Abstract:
The invention involves isolation of a new class of proteins, referred to hereafter as the cryopyrins. Also described are the isolated nucleic acid molecules involved in their expression. The inventive molecules are useful in diagnosing and treating inflammatory diseases, such as FCU/FCAS and MWS.

Description:
RELATED APPLICATION  
       [0001]    This application claims priority of provisional application Serial No. 60/327,728, filed Oct. 5, 2001, incorporated by reference in its entirety. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    This invention relates to nucleic acid molecules associated with familial cold urticaria, or familial cold autoinflammatory syndrome, as well as Muckle Wells Syndrome, the proteins encoded thereby, and uses thereof. Various aspects of the invention are disclosed.  
         BACKGROUND AND PRIOR ART  
         [0003]    Familial cold urticaria (“FCU” hereafter), was first described by Kile, et al.,  JAMA  114:1067-1068 (1940). It has been referred to as familial polymorphous cold eruption, cold hypersensitivity, cold pathergy and cold specific vasomotor neuropathy.  
           [0004]    Clinical features of FCU include recurrent attacks of a nonpruritic, nonurticarial maculopapular exanthem associated with arthralgia, fever and chills, conjunctivitis, myalgia, headaches, fatigue and swelling of the extremities. See Tindall, et al.,  Arch. Intern Med  124:129-134 (1969); Zip, et al.,  Clin. Exp. Dermatol  18:338-341 (1993).  
           [0005]    Onset of symptoms of FCU is generally delayed 30 minutes to 3 hours after exposure to cold, and these persist for 24-48 hours. The most consistent laboratory finding is marked, polymorphonuclear leukocytosis, which is found during attacks. See Tindall, et al., supra; Doeglas, et al.,  Arch. Dermatol  110:382-388 (1974).  
           [0006]    Pathological changes which occur to the skin of patients during attacks include a primarily polymorphonuclear leukocyte perivascular infiltrate, increased vascularity, and dermal edema; however, no vasculitis is observed. See Martin, et al.,  Cutis  27:173-175 (1981); Tomesen, et al.,  Clin. Res.  33:690 (1985); Zip, et al., supra.  
           [0007]    FCU manifests as early as birth, but generally not later than childhood. The condition persists through the subject&#39;s life. Generally, afflicted individuals have normal longevity, but some exhibit late-onset renal amyloidosis. Some patients also exhibit variants of Muckle-Wells syndrome, which is a condition that has variable expression, classically including recurrent rash, late-onset progressive nerve deafness, and late onset renal amyloidosis. See Muckle, et al.,  Br. J. Dermatol  100:87-92 (1979). Muckle-Wells syndrome patients exhibit phenotypes similar to FCU patients, but the symptoms are not precipitated by cold. See Muckle, supra; Jung, et al.,  Am. J. Hum. Genet.  59:A223 (1996).  
           [0008]    With respect to inflammatory mediators, the only significant abnormalities in FCU patients are elevations of G-CSF and IL-6 (Urano, et al.,  Br. J. Dermatol  139:504-507 (1998)), and acute phase reactants such as C-reactive protein (Tonesen, et al.,  Aspen Allergy Conference Presentation,  1985).  
           [0009]    Previously, Cuisset, et al.,  J. Hum. Genet  65:1054-1059 (1999), mapped Muckle-Wells Syndrome to chromosome 1q44. Hoffman, et al.,  Am. J. Hum. Genet  66:1693-1698 (2000), incorporated by reference, linked FCU to a locus on 1q44 as well. They describe a 10 cM region in which the gene is located.  
           [0010]    None of these references describe the isolation and/or identification of a nucleic acid molecule associated with FCU or as it is also referred to, familial cold autoflammatory syndrome, or FCAS, or a nucleic acid molecule associated with Muckle-Wells Syndrome. An aspect of the invention is the isolation and identification of such nucleic acid molecules, and the ramifications thereof. Also disclosed are mutations within this gene which are associated with the disorder.  
           [0011]    These will be elaborated upon in the detailed description which follows. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0012]    [0012]FIG. 1 set forth analysis of one family which includes individuals that suffer from FCAS.  
         [0013]    [0013]FIG. 2 presents data on additional families which present heterozygous missense mutations.  
         [0014]    [0014]FIG. 3 shows the structure of the CIAS 1 gene.  
         [0015]    [0015]FIG. 4 details information on additional families analyzed for FCAS.  
         [0016]    [0016]FIG. 5 presents haplotype analysis of selected individuals for the families analyzed.  
         [0017]    [0017]FIGS. 6 a - 6   d  present variations on the physical map of the region relevant to the gene in question.  
     
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS  
     EXAMPLE 1  
       [0018]    Analysis of DNA was carried out on samples taken from members of 3 families which included at least two individuals who suffered from FCAS, and one family which included members suffering from MWS. The diagnosis of these individuals was based on a history of recurrent episodes of rash, arthralgia, and fever after generalized cold exposure. MWS subjects also suffered from sensoryneural hearing loss; however, the symptoms were not associated with exposure to cold.  
         [0019]    Genomic DNA was isolated from peripheral blood samples taken from each subject. The DNA was then amplified, via PCR, and subjected to automated, fluorescent genotyping following Hoffman, et al.,  Am J. Hum Genet.  66:1693-1698 (2000), incorporated by reference, and microsatellite markers available in public databases.  
         [0020]    [0020]FIG. 1 presents the analysis of one such family. In this figure, the filled in figures represent individuals suffering from FCAS, while empty figures are unaffected individuals. The top of the figure indicates the microsatellite markers used, together with the allele numbers for each marker, as observed for each of the chromosome 1q44 haplotypes. Marker order was derived from several databases, and was confirmed by standard radiation hybrid mapping.  
         [0021]    The boxed haplotype is the haplotype associated with FCAS. It is shared by all of the diseased individuals; however, note that it is also shared by a non-diseased individual. This suggests that a new mutation arose in individual “4,” which was also passed to descendants.  
       EXAMPLE 2  
       [0022]    These experiments describe further work on identifying and analyzing the coding region associated with FCAS and MWS.  
         [0023]    Given the association of locus 1q44 with the disease, public data bases were screened to identify ESTs associated with this region. Once they were identified, these CDNA molecules were sequenced, assembled into longer sequence contigs to the extent possible, based upon overlap, extended, and then had their sequences confirmed, using a commercially available computer algorithm, i.e., “SEQUENCER 3.1.” 
         [0024]    Primers were designed to be used in PCR amplification, based upon comparison of isolated cDNA sequences and the sequence of 1q44 in public data bases. The GENSCAN prediction program was used to analyze the publicly available information regarding 1q44. Some of the primers used to amplify regions of transcribed sequences in genomic DNA, were as follows:  
                               5′-ggctggtcttgaattcctca-3′,   (SEQ ID NO: 1)                   5′-aggttgcagtgagccaagat-3′,   (SEQ ID NO: 2)               5′-gctcccaaccagacttttga-3′,   (SEQ ID NO: 3)               5′-gactgacaagagccacacaaa-3′,   (SEQ ID NO: 4)               5′-gttaccactcgcttccgatg-3′,   (SEQ ID NO: 5)               5′-cctcgttctcctgaatcagac-3′,   (SEQ ID NO: 6)               5′-catgtggagatcctgggttt-3′,   (SEQ ID NO: 7)               5′-gctgtggcaacagtatttgg-3′,   (SEQ ID NO: 8)               5′-cggaaggcatttctctgaac-3′,   (SEQ ID NO: 9)               5′-aagaaaccacaccagcaacc-3′,   (SEQ ID NO: 10)               5′-aggtgtgtcctgatgcttcc-3′,   (SEQ ID NO: 11)               5′-cctcactgaagccagagtgc-3′,   (SEQ ID NO: 12)               5′-gagtagaggcagtggcaggt-3′,   (SEQ ID NO: 13)               5′-cctccagtccttcaaagcat-3′,   (SEQ ID NO: 14)               5′-ttggctctttctgtcggact-3′,   (SEQ ID NO: 15)               5′-tccagcttagccttggtgat-3′,   (SEQ ID NO: 16)               5′-agtgcaacccaggctttcta-3′,   (SEQ ID NO: 17)               5′-tcacagagctgtggtcttgg-3′,   (SEQ ID NO: 18)          
 
         [0025]    These primers were designed to amplify exons with flanking intronic sequences. Amplification was carried out in 20 μl PCR reactions, using Taq DNA polymerase (0.8U), dNTPs (250 μM), MgCl 2  (2.5 mM), PE Buffer I (1×), primers (500 nM) and 20-80 ng of DNA taken from afflicted individuals, and a control who did not suffer from the syndrome, using commercially available reagents. The conditions used included initial denaturation at 94° C. for 4 minutes followed by 10 cycles at 94° C., 30 seconds per cycle, touchdown annealing (1° C. decrease per cycle) between 65-55° C., for 30 seconds, extension at 74° C. for 1 minute, and 25 additional cycles. The annealing temperature was 55° C., followed by a final extension at 72° C., for 7 minutes.  
         [0026]    Ninety genomic fragments, containing more than 80 exons resulted, in total, using oligonucleotide primers in addition to those given, supra. These were purified, and sequenced via automated sequence techniques, in accordance with Kolodner, et al.,  Canc. Res.  59:5068-5074 (1999), incorporated by reference. Both the forward and reverse strands of PCR products were sequenced.  
         [0027]    Sequencing of the amplified genonic DNA revealed 4 different missense mutations. Particulars of the position and specific mutations are presented infra. In terms of the individuals and families under analysis, the mutation which occurred in individual “4” appears to have arisen “de novo,” as only affected members of subsequent generations of the family inherited it, i.e., subjects 5, 9 and 11 in FIG. 1. Neither parent of subject “4” had the mutation, notwithstanding the presence of the disease haplotype in the mother. Three additional families, as shown in FIG. 2, possessed different, heterozygous missense mutations present in all afflicted subjects. One of the families had members diagnosed with Muckle-Wells Syndrome, and both suffered from sensorineural hearing loss. The family had a mutation in the same exon as the FCU/FCAS families. In a fourth family, individuals of which suffered from MWS, the phenotype appeared to arise as a result of a de novo mutation.  
         [0028]    Following these results, controls were run on over 100 samples taken from unaffected family members, and taken from random North American blood banks. The mutations were not found in any of these samples. This absence supports the conclusion that the mutations described herein cause FCU/FCAS, and Muckle-Wells Syndrome.  
         [0029]    The mutations referred to supra all appeared in the same exon, i.e., one consisting of 1753 base pairs. BLAST searching of public data bases identified two ESTs containing all or a part of this exon, i.e., AK 027194, and AF 054176. In all, analysis of publicly available genome sequences and sequences of BACs containing region 1q44 resulted in identification of 7 exons.  
       EXAMPLE 3  
       [0030]    The experiments described herein were designed to analyze the nucleic acid molecules more fully.  
         [0031]    RT-PCR was carried out, via standard methods. Following sequencing, two additional exons and extensive alternative slice variants were identified, in the C-terminal region.  
         [0032]    PCR products were obtained via amplifying human genomic DNA, chromosome 1q44, and RPI11 BAC clones (433k2, 978I15 AND 482N10), using standard methods, in order to characterize exons and flanking intron sequences.  
         [0033]    The experiments led to a predicted structure to the gene, as set forth in FIG. 3. The structure indicates that the gene contains 9 exons, encoding a 3105 base pair ORF. Two alternative start codons were found in the first exon, with the second one satisfying more Kozak criteria than the first. A stop codon was found in exon 9.  
       EXAMPLE 4  
       [0034]    These experiments describe Northern Blotting work. Probes were prepared corresponding to nucleotides-877 to 267, 1093 to 2150 and 2353-2970 of the ORF. A β actin control probe was also used. Probes were labeled with  α32 PdCTP, using commercially available materials and the instructions provided therein. Commercially available, multiple tissue blots were used.  
         [0035]    The results identified a broad band of about 4 kb mRNA, expressed at low levels in peripheral blood lymphocytes, and with little or no expression in other tissue. This expression pattern is identical to that described for the “MEFV” gene, which is associated with Familial Medittaranean Fever. See  Cell  90:797-807 (1997).  
         [0036]    In experiments designed to verify the size of the mRNA, peripheral blood leukocyte mRNA was amplified, using primers designed from the genomic coding sequence, and the ends of the mRNA were amplified via RACE, using primers:  
                                           ttgtgacaca gaggagcctg   (SEQ ID NO: 19)               and                       cctcgttctc ctgaatcagac,   (SEQ ID NO: 20)          
 
         [0037]    in accordance with the instructions from a commercially available product. The sequences were analyzed, and revealed that there was extensive alternative splicing of exons 4-8 of FIG. 3. The mRNA ranged in size from 3315 to 4170 base pairs. The pattern of distribution of the exons in the splice variants is set forth below:  
         [0038]    1, 2, 3, 7, 8, 9-26.9%  
         [0039]    1, 2, 3, 4, 5, 6, 7, 8, 9-16%  
         [0040]    1, 2, 3, 5, 7, 8, 9-14.6%  
         [0041]    1, 2, 3, 5, 6, 7, 8, 9-13.1%  
         [0042]    1, 2, 3, 4, 5, 7, 8, 9-3.1%  
         [0043]    1, 2, 3, 9-3.1%  
         [0044]    1, 2, 3, 5*, 7, 8, 9-3.1%  
         [0045]    1, 2, 3, 4, 7, 8, 9-2.4%  
         [0046]    1, 2, 3, 5, 7, 9-1.5%  
         [0047]    1, 2, 3, 8, 9-1.5%  
         [0048]    1, 2, 3, 4, 9-1.5%  
         [0049]    1, 2, 3, 4, 7, 9-0.8%  
         [0050]    1, 2, 3, 6, 7, 8, 9-0.8%  
         [0051]    1, 2, 3, 5, 8, 9-0.8%  
         [0052]    1, 2, 3, 5, 6, 8, 9-0.8%  
         [0053]    1, 2, 3, 4, 5, 6, 8, 9-0.8%  
         [0054]    1, 2, 3, 4, 6, 7, 8, 9-0.8%  
         [0055]    1, 2, 3, 4, 5*, 6, 7, 8, 9-0.8%  
         [0056]    1, 2, 3, 5*, 6, 7, 8, 9-0.8%  
         [0057]    1, 2, 3, 5, 6, 7**, 8, 9-0.8%  
         [0058]    with reference to the open reading frame, these exons are encoded by nucleotides in parenthesis:  
         [0059]    1 (1-277), 2 (278-397), 3 (298-2150), 4 (2151-2321), 5 (2322-2492), 6 (2493-2663), 7 (2664-2834), 8 (2835-3005) and 9 (3006-3105).  
         [0060]    5* is a splicing event where the upstream exon is spliced to a site 57 bp downstream of exon 5. 7** is a splicing event where the upstream exon is spliced to a site 140 bp upstream of exon 7, resulting in an in frame stop codon downstream of the splice junction.  
       EXAMPLE 5  
       [0061]    One of the splice forms identified in the examples described supra is set forth at SEQ ID NO: 21. It contains exons 1-3, 5, 7 and 9. The deduced amino acid sequence (SEQ ID NO.: 22) consists of 920 amino acids, a size of about 105.7 kilodaltons as determined by SDS-PAGE, and a pI of about 6.16. Additional sequence information is also provided.  
         [0062]    The sequence contains several distinct motifs. Specifically, a pyrin domain is found at amino acids 13-83 (see Martinin, et al.,  Curr. Biol  11:R118-R120 (2001); Bertin, et al,  Cell Death Differ  7:1273-1274 (2000); Pawlowski, et al.,  Trends Biochem Sci  26:85-97 (2001)), a central NBS from the NACHT subfamily (Koonin, et al.,  Trends Biochem Sci  25:223-224 (2000)), in exon 3 at amino acids 217-533, and a C-terminal LRR domain, with 7 LRRs contained within amino acids 697-920. “LRRs” are known to the skilled artisan as domains of proteins which bind to other proteins. See Kobe, et al.,  Nature  374:183-186 (1995). No nuclear localization signals were identified, nor were any clear transmembrane regions found.  
         [0063]    If all 9 exons are transcribed, the largest protein produced contains 1034 amino acids, is about 117.8 kD in size, and contains 11 LRRs. These features are consistent with the protein being a signaling protein, involved in regulation of inflammation and apoptosis. The proteins will be referred to collectively as cropyrins, and their encoding molecules as forms of the CIAS1, (cold induced autoinflammatory syndrome 1) gene.  
       EXAMPLE 6  
       [0064]    In further experiments, informed consent was obtained, and a total of 179 individuals, from four families which included sufferers from FCAS, were analyzed. Of these 179 individuals, 105 were unaffected, and 74 were affected.  
         [0065]    Pedigree information and family history information were obtained from multiple sources within each family. Detailed information on pedigrees can be found at Hoffman, et al, J. Allergy Clin. Immunol 108:615-620 (2001), incorporated by reference. The individual names and pedigree structures were evaluated extensively for relatedness between families. No relationship was ascertained.  
         [0066]    Representative segments of the pedigrees are presented in FIG. 4. Filled squares represent afflicted males, and filled circles afflicted females. Unaffected individuals are represented by open figures.  
         [0067]    The pedigrees demonstrate an autosomal dominant inheritance pattern, with near complete, penetrance, and a transmission rate of 50%. The earliest affected ancestors lived in the 17 th  century.  
         [0068]    As noted, supra, there was no evidence of relatedness between families based upon names or pedigree structure; however, members of the first three families share a legend concerning the origin of the disorder, relating to a healthy man who had a prolonged, life threatening exposure to cold water. Following the exposure, the individual developed symptoms of FCAS, and passed them onto offspring.  
       EXAMPLE 7  
       [0069]    Following the results discussed supra, blood samples were taken from 179 members of the four families described above. Of these, 74 were afflicted individuals, and 105 were unafflicted. Standard venipuncture and genomic DNA isolation techniques were used.  
         [0070]    Clones from the FCAS locus were identified, using oligonucleotides derived from genetic markers that had been marked previously. These oligonucleotides were labeled with  32 P, and were hybridized to RPCI-11BAC, in accordance with Shizuya, et al, Proc. Natl. Acad. Sci. USA 89:8794-8797 (1992), and RPCI-4PAC, in accordance with Ioanormu, et al, Nat. Genet. 6:84-89 (1994).  
         [0071]    Further oligonucleotides were designed, using clone end sequences, labeled with  32 P, and then hybridized to filters, in order to identify overlapping clones, via chromosome walking.  
         [0072]    A group of 31 clones, covering over 60% of the region, were sent to Wellcome Trust Sanger Institute for incorporation into their chromosome 1 physical map.  
         [0073]    Restriction digest fingerprints of the clones were prepared, in accordance with Gregory, et al, Genome Res. 7:1162-1168 (1997), incorporated by reference, and these were compared to chromosome 1 data set using fingerprint contigs, in accordance with Soderland, et al, Comput. Appl. Biosci. 13:523-535 (1997), incorporated by reference. When fingerprint overlaps were found to be statistically significant, they were assimilated into the map.  
         [0074]    Two clones formed the basis of the sequence tiling map, discussed infra, and one facilitated the cloning of a gap in the map of chromosome 1.  
       EXAMPLE 8  
       [0075]    All 179 subjects were then genotyped in the 10 cM region between markers D1S423 and D1S2682, in order to identify rare cross over events. Ten of the oldest affected and available family members from these families were also genotyped in the 50 cM region between markers D1S549 and D1S2682, in order to evaluate shared haplotypes. Microsatellite markers were identified in a series of public databases, including www.chlc.org; www.genethon.fr; www.cedargenetics.soton.ac.uk; www.gdb.org; www.morshmed.org/genetics; and www.genome.wi.mit.edu. Additional microsatellite markers were designed by searching the public genome database in the region (www.ncbi.nlm.nih.gov/entrez), for short, tandem repeat sequences greater than 20 base pairs long. Flanking oligonucleotides primers were designed, and PCR was carried out as described by Hoffman, et al, Am. J. Hum. Genet. 66:1693-1698 (2000), incorporated by reference. Haplotypes were constructed using ordered genotypes from multiple family members, in order to identify recombination events and to recognize shared alleles, using the commercially available program Cyrillic 3.1.  
         [0076]    The primer sequences designed and used for markers were:  
                                           gtgaattctg cagctgttgg   (SEQ ID NO: 23)               and           tgtaatccct gcactgagga   (SEQ ID NO: 24)           for D1S3770;                       ctcaactcct gcccagtgaa   (SEQ ID NO: 25)           and           tgagctgaga tcatgcact   (SEQ ID NO: 26)           for D1S3771;                       tcatttcacc tccctaaatt gaa   (SEQ ID NO: 27)           and           ccctttggaa ggaaattctg   (SEQ ID NO: 28)           for D1S3772; and,                       cccctggtat ataacccctt aca,   (SEQ ID NO: 29)           and           cctgcctgat aaagttgttt tg   (SEQ ID NO: 30)           for D153773.          
 
         [0077]    These four markers, together with known markers, were used to study haplotypes. The results, set forth in Table 1, which follows, show that families 1-3 share a pattern of multiple alleles for polymorphic microsatellite markers at 1q44, indicating a common serial haplotype of approximately 10 cM between D1S423 and D1S2682. In this table, critical mapped regions are shaded. “MB” stands for the USCS Golden Path genomic map, while “cM” is based upon the Genethon genetic map.  
                         TABLE 1                       Microsatellite markers in the FCAS locu                                                                                                                                                 
 
         [0078]    Several individuals, marked by asterisks in FIG. 4, were genotyped over a 50 cM region. These individuals are marked by a thatch (“#” mark). They shared a much longer haplotype of approximately 40 cM, between D1S3462, and D1S2682.  
         [0079]    Further pedigree analysis and family history did not uncover a common ancestor, suggesting the familial connection must extend beyond current data, to at least 8 generations in the past. Family 4 was not seen to share the hapoltype.  
       EXAMPLE 9  
       [0080]    Haplotype analysis was carried out on all 4 families, as described, supra. Informative recombination information was found in some individuals, and this information is set forth in FIG. 5. Ordered STS markers were used, and are presented to the left of subject 2A. The allele numbers for each microsatellite marker are presented for the 2 chromosomes 1q44 haplotypes which were inherited by each individual. FIG. 2 shows the haplotype associated with FCAS, boxed.  
         [0081]    There were key, crossover events found in unaffected patient 2D, where there was a crossover between D1S2836 and AFM 207xa7, one in affected patient 3D, between AFMb005wh9 and AFM155xc11, and one in affected patient 4C, between D1S3772 and D1S3773. Recombinational analysis of all of the family data suggests a centromeric limit at D1S2836 and a telomeric limit at D1S3773.  
       EXAMPLE 10  
       [0082]    A complete physical map of the region extending from D1S423 through D1S2682 was constructed, using standard chromosomal working and BAC fingerprinting methods. This map is set forth in FIGS. 6 a - 6   d . It shows the filing of 34 BACs covering the region. FIG. 6 a  shows the location of 13 ordered microsatellite markers on the Genethon genetic map, which can be found at www.genethon.fr, and is incorporated by reference. This covers about 10 cM. FIG. 6C shows corresponding BACs, while FIG. 6 d  shows a map, based upon USC Genome Bioinformatics (www.genome.USC.edu), which covers about 3 Mb. The critical region, i.e., that discussed supra, between D1S8236 and D1S3773, in less than 1 Mb in size.  
       EXAMPLE 11  
       [0083]    Examples 1-5, supra, describe the isolation and identification of the CIAS1 gene. An intron/exon map is set forth at FIG. 6 e . Exons 1-6 are found on BAC-RPCI-11 433K2, while exons 7-9 are found on BAC-RPCI-11 97815.  
       EXAMPLE 12  
       [0084]    This example describes experiments which were carried out to determine if any additional mutations within the CIAS1 gene were connected to FCAS.  
         [0085]    The primers set forth in example 2 were used, as was an additional primer based upon exon 3, i.e.:  
         [0086]    ttgtgacaca gaggagcctg (SEQ ID NO: 31).  
         [0087]    PCR was carried out as described in example 2, as was purification and sequencing. Normal controls were unaffected spouses, over 100 normal blood bank control DNA samples, and 50 DNA samples of rheumatoid arthritis patients. Allele frequencies were calculated by dividing the number of base changes found, by the total number of chromosome sequenced, which was about 400.  
         [0088]    A total of 11 single nucleotide polymorphisims, or “SNPs” were found, as shown in Table 2. Two are rare (less than 1%) variations, and all but one were located in exon 3. All disease causing mutations were found in exon 3. With the exception of a change at nucleotide 2107, all of these mutations are silent. Three of the mutations, i.e., G780A, C930T, and C1302T, are located within a few base pairs of previously identified disease causing mutations, i.e., C778T, T926C, C1307A. See Dode, et al, Am. J. Hum. Genet. 71:198-203 (2002). Dode, et al, had reported four of these SNPs previously, with similar allele frequency, in a European population.  
                                                                   TABLE 2                           Single nucleotide polymorphisims within CIAS1                Amino       Frequency       Frequency       Nucleotide   Acid   Allele   (%)   Allele   (%)*                     657   219   C   92.4   T   7.6        726   242   A   56.0   G   44.0        780#   260   G   97.7   A   2.3        930#   310   C   99.5   T   0.5       1231   411   C   98.5   T   1.5        1302#   434   C   84.5   T   15.5       1383   461   C   99.0   T   1.0       1389   463   C   99.5   T   0.5       1600   534   C   99.7   T   0.3       2107   Q703K   C   97.0   A   3.0       Intron + 59   —   G   96.5   A   3.5                                  
 
         [0089]    The foregoing examples set forth variants features of the invention, including various members of the cryopyrin protein family and nucleic acid molecules which encode them. “Cryopyrin” as used herein, refers to any and all forms of the protein described herein. As used herein “cryopyrin” refers to any protein which comprises, in its amino acid sequence, at least the amino acids encoded by exons 1, 2 and 3 of the CISA1 gene, as described herein. In the alternative, with reference to the open reading frame, these proteins include amino acids encoded by nucleotides 2150, in SEQ ID NO: 21. Additional embodiments relate to proteins which are encoded by nucleotide sequences which comprise nucleotides 1-2150, followed by additional nucleotides, and ending with nucleotides 3006-3105. An especially preferred embodiment is a protein encoded by nucleotides 1-2150, concatenated to nucleotides 2834-3105 of SEQ ID NO: 21. The various splice variants described herein, together with the portions of the ORF which encode them, are described supra. All of these, as well as the encoded proteins, expression vectors which comprise the nucleic acid molecule splice variants operably linked to a promoter, and cells transformed or transfected thereby, are part of the invention.  
         [0090]    While human molecules are described, other species are included in the invention, such as mammalian molecules. Exemplary, but by no means exclusive examples of such molecules include murine, bovine, and other species. Especially preferred are molecules which exhibit at least 60%, preferably at least 70%, and most preferably, at least 80% homology with the human splice variants described herein.  
         [0091]    It will be understood by the skilled artisan that the splice variants described above are not limiting, and other splice variants should be expected. While it can perhaps be assumed that most of these will include exons 1, 2, and 3, this is not a requirement, and the inventors do not wish to be bound to such a requirement.  
         [0092]    Similarly, nucleic acid molecules which encode the missense forms described herein are a feature of the invention, as are the resulting mutants. As pointed out, supra, mutations associated with various positions, such as 592, 657, 726, 780, 930, 1055, 1231, 1302, 1316, 1383, 1389, 1600, 1880 and 2107 of the open reading frame are in turn associated with FCU/FCAS and MWS. Hence, any variant nucleic acid molecule and/or protein with a mutation relative to the wild type molecules is a feature of this invention, including the specific mutations described herein.  
         [0093]    Also a feature of this invention are methods for diagnosing disorders. As has been shown, supra, missense and silent mutations within the CIAS1 gene are associated with disorders such as FCU/FCAS and MWS. One of ordinary skill in the art can envision other disorders, associated with the mutations described herein, or others in the CIAS1 gene. Various methodologies for identifying mutations in genes, transcripts and proteins, are all well known to the art and need not be elaborated upon in detail. Exemplary of such assays are hybridization assays, such as assays based upon labelled oligonucleotide probes, PCR, other assays based upon nucleic acid amplification, assays based upon enzymatic cleavage with restriction endonucleases, and so forth. Proteins based assays, such as immunoassays, electrophoretic assays, and so forth, are also features of the invention.  
         [0094]    As noted, supra, the mutations in the proteins and nucleic acid molecules are associated with FCU/FCAS and MWS, while the wild type proteins are not. Both FCU/FCAS and MWS are inflammation related disorders. Hence, a further aspect of the invention is the treatment of inflammatory disorders, including FCU/FCAS and MWS, via administration of appropriate forms of wild type protein. Such forms of wild type protein include an amino acid sequence including at least the wild type amino acids corresponding to the relevant mutation or mutations. These can be determined via standard methodologies for determining the nucleotide sequence of the gene from the afflicted individuals. The proteins may be administered in any of the standard methods used for administration of proteins, such as intravenous administration, subcutaneous administration, etc. The proteins can be combined with various substances to optimize their delivery and/or efficacy, such as standard pharmaceutical carriers. They can be formulated for timed or delayed release, etc.  
         [0095]    The multiple splice patterns described supra include molecules with varying numbers of LRRs. As noted, supra, LRRs are motifs involved in protein-protein interaction and binding. Differences in LRR content suggest that the different proteins interact with a variety of other molecules, and such interactions may be involved in the development or prevention of pathological processes, including inflammation. One aspect of the invention is a method for identifying molecules which interact with one or more of the splice variants of the invention, by contacting the molecule of interest with a splice variant, and determining binding there between. One can identify those molecules which bind specifically to one or less than all variants, by screening against a plurality of variants. Such a method is especially useful in situations where, e.g., a variant is expressed in particular tissue types, at development stages, in connection with particular disorders, etc. It has also been found that when CIAS1 patients are heat induced, extraordinarily high levels of interleukin-6 (“IL-6”) are released, suggesting a therapeutic role for this molecule in treatment of the disorders set forth herein.  
         [0096]    Further features of the invention include antibodies, such as monoclonal antibodies, humanized antibodies, fragments of antibodies which bind to the proteins of the invention, hybridimas which produce the antibodies, and so forth are also a part of the invention.  
         [0097]    Other features of the invention will be clear to the artisan and need not be set forth herein.  
     
       
       
         1 
         
           
             31  
           
           
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             Homo sapiens  
             
 
           
            1 

ggctggtctt gaattcctca                                                 20 

 
           
             2  
             20  
             DNA  
             Homo sapiens  
             
 
           
            2 

aggttgcagt gagccaagat                                                 20 

 
           
             3  
             20  
             DNA  
             Homo sapiens  
             
 
           
            3 

gctcccaacc agacttttga                                                 20 

 
           
             4  
             21  
             DNA  
             Homo sapiens  
             
 
           
            4 

gactgacaag agccacacaa a                                               21 

 
           
             5  
             20  
             DNA  
             Homo sapiens  
             
 
           
            5 

gttaccactc gcttccgatg                                                 20 

 
           
             6  
             21  
             DNA  
             Homo sapiens  
             
 
           
            6 

cctcgttctc ctgaatcaga c                                               21 

 
           
             7  
             20  
             DNA  
             Homo sapiens  
             
 
           
            7 

catgtggaga tcctgggttt                                                 20 

 
           
             8  
             20  
             DNA  
             Homo sapiens  
             
 
           
            8 

gctgtggcaa cagtatttgg                                                 20 

 
           
             9  
             20  
             DNA  
             Homo sapiens  
             
 
           
            9 

cggaaggcat ttctctgaac                                                 20 

 
           
             10  
             20  
             DNA  
             Homo sapiens  
             
 
           
            10 

aagaaaccac accagcaacc                                                 20 

 
           
             11  
             20  
             DNA  
             Homo sapiens  
             
 
           
            11 

aggtgtgtcc tgatgcttcc                                                 20 

 
           
             12  
             20  
             DNA  
             Homo sapiens  
             
 
           
            12 

cctcactgaa gccagagtgc                                                 20 

 
           
             13  
             20  
             DNA  
             Homo sapiens  
             
 
           
            13 

gagtagaggc agtggcaggt                                                 20 

 
           
             14  
             20  
             DNA  
             Homo sapiens  
             
 
           
            14 

cctccagtcc ttcaaagcat                                                 20 

 
           
             15  
             19  
             DNA  
             Homo sapiens  
             
 
           
            15 

ttggctcttt cgtcggact                                                  19 

 
           
             16  
             20  
             DNA  
             Homo sapiens  
             
 
           
            16 

tccagcttag ccttggtgat                                                 20 

 
           
             17  
             20  
             DNA  
             Homo sapiens  
             
 
           
            17 

agtgcaaccc aggctttcta                                                 20 

 
           
             18  
             20  
             DNA  
             Homo sapiens  
             
 
           
            18 

tcacagagct gtggtcttgg                                                 20 

 
           
             19  
             20  
             DNA  
             Homo sapiens  
             
 
           
            19 

ttgtgacaca gaggagcctg                                                 20 

 
           
             20  
             21  
             DNA  
             Homo sapiens  
             
 
           
            20 

cctcgttctc ctgaatcaga c                                               21 

 
           
             21  
             4193  
             DNA  
             Homo sapiens  
             
 
           
            21 

gtagatgagg aaactgaagt gaggaatagt gaagagtttg tccaatgtca tagccccgta     60 

atcaacggga caaaaatttt cttgctgatg ggtcaagatg gcatcgtgaa gtggttgttc    120 

accgtaaact gtaatacaat cctgtttatg gatttgtttg catatttttc cccccatagg    180 

gaaacctttt ttccatggct caggacacac tcctggatcg agccaacagg agaactttct    240 

ggtaagcatt tggctaactt tttttttttt gagatggagt cttgctgtgt cgcctaggct    300 

ggagtgcagt ggcgtgatct tggctcactg cagcctccac ctcccgggtt caatcaattc    360 

tcctacctca acttcctgag tagctgggat tacaggcgcc cgccaccaca cccggctcat    420 

ttttgtactt ttagtagaga cacagttttg ccatgttggc caggctggtc ttgaattcct    480 

cagctcaggt gatatgcctg ccttggcctc tcaaagtgct gggattacag gcgtgagcca    540 

ctgtgcccgg ccttggctaa cttttcaaaa ttaaagattt tgacttgtta cagtcatgtg    600 

acattttttt ctttctgttt ggtgagtttt tgataattta tatctctcaa agtggagact    660 

ttaaaaaaga ctcatctgtg tgccgtgttc actgcctggt atcttagtgt ggaccgaagc    720 

ctaaggaccc tgaaaacagc tgcagatgaa gatggcaagc acccgctgca agctggccag    780 

gtacctggag gacctggagg atgtggactt gaagaaattt aagatgcact tagaggacta    840 

tcctccccag aagggctgca tccccctccc gaggggtcag acagagaagg agaccatgtg    900 

gatctagcca cgctaatgat cgacttcaat ggggaggaga aggcgtgggc catggccgtg    960 

tggatcttcg ctgcgatcaa caggagagac ctttatgaga aagcaaaaag agatgagccg   1020 

aagtggggtt cagataatgc acgtgtttcg aatcccactg tgatatgcca ggaagacagc   1080 

attgaagagg agtggatggg tttactggag tacctttcga gaatctctat ttgtaaaatg   1140 

aagaaagatt accgtaagaa gtacagaaag tacgtgagaa gcagattcca gtgcattgaa   1200 

gacaggaatg cccgtctggg tgagagtgtg agcctcaaca aacgctacac acgactgcgt   1260 

ctcatcaagg agcaccggag ccagcaggag agggagcagg agcttctggc catcggcaag   1320 

accaagagtg tgagagcccc gtgagtccca ttaagatgga gttgctgttt gaccccgatg   1380 

atgagcattc tgagcctgtg cacaccgtgg tgttccaggg ggcggcaggg attgggaaaa   1440 

caatcctggc caggaagatg atgttggact gggcgtcggg gacactctac caagacaggt   1500 

ttgactatct gttctatatc cactgtcggg aggtgagcct tgtgacacag aggagcctgg   1560 

gggacctgat catgagctgc tgccccgacc caaacccacc catccacaag atcgtgagaa   1620 

aaccctccag aatcctcttc ctcatggacg gcttcgatga gctgcaaggt gcctttgacg   1680 

agcacatagg accgctctgc actgactggc agaaggccga gcggggagac attctcctga   1740 

gcagcctcat cagaaagaag ctgcttcccg aggcctctct gctcatcacc acgagacctg   1800 

tggccctgga gaaactgcag cacttgctgg accatcctcg gcatgtggag atcctgggtt   1860 

tctccgaggc caaaaggaaa gagtacttct tcaagtactt ctctgatgag gcccaagcca   1920 

gggcagcctt cagtctgatt caggagaacg aggtcctctt caccatgtgc ttcatccccc   1980 

tggtctgctg gatcgtgtgc actggactga aacagcagat ggagagtggc aagagccttg   2040 

cccagacatc caagaccacc accgcggtgt acgtcttctt cctttccagt ttgctgcagc   2100 

cccggggagg gagccaggag cacggcctct gcgcccacct ctgggggctc tgctctttgg   2160 

ctgcagatgg aatctggaac cagaaaatcc tgtttgagga gtccgacctc aggaatcatg   2220 

gactgcagaa ggcggatgtg tctgctttcc tgaggatgaa cctgttccaa aaggaagtgg   2280 

actgcgagaa gttctacagc ttcatccaca tgactttcca ggagttcttt gccgccatgt   2340 

actacctgct ggaagaggaa aaggaaggaa ggacgaacgt tccagggagt cgtttgaagc   2400 

ttcccagccg agacgtgaca gtccttctgg aaaactatgg caaattcgaa aaggggtatt   2460 

tgatttttgt tgtacgtttc ctctttggcc tggtaaacca ggagaggacc tcctacttgg   2520 

agaagaaatt aagttgcaag atctctcagc aaatcaggct ggagctgctg aaatggattg   2580 

aagtgaaagc caaagctaaa aagctgcaga tccagcccag ccagctggaa ttgttctact   2640 

gtttgtacga gatgcaggag gaggacttcg tgcaaagggc catggactat ttccccaaga   2700 

ttgagatcaa tctctccacc agaatggacc acatggtttc ttccttttgc attgagaact   2760 

gtcatcgggt ggagtcactg tccctggggt ttctccataa catgcccaag gaggaagagg   2820 

aggaggaaaa ggaaggccga caccttgata tggtgcagtg tgtcctccca agctcctctc   2880 

atgctgcctg ttctcatgga ttggtgaaca gccacctcac ttccagtttt tgccggggcc   2940 

tcttttcagt tctgagcacc agccagagtc taactgaatt ggacctcagt gacaattctc   3000 

tgggggaccc agggatgaga gtgttgtgtg aaacgctcca gcatcctggc tgtaacattc   3060 

ggagattgtg gttggggcgc tgtggcctct cgcatgagtg ctgcttcgac atctccttgg   3120 

tcctcagcag caaccagaag ctggtggagc tggacctgag tgacaacgcc ctcggtgact   3180 

tcggaatcag acttctgtgt gtgggactga agcacctgtt gtgcaatctg aagaagctct   3240 

ggttggtcag ctgctgcctc acatcagcat gttgtcagga tcttgcatca gtattgagca   3300 

ccagccattc cctgaccaga ctctatgtgg gggagaatgc cttgggagac tcaggagtcg   3360 

caattttatg tgaaaaagcc aagaatccac agtgtaacct gcagaaactg gggttggtga   3420 

attctggcct tacgtcagtc tgttgttcag ctttgtcctc ggtactcagc actaatcaga   3480 

atctcacgca cctttacctg cgaggcaaca ctctcggaga caaggggatc aaactactct   3540 

gtgagggact cttgcacccc gactgcaagc ttcaggtgtt ggaattagac aactgcaacc   3600 

tcacgtcaca ctgctgctgg gatctttcca cacttctgac ctccagccag agcctgcgaa   3660 

agctgagcct gggcaacaat gacctgggcg acctgggggt catgatgttc tgtgaagtgc   3720 

tgaaacagca gagctgcctc ctgcagaacc tggggttgtc tgaaatgtat ttcaattatg   3780 

agacaaaaag tgcgttagaa acacttcaag aagaaaagcc tgagctgacc gtcgtctttg   3840 

agccttcttg gtaggagtgg aaacggggct gccagacgcc agtgttctcc ggtccctcca   3900 

gctgggggcc ctcaggtgga gagagctgcg atccatccag gccaagacca cagctctgtg   3960 

atccttccgg tggagtgtcg gagaagagag cttgccgacg atgccttcct gtgcagagct   4020 

tgggcatctc ctttacgcca gggtgaggaa gacaccagga caatgacagc atcgggtgtt   4080 

gttgtcatca cagcgcctca gttagaggat gttcctcttg gtgacctcat gtaattagct   4140 

cattcaataa agcactttct ttattttaaa aaaaaaaaaa aaaaaaaaaa aaa          4193 

 
           
             22  
             1034  
             PRT  
             Homo sapiens  
             
 
           
            22 

Met Ala Ser Thr Arg Cys Lys Leu Ala Arg Tyr Leu Glu Asp Leu Glu 
                5                   10                  15 

Asp Val Asp Leu Lys Lys Phe Lys Met His Leu Glu Asp Tyr Pro Pro 
            20                  25                  30 

Gln Lys Gly Cys Ile Pro Leu Pro Arg Gly Gln Thr Glu Lys Ala Asp 
        35                  40                  45 

His Val Asp Leu Ala Thr Leu Met Ile Asp Phe Asn Gly Glu Glu Lys 
    50                  55                  60 

Ala Trp Ala Met Ala Val Trp Ile Phe Ala Ala Ile Asn Arg Arg Asp 
65                  70                  75                  80 

Leu Tyr Glu Lys Ala Lys Arg Asp Glu Pro Lys Trp Gly Ser Asp Asn 
                85                  90                  95 

Ala Arg Val Ser Asn Pro Thr Val Ile Cys Gln Glu Asp Ser Ile Glu 
            100                 105                 110 

Glu Glu Trp Met Gly Leu Leu Glu Tyr Leu Ser Arg Ile Ser Ile Cys 
        115                 120                 125 

Lys Met Lys Lys Asp Tyr Arg Lys Lys Tyr Arg Lys Tyr Val Arg Ser 
    130                 135                 140 

Arg Phe Gln Cys Ile Glu Asp Arg Asn Ala Arg Leu Gly Glu Ser Val 
145                 150                 155                 160 

Ser Leu Asn Lys Arg Tyr Thr Arg Leu Arg Leu Ile Lys Glu His Arg 
                165                 170                 175 

Ser Gln Gln Glu Arg Glu Gln Glu Leu Leu Ala Ile Gly Lys Thr Lys 
            180                 185                 190 

Thr Cys Glu Ser Pro Val Ser Pro Ile Lys Met Glu Leu Leu Phe Asp 
        195                 200                 205 

Pro Asp Asp Glu His Ser Glu Pro Val His Thr Val Val Phe Gln Gly 
    210                 215                 220 

Ala Ala Gly Ile Gly Lys Thr Ile Leu Ala Arg Lys Met Met Leu Asp 
225                 230                 235                 240 

Trp Ala Ser Gly Thr Leu Tyr Gln Asp Arg Phe Asp Tyr Leu Phe Tyr 
                245                 250                 255 

Ile His Cys Arg Glu Val Ser Leu Val Thr Gln Arg Ser Leu Gly Asp 
            260                 265                 270 

Leu Ile Met Ser Cys Cys Pro Asp Pro Asn Pro Pro Ile His Lys Ile 
        275                 280                 285 

Val Arg Lys Pro Ser Arg Ile Leu Phe Leu Met Asp Gly Phe Asp Glu 
    290                 295                 300 

Leu Gln Gly Ala Phe Asp Glu His Ile Gly Pro Leu Cys Thr Asp Trp 
305                 310                 315                 320 

Gln Lys Ala Glu Arg Gly Asp Ile Leu Leu Ser Ser Leu Ile Arg Lys 
                325                 330                 335 

Lys Leu Leu Pro Glu Ala Ser Leu Leu Ile Thr Thr Arg Pro Val Ala 
            340                 345                 350 

Leu Glu Lys Leu Gln His Leu Leu Asp His Pro Arg His Val Glu Ile 
        355                 360                 365 

Leu Gly Phe Ser Glu Ala Lys Arg Lys Glu Tyr Phe Phe Lys Tyr Phe 
    370                 375                 380 

Ser Asp Glu Ala Gln Ala Arg Ala Ala Phe Ser Leu Ile Gln Glu Asn 
385                 390                 395                 400 

Glu Val Leu Phe Thr Met Cys Phe Ile Pro Leu Val Cys Trp Ile Val 
                405                 410                 415 

Cys Thr Gly Leu Lys Gln Gln Met Glu Ser Gly Lys Ser Leu Ala Gln 
            420                 425                 430 

Thr Ser Lys Thr Thr Thr Ala Val Tyr Val Phe Phe Leu Ser Ser Leu 
        435                 440                 445 

Leu Gln Pro Arg Gly Gly Ser Gln Glu His Gly Leu Cys Ala His Leu 
    450                 455                 460 

Trp Gly Leu Cys Ser Leu Ala Ala Asp Gly Ile Trp Asn Gln Lys Ile 
465                 470                 475                 480 

Leu Phe Glu Glu Ser Asp Leu Arg Asn His Gly Leu Gln Lys Ala Asp 
                485                 490                 495 

Val Ser Ala Phe Leu Arg Met Asn Leu Phe Gln Lys Glu Val Asp Cys 
            500                 505                 510 

Glu Lys Phe Tyr Ser Phe Ile His Met Thr Phe Gln Glu Phe Phe Ala 
        515                 520                 525 

Ala Met Tyr Tyr Leu Leu Glu Glu Glu Lys Glu Gly Arg Thr Asn Val 
    530                 535                 540 

Pro Gly Ser Arg Leu Lys Leu Pro Ser Arg Asp Val Thr Val Leu Leu 
545                 550                 555                 560 

Glu Asn Tyr Gly Lys Phe Glu Lys Gly Tyr Leu Ile Phe Val Val Arg 
                565                 570                 575 

Phe Leu Phe Gly Leu Val Asn Gln Glu Arg Thr Ser Tyr Leu Glu Lys 
            580                 585                 590 

Lys Leu Ser Cys Lys Ile Ser Gln Gln Ile Arg Leu Glu Leu Leu Lys 
        595                 600                 605 

Trp Ile Glu Val Lys Ala Lys Ala Lys Lys Leu Gln Ile Gln Pro Ser 
    610                 615                 620 

Gln Leu Glu Leu Phe Tyr Cys Leu Tyr Glu Met Gln Glu Glu Asp Phe 
625                 630                 635                 640 

Val Gln Arg Ala Met Asp Tyr Phe Pro Lys Ile Glu Ile Asn Leu Ser 
                645                 650                 655 

Thr Arg Met Asp His Met Val Ser Ser Phe Cys Ile Glu Asn Cys His 
            660                 665                 670 

Arg Val Glu Ser Leu Ser Leu Gly Phe Leu His Asn Met Pro Lys Glu 
        675                 680                 685 

Glu Glu Glu Glu Glu Lys Glu Gly Arg His Leu Asp Met Val Gln Cys 
    690                 695                 700 

Val Leu Pro Ser Ser Ser His Ala Ala Cys Ser His Gly Leu Val Asn 
705                 710                 715                 720 

Ser His Leu Thr Ser Ser Phe Cys Arg Gly Leu Phe Ser Val Leu Ser 
                725                 730                 735 

Thr Ser Gln Ser Leu Thr Glu Leu Asp Leu Ser Asp Asn Ser Leu Gly 
            740                 745                 750 

Asp Pro Gly Met Arg Val Leu Cys Glu Thr Leu Gln His Pro Gly Cys 
        755                 760                 765 

Asn Ile Arg Arg Leu Trp Leu Gly Arg Cys Gly Leu Ser His Glu Cys 
    770                 775                 780 

Cys Phe Asp Ile Ser Leu Val Leu Ser Ser Asn Gln Lys Leu Val Glu 
785                 790                 795                 800 

Leu Asp Leu Ser Asp Asn Ala Leu Gly Asp Phe Gly Ile Arg Leu Leu 
                805                 810                 815 

Cys Val Gly Leu Lys His Leu Leu Cys Asn Leu Lys Lys Leu Trp Leu 
            820                 825                 830 

Val Ser Cys Cys Leu Thr Ser Ala Cys Cys Gln Asp Leu Ala Ser Val 
        835                 840                 845 

Leu Ser Thr Ser His Ser Leu Thr Arg Leu Tyr Val Gly Glu Asn Ala 
    850                 855                 860 

Leu Gly Asp Ser Gly Val Ala Ile Leu Cys Glu Lys Ala Lys Asn Pro 
865                 870                 875                 880 

Gln Cys Asn Leu Gln Lys Leu Gly Leu Val Asn Ser Gly Leu Thr Ser 
                885                 890                 895 

Val Cys Cys Ser Ala Leu Ser Ser Val Leu Ser Thr Asn Gln Asn Leu 
            900                 905                 910 

Thr His Leu Tyr Leu Arg Gly Asn Thr Leu Gly Asp Lys Gly Ile Lys 
        915                 920                 925 

Leu Leu Cys Glu Gly Leu Leu His Pro Asp Cys Lys Leu Gln Val Leu 
    930                 935                 940 

Glu Leu Asp Asn Cys Asn Leu Thr Ser His Cys Cys Trp Asp Leu Ser 
945                 950                 955                 960 

Thr Leu Leu Thr Ser Ser Gln Ser Leu Arg Lys Leu Ser Leu Gly Asn 
                965                 970                 975 

Asn Asp Leu Gly Asp Leu Gly Val Met Met Phe Cys Glu Val Leu Lys 
            980                 985                 990 

Gln Gln Ser Cys Leu Leu Gln Asn  Leu Gly Leu Ser Glu  Met Tyr Phe 
        995                 1000                 1005 

Asn Tyr  Glu Thr Lys Ser Ala  Leu Glu Thr Leu Gln  Glu Glu Lys Pro 
    1010                 1015                 1020 

Glu  Leu Thr Val Val Phe  Glu Pro Ser Trp 
1025                 1030 

 
           
             23  
             20  
             DNA  
             Homo sapiens  
             
 
           
            23 

gtgaattctg cagctgttgg                                                 20 

 
           
             24  
             20  
             DNA  
             Homo sapiens  
             
 
           
            24 

tgtaatccct gcactgagga                                                 20 

 
           
             25  
             20  
             DNA  
             Homo sapiens  
             
 
           
            25 

ctcaactcct gcccagtgaa                                                 20 

 
           
             26  
             19  
             DNA  
             Homo sapiens  
             
 
           
            26 

tgagctgaga tcatgcact                                                  19 

 
           
             27  
             23  
             DNA  
             Homo sapiens  
             
 
           
            27 

tcatttcacc tccctaaatt gaa                                             23 

 
           
             28  
             20  
             DNA  
             Homo sapiens  
             
 
           
            28 

ccctttggaa ggaaattctg                                                 20 

 
           
             29  
             23  
             DNA  
             Homo sapiens  
             
 
           
            29 

cccctggtat ataacccctt aca                                             23 

 
           
             30  
             22  
             Dna  
             Homo sapiens  
             
 
           
            30 

cctgcctgat aaagttgttt tg                                              22 

 
           
             31  
             20  
             DNA  
             Homo sapiens  
             
 
           
            31 

ttgtgacaca gaggagcctg                                                 20