Patent application title: NUCLEIC ACID MOLECULES ENCODING BANK1 SPLICE VARIANTS
Inventors:
Hadi Abderrahim (Divonne Les Bains, FR)
Sergei V. Kozyrev (Uppsala, SE)
Assignees:
Merck Serono S.A.
IPC8 Class: AC12Q168FI
USPC Class:
4241721
Class name: Drug, bio-affecting and body treating compositions immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material binds eukaryotic cell or component thereof or substance produced by said eukaryotic cell (e.g., honey, etc.)
Publication date: 2013-01-03
Patent application number: 20130004512
Abstract:
The present invention relates to a new splice variant of BANK1, the use
of SNPs in BANK1 for diagnostics and the use of antagonists to modulate
BANK1 and/or the BANK1 pathway.Claims:
1. A method for genotyping comprising the steps of: a) isolating a
nucleic acid from a sample of an individual; and b) determining in B-cell
scaffold protein with ankyrin repeats (BANK1) encoding nucleic acid
whether: biallelic marker rs10516487 is present as a guanine or an
adenine, biallelic marker rs17266594 is present as a thymine or a
cytosine, and/or biallelic marker rs3733197 is present as an adenine or a
guanine.
2. The method according to claim 1, wherein the nucleotides at said biallelic markers are determined for both copies of said biallelic markers present in said individual's genome.
3. The method according to claim 1, wherein said determining is performed by a microsequencing assay.
4. The method according to claim 1, further comprising amplifying a portion of a sequence comprising the biallelic marker prior to said determining step.
5. The method according to claim 4, wherein said amplifying is performed by PCR.
6. The method according to claim 1, further comprising the step of correlating the result of the genotyping steps with a risk of suffering or a predisposition for an auto-immune disease or inflammatory disease.
7. The method according to claim 1, wherein the presence of a guanine in rs10516487, a thymine in rs17266594 and a thymine in rs3733197 in said individual indicates that said individual suffers from, has a predisposition for or is at risk of suffering from said auto-immune disease or inflammatory disease.
8. The method according to claim 7, wherein the disease is Systemic Lupus Erythrematosus or Multiple Sclerosis.
9. A method for detecting whether an individual has a predisposition for or is at risk of an auto-immune disease or inflammatory disease comprising the steps: a) isolating the nucleic acid of an individual; b) detecting and quantifying the BANK1 full length nucleic acid; c) detecting and quantifying the BANK1 delta 2 nucleic acid; and d) determining the ratio b./c. and/or c./b. of the results of step b) and c).
10. The method according to claim 9, wherein the nucleic acid is a mRNA, cRNA or cDNA.
11. A method for the treatment of diseases selected from auto-immune or inflammatory diseases comprising the administration of an antagonist targeting BANK1, the biological pathway of BANK1 and/or factors connected to the BANK1 pathway.
12. The method according to claim 11, wherein the disease is Systemic Lupus Erythrematosus or Multiple Sclerosis.
13. The method according to claim 11, wherein the antagonist targets BANK1, LYN and/or IP3R or their interaction.
14. The method according to claim 13, wherein the antagonist targets the nucleic acid of BANK1.
15. The method according to claim 11, wherein the antagonist is an anti-sense RNA, siRNA, an aptamer, a peptide, an antibody or fragment thereof or a small molecule.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser. No. 12/738,418, filed Apr. 16, 2010, which is the U.S. national stage application of International Patent Application No. PCT/EP2008/065980, filed Nov. 21, 2008, which claims the benefit of U.S. Provisional Patent Application No. 61/004,480, filed Nov. 28, 2007, the disclosures of which are hereby incorporated by reference in their entirety, including all figures, tables and amino acid or nucleic acid sequences.
[0002] The Sequence Listing for this application is labeled "Seq-List.txt" which was created on Mar. 25, 2010 and is 29 KB. The entire contents of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to a new splice variant of BANK1, the use of SNPs associate with BANK1 for diagnostics and the use of antagonists to modulate BANK1 and/or the BANK1 pathway.
BACKGROUND OF THE INVENTION
[0004] Genetic techniques allow the identification of single nucleotide polymorphisms (SNPs) in individuals. SNPs are changes in a gene in one single nucleotide. Identification of SNPs can be correlated with a biological pathway having implications for a particular disease. The polymorphisms may be correlated also with a predisposition or risk for a disease by application of statistical analyses. Accordingly, targeting a particular biological pathway related to a disease is a means to treat such disease.
[0005] B-cell scaffold protein with ankyrin repeats (BANK1) is expressed in B cells and is tyrosine phosphorylated upon B-cell antigen receptor (BCR) stimulation. The BANK1 gene has 284 kb. BANK1 is an adaptor protein (6, 7) expressed mainly in B cells. The two full length isoforms of 785 and 755 amino acids, differ by 30 amino acids in the N-terminal region coded by the alternative exon 1A (FIG. 1e) and contain ankyrin repeat motifs and coiled-coil regions--structures highly similar between BANK1, BCAP and Dof adaptor proteins (8). B cell activation through BCR engagement leads to tyrosine phosphorylation of BANK1, which in turn promotes its association with the protein tyrosine kinase Lyn and the calcium channel IP3R (3). BANK1 serves as a docking station bridging together and facilitating phosphorylation and activation of IP3R by Lyn and the consequent release of Ca2+ from endoplasmic reticulum stores (3, 9). It was previously found that IP3R associates with the SNP rs10516487 lying within a region essential for binding of IP3R.
[0006] The BANK1 SNPs rs17266594 and rs3733197 have also been described in the literature.
[0007] None of the above SNPs have been described in the literature to be useful for the prediction of an inflammatory, auto-immune or neurological disease.
[0008] BANK1 and the pathway it is involved in, is considered to have implications for inflammatory and auto-immune disorders. In particularly, BANK1 is expressed in B-cells and therefore the pathway wherein BANK1 is involved has an implication for diseases associated with B-cells, e.g. Systemic Lupus Erythematosus (SLE). Multiple Sclerosis (MS) is related to T-cells, however, also the role of B-cells has been discussed in this disease. Accordingly, polymorphisms in the BANK1 gene may be used to diagnose a predisposition or risk for MS. Moreover, the BANK1 pathway may have implications for MS. In consequence, targeting this pathway and its modulation may represent a means to prevent or treat MS.
SUMMARY OF THE INVENTION
[0009] According to one aspect of the invention, a novel splice variant of BANK1 is provided.
[0010] According to another aspect of the invention, a method is provided for diagnosing an individual for the predisposition of, the risk of developing or suffering from an auto-immune or inflammatory disease wherein the pathway of BANK1 is involved.
[0011] According to another aspect of the invention, a method for the treatment and/or prevention of an auto-immune or inflammatory disease is provided using an antagonist targeting BANK1, the biological pathway of BANK1 and/or factors connected to the BANK1 pathway.
[0012] According to another aspect of the invention, a method of treating diseases is provided wherein the pathway of BANK1 is involved using an antagonist targeting BANK1, the biological pathway of BANK1 and/or factors connected to the BANK1 pathway.
BRIEF DESCRIPTION OF THE SEQUENCES AND DRAWINGS
[0013] SEQ ID NO: 1, 3, 5 are the nucleic acid sequences of the BANK1 delta 2 splice variant of human, chimpanzee and mouse, respectively.
[0014] SEQ ID NO: 2, 4, 6 are the amino acid sequences of the BANK1 delta 2 splice variant of human, chimpanzee and mouse, respectively.
[0015] FIGS. 1a-1e. Association of rs17266594 with increased levels of the full-length isoform of BANK1. (a) Total expression of BANK1 gene in separated mononuclear cell subpopulations. (b) RT-PCR of the coding part of BANK1 amplified from total human spleen cDNA reveals two bands on a gel. 1 kb ladder (New England Biolabs) is shown on the left. The identity of both bands, 2.3 kb upper band and 1.9 kb smaller band, was confirmed by sequencing analysis. (c) Relative mRNA expression levels of the full-length and delta 2 isoforms of BANK1, as determined by quantitative real-time RT-PCR on total RNA purified from human PBMCs. Data represent mean±S.D. 39 individuals with TT for the branch point site SNP, 34 with TC and 10 with CC genotype were analysed. Full-length transcript: TT versus CC, P=0.0004 (Student's t-test); delta 2 transcript: TT versus CC, P=0.0088. (d) Total BANK1 expression was not significantly affected by SNP rs17266594. (e) Schematic structure of the 5'-end of the gene. SNP rs17266594 located in the branch point site of intron 1 alters splicing efficiency of the full-length and delta 2 transcripts. SNP rs10516487 results in non-synonymous substitution of Arg61 to His. Alternative splicing gives rise to two isoforms, full-length and delta 2 with in-frame deletion of entire exon 2 of BANK1. Thus, the short protein isoform lacks the putative domain for IP3R binding and could function as a dominant negative isoform attenuating signaling from the full-length protein.
[0016] IP3R BD-inositol 1,4,5-triphosphate receptor binding domain, Lyn BD-tyrosine kinase Lyn binding domain.
[0017] FIG. 2a. Linkage disequilibrium and haplotype block structure across BANK1. Data calculated with Haploview analysis of our data using the Swedish cases and controls run for 30 SNPs across the gene.
[0018] FIG. 2B. R2 for all SNPs across BANK1.
[0019] FIG. 2c FIGS. 2a and 2b (combined)
[0020] FIG. 3. Frequencies of the haplotypes constructed with rs17266594 and rs10516487 (74.1% TG, 24.2% CA), and allele frequencies for rs3733197 (68.0% G, 32.0% A). The figure also shows the frequencies of the haplotypes when including all three SNPs (64.1% TGG, 10.1% TGA, 20.3% CAA, 3.8% CAG). Data is calculated using all populations, combined.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The following paragraphs contain definitions used according to the invention and are intended to apply uniformly throughout the specification and claims unless otherwise expressly set out definition provides a broader definition.
[0022] The present invention is directed to an isolated nucleic acid sequence comprising the sequence of BANK1 lacking exon 2. In a preferred embodiment the nucleic acid is of human, chimpanzee, or mouse origin. As a reference for the BANK1 sequence one may refer to Nature 431 (7011), 931-945 (2004).
[0023] In the human BANK1 sequence as described in NCBI's human genome assembly build 36, chromosome 4 the exons/introns are as follows:
Exon1: 102930919-102931130
Intron 1: 102931131-102969987
Exon2: 102969988-102970386
Intron2: 102970387-102995214
Exon3: 102995215-102995369
Intron3: 102995370-103002705
Exon4: 103002706-103002844
Intron4: 103002845-103010684
Exon5: 103010685-103010824
Intron5: 103010825-103035484
Exon6: 103035485-103035590
Intron6: 103035591-103058172
Exon7: 103058173-103058369
Intron7: 103058370-103161693
Exon8: 103161694-103161772
Intron8: 103161773-103165380
Exon9: 103165381-103165689
Intron9: 103165690-103170139
Exon10: 103170140-103170445
Intron10: 103170446-103184018
Exon11: 103184019-103184087
Intron11: 103184088-103200390
Exon12: 103200391-103200569
Intron12: 103200570-103203254
Exon13: 103203255-103203318
Intron13: 103203319-103211454
Exon14: 103211455-103211484
Intron14: 103211485-103212524
Exon15: 103212525-103212580
Intron15: 103212581-103213863
Exon16: 103213864-103213928
Intron16: 103213929-103214184
Exon17: 103214185-103214918
[0024] It is preferably possible that only part of the BANK1 exon 2 is deleted. Such a molecule is equally useful according to the invention.
[0025] In one embodiment the isolated nucleic acid comprises SEQ ID NO: 1, 3, or 5, or the complement of said nucleic acid sequence.
[0026] In one embodiment the invention relates to an isolated nucleic acid which: [0027] a) hybridizes under high stringency conditions; or [0028] b) exhibits at least about 85%, preferably at least about 90% and more preferably at least 95% identity over a stretch of at least about 30 nucleotides with a nucleic acid selected from the group consisting of SEQ ID NO: 1, 3, or 5, or a complement of said nucleic acid sequence.
[0029] Another embodiment of the invention is a polypeptide encoded by any of the nucleic acid sequences as mentioned above.
[0030] Another embodiment is a vector comprising a nucleic acid as described above, preferably a nucleic acid selected from the group consisting of SEQ ID NO: 1, 3, or 5, or a complement of said nucleic acid sequence.
[0031] Preferably the vector containing said nucleic acid molecule is operatively linked to at least one expression control sequence allowing expression in prokaryotic or eukaryotic host cells of the encoded polypeptide.
[0032] Another embodiment is a host cell transformed with a vector or a nucleic acid as described above.
[0033] Yet another embodiment of the invention is a method for making a polypeptide as described above comprising culturing a host cell as defined above under conditions in which the nucleic acid is expressed, and recovering the polypeptide encoded by said nucleic acid from the culture.
[0034] Another embodiment is a method for genotyping comprising the steps of: [0035] a. Isolating a nucleic acid from a sample of an individual; and [0036] b. Determining whether in rs10516487 a guanine or an adenine is present, in rs17266594 a thymine or a cytosine is present, in rs3733197 an adenine or a guanine is present in the biallelic marker.
[0037] In a preferred method the identity of the nucleotides at said biallelic markers is determined for both copies of said biallelic markers present in said individual's genome.
[0038] The method for genotyping according to the invention is preferably performed by a microsequencing assay. The method preferably further comprises amplifying a portion of a sequence comprising the biallelic marker prior to said determining step. Preferably said amplifying is performed by PCR. The method according to the invention further comprises the step of correlating the result of the genotyping steps with a risk of suffering or a predisposition for an auto-immune disease or inflammatory disease.
[0039] In a preferred embodiment the method is performed, wherein the presence of a guanine in rs10516487, a thymine in rs17266594 and an adenine in rs3733197 in said individual indicates that said individual suffers from, has a predisposition for or is at risk of suffering from said auto-immune disease or inflammatory disease.
[0040] The method of the invention preferably is applied wherein the disease is Systemic Lupus Erythrematosus or Multiple Sclerosis.
[0041] Now that the inventors have established the association between BANK1 and SLE and MS or related diseases, it should be understood that additional susceptibility alterations can be identified within said gene or polypeptide, e.g., following the methodology disclosed in the examples.
[0042] The presence of an alteration in the BANK1 gene may be detected by any technique known per se to the skilled artisan, including sequencing, pyrosequencing, selective hybridisation, selective amplification and/or mass spectrometry including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Gut et al., 2004). In a particular embodiment, the alteration is detected by selective nucleic acid amplification using one or several specific primers. The alteration is detected by selective hybridization using one or several specific probes.
[0043] Further techniques include gel electrophoresis-based genotyping methods such as PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and mini sequencing; fluorescent dye-based genotyping technologies such as oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan, and molecular beacon genotyping; rolling circle amplification and Invader assays as well as DNA chip-based microarray and mass spectrometry genotyping technologies (Shi et al., 2001).
[0044] Furthermore, RNA expression of altered genes can be quantified by methods known in the art such as subtractive hybridisation, quantitative PCR, TaqMan, differential display reverse transcription PCR, serial, partial sequencing of cDNAs (sequencing of expressed sequenced tags (ESTs) and serial analysis of gene expression (SAGE)), or parallel hybridization of labeled cDNAs to specific probes immobilized on a grid (macro- and microarrays and DNA chips. Particular methods include allele-specific oligonucleotide (ASO), allele-specific amplification, fluorescent in situ hybridization (FISH) Southern and Northern blot, and clamped denaturing gel electrophoresis.
[0045] Protein expression analysis methods are known in the art and include 2-dimensional gel-electrophoresis, mass spectrometry and antibody microarrays (Freeman et al., 2004 and Zhu et al., 2003).
[0046] Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing may be performed on the complete gene or, more preferably, on specific domains thereof, typically those known or suspected to carry deleterious mutations or other alterations.
[0047] Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand displacement amplification (SDA). These techniques can be performed using commercially available reagents and protocols. A preferred technique is allele-specific PCR.
[0048] Nucleic acid primers useful for amplifying sequences from the BANK1 gene are able to specifically hybridize with a portion of the BANK1 gene that either flanks or overlaps with a susceptibility alteration. The primer sequence overlaps with the alteration when said alteration is contained within the sequence of the BANK1 gene to which the primer hybridizes. The primer sequence flanks the alteration when the primer hybridizes with a portion of the BANK1 gene that is preferably located at a distance below 300 by of said alteration, even more preferably below 250, 200, 150, 100, 50, 40, 30 or 20 by from said alteration. Preferably, the primer hybridizes with a portion of the BANK1 gene that is at 5, 4, 3, 2, 1 by distance or immediately adjacent to said alteration.
[0049] In another embodiment the method for detecting whether an individual has a predisposition for or is at risk of an auto-immune disease or inflammatory disease according to the invention comprises the steps: [0050] a. Isolating the nucleic acid of an individual; [0051] b. Detecting and quantifying the BANK1 full length nucleic acid; [0052] c. Detecting and quantifying the BANK1 delta 2 nucleic acid; [0053] d. Determining the ratio b./c. and/or c./b. of the results of step b. and c.
[0054] In this method the nucleic acid is preferably a mRNA, cRNA or cDNA.
[0055] In step d. of the above method the determined ratio is an indication of the disease or its increased susceptibility. The more full length mRNA and the less delta 2 splice variant the more risk of disease an individual has. In particular, the higher this ratio is in the b./c correlation and the lower this ratio is in the c./b. correlation the higher is the risk to develop an auto-immune or inflammatory diseases, in particular SLE or MS.
[0056] The inventors have found that the total BANK1 mRNA is not influenced by the presence of particular SNPs. IN particular SNPs rs10516487, rs17266594 and rs3733197 do not change the total amount of BANK1 mRNA content. Accordingly the ratio of full length to delta 2 splice variant of BANK1 mRNA or cDNA is not influenced by the presence of the SNPs of the invention. Preferably the ratio b./c. or c./b is about 1. The ratios useful in the invention are as described above either b./c. or c./b.
[0057] A change in rs17266594 from TT to TC to CC has an influence in the amount of delta 2 BANK1 splice variant mRNA detectable. A ration of b./c. greater than 1, preferably significantly greater than 1 is indicative of a suffering from, or a predisposition for auto-immune or inflammatory diseases, preferably Systemic Lupus Erythrematosus or Multiple Sclerosis. A ration of c./b. less than 1, preferably significantly less than 1 is indicative of a suffering from, or a predisposition for auto-immune or inflammatory diseases, preferably Systemic Lupus Erythrematosus or Multiple Sclerosis. A change in this SNP from TT to CC may be most reliably be used to make this prediction. The suffering or predisposition may be expressed by calculation of the odd ration (OD). It will be appreciated by the skilled person that any method detecting and/or calculating a change in the SNP rs17266594 and/or mRNA or cDNA of BANK1 full length and/or delta 2 splice variant may be used to detect a predisposition for auto-immune or inflammatory diseases. In one embodiment the invention may be applied by comparing the mRNA of the BANK1 delta 2 splice variant of a sample with a control. The control may be chosen from one sample or a number of pooled samples.
[0058] The SNPs rs10516487 and rs3733197 can also be used to predict a suffering or predisposition and may serve as indirect markers. According to the invention also other SNPs may be used as predictive markers if a linkage with the above markers can be detected. Such a linkage, preferably strong linkage, is expressed by the LD and is preferably D' 0.7, preferably D' 0.8, more preferably D' 0.9. Such markers can be identified by standard techniques known in the art.
[0059] In another embodiment the invention relates to a method for the treatment and/or prevention of diseases selected from auto-immune or inflammatory diseases using an antagonist targeting BANK1, the biological pathway of BANK1 and/or factors connected to the BANK1 pathway. Preferably disease is Systemic Lupus Erythrematosus or Multiple Sclerosis.
[0060] The antagonist may be any molecule that antagonizes partly or essentially completely the targets of interest. Preferably the antagonist targets BANK1, LYN and/or IP3R or their interaction. Preferably the antagonist targets the nucleic acid of BANK1. In one embodiment the antagonist is an anti-sense RNA, siRNA, an Aptamer, a peptide or a small molecule. In another embodiment the antagonist is an antibody or antibody fragment specifically binding to the targets BANK1, LYN and/or IP3R. Particularly preferred is an antagonist that binds specifically to IP3R or interferes with the function of IP3R. In this manner it can be preferably achieved that the impact of B-cells involved in the disease development or manifestation of the disease is positively modulated, preferably inhibited.
[0061] The preferred SNPs as used in the invention are as follows:
TABLE-US-00001 Biallelic marker Alternative nucleotides rs10516487 G/A rs17266594 T/C rs3733197 A/G
[0062] The risk allel of rs10516487 is G. The risk allel of rs17266594 is the T and of rs3733197 is A. It will be understood that also other SNPs in Linkeage Disequilibrium (LD) may be used in the sense of the invention as described herein.
[0063] All references cited in this application are herewith incorporated by reference. In the following the present invention shall be illustrated by means of the following examples, which are not construed to be viewed as limiting the scope of the invention.
EXAMPLES
[0064] A set of 279 Swedish cases with SLE and 515 Swedish controls were genotyped for the 100 k Affymetrix SNPs array. After filtering, data from 85042 SNPs was used. As our purpose was to identify non-MHC genes and important functional polymorphisms, we proceeded to perform an analysis of the genomic location of the associated SNPs within known genes, discarding genomic deserts. Analysis of the data showed that among all the non-MHC-associated SNPs, one (rs10516487) was a non-synonymous substitution of arginine to histidine (triplet cGc->cAc, Arg->His) at amino-acid position 61 (from exon1A) of the BANK1 translated protein (allelic association, P=6.4×10-3; genotypic association, P=2.01×10-2). This SNP was ranked as #679 across the whole genome scan in the allelic association analysis and as #2148 in the genotypic test. The estimated FDR (False Discovery Rate) was 71.1% and 77.5% for these selections, respectively (2). Four more SNPs within BANK1 showed also association with SLE in the Affymetrix scan (Supplementary Table 1). The described B cell-specific expression of BANK1 and its potential role in B cell receptor-mediated activation led us to pursue this gene (3, 4).
[0065] We genotyped 30 SNPs in Swedish cases and 352 controls including the Affymetrix SNPs covering the complete 284 kb of the BANK1 gene. Two SNPs were not polymorphic in our population. Individual SNP analysis showed that 9 SNPs including rs10516487 were associated (Table 1). Using the solid-spine LD (Linkage Disequilibrium) haplotype block definition available from Haploview, 5 LD blocks could be recognized. All of the SNPs showing genetic association were lying on block 2, 3 and 4. No genetic association was detected for SNPs located in block 5 (Table 1, Supplementary Table 2 and FIG. 2a).
[0066] To confirm the genetic association, we genotyped four more sets of cases and controls from Germany, Spain, Italy and Argentina for rs10516487. We could corroborate the genetic association with all the European sets, although the Argentine set showed a clear tendency without reaching significance (Table 2). We performed homogeneity and combinability analysis of the sets using the Breslow-Day method. As the data could be combined, a meta-analysis was performed on all the sets comprising 3971 individuals. The Mantel-Haenzel (MH) test revealed a P value reaching genome-wide significance and a pooled odds ratio of 1.38 (X2=39.243, P=3.74×1010, 95% CI 1.25-1.53) for the allelic association. A significant genotypic association was also observed (Table 2).
[0067] We initiated a detailed analysis of BANK1 expression and structure. We observed that indeed and as described, BANK1 is primarily expressed in CD19+ B cells at high levels, while very low expression could be detected in CD4+, CD8+ and CD14+ cells (FIG. 1a). We then sequenced the proximal promoter region, exon1A, exon1B, and exon2 (where haploblock 2 is located) and 500 by up and downstream of these exons in 24 SLE patients and 8 controls. No novel SNPs were found for these regions. In order to clone BANK1 cDNA in an expression vector for functional analysis, we amplified full-length cDNA with distal primers. Surprisingly, two bands were detected on a gel after PCR (FIG. 1b). Subsequent cloning and sequencing revealed a new isoform with an in-frame deletion of the entire exon 2 (delta 2 isoform of BANK1). We analyzed cDNA from 83 healthy individuals and 30 SLE patients and found that this isoform was present in each sample, indicating that it is constitutively spliced. Moreover, this isoform was detected by PCR amplification of cDNA from chimp and mouse spleen as well, suggesting its conserved expression across species. Thus, we detected transcripts for three BANK1 isoforms, two full-length using exon1A or exon1B and a delta 2 isoform.
[0068] We next performed quantitative analysis of isoform expression in peripheral blood mononuclear cells. First, the relative levels of the two full-length isoforms, beginning with exon 1A and exon 1B, were determined. Since the latter transcript was present at very low levels, we continued the analysis measuring common full-length isoform levels. We noticed that the ratio of the full-length (FL) isoform to delta2 was not constant, which would be expected if delta 2 were equally expressed regardless of the genotypes of the analyzed samples. On the contrary, samples could be divided into groups according to the FL/delta 2 isoform ratio. After close examination of the genomic sequences surrounding exon 2 where putative signals affecting splicing could be located, one SNP, rs17266594, was found to lie in the putative branch point site and could potentially affect splicing. When expression data was re-grouped according to this SNP, a clear difference between the genotypes could be observed (FIG. 1c). Individuals homozygous for the T allele and thus having the classical structure of the branch point site (5) (YNYTGAYYN), showed equal expression of both isoforms, while expression of the full-length transcript was significantly suppressed (up to 40%) with concomitant upregulation of delta 2 isoform expression in individuals homozygous for the minor allele C. Total BANK1 transcription level was not significantly affected by the SNP (FIG. 1d). Genotyping of all of our sets of cases and controls for rs17266594 showed that the T allele was associated with SLE (Table 2; P=4.74×10-11, OR=1.42; 95% CI 1.28-1.58).
[0069] Both SNPs, rs17266594 and rs10516487, are separated by 153 nucleotides (nt) and are in strong LD (D'=0.95; R2=0.90; FIG. 2B). The T allele of the first SNP and the G allele of second one were found in the same risk haplotype associated with SLE (Table 2, bottom; P=4.75×10-6; OR 1.30, 95% CI 1.16-1.45) and FIG. 3.
[0070] We identified five non-synonymous substitutions in the databases. While most SNPs were non-polymorphic, one, rs3733197, an alanine to threonine substitution in amino acid position 383 (triplet Gca->Aca) in exon 7 coding for the ankyrin repeat-like motif, showed association in the combined sample (X2=16.576; P=4.67×10-5 (OR=1.23, 95% CI 1.11-1.36;) although it had not shown association in our first analysis on Swedish individuals nor in the whole Scandinavian set (Table 1 and Supplementary Table 3). This SNP is in haploblock 4 (FIG. 2a) 88211 bp apart from rs10516487 (D'=0.72; R2=0.39) and rs17266594 (R2=0.27), could segregate with the risk haplotype composed of the other two SNPs in some cases (FIG. 3) and could be a minor functional polymorphism.
[0071] Thus, herein we identify three functional polymorphisms in BANK1 associated with SLE.
[0072] The associated T allele of rs17266594 correlates with increased levels of the full-length isoform of BANK1. Thus, both polymorphisms in combination would lead to the achievement of one effect--high expression of a "more active" protein--through more efficient splicing of the full-length transcript that encodes a protein with an arginine residue in the IP3R binding domain. Since the delta 2 isoform lacks the entire exon 2 coding for IP3R binding and PH domains, it possibly functions as a dominant negative isoform thereby attenuating BANK1-mediated signaling (FIG. 1e).
[0073] Importance of mutations in ankyrin motifs for interaction with IP3R was recently highlighted by the discovery linking single amino acid substitutions in the adaptor protein ankyrin-B with cardiac arrhythmia and sudden cardiac death (10). While the alanine is associated with SLE, the rare allele A of rs3733197 might create a potential site for threonine kinases (11).
[0074] B cells are the major cell type affected in SLE. Novel therapies are aimed at depleting hyperactivated B cells that may function not solely as autoantibody producing cells, but also as important regulators of the innate and adaptive immune responses through antigen presentation and cytokine-mediated signaling (12). Functional and expression abnormalities of signaling molecules in B cells have been described in lupus. Of particular interest is the fact that Lyn, a binding partner of BANK1 is of key importance in human and mouse lupus autoimmune disease (13-18).
[0075] B cell hyperresponsiveness or a lack of control of B cell activation during immune responses. The precise role of BANK1 in BCR-mediated signaling remains unclear since two reports published so far contain conflicting data regarding the stimulatory or inhibitory role of BANK1 on B cell activation. Given the previously unreported existence of the alternative splicing of exon 2 we can speculate that the negative role for BANK1 assigned for the KO model was in part because of the remaining expression of the delta 2 isoform, as this exon was targeted by the KO-construct (4).
DNA samples
[0076] 279 cases and 515 controls were genotyped for the 100 k array. Of these individuals 279 cases and 352 controls were typed for the BANK1 coverage shown in Table 1.
[0077] For the functional polymorphisms an additional 185 Swedish patients were genotyped and 465 of the controls were available for genotyping of rs17266594 and rs3733197. We also added for the final MH (Mantel Haentsel) analysis and OR (Odds Ratio) estimation 84 Danish cases with the Swedish cases comprising the Scandinavian set shown in Table 2. The replication sets included 384 North German patients and 374 controls, 288 Argentine patients and 372 controls, 286 Italian patients and 252 controls. The Spanish cohort included 799 patients and 542 controls from several regions in Spain. 707 of the patients and 469 of the controls were genotyped for rs10516487 and rs3733197, and 678 of the patients and 457 of the controls for rs17266594. The reason for this is that DNA from a number of controls was not available. The German, Spanish and Argentine patients have all been previously described (19). The Italian cases are a multicenter collection of patients and their matched controls from Rome, Siena, Milan and Naples, that is North and Mid-Italy. All patients fulfil the 1982 ACR (American College of Rheumatology) criteria for the classification of SLE (20).
Genotyping
[0078] Genotyping of the 100 k Affymetrix array was performed according to the manufacturers instructions. Fine mapping and replication for SNPs rs10516487, rs17266594 and rs3733197 were done using TaqMan SNP genotyping assays (Applied Biosystems, Foster City, Calif.). The Affymetrix genotyping and fine mapping were performed at Serono Genetics Institute in Evry, France (now MerckSerono SA). The functional polymorphism replications were done. One hundred and six of samples were genotyped twice for verification showing 100% concordance. Genotyping success rate for all the samples was over 92%.
Statistical Analysis
[0079] For the 100K Affymetrix whole-genome scan analysis, pre-processing filters have been applied: SNPs have been discarded if (i) the proportion of missing genotypes is higher than 5%, (ii) the relative minor allele frequency is lower than 1% or (iii) the probability that the observed genotype distribution results from sampling a SNP which follows the Hardy-Weinberg equilibrium is lower than 0.02. Only SNPs from autosomal chromosomes have been kept for the sake of homogeneity between male and female individuals. SNP sequences have been mapped onto NCBI 36 human genome assembly and SNPs with multiple localizations have been discarded. For each remaining SNP, genotypic and allelic frequencies in cases and controls are calculated and the corresponding probability values are computed using exact (non-asymptotic) and unbiased algorithms (21). The False-Discovery Rate (FDR) is then estimated using the method described by Former, et al. (2).
[0080] For fine mapping analyses, genetic association, haplotype estimation, LD and R2 were all estimated using Haploview (v4.0RC2). The Breslow-Day test of combinability and the Mantel-Haenzel test were performed using the StatsDirect software (v2.4.6). As the Breslow-Day test showed combinability of the strata, the MH test for fixed effects was used in the analysis. Haplotypes were estimated using the PHASE software (v2.1) (22, 23). Genotypic odds ratios were calculated using the Unphased software (v3.0.9) (24).
Sequencing
[0081] DNA fragments for sequencing were amplified with the corresponding primers (see Supplementary Table 4), purified from agarose gel with QIAquick gel extraction kit (Qiagen) and sequenced using BigDye Terminator 3.1 (Applied Biosystems) at the Uppsala Genome Center.
RNA Purification and BANK1 Expression Analysis
[0082] Total RNA was purified with TRIZOL Reagent (Invitrogen) from peripheral blood mononuclear cells (PBMCs) obtained with agreed consent from healthy donors and lupus patients. 2 μg of RNA were reverse-transcribed with 2 U of MultiScribe transcriptase in PCR buffer II containing 5 mM MgCl2, 1 mM dNTPs, 0.4 U of RNase inhibitor and 5 μM oligo-dT. All reagents were purchased from Applied Biosystems. cDNA synthesis was performed at 42° C. for 80 min, and then the reaction was terminated at 95° C. for 5 μM. All cDNA samples were diluted to 15 ng/μl.
[0083] BANK1 expression was determined by real-time PCR on an ABI PRISM 7700 Sequence Detector (Applied Biosystems) with SDS 1.9.1 software. Total Bank1, both alternative full-length isoforms and delta2 isoform were quantified with SYBR Green and relevant primers (see Supplementary Table 4). We performed initial denaturation at 95° C. for 5 mM followed by 45 cycles of PCR (95° C. for 15 s, 62° C. for 15 s and 72° C. for 30 s). PCR buffer provided with enzyme was supplemented with 3 mM MgCl2, 200 μM of each of dNTPs, primers, SYBR Green (Molecular Probes), 15 ng of cDNA and 0.5 U of Platinum Taq polymerase (Invitrogen). Expression levels were normalized to the levels of TBP in the same samples amplified with commercial reagents (Applied Biosystems). All experiments were run in triplicate. Independent cDNA synthesis was carried out twice.
Cloning of Human, Mouse and Chimpanzee BANK1 Delta 2 Isoform
[0084] Purification of total RNA from mouse spleen and cDNA synthesis were conducted as described above for the human PBMCs. Total RNA from chimpanzee (Pan troglodytes) spleen was kindly provided by Drs. Tomas Bergstrom and Lucia Cavelier, Uppsala University. Human gene was amplified from Human Spleen BD Marathon-Ready cDNA (Clontech). After initial denaturation at 95° C. for 5 min, 35 cycles (95° C. for 20 s, 60° C. for 15 and 72° C. for 2 min 30 s) were performed in PCR buffer containing 2 mM MgSO4, 200 μM of each of dNTPs, 0.4 μM of each of the corresponding primers (see Supplementary Table 4), and 0.5 U of Platinum Taq-High Fidelity enzyme (Invitrogen). Chimp cDNA was amplified with human-specific primers. PCR products were purified from agarose gel and cloned in pCR 4-TOPO vector (Invitrogen) according to the manufacturer's instructions. Plasmid DNA from positive clones was purified with QIAprep Spin Miniprep kit (Qiagen) and verified by sequencing.
Accession Codes
[0085] BANK1 delta 2 transcripts were deposited in Genbank under the following accession numbers EU051376 for human, EU051377 for chimpanzee and EU051378 for mouse.
[0086] URLs. Haploview: www.broad.mit.edu/mpg/haploview/; GraphPad Software: http://www.graphpad.com; Protein analysis: http://www.ebi.ac.uk/saps/; http://smart.embl-heidelberg.de/, http://ca.expasy.org/prosite/, http://www.cbs.dtu.dk/services/NetPhos/.
TABLE-US-00002 TABLE 1 Association of SNPs in BANK1 in Swedish SLE SNP rs Associated name allele Chi Sq P Value rs7675129 T 0.147 0.701 rs11726012 G 0.495 0.4963 rs11097755 C 0.406 0.524 rs4522865 A 4.758 0.0292 rs4496585 A 1.933 0.1644 rs4572885 T 4.442 0.0355 rs10516487 G 7.185 0.0074 rs10516486 C 10.041 0.0015 rs17200824 A 2.780 0.0955 rs6849308 C 7.347 0.0067 rs10516482 C 8.709 0.0032 rs10516483 C 9.121 0.0025 rs10516484 A 0.577 0.4476 rs4493533 C 0.833 0.3614 rs3733197 A 0.006 0.9402 rs2631271 G 6.793 0.0092 rs2850390 C 1.032 0.3096 rs2631265 T 0.001 0.9815 rs2631267 G 0.048 0.827 rs2631268 T 1.375 0.2409 rs10516491 C 2.388 0.1223 rs1872701 G 1.454 0.2278 rs2850393 T 0.313 0.5759 rs2850396 C 0.344 0.5575 rs10516490 G 0.311 0.5769 rs10516489 T 0.312 0.5712 rs10516488 G 0.537 0.4635 rs1395306 T 1.739 0.1872
TABLE-US-00003 SUPPLEMENTARY TABLE 1 BANK1 SNPs in the 100k Array SNP rs number Position (-log) P value SNP_A-1701374 rs10516487 103108254 2.27 SNP_A-1701494 rs10516486 103108454 2.79 SNP_A-1664926 rs6849308 103133261 2.22 SNP_A-1706628 rs10516482 103137348 2.52 SNP_A-1744756 rs10516483 103149083 3.25 SNP_A-1683131 rs2631271 103271574 n.s. SNP_A-1697391 rs10516489 103331537 n.s.
TABLE-US-00004 TABLE 2 Genotypic, Allelic and Haplotypic Association of rs10516487 (R61H) and rs17266594 in five sets of SLE cases and controls and joint analysis with Mantel-Haenz Population GG GA AA Chi square P-Value Odds ratio (CI) a Allele G Allele A P-Value Odds ratio (CI) rs10516487 Scandinavian SLE Cases (536) 309 (57.6%) 200 (37.3%) 27 (5.0%) 11.7874 0.0028 GG: 2.12 (1.29-3.47) 818 (76.3%) 254 (23.7%) 7.27E-04 1.39 (1.14-1.68) Controls (565) 276 (48.8%) 238 (42.1%) 51 (9.0%) GA: 1.59 (0.96-2.63) 790 (69.9%) 340 (30.1%) Argentina SLE Cases (255) 164 (64.3%) 75 (29.4%) 16 (6.3%) 3.8013 0.1495 GG: 1.41 (0.73-2.72) 403 (79%) 107 (21%) 0.0564 1.31 (0.98-1.74) Controls (337) 190 (56.4%) 121 (35.9%) 26 (7.7%) GA: 1.01 (0.51-2.00) 499 (74.3%) 173 (25.7%) Germany SLE Cases (312) 181 (58.0%) 118 (37.8%) 13 (4.2%) 11.8503 0.0027 GG: 2.60 (1.32-5.14) 480 (76.9%) 144 (23.1%) 8.13E-04 1.52 (1.18-1.95) Controls (360) 166 (46.1%) 163 (45.3%) 31 (8.6%) GA: 1.73 (0.87-3.44) 495 (68.8%) 225 (31.2%) Italy SLE Cases (279) 166 (59.5%) 100 (35.8%) 13 (4.7%) 7.5139 0.0234 GG: 2.49 (1.22-5.09) 432 (77.4%) 126 (22.6%) 0.0078 1.46 (1.09-1.94) Controls (245) 123 (50.2%) 98 (40.0%) 24 (9.8) GA: 1.88 (0.91-3.91) 344 (70.2%) 146 (29.8%) Spain SLE Cases (702) 414 (59.0%) 243 (34.6%) 45 (6.4%) 11.3579 0.0034 GG: 1.26 (0.77-2.06) 1071 (76.3%) 333 (23.7%) 0.0065 1.30 (1.07-1.58) Controls (446) 219 (49.1%) 197 (44.2%) 30 (6.7%) GA: 0.82 (0.50-1.35) 635 (71.2%) 257 (28.8%) Pooled Cases (2003) 1187 (59.3%) 706 (35.2%) 110 (5.5%) 3080 (76.9%) 926 (23.1%) 3.74E-10 1.38 (1.25-1.53) c Controls (1968) 974 (49.9%) 817 (41.8%) 162 (8.3%) 2763 (70.8%) 1141 (29.2%) Population TT CT CC Chi square P-Value Odds ratio (CI) Allele T Allele C P-Value Odds ratio (CI) rs17266594 Scandinavian SLE Cases (511) 296 (57.9%) 189 (37.0%) 26 (5.1) 9.4399 0.0089 TT: 2.17 (1.28-3.66) 781 (76.4%) 241 (23.6%) 0.0036 1.36 (1.10-1.68) Controls (416) 210 (50.5%) 166 (39.9%) 40 (9.6%) CT: 1.75 (1.03-2.99) 586 (70.4%) 246 (29.6%) Argentina SLE Cases (274) 188 (68.6%) 77 (28.1%) 9 (3.3%) 14.1697 8.38E-04 TT: 3.26 (1.51-7.06) 453 (82.7%) 95 (17.3%) 1.06E-04 1.73 (1.30-2.31) Controls (346) 192 (55.5%) 124 (35.8%) 30 (8.7%) CT: 2.07 (0.93-4.59) 508 (73.4%) 184 (26.6%) Germany SLE Cases (241) 132 (54.8%) 98 (40.7%) 11 (4.6%) 7.7164 0.0211 TT: 2.46 (1.19-5.09) 362 (75.1%) 120 (24.9%) 0.0080 1.43 (1.09-1.87) Controls (335) 151 (45.1%) 153 (45.7%) 31 (9.3%) CT: 1.81 (0.87-3.76) 455 (67.9%) 215 (32.1%) Italy SLE Cases (231) 130 (56.3%) 87 (37.7%) 14 (6.1%) 10.1706 0.0062 TT: 2.42 (1.19-4.93) 347 (75.1%) 115 (24.9%) 0.0016 1.59 (1.18-2.14) Controls (219) 92 (42.0%) 103 (47.0%) 24 (11.0%) CT: 1.45 (0.71-2.97) 287 (65.5%) 151 (34.5%) Spain SLE Cases (678) 404 (59.6%) 231 (34.1%) 43 (6.3%) 14.8617 5.93E-04 TT: 1.04 (0.62-1.76) 1039 (76.6%) 317 (23.4%) 0.010 1.29 (1.06-1.56) Controls (458) 225 (49.1%) 208 (45.4%) 25 (5.5%) CT: 0.65 (0.38-1.09) 658 (71.8%) 258 (28.2%) Pooled Cases (1856) 1102 (59.4%) 655 (35.3%) 99 (5.3%) 2859 (77.0%) 853 (23.0%) 4.74E-11 1.42 (1.28-1.58) c Controls (1774) 870 (49.0%) 754 (42.5%) 150 (8.5%) 2494 (70.3%) 1054 (29.7%) Population TG/TG TG/other other/other Chi square P-Value TG other P-Value Odds ratio (CI) Haplotype Scandinavian SLE Cases (509) 293 (57.6%) 190 (37.3%) 26 (5.1%) 4.6600 0.0973 776 (76.3%) 242 (23.8%) 0.22738 1.14 (0.91-1.43) Controls (365) 205 (56.2%) 128 (35.1%) 32 (8.8%) 538 (73.8%) 192 (26.4%) Argentina SLE Cases (260) 187 (71.9%) 65 (25.0%) 8 (3.1%) 11.8483 0.0027 439 (84.4%) 81 (15.6%) 0.00032 1.72 (1.27-2.36) Controls (317) 189 (59.6%) 103 (32.5%) 25 (7.9%) 481 (75.9%) 153 (24.1%) Germany SLE Cases (237) 131 (55.3%) 94 (39.7%) 12 (5.1%) 6.6099 0.0367 356 (75.1%) 118 (24.9%) 0.01228 1.40 (1.07-1.85) Controls (331) 151 (45.6%) 150 (45.3%) 30 (9.1%) 452 (68.3%) 210 (31.7%) Italy SLE Cases (230) 130 (56.5%) 87 (37.8%) 13 (5.7%) 9.4922 0.0087 347 (75.4%) 113 (24.6%) 0.00225 1.57 (1.16-2.13) Controls (214) 92 (43.0%) 99 (46.3%) 23 (10.7%) 283 (66.1%) 145 (33.9%) Spain SLE Cases (589) 324 (55.0%) 217 (36.8%) 48 (8.1%) 5.4954 0.0641 865 (73.4%) 313 (26.6% 0.43109 1.09 (0.88-1.34) Controls (374) 186 (49.7%) 165 (44.1%) 23 (6.1%) 537 (71.8%) 211 (28.2%) Pooled Cases (1825) 1065 (58.4%) 653 (35.8%) 107 (5.9%) 2783 (76.2%) 867 (23.8%) 4.75E-06 1.30 (1.16-1.45) Controls (1601) 823 (51.4%) 645 (40.3%) 133 (8.3%) 2291 (71.5%) 911 (28.5%) a Genotypic odds ratio calculated using homozygosity for the protective allele as reference with OR = 1 bMantel-Haenzel Chi square using fixed effects c Using the Robins, Breslow and Greenland method
TABLE-US-00005 SUPPLEMENTARY TABLE 2 SNP rs number MB Build 36 Location in BANK1 rs7675129 102894046 intergenic rs11726012 102925041 promoter rs11097755 102928331 5'UTR rs4522865 102934911 intronic rs4496585 102937309 intronic rs4572885 102954536 intronic rs10516487 102970099 exon coding (NS)* rs10516486 102970299 exon 2 (synonymous) rs17200824 102971612 intronic rs6849308 102995106 intronic rs10516482 102999193 intronic rs10516483 103010928 intronic rs10516484 103011108 intronic rs4493533 103039707 intronic rs3733197 103058310 exon coding NS rs2631271 103133419 intronic rs2850390 103163019 intronic rs2631265 103164099 intronic rs2631267 103167495 intronic rs2631268 103167753 intronic rs10516491 103171889 intronic rs1872701 103172704 intronic rs2850393 103174239 intronic rs2850396 103187471 intronic rs10516490 103193084 intronic rs10516489 103193382 intronic rs10516488 103196800 intronic rs1395306 103204873 intronic *NS: non-synonymous substitution
TABLE-US-00006 SUPPLEMENTARY TABLE 3 Genotypic and Allelic Association of rs3733197 in five sets of SLE cases and controls and joint analysis with Mantel-Haenzel test. Population GG GA AA Chi square P-Value Odds ratio (CI) a Scandinavian SLE Cases (419) 167 (39.9%) 192 (45.8%) 60 (14.3%) 1.2365 0.5389 GG: 1.04 (0.69-1.58) Controls (444) 163 (36.7%) 220 (49.6%) 61 (13.7%) GA: 0.89 (0.59-1.33) Argentina SLE Cases (287) 177 (61.7%) 97 (33.8%) 13 (4.5%) 9.6496 0.0080 GG: 2.36 (1.20-4.66) Controls (363) 184 (50.7%) 147 (40.5%) 32 (8.8%) GA: 1.62 (0.81-3.25) Germany SLE Cases (272) 128 (47.1%) 112 (41.2%) 32 (11.8%) 4.1431 0.1260 GG: 1.65 (1.01-2.69) Controls (362) 148 (40.9%) 153 (42.3%) 61 (16.9%) GA: 1.40 (0.85-2.28) Italy SLE Cases (253) 131 (51.8%) 102 (40.3%) 20 (7.9%) 8.2595 0.0161 GG: 1.74 (0.92-3.29) Controls (251) 98 (39.0%) 127 (50.6%) 26 (10.4%) GA: 1.04 (0.55-1.98) Spain SLE Cases (588) 307 (52.2%) 234 (39.8%) 47 (8.0%) 3.4580 0.1775 GG: 1.14 (0.72-1.82) Controls (455) 212 (46.6%) 206 (45.3%) 37 (8.1%) GA: 0.89 (0.56-1.43) Pooled Cases (1819) 910 (50.0%) 737 (40.5%) 172 (9.5%) Controls (1875) 805 (42.9%) 853 (45.5%) 217 (11.6%) Population Allele G Allele A Chi square P-Value Odds ratio (CI) Scandinavian SLE Cases (419) 526 (62.8%) 312 (37.2%) 0.301 0.5832 1.06 (0.87-1.29) Controls (444) 546 (61.5%) 342 (38.5%) Argentina SLE Cases (287) 451 (78.6%) 123 (21.4%) 9.787 0.0018 1.15 (0.95-1.40) Controls (363) 515 (70.9%) 211 (29.1%) Germany SLE Cases (272) 368 (67.6%) 176 (32.4%) 4.297 0.0382 1.28 (1.00-1.63) Controls (362) 449 (62.0%) 275 (38.0%) Italy SLE Cases (253) 364 (71.9%) 142 (28.1%) 6.696 0.0097 1.42 (1.08-1.87) Controls (251) 323 (64.3%) 179 (35.7%) Spain SLE Cases (588) 977 (72.1%) 379 (27.9%) 2.099 0.1474 1.50 (1.15-1.96) Controls (455) 630 (69.2%) 280 (30.8%) Pooled Cases (1819) 2686 (70.4%) 1132 (29.6%) 16.5763 4.67E-05 1.23 (1.11-1.36) Controls (1875) 2463 (65.7%) 1287 (34.3%)
TABLE-US-00007 SUPPLEMENTARY TABLE 4 PRIMER SEQUENCES SEQ SEQ Gene/gene ID ID fragment/isoform Forward NO Reverse NO hBANK cDNA CACCTCAACCGCCAC 7 ATAATAACCTTCT 8 amplification AATGCTGCCAGCA TTAATGATCTTTC TTGC Total BANK1 AGAGGAAACTACACC 9 GATGAGTTCTTCC 10 qRT-PCR TTACATAGCTC TGACCATCAG Total full-length TCAAAGCAGATGGGA 11 isoforms GATCTCAAC Delta2 isoform CAGCGCCCCCAGATT 12 CTGAAG Exon1A full-length CAGCGCCCCCAGGAA 13 isoform ATACA Alternative GCCTATTCTTTGTTTT 14 exon1 full-length GGAAATACA isoform Common reverse primer for all isoforms for qRT- PCR CACATGGAATTTC 15 AGTGGGAAGCAC Common reverse primer for gel-analysis ATCACAGTAGACA 16 TTGACATGGAC
For Genomic Sequencing:
TABLE-US-00008 [0087] Gene/gene SEQ ID SEQ ID fragment/isoform Forward NO Reverse NO promoter, exon TTGGAGAGGGTAT 17 AAGCAGGGCTAC 18 1A and 5'-part of TTAGAGCCATA CAATTCACCAG intron1 Alternative CTATGATACTGGA 19 AGCATATGACCA 20 exon1B AATACTGTCAGT GCTGATCAG Exon2 TTGATTTACTATG 21 TTACATAAGAAA 22 AAAATATCAAGC CCAGCTTCCAG mouse BANK1 ACCTCCCGCAATG 23 ACATGGAATTTCC 24 Cdna CTTCCTGT CCAGGAAGCAC
REFERENCE LIST
[0088] 1. Sherer, Y., Gorstein, A., Fritzler, M. J. & Shoenfeld, Y. (2004) Semin Arthritis Rheum 34, 501-37. [0089] 2. Former K, L. M., Guedj M, Dauvillier J and Wojcik J. Hum Hered, In Press. [0090] 3. Yokoyama, K., Su Ih, I. H., Tezuka, T., Yasuda, T., Mikoshiba, K., Tarakhovsky, A. & Yamamoto, T. (2002) Embo J 21, 83-92. [0091] 4. Aiba, Y., Yamazaki, T., Okada, T., Gotoh, K., Sanjo, H., Ogata, M. & Kurosaki, T. (2006) Immunity 24, 259-68. [0092] 5. Burge, C. B., Tuschl, T. & Sharp, P. A (1999), ed. Gesteland, R. F., Cech, T. R. & Atkins, J. F (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), pp. 525-560. [0093] 6. Jordan, M. S., Singer, A. L. & Koretzky, G. A. (2003) Nat Immunol 4, 110-6. [0094] 7. Kurosaki, T. (2002) Nat Rev Immunol 2, 354-63. [0095] 8. Okada, T., Maeda, A., Iwamatsu, A., Gotoh, K. & Kurosaki, T. (2000) Immunity 13, 817-27. [0096] 9. Patterson, R. L., Boehning, D. & Snyder, S. H. (2004) Annu Rev Biochem 73, 437-65. [0097] 10. Mohler, P. J., Schott, J. J., Gramolini, A. 0., Dilly, K. W., Guatimosim, S., duBell, W. H., Song, L. S., Haurogne, K., Kyndt, F., Ali, M. E., Rogers, T. B., Lederer, W. J., Escande, D., Le Marec, H. & Bennett, V. (2003) Nature 421, 634-9. [0098] 11. Blom, N., Gammeltoft, S. & Brunak, S. (1999) J Mol Biol 294, 1351-62. [0099] 12. Anolik, J., Sanz, I. & Looney, R. J. (2003) Curr Rheumatol Rep 5, 350-6. [0100] 13. Liossis, S. N., Kovacs, B., Dennis, G., Kammer, G. M. & Tsokos, G. C. (1996) J Clin Invest 98, 2549-57, [0101] 14. Huck, S., Le Corre, R., Youinou, P. & Zouali, M. (2001) Autoimmunity 33, 213-24. [0102] 15. Liossis, S. N., Solomou, E. E., Dimopoulos, M. A., Panayiotidis, P., Mavrikakis, M. M. & Sfikakis, P. P. (2001) J Investig Med 49, 157-65. [0103] 16. Hibbs, M. L., Harder, K. W., Armes, J., Kountouri, N., Quilici, C., Casagranda, F., Dunn, A. R. & Tarlinton, D. M. (2002) J Exp Med 196, 1593-604. [0104] 17. Flores-Borja, F., Kabouridis, P. S., Jury, E. C., Isenberg, D. A. & Mageed, R. A. (2005) Arthritis Rheum 52, 3955-65. [0105] 18. Cornall, R. J., Cyster, J. G., Hibbs, M. L., Dunn, A. R., Otipoby, K. L., Clark, E. A. & Goodnow, C. C. (1998) Immunity 8, 497-508. [0106] 19. Kozyrev, S. V., Lewen, S., Ling a Reddy, M.V.P., Pons-Estel, B. A., The Argentine Collaborative Group, Witte, T., The German Collaborative Group, Junker, P., Laustrup, H., Gutierrez, C., Suarez, A., Gonzalez-Escribano, M. F., Martin, J., The Spanish Collaborative Group and Alarcon-Riquelme, M. E. (2007) Arthritis and Rheumatism 56, 1234-41. [0107] 20. Tan, E. M., Cohen, A. S., Fries, J. F., Masi, A. T., McShane, D. J., Rothfield, N. F., Schaller, J. G., Talal, N. & Winchester, R. J. (1982) Arthritis Rheum 25, 1271-7. [0108] 21. Guedj, M., Wojcik, J., Della-Chiesa, E., Nuel, G. & Former, K. (2006) Hum Hered 61, 210-21. [0109] 22. Stephens, M. & Donnelly, P. (2003) Am J Hum Genet 73, 1162-9. [0110] 23. Stephens, M., Smith, N. J. & Donnelly, P. (2001) Am J Hum Genet 68, 978-89. [0111] 24. Dudbridge, F. (2003) Genet Epidemiol 25, 115-21. [0112] 25. Freeman, W. M. and S. E. Hemby (2004). "Proteomics for protein expression profiling in neuroscience." Neurochem Res 29(6): 1065-81. [0113] 26. Gut, I. G. (2004). "DNA analysis by MALDI-TOF mass spectrometry." Hum Mutat 23(5): 437-41. [0114] 27 Shi, M. M. (2001). "Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies." Clin Chem 47(2): 164-72. [0115] 28. Zhu, H. and M. Snyder (2003). "Protein chip technology." Curr Opin Chem Biol 7(1): 55-63.
Sequence CWU
1
2411984DNAHomo sapiens 1tcaaccgcca caatgctgcc agcagcgcca ggcaaggggc
ttgggagccc ggaccccgcc 60ccctgcggcc cagcgccccc agattctgaa gactactttg
aggtcaacat tccaacagac 120ctacgagcaa aacattctgg ggaaataagt gagagaaagg
aaattgaaga actatcagaa 180gcttcaagaa acaccatacc actagcagtg gtgcttccca
ctgaaattcc atgtgagaat 240cctggtgaaa tattcataat tttgagagat gaagtaattg
gtgatactgt agaggttgaa 300tttacatcaa gtaataagcg cattagaaca cggccagccc
tttggaataa gaaagtctgg 360tgcatgaaag ctttagagtt tcctgctggt tcagtccatg
tcaatgtcta ctgtgatgga 420atcgttaaag ctacaaccaa aattaagtac tacccaacag
caaaggcaaa ggaatgccta 480ttcagaatgg cagattcagg agagagtttg tgccagaata
gcattgaaga acttgatggt 540gtccttacat ccatattcaa acatgagata ccatattatg
agttccagtc tcttcaaact 600gaaatttgtt ctcaaaacaa atatactcat ttcaaagaac
ttccaactct tctccactgt 660gcagcaaaat ttggcttaaa gaacctggct attcatttgc
ttcaatgttc aggagcaacc 720tgggcatcta agatgaaaaa tatggagggt tcagaccccg
cacatattgc tgaaaggcat 780ggtcacaaag aactcaagaa aatcttcgaa gacttttcaa
tccaagaaat tgacataaat 840aatgagcaag aaaatgatta tgaagaggat attgcctcat
tttccacata tattccttcc 900acacagaacc cagcatttca tcatgaaagc aggaagacat
acgggcagag tgcagatgga 960gctgaggcaa atgaaatgga aggggaagga aaacagaatg
gatcaggcat ggagaccaaa 1020cacagcccac tagaggttgg cagtgagagt tctgaagacc
agtatgatga cttgtatgtg 1080ttcattcctg gtgctgatcc agaaaataat tcacaagagc
cactcatgag cagcagacct 1140cctctccccc cgccgcgacc tgtagctaat gccttccaac
tggaaagacc tcacttcacc 1200ttaccaggga caatggtgga aggccaaatg gaaagaagtc
aaaactgggg tcatcctggt 1260gttagacaag aaacaggaga tgaacccaaa ggagaaaaag
agaagaaaga agaggaaaaa 1320gagcaggagg aggaagaaga cccatatact tttgctgaga
ttgatgacag tgaatatgac 1380atgatattgg ccaatctgag tataaagaaa aaaactggga
gtcggtcttt cattataaat 1440agacctcctg cccccacacc ccgacccaca agtatacctc
caaaagagga aactacacct 1500tacatagctc aagtgtttca acaaaagaca gccagaagac
aatctgatga tgacaagttc 1560cgtggtcttc ctaagaaaca agacagagct cggatagaga
gtccagcctt ttctactctc 1620aggggctgtc taactgatgg tcaggaagaa ctcatcctcc
tgcaggagaa agtaaagaat 1680gggaaaatgt ctatggatga agctctggag aaatttaaac
actggcagat gggaaaaagt 1740ggcctggaaa tgattcagca ggagaaatta cgacaactac
gagactgcat tattgggaaa 1800aggccagaag aagaaaatgt ctataataaa ctcaccattg
tgcaccatcc aggtggtaag 1860gaaactgccc acaatgaaaa taagttttat aatgtacact
tcagcaataa gcttcctgct 1920cgaccccaag ttgaaaagga atttggtttc tgttgcaaga
aagatcatta aagaaggtta 1980ttat
19842652PRTHomo sapiens 2Met Leu Pro Ala Ala Pro
Gly Lys Gly Leu Gly Ser Pro Asp Pro Ala 1 5
10 15 Pro Cys Gly Pro Ala Pro Pro Asp Ser Glu Asp
Tyr Phe Glu Val Asn 20 25
30 Ile Pro Thr Asp Leu Arg Ala Lys His Ser Gly Glu Ile Ser Glu
Arg 35 40 45 Lys
Glu Ile Glu Glu Leu Ser Glu Ala Ser Arg Asn Thr Ile Pro Leu 50
55 60 Ala Val Val Leu Pro Thr
Glu Ile Pro Cys Glu Asn Pro Gly Glu Ile 65 70
75 80 Phe Ile Ile Leu Arg Asp Glu Val Ile Gly Asp
Thr Val Glu Val Glu 85 90
95 Phe Thr Ser Ser Asn Lys Arg Ile Arg Thr Arg Pro Ala Leu Trp Asn
100 105 110 Lys Lys
Val Trp Cys Met Lys Ala Leu Glu Phe Pro Ala Gly Ser Val 115
120 125 His Val Asn Val Tyr Cys Asp
Gly Ile Val Lys Ala Thr Thr Lys Ile 130 135
140 Lys Tyr Tyr Pro Thr Ala Lys Ala Lys Glu Cys Leu
Phe Arg Met Ala 145 150 155
160 Asp Ser Gly Glu Ser Leu Cys Gln Asn Ser Ile Glu Glu Leu Asp Gly
165 170 175 Val Leu Thr
Ser Ile Phe Lys His Glu Ile Pro Tyr Tyr Glu Phe Gln 180
185 190 Ser Leu Gln Thr Glu Ile Cys Ser
Gln Asn Lys Tyr Thr His Phe Lys 195 200
205 Glu Leu Pro Thr Leu Leu His Cys Ala Ala Lys Phe Gly
Leu Lys Asn 210 215 220
Leu Ala Ile His Leu Leu Gln Cys Ser Gly Ala Thr Trp Ala Ser Lys 225
230 235 240 Met Lys Asn Met
Glu Gly Ser Asp Pro Ala His Ile Ala Glu Arg His 245
250 255 Gly His Lys Glu Leu Lys Lys Ile Phe
Glu Asp Phe Ser Ile Gln Glu 260 265
270 Ile Asp Ile Asn Asn Glu Gln Glu Asn Asp Tyr Glu Glu Asp
Ile Ala 275 280 285
Ser Phe Ser Thr Tyr Ile Pro Ser Thr Gln Asn Pro Ala Phe His His 290
295 300 Glu Ser Arg Lys Thr
Tyr Gly Gln Ser Ala Asp Gly Ala Glu Ala Asn 305 310
315 320 Glu Met Glu Gly Glu Gly Lys Gln Asn Gly
Ser Gly Met Glu Thr Lys 325 330
335 His Ser Pro Leu Glu Val Gly Ser Glu Ser Ser Glu Asp Gln Tyr
Asp 340 345 350 Asp
Leu Tyr Val Phe Ile Pro Gly Ala Asp Pro Glu Asn Asn Ser Gln 355
360 365 Glu Pro Leu Met Ser Ser
Arg Pro Pro Leu Pro Pro Pro Arg Pro Val 370 375
380 Ala Asn Ala Phe Gln Leu Glu Arg Pro His Phe
Thr Leu Pro Gly Thr 385 390 395
400 Met Val Glu Gly Gln Met Glu Arg Ser Gln Asn Trp Gly His Pro Gly
405 410 415 Val Arg
Gln Glu Thr Gly Asp Glu Pro Lys Gly Glu Lys Glu Lys Lys 420
425 430 Glu Glu Glu Lys Glu Gln Glu
Glu Glu Glu Asp Pro Tyr Thr Phe Ala 435 440
445 Glu Ile Asp Asp Ser Glu Tyr Asp Met Ile Leu Ala
Asn Leu Ser Ile 450 455 460
Lys Lys Lys Thr Gly Ser Arg Ser Phe Ile Ile Asn Arg Pro Pro Ala 465
470 475 480 Pro Thr Pro
Arg Pro Thr Ser Ile Pro Pro Lys Glu Glu Thr Thr Pro 485
490 495 Tyr Ile Ala Gln Val Phe Gln Gln
Lys Thr Ala Arg Arg Gln Ser Asp 500 505
510 Asp Asp Lys Phe Arg Gly Leu Pro Lys Lys Gln Asp Arg
Ala Arg Ile 515 520 525
Glu Ser Pro Ala Phe Ser Thr Leu Arg Gly Cys Leu Thr Asp Gly Gln 530
535 540 Glu Glu Leu Ile
Leu Leu Gln Glu Lys Val Lys Asn Gly Lys Met Ser 545 550
555 560 Met Asp Glu Ala Leu Glu Lys Phe Lys
His Trp Gln Met Gly Lys Ser 565 570
575 Gly Leu Glu Met Ile Gln Gln Glu Lys Leu Arg Gln Leu Arg
Asp Cys 580 585 590
Ile Ile Gly Lys Arg Pro Glu Glu Glu Asn Val Tyr Asn Lys Leu Thr
595 600 605 Ile Val His His
Pro Gly Gly Lys Glu Thr Ala His Asn Glu Asn Lys 610
615 620 Phe Tyr Asn Val His Phe Ser Asn
Lys Leu Pro Ala Arg Pro Gln Val 625 630
635 640 Glu Lys Glu Phe Gly Phe Cys Cys Lys Lys Asp His
645 650 31983DNAPan troglodytes
3tcaaccgcca caatgctgcc agcagcgcca ggcaaggggc ttgggagccc ggacccggcc
60ccctgcggcc cagcgccccc agattctgaa gactactttg aggtcaacat tccaacagac
120ctacgagcaa aacattctgg ggaaataagt gagagaaagg aaattgaaga actatcagaa
180gcttcaagaa acaccatacc actagcagtg gtgcttccca ctgaaattcc atgtgagaat
240cctggtgaaa tattcataat tttgagagat gaagtaattg gtgatactgt agaggttgaa
300tttacatcac gtaataagcg cattagaaca cggccagccc tttggaataa gaaagtctgg
360tgcgtgaaag ctttagagtt tcctgctggt tcagtccatg tcaatgtcta ctgtgatgga
420atcgttaaag ctacaaccaa aattaagtac tacccaacag caaaggcaaa ggaatgccta
480ttcagaatgg cagattcaag agagagtttg tgccagaata gcattgaaga acttgatggt
540gtccttacat ccatattcaa acatgagata ccatattatg agttccaatc tcttcaaact
600gaaatttgtt ctcaaaacaa atatactcat ttcaaagaac ttccaactct tctccactgt
660gcagcaaaat ttggcttaaa gaacctggct attcatttgc ttcaatgttc aggagcaacc
720tgggcatcta agatgaaaaa tacggagggt tcagaccccg cacatattgc tgaaagacat
780ggtcacaaag aactcaagaa aatcttcgaa gacttttcaa tccaagaaat tgacataaat
840aatgagcaag aaaatgatta tgaagaggat attgcctcat tttccacata tattccttcc
900acacagaacc cagcatttca tcatgaaagc aggaagacat acgggcagag tgcagatgga
960gctgaggcaa atgaaatgga aggggaagga aaacagaatg gatcaggcat ggagaccaaa
1020cacagcccac tagaggttgg cagtgagagt tctgaggacc agtatgatga cttgtatgtg
1080ttcattcctg gtgctgatcc agaaaataat tcacaagagc cactcatgag cagcagacct
1140cctctccccc cgccgcgacc tgtagctaat gccttccaac tggaaagacc tcacttcacc
1200ttaccaggga caacggtgga aggccaaatg gaaagaagtc aaaactgggg tgatcctggt
1260gttagacaag aaacaggaga tgaacccaaa ggagaaaaag agaagaaaga agacgaaaaa
1320gagcaggagg aggaagaaga cccatatact tttgctgaga ttgatgacag tgaatatgac
1380atgatattgg ccaatctgag tataaagaaa aaaactggga gtcggtcttt cattataaat
1440agacctcctg cccccacacc ccgacccaca agtaaacctc caaaagagga aactacacct
1500tacatagctc aagtgtttca acaaaagaca gccagaagac aatctgatga tgacaagttc
1560cgtggtcttc ctaagaaaca agacagagct cggatagaga gtccagcttt ttctactctc
1620aggggctgtc taactgatgg tcaggaagaa ctcatcctcc tgcaggagaa agtcaagaat
1680gggaaaatgt ctatggatga agctctggag aaatttaaac actggcagat gggaaaaagt
1740ggcctggaaa tgattcagca ggagaaatta cggcaactac gagactgcat tattgggaaa
1800aggccagaag aagaaaatgt ctataataaa ctcaccattg tgcaccatcc aggtggtaag
1860gaaactgccc acaatgaaaa taagttttat aatgtacact tcagcaataa gcttcctgct
1920cgaccccaag ttgaaaagga atttggtttc tgttgcaaga aagatcatta aaggaggtta
1980tta
19834652PRTPan troglodytes 4Met Leu Pro Ala Ala Pro Gly Lys Gly Leu Gly
Ser Pro Asp Pro Ala 1 5 10
15 Pro Cys Gly Pro Ala Pro Pro Asp Ser Glu Asp Tyr Phe Glu Val Asn
20 25 30 Ile Pro
Thr Asp Leu Arg Ala Lys His Ser Gly Glu Ile Ser Glu Arg 35
40 45 Lys Glu Ile Glu Glu Leu Ser
Glu Ala Ser Arg Asn Thr Ile Pro Leu 50 55
60 Ala Val Val Leu Pro Thr Glu Ile Pro Cys Glu Asn
Pro Gly Glu Ile 65 70 75
80 Phe Ile Ile Leu Arg Asp Glu Val Ile Gly Asp Thr Val Glu Val Glu
85 90 95 Phe Thr Ser
Arg Asn Lys Arg Ile Arg Thr Arg Pro Ala Leu Trp Asn 100
105 110 Lys Lys Val Trp Cys Val Lys Ala
Leu Glu Phe Pro Ala Gly Ser Val 115 120
125 His Val Asn Val Tyr Cys Asp Gly Ile Val Lys Ala Thr
Thr Lys Ile 130 135 140
Lys Tyr Tyr Pro Thr Ala Lys Ala Lys Glu Cys Leu Phe Arg Met Ala 145
150 155 160 Asp Ser Arg Glu
Ser Leu Cys Gln Asn Ser Ile Glu Glu Leu Asp Gly 165
170 175 Val Leu Thr Ser Ile Phe Lys His Glu
Ile Pro Tyr Tyr Glu Phe Gln 180 185
190 Ser Leu Gln Thr Glu Ile Cys Ser Gln Asn Lys Tyr Thr His
Phe Lys 195 200 205
Glu Leu Pro Thr Leu Leu His Cys Ala Ala Lys Phe Gly Leu Lys Asn 210
215 220 Leu Ala Ile His Leu
Leu Gln Cys Ser Gly Ala Thr Trp Ala Ser Lys 225 230
235 240 Met Lys Asn Thr Glu Gly Ser Asp Pro Ala
His Ile Ala Glu Arg His 245 250
255 Gly His Lys Glu Leu Lys Lys Ile Phe Glu Asp Phe Ser Ile Gln
Glu 260 265 270 Ile
Asp Ile Asn Asn Glu Gln Glu Asn Asp Tyr Glu Glu Asp Ile Ala 275
280 285 Ser Phe Ser Thr Tyr Ile
Pro Ser Thr Gln Asn Pro Ala Phe His His 290 295
300 Glu Ser Arg Lys Thr Tyr Gly Gln Ser Ala Asp
Gly Ala Glu Ala Asn 305 310 315
320 Glu Met Glu Gly Glu Gly Lys Gln Asn Gly Ser Gly Met Glu Thr Lys
325 330 335 His Ser
Pro Leu Glu Val Gly Ser Glu Ser Ser Glu Asp Gln Tyr Asp 340
345 350 Asp Leu Tyr Val Phe Ile Pro
Gly Ala Asp Pro Glu Asn Asn Ser Gln 355 360
365 Glu Pro Leu Met Ser Ser Arg Pro Pro Leu Pro Pro
Pro Arg Pro Val 370 375 380
Ala Asn Ala Phe Gln Leu Glu Arg Pro His Phe Thr Leu Pro Gly Thr 385
390 395 400 Thr Val Glu
Gly Gln Met Glu Arg Ser Gln Asn Trp Gly Asp Pro Gly 405
410 415 Val Arg Gln Glu Thr Gly Asp Glu
Pro Lys Gly Glu Lys Glu Lys Lys 420 425
430 Glu Asp Glu Lys Glu Gln Glu Glu Glu Glu Asp Pro Tyr
Thr Phe Ala 435 440 445
Glu Ile Asp Asp Ser Glu Tyr Asp Met Ile Leu Ala Asn Leu Ser Ile 450
455 460 Lys Lys Lys Thr
Gly Ser Arg Ser Phe Ile Ile Asn Arg Pro Pro Ala 465 470
475 480 Pro Thr Pro Arg Pro Thr Ser Lys Pro
Pro Lys Glu Glu Thr Thr Pro 485 490
495 Tyr Ile Ala Gln Val Phe Gln Gln Lys Thr Ala Arg Arg Gln
Ser Asp 500 505 510
Asp Asp Lys Phe Arg Gly Leu Pro Lys Lys Gln Asp Arg Ala Arg Ile
515 520 525 Glu Ser Pro Ala
Phe Ser Thr Leu Arg Gly Cys Leu Thr Asp Gly Gln 530
535 540 Glu Glu Leu Ile Leu Leu Gln Glu
Lys Val Lys Asn Gly Lys Met Ser 545 550
555 560 Met Asp Glu Ala Leu Glu Lys Phe Lys His Trp Gln
Met Gly Lys Ser 565 570
575 Gly Leu Glu Met Ile Gln Gln Glu Lys Leu Arg Gln Leu Arg Asp Cys
580 585 590 Ile Ile Gly
Lys Arg Pro Glu Glu Glu Asn Val Tyr Asn Lys Leu Thr 595
600 605 Ile Val His His Pro Gly Gly Lys
Glu Thr Ala His Asn Glu Asn Lys 610 615
620 Phe Tyr Asn Val His Phe Ser Asn Lys Leu Pro Ala Arg
Pro Gln Val 625 630 635
640 Glu Lys Glu Phe Gly Phe Cys Cys Lys Lys Asp His 645
650 52849DNAMus musculus 5agagtaggaa gcaagctgca
gggccacaga ccatcggaac taaaggtctg caggggaagt 60tgcaggcagg gctggccact
gcagctccca ggctccaacc tcccgcaatg cttcctgtgg 120cttctggcac taggggtagc
acccaggatc tgttccaggt tggcctagca cctccaggtc 180ctgaagacta ccttgaggtc
agcattccaa cagactcaag agccaagtat cctgaggaca 240caagtggaca gaagggaact
gacgtcctag catctctgag accatctgtg cctcgggtac 300tagtgcttcc tggggaaatt
ccatgtgaga aacctggtga gatattcatt ctgttgaaag 360atgaactaat tggcgaaatt
ctagaggttg aatttatatc aaccaacaag cgcctcagag 420cacggccagc acgttggaat
aagagtgtct ggcatatgaa agctgcagat tttccagctg 480gctcggtcac tgtcaatatc
cactgtgatg gaatcatcaa ggccacaaca gagattaaat 540actgttcagc agcaaaagca
acagaaagtc catttagagt gtcagacccg ggcaagagtt 600tgtgccagaa aagcatcgaa
gaacttgata atgttcttgc atctatattc aagcgtgaga 660taccatatta tgaattcaaa
catctccaag ctgaaactta ccctcaaaaa gaacgtactc 720acaccacaga gctcccaaca
cttcttcact gtgcagcaaa atttggctta aagaatctgg 780ctcttcatct gctgcagtgt
tcaggagcaa ccagggcagc tagaatgaag gcgacagatg 840gttcagacct gctgcatatt
gctgaaaggc atggtcatga agaactcaag gaagtctttg 900aagactttct cagccaaaac
actggcagaa atagcaagca agaaaatgac tatgaagaag 960atgtaatctc attttccaca
tattcaccct ccatgccgtc tccggcatcc cttcatgaac 1020tcaggaagac acacaggcgg
aacacagaca gatctgagga gcctgaaagg tctgtggaga 1080tgaaggagga agaagcaggt
gctgaggcaa gacgcagcct gtcagagggt gaaagggaaa 1140gctccgagaa ccagtatgac
gatctgtatg ttttcatccc tgggtttgac accgaaggca 1200actctgaaga gcctctccca
cactgcaggc cacctctgct gccaccacga ccaggcactg 1260ctgcctccca gctagaaaga
cctcacttta cctcacaagg aaaagtactg gaagaccaaa 1320tggaaagaag tcaaaactgg
aatgatctca atgcaagacc agagacaaga gaggaatcca 1380gcagagaaga aaagaaagaa
gaagcccagg aggaggagga agaagaagaa aacccatatg 1440catttgcaga gactgaagac
aatgagtatg acctgatact ggccagtaag agtgtcaaga 1500aaagaactgg aaatcggtct
ttcattataa acagaccacc ggctcccaca ccccggccca 1560cgcacatccc tcccaaagaa
gaaacaacac cttacatagc tcaagtgttc caacaaaagg 1620cagcccgaag acaatctgat
ggtgataagt tctacagtct acctaagaaa cccgacaaaa 1680ctcggatgga gggcccaacc
ttccctagta caagggatta tctgactact gggcaggaag 1740aactgatcct cctgcaggag
agagtcaaga atgggaaaat gtctgtggat gaagctctgg 1800agaaatttaa acattggcag
atgggaaaga gtggtctgga aatgattcag caggaaaagc 1860tacggcaact acgagacaac
attattggga aaaggccaga agatgaaaat gcctatgata 1920aactgaccat tgtgcaccat
ccaagtggta atactgccca caatgaaaat atgttgtaca 1980acagtccatt caacagtaag
tttcctgctc gaatccaagt tgaaaaggag tttggtttct 2040gctgcaaaaa agatcattaa
aaaagactat tataatcaaa ctcaagaatc tgccaacatg 2100ttgcgcctcg gtgaagccag
cctgcttctg gaatacctgg tctccagggc taatctgcat 2160ggacacagga cacaagtgtg
cctttggatt tcaaagtgtg ttagcaccac aatttattgg 2220tactgtacca cttcagatgg
atacaacaaa agatggagac tcatagcatt ctctgaaaat 2280ccattcattt ttaccacaac
ttttgccacc agagcacctc attctcccat cttgaaaatt 2340aaagaaaaaa aatcagcaaa
gttaaatgca gaatagcaaa attaaggacc caaactatat 2400aggttattct tcctattctt
cctccttcaa ctaagaacgt tttgcatatt tgctctttaa 2460atgaccatct tctgtctgcc
tttctcacat tcagagccat aatgttcttg tgatgccatg 2520ttttcagata gcttctttta
tttactgcct atttgcatgt acctttgaaa tgtactttta 2580ttcgcagttt tcgttagttg
atggtgtttt gtatttgggt gataagcaag cactctacca 2640gtgcccaagt cttcagccct
ctacacggat tcctgaagtc atgttaattc tatcaattaa 2700tgacgtgcac agtaatattt
cagaacatgg atgcctcgca cattgcctgt gctcatcctc 2760tgctcttctg gaaggtattt
ccccatgctt ccctgtcccc aaggacttac taaatgtctt 2820tctctcaaat taaaaagtac
ttttgcaaa 28496650PRTMus musculus
6Met Leu Pro Val Ala Ser Gly Thr Arg Gly Ser Thr Gln Asp Leu Phe 1
5 10 15 Gln Val Gly Leu
Ala Pro Pro Gly Pro Glu Asp Tyr Leu Glu Val Ser 20
25 30 Ile Pro Thr Asp Ser Arg Ala Lys Tyr
Pro Glu Asp Thr Ser Gly Gln 35 40
45 Lys Gly Thr Asp Val Leu Ala Ser Leu Arg Pro Ser Val Pro
Arg Val 50 55 60
Leu Val Leu Pro Gly Glu Ile Pro Cys Glu Lys Pro Gly Glu Ile Phe 65
70 75 80 Ile Leu Leu Lys Asp
Glu Leu Ile Gly Glu Ile Leu Glu Val Glu Phe 85
90 95 Ile Ser Thr Asn Lys Arg Leu Arg Ala Arg
Pro Ala Arg Trp Asn Lys 100 105
110 Ser Val Trp His Met Lys Ala Ala Asp Phe Pro Ala Gly Ser Val
Thr 115 120 125 Val
Asn Ile His Cys Asp Gly Ile Ile Lys Ala Thr Thr Glu Ile Lys 130
135 140 Tyr Cys Ser Ala Ala Lys
Ala Thr Glu Ser Pro Phe Arg Val Ser Asp 145 150
155 160 Pro Gly Lys Ser Leu Cys Gln Lys Ser Ile Glu
Glu Leu Asp Asn Val 165 170
175 Leu Ala Ser Ile Phe Lys Arg Glu Ile Pro Tyr Tyr Glu Phe Lys His
180 185 190 Leu Gln
Ala Glu Thr Tyr Pro Gln Lys Glu Arg Thr His Thr Thr Glu 195
200 205 Leu Pro Thr Leu Leu His Cys
Ala Ala Lys Phe Gly Leu Lys Asn Leu 210 215
220 Ala Leu His Leu Leu Gln Cys Ser Gly Ala Thr Arg
Ala Ala Arg Met 225 230 235
240 Lys Ala Thr Asp Gly Ser Asp Leu Leu His Ile Ala Glu Arg His Gly
245 250 255 His Glu Glu
Leu Lys Glu Val Phe Glu Asp Phe Leu Ser Gln Asn Thr 260
265 270 Gly Arg Asn Ser Lys Gln Glu Asn
Asp Tyr Glu Glu Asp Val Ile Ser 275 280
285 Phe Ser Thr Tyr Ser Pro Ser Met Pro Ser Pro Ala Ser
Leu His Glu 290 295 300
Leu Arg Lys Thr His Arg Arg Asn Thr Asp Arg Ser Glu Glu Pro Glu 305
310 315 320 Arg Ser Val Glu
Met Lys Glu Glu Glu Ala Gly Ala Glu Ala Arg Arg 325
330 335 Ser Leu Ser Glu Gly Glu Arg Glu Ser
Ser Glu Asn Gln Tyr Asp Asp 340 345
350 Leu Tyr Val Phe Ile Pro Gly Phe Asp Thr Glu Gly Asn Ser
Glu Glu 355 360 365
Pro Leu Pro His Cys Arg Pro Pro Leu Leu Pro Pro Arg Pro Gly Thr 370
375 380 Ala Ala Ser Gln Leu
Glu Arg Pro His Phe Thr Ser Gln Gly Lys Val 385 390
395 400 Leu Glu Asp Gln Met Glu Arg Ser Gln Asn
Trp Asn Asp Leu Asn Ala 405 410
415 Arg Pro Glu Thr Arg Glu Glu Ser Ser Arg Glu Glu Lys Lys Glu
Glu 420 425 430 Ala
Gln Glu Glu Glu Glu Glu Glu Glu Asn Pro Tyr Ala Phe Ala Glu 435
440 445 Thr Glu Asp Asn Glu Tyr
Asp Leu Ile Leu Ala Ser Lys Ser Val Lys 450 455
460 Lys Arg Thr Gly Asn Arg Ser Phe Ile Ile Asn
Arg Pro Pro Ala Pro 465 470 475
480 Thr Pro Arg Pro Thr His Ile Pro Pro Lys Glu Glu Thr Thr Pro Tyr
485 490 495 Ile Ala
Gln Val Phe Gln Gln Lys Ala Ala Arg Arg Gln Ser Asp Gly 500
505 510 Asp Lys Phe Tyr Ser Leu Pro
Lys Lys Pro Asp Lys Thr Arg Met Glu 515 520
525 Gly Pro Thr Phe Pro Ser Thr Arg Asp Tyr Leu Thr
Thr Gly Gln Glu 530 535 540
Glu Leu Ile Leu Leu Gln Glu Arg Val Lys Asn Gly Lys Met Ser Val 545
550 555 560 Asp Glu Ala
Leu Glu Lys Phe Lys His Trp Gln Met Gly Lys Ser Gly 565
570 575 Leu Glu Met Ile Gln Gln Glu Lys
Leu Arg Gln Leu Arg Asp Asn Ile 580 585
590 Ile Gly Lys Arg Pro Glu Asp Glu Asn Ala Tyr Asp Lys
Leu Thr Ile 595 600 605
Val His His Pro Ser Gly Asn Thr Ala His Asn Glu Asn Met Leu Tyr 610
615 620 Asn Ser Pro Phe
Asn Ser Lys Phe Pro Ala Arg Ile Gln Val Glu Lys 625 630
635 640 Glu Phe Gly Phe Cys Cys Lys Lys Asp
His 645 650 728DNAArtificial
Sequenceprimer7 7cacctcaacc gccacaatgc tgccagca
28830DNAArtificial Sequenceprimer8 8ataataacct tctttaatga
tctttcttgc 30926DNAArtificial
Sequenceprimer9 9agaggaaact acaccttaca tagctc
261023DNAArtificial Sequenceprimer10 10gatgagttct tcctgaccat
cag 231124DNAArtificial
Sequenceprimer11 11tcaaagcaga tgggagatct caac
241221DNAArtificial Sequenceprimer12 12cagcgccccc
agattctgaa g
211320DNAArtificial Sequenceprimer13 13cagcgccccc aggaaataca
201425DNAArtificial Sequenceprimer14
14gcctattctt tgttttggaa ataca
251525DNAArtificial Sequenceprimer15 15cacatggaat ttcagtggga agcac
251624DNAArtificial Sequenceprimer16
16atcacagtag acattgacat ggac
241724DNAArtificial Sequenceprimer17 17ttggagaggg tatttagagc cata
241823DNAArtificial Sequenceprimer18
18aagcagggct accaattcac cag
231925DNAArtificial Sequenceprimer19 19ctatgatact ggaaatactg tcagt
252021DNAArtificial Sequenceprimer20
20agcatatgac cagctgatca g
212125DNAArtificial Sequenceprimer21 21ttgatttact atgaaaatat caagc
252223DNAArtificial Sequenceprimer22
22ttacataaga aaccagcttc cag
232321DNAArtificial Sequenceprimer23 23acctcccgca atgcttcctg t
212424DNAArtificial Sequenceprimer24
24acatggaatt tccccaggaa gcac
24
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