bacteria may play an etiological role in Crohn's disease (McKay, 1999). Third, NF-kB plays a pivotal role in inflammatory bowel diseases and is activated in lamina propria mononuclear cells in Crohn's disease (Schreiber et al., 1998). Fourth, the treatment of Crohn's disease is based on the use of sulfasalazine and glucocorticoids, which are both known to be NF-kB inhibitors (Auphan et al., 1995 and Wahl et al., 1998).
[0210] Even more recently, it has been shown that the IBD1 candidate gene encodes a protein very similar to NOD2, a member of the CED4/APAF1 superfamily (Ogura et al., 2000). The nucleotide and protein sequences of IBD1 and NOD2 in reality only diverge for a small portion right at the start of the two reported sequences. The tissue expressions of Nod2 and IBD1 can, in addition, be superimposed. These two genes (proteins) can therefore be considered to be identical. It has been demonstrated that the LRR domain of Nod2 has binding activity for bacterial lipopolysaccharides (LPS) (Inohara et al., 2000) and that deletion thereof stimulates the NFkB pathway. This result confirms the data of the invention.
[0211] The tissue expression of IBD1 was then studied by Northern blotting. A 4.5 kb transcript is visible in most human tissues. The size of the transcript is in accordance with the size predicted by the cDNA. The 4.5 kb transcript appears to be very poorly abundant in the small intestine and the colon. It is, on the other hand, very strongly expressed in white blood cells. This is in agreement with clinical data on transplants which suggest that Crohn's disease is potentially a disease associated with circulating immune cells. In fact, bowel transplantation does not prevent recurrence on the transplant in Crohn's disease, whereas bone marrow transplantation appears to have a beneficial effect on the progression of the disease.
[0212] Certain data also call to mind alternative splicing, which may prove to be an important element in the possibility of generating mutants which may play a role in the development of inflammatory diseases.
[0213] The promoter of the IBD1 gene has not currently been identified with precision. It is, however, reasonable to think, by analogy with a very large number of genes, that this promoter lies, at least partly, immediately upstream of the gene, in the 5' portion thereof. This genetic region contains transcribed sequences, as witnessed by the presence of ESTs (HUMGS01037, AA835524, hs.105242, SHGC17274, hs.146128, hs.122983, hs.87280). The ATCC clones containing these sequences were sequenced and analyzed in the laboratory, making it possible to demonstrate an exon and intron organization with possible alternative splicings. These data suggest the existence of another gene (named IBD1prox due to its proximity to IBD1). The partial sequence of the complementary DNA of IBD1prox is reported (SEQ ID No. 4), as is its intron-exon structure, on SEQ ID No. 6.
[0214] Translation of the cDNAs corresponding to IBD1prox results in a protein containing a homeobox. Analysis of several cDNAs of the gene suggests, however, the existence of alternative splicings. IBD1prox, according to one of the possible alternative splicings, corresponds to the anonymous EST HUMGS01037, the RNA of which is expressed more strongly in differentiated leukocytic lines than in undifferentiated lines.
[0215] Thus, it is possible that this gene may have a role in inflammation and cell differentiation. It may therefore also, itself, be considered to be a good candidate for susceptibility to IBD. The association between CD and the polymorphism ctg35ExC located on the coding sequence of IBD1prox supports this hypothesis even though this polymorphism does not cause any sequence variation at the protein level.
[0216] Finally, more recently, the existence of a genetic linkage in families suffering from Crohn's disease and not comprising any mutation in the IBD1 gene also, itself, suggests that IBD1prox has a role in addition to IBD1 in genetic predisposition to the disease.
[0217] The functional relationship between IBD1 and IBD1prox is not currently established. However, the considerable proximity between the two genes may reflect an interaction between them. In this case, the "head-to-tail" location of these genes suggests that they may have common or interdependent methods of regulation.
Example 6
Identification of IBD1 Gene Mutations in Inflammatory Diseases
[0218] In order to confirm the role of IBD1 in inflammatory diseases, the coding sequence and the intron-exon junctions of the gene were sequenced from exon 2 to exon 12 inclusive, in 70 independent individuals, namely: 50 sick individuals suffering from CD, 10 sick individuals suffering from UC, 1 sick individual suffering from Blau's syndrome and 9 healthy controls. The sick individuals studied were mostly familial forms of the disease and were often carriers of the susceptibility haplotype defined by the transmission disequilibrium studies. The healthy controls were of Caucasian origin.
[0219] It was thus possible to identify 24 sequence variants on this group of 70 unrelated individuals (table 3).
[0220] The nomenclature of the mutations reported refers to the initial sequence of the protein comprising 1 041 amino acids. The more recently proposed nomenclature is easily deduced by removing 28 amino acids from the initial sequence, and therefore corresponds to a protein comprising 1 013 amino acids (cf. example 5).
TABLE-US-00004 TABLE 4 Mutations observed in the IBD1 gene Nucleotide Protein Crohn's Ulcerative Health Exon variant variant disease colitis controls 1 Not tested 2 G417A Silent 2 C537G Silent 3 None 4 T805C S269P 48/100 6/20 3/18 4 A869G N290S 0 0 1/18 4 C905T A302V 1/100 0 0 4 C1283T P428L 1/100 0 0 4 C1284A Silent 4 C1287T Silent 4 T1380C Silent 4 T1764G Silent 4 G1837A A613T 1/100 0 0 4 C2107T R703W 10/10 1/20 1/18 4 C2110T R704C 4/10 1/20 0 5 G2365A R792Q 1/100 0 0 5 G2370A V794M 0 1/20 0 5 G2530A E844K 1/10 0 0 6 A2558G N853S 1/100 0 0 6 A2590G M864V 1/100 0 0 7 None 8 G2725C G909R 7/100 0 0 8 C2756A A919D 1/100 0 0 9 G2866A V9561 2/100 1/20 3/18 10 C2928T Silent 11 3022insC Stop 20/100 0 0 12 none
[0221] The mutations other than silent mutations observed in each exon are reported. They are indicated by the variation in the peptide chain. For each mutation and for each phenotype studied, the number of times where the mutation is observed, related to the number of chromosomes tested, is indicated.
[0222] No functional sequence variant was identified in exons 1 to 3 (corresponding to the CARD domain of the protein). Exons 7 and 12 did not show any sequence variation either. Certain variants corresponded to polymorphisms already identified and typed for transmission disequilibrium studies, namely: [0223] Snp3-2931: nucleotide variant T805C, protein variant S269P [0224] ctg2931-5ag/ola: nucleotide variant T1380C (silent) [0225] ctg2931-3ac/ola: nucleotide variant T1746G (silent) [0226] SNP1: nucleotide variant C2107T, protein variant R703W.
[0227] Several sequence variations were silent (G417A, C537G, C1284A, C1287T, T1380C, T1764G and C2928T) and did not lead to any modification of the protein sequence. They were not studied further here.
[0228] For the 16 non-silent sequence variations, protein sequence variants were observed in 43/50 CD versus 5/9 healthy controls, and 6/10 UC. The existence of one or more sequence variation(s) appeared to be associated with the CD phenotype. Several sequence variations often existed in the same individual suffering from CD, suggesting a sometimes recessive effect of the gene for CD. On the other hand, no composite heterozygote or homozygote was observed among the patients suffering from UC or among the healthy controls.
[0229] Some non-silent variants were present both in the sick individuals suffering from UC or from CD and in the healthy individuals. They were the variants S269P, N290S, R703W and V956I located in exons 2, 4 and 9. Further information therefore appears to be necessary before selecting a possible functional role for these sequence variants.
[0230] V956I is a conservative sequence variation (aliphatic amino acids).
[0231] The sequence variant S269P corresponds to a variation in amino acid class (hydroxylated to immuno acid) at the beginning of the nucleotide-binding domain. This sequence variant and CD are in transmission disequilibrium. It is in fact the polymorphism Snp3 (cf. above).
[0232] R703W results in a modification of the amino acid class (aromatic instead of basic). This modification occurs in the intermediate region between the NBD and LRR domains, which is a region conserved between IBD1 and CARD4/NOD1. A functional role may therefore be suspected for this polymorphism. This sequence variation (corresponding to the polymorphic site Snpl) is transmitted to sick individuals suffering from CD more often than at random (cf. above), confirming that this polymorphism is associated with CD. It is possible that the presence of this mutant in healthy individuals reflects incomplete penetration of the mutation as is expected for complex genetic diseases such as chronic inflammatory bowel diseases.
[0233] The variant R704C, located immediately next to R703W, could be identified in both CD and UC. It also, itself, corresponds to a nonconservative variation of the protein (sulfur-containing amino acid instead of basic amino acid) on the same protein region, suggesting a functional effect for R704C which is as important as that for R703W.
[0234] Other sequence variations are specific for CD, for UC or for Blau's syndrome.
[0235] Some sequence variations are, on the contrary, rare, present in one or a few sick individuals (A613T, R704C, E844K, N853S, M864V, A919D). They are always variations leading to nonconservative modifications of the protein in leucine-rich domains, at positions which are important within these domains. These various elements suggest that these variations have a functional role.
[0236] Two sequence variations (G909R and L1008P*) are found in quite a large number of Crohn's diseases (respectively 7/50 and 16/50) whereas they are not detected in the controls or in the individuals suffering from UC.
[0237] The deletion/insertion of a guanosine at codon 1008 results in transformation of the third leucine of the alpha helix of the last LRR to proline followed by a STOP codon (L1008P*). This sequence variation therefore leads to an important modification of the protein: decrease in size of the protein (protein having a truncated LRR domain) and modification of a very conserved amino acid (leucine). This sequence modification is associated with CD, as witnessed by a transmission disequilibrium study in 16 families carrying the mutation (P=0.008).
[0238] The mutation G909R occurs on the last amino acid of the sixth LRR motif. It replaces an aliphatic amino acid with a basic amino acid. This variation is potentially important given the usually neutral or polar nature of the amino acids in the terminal position of the leucine-rich motifs (both for IBD1 and for NOD1/CARD4) and the conserved nature of this amino acid on the IBD1 and NOD1/CARD4 proteins.
[0239] In Blau's syndrome, the sick individuals (n=2) of the family studied carried a specific sequence variation (L470F) located in exon 4 and corresponding to the NBD domain of the protein. In this series, this sequence variant was specific for Blau's syndrome.
[0240] In UC, several sequence variants not found in healthy individuals were also identified. The proportion of sick individuals carrying a mutation was smaller than for CD, as expected given the less strongly established linkage between IBD1 and UC, and the supposedly less genetic nature of the latter disease. Sequence variations were common to CD and to UC (R703W, R704C). Others, on the other hand, appeared to be specific for UC (V794M). This observation makes it possible to confirm that CD and UC are diseases which, at least partly, share the same genetic predisposition. It lays down the foundations of a nosological classification for IBDs.
[0241] The study of the sequence variants of the IBD1 gene has therefore made it possible to identify several variants having a very probable functional effect (for example: truncated protein) and associated with Crohn's disease, with UC and with Blau's syndrome.
[0242] The promoter of the gene is not currently determined. In all probability, however, it is likely to be located in the 5' region upstream of the gene. According to this hypothesis, the sequence variants observed in this region may have a functional effect. This may explain the very strong association between CD and certain polymorphic loci, such as ctg35ExC or Ctg25Ex1.
[0243] The invention thus provides the first description of mutations in the family of genes containing a CARD domain in humans. The frequency of these mutations in various inflammatory diseases shows that the IBD1 gene has an essential role in normal and pathological inflammatory processes. This invention provides new paths of understanding and of research in the field of the physiopathology of normal and pathological inflammatory processes. As a result, it makes it possible to envision the development of new pharmaceutical molecules which regulate the effector pathways controlled by IBD1 and which are useful in the treatment of inflammatory diseases and in the regulation of inflammatory processes in general.
Example 7
Bases for a Biological Diagnosis of Susceptibility to Crohn's Disease
[0244] More recently, 457 independent patients suffering from Crohn's disease, 159 independent patients suffering from ulcerative colitis and 103 healthy controls were studied in the search for mutations. This study made it possible to confirm the mutations previously reported and to identify additional mutations, reported in FIG. 4. The main mutations were then genotyped in 235 families suffering from Crohn's disease. This more recent study is reported using, as reference, the shorter protein sequence (1 013 amino acids, see example 5), but the prior nomenclature for the mutations is easily deduced from the latter by adding 28 to the number indicating the position of the amino acids.
[0245] Among the 5 most common mutations, the conservative mutation V928I (formerly V956I) is not significantly associated with one or the other of the inflammatory bowel diseases, and does not therefore appear to have an important role in the disease.
[0246] The mutation S241P (formerly S269P) is in linkage disequilibrium with the other main mutations and does not appear to play an important role, by itself, in susceptibility to inflammatory bowel diseases (data not shown).
[0247] Conversely, the other 3 mutations, R675W (formerly R703W), G881R (formerly G909R) and 980fs (formerly L1008P*), are significantly associated with Crohn's disease but not with ulcerative colitis (cf. below). The location in the LRR, or in its immediate proximity, of the 3 common mutations pleads very strongly in favor of a functional mechanism involving this protein domain, probably via a defect in negative regulation of NFkB by the mutated protein. The other mutations are more rare (FIG. 4). These cumulative mutations are present in 17% of the individuals suffering from Crohn's disease versus, respectively, 4% and 5% of the healthy individuals or individuals suffering from ulcerative colitis. A large number of rare mutations are also located in the LRR.
[0248] The intrafamily studies of the three polymorphisms most common in Crohn's disease show that all three are associated with the disease (table 5). As expected, for a mutation supposed to be very deleterious, the polymorphism most strongly associated is the truncating mutation. These three polymorphisms are independently associated with Crohn's disease, since it was not possible to identify, on 235 families, chromosomes carrying more than one of these three mutations. The independent nature of these associations considerably supports the hypothesis that the IBD1 gene is clearly involved in genetic predisposition to Crohn's disease.
TABLE-US-00005 TABLE 5 Study of the 3 common polymorphisms of IBD1 in 235 families suffering from Crohn's disease MUTATION VALUE p OF THE PDT TEST R675W 0.001 G881R 0.003 980fs 0.000006
[0249] The case-control studies confirm this association (table 6). They show that the mutations most common in Crohn's disease are not common in ulcerative colitis.
TABLE-US-00006 TABLE 6 Case-control study of the 3 common polymorphisms of IBD1 in inflammatory bowel diseases No. OF FREQUENCY OF FREQUENCY OF FREQUENCY OF TOTAL CHROMOSOMES THE ALLELE AT THE ALLELE AT THE ALLELE AT ALLELES MUTATION STUDIED RISK R675W RISK G881R RISK 980fs AT RISK Healthy 206 0.04 0.01 0.02 0.07 controls Ulcerative 318 0.03 0.00 0.01 0.05 colitis Crohn's 936 0.11 0.06 0.12 0.29 disease
[0250] The study of the dose-effect of these mutations shows that individuals carrying a mutation in the homozygous or composite heterozygous state exhibit a much greater risk of developing the disease than individuals who are not carrying or are heterozygous for these mutations (table 7).
TABLE-US-00007 TABLE 7 Relative and absolute risk of Crohn's disease attributable as a function of the genotype of IBD1 In the general population, a risk of Crohn's disease of 0.001 has been taken as a reference, and it has been presumed that the mutations are in Hardy-Weinberg equilibrium. Distribution GENOTYPE SIMPLE COMPOSITE No HETERO- HOMO- HETERO- VARIANT ZYGOTE ZYGOTE ZYGOTE Healthy 88 15 0 0 Ulcerative 145 13 1 0 colitis Crohn's 267 133 28 40 disease Attributable risk of CD: Relative risk 1 3 38 44 Absolute risk 0.0007 0.002 0.03 0.03
[0251] The studies mentioned above confirm the prior preliminary data and provide the detailed bases for a biological diagnosis of Crohn's disease by studying the IBD1 variants. In fact, this work: [0252] 1) defines the mutations, the frequency of which is greater than 0.001 in a mixed Caucasian population; [0253] 2) defines the frequency of the mutations observed and makes it possible to define 3 main mutations associated with Crohn's disease. Thus, it is possible, by virtue of this work, to define a strategy for studying the gene in order to search for morbid variants, namely: firstly, typing the 3 main mutations; secondly, searching for mutations in the last 7 exons; thirdly, searching for other sequence variants; [0254] 3) defines the practical modalities for searching for these mutations by pointing out their position and their nature. In fact, it is then easy for those skilled in the art to develop typing and sequencing methods according to their personal expertise. Mention may in particular be made of the possibility of genotyping the three main mutations by PCR followed by enzymatic digestion and electrophoresis, study of the migration profiles by dHPLC, DGGE or SSCP, oligoligation, microsequencing, etc.; [0255] 4) demonstrates the independence of the most common mutations which are not observed on the same chromosome in this extended and varied population. This information makes it possible to reliably classify the individuals who are composite heterozygotes (having two mutations) as carriers with a double dose of intragenic variations; [0256] 5) demonstrates that the great majority of the mutations only lead to a null or minimal effect on the risk of ulcerative colitis. This result makes it possible to envision assisting the clinician in the differential diagnosis between these two diseases. In fact, in approximately 10% of cases, inflammatory bowel diseases remain unclassified despite biological, radiological and endoscopic examination; [0257] 6) defines a relative and absolute risk of disease for the most common genotypes. This result lays down the foundations of a predictive diagnosis potentially useful in an approach of preventive monitoring and intervention in populations at risk, in particular the relatives of sick individuals; [0258] 7) demonstrates the existence of a dose-effect for the IBD1 gene and confirms the partly recessive nature of genetic predisposition to Crohn's disease. It therefore makes it possible to lay the foundations for genetic counseling and for intra-familial preclinical diagnosis.
[0259] Finally, it should be noted that an additional mutation of the NBD domain was isolated in a second family carrying Blau's syndrome. The rareness of the two events in 2 different families is sufficient to confirm the involvement of this gene in Blau's syndrome and in granulomatous diseases in general.
[0260] All of these data provide a diagnostic tool which is directly applicable and of use to the practitioner in his or her daily practice.
[0261] The IBD1prox gene, located in the promoter region of IBD1, and the partial sequence of which is disclosed in the present invention, may also, itself, have an important role in the regulation of cellular apoptosis and of the inflammatory process, as suggested by its differential expression in mature cells of the immune system. The strong association reported in this work between the polymorphism marker ctg35ExC (located in the transcribed region of the gene) and Crohn's disease also pleads very strongly in favor of this hypothesis.
[0262] Inflammatory bowel diseases are complex genetic diseases for which, until now, no susceptibility gene had been identified with certainty. The invention has made it possible to identify the first gene for susceptibility to Crohn's disease, using a positional cloning (or reverse genetics) approach. This is the first genetic location obtained using such an approach for a complex genetic disease, which demonstrates its usefulness and its feasibility, at least in certain cases in complex genetic diseases.
[0263] The present invention also relates to a purified or isolated nucleic acid, characterized in that it encodes a polypeptide possessing a continuous fragment of at least 200 amino acids of a protein chosen from SEQ ID No. 2 and SEQ ID No. 5.
REFERENCES
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Sequence CWU
1
9014374DNAHomo sapiensIBD1 cDNA 1atg gag aag aga agg ggt cta acc att gag
tgc tgg ggc ccc caa agt 48Met Glu Lys Arg Arg Gly Leu Thr Ile Glu
Cys Trp Gly Pro Gln Ser 1 5 10
15 ccc tca ctg acc ttg ttc tcc tcc cca ggt tgt gaa atg tgc tcg
cag 96Pro Ser Leu Thr Leu Phe Ser Ser Pro Gly Cys Glu Met Cys Ser
Gln 20 25 30 gag gct
ttt cag gca cag agg agc cag ctg gtc gag ctg ctg gtc tca 144Glu Ala
Phe Gln Ala Gln Arg Ser Gln Leu Val Glu Leu Leu Val Ser 35
40 45 ggg tcc ctg gaa ggc ttc gag
agt gtc ctg gac tgg ctg ctg tcc tgg 192Gly Ser Leu Glu Gly Phe Glu
Ser Val Leu Asp Trp Leu Leu Ser Trp 50 55
60 gag gtc ctc tcc tgg gag gac tac gag ggc ttc cac
ctc ctg ggc cag 240Glu Val Leu Ser Trp Glu Asp Tyr Glu Gly Phe His
Leu Leu Gly Gln 65 70 75
80cct ctc tcc cac ttg gcc agg cgc ctt ctg gac acc gtc tgg aat aag
288Pro Leu Ser His Leu Ala Arg Arg Leu Leu Asp Thr Val Trp Asn Lys
85 90 95 ggt act tgg gcc
tgt cag aag ctc atc gcg gct gcc caa gaa gcc cag 336Gly Thr Trp Ala
Cys Gln Lys Leu Ile Ala Ala Ala Gln Glu Ala Gln 100
105 110 gcc gac agc cag tcc ccc aag ctg cat
ggc tgc tgg gac ccc cac tcg 384Ala Asp Ser Gln Ser Pro Lys Leu His
Gly Cys Trp Asp Pro His Ser 115 120
125 ctc cac cca gcc cga gac ctg cag agt cac cgg cca gcc att
gtc agg 432Leu His Pro Ala Arg Asp Leu Gln Ser His Arg Pro Ala Ile
Val Arg 130 135 140 agg
ctc cac agc cat gtg gag aac atg ctg gac ctg gca tgg gag cgg 480Arg
Leu His Ser His Val Glu Asn Met Leu Asp Leu Ala Trp Glu Arg145
150 155 160ggt ttc gtc agc cag tat
gaa tgt gat gaa atc agg ttg ccg atc ttc 528Gly Phe Val Ser Gln Tyr
Glu Cys Asp Glu Ile Arg Leu Pro Ile Phe 165
170 175 aca ccg tcc cag agg gca aga agg ctg ctt gat
ctt gcc acg gtg aaa 576Thr Pro Ser Gln Arg Ala Arg Arg Leu Leu Asp
Leu Ala Thr Val Lys 180 185
190 gcg aat gga ttg gct gcc ttc ctt cta caa cat gtt cag gaa tta
cca 624Ala Asn Gly Leu Ala Ala Phe Leu Leu Gln His Val Gln Glu Leu
Pro 195 200 205 gtc cca
ttg gcc ctg cct ttg gaa gct gcc aca tgc aag aag tat atg 672Val Pro
Leu Ala Leu Pro Leu Glu Ala Ala Thr Cys Lys Lys Tyr Met 210
215 220 gcc aag ctg agg acc acg gtg
tct gct cag tct cgc ttc ctc agt acc 720Ala Lys Leu Arg Thr Thr Val
Ser Ala Gln Ser Arg Phe Leu Ser Thr225 230
235 240tat gat gga gca gag acg ctc tgc ctg gag gac ata
tac aca gag aat 768Tyr Asp Gly Ala Glu Thr Leu Cys Leu Glu Asp Ile
Tyr Thr Glu Asn 245 250
255 gtc ctg gag gtc tgg gca gat gtg ggc atg gct gga tcc ccg cag aag
816Val Leu Glu Val Trp Ala Asp Val Gly Met Ala Gly Ser Pro Gln Lys
260 265 270 agc cca gcc acc
ctg ggc ctg gag gag ctc ttc agc acc cct ggc cac 864Ser Pro Ala Thr
Leu Gly Leu Glu Glu Leu Phe Ser Thr Pro Gly His 275
280 285 ctc aat gac gat gcg gac act gtg ctg
gtg gtg ggt gag gcg ggc agt 912Leu Asn Asp Asp Ala Asp Thr Val Leu
Val Val Gly Glu Ala Gly Ser 290 295
300 ggc aag agc acg ctc ctg cag cgg ctg cac ttg ctg tgg
gct gca ggg 960Gly Lys Ser Thr Leu Leu Gln Arg Leu His Leu Leu Trp
Ala Ala Gly305 310 315
320caa gac ttc cag gaa ttt ctc ttt gtc ttc cca ttc agc tgc cgg cag
1008Gln Asp Phe Gln Glu Phe Leu Phe Val Phe Pro Phe Ser Cys Arg Gln
325 330 335 ctg cag tgc atg
gcc aaa cca ctc tct gtg cgg act cta ctc ttt gag 1056Leu Gln Cys Met
Ala Lys Pro Leu Ser Val Arg Thr Leu Leu Phe Glu 340
345 350 cac tgc tgt tgg cct gat gtt ggt caa
gaa gac atc ttc cag tta ctc 1104His Cys Cys Trp Pro Asp Val Gly Gln
Glu Asp Ile Phe Gln Leu Leu 355 360
365 ctt gac cac cct gac cgt gtc ctg tta acc ttt gat ggc ttt
gac gag 1152Leu Asp His Pro Asp Arg Val Leu Leu Thr Phe Asp Gly Phe
Asp Glu 370 375 380 ttc
aag ttc agg ttc acg gat cgt gaa cgc cac tgc tcc ccg acc gac 1200Phe
Lys Phe Arg Phe Thr Asp Arg Glu Arg His Cys Ser Pro Thr Asp385
390 395 400ccc acc tct gtc cag acc
ctg ctc ttc aac ctt ctg cag ggc aac ctg 1248Pro Thr Ser Val Gln Thr
Leu Leu Phe Asn Leu Leu Gln Gly Asn Leu 405
410 415 ctg aag aat gcc cgc aag gtg gtg acc agc cgt
ccg gcc gct gtg tcg 1296Leu Lys Asn Ala Arg Lys Val Val Thr Ser Arg
Pro Ala Ala Val Ser 420 425
430 gcg ttc ctc agg aag tac atc cgc acc gag ttc aac ctc aag ggc
ttc 1344Ala Phe Leu Arg Lys Tyr Ile Arg Thr Glu Phe Asn Leu Lys Gly
Phe 435 440 445 tct gaa
cag ggc atc gag ctg tac ctg agg aag cgt cat cat gag ccc 1392Ser Glu
Gln Gly Ile Glu Leu Tyr Leu Arg Lys Arg His His Glu Pro 450
455 460 ggg gtg gcg gac cgc ctc atc
cgc ctg ctc caa gag acc tca gcc ctg 1440Gly Val Ala Asp Arg Leu Ile
Arg Leu Leu Gln Glu Thr Ser Ala Leu465 470
475 480cac ggt ttg tgc cac ctg cct gtc ttc tca tgg atg
gtg tcc aaa tgc 1488His Gly Leu Cys His Leu Pro Val Phe Ser Trp Met
Val Ser Lys Cys 485 490
495 cac cag gaa ctg ttg ctg cag gag ggg ggg tcc cca aag acc act aca
1536His Gln Glu Leu Leu Leu Gln Glu Gly Gly Ser Pro Lys Thr Thr Thr
500 505 510 gat atg tac ctg
ctg att ctg cag cat ttt ctg ctg cat gcc acc ccc 1584Asp Met Tyr Leu
Leu Ile Leu Gln His Phe Leu Leu His Ala Thr Pro 515
520 525 cca gac tca gct tcc caa ggt ctg gga
ccc agt ctt ctt cgg ggc cgc 1632Pro Asp Ser Ala Ser Gln Gly Leu Gly
Pro Ser Leu Leu Arg Gly Arg 530 535
540 ctc ccc acc ctc ctg cac ctg ggc aga ctg gct ctg tgg
ggc ctg ggc 1680Leu Pro Thr Leu Leu His Leu Gly Arg Leu Ala Leu Trp
Gly Leu Gly545 550 555
560atg tgc tgc tac gtg ttc tca gcc cag cag ctc cag gca gca cag gtc
1728Met Cys Cys Tyr Val Phe Ser Ala Gln Gln Leu Gln Ala Ala Gln Val
565 570 575 agc cct gat gac
att tct ctt ggc ttc ctg gtg cgt gcc aaa ggt gtc 1776Ser Pro Asp Asp
Ile Ser Leu Gly Phe Leu Val Arg Ala Lys Gly Val 580
585 590 gtg cca ggg agt acg gcg ccc ctg gaa
ttc ctt cac atc act ttc cag 1824Val Pro Gly Ser Thr Ala Pro Leu Glu
Phe Leu His Ile Thr Phe Gln 595 600
605 tgc ttc ttt gcc gcg ttc tac ctg gca ctc agt gct gat gtg
cca cca 1872Cys Phe Phe Ala Ala Phe Tyr Leu Ala Leu Ser Ala Asp Val
Pro Pro 610 615 620 gct
ttg ctc aga cac ctc ttc aat tgt ggc agg cca ggc aac tca cca 1920Ala
Leu Leu Arg His Leu Phe Asn Cys Gly Arg Pro Gly Asn Ser Pro625
630 635 640atg gcc agg ctc ctg ccc
acg atg tgc atc cag gcc tcg gag gga aag 1968Met Ala Arg Leu Leu Pro
Thr Met Cys Ile Gln Ala Ser Glu Gly Lys 645
650 655 gac agc agc gtg gca gct ttg ctg cag aag gcc
gag ccg cac aac ctt 2016Asp Ser Ser Val Ala Ala Leu Leu Gln Lys Ala
Glu Pro His Asn Leu 660 665
670 cag atc aca gca gcc ttc ctg gca ggg ctg ttg tcc cgg gag cac
tgg 2064Gln Ile Thr Ala Ala Phe Leu Ala Gly Leu Leu Ser Arg Glu His
Trp 675 680 685 ggc ctg
ctg gct gag tgc cag aca tct gag aag gcc ctg ctc cgg cgc 2112Gly Leu
Leu Ala Glu Cys Gln Thr Ser Glu Lys Ala Leu Leu Arg Arg 690
695 700 cag gcc tgt gcc cgc tgg tgt
ctg gcc cgc agc ctc cgc aag cac ttc 2160Gln Ala Cys Ala Arg Trp Cys
Leu Ala Arg Ser Leu Arg Lys His Phe705 710
715 720cac tcc atc ccg cca gct gca ccg ggt gag gcc aag
agc gtg cat gcc 2208His Ser Ile Pro Pro Ala Ala Pro Gly Glu Ala Lys
Ser Val His Ala 725 730
735 atg ccc ggg ttc atc tgg ctc atc cgg agc ctg tac gag atg cag gag
2256Met Pro Gly Phe Ile Trp Leu Ile Arg Ser Leu Tyr Glu Met Gln Glu
740 745 750 gag cgg ctg gct
cgg aag gct gca cgt ggc ctg aat gtt ggg cac ctc 2304Glu Arg Leu Ala
Arg Lys Ala Ala Arg Gly Leu Asn Val Gly His Leu 755
760 765 aag ttg aca ttt tgc agt gtg ggc ccc
act gag tgt gct gcc ctg gcc 2352Lys Leu Thr Phe Cys Ser Val Gly Pro
Thr Glu Cys Ala Ala Leu Ala 770 775
780 ttt gtg ctg cag cac ctt cgg cgg ccc gtg gcc ctg cag
ctg gac tac 2400Phe Val Leu Gln His Leu Arg Arg Pro Val Ala Leu Gln
Leu Asp Tyr785 790 795
800aac tct gtg ggt gac att ggc gtg gag cag ctg ctg cct tgc ctt ggt
2448Asn Ser Val Gly Asp Ile Gly Val Glu Gln Leu Leu Pro Cys Leu Gly
805 810 815 gtc tgc aag gct
ctg tat ttg cgc gat aac aat atc tca gac cga ggc 2496Val Cys Lys Ala
Leu Tyr Leu Arg Asp Asn Asn Ile Ser Asp Arg Gly 820
825 830 atc tgc aag ctc att gaa tgt gct ctt
cac tgc gag caa ttg cag aag 2544Ile Cys Lys Leu Ile Glu Cys Ala Leu
His Cys Glu Gln Leu Gln Lys 835 840
845 tta gct cta ttc aac aac aaa ttg act gac ggc tgt gca cac
tcc atg 2592Leu Ala Leu Phe Asn Asn Lys Leu Thr Asp Gly Cys Ala His
Ser Met 850 855 860 gct
aag ctc ctt gca tgc agg cag aac ttc ttg gca ttg agg ctg ggg 2640Ala
Lys Leu Leu Ala Cys Arg Gln Asn Phe Leu Ala Leu Arg Leu Gly865
870 875 880aat aac tac atc act gcc
gcg gga gcc caa gtg ctg gcc gag ggg ctc 2688Asn Asn Tyr Ile Thr Ala
Ala Gly Ala Gln Val Leu Ala Glu Gly Leu 885
890 895 cga ggc aac acc tcc ttg cag ttc ctg gga ttc
tgg ggc aac aga gtg 2736Arg Gly Asn Thr Ser Leu Gln Phe Leu Gly Phe
Trp Gly Asn Arg Val 900 905
910 ggt gac gag ggg gcc cag gcc ctg gct gaa gcc ttg ggt gat cac
cag 2784Gly Asp Glu Gly Ala Gln Ala Leu Ala Glu Ala Leu Gly Asp His
Gln 915 920 925 agc ttg
agg tgg ctc agc ctg gtg ggg aac aac att ggc agt gtg ggt 2832Ser Leu
Arg Trp Leu Ser Leu Val Gly Asn Asn Ile Gly Ser Val Gly 930
935 940 gcc caa gcc ttg gca ctg atg
ctg gca aag aac gtc atg cta gaa gaa 2880Ala Gln Ala Leu Ala Leu Met
Leu Ala Lys Asn Val Met Leu Glu Glu945 950
955 960ctc tgc ctg gag gag aac cat ctc cag gat gaa ggt
gta tgt tct ctc 2928Leu Cys Leu Glu Glu Asn His Leu Gln Asp Glu Gly
Val Cys Ser Leu 965 970
975 gca gaa gga ctg aag aaa aat tca agt ttg aaa atc ctg aag ttg tcc
2976Ala Glu Gly Leu Lys Lys Asn Ser Ser Leu Lys Ile Leu Lys Leu Ser
980 985 990 aat aac tgc atc
acc tac cta ggg gca gaa gcc ctc ctg cag gcc ctt 3024Asn Asn Cys Ile
Thr Tyr Leu Gly Ala Glu Ala Leu Leu Gln Ala Leu 995
1000 1005 gaa agg aat gac acc atc ctg gaa gtc
tgg ctc cga ggg aac act ttc 3072Glu Arg Asn Asp Thr Ile Leu Glu Val
Trp Leu Arg Gly Asn Thr Phe 1010 1015
1020 tct cta gag gag gtt gac aag ctc ggc tgc agg gac acc
aga ctc ttg 3120Ser Leu Glu Glu Val Asp Lys Leu Gly Cys Arg Asp Thr
Arg Leu Leu1025 1030 1035
1040ctt tga agtctccggg aggatgttcg tctcagtttg tttgtgagca ggctgtgagt
3176Leu
ttgggcccca gaggctgggt gacatgtgtt ggcagcctct tcaaaatgag ccctgtcctg
3236cctaaggctg aacttgtttt ctgggaacac cataggtcac ctttattctg gcagaggagg
3296gagcatcagt gccctccagg atagactttt cccaagccta cttttgccat tgacttcttc
3356ccaagattca atcccaggat gtacaaggac agcccctcct ccatagtatg ggactggcct
3416ctgctgatcc tcccaggctt ccgtgtgggt cagtggggcc catggatgtg cttgttaact
3476gagtgccttt tggtggagag gcccggccct ctcacaaaag accccttacc actgctctga
3536tgaagaggag tacacagaaa cataattcag gaagcagctt tccccatgtc tcgactcatc
3596catccaggcc attccccgtc tctggttcct cccctcctcc tggactcctg cacacgctcc
3656ttcctctgag gctgaaattc agaatattag tgacctcagc tttgatattt cacttacagc
3716acccccaacc ctggcaccca gggtgggaag ggctacacct tagcctgccc tcctttccgg
3776tgtttaagac atttttggaa ggggacacgt gacagccgtt tgttccccaa gacattctag
3836gtttgcaaga aaaatatgac cacactccag ctgggatcac atgtggactt ttatttccag
3896tgaaatcagt tactcttcag ttaagccttt ggaaacagct cgactttaaa aagctccaaa
3956tgcagcttta aaaaattaat ctgggccaga atttcaaacg gcctcactag gcttctggtt
4016gatgcctgtg aactgaactc tgacaacaga cttctgaaat agacccacaa gaggcagttc
4076catttcattt gtgccagaat gctttaggat gtacagttat ggattgaaag tttacaggaa
4136aaaaaattag gccgttcctt caaagcaaat gtcttcctgg attattcaaa atgatgtatg
4196ttgaagcctt tgtaaattgt cagatgctgt gcaaatgtta ttattttaaa cattatgatg
4256tgtgaaaact ggttaatatt tataggtcac tttgttttac tgtcttaagt ttatactctt
4316atagacaaca tggccgtgaa ctttatgctg taaataatca gaggggaata aactgttg
437421041PRTHomo sapiensIBD1 plus putative additional 5' exon 2Met Glu
Lys Arg Arg Gly Leu Thr Ile Glu Cys Trp Gly Pro Gln Ser 1
5 10 15 Pro Ser Leu Thr Leu Phe
Ser Ser Pro Gly Cys Glu Met Cys Ser Gln 20
25 30 Glu Ala Phe Gln Ala Gln Arg Ser Gln Leu
Val Glu Leu Leu Val Ser 35 40
45 Gly Ser Leu Glu Gly Phe Glu Ser Val Leu Asp Trp Leu Leu
Ser Trp 50 55 60
Glu Val Leu Ser Trp Glu Asp Tyr Glu Gly Phe His Leu Leu Gly Gln 65
70 75 80 Pro Leu Ser His
Leu Ala Arg Arg Leu Leu Asp Thr Val Trp Asn Lys 85
90 95 Gly Thr Trp Ala Cys Gln Lys Leu
Ile Ala Ala Ala Gln Glu Ala Gln 100 105
110 Ala Asp Ser Gln Ser Pro Lys Leu His Gly Cys Trp
Asp Pro His Ser 115 120 125
Leu His Pro Ala Arg Asp Leu Gln Ser His Arg Pro Ala Ile Val Arg
130 135 140 Arg Leu
His Ser His Val Glu Asn Met Leu Asp Leu Ala Trp Glu Arg 145
150 155 160 Gly Phe Val Ser Gln Tyr
Glu Cys Asp Glu Ile Arg Leu Pro Ile Phe 165
170 175 Thr Pro Ser Gln Arg Ala Arg Arg Leu Leu
Asp Leu Ala Thr Val Lys 180 185
190 Ala Asn Gly Leu Ala Ala Phe Leu Leu Gln His Val Gln Glu
Leu Pro 195 200 205
Val Pro Leu Ala Leu Pro Leu Glu Ala Ala Thr Cys Lys Lys Tyr Met 210
215 220 Ala Lys Leu Arg
Thr Thr Val Ser Ala Gln Ser Arg Phe Leu Ser Thr 225 230
235 240 Tyr Asp Gly Ala Glu Thr Leu Cys
Leu Glu Asp Ile Tyr Thr Glu Asn 245 250
255 Val Leu Glu Val Trp Ala Asp Val Gly Met Ala Gly
Ser Pro Gln Lys 260 265 270
Ser Pro Ala Thr Leu Gly Leu Glu Glu Leu Phe Ser Thr Pro Gly His
275 280 285 Leu Asn
Asp Asp Ala Asp Thr Val Leu Val Val Gly Glu Ala Gly Ser 290
295 300 Gly Lys Ser Thr Leu Leu
Gln Arg Leu His Leu Leu Trp Ala Ala Gly 305 310
315 320 Gln Asp Phe Gln Glu Phe Leu Phe Val Phe
Pro Phe Ser Cys Arg Gln 325 330
335 Leu Gln Cys Met Ala Lys Pro Leu Ser Val Arg Thr Leu Leu
Phe Glu 340 345 350
His Cys Cys Trp Pro Asp Val Gly Gln Glu Asp Ile Phe Gln Leu Leu
355 360 365 Leu Asp His Pro
Asp Arg Val Leu Leu Thr Phe Asp Gly Phe Asp Glu 370
375 380 Phe Lys Phe Arg Phe Thr Asp Arg
Glu Arg His Cys Ser Pro Thr Asp 385 390
395 400 Pro Thr Ser Val Gln Thr Leu Leu Phe Asn Leu Leu
Gln Gly Asn Leu 405 410
415 Leu Lys Asn Ala Arg Lys Val Val Thr Ser Arg Pro Ala Ala Val Ser
420 425 430 Ala Phe
Leu Arg Lys Tyr Ile Arg Thr Glu Phe Asn Leu Lys Gly Phe 435
440 445 Ser Glu Gln Gly Ile Glu
Leu Tyr Leu Arg Lys Arg His His Glu Pro 450 455
460 Gly Val Ala Asp Arg Leu Ile Arg Leu Leu
Gln Glu Thr Ser Ala Leu 465 470 475
480 His Gly Leu Cys His Leu Pro Val Phe Ser Trp Met Val Ser
Lys Cys 485 490 495
His Gln Glu Leu Leu Leu Gln Glu Gly Gly Ser Pro Lys Thr Thr Thr
500 505 510 Asp Met Tyr Leu
Leu Ile Leu Gln His Phe Leu Leu His Ala Thr Pro 515
520 525 Pro Asp Ser Ala Ser Gln Gly Leu
Gly Pro Ser Leu Leu Arg Gly Arg 530 535
540 Leu Pro Thr Leu Leu His Leu Gly Arg Leu Ala Leu
Trp Gly Leu Gly 545 550 555
560 Met Cys Cys Tyr Val Phe Ser Ala Gln Gln Leu Gln Ala Ala Gln Val
565 570 575 Ser Pro
Asp Asp Ile Ser Leu Gly Phe Leu Val Arg Ala Lys Gly Val 580
585 590 Val Pro Gly Ser Thr Ala
Pro Leu Glu Phe Leu His Ile Thr Phe Gln 595 600
605 Cys Phe Phe Ala Ala Phe Tyr Leu Ala Leu
Ser Ala Asp Val Pro Pro 610 615 620
Ala Leu Leu Arg His Leu Phe Asn Cys Gly Arg Pro Gly Asn
Ser Pro 625 630 635 640
Met Ala Arg Leu Leu Pro Thr Met Cys Ile Gln Ala Ser Glu Gly Lys
645 650 655 Asp Ser Ser Val
Ala Ala Leu Leu Gln Lys Ala Glu Pro His Asn Leu 660
665 670 Gln Ile Thr Ala Ala Phe Leu Ala
Gly Leu Leu Ser Arg Glu His Trp 675 680
685 Gly Leu Leu Ala Glu Cys Gln Thr Ser Glu Lys Ala
Leu Leu Arg Arg 690 695 700
Gln Ala Cys Ala Arg Trp Cys Leu Ala Arg Ser Leu Arg Lys His Phe
705 710 715 720 His Ser
Ile Pro Pro Ala Ala Pro Gly Glu Ala Lys Ser Val His Ala
725 730 735 Met Pro Gly Phe Ile Trp
Leu Ile Arg Ser Leu Tyr Glu Met Gln Glu 740
745 750 Glu Arg Leu Ala Arg Lys Ala Ala Arg Gly
Leu Asn Val Gly His Leu 755 760
765 Lys Leu Thr Phe Cys Ser Val Gly Pro Thr Glu Cys Ala Ala
Leu Ala 770 775 780
Phe Val Leu Gln His Leu Arg Arg Pro Val Ala Leu Gln Leu Asp Tyr 785
790 795 800 Asn Ser Val Gly
Asp Ile Gly Val Glu Gln Leu Leu Pro Cys Leu Gly 805
810 815 Val Cys Lys Ala Leu Tyr Leu Arg
Asp Asn Asn Ile Ser Asp Arg Gly 820 825
830 Ile Cys Lys Leu Ile Glu Cys Ala Leu His Cys Glu
Gln Leu Gln Lys 835 840 845
Leu Ala Leu Phe Asn Asn Lys Leu Thr Asp Gly Cys Ala His Ser Met
850 855 860 Ala Lys
Leu Leu Ala Cys Arg Gln Asn Phe Leu Ala Leu Arg Leu Gly 865
870 875 880 Asn Asn Tyr Ile Thr Ala
Ala Gly Ala Gln Val Leu Ala Glu Gly Leu 885
890 895 Arg Gly Asn Thr Ser Leu Gln Phe Leu Gly
Phe Trp Gly Asn Arg Val 900 905
910 Gly Asp Glu Gly Ala Gln Ala Leu Ala Glu Ala Leu Gly Asp
His Gln 915 920 925
Ser Leu Arg Trp Leu Ser Leu Val Gly Asn Asn Ile Gly Ser Val Gly 930
935 940 Ala Gln Ala Leu
Ala Leu Met Leu Ala Lys Asn Val Met Leu Glu Glu 945 950
955 960 Leu Cys Leu Glu Glu Asn His Leu
Gln Asp Glu Gly Val Cys Ser Leu 965 970
975 Ala Glu Gly Leu Lys Lys Asn Ser Ser Leu Lys Ile
Leu Lys Leu Ser 980 985 990
Asn Asn Cys Ile Thr Tyr Leu Gly Ala Glu Ala Leu Leu Gln Ala Leu
995 1000 1005 Glu Arg
Asn Asp Thr Ile Leu Glu Val Trp Leu Arg Gly Asn Thr Phe 1010
1015 1020 Ser Leu Glu Glu Val Asp
Lys Leu Gly Cys Arg Asp Thr Arg Leu Leu 1025 1030
1035 1040 Leu
337443DNAHomo sapiensIBD1 genomic sequence
3tcaccatata actggtattt aaagccacaa gagcaggtgg gctcatctag ggatggagtg
60atatggagaa gagaaggggt ctaaccattg agtgctgggg cccccagtgt taggaaccag
120ccaagaagac agaaagagtg aaaatcagag agttggggtg tcctggagga aatgaagaaa
180atgccccaaa gaggaaggag ggaacaaata tgaccaatgc ccctggcaga gcaagcaggc
240tgagggctga ggattgagca atgggaggtc actggtgaca gtttcactgg agctggatgg
300ggaactagag ggaatgggag gggatgggag gacttgggga cagcagtaca ggcaacagac
360aagggggcct gctgtaaagg gagcagataa atgggattgg agccaaatga agaaggggag
420tgtcaagaga gtgctttact tttacaatgg agaattagag tgcattgtgc actggtgggg
480ggatttgatc tcttagggag agaacagtgt tagggaggga gaatgcagga tagctggggg
540agggtggggg gcttggcccc agcagagact caggacactt gggaagttga gcttccctgg
600gcttcccctc ctctcctgtc tgcaaggggt cagtgggctg agatttcagc acttaagcaa
660agcatttgct cttggcccca gagaaaccgg gctggctgtg gtctcaggaa ggaaggaggt
720gtccaggctc aggcctgggc ctgggtttca gggagggccc acgtgggtca ccccttgacc
780ctctctttca gcaaggaagt gatcctttct ctacatgggc ctcaccttgg ggaggacaat
840ggtgtctttg aagttgtagt aactgaagta gagatcaaaa ggcaatgcag atagactgac
900agatttcgcc tgaagagggg aagcccgacc aggtaataaa ggagtaagag gaaggatgtt
960aaggacaatt ttaggaaaca gataatgagt gaatattttt tctctctctt tcccaattta
1020aactgaagca ggagaaactg aagctagaca taatgattaa cttcccaagc tggtgagctt
1080cctgagctgg ttagtgagaa cagcactaag gccaggttct cctccccaga tgtttaagat
1140gagacaggac aatgcctgct cagagacagg gcctggctga attggccctc aggattctct
1200ctgctctgag gtttctggaa gaaggccagg gcagaggtgt ggtgatgtag ctgctgggag
1260gacagagctc cgagtcacgt ggcttgggcg ggcctcccct tcctggtgtc cacagaagcc
1320caacgtcact agctggggtg tgtatggctc acacgtaggc caggctgccc taggcttggt
1380gtgcaaggga ggggccccta cttacttgtg gcctgtcccc tcgtgaatgt gtctcatgtc
1440cccagtgggg tttttcagtg agggtcatgg tctccaggat gcacaaggct ttgtgccaga
1500attgcttgga attgcctagt tctggaaggc tggttggcca actctggcct ccggcttttc
1560ctttgggaat ttcccttgaa ggtggggttg gtagacagat ccaggctcac cagtcctgtg
1620ccactgggct tttggcattc tgcacaaggc ctacccgcag atgccatgcc tgctccccca
1680gcctaatggg ctttgatggg ggaagagggt ggttcagcct ctcacgatga ggaggaaaga
1740gcaagtgtcc tcctcggaca ttctccgggt aagaggagca ggcattgtcc cgtcccagct
1800tgatcctcag ccttctttca tccttggccg cgacatgctc ccaggcctgg ggtcagatgg
1860ggagtgctga ctctgtttct gggctgtttt ctggggagaa tgggtcggcg ggtttttttc
1920cccaggacct gggcagggtc aatggtgggg gccgctgtcg catccttggc tggtgtttcc
1980acagctgaga accactccag ggccaagccc agagcttatt ctaccctttt ttgtcctctc
2040ttcccctgtc ctcggccacc ccaccctctt ggctcctctg cttagatgtg ggcacaagga
2100ggagaactcc ttggcctgag agaactacct tagatcctgg cttccagtgg cctctgcagg
2160ggggtacacc ctctctccca agcagccaga cacacaagta acctcattgc ctcagtttcc
2220ccatctgacc agcacagggc cccctgtgcc ccagcagcgt tctgagagat tggagctttc
2280tccttttgct taccttggct accgtatgag gacggataca gagtgttccc cccaccccca
2340gcccagggga tatttgattc atgaacattc cctcagtgtc tttgtggggg acaatgctgt
2400gccaggctca gggatgccag gacgagtaag acccaggctc ccacgtggcc caggcaggga
2460gagagacaca taaacaacca tcaggaaaga ggtaaaatcc ccaggccact tggcatctgc
2520tcccttgagt gtctgggaat gtccctgatt tataaaaaga agctgacggc cctctttgtt
2580gtccatgcct acaccctttc actttcgttt cttcggggca ctgcagcagc ccttgtccac
2640agaccccatg acaatcgcag aactgaccat gctgagagat tttcttggct gctcagggac
2700cctgccaggg cttgaagctc ctggagggtc acttgccctc aaattcccag aacgcacagc
2760aggtcactga tgatagcagt ggcagcagtc tgtgcacggt ggtttcgagg gcgtgggagg
2820gaggtgaggg ccctagggca agtgtgtgtg ggaagtgttg atgggggaca aggcaccaga
2880acgctcggaa acaacttagt ttgcaccgta atttttcact tcgcctagga caggaccttt
2940agagcaatat tctgagtcta ccccttggag tagcagtgtg caaaacacac agcacgggct
3000tggggccccc gtggggaacc caaatgtaag agttagagac atgcattccg gagtcataca
3060tggctcgtgt tgaaatcctg actctgcctg tctagctgtg acacatcgta caaatcactt
3120agcttcttgg tgcctcagtg tcttcctctg tagaatgggt agatcatagg cactacttca
3180gagtggctgg gagggttcag tgaattcctg caggagagca cttagaatgg cacttggtgt
3240gtagtttatg cttaattaat attagccgtt actgaaactg ctgtagcctg aatccagcca
3300gcatgaaaga gcccctctca ccctgcttcg aagagaatga attccctgat tgtttggaag
3360atctctctct ctctctctgt cttttttttt tttttttgag aaacggtctt gctctcttgc
3420ccaggctgga gcgcaatggt gccatcttgg ctcactgcaa cctctgcctc ccgggttcaa
3480gtgattctcc tgtctcagcc tcctgagtag ctgggattac aggcgctcgc caccacgcct
3540ggctaatttt tgtattttta gtagagacag cgtttcaccg tgttggccgg gctggtctag
3600cgctcctgat ctcaagtgac cttgggagat ctcttgctcc taatattacc tcaagccttt
3660ttaaacgttt taagccggag accaagcatg gatatgggag ttaggggtct tgatttaatt
3720cttggttgct tcaaactctg tggaaccttg aggtgtttct tgccttctct gggtctcaat
3780tttcacatct atatggtggg gagcttggat tgggtaatgt ctgaggctag aaccatggcc
3840aactcgggtt ctgctggggc tgacttgccc tggccttccc tgaccaccct gcatctggct
3900tctggagaag tccctcactg accttgttct cctccccagg ttgtgaaatg tgctcgcagg
3960aggcttttca ggcacagagg agccagctgg tcgagctgct ggtctcaggg tccctggaag
4020gcttcgagag tgtcctggac tggctgctgt cctgggaggt cctctcctgg gaggactacg
4080agggcttcca cctcctgggc cagcctctct cccacttggc caggcgcctt ctggacaccg
4140tctggaataa gggtacttgg gcctgtcaga agctcatcgc ggctgcccaa gaagcccagg
4200ccgacagcca gtcccccaag ctgcatggct gctgggaccc ccactcgctc cacccagccc
4260gagacctgca gagtcaccgg ccagccattg tcaggaggct ccacagccat gtggagaaca
4320tgctggacct ggcatgggag cggggtttcg tcagccagta tgaatgtgat gaaatcaggt
4380tgccgatctt cacaccgtcc cagagggtga ggcactcctg gtgtgcatca cagagttctc
4440aggaaagggg tgcttagtca ccaagactga tttgtcctca tgaagtcagc ctgtggggta
4500acttggtccg tgggatttcc cctaaaaagg tagccaggca ggtaaaattt gctcttgact
4560cttggcagga aacatacaac tctttctttc ttcttttctt ttctttttct cactctgtta
4620ccctggctag aatgcagtgg cacaatcata gctcactgta gccttgaatt cctgcgctca
4680agtgatcttc tggccttaga gtagctggga ctacggctgc tgtaccacca tgaacagcta
4740attttttttt tttcttttag agatggggtg ttgctatgtt gcccaggctg gtctccagct
4800cctggcttta agcaatcctc ccgccttggc ctcccaaact gttgggattg caggcatgag
4860ccactttgcc tggccaacag aacacttctg ccgagaggaa gtgtgtggtg gccaggaact
4920cagattctgg agccagaatg gtgcaggctc aaggtcaacc ctgtgtgatc tcaggcttcc
4980ctatggagcc tctccagcct cagtctccct tgtttcagtt tcctcatcta caaaacaatg
5040ttaatagtca aatggtgcct atcctataag gctcttggga ggattcagtg agttaatttg
5100agtaatgctt aggatagtgt ctattaccac tggctgctat ttattatttc tgttatgagt
5160gatactctgt acttgtacac ttttatttct gtctgtttta aattaacagc acaacagacc
5220ataacactgc agtatattga atttatttta taattaacat agcatattat aaactaatat
5280agcttaaatg tttatgtagg atttctgaca tgaaattgca ttagatcata gatgttcaga
5340gttggtatat aacagcccct gagaatgtag taactcagca gagaccagaa ggtcagagaa
5400atgaccactg agtatttttg aaactctttt gttttcttcc aaatagtgat tcttagggct
5460cctgagaggc agatggaaca atcattaaca ttccacttta taaatcggga agttgagacc
5520aaggaaagta gtttgaataa gctcacagta gttaatgagg gggccagtgc tggaccaatt
5580ggccagcact ggtcattgac ttattcatcc atcattcatt tattcagcca gaatctatta
5640ggtgcttcat acatatttgc ttaaagtttg ttgtgttcat agagctttgc acacggtagg
5700tactccataa acatttgttg atgaaataag tgagttactg aatgaatgat tgaattagaa
5760tgacactgca gtgttaaaat gggctgggtt ggggaacatt ttagtttttg tttttgtctg
5820ttttccaaaa atgtatgtgt tgttcacatg agtctggata accctagatt gagattgatg
5880acataaataa atttgtcttc aaggctgcac taaagctggc tcacatggct aggtatttac
5940agagcagaag tggtgcagtc ctctctgatt agttgcacgt acagaagaca tattcgttat
6000tggactgacc ttagtttctc ttataatttg ttaggggaat tgaatcagcc catctgagaa
6060gttacaagat tgtgtcttgt catctttaaa agttcagcaa tgtgatgtgg tacagatggt
6120ctgaggggtt tggagaaggt agcctagatc cctagggccc agagaagaca ggatgtgaac
6180agaggaagta catggattgg tgaagaaaag aaatgggata actcatgggt caaagaagaa
6240atcatgatgg aaatcagaaa atattcagaa ccatacaata atgagaatat tatttatcaa
6300aatctattgg atgcagctaa agcaggacat agggggaaat ttacaacctt aggtgcctag
6360attaggaaag aaggaaggca tttgtttatt tatttgttta tttatttatt tgagatgggg
6420gtctcactgt gtcacccagg ctgctggagt gcagtagcac gatcataaat cactgaagtc
6480tcgaacttct gggctgaagt gatcctcccg cctcagcctt ccaagtaggt gggacacagg
6540ctagcaccac cataccaggc taattttttt tttgtagaca cagggtcttg ctatgttgag
6600gtctcaaact cctgggctca agtaatcctc ctccctcggc ttcccaaagt gctgggatta
6660caggcatgag ccactgcgcc catctaaggc tgaattttaa tgagctaaga attcatctta
6720agaaagggct aaatagacag caaaagcaaa cattgaaggt tgggactgag ctgagtgggt
6780agcagggatg ggagacaaca gatctgagga gagcaggaga ttttgaaagg attgcactgc
6840ctgaggttta agcctttaga atccagctct ctctgagctc cctttgagct ctgacattct
6900gtgactctga tttggtggcc ttcccttagt ggccttactg atttcatttg gatggtgctt
6960gtggtatatc caaccaacat gtcttcccaa atggcctttt aatttcctat aaagaagtag
7020ttgtcattga ttgcaggtta gggacagaaa atgctgtgga atgaaacaaa atgcaagtta
7080aagaactaaa ttccaaaaat acccattgct actattgact gagtgaattc ctactgtgtg
7140ccagacactg tacccagtcc attccctgta ttgttttatt taagcctcac aagggtatag
7200tgtgactaca ctgtttctta acaatgaaga aactgcccaa atcgcccatc tgggaagcgg
7260cccagctaga atttgaatcc aggcctgttt tcctccagag cttgtgctat tctctgtctg
7320tcataaaatg tgggggcttt gtgtggtaaa cttgctcagt tgggcatagc agttgttagg
7380aaacctgagg ctggtaacac cagctgtaat accagctgtc cgtctgactc atgcaactgt
7440taaagttgat agggctgagg tgtcagactg agctctgaat tgcctgattc ctataacaat
7500attaacttaa acatttttta aattgggaaa tgcaccatgc atacagaaga gtgtgtatat
7560ttcatatgta tagtgtaaac tgttcccatc acccaggtta aaaaacagga tgttgccagt
7620acctggggcc ttctttaact gcaactgcta gaggtaaaca ctggcttgac ttttgtgtaa
7680atcatctctt tgcctttctt taatgtttta gcatctttta aaataaatcc ccaaataatg
7740tattgttcta ttttgaaaaa ctgagtagca agccaaaaat agctgtgtaa agaaaggtca
7800cttaaattag gctgggtgca gtggctcaag cctttaatcc cagtactttg ggaggctgag
7860gcaggtggat cacaaggtca ggagatcgag accatcctgg ccaacatgga gaaaccccgt
7920ctctactaaa aatacaaaaa attagccaag aatagtggca tgtgcctgta gtcccagcta
7980ctcgggaggc tgaggcagga gaatcgcttg aacccgggag gcagatgttg cagtgagctg
8040agatcgcact gcttgaaccc gggaggcaga ggttgcagtg agccaagatc gcaccactgc
8100actctagcct gggtcacaga gcaagactct gtctcaaaaa aaaaaaaaaa aaaaagaaag
8160gttactattg ccttttctta gatgaaggtt cccaaggcag ggaaagctaa gtggagtctc
8220agggacttgg tctggctttt ccttccctgg gaatttataa ggacctcttc tgggaagtca
8280gtcggcaatg ccatgaatga gtctggggaa atattgggct cattgcaact ggagggtctg
8340gtaggactga tgtgaattag gtgctgtgtc cggaggaaaa tggccagagg aagtgggctg
8400ctttgtacag tcagtggtaa agttgccaaa ggctattata gctcacagga atgggccaag
8460gctaaacact cctgtggagt gaaatgaatg tcctcagctg actgaggcag cgggagttga
8520gaagaaacga tattagttca tggtgaagac aagtcaaata tagataaagg ttagggtcag
8580gcttgcctgg acatctagga gataactgcc ctcaacttgt ttgaatcttg agtcactgct
8640ccattttgtt tgaactggtg gccatctact tatagtatac agccatcaac ctgagatttc
8700cctacatggt cttcctgcct tggtctcctg tatcctgaat cctatggcct cttcttccct
8760ggtttactac attttgctag accgtatcct ccagtcaatt ccttagaatg aatgtatgaa
8820agttaaaatt tctgaggtct cacatgtctt aaagttccct catactggat tgatagtttg
8880gctgggtata aaattctggg ctggccatca ttttccttca gaattttgat tgcattattc
8940cattatcctc tcttttcaat attgcttcta agaattccaa aacctttttt tttttttctt
9000tttgagacag tgtctcactc tgtcacccag gctggaatgc agtagtgtga tctcagctca
9060ctgcaacctc cacctcctgg gtttaagcga ttcttcttcc tcagcctcct gagcagctgg
9120gattacaggc acccaccacc acacccttta gtagagatgg ggttttgcta tgttggccag
9180gctggtcttg aacttctgac tttaggtgat ctgcctactt cggcctccca aagtgctggg
9240attaaaggcg tgagccacca cacccagcct ccaaaaccat tttaaaactc tttctggaag
9300cttttaaaat tttcttttag tccccagaat tttaaaattt caattatgtg ccttggtgtt
9360cttccattat attagtcacc caagaggtac tttcaatctg gaaacttctc tatgttttgg
9420gaaatgttct tgattagttt acaggtgatt tcttcctctc cattttatct cttctctttt
9480catgaaacta ctattaattc aatgttagaa ttccttgact gatcatttaa ttttcttcta
9540ttttccatct ctgtgtcttt ttgctctact tttctatgat agtcacagct ctatctttaa
9600actcttgagt ttttcatttt tgatgtcatg attttaattt gcaagaggta ggtttgactg
9660attctttttt gtagtatctt actcttgttt tatggatgca acatcttctt tgacttaagg
9720atcataagat aggtgggttc tttgtttgtt tgtttgactg tttttcaccc tatgtaaact
9780ttttctacaa gtttctttcc ccttcccccc tttttggctt ctatctccca cattagatgc
9840tttctctggg ctcatgatac tctttggttt tctttctcaa gattgacagg taggacttta
9900aaacttgttg agcatgcggg tgaaacttgt ctaccatgaa tttcactgta gatattttgg
9960agattgacag tgtttatatc tttagatctc acctcctggg ttgatcaagt tatctgagta
10020caccacagac cttttgcctg gggataaacc agaaatctgt ttcagaaacc actttgattc
10080agtcttcctt gttttagtca tttccttcag ttccggaggt ccgtcatgct gatcattcca
10140gagcccttta cagatcctag ggtacacact gcatggtttt caactttctt gttttggggt
10200taagatttgg ctttcaggag tctcctcagt ccgttactat tcattcaatc agcaagtcct
10260tgagcacctg atttgtgcca gacattcttc taggtgttag ggatacctca gtgaacaaaa
10320cagacaaaaa tctttgtctt ggaaatacac acactccagt caggggagag ggacaataag
10380ccaaaggaag gaaattacag cgtgtgctag aaggtgataa gtgctgtaga aagtaagtaa
10440agtgggtttg ggagttgaga gtttgggaag gggataaatg atggcaattg taaatagagt
10500agtcagagtt ctcacttaga aggtgaaatt caagtaaaga cttgaaggag gacagggaat
10560tagccacatg gatggctagg ggaaggcttc caagctgaga ggacagccag agccaaggcc
10620cagaggcagg agcatacctg gtagttttag gaaacaggag gccaggatgc tgagtggagt
10680aagagggggc atgaaaggag aaacttgggt ccacgtggtt ctagacaggt atttttgtct
10740gttttgggcc ctgaaggtta ctattggact tggactctta ctctgaggaa atagggacgc
10800tattgggacg tttgtacagg agcaatgtga cctgagtttt gtttgtaaag gattagactc
10860tggctgtggc attaaggcta ggctgtgggg gcaggaacag aagcaggggg accagttttg
10920cagcctgtgc agctttccag ataagcaggg attgtggctt ggaggaggat ggtatagagg
10980aggtgacaag aaatgactct atgtctggta tgtagatatt ggccacagat ggcatttgag
11040cactagagac ctggctggtc cacatggagt ttccataagc acataataca catcagattt
11100caaagactta atatgaaaaa aaaaatttaa cgggccccgg gaattttttt cttttttttt
11160ttttttgaga cccagtcttg ctctgtcacc caggctggag tgcagtggtg tgatctcggc
11220tcactgcaac ctccgcctcc caggttcaag tgattctcct gcctcagcct cctgagtacc
11280tgggactaca ggcacctgcc accacgcctg gctaattttt tgtattttta gtagtgatgg
11340ggtttcacca tgttgtccag gctggtctgg aactccggac cttaggggat ctacccgcct
11400tggcctccca aattgctggg attacaggca tgagccacca tgctcagcca tatcttgcta
11460ttttctacat ggattacatg ttgaaatggt aatgttttgg ctattgtgga ttaaatagaa
11520tatatgatta aagttgattt catctatttc ttttaacttt aaaaaatatg tctgttagag
11580gatttgaaat tccacatgcg gcttgcattt gtgacctgca tttcatttct gtggaacagt
11640gccctttttg ggacatgctt tgaaggtgga gtcaacagga tttggcagat tacagacgag
11700aggcttcaag ggtgactcca agacttcggg gcagagcacc tggaagaaag gggttaatat
11760tagccaagat gaggaaggct gtcggtttgg caggtgcatg ggcaggttag gagtttagtt
11820ttgaatatgt tggaggtgtt tatgaaactt ttaagtggag atggaaaata ggcagttgga
11880tgtgcaagtc cagggttcag ggagacagtt caggctggag atgaagatgt gggagtctga
11940ggagagattg tattcaaata ttcaatccat gagacttgat gaaatcactt ctcttccaaa
12000tgatttacag cctgcagaat cattttccct atctttgtag gtttatgtct tcattttgtt
12060tcatttattt ttcagttatt cactgtttta gtgagttttg agtaggagcc agattggatg
12120catgcgttca attcaccatc caacactgta ttaactactt gaaactcatg tggttgttcg
12180gttgtttttt tgacctttta ttctggatgg aagagagatg cttatgaagt tgcagtaatc
12240agtaagcctt cccacattgc tccatcagcc ttcctggaag aataatgtct tctgcctttc
12300ctgtaggcaa gaaggctgct tgatcttgcc acggtgaaag cgaatggatt ggctgccttc
12360cttctacaac atgttcagga attaccagtc ccattggccc tgcctttgga aggtaggtgt
12420atgttctcag ttaatcagaa agggaagggc agtcagtgca gatccatggt taagagcaga
12480acacacctcg gttaacatcc catatgctgg cagtatagcc tccctatgac tcaatttcct
12540tgttttaagg ctagcaccac cccgtctcat tgggattttg ggagcattaa aaggacaaaa
12600gcgtgtaatg ttagctatta gctttcatta tctcccacac agtatactga caattgggct
12660accatatatt gagggctaac taaaggtgtt acttaccatc caaactctca ttatctgtac
12720cgaaaagata tggacacatg ttttgagtta gggctggtat ctcttgatct ctgaaattta
12780gcagctcaca atgggaaact caagaaccaa gtggatctag agactctggt atccctcagt
12840gcccagggtc accacccaaa ctcaggaaca ggaggggctt ggaccgcacc acttgaacat
12900accaggcatc ctgccaggtg ctttatggac aatgtctacc ctttgcaaca accctgagaa
12960gtaggtggtg tttttttcca ccttatagat gtggaaactg ggcagggagg ttaagtgacg
13020agggagggga agatgggtct gattgtaaat tgtccccacc tacactttct cttttcttgg
13080gagaagaaat gtcagttgta aagagagagt gcaagcctgg cactctttag ggcttgttcc
13140tacaccactg tagggaaagc tcattggcac tgaagccccc tgagctgtgt gtggtgctgg
13200cagatgggtc tatcaccctg gactgtgtcc tctgggcagc aagcaagcct gtgggcgggg
13260tggctggaag tctgtgcctg gcactcgcga gtgcaccgtc tcattgaaga acaggatcta
13320aacatcagtg cgccacagca gggtgcgcgg cacggagtgc aggccctggt ttggcccttg
13380gttgaggttt gctgttgaca tcatcaagca cagctagtca ctgtaagacc aggccagggt
13440gcaagattcc ccacacttct aaaggtgaca attggtgtat ttatttctct ataaaatgac
13500attttttttt tctggagaat tttagtatca ttggtgatga ctggaaaacc tgcatcagaa
13560atcaggtcgg aagaggaaga tatatatctg atatgtactg gagaggaaga tatctatctt
13620atggtctaag ttcagggatc ctggtatatt cagagggcag aaagctcagc aataatcatc
13680aactctggga acagaggtga cataaacaca gggcgtcccc tttgtgtgac tgcagatagt
13740catcagtgag ctcagagctc tatgaaaatt acttgctagt ttttgggttg aaaatagtgg
13800gccagtgttt ggttgggggc agtgaggctg tgatggcggg ggaccatgcc aagctcctac
13860cagcctggga cgctaaacca gcacttcccc atttcctgaa aggggaacta aactctgaca
13920caggaaatgg tttgcttgca ttactttcag gatgagaaag gaagagcact ggccttccaa
13980acacaccccg tgcatgaaaa ctctccctgc atggggtgca tggggaggat ggggaagtgg
14040aggcaggatc acagactctt gttcgagtgc tcagctgggg caccccggtg accccgaggc
14100cttcccttgc taggtccacc cagatcaatc aggatcatct ccccatctcg aagtttaact
14160ttatcacatc tcagagttcc ttttgccacg taaggtaaca tattcacagg ttctgagaat
14220ccggacatgg acatctttga gggtctattg ttgtgcctac tatatccatg aataataatg
14280ataataagca ccattttttg agagtttgcc atgtcagata ttcttttaaa ctgtatttta
14340tctcgctgcc tcctgaaaaa atccttccag gtgtatattg tccccatttt tacagatgag
14400agaactgagg cccagaaagg ctaaatggct tgcccaagtg tatggtggac ccaggttttc
14460aaactcaggt gtgtctggct tcagagactg ggctcctgag cccttaagcc ctttgttccc
14520ctttagaaaa agtcacctga ggctgagtgg tgaagggatt tatccaaagc cacccggcca
14580ctatggcagg acagatatca gaatacaggt cttccgatcc cagcccagag ccccttcccg
14640tcatctagaa ctcctcctgg tgtcagtaat gataacggca gtcactgatg tcttttgagc
14700acttactttg tgttgagcac ttacactgtg ctaagcactt gacataggtc atcttagttg
14760atccgtgtaa aactctgtga ggtagtgacc aacatttctc ccaccttaca gaggtggaaa
14820ctgagggtta ggaagtttcc ttgactgtcc tcaaagtgca cagcttgtga atggaggagc
14880caggatgggc gcccgctggc tctcctatcc cttcagttat gtcagcgtcc cccgcagcag
14940cccattgtct ggttaggtcc cgtcttcacc atggtgccac cttcatctgc ctcttcttct
15000gccttccagc tgccacatgc aagaagtata tggccaagct gaggaccacg gtgtctgctc
15060agtctcgctt cctcagtacc tatgatggag cagagacgct ctgcctggag gacatataca
15120cagagaatgt cctggaggtc tgggcagatg tgggcatggc tggatccccg cagaagagcc
15180cagccaccct gggcctggag gagctcttca gcacccctgg ccacctcaat gacgatgcgg
15240acactgtgct ggtggtgggt gaggcgggca gtggcaagag cacgctcctg cagcggctgc
15300acttgctgtg ggctgcaggg caagacttcc aggaatttct ctttgtcttc ccattcagct
15360gccggcagct gcagtgcatg gccaaaccac tctctgtgcg gactctactc tttgagcact
15420gctgttggcc tgatgttggt caagaagaca tcttccagtt actccttgac caccctgacc
15480gtgtcctgtt aacctttgat ggctttgacg agttcaagtt caggttcacg gatcgtgaac
15540gccactgctc cccgaccgac cccacctctg tccagaccct gctcttcaac cttctgcagg
15600gcaacctgct gaagaatgcc cgcaaggtgg tgaccagccg tccggccgct gtgtcggcgt
15660tcctcaggaa gtacatccgc accgagttca acctcaaggg cttctctgaa cagggcatcg
15720agctgtacct gaggaagcgt catcatgagc ccggggtggc ggaccgcctc atccgcctgc
15780tccaagagac ctcagccctg cacggtttgt gccacctgcc tgtcttctca tggatggtgt
15840ccaaatgcca ccaggaactg ttgctgcagg agggggggtc cccaaagacc actacagata
15900tgtacctgct gattctgcag cattttctgc tgcatgccac ccccccagac tcagcttccc
15960aaggtctggg acccagtctt cttcggggcc gcctccccac cctcctgcac ctgggcagac
16020tggctctgtg gggcctgggc atgtgctgct acgtgttctc agcccagcag ctccaggcag
16080cacaggtcag ccctgatgac atttctcttg gcttcctggt gcgtgccaaa ggtgtcgtgc
16140cagggagtac ggcgcccctg gaattccttc acatcacttt ccagtgcttc tttgccgcgt
16200tctacctggc actcagtgct gatgtgccac cagctttgct cagacacctc ttcaattgtg
16260gcaggccagg caactcacca atggccaggc tcctgcccac gatgtgcatc caggcctcgg
16320agggaaagga cagcagcgtg gcagctttgc tgcagaaggc cgagccgcac aaccttcaga
16380tcacagcagc cttcctggca gggctgttgt cccgggagca ctggggcctg ctggctgagt
16440gccagacatc tgagaaggcc ctgctccggc gccaggcctg tgcccgctgg tgtctggccc
16500gcagcctccg caagcacttc cactccatcc cgccagctgc accgggtgag gccaagagcg
16560tgcatgccat gcccgggttc atctggctca tccggagcct gtacgagatg caggaggagc
16620ggctggctcg gaaggctgca cgtggcctga atgttgggca cctcaagttg acattttgca
16680gtgtgggccc cactgagtgt gctgccctgg cctttgtgct gcagcacctt cggcggcccg
16740tggccctgca gctggactac aactctgtgg gtgacattgg cgtggagcag ctgctgcctt
16800gccttggtgt ctgcaaggct ctgtagtgag tgttactggg cattgctgtt caggtatggg
16860ggagcaccat caaggctaag tgtgggagca ccgagctggg ctctagaagt ctgggcccag
16920cttcgcctct gccaccctgc tttgcaacac tgcccagatc ccttcccttc tgggccttaa
16980tttcaatatg tgatgatgac agccacactt tattgactgg cctatgtgct gggtctggtg
17040ctatgctttc cggaatgacc tcatctaatc tctacaacca ccctgggggg taggcaggaa
17100tgttattatc tccattatcc ttgacttgag gctcagagaa gtgaagtaac ttgtccagga
17160aatggcagag ctggggttca caaattgcat cattctgatt acaggttttc tgcctcccac
17220cagtctatgg atacacttca gaggctccct gaaaaccttg aggtcacttg cagaaagttt
17280tgtgtagtat gtgtccgtat caggaacaac accaaatcag aggtgacttg tgccccatca
17340gagactttaa caccccaacc agatgggaat ttcaggaccc aagaaataga aagtggctgc
17400agggttacaa ctactgttgg attcctgagg tagcacagtg tccaaacagg atttcagcac
17460tacccgtatt gcttagagcc ccagccaaag atgtgaggtt ttgccctttg gagaatctgt
17520gcccctgaac tcgggggcct ctttccacat cttgggggca ggcaagggca gagggtgtgc
17580ctaggcctgc ggatcagcat gcgacagatt ccccaacatc cttccagctt gaaaggggat
17640tgccctgctt ctatttagaa cctataggaa agcagaagtt ctagattgaa gttaaaattg
17700attcccagcc tccaggggct ttgggctaca cctggatgac cttaattgac cctaagcatg
17760ggacaaacca cttcctgaga gtattaggat ggtatacatc ttctctgggg gcaaagcaac
17820aagatttatt tttcatcatg gaccaaacac atggataccc actagaaact gtgtagtgaa
17880ttttgttaac cctgacatag ggaccatggt ctttaggtta aagcataata acaacataat
17940acataacata tatagcgaat atatatatgt attatatgca atgaatgtaa atatgattat
18000acccatcatg gtcttggagg aaacagatga cacacttaaa atgggtgttt tgaggagagt
18060ttgaaaaaca gattgtttac aagccatggg caggagttag gaagagtgag agggttggtg
18120caggggcctg gggttagtaa cagctggggg agggtagact tgaaggggga aggggaggga
18180gactaattag ctggggggaa ggtatggaga cggctgcctg agcttctgca aagtggaaga
18240atactgcttg gccctaactc ctcaccccaa ctcttgctcg tggccagcgc cttccaccag
18300ctggacccat cagggaggcc gagtgggctg tctgctggag tagtccccag gcatcagcct
18360cccaggagcc agggacgggt agagaagggg gagagtggat ctggccaggc aaatggaaaa
18420cagccagcac caaactctat ttccctagga gggaggatca tgatactttg agtgggaatt
18480tggaaacctg tctgttggag caatttccct gatagaaata agaatgtgca ttttcctggg
18540tagtagactc agtttttacc ccaagaggcc aggcatcact ggcctgtgtg atcctcatag
18600gccagtccat ctctggaatt cttgaatgga tcatccatcc ttgattaggg atgtccccgt
18660gattaccagg gtgtgcagaa gggctctggg aaacctgtgg gtctgtctct gtgttcagag
18720aaaggtgagg gtggcctggt tctagctcat ggtgctcaga ctgtggtgtg taaaggcact
18780cgtggcaatg cagattcctg ggcctgcctc tagtgattcc cattcagtag gtttggggtg
18840gggcccagga aatctatatt tttcacagac acccctggtg attctgatac aagtggtctc
18900gccctgggag aactactggt ctgcagcaac cagcttggtt ttccattagc aattactgtc
18960cttgagcgag ttttactgct cttcacctta cacacactaa aactgccaag gccgtagggg
19020aggggaagca accatgaggt tgctgtgagt gcactgtgtg tgtgtgtgtg tgtgtgtgtg
19080tgtgtgtgtg tgtatgagag agagagagag attgagaaag agaggaaggg aggaaggggg
19140agggcacagg ctcctctccc acagtgccaa cctgcctctc tcccacttga agcgtttcca
19200tgccaactga aatcctcagc ctctaggaaa ccctatatac acagtgcccc tatataggtt
19260tctttagact ctggctctct cagactctag agtgatggct ttaaaagttt tatgttaccc
19320acagagagag agcacgcacc accatgtaaa catggaacct aagtttcaca aaatgacttc
19380gctttatgaa ctctgagaca ctctgctctc ttctgttctg ttctatttcc attttagaaa
19440tgctgctcag gaccttcaaa atgatttgca tgacctgcaa cctgcagtct gaaaaatcac
19500tgcactacag aagtggccat aagaggccct gagggagaag ctgcacaatg tcatggttaa
19560gagtggggtt tggagccaag ccgcctaggc tcaaagcctt tatgtgccgt acaaccttgg
19620caaagtcact tcgcttgtct gtgcctcagt ttctttctca cgaatgctca taataatggt
19680tcccatttca ctggcttgtt gtgaggatga aatagtgtta ttattgagaa gtggtaaggg
19740tagtgatcag tgctagcgat catgattcta ggtgactttt actgtgtacc gggtgctcac
19800aaggctttat gtgcacagcc tggtgaggct gataatacta ttgttccctc tttttttttt
19860ttggaaacgg agtctcgttc tgttgcccag gctgggggta cagtggcaca atctcggctc
19920atgcaatctc tgcctcccgg gttcacgcca ttctcctgcc tcagcctccc aagtagctgg
19980gactacaggc gcctgccacc acgcccggct aatttttttg tatttttggt agcgacaggg
20040tttcactgtg ttaaccagga tggtctcgat ctcctgacct cgtgatccgc ccgcctcggc
20100ctcccaaagt gctgggatta caggcgtgag ccaccgtgcc cggcctgttc cctcttttat
20160agatgaagag accagcaaat aactagtaag tcgctgatca ggatcacaat atccagctga
20220ggcactccag agcctgagct gttaaccatt cagtcagggc ctcccaagtt tgcctaaaga
20280taaagaatca tgtgcacagt tgttaaaata tacagattcc tgggccccac cccgcagata
20340cttgattgcc agctccaggg tatgggcctg agaatctgtc ttttagggaa gctttcagat
20400gatgttgtga tcaggtgagt tttgggaatg gtgccccaag aggagtggca gacagggctt
20460gctcggcagg gactagcctg ttggagtggt gccattgggg ttaaggactg ggcagcaggg
20520cctcactaac cacagcctat atgcctgttt ctgaagtttt ggccactctc atccagctgg
20580tctactgtct gctgacctag atgatggtaa attgtcccca ggggtagcct gtctagttca
20640ggctgcacct ttcgcatata tcagctcctt tccaccatca tcccctttgt gaggctgctg
20700tgattatcat gttccttttg cagagatgga aacattgcct caaattagct ctgtcatttc
20760ctaaggattc cagggttctt tagtaggggg tctggatcct acgtcctggg ccatccccat
20820catagtgcac cacgtcacct ccctggccag ggaccgtggg gtctccactt ttttggggtg
20880ctccatctat gcagggtttc ctggaagcac agatgctggc acttcaggga tgaatgaaag
20940tctttttggg ggatttgtag atttttttct tgtcttacta gctccatttt caaatgtatt
21000tattttgtct ctttagtttg cgcgataaca atatctcaga ccgaggcatc tgcaagctca
21060ttgaatgtgc tcttcactgc gagcaattgc agaagttagc gtaagtcagc ctgggctgtg
21120gacaatgggc tccaagtgcc ctggtctcac cccaggtcgt gcagcctggg aagctgtgag
21180tgatgggctg gggcaggggc tgtttgcatg atggggggtg caggtgattc ctgcccagag
21240gggaagggca accctgggat ttggtgctca ctgtccaatg tgctttgctt ctgtgtctcc
21300tctcttctgg aactgaacag tctattcaac aacaaattga ctgacggctg tgcacactcc
21360atggctaagc tccttgcatg caggcagaac ttcttggcat tgaggtgagc ccaggttttc
21420cttattccct ggaaactatt ttttgcccca ttcctgagtc agtctgatct ggtcttggcc
21480tggcactgcc cacactggct cctgacctcc tgattgaatg cagggacagt gtctcatttt
21540aagcaggggt tctctaatgc tgtgatctcc ccagtaaact ctggactagc tctgctgagg
21600acttcctgtc ttttgacctt tagcccgtag ggcaagaaag cttttctagg cccctttcct
21660tttctgtgtc taagagtgtc acagctttct ggggttactg agttccacga tgcatgttga
21720gctcgtcctg gtgggggagg catacacagt tacttgccac cccagctgtg gcagcgagtt
21780gctgcaacac tcccaggagg tcctttcacc actcagagca tgcaaggttt gcagtccatc
21840tggttctgca tttctgctac tccagtgtct cccagtttca acaggagtct ctctctctcc
21900tacctgatgc ctttaaattg cccctctagc tggccgctgg gttggcctgg cttctctctc
21960cttctctctc tctcagatat tcttgcctcc tgtgatttgt gaggcagtaa aaaaagacaa
22020agtaaagaat tgcttccatc tattctttta cctcttgggc tgggtttgtg gatgggagcc
22080gccattttaa aatggcgggc cacatagctc agtctcggca agggctactg agatcagaac
22140cacaggtgcc aatttgtaca aaggactcag tcctgctacc actgcctgat ccctcagact
22200cacaagcctg gaataggctg tggccagacc tggctggccc atccctgaga agggtgctag
22260tttcagaaat ggaggctgag tttgtggcca acacagtagt cctccggtat gtgcaggaga
22320gatgttctaa gaccccagtg gatgcctgaa accatggaga gtatcaagcc ctacacatac
22380catgcttttc ccaataccta cacacctgca ataaagtgta gtttataaat taggctcagt
22440aagagagtaa tagcaactca taataaaata gaacaattat aacaatcaat atactataat
22500aacactatgt gaatgtggac tctctccatc tccctcaaaa tatcttcttg tactgtactc
22560acccttcttc ttgggaagat gtgtggtggt aaaatgcctg tgtgatggga ggaagtgagg
22620tggatgacgc atgcagcact gtgctctagc gctgggctgc tgttgacctg accacacttc
22680agaaggagaa tcatctgctc ccagagatcc ctaatctttg agcaacaatg aggtcggcag
22740ctggatgtca ggagcagacg atcttgatga ttaccaaatg ggagcgtata gagcgtggat
22800gcgctggacg gggggctgat tcacgtcctg ggtgggatgg agctggatgg cacgtgatca
22860gaatagcatg caatttaaaa tgtatgaatt gtttatctct agaattttcc atttaatatt
22920tttggactgc agttgatttc agataactga aaccatagaa ggcgaagctg cggataagca
22980gggggcaggg attaccgtat atcattgtaa tagagagcac aggctctgga gccagactgc
23040ccgaggtttg aaccctcatt agctgcgtga cctcaggtca gcccaatgtc tgtgtgcctc
23100cgtttcccct tctgtagaat ggaggtaata accctggcta cctcacaggc tgtagtgatg
23160agcaagcaag ttaatccaca tgaagggctg caccgtctgg caggggcttt atatagtaag
23220cgagtggctg aaagatgatg ggtaaatcac acaagcactc agcttgtttc tccttatgtg
23280agtccggtcc tccaagcagg gattcaatgt gccacccatt tattggggaa aagtcctaaa
23340aggggaagtg gggaagggag ctgggggagg ctgggaggtg tgtccctgag tgaaggagag
23400agggaaggaa ggaaggttga gactgggcac cttggacttc agtgcagtcc taagacatct
23460tggcaaggct gatgaggagt tcttgaacca aattcaccag gcaggggagc ctgatgtctc
23520aggcaggggc tggcaagtgc agatgcgagg atgttagatt ttggagcaca gcagctgggg
23580cccttggcta cctccaagga gctgaggctg gagacctgaa aggcgagttc tcctagctgc
23640cacacccctt ctccaaggat acaataatat ctgccttata ggattgttgt gagctgagtg
23700gcttgacgtt ccttgaaaga atgaaagcgt atagttatcc caggaagcct agggttgcag
23760gtgagagctc tggggcttct ccgaagctct ccgaggtgtc tggattcagt tgcagcagga
23820gccttccttg ctgggatctt cccccacccc tagccttggc cctccctctc tccttccttt
23880ctggaaggct cagtgggccc cacccctccc tccagccacc tggacctgcc cagcgctctt
23940gtgcaacagg taaagcctac ctgtagcaac aacagatctg ggaaggctgc agagggcacg
24000atggggtctg gatcgagggc ggctgagacc agagggaaag gtgtgaccct gagtcaccct
24060cgctgtcccg gggaaaccac ctcccaggac agctgcctac tgtggctcct gcctggaatt
24120gtcacactgc tgtgcaaaca gcgtcccgct gcccctttcc ctttgctggg ggaaaatgaa
24180gttgtgggag ccgctgagta aactagacct agcagcgagg gcacctgatg tggctgctgc
24240ctcccgggca ggtcttcaat gctttcttcc tgtgtttccc tggccagggc acagacggcc
24300ctccttttct gcctgccgct gtgttctctc agcctcctct gtcttccctt ccaggctggg
24360gaataactac atcactgccg cgggagccca agtgctggcc gaggggctcc gaggcaacac
24420ctccttgcag ttcctggggt aggttggatt ccaggaagag ggacctgcat ggaggggctt
24480gggacttttg aggatttagg ggcaggtgaa actcttcagc caggaggccc cagaggcagc
24540ccagctccag tggggaggac aagccaggga gagagtgggc ggcccttgac tgccaccttc
24600atacttggtc tatgcctgac aaacaggaag tttgggatgt tggggctagg ggaggacagt
24660gcccacgagc tggtgacagg aagccctctg atcctcaggg ggcgctaggg ctgtacttta
24720gctgcatatt aaaaccacct ggaagcttct aaacactatt gccaggcctc ccaccccaga
24780ctgatgaaat gcaaatatct aggtgcaagg cccaggtatc aggagtttta aaaagcttcc
24840caggggatgt acagccaggg gtgaggaccc ctgacctaag aaagagaagg aaatggggaa
24900ggataggaag gcacccagga taagaggggc tgtgctaggt ccctcggagc tcttgctccc
24960tgtaggacca tgctagggcc tgccagggag gggagtaccc caacctgcag ccccagggtg
25020ggcttcctct gtttgctagg cacccaggct tgcacctgtg ctgtttccag cagcctctct
25080cctatcctgt catgccctag tgtgaactgg agtccatttg acaagaactg ggagttttag
25140aacctgggac tgtaggaaga gagaataacc ttagggccta ggtgttccag cccatttcac
25200agggaggcaa gttgccccca agctcagttt tttgttttgt tttgttttgt ttgagatgta
25260gtctcactct gttgcccagg ctagagtgca gtggcacgat cttggctcac tgcaacctcc
25320gcctccttgg ttcaagcgat tcacctgcct cagcttctca agtagctggg attataggca
25380cccaccacca cgcccagcta atttttgtat ttttagtaga gacagggttt caccatgttg
25440gcccggctgg tcttgaactc ctgatctcag atgatccgcc cgcctcggcc tcccaaagtg
25500ctgggattac aggtgtgagc caccgcaccc ggcccccaag ctcagtttga gccacaaatg
25560ggactatgtt gctctagaaa tcaacatctt ttccacactg cattagtagc aacagagtct
25620agaacaaagg aggccacagc cccactgaac tctcttctgc ttgaggtcac atctgccaca
25680tcaggggtat ttacctcttt caacacatat ttattagggc acctgtctgg gccaggcgtt
25740gtgctaaaac ccccaaacgc tgtcatatga tacaaagtgt tctgtaactt gcttggtttt
25800tttttttgtt tgtttgtttg ttttgttttg tttttgttgt tgtttttttt tgcttcgcca
25860tatattatag gaattttttt aggtcattat gacctcttta tttacttaat tatctattta
25920tttattttac taatatttac agaaagggtc tcactctgtc acccaggctg gagtgcagtg
25980gttgcaatca tagctcattg tagccttgaa ctcctgagct caagtgatct tcctacctcg
26040gcctcctgag tagctgggac tacaggcaca agccaccatg cctggccgat atttttatgt
26100tttgtagaga cggggtctca ctatgttgcc caggctggtc tcaaactcct gggctcaggt
26160gatcctccct cctttgcctc ccaaagtatt gggattacac aagtgagcca ccttgctcag
26220cctgacctca tttttcaaag agctgcagag tgttacataa tgtatttaac tggtcacttt
26280ttgatgacta ttaagttgtt ttcaggtttt ttgttattac agtgtcatat ccctggggca
26340cagagcagtg ctggcacata gccagagctc aatcgataca tacctaatga atgaaagtac
26400agtggacatc ctaattcagc cattctttgc taacttgtgt acatacctgt ccagggtagg
26460tccctagaat acagtcaata agtcagaagg tgtgagttgg gatctacctt ttggaaaggg
26520atgttttcaa actacagtga gtcagaggag gatggcccag aagctggggg agttgaagct
26580gatggcgtga aggaattagg ggtgttagga agaagcagga gataaagagc tagcttgcag
26640aagaagtgtt agacttgtta tgggcaggta ctggagggta gctaaggact tgtgggtggc
26700agttaccagg aagcgtatct gaactaagtg tcagaaaaag tgtcacaact gtaaattact
26760cttgtcagtg agttcctgtc cttaagggtt agggctgggt agccctctac tattctctaa
26820gtctgtaatg taaagccact gaaaactctt gggttaagtt tggccatccc acccaaaaga
26880tggaggcagg tccactttgc tgggaccagg agccccagtg aggccactct gggattgagt
26940ggtcctgccc ctctggctgg gactgcagag ggaggaggac tgttagttca tgtctagaac
27000acatatcagg tactcactga cactgtctgt tgactctttt ggccttttca gattctgggg
27060caacagagtg ggtgacgagg gggcccaggc cctggctgaa gccttgggtg atcaccagag
27120cttgaggtgg ctcaggtaag cttcagagtc tatcctgcag ttttcttggg gagatcaggt
27180gaagagggag gagctggggc cagttctgaa ggtctttgaa ctttatttct accccacaat
27240gttaggcaat ggagtaagga aaaaagacca ttggatttca agagaggaca cttgagtctt
27300tctgggtgac ttggaaatgt cccttgtcct ctcagggttt tgatacagta tctgtaaatt
27360gaagatattg ggctggatca ggtacatttt atcttaaggg ccaattccaa tccattggta
27420gtgggtgccc agtgcaccac attaaaaaga attctaaggc tgcacctggg cttaaagaag
27480agcactataa tcaattagtg atgtctaaaa aagctaaaaa aaaaaaaaaa gagcactgca
27540ttcaattagt gatgtctaaa aagggtagaa aaaaaaaaaa aaagaaaaaa gaaagagcac
27600cgcaatcaat tagtgatgtc tgaaatggag cagaccagga gagcaccacg aattttgccc
27660tccataggtt agctcatctc tgaggtcttt ccctgctctg acatactttt gttccatgat
27720tacctccagc ctggtgggga acaacattgg cagtgtgggt gcccaagcct tggcactgat
27780gctggcaaag aacgtcatgc tagaagaact ctggtgagtt tgggggattc tctgctctgg
27840ggaagtggat cacaatctct gttgatcccc tggcctcatc cataggagcg gttgtgtgga
27900cagacaaagg tggatgattg agtgattgac tgattgattg attgtgtttg tctttatatg
27960tactgagtgg tatgaagctt atagagcctg gtatgtacat gctaattttt ttatttaata
28020aaatatatgg gtttgctggt ttggtgactg cctccacatg gcataagtgt taagagcaca
28080gactctgtaa tcaagcaggc cgtgatctta ggcaagttaa ataacaattt cagaatctca
28140agtttcatgt ctgtaaaatg agggtaagaa tacttccaac cataaaggat ttttgcaaga
28200attagataaa gtagtgcctg tgaagacctt aatatagtgc ctggcatatt tgtaagtgct
28260ccataaatgt taaattagaa taatggcagg gttactacta ctattactgc tgctgctgct
28320gctgctgctg ctacaactac tatagtactg tgactactac tactaataaa gttttgttat
28380tttaaagtga ttttgagttc ctaggagcac tgggtattca agtcttaggt cattttggaa
28440ggtgtaatgg agttttgata gttgaaagag gaaccatgaa tcatgcttat actgttgacc
28500tgaagcagat tctaagtttc tcatccttta gatgccacta gtatagtttt ctgacatgtt
28560ctgggcagct tcagattatg tcagggagat aaaatactga atgtttgatt ttcccgggaa
28620gcagaaaggc actgcaacat atgggcattg ccataaacag attttatgga tggaccttgg
28680ctgttgcagg gcttactagc tctactcaag tatgattgat tctatcctga ctggattttg
28740ccacttggaa tttcttagta gaggagaacc ttgttatgag agcatcagtt atgattactg
28800ttaaaagaaa aactttaggc aaattaaatt tagcagaact ggtttgaaca tacagcaatt
28860tatgaattgg gcagcattca gaactgggag tgctccaccc agcaaggtag gcaagcagta
28920tctatagaca ggaaaaggaa gtgatgtaca aaacagcttg attggttgca gctgggcatt
28980tgccttatat gggcatggtg tgatgaggca ttttctttat atggatatag actgatcagc
29040tggtagactg tgactgactg aagcctggct gctgtgattg gctaagactt agctgtttgt
29100tataaggata tgttgttagg ttgcagtttg ctacatagga actcaaagta cagaggcagt
29160ctcaggccaa atttagttta actatatgtt aagctgcagg tgacagaata cctccatcta
29220tagaggttta aacaaggaaa gggtttattt tttcctgtat aggcagctgg atgtaggcag
29280tgtagggttt gtacagtggc tacaagaggc caggaggggt ctcagctctg tctcattctc
29340ttcctgttcc atcatcctta gcctgtaact tcattcacat ggttggttgt ctcatgatca
29400caggatggct gctccaggtg cagcactact tctgtattcc cggattcgat ctatataccc
29460aggaaagcca tctgggttct ctcctttaaa aagcattcct ggaagcccca cctgtcgact
29520tccccttatg tatcaaccat gtgtatgtca cttgaccaac ccacttgtat gttgtttgac
29580cagccctggc tgcaatggag agtgggaaat acagtttttt caccaagtgc atggctgtcc
29640aaatgaaatg agacttccat taataaggaa gaaaggaaag atggagatca ggaagctggg
29700ggatcaggga acttattaca ttgagagccc ttggagtgaa ttctcttgca aatatgtccc
29760tggaattgag aatccccaca acgtctttat ctgttctttc tttatccatg agtttgggtt
29820ttcagatgtt ggatttccta tatggggggc atgtgagttc atcatcttcc ataatcaatg
29880ttgtatcaac tggattttct ctcttcttct caccagcctg gaggagaacc atctccagga
29940tgaaggtgta tgttctctcg cagaaggact gaagaaaaat tcaagtttga aaatcctgaa
30000gtaaggaacc cataagcagg aaacaggaca ataattgctg gcctttggaa ggggcatttc
30060tgattaagat ctgggccgct ctccgctggg ctaactcatg tgaggtggcc tggtagaaca
30120gcttgccttg gtctaggtgg acaaggattc cagtgcaagt tgtttatctg ggaggtggtc
30180ccagtaaatg ctgataggag agtggtgaag tgagatgggg aagtgaaggt aaccaataaa
30240ggggagttat caagccagtt atcaatgagg gaaattggag ctcagtactc tggggcactc
30300ctggagccag tgcagaacac acatggtcac ctacccaacc aatgggcaag aaagccatgg
30360catttatcca ccaaccctct gtccttccta tgttgatgtg cgctcatggg gcactgattc
30420tccagcactt ccagctcacc ctcacccagc tgaacatgct tctggggtca ggagaatggc
30480ctcaggcaga gagtggcagg tcttctctgc aagcagtggc tggggaggtg atgtgatggg
30540gagtactgtg gcctcctcca gtggctgact cagtggcttg ggacttgtgc cacaaagaga
30600tggacagctc aggtgaacat gaacccacct agtgaccatc atgggtttgt cagggtgctc
30660tctgaggctg atgccaaaat tcttatttca agtagacctc aggaacccca tcagatggct
30720ccttttgctg gaggaaagtg gcatctgcct aggcaaatgt ggtcctagga aaacgcttgc
30780ctttagagac agacagacag acagctgcct ctgtgagtgc cagctttgct gccaggctgc
30840tacccactct ggcgacactc atttgtgttg ctttcacaag ctaggaagtt tccaaatatt
30900tggagaaaac acttccacta attatttggg tggaaatggg ctgggaagtt ggggtgaagc
30960ccggatgtgt ctgagccaga tgccagcttt gcactgaggg tcggcctttg ggaataccaa
31020gcccattatc aaccaggtgt ggatatggca ggtttgtctt ccctccttgt cacagcctta
31080ctccacttga ctcccatgga tgccaggcaa tgaggctggg gttggtccca tgccaccctg
31140tcatcagcct tatttttcag catcctaaac tatatcatcc cccacaaaaa ttgaacttct
31200gatatatctt ttataaaaaa gagaaatgcc tacatctttc ttttccagga ttagtttctg
31260ccaagagttg gttgagagcc caggcttgct gggtgcagtg gctcacacct gtaatcccag
31320cactttggga ggctgaggcg ggtggatcac ctgaggtggg gagttccata ccagcctgac
31380caacatggag aaaccccatc tctactaaaa atacaaaatt agccgggcgt ggtggcatac
31440acctgtaatc ccatctactc aggaagctga ggcaggagaa tcacttgaac ctgggaggtg
31500gaggttgcca tgagccaaga tcacaccatt gcaccctaga ctggacaaga gagaaacttc
31560catctcaaaa aaaaaaaaaa ggatgagaaa aataataatt taaaaaaaag agtccaggct
31620ctggaaccag acagcctggg tcttacccct gctccaccat taccagccag ttcttcttgg
31680atgagtgcct cagttgcctc aagtgtaaat ggagataatg gctggacctt cattataggc
31740catgagcatt cactgagaga atgtagctaa caaaagtgag ttgtaggttg gagcaaaagt
31800aattgtggtt tcagaccatg aactttaaat tattataact aggctaaaat acatctttat
31860taatcaaaat aggaaccatt aaaatcaaca catttttgcc aataagaaat aagtttgttt
31920attcctgtag cataaaaatt catgcttcgg gattcaacaa actcttggaa agcattttct
31980gcatcctcct ggttgtggaa gcatttttcc tgcagaaagt tgtcaagatt cttgaagaaa
32040tggtagtcag ttggctagag gtcaggtaaa tatggcggat gaggcaaaac ttcatagtcc
32100aattcattca acttttgaag ctttggttgt gtgacatgca gtccggttgt tgtcgcggag
32160aattggaccc tttctgttga cgaatgccgg ttgcaggtgt tgcagttttc agtgcatctc
32220attgacttgc cgagcatact tctcatatgt aatggtttcg cagggattca gaaagctgta
32280ggggatcaga ctagcagcag accaccagtg accatgacct tttttttttg gtgcgaattt
32340gcctttggga agtgctttgg agcttcttct cggtccaacc actgagctag tcattgccag
32400ttgtataaaa tccacttttc atcgcacgtc acaatcagat caagaaatgg ttcgctgttg
32460ttgtgtagaa taagagaaga tgacacttca aaatgacgat tttcttggtt ttcactcagc
32520tcatgaggca cacacttatc gaggtttttc acctttccaa tttgcttcaa atgctgaatg
32580accatggaat ggtcgatgtt gagttctcaa gtagttgtaa gaaaatcagc tttgatgatt
32640gctctcaatt ggtcattgtc agcttctgat ggcctgccag tacactcctc atcttcaagg
32700ctcttatctc cttcgcaaaa cttcttgaac caccactgca ctatacgtta gttagcagtt
32760cctgggccaa atgcattgct gatgttgtga gttgtctccg ctgctttaca acccattttg
32820aattcaaata agaaaattgc ttgaatttgc tttttgtcta acatcatttt catagtctaa
32880aataaatata aaataaacag aaagtattaa gtcattagca aaaaatcata aagtgagaat
32940tgtgcattaa aatgatgtat agcataacca catttattta agaatgtatt ccaatatcaa
33000atggcaaatt tcaacaatgc aaaaactgca attacttttg caccaatcta atagaagttc
33060aataaatact ggcaattaca attggcattg ccttagggtc aacttgtaag acattcctga
33120aattgtggga aagggggagg acctggagtg gacattattg gaaggcaaag ctgtaaccaa
33180aagagcaacc tgggaaacac atgactcctc tgttgctgtc cctggcccta tcctgtctcc
33240cctccctgtt gtcagctacc tcatatgttc tctaatctct gtctctgtgc cctcaaagac
33300ccccctgaaa atagaaatat tactgctcat tggttatttt ctatcaatta agtactgtat
33360tagtccgttt tcatgctgat gataaagata tacccaagac tgggcacttt atgaaagaaa
33420gagttttatt gaacttacag ttccacgtgg ctggggaggt ctcacaatca tggctgaagg
33480tgaaaggcac atctcacatg gcagcagaca ggagaagagg gcttgttcag ggaaactccc
33540ctttttaaaa ccatcagata tcatgaaact tatttactgt aatgagaaca ggatgggatt
33600caattacctt ccactgggtc gctcccacaa cacgtgggaa ttcaagagat ttgggtgggg
33660acacagccaa accatatcaa gtactgtgca agtgttttag gcatgcagag agtggtgggt
33720cttcccagca agcagagtgt ggggaggtaa tgggggactg gtggctgact taatggccca
33780ggacccatgc cacaaggaga tggatggtgg atgtgaatag gagcctgctt acacccatca
33840caatttagat tcttatgctc gatggcacgg gtactctttt aggcccattt taccaatgag
33900gagattggga ctaatttgct cgagatcaaa aaagaagtgg tgtaggtggg atttaaaccc
33960aggatgtcta gcactaaaat gcaggtactt aaccactatc ctaagggagt ggctacttaa
34020tttgataaac tcatctagtg aatggaagag agacggttac atttcactga tggtactgag
34080cctttgttga tgagctcatt gggaatctca gacatgagca ggatgtgtct aagggacagg
34140tgggcttcag tagactggct aactcctgca gtctctttaa ctggacagtt tcaagaggaa
34200aaccaagaat ccttgaagct caccattgta tcttcttttc caggttgtcc aataactgca
34260tcacctacct aggggcagaa gccctcctgc aggcccttga aaggaatgac accatcctgg
34320aagtctggta aggcccctgg gcaggcctgt tttagctctc cgaacctcag tttttctatc
34380tgtaaaatgg ggtgacggga gagaggaatg gcagaatttt gaggatccct tctgattctg
34440acattcagtg agaatgattc tgcatgtgaa ggatctgatt ctctgtctaa gaaagaagtc
34500tttacctctt taagtaggga gcaatgattt catttttaaa ccttgactat ttattcagca
34560acttctctgc tctatgagat agtgtaggaa tggggatgtg gttgaagaat gaaaagaaaa
34620gtcagctccc gccctcctag aaattgcatc tgccttcaca ggtcaaggat attggatcag
34680accttctgcg gttctgaatg gagattacac aggttaggag caggttgcac agtgtttcca
34740attctctata attaaagcca tagactttca tgtattgaaa aaagcaagaa ttgcattctt
34800gacagattct ttcattgcct taaaaagaat gactagcctt gggagtctgg gcagctgggt
34860ccagtgttgt agactttctc tctgctgagc cacagcttca aagatttgtc cttcttgttt
34920ccagggatct atttctcaga caataagtaa aggctttccc tggcctaatg tgctgtaagt
34980gaatgctact atatatgttc caggcactgg gctagagact aatatttaaa agccaggaaa
35040tttcctatag aaaatctata tctcagggtt ttctcaaaag agctgggaac tctggatgcc
35100cattcatgat tccagtagtt aaccagagta caagaagggc tgagtcttct cagatgggca
35160aacccactct ggctgactgc agatccacca agcctattgt cttagaccag gaccctttgg
35220caactcattc ccataagcct gtgacccttg ctttaaatat gcaggccttg tcttctctca
35280aaaagcacat caaggctgca gcgaatgcag atatcaaatg atgaagttaa aaacaaaagc
35340tttgctgggc gtggcagctc acacctgtaa tcctagcact ttgggaggct gaggcaggag
35400gatcacttta ggccagaggt tcaacaccag accttgtctc tcaaaaaata aaaaattcag
35460ctgggtgcgg tgtagttcct agccacttgg gaggctggga tggaaggatc ccttgaaccc
35520aggagttcaa ggctgcagtg ggccatgatt gcatcactgc acaggcgaca gaattagatc
35580ccatctctta aaaaaataaa aaatttaaaa gtgacttcaa aaatctatgc tgtgatggag
35640agatttttcc ttctgtatga ttgtgatagc tctgtggcct atgacgtcat caggttctgg
35700gcaaagtgta ggttttctgt ttctttgttt ttgaaaccat tgcacagtcc taagaaacat
35760cacattctgg gtcctgggca ccagccaaca tgaggtgagg gcaccagggt ttgctcattg
35820cattcttgac agattctctt attgccttaa aaagaatcac tggccttggg gagtctgtgg
35880ctggctgggt gcagtgttgt ggactctctc tgcagagtca tggagccttg ttcagaatgc
35940ttcctgagct gccctggttg gccaagggta aaaacagccc tgacttccct gcaagaaaca
36000ctgcagctgg gccagagagt cagcccatcc caggcatggg tttaaaaagt ggaggctttt
36060gtttgaaagc cctgctctaa ttttgtcctc actcaaacct ctgttcactt gatctgcttt
36120aggctccgag ggaacacttt ctctctagag gaggttgaca agctcggctg cagggacacc
36180agactcttgc tttgaagtct ccgggaggat gttcgtctca gtttgtttgt gagcaggctg
36240tgagtttggg ccccagaggc tgggtgacat gtgttggcag cctcttcaaa atgagccctg
36300tcctgcctaa ggctgaactt gttttctggg aacaccatag gtcaccttta ttctggcaga
36360ggagggagca tcagtgccct ccaggataga cttttcccaa gcctactttt gccattgact
36420tcttcccaag attcaatccc aggatgtaca aggacagccc ctcctccata gtatgggact
36480ggcctctgct gatcctccca ggcttccgtg tgggtcagtg gggcccatgg atgtgcttgt
36540taactgagtg ccttttggtg gagaggcccg gcctctcaca aaagacccct taccactgct
36600ctgatgaaga ggagtacaca gaacacataa ttcaggaagc agctttcccc atgtctcgac
36660tcatccatcc aggccattcc ccgtctctgg ttcctcccct cctcctggac tcctgcacac
36720gctccttcct ctgaggctga aattcagaat attagtgacc tcagctttga tatttcactt
36780acagcacccc caaccctggc acccagggtg ggaagggcta caccttagcc tgccctcctt
36840tccggtgttt aagacatttt tggaagggga cacgtgacag ccgtttgttc cccaagacat
36900tctaggtttg caagaaaaat atgaccacac tccagctggg atcacatgtg gacttttatt
36960tccagtgaaa tcagttactc ttcagttaag cctttggaaa cagctcgact ttaaaaagct
37020ccaaatgcag ctttaaaaaa ttaatctggg ccagaatttc aaacggcctc actaggcttc
37080tggttgatgc ctgtgaactg aactctgaca acagacttct gaaatagacc cacaagaggc
37140agttccattt catttgtgcc agaatgcttt aggatgtaca gttatggatt gaaagtttac
37200aggaaaaaaa attaggccgt tccttcaaag caaatgtctt cctggattat tcaaaatgat
37260gtatgttgaa gcctttgtaa attgtcagat gctgtgcaaa tgttattatt ttaaacatta
37320tgatgtgtga aaactggtta atatttatag gtcactttgt tttactgtct taagtttata
37380ctcttataga caacatggcc gtgaacttta tgctgtaaat aatcagaggg gaataaactg
37440ttg
3744341315DNAHomo sapiensIBD1prox cDNA 4cgatcagaag caggtcacac agcctgtttc
ctgttttcaa acggggaact tagaaagtgg 60cagcccctcg gcttgtcgcc ggagctgaga
accaagagct cgaaggggcc atatga cac 119
His
1tcc tcc cgg acc cct gga cac aca cag ccc tgg aga ctg
gag cct tgg 167Ser Ser Arg Thr Pro Gly His Thr Gln Pro Trp Arg Leu
Glu Pro Trp 5 10 15
agc atg gca agt cca gag cac cct ggg agc cct ggc tgc atg gga ccc
215Ser Met Ala Ser Pro Glu His Pro Gly Ser Pro Gly Cys Met Gly Pro
20 25 30 ata acc cag tgc
acg gca agg acc cag cag gaa gca cca gcc act ggc 263Ile Thr Gln Cys
Thr Ala Arg Thr Gln Gln Glu Ala Pro Ala Thr Gly 35
40 45 ccc gac ctc ccg cac cca gga cct gac
ggg cac tta gac aca cac agt 311Pro Asp Leu Pro His Pro Gly Pro Asp
Gly His Leu Asp Thr His Ser 50 55 60
65ggc ctg agc tcc aac tcc agc atg acc acg cgg gag ctt cag
cag tac 359Gly Leu Ser Ser Asn Ser Ser Met Thr Thr Arg Glu Leu Gln
Gln Tyr 70 75 80 tgg
cag aac cag aaa tgc cgc tgg aag cac gtc aaa ctg ctc ttt gag 407Trp
Gln Asn Gln Lys Cys Arg Trp Lys His Val Lys Leu Leu Phe Glu 85
90 95 att gct tca gct cgc atc
gag gag aga aaa gtc tct aag ttt gtg gtg 455Ile Ala Ser Ala Arg Ile
Glu Glu Arg Lys Val Ser Lys Phe Val Val 100 105
110 tac caa atc atc gtc atc cag act ggg agc ttt
gac aac aac aag gcc 503Tyr Gln Ile Ile Val Ile Gln Thr Gly Ser Phe
Asp Asn Asn Lys Ala 115 120 125
gtc ctg gaa cgg cgc tat tcc gac ttc gcg aag ctc cag aaa gcg ctg
551Val Leu Glu Arg Arg Tyr Ser Asp Phe Ala Lys Leu Gln Lys Ala Leu130
135 140 145ctg aag acg ttc
agg gag gag atc gaa gac gtg gag ttt ccc agg aag 599Leu Lys Thr Phe
Arg Glu Glu Ile Glu Asp Val Glu Phe Pro Arg Lys 150
155 160 cac ctg act ggg aac ttc gct gag gag
atg atc tgt gag cgt cgg cgc 647His Leu Thr Gly Asn Phe Ala Glu Glu
Met Ile Cys Glu Arg Arg Arg 165 170
175 gcc ctg cag gag tac ctg ggc ctg ctc tac gcc atc cgc tgc
gtg cgc 695Ala Leu Gln Glu Tyr Leu Gly Leu Leu Tyr Ala Ile Arg Cys
Val Arg 180 185 190 cgc
tcc cgg gag ttc ctg gac ttc ctc acg cgg ccg gag ctg cgc gag 743Arg
Ser Arg Glu Phe Leu Asp Phe Leu Thr Arg Pro Glu Leu Arg Glu 195
200 205 gct ttc ggc tgc ctg cgg
gcc ggc cag tac ccg cgc gcc ctg gag ctg 791Ala Phe Gly Cys Leu Arg
Ala Gly Gln Tyr Pro Arg Ala Leu Glu Leu210 215
220 225ctg ctg cgc gtg ctg ccg ctg cag gag aag ctc
acc gcc cac tgc cct 839Leu Leu Arg Val Leu Pro Leu Gln Glu Lys Leu
Thr Ala His Cys Pro 230 235
240 gcg gcc gcc gtc ccg gcc ctg tgc gcc gtg ctg ctg tgc cac cgc gac
887Ala Ala Ala Val Pro Ala Leu Cys Ala Val Leu Leu Cys His Arg Asp
245 250 255 ctc gac cgc ccc
gcc gag gcc ttc gcg gcc gga gag agg gcc ctg cag 935Leu Asp Arg Pro
Ala Glu Ala Phe Ala Ala Gly Glu Arg Ala Leu Gln 260
265 270 cgc ctg cag gcc cgg gag ggc cat cgc
tac tat gcg cct ctg ctg gac 983Arg Leu Gln Ala Arg Glu Gly His Arg
Tyr Tyr Ala Pro Leu Leu Asp 275 280
285 gcc atg gtc cgc ctg gcc tac gcg ctg ggc aag gac ttc
gtg act ctg 1031Ala Met Val Arg Leu Ala Tyr Ala Leu Gly Lys Asp Phe
Val Thr Leu290 295 300
305cag gag agg ctg gag gag agc cag ctc cgg agg ccc acg ccc cga ggc
1079Gln Glu Arg Leu Glu Glu Ser Gln Leu Arg Arg Pro Thr Pro Arg Gly
310 315 320 atc acc ctg aag
gag ctc act gtg cga gaa tac ctg cac tga 1121Ile Thr Leu Lys
Glu Leu Thr Val Arg Glu Tyr Leu His 325
330 gccggcctgg gaccccgcag ggacgctgga gatttggggt
caccatggct cacagtgggc 1181tgtttggggt tctttttttt tatttttcct tttctttttt
gttatttgag acagtcttgc 1241tctgtcaccc agactgaagt gcagtggctc aattatgtct
cactgcagcc tcaaactcct 1301gggcacaagc aatc
13155334PRTHomo sapiensIBD1prox 5His Ser Ser Arg
Thr Pro Gly His Thr Gln Pro Trp Arg Leu Glu Pro 1 5
10 15 Trp Ser Met Ala Ser Pro Glu His
Pro Gly Ser Pro Gly Cys Met Gly 20 25
30 Pro Ile Thr Gln Cys Thr Ala Arg Thr Gln Gln Glu
Ala Pro Ala Thr 35 40 45
Gly Pro Asp Leu Pro His Pro Gly Pro Asp Gly His Leu Asp Thr His
50 55 60 Ser Gly
Leu Ser Ser Asn Ser Ser Met Thr Thr Arg Glu Leu Gln Gln 65
70 75 80 Tyr Trp Gln Asn Gln Lys
Cys Arg Trp Lys His Val Lys Leu Leu Phe 85
90 95 Glu Ile Ala Ser Ala Arg Ile Glu Glu Arg
Lys Val Ser Lys Phe Val 100 105
110 Val Tyr Gln Ile Ile Val Ile Gln Thr Gly Ser Phe Asp Asn
Asn Lys 115 120 125
Ala Val Leu Glu Arg Arg Tyr Ser Asp Phe Ala Lys Leu Gln Lys Ala 130
135 140 Leu Leu Lys Thr
Phe Arg Glu Glu Ile Glu Asp Val Glu Phe Pro Arg 145 150
155 160 Lys His Leu Thr Gly Asn Phe Ala
Glu Glu Met Ile Cys Glu Arg Arg 165 170
175 Arg Ala Leu Gln Glu Tyr Leu Gly Leu Leu Tyr Ala
Ile Arg Cys Val 180 185 190
Arg Arg Ser Arg Glu Phe Leu Asp Phe Leu Thr Arg Pro Glu Leu Arg
195 200 205 Glu Ala
Phe Gly Cys Leu Arg Ala Gly Gln Tyr Pro Arg Ala Leu Glu 210
215 220 Leu Leu Leu Arg Val Leu
Pro Leu Gln Glu Lys Leu Thr Ala His Cys 225 230
235 240 Pro Ala Ala Ala Val Pro Ala Leu Cys Ala
Val Leu Leu Cys His Arg 245 250
255 Asp Leu Asp Arg Pro Ala Glu Ala Phe Ala Ala Gly Glu Arg
Ala Leu 260 265 270
Gln Arg Leu Gln Ala Arg Glu Gly His Arg Tyr Tyr Ala Pro Leu Leu
275 280 285 Asp Ala Met Val
Arg Leu Ala Tyr Ala Leu Gly Lys Asp Phe Val Thr 290
295 300 Leu Gln Glu Arg Leu Glu Glu Ser
Gln Leu Arg Arg Pro Thr Pro Arg 305 310
315 320 Gly Ile Thr Leu Lys Glu Leu Thr Val Arg Glu Tyr
Leu His 325 330
68135DNAHomo
sapiensexon(1)..(161)exon(3812)..(3950)exon(5426)..(5577)exon(7273)..(813-
5) 6cgatcagaag caggtcacac agcctgtttc ctgttttcaa acggggaact tagaaagtgg
60cagcccctcg gcttgtcgcc ggagctgaga accaagagct cgaaggggcc atatgacact
120cctcccggac ccctggacac acacagccct ggagactgga ggtcagtatt tgatcccaag
180ctcagctgtc ctctgcctgc tgtggcctga gtccccttct cctggggccc tgcctggcac
240ctgctggggg cagggtggga gggggaagag ttagtgacag ccgctgtgtc tggagctctc
300cttagcacac tgaggcagag gaagggacag ctcctggacc ttccatcacc tccattcctt
360ttgaaatgct aggcgcttgt acaacccatc ttgggcctgg agaataagtc accacacctg
420tgtttctcaa aagaacagtg tcagggaacc cctgcctcag cacagcctta gaggactcat
480ggaaaatgca gaatccaggc ctgttcaatg gcaccttcct atgttagcag ccaggaaacc
540tgctcttgga caagcccctg ggatcccacc cccaccccac caggggattc ttacacacac
600tgggttggga gcccctggct ttggcaaggc ttctcaggtg agcgtccagt tgttggaggg
660tacccaccct ttccccaaga gaggcagcca cacatccaac atcctgggat ctctgtctcc
720cagcgtgggc catgtgcttt atttcacccc ctagaggctc atcccccatg aaaagtcctc
780cgcaggccct cagaaagata gtgtggcctc tgtgtgccca gcagaagaag gactggactt
840ggcagtcagc tcttggagag ggggtggtta ggacacctgg ggacaggagg aggagaatga
900ctgtctgtgc acacacggct ggaaggtaca ggaggctggg aagctgctct gtcccctggg
960ccaactacag gcccccaggc caacagcaac aacactttta gtattttgtt ataaagtcaa
1020gaaatctttg ctacagaggg tgaggagagg gaaggaaagg gccatggaac cgtctatgtg
1080gctatcccca gagagctttt agagtgacag gattgctttc ccatttcaca gatgaggaaa
1140ctgaggcctg gagagggatg ggaagctacc caaggcccca tggatacacc agtgcacaac
1200tctttccttc cccctcctct ttaaatgggt gattcccaat gaaacctgta agagacaacc
1260ataagggagc tgactgtggc tgctgaattt gattttattc taaggcctgg ttttataatc
1320agctttctca gtctttactg gagtgtcaag ccgaggcatc atttctaggg tcttacaggg
1380tctctgggcc aatagtgccc tgcttctgac ctggagccag ctgcctggtc atgaaagcag
1440atctgcaaag gctggggccc ctgaggccaa ggccactcgc catcacccat tttacagaag
1500tgctgagcat aggagtgccc tgggccccca agaatcccag ccaccaagaa tcacgtaaac
1560catccactgt ctcacttagg caccagtcag aatgtaggga acccacccct agtcatccat
1620catcttatca acaggacggg gcttgtagcc acatttatca ggtagggaaa ctgaagccta
1680gagatattaa agcacttgct taaggacaca cggttggtca ggatggaagg cgatgtctcc
1740tgactccctg acaggcacaa gagacaagcg agaggtgccc gtgacggcat gctcaagaac
1800gtgcagccct gggccagcca ggcccctgct ccgtgcctct gtttgcccat ctgtaaaagg
1860tgaggttgga tcgagggtcc ctgagggccg cccactggat ggctgtgcag agccaaacgg
1920agaaggcccc agggttcctt tcacccgaca cagcaagcac ttccccctga agtgcaggct
1980ccaggcccca gctgacctcc cctctcccag gccagcggct ctcacccctg gagcaaggga
2040caggcgctgg ctgtgctcag ggacatgcat gactcccgcc cccatctgtg ctcagggggt
2100gccagggagg cactggctct atctttctct aggccgtagt cagcccaggg gttcagacca
2160agagcccaga atccaacaga tcagagttca agtcccagct ctacctctat gttccactgg
2220cagcttcctc aggtcatttg caccttcctt gtcttgaatt tccatgccta accagtatac
2280cagctactcc ctccagccga tctaatgttt taattgtccc tttctctaag ttgtctcaaa
2340catttgtaat tctattccaa tccaccttaa tttagtcatt tatttcacaa atatttctgg
2400aaacatctag cacttaacag acactaaaag cgggggtact acacagtccc tgggatggac
2460agggccctga gctgaggctt cagagtctgc ctgactgaat cctcacccca gccttgtgaa
2520cgtgggttct gttattatcc ccaatttata ggaaacagaa gcacagagaa gttgagtcac
2580ttgccagcta ccaggtcatc ccttccactt atccgggtca cagacagagt tattatgtaa
2640accagatccc agctgcctgt tctccctccc tgagtaaggt ggagagaatt ctgaagtcag
2700cccagcctgg gtctgtatcc tgcccaccac tcaccagctc ctcatctttg gcaactctaa
2760gtctcagttc ccttatcata aaagggagat gtaaacagtc ctgagtgcag acagtgttca
2820ggttagtgca agagtgtgtg ctgggtgtga agtgcacagc cagcacgtca caagcactgg
2880agacaaattc agctttgctt gttgcgcaca ctcaccagct gcgtgacttt agacctcagt
2940tttctcatct gttatgtggt ggtaatgata gacttttgtg agcattaaac tagattaggg
3000gctatggaga acctagatgg gtatgaagtg ggtataataa gctatcagtt aattttgctg
3060atagatagat tattgattga ttgatcgata gaagattcat accagtatct acctgctctg
3120aacactgacc tttctttttt tctttttgag atggtcttgt tctgtcaccc agactggagt
3180gcagtggcat catcatagct cactgcagcc tcagtctctt gggcttaagg gatcctcctg
3240tctcagcctc ccaagtagct gggaccacag gcgtgcatcc tggataattt ttttttattt
3300tttctagaga cggggtctca ctacattggc caggctggtc tcaaattcct gggctcaagt
3360gatccttcta acccagcctc ccaaagcgct gggattacag gcatgagtgg ccatgttcaa
3420cttgaacact gagacttcat tcgcatgtgt aacataaaac tgagtatcta gacaagccag
3480catctttctt tcaagtaatc actaaagcca atacttttac ttgaaatcat ctcatttaaa
3540actctgagca atacgtaagg atcacctcaa taacatatgg atcatcgcaa taggtgaagg
3600gtcttctctg ccttggagta acctgcccag caaaggggca gacccagatt tgggatctgg
3660cagctgggag agtggggaag gttgagccgt ggggcccttg tcattccctc tgcctgccag
3720gagggggcat gacacagctc ctaggcaccc caggagccac cgggaacccc aactggagtg
3780ggtcctcact gttctctttt tcctctggca gccttggagc atggcaagtc cagagcaccc
3840tgggagccct ggctgcatgg gacccataac ccagtgcacg gcaaggaccc agcaggaagc
3900accagccact ggccccgacc tcccgcaccc aggacctgac gggcacttag gtgggcttga
3960ggcttgagac tcggtctggg ggagaggtct gaagacattc aaagtacaaa tgtgggtcac
4020tttgggggat gcagcaagag gcccgggcag ctcttgtaac ttgggttatc ccaaaacaga
4080cactgagaca cagatctagt gcaagctgtt tatccgggag acggtcctag gagtcatggc
4140aggggagtgg gaatggaagg aaagggcaag aggccagggc aggacatcag tgaacagata
4200ggcacggtag gtggctgaag ctcaacccca gcgggggtct tctgggagac cctggaacat
4260atctctgggt tgtcctatcc taggggtgag gaagccgggc tgttatctac cagtcctgcc
4320ctgcatagga gaagggacgc tcctgggcct gctgctatgg ccctagaaag ccctcaggga
4380agccagtggc atgttctgga aaagtgggtg ccaagagggc acggtccagc ctggggcatg
4440gacagcatct gctgtagtgc catctcctgg aacagatctt ttcttacagt ccttcgagat
4500gccctattca atacctgctc tgttcctggc cctatgcagg gcactggaga aacagaaaca
4560ggaagaaatc aaacactgca ctagtcctga ggtttggtag agaaacagat cagtgagaaa
4620cagttacacg tgccacgaga aataaataaa taaaatgaaa aacctgtagg aacaaggtgg
4680gaagctctta ctctaatgcc aaggggcatt tgcagtgatg tgggggctgg gtcttgaagg
4740gtagactgga aaagggctgg gacccatgcc ctttgcaata aaatgcacaa ttatttgtgc
4800ttcttaagaa cctcagagtg gcgcagggct caagtggggt ttaagaaaca ctgtgttcgt
4860tttccaggcg tggaaataga gggttggatg caaggcagag cagtgcacgt ccgagaagag
4920cccggcatgt gggcagttag atgagaaggt taggaagggc cagcccgctg aggctggaac
4980ataacatcct cctcactgcc tcccctgccc actgatgtgt gctcaaggag tcgtggcaac
5040agtcacgaag tcagggctgc agggagcaca gaaacacaca agccaccgtc tctgcttgtc
5100cagagcaggg atttcaccat ggccaatcta cagaccagaa gtggacgatg caaagtgccc
5160gcaccgcatt ccaaagctgt gaaaccactt gggggtgatg ggctatttgg gattgtcggt
5220ggtagggtgg attctgccag gctgggcaca gaggtctgtc tgatgcccca attgggccta
5280taaatggcgg ggtgggagag agggatattc aatactcttc aggagttctg atatgccatc
5340tcagatagac ccagccatct ccccaagccc atgcctcgga agtgcactga cagggtgcag
5400atccttaagg gtgttgtcct tccagacaca cacagtggcc tgagctccaa ctccagcatg
5460accacgcggg agcttcagca gtactggcag aaccagaaat gccgctggaa gcacgtcaaa
5520ctgctctttg agatcgcttc agctcgcatc gaggagagaa aagtctctaa gtttgtggta
5580agcagagatt gggaaatggt ggagcctctt tcactctgct tccttcctgg ccctgaataa
5640gtcttgtaga gcctcaggtt tcccaactat gaaatgggtc aacacactaa ctcacagctt
5700tcttctggag aaaatggcca aagagcaaga tttcaggctc agcacctgct agggtctgtg
5760aggattcgaa ccatataagt catatttctt ggtcccaaga aggaaatagc ccagtttaat
5820cccatcttat caggtgtcag tcacctgtgt cctttcttca ccaattttgc catatcactg
5880tatctgttct aattattatt acttattttt ttctttaaat tggatcactt tttaaaaaca
5940tgaagcacat ttatttcaaa gagaaatacc ttaaatggaa aaccaatatc acatggcaca
6000aagcaaaagt aacatactag aaaagtcgat acaaggaaag tcaatacaag gaaagctatg
6060tgctgttatt aaattctagc tggttactgt ggcttcggga aagccctgtg cctgggagct
6120gctcctctcc ctgttagaat ggaattttag cttgtgttaa gggatgttaa agactgccta
6180agagccacac ttcatccttc tccttcactt acctgggacc gggataaata acatagctac
6240cactgaatgc caatggcatg ccgggcacag ctccatgtgg tttcagtgca ttaactcatt
6300taatcctcac tgggtgaggt aggcactatg cctatccttg ttttatgaat gagaaaagtg
6360agactcggag aggttaaatt actcatctaa aaccacacag ctagaccatg gtagggctat
6420aattacaacc catgcaatct ggctctggag tcagatgcat gggttataat tgcccttaat
6480atataattgc ccgtaatcag gattctcttg aaagatgatt gaaaaggatt gattttctta
6540ccatataacg gcatcaccag tgtacctaaa tgatgttata ttgtacgtaa aactaattcc
6600caagtgtgaa acatttggaa aacacagcat ctcagttcag aaaacagagg cccagtttta
6660gcaagtaaag ccaagaggga ccccagcagc ctgcagggca ggaccctctg ccctttctcc
6720tcccagatgt ccccaccttg ctgtgttgtt gttccagggt tgactcagct gatgccaata
6780gcaatttaaa acagaattgg gccaggtgca gtggctcatg cctgtaatcc cagcactttg
6840ggaggcccag gtaggaggat cgcttgagcc caggagttgg agaccagcct gggcaacaca
6900gccagacccc atcttttaaa aagaatcaaa aaatctgcca ggtagtgggt gtgcctgtag
6960tcccagctac tcaggaggct caggtgggca ggtcaattga gcccataagt tcaaggttgc
7020agtgaggtat gatcgcatca ctgtactcca gcctgggtaa cagtgcgaga ccctgtctct
7080aaaaataaat aaataaataa ataaataaat aaataaacaa acaaacaaac aaacaaacaa
7140tcaattgcat ataaggatcg cccgttttca gggcatgctt tacaccggcc tggttaactt
7200tactctgggt gtgctccgtc cgccgcagcc cccgccggga ggtggccaca gctctctctg
7260gttgcgccct aggtgtacca aatcatcgtc atccagactg ggagctttga caacaacaag
7320gccgtcctgg aacggcgcta ttccgacttc gcgaagctcc agaaagcgct gctgaagacg
7380ttcagggagg agatcgaaga cgtggagttt cccaggaagc acctgactgg gaacttcgct
7440gaggagatga tctgtgagcg tcggcgcgcc ctgcaggagt acctgggcct gctctacgcc
7500atccgctgcg tgcgccgctc ccgggagttc ctggacttcc tcacgcggcc ggagctgcgc
7560gaggctttcg gctgcctgcg ggccggccag tacccgcgcg ccctggagct gctgctgcgc
7620gtgctgccgc tgcaggagaa gctcaccgcc cactgccctg cggccgccgt cccggccctg
7680tgcgccgtgc tgctgtgcca ccgcgacctc gaccgccccg ccgaggcctt cgcggccgga
7740gagagggccc tgcagcgcct gcaggcccgg gagggccatc gctactatgc gcctctgctg
7800gacgccatgg tccgcctggc ctacgcgctg ggcaaggact tcgtgactct gcaggagagg
7860ctggaggaga gccagctccg gaggcccacg ccccgaggca tcaccctgaa ggagctcact
7920gtgcgagaat acctgcactg agccggcctg ggaccccgca gggacgctgg agatttgggg
7980tcaccatggc tcacagtggg ctgtttgggg ttcttttttt ttatttttcc ttttcttttt
8040tgttatttga gacagtcttg ctctgtcacc cagactgaag tgcagtggct caattatgtc
8100tcactgcagc ctcaaactcc tgggcacaag caatc
8135716DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S3120 (AFM326vc5) polymorphism marker 7ctgggtgcga ttgctc
16816DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S3120
(AFM326vc5) polymorphism marker 8ccaggcccca tgacag
16925DNAArtificial SequenceDescription of
Artificial SequencePCR primer for D16S298 (AFMa189wg5) polymorphism
marker 9tggtcccggc ccaatcccaa tgctt
251028DNAArtificial SequenceDescription of Artificial SequencePCR
primer for D16S298 (AFMa189wg5) polymorphism marker 10ttcctcatgt
ataaattggg tgtggcca
281125DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S299 polymorphism marker 11acagagtgag gaccccatct ctatc
251225DNAArtificial SequenceDescription
of Artificial SequencePCR primer for D16S299 polymorphism marker
12tccaactgct gggattacag gcaca
251322DNAArtificial SequenceDescription of Artificial SequencePCR primer
for SPN polymorphism marker 13agtccccgag accagggcaa ac
221423DNAArtificial SequenceDescription of
Artificial SequencePCR primer for SPN polymorphism marker
14tccatttctg cagtacacat gca
231520DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S383 polymorphism marker 15ctctccccat agaaggcatc
201620DNAArtificial SequenceDescription
of Artificial SequencePCR primer for D16S383 polymorphism marker
16ggatagagac gttctcttaa
201720DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S753 (GGAA3G05) polymorphism marker 17caggctgaat gacagaacaa
201820DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S753
(GGAA3G05) polymorphism marker 18attgaaaaca actccgtcca
201925DNAArtificial SequenceDescription of
Artificial SequencePCR primer for D16S3044 (AFMa222za9) polymorphism
marker 19atactcactt ttagacagtt caggg
252021DNAArtificial SequenceDescription of Artificial SequencePCR
primer for D16S3044 (AFMa222za9) polymorphism marker 20ggctcagttc
ctaaccagtt c
212120DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S409 (AFM161xa1) polymorphism marker 21agtcagtctg tccagaggtg
202220DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S409
(AFM161xa1) polymorphism marker 22tgaatcttac atcccatccc
202317DNAArtificial SequenceDescription of
Artificial SequencePCR primer for D16S3105 (AFMb341zc5) polymorphism
marker 23gatcttccca aagcgcc
172417DNAArtificial SequenceDescription of Artificial SequencePCR
primer for D16S3105 (AFMb341zc5) polymorphism marker 24tcccgtcagc
caagcta
172520DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S261 (MFD24) polymorphism marker 25aagcttgtat ctttctcagg
202620DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S261
(MFD24) polymorphism marker 26atctaccttg gctgtcattg
202720DNAArtificial SequenceDescription of
Artificial SequencePCR primer for D16S540 (GATA7B02) polymorphism
marker 27cctccataat catgtgagcc
202820DNAArtificial SequenceDescription of Artificial SequencePCR
primer for D16S540 (GATA7B02) polymorphism marker 28aatctcccca
actcaagacc
202920DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S3080 (AFMb068zb9) polymorphism marker 29ggatgcctgc tctaaatacc
203019DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S3080
(AFMb068zb9) polymorphism marker 30cccaggggtc aaacttaat
193121DNAArtificial SequenceDescription of
Artificial SequencePCR primer for D16S517 (AFMa132we9) polymorphism
marker 31ggtttgaaag tatctccagg g
213221DNAArtificial SequenceDescription of Artificial SequencePCR
primer for D16S517 (AFMa132we9) polymorphism marker 32ggtttgaaag
tatctccagg g
213320DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S411 (AFM186xa3) polymorphism marker 33gtgcatgtgt tcgtatcaac
203420DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S411
(AFM186xa3) polymorphism marker 34tcatctccaa aggagtttct
203518DNAArtificial SequenceDescription of
Artificial SequencePCR primer for D16S3035 (AFMa189wg5) polymorphism
marker, PCR or ligation oligonucleotide primer for D16S3035
biallelic polymorphism marker 35aaagccaacc ttgcttca
183620DNAArtificial SequenceDescription
of Artificial SequencePCR primer for D16S3035 (AFMa189wg5)
polymorphism marker, PCR or ligation oligonucleotide primer for
D16S3035 biallelic polymorphism marker 36tcttggaaac aggtaagtgc
203718DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S3136
(AFMa061xe5) polymorphism marker, PCR or ligation oligonucleotide
primer for D16S3136 biallelic polymorphism marker 37attgccctca
agaacagc
183817DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S3136 (AFMa061xe5) polymorphism marker, PCR or ligation
oligonucleotide primer for D16S3136 biallelic polymorphism marker
38gtgctatgcc atcccag
173920DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S541 (GATA7E02) polymorphism marker 39ccacaccagc gtttttctaa
204024DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S541
(GATA7E02) polymorphism marker 40cacactttac acacacctat accc
244122DNAArtificial SequenceDescription of
Artificial SequencePCR primer for D16S3117 (AFM288wb1) polymorphism
marker 41aagccatatt aggtctgtcc at
224219DNAArtificial SequenceDescription of Artificial SequencePCR
primer for D16S3117 (AFM288wb1) polymorphism marker 42gcttgggtta
aatgcgtgt
194320DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S416 (AFM210yg3) polymorphism marker 43agcagtttgg gtaaacattg
204420DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S416
(AFM210yg3) polymorphism marker 44aaatatgcct tctggaggtg
204520DNAArtificial SequenceDescription of
Artificial SequencePCR primer for D16S770 (GGAA20G02) polymorphism
marker 45ggaggatcag gggagtttat
204624DNAArtificial SequenceDescription of Artificial SequencePCR
primer for D16S770 (GGAA20G02) polymorphism marker 46caaagtaaat
gaatgtctac tgcc
244723DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S2623 (GATA81B12) polymorphism marker 47ccaactctgt agtttcaaag
agc 234820DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S2623
(GATA81B12) polymorphism marker 48tcacagccta cttgcttggt
204925DNAArtificial SequenceDescription of
Artificial SequencePCR primer for D16S390 polymorphism marker
49gacagcctca aatgaaatat aacac
255025DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S390 polymorphism marker 50gctctcagct agggtagttg tttat
255125DNAArtificial SequenceDescription
of Artificial SequencePCR primer for D16S419 (AFM225zf2)
polymorphism marker 51atttttaagg aatgtaaagn acaca
255220DNAArtificial SequenceDescription of Artificial
SequencePCR primer for D16S419 (AFM225zf2) polymorphism marker
52gaccaggagt cagtaaaagg
205320DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S771 (GGAA23C09) polymorphism marker 53gtccaaaaca ccaccctcta
205424DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S771
(GGAA23C09) polymorphism marker 54gaagtagatc agtcatcttg ctgc
245519DNAArtificial SequenceDescription of
Artificial SequencePCR primer for D16S408 (AFM137xf8) polymorphism
marker 55tcctctgggg gattcactc
195620DNAArtificial SequenceDescription of Artificial SequencePCR
primer for D16S408 (AFM137xf8) polymorphism marker 56gggacatcac
caagcacaag
205725DNAArtificial SequenceDescription of Artificial SequencePCR primer
for D16S508 (AFM304xf1) polymorphism marker 57caggaaaata aatctaacac
acata 255820DNAArtificial
SequenceDescription of Artificial SequencePCR primer for D16S508
(AFM304xf1) polymorphism marker 58cctgtgggca ctgataaata
205919DNAArtificial SequenceDescription of
Artificial SequencePCR or ligation oligonucleotide primer for
ADCY7int7 biallelic polymorphism marker 59cccagccccc atctcaccg
196019DNAArtificial
SequenceDescription of Artificial SequencePCR or ligation
oligonucleotide primer for ADCY7int7 biallelic polymorphism marker
60cccagccccc atctcacca
196119DNAArtificial SequenceDescription of Artificial SequencePCR or
ligation oligonucleotide primer for ADCY7int7 biallelic polymorphism
marker 61ctgcggagga ggctgctgg
196219DNAArtificial SequenceDescription of Artificial SequencePCR or
ligation oligonucleotide primer for hb133D1f biallelic
polymorphism marker 62tcactcccac caccctttc
196320DNAArtificial SequenceDescription of Artificial
SequencePCR or ligation oligonucleotide primer for hb133D1f
biallelic polymorphism marker 63agaagtttag tgtggcgtgg
206417DNAArtificial SequenceDescription
of Artificial SequencePCR or ligation oligonucleotide primer for
ctg35ExC biallelic polymorphism marker 64gccatctccc caagccc
176518DNAArtificial
SequenceDescription of Artificial SequencePCR or ligation
oligonucleotide primer for ctg35ExC biallelic polymorphism marker
65tcgatgcgag ctgaagcg
186618DNAArtificial SequenceDescription of Artificial SequencePCR or
ligation oligonucleotide primer for ctg35ExC biallelic polymorphism
marker 66tcgatgcgag ctgaagca
186720DNAArtificial SequenceDescription of Artificial SequencePCR or
ligation oligonucleotide primer for CTG35ExA biallelic
polymorphism marker 67tgaatgttaa agggctctgg
206819DNAArtificial SequenceDescription of Artificial
SequencePCR or ligation oligonucleotide primer for CTG35ExA
biallelic polymorphism marker 68ttggttctca gctccggcg
196919DNAArtificial SequenceDescription
of Artificial SequencePCR or ligation oligonucleotide primer for
CTG35ExA biallelic polymorphism marker 69ttggttctca gctccggca
197019DNAArtificial
SequenceDescription of Artificial SequencePCR or ligation
oligonucleotide primer for Ctg25Ex1 biallelic polymorphism marker
70agaaaccggg ctggctgtg
197121DNAArtificial SequenceDescription of Artificial SequencePCR or
ligation oligonucleotide primer for Ctg25Ex1 biallelic polymorphism
marker 71gcattgcctt ttgatctcta c
217218DNAArtificial SequenceDescription of Artificial SequencePCR or
ligation oligonucleotide primer for SNP3-2931 biallelic
polymorphism marker 72tgggctcttc tgcgggga
187318DNAArtificial SequenceDescription of Artificial
SequencePCR or ligation oligonucleotide primer for SNP3-2931
biallelic polymorphism marker 73tgggctcttc tgcggggg
187420DNAArtificial SequenceDescription
of Artificial SequencePCR or ligation oligonucleotide primer for
SNP3-2931 biallelic polymorphism marker 74tgcctcttct tctgccttcc
207522DNAArtificial
SequenceDescription of Artificial SequencePCR or ligation
oligonucleotide primer for ctg2931-5ag/ola biallelic polymorphism
marker 75cgagctgtac ctgaggaagc gt
227624DNAArtificial SequenceDescription of Artificial SequencePCR or
ligation oligonucleotide primer for ctg2931-5ag/ola biallelic
polymorphism marker 76cctgagctgt acctgaggaa gcgc
247720DNAArtificial SequenceDescription of Artificial
SequencePCR or ligation oligonucleotide primer for ctg2931-5ag/ola
biallelic polymorphism marker 77catcatgagc ccggggtggc
207823DNAArtificial SequenceDescription
of Artificial SequencePCR or ligation oligonucleotide primer for
ctg2931-3ac/ola biallelic polymorphism marker 78tttctcttgg
cttcctggtg cgt
237925DNAArtificial SequenceDescription of Artificial SequencePCR or
ligation oligonucleotide primer for ctg2931-3ac/ola biallelic
polymorphism marker 79accttctctt ggcttcctgg tgcgg
258026DNAArtificial SequenceDescription of Artificial
SequencePCR or ligation oligonucleotide primer for ctg2931-3ac/ola
biallelic polymorphism marker 80gccaaaggtg tcgtgccagg gctcca
268120DNAArtificial SequenceDescription
of Artificial SequencePCR or ligation oligonucleotide primer for
SNP1 biallelic polymorphism marker 81atctgagaag gccctgctct
208220DNAArtificial
SequenceDescription of Artificial SequencePCR or ligation
oligonucleotide primer for SNP1 biallelic polymorphism marker
82atctgagaag gccctgctcc
208319DNAArtificial SequenceDescription of Artificial SequencePCR or
ligation oligonucleotide primer for SNP1 biallelic polymorphism
marker 83cccacactta gccttgatg
198419DNAArtificial SequenceDescription of Artificial SequencePCR or
ligation oligonucleotide primer for Ctg22Ex1 biallelic
polymorphism marker 84atgagttagc ccagcggag
198519DNAArtificial SequenceDescription of Artificial
SequencePCR or ligation oligonucleotide primer for Ctg22Ex1
biallelic polymorphism marker 85attgagagcc cttggagtg
198619DNAArtificial SequenceDescription
of Artificial SequencePCR or ligation oligonucleotide primer for
hb27G11F biallelic polymorphism marker 86tgatttcgta agacaagtg
198720DNAArtificial
SequenceDescription of Artificial SequencePCR or ligation
oligonucleotide primer for hb27G11F biallelic polymorphism marker
87agcaaattct aggagttatg
208819DNAArtificial SequenceDescription of Artificial SequencePCR or
ligation oligonucleotide primer for KIAA0849ex9 biallelic
polymorphism marker 88agctgagatg tccggatcg
198918DNAArtificial SequenceDescription of Artificial
SequencePCR or ligation oligonucleotide primer for KIAA0849ex9
biallelic polymorphism marker 89agctgagatt ccggatca
189020DNAArtificial SequenceDescription
of Artificial SequencePCR or ligation oligonucleotide primer for
KIAA0849ex9 biallelic polymorphism marker 90gtcctcttaa cttcccttcc
20
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