US 2003O1622O2A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0162202 A1 Becker et al. (43) Pub. Date: Aug. 28, 2003

(54) SINGLE NUCLEOTIDE POLYMORPHISMS Related U.S. Application Data AND MUTATIONS ON ALPHA-2-MACROGLOBULIN (60) Provisional application No. 60/337,434, filed on Nov. 9, 2001. (76) Inventors: Kenneth David Becker, San Diego, CA (US); Gonul Velicelebi, San Diego, CA Publication Classification (US); Xin Wang, San Diego, CA (US); Lars Bertram, Brighton, MA (US); (51) Int. C.7 ------C12O 1/68 Aleister J. Saunders, Philadelphia, PA (52) U.S. Cl...... 435/6 (US); Rudolph E. Tanzi, Hull, MA (US) (57) ABSTRACT Correspondence Address: The present invention is related to the discovery of several Stephanie Seidman single nucleotide polymorphisms (SNPs) and/or mutations HELLER EHRMAN WHITE & McAULIFFE in the Alpha-2-Macroglobulin gene (A2M), which are risk LLP factors for Alzheimer's Disease (AD). More specifically, 7th Floor aspects of the invention concern nucleic acids corresponding 4350 La Jolla Village Drive to the A2M gene or fragments thereof, which contain one or San Diego, CA 92122-1246 (US) more of the SNPs and/or mutations described herein, pep tides or proteins encoded by Said nucleic acids, antibodies to (21) Appl. No.: 10/292,081 Said peptides or proteins and methods of making Said compositions, diagnostic methods, methods of data analysis, (22) Filed: Nov. 8, 2002 and pharmaceutical discovery and preparation methods.

Patent Application Publication Aug. 28, 2003 Sheet 8 of 22 US 2003/0162202 A1

3 Af2. CCATGGATGAAAACTTTCACCCCCTGAATGAGTTGGTGAGTTTTCTATTATCTACATAAAATGATTGTCTGTATAAACAG 3 A8O1 GCTGGGAACCTGTTTTTTGTGCTGAGGGAAGACCAGGGAGAGGAAGAATCTGGTATCATTAACAGTAACTTCTGGCATTA 3 A881 CAACAGCACAAGATCCTAATCTAAAACATCATTCCAGGTAAAGAAAGTAGGTAATTCTTTCTGTCTTGGTGCTGGTACTC 3496. AGTCAGI"IGICACACAATTAAATTTACTTTTCGGATGGTTCTTAATTAGGACAATTAGAAAGATACATTCAATAGCAGAC 35041 ACAGAAAAATCCCAAAGAACCTAAGCTCAAAAAACATTTTAAAGATTTAGATTTTTTCTATACACATCTACCAAAATCT 352 TCACTATAAAGGAAAGTCAGGGTAATTAATTTGTTCCTCAAGACTAACTCTTGGTATCTGTGATAAGAAACAGTTCTTTC 3520 TATTGTAATGCAGACATCAACCCAAAGTCTTCAT'''I'''CTCTCCCAAATTAACTTCTTCACATTTTCTTATCT CAAAAAG 3528 AGGCAACTCTTCTCTCTAGCTCCAAAGACAAAAGATTATGGCCTAGTTCTTGTTTCTCTTTCTCTCATACCCACATCCA 353 61 CTTCACTGGAAAATCATGTTGGCTTAAAATATATTCAGACTATTTCTTATCATCTGAACTACTGCTCCAAGCTAGTCCTA 35. A 4 GT CAATGTCATCT CAAATAAGATCATTACAATAACCTTCAAAGTGGTCTCCCAGCTTCTACTTTCACTCCTCTGTCTAA 35.52 AATGAGGCACCACACCCATCACTCTGTCAGCCTAACTTGGCTTTGTTTTTCTATTTGCACTTACCACCATTCTATACGTA 3560 TTGATTTTTAAAAATCTGCCTATTTCTATTATAACAIAAGCTCCAAAAACATGGTTTATTGTTCTTTTGTGCGTGGTT 35581 ATATTCT CAATACCTAGAATGATACACAGCGCATGAGAAGGTACTTAATAAAGATTAGTTTTTAAAAATGAATAAACATT 35.81 CAAGTAGCTCTTATCAATGTTTATTCATTTTTAAAAGTAATATTTATTAAACTAAATTTATTAATGAAATATGTCCIT rS 226384 A. 384 TCCTCCCATTC'''AGAACTACGTAAGAATTCTATGACCCATCTTAATGATTACATCTGAACATATFTACTTATACTTAC 3592 ATAAGTGTTTATTTCTCAACCTGTAAAATTGCATAGCAAAGGATTGATCTTCACTAGGATGCTATAAGCACTTAAGACAT 36OOL GITAATCCATTTTTAGTAAATGGCACTTTACATGTATATTTGTTCCTGAAGGCCTAGTAAGTTCTTAACATTATTTATTA 36,081 ATTGCTTAAAATAGATTAATGAAAAGTTCTATAAAATTTAATCTGGAATATTTCTGTATTCACTATAGAGGGAATTATE 3661 CTATATCAAACTAAFTTAGATTTTTTAAACTTTTTATGTTTTTAATTTTTGTGGAAACAFAGCAGGTATATATATTTAT rS226385 C 36,241 GGGTTACATGACAATTTTGAACAGGCATGCAGTGCAGATAATGATTATGGATGATAATCATTATCATTATCCATATC 36321 ATTATCCAATCATGGATAATGATTATTATCATGCATTGCATGCCTGTATCAATAAATGGAGTACCCATCCCCT CAAACAT 364 Ol TIATTCTTGTGTEACGAACAAATCCAATTATACTTTTAGTTATTTTTAAATGTACAATTAAATTATTTTTTACTATACT 36A8. CACCCGTTATGCTAGCAAATACTAGCTGCTTTGCITATAAATGAGATTTAACAATATTTGAAAATAATTATAAACTTCT Exon 5

385 6. TTTTTCTTTTGTCTTTCAGATTCCACTAGTATACATTCAGGTAAGCAACATGAAACATTCCATATTAAAAGGAAAGCAAT 3664 ACATATAGGGAAAATGTTCTTATTTCAGAGGTTTTTACAATATCTCAGAAACTTGTCATTAAAGGAGAAGCCTTCAAACT is 226386 C 3672 CCCATAGAGCTAGATGGCTATAACT CATCTCCTCTACT CACCCTTTACTACTACCCCATTTGACCTTTTTGTAGATAACT Exon 6

368O TAGGGTTTCCATAGATATCTTTTATTAGCTCCAATGCTCCAGGTGCTTTTGTAAGTTATAATTAATTTACTCATATAGGA ExOn 6

36881 TCCCAAAGGAAATCGCATCGCACAATGGCAGAGTTTCCAGTAGAGGGTGGCCTCAAGCAATTTTCTTTTCCCCTCTCAT Exon 6

36.961 CAGAGCCCTTCCAGGGCTCCTACAAGGTGGTGGTACAGAAGAAATCAGGTGGAAGGACAGAGCACCCTTTCACCGTGGAG Exon 6

3704. GAATTGGTATGGATCATGAAAAGTCATCAAGCATTATTTTTCTTCATATTTAAACTCTTAGGTCCTGGAATTTAAGTTC 3t 12 ATTGGAGTCTTTCCATTTCCCATGGGTGACATTGGGCTGGAGTAGAATTAATTACACCTAAGICCAAGAGGACATCA A2M 6i A. 372O1 GrgATCTGTGAATAGGACTTCACATAGCTTCGTTATTTTCTGTAGCAATATTTAATACCAACCCCCAAAATTAAAACATT 37281 CGTTTTAATGGAGTTTTCCATAATAATTAAGCACACAGTGATCTCTCATAGTCCCTCAACTGAAATCTTCATTTGAG 3.361 AGGAGGATAGAAAGAATAGATTGGAGAGCAGAGCTACCTTTCAGAGCCCTAAAAATTALIAGGGAACTGITACAGGG Exon 7

374. A AACCTGAAAATAGGAATTCCCCCAAAGTTGAAAACCAATCACCAACCTTCTTTATCACCAATCAACAGTTCTTCCCAAGT Exon 7

37 521 TTGAACTACAAGTAACAGFGCCAAAGATAATCACCATCTTGGAAGAAGAGATGAATGTATCAGTGTGTGGCCTGTGAGEF 3. 801. CATTTTTAAAAATCTTTTGTGGGGGATTATTFAAAAGAGACCACCATTTGGGATATTTTAACTACTCTCTCCGGGAGC 3i 681 AGTGGCAACACAAAAATTTTAAGTGCTTTGACAGCATCCTCATCTGTAGAATGTTATTCTCCTGTTGCTTTTCTATTTTT 377 61 ATTTTCTTTCACGT''C'IAICAGTATTATTCTATCATGAGGTAAGAAACTGTTTCTAGAGAGGTTATGCTAATAGGATTG 378 41 ATCTGAAAGTGACAAAACTGCACACACACACACACACAAAAGGGAAGGGTGGATAGGTTGAAGGGTATTGGTTCIGC 37921 CTAACCAAAAATAACCAAACGTATATTAGGGAGATAATTAACACATGGCTATAGGGGAAATCAATCATCAACAGATCA 38 OO1 TTTACCTGACCTTGCAIGCTTACTGGAAAAATCACTTAGATTCAGAATTTGTAGAGATAGGCATACAACTGAAATCTCAT 38O81 CTAACT CACTAGTCCACCTAAGGCGGGTCTACATACCFGTCTGCACTGACATTTTTACGAAGAGACTGAACGTGATTTCT 388 CAAGACAGCIGTTCCAACCTTGAAAAGTCTTGTTTCCTGGATCTTATGTTCCCATCCATGGGGCACACAGTAGCAGTA rS22.638 C

Patent Application Publication Aug. 28, 2003 Sheet 20 of 22 US 2003/0162202 A1

Exon 32

7 6321 CCCCTGGATCCTCAGTTACACAGGGAGCCGCTCTGCCTCCAACATGGCGATCGTTGATGTGAAGATGGTCTCTGGCTTCA Exon 32 764O1 TTCCCCTGAAGCCAACAGTGAAAATGGTAGGTTTATCATAACCCCAGACTGCCCTATTTTATTTAATGATGTATGTATCC 76481. CCAGCATAAGACAATACTAATATCAAAATACTATAAAGT CAATCTCTATCAAAGCCTTATCCTTTTTCCAGCTCAGAAA 7656. TATAATCACATGTGTTTGTATGAATGCTGACCATGTGCAGAGCACTGTGCTAGGACCCAIGACTACAAGAAAAAGATTGT 766A1 CAGCAGGTTCCTGCTTTTCAATTTCTTCTTAGCTTAGAATTTTGCTAAGAAGATAAAAGATATGAACACGAACCAGTGAA 76721 AAATATGAAAATGACTGATTGGCATAAACTATAAGTATTACAGAAGTTAAAAGAAAAATAGAGCAAGCAAAACAGGAAAA 768 Ol AAACTCCTTATGAAGAAATAGAAACTGAATTGAACTTTGAAATATGAGTAAC'IACCAAGTTTAGGATACTTAGCTGTCTT 76881 TTCTTCAGATAAATAACTTTACACATTAGTCGTGTGTTATACTAATAGTAAACCCTTTATGCCTTTCATTTTTAATTGTA 7696.1 TTACATTATATATTTCCTTACAAAAAGCATTTGAAGAATTCTACCCTCAGGGTTATTTTGGCAATACAAAGATTTTTTCT Exon 33

77041 CTGGATCCCCCAGGGGTTTCATCTATTTATTAACATTTGTGGTATTTCAATTTTCTTCAGCTTGAAAGATCTAACCATGT Exon 33 771.21 GAGCCGGACAGAAGTCAGCAGCAACCATGTCTTGATTTACCTTGATAAGGTAAGAGAACTTCCAGTCTATTTGCAAAAAA rs 1805,664 A. 772.01 ACGTAGATAATAATCCTCTAAGGGAACATCTGGGAAGGTAAATGCATTTTAGAAACATCACTTCCATGCTAGAAATTTGA 7 723 GAATTCTAATGTTAACTCTAAAAGAATGTTCTTCTCTCCTTTATTTATATTCACCAGGGATTACAGGTAGAAATGGCTT S1805,665 rs1805 666 G G 77.361 ATTATGATCTTGGGATATGAATATTCCTAAAATCCCATAAGCAAGAAATCTTCACAAAATGTGTTTATATGTTGACAAG rS302622.6 C l, 441 TTTTTTGGALACCCAGTAATAAAGGAAGTAGCCCTTGTGATTAGTCAATTATTAGTTAATTATCAACATACTCAACAAC 77 521 AATATGAAAGGGAAAAAAAACTGTCAGTCTCCACAAGGACTTGAACCATAAAATAATAAGACCAGTTCACCAGTAAACCA 77 601 ATCTGATTTTATAGATATGTGTGGTAGGAGAGTTTGTTCATGCATAAGTTGATGGGAATTATAGTTTACAAATTTTATGA rs3O26227 A/C 77 681 AACTTAAGCCTGGGAAGATCAACCTTTTAGATGCCTCTTTGAGTCTACGCAAGTATTCCTGCAAGACAGAGAAGTCAAAC rs1805 686 G 7776 TATACCAAATCTCTGGATATTAAAAAATGAACACAGTTAGTCATCCAATAAAAAGTATATATCATTTACCCCCATGAACA rs3O26228 A 77.841 GAGCTATCTATTGGCATTGACAGAGGTATATGCGTTATGTTAGTTATTTAAGAAATAATCTGGAGAATTTATCATCCCCT 77921 CTGAGAGATTTCTGCACAATTTAATTAAGGACCCTATAGTGTGCTGTAGGATAATAAAGCTTTTCCCCCAAAAAACAGG ------Exon 34 -

78OO1 GAATACTTAAACTAATTCAAAGAGAGAAGAAAGCTTCCTGAAAGGTCATT'TAATTGACTTTTGCTTTCCAGGTGTCAAAT rs 1802965 C (F1431L) Exon 34 78O81 CAGACACTGAGCTTGTTCTTCACGGTTCTGCAAGATGTCCCAGTAAGAGATCTGAAACCAGCCATAGTGAAAGTCTATGA Exon 34 8161. TTACTACGAGACGGGTGAGTGAGAGTGATTTTCACG''AGAAATATTTAATTCCTGATCACAGAAATTCAGGTTTAGGAGA 78241 TGTGTTGGGGTTATTTATTACATTAAGTAATTACATTATCACTTCATTTTGTCTCCATCAAGTCTGATGCCCCTCTTTTT 78.321 GTCTCTTATACATACATTATAGAAACAACCTACATTATAAATTTATCAACTACTAATACAAAACACCTGTGGGATATTTA 784 ()1 GTTCCCTTTTCATCAGATAAATGGACTGIATGACAATATGAGATTTAAGTAAGTAGAACATCTGAAGAGTCCTTCAGGAG 78.481. TTTGGGATAAAAGAATATATAAAACACTATATTGAAAGGAGAATATAAGGTAGCAAGCAACACATCAGATGAATGATGC 85 61 TTATGTTTCTGGTACAATACTGTTCTTCCCACAACAAACTCCTTCCTTGGCCTGTATCCCACAGATGTTTGCITICTTTC k ExOI) 35

7864 TCACTTCATGTAATGATTTCTGGTTTTTTGTTGGTTTTTTTTTTTTCAGATGAGTTTGCAATTGCTGAGTACAATGCTCC Exon 35

1W awm a m MW an M. M. M. 78721 TTGCAGCAAAGGTAAGCCACTCACACTCCTCCAAAAGGCAGTCAGAGCTCCT"I'CAGCTTGCCCCCCAAACCTTCTCCTTC 788O ATAAAACGCTGGGTAAATATTTGTCAAAAACATCAAATTACT CACACTGCACATTATTATAGAAAAACACATTTATTGGA T888 GAGGGCCGCTGACTCGTCAAACCTCAGAGAGTCCATAGGATTGCTTATGGGTAATGATTTGGAATAGATTTGGTTTCCC ris3180682 rs 1805,687 T C 789 61 ACTGTACTGATTAGGTTTCCTTGGGCACTATGCTACCCAGAACTAAGGGAAAGAATACTCTCTGCTCATGGAGACCCAAA rS130840

Patent Application Publication Aug. 28, 2003 Sheet 22 of 22 US 2003/0162202 A1

TACACGACAGGAAATGAAATGGATACTTGTTGGGGTCAGTTTTATGTATAGTTTGTATTTTATTTTGAAATATGAI"ACAC TGCTATTCTCTTGCATTTTCTTATATGTGACT CACCACTAACCCTATATTCCCCCATTTCAGGCCAGTTGGTCATAAGCA TCCATTTGCCTCAGAGAATACTGGGTTGTTATGACAAGAATATAAAGTTGCAAAGAAATAGAATATTTGAGTCTACCCTG TAAGAATAAAAAGAATAAAAGGGGTTTAAATTTAI"TTAGACCCTATTGTTTAATCAAGAATTCTGGCCAGGAGCAGTGGT TCATGCCTATAATCCCAATGCTTTAGGAGGCCAAGGCAGGAGGATCATTTGAGGCCAAGAGTTTGAGACCAGCCTGGGCA AATTATTGCTCGGGAAAAAAAGGTCTTATTTAGTATTTTAGTCTTTACAATGTTTTTTTCTATTATGCAATATTCTCTCA AATACTTTATG''CCATCCACGTTGTCTGAGACATGCCACTTTAACATTCTAGT"TATGCTGTAGCTGTCATTTTACCCTAA GCCGTTAGTAGTACTGATCCACATAAAATTGGGCTGTTTAGGTGTTTAACTGTTTAAATGTATAATATATCTGATATATT TATATATTGTATAAAAAATCACCTAACACAATAGATATTTACTATGTCTGCTATAAATATATATCATATAACAATAACA ATATATTATAAATGATAATATACTATAATAAAAATATAATAAATATATTATAAATGTACAATATATCCGATATATATAT ATAAAATGTCAACTATATTATACAATAAATGTATATGTATAATATATACATATATGTATAATACAATGTATTTATTAT GTATATATATAAATGTATATGTATAATATATAAATGTATAATATATCTGATATAAACATATCAGATATATTATCCATCA TTGTGTCAGGTGATTCTTTATACAATATATAAATATATCAGATATATTAFCCATCTATTGTGTCAGGTGATTTTTTATAC AATATATAAATATATCAGATACATTATCCATCTATTGTGTCAGGTGATTTTTTATACAATAATAAATATATCAGATATA TTATCCATCTATTGTGTCAGGTGATTTTTTATACAATATACAAATATATCAGATATATTATCCATCTATTGTGTCACGTG ATTTTTTATACAATATATAAATATATCAGATACATTATCCATCTATTGTGTCAGGTGATTTTTTATACAATATATAAATA TATCAGATATATTATCCATCTATTGTGTCAGGTGATTTTTTATACAATATATAAATATATCAGATATATTATCCATCTAT TGTGTCAGGTGATTTTTTATACAATATATAAATATATCAGATATATTACATATCTATTGTGTCAGGTGATTTCTTATACG ATATATAAATATATCAGATATATTATCCATCTATTGTGTCAGGTGATTTTTTATACACTATATAAATATATCAGATATAC TATACAGTTCAGCCCATCAAAGCACCATATTTTGGGGGGTTGGTTTCCGTGTCCCAACACTAGCTACGAAAATATTAGCT ATTAGCTACCTATAACTCTTCAGTAGTAAATTCAAGAAACGTAAAGTAATTCTCTTCATTAACTTCTTGCCTTGTCTAC AAAAAAATGGTCAT CACCGATGTGGACAATGAAGACCTGTGGGGITAAAAGCTCTAACTAGTATGCCTCCAAGATTCTTT GATTGCCTGCCATCATGATCGAAGAATAAATAACTTCTTTTTCATCTTATTTATTTATTTTTTGTAGAGATAGGGTCTCG CTATGCTGCCCAGGCTGGTCTCAAACTCCTGGGCTCAAGAGATCCTTCTACTTAAGCCTCTCAAAGTGCTGGAATTACAG GGGTGAGTCACCACGACTGACCATTAATAATTTCTTTCAATGACACTTTAACATAGGCTCATTCATCTTTACCTCTAAAG AAAAGTCTTTCTGGTCTTTTTAAAATTATATTTTTGGCCAGGCACAATGGCCAGGTGCGGTGGCTGACACCTGTAATCC TNGCACTTTGGGAGGCCAAGGTAGGAAGATTGCTTGAGGCCAGGAGTGCAAGACCAACCTGGCAAACATCTGGAAAACAT AGCAAGGCCCCATCTCTATTAAAAAAAAATTAATTCTATTTTTCTAAGAGAAAAAACATTCCCNATTCAACAACACTTTT CAAAAACTTTATCTGGCAGCTACTCAGGAGATTGAGA"I'GGGAGGATCATATGAAGCCCAGGAATTCAAAACCAGTGTGGG CAACATAGTGAGATCCTATCTCATAAAAAAATAAAAAATAAAAAAAGCTTTACTTGGAATACAACCCATGACTCTGGTTA TAAATACAAAATTCTTCAAATTCATTTAAAGGAATTTAATCCTAGCTTCTCGGATGAAAAAAGGAAATAATATTCACAAT TTGA"I'CCATCATCAGTAGACAAGTTAAAGTGTTTCACAAAAGCAAGACATATTAATTAAGCAAAATCATATTCGAGTAA CCACAGGAAATATAAATATACTGTCTCTTACCTAGAGAAATCTTATAGTCTAATTGTGAAGATAGTC'''CACGIGACGAA AAAGATCATCATTAATCCAAAACATATAAGTTATAAAGAAGCGACATATACCAGCAATTCTAAAATCTGGTTAGCATCCT TTGTAGAATTTATTTTAAAATGCAGATATCCAGGTCTCATCAATAAAGATT'TAATTAATTATTTTTGGGGATGTGCTCAG ACATCTGCGTTTTTTGTTTTTTGTTTTCGT"""TTTTGTTTTTTGAGATGGAGTCTCACTCTGTTGCCCAGGCTGGAGTGC AATGGCGCAATCTCAGCTCACTGCAACCTCTGCTCCCAGGTTCAAGCAATTCTTCTGCCTCAGCCTCCCTAGGAGCTGGA 864 O1 ACTATAGGCGCCCACCACCACGTTGGGCTAACAGGCATCTATGTTTTTAATGAACTCTGTAGGTGGTTCTATCATGCAGT 86481. TAGTTTTCAGAACCATTCACACTGACAGTAAAGGCTATTTATTCCCAGCAGTTGAAAGACCACTAAGGACACAGGAATAG 865 61 TTAGCAAAGCTACTTAAAGATGCCAGGGCTGGGGCCGGGTGCGATGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCC 8664 AAGGTGGGCAGATCATGACGTCAGGAGATCGAGACCATCCTGGCTAACACAATGAAGCCCCCTCTCTACAAACAAACAAA 8612T CAAACAAACAAAATACAAAAAATTAGCCGGATGTGGTGGCGGGCACCTGTAGTCCCAACTACTCGGGAGGCTGAGGCAGC 86801 AGAATGGCTTGAACCCAGGAGGCGGAGCTTGCAGTGAGCCGAGATCACGCCACTGCACTCCAGCCTGGGCGACAGAGCGA 86881 AACTCCATCT CAAAAAAAAAAAAAAAAAAAAAAAAAAGATGCCAGGGCTGGCTGGGCACAGTGGCTCACACCGTAACCC 86.961 CAACACTTTGGTTGGGAGGCCAAGGCGGATGGATTGCTTGAGTTCAGGGGTTCAAGACCAGCCCAGGAAACATGGCAAA 87 (A1 ACCTCATCTCTACCAAAAACACAAAAATTAGCCGGGCATAGTGGCATGCACCTGTGGTCCCAGCTACT CAGGAGGCTGAG 87121 GTGGGAGGATAGCTGGAGCCTGGGAAGCTGCAGTGATCAGTGATCATGICACCACACTCCAGCCTCGGTGACAGAGCAAG 872O1 AACCTGTCCAACATACATACATGCATATATAAAATTAAACATAAAAACAAAAATAAATAAAGATGTCAGGGCTTATGTT 87281 GAACCT"TAACTGAGAGCAAGATTCAAAAGACACTGAGGCTTATT'I'''CTTTCTTATATCTATAGTTACACAGGGAGCTGT 873 61. CTAATCTTGGATGTATCCAAGTTGATATCTGGTTTTATCCATTGAAACCCACAGTGAAAATCGTAAATAGGGCTAGGG 874. 41 TTTGGATTTTTTTAATCCAATGTAAGAATAAAACAATGGTATCCTAATAATGTCAAAGCAACATTGGTCATAATCTAAGG 87521 AAATTGAATTCATATAGl'ACCAAATATATATTTAGCATTGTGCTAGGTGCTGATACATTCTAGATAAAAATATTACACAT 87 60 GGGTAACCAAAACTGTCAAATGACATTTCAGGGCAAGATATAATTAAGTACCAAAATCATTGGCATAGTCTTTAAGTACT 87 681 GTGAAATCTAGAGAAAGCTGAGATGAATGGCAGTGTAGAGACAGATGGCTTTGCAAATCATCTCAGATGACTAGCTATCC 877 61 AATGTGAGGACACTTCTCCCT CACCTTCAAACAAATGCTAAAGACGCCTGTTACTTAATCATATGAATATTCAATCTTGT 87.841 ATCTAATGTGGTGGTA'''CATAATACTCTGTATATGTTTTCATCTTACTGGACAAGTGICTTCGAACT CATTTAAATGAA 8792 TTTAACCCCAGCTTTGTTTATGTATAGACTTCTTCAATCTCATAGTCTATTGTCTCTTTGTACCCCACAGGCTTTCTTT 88OO1 ATTCAGTAACACTGGTGATTTCTCTTATTTTCCTTAGCTTGAAACATCTAGCCACGTGAGCAGGACAGAAGTGCACAACA 8808 ACCATATCTTGATTTCTGTGGACCAGGTGGGGCCCCTGCCAGCCTTGCTAGACAGACCCAGGTGAACAGTCCTTCTAGGC 88: 61 GATCTCATCACCAGGCAAGCACGTGGTACGAGAAGAGCAGTCATAGGAAGGCCAT"T"I'GGAAAAGCACATCCTCTCTGTT 882.41. CACGTGAGATATTTTACATCCTCATTCCTCATCGCAAGCTTCCTGGGATTTGGAGTGTCACAGACAAGAGGGTTGGGGGA 88.32 GGCCAGTAGGTATGGATTTGTTTATATTAAAATGAGCATATGAATATTTATATGTTTATATTAAAACATATATGTTTGT 884 Ol TATATTACAATGAGCATATGAATATTTATAGITTATATTAAAACATATATGTTTGTTTATATTAAAATGAGCATATGAA 884.81 TATTTCTGTATACTTCAGATAAACATTCTTTTCCATAAATAAGCTTCATCATCCAGAAGCCATGTTGAAAGTTGGTAATC 885 61 AAGGATAGGAAGTGTTTCCNAGGGTTGTCAGTGATTAAATCAACCTTACCTTAGCATACATGTA US 2003/0162202 A1 Aug. 28, 2003

SINGLE NUCLEOTDE POLYMORPHSMS AND ment of proteases and the exposure of the four receptor MUTATIONS ON ALPHA-2-MACROGLOBULIN binding domains to the extracellular environment. 0006. In the human A2M tetramer, each subunit contains RELATED APPLICATIONS at least five binding sites: the bait region, the internal thiol 0001. This application claims priority to U.S. Provisional ester, the receptor binding site, the AB binding site, and the Patent Application No. 60/337,434, entitled SINGLE Zinc binding Site. The bait region, the internal thiol ester and NUCLEOTIDE POLYMORPHISMS AND MUTATIONS the receptor binding site have a pivotal role in the activation ON ALPHA-2 MACROGLOBULIN, filed Nov. 9, 2001, the and internalization of A2M. The bait region in each mono disclosure of which is incorporated herein by reference in its mer is located between amino acids 666 to 706, at the center entirety. This application is also related to the Patent Coop of each molecule, and it binds any known protease. The four eration Treaty Application having the Attorney Docket bait regions in the tetramer are in close contact and are Number NEURINC,009VPC, entitled SINGLE NUCLE cleaved by the bound proteases, which triggerS activation of OTIDE POLYMORPHISMS AND MUTATIONS ON A2M. This conformational change results in a Sudden expo ALPHA-2-MACROGLOBULIN, filed on Nov. 8, 2002, the sure of the four thiol esters between Cys949 and Glu952, disclosure of which is incorporated herein by reference in its and of the four receptor binding sites, to the extracellular entirety. environment. 0007. The A2M region of chromosome 12 has first been GOVERNMENTAL INTERESTS associated with AD in genetic linkage analyses. (See e.g., Scott et al., JAMA, 281:513-514 (1999)). Two specific 0002. Subject matter of this application was made in part AD-associated polymorphisms have been reported in the with government support. The United States Government A2M gene: an intronic deletion at exon 18 (18i, see e.g., may retain certain rights in this Subject matter. Matthijs and Marynen, Nucleic Acids Res., 19:5102 (1991)) and a single amino acid substitution at position 1000 (1000 FIELD OF THE INVENTION V/I; see e.g., Liao et al., Hum. Mol. Genet., 7:1953-1956 0003) The present invention is related to the field of (1998)). Both of these polymorphisms were found to be disease diagnosis and treatment. More specifically, the associated with increased f-amyloid deposition (Myllykan invention is related to the discovery of Single nucleotide gas et al., Ann. Neurol., 46:382-390 (1999)). polymorphisms (SNPs) and/or mutations in the Alpha-2- 0008 Alzheimer's disease is a devastating neurodegen Macroglobulin gene (A2 uM). Included among the A2M erative disorder that affects more than 4 million people per polymorphisms and/or mutations are those that can be year in the US (Döbeli, H., Nat. Biotech. 15:223-24 (1997)). indicative of an altered risk for Alzheimer's Disease (AD). It is the major form of dementia occurring in mid to late life: approximately 10% of individuals over 65 years of age, and BACKGROUND OF THE INVENTION approximately 40% of individuals over 80 years of age, are 0004 Alpha-2-Macroglobulin (A2M) is an abundant symptomatic of AD (Price, D. L., and Sisodia, S. S., Ann. plasma protein Similar in Structure and function to a group Rev. Neurosci. 21:479-505 (1998)). The need for diagnostics of proteins called C-macroglobulins. A2M is also produced and therapeutics for AD is manifest. in the brain where it binds multiple extracellular ligands and SUMMARY OF THE INVENTION is internalized by neurons and astrocytes. In the brain of Alzheimer's disease (AD) patients, A2M has been localized 0009. Some aspects of the present invention are described to diffuse amyloid plaques. A2M also binds soluble B-amy in the numbered paragraphs below. loid and mediates its degradation. An exceSS of A2M, however, can have neurotoxic effects. Kovacs, Experimental 0010) 1. A method for identifying a polymorphism or Gerontology, 35:473–479 (2000). Based on genetic evi combination of polymorphisms associated with an A2M dence, A2M is now recognized as one of the two confirmed mediated disease or disorder, comprising testing one or more late onset AD genes. AS for the three early onset genes (the polymorphisms in an A2M gene individually and/or in amyloid B-protein precursor and the two presenilins) and for combinations for genetic association with an A2M-mediated the other late onset gene (ApoB), DNA polymorphisms in disease or disorder, wherein the one or more polymorphisms the A2M gene associated with AD result in Significantly is/are Selected from the group consisting of 6i, 12i.1, 12i.2, increased accumulation of amyloid plaques in AD brains. 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e. These data Support an important role for A2M in AD 0011) 2. A method for identifying a polymorphism or etiopathology. combination of polymorphisms associated with a neurode 0005 Human A2M is a 720 kDa soluble glycoprotein generative disease or disorder, comprising testing one or composed of four identical 180 kDa (1451 amino acid) more polymorphisms in an A2M gene individually and/or in Subunits, each of which is encoded by a single-copy gene on combinations for genetic association with a neurodegenera chromosome 12. Disulfide bonds and noncovalent interac tive disease or disorder, wherein the one or more polymor tions connect the Subunits within the tetramer. A2M is often phisms is/are Selected from the group consisting of 6i, 12i.1, referred to as a panprotease inhibitor, because it entraps and 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e. isolates virtually any protease from the extracellular envi 0012. 3. The method of Paragraph 1, wherein the nucle ronment followed by its degradation. Activation of A2M otide at 6i is A, the nucleotide at 12i. 1 is G, the nucleotide involves a complex conformational change of the tetramer, at 12i.2 is T, the nucleotide at 12e is T, the nucleotide at 14e triggered either by protease cleavage of A2M or by methy is C, the nucleotide at 14i.2 is C, the nucleotide at 17i.1 is lamine treatment. Activation of A2M results in the entrap G, the nucleotide at 20e is T, the nucleotide at 20i is G, the US 2003/0162202 A1 Aug. 28, 2003

nucleotide at 21i is C, the nucleotide at 28i is T and the 0029. 18. The method of Paragraph 17, further compris nucleotide at 30e is C, or the complementart nucleotide ing determining the identity of one or more nucleotides at a thereof. position Selected from the group consisting of 18i and 24e. 0013 4. The method of Paragraph 2, wherein the nucle 0030) 19. The method of Paragraph 6, comprising deter otide at 6i is A, the nucleotide at 12i. 1 is G, the nucleotide mining the identity of one or more nucleotides at a position at 12i.2 is T, the nucleotide at 12e is T, the nucleotide at 14e Selected from the group consisting of 6i, 12e, 14i.1 and 21i. is C, the nucleotide at 14i.2 is C, the nucleotide at 17i.1 is 0031, 20. The method of Paragraph 19, further compris G, the nucleotide at 20e is T, the nucleotide at 20i is G, the ing determining the identity of one or more nucleotides at a nucleotide at 21i is C, the nucleotide at 28i is T and the position Selected from the group consisting of 18i and 24e. nucleotide at 30e is C, or the complementart nucleotide thereof. 0032) 21. A method of genotyping a cell comprising: 0.014) 5. The method of Paragraph 2, wherein the disease 0033 obtaining from an individual a biological is Alzheimer's disease. Sample containing an alpha-2-macroglobulin polypeptide or portion thereof, and 0.015 6. A method of genotyping a cell comprising: 0034) determining the identity of one or more amino 0016 obtaining from an individual a biological acids in Said alpha-2-macroglobulin polypeptide or Sample containing an alpha-2-macroglobulin nucleic portion thereof wherein Said one or more amino acid or portion thereof, and acids are located at a position Selected from the 0017 determining the identity of one or more nucle group consisting of 14e, 20e and 30e. otides in Said alpha-2-macroglobulin nucleic acid or 0035) 22. A method of identifying a subject at risk for portion thereof wherein Said one or more nucleotides Alzheimer's Disease, Said method comprising: are located at a position Selected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 0036 obtaining from said subject a biological 17i.1, 20e, 20i, 21i, 28i and 30e. Sample containing an alpha-2-macroglobulin nucleic acid or portion thereof, and 0.018 7. The method of Paragraph 6, wherein said alpha 2-macroglobulin nucleic acid is genomic DNA. 0037 determining the presence or absence of one or more polymorphisms or mutations in Said alpha-2- 0.019 8. The method of Paragraph 6, wherein said alpha macroglobulin nucleic acid or portion thereof 2-macroglobulin nucleic acid is RNA. wherein Said one or more polymorphisms or muta 0020. 9. The method of Paragraph 6, comprising deter tions occur at a position Selected from the group mining the identity of one or more nucleotides at a position consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, Selected from the group consisting of 6i, 12e, 14i.1 and 20e. 17i.1, 20e, 20i, 21i, 28i and 30e. 0021 10. The method of Paragraph 9, further comprising 0038 23. The method of Paragraph 22, wherein said determining the identity of one or more nucleotides at alpha-2-macroglobulin nucleic acid is genomic DNA. position 18i. 0039. 24. The method of Paragraph 22, wherein said 0022 11. The method of Paragraph 6, comprising deter alpha-2-macroglobulin nucleic acid is RNA. mining the identity of one or more nucleotides at a position 0040 25. The method of Paragraph 22, wherein the Selected from the group consisting of 6i, 12e, 14i.1 and 21i. nucleotide at 6i is A, the nucleotide at 12i. 1 is G, the nucleotide at 12i.2 is T, the nucleotide at 12e is T, the 0023) 12. The method of Paragraph 11, further compris nucleotide at 14e is C, the nucleotide at 14i.2 is C, the ing determining the identity of one or more nucleotides at nucleotide at 17i. 1 is G, the nucleotide at 20e is T, the position 18i. nucleotide at 20i is G, the nucleotide at 21i is C, the 0024 13. The method of Paragraph 6, comprising deter nucleotide at 28i is T and the nucleotide at 30e is C or the mining the identity of one or more nucleotides at a position complemtary nucleotides thereof. Selected from the group consisting of 12e, 14i.1 and 21i. 0041. 26. The method of Paragraph 22, comprising deter 0.025 14. The method of Paragraph 13, further compris mining the presence or absence of one or more polymor ing determining the identity of one or more nucleotides at a phisms at a position Selected from the group consisting of 6i, position Selected from the group consisting of 18i and 24e. 12e, 14i.1 and 20e. 0042. 27. The method of Paragraph 26, further compris 0.026 15. The method of Paragraph 6, comprising deter ing determining the presence or absence of one or more mining the identity of one or more nucleotides at a position polymorphisms at position 18i. Selected from the group consisting of 14i.1, 20e and 21i. 0043. 28. The method of Paragraph 22, comprising deter 0027. 16. The method of Paragraph 15, further compris mining the presence or absence of one or more polymor ing determining the identity of one or more nucleotides at a phisms at a position Selected from the group consisting of 6i, position Selected from the group consisting of 18i and 24e. 12e, 14i.1 and 21i. 0028 17. The method of Paragraph 6, comprising deter 0044) 29. The method of Paragraph 28, further compris mining the identity of one or more nucleotides at a position ing determining the presence or absence of one or more Selected from the group consisting of 20e, 21i and 28e. polymorphisms at position 18i. US 2003/0162202 A1 Aug. 28, 2003

0.045 30. The method of Paragraph 22, comprising deter 0061 contacting said cells with an alpha-2-macro mining the presence or absence of one or more polymor globulin polypeptide comprising at least one poly phisms at a position Selected from the group consisting of morphism or mutation having a position Selected 12e, 14i.1 and 21i. from the group consisting of 14e, 20e, and 30e, and 0.046 31. The method of Paragraph 30, further compris 0062) identifying a compound that modulates an ing determining the presence or absence of one or more alpha-2-macroglobulin activity. polymorphisms at a position Selected from the group con Sisting of 18i and 24e. 0063 42. The method of Paragraph 41, wherein said alpha-2-macroglobulin activity is an interaction of Said 0047 32. The method of Paragraph 22, comprising deter alpha-2-macroglobulin polypeptide with the LRP receptor. mining the presence or absence of one or more polymor phisms at a position Selected from the group consisting of 0064. 43. The method of Paragraph 41, wherein said 14i.1, 20e and 21i. alpha-2-macroglobulin activity is the degradation of Said 0.048 33. The method of Paragraph 32, further compris alpha-2-macroglobulin polypeptide. ing determining the presence or absence of one or more 0065. 44. The method of Paragraph 41, wherein said polymorphisms at a position Selected from the group con alpha-2-macroglobulin activity is a protease inhibitor activ Sisting of 18i and 24e. ity. 0049 34. The method of Paragraph 22, comprising deter 0066 45. The method of Paragraph 41, wherein said mining the presence or absence of one or more polymor alpha-2-macroglobulin activity is the clearance of Said phisms at a position Selected from the group consisting of alpha-2-macroglobulin polypeptide. 20e, 21i and 28e. 0050) 35. The method of Paragraph 34, further compris 0067 46. The method of Paragraph 41, wherein said cells ing determining the presence or absence of one or more are contacted with an alpha-2-macroglobulin polypeptide in polymorphisms at a position Selected from the group con the presence of amyloid B. Sisting of 18i and 24e. 0068 47. The method of Paragraph 46, wherein said 0051 36. The method of Paragraph 22, comprising deter alpha-2-macroglobulin activity is an interaction of amyloid mining the presence or absence of one or more polymor B or said alpha-2-macroglobulin polypeptide with the LRP phisms at a position Selected from the group consisting of 6i, receptor. 12e, 14i.1 and 21i. 0069 48. The method of Paragraph 47, wherein said 0.052 37. The method of Paragraph 36, further compris alpha-2-macroglobulin mediates clearance of amyloid B. ing determining the presence or absence of one or more polymorphisms at a position Selected from the group con 0070 49. A method of identifying a compound that sisting of 18i and 24e modulates an alpha-2-macroglobulin activity comprising: 0.053 38. The method of Paragraph 22, comprising deter 0071 providing an alpha-2-macroglobulin polypep mining the presence or absence of one or more polymor tide comprising at least one of the polymorphisms or phisms at a position Selected from the group consisting of mutations having a position Selected from the group 12e, 12i and 28i. consisting of 14e, 20e, and 30e, 0054 39. The method of Paragraph 38, wherein the 0072 contacting said alpha-2-macroglobulin nucleotide at position 12e is T, or the complement thereof, polypeptide with Said compound; the nucleotide at position 21i is A, or the complement thereof and the nucleotide at position 28i is A, or the 0073 contacting said alpha-2-macroglobulin complement thereof. polypeptide with methylamine; and 0.055 40. A method of identifying a subject at risk for 0074 identifying a compound that modulates an Alzheimer's Disease, Said method comprising: alpha-2-macroglobulin activity by detecting a modul lation in the activation of Said alpha-2-macroglobu 0056 obtaining from said subject a biological lin polypeptide. 50. A method of identifying a com Sample containing an alpha-2-macroglobulin pound that modulates an alpha-2-macroglobulin polypeptide or portion thereof, and activity comprising: 0057 determining the presence or absence of one or more polymorphisms or mutations in Said alpha-2- 0075 providing an alpha-2-macroglobulin polypep macroglobulin polypeptide or portion thereof tide comprising at least one of the polymorphisms or wherein Said one or more polymorphisms or muta mutations having a position Selected from the group tions occur at a position Selected from the group consisting of 14e, 20e, and 30e, consisting of 14e, 20e and 30e. 0076 contacting said alpha-2-macroglobulin 0.058 41. A method of identifying a compound that polypeptide with Said compound; modulates an alpha-2-macroglobulin activity comprising: 0059 providing a plurality of cells that express the 0077 contacting said alpha-2-macroglobulin LRP receptor; polypeptide with amyloid 3; and 0060 contacting said cells with a candidate com 0078 identifying a compound that modulates an pound; alpha-2-macroglobulin activity by detecting a modul US 2003/0162202 A1 Aug. 28, 2003

lation in the formation of a complex of amyloid B 0093 64. A cultured cell comprising the nucleic acid of and Said alpha-2-macroglobulin polypeptide. any one of Paragraphs 52-58 or the polypeptide of any one of Paragraphs 59-62. 0079 51. A method of making a pharmaceutical com prising: 0094 65. A cultured cell comprising the recombinant vector of Paragraph 63. 0080 identifying a compound by a method of any one of Paragraphs 41, 49 and 50 incorporating Said 0095 66. An isolated or purified antibody that specifi compound into a pharmaceutical. cally binds to the polypeptide of any one of Paragraphs 59-62. 0.081 52. A purified or isolated nucleic acid comprising 0096) 67. The antibody of Paragraph 66, wherein said an alpha-2-macroglobulin Sequence having a polymorphism antibody is monoclonal. 68. A method of expressing an or mutation at a position Selected from the group consisting alpha-2-macroglobulin polypeptide comprising: of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i , 28i and 30e, wherein the nucleotide or nucleotide Sequence 0097 providing a construct comprising a promoter at Said position is other than an A2M-1. operably linked to an alpha-2-macroglobulin nucleic acid having a polymorphism or mutation at a posi 0082) 53. The purified or isolated nucleic acid of Para tion Selected from the group consisting of 14e, 20e graph 52, wherein Said alpha-2-macroglobulin Sequence is and 30e, wherein the nucleotide at Said position is SEQ ID NO: 1 or a sequence complementary thereto. other than an A2M-1, and 0083) 54. The purified or isolated nucleic acid of Para graph 53, wherein the nucleotide or nucleotide Sequence at 0098 expressing said alpha-2-macroglobulin from said position is A2M-2. Said construct. 0084. 55. The purified or isolated nucleic acid of Para 0099 69. The method of Paragraph 68, wherein said graph 52, wherein Said alpha-2-macroglobulin Sequence is nucleotide at Said position is A2M-2. selected from the group consisting of SEQID NOS: 2-8 and Said polymorphism of mutation is at a position Selected from BRIEF DESCRIPTION OF THE DRAWINGS the group consisting of 14e, 20e and 30e. 0100. The Figure shows a nucleotide sequence of a portion of chromosome 12 that includes the genomic 0085 56. The purified or isolated nucleic acid of Para Sequence of A2M that has been annotated to include the graph 55, wherein the nucleotide or nucleotide sequence at locations of exons as well as the names and locations of the said position is A2M-2. polymorphisms and/or mutations described herein. The 0.086 57. The purified or isolated nucleic acid comprising name of the polymorphism and/or mutation as well as the a fragment of at least 16 consecutive nucleotides of SEQ ID corresponding nucleotide change(s) are indicated at posi NO: 1 having a polymorphism or mutation at a position tions above the A2M gene Sequence. The nucleotide Selected from the group consisting of 6i, 12i.1, 12i.2, 12e, Sequence provided in the Figure is from the University of 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, wherein California at Santa Cruz draft human genome Sequence the nucleotide or nucleotide at Said position is other than an build 12 for chromosome positions 9007566-8918942 as is A2M-1 or a Sequence complementary thereto. available at www.genome.ucSc.edu. The Sequence presented is that of the “minus' strand in the sense that it is the 0087) 58. The purified or isolated nucleic acid of Para complement of the strand that extends 5'-->3' from the p graph 56, wherein the nucleotide or nucleotide Sequence at terminus to the centromere of chromosome 12. The said position is A2M-2. Sequence is, however, presented as the “Sense' Strand for the A2M gene. The sense strand refers to that strand of a double 0088 59. A purified or isolated polypeptide comprising Stranded nucleic acid molecule associated with a gene that an amino acid Sequence Selected from the group consisting has the Sequence of the mRNA that encodes the amino acid of SEQ ID NOs: 9-15 having a polymorphism or mutation Sequence. This sequence also corresponds to nucleotides at a position Selected from the group consisting of 14e, 20e 1-88624 of NCBI Accession Number AC007436 (SEQ ID and 30e, wherein the amino acid at Said position is other than A2M-1. NO: 1). 0089) 60. The purified or isolated polypeptide of Para DETAILED DESCRIPTION OF THE graph 59, wherein the amino acid at Said position is A2M-2. INVENTION 0101. Several single nucleotide polymorphisms (SNPs) 0090 61. A purified or isolated polypeptide comprising a and/or mutations of A2M gene have been discovered. Spe fragment of an amino acid Sequence Selected from the group cifically, several novel SNPs and/or mutations were found in consisting of SEQ ID NOs: 9-15 having a polymorphism or patients suffering from Alzheimer's Disease (AD). These mutation at a position Selected from the group consisting of SNPs and/or mutations are referred to as: 6i, 12i.1, 12i.2, 14e, 20e and 30e, wherein the amino acid mutation at Said 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e. The position is other than A2M-1. location of each of these SNPs and/or mutations on the A2M 0.091 62. The purified or isolated polypeptide of Para gene (Human Genome Project Gene Locus chr12: 9007566 graph 61, wherein the amino acid at Said position is A2M-2. 8918942 (minus strand); including a section of human chromosome 12 the Sequence of which is provided in 0092 63. A recombinant vector comprising the nucleic National Center for Biotechnology Information (NCBI) acid of any one of Paragraphs 52-58. Accession Number NTO09702, incoporated herein by ref US 2003/0162202 A1 Aug. 28, 2003

erence, and also present as nucleotides 1-88624 of NCBI column 4 represents the nucleotide present in SEQ ID NO: Accession Number AC007436, incorporated herein by ref 1 at the position indicated in column 2 of Table 1 (A2M-1). erence) (SEQ ID NO: 1) is identified in Table 1 and the The nucleotide to the right of the arrow represents the Figure. Provided herein are polymorphisms in the region of nucleotide substitution that occurs at this position (A2M-2). chromosome 12 Surrounding and including the A2M gene. For example, the A2M-1 allele of SNP 6i comprises a C at Thus, the polymorphisms provided herein include polymor nucleotide position 37221 of NCBI Accession Number phisms in exons, introns or intervening Sequences, inter AC007436. The A2M-2 allele of SNP 6i comprises an A at genic regions and gene upstream and downstream regions, nucleotide position 37221 of NCBI Accession Number Such as, for example, gene expression regulatory regions. ACO07436. For the 14i.1 mutation, the A2M-2 allele com 0102) A particular polymorphism, depending on the prises an insertion of the nucleotides “AAG” immediately nature and location of the polymorphism(s) in a gene allele, following the nucleotide position indicated in column 2 of can play various roles in the manifestation of a disease Table 1. condition or disorder. A polymorphism that gives rise to a 0104. When reference is made herein to a SNP or muta particular variant phenotype can produce its effect(s), for tion (as designated in column 1) with respect to a cDNA or example, at the level of RNA or protein. Effects on RNA any other contiguous nucleic acid Sequence which encodes include altered splicing, Stability, editing and expression. A2M, the location of the SNP or mutation with respect to a Effects on the protein include altered protein function, Specific cDNA or A2M coding Sequence is Set out in column folding, transport, localization, Stability and expression. 3 of Table 1. Accordingly, the location of a SNP and or Polymorphisms located in the 5' untranslated region of the mutation in a particular cDNA or A2M coding Sequence can gene may alter the activity of an element of the gene promoter and change the expression of the mRNA (e.g., be determined with reference to-Table 1, column 3. level, pattern and/or timing of expression). Polymorphisms 0105. In cases where the SNP or mutation results in an located in introns may alter RNA Stability, editing, Splicing, amino acid change, the amino acid change and position are etc. Polymorphisms located in the 3' untranslated region noted. The amino acid to the left of the arrow in column 5 may influence polyadenylation, transcription and/or mRNA represents the A2M-1 amino acid at the position indicated. Stability. Silent alterations in the coding region of a gene The amino acid to the right of the arrow represents the may affect codon usage and/or splicing. Changes in an A2M-2 amino acid at the position indicated. The Figure encoded amino acid Sequence, e.g., deletions and insertions, provides an annotated A2M gene Sequence which shows may affect protein function by increasing or decreasing a each of the SNPs and/or mutations listed in Table 1, includ native function or bringing about an altered function. ing both the A2M-1 alleles, represented by the nucleotides of SEQID NO: 1, and the A2M-2 alleles, represented by the 0103) The first column of Table 1 provides a name for nucleotides listed immediately above SEQ ID NO: 1. each of the novel SNPs or mutations described herein. The Accordingly, the locations of nucleotide or amino acid name of the SNP or mutation (i.e., the polymorphism Sequence polymorphisms Set forth in Table 1 are referred to designation) corresponds to its general location in the A2M by the polymorphism designation (i.e., as set forth in column gene. For example, 14e refers to a SNP present in exon 14 1 of Table 1) with reference to a location corresponding to of the A2M gene whereas 12i.1 refers to a SNP present in the nucleotide or amino acid position as Set forth in columns intron 12 of the A2M gene. The number to the right of the 2 and 5 of Table 1, respectively. decimal point in 12i.1 indicates that this SNP is one of multiple SNPs found in intron 12. Table 1 also provides the 0106 Generally, when a polymorphism designation, for location of each SNP with reference to SEQID NO: 1 (SEQ example, 6i, is referred to herein, it is used to specify a ID NO: 1 is the sequence of nucleotides 1-88624 of NCBI position or location within an A2M gene, cDNA, mRNA, Accession Number AC007436, which contains the sequence hnRNA or protein Sequence, without regard to the particular of an A2M gene) and the nucleotide change(s) caused by nucleotide or amino acid that may be present at the position. each SNP or mutation. In particular, for each of the poly The nucleotide or amino acid at the Specified location of the morphisms and/or mutations Set out in Table 1, except for A2M gene or A2M protein can be any nucleotide or amino the 14i.1 mutation, the nucleotide to the left of the arrow in acid unless a particular nucleotide or amino acid is specified.

TABLE 1.

Novel SNPs and Mutations Associated with Alzheimer's Disease

Location with reference to NCBI SNP? Accession Number AC007436 Location with reference to coding Nucleotide Amino Acid Change (with Mutation (SEQ ID NO: 1) nucleotide sequences (e.g. cDNAs) Change(s) reference to SEQ ID NO: 9) 6i 174 bp downstream of exon 6 C->A nucleotide position 37221 12e exon 12 Nucleotide positions: 1339 of SEQ ID NOs: 3 C-T Y-sy nucleotide position 45269 and 5; and 1338 of SEQ ID NO: 7 Silent effect 12i.1 152 bp upstream of exon 12 C-sG nucleotide position 45088 121.2 115 bp upstream of exon 12 A->T nucleotide position 45125 14e exon 14 Nucleotide positions: 1730 of SEQ ID NOs: 3 T-C C-R nucleotide position 47519 and 5; and 1729 of SEQ ID NO: 7 Amino acid position 563 US 2003/0162202 A1 Aug. 28, 2003

TABLE 1-continued

Novel SNPs and Mutations Associated with Alzheimer's Disease

Location with reference to NCBI SNP? Accession Number AC007436 Location with reference to coding Nucleotide Amino Acid Change (with Mutation (SEQ ID NO: 1) nucleotide sequences (e.g. cDNAs) Change(s) reference to SEQ ID NO: 9) 14i.1 136 bp downstream of exon 14 insertion nucleotide position 47669 of AAG 14i.2 151 bp downstream of exon 14 A->C nucleotide position 47684 17.1 240 bp upstream of exon 18 C-sG nucleotide position 53095 2Oe exon 20 Nucleotide positions: 2574 of SEQ ID NOs: 3 C-T A->V nucleotide position 56493 and 5; 2573 of SEQ ID NO: 7; and 38 of SEQ Amino acid position 844 ID NO: 4 2O 27 bp downstream of exon 20 C-sG nucleotide position 56586 21i 2 bp upstream of exon 21 T-C nucleotide position 56887 28 55 upstream of exon 29 G-T nucleotide position 72076 3Oe exon 30 Nucleotide positions: 3912 of SEQ ID NOs: 3 T-C F-L nucleotide position 74154 and 5; 3911 of SEQ ID NO: 7; and 1376 of SEQ Amino acid position 1290 ID NO: 4

0107 Table 2 provides a list of additional SNPs and mutations and their position on the A2M gene. The Figure TABLE 2-continued also shows the positions of each of the SNPs and mutations listed in Table 2 as well as the nucleotide change (A2M-2) oAdditional SNPs and Mutations Associated with Alzheimer's Disease that is associated with the SNP and/or mutation. A2M Gene Sequence Database Chromosome 12 Coordinate NCBI TABLE 2 SNP Identifier Coordinate Accession AC007436 rS2264O7 89.53836 53731 Additional SNPs and Mutations Associated with Alzheimer's Disease rS2264-08 895.3258 54309 rS226409 89.53062 54505 A2M Gene Sequence rS2264-10 89527OO 54867 Database Chromosome 12 Coordinate NCBI rS113973 89.52324 55.243 SNP Identifier Coordinate Accession AC007436 rS2277412 89.52004 55.563 rS104.9143 8951935 55632 rS226379 8976642 30925 rS2277413 8951903 55664 rS22638O 8976530 31037 rs318O392 8951879 55688 rS226381 8975616 31951 rS32101.07 8951879 55688 rS3080605 897539 32176 rs226411 89511.78 56389 5. .. SE rS226412 8949081 584.86 S rS226413 89.48804 58763 rS2477 897.3853 33714 rS28897O6 89.48741 58826 rS226383 8973OO3 34564 rs2111023 89.48292 59275 rS226384 897 1704 35863 rs226414 8947972 59595 rS226385 897.1288 36.279 rS2193OO6 894.4647 6292O rS226386 89.70784 36.783 rS1800433 894O4.08 67159 rS226.387 89693O2 38265 rs3168556 89.403.25 67242 : ... 5. rS1805651 89.39695 67872 rS1049134 8964919 42648 E. S. rS226390 8964765 428O2 rS2377682 89.38095 69472 rS226391 896441 43156 rS1805654 89.376.86 69881 rS226392 8964312 4.3255 rS1805678 8937227 70340 rS226393 896.3888 43679 rS1805655 8936.701 70866 rS226394 896309 44476 rS1805656 8936688 70879 rS226395 896.2840 44727 rS1805679 8936686 70881 rS226396 8962.283 45284 rS3O26223 8936527 71040 rS226397 896195 45616 rS1805657 8936491 71076 2. .. 3. rS1805680 8936426 71141 rS226400 89.58524 49043 rS1805658 8936355 71212 rS2264O1 8958,516 49051 rS1805659 8936312 71255 rS2264O2 8957932 4963.5 rS3O26224 89.362O5 71362 rS22.6403 8957810 49757 rS23OO147 8936088 714.79 rS2264.04 895.6453 51114 rS23OO148 893.6081 714.86 rS2264-05 8956290 51277 rS1805681 89.35925 71642 rS1800434 89.55640 51927 rS1805682 89.35844 71723 rS2264O6 8954411 53156 rS1805683 8935145 72422 US 2003/0162202 A1 Aug. 28, 2003

for A2M are contemplated by the present invention. Fur TABLE 2-continued thermore, A2M genes that have one or more of the SNPs and/or mutations described herein and which are altered Additional SNPs and Mutations Associated with Alzheimer's Disease from SEQID NO: 1 (as well as SEQID NOS:2-15) or known A2M Gene Sequence variants thereofas result from one or more Sequencing errors Database Chromosome 12 Coordinate NCBI are also contemplated by the present invention. AS used SNP Identifier Coordinate Accession AC007436 herein, the term "mutation' means nucleotide variations that rS1805660 8935115 72452 are not limited to Single nucleotide Substitution. For rS1805661 8935O18 72549 example, a mutation includes, but is not limited to, the rS3O80599 8934757 72810 insertion of one or more bases, the deletion of one or more rS1805684 89.34307 7326O rS3O26225 8934282 73285 bases, or an inversion of multiple bases. rS1805662 8934281 73286 0111. In view of the above, as used herein, “A2M', rS1805685 893.3979 73588 rS1805663 89.32O10 75557 “A2M gene' or “A2M genomic nucleic acid”, when used rS1805664 893O343 77224 with reference to SEQ ID NO: 1, means the nucleic acid rS1805665 893016O 774O7 sequence of SEQID NO: 1 or portions thereofas well as any rS1805666 893O154 77413 nucleic acid variants which include one or more SNPs rS3O26226 893O105 77462 rS3O26227 892.985.5 77712 and/or mutations, Such as those described in Table 2 and the rS1805686 8929764 77803 Figure. Similarly, “A2M cDNA”, “A2M coding sequence” rS3O26228 8929693 77874 or “A2M coding nucleic acid”, when used with reference to rs3180682 892.8606 78961 SEQ ID NOS: 2-8, means the nucleic acid sequences of SEQ rS1805687 892.8558 79009 rS1049985 8928436 791.31 ID NOS: 2-8 or portions thereof as well as nucleic acid rs3190224 8928425 79142 variants which include one or more SNPs and/or mutations, rS1805688 8928157 79.410 such as those described in Table 2 and the Figure. With rS1805667 8928O23 79544 respect to polypeptides “A2M”, “A2M polypeptide' or rS3O26229 8927957 7961O “A2M protein', when used with reference to SEQ ID NOs: 9-15, means the amino acid sequence of SEQ ID NOs: 9-15 or portions thereof as well as amino acid Sequence variants 0108. It will be appreciated that the nomenclature for the which are encoded by nucleic acids which include one or polymorphisms and/or mutations used in the Figure and in more SNPs and/or mutations, Such as those described in Tables 1 and 2 refers to the location of the polymorphism Table 2, and the Figure and which effect the polypeptide and/or mutation disclosed herein. Accordingly, the use of a encoded by the A2M coding Sequence. polymorphism or mutation name (or designation), Such as 6i, 14e, or rS226381 indicates a polymorphic position in the 0112 According to Some aspects of the present invention, reference nucleotide or amino acid Sequence and not nec A2M includes nucleotide sequences having at least 99%, essarily the identity of the nucleotide or amino acid change. 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, The nucleotide and amino acid changes indicated in the 88%, 87%, 86%, or 85% sequence identity to SEQ ID NO: Figure and in Table 1 correspond to one of many changes 1 as determined by BLASTN with default parameters (Alts which can occur at the location of the polymorphism and/or chuletal, (1990).J. Mol. Biol. 215: 403, incorporated herein mutation. by reference in its entirety). In other aspects of the present invention, A2M coding Sequence includes nucleotides 0109 The reference nucleic acid sequence is provided by sequences having at least 99%, 98%, 97%, 96%, 95%, 94%, SEQ ID NO:1 which corresponds to nucleotides 1-88624 of 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85% NCBI Accession Number AC007436. It will be appreciated sequence identity to any one of SEQ ID NOS: 2-8 as that a nucleic acid corresponding to an A2M coding determined by BLASTN version 2.0 with default parameters sequence (SEQ ID NO: 2) can be constructed by joining the (Altschul et al., (1990).J. Mol. Biol. 215: 403, incorporated exons at the Splice Sites listed for nucleotide Sequence region herein by reference in its entirety). In still other aspects of 1-88624 as provided in the header section of NCBI Acces the present invention, A2M includes polypeptide Sequences sion Number AC007436. Additionally, a number of cDNA having at least 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, variants of A2M are also available. These cDNAS, Some of 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence which encode variant polypeptides, are provided as SEQ ID identity or similarity to any one of SEQ ID NOs: 9-15 as NOs: 3-8.Variant A2M polypeptide sequences are provided determined by FASTA version 3.0t78 with default param as SEO ID NOS: 9-15. eters (Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA, 85: 2444, incorporated herein by reference in its 0110. In view of the above, it will be appreciated that, entirety). although each of the novel SNPs and/or mutations disclosed herein are described with reference to SEQ ID NO: 1 (as 0113 AS used in connection with any one of the poly well as SEQ ID NOS:2-15), each of these SNPs and/or morphisms and/or mutations disclosed herein, A2M-1 refers mutations can occur in the context of nucleic acid Sequence to the nucleotide or nucleotide sequence of SEQ ID NO: 1 variants. For example, in addition to one or more of the which is present at the location of the polymorphism or SNPs disclosed herein, SNPs and/or mutations previously mutation. AS used in connection with any one of the poly described for A2M (e.g. SNPs and/or mutations described in morphisms and/or mutations disclosed herein, A2M-2 refers Table 2) may occur within SEQ ID NO: 1 (as well as SEQ to the nucleotide change, nucleotide insertion or nucleotide ID NOS:2-15). Such nucleic acids having both one or more deletion indicated in the Figure and/or in Table 1 which is of the SNPs and/or mutations described herein and one or present at the location of the polymorphism or mutation. AS more known or previously described SNPs and/or mutations used in connection with any one of the polymorphisms US 2003/0162202 A1 Aug. 28, 2003

and/or mutations disclosed herein, A2M-1 refers to the and in particular the polymorphisms provided herein, indi amino acid of SEQID NO:9 which is present at the location vidually or in combination, e.g., haplotypes, for association of the polymorphism or mutation. AS used in connection with a disease or disorder. For example, the polymorphisms with any one of the polymorphisms and/or mutations dis provided herein can be tested individually, in combinations closed herein, A2M-2 refers to the amino acid change of the provided polymorphisms, or in combinations with indicated in the Figure and/or in Table 1 which is present at other previously described polymorphisms (e.g., polymor the location of the polymorphism or mutation. phisms listed in Table 2). The analysis or testing may 0114 Polymorphisms can serve as genetic markers. A involve genotyping DNA from individuals affected with the genetic marker is a DNA segment with an identifiable disease or disorder, and possibly also from related or unre location in a chromosome. Genetic markers may be used in lated individuals, with respect to the polymorphic marker a variety of genetic Studies Such as, for example, locating the and analyzing the genotyping data for association with the chromosomal position or locus of a DNA sequence of disease or disorder using methods described herein and/or interest, identifying genetic associations of a disease, and known to those of skill in the art. For example, Statistical determining if a Subject is predisposed to or has a particular analysis of the data may involve a chi-squared or Fisher's disease. Because DNA sequences that are relatively close exact test and may be conducted in conjunction with a together on a chromosome tend to be inherited together, number of programs, Such as the transmission disequilib tracking of a genetic marker through generations in a family rium test (TDT), affected family based control test (AFBAC) and comparing its inheritance to the inheritance of another and the haplotype relative risk test (HRR). Case-control DNA sequence of interest can provide information useful in Strategies can be applied to the testing, as can, for example, determining the relative position of the DNA sequence of TDT approaches. interest on a chromosome. Genetic markers particularly useful in Such genetic Studies are polymorphic. Such mark 0118 Several embodiments of the invention have bio erS also may have an adequate level of heterozygosity to technological, diagnostic, and therapeutic use. For example, allow a reasonable probability that a randomly Selected the nucleic acids and proteins described herein can be used person will be heterozygous. as probes to isolate more polymorphic and/or mutant A2M genes, to detect the presence or absence of wild type or 0115 The polymorphisms provided herein in the region polymorphic and/or mutant A2M proteins in an individual, of chromosome 12 Surrounding and including the A2M gene and these molecules can be incorporated into constructs for include single nucleotide polymorphisms (SNPs). SNPs preparing recombinant polymorphic and/or mutant A2M have use as genetic markers, for example, in fine genetic proteins or used in methods of Searching or identifying mapping and genetic association analysis, as well as linkage agents that modulate A2M levels and/or activity, for analysis see, e.g., Kruglyak (1997) Nature Genetics 17:21 example, candidate therapeutic agents. The Sequences of the 24). Combinations of SNPs (which individually occur about nucleic acids and/or proteins described herein can also be every 100-300 bases) can also yield informative haplotypes. incorporated into computer Systems, used with modeling Also provided herein, are polymorphisms of the A2M gene Software So as to enable rational drug design. Information and Surrounding region of chromosome 12 that are associ obtained from genotyping methods provided herein can be ated, individually and/or in combination, with a neurode used, for example, in computer Systems, in pharmacoge generative disease, Such as, for example, Alzheimer's dis nomic profiling of therapeutic agents to predict effectiveness CSC. of an agent in treating an individual for a neurodegenerative disease Such as AD. The nucleic acids and/or proteins 0116 Based on the discovery of association between described herein can also be incorporated into pharmaceu SNPs described herein, individually and/or in combinations ticals and used for the treatment of neuropathies, Such as (haplotypes), with AD, additional markers associated with Alzheimer's Disease (AD). AD may now be identified using methods as described herein and known in the art. The availability of additional 0119) Accordingly, some embodiments of the invention markers is of particular interest in that it will increase the include isolated or purified nucleic acids comprising, con density of markers for this chromosomal region and can Sisting essentially of, or consisting of an A2M gene, cDNA provide a basis for identification of an AD DNA segment or or mRNA with one or more of the SNPs and/or mutations gene in the region of chromosome 12. An AD DNA segment described in Table I or a fragment of said A2M gene, cDNA or gene may be found in the vicinity of the marker or Set of or mRNA, wherein Said fragment contains at least 9, at least markers showing the highest correlation with AD. Further 16 or at least 18 consecutive nucleotides of the polymorphic more, the availability of markers associated with AD makes or mutant A2M gene, cDNA or mRNA but including at least possible genetic analysis-based methods of determining a one of the SNPs and/or mutations in Table 1. Isolated or predisposition to or the occurrence of AD in an individual by purified nucleic acids that are complementary to Said A2M detection of a particular allele. nucleic acids and fragments thereof are also embodiments. 0117 Polymorphisms of the A2M gene region of chro 0120) Some nucleic acid embodiments for example, moSome 12 provided herein may be analyzed individually include genomic DNA, RNA, and cDNA encoding the and in combinations, e.g., haplotypes, for genetic associa polymorphic and/or mutant A2M proteins or fragments tion with any disease or disorder. In a particular example, the thereof. Methods for obtaining Such nucleic acid Sequences disease is a neurodegenerative disease, Such as, for example, are also embodiments. The nucleic acid embodiments can be AD. Thus, also provided herein are methods of identifying altered, mutated, or changed Such that the alteration, muta polymorphisms associated with diseases and disorders. The tion, or change results in a conservative amino acid replace methods involve a step of testing polymorphisms of the ment. These altered or changed nucleic acids are equivalent A2M gene, and/or Surrounding region of chromosome 12, to the nucleic acids described herein. In Some contexts, the US 2003/0162202 A1 Aug. 28, 2003 term “consisting essentially of is used to include nucleic the polymorphic and/or mutant A2M proteins or fragments acids having the changes or alterations above. thereof can include, but are not limited to, the ability to bind proteases, bind amyloid-fi, bind a receptor (e.g., the LRP 0121 Vectors having the nucleic acids above, including receptor), bind Zinc, and the ability to form a tetramer. expression vectors, and cells containing Said nucleic acids Several computer-based methodologies are discussed, which and vectors are also embodiments. Methods of making these involve three-dimensional modeling of the polymorphic constructs and cells are aspects of the invention, as well. and/or mutant A2M proteins or fragments thereof and Sus Other embodiments of the invention include genetically pected binding partners (e.g., antibodies, proteases, amy altered organisms that express the polymorphic and/or mutant A2M transgenes or polymorphic portions thereof loid-P, zinc, and the LRP receptor). (e.g., mutant A2M transgenic or knockout animals). Meth 0.125 Several A2M characterization assays are also ods of making Such organisms are also aspects of the described. These assays test the functionality of a polymor invention. Transgenic animals that are contemplated (par phic and/or mutant A2M protein or fragment thereof and can ticularly non-human animals) can be used, for example, in identify agents that modulate the activity and/or expression elucidating disease processes and/or identifying therapeutic of Such proteins, including, for example, binding partners agents. that interact with Said molecules. Agents that modulate the activity of a wild-type or polymorphic or mutant A2M, for 0122) Some polypeptide embodiments of the invention example, can be identified using an A2M characterization include isolated, enriched, recombinant or purified polypep assay and molecules identified using these methods can be tides consisting of, consisting essentially of, or comprising incorporated into medicaments and pharmaceuticals, which the complete amino acid sequences (or portions thereof can be provided to Subjects in need of treatment or preven containing the polymorphic amino acid change) of the tion of neuropathies, including AD. polymorphic and/or mutant A2M proteins described herein. (See Table 1, which includes the nucleotide polymorphisms 0.126 Some functional assays involve the use of multi of the A2M gene coding Sequence that result in correspond meric polymorphic and/or mutant A2M proteins or frag ing amino acid changes in the A2M polypeptide Sequence. ments thereof and/or binding partners, which are disposed Additionally, Table 1 sets out the identity and location of the on a Support, Such as a resin, bead, lipid vesicle or cell amino acid Substitution with respect to a reference A2M membrane. These multimeric agents are contacted with polypeptide sequence). Other polypeptide embodiments are candidate binding partners and the association of the binding equivalents to the polymorphic and/or mutant A2M proteins partner with the multimeric agent is determined. Successful described herein in that Said equivalent molecules have binding agents can be further analyzed for their effect on conservative amino acid Substitutions. In Some contexts, the A2M function in other types of cell based assays. One such term “consisting essentially of is used to include polypep assay evaluates internalization of a protease or amyloid B. tides having Such conservative amino acid Substitutions. Other types of characterization assays involve molecular Embodiments also include isolated, enriched, recombinant biology techniques designed to identify protein-protein or purified fragments of the polymorphic and/or mutant interactions (e.g., two-hybrid Systems). A2M proteins at least 3 amino acids in length So long as Said fragments contain at least one of the amino acid polymor 0127. The diagnostic embodiments of the invention phisms and/or mutants described herein (See Table 1). (including diagnostic kits) are designed to identify individu Additional embodiments concern methods of preparing the als at risk of acquiring AD or individuals that have a polypeptides and peptides described herein and, in Some predilection for AD. Nucleic acid and protein based diag preparative methods, chemical Synthesis and/or recombinant nostics are provided. Some of these diagnostics identify techniques are used. individuals at risk for acquiring AD by detecting a particular nucleotide or amino acid polymorphism and/or mutation or 0123 Embodiments of the invention also include anti combinations of polymorphisms and/or mutations, for bodies directed to the mutant and/or polymorphic A2M example a haplotype, in an A2M gene or A2M protein. Other proteins. Preferably, said antibodies Specifically interact diagnostic approaches are concerned with the detection of with the mutant and/or polymorphic A2M proteins and can aberrant amounts or levels of expression of polymorphic or be used to differentiate wild-type A2M proteins (e.g., A2M mutant A2M RNA or A2M protein. The polymorphisms proteins having a reference Sequence of amino acids and/or and/or mutations, levels of expression of polymorphic or that are most prevalent in the population or in a particular mutant A2M RNAS or proteins can be recorded in a data study) from polymorphic and/or mutant A2M proteins. The base, which can be accessed to identify a type of AD, a antibody embodiments can be monoclonal or polyclonal and Suitable treatment., and Subjects for which further genotyp approaches to manufacture both types of antibodies, which ing should be investigated. It is contemplated that many are specific for the polymorphic and/or mutant A2M proteins other SNPs and/or mutations, which are predictive of AD, are disclosed. can be found in Subjects identified as already having at least 0.124 Approaches to rational drug design are also pro one SNP and/or mutation described herein. Vided in this disclosure, and these methods can be used to 0128. Accordingly, a method of identifying an individual identify molecules that interact with the polymorphic and/or having an altered risk for AD is provided, wherein a bio mutant A2M proteins or fragments thereof. Molecules that logical Sample containing nucleic acid is obtained from an interact with the polymorphic and/or mutant A2M proteins individual, and the Sample is analyzed to determine the or fragments thereof are referred to as “binding partners'. nucleotide identity of at least one novel SNP and/or muta Preferred binding partners modulate (e.g., increase or tion, Such as at least one SNP and/or mutation selected from decrease) the activity of the polymorphic and/or mutant the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, A2M proteins or fragments thereof The various activities of 17i.1, 20e, 20i, 21i, 28i and 30e. The presence or absence of US 2003/0162202 A1 Aug. 28, 2003 a particular nucleotide or nucleotide Sequence at the location and pharmacogenetic-based treatment programs and in the of any one of these SNPs and/or mutations can indicate an Search for a gene responsible for AD or other AD-associated altered risk of AD. Additionally, the nucleotide identity geneS. information obtained from the analysis of combinations of SNPs and/or mutations can further indicate an altered risk of 0.132. Also provided herein are methods of genotyping an AD. The biological Sample can also be analyzed to deter individual comprising obtaining a nucleic acid Sample from mine the nucleotide identity of publicly available SNPs an individual and determining the nucleotide identity of at and/or mutations. Nucleotide identity information obtained least one novel SNP and/or mutation described herein, Such from the analysis of publicly available SNPs and/or muta as at least one SNPs and/or mutations selected from the tions in combination with novel SNPs disclosed herein, Such group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, as at least one SNP and/or mutation selected from the group 17i.1, 20e, 20i, 21i, 28i and 30e. In a particular embodiment, consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, the nucleotide identity of more than one novel polymor 20e, 20i, 21i, 28i and 30e, can indicate an altered risk for phism and/or mutation is determined. Accordingly, a Set of AD. The analysis can include an association Study (e.g., a novel polymorphisms and/or mutations can be analyzed to family study) and/or haplotype analysis. determine the nucleotide identity for each polymorphism and/or mutation in the entire Set. The Set of polymorphisms 0129. Also provided are methods of identifying polymor and/or mutations can also include polymorphisms and/or phisms associated with a disease or disorder. The novel mutations that are publicly available as well as novel poly SNPs and/or mutations described herein, Such as a SNP morphisms and/or mutations. Determination of the nucle and/or mutation Selected from the group consisting of 6i, otide identities for Sets of polymorphisms and/or mutations 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i as described above provides a method for determining the and 30e, can be analyzed separately or in combinations to haplotype of an individual. identify association with any A2M-mediated disease or disorder. The polymorphisms can be analyzed to identify 0.133 Also provided herein are methods of confirming a asSociation with neurodegenerative diseases. For example, a phenotypic diagnosis of a disease or disorder which include single or combinations of novel SNPs and/or mutations can a step of detecting in nucleic acid obtained from a Subject be checked for association with neurodegenerative disorders diagnosed with a disease or disorder the presence or absence or other diseases having a relationship to the A2M gene of one or more polymorphisms and/or Selected from the using methods well known in the art, Such as those described group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, herein. 17i.1, 20e, 20i, 21i, 28i and 30e, wherein the presence of the one or more polymorphisms, individually and/or in combi 0130 For example, the genotype of individuals with nation, confirms a phenotypic diagnosis of the disease or respect to one or more polymorphisms and/or mutations disorder. In a particular embodiment of these methods, the Selected from the group consisting of 6i, 12i.1, 12i.2, 12e, disease or disorder is an A2M-mediated disease disorder. In 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e can be one embodiment, the disease or disorder is a neurodegen compared between individuals that have AD or a particular erative disease or disorder, Such as, for example, AD. For disease or a family history of the disease and individuals that example, the disease may be Alzheimer's disease with an do not have the disease or a family history of the disease So onset age of greater than or equal to about 50 years, or as to identify a polymorphism or combination of polymor greater than or equal to about 60 years, or greater than or phisms that associate with a disease or disorder, Such as a equal to about 65 years. In another embodiment of the neurodegenerative disease or disorder, for example AD. methods of confirming a phenotypic diagnosis of a neuro Additionally, Since there are many different genotypes that degenerative disease or disorder, the method further includes can be associated with AD, individuals with AD having one a step of detecting in nucleic acid obtained from the Subject genotype can be compared with individuals with AD having the presence or absence of one or more polymorphisms of at another genotype to identify the presence of a novel SNP least one different gene allele associated with neurodegen and/or mutation. In one embodiment of the invention, the erative disease. In a particular embodiment, the at least one information and analysis above can be recorded on a data different gene allele is an APOE4 allele. base and the comparisons can be performed by a computer System accessing Said database. Thus, by Virtue of the fact 0134) Further provided are methods of treating a subject that at least one SNP and/or mutation selected from the manifesting an Alzheimer's disease phenotype. Certain group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, ambiguous phenotypes, e.g., dementia, manifested in AD 17i.1, 20e, 20i, 21i, 28i and 30e has been identified in an also occur in connection with other diseases and conditions individual or a family, the nucleic acids and proteins isolated which may be treated using drugs and other treatments that or purified from said individuals becomes a novel tool with are different from drugs and methods used to treat AD. which more SNPs and mutations associated with AD can be Genotyping of polymorphisms of the A2M gene region identified. described herein, and optionally other AD-associated mark ers, in Subjects manifesting Such an AD phenotype(s) per 0131. In yet another aspect of the present invention, the mits confirmation of AD phenotypic diagnoses and assists in information gained from analyzing biological Samples distinguishing between AD and other possible diseases or obtained from one or more individuals to determine the disorders. Once an individual is genotyped as having or nucleotide identity of at least one novel SNP and/or mutation being predisposed to AD, he or she may be treated with any described herein, Such as the SNPs and/or mutations known methods effective in treating AD. Selected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, can be 0.135 Accordingly, methods of treating a subject mani used in fine chromosome mapping of chromosome 12, in festing an Alzheimer's disease phenotype provided herein genetic association Studies, in pharmacogenetic profiling include Steps of US 2003/0162202 A1 Aug. 28, 2003 11

0136 (a) determining the nucleotide identity, in a and/or the Sequence of AAG occurs between nucleotides at nucleic acid obtained from the Subject, of one or positions corresponding to positions 47669 and 47670; and more polymorphisms Selected from the group con (d) the nucleotide sequence provided in SEQ ID NO: 1, or Sisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, a portion(s) thereof, wherein the nucleotide at a position 17i. 1, 20e, 20i, 21i, 28i and 30e, wherein the pres corresponding to 37221 is A, the nucleotide at a position ence of a particular nucleotide or nucleotides at the corresponding to 45269 is T, the nucleotide at a position one or more polymorphisms, individually and/or in corresponding to 45088 is G, the nucleotide at a position combination, is indicative of the occurrence of corresponding to 45125 is T, the nucleotide at a position Alzheimer's disease in a Subject; and corresponding to 47519 is C, the nucleotide at a position corresponding to 47684 is C, the nucleotide at a position 0137 (b) selecting and/or administering a treatment corresponding to 53095 is G, the nucleotide at a position that is effective for treatment of Alzheimer's disease. corresponding to 56493 is T, the nucleotide at a position 0.138. The pharmaceutical embodiments of the invention corresponding to 56586 is G, the nucleotide at a position include medicaments containing an agent, for example, a corresponding to 56887 is C, the nucleotide at a position binding partners that modulates the activity of wild-type or corresponding to 72076 is T, the nucleotide at a position polymorphic or mutant A2M. These medicaments can be corresponding to 74154 is C, and/or the Sequence of AAG prepared in accordance with conventional methods of occurs between nucleotides corresponding to positions galenic pharmacy for administration to organisms in need of 47669 and 47670. treatment. A therapeutically effective amount of agent, for 0.141. Additionally, aspects of the present invention example, a binding partner (e.g., an amount Sufficient to include the A2M coding sequences and cDNAs of SEQ ID modulate the function of a wild-type or polymorphic or NOs: 2-8 as modified by a nucleotide(s) change at least one mutant A2M) can be incorporated into a pharmaceutical SNP and/or mutation selected from the group consisting of composition with or without a carrier. Routes of adminis 12e, 14e, 20e, and 30e. More embodiments concern the tration of the pharmaceuticals of the invention include, but nucleic acids of SEQ ID NOs: 1-8 having nucleotide(s) are not limited to, topical, transdermal, parenteral, gas variations at one or more previously described SNPs and/or trointestinal, transbronchial, and transalveolar. These phar mutations for A2M (e.g. SNPs and/or mutations provided in maceuticals can be provided to Subjects in need of treatment Table 2) in addition to a nucleotide(s) change at least one for neurodegenerative diseases, in particular AD. The Sec SNP and/or mutation selected from the group consisting of tion below describes several of the nucleic acid embodi 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, ments of the invention. 28i and 30e. 0142. In this regard, the nucleic acid embodiments 0139 A2M Nucleic Acids described herein can have from 9 to approximately 88,624 0140. The A2M nucleotide sequences of the invention consecutive nucleotides So long as the Sequence contains include: (a) the nucleotide sequence provided in NCBI nucleotide(s) variation at a SNP and/or mutation selected Accession Number AC007436 nucleotide positions from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 1-88624, incorporated herein by reference in its entirety 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, for example, or the (SEQ ID NO: 1), or a portion thereof, as modified by a nucleotides specified for the particular locations within SEQ nucleotide(s) change at least one SNP and/or mutation ID NO: 1 as set forth in (c) and (d) immediately above. Some Selected from the group consisting of 6i, 12i.1, 12i.2, 12e, of these compositions, for example, include nucleic acids 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e as having any number between 9-50, 16-50, 17-50, 18-50, indicated in the Figure and/or in Table 1; (b) nucleotide 19-50, 50-100, 100-500, 500-1000, 1000-10,000, 10,000 Sequences encoding amino acid Sequences (a sequence 50,000, or 50-88,634 consecutive nucleotides of SEO. ID. formed by the joining the exons of the genomic Sequence NO. 1, wherein said nucleic acid contains a SNP and/or provided in NCBI Accession Number AC007436 between mutation Selected from the group consisting of 6i, 12i.1, nucleotide positions 31033 and 79.197 (SEQ ID NO: 2), or 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e A2M, mRNA or cDNA sequences (e.g., SEQID NOs: 3-8) (e.g., greater than or equal to 9, 10, 11, 12, 13, 14, 15, 16, as modified by a nucleotide(s) change at least one SNP 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and/or mutation Selected from the group consisting of 12e, 33, 34, 35,36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 14e, 20e, and 30e as indicated in the Figure and/or in Table 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 1; (c) the nucleotide sequence provided in SEQ ID NO: 1, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77,78, 79,80, 81, or a portion(s) thereof, wherein the nucleotide at a position 82, 83, 84, 85, 86, 87, 88, 89,90,91, 92,93, 94, 95, 96, 97, corresponding to 37221 is A, T or G, the nucleotide at a 98, 99, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, position corresponding to 45269 is T, A or G, the nucleotide 600, 700, 800, 900, 1000, 1300, 1400, 1500, 1600, 1700, at a position corresponding to 45088 is G, A or T, the 1800, 1900, 2000, 3000, 5000, 10,000, 25,000, 50,000, nucleotide at a position corresponding to 45125 is T, C or G, 75,000 and 88,624 consecutive nucleotides of a sequence of the nucleotide at a position corresponding to 47519 is C, A SEQ ID NO:1 or portions of the above nucleotide list for or G, the nucleotide at a position corresponding to 47684 is SEO ID NOS: 2-8, wherein said nucleic acid contains a C, G or T, the nucleotide at a position corresponding to nucleotide(s) variation at a SNP and/or mutation selected 53095 is G, A or T, the nucleotide at a position correspond from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, ing to 56493 is T, A or G, the nucleotide at a position 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e). In one embodiment, corresponding to 56586 is G, A or T, the nucleotide at a the nucleic acids comprise at least 12, 13, 14, 15, 16, 17, 18, position corresponding to 56887 is C, G or A, the nucleotide 19, 20 consecutive nucleotides of a sequence of SEQ ID at a position corresponding to 72076 is T, A or C, the NO:1 or SEQ ID NOS: 2-8, wherein said nucleic acid nucleotide at a position corresponding to 74154 is C, A or G, contains a nucleotide(s) variation SNP and/or mutation US 2003/0162202 A1 Aug. 28, 2003

Selected from the group consisting of 6i, 12i.1, 12i.2, 12e, 212:351 (1993); Huber, et al., Anal. Chem, 67:578 (1995); 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, for O'Donovan et al., Genomics 52:44 (1998), Am J Hum example, or the nucleotides Specified for the particular Genet. Dec;67(6): 1428-36 (2000); Ann Hum Genet. Sep:63 locations within SEQ ID NO: 1 as set forth in (c) and (d) (Pt. 5):383-91 (1999); Biotechniques, Apr;28(4):740-5 immediately above, or a complement thereof. In another (2000); Biotechniques. Nov;29(5):1084-90, 1092 (2000); embodiment, the nucleic acid embodiments comprise at Clin Chem. Aug:45(8 Pt 1): 1133-40 (1999); Clin Chem. least 20-30 consecutive nucleotides of a sequence of SEQID Apr;47(4):635-44 (2001); Genomics. Aug 15:52(1):44-9 NO: 1 or SEQ ID NOS: 2-8, wherein said nucleic acid (1998); Genomics. Mar 15:56(3):247-53 (1999); Genet contains a nucleotide(s) variation at a SNP and/or mutation Test.;1(4):237-42 (1997-98); Genet Test.:4(2):125-9 (2000); Selected from the group consisting of 6i, 12i.1, 12i.2, 12e, Hum Genet. Jun; 106(6):663-8 (2000); Hum Genet. 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, for Nov;107(5):483-7 (2000); Hum Genet. Nov;107(5):488-93 example, or the nucleotides Specified for the particular (2000); Hum Mutat. Dec;16(6):518-26 (2000); Hum Mutat. locations within SEQ ID NO:1 as set forth in (c) and (d) 15(6):556-64 (2000); Hum Mutat. Mar;17(3):210-9 (2001); immediately above, or complement thereof. J Biochem Biophys Methods. Nov 20:46(1-2):83-93 (2000); 0143. Several embodiments also include the above-de J Biochem Biophys Methods. Jan 30:47(1-2):5-19 (2001); scribed fragments of the nucleic acids of SEQ ID NOs: 1-8 Mutat Res. Nov 29;430(1): 13-21 (1999); Nucleic Acids Res. having a nucleotide(s) variation at one or more previously Mar 1 ;28(5):E13 (2000); and Nucleic Acids Res. Oct described SNPs and/or mutations for A2M (e.g. SNPs and/or 15:28(20):E89 (2000), all of which, including the references mutations provided in Table 2) in addition to a nucleotide(s) contained therein, are hereby expressly incorporated by variation at least one SNP and/or mutation selected from the reference in their entireties. group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 0146 Also provided herein are oligonucleotides that can 17i.1, 20e, 20i, 21i, 28i and 30e, for example, the nucle Serve as primerS. Such oligonucleotides can be made, for otides specified for the particular locations within SEQ ID example, by conventional oligonucleotide Synthesis for use NO:1 as set forth in (c) and (d) immediately above. in isolation and diagnostic procedures that employ the 0144. The nucleic acid embodiments described herein Polymerase Chain Reaction (PCR) or other enzyme-medi can also be altered by mutation Such as Substitutions, addi ated nucleic acid amplification techniqueS or primer exten tions, or deletions that provide for Sequences encoding sion techniques. For a review of PCR technology, see equivalent molecules. Due to the degeneracy of nucleotide Molecular Cloning to Genetic Engineering White, B. A. Ed. coding Sequences, other DNA sequences that encode Sub in Methods in Molecular Biology 67: Humana Press, Totowa Stantially the same polymorphic/mutant A2M amino acid (1997), the disclosure of which is incorporated herein by Sequence can be made. These include, but are not limited to, reference in its entirety and the publication entitled “PCR nucleic acid Sequences comprising all or portions of SEQID Methods and Applications” (1991, Cold Spring Harbor NO: 1 or SEQ ID NOS: 2-8, wherein said nucleic acid Laboratory Press), the disclosure of which is incorporated Sequences contain a a nucleotide(s) variation at a SNP and/or herein by reference in its entirety. mutation Selected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, 0147 Oligonucleotide primers provided herein can con or complements thereof, which have been altered by the tain a Sequence of nucleotides that Specifically hybridizes substitution of different codons that encode a functionally adjacent to or at a polymorphic region of the A2M gene equivalent amino acid residue within the Sequence, thus Spanning a nucleotide position corresponding to any of the producing a Silent change. following nucleotide positions of SEQ ID NO: 1: 37221, 45269, 45088,45125,47519,47684, 53095,56493, 56586, 0145 The nucleic acid sequences described above have 56887, 72076, 74154 and 47669, or the complementary biotechnological and diagnostic use, e.g., in nucleic acid positions thereof adjacent to or at a polymorphic region of hybridization assays, Southern and Northern Blot analysis, an A2M cDNA spanning a nucleotide position correspond etc. and the prognosis of neuropathies, Such as Alzheimer's ing to any of the following positions: 1339, 1730, 2574 and Disease (AD). By using the nucleic acid Sequences 3912 of SEQ ID NOs: 3 and 5; 1338, 1729, 2573 and 3911 described herein, for example, probes that complement the of SEQ ID NO: 7; and 38 and 1376 of SEQ ID NO: 4. In polymorphic and/or mutant A2M genes or cDNAs can be particular embodiments, the oligonucleotides hybridize to a designed and manufactured by oligonucleotide Synthesis. polymorphic region of the A2M gene under conditions of Desirable probes comprise a nucleic acid Sequence that is moderate or high Stringency. Also provided are oligonucle unique to the polymorphic and/or mutant A2M genes or otides, Such as primers and probes that are the complements cDNAS. These probes can be used to Screen nucleic acids of these primerS and probes. In particular embodiments, the isolated from tested individuals So as to identify the presence probes or primerS contain a number of nucleotides Sufficient or absence of a polymorphism or combination of polymor to allow Specific hybridization to the target nucleotide phisms indicative of an altered, for example increased, risk Sequence. In particular embodiments of the probes and of AD. Analysis can involve denaturing gradient gel elec primerS provided herein, the molecules are of Sufficient trophoresis or denaturing HPLC methods, for example. For length to Specifically hybridize to portions of an A2M gene guidance regarding probe design and denaturing gradient gel at polymorphic Sites. Typically Such lengths depend upon electrophoresis or denaturing HPLC methods See, e.g., the complexity of the Source organism genome. For humans Ausubel et al., 1989, Current Protocols in Molecular Biol such lengths generally are at least 14, 15, 16, 17, 18 or 19 Ogy, Green Publishing ASSociates and Wiley InterScience, nucleotides, and typically may be at least 20, 30, 40, 50, 60, N.Y., including updated materials, U.S. Pat. Nos. 5,795,976; 70, 80, 90, 100, 200, 300, 400 or 500 or more nucleotides. 5,585,236; 6,024,878; 6,210,885; Huber, et al., Chro In other embodiments, Such lengths of the probes and matographia 37:653 (1993); Huber, et al., Anal. Biochem. primers provided are not more than 14, 15, 16, 17, 18 or 19 US 2003/0162202 A1 Aug. 28, 2003

nucleotides, and further may be not more than 20, 30, 40, 50, 50-1000 bp, and fragments that are any number from 100 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 600 bp are common. It will be appreciated that amplification or 1000 nucleotides in length. primers can be of any Sequence that allows for Specific amplification of a region of a polymorphic and/or mutant 0148 For amplification of mRNAS, it is within the scope of the invention to reverse transcribe mRNA into. cDNA A2M gene and can, for example, include modifications Such followed by PCR (RT-PCR); or, to use a single enzyme for as restriction Sites to facilitate cloning. both steps as described in U.S. Pat. No. 5,322,770, the 0151. The PCR product can be subcloned and sequenced disclosure of which is incorporated herein by reference in its to ensure that the amplified Sequences represent the entirety. Another technique involves the use of Reverse Sequences of polymorphic and/or mutant A2M gene. The Transcriptase Asymmetric Gap Ligase Chain Reaction (RT PCR fragment can then be used to isolate a full length cDNA AGLCR), as described by Marshall R. L. et al. (PCR clone by a variety of methods. For example, the amplified Methods and Applications 4:80-84, 1994), the disclosure of fragment can be labeled and used to Screen a cDNA library, which is incorporated herein by reference in its entirety. In Such as a bacteriophage cDNA library. Alternatively, the each of these amplification procedures, primers on either labeled fragment can be used to isolate genomic clones via Side of the Sequence to be amplified are added to a Suitably the Screening of a genomic library. prepared nucleic acid Sample along with dNTPs and a 0152 Aspects of the invention also encompass (a) DNA thermostable polymerase, Such as Taq polymerase, Pfu poly vectors that contain any of the foregoing nucleic acid merase, or Vent polymerase. The nucleic acid in the Sample Sequences; (b) DNA expression vectors that contain any of is denatured and the primers are specifically hybridized to the foregoing nucleic acid Sequences operatively associated complementary nucleic acid Sequences in the Sample. The with a regulatory element that directs the expression of the hybridized primers are then extended. Thereafter, another coding sequences; and (c) genetically engineered host cells cycle of denaturation, hybridization, and extension is initi that contain any of the foregoing nucleic acid Sequences ated. The cycles are repeated multiple times to produce an operatively associated with a regulatory element that directs amplified fragment containing the nucleic acid Sequence the expression of the coding Sequences in the host cell. between the primer sites. PCR has further been described in These recombinant constructs are capable of replicating several patents including U.S. Pat. Nos. 4,683,195, 4,683, autonomously in a host cell. Alternatively, the recombinant 202 and 4,965,188, the disclosure of which is incorporated constructs can become integrated into the chromosomal herein by reference in their entirety. DNA of a host cell. 014.9 The primers are selected to be substantially 0153. As used herein, regulatory elements include, but complementary to a portion of the nucleic acid sequence of are not limited to, inducible and non-inducible promoters, SEO ID NO: 1 or SEO ID NOS: 2-8 that is downstream and enhancers, operators and other elements known to those upstream of the SNP and/or mutation to be detected such skilled in the art that drive and regulate expression. Such that the fragment produced by the amplification or extension regulatory elements include, but are not limited to, the reaction contains the SNP and/or mutant. Preferably, primers cytomegalovirus hCMV immediate early gene, the early or are designed to be downstream and upstream of at least one late promoters of SV40 adenovirus, the lac system, the trp of 6i, 12i. 1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, System, the TAC System, the TRC System, the major opera 28i and 30e, for example downstream or upstream of a tor and promoter regions of phage A, the control regions of nucleotide position corresponding to any of the following fal coat protein, the promoter for 3-phosphoglycerate kinase, positions: 1339, 1730, 2574 and 3912 of SEQID NOs: 3 and the promoters of acid phosphatase, and the promoters of the 5; 1338, 1729, 2573 and 3911 of SEQ ID NO: 7; and 38 and yeast C.-mating factors. 1376 of SEQ ID NO: 4, thereby allowing the sequences 0154) In addition, recombinant polymorphic and/or between the primers to be amplified or extended. Primers are mutant A2M-encoding nucleic acid Sequences can be engi desirably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, neered So as to modify processing or expression of the 29 and 30 nucleotides in length. The formation of stable protein. For example, and not by way of limitation, the hybrids depends on the melting temperature (Tm) of the polymorphic and/or mutant A2M genes can be combined DNA. The Tm depends on the length of the primer, the ionic with a promoter Sequence and/or ribosome binding site, or strength of the solution and the G+C content. The higher the a signal Sequence can be inserted upstream of A2M-encod G+C content of the primer, the higher is the melting tem ing Sequences to permit Secretion of the A2M protein and perature because G:C pairs are held by three H bonds thereby facilitate harvesting or bioavailability. Additionally, whereas A:T pairs have only two. The G+C content of the a given polymorphic and/or mutant A2M nucleic acid can be amplification primers of the present invention preferably mutated in vitro or in Vivo, to create and/or destroy trans ranges between 10 and 75%, more preferably between 35 lation, initiation, and/or termination Sequences, or to create and 60%, and most preferably between 40 and 55%. The variations in coding regions and/or form new restriction Sites appropriate length for primers under a particular set of assay or destroy preexisting ones, or to facilitate further in Vitro conditions can be empirically determined by one of skill in modification. Any technique for mutagenesis known in the the art. art can be used, including but not limited to, in Vitro 0150. The spacing of the primers relates to the length of Site-directed mutagenesis. (Hutchinson et al., J. Biol. Chem., the Segment to be amplified. In the context of the present 253:6551 (1978), herein incorporated by reference). invention, amplified Segments carrying nucleotides corre 0.155) Further, nucleic acids encoding other proteins or sponding to a nucleotide location of 6i, 12i.1, 12i.2, 12e, domains of other proteins can be joined to nucleic acids 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and/or 30e can encoding polymorphic and/or mutant A2M proteins or frag range in size from at least about 25 bp to 35 kb. Amplifi ments thereof So as to create a fusion protein. Nucleotides cation fragments that are any number from 25-1000 bp, encoding fusion proteins can include, but are not limited to, US 2003/0162202 A1 Aug. 28, 2003 a full length polymorphic and/or mutant A2M protein, a polymorphic A2M gene or portion(s) as described herein truncated polymorphic and/or mutant A2M protein or a thereof into a cell. The cell may be any transfectable cell. peptide fragment of a polymorphic and/or mutant A2M Such cells, and methods of introducing heterologous nucleic protein fused to an unrelated protein or peptide, Such as for acids into the cells, are known to those of skill in the art. example, a transmembrane Sequence, which anchors the A2M peptide fragment to the cell membrane, an Ig Fc 0160 The exogenous nucleic acid containing a polymor domain which increases the stability and half life of the phic A2M gene or portion(s) thereof that is used in the resulting fusion protein (e.g., A2M-Ig); or an enzyme, generation of recombinant cells provided herein contains, in fluorescent protein, luminescent protein which can be used particular embodiments, a Sequence of nucleotides that as a marker (e.g., an A2M-Green Fluorescent Protein ultimately provides for a product upon transcription of the (“A2M-GFP) fusion protein). The fusion proteins are use A2M gene or portion(s) thereof. The product can be, for ful as biotechnological tools or pharmaceuticals or both, as instance, RNA and/or a protein translated from a transcript. will be discussed infra. The section below describes several For example, the product can be A2M mRNA and/or an of the polypeptides of the invention and methods of making A2M protein or a reporter molecule Such as a reporter these molecules. protein. If the polymorphic A2M gene or portion(s) thereof being used in the generation of recombinant cells provided 0156 The disclosed nucleic acids and others that can be herein does not contain Sequences that provide for transcip obtained using methods described herein may be transferred tion of the A2M gene or portion(s) thereof, any appropriate into a host cell Such as bacteria, yeast, insect, mammalian, transcription control Sequences, Such as a promoter, from or plant cell for recombinant expression therein. Thus, any appropriate Source which will provide for transcliption of provided herein are recombinant cells containing an A2M the A2M gene or portion(s) thereof in the cell can be used. gene or a portion or portions thereof, Such as, for example, If the polymorphism(s) occur in a transcription control a transcriptional control region (including, for example, a region of an A2M gene, the polymorphic control region of promoter and 3' untranslated (UTR) sequences) and/or a the gene can be isolated or Synthesized and operatively coding Sequence of an A2M gene. The A2M gene or linked to nucleic acid encoding a reporter molecule, e.g., portion(s) thereof contains at least one polymorphic region galactosidase, a fluorescent protein Such as green fluorescent and is thus referred to as a polymorphic A2M gene or protein, or Some other readily detectable molecule, or portion(s) thereof. An "A2M gene or a portion or portions nucleic acid encoding an A2M protein. The resultant fusion thereof includes an A2M cDNA or portion(s) thereof gene can be used as the transgene that is introduced into a O157 Cells containing nucleic acids encoding polymor host cell for use in development of recombinant cells phic A2M proteins, and vectors and cells containing the therefrom. The patterns and levels of expression of the nucleic acids as provided herein permit production of the reporter or other molecule in the recombinant cells can be polymorphic proteins, as well as antibodies to the proteins. analyzed and compared to those in cells containing a fusion This provides a means to prepare Synthetic or recombinant gene in which a wild-type or reference A2M transcription polymorphic proteins and fragments thereof that are Sub control region Sequence is operatively linked to nucleic acid Stantially free of contamination from other proteins, the encoding a reporter or other molecule. presence of which can interfere with analysis of the poly morphic proteins. In addition, the polymorphic proteins may 0.161 Polymorphic and/or mutant A2 M Polypeptides be expressed in combination with Selected other proteins 0162) Isolated or purified polymorphic and/or mutant that the protein of interest may associate with in cells. The A2M polypeptides and fragments of these molecules at least ability to Selectively express the polymorphic proteins alone 3 amino acids in length, which contain at least one of the or in combination with other Selected proteins makes it mutations identified in Table 1, are embodiments of the possible to observe the functioning of the recombinant invention. In Some contexts, the term “polymorphic and/or polymorphic proteins within the environment of a cell. mutant A2M polypeptides' refers not only to the full-length 0158 Recombinant cells provided herein may be used for polymorphic and/or mutant A2M proteins but also to frag numerous purposes. For example, the cells may be used in ments of these molecules at least 3 amino acids in length but testing polymorphic A2M genes or portion(s) thereof for containing at least one of the mutations identified in Table 1. characterization of phenotypic outcomes correlated with the 0163 The nucleic acids encoding the A2M polypeptides particular polymorphisms. The cells may also be used in the or fragments thereof, described in the previous Section, can production of recombinant A2M protein. Such protein may be manipulated using conventional techniques in molecular be used, for example, in assays for molecules that bind to, biology So as to create recombinant constructs that express and in particular affect the activity of, A2M. The proteins polymorphic and/or mutant A2M polypeptides. The poly may also be used in the production of antibodies Specific for morphic and/or mutant A2M polypeptides or fragments the protein. Additionally, the recombinant A2M protein may thereof of the invention, include but are not limited to, those be used as a Source of a protease inhibitor. Recombinant containing as a primary amino acid Sequence all or part of cells containing polymorphic A2M genes or portion(s) the amino acid sequence encoded by SEQ ID NO: 1, SEQ thereof may also be used in methods of identifying agents ID NO: 2 (encoding SEQ ID NO: 9) or SEQ ID NOs: 3-8 that modulate A2M gene and protein expression and/or (encoding SEQ ID NOS: 10-15), as modified by a SNP activity or that modulate a biological event characteristic of and/or mutation described in Table 1 (for example, 14e, 20e a disease or disorder involving altered A2M gene and/or and 30e), and fragments of these proteins at least three protein expression or function which may be candidate amino acids in length but including at least one of the treatments for a disease or disorder. mutations listed in Table 1, including altered Sequences in 0159. Also provided herein are methods of producing which functionally equivalent amino acid residues are Sub recombinant cells by introducing nucleic acid containing a Stituted for residues within the Sequence resulting in a Silent US 2003/0162202 A1 Aug. 28, 2003 change. The A2M peptide fragments of the invention can be, et al., J. Am. Chem. Soc. 85:2149 (1964), Houghten et al., for example, any number of between 4-20, 20-50, 50-100, Proc. Notl. Acad. Sci. USA, 82:51:32 (1985), Stewart and 100-300, 300-600, 600-1000, 1000-1450 consecutive amino Young (Solid phase peptide Synthesis, Pierce Chem Co., acids of SEQ. ID NOS. 9-15 (e.g., less than or equal to 4, 5, Rockford, Ill. (1984), and Creighton, 1983, Proteins: Struc 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, tures and Molecular Principles, W. H. Freeman & Co., N.Y., 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37,38, all of which are hereby incorporated by reference in their 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, entireties. Such polypeptides can be Synthesized with or 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, without a methionine on the amino terminus. Chemically 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, Synthesized polypeptides can be oxidized using methods Set 88, 89,90,91, 92,93, 94, 95, 96, 97,98, 99, 100, 125, 150, forth in these references to form disulfide bridges. 175, 200, 250, 300,350, 400, 500, 600, 700, 800, 900, 1000, 0167 While the polymorphic and/or mutant A2M and 1450 amino acids in length of SEQ ID NOs: 9-15). polypeptides and fragments thereof can be chemically Syn Polypeptides of the present invention also contemplate the thesized, it can be more effective to produce these molecules polypeptides of SEQ ID NOs: 9-15 or fragments thereof by recombinant DNA technology using techniques well encoded by the nucleic acids of SEQ ID NOS: 2-8 having known in the art. Such methods can be used to construct one or more previously described SNPs and/or mutations for expression vectors containing the polymorphic and/or A2M which affect the A2M polypeptide (e.g. some SNPs mutant A2M nucleotide Sequences, for example, and appro and/or mutations provided in Table 2) in addition to at least priate transcriptional and translational control signals. These one SNP and/or mutation Selected from the group consisting methods include, for example, in vitro recombinant DNA of 14e, 20e and 30e. techniques, Synthetic techniques, and in Vivo genetic recom 01.64 Embodiments also include isolated or purified bination. Alternatively, RNA capable of encoding an poly polymorphic and/or mutant A2M polypeptides that have one morphic and/or mutant A2M polypeptide Sequences and or more amino acid residues within the polypeptide that are fragments thereof can be chemically Synthesized using, for Substituted by another amino acid of a similar polarity that example, Synthesizers. See, for example, the techniques acts as a functional equivalent, resulting in a Silent alter described in Oligonucleotide Synthesis, 1984, Gait, M. J. ation. Substitutes for an amino acid within the Sequence can ed., IRL Press, Oxford, which is incorporated by reference be selected from other members of the class to which the herein in its entirety. amino acid belongs. For example, the non-polar (hydropho bic) amino acids include alanine, leucine, isoleucine, Valine, 0.168. In several embodiments, polymorphic and/or proline, phenylalanine, tryptophan, and methionine. The mutant A2M nucleic acids and polypeptides are expressed in polar neutral amino acids include glycine, Serine, threonine, a cell line. For example, Some cells are made to express the cysteine, tyrosine, asparagine and glutamine. The positively a polymorphic and/or mutant A2M polypeptide having the sequence encoded by SEQID NOS: 2-8 or such nucleic acids charged (basic) amino acids include arginine, lysine, and having one or more previously described SNPs and/or histidine. The negatively charged (acidic) amino acids mutations for A2M which affect the A2M polypeptide in include aspartic acid and glutamic acid. The aromatic amino addition to at least one SNP and/or mutation selected from acids include phenylalanine, tryptophan, and tyrosine. the group consisting of 14e, 20e and 30e. A variety of 0.165. The Sequences, constructs, vectors, clones, and host-expression vector Systems can be utilized to express the other materials comprising the embodiments of the present polymorphic and/or mutant A2M nucleic acids and polypep invention can be in enriched or isolated form. AS used tides of the invention. The expression Systems that can be herein, "enriched” means that the concentration of the used include, but are not limited to, microorganisms. Such as material is at least about 2, 5, 10, 100, or 1000 times its bacteria (e.g., E. coli or B. Subtilis) transformed with recom natural concentration (for example), advantageously 0.01%, binant bacteriophage DNA, plasmid DNA or cosmid DNA by weight, preferably at least about 0.1% by weight. expression vectors containing polymorphic and/or mutant Enriched preparations from about 0.5%, 1%, 5%, 10%, and A2M nucleotide sequences; yeast (e.g., Saccharomyces, 20% by weight are also contemplated. The term “isolated” Pichia) transformed with recombinant yeast expression vec requires that the material be removed from its original tors containing the polymorphic and/or mutant A2M nucle environment (e.g., the natural environment if it is naturally otide Sequences, insect cell Systems infected with recombi occurring). For example, a naturally-occurring polynucle nant virus expression vectors (e.g., Baculovirus) containing otide present in a living animal is not isolated, but the same the polymorphic and/or mutant A2M Sequences, plant cell polynucleotide, Separated from Some or all of the coexisting Systems infected with recombinant virus expression vectors materials in the natural System, is isolated. It is also advan (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic tageous that the Sequences be in purified form. The term virus, TMV) or transformed with recombinant plasmid “purified” does not require absolute purity; rather, it is expression vectors (e.g., Ti plasmid) containing polymor intended as a relative definition. Isolated proteins have been phic and/or mutant A2M nucleotide Sequences, or mamma conventionally purified to electrophoretic homogeneity by lian cell systems (e.g., COS, CHO, BHK, 293, 3T3) har Coomassie Staining, for example. Purification of Starting boring recombinant expression constructs containing material or natural material to at least one order of magni promoters derived from the genome of mammalian cells tude, preferably two or three orders, and more preferably (e.g., metallothionein promoter) or from mammalian viruses four or five orders of magnitude is expressly contemplated. (e.g., the adenovirus late promoter; the vaccinia virus 7.5K 0166 The polymorphic and/or mutant A2M polypeptides promoter). described herein can be prepared by chemical Synthesis 0169. In bacterial systems, a number of expression vec methods (such as Solid phase peptide Synthesis) using tech tors can be advantageously Selected depending upon the use niques known in the art such as those set forth by Merrifield intended for the polymorphic and/or mutant A2M gene US 2003/0162202 A1 Aug. 28, 2003 product being expressed. For example, when a large quantity 0172 However, in cases where only a portion of the of Such a protein is to be produced, for the generation of polymorphic and/or mutant A2M coding Sequence is pharmaceutical compositions of polymorphic and/or mutant inserted, exogenous translational control Signals, including, A2M polypeptide or for raising antibodies to the polymor perhaps, the ATG initiation codon, may be provided. Fur phic and/or mutant A2M polypeptide, for example, vectors thermore, the initiation codon is desirably in phase with the which direct the expression of high levels of fusion protein reading frame of the desired coding Sequence to ensure products that are readily purified can be desirable. Such translation of the entire insert. These exogenous transla vectors include, but are not limited, to the E. coli expression tional control Signals and initiation codons can be of a vector puR278 (Ruther et al., EMBO.J., 2:1791 (1983), in variety of origins, both natural and Synthetic. The efficiency which the polymorphic and/or mutant A2M nucleic acids of expression can be enhanced by the inclusion of appro can be ligated individually into the vector in frame with the priate transcription enhancer elements, transcription termi lac7, coding region So that a fusion protein is produced; plN nators, etc. (See Bittner et al., Methods in Enzymol, 153:516 vectors (Inouye & Inouye, Nucleic Acids Res., 13:3101 544 (1987)). 3109 (1985); Van Heeke & Schuster, J. Biol. Chem., 0.173) In addition, a host cell strain can be chosen which 264:5503–5509 (1989)); and the like, herein expressly incor modulates the expression of the inserted Sequences, or porated by reference. pCEX vectors can also be used to modifies and processes the gene product in the Specific express foreign polypeptides as fusion proteins with glu fashion desired. Such modifications (e.g., glycosylation) and tathione S-transferase (GST). In general, Such fusion pro processing (e.g., cleavage) of protein products are important teins are soluble and can be purified from lysed cells by for the function of the protein. Different host cells have adsorption to glutathione-agarose beads followed by elution characteristic and Specific mechanisms for the post-transla in the presence of free glutathione. The PGEX vectors are tional processing and modification of proteins and gene designed to include thrombin or factor Xa protease cleavage products. Appropriate cell lines or host Systems can be Sites So that the cloned target gene product can be released chosen to ensure the correct modification and processing of from the GST moiety. the foreign protein expressed. To this end, eukaryotic host 0170 In an insect system, Autographa californica cells that possess the cellular machinery for proper proceSS nuclear polyhedrosis virus (ACNPV) is used as a vector to ing of the primary transcript, glycosylation, and phospho express foreign genes. The virus grows in Spodoptera fru rylation of the gene product can be used. Such mammalian giperda cells. The polymorphic and/or mutant A2M nucleic host cells include, but are not limited to, CHO, VERO, BHK, acid Sequences can be cloned individually into non-essential HeLa, COS, MDCK, 293, 3T3, and W138. regions (for example the polyhedrin gene) of the virus and 0.174 For long-term, high-yield production of recombi placed under control of an AcNPV promoter (for example nant proteins, Stable expression is preferred. For example, the polyhedrin promoter). Successful insertion of polymor cell lines that Stably express the polymorphic and/or mutant phic and/or mutant A2M nucleic acid Sequence will result in A2M Sequences described herein can be engineered. Rather inactivation of the polyhedrin gene and production of non than using expression vectors that contain Viral origins of occluded recombinant virus, (i.e., virus lacking the proteina replication, host cells can be transformed with DNA con ceous coat coded for by the polyhedrin gene). These recom trolled by appropriate expression control elements (e.g., binant viruses are then used to infect Spodoptera frugiperda promoter, enhancer Sequences, transcription terminators, cells in which the inserted gene is expressed. (E.g., See polyadenylation sites, etc.), and a Selectable marker. Fol Smith et al., J. Virol. 46: 584 (1983); and Smith, U.S. Pat. lowing the introduction of the foreign DNA, engineered No. 4,215,051, all of which are hereby expressly incorpo cells are allowed to grow for 1-2 days in an enriched media, rated by reference in their entireties). and then are Switched to a Selective media. The Selectable marker in the recombinant plasmid conferS resistance to the 0171 In mammalian host cells, a number of viral-based Selection and allows cells to stably integrate the plasmid into expression Systems can be utilized. In cases where an their chromosomes and grow to form foci which in turn are adenovirus is used as an expression vector, the polymorphic cloned and expanded into cell lines. This method is advan and/or mutant A2M nucleotide Sequence of interest can be tageously used to engineer cell lines which express the ligated to an adenovirus transcription/translation control polymorphic and/or mutant A2M gene product. Such engi complex, e.g., the late promoter and tripartite leader neered cell lines are particularly useful in Screening and Sequence. This chimeric gene can then be inserted in the evaluation of compounds that affect the endogenous activity adenovirus genome by in vitro or in Vivo recombination. Insertion in a non-essential region of the viral genome (e.g., of the polymorphic and/or mutant A2M gene product. region E1 or E3) will result in a recombinant virus that is 0.175. A number of selection systems can be used, includ viable and capable of expressing the polymorphic and/or ing but not limited to the herpes simplex virus thymidine mutant A2M gene product in infected hosts. (E.g., See kinase (Wigler, et al., Cell 11:223 (1977), hypoxanthine Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659 guanine phosphoribosyltransferase (Szybalska & Szybalski, (1984), herein expressly incorporated by reference in its Proc. Natl. Acad. Sci. USA 48:2026 (1962), and adenine entirety). Specific initiation signals can also be required for phosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980) efficient translation of inserted nucleotide Sequences. These genes can be employed in th, hgprt or aprt cells, respec Signals include the ATG initiation codon and adjacent tively. Also, antimetabolite resistance can be used as the Sequences. In cases where an entire polymorphic and/or basis of selection for the following genes: dhfr, which mutant A2M gene or cDNA, including its own initiation confers resistance to methotrexate (Wigler, et al., Proc. Natl. codon and adjacent Sequences, is inserted into the appropri Acad. Sci. USA 77:3567 (1980); O'Hare, et al., Proc. Natl. ate expression vector, no additional translational control Acad. Sci. USA 78:1527 (1981); gpt, which confers resis Signals are needed. tance to mycophenolic acid (Mulligan & Berg, Proc. Natl. US 2003/0162202 A1 Aug. 28, 2003 17

Acad. Sci. USA 78:2072 (1981); neo, which confers resis head-to-tail tandems. The transgene can also be Selectively tance to the aminoglycoside G-418 (Colberre-Garapin, et al., introduced into and activated in a particular cell type by J. Mol. Biol. 150:1 (1981); and hygro, which confers resis following, for example, the teaching of Lasko et al. (Lasko, tance to hygromycin (Santerre, et al., Gene 30:147 (1984)). M. et al., Proc. Natl. Acad. Sci. USA 89: 6232-6236 (1992), herein expressly incorporated by reference in its entirety). 0176 Alternatively, any fusion protein can be readily The regulatory Sequences required for Such a cell-type purified by utilizing an antibody Specific for the fusion Specific activation will depend upon the particular cell type protein being expressed. For example, a System described by Janknecht et al. allows for the ready purification of non of interest, and will be apparent to those of skill in the art. denatured fusion proteins expressed in human cell lines. 0180. When it is desired that the polymorphic and/or (Janknecht, et al., Proc. Natl. Acad. Sci. USA 88: 8972-8976 mutant A2M gene transgene be integrated into the chromo (1991)). In this system, the gene of interest is subcloned into Somal Site of the endogenous A2M gene, gene targeting is a Vaccinia recombination plasmid Such that the gene's open preferred. Briefly, when Such a technique is to be utilized, reading frame is translationally fused to an amino-terminal vectors containing Some nucleotide Sequences homologous tag consisting of Six histidine residues. Extracts from cells to the endogenous A2M gene are designed for the purpose infected with recombinant vaccinia virus are loaded onto of integrating, via homologous recombination with chromo Ni" nitriloacetic acid-agarose columns and histidine-tagged Somal Sequences, into and disrupting the function of the proteins are Selectively eluted with imidazole-containing nucleotide Sequence of the endogenous A2M gene. The buffers. transgene can also be selectively introduced into a particular cell type, thus inactivating the endogenous A2M gene in 0177. The polymorphic and/or mutant A2M nucleic acids only that cell type, by following, for example, the teaching and polypeptides can also be expressed in plants, insects, of Gu et al. (Gu, et al., Science 265: 103-106 (1994), herein and animals So as to create a transgenic organism. Plants and expressly incorporated by reference in its entirety). The insects of almost any Species can be made to express the regulatory Sequences required for Such a cell-type specific polymorphic and/or mutant A2M nucleic acids and/or inactivation will depend upon the particular cell type of polypeptides, described herein. Desirable transgenic plant interest, and will be apparent to those of skill in the art. Systems having one or more of these Sequences include Arabadopsis, Maize, and Chlamydomonas. Desirable insect 0181. Once transgenic animals have been generated, the Systems having one or more of the polymorphic and/or expression of the recombinant A2M gene can be assayed mutant A2M nucleic acids and/or polypeptides include, for utilizing Standard techniques. Initial Screening can be example, D. melanogaster and C. elegans. Animals of any accomplished by Southern blot analysis or PCR techniques Species, including, but not limited to, amphibians, reptiles, to analyze animal tissues to assay whether integration of the birds, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, transgene has taken place. The level of mRNA expression of goats, dogs, cats, and non-human primates, e.g., baboons, the transgene in the tissueS of the transgenic animals can also monkeys, and chimpanzees can be used to generate poly be assessed using techniques which include, but are not morphic and/or mutant A2M containing transgenic animals. limited to, Northern blot analysis of tissue samples obtained Transgenic organisms of the invention desirably exhibit from the animal, in situ hybridization analysis, and RT-PCR. germline transfer of polymorphic and/or mutant A2M The section below describes antibodies of the invention and nucleic acids and polypeptides. Still other transgenic organ methods of making these molecules. isms of the invention exhibit complete knockouts or point 0182 Cells and transgenic animals containing nucleic mutations of one or more of the A2M genes described acids that include variant A2M gene or cDNA sequences as herein. described herein have numerous uses. For example, Such 0.178 Any technique known in the art is preferably used cells and animals can be used in methods of assessing to introduce the polymorphic and/or mutant A2M transgene candidate agents that modulate A2M activity and/or expres into animals to produce the founder lines of transgenic Sion, and candidate therapeutic agents for the treatment of animals or to knock out or replace existing A2M genes. Such diseases, Such as neurodegenerative diseases, e.g., AD. Such techniques include, but are not limited to pronuclear micro cells and animals can also be used to assess the effects of a injection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. particular variant of a polymorphism. For example, trans No. 4,873,191); retrovirus mediated gene transfer into germ genic animals in which nucleic acid containing a particular lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA variant of a polymorphism has been introduced may be 82:6148-6152 (1985); gene targeting in embryonic stem analyzed for a particular phenotype. The transgenic animal cells (Thompson et al., Cell 56.313-321 (1989); electropo may be one in which the wild-type gene or predominant ration of embryos (Lo, Mol Cell. Biol. 3:1803-1814 (1983); allele may have been knocked out. RNA and/or protein is and Sperm-mediated gene transfer (Lavitrano et al., Cell compared in the transgenic animal harboring the allelic 57:717-723 (1989); etc. For a review of such techniques, see variant with an animal harboring a different allele, e.g., a Gordon, Transgenic Animals, Intl. Rev. Cytol. 115:171-229 predominant or reference allele. For example, the variant (1989), which is incorporated by reference herein in its may result in alterations of RNA levels or RNA stability or entirety. in increased or decreased Synthesis of the associated protein and/or aberrant tissue distribution or intracellular localiza 0179 Aspects of the invention also concern transgenic tion of the associated protein, altered phosphorylation, gly animals that carry a polymorphic and/or mutant A2M trans cosylation and/or altered activity of the protein. Further gene in all their cells, as well as animals that carry the more, various molecular, cellular and organismal transgene in Some, but not all their cells, i.e., mosaic manifestations of a disease can be monitored. For example, animals. The transgene can be integrated as a Single trans to assess a polymorphism for an effect that may be related gene or in concatamers, e.g., head-to-head tandems or to Alzheimer's disease, certain characteristic features of the US 2003/0162202 A1 Aug. 28, 2003

disease, Such as APP gene products, particularly A protein, device, to the level of a few micrograms/ml. Monoclonal or neurite plaques, deficits of memory and learning and neu polyclonal antibody to the polypeptide of interest can then rodegeneration of Specific Systems of cells may be evaluated be prepared as follows: in a transgenic animal containing nucleic acid containing the 0188 Monoclonal antibodies to polymorphic and/or polymorphism. Such analysis could also be performed in mutant A2M polypeptides can be prepared using any tech cultured cells into which the variant allele gene or portion nique that provides for the production of antibody molecules thereof is introduced. If the host cell contains a different by continuous cell lines in culture. These include, but are not allele of the same gene, it is possible to replace the endog limited to, the hybridoma technique originally described by enous gene with the variant gene in the cell, if desired. These Koehler and Milstein (Nature 256:495-497 (1975), the effects can be determined according to methods known in human B-cell hybridoma technique (Kosbor et al. Immunol the art and as described below. Particular variants of a Today 4:72 (1983); Cote et al Proc Natl AcadSci 80:2026 polymorphism can be assayed individually or in combina 2030 (1983), and the EBV-hybridoma technique Cole et al. tion. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss 0183 Antibodies Specific for Polymorphic and/or mutant Inc, New York N.Y., pp 77-96 (1985), all of which are A2M Polypeptides hereby incorporated by reference in their entireties. In addition, techniques developed for the production of “chi 0184. Following synthesis or expression and isolation or meric antibodies', the Splicing of mouse antibody genes to purification of the A2M protein or a portion thereof, the human antibody genes to obtain a molecule with appropriate isolated or purified protein can be used to generate antibod antigen Specificity and biological activity can be used. ies and tools for identifying agents that interact with poly (Morrison et al. Proc Natl AcadSci 81:6851-6855 (1984); morphic and/or mutant A2M polypeptides. Depending on Neuberger et al. Nature 312:604-608(1984); Takeda et al. the context, the term “antibodies' can encompass poly Nature 314:452-454(1985), all of which are hereby incor clonal, monoclonal, chimeric, Single chain, Fab fragments porated by reference in their entireties. Alternatively, tech and fragments produced by a Fab expression library. Anti niques described for the production of Single chain antibod bodies that recognize polymorphic and/or mutant A2M ies (U.S. Pat. No. 4,946,778) can be adapted to produce polypeptides have many uses including, but not limited to, Specific Single chain antibodies, hereby incorporated by biotechnological applications, therapeutic/prophylactic reference. Antibodies can also be produced by inducing in applications, and diagnostic applications. Vivo production in the lymphocyte population or by Screen ing recombinant immunoglobulin libraries or panels of 0185. For the production of antibodies, various hosts highly specific binding reagents as disclosed in Orlandi et including goats, rabbits, rats, mice, etc. can be immunized al., Proc Natl AcadSci 86: 3833-3837 (1989), and Winter G. by injection with polymorphic and/or mutant A2M polypep and Milstein C; Nature 349:293–299 (1991), all of which are tides, in particular, any portion, fragment or oligopeptide hereby incorporated by reference in their entireties. that retains immunogenic properties. Depending on the host Species, various adjuvants can be used to increase immuno 0189 Antibody fragments that contain specific binding logical response. Such adjuvants include, but are not limited Sites for polymorphic and/or mutant A2M polypeptides can to, Freunds, mineral gels Such as aluminum hydroxide, and also be generated. For example, Such fragments include, but Surface active Substances Such as lySolecithin, pluronic are not limited to, the F(ab')2 fragments that can be produced polyols, polyanions, peptides, oil emulsions, keyhole limpet by pepsin digestion of the antibody molecule and the Fab hemocyanin, and dinitrophenol. BCG (Bacillus Calmette fragments that can be generated by reducing the disulfide Guerin) and Corynebacterium parvum are also potentially bridges of the F(ab')2 fragments. Alternatively, Fab expres useful adjuvants. Sion libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired 0186 Peptides used to induce specific antibodies can specificity. (Huse W. D. et al. Science 256: 1275-1281 have an amino acid Sequence consisting of at least three (1989)). amino acids, and preferably at least 10 to 15 amino acids. Preferably, short Stretches of amino acids encoding frag 0190. By one approach, monoclonal antibodies to poly ments of polymorphic and/or mutant A2M polypeptides morphic and/or mutant A2M polypeptides are made as containing one or more of the mutations described in Table follows. Briefly, a mouse is repetitively inoculated with a 1 are fused with those of another protein Such as keyhole few micrograms of the Selected protein or peptides derived limpet hemocyanin Such that an antibody is produced therefrom over a period of a few weeks. The mouse is then against the chimeric molecule. While antibodies capable of Sacrificed, and the antibody producing cells of the Spleen Specifically recognizing polymorphic and/or mutant A2M isolated. The Spleen cells are fused in the presence of polypeptides can be generated by injecting Synthetic 3-mer, polyethylene glycol with mouse myeloma cells, and the 10-mer, and 15-mer peptides that correspond to a protein exceSS unfused cells destroyed by growth of the System on Sequence of polymorphic and/or mutant A2M polypeptides Selective media comprising aminopterin (HAT media). The into mice, a more diverse Set of antibodies can be generated Successfully fused cells are diluted and aliquots of the by using recombinant polymorphic and/or mutant A2M dilution placed in Wells of a microtiter plate where growth polypeptides. of the culture is continued. Antibody-producing clones are identified by detection of antibody in the Supernatant fluid of 0187 To generate antibodies to polymorphic and/or the Wells by immunoassay procedures, Such as ELISA, as mutant A2M polypeptides, Substantially pure polypeptides originally described by Engvall, E., Meth. Enzymol. 70:419 are isolated from a transfected or transformed cell. The (1980), and derivative methods thereof. Selected positive concentration of the polypeptide in the final preparation is clones can be expanded and their monoclonal antibody adjusted, for example, by concentration on an Amicon filter product harvested for use. Detailed procedures for mono US 2003/0162202 A1 Aug. 28, 2003 19 clonal antibody production are described in Davis, L. et al. identification of individuals at risk of acquiring a neuropa Basic Methods in Molecular Biology Elsevier, New York. thy, Such as AD, are contemplated. The diagnostic kits can Section 21-2, herein expressly incorporated by reference in include a nucleic acid probe or an antibody or combinations its entirety. thereof, which Specifically detect a polymorphic and/or 0191 Polyclonal antiserum containing antibodies to het mutant A2M polypeptide or nucleic acid or a nucleic acid erogenous epitopes of a single protein can be prepared by probe or an antibody or combinations thereof, which can be immunizing Suitable animals with the expressed protein or used to determine the level of RNA or protein expression of peptides derived therefrom described above, which can be one or more polymorphic and/or mutant A2M nucleic acids unmodified or modified to enhance immunogenicity. Effec or polypeptides. The detection component of these kits will tive polyclonal antibody production is affected by many typically be Supplied in combination with one or more of the factorS related both to the antigen and the host species. For following reagents. A Support capable of absorbing or oth example, Small molecules tend to be leSS immunogenic than erwise binding DNA, RNA, or protein will often be Sup others and can require the use of carriers and adjuvant. Also, plied. Available Supports include membranes of nitrocellu host animals vary in response to Site of inoculations and lose, nylon or derivatized nylon that can be characterized by dose, with both inadequate or excessive doses of antigen bearing an array of positively charged Substituents. One or resulting in low titer antisera. Small doses (ng level) of more restriction enzymes, control reagents, buffers, ampli antigen administered at multiple intradermal Sites appears to fication enzymes, and non-human polynucleotides like calf be most reliable. An effective immunization protocol for thymus or Salmon-Sperm DNA can be Supplied in these kits. rabbits can be found in Vaitukaitis, J. et al. J. Clin. Endo 0.196 Useful nucleic acid-based diagnostic techniques crinol. Metab. 33:988-991 (1971),herein expressly incorpo include, but are not limited to, direct DNA sequencing, rated by reference in its entirety. Southern Blot analysis, Single-Stranded confirmation analy 0.192 Booster injections can be given at regular intervals, sis (SSCA), RNAse protection assay, dot blot analysis, and antiserum harvested when antibody titer thereof, as nucleic acid amplification, and combinations of these determined Semi-quantitatively, for example, by double approaches. The Starting point for these analysis is isolated immunodiffusion in agar against known concentrations of or purified nucleic acid from a biological Sample. If the the antigen, begins to fall. See, for example, Ouchterlony, 0. diagnostic assay is designed to determine the presence of a et al., Chap. 19 in: Handbook of Experimental Immunology polymorphic and/or mutant A2M nucleic acid, any Source of D. Wier (ed) Blackwell (1973). Plateau concentration of DNA including, but not limited to hair, cheek cells and blood antibody is usually in the range of 0.1 to 0.2 mg/ml of Serum can be used as a biological Sample. The nucleic acid is (about 12 uM). Affinity of the antisera for the antigen is extracted from the sample and can be amplified by a DNA determined by preparing competitive binding curves, as amplification technique Such as the Polymerase Chain Reac described, for example, by Fisher, D., Chap. 42 in: Manual tion (PCR) using primers that correspond to regions flanking of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) DNA recognized as a SNP and/or mutation in the A2M gene Amer. Soc. For Microbiol., Washington, D.C. (1980). Anti (See Table 1). body preparations prepared according to either protocol are 0197) Once a sufficient amount of DNA is obtained from useful in quantitative immunoassays that determine concen an individual to be tested, Several methods can be used to trations of antigen-bearing Substances in biological Samples, detect a polymorphism and/or mutation. Direct DNA they are also used Semi-quantitatively or qualitatively (e.g., Sequencing, either manual Sequencing or automated fluo in diagnostic embodiments that identify the presence of rescent Sequencing can detect Such Sequence Variations. polymorphic and/or mutant A2M polypeptides in biological Another approach is the Single-Stranded confirmation poly Samples). In the discussion that follows, several methods of morphism assay (SSCA) (Orita et al., Proc. Natl. Acad. Sci. molecular modeling and rational drug design are described. USA 86:2776-2770 (1989), herein incorporated by refer These techniques can be applied to identify molecules that ence). This method, however, does not detect all sequence interact with polymorphic and/or mutant A2M polypeptides changes, especially if the DNA fragment size is greater than and, thereby modulate their function. 200 base pairs, but can be optimized to detect most DNA 0193 Diagnostic Embodiments Sequence variation. 0198 The reduced detection sensitivity is a disadvantage, 0194 Generally, the diagnostics of the invention can be but the increased throughput possible with SSCA makes it classified according to whether the embodiment is a nucleic an attractive, variable alternative to direct Sequencing for acid or protein-based assay. Some diagnostic assays detect mutation detection. The fragments that have shifted mobility mutations or polymorphisms in A2M nucleic acids or A2M on SSCA gels are then Sequenced to determine the exact proteins, which contribute to or place individuals at risk of nature of the DNA sequence variation. Other approaches acquiring neuropathies, Such as AD. Other diagnostic assays based on the detection of mismatches between the two identify and distinguish defects in A2M activities by detect complimentary DNA Strands include clamped denaturing ing a level of polymorphic and/or mutant A2M RNA or A2M gel electrophoresis (CDGE) (Sheffield et al., Am. J. Hum. protein in a tested subject that resembles the level of Genet. 49:699-706 (1991)), heteroduplex analysis (HA) polymorphic and/or mutant A2M RNA or A2M protein in a (White et al., Genomics 12:301-306 (1992)), and chemical Subject Suffering from a neuropathy (e.g., AD) or by detect mismatch cleavage (CMC) (Grompe et al., Proc. Natl. Acad. ing a level of RNA or protein in a tested subject that is Sci. USA 86:5855-5892 (1989), all of which, including the different than a Subject not Suffering from a disease. references contained therein, are hereby expressly incorpo 0.195 Additionally, the manufacture of kits that incorpo rated by reference in their entireties). A review of currently rate the reagents and methods described in the following available methods of detecting DNA sequence variation can embodiments So as to allow for the rapid detection and be found in Grompe, Nature Genetics 5:111-117 (1993). US 2003/0162202 A1 Aug. 28, 2003 20

0199. Seven well-known nucleic acid-based methods for techniques are leSS Sensitive than Sequencing, they are easily confirming the presence of a polymorphism are described performed on a large number of biological Samples and are below. Provided for exemplary purposes only and not amenable to array technology. intended to limit any aspect of the invention, these methods 0208. In some embodiments, nucleic acid probes that include: differentiate polynucleotides encoding wild type A2M from 0200 (1) single-stranded confirmation analysis polymorphic and/or mutant A2M are attached to a Support in an ordered array, wherein the nucleic acid probes are (SSCA) (Orita et al.); attached to distinct regions of the Support that do not overlap 0201 (2) denaturing gradient gel electrophoresis with each other. Preferably, such an ordered array is (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699 designed to be “addressable” where the distinct locations of 2705 (1990) and Sheffield et al., Proc. Natl. Acad. the probe are recorded and can be accessed as part of an Sci. USA 86:232-236 (1989)), both references herein assay procedure. These probes are joined to a Support in incorporated by reference; different known locations. The knowledge of the precise location of each nucleic acid probe makes these "address 0202 (3) RNAse protection assays (Finkelstein et able' arrays particularly useful in binding assays. The al., Genomics 7:167-172 (1990) and Kinszler et al., nucleic acids from a preparation of Several biological Science 251: 1366-1370 (1991)) both references Samples are then labeled by conventional approaches (e.g., herein incorporated by reference; radioactivity or fluorescence) and the labeled Samples are 0203 (4) the use of proteins which recognize nucle applied to the array under conditions that permit hybridiza otide mismatches, Such as the E. ColimutS protein tion. (Modrich, Ann. Rev. Genet. 25:229-253 (1991), 0209 If a nucleic acid in the samples hybridizes to a herein incorporated by reference; probe on the array, then a signal will be detected at a position on the Support that corresponds to the location of the hybrid. 0204 (5) allele-specific PCR (Rano and Kidd, Nucl. Since the identity of each labeled sample is known and the Acids Res. 17:8392 (1989), herein incorporated by region of the Support on which the labeled Sample was reference), which involves the use of primers that applied is known, an identification of the presence of the hybridize at their 3' ends to a polymorphism and, if polymorphic variant can be rapidly determined. These the polymorphism is not present, an amplification approaches are easily automated using technology known to product is not observed; and those of Skill in the art of high throughput diagnostic or 0205 (6) Amplification Refractory Mutation Sys detection analysis. tem (ARMS), as disclosed in European Patent Appli 0210 Additionally, an opposite approach to that pre cation Publication No. 0332435 and in Newton et al., Sented above can be employed. Nucleic acids present in Nucl. Acids Res. 17:2503-2516 (1989), both refer biological Samples can be disposed on a Support So as to ences herein incorporated by reference, and create an addressable array. Preferably, the Samples are disposed on the Support at known positions that do not 0206 (7) temporal temperature gradient gel electro overlap. The presence of nucleic acids having a desired phoresis (TTGE), as described by Bio-Rad in U.S./ polymorphism in each Sample is determined by applying E.G. Bulletin 2103, herein incorporated by refer labeled nucleic acid probes that complement nucleic acids CCC. that encode the polymorphism and detecting the presence of 0207. In SSCA, DGGE, TTGE, and RNAse protection a Signal at locations on the array that correspond to the assay, a new electrophoretic band appears when the poly positions at which the biological Samples were disposed. morphism is present. SSCA and TTGE detect a band that Because the identity of the biological Sample and its position migrates differentially because the Sequence change causes on the array is known, the identification of the polymorphic a difference in Single-Strand, intramolecular base pairing, variant can be rapidly determined. These approaches are also which is detectable electrophoretically. RNASe protection easily automated using technology known to those of skill in involves cleavage of the mutant polynucleotide into two or the art of high throughput diagnostic analysis. more smaller fragments. DGGE detects differences in 0211) Any addressable array technology known in the art migration rates of Sequences using a denaturing gradient gel. can be employed with this aspect of the invention. One In an allele-specific oligonucleotide assay (ASOS) (Conner particular embodiment of polynucleotide arrays is known as et al., Proc. Natl. Acad. Sci. USA 80:278-282 (1983)), an GenechipsTM, and has been generally described in U.S. Pat. oligonucleotide is designed that detects a Specific Sequence, No. 5,143,854; PCT publications WO 90/15070 and and an assay is performed by detecting the presence or 92/10092. These arrays are generally produced using absence of a hybridization Signal. In the mutS assay, the mechanical Synthesis methods or light directed Synthesis protein binds only to Sequences that contain a nucleotide methods, which incorporate a combination of photolitho mismatch in a heteroduplex between polymorphic and non graphic methods and Solid phase oligonucleotide Synthesis. polymorphic Sequences. Mismatches, in this Sense of the (Fodor et al., Science, 251:767-777, (1991)). The immobi word refers to hybridized nucleic acid duplexes in which the lization of arrays of oligonucleotides on Solid Supports has two strands are not 100% complementary. The lack of total been rendered possible by the development of a technology homology results from the presence of one or more poly generally identified as “Very Large Scale Immobilized Poly morphisms in an amplicon obtained from a biological mer Synthesis” (VLSPISTM) in which, typically, probes are Sample, for example, that has been hybridized to a non immobilized in a high density array on a Solid Surface of a polymorphic Strand. Mismatched detection can be used to chip. Examples of VLSPISTM technologies are provided in detect point mutations in DNA or in an mRNA. While these U.S. Pat. Nos. 5,143,854 and 5,412,087 and in PCT Publi US 2003/0162202 A1 Aug. 28, 2003

cations WO 90/15070, WO 92/10092 and WO95/11995, phoretic ability of mismatched duplexes relative to matched which describe methods for forming oligonucleotide arrayS duplexes. (See, e.g., Cariello, Human Genetics 42:726 through techniques Such as light-directed Synthesis tech (1988), herein incorporated by reference). With any of the niques. In designing Strategies aimed at providing arrays of techniques described above, the mRNA or DNA from a nucleotides immobilized on Solid Supports, further presen tested organism that corresponds to regions of an A2M gene tation Strategies were developed to order and display the having a polymorphism Selected from the group consisting oligonucleotide arrays on the chips in an attempt to maxi of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, mize hybridization patterns and diagnostic information. 28i and 30e can be amplified by PCR before hybridization. Examples of Such presentation Strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 0215. The presence of polymorphic and/or mutant A2M 97/29212, and WO 97/31256, all of which are hereby polypeptides in a protein Sample can also be detected by incorporated by reference in their entireties. using conventional assayS. For example, antibodies immu noreactive with a polymorphic and/or mutant A2M polypep 0212. A wide variety of labels and conjugation tech tide can be used to Screen patient biological Samples to niques are known by those skilled in the art and can be used determine if Said patients are at risk of acquiring AD or have in various nucleic acid assays. There are Several ways to a predilection to acquire AD. Additionally, antibodies that produce labeled nucleic acids for hybridization or PCR differentiate the wild type A2M from polymorphic and/or including, but not limited to, oligolabeling, nick translation, mutant A2M polypeptides can be used to determine that an end-labeling, or PCR amplification using a labeled nucle organism does not have a risk of acquiring AD or a predi otide. Alternatively, a nucleic acid encoding a polymorphic lection to acquire AD. and/or mutant A2M polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are 0216) In preferred embodiments, antibodies are used to known in the art, are commercially available, and can be immunoprecipitate the polymorphic and/or mutant A2M used to synthesize RNA probes in vitro by addition of an polypeptides from Solution or are used to react with the appropriate RNA polymerase such as T7, T3 or SP6 and polymorphic and/or mutant A2M polypeptides on Western labeled nucleotides. A number of companies Such as Phar or Immunoblots. Favored diagnostic embodiments also macia Biotech (Piscataway N.J.), Promega (Madison Wis.), include enzyme-linked immunosorbant assays (ELISA), and U.S. Biochemical Corp (Cleveland Ohio) supply com radioimmunoassays (RIA), immunoradiometric assays mercial kits and protocols for these procedures. Suitable (IRMA) and immunoenzymatic assays (IEMA), including reporter molecules or labels include those radionuclides, Sandwich assays using monoclonal and/or polyclonal anti enzymes, fluorescent, chemiluminescent, or chromogenic bodies. Exemplary sandwich assays are described by David agents, as well as, Substrates, cofactors, inhibitors, magnetic et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby particles and the like. incorporated by reference. Other embodiments employ aspects of the immune-Strip technology disclosed in U.S. 0213 The RNASe protection method, briefly described Pat. Nos. 5,290,678; 5,604,105; 5,710,008; 5,744,358; and above, is an example of a mismatch cleavage technique that 5,747,274, herein incorporated by reference. is amenable to array technology. Preferably, the method involves the use of a labeled riboprobe that is complemen 0217. In another preferred protein-based diagnostic, anti tary to polymorphic and/or mutant A2M nucleic acid bodies of the invention are attached to a Support in an Sequences Selected from the group consisting of 6i, 12i.1, ordered array wherein a plurality of antibodies are attached 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e. to distinct regions of the Support that do not overlap with The riboprobe and either mRNA or DNA isolated and each other. AS with the nucleic acid-based arrays, the amplified from a biological Sample are annealed (hybrid protein-based arrays are ordered arrays that are designed to ized) and Subsequently digested with the enzyme RNASe A, be “addressable” Such that the distinct locations are recorded which is able to detect mismatches in a duplex RNAse and can be accessed as part of an assay procedure. These structure. If a mismatch is detected by RNASe A, the probes are joined to a Support in different known locations. polymorphic variant is not present in the Sample and the The knowledge of the precise location of each probe makes enzyme cleaves at the Site of the mismatch and destroys the these “addressable' arrays particularly useful in binding riboprobe. Thus, when the annealed RNA is separated on a assayS. For example, an addressable array can comprise a electrophoretic gel matrix, if a mismatch has been detected Support having Several regions to which are joined a plu and cleaved by RNASe A, an RNA product will be seen rality of antibody probes that Specifically recognize a par which is much smaller than the full length duplex RNA for ticular A2M and differentiate the polymorphic and/or mutant the riboprobe and the mRNA or DNA. A2M polypeptides from wild type A2M. 0214 Complements to the riboprobe can also be dis 0218. Proteins are obtained from biological samples and persed on an array and Stringently probed with the products are labeled by conventional approaches (e.g., radioactivity, from the Rnase A digestion after denaturing any remaining calorimetrically, or fluorescently). The labeled samples are hybrids. In this case, if a mismatch is detected and probe then applied to the array under conditions that permit destroyed by Rnase A, the complements on the array will not binding. If a protein in the Sample binds to an antibody probe anneal with the degraded RNA under Stringent conditions. In on the array, then a signal will be detected at a position on a similar fashion, DNA probes can be used to detect mis the Support that corresponds to the location of the antibody matches, through enzymatic or chemical cleavage. See, e.g., protein complex. Since the identity of each labeled Sample Cotton, et al., Proc. Natl. Acad. Sci. USA 85.4397 (1988); is known and the region of the Support on which the labeled Shenk et al., Proc. Natl. Acad. Sci. USA 72:989 (1975); and Sample was applied is known, an identification of the Novack et al., Proc. Natl. Acad. Sci. USA 83:586 (1986). presence, concentration, and/or expression level can be Mismatches can also be detected by shifts in the electro rapidly determined. That is, by employing labeled Standards US 2003/0162202 A1 Aug. 28, 2003 22 of a known concentration of polymorphic and/or mutant nucleic acid or A2M polypeptide, Some diseases may result A2M polypeptide or wild-type A2M, an investigator can from skewed levels of wild-type A2M as compared to accurately determine the protein concentration of the par polymorphic and/or mutant A2M. By monitoring the level of ticular A2M in a tested Sample and can also assess the expression of Specific A2M polypeptides, for example, a expression level of the A2M. Conventional methods in diagnosis can be made or a disease State can be identified. densitometry can also be used to more accurately determine Similarly, by determining ratios of the level of expression of the concentration or expression level of the A2M. These various A2M polypeptides a prognosis of health or disease approaches are easily automated using technology known to can be made. The levels of expression of different types of those of skill in the art of high throughput diagnostic A2M in various healthy individuals, as well as, individuals analysis. Suffering from AD can be determined, for example. These 0219. In another embodiment, an opposite approach to values can be recorded in a database and can be compared that presented above can be employed. Proteins present in to values obtained from tested individuals. Additionally, the biological Samples can be disposed on a Support So as to ratioS or patterns of expression of various A2M polypeptides create an addressable array. Preferably, the protein Samples from both healthy and diseased individuals is recorded in a are disposed on the Support at known positions that do not database. These analyses are referred to as “disease State overlap. The presence of a protein encoding a polymorphic profiles' and by comparing one disease State profile (e.g. and/or mutant A2M polypeptide in each Sample is then from a healthy or diseased individual) to a disease State determined by applying labeled antibody probes that recog profile from a tested individual, a clinician can rapidly nize epitopes Specific for the polymorphic and/or mutant diagnose the presence or absence of disease. . A2M polypeptide. Because the identity of the biological 0223) The nucleic acid and protein-based diagnostic tech Sample and its position on the array is known, an identifi niques described above can be used to detect the level or cation of the presence, concentration, and/or expression amount or ratio of expression of a particular A2M RNAS or level of a particular polymorphism can be rapidly deter A2M proteins in a tissue. Through quantitative Northern mined. hybridizations, In Situ analysis, immunohistochemistry, 0220 That is, by employing labeled standards of a known ELISA, genechip array technology, PCR, and Western blots, concentration of polymorphic and/or mutant A2M polypep for example, the amount or level of expression of RNA or tides, an investigator can accurately determine the concen protein for a particular A2M (wild-type or mutant) can be tration of A2M in a sample and from this information can rapidly determined and from this information ratios of A2M assess the expression level of the particular form of A2M. expression can be ascertained. Preferably, the expression Conventional methods in densitometry can also be used to levels of A2M genes having one or more of a polymorphism more accurately determine the concentration or expression and/or mutation Selected from the group consisting of 6i, level of the A2M. These approaches are also easily auto 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i mated using technology known to those of skill in the art of and 30e are measured to determine the ratioS. high throughput diagnostic analysis. AS detailed above, any 0224. Once the levels of various A2M polypeptides or addressable array technology known in the art can be nucleic acids are determined, the information can be employed with this aspect of the invention and display the recorded onto a computer readable media, Such as a hard protein arrays on the chips in an attempt to maximize drive, floppy disk, DVD drive, Zip drive, etc. After recording antibody binding patterns and diagnostic information. and the generation of a database comprising the levels of 0221 AS discussed above, the presence or detection of expression of the various A2M polypeptides or nucleic acids one or more of the mutations and/or polymorphisms pro Studied, a comparing program is used which compares the Vided in Table 1 can provide a diagnosis that the tested levels of expression of the various A2M polypeptides or Subject is at risk of acquiring AD or has a predilection to nucleic acids So as to create a ratio of expression. The acquire AD. Additional embodiments include the prepara following Section describes the preparation of pharmaceu tion of diagnostic kits comprising detection components, ticals having polymorphic and/or mutant A2M polypeptides Such as antibodies, Specific for one or more of the particular or binding partners, which can be administered to organisms polymorphic variants of A2M or A2M described herein. The in need to modulate A2M activities. detection component will typically be Supplied in combina tion with one or more of the following reagents. A Support 0225 Pharmacogenomics capable of absorbing or otherwise binding RNA or protein 0226. It is likely that subjects having one or more differ will often be Supplied. Available Supports for this purpose ent allelic variants of the A2M gene will respond differently include, but are not limited to, membranes of nitrocellulose, to drugs to treat associated diseases or disorders. For nylon or derivatized nylon that can be characterized by example, alleles of the A2M gene that associate with neu bearing an array of positively charged Substituents, and rodegenerative disease will be useful alone or in conjunction GenechipsTM or their equivalents. One or more enzymes, with other genes associated with the development of neu Such as Reverse Transcriptase and/or Taq polymerase, can rodegenerative disease (e.g., APOE4) to predict a Subjects be furnished in the kit, as can dNTPs, buffers, or non-human response, either positive or negative, to a therapeutic drug. polynucleotides like calf-thymus or Salmon-Sperm DNA. Multiplex primer eXtension assays or microarrays compris Results from the kit assays can be interpreted by a healthcare ing probes for Specific alleles are useful formats for deter provider or a diagnostic laboratory. Alternatively, diagnostic mining drug response. A correlation between drug responses kits are manufactured and Sold to private individuals for and Specific alleles or combinations of alleles (haplotypes) Self-diagnosis. of the A2M gene and other genes that associate with disease 0222. In addition to diagnosing disease according to the can be shown, for example, by clinical Studies wherein the presence or absence of a polymorphic and/or mutant A2M response, either positive or negative, to specific drugs of US 2003/0162202 A1 Aug. 28, 2003

Subjects having different allelic variants of polymorphic is indicative of an increased or decreased likelihood that the regions of the A2M gene alone or in combination with allelic treatment will be effective. Also provided are methods for variants of other genes are compared. Such studies can also predicting a response of a Subject to an agent used to treat be performed using animal models, Such as mice having a neurodegenerative disease or disorder which include a step various alleles and in which, e.g., the endogenous uPA gene of determining in nucleic acid obtained from the Subject, the has been inactivated Such as by a knock-out mutation. Test identity of nucleotide(s) at one or more polymorphisms of an drugs are then administered to the mice having different A2M gene that occur at positions corresponding to 6i, 12i.1, alleles and the response of the different mice to a specific 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i, and compound is compared. Accordingly, assays, microarrayS 30e, wherein the presence or absence of a particular nucle and kits are provided for determining the drug which will be otide(s) at the one or more polymorphisms, individually best Suited for treating a specific disease or condition in a and/or in combination, is indicative of an increased or Subject based on the individual's genotype. For example, it decreased likelihood that the treatment will be effective. will be possible to select drugs which will be devoid of 0230. Also provided are any of the above methods toxicity, or have the lowest level of toxicity possible for wherein the neurodegenerative disease or disorder is Alzhe treating a Subject having a disease or condition, e.g., neu imer's disease. In particular methods, the neurodegenerative rodegenerative disease or Alzheimer's disease. disease or disorder is Alzheimer's disease wherein the age of 0227 For example, therapeutic agents for treatment of onset is greater than or equal to about 50 years, or greater neurodegenerative disease that can be genetically profiled than or equal to about 60 years, or greater than or equal to include, but are not limited to, ALCAR, Alpha-tocopherol about 65 years. (Vitamin E),), Ampalex, AN-1792 (AIP-001), Cerebrolysin, Daposone, Donepezil (Aricept), ENA-713 (Exelon), Estro 0231. Also provided are any of the above methods which gen replacement therapy, Galanthamine (Reminyl), Ginkgo include a step of determining the identity of a nucleotide(s) Biloba eXtract, Huperzine A, Ibuprofen, Lipitor, Naproxen, at a position corresponding to the position of at least one Nefiracetam, Neotrofin, Memantine, Phenserine, Rofecoxib, polymorphism of at least one different gene, wherein the Selegiline (Eldepryl), Tacrine (Cognex), Xanomeline (skin different gene is associated with a neurodegenerative disease patch), Resperidone (RisperidolTM), Neuroleptics, Benzodi or disorder. For example, the at least one different gene can aZepenes, Valproate, Serotonin reuptake inhibitors (SRIs), be APOE4. Beta and Gamma Secretase Inhibitors, CX-516 (Ampalex), 0232. As set forth above, the ability to predict whether a Statins and AF-102B (Evoxac). person will respond to a particular therapeutic agent or drug 0228. Other therapeutic agents for treatment of neurode is useful, among other things, for matching particular drug generative disease include those that are neuroprotective. treatments to particular patient population to thereby elimi Drugs with anti-oxidative properties, e.g., flupirtine, N-ace nate from a treatment protocol drugs that may be leSS tylcysteine, idebenone, melatonin, and also novel dopamine efficacious in particular patients. agonists (ropinirole and pramipexole) have been shown to 0233 Provided herein is a computer-assisted method of protect neuronal cells from apoptosis and thus have been identifying a proposed treatment for a disease, Such as, for Suggested for treating neurodegenerative disorders like AD example, a neurodegenerative disease. The method involves or PD. Also, free radical Scavengers, calcium channel block the steps of (a) storing a database of biological data for a erS and modulators of certain Signal transduction pathways plurality of Subjects, the biological data that is being Stored that might protect neurons from downstream effects of the include for each of the plurality of Subjects (i) treatment accumulation of A-Beta intracellularly and/or extracellu type, (ii) the presence or absence of a particular nucle larly. Also, other agents like non-Steroidal anti-inflammatory otide(s) at one or more polymorphisms of the A2M gene drugs (NSAIDs) partly inhibit cyclooxygenase (COX) Selected from the group consisting of 6i, 12i.1, 12i.2, 12e, expression, as well as having a positive influence on the 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i, and 30e, and (iii) clinical expression of AD. Distinct cytokines, growth factors at least one disease progression measure for the neurode and related drug candidates, e.g., nerve growth factor generative disease (e.g., AD), or other disease, from which (NGF), or members of the transforming growth factor-beta treatment efficacy may be determined; and then (b) querying (TGF-beta) Superfamily, like growth and differentiation fac the database to determine the dependence on the one or more tor 5 (GDF-5), are shown to protect tyrosine hydroxylase or polymorphisms of the effectiveness of a treatment type in dopaminergic neurones from apoptosis. CRIB (cellular treating the disease, to thereby identify a proposed treatment replacement by immunoisolatory biocapsule) is a gene as an effective treatment for a Subject carrying a particular therapeutical approach for human NGF Secretion, which has polymorphism (or combination of polymorphisms) for the been shown to protect cholinergic neurones from cell death disease, Such as AD. The polymorphisms entered into the when implanted in the brain ((2000) Expert Opin Investig database can also include previously known polymorphisms, Drugs 9(4):747-64). including, for example, polymorphisms included in Table 2. 0229 Provided herein is a method for predicting a 0234 Any Suitable disease progression measure can be response of a Subject to an agent used to treat an A2M used. For example, for neurodegenerative disease, measures mediated disease which includes a step of determining in of motor function, cognitive function, dementia and com nucleic acid obtained from the Subject the identity of nucle binations thereof can be used as measures of disease pro otide(s) at one or more polymorphisms of an A2M gene that gression. The measures can be Scored in accordance with occur at positions corresponding to 6i, 12i.1, 12i.2, 12e, 14e, Standard techniques for entry into the database. Measures 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i, and 30e, wherein the can be taken at the initiation of the Study, and then during the presence or absence of a particular nucleotide(s) at the one course of the study (that is, treatment of the group of patients or more polymorphisms, individually and/or in combination, with the experimental and control treatments), and the US 2003/0162202 A1 Aug. 28, 2003 24

database can incorporate a plurality of these measures taken available and virtually all pharmaceutical companies have over time So that the presence, absence or rate of disease computer modeling groups devoted to rational drug design. progression in particular individuals or groups of individuals Molecular Simulations Inc., for example, Sells Several may be assessed. The database can be queried for the Sophisticated programs that allow a user to Start from an effectiveness of a particular treatment in patients carrying amino acid Sequence, build a two or three-dimensional any of a variety of polymorphisms, or combinations of model of the protein or polypeptide, compare it to other two polymorphisms, or who lack particular polymorphisms. and three-dimensional models, and analyze the interactions Computer Systems used to carry out these methods may be of compounds, drugs, and peptides with a three dimensional implemented as hardware, Software, or both hardware and model in real time. Accordingly, in Some embodiments of Software. Systems that may be used to implement these the invention, Software is used to compare regions of poly methods are known and available. See, e.g., U.S. Pat. No. morphic and/or mutant A2M polypeptides and molecules 6,108,635 and Eas, M. A.: A program for the meta-analysis that interact with polymorphic and/or mutant A2M polypep of clinical trials, Computer Methods and Programs in Bio tides (collectively referred to as “binding partners”) with medicine, vol.53, no.3 (July 1997); D. Klinger and M. Jaffe, other molecules, Such as peptides, peptidomimetics, and An Information Technology Architecture for Pharmaceutical chemicals, So that therapeutic interactions can be predicted Research and Development, 14" Annual Symposium on and designed. (See Schneider, Genetic Engineering News Computer Applications in Medical Care, Nov. 4-7, pp. December: page 20 (1998), Tempczyk et al., Molecular 256-260 (Washington D.C., 1990); M. Rosenberg, “ClinAc Simulations Inc. Solutions April (1997) and Butenhof, ceSS: An integrated client/server approach to clinical data Molecular Simulations Inc. Case Notes (August 1998) for a management and regulatory approval.” Proc. Of the 21" discussion of molecular modeling). Annual SAS Users Group International Conference (Cary, 0238 For example, the protein sequence of a polymor N.C., Mar. 10-13, 1996). Querying of the database may be phic and/or mutant A2M polypeptide or binding partner, or carried out in accordance with known techniques Such as domains of these molecules (or nucleic acid sequence regression analysis or other types of comparisons Such as encoding these polypeptides or both), can be entered onto a with simple normal or t-tests, or with non-parametric tech computer readable medium for recording and manipulation. niques. Such querying may be carried out prospectively or It will be appreciated by those skilled in the art that a retrospectively on the database by any Suitable means, but is computer readable medium having these Sequences can generally done by Statistical analysis in accordance with interface with Software that converts or manipulates the known techniques. Sequences to obtain Structural and functional information, 0235 Rational Drug Design Such as protein models. That is, the functionality of a 0236 Rational drug design involving polypeptides Software program that converts or manipulates these requires identifying and defining a first peptide with which Sequences includes the ability to compare these Sequences to the designed drug is to interact, and using the first target other Sequences or structures of molecules that are present peptide to define the requirements for a Second peptide. With on publicly and commercially available databases So as to Such requirements defined, one can find or prepare an conduct rational drug design. appropriate peptide or non-peptide that meets all or Substan 0239). The polymorphic and/or mutant A2M polypeptide tially all of the defined requirements. Thus, one goal of or binding partner polypeptide or nucleic acid Sequence or rational drug design is to produce Structural or functional both can be Stored, recorded, and manipulated on any analogs of biologically active polypeptides of interest or of medium that can be read and accessed by a computer. AS Small molecules with which they interact (e.g., agonists, used herein, the words “recorded” and “stored” refer to a antagonists, null compounds) in order to fashion drugs that process for Storing information on computer readable are, for example, more or less potent forms of the ligand. medium. A skilled artisan can readily adopt any of the (See, e.g., Hodgson, Bio. Technology 9:19-21 (1991)). An presently known methods for recording information on a example of rational drug design is shown in Erickson et al., computer readable medium to generate manufactures com Science 249:527-533 (1990). Combinatorial chemistry is the prising the nucleotide or polypeptide Sequence information Science of Synthesizing and testing compounds for bioac of this embodiment. A variety of data Storage Structures are tivity en masse, instead of one by one, the aim being to available to a skilled artisan for creating a computer readable discover drugs and materials more quickly and inexpen medium having recorded thereon a nucleotide or polypep Sively than was formerly possible. Rational drug design and tide Sequence. The choice of the data Storage Structure will combinatorial chemistry have become more intimately generally be based on the component chosen to access the related in recent years due to the development of approaches Stored information. Computer readable media include mag in computer-aided protein modeling and drug discovery. netically readable media, optically readable media, or elec (See e.g., U.S. Pat. No. 4,908,773; 5,884.230; 5,873,052; tronically readable media. For example, the computer read 5,331.573; and 5,888,738). able media can be a hard disc, a floppy disc, a magnetic tape, 0237) The use of molecular modeling as a tool for ratio Zip disk, CD-ROM, DVD-ROM, RAM, or ROM as well as nal drug design and combinatorial chemistry has dramati other types of other media known to those skilled in the art. cally increased due to the advent of computer graphics. Not The computer readable media on which the Sequence infor only is it possible to view molecules on computer Screens in mation is Stored can be in a personal computer, a network, three dimensions but it is also possible to examine the a Server or other computer Systems known to those skilled in interactions of macromolecules Such as enzymes and recep the art. tors and rationally design derivative molecules to test. (See 0240 Embodiments of the invention utilize computer Boorman, Chem. Eng. News 70:18-26 (1992). A vast based Systems that contain the Sequence information amount of user-friendly Software and hardware is now described herein and convert this information into other US 2003/0162202 A1 Aug. 28, 2003 types of usable information (e.g., protein models for rational file, a html file, or a pdf file in a variety of database programs drug design). The term "a computer-based System” refers to familiar to those of skill in the art, Such as DB2, SYBASE, the hardware, Software, and any database used to analyze an or ORACLE. polymorphic and/or mutant A2M or a binding partner 0243 A“search program” refers to one or more programs (nucleic acid or polypeptide sequence or both), or fragments that are implemented on the computer-based System to of these biomolecules So as to construct models or to compare a polymorphic and/or mutant A2M or binding conduct rational drug design. The computer-based System partner (nucleotide or polypeptide sequence) with other preferably includes the Storage media described above, and nucleotide or polypeptide Sequences and agents including a processor for accessing and manipulating the Sequence but not limited to peptides, peptidomimetics, and chemicals data. The hardware of the computer-based systems of this Stored within a database. A Search program also refers to one embodiment comprise a central processing unit (CPU) and or more programs that compare one or more protein models a database. A skilled artisan can readily appreciate that any to Several protein models that exist in a database and one or one of the currently available computer-based Systems are more protein models to Several peptides, peptidomimetics, Suitable. and chemicals that exist in a database. A Search program is used, for example, to compare one polymorphic and/or 0241. In one particular embodiment, the computer system mutant A2M functional profile to one or more polymorphic includes a processor connected to a bus that is connected to and/or mutant A2M functional profiles that are present in a a main memory (preferably implemented as RAM) and a database So as to determine an appropriate treatment proto variety of Secondary Storage devices, Such as a hard drive col, for example. Still further, a Search program can be used and removable medium Storage device. The removable to compare values or results from A2M functional assays medium Storage device can represent, for example, a floppy and agents that modulate A2M-mediated activities. disk drive, a DVD drive, an optical disk drive, a compact 0244. A “retrieval program” refers to one or more pro disk drive, a magnetic tape drive, etc. A removable Storage grams that can be implemented on the computer-based medium, Such as a floppy disk, a compact disk, a magnetic System to identify peptides, peptidomimetics, and chemicals tape, etc. containing control logic and/or data recorded that interact with a polymorphic and/or mutant A2M therein can be inserted into the removable Storage device. polypeptide Sequence, or a polymorphic and/or mutant A2M The computer System includes appropriate Software for polypeptide model Stored in a database. Further, a retrieval reading the control logic and/or the data from the removable program is used to identify a specific agent that modulates medium Storage device once inserted in the removable A2M-mediated activities to a desired set of values, results, medium storage device. The polymorphic and/or mutant or profile. That is, a retrieval program can also be used to A2M or binding partner (nucleic acid or polypeptide obtain “a binding partner profile' that is composed of a Sequence or both) can be stored in a well known manner in chemical Structure, nucleic acid Sequence, or polypeptide the main memory, any of the Secondary Storage devices, Sequence or model of an agent that interacts with a poly and/or a removable Storage medium. Software for accessing morphic and/or mutant A2M polypeptide and, thereby and processing these sequences (Such as Search tools, com modulates (inhibits or enhances) an A2M activity, Such as pare tools, and modeling tools etc.) reside in main memory binding to a receptor, amyloid B, a protease. Zinc, or tetramer during execution. formation. Further, a binding partner profile can have one or 0242. As used herein, “a database' refers to memory that more Symbols that represent these molecules and/or models, can Store a polymorphic and/or mutant A2M or binding an identifier that represents one or more agents including, partner nucleotide or polypeptide Sequence information, but not limited to peptides and peptidomimetics (referred to protein model information, information on other peptides, collectively as "peptide agents”) and chemicals, and a value chemicals, peptidomimetics, and other agents that interact or result from a functional assay. with polymorphic and/or mutant A2M polypeptides, and 0245. As a starting point to rational drug design, a two or values or results from functional assayS. Additionally, a three dimensional model of a polypeptide of interest is "database' refers to a memory access component that can created (e.g., polymorphic and/or mutant A2M polypeptide, access manufactures having recorded thereon polymorphic or a binding partner, Such as the LRP receptor, amyloid B, a and/or mutant A2M or binding partner nucleotide or protease, or an antibody). In the past, the three-dimensional polypeptide Sequence information, protein model informa Structure of proteins has been determined in a number of tion, information on other peptides, chemicals, peptidomi ways. Perhaps the best known way of determining protein metics, and other agents that interact with polymorphic Structure involves the use of X-ray crystallography. A general and/or mutant A2M polypeptides, and valueS or results from review of this technique can be found in Van Holde, K. E. functional assays. In other embodiments, a database Stores a Physical Biochemistry, Prentice-Hall, N.J. pp. 221-239 "polymorphic and/or mutant A2M polypeptide functional (1971). Using this technique, it is possible to elucidate profile' comprising the values and results (e.g., ability to three-dimensional Structure with good precision. Addition asSociate with a receptyor, amyloid, B, a protease, Zinc, or ally, protein Structure can be determined through the use of the ability to form a tetramer) from one or more “A2M techniques of neutron diffraction, or by nuclear magnetic functional assays, as described herein or known in the art, and relationships between these values or results. The resonance (NMR). (See, e.g., Moore, W.J., Physical Chem Sequence data and values or results from these functional istry, 4" Edition, Prentice-Hall, N.J. (1972)). assays can be Stored and manipulated in a variety of data 0246 Alternatively, protein models of a polypeptide of processor programs in a variety of formats. For example, the interest can be constructed using computer-based protein Sequence data can be Stored as text in a word processing file, modeling techniques. By one approach, the protein folding such as Microsoft WORD or WORDPERFECT, an ASCII problem is Solved by finding target Sequences that are most US 2003/0162202 A1 Aug. 28, 2003 26 compatible with profiles representing the Structural environ bases; AMPAC/MOPAC and QCPE programs; Molecular ments of the residues in known three-dimensional protein Design Limited Molfile and SD files, Sybel Mol2 files, structures. (See, e.g., U.S. Pat. No. 5,436.850). In another VRML, and Pict files. technique, the known three-dimensional Structures of pro teins in a given family are Superimposed to define the 0250 Additionally, the techniques described above can Structurally conserved regions in that family. This protein be Supplemented with techniques in molecular biology to modeling technique also uses the known three-dimensional design models of the protein of interest. For example, a Structure of a homologous protein to approximate the Struc polypeptide of interest can be analyzed by an alanine Scan ture of a polypeptide of interest. (See e.g., U.S. Pat. Nos. (Wells, Methods in Enzymol. 202:390-411 (1991)) or other 5,557,535; 5,884.230; and 5,873,052). Conventional homol types of Site-directed mutagenesis analysis. In alanine Scan, ogy modeling techniques have been used routinely to build each amino acid residue of the polypeptide of interest is models of proteases and antibodies. (Sowdhamini et al., Sequentially replaced by alanine in a step-wise fashion (i.e., Protein Engineering 10:207, 215 (1997)). Comparative only one alanine point mutation is incorporated per molecule approaches can also be used to develop three-dimensional Starting at position #1 and proceeding through the entire protein models when the protein of interest has poor molecule), and the effect of the mutation on the peptide's Sequence identity to template proteins. In Some cases, pro activity in a functional assay is determined. Each of the teins fold into Similar three-dimensional Structures despite amino acid residues of the peptide is analyzed in this manner having very weak Sequence identities. For example, the and the regions important for A2M activities, are identified. three-dimensional Structures of a number of helical cytok These functionally important regions can be recorded on a ines fold in Similar three-dimensional topology in spite of computer readable medium, Stored in a database in a com weak Sequence homology. puter System, and a Search program can be employed to 0247 The recent development of threading methods and generate a protein model of the functionally important “fuzzy' approaches now enables the identification of likely regions. folding patterns and functional protein domains in a number 0251 Once a model of the polypeptide of interest is of Situations where the Structural relatedness between target created, a candidate binding partner can be identified and and template(s) is not detectable at the Sequence level. By manufactured as follows. First, a molecular model of one or one method, fold recognition is performed using Multiple more molecules that are known to interact with A2M or Sequence Threading (MST) and structural equivalences are portions thereof are created using one of the techniques deduced from the threading output using the distance geom discussed above or as known in the art. Next, chemical etry program DRAGON that constructs a low resolution libraries and databases are searched for molecules similar in model. A full-atom representation is then constructed using Structure to the known molecule. That is, a Search can be a molecular modeling package Such as QUANTA. made of a three dimensional data base for non-peptide 0248. According to this 3-step approach, candidate tem (organic) structures (e.g., non-peptide analogs, and/or dipep plates are first identified by using the novel fold recognition tide analogs) having three dimensional Similarity to the algorithm MST, which is capable of performing simulta known Structure of the target compound. See, e.g., the neous threading of multiple aligned Sequences onto one or Cambridge Crystal Structure Data Base, Crystallographic more 3-D Structures. In a Second step, the Structural equiva Data Center, Lensfield Road, Cambridge, CB2 1EW, lences obtained from the MST output are converted into England; and Allen, F. H., et al., Acta Crystallogr., B35: interresidue distance restraints and fed into the distance 2331-2339 (1979). The identified candidate binding partners geometry program DRAGON, together with auxiliary infor that interact with A2M can then be analyzed in a functional mation obtained from Secondary Structure predictions. The assay (e.g., binding assays with amyloid?, the LRP receptor, program combines the restraints in an unbiased manner and Zinc, protease, or tetramer formation) and new molecules rapidly generates a large number of low resolution model can be modeled after the candidate binding partners that confirmations. In a third Step, these low resolution model produce a desirable response. Preferably, these interactions confirmations are converted into full-atom models and orga are studied with both wild-type A2M and polymorphic nized to energy minimization using the molecular modeling and/or mutant A2M polypeptides. By cycling in this fashion, package QUANTA. (See e.g., ASZódi et al., Proteins:Struc libraries of molecules that interact with A2M, preferably ture, Function, and Genetics, Supplement 1:38-42 (1997)). polymorphic and/or mutant A2M polypeptides, and produce a desirable or optimal response in a functional assay can be 0249. In a preferred approach, the commercially avail Selected. able “Insight II 98” program (Molecular Simulations Inc.) and accompanying modules are used to create a two and/or 0252) It is noted that search algorithms for three dimen three dimensional model of a polypeptide of interest from an Sional database comparisons are available in the literature. amino acid Sequence. Insight II is a three-dimensional See, e.g., Cooper, et al., J. Comput. Aided Mol. Design, 3: graphics program that can interface with Several modules 253-259 (1989) and references cited therein; Brent, et al., J. that perform numerous structural analysis and enable real Comput.-Aided Mol. Design, 2: 311-310 (1988) and refer time rational drug design and combinatorial chemistry. ences cited therein. Commercial Software for Such Searches Modules such as Builder, Biopolymer, Consensus, and Con is also available from Vendors Such as Day Light Informa verter, for example, allow one to rapidly create a two tion Systems, Inc., Irvine, Calif. 92714, and Molecular dimensional or three dimensional model of a polypeptide, Design Limited, 2132 Faralton Drive, San Leandro, Calif. carbohydrate, nucleic acid, chemical or combinations of the 94577. The searching is done in a systematic fashion by foregoing from their Sequence or Structure. The modeling Simulating or Synthesizing analogs having a Substitute moi tools associated with Insight II Support many different data ety at every residue level. Preferably, care is taken that file formats including Brookhaven and Cambridge data replacement of portions of the backbone does not disturb the US 2003/0162202 A1 Aug. 28, 2003 27 tertiary Structure and that the Side chain Substitutions are partner can be modified and the Structural effects of modi compatible to retain the receptor Substrate interactions. fication can be determined using the experimental and computer modeling methods described above applied to the 0253). By another approach, protein models of binding new composition. The altered Structure is then compared to partners that interact with A2M, preferably polymorphic the active Site Structure of the compound to determine if an and/or mutant A2M polypeptides, can be made by the improved fit or interaction results. In this manner Systematic methods described above and these models can be used to variations in composition, Such as by varying Side groups, predict the interaction of new molecules. Once a model of a can be quickly evaluated to obtain modified modulating binding partner is identified, the active sites or regions of compounds or ligands of improved Specificity or activity. interaction can be identified. Such active sites might typi cally be ligand binding Sites. The active site can be identified 0257) A number of articles review computer modeling of using methods known in the art including, for example, from drugs interactive with Specific-proteins, Such as Rotivinen, the amino acid Sequences of peptides, from the nucleotide et al., 1988, Acta Pharmaceutical Fennica 97:159-166; Sequences of nucleic acids, or from Study of complexes of Ripka, New Scientist 54-57 (Jun. 16, 1988); McKinally and the wild-type and/or polymorphic and/or mutant A2M Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol. 29:111 polypeptides with a ligand. In the latter case, chemical or 122; Perry and Davies, OSAR: Quantitative Structure-Ac X-ray crystallographic methods can be used to find the tivity Relationships in Drug Design pp. 189-193 (Alan R. active Site by finding where on the wild-type and/or poly Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc. Lond. morphic and/or mutant A2M polypeptides the complexed 236:125-140 and 141-162; and, with respect to a model ligand is found. Next, the three dimensional geometric receptor for nucleic acid components, Askew, et al., 1989, J. Structure of the active Site is determined. This can be done Am. Chem. Soc. 111:1082-1090. Other computer programs by known methods, including X-ray crystallography, which that Screen and graphically depict chemicals are available can determine a complete molecular structure. On the other from companies Such as BioDesign, Inc. (Pasadena, Calif.), hand, solid or liquid phase NMR can be used to determine Allelix, Inc. (Mississauga, Ontario, Canada), and Hyper certain intra-molecular distances. Any other experimental cube, Inc. (Cambridge, Ontario). Although these are prima method of Structure determination can be used to obtain rily designed for application to drugs specific to particular partial or complete geometric Structures. The geometric proteins, they can be adapted to design of drugs specific for Structures can be measured with a complexed ligand, natural the modulation of A2M activities. or artificial, which may increase the accuracy of the active 0258 Many more computer programs and databases can Site Structure determined. be used with embodiments of the invention to identify new binding partners that modulate A2M function. The following 0254. If an incomplete or insufficiently accurate structure list is intended not to limit the invention but to provide is determined, the methods of computer based numerical guidance to programs and databases that are useful with the modeling can be used to complete the Structure or improve approaches discussed above. The programs and databases its accuracy. Any recognized modeling method can be used, that can be used include, but are not limited to: MacPattern including parameterized models Specific to particular (EMBL), DiscoveryBase (Molecular Applications Group), biopolymerS Such as proteins or nucleic acids, molecular GeneMine (Molecular Applications Group), Look (Molecu dynamics models based on computing molecular motions, lar Applications Group), MacLook (Molecular Applications Statistical mechanics models based on thermal ensembles, or Group), BLAST and BLAST2 (NCBI), BLASTN and combined models. For most types of models, Standard BLASTX (Altschul et al., J. Mol. Biol. 215: 403 (1990), molecular force fields, representing the forces between con herein incorporated by reference), FASTA (Pearson and Stituent atoms and groups, are necessary, and can be Selected Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988), herein from force fields known in physical chemistry. The incom incorporated by reference), Catalyst (Molecular Simulations plete or less accurate experimental Structures can Serve as Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), constraints on the complete and more accurate Structures Cerius. DBAccess (Molecular Simulations Inc.), HypoGen computed by these modeling methods. (Molecular Simulations Inc.), Insight II, (Molecular Simu 0255 Finally, having determined the structure of the lations Inc.), Discover (Molecular Simulations Inc.), active Site of the known binding partner, either experimen CHARMm (Molecular Simulations Inc.), Felix (Molecular tally, by modeling, or by a combination, candidate binding Simulations Inc.), DelPhi, (Molecular Simulations Inc.), partners can be identified by Searching databases containing QuanteMM, (Molecular Simulations Inc.), Homology compounds along with information on their molecular struc (Molecular Simulations Inc.), Modeler (Molecular Simula ture. Such a Search SeekS compounds having structures that tions Inc.), Modeller 4 (Sall and Blundell J. Mol. Biol. match the determined active site Structure and that interact 234:217-241 (1997)), ISIS (Molecular Simulations Inc.), with the groups defining the active site. Such a Search can be Quanta/Protein Design (Molecular Simulations Inc.), manual, but is preferably computer assisted. One program WebLab (Molecular Simulations Inc.), WebLab Diversity that allows for Such analysis is Insight II having the Ludi Explorer (Molecular Simulations Inc.), Gene Explorer module. Further, the Ludi/ACD module allows a user access (Molecular Simulations Inc.), SeqFold (Molecular Simula to over 65,000 commercially available drug candidates tions Inc.), Biopendium (Inpharmatica), SBdBase (Struc (MDL's Available Chemicals Directory) and provides the tural Bioinformatics), the EMBL/Swissprotein database, the ability to Screen these compounds for interactions with the MDL Available Chemicals Directory database, the MDL protein of interest. Drug Data Report database, the Comprehensive Medicinal Chemistry database, Derwents's World Drug Index data 0256 Alternatively, these methods can be used to iden base, and the BioByte MasterFile database. Many other tify improved binding partners from an already known programs and data bases would be apparent to one of skill binding partner. The composition of the known binding in the art given the present disclosure. US 2003/0162202 A1 Aug. 28, 2003 28

0259 Once candidate binding partners have been identi attach the polymorphic and/or mutant A2M polypeptides. fied, desirably, they are analyzed in a functional assay. For example, cyanogen bromide activated matrices, epoxy Further cycles of modeling and functional assays can be activated matrices, thio and thiopropyl gels, nitrophenyl employed to more narrowly define the parameters needed in chloroformate and N-hydroxy succinimide chlorformate a binding partner. Each binding partner and its response in linkages, or oxirane acrylic Supports are used. (Sigma). a functional assay can be recorded on a computer readable 0264. Furthermore, in some embodiments, a liposome or media and a database or library of binding partners and lipid bilayer (natural or Synthetic) is contemplated as a respective responses in a functional assay can be generated. Support and polymorphic and/or mutant A2M polypeptides, These databases or libraries can be used by researchers to or binding partners are attached to the membrane Surface or identify important differences between active and inactive are incorporated into the membrane by techniques in lipo molecules So that compound libraries are enriched for bind Some engineering. Carriers for use in the body, (i.e. for ing partners that have favorable characteristics. The Section prophylactic or therapeutic applications) are desirably below describes several A2M functional assays that can be physiological, non-toxic and preferably, non-immunore used to characterize A2M interactions with candidate bind sponsive. Suitable carriers for use in the body include ing partners. poly-L-lysine, poly-D, L-alanine, liposomes, and Chro 0260 A2M Characterization Assays mosorb(R) (Johns-Manville Products, Denver Co.). Ligand conjugated Chromosorb(R) (Synsorb-Pk) has been tested in 0261) The term “A2M characterization assay” or “A2M humans for the prevention of hemolytic-uremic Syndrome functional assay” or “functional assay” the results of which and was reported as not presenting adverse reactions. (Arm can be recorded as a value in a “A2M functional profile', strong et al. J. Infectious Diseases 171:1042-1045 (1995)). include assays that directly or indirectly evaluate the pres ence of an A2M nucleic acid or protein in a cell and the 0265 The insertion of linkers, such as linkers (e.g., “). ability of a particular type of A2M polypeptide, in particular linkers' engineered to resemble the flexible regions of 2. polymorphic and/or mutant A2M polypeptides, to associate phage) of an appropriate length between the polymorphic with a receptor, a protease, amyloid B, zinc, or to form a and/or mutant A2M polypeptides and the Support are also tetramer. contemplated So as to encourage greater flexibility and thereby overcome any Steric hindrance that can be presented 0262 Some functional assays involve binding assays that by the Support. The determination of an appropriate length utilize multimeric agents. One form of multimeric agent of linker that allows for an optimal cellular response or lack concerns a manufacture comprising an polymorphic and/or thereof, can be determined by Screening the polymorphic mutant A2M polypeptide disposed on a Support. These and/or mutant A2M polypeptides with varying linkers in the multimeric agents provide the polypeptide in Such a form or assays detailed in the present disclosure. in Such a way that a Sufficient affinity for its ligand is achieved. A multimeric agent having an polymorphic and/or 0266. A composite Support comprising more than one mutant A2M polypeptide is obtained by joining the desired type of polymorphic and/or mutant A2M polypeptides is polypeptide to a macromolecular Support. A “Support' can also envisioned. A “composite Support' can be a carrier, a be a termed a carrier, a protein, a resin, a cell membrane, or resin, or any macromolecular Structure used to attach or any macromolecular structure used to join or immobilize immobilize two or more different binding partners or poly Such molecules. Solid Supports include, but are not limited morphic and/or mutant A2M polypeptides. In Some embodi to, the walls of Wells of a reaction tray, test tubes, polySty ments, a liposome or lipid bilayer (natural or synthetic) is rene beads, magnetic beads, nitrocellulose Strips, mem contemplated for use in constructing a composite Support branes, microparticles Such as lateX particles, animal cells, and polymorphic and/or mutant A2M polypeptides or bind Duracyteo (E), artificial cells, and others. A polymorphic ing partners are attached to the membrane Surface or are and/or mutant A2M polypeptide can also be joined to incorporated into the membrane using techniques in lipo inorganic carriers, Such as Silicon oxide material (e.g., Silica Some engineering. gel, Zeolite, diatomaceous earth or aminated glass) by, for 0267 As above, the insertion of linkers, such as 2 linkers, example, a covalent linkage through a hydroxy, carboxy or of an appropriate length between the polymorphic and/or amino group and a reactive group on the carrier. mutant A2M polypeptides or binding partner and the Support is also contemplated So as to encourage greater flexibility in 0263. In several multimeric agents, the macromolecular Support has a hydrophobic Surface that interacts with a the molecule and thereby overcome any Steric hindrance that portion of the polymorphic and/or mutant A2M polypeptides can occur. The determination of an appropriate length of by a hydrophobic non-covalent interaction. In Some cases, linker that allows for an optimal cellular response or lack the hydrophobic Surface of the Support is a polymer Such as thereof, can be determined by Screening the polymorphic plastic or any other polymer in which hydrophobic groups and/or mutant A2M polypeptides or binding partners with have been linked Such as polystyrene, polyethylene or varying linkers in the assays detailed in the present disclo polyvinyl. Additionally, polymorphic and/or mutant A2M SUC. polypeptides can be covalently bound to carriers including 0268. In other embodiments of the invention, the multi proteins and oligo/polysaccarides (e.g. cellulose, starch, meric and composite Supports discussed above can have glycogen, chitosane or aminated Sepharose). In these later attached multimerized polymorphic and/or mutant A2M multimeric agents, a reactive group on the molecule, Such as polypeptides, or binding partners So as to create a “multi a hydroxy or an amino group, is used to join to a reactive merized-multimeric Support' and a "multimerized-compos group on the carrier So as to create the covalent bond. ite Support', respectively. A multimerized ligand can, for Additional multimeric agents comprise a Support that has example, be obtained by coupling two or more binding other reactive groups that are chemically activated So as to partners in tandem using conventional techniques in molecu US 2003/0162202 A1 Aug. 28, 2003 29

lar biology. The multimerized form of the polymorphic 0273 (2) reverse two-hybrid system (Leanna & and/or mutant A2M polypeptides, or binding partner can be Hannink, Nucl. Acid Res. 24:3341-3347 (1996), advantageous for many applications because of the ability to herein incorporated by reference); obtain an agent with a higher affinity for A2M, for example. 0274 (3) repressed transactivator system (Sadowski The incorporation of linkers or Spacers, Such as flexible 2. et al., U.S. Pat. No. 5,885,779), herein incorporated linkers, between the individual domains that make-up the by reference); multimerized agent can also be advantageous for Some embodiments. The insertion of 2 linkers of an appropriate 0275 (4) phage display (Lowman H B, Annu. Rev. length between protein binding domains, for example, can Biophys. Biomol. Struct. 26:401-424 (1997), herein encourage greater flexibility in the molecule and can over incorporated by reference); and come Steric hindrance. Similarly, the insertion of linkers 0276 (5) GST/HIS pull down assays, mutant opera between the multimerized binding partner or polymorphic tors (Granger et al., WO 98/01879) and the like (See and/or mutant A2M polypeptides and the Support can also Mathis G., Clin. Chem. 41:139-147 (1995); Lam encourage greater flexibility and limit Steric hindrance pre K. S. Anticancer Drug Res., 12:145-167 (1997); and Sented by the Support. The determination of an appropriate Phizicky et al., Microbiol. Rev. 59:94-123 (1995), all length of linker can be determined by Screening the poly references herein expressly incorporated by refer morphic and/or mutant A2M polypeptides and binding part ence). ners with varying linkers in the assays detailed in this 0277. An adaptation of the system described by Chien et disclosure. al., 1991, Proc. Natl. Acad. Sci. USA, 88: 9578-9582, herein incorporated by reference), which is commercially available 0269. Thus, several approaches to identify agents that from Clontech (Palo Alto, Calif.) is as follows. Plasmids are interact with a polymorphic and/or mutant A2M polypep constructed that encode two hybrid proteins: one plasmid tide, employ a polymorphic and/or mutant A2M polypeptide consists of nucleotides encoding the DNA-binding domain joined to a Support. Once the Support-bound polypeptide is of a transcription activator protein fused to a nucleotide obtained, for example, candidate binding partners are con Sequence encoding a polymorphic and/or mutant A2M tacted to the Support-bound polypeptide and an association polypeptide, and the other plasmid consists of nucleotides is determined directly (e.g., by using labeled binding part encoding the transcription activator protein's activation ner) or indirectly (e.g., by using a labeled antibody directed domain fused to a cDNA encoding an unknown protein that to the binding partner). Candidate binding partners are has been recombined into this plasmid as part of a cDNA identified as binding partners by virtue of the association library. The DNA-binding domain fusion plasmid and the with the Support-bound polypeptide. The properties of the cDNA library are transformed into a strain of the yeast binding partners are analyzed and derivatives are made Saccharomyces cerevisiae that contains a reporter gene (e.g., using rational drug design and combinatorial chemistry. HBS or lacz) whose regulatory region contains the tran Candidate binding partners can be obtained from random Scription activator's binding Site. Either hybrid protein alone chemical or peptide libraries but, preferably, are rationally cannot activate transcription of the reporter gene: the DNA Selected. For example, monoclonal antibodies that bind to binding domain hybrid cannot because it does not provide polymorphic and/or mutant A2M polypeptides can be cre activation function and the activation domain hybrid cannot ated and the nucleic acids encoding the VH and VL domains because it cannot localize to the activator's binding sites. of the antibodies can be sequenced. These Sequences can Interaction of the two hybrid proteins reconstitutes the then be used to Synthesize peptides that bind to the poly functional activator protein and results in expression of the morphic and/or mutant A2M polypeptides. Further, pepti reporter gene, which is detected by an assay for the reporter domimetics corresponding to these Sequences can be cre gene product. ated. These molecules can then be used as candidate binding partners. 0278. The two-hybrid system or related methodology can be used to Screen activation domain libraries for proteins 0270 Additionally, a cell based approach can be used that interact with the “bait' gene product. By way of characterize polymorphic and/or mutant A2M polypeptides example, and not by way of limitation, polymorphic and/or or to rapidly identify binding partners that interact with Said mutant A2M polypeptides can be used as the bait gene polypeptides and, thereby, modulate A2M activities. Pref product. Total genomic or cDNA sequences are fused to the erably, molecules identified in the Support-bound A2Massay DNA encoding an activation domain. This library and a described above are used in the cell based approach, how plasmid encoding a hybrid of a bait gene encoding the ever, randomly generated compounds can also be used. polymorphic and/or mutant A2M polypeptide fused to the DNA-binding domain are cotransformed into a yeast 0271 Many A2M characterization assays take advantage reporter Strain, and the resulting transformants are Screened of techniques in molecular biology that are employed to for those that express the reporter gene. For example, and discover protein-protein interactions. One method that not by way of limitation, a bait gene Sequence encoding a detects protein-protein interactions in Vivo, the two-hybrid polymorphic and/or mutant A2M polypeptide can be cloned System, is described in detail for illustration only and not by into a vector such that it is translationally fused to the DNA way of limitation. Other similar assays that can be can be encoding the DNA-binding domain of the GAL4 protein. adapted to identify binding partners include: These colonies are purified and the library plasmids respon 0272 (1) the two-hybrid systems (Field & Song, Sible for reporter gene expression are isolated. DNA Nature 340:245–246 (1989); Chien et al., Proc. Natl. Sequencing is then used to identify the proteins encoded by Acad. Sci. USA 88: 9578-9582 (1991); and Young K the library plasmids. H, Biol. Reprod. 58:302-311 (1998), all references 0279 AcDNA library of the cell line from which proteins herein expressly incorporated by reference); that interact with bait polymorphic and/or mutant A2M US 2003/0162202 A1 Aug. 28, 2003 30 polypeptides are to be detected can be made using methods 0283 The effective dose and method of administration of routinely practiced in the art. According to the particular a particular pharmaceutical formulation having polymorphic System described herein, for example, the cDNA fragments and/or mutant A2M polypeptides or nucleic acids or binding can be inserted into a vector Such that they are translationally partners, or fragments thereof can vary based on the indi fused to the transcriptional activation domain of GAL4. This vidual needs of the patient and the treatment or preventative library can be co-transformed along with the bait polymor measure Sought. Therapeutic efficacy and toxicity of Such phic and/or mutant A2M gene-GAL4 fusion plasmid into a compounds can be determined by Standard pharmaceutical yeast Strain which contains a lacZ gene driven by a promoter procedures in cell cultures or experimental animals, e.g., which contains GAL4 activation Sequence. A cDNA ED50 (the dose therapeutically effective in 50% of the encoded protein, fused to GAL4 transcriptional activation population). The data obtained from these assays is then domain, that interacts with bait A2M gene product will used in formulating a range of dosage for use with other reconstitute an active GAL4 protein and thereby drive organisms, including humans. The dosage of Such com expression of the lacZ gene. Colonies that express lac7, can pounds lies preferably within a range of circulating concen be detected and the cDNA can then be purified from these trations that include the ED50 with no toxicity. The dosage Strains, and used to produce and isolate the binding partner varies within this range depending upon type of polymor by techniques routinely practiced in the art. The examples phic and/or mutant A2M polypeptide or nucleic acid or below describe preferred A2M characterization assayS. binding partner, or fragment thereof, the dosage form employed, Sensitivity of the organism, and the route of 0280 Pharmaceutical Preparations and Methods of administration. Administration 0284. Normal dosage amounts of various polymorphic 0281. The polymorphic and/or mutant A2M nucleic acids and/or mutant A2M polypeptide or nucleic acid or binding and polypeptides and their binding partners are Suitable for partner, or fragment thereof can vary from any number incorporation into pharmaceuticals that treat or prevent between approximately 1 to 100,000 micrograms, up to a neuropathies, Such as AD. These pharmacologically active total dose of about 10 grams, depending upon the route of compounds can be processed in accordance with conven administration. Desirable dosages include, for example, 250 tional methods of galenic pharmacy to produce medicinal tig, 500 lig, 1 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, agents for administration to organisms, e.g., plants, insects, .300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mold, yeast, animals, and mammals including humans. The mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1 active ingredients can be incorporated into a pharmaceutical g, 1.1 g, 1.2g, 1.3 g, 1.4g, 1.5 g, 1.6 g., 1.7g, 1.8g, 1.9 g, product with and without modification. Further, the manu facture of pharmaceuticals or therapeutic agents that deliver 2g, 3 g, 4 g., 5, 6 g, 7 g., 8 g, 9 g, and 10 g. the pharmacologically active compounds of this invention 0285) In some embodiments, the dose of polymorphic by Several routes are aspects of the invention. For example, and/or mutant A2M polypeptide or nucleic acid or binding and not by way of limitation, DNA, RNA, and viral vectors partner, or fragment thereof preferably produces a tissue or having Sequence encoding the polymorphic and/or mutant blood concentration or both from approximately any number A2M polypeptides, binding partners, or fragments thereof between 0.1 M to 500 mM. Desirable doses produce a are used with embodiments. Nucleic acids encoding poly tissue or blood concentration or both of about any number morphic and/or mutant A2M polypeptides or binding part between 1 to 800 uM. Preferable doses produce a tissue or ners can be administered alone or in combination with other blood concentration of greater than about any number active ingredients. between 10 uM to about 500 uM. Preferable doses are, for 0282. The compounds of this invention can be employed example, the amount of active ingredient required to achieve in admixture with conventional excipients, i.e., pharmaceu a tissue or blood concentration or both of 10 uM, 15 uM, 20 tically acceptable organic or inorganic carrier Substances uM, 25uM, 30 uM, 35uM, 40 uM, 45uM, 50 uM, 55uM, Suitable for parenteral, enteral (e.g., oral) or topical appli 60 uM, 65uM, 70 uM, 75 uM, 80 uM, 85 uM, 90 uM, 95 cation that do not deleteriously react with the pharmaco uM, 100 uM, 110 uM, 120 uM, 130 uM, 140 uM, 145 uM, logically active ingredients of this invention. Suitable phar 150 uM, 160 uM, 170 uM, 180 uM, 190 uM, 200 uM, 220 maceutically acceptable carriers include, but are not limited uM, 240 uM, 250 uM, 260 uM, 280 uM, 300 uM, 320 uM, to, water, Salt Solutions, alcohols, gum arabic, vegetable oils, 340 uM, 360 uM, 380 uM, 400 uM, 420 uM, 440 uM, 460 benzyl alcohols, polyetylene glycols, gelatine, carbohy LiM, 480 uM, and 500 uM. Although doses that produce a drates Such as lactose, amylose or Starch, magnesium Stear tissue concentration of greater than 800 uM are not pre ate, talc, Silicic acid, Viscous paraffin, perfume oil, fatty acid ferred, they can be used with some embodiments of the monoglycerides and diglycerides, pentaerythritol fatty acid invention. A constant infusion of the polymorphic and/or esters, hydroxy methylcellulose, polyvinyl pyrrollidone, etc. mutant A2M polypeptide or nucleic acid or binding partner, Many more suitable vehicles are described in Remmington's or fragment thereof can also be provided So as to maintain Pharmaceutical Sciences, 15th Edition, Easton: Mack Pub a stable concentration in the tissueS as measured by blood lishing Company, pages 1405-1412 and 1461-1487(1975) levels. and The National Formulary XIV, 14th Edition, Washington, 0286 The exact dosage is chosen by the individual American Pharmaceutical Association (1975), herein incor physician in View of the patient to be treated. Dosage and porated by reference. The pharmaceutical preparations can administration are adjusted to provide Sufficient levels of the be sterilized and if desired mixed with auxiliary agents, e.g., active moiety or to maintain the desired effect. Additional lubricants, preservatives, Stabilizers, wetting agents, emul factors that can be taken into account include the Severity of sifiers, Salts for influencing OSmotic preSSure, buffers, col the disease, age of the organism, and weight or size of the oring, flavoring and/or aromatic Substances and the like that organism; diet, time and frequency of administration, drug do not deleteriously react with the active compounds. combination(s), reaction Sensitivities, and tolerance/re US 2003/0162202 A1 Aug. 28, 2003

Sponse to therapy. Short acting pharmaceutical compositions 0292 Having now generally described the invention, the are administered daily whereas long acting pharmaceutical following examples are offered to illustrate, but not to limit compositions are administered every 2, 3 to 4 days, every the claimed invention. week, or once every two weekS. Depending on half-life and clearance rate of the particular formulation, the pharmaceu EXAMPLES tical compositions of the invention are administered once, 0293. The nucleic acid embodiments of the invention twice, three, four, five, Six, Seven, eight, nine, ten or more include isolated or purified nucleic acids comprising, con times per day. sisting essentially of, or consisting of an A2M gene (e.g., 0287 Routes of administration of the pharmaceuticals of SEQ ID NO: 1) with one or more of the SNPs and/or the invention include, but are not limited to, topical, trans mutations described in Table 1. Other embodiments include dermal, parenteral, gastrointestinal, transbronchial, and tran isolated or purified nucleic acids comprising, consisting Salveolar. Transdermal administration is accomplished by essentially of, or consisting of an A2M gene having at least application of a cream, rinse, gel, etc. capable of allowing one SNP and/or mutation described in Table 1 along with the pharmacologically active compounds to penetrate the other SNPs, such as those described in Table 2. Still other skin. Parenteral routes of administration include, but are not embodiments relate to isolated or purified nucleic acid limited to, electrical or direct injection Such as direct injec fragments of the A2M gene which include at least one of the tion into a central venous line, intravenous, intramuscular, SNPs described in Table 1. Such fragments can range in intraperitoneal, intradermal, or Subcutaneous injection. Gas length from at least 10, at least 15, at least 20, at least 25, at trointestinal routes of administration include, but are not least 30, at least 40, at least 50, at least 75, at least 100, a limited to, ingestion and rectal. Transbronchial and transal least 150, at least 200, at least 250, at least 300, at least 400, Veolar routes of administration include, but are not limited at least 500, at least 750, at least 1000, at least 2500, at least to, inhalation, either via the mouth or intranasally. 5000, at least 7500, at least 10,000, at least 20,000, at least 0288 Compositions having the pharmacologically active 30,000, at least 40,000, at least 50,000 or greater than 50,000 compounds of this invention that are Suitable for transdermal nucleotides and include both exons and introns of the A2M or topical administration include, but are not limited to, gene. Isolated or purified nucleic acid fragments of the A2M pharmaceutically acceptable Suspensions, oils, creams, and gene having at least one SNP and/or mutation described in ointments applied directly to the skin or incorporated into a Table 1 along with other SNPs, such as those described in protective carrier Such as a transdermal device ("transder Table 2, are also contemplated. Such fragments can range in mal patch'). Examples of Suitable creams, ointments, etc. length from at least 10, at least 15, at least 20, at least 25, at can be found, for instance, in the Physician's Desk Refer least 30, at least 40, at least 50, at least 75, at least 100, a ence. Examples of Suitable transdermal devices are least 150, at least 200, at least 250, at least 300, at least 400, described, for instance, in U.S. Pat. No. 4,818,540 issued at least 500, at least 750, at least 1000, at least 2500, at least Apr. 4, 1989 to Chinen, et al., herein incorporated by 5000, at least 7500, at least 10,000, at least 20,000, at least reference. 30,000, at least 40,000, at least 50,000 or greater than 50,000 0289 Compositions having the pharmacologically active nucleotides and include both exons and introns of the A2M compounds of this invention that are Suitable for parenteral gene. Other embodiments of the present invention include administration include, but are not limited to, pharmaceuti fragments of the A2M gene, wherein the fragments contains cally acceptable Sterile isotonic Solutions. Such Solutions at least 9, at least 16, or at least 18 consecutive nucleotides include, but are not limited to, Saline and phosphate buffered of the polymorphic or mutant A2M gene but including at Saline for injection into a central venous line, intravenous, least one of the SNPs and/or mutations in Table 1. Isolated intramuscular, intraperitoneal, intradermal, or Subcutaneous or purified nucleic acids that are complementary to Said injection. A2M nucleic acids and fragments thereof are also embodi ments. Some embodiments also concern genomic DNA, 0290 Compositions having the pharmacologically active RNA, and cDNA corresponding to polymorphic and/or compounds of this invention that are Suitable for transbron mutant A2M genes, described herein. Accordingly, in Some chial and transalveolar administration include, but not lim contexts, the term “polymorphic and/or mutant A2M nucleic ited to, various types of aerosols for inhalation. Devices acids’ refers not only to the full-length polymorphic and/or Suitable for transbronchial and transalveolar administration mutant A2M nucleic acids (e.g., SEQ ID NOs: 1) but also to of these are also embodiments. Such devices include, but are fragments of these molecules at least 9, at least 16, or at least not limited to, atomizers and vaporizers. Many forms of 18 nucleotides in length but containing at least one of the currently available atomizers and vaporizers can be readily SNPs and/or mutations identified in Table 1, nucleic acids adapted to deliver compositions having the pharmacologi that are complementary to Said full-length Sequences and cally active compounds of the invention. fragments thereof, and genomic DNA, RNA, and cDNA 0291 Compositions having the pharmacologically active corresponding to Said Sequences. compounds of this invention that are Suitable for gastrointes 0294 The discovery of SNPs and/or mutations in the tinal administration include, but not limited to, pharmaceu A2M gene was made while analyzing the Sequences of the tically acceptable powders, pills or liquids for ingestion and A2M gene obtained from patients suffering from AD. The Suppositories for rectal administration. Due to the ease of use, gastrointestinal administration, particularly oral, is a approaches used in these experiments is described in preferred embodiment. Once the pharmaceutical comprising EXAMPLE 1. the polymorphic and/or mutant A2M polypeptide or nucleic Example 1 acid or binding partner, or fragment thereof has been obtained, it can be administered to a organism in need to 0295 Methods of Identifying SNPs and Other Mutations treat or prevent a neuropathy, Such as AD. in the A2M Gene The following protocol that was used to US 2003/0162202 A1 Aug. 28, 2003 32 identify the SNPs and/or mutations described herein in manufacturers instructions. Thermocycling included 30 patients from the National Intstitute of Mental Health cycles of (a) 96° C. for 10 seconds; (b) 50° C. for 5 seconds; (NIMH) AD Genetics Initiative Sample. However, it will be and (c) 60° C. for 4 minutes. Reaction products were purified appreciated that this protocol has general applicability to any using CentriSep 96 well plates (Princeton Separations) human Subject. according to manufacturer's instructions. Data was collected 0296. The A2M gene was identified as a candidate gene from purified reaction products using an ABI 3700 DNA linked to AD based both on its known function and available Analyzer. linkage data. Sample Sets of DNA showing Strong linkage 0301 Using the above amplification and sequencing pro disequilibrium and/or association in the A2M region were tocol, several SNPs and/or mutations were found in the A2M chosen for further study. gene, including both exon and intron regions, in individuals 0297. The genomic DNA sequence of the A2M gene was having AD. These results are set out in Table 1 herein. obtained as a part of the draft Sequence of chromosome 12 0302) In view of the fact that the presence of one or more from a Human Genome Project information database located of SNPs and/or mutations in an individual can present a risk at the University of California Santa Cruz available at that the individual will acquire AD, it is contemplated that genome.ucSc.edu. The full-length A2M coding Sequence the SNPs and/or mutations described in Table 1 (i.e., 6i, (SEQ ID NO: 2) and A2M protein (SEQ ID NO: 9) 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i Sequences were also obtained. The coordinates of publicly and 30e) can be indicative for altered risk for AD. As a available SNPs in the A2M gene were obtained from bio preliminary evaluation of the risk associated with possessing chip.org. The program SNPer (available at bio.chip.org) one or more of these SNPs, an association analysis in was used to place the publicly available SNPs in relation to families and individuals having AD was performed. That is, the exons of the A2M gene. Exon positions generated by the nucleotide identities at the position of one or more of SNPer were verified by comparing the cDNA sequence SNPs and/or mutations included in Table 1 (i.e., 6i, 12i.1, (SEQ ID NO: 2) to the genomic database at the NCBI using 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e) (Basic Local Alignment Search Tool) BLASTN with the in individuals and families with AD were determined and default filter (Altschul, et al. (1990).J. Mol. Biol. 215:403 tested by both single SNP association analyses and haplo 410). Alternatively, the A2M cDNA sequence was queried type analyses. EXAMPLE 2 describes these experiments. against the High Throughput Genomic Sequence (HTGS) database using BLASTN. Example 2 0298 Subsequent to exon verification, specific regions of the A2M gene were Selected for Sequencing. Regions 0303 Association of A2M SNPs and Haplotypes with Selected for Sequencing were as follows: (1) a region begin Alzheimer's Disease ning approximately 1000 base pairs upstream of the nucleic 0304. The polymorphisms listed in Table 1 can be acid Sequence corresponding to the Start codon and extend detected from biological Samples provided by families hav ing about 150-200 base pairs beyond last nucleotide of the ing members afflicted with AD using the methods described first exon; (2) a region beginning approximately 150-200 below as well as methods known to those having ordinary base pairs upstream of the nucleic acid Sequence corre skill in the art. Furthermore, association of one or more sponding to the beginning of the least exon of the A2M gene polymorphisms listed in Table 1 with an altered risk of AD and extending about 700 base pairs beyond last nucleotide of can be determined using the methods described below as this exon; and (3) a nucleic acid region Surrounding each well as those described in U.S. Pat. No. 6,265,546, the exon which begins approximately 150-200 base pairs disclosure of which is incorporated herein by reference in its upstream and ends approximately 150-200 base pairs down entirety, and those methods known to those having ordinary Stream of each remaining eXon. skill in the relevant art. AS described in Example 1, for each 0299. Within the selected regions, 500-800 base pair of the polymorphisms listed in Table 1, the A2M-1 allele fragments were amplified by using amplification primers corresponds to the allele represented in SEQ ID NO: 1. The flanking specific regions of interest (forward and reverse A2M-2 allele corresponds to an allele having the polymor primers). In general, primers used for amplification ranged phic change (nucleotide Substitution or mutation) as indi from 20 to 24 nucleotides and had an annealing temperature cated in column 3 of Table 1 at the Sequence position between 54-60C. Amplification was performed using about Specified in column 2 of Table 1 (the positions and nucle 30 ng of human genomic DNA, 5 umol of each primer, and otides affected by each polymorphism and/or mutation are HotStarTaq Mix (Qiagen). Thermocycling was initiated by also provided in the Figure). heating for 15 minutes at 95 C. followed by 35 cycles of (a) 0305) To test for a link between the polymorphisms 94 C. for 30 seconds; (b) primer annealing temperature for described herein and AD, Samples from families having 45 seconds; and (c) 72° C. for 1 minute. The cycling was members afflicted with AD were used. An example of an followed by a final 7 minute extension at 72 C. Subsequent appropriate population is the National Institute of Mental to thermocycling, PCR products were purified then quanti Health (NIMH) Genetics Initiative AD sample, a large tated. Sample of affected Sibling pairs and other Small families with 0300 Both strands of each amplified fragment were AD. It should be noted, however, that any population of Sequenced using Sequencing primers complementary to a families having members meeting the criteria described region near the 3'-end of each Strand. Approximately, 3.2 below can be used for association and haplotype analyses. pmol of Sequencing primer and 12 ng of amplified fragment 0306 Participants in the NIMH sample were recruited were added to Sequencing buffer including Big Dye Termi from local memory disorder clinics, nursing homes, and the nator Mix (Applied Biosystems-ABI) according to the Surrounding communities with the only requirement for US 2003/0162202 A1 Aug. 28, 2003

inclusion in the Sample being that each family member final concentration is 200 mM each of dATP, dCTP, and include at least two living blood relatives with memory dTTP; and 2 mM dGTP), 1 mCi alpha-32PdGTP or P problems. They were evaluated following a Standardized dGTP, 15 pM of each flanking primer and 0.25 units of Taq protocol (Blacker, D., et al., Arch. Neurol. 51:1198-1204 polymerase in a total Volume of 10 it. The reaction are (1994)) to assure that they met NINCDS/ADRDA criteria denatured at 94 C. for 4 minutes, followed by 25-30 cycles for Probable AD (or in the case of secondary probands, of 1 minute denaturing at 94 C., 0.5-1 minute annealing Possible AD) (McKhann, G., et al., Neurology 34:939-944 (variable temperature, usually 55-65 C.) and extension for (1984)), or research pathological criteria for Definite AD 1 minute at 72° C. Forty-eight (48) experimental and two (Khachaturian, Z., Arch. Neurol. 42:1005 (1985)). Among control (for standardization of size) samples are loaded on a the affected individuals, 142 (22.2%) had autopsy confir gel at one time, thereby increasing the amount of informa mation of the diagnosis of AD. Unaffected relatives, gener tion per gel. Whenever possible (e.g., if maker background ally Siblings, were included when they were available and is sufficiently low) multiple markers (two to four markers) Willing to participate. are multiplexed, or are temporally staggered (30-45 min 0307 There were a total of 239 unaffected subjects from utes) two to three mm on a single gel. Allele sizes for CEPH 131 families (45.6%). An additional 22 study subjects with individuals 1331-01 and 1331-02 are used as standards. In blood available who had unclear phenotypes were consid the rare event that no Standards are available for a marker, ered phenotype unknown, as were 5 unaffected Subjects with an initial gel is run, which includes a Sequencing ladder, to unknown ages, and 19 unaffected subjects below 50 years of determine allele sizes in these individuals. Two till of Sample age (primarily children of affected participants). There were are mixed with loading dye and size-fractionated on a 6% a total of 639 individuals affected with AD, from 286 denaturing polyacrylamide gel. The gels are then dried and families. The majority of the affected individuals were placed on X-ray film for 2-24 hrs. at -80 C. and read by two sibling pairs (202 families, 71%), but there were 46 larger independent readers. Sibships (16%), and 38 families with other structures (13%; 0312. It will be apprciated that the manual geneotyping e.g., parent-child, first cousin, avuncular, extended). All method described above is only one method that is available Subjects (or, for significantly cognitively impaired individu for detecting Specific alleles at polymorphic loci. Several als, their legal guardian or caregiver with power of attorney) other methods that are useful for detecting Specific alleles at gave informed consent. polymorphic loci, in particular human polymorphic loci. The 0308 The full NIMH sample can be used in the descrip preferred method for detecting a particular polymorphism, tive Statistics for genotype counts and allele frequencies, for depends on the nature of the polymorphism. Several meth the analyses of age of onset in affected individuals, and for ods of determining the presence or absence of allelic vari all of the genetic linkage analyses (except ASPEX, which ants of a gene are provided below. Methods that are useful uses sibships only). However, because the Mantel-Haenzel are not limited to those described below, but include all test, conditional logistic regression, and Sibship DiSequal available methods. ibrium Test and EV-FBAT depend on comparisons of closely 0313 Generally, these methods are based in sequence related affected and unaffected individuals, they are per Specific polynucleotides, oligonucleotides, probes and prim formed on a Subsample including all families in which there ers. Any method known to those of skill in the art for is at least one affected and at least one unaffected Sibling detecting a specific nucleotide within a nucleic acid with A2M data available: 104 families with 217 affected and Sequence or for determining the identity of a specific nucle 181 unaffected siblings. otide in a nucleic acid Sequence is applicable to the methods of determining the presence or absence of an allelic variant 0309. In order to avoid examining very early onset AD, of these genes on chromosome 12. Such methods include, which appears to have a distinct genetic etiology (Blacker, but are not limited to, techniques utilizing nucleic acid D. & Tanzi, R. E., Arch Neurol 55:294-296 (1998)), only hybridization of Sequence-specific probes, nucleic acid those families in which all examined affected individuals Sequencing, Selective amplification, analysis of restriction experienced the onset of AD at age 50 of later are included. enzyme digests of the nucleic acid, cleavage of mismatched Although Late Onset Alzheimer's Disease (LOAD) is con heteroduplexes of nucleic acid and probe, alterations of ventionally identified based on onset after age 60, families electrophoretic mobility, primer Specific extension, oligo with onsets between 50 and 60 are included because onset in nucleotide ligation assay and Single-Stranded conformation this decade is only partly explained by the known AD genes. polymorphism analysis. In particular, primer extension reac Age of onset is determined based on an interview with a tions that specifically terminate by incorporating a dideoxy knowledgeable informant and review of medical records. nucleotide are useful for detection. Several Such general 0310. The polymorphisms described herein can be manu nucleic acid detection assays are known (see, e.g., U.S. Pat. ally genotyped according to, for example, the protocol No. 6,030,778). described in Matthijs et al. (Matthijs, G., & Marynen, P., Nuc. Acid Res. 19:5102 (1991)). Alternatively, an appropri 0314. Any cell type or tissue may be utilized to obtain ate fragment of the A2M gene corresponding to the region nucleic acid Samples, e.g., bodily fluid Such as blood or of a polymorphism and/or mutation described herein is Saliva, dry Samples Such as hair or skin. amplified and Sequenced using the methods described in 0315) a. Primer Extension-Based Methods Example I. 0316 Several primer extension-based methods for deter 0311. In one example, manual genotyping is carried out mining the identity of a particular nucleotide in a nucleic using a 96-well microtiter dish format as follows. Three to acid Sequence have been reported (see, e.g., PCT Applica 10 nanograms of human DNA is mixed with a reaction tion Nos. PCT/US96/03651 (WO96/29431), PCT/US97/ buffer, deoxynucleotide mix (e.g. for a poly-dGdTISTR, the 20444 (WO 98/20166), PCT/US97/20194 (WO 98/20019), US 2003/0162202 A1 Aug. 28, 2003 34

PCT/US91/00046 (WO91/13075), and U.S. Pat. Nos. 5,547, 0318 b. Polymorphism-Specific Probe Hybridization 835, 5,605,798, 5,622,824, 5,691,141, 5,872,003, 5,851,765, 03.19. Another detection method is allele specific hybrid 5,856,092, 5,900,481, 6,043,031, 6,133,436 and 6,197.498.) ization using probes overlapping the polymorphic Site and In general, a primer is prepared that Specifically hybridizes having about 5, 10, 15, 20, 25, or 30 nucleotides around the adjacent to a polymorphic Site in a particular nucleic acid polymorphic region. The probes can contain naturally occur molecule. The primer is then extended in the presence of one ring or modified nucleotides (see U.S. Pat. No. 6,156,501). or more dideoxynucleotides, typically with at least one of For example, oligonucleotide probes may be prepared in the dideoxynucleotides being the complement of the nucle which the known polymorphic nucleotide is placed centrally otide that is polymorphic at the Site. The primer and/or the (allele-specific probes) and then hybridized to target DNA dideoxynucleotides may be labeled to facilitate a determi under conditions which permit hybridization only if a per nation of primer extension and identity of the extended fect match is found (Saiki et al. (1986) Nature 324:163; nucleotide. Saiki et al. (1989) Proc. Natl. Acad. Sci U.S.A. 86:6230; and Wallace et al. (1979) Nucl. Acids Res. 6:3543). Such allele 0317. A preferred method of genotyping or determining Specific oligonucleotide hybridization techniques may be the presence of an allelic variant two-dye fluorescence used for the Simultaneous detection of Several nucleotide polarization detected single base extension (FP-SBE (12)) changes in different polymorphic regions. For example, on an LJL-Biosystems Criterion Analyst AD (Molecular oligonucleotides having nucleotide Sequences of Specific Devices, Sunnyvale, Calif.). PCR primers are designed to allelic variants are attached to a hybridizing membrane and yield products between 200-400 bp in length, and are used this membrane is then hybridized with labeled sample at a final concentration of 100-300 nM (Invitrogen Corp., nucleic acid. Analysis of the hybridization signal will then Carlsbad, Calif.) along with Taq polymerase (0.25 U/reac reveal the identity of the nucleotides of the Sample nucleic acid. In a preferred embodiment, Several probes capable of tion; Qiagen, Valencia, Calif. and Roche, Indianapolis, Ind.) hybridizing Specifically to allelic variants are attached to a and dNTPs (2.5uM/rxn; Amersham-Pharmacia, Piscataway, Solid phase Support, e.g., a “chip'. Oligonucleotides can be N.J.). All PCR reactions are performed from -10 ng of bound to a Solid Support by a variety of processes, including DNA. General PCR thermo-cycling conditions are as fol lithography. For example a chip can hold up to 250,000 lows: initial denaturation 3 minutes at 94EC, followed by oligonucleotides (GeneChip, Affymetrix, Santa Clara, 30-35 cycles of denaturation at 94EC for 45 seconds, Calif.). Mutation detection analysis using these chips com primer-specific annealing temperature (see below) for 45 prising oligonucleotides, also termed "DNA probe arrays” is seconds, and product extension at 72EC for 1 minute. Final described e.g., in Cronin et al. (1996) Human Mutation extension at 72EC for six minutes. PCR products can be 7:244 and in Kozal et al. (1996) Nature Medicine 2:753. In Visualized on 2% agarose-gels to confirm a single product of one embodiment, a chip includes all the allelic variants of at the correct size. PCR primers and unincorporated dNTPs least one polymorphic region of a gene. The Solid phase can be degraded by adding exonuclease I (Exol, 0.1-0.15 Support is then contacted with a test nucleic acid and U/reaction; New England Biolabs, Beverly, Mass.) and hybridization to the Specific probes is detected. Accordingly, shrimp alkaline phosphatase (SAP, 1U/reaction; Roche, the identity of numerous allelic variants of one or more Indianapolis, Ind.) to the PCR reactions and incubating for genes can be identified in a simple hybridization experiment. 1 hour at 37EC, followed by 15 minutes at 95EC to 0320 C. Nucleic Acid Amplification-Based Methods inactivate the enzymes. The Single base extension Step is 0321) In other detection methods, it is necessary to first performed by directly adding SBE primer (100 nM; Invit amplify at least a portion of a gene prior to identifying the rogen Corp., Carlsbad, Calif.), ThermoSequenase (0.4 U/re allelic variant. Amplification can be performed, e.g., by PCR action; Amersham-Pharmacia, Piscataway, N.J.), and the and/or LCR, according to methods known in the art. In one appropriate mixture of R110-ddNTP, TAMRA-ddNTP (3 embodiment, genomic DNA of a cell is exposed to two PCR uM; NEN, Boston, Mass.), and all four unlabeled ddNTPs primerS and amplification is performed for a number of (22 or 25 uM; Amersham-Pharmacia, Piscataway, N.J.) to cycles Sufficient to produce the required amount of amplified the Exol/SAP treated PCR product. Acycloprime-FP SNP detection kits (G/A)(Perkin-Elmer, Boston, Mass.) may also DNA. In another embodiment, the primers are located be used for the SBE reaction. Incorporation of the SNP between 150 and 350 base pairs apart. specific fluorescent ddNTP is achieved by subjecting 0322. Alternative amplification methods include: self samples to 35 cycles of 94EC for 15 seconds and 55EC for sustained sequence replication (Guatelli, J. C. et al. (1990) 30 seconds. The length of the SBE primers are designed to Proc. Natl. Acad. Sci. U.S.A. 87: 1874-1878), transcriptional yield a melting temperature T of 62-64EC. Fluorescent amplification system (Kwoh, D. Y. et al. (1989) Proc. Natl. ddNTP incorporation is detected using the AnalystTM AD Acad. Sci. U.S.A. 86: 1173-1177), Q-Beta Replicase (Lizardi, System (Molecular Devices, Sunnyvale, Calif.) and measur P. M. et al. (1988) Bio/Technology 6:1197), or any other ing fluorescent polarization for R110 (excitation at 490 nm, nucleic acid amplification method, followed by the detection emission at 520 nm) and TAMRA (excitation at 550 nm, of the amplified molecules using techniques well known to emission at 580 nm). Genotypes are called manually or those of skill in the art. These detection Schemes are espe automatically using the manufacturer's Software ( Allele cially useful for the detection of nucleic acid molecules if caller vers. 1.0, Molecular Devices, Sunnyvale, Calif.). In Such molecules are present in very low numbers. view of the polymorphic regions provided herein, SNP 0323 Alternatively, allele specific amplification technol Specific PCR primers (5' to 3' sequences), annealing tem ogy, which depends on Selective PCR amplification may be perature, product length, SBE primer Sequence, SNP loca used in conjunction with the alleles provided herein. Oligo tion and reference Sequence position, can readily be deter nucleotides used as primers for Specific amplification may mined by those of skill in the art using well-known methods. carry the allelic variant of interest in the center of the US 2003/0162202 A1 Aug. 28, 2003

molecule (So that amplification depends on differential a nucleotide Sequence of an allelic variant with a Sample hybridization) (Gibbs et al. (1989) Nucleic Acids Res. nucleic acid, e.g., RNA or DNA, obtained from a tissue 17:2437-2448) or at the extreme 3' end of one primer where, Sample. The double-Stranded duplexes are treated with an under appropriate conditions, mismatch can prevent, or agent, which cleaves Single-Stranded regions of the duplex reduce polymerase extension (Prossner (1993) Tibtech Such as duplexes formed based on basepair mismatches 11:238; Newton et al. (1989) Nucl. Acids Res. 17:2503). In between the control and sample strands. For instance, RNA/ addition it may be desirable to introduce a restriction site in DNA duplexes can be treated with RNase and DNA/DNA the region of the mutation to create cleavage-based detection hybrids treated with S1 nuclease to enzymatically digest the (Gasparini et al. (1992) Mol. Cell Probes 6:1). mismatched regions. 0324 d. Nucleic Acid Sequencing-Based Methods 0330. In other embodiments, either DNA/DNA or RNA/ DNA duplexes can be treated with hydroxylamine or 0325 Any of a variety of sequencing reactions known in oSmium tetroxide and with piperidine in order to digest the art can be used to directly Sequence at least a portion of mismatched regions. After digestion of the mismatched a gene and to detect allelic variants, e.g., mutations, by regions, the resulting material is then Separated by Size on comparing the Sequence of the Sample Sequence with the denaturing polyacrylamide gels to determine whether the corresponding wild-type (control) sequence. Exemplary control and Sample nucleic acids have an identical nucle Sequencing reactions include those based on techniques otide sequence or in which nucleotides they differ (See, for developed by Maxam and Gilbert (1977) Proc. Natl. Acad. example, Cotton et al. (1988) Proc. Natl Acad Sci U.S.A. Sci. U.S.A. 74:560) or Sanger et al. (1977) Proc. Natl. Acad. 85:4397; Saleeba et al. (1992) Methods Enzymod. 217:286 Sci 74:5463. It is also contemplated that any of a variety of 295). The control or sample nucleic acid is labeled for automated Sequencing procedures may be used when per detection. forming the subject assays (1995) Biotechniques 19:448), including Sequencing by mass spectrometry (see, for 0331 g. Electrophoretic Mobility Alterations example, U.S. Pat. Nos. 5,547,835, 5,691,141, and Interna 0332. In other embodiments, alteration in electrophoretic tional PCT Application No. PCT/US94/00193 (WO mobility is used to identify the type of allelic variant of a 94/16101), entitled “DNA Sequencing by Mass Spectrom gene of interest. For example, Single-Strand conformation etry” by H. Koster; U.S. Pat. Nos. 5,547,835, 5,622,824, polymorphism (SSCP) may be used to detect differences in 5,851,765, 5,872,003, 6,074,823, 6,140,053 and Interna electrophoretic mobility between mutant and wild type tional PCT Application No. PCT/US94/02938 (WO nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. 94/21822), entitled “DNA Sequencing by Mass Spectrom U.S.A. 86:2766, see also Cotton (1993) Mutat Res 285:125 etry Via Exonuclease Degradation” by H. Koster, and U.S. 144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79). Pat. Nos. 5,605,798, 6,043,031, 6,197,498, and International Single-Stranded DNA fragments of Sample and control Patent Application No. PCT/US96/03651 (WO 96/29431) nucleic acids are denatured and allowed to renature. The entitled “DNA Diagnostics Based on Mass Spectrometry” Secondary Structure of Single-Stranded nucleic acids varies by H. Koster; Cohen et al. (1996) Adv Chromatogra6:127 according to Sequence, the resulting alteration in electro 162; and Griffin et al. (1993) Appl Biochem Biotechnol phoretic mobility enables the detection of even a Single base 38:147-159). It will be evident to one skilled in the art that, change. The DNA fragments may be labeled or detected for certain embodiments, the occurrence of only one, two or with labeled probes. The sensitivity of the assay may be three of the nucleic acid bases need be determined in the enhanced by using RNA (rather than DNA), in which the Sequencing reaction. For instance, A-track Sequencing or an Secondary Structure is more Sensitive to a change in equivalent, e.g., where only one nucleotide is detected, can Sequence. In another embodiment, the Subject method uses be carried out. Other Sequencing methods are known (see, heteroduplex analysis to Separate double Stranded heterodu e.g., in U.S. Pat. No. 5,580,732 entitled “Method of DNA plex molecules on the basis of changes in electrophoretic Sequencing employing a mixed DNA-polymer chain probe' mobility (Keen et al. (1991) Trends Genet 7:5). and U.S. Pat. No. 5,571,676 entitled “Method for mismatch directed in vitro DNA sequencing”). 0333 h. Polyacrylamide Gel Electrophoresis e. Restriction Enzyme Digest Analysis 0334. In yet another embodiment, the identity of an 0326 allelic variant of a polymorphic region of an gene is obtained 0327 In some cases, the presence of a specific allele in by analyzing the movement of a nucleic acid comprising the nucleic acid, particularly DNA, from a Subject can be shown polymorphic region in polyacrylamide gels containing a by restriction enzyme analysis. For example, a specific gradient of denaturant is assayed using denaturing gradient nucleotide polymorphism can result in a nucleotide gel electrophoresis (DGGE) (Myers et al. (1985) Nature Sequence containing a restriction site which is absent from 313:495). When DGGE is used as the method of analysis, the nucleotide Sequence of another allelic variant. DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approxi 0328 f. Mismatch Cleavage mately 40 bp of high-melting GC-rich DNA by PCR. In a 0329 Protection from cleavage agents, such as, but not further embodiment, a temperature gradient is used in place limited to, a nuclease, hydroxylamine or oSmium tetroxide of a denaturing agent gradient to identify differences in the and with piperidine, can be used to detect mismatched bases mobility of control and sample DNA (Rosenbaum and in RNA/RNA DNA/DNA, or RNA/DNA heteroduplexes Reissner (1987) Biophy's Chem 265:1275). (Myers, et al. (1985) Science 230:1242). In general, the technique of "mismatch cleavage' starts by providing het 0335) i. Oligonucleotide Ligation Assay (OLA) eroduplexes formed by hybridizing a control nucleic acid, 0336. In another embodiment, identification of the allelic which is optionally labeled, e.g., RNA or DNA, comprising variant is carried out using an oligonucleotide ligation assay US 2003/0162202 A1 Aug. 28, 2003 36

(OLA), as described, e.g., in U.S. Pat. No. 4,998,617 and in diately 3' to a polymorphic site. The method determines the Landegren, U. et al. (1988) Science 241:1077-1080. The identity of the nucleotide of that site using labeled dideoxy OLA protocol uses two oligonucleotides which are designed nucleotide derivatives, which, if complementary to the to be capable of hybridizing to abutting Sequences of a Single nucleotide of the polymorphic Site will become incorporated Strand of a target. One of the oligonucleotides is linked to a onto the terminus of the primer. Separation marker, e.g. biotinylated, and the other is detect ably labeled. If the precise complementary Sequence is 0342 k. Genetic Bit Analysis found in a target molecule, the oligonucleotides will hybrid ize Such that their termini abut, and create a ligation Sub 0343 An alternative method, known as Genetic Bit Strate. Ligation then permits the labeled oligonucleotide to Analysis or GBATM is described by Goelet, et al. (U.S. Pat. be recovered using avidin, or another biotin ligand. Nick No. 6,004,744, PCT Application No.92/15712). The method erSon, D. A. et al. have described a nucleic acid detection of Goelet, et al. uses mixtures of labeled terminators and a assay that combines attributes of PCR and OLA (Nickerson, primer that is complementary to the Sequence 3' to a D. A. et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:8923 polymorphic Site. The labeled terminator that is incorporated 8927). In this method, PCR is used to achieve the exponen is thus determined by, and complementary to, the nucleotide tial amplification of target DNA, which is then detected present in the polymorphic Site of the target molecule being using OLA. evaluated. In contrast to the method of Cohen et al. (French 0337. Several techniques based on this OLA method have Patent 2,650,840; PCT Application No. WO91/02087), the been developed and can be used to detect specific allelic method of Goelet, et al. is preferably a heterogeneous phase variants of a polymorphic region of a gene. For example, assay, in which the primer or the target molecule is immo U.S. Pat. No. 5,593.826 discloses an OLA using an oligo bilized to a Solid phase. nucleotide having 3'-amino group and a 5'-phosphorylated 0344) 1. Other Primer-Guided Nucleotide Incorporation oligonucleotide to form a conjugate having a phosphorami Procedures date linkage. In another variation of OLA described in Tobe et al. (1996) Nucl. Acids Res. 24:3728, OLA combined with 0345) Other primer-guided nucleotide incorporation pro PCR permits typing of two alleles in a single microtiter well. cedures for assaying polymorphic Sites in DNA have been By marking each of the allele-Specific primers with a unique described (Komher, J. S. et al. (1989) Nucl. Acids Res. hapten, i.e. digoxigenin and fluorescein, each OLA reaction 17:7779-7784; Sokolov, B. P. (1990) Nucl. Acids Res. can be detected by using hapten Specific antibodies that are 18:3671; Syvanen, A. C., et al. (1990) Genomics 8:684-692, labeled with different enzyme reporters, alkaline phos Kuppuswamy, M. N. et al. (1991) Proc. Natl. Acad. Sci. phatase or horseradish peroxidase. This System permits the (U.S.A.) 88:1143-1147; Prezant, T. R. et al. (1992) Hum. detection of the two alleles using a high throughput format Mutat. 1:159-164; Ugozzoli, L. et al. (1992) GATA 9:107 that leads to the production of two different colors. 112; Nyren, P. et al. (1993) Anal. Biochem. 208:171-175). These methods differ from GBATM in that they all rely on 0338 j. SNP Detection Methods the incorporation of labeled deoxynucleotides to discrimi 0339. Several methods have been developed to facilitate nate between bases at a polymorphic Site. In Such a format, the analysis of Single nucleotide polymorphisms. Since the Signal is proportional to the number of deoxy 0340. In one embodiment, the single base polymorphism nucleotides incorporated, polymorphisms that occur in runs can be detected by using a specialized exonuclease-resistant of the same nucleotide can result in Signals that are propor nucleotide, as disclosed, e.g., in Mundy, C. R. (U.S. Pat. No. tional to the length of the run (Syvanen, A. C., et al. (1993) 4,656,127). According to the method, a primer complemen Amer. J. Hum. Genet. 52:46-59). tary to the allelic Sequence immediately 3' to the polymor 0346 For determining the identity of the allelic variant of phic Site is permitted to hybridize to a target molecule a polymorphic region located in the coding region of a gene, obtained from a particular animal or human. If the polymor yet other methods than those described above can be used. phic Site on the target molecule contains a nucleotide that is For example, identification of an allelic variant which complementary to the particular exonuclease-resistant encodes a mutated protein can be performed by using an nucleotide derivative present, then that derivative will be antibody Specifically recognizing the mutant protein in, e.g., incorporated onto the end of the hybridized primer. Such immunohistochemistry or immunoprecipitation. Binding incorporation renders the primer resistant to exonuclease, assays are known in the art and involve, e.g., obtaining cells and thereby permits its detection. Since the identity of the from a Subject, and performing binding experiments with a exonuclease-resistant derivative of the Sample is known, a labeled lipid, to determine whether binding to the mutated finding that the primer has become resistant to exonucleases form of the protein differs from binding to the wild-type reveals that the nucleotide present in the polymorphic Site of protein. the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has 0347) m. Molecular Structure Determination the advantage that it does not require the determination of 0348 If a polymorphic region is located in an exon, either large amounts of extraneous Sequence data. in a coding or non-coding region of the gene, the identity of 0341 In another embodiment, a solution-based method the allelic variant can be determined by determining the for determining the identity of the nucleotide of a polymor molecular structure of the mRNA, pre-mRNA, or cDNA. phic site is employed (Cohen, D. et al. (French Patent The molecular structure can be determined using any of the 2,650,840; PCT Application No. WO91/02087)). As in the above described methods for determining the molecular Mundy method of U.S. Pat. No. 4,656,127, a primer is Structure of the genomic DNA, e.g., Sequencing and Single employed that is complementary to allelic Sequences imme Strand conformation polymorphism. US 2003/0162202 A1 Aug. 28, 2003 37

0349 n. Mass Spectrometric Methods to centromeric markers on chromosome 12. Wherever pos 0350 Nucleic acids can also be analyzed by detection sible, APOE-e4 effects are controlled for by stratification or methods and protocols, particularly those that rely on mass by including APOE-eA as a covariate in multivariate analy spectrometry (see, e.g., U.S. Pat. Nos. 5,605,798, 6.043,031, SeS. Except as otherwise noted, the analyses reported here 6,197,498, and International Patent Application No. WO can be performed using Statistical analysis Software Such as, 96/29431, International PCT Application No. WO the SAS statistical analysis package (SAS Institute, SAS 98/20019). Program Guide, Version 6, Cary, N.C. (1989)). 0358 For all types of analysis, allele frequencies are 0351 Multiplex methods allow for the simultaneous computed from the data, but rare alleles can be adjusted up detection of more than one polymorphic region in a particu to a frequency of 0.01 (with a compensatory Small decrease lar gene. This is the preferred method for carrying out in the frequency of the most common alleles) in order to haplotype analysis of allelic variants of a gene. minimize the possibility of a false positive result. All analy 0352 Multiplexing can be achieved by several different SeS are repeated using the uncorrected frequencies. methodologies. For example, Several mutations can be 0359 For descriptive purposes, A2M genotype counts Simultaneously detected on one target Sequence by employ and allele frequencies are examined in affected and unaf ing corresponding detector (probe) molecules (e.g., oligo fected Subjects in study families. Unaffected individuals in nucleotides or oligonucleotide mimetics). Variations in addi AD families are not genetically independent of their affected tions to those set forth herein will be apparent to the skilled relatives, of course, and thus would be expected to Show artisan. higher frequencies of AD-associated alleles compared to the general population. However, given an increased risk of AD 0353 A different multiplex, detection format is one in with a given allele, its frequencies would be expected to be which differentiation is accomplished by employing differ higher among affected individuals than among their unaf ent Specific capture Sequences which are position-specifi fected relatives. However, Since these frequencies are cally immobilized on a flat Surface (e.g., a chip array). pooled acroSS families, they are neither as accurate nor as 0354) o. Other Methods powerful an indicator of genetic association as the SDT. 0355 Additional methods of analyzing nucleic acids 0360 A2M genotype counts and allele frequencies for include amplification-based methods including polymerase each polymorphism described herein are reported Separately chain reaction (PCR), ligase chain reaction (LCR), mini for primary and Secondary probands, with primary probands PCR, rolling circle amplification, autocatalytic methods, Serving as the primary Subject population, and Secondary Such as those using QJ replicase, TAS, 3SR, and any other probands as a confirmation Sample. Allele frequencies in the Suitable method known to those of skill in the art. probands are compared to those for unaffected individuals based on the oldest unaffected individuals from each of the 0356. Other methods for analysis and identification and 105 families in which one or more unaffected subjects with detection of polymorphisms, include but are not limited to, A2M data is available. In addition, the analyses are repeated allele Specific probes, Southern analyses, and other Such using an unaffected Sample that had passed through a analyses. majority of the age of risk, the “stringent' unaffecteds, those who are at least as old as the age of onset of the latest 0357 Five groups of statistical analyses can be used to onsetting affected family member, again Selecting the oldest explore the relationship between A2M and AD in study Such individual in each family. Because age of onset is families. First, the A2M genotype and allele frequencies for correlated in families (Farrer, L. A., et al., Neurology affected and unaffected individuals are calculated. Second, 40.395-403 (1990)), using onset ages in the subjects own stratified on families, Mantel-Haenzel odds ratios (see Man families is preferable to Setting an arbitrary cutoff. tel, H. & Haenszel, W. J. Natl. Cancer InSt. 22:719-748 (1959), the disclosure of which is incorporated by reference 0361 Initial genotype counts and allele frequencies for in its entirety) are calculated for the effect of possessing an each polymorphism and/or mutation described herein are allele for each polymorphism and/or mutation described determined (Matthijs, G., Marynen, P., Nuc. Acid. Res. herein on altering the risk for AD, and conditional logistic 19:5102 (1991)) in primary probands, secondary probands, regression, conditioning on family, is used to control for the unaffected individuals (oldest in family), and “stringent” effect of APOE-eA. Third, association for each polymor unaffecteds, (those who have reached the onset age of the phism and/or mutation described herein is tested for using latest-onsetting affected, again using the oldest Such indi the Sibship Disequilibrium Test (SDT) of Horvath and Laird vidual), stratified on individual APOE dose. (Horvath, S. & Laird, N., Am. J. Hum. Genet. 63:1886-1897 0362 Mantel-Haenzel odds ratios (see Mantel, H. & (1998), the disclosure of which is incorporated by reference Haenszel, W. J. Natl. Cancer Inst. 22:719-748 (1959), the in its entirety), a variation of the Transmission Disequilib disclosure of which is incorporated by reference in its rium Test (TDT) that is able to detect linkage and association entirety) can be calculated for the odds of being affected in the absence of parental data or the FBAT or EV-FBAT given the possession of at least one allele of a polymorphism developed by Rabinowitz and Laird (Rabinowitz, D & described herein. These analyses are preformed Stratified on Laird, N., Hum. Hered. 50:211-23 (2000), the disclosure of family using n-to-m matching, So all members of a Sibship which is incorporated by reference in its entirety). Fourth, a can be used and intercorrelations among Siblings can be variety of techniques are used to assess whether any A2M taken into account. Spielman and Ewens (Spielman, R. S., effect occurs via a change in age of onset. Fifth, Several and Ewens, W. J. Am. J. Hum. Genet. 62:450-458 (1998)) genetic association methods can be used to assess the have Suggested the use of a similar analysis to test for relationship between A2M and AD, and whether any allelic linkage. The analyses are performed first using all unaffected asSociation might be related to the recent report of linkage Siblings, and then only the Stringent unaffected Siblings. US 2003/0162202 A1 Aug. 28, 2003 38

0363 Conditional logistic regression is used to control in an examination of 150 sibships with 286 affected and 242 the Mantel-Haenzel odds ratio for the effect of APOE-eA. on unaffected individuals from the sample, the SDT was able to AD risk. Here, the outcome is disease Status of each Sibling, detect not only the deleterious APOE-eA effect but also the conditioning on family using an n-to-m matching paradigm, more difficult to detect APOE-2 protective effect (Farrer, L. and including APOE-eA/e4 homozygosity as a covariate, A., et al., JAMA 278:1349-1356 (1997); Corder, E. H., et al., along with a term for the interaction between APOE-eA and Nature Genet. 7:180-184 (1994)) not previously detected in A2M alleles of polymorphisms described herein. Like the these data (Blacker, D., et al., Neurology 48:139-147 Mantel-Haenzel Odds ratio, conditional logistic regression is (1997)). a Standard method for analysis of data from matched Sets, and can control for clustering of genotypes within families 0367 The primary analysis of the association of A2M of arbitrary size. These analyses are performed using the polymorphisms with AD examines the probability of pass PHREG procedure in SAS (SAS Institute, SAS Program ing along an A2M polymorphic allele as a function of Guide, Version 6, Cary N.C. (1989)). These analyses are affection Status. In order to increase the likelihood of correct repeated using only the “stringent' unaffected Siblings classification of unaffected Status, the analyses are repeated (those who were as least as old as the onset age of the including only "stringent' unaffected Siblings, those who oldest-onsetting affected Sibling) in order to minimize the were at least as old as the latest on Setting affected Siblings, effect of misclassification of unaffected siblings. These a Sample of 60 families. In addition, in order to assess analyses can also be performed coding APOE-eA as gene whether the effect differed in different APOE genotypes dosage, and including a term for the possession of an persists in individuals with similar APOE genotypes, the APOE-2 allele, previously shown to decrease disease risk analyses are repeated within Strata defined by matching affected and unaffected siblings for APOE-eA gene dose. To (Corder, E. H., et al., Nat. Genet. 7:180-184 (1994); Farrer, provide further validation of the SDT, the Sibling TDT L. A., et al. JAMA 278;1349-1356 (1997)). (Spielman, R. S. and Ewens, W. J., Am. J. Hum. Genet. 0364. Mantel-Haenzel odds ratios and p-values for the 62:450-458 (1998), the disclosure of which is incorporated association of A2M alleles for each polymorphism described herein by reference in its entirety) (S-TDT) is applied. herein with risk of AD will be greater than 2 and less than 0.05, respectively. Conditional logistic regression analyses, 0368. The SDT Z values and p-values for the association which allow for the calculation of Mantel-Haenzel odds of A2M alleles for each polymorphism described herein with ratios adjusted for the effect of APOE-e4 on AD risk, are also risk of AD will be greater than 2 and less than 0.05, expected to generate Statistically significant p-values (less respectively. The SDT values are expected to be confirmed than 0.05) for association of A2M alleles for each polymor by the S-TDT. phism described herein with risk of AD. Interaction between 0369 The general approach to family-based examina A2M alleles for each polymorphism described herein and tions described by Rabinowitz and Laird (Rabinowitz, D & APOE-e4 are not expected to be Statistically Significant. Laird, N., Hum. Hered 50:211-23 (2000), the disclosure of 0365. The Sibship Disequilibrium Test (SDT) (Horvath, which is incorporated by reference in its entirety) (FBAT and S. & Laird, N., Am. J. Hum. Genet. 63:1886-1897 (1998), EV-FBAT) can also be used to test the association between the disclosure of which is incorporated by reference in its the A2M alleles of the polymorphisms described herein and entirety) is a non-parametric sign test developed for use with risk of AD. This approach is based on computing p-values by Sibling pedigree data that compares the average number of comparing test Statistics for association to their conditional candidate alleles between affected and unaffected Siblings. distributions given the minimal Sufficient Statistic under the The SDT is similar to the S-TDT, a recently developed test null hypothesis for the genetic model, Sampling plan and that also does not require parental data (Spielman, R. S., and population admixture. The approach can be applied with any Ewens, W.J., Am. J. Hum. Genet. (Suppl.) 53:363 (1993) the test Statistic, So any kind of phenotype and multi-allelic disclosure of which is incorporated herein by reference in its markers may be examined, and covariates may be included entirety), but has the advantage of being able to detect in analyses. By virtue of the conditioning, the approach association in sibships of an arbitrary size. Like the TDT, results in correct type I error probabilities regardless of S-TDT, and other family-based association tests, the SDT population admixture, the true genetic model and the Sam offers the advantage of not being Susceptible to errors due to pling strategy. The EV-FBAT test statistics and p-values for admixture. Another advantage of these methods is that the association of A2M alleles for each polymorphism misclassification of affection status (e.g., due to the unaf described herein with risk of AD will be greater than 2 and fected Siblings not having passed through the age of risk) less than 0.05, respectively. decreases the power of the test, but does not lead to invalid results. The SDT can test for both linkage and linkage 0370. In order to see if A2M effects appear to operate via disequilibrium; it can only detect linkage disequilibrium in changes in age of onset, affected individuals are examined the presence of linkage, hence there is no confounding due according to A2M genotype, Stratifying on or controlling for to admixture. The null hypothesis of the SDT is that 0=% the powerful effect of APOE-e4. First, this is examined (no linkage) or Ö=0 (no disequilibrium), i.e., Ho:ö(0)-/2)=0. graphically using Kaplan Meier curves including all affected The SDT program (for several platforms) and documenta and unaffected individuals, first Stratifying on A2M geno tion may be found at ftp://sph70-57.harvard.edu/XDT/. type alone, and then on A2M risk allele carrier Status for each polymorphism describe herein and APOE-ea dose. 0366 Because the SDT does not require parental data, Second, the mean ages of onset of primary and Secondary and can use all information from Sibships of arbitrary size, probands are compared by A2M genotype overall, and it is well-suited to the analysis of the NIMH AD data. Before stratified on APOE-ea gene dose. Third, analysis of variance using it to detect novel AD genes, the SDT is validated with (performed separately for primary and Secondary probands) the known AD gene APOE-eA. in the sample. For example, is used, including first only A2M genotype (defined as any US 2003/0162202 A1 Aug. 28, 2003 39

2 vs. none), then only APOE genotype (defined as APOE-e4 A2M-2 allele of 18i showed Strong Statistical significance gene dose or APOE-e4/ea vs. not), then both, and then both for association with AD and the A2M-2 allele of 24e plus an interaction term. displayed a trend for association. 0371 Analyses of haplotypes that are associated with AD 0375. The 21i polymorphism described herein was tested can be performed using software such as TRANSMIT for association with AD using the Sibling TDT and EV version 2.5 (Clayton, (1999) Am. J. Hum. Genet. 65: 1170 FBAT as above. The population that was sampled has an 1177, see also Clayton et al., (1999) Am. J. Hum. Genet. 65: effective size of 92 individuals. The frequency of the minor 1161-1169, the disclosures of which are incorporated herein allele in this population was 0.22. The p-value calculated by reference in their entireties). This approach is a gener using the S-TDT was 0.001 whereas the p-value calculated alization of the TDT and uses an expectation-maximization using the EV-FBAT was 0.004. Each of these values are (EM) algorithm to reconstruct haplotypes with missing Statistically significant and provide evidence that the 21i parental genotypes. Nominal global p-values are estimated polymorphism is associated with an increased risk of incur using the empirical variance function. ring AD. 0372 For all types of analyses, allele frequencies are 0376 Table 3 displays the results of similar analyses that computed from the data, but rare alleles are adjusted up to were performed for 21 i from other Sample populations and a frequency of 0.01 (with a compensatory Small decrease in for other SNPs and/or mutations described in Table 1. In the frequency of the most common alleles) in order to particular, Table 3 lists the size of the population of AD minimize the possibility of a false positive result. All analy patients sampled for each SNP and/or mutation and the SeS are repeated using the uncorrected frequencies. frequency of the minor allele in that population. The p-val 0373 The association analysis and haplotype analysis ues (based on EV-FBAT statistics) for each of these SNPs can be performed for the SNPs and/or mutations described and/or mutations Samples are also provided in Table 3. In herein using the methodology employed in U.S. Pat. Nos. Some cases, the population was made up entirely of affected 6,265,546; 6,090,620; 6,201,107; or 6,303,307; all of which individuals over the age of 65. In these cases, a Separate are hereby expressly incorporated by reference in their p-value is included that represents the Significance of the entireties. The p-values for the association of haplotypes, association of the examined SNP and/or mutation with the which include A2M alleles for polymorphism and/or muta development of Late Onset AD (LOAD). EVA-BAT-based p-values that are less than or equal to 0.05 indicate Statistical tions described herein, with risk of AD will be less than 0.05. significance. Additionally, for each SNP and/or mutation 0374 SNP 18i (the site of a five base pair deletion of the that was investigated, Table 3 provides an odds ratio (OR) Sequence ACCAT located 1 base pair upstream of eXOn 18, and the corresponding 95% confidence interval, which See the Figure) and 24e polymorphism (site of a nucleotide describes the association with AD for both heterozygous and substitution of A to G at nucleotide position 145 within exon homozygous genotypes.

TABLE 3

Genetic Association of Individual SNPs and for Mutations with Alzheimer's Disease Odds Ratio (95% Odds Ratio (95% Minor Confidence Confidence Sample Allele p-value Interval) for a Interval) for two Mutation Size Frequency (EV-FBAT) single minor allele minor alleles 12e 37 O.O6 O.OOO9 3.62 (1.79, 7.34) 12.9 (0.94, 176) 12e 39 O.O7 O.OO18 3.18 (1.69, 5.99) 11.6 (0.88, 154) 12e 31* O.O7 O.OO31* ND ND 21i 92 O.22 O.OO)4 2.00 (1.34, 3.02) 4.01 (1.27, 11.8) 21i 71 0.17 O.O41 1.72 (1.16, 2.56) 1.84 (0.55, 6.11) 21i 50* 0.17 O.OO39* ND ND * All individuals sampled were over the age of 65. ND Not determined.

24 which results in an isoleucine to valine Substitution in the 0377 Haplotype analyses were performed for groups of A2M polypeptide (SEQ ID NO: 9) at amino acid position either five or six SNPs and/or mutations described in Table 1000, see the Figure) were examined for association with 1. The nominal p-value for each haplotype as calculated AD using some of the above-described methods. Specifi using TRANSMIT ver 2.5 is provided below in Table 4. In cally, the Sibling TDT described by Spielman and Ewens Some cases, the population was made up entirely of affected and the EV-FBAT described by Rabinowitz and Laird were determined. For 18i the population sample size was 76 and individuals over the age of 65. In these cases, a Separate for 24e the sample size was 110. The p-value for the p-value is included that represents the Significance of the association of the 18i deletion with AD was 0.0002 using association of the examined SNP and/or mutation with the EVA-BAT and 0.0015 using S-TDT whereas the p-value for development of Late Onset AD (LOAD). Nominal p-values the association of the 24e polymorphism with AD was 0.09 that are less than or equal to 0.05 indicate Statistical signifi using EV-FBAT and 0.14 using S-TDT. Accordingly, the CaCC. US 2003/0162202 A1 Aug. 28, 2003 40

incubated in the dark for 5 minutes. The luminescence of TABLE 4 each well is then quantitated using an ABI TR717 luminom eter. Association of Haplotypes with Alzheimer's Disease 0383 Compounds that enhance the binding of AB to Haplotype Nominal p-value A2M promote the subsequent clearance of A2M/AB com 6i, 12e, 14i.1, 18i, 20e O.O7 plexes from the medium Via LRP. Accordingly, decreased 6i, 12e, 14i.1, 18i, 21i O.OO32 luminescence indicates compounds that enhance the binding 6i, 12e, 14i.1, 18i, 21i O.O60 of AB to A2M. 12e, 14i.1, 18i, 21i, 24e O.OO31 12e, 14i.1, 18i, 21i, 24e O.O33 Example 4 14i.1, 18i, 20e, 21i, 24e O.O40 18i, 20e, 21i, 24e, 28i O.OO16 Screening Potential Therapeutics by Analyzing the 6i, 12e, 14i.1, 18i, 21i, 24e O.OOO23 Binding of Polymorphic A2M to Cells Expressing 6i, 12e, 14i.1, 18i, 21i, 24e O.O14 LRP 0384) To screen for therapeutic compounds capable of * All individuals samples were over the age of 65. modulating the binding of polymorphic A2M to LRP, A2M 0378. The results demonstrate that haplotypes that from the media and extracts of the transfected cells are include polymorphisms of the A2M gene provided herein labeled with 'I then treated with 5, 20, 50 or 100 ug of test associate with risk for AD. Furthermore, the results indicate compound in Tris/HCl or sodium phosphate buffer at 37 C. that at least a few of the tested haplotypes can be associated for 2 hours. Untreated polymorphic A2M and wildtype A2M with an increased risk of LOAD. The nucleotide identities of labeled with 'I are used as controls. A2M can be labeled the haplotypes are the three most common combinations of with 'I using kit for radiolabeling proteins obtainable from genotypes as determined in the NIMH Sample Set using the Pierce according to the manufacturers instructions. TRANSMIT analysis program. Thus, in methods provided 0385) HEK 293 cells expressing LRP (LRP:TCR9 chi herein which include genotyping an individual for the poly mera) and HEK 293 cells lacking LRP (IL-2:TCR0 chime morphisms included in the haplotypes, a Step can be deter ras) are seeded in 96 well microplates and grown for 18 mining the identity of the nucleotide(s) to see if it is hours in DMEM. Subsequent to growth, the cells are washed consistent with any of these three most common haplotypes. with 0.2 mL DMEM then pre-incubated for 30 minutes with 0.2 mL of assay medium comprising DMEM, 1.5% BSA, 0379 It will be appreciated that other haplotypes which and 20 mM Hepes at pH 7.4. After the pre-incubation, the include one or more the SNPs and/or mutations described in assay medium is removed and about 0.1 pmol of the Table 1 in combination with SNPs and/or mutations that are 'I-labeled A2M samples described above are added to described in Table 2 are likely to be implicated with an duplicate wells in 0.1 mL of assay medium. To control for increased risk of AD. nonspecific background, Wells to which no cells are added and wells to which no compounds are added are also Example 3 included. Additional controls for binding Specificity include wells to which 100-fold excess cold wildtype A2M or cold 0380 Screening Potential Therapeutics by Analyzing receptor associated protein (RAP) is added. Both RAP and Clearance of AB by Polymorphic A2M cold wildtype A2M act inhibitors of labeled A2M binding. 0381. The activation of polymorphic and/or mutant A2M 0386. After a 1 hour incubation at 4 C., the media layer (A2M) by AB (amyloid B) can be detected by monitoring the is removed and the cells are washed twice with 1 mL of LRP-mediated clearance of AB. HE 293 cells expressing isotonic phosphate buffered saline (PBS). The cell layer is LRP (LRP:TCRichimera) are seeded in 384 well micro then solubilized using 0.5 mL of 10 N. NaOH. The cell plates and grown in DMEM. HEK 293 cells not expressing bound 'I-labeled A2M is quantified using a gamma LRP (IL-2:TCR chimeras) are used as negative controls. To COunter. each well is added 5, 20, 50 or 100 lug of test compound in DMEM. After an hour incubation at 37° C., unlabeled AB Example 5 and polymorphic A2M from the media and extracts of the Screening Potential Therapeutics by Analyzing the transfected cells are added. Unlabeled AB together with Internalization and Degradation of Polymorphic wildtype A2M (Sigma) are also tested as a positive control. A2M After 3 days, the Supernatant is removed from each well and 0387 To screen for therapeutic compounds capable of A? levels are determined by ELISA. promoting the internalization and degradation of polymor 0382 To monitor the clearance of AB by ELISA, each phic A2M, A2M from the media and extracts of the trans well of the microplate is blocked with 200 till of 1% BSA in fected cells are labeled with 'I then treated with 5, 20, 50 Tris buffered saline pH 7.4 (TBS) for 1 hour. After the or 100 tug of test compound in Tris/HCl or sodium phosphate incubation, the Supernatant is removed and each well is buffer at 37 C. for 2 hours. Untreated polymorphic A2M washed three times with 200 till of TBS containing 0.1% and wildtype A2M labeled with 'I are used as controls. Tween-20. 50 uL of a 1:3000 dilution of AB1-12 alkaline A2M can be labeled with an 'I labeling kit for radiola phosphatase conjugated monoclonal antibody 436 in TBS beling proteins obtainable from commercial Suppliers, containing 1% BSA is added to each well and the microplate according to the manufacturers instructions. is incubated at room temperature for 1 hour. After the 0388 HEK 293 cells expressing LRP (LRP:TCR0 chi incubation, the Supernatant is removed and each well is mera) and HEK 293 cells lacking LRP (IL-2:TCR0 chime washed as described above. 50 uL of CDP-Star (Sapphire) ras) are seeded in 48 well microplate and grown for 10 days luminescence SubStrate is added to each well and the plate is in DMEM. Subsequent to growth, the cells are washed with US 2003/0162202 A1 Aug. 28, 2003

1 mL DMEM then pre-incubated for 30 minutes with 0.5 mL I-AR can be detected by gamma counter at two peaks, one of assay medium comprising DMEM, 1.5% BSA, and 20 corresponding to the molecular weight of the A2M/I-AB mM Hepes at pH 7.4. After the pre-incubation, the assay complex (about 724 kD depending on the polymorphism), medium is removed and about 0.1 pmol of the I-labeled and one corresponding to the molecular weight of unbound A2M samples described above are added to duplicate wells I-AB (4.5 kD). in 0.4 mL of assay medium. To control for nonspecific 0393. In some embodiments of the present invention, background, Wells to which no cells are added and wells to immunoblotting may be performed. For example, immuno which no compounds are added are also included. Addi blotting may be used to confirm the results of the gel tional controls for binding Specificity include Wells to which filtration analysis. In immunoblot experiments, unlabeled 100-fold excess cold wildtype A2M or cold receptor asso A? with A2M samples as described above. After incubation, ciated protein (RAP) is added. Both RAP and cold wildtype the samples are electrophoresed on a 5% SDS-PAGE, under A2M act as inhibitors of labeled A2M binding. non-reducing conditions, and transferred to polyvinyl dif 0389. After a 2 hour incubation at 37°C., the media layer luoride nitrocellulose membrane (Immobilon-P). Two mem is removed and added to 50% trichloroacetic acid (TCA). branes having parallel Samples are then probed with poly The nondegraded material in the Sample is precipitated by clonal anti-A2M IgG and monoclonal anti-AB IgG. centrifugation at 14,000 g. The amount of degraded material Immunoreactive proteins are visualized using ECL and present in each Sample is determined by counting 0.3 mL peroxidase conjugated anti-rabbit IgG. Molecular mass using a gamma counter. The cell layer is washed twice with markers are used to determine if the immunoreactive pro 1 mL of isotonic phosphate buffered saline (PBS). The cell teins from the anti-A2M and anti-AB blots for corresponding layer is then solubilized using 0.3 mL of 10 N NaOH. This lanes display the same mobility. If the immunoreactive layer represents the cell-bound and internalized 'I-labeled proteins display the same mobility then it will be concluded A2M is quantified using a gamma counter. that AB binds the A2M sample. Example 6 Example 7 Screening Potential Therapeutics by Analyzing AB Screening Potential Therapeutics by Analyzing the Binding of Polymorphic A2M Activation of Polymorphic A2M 0390 To screen for therapeutic compounds capable of 0394. To screen for therapeutic compounds capable of modulating the ability of polymorphic A2M to bind AB, activating polymorphic A2M, unactivated tetrameric A2M A2M from the media and extracts of the transfected cells are from the media and extracts of the transfected cells is treated treated with 5, 20, 50 or 100 lug of test compound in Tris/HCl with 5, 20, 50 or 100 lug of test compound in Tris/HCl or or sodium phosphate buffer at 37 C. for 2 hours. Untreated Sodium phosphate buffer at 37 C. for 2 hours. Untreated A2M and untreated A2M that has been activated with unactivated A2M, and untreated A2M activated with methy methylamine are used as controls. lamine or trypsin are used as controls. For example, A2M positive controls can be activated by Stirring A2M in a 0391) One method of detecting the binding of AB to A2M solution of 100 mM methylamine at room temperature in the is through an assay based on gel-filtration chromatography. dark for 30 minutes. The methylamine solution is then A second method is by immunoblot analysis. Both of these eXchanged for Tris buffer using a desalting column accord methods have been used Successfully by other investigators ing to the manufacturers instructions. After the incubation to investigate AB binding to wild type and variant A2M with the test compounds, the activation of A2M can be (Narita, M., et al., J. Neurochem. 69:1904-1911 (1997); Du, determined by methods such as ELISA assay or gel mobility Y., et al., J. Neurochem. 69:299-305 (1997)). shift analysis. 0392 For the gel-filtration assay, AB1-42 is iodinated 0395. An analysis of A2M activation by ELISA is as with 'I, following the procedure of Narita et al. (Narita, follows. Microtiter plates are incubated for 2 hours at 37 C. M., et al., J. Neurochem. 69:1904-1911 (1997)). 125I-AB (5 with 50 ul of LRP (10 ug)/well, and then rinsed with mmol) then is incubated separately with treated and deionized water. The plates are then filled with blocking untreated A2M Samples as well as treated and untreated buffer and rinsed. 50 ul of treated A2M, untreated unacti A2M samples that have been activated with methylamine vated A2M, or untreated A2M activated with methylamine according to the method described above. Activated A2M or trypsin is added to each well and incubated for 2 hours at (Sigma) is also incubated with 'I-AB as a positive control. room ternperature. After rinsing, 50 ul anti-A2M IgG con A ten fold molar excess of AB is used and the Samples are jugated with MUP in blocking buffer is added to the wells incubated in 25 mM Tris-HCl, 150 mM NaCl, pH 7.4 for two and incubated for 2 hours at room temperature. After rinsing, hours at 37° C. Controls containing only 'I-AB are also MUP Substrate is added to the wells, and incubated for 1 incubated. The A2M/' I-AB complex is then separated hour at room temperature. The amount of A2M bound is from unbound 'I-AB using a Superose 6 gel-filtration quantitated with a spectrofluorometer with a 365 nm exci column (0.7x20 cm) under the control of an FPLC (Phar tation filter and 450 nm emission filter. macia). 25 MM Tris-HCl, 150 mM NaCl, pH 7.4 are used to equilibrate the column and elute the Samples. Using a flow 0396 Alternatively, the activation of A2M can be moni rate of 0.05 ml/minute, 200 till fractions are collected. tored using a gel shift assay. Activation of A2M increases its Having Standardized the column with molecular weight electrophoretic mobility on a native polyacrylamide gel. To markers ranging from 1000 kD to 4 kD, A2M/25I-AP determine electrophoretic mobility, the A2M samples that fractions are counted in a Y counter to determine the elution were incubated with test compounds and A2M activated and profile of 'I-AB. If treated samples of A2M bind -A?3, unactivated controls are run on a native 3-8% polyacryla US 2003/0162202 A1 Aug. 28, 2003 42 mide gel (Novex) at 75 V for a sufficient time to allow A2M antibody (Sigma) using standard Western blotting Separation of activated and unactivated forms. The gel is techniques known to those of ordinary skill in the art. An then Stained with Colloidal Blue using that procedure rec A2M Sample treated with a compound capable of inducing ommended by Novex. Activation of A2M by test com tetramer formation produces a band at 720 kD. pounds can be determined by comparing the electrophoretic mobility of activated and unactivated controls with the 0399. The ability of the test compound to modulate dimer electrophoretic mobility of A2M incubated with test com formation can also be determined using the above method pounds. except treated and untreated A2M Samples are run on a denaturing 3-8% polyacrylamide gel (Novex) under nonre Example 8 ducing conditions, at 75 V for a sufficient time to allow Separation of the dimer from monomers. An A2M Sample Screening Potential Therapeutics by Analyzing treated with a compound capable of inducing dimer forma Multimer Formation of Polymorphic A2M tion produces a band at 360 kD. Monmeric A2M produces a band at 180 kD. In the disclosure below, several diagnostic 0397) To screen for therapeutic compounds capable of embodiments of the invention are described. modulating the ability of polymorphic A2M to form multi mers, A2M from the media and extracts of the transfected 0400 Although the invention has been described with cells is treated with 5, 20, 50 or 100 tug of test compound in reference to embodiments and examples, it should be under Tris/HCl or sodium phosphate buffer at 37 C. for 2 hours. stood that various modifications can be made without Untreated A2M and wildtype A2M are used as a control. departing from the Spirit of the invention. Accordingly, the 0398. To assess the ability of the test compound to invention is limited only by the following claims. modulate tetramer formation, treated and untreated A2M 04.01 All references cited herein are hereby expressly Samples are run on a native 3-8% polyacrylamide gel incorporated by reference in their entireties. Where refer (Novex) under nonreducing conditions, at 75 V for a suffi ence is made to a uniform resource locator (URL) or other cient time to allow Separation of the tetramer from other Such identifier or address, it is understood that Such identi multimeric forms. 10 till of prestained molecular weight fiers can change and particular information on the internet markers (Biorad) are also run. The proteins are then trans can be added, removed, or Supplemented, but equivalent ferred from the gel to a polyvinyl difluoride nitrocellulose information can be found by Searching the internet. Refer membrane (Immobilon-P) by electroblotting at 100 V for 1 ence thereto evidences the availability and public dissemi hour. The A2M samples are then detected with polyclonal nation of Such information.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS : 15 <210> SEQ ID NO 1 &2 11s LENGTH 88 624 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 1 totttgcatc caatacticca acttctotgt ggct gaccaa agaattggca cotatottgc 60 cagtcaggta gttctgatgg gtc.ca.gcaca gactggctgc citgggggaga aaga cago at 120 tgatttgaag toggtgaacac tataacticcic ctagotcatc acaaaacaag cagacaagaa 18O ccacagottc. cit gcttctoc ct gagaagag aaaggattgt tagaat citcc caca acctico 240 aacaaggct g attgatagga accittct cot atacaag act agtctgttgaa gaatgg gaga 3OO ggtgcct tcc tttgtctaat gcagaggcaa caacacagag agtcaaagaa aatgaagaat 360 taggcaaaga tattoctitta aa gaggaaca aaatacattc tagaaattaa cactaatgaa 420 atggaattat gtgatttact titatggagaa ttcaaaataa ttctdataaa gatgctcact 480 gaagttcaaaa gaacaatgta toagcagtga gaatttcaac aaaaccacaa aaagtatcaa 540 aaggtaccala gcagaaatca ttgagctgaa galacacagta acttaaaaat to actataag 600 agttcaatag caaactagat aaag.ca.gaag aaaagat cag ttaatttgaa caccagtcat 660 tggaagtagt to agt cagaa gagaaaaaaa gacaaagaaa taaaaagtgt agaaaaccita 720

aggaactitat gtag caccat caaattgacc attatacaaa ttatga gagt cagaaaagga 78O US 2003/0162202 A1 Aug. 28, 2003 43

-continued gaatagaaag agaaagaaag aaaaaactta ttcatagaaa taatgacitaa aacct tcto a 840 acctgaaaaa goaaatggaa toc aggttca aaaataacta agtaagatga accolaatgaa 9 OO atccacataa aaatacataa to attaaatt atcaaaagta aaaga gaatt ttcaaagcaa. 96.O taag agaaca gtgacittgta agatagacaa gatgcctgat aagatgatca gctggtttitt O20 cago agaaat ttgcagtcca gaagg cagtg aaattattoa cagtggtaaa ataatacaaa O8O cctgctaacc aagaatacta tacctgggaa acctgtc.cat caaaaatgga ggagtaataa 14 O agacitttcto agacaaacga aagct gaggg agttcatcac citctagattt gttcttaccag 200 aaatgctaaa gagagtttitt caactgaaag aaaaggacac taalacagdala cacaatato a 260 agagaaggta toaaactgat td.gcaaaggc aaatataaag aaaaacacat gatactgitat 320 tact gtaatg citagtaagttc actitt tactt coagttaaaa gttaaaagag aaaagtatta 38O aaaataacaa actaaaatat gttittaaaac ataaaataga tatcaattitt cacaaaaata 4 40 aagtgtgtag ggacagatga taaagggcaa agtttttgta totgattaaa citgaagttgt 5 OO tatcagotta aaacagacitg citatalactac aagatattitt gtgtag actic caagg talacc 560 ccaaaaagtc. tataaaagtt acacaaaaga cagagattta aaaatcaaag tatattggta 62O cacaaaaaaa caataaaa.ca caaggaagac aggaaga gag gaaaaga cag acaaaataat 680 tacaaggcta acataaaaca actaacatgg caaaaataaa tottccccta toaacaatta 740 citttaaatgc aaattgagta aacct cocaa taaaacaca tatagtgact gaaaagttaa 800 aaaaacagac ccaaatatat totatataca agg tacttac tittagattta agaacacaca 860 taggctgaaa gtgaagggat gaaaaagata toccatgcaa atggtaatca caaga gagta 920 ggtgacaggg caggagtatc atcatcttgg acaag cactg gcattittaaa gttcc cctta 98O atcaaaaact gcc.ccaaagg gcattggcct aatggctaac gtcagoatga ccataalacca 20 40 caaatgacat citctgaccag aaa.cattcca acacgaaaat aaa.ccct coc cqaccagaga 2100 tatgcctgcc ccalagataac citc.cccitc.cg gccagagaga tigtoagcc.cc aagataactt 216 O toccitctgac cagagacatt coaaccocac aataaactitc toctocacac agaaacattc 2220 caagcctgtg ataagctcitc. tcaccctaaa acccittaaat act cittagtic totaaga gag 228O agtggtocto actaaaattg gcc agaagcc cct citcaggit ttattotcca aaataaacct 234. O gtotttgact gttgagccac taatcgtgtt totttccitct ttctittaact cittacatttg 24 OO gtgccaaaac ccaggacggg togttgttgggit agaggct citc ttgcaa.ccca ggaag cagtg 2460 ggcagtggca gct catcc.ca citggatcc to agagt citctg gccalaccacc ccatcttgcc 252O tott acttica cittittcaagt gatttacatg agcaggacaa cita acctgaa goggaactgtg 258O aggcto aggc tiggggctact citccagtggg citcto agagt cct gag acct gaccacttct 264 O gaccaccoac agtggg tatt ttgctcitcta acacttgtcc cctoctocto cotcatcctc 27 OO acttitcctitt citctotgtct citctotgtct citctotcitct citctotctitc citcatgttggc 276 O. totggttcaa gaggcc ctitt gccaatticca actggaacat coaatattgg acactaatcc 282O agccaactgg taagatctgc citt.cccct ga citttcto atg gtaccc.ggga aagttcaggta 2880 tgccatcc to atccitcagag gaccagtggg act aggctag aagaaatctt goggacaccc 2.940 agtttcttct cagottaatt gttctottta gaaag aggat tctgggtotc tdtcttttgt 3OOO citgggg acac ctacaacaaa alacagacacc citaggcttct tcttaccagt coacataggit 3060 US 2003/0162202 A1 Aug. 28, 2003 44

-continued gctdaacaat coaaaattcc tatgtc.ctct coactgagct gtctoctitca cagocttgcc 312 O aaacttggct tatgggaagc ataaag.ccaa agtgtttggc tittittattgc aacgtggcc.g 318O ggcc.ccagtg caaattagat aatgacagct aatggcc.cga aaatggcacc tittgatttitt 324 O caaattctica ggg accittga caactittata accaggaaca gcaaatggca ggaggttctt 33OO gcctgaatat tcaggcttitc ttctacctaa gatcctgacc citccctgttgt caagcttgca 3360 ccccitcatga aatccttctt aataaaacco tocccaggitt totcctitcct citgaaatticc 342O titccttctaa accoctitcct citgaaaccoc titttgaccot acagatgaat gcc.ctcitcta 3480 toctoatccc cct ggatcca cittcttgcct gtc.cgaacco toag.ccccaa acticcactct 354. O toctocittct coacctgtta citcattcaaa aactgctitca accagtcaaa ccaccitctg.c 3600 citttct cota citctgggaag togctagggit cqaagg tatt gcttgttgttc atgtc.cctitt 3660 citccatgtct gatttgtcac agattaaaca gcaccittgga totttctotg aaaatcc citc 372 O toattatcac agg gaattcc td cacataac ccaatcctitt aatttaactt ggtatattat 378 O. ttacataatt citaaccitcaa cccitcacccc tdatgaaaaa gag cacticag cittaactaaa 384 O atcgatgtcc aagctatgag tatattoaaa ggcctittatgtttittctott cataaatctt 39 OO gtttitcctgg aaaaggittitt titcccagtca actgaattac titttcto cat tctittcttgc 396 O cact cittggit gcagg tatta aag accotaa aatgacittct ggtggcc to g g attccttgg 4020 gaaaacagaa aagttgccac aaatcc.catt togggaaaaa cctttgttitt cqttgtggaa 408 O cc.cctggaat tag aggtaaa taagtacctc. tcaaaatctg. tctttgttcto tcagotttac 4140 ttgtttatta gg.ccctggaa attatttitcc tag coctgtt cittaaagggc citcaccoaaa 4200 ggcc-aataat coaattggaa aattagaaaa aaaatctitat alactactgga ttittcttctg 4260 gttgttctgtg toggctatata totgttaggt gtgcaatgtc. tatttaaaaa gotctaattg 4320 actggccitaa gaaaaataag to cittaaatc aaatatttitt agaggaaaag taaaagctat 4.380 ggg accttitc agttcacgtg actittaatct ttaaaactta citggcacagt aaaattagaa 4 440 atgttittaag agttgccago atacatttitt gtttgcattt attaatcaag caattitcata 4500 cittatctotg ccaaatacta ttaggtgtca aaatttggca tagagacitac aaaactataa 45 60 citcagoccaa acagaataat citttgcttgt gtaattittitt aataaatgaa acattaatat 462O tggitttaata aagatagott catcttgaac tatttagtga aatacccitaa cittctaattit 4680 tgtggc citta ggcagtictag to cacagaca toaaggaagt ttgctittggg aaagg actot 474. O tatcatctitt gat attaaag aaaagaga at ttatacaaaa aagaatcata tatggtaaat 4800 to citgtcc td aagtaaatta act agttgtt taaagagagg gatgtttaca acaaagttcga 4860 ggcatgtcag agacitgtc.ca totaagttcat gaaaaaattt ataaaaggga atttatgcaa. 4920 gaaatgttgt acaatttalaa agtgattagg acticcitgaat gctittataaa atgccatata 4.980 actcittagct gtacaacttg cct gctttgc agctaggtaa gaccitaggac acatggagtt 5040 aaatgctgga ataagttcgga ccttatctga acttctgtct gggtoctagg citctocacct 51OO agtacataat taaaatcc.ca aacttaccala caaaagtaaa gottgctaaa agitta acagt 5 160 gtaacatgta tittaagacita ttgaaaaaac agtttacata tacttittggit aaaaagatta 5220 taaggaggca tagaatgttg gattitttacc tag attaaaa gottaaagaa ttgttittaag 528 O ttgaataaaa taaaaatgaa gotttalagca agttittggaa gotta attgt aaaggaaatt 5340 US 2003/0162202 A1 Aug. 28, 2003 45

-continued citgttgttgtaa atatattggc taaagttgaa gaagitat cat coagtttittctgtaaactoga 5 400 cattaaaata aaag cacagt g g gtttggitt totcttaaag cacta acct g citctittaaca 546 O aaaattataa agggittaaaa agggtotata gaaatcttac cittatggtoa aac attaaaa 552O togggtaaat gitatctacaa gottctatta aaaattgagt ttaac attag tag cacacta 558 O atataaaggt ttagct tatt togtataaaa toatacagga agcattgtca aatataaaat 5640 ggtgtttggc tittctttggg citatatttgc atacatatgt tattgg tatg tdttccaaag 5700 ttataagaga citcctatatt totgatatat cittagtgitac gittatcagta ataattataa 576 O. ttgttatgtt aaaatattgc atgccacaaa ggtaacagat attcttgtca attgtaactt 582O tatggctact ataaaactitt ttgtcatcca taaacaattg ttgttcttgtt tittggtoccc 588 O tagagactga agtaatctitt tttactttitt gagtatattt aagttatggc aatatagitta 594 O tittccatcag td caataaga atctgttitta ttttgtaaca gaacatgatt tdaaaaactg 6 OOO gttattittac caaggctttg actggagagg totgctdtcc tittaaggaat caaacttgac 6060 titatggagcc aataaaactc. tcgggaaact ggcct catat tittatgtgca cagtc.cctgt 61.20 acagggitttctgacct gtgg taagtaaaga atgtcactitt citgacaggcc agtaccc.cca 618O agittatcttg galacct cagg aggagaggaa ttcacccaac toataggitat ttaatggtac 624 O aatticcatga citgggctcag ctittaaaagg ccttatctoa gattoctitct atggaacaaa 6300 attccatcaa taccagttta aaaggcctag gtaacaaata attattottg citgcactgta 6360 tgcaaataat taaaccaagt ataataatgcaaaccattcc taccatgatt tatttitttaa 6 420 taacggttac togcagaaaa talacacgtgg cccttitccaa acatgtgcct citgccitcto a 64.80 ttaggtaagg aatgttgctt citatctoaac caattgggcc gagtaagaaa cactgctaaa 654. O taacttaaag aaagggccaa agagctaagg gaatticcaaa acaaccalaat ggattcttga 6600 tittgggaaaa aaaccatago atgggtocatc ccattcc togg gcc.ccc.ccct act actatoc 6660 citagg actaa tagttcttacc citgcctaatt aatcttittcc agagatttitt aactgacagg 672O atcatggcca titt cacagac alactacccaa aaact gcc.cc aaagggcatc agcctaatgg 678 O. citaatgtcag catgaccata aaccacaaat gacatctotg accagaaa.ca titcca acacg 6840 aaaataalacc cctocc cago cagagacatg cccatcc caa gatalaccitcc totcc.ggcca 69 OO gagagatgca gcc.ccaagat aacct cocct citgaccagag acattccaac cccacaataa 696 O acttctocto cacacagaaa cattccaagc citgttgataag citctottacc ctaaaaccot 7 O2O taaatactict tagtctgtaa gagagagtgg toctoaccala aaattgg to a gaatc.ccctic 708O tdaggttitat tctocaaaat aaacctgtct titcactgttg agccatttitt catgtttctt 714. O to citctttct tta acticitta caggaggagt ggctacactt atato agata aaatagacitg 72OO agittaaaaac tottacaaga gaccaaaagg gaaattittat aatgataaaa gogtcaattic 726 O aacaggaata tataacaatt acaattatat atgcatccala cagtagaa.ca cctaaatata 732O taaa.gcaaac attggaaaaa cagaaaagag aaatagg gag caataaaata atagtaggaa 738O acttcaatac tatttgcaat aatggataca toattcagac agaaatctgt atccataaga 440 aaac agcaga cittgaataac acaataga.cc aaatggacca aacagacata toc agaatat 7500 tocaccaaac agcagotgaa tacatactct tctdatgtgc acticagatcc titccccagga 756 O taagtcacat attaagtcac aaaataagtt ataacaaatt tagaaagatt gaaatcacac 762O US 2003/0162202 A1 Aug. 28, 2003 46

-continued caagtgtc.tt totggccaca acaaaattaa actataaatc aataacagaa goaag actogg 768O aaaaataaaa aatacataga tattaaacaa cagacitcttgaaaagttcatt gagtgaaaga 774. O aggaatcaag aaggaattta aagtgccitta atacaaatga aaacaaaaat aaaaaac act 7800 aaaattatgg tattoagcaa aag cactatt aagaaggagg titt actotga taaataccta 786 O cattaacaaa gaacaaagat citaaaataat caa.cccaaat ttatactitca agaggctaga 7920 aaaagaacaa actaagctica aaattagcag agaaagaaat aacaaaaatt agagaagaaa 798O taaataaaac aagaaaagaa aaaaatcaaa gaggctcagt gggttttittgaaaaaaataa 804. O ttalacacacc cittatttaga citaaaaaaaa agaaatgaaa gaagatacat tataactgct 8100 cittittagaaa taaaaatgat cataagtgaa caaatatgtc. alactaatcga atalaccitaga 81 60 agaattgttgt aaattic ccaa atacacaaaa catgtcaaaa gtaaaaatca agaaagtttg 8220 aacagaccita to attagtat ggagatttaa taataacaa aaatcct tot aacaaataaa 828O aaccoaggat cagatagott cacaggtaga ttctaataca catttittitta aaaaagtgcc 8340 aatcagtc.tc. aaatgctitcc aaaaagtgag agaac actitc caaactcatt ttata aggcc 84 OO agtag cacac togctaccalaa goc agacaag gacactacaa gaaaaaaaaa atgacaggcc 84 60 aatatotctg atgaatatag atgcaaaaaa tattittaaaa at attaggaa atagaatcca 852O acagdacatt aaagggat.ca tacat catga ccalagtagta tittattocca ggatgcaagg 858O atggttcagt atgtataa at ttgtaccaca ttcacagaat aaagggaaaa aaaatcacat 864. O aattatataa atacaag cag agaaag catt to acaaaatt caacattctt toatgttaaa 87 OO aact citcaag aaactataaa taggagagta totcaa.cata atacaggcta tatatgaaag 876O gcc catagat aatato agac toaacggtga aaagttgaaa gotttitcctt taagaac agg 882O agcaaggcaa tatgcc.cac tottgccact tittattocaat atagalaccala agcc.ctagoc 888O agaacagtta ggtaagaaaa ataaattaaa goctaccalaa totagaaagga agagataaac 894 O tttittcctgt ttgctgatgc cattatatta totattaaaa atcccaaagg citccattitta 9 OOO aaaaactgtt aaaactaata cacaaataca gtaagattgc aagctacaaa atcaacttac 9 O60 aaaaatcagt togcatttcta tacactagoa ataaactctgaaaaggaaat talagacaa.ca 912 O atcc cattta caatagocaca aaaaagaatt aaatacttaa goaaaaactt titccaaggaa 918O gtgaaag acc totgtctgga aacaaaaa.ca ttgatgaaag aaattalagac acaattaaat 924 O aaaaatatat accatgttca ttgattggaa gatttacitat tigtoaaaata accataatat 93OO caaaag caat citatagattcaatgcaa.ccc citgtcaaaat cacattggta ttgtttaaaa 936 O aaatagaaaa goaaatccita aaatttatag g gaatgagaa alacaccacaa ataacaaaat 9420 caatcttgag aaagaagaag aaagctggag gacticacact tcc taattitc aaaatttagt 94.80 acaaaaccac agtaatcaaa acagtatggit gctgg cataa agacagataa acatcaatga 954. O cagaatagag atctoaggaa taaatgcacc cataaaaggit caactggtot ttgacaaggg 96.OO taccaagaat acactagggg gaatggatag toccittcaac aaatggtgttg gagaaaactg 9 660 tatato cata agcaaaacaa taaaatttga tigtttatctt acaccataca caagaattaa 972 O citcaaagtgg attaaagaca taaaagtaag gCCtgaaact gtaaaactga tataagaaaa 978O aataaaagac atgctittatg atcttggtct togcaatgat ttcttggata toacaccalaa 984 O atcacagaca acgaaaacaa aaacaaataa gttgaactat gtcaaactgg aaagctottg 9900

US 2003/0162202 A1 Aug. 28, 2003 48

-continued atacgacaaa actaatgaat citt gaggaca ttatgcticag togaaataagt cacagaaaga 2240 caaatactg.c atgattccac ttacatgatt tatctaaaac agccaaattic atagaatcaa 2300 agagtaaaac agtggittatc aggggctaaa citgaagaggg aacggggagt ttgtaatcaa 2360 tgga catalaa atttcagtga agcaaggtaa ataagctctg gagatttgct citacaatgtt 2420 gtacttatag togacaataa tatattgtac acttacaatt tottaaaagg gagatctoat 24.80 gttaaatgtt cittaccacaa taaaataaaa tttittaaaag aaaaaagaca cct ggtgtct 2540 ttittcttgta gcaa.ccgaag taaatagaga tigtatgggtg tataggcatt agtttgattit 2600 totcittacga agaggtotgg attatago ct cocatcttgag accggagtoa agc actt gag 2660 gtggggaatg atggagagat tdgaagtggc attcttctoc acaacac cat gggttctgta 2720 gtoccittgtg caattctgca attctgcc at gatagittatt gagaccalaga caggcaaagt 2780 citaaccaggg aaagaagagt totgtag act tcc titccaat totaccalagg ttgatattga 284 O gag caggctt cittgcc agca gcattaalacc caacaatcta tact tcc tag gacaatctot 29 OO acctittgatt totgtaaaat aac acttittctgtttittctt citgagg.cccc toctocacag 2960 accagtttct ggatatottc ttctittatca gatctotaaa tagttgagatt cittctagatt 3020 tagatctatg citgcttgttc tittgcactict acattttctt cattcatttc tacaacttitt 3O8O aatagtgttgt ataggtagct cocag attgc tattittcacc gtgattctot tacctcatac 314 O tttitcc ccto tttggtgatg atgtgtcago acattggccc totctotaat cittgaatact 3200 ttatttctgt citggggagitt tag cacatgc titcccitctgc ctataatgct gtataacttg 326 O ccctcitccct ttittgcaagg ataaatticco agcttaaatg ccatatoata agagaggtot 3320 tgactogaatg cacaatctat gttggatcto cotggatgtt cagggaagaa aagaaac att 3380 tttittcttitc acago attta toatcatatg gaattacctt atttaattta tttgtttata 34 40 tactittgatc totattottc. cacaggaatg tataatc.ccc aatgg cagga acatgtggitt 3500 tttgttaacc acagaacagt atctgataca aaatatgcat taaaattctt gtgaactgac 356 O taaataatat coatctotcc acttgattgc tittacttgcc acacatccoc agtacctaat 362O tdaag agctt ggctoattgt cagoctittaa atattgactg aaccoaatta atcactttitt 3 680 totttaactt citgaactctg td.cccagotc cctactitgaa tagcttcact ggatatotca 3740 aaagaaccitc aaattcaata totctactitt coctatotgt tattagcaac aaaccoag.ca 38 OO cittgccctag taggaaatct acaatcatcc tttacittatc. tctaaaatga aaaaaaaacc 3860 cacaccittitt toataaagtt ttgagttcac taagtgaaat aag caaggtg cct atcacaa 392 O tgcctgg cat acagtatcta taaatattag tittagctata coagttittca ttcattcaaa 398O gagg cacatc tttitcacatc. tctgaaatca agatgttgttt totgatcagt agtgttt cat 4040 aattggatat acatctaatc attaatcagg taggaaattitt ttctittaaat actittattag 41 OO aaaattgttgt cotttacctd aatggtgctt cagatgttgat aatttgcagt aacacaactt 416 O citctotccaa tatgcc cata tatattaaag tagittattag gtcacatcac gitacatttga 4220 aatccatatt ttittctoact cittcc cactg ttgtaatctt actittgggcc citcattttct 428O to caactgaa cactgaatta gcc.ccctgaatggtottcct gctgactata ttgcccttct 434 O ccaatgtgtg tdtcatatto atgccagagt aatcatgttt citctoatgcc taactcactt 4 400 cattatctac acaact gcct gcagaatgga aaccagatto titt cacatgg cacacagggc 4 460 US 2003/0162202 A1 Aug. 28, 2003 49

-continued totgacitcto tctotctitta gcctaatccc taagccacag toaagttgac citcctttctg 4520 tacttgcaga tigacittaaca citgitatgttg tittcacaacc catgtcgttt atcacactga 4580 ccitatcttga aaatattittg tat cagotgg aatcc citctg gcaataaata acagaaaag.c 4 640 aactagoagc tigcttaaa.ca aataggaaag to cataggitt gotgttccala gqttgataga 47 OO attgcc caat aatgctgatg agg accaagg cccatttitat citttcag act tccittaacat 476 O gcag acctitc atccccctg.c ttgttactitt gctgtctdaa gatggtoatt gaaatattaa 482O atat cacatc tatttitcc at taagtaagaa caaggaagga cattcagggg tatgcc.cact 488 O acaactctitc tttittaagaa agcaaatgtt toccataagc cctacccago agittittctac 494. O ttatat citca ttgcc.caaac agcaccatag to actctggit ttgaagatgc citagaga gag 5 OOO ggatgttgtg g g tagtttag ccaatgagca gtgtctgcca cagtotgctt goaaaat atc 5060 ttctoattitt ttgtgagatt caactcaaga gttatatott agaaatttag aaatgtgitat 5 120 atatatgtat ataaactata tatgtatata aattatattg atatttatat aag catacat 5 18O aaaacacaaa tatataaacg tatatatagt atgtaccctt toatcattaa citgaatattt 5240 atcaaacacc totacacagt gcgtgctagg tactgttcta gg cactggga atacagoaat 53OO gaacaacatc titatcctttg toggaatttac atcctagtgt ttaccctittg atctaactitt 536 O ttcaccitcta gtaacttacc titacagacita tacctcaaaa acaag gatgc ticattgaagt 542O gatalactaga ataacaaaaa totgattaaa tat cactato ggattgatta taatatacat 54.80 aaaattataa aatgtaatta ttcaaattac citcttaatag agatatacta acatggaaag 554. O citgtc.cccaa catatattitt aatttaaata atggaag acc aaaatgtata gitaaacticto 5 6.OO acttittaaaa agaaaatcca tocccaaatt catacggaat citc gaggaat citgaaatago 566 O caaaacaact ttggaagaag aataaaggta ttagacticac actitcct gat ttcaaag cat 572O atacaaagac acaatgatca aag cagtgtg gcactgg cat aaagacagac aaataalacca 578O togtaataga ataaagagcc gagaaataag cct gtgttgta tatggtoaaa to atcttcaa 584 O caagggtgcc aagaccactc attgagaaaa tatagt citc citcaacaaac ggtgttggga 59 OO acaatggaaa tagaaagttaa acctittattt tacactatot acaaaattta acttataata 596 O tattaaagac ataagcataa gacctaaaac tatgaaattt citagaagaaa acagacagga 6O20 taatctoatg acatggggitt totcaatgac ttcttagata toacaccalaa agcacaggta 608 O acaaaagcaa aaaaagataa atgggactac atcaaacttgaaaac atttg togcatcaaag 614 O gacacaatca aaagagtgaa go.gcaa.cata caaaatggga aaaaaaaatt tacaaagaat 62OO atatttagaa gttattatcc acaatatata aagaactccc acaactaaac aatgaaaaaa 6260 aaatcaaata act agattitt aaagtgtgca aaatatttga atagatattt citctaaagaa 632O tatatacaaa togctaataa acattgaaaa gqtgctcaac attactaatc accagagaaa 6,380 tgcaaatcaa aatcacaatg acatactact tca catctgt gaggatggct gctataaaaa 6 440 aaaaacagaa agtaacaagt gttggcaagg atggggacaa attgaaacco ttgttgcgcag 6500 ttggtoggat totaaaatgg tittaactgct atggaaaa.ca cc.gtcaagtt to citcaaaaa 656 O attcaaatag alactaccata to atcCagga attitt actta toggtgtata tocaaaagaa 662O ttgaaaa.cat ggtottgaag agatatttat ataccacagt catagdacta gtc.ccaataa 6680 ccaagaagta aaag.cgagcc aaatgtc.cat caatagaaga atgaatacat acaatcaaat 6740

US 2003/0162202 A1 Aug. 28, 2003 51

-continued tottgaagta gtggct gtaa ttgcc.ggggc ccc.gggg.ccc agtc.gcagat citt caccittc 9 O8O agct cacccg gccitccitcca gag cocqcac cittgtcCagg taggaaccala gtggtcgtgg 914 O atgttctg.cg toggtgatctt gotgc.ccg to agcagoctot cqgacct go a gaggacgctg 920 O gCatcgc.cgc tigC Catagcc ccagaggagg atgaggg cac aaagtgggtg gaggacacag 9260 acaggctacg gctitcggagc cccagtggat gctgggctot aggaaggcgc ccc.cgacitcc 932O aaagtgcacg gagcc.gctgc ccagoccc.ca aaggactaag togccaatga citggc gaaag 9380 Ctgcaggaag ttagggtgag gcagagaacg gttcaaggag Ctgcaatcgg Caggaggaat 944. O cggaaattitt aattgcatta ttaccaatta taagaattitt tatgctato a gtgggittittg 95 OO tgctatagg ggacccaaaa aagggctgat aaaacaccaa alaccactggit gaggagaact 956 O taagagggaa ccaggctt.ca agctoctaaa citcac catgg act gccacag aatttaaag.c 962O acagtccata titccitcagaa agctalaccala gcc caccact gggtgtc.cta ccaaaggcc.g 968O titcc citggca ttattotaga cagaacaa.cc cagacggcaa aaatgtact g c totggagag 974. O atgaatcaat toctaggagg actggacaga cattcto aaa agtttgttgg aaagatagoa 98OO gaga agagaa gaatgaggtg ctittaataaa gqgataggaa galaggtgaga acaaggaact 986 O gagattagga gctgagtgtg to catgacag accgc.ctaga aagaatgtag tdtctagagc 992 O ttttacitcat agaccg.cggg acaaggtata tttaccatat agctotgitat tittccctcitt 998O gtgttataat gttittctgta attaattact citaaccottt tag caaagttc taatacactt 20040 cago attgtc. tcgtgatgat atacagacita aatgg acagg tagtaaaag aaaaaattaa 20100 ggcgtoctoa citggtgg gag cagaatcc.ca ggcagagaac tatgcacaga agtttcaggc 20160 agtgat cago togaaagtcag attgcagtcc attgtattoc atggtggaga caagaac agg 20220 gagggagcac ccatacittaa aagaaactitc taggcttgga gcagtatggt attittataca 20280 catcaccitct gttagt caag citttggatgg g g gctagata gcagoccact gaaag acct g 20340 gagtcc citgt attcttaatt gctatotcgg aaagttc.cat atttagaaat cagatgggca 20 400 gcaggtaagt tagg cattct tctgaaaaaa attaa.gc.citc taccagg act citc.gcttggt 20460 ttataaaatg agccacagaa gaalacago aa acticaagttt to citctoaaa aagttcct gtg 20520 toactitgaaa agttgcttca tacacattgt gtcctaaata tdaaatctaa agttgttact 20580 gttittgaaat aaagtttcaa gagaatatat tittatataac attcaaatga gittatagitaa 20640 aaatgctagg tottaa.gaga titcaccggat gaaataa.cat totcc acttic agaaaatgaa 20700 actagtacaa tattaaatac tatgattaat aataatctot aatatttcta catacttittg 20760 citcctaacag titgagttata tacticaccct gtatgattgt ggtatattot gttatgccag 20820 ttgcattatt gtaattataa atttgaggaa taaagtacac gaacttataa aatataaaca 20880 tggc.caggca cagtggctica cqcct gtaat cocago actt togg gaggcc g aggtgggtgg 20940 atcacaagct caggagtttg agaccago cit gcc caatatg gtgaaac coc gttctotact a 21000 aaaatacaaa aattagccag goatggtggit gag caccitgttgtcc cagot acttgggagg 21060 citgagg cagg agaatcattt galaccc.ggga ggtggaggitt gcagtgagcc aagattgttgc 21120 cactgcactc. cagoctdggc gttagagcaa gattc catct caaaaaacaa acaaacaaac 21 180 aaacaaaaaa tatacataca cqcactattt taaaactcag tttittatcta aaaccitagat 21240 taaaagttctt toggaaagagt ccato gagag gaatacgitta aaaatgc.cat tdaagccatt 21300 US 2003/0162202 A1 Aug. 28, 2003 52

-continued citgcaatgta taatatttca gaatgacatg ttgcacacaa tdtctacata atttittcgtc. 21360 aattgaaaat aatgttgatca attggattaa aaatatatoa ttaactcatgaatttaaata 21420 tatttaatga gcttaagttg attgtattitt citgitatttct taaactcaag gtattaccta 21480 gtgctttgag gtgtcgattt totggccaga aacttctg.cg gctgg.cgaca agctottgtc. 21540 citgcatccag gaagaatgac aggaagaatg aggtacacga acaagtgaag g g to cacaat 21600 aagaatgagg tacccagaca agtgaaggac aagatgaaga tigagctttac taaattittag 21660 aacagotcag aggaga.ccca cagtgggtag citcct citcc.g. taggcagg to titc.ccaa.cat 21720 citactgctict cagoagagag gaggcc.ctgg agcgggtgct cottgct colt citctgcact g 21780 tagt cocqaa gtc.tctgcag gtc.tctgaag citctoag cag agagggtagt to citctgtgc 21840 agctggttgt cocatcgtct cotgctatta gcagagaggg cagottctot ctogcaactogg 21900 tottcctgtc. cctocatcct citctottgct citgcctgagc cc.gaggcttt tatggggtgg 21960 aggaaatgcc togctgattga to catgggca gccataggca ggccaaagga ggcaccacaa 22020 gtoctaggga citgactg.ccg g gatc.ccaac cittcaggtoc toccitggcct galaggtgggg 22080 ccttacaggg gacctg.cccc cittctg.ccca ggagtctgcc toccitccc.gc tigc cattcat 22140 ggcc cctogg citcagoccca agattggagc aggctctggg agaggagaaa goccaggcaa 22200 tgggag caga caccc.ctgag cct gcagggit cqagaga agg ggagggtott cocggct citc. 22260 gagggtgcag gCtgcagaga tigcctggacc tocaccitgtg agggit gcc.gc agctgcacca 22320 gggaatctoc cqcgcago.ca actgg gaatg ggcaggtotc cc.gcttgtcc cgggctotct 22380 citggcticagt ggagcagtag goccaggtot gcago cactg gtcggggtgc tigcagcc.gca 22440 cggggagtgt agatcttgcc toc toccago coctitccaag agcacaggga ggctoagat c 22500 cacago cqca gtttggg.cgg ggctoctacc toc to catag agcaggaggc cqcggtotgc 22560 agccitcggitt toggcagotg cagoggcacc ggggagctot goccc.caact cagaagg gac 22620 ggagcticcca ccggcticcac to cagccaac attatgg cag cagoggctac catcaatggt 22680 atcc catctt togcaagtgg aaacatctta atgaattitcc cgtttgccoc gagaatcctc. 22740 ttgtc.tctga toctaaggta acatcacata catgtctgtt acactaggat tagagacaag 22800 ttctgtttag aaataactcc aagaacagot tittatattitt attittcacat tigaaaatcag 22860 toagatttgc ticcago citca aagaatgtgt titactaaaat taaatgaatg citggcaggga 22920 gctgcactitt tttitttctaa ataggaaatg g gtta agggc ggcagotgag toctittcgac 22980 ataaccotaa tagtotttgg cagoatccitt totgtttggit aggataagat attctaggct 23040 catcttgcat titt.ccg.cgaa aaaaaaaaag ccattgaatt agg catagag galaattact a 23100 taaagtttaa gqgataatgt aaaggatcta ggaaaatact citaattgctt cacaaacaag 23160 gaagaaactgaaactagaaa totagtcaat tag tattgct gatgtgctitt atgcaagaaa 23220 gatagtatat ttgttctotc titatctgcag cittcactittctgtggitttca gttaccottig 23280 gtocatcCagg gtccaaaaat attaaatgga aaatticciaga aataaacgat tdataaattit 23340 tdaattgttgt gcc attctga gtggtgttgat aaaatcttga caccc.gggat atgaatcatt 23 400 cctittctoca gcgitaticcat gctgtatatg citgctgttat tcc.ccctaac ccccacccca 23460 gtoacttagt agctatotca atactgcagg gctitatgttcaaataatcct tatttitactt 23520 aataatgg to coaaagctca agagtagtga tigttgg cata ttgttataat tigttcttcta 2.3580 US 2003/0162202 A1 Aug. 28, 2003 53

-continued ttatcagttt tattaatcto ttactaagcc taatttataa attaaacttic atcctaagta 23640 tgitatgtata ggaaaaaa.ca tagtatatat agggittaggit act atctgttg gtttcaggtg 23700 to cactgggg atcttggaac atatgcc.cct c ggataaggg galactacitat atacaagatt 23760 aaataagg to acttaaaatt catttataaa attatgtcct aaaatatttg taccagtaac 23820 aagatatatt caaattattt tatctaataa aaattaacat gttittattitt citaataaaag 23880 ttaaaatatgttagcagtct ataccacttic tittaattaca toggaaaaaat tacattatga 23940 tgtctaatta ataaatagitt atttataatt actg.ccaaag toaaaaacca gtttgatact 24000 tacatcttaa ataccatgcc tittittatacc cittittctitta taaataactt gtaatttaac 24060 cittcaaatta tactgcatta citttaaatta aataatgaga gtacgitatgt ataacttgct 24 120 ttgaaaataa atgaattatgaaagctacac tatttaaaac ttgagaactt toaataatac 24 180 cittatacaat atatto cata attactttitt aataaattitt tataatatta citgitaaattg 24240 tgggtttagg gaccgacagt toaactatta gcaaaataat cittittagcat aacaaaaatt 24.300 ggaacatcta aatctottag aacttgttctt agcc.cattta tatagtaagt actacgtaat 24360 tgctag ctgt cattacctgt tactgttgta gctattgttg ttagcctcat tatcatcago 24420 atcatctgag taattaagta aagttggcaa acaacttitat cittitcctact tittaaaattit 24480 aacco cagtg tittgcticcca aactalagtag aactcitctaa aatgaaagtt citgacactogg 24540 aaagttccac agc galacatg agcactacag tdaacaaagg ggcto citat g gttaagggit c 24 600 atgaaccaac cittgttgcaaa cccacacaca aacacgcata cacttattitt citgttcatca 24 660 aaggaaaagt aatcctgitaa catttccc.cc accittgctitc ctattocaaa ataaactgca 24720 gatgaagatt aaaatctaaa cacaaatact taatgtagga aaaaatgcag gataatattt 24780 ttacaattitc agtatgcata togcagaagga tittcttaatc agaaaataaa gatacagtat 24840 aaacaaaaag aaagttaaac aaagataa at tataagcaac agg gttccta caagtgacaa 24900 tgatgctgaa catcaccagt aatcatgaac ttgaaaaaga aaagacaaaa tagat catgc 24960 tocc citcc cc atcagattgg taaaattitta aatgagtgct g g gaaaatat atacacataa 25020 gcattgctag tdagaaggta agttggcatt ttagaaagtt gatgagatag tat citataaa 25 080 tittgaaatgt atatacatag caaattcaca atggggacag attcattata caaaaatact 25 140 togg catgcc ccaaagtata aatatgttag gacago catt gaattictatt gtttgtagta 25200 aattgttitta gtccaaacac taatticcitct gtag caaaca taggatctaa taaaatggat 25260 tatgtctgga aatcagtcct citttagaaac ctaaagg acc aagtgitatcc to attaaaaa 25320 gataaaacgc tittctttctt totttttgtt tttgttttitt tatttgtttgtttc.gagaca 25380 gaggctcqct citgttgccag gotggagtgc agtgg.cgtga totcggcto a citgcaacct c 25440 tgccitc.ccgg gtttaa.gcga ttctogtgca totagt citccc gtgcagotgg gactacaggc 25500 gcacgccacc acacco agct aattitttgta gtttaagtag agacggggitt toaccatgtt 25560 ggccaggatg gtctoaatct cittgacct catcatccacct gccitcagtct cocaaagtgc 25 620 tttittgataa ttittgagaaa togatggaggc at attagaat gaaaacaa.cc tdaggatgtg 25 680 cittittatctt totatattoa aatattttitt citcattaaaa agcagaaagt coggg tatga 25740 tggttcatgc cittaa.ccct aac actittgc ggggcc.gaga taggaagat.c cc.gtgaggcc 25800 agg actittga ggctagocto agcaa.catgg taggaccctd to tccatalaa aag cittaaga 25860 US 2003/0162202 A1 Aug. 28, 2003 54

-continued aaaaaattag cqggg.cgtgg toggagtgcac citgtagt citt agctatttgg gaggctgaga 25 920 tgggaggatc agttgagcct aggagttcaa gqctgcactg agctato atc talaccactgt 25.980 actc.ca.gc.ct gggcaacaga gcaag accct gtc.tctgaaa aaaaaaaata cacacacaca 26040 cacacacaca cacacacaca cqttagtggg atago acaaa to agaaaaac totgctottt 26100 gatcactgag tacatctotg tagatatata tittccittcac tacagattitt gcc.caagata 26160 cittcgtcaaa gacaaag.cca gtacacccitc taatagg gtg aatatggitta toccaccitac 26.220 tgagcttgtt tittgatacta gittaatatgt aaccagatga aattgtcatt atcgtoactg. 26280 to aggacitat gggaagctta agtgttct ct tittcaaggac aatgtgcgct aactgtacaa 26340 ttggtaca at taaataagtt atattoagtt cotgggaagc actatagdala tacaaggaga 26400 aaatttgatt citatttattt ttgttaaggc ccaccitacct cotaatccta atttctotca 26460 tittcccaaat attccttgtt tattottact gttatgttgtt titcctgtatt ttgctcittct 26520 actittcttitt coatgg acta totttittccc titcctttittt togctctacc cctttacct c 26580 agctittctag cagtatttgc taaatactitc aaaactgtat agaactggitt caaattgtgt 26640 gctoccittitt citgtcaagaa cittgctactic aggtaaccoa attggtgatt tittcctggaa 26700 acactgatgg atgctgttcc tatagogalaa cccagaacag agatgaaata gatgtcatcc 26760 tdag coatta gcattcaaac tataaaaatt aatttacact g gtatagitaa ggatcagaat 26820 gtoaaagctg tdttacacct agcatcttgt atgaaactac cocattaagg tdagaccaca 26880 gatattattg ccccactatt gg catgaaag citgaggctoa gag cagittaa citgagttacc 26940 caggaccaca cagotaagtt agaagtaggg citcaggtgtc. citggcaacta actgg to cag 27000 ttatttitttc. tctdaagcto gtttitcccitc. tcctaaagaa taggaggctc tdtcgtggtg 27060 aaagg.cgatt ttagtaatac tittcctttitt atctgttgatt ataatgaatg cgg catctot 27 120 cccattaagg atcattccitc. caccoacatt cittaatacat citgctgcatg catccttcag 27 180 agacctcc ct citgggatcat cocttctoac tocaaaaag.c tdaacttcto coct9tcatt 27240 tgtacctocc acticagoatt tittagaagica atatttcatt caaacttatt caagtttatt 27.300 tocaccitaaa gaaatattoc tittcaccotg gcatctocqt caggtactgc tictattgttt 27360 ttctoccott cag acaaact gccaaactgg citctagttcc toacattccc catcaccctc 27420 agcaa.gcttctg.ccccacac cqg cactgaa acagotgaat cocaatgtcc ttgtc.cittaa 27480 acco agcaga aaaaaaaaat caatcaatta tittgattitca cagoggcact togacatgggt 275 40 agccaggaat ttatcaatga caacctttac agatcatctt totaattitat catgagg cat 27600 caaatgaatg citattaac at taatccctcc tattittaagt cattaatcca agtaaatgct 27660 cacttattitc tag.cgtctta gaaac cattt aaattatgtt acattatgaatcaatacatt 27720 ataaaattat accatcattt gtaataattt tittaaaatgttgttgttgctat taacattgat 27780 gccttggitat aaagttcatga toattctggit citagtag caa tottctattg actattotct 27840 tactaaag.cg gtc.cctitcc.g. tagg actoag agaccitcaca citctoctgcc tatgtttctt 27900 cctotctaat tigg.cccttct tactccactt g g g tacticct gcc cattgcc tag acaagag 27960 cattccctgt aactctgtct togggctottt ttctotttitc atcaacatct tctacgtggg 28020 tattatcatc catttccatg gcatcagott gcc caataaa citgataaatc catag totct 28080 ataagtacag cagatctoat caagctagtg gcattcagac toctittaact ttalaccaaaa 28140 US 2003/0162202 A1 Aug. 28, 2003 55

-continued ataagggatt ttgtacatgttcaataag ca gttcc cactg tdacactgta atcacatttit 28200 caca attgtg accitaggaca cittagagtaa aggatacaga tigattgagac agaaatagt g 28260 acaaagaaaa ataaggittag gatatagatt ttaatgctgt alacagacctic aaaatacaat 28320 ggcttaacta agagaatgca tttctotgtc. acataaaggit cocaactggc gtag acttitt 28380 gatgacitcaa gogcto aggc tigt gcctggit ttgtggttct gcct tcc tta acacatggct 28440 to catctgat gagctacago agtacctato actag to agc atgtccacat tccagoctogg 28500 gcaaggaaga aaggggaagc gcagaactgt accct tccitt ttttalagtoa togaactgaaa 285 60 gttgcatgita toactitccac ttgcc citcca gtcaccagaa cittagtcata taccataccc 28 620 agcttcaagg gagtgggitta aaaacataga agt caac tag goagtctgca cccagcaaag 28680 gatcgg gagt totatt atta aag cagaatt gagaagtgg taa.caggaaa caaccaccag 28740 cctotgctg.c atgtatatga aacagatgtt toccaaatca citattotcac titattotgtc. 28800 tgatacactg. tattttittat tatattotct titcatttittt aaaatcctgg toatgactica 28.860 caggg.catga tigttacaacc cacttagatg citaacaccat aatctgaaaa atattaccta 28920 tattatgtct aatattggcc acttgaagta tdgctag cot aaattgatct atgttgtaag 28.980 tataaaattic acaccagott gtgaaaacaa attatgaaaa aaaagttctitt aagatatoat 29040 taacaattitt atattggcta aatgttgaaa tdatcatatt ttggatatat tiggattaaat 29 100 aaaatacact attaaaatta atttaatgtt totctittatg tdgttactag aaaatttaaa 29160 atttaaaatt acacaggg.cg atcacattct atttctagta gaccacactg. citgitaagctc. 29220 aag attcaaa tdtcaaactc. citgttgaatat taatacgtga atatoccaca agcacttact 29280 ccatctitccc aaccotcago cottctgtcc toctitctgct cocaccaatc tatgtttctt 29340 citgttt cact cacccagota aaggcaacac aattcactcc gtgacgagcc aggaaaatgg 29 400 aaag acacat tittcctittat toctoacatt gatatattoa citgag cacta taattacctc 2.9460 ttaaatatga tataaatctg caagctottt toaataccac cacaaattcc atagttcaaa 29520 atgccatcag citttcaccita tattattaca ccagotccca totggtottc citgcatcct g 29580 gatcaccitct ttctag ctgc cctittcaaat ttcaataaga gcaa.gctttc caggaaacaa 29640 acctgaagttcaatccactga gtacticcitct gaatacctta atattgttga caaatticcitt 297.00 totgatttga agitatcagaa aggaatattt cotccatacc aaatagittitt catttcatgc 29760 atgttgcc.gtg attcttctoc citcctittgcatctgtcattc gttatgctta gaaagctcitt 29820 ttcatctott tattottcga gacaaccact acticatactt cagagcttaa tttacatttit 29880 gcttitcccitc aaaattittitt taaaaggttc caggtotggg titatgtgctc. tctitatgtgc 29940 toccagag catcctgaactt citgcaataat atgtttggct actgtattitt atacagtagt 30000 tittatattgt attittatact gtattittata cagtaggtot tatattgitat tittatacagt 30 060 agttgtttitt citgtctgttt ttgcc.ccaac aagaatgitaa aatctittaag tacctgttitt 30 120 catacttatt to accaccct atctotagaa tottgcatga tigtotag coc tag tagg atc 30 180 aaaaaatact tacaaa.gcaa citgaatagct acatgaatag atggatgaat aaatgcatog 30240 gtggatggat ggattaatga aatcatttat atgacittaaa gtttgcagag gagtatcata 30300 tittggaaggc agtaaggaag totgtgtagt cqatggtaaa gocaattggg aagtttgtta 30360 ggcacaatag gtcaaaattt gtttittgaag toct9ttact tcacgtttct ttgtttcact 30420 US 2003/0162202 A1 Aug. 28, 2003 56

-continued ttcttaaaac aggaaactct tittctatgat cattctitcca g g g cctogct cittcatctgc 30480 aaccoagtaa tatccctaat gtoaaaaag.c tactggittta attcgtgcca ttttcaaaga 30540 ggacitact ga attctgatgt ggcttcaaac atttaggitta gg catat cita atggagaact 30 600 tgcagocaca citg acttgta gtgaaatato tattittgagc ctg.cccagtg ttgcttaaat 30660 tgtagtttitc cittgcc agct attcatacaa gagatgtgag aag caccata aaaggcgttg 30720 tgaggagttg togggggagtg agg gagagaa gaggttgaaa agctt attag citgct gtacg 30780 gtaaaagtga gct cittacgg gaatgggaat gtagttittag cccitcCaggg attctattta 30840 gccc.gc.cagg aattaaccitt gactataaat aggccatcaa tdaccittitcc agagaatgtt 30900 cagagaccitc aactttgttt agagatcttg totgggtgga actitcctgtt to cacacaga 30960 gcagoataaa goccagttgc tittgggaagt gtttggg acc agatggattig tagggagtag 31020 ggtacaatac agtctgttct cotccagotc cittctittctg caacatgggg aagaacaaac 31080 toctitcatcc aagttctggitt cittctoctot taggtoctocit gcc cacagac goctoagtct 31140 citggaaaacc gtgagttcca cacagaga.gc gtgaag catg aaccitagagt cottcattta 31200 ttgcag attt ttctittatat cattccttitt totttccitat gatactgtca tottcttatc. 31260 totaag attc ctitccagatt ttacaaatct agtttacitca ttacttgctt acttittaatc. 31320 attctitcc cc aactcitctga agctotaata tagcaaag.cct tcctaagggg tdtcagaaat 31380 ttittagcttt ttaaaagaat aaattittaga tatto acatt catattgatc tacttgagac 31440 catgctattt atcttittctt attitcctott totcaagggit coattittcta ttittataaaa 31500 ataaag acaa ttctotcc.ca caaccaaaca taggaacaatig ccctggagta taaaaatcta 31560 tagagtgcca aataaaggaa caatttgaaa tactggtgtt gatattgaaa aag caaggga 31620 citctaatgtc. agaagagaaa toctitttgca gatgaggtgg to atgaattic tttgtttcaa 31 680 cacaactgaa gaggaactg aaggaaatac cagct gatga gtgatgaga a gggattcttg 31740 ataatagagt act agg to at ttittggcatg taatgcagaa gttgcaagaa gtggtaacaa 31800 tgatgcaatt gttttacctg. ccatttattt acttittatgt gag coattct tcttago act 31860 tatagotaca caaaacaaaa atagtaacag aattaatgtt gtttaattct td caatccat 31920 ggatgcataa attcactggg ggaaaaaa.ca gct catcatt citcattaaag atgtgct tca 3 1980 aaagtattitt aattittatat citaatatgta tdaatcatac tttgtattta titttgttittg 32040 atcagttata tacaagtatt tittgaacata gctcagtcag aaggaaatgt ttaatattta 32100 taaattitatg gttacattct atttaaaaga ggagittaaag ttaaatttac citacccacat 32160 atgttacata tatatgtatt tatgtatatg tattoatata totatatatg tdaacataag 32220 tatacatacg tatatgtata gatgcttgac aataaagaag taagaataat tcacaacatt 32280 ttittgaaata taaaaattta ggataaattt citgitatggta attgg catgg aaattcaaat 32340 toaaaaagga aaaaagaaga gaaagatatt aaatatoaga ccattaaaag aattittittaa 32400 tgtacttitta aatagtgata gtaggitatct tatactacag tatttattat tcatgagaaa 32460 attgtaaaag taatctaagt attaatttaa aatatoatca aaaataatat cittittgctat 32520 tacttaaaat catgataaaa atatgtttac ttgaaaatat gtaaggagtig cacagagtcc 32580 aaaaattatt ttaggagttctgtgagcaaa aatgtataaa alactacaggg ttgat cittaa 32640 attacatgtc. aggg tact ga gaaagtttct gtactgcaca toagttacca aggtotaaag 32700 US 2003/0162202 A1 Aug. 28, 2003 57

-continued tdaatcacca gaggaccatt tittggatgga gcc attgttct aatcatgagc tigaaaggcaa 32760 atatttaaaa tdcaaacatc catggittagg taacactictt aag acctitat tagctgctta 32820 ccacaactga gactgtgaag taatggctca citttctittga ggctoagatt coatatotgt 32880 ggagtggaag cqagtgctta ccatacaatt ttcacagggc tigctdaatgt gtgitatgtat 32940 gtatgttgttgt atatatattt taatgaaaat tctataattt gattagttitt totaatgtcc 33000 gcatgactga gagcttgctt acttitttaca gcaacttgaa ggtaaaaata gattittacaa 33060 catgaacaaa totaactaca tatttittatt tdaattcaga tigttcacaaa ttgttccitta 33 120 aagttgaagica tacctacaag ttittaatctg tittaag acct accitcaagta aaatgttcac 33 180 tgcc atggca totgagggala aagggaaata attctitatgc atggc ctitca acggccaaat 33240 ttcatgctca toagtacatc ttctottggit gtagaactga tigatgataat tatgatgatg 33300 gaaaaaagtg citgttgatag caatgcctct cittccittcac tittccitctaa citgaaccgtc. 33.360 to attcc.cag gCagtatatg gttctggtoc cct coct gct coacact gag accactgaga 33 420 agggctgttgt cottctgagc tacctgaatg agacagtgac totalagtgct tcc ttggagt 33480 citgtcagggg aaa.caggagc citctt cactg acctggaggc ggagaatgac gtacticcact 33540 gtgtc.gc.citt cqctgtgagt gtggctgttt gacittaatat acttggttct tittagtcagg 33600 gtoataggga totagtatto tdtcagatga ggctttggga ggttggtaag aactgcagga 33660 aggaatccaa atgtag caaa citaagtatag aaattaagga gcaatgcatg act citccago. 33.720 catgg gagac aaatatttac aggcaaacta aaagttcagot taataatcac atagaaacco 33780 tattagccag aaaggaaaaa aaaaaaaaga aaatgtgact tcttaaaatt aagatggaaa 33840 aaat attaat aaa.gcaaact agaccaaggc tica acatgag ccaccc.gtgg togg actdaca 33900 ggaacacatc acticcacact acttctaaga gtaaaaattig tagaaattac cct gagcaaa 33960 ggtgtaatat citcttcagag ttgtccacat gtgagatgct tacagoctag tdtcagtaaa 34020 atacggggitt tttittccata gcctgtaaaa catgctitcga ccatgcc citt ttagoatgta 34080 atat cago cit ggtaaagcto agcgtata at tdaatcaaga citgtttctgt gagct gitat g 34140 aaggtotgaa totcacttac aacctcitctt actgattitat tittccitccac cittgttgtcct 34200 gtotcc cc at ttgtaaaatg gcagaagttga titcctgtcca toccaactitt coctogctgag 34260 aacaagggag tdaagttaag taagaagggc aagat citcca gggaagagtg caa.cago aaa 34320 taggggagga gacctg.cggg togctdaatgg ggtttcagga toggtoctogt atttcagg to 34380 ccaaagttctt catccaatga ggaggtaatg titcct cactg. tccaagtgaa agg accalacc 34440 caagaattta agaag.cggac cacagtgatg gttaagaacg agg acagtot got citttgtc. 34500 cagacagaca aatcaatcta caaaccaggg cagacaggta toaagaa.gcc tacagacagg 345 60 acaacttcaa aaaggaaaga tottctitccc citggatgttc cccaggcaaa gttcctataa 34620 tottggttcc ttaatagott gtottaccag cctacaggcc tactittgggit ttgggggctc. 34680 atgaaaaata tittctgtttc agtgaaattt cqtgttgttct coatggatga aaactittcac 34740 cccctgaatg agttggtgag titttctatta totacataaa atgattgttct gtataaacag 34800 gctgggalacc tdtttitttgt gct gagggaa gaccagg gag aggaagaatc togtaltcatt 34.860 aacagtaact tctggcatta caacagdaca agatcctaat citaaaacatc attccaggta 34920 aagaaagtag gtaattctitt citgtcttggit gctgg tactic agtcagttgt cacacaatta 34980 US 2003/0162202 A1 Aug. 28, 2003 58

-continued aatttactitt toggatggitt cittaattagg acaattagaa agatacattcaatagoagac 35040 acagaaaaat cotcaaagaa cotaagctca aaaaacattt taaag attta gatttitttct 35 100 atacacatct accaaaatct tcactataaa ggaaagttcag g gtaattaat ttgttccitca 35160 agacitaactc ttggitatctg tdataagaaa cagttctittc tattgtaatg cag acatcaa 35220 cccaaagttct tcatttittct citcccaaatt aacttcttca cattttctta totcaaaaag 35280 aggcaactct tctottctag citccaaagac aaaag attat ggcctagttc ttgtttctot 35340 ttctotcata cocacatcca cittcactgga aaatcatgtt ggcttaaaat atattoagac 35.400 tatttctitat catctgaact actgctgcaa gottagtocta gtcaatgtca totctaaata 35460 agatcattac aataacctitc aaagtggtot cocag cittct actitt cactic citctgtctaa 35520 aatgaggcac cacacccatc actctgtcag cctaacttgg citttgtttitt citatttgcac 35580 ttaccaccat tctatacgta ttgatttitta aaaatctg.cc tatttctatt ataacataag 35640 citccattaaa acatggittta ttgttcttitt gtgcgtggitt atattotcaa tacctagaat 35700 gatacacago goatgagaag gtacttaata aagattagtt tittaaaaatgaataaacatt 35760 catagtagct tcttatcaat gtttatto at ttittaaaagt aatatttatt aaactaaatt 35820 tattaatgaa atatgtc.cat tccitccto at tcttagaact acgtaagaat tctato acco 35880 atcttaatga ttacatctga acatatttac titatagittac ataagttgttt atttctoaac 35.940 citgtaaaatt gcatagoa aa goattgatct tcactaggat gctataag.ca cittaaga cat 36000 gttaatcc at ttittagtaaa tag cactitta catgtatatt tattgctgaa ggcctagtaa 36060 gttcttaaca ttatttatta attgcttaaa atagattaat gaaaagttct ataaaattta 36120 atctggaata tittctgtatt toactataga g g gaattatt citatatgaaa citaatttaga 36180 ttttittaaac tttittattgt ttittaattitt totggaaaca tag caggitat atatattitat 36240 gggitta catg agatattittg atacaggcat gcagtgcatc. ataatgatta toggatgataa 36300 toattatcat tatccatato attatccaat catggataat gattattatc atgcattgca 36360 tgcctgitatc aataaatgga gitacccatcc cctoaaacat ttattotttg tattacgaac 36420 aaatccaatt atacttittag titatttittaa atgtacaatt aaattattitt ttactatact 36480 caccctgtta toctag caaa tactagotgc tittgcttata aatgagattit aagaatattt 36540 gaaaataatt ataaacttct tttittcttitt gtctittcaga titccactagt atacattcag 36600 gtaagcaa.ca toaaac attc cat attaaaa gqaaa.gcaat acatataggg aaaatgttct 36 660 tatttcagag gtttittacaa tatctoagaa acttgtcatt aaag.gagaag ccttcaaact 36720 cccatagagc tagatggcta taacticatct cotctactica coctittacta citaccccatt 36780 tgacctttitt gtagataact tagggitttcc atagatatot tittattagot coaatgcto c 36840 aggtgcttitt gtaagttata attaatttac toatatagga toccaaagga aatc.gcatcg 36900 cacaatggca gagtttccag titagagggitg gccitcaag.ca attittcttitt coccitctdat 36960 cagagcc.citt coagggctico tacaaggtgg togtacagaa gaaatcaggt ggaaggacag 37020 agcaccctitt caccgtggag gaatttggta toggat catga aaagt catca agcattattt 37080 ttcttcatat ttaaactcitt aggtoctoga atttaagttc atttggagtc tittccatttc. 37140 ccatgggtga cattgggctt gagtagaat taattacacc taagtccaat gagga catca 37200 gtgatctgtg aatagg actt cacatagott cqttattittctgtag caata tittaatacca 37260 US 2003/0162202 A1 Aug. 28, 2003 59

-continued accoccaaaa ttaaaacatt cittgttittaa taggagitttitc cataattaat taag cacaca 37320 gtgatctotc atagtc.ccitc aactgaaatc titcatttgag aggaggatag ataagaatag 37380 attggaga.gc agagctacct titt cagagcc ctaaaatatt attagggaac tottacaggg 37 440 aacctgaaaa taggaattcc cccaaagttgaaaaccaatc accaacctitc tittatcacca 37500 atcaacagtt cittcccaagt ttgaagtaca agtaacagtg ccaaagataa toaccatctt 37560 ggaagaagag atgaatgitat cagtgttgttgg cct gtgagtt cattttittaa aaatcttittg 37620 tgggggatta tittaaaagag acticaccatt toggatattt taact actot citc.cgggagc 37680 agtggcaa.ca caaaaattitt aagtgctttg acago atcct catctgtaga atgttattot 37740 cctgttgctt ttctatttitt attittcttitc acgttctitat cagtattatt citatcatgag 37800 gtaagaaact gtttctagag aggttatgct aataggattg attctgaaag togacaaaact 37860 gcacacacac acacacacaa aatgggaagg gtggatagtg ttgaagg gta ttggttctgc 37920 cittalaccalaa aataac caaa cqtat attag g gagataatt aacacatggc tataggggaa 37.980 attcaatcat caacagatca tttacctgac cittgcatgct tactggaaaa atcacttaga 38040 ttcagaattt gtagagatag goatacaact gaaatctoat citaactcact agtccaccita 38100 aggcgggtot acatacct gt citgcactgac atttittacga agagactgaa cqtgatttct 381.60 caag acagtc tagttccaacc titgaaaagttc ttgttitcctg gatctitatgttcc catccat 38220 ggtggcacac agtag cagta gcaggaagag caggtatagt cct gtccaaa gat cacacat 38280 gtaatacacc tactattoag citagg.cccct gctccagttctgtctoacag tdatggccaa 38340 gttcctggitt cotcataact ggtgttgatct tdcaaataga catacagagg acattctdaa 38400 ataggaaagg agacaaatgc acttggaaac ccagoaattg totatgacat atttacgaca 38 460 caatcc cact tttgtaaaaa agtaalacagg ccc.ggcgtgg togct cacgc ctotaatcc c 38520 agcactittgg gaggct gagg toggcagatc acgaggtoag gagatcaaga ccatcctggc 38580 taacatggtg aaacco cqtc. tctactaaaa aatacaaaaa attagcc agg catggtggcc 38640 agc.gc.ctgta gtc.ccagota cittgggaagc tigagg cagga gaatggcatg aacco aggag 38700 gtggaggttg cagtgagcca agatcgcacc actgcactcc agcctggg.cg acagagc gag 38760 actccatcto aaaaataaaa taaaataaaa taaaataaaa taaaataaaa taaacaagtig 38820 aaactcgata tagagatggc tigcacatggg gagaggatga tiggaagaaga gacactato g 38880 tactaatagt ttgtattoct aggtag actt gaggg.cgittt g g gttitttitt tattotgttt 38940 tgttttgttt gtttcgtott gctgg tattg tittctotttit aagaacgggit ttaagttitta 39000 taataagaaa gacittttitta agataaaact aaaaaaaaac gaggaaaaaa aagaaatgat 39 060 aaaagaagaa totaaattitc agcatgttgc aatggagatt totataagag ccattagtga 39120 citcttgtc.tt caatattgttg tdagcc.ca.gc agaga.gcaga gggaagtaca gacagggaat 39180 atactgtagc taaaggggag atataaatag citagaaaatg gg gaggttca gtgtctgct c 39240 tgattoctitt go gaatgttc. tcatcacaga tacacatatg ggaagcctgt ccctggacat 39.300 gtgactgttga gcatttgcag aaagtatagt gacgctt.ccg act gccacgg togalagatto a 39360 caggctttct gtgagaaatt cagtggacag gtaggttgaa cactatttitt totagagaat 39420 agcgataaag gCattgttga aaag.cagtga gttgcagoat ttittctgacg caggaagaga 394.80 acaatctaga agagaattcc atgttggcta ttgtaattitt toaaaaaaaa toatgaactt 395.40 US 2003/0162202 A1 Aug. 28, 2003 60

-continued agcacaatgg gaattattta tittctogtaa ttgcc cattg tdagtgtttc agaacgatag 39600 acactgagcc atctaaagcc to catgggca ttcacttcta caaaggaagg aaaaaac cat 39 660 acaccitctta attgccittag citggccaggc catcagttct gctcitctotg tdaacaagaa 39.720 ccitatcacat ggc.cccacca agatgccaga gagttgacaa acacagt coc catatagaag 39780 gcc.gct tccc agccacagot gaatattatg gaggaggaac cag actittga tigaggagttc. 3984 O taatgg to aa gag cagatgt actgtgtatt toaaaatago aagtggacag gacittgaact 39900 attitccaaca catagaaatg atacatactt gagttggtag goaccctaaa toccg.cgatg 39960 tgatcattac acattctotg catgtaacaa aatatotcgt gtaccccata aatatgtata 40020 aatattatgc atcctttgta caaaaaaaat tacticcitcaa attittaagac atttctatog 40080 caatatat ct agg gaattitc agataccalag gaata catct gtcaattata catgagatat 40140 tgttgttgaaa tittaatattt agttgctaga gaatattitat tigtgtccitcg tdatagaaaa. 40200 tgcctacatg atgttgtc.cc cccacaaaaa toacaccggit tatctgacca citgatctatt 40260 tagtataata to cattatat titttgtattt tattittctoc cittcttagot aaacago cat 40320 ggctgcttct atcago aagt aaaaaccalag gtc.ttccago togalagaggaa gagtatgaa. 40380 atgaaactitc acactgaggc ccagatccaa gaagaaggaa caggtttgttg tactacatgg 40 440 gtataagaga aaacacaa.ca ggcattgatt ttcttagcca aataatgaat totaagttgg 40500 ggggaggtga tagaattitta gag acatcat tctitcccaaa aataacagat titttittctot 40560 tttittcagtg gtggaattga citggaaggca gtccagtgaa atcacaagaa ccataaccaa 40620 actcitcattt gtgaaagtgg acticacactt togacaggga attcc cittct ttggg caggt 40 680 ggagtattitt coagttcact catcaaccoa totactgtta cotaattagc acaatagitta 40740 tggitttgttgc taaaac catg cct ggittaat gttatcattt aatataacca aaagtataaa. 40800 atatoaccaa ggcttgatta gtataaccaa aggtataaaa citacataaaa atagattitat 40860 tottctgtaa atttgttgtat gaaatgitatg taattatcct aaggcctitat taaaattagt 40920 agagitttitcc ccccttctitt togaaacagoa ttgtacaagt cactcaatct cottctoatg 40980 gtttaccagg cagtctdagg tttittatgac attittctoac aagaatctoa aaatticatgc 41040 tgaccagttt cattacgatg cita acactaa citttgtatgg aaa.caggtgg gtaggtggitt 41100 ttaatttitta ttittgaagta ttaaagattc tacaataatg tittatttcat ggatagtata 41160 tttacactat ttittctataa caagtatatt totaaaacag tdaacatgg g g taaaac act 41220 gctatttaag gttctdag at ttittgaatta tdaattittca tottatctac caaaaaaatc. 41280 ttctottaca atttittctgttgtcagoaga tigtgcaaatg gatctittatg actictagaag 41340 totgag atca cagttacatg tacctcaatg totatttact gtgitatcatt ttcttaatgt 41400 aaatgttaca ggatttcago tatgaagggit gtaaaag agg catagacitca acaaagtgga 41460 gtacattcta gaaggcttgg titatgtcatg acaccaaaat gtattoaact tcctaaaatg 41520 aaagtgtagc ticatttgtaa atttctdaga aagatagtgt actttgtaag tacatttitat 41580 tgctgttaaa tatcctattt gtoataag ac totctag cta gaagaaaatc agaattatgt 41640 gcctattott gtctttgtac atgcagocaa catttcactg goaaacttag attttgtaaa 41700 tdaattatga gtactgatgg g g g to agcct attgttctitc ctgctaccaa citgtag citta 41760 tactgaaaaa gaattgccac citttacaatc tdaactggac tactcaact g c caatacaat 41820 US 2003/0162202 A1 Aug. 28, 2003 61

-continued attagoagta aattagattt ggcaactitat ttaatactgt gtgccitcttt toaattittat 41880 atttitt caca toggaagtgt gtcataaaat citgccitacct tccaaaaggc tigaagat cita 4 1940 ggttgagaag cagagaccat gtctaaggca actggagaaa cacacaggac aatgtattgg 42000 caattgttta cittgttgcact tatgagacitt cag acactaa totataggaa gttaatgg to 42060 cacticcaaaa taggtgtttg g g g cacaaat aaatttagtt aatagattaa tag attaaaa. 42120 tatttcatta toatattact citagtgctct gtgacticitot aag agittata tataataata 42180 cacago actt coaaaatato tagaagacat ttittcagg to acticttgtta ttatcccitat 42240 cactictotat coctitcacac tactctgttt ttcttcttag gattgattac tactaaatta 42300 gtgitatgtta totatatatt tatttagcat citatctottt cactagacag taagctotgt 42360 gatggaagaa actitcgttitt ttittcactgc tigtgtc.citca gtgcctggaa ccatgttcaa. 42420 catagaggag gCactaaaaa atgtgataaa taatatgtt togtoccitat tagtt attac 42.480 caatataact aatccacago ttittaatctt caggtgcgcc tagtagatgg gaaaggcgtc. 42540 cctataccaa ataaagttcat attcatcaga ggaaatgaag caaactatta citccaatgct 42600 accacggatg agcatggcct totacagttc. tctatocaa.ca ccaccalatgt tatgggtacc 42660 totcittactg. ittagggtaag tittggaaaga aattaccaat gacatgaagt agccttggaa. 42720 acaaggttgc aacctaaggg to agaaaatt to caaactgt gtctagttct atggagagaa. 42780 aaaaactago aattagaaac cqattgaagg tta acttittt taaagttitat gaaaagaagg 42840 cagtatattg to attaaaag togcgggittag actittagcag togttgctggg aacgtaaaat 42900 ggcggagcca citatgaaaaa cagtatagta gttcc to aaa aaattaaaaa atagaattac 42960 caaatgatcc agtaatccita cittctggaca tatatto aaa agaatcgaaa acggggtotc. 43020 aaagagct at ttgcacacco gtgttcatag cc.gcactatt cacaatagot gagagat.cga 43 080 ggct accolaa atgtc.catca agg gatgaac aggtaaacaa aatgtggitat ataaatacaa 43140 cagaat atta togcagoctoga gaagggaaga aaatcctgtc. acatgctaca gcatgaatga 43200 to cittgagga cqttatggta agtgaaataa got agtcaca aaaag accala tactgtatga 43260 ttcacgtaca togggtttct aaagtagt ca aagttcataga aacagaaaac aggatggtgg 43320 ttgcca aggg citagggaaag agagaaatgg ggaattic citc ttattggitat to aggtttag 43380 ttttacaaga tigaaaagttctggagatctg ttgcacaa.ca gatatacitta atactaccala 434 40 actgtacact aaagaataat galagatggta attittatgtt gtgttgttgtgt gtgttgttitat 43500 aataacttitt ttgaaaagtg tdaatticcag aatctgaatc agataaagtg gttaaaaata 43560 citggctcaac cotattittaa ggaattaact aaaacctctg td cittcattt cottatctgt 43620 aaaatgattg caatactaac acctgactict toggg to gtt gtgaagatta agtgaaatca 43 680 tacatgttga atcacttagt aag coccott taactgttag atacttittac tataaaag.cc 43740 aattctaaca taattagcat ttagttittaa atatatatac tocaaaaatt attaccttac 4.3800 tittattttgt cittgctatto taattittcto cagatgacct gttcatacca atataagtta 43860 titcggtatag catgcagtcc totattocatc aagagagatg tag acatgtc. tatagataca 43920 tggatataca atacatattt aatatatatt acataatcat ggtaatggaa aacgcctgcc 43980 totttagagt tttaccttgt tatagagaaa taaataatac attittattta tittagctitta 44040 atagatggca taaaag cacc toccitctaat ttgaatgttt acticatttcaaaaagtgtct 44 100 US 2003/0162202 A1 Aug. 28, 2003 62

-continued tgaatgctitt coacgtgcca gcactgtgct agtcttgaca atgaaattat tatttctacc 44 160 tgttcc citgt ttggggtgat catatatgtg ttgtcaccala aatatacatt aagatatgaa. 44220 caaaatgttt togctcaagga titcactagtg citattgg gag citggggatga aggtagagga 4428O agcctgaaag gagctaagga ataacttcta caggaagaaa agttggaaat atalacccaaa. 44340 tagg gccitct gagg tattag aatccagatt gcagg gag.ca ttaaaaatag to agccitagg 44 400 ccaggcacgg tag citcacgc citgtaatc.cc agcactittgg gaggctgagg toggtggatt 44 460 gcqtgaggcc aggagatcga gacgagccita ggcaacgtgg toga aaccotg totctacaaa. 44520 atatacaaaa aattagctgg gtggggtggc atgcaccitgt agticcitagca acticaggagg 44580 citgactggga ggatcactitg agc.cccagag gCagagattg cactgagcca agattgttgcc 44640 actgtact.cc agcatggg.cg ccagagcaaa accctgtc.to aaaaaaataa taataatago. 44700 aatcagccita agatagocca gaa gaggtag actitcagota attcatcago totagotcitta 44.760 aaccaatgct ttctaccaat gtottctdag accoctaagg ttacaatatt ttatttatto 44820 ataataccca ttcaaaatcc accotaaaag agg cagotgc tittctgaaag cacagttctt 44880 ccatttctag agattattta citctoctoaa tdaagtttca tagctccagt gitotictaatt 44940 gcacaggtaa agcagtcaaa gaaatttcaa goaa.gctaat cagagcaaag gatgcct cot 45 000 tittatgctct taagaaatat aaatctoaat cocaggaggc tictagoagtgt aaagttcacaa 45060 agcatgccita catttgaagc agagaaacaa aatcaggggit cottctocca cittitt cattg 45120 tggaacaaaa gatttctago tactagittaa gg tagg acag taaacttacg tagttttgttg 45180 agaacattaa totttatgac gtataatcta aaaataatat aatttittcta attcattagg 45240 tdaattacaa gqatcgtagt ccctdttacg gctaccagtg ggtgtcagaa galacacgaag 45.300 agg cacatca cactgctitat cittgttgttct coccaa.gcaa gagctttgtc. caccittgagc 45360 ccatgtctoa togaactaccc tdtggccata citcag acagt coagg cacat tatattotga 45420 atggaggcac cct gctgggg citgaagaagc tictocittcta ttatctggtg agaagggagg 45 480 ttactg.cgitt gacittcactd tag acaaaag citctotgtgg agcaagtaat catgaagctic 455 40 tittagatgtc attacttcaa cittcttatcc atgttccttt tdaaagtttg atttctottg 45600 aggtgaatta ttgcc.ggcag g g acticaata aaacaagtat attgaagtga gactgaag.cg 45 660 tgttctotct g g cacattta atttctttitt attitccttitt ttgcagataa tagg caaaggg 45720 aggcattgtc. c galactggga citcatggact gcttgttgaag caggaagaca gtgagtattt 45780 ccatcatcto tgcattgctg. ccccattctg accoattcag cottacacca cqgaagitatc. 45840 agaatactitt coctittttitt coataggittt ttgggggaac aggtggtgtt tag to acatg 45900 aatgaataaa ttctittggta gtgattitccg agattittggit gcacccatca ccc.gagcagt 45960 atacattgta cocaatttgc agtcttittat cocto accoc ctitccaccot toccoctgaa 4.6020 tocacaaagt coattgitatc attctitatgc ctittgcatcc tdata actta gctcc cactt 46080 cctaatagot tagctgccac titcctaatag cittagctcct acttctgagt gagaagatat 46140 gatgtttggt titt.ccattcc tdagctggga atato aggaa tactittcaag taacgagaga 4620 0 ccatttcc at citaatattaa ttaaaagaaa gttgagg gag ggaaaacaat aatggtttitt 46260 ttctoaccct ttatcaaata tatgttgttct tttittittcag cotattittct ttgcgittatt 46320 taaaatattg gtgtgggcca gctatatgg cittacgc.ctg taatcc.cagt actittgg gag 46.380 US 2003/0162202 A1 Aug. 28, 2003 63

-continued aacgggatgg gaggatctot to aggtoagg agtttgagac cagoctogto: aac at agtga 46.440 gaccc.catct citataaaagt aaataaataa ataaataaat aaataagaac aaagggggaa. 46500 aaataaaata cataaaatat tdttgtgitat aatctatact tatto.cagat ttittaaacca 46560 taaatattot talaccitctt citcaaaatga gaattgttgtt agagaatggg gtggagaaaa. 46 620 tgtacgattt caagg tatgttgtatatacc aggtottgttg citgagtcatt tatgtcattt 46680 atattattat aaatatottt tttcc ctittcaagatagitta to atttatga agcattaaat 46740 tggg tactitc taccgact ga agcgg actict toccatgagc aaatatgtca taatataaac 46800 citttgaaggit togctcc tagt citagtag act citaaaagata acttgctaga aaagtaticag 46860 taatctactc. tct cattagt tottgtag ca acacagaagc accatagoag actgaaggaa. 46920 acttaatgca accitctaaag aatctacata ggcatcaaaa aataacttct gaaggag cat 46980 ggtgttcc to titatggagaa agaac caaat attggct cat agaaggittaa agagcatgta 47040 gcatgaatac aatgttgttga acticttactic ttacatctoa taactitcagg ttctattitcc 47100 citcttctagt atgagg ccta gatcaaatgg tagtgaattig gatgtcttca taccottaac 47160 tagcatcaga acattagg to ggtgagctga agcttaaagc tiggtgaaaaa toaacagagt 47220 atatgattta toga gaggaga gagggctttgatagg gttitt tittctaaag.c tagagtc.citt 47280 agaggtaatc atccatttgt tacatctact gtttattatgtctgttacat citactaatgg 47340 aaatag to at taacctatga taggctitccitc. tcattctott tatgcc.ccag tdaagggcca 47400 tttittccatc. tcaatccctg tdaagttcaga cattgctcct g togctogg't tacticatcta 47460 tgctgttitta cct accgggg acgtgattgg ggattctgca aaatatgatgttgaaaattg 47520 totggccaac aaggtgttgttg ttittagatca taaaatcttcaacatgtaaa actagaagtt 47580 actattgtta toatttgttt tacacatgtg aac attaggg ccaaaagggit taaacaaatt 47640 ccc.ca.gagac attcagdaag gtagtggcag agt cacacta agaag cagaa toactitgatt 47700 ccittatacaa aactcitcaaa cittctoctag cagtgccitct taccaattac acagttcaga 47760 gtatgttatt cocttcttct atgatgaaac gttgggaaat atgtaag caa gtttittaaga 47820 citactaagga gccaaaagaa aaatgcaaga gcaccitagac ccato catgt coccaccacaa 47880 ttaatgagca cottgcc.gtg tatcaaag at aaaaatgg catttcatgact agtaattitat 47 940 aagaataatt agaaataatt citactggctd aagtgattct to agaatgaa agagaagtgt 48000 agggaacaaa aagcttcagg attgacacaa toccaa.ccct coacgaagttcaag cagagtg 48060 gttata catt gtacatgagg aac actotgc aaatgcgagt gaatgcatgg gtaaacct g g 48120 aaatttgctt ttctgaccitt ttgttccaat titcacaggtg gatttgagct tcagoccatc. 4818O acaaagttcto coagccitcac acgc.ccacct gcgagtcaca gcggcto citc agtcc.gtctg. 48240 cgcc citcc.gt gctgtgg acc aaag.cgtgct gct catgaag cct gatgct g agctotcggc 48300 gtoctoggtg agttcc togc agccticagga atcaagaagg gcc.gtgc.cag gggcticagag 48360 caaggaaaaa tactggata agtaggataa gatcaattaa aattatattt atctaggaat 48420 taattgctitt gagctotcitt gtgggcttitc attggtaagg aattatatat atatatatat 484.80 ataactactic acttctgcag ttaaaaaaat aaacaaaata caatatattgaatcaaataa 48540 aagaatttitt aaaaggacaa aatgttgatga aaattaacaa ccaaaaaagg acticgittatt 486.00 cag acacgtt aagcttcttig citcaagagca catag caata caaattcaaa tottctgatt 48660 US 2003/0162202 A1 Aug. 28, 2003 64

-continued gtgtcaggac atc.cgtatat gcaaggctgg agagaataca gag cagattg tdaaaagtgc 48720 tatatgagaa gtgcagttat gcaaaataaa agaaaggatt aactgagcaa ccggggaagc 48780 cgttctgaaaa ttatatattt aaaatgtaaa aag accatac agtacccaaa gtoctitaaaa 48840 toccagagct coatgcaacc agtaatagga gttgttcaaac tagtttcaac atttcaaaaa. 48900 gcctaacaaa agtgatacat atacctgcac taggctata caggctaaat gaaatat cag 48960 attitcc.gttt ttataagaat tatggctggg cct g g toggct cacgcctgta atcccaacac 49020 tgggaggcca agg caggcag ataacttgag atcaggagtt toga gaccagt citggccaata 490.80 taatgaaacc cc.gitat citac taaaaataca aaaattagtc. agacgtggtg gcggg.cgcct 491 40 gtocatcc.cag citacticagga ggctgaggca ggagaatcac ttgaaccoag gaggcagagg 49200 ttgagtgagc cqagat.cgtg ccactgcact coagcctggg caatagagca agacittggtg 49260 toaaaaaaaa aaaaaaaaaa agaattacat cagttgaaat aattgctgtc. ccaagctaca 49320 cataatttct gagggaac at atgtattatc. tctgaggaac aaaacttagg galaattgaac 49380 tactatataa ttgacattga taactgaact citttgaaaat atggagctaa tagaagaaat 49440 accaaaagga tatgctgaat tdgaacaa.ca aaaactagtt aatagoatgt actaatagta 4.9500 gtttgctoac atcttcaaat ttittaatatg agaccaattig taataacct c titactgaagic 49560 tttittctact accataattit aacaaagaga aatttatatt tittatttitcc tdaaatagaa. 49 620 actitataaaa aaatgtttct tacttgtttgtc.cttctaat gggattittaa citgaaaatat 49680 tagaatactt citaaaggaag catcaagtac atttcaacag taggatctoa agaggatatg 49740 tgggaaaata atatacatgt tittatactitt tataatataa ttittataatt citcaaagaat 49800 ttittcaaatt ataacaacco tagatgggtag ataagg caga cagtcctatt tattaataat 49860 aaag cagaag citttcaggga tittagtagta gacctgagtt aaaacaccala citcttct caa 4.9920 gctittgatta gtgtcttacc aatgaaacac tittgctgcta citagtgtagg to attcattc. 49.980 aacacattta tittaatgccc actgtgttct agg tattata citaagtgcta gtagagatca 500 40 agcagtgagc tactggaaag ataaaaatgt atgtc.tcato gaacttacat tdtctgtc.cc 50100 atagatgaga cagacaataa titatgcaata toccacaata aaag.caggga gaggaaatga 50160 gaaatgttaa gatactittga gaaagtgtct aattitcatca caccacticac tittgctdatc. 50220 tgttcttgtc. aatcagttitt aaacticcitac gaatataatg caatgtaact atcacaattit 50280 titatgtctgt cittacttcto accittcaaat g g acttgaaa goatcatgcc tagaattitta 50340 cggittaaagt totatgtatt atatgaagat citggag catt ttgtttccac taataatacc 50 400 taagaaaatg ccatcgtgtc. citgtggagag aggatattoc tattogtgtg cct gtttaga 50 460 acatgcacco attaactittg citatatactg agtcagttgc ticaccacaag ataag cacaa 50520 aactatoatt toctitctato atctoaaag.c tttgttgcaat gtcacaaata cagoagacct 50580 cgatttittca attaataaag ttittatttca titccagtgtt gag totagt g g toggcctctg. 50640 aactgtgtaa cqaagtagta cittagtactt agatgagtac ttagatggag tdtttggttt 50700 titcctaaaat tigittaaacat cittcaaaatgaaaac actot gtcaagaaaa tdatccatac 50760 cctotataaa toatcaaag.c aatgagag cq citcaaagaaa gacggatgtt cattattoct 50820 gttcttittct cottgaactt aaaaaatgtc. acaaaggcc.g. g.gc.gcggtgg citcacgc.ctg. 50880 taatcc cago actittgggag goc gaggcgg gtggatcatg agg to aggag atc.gagacca 50940 US 2003/0162202 A1 Aug. 28, 2003 65

-continued to citggctaa caaggtgaaa ccc.cgtotct actaaaaata caaaaaatta gcc.ggg.cgcg 51 000 gtgg.cggg.cg cct gtagt co cagot actic g g gaggct gag goaggagaat gg.cgtgaacc 51060 cgggaag.cgg agcttgcagt gagco gagat tdcgc.cactg cag to cqcgg toc ggcc togg 51 120 gcqacagagc gagacitcc.gt citcaaaaaaa aaaaaaaaaa aaaaaaaaaa agt cacaaat 51180 aagtttgcct ttttgttctitt cqtatttgta caggtttaca acctgctacc agaaaaggac 51240 citcactggct tcc ctdggcc tittgaatgac caggacaatg aag acto cat caatcgtcat 51300 aatgtctata ttaatggaat cacatatact coagtatcaa gtacaaatga aaaggatatg 51360 tacagottcc tag aggtaaa citccittatgttgcagatggit citgatcttaa gottcttaaa 51420 atattacaca toggaaaagag totgtatttgaatgcct tca totcc tagtt gagggitaatg 51.480 ggatataaag aggtaagtgg cittctic tact aatagoagca gatctg.cgaa aagctgctda 51540 actaagctgg aactttittgg agtattatto aagttittctt ttittcacagg aattaattgc 51600 totgttgatgt ttattaaaat cacatataca aaataattac tattgaaaga gtttaatgaa 51660 aagaataaaa ttacatccitt aagtatataa atatoccittc tacagatgttgaatgttaaaa 51720 gtttaattta agittaactgg attgcttgtc. aaattcaata aaaagaagta gctacctatg 51780 tatattittat aatatatatt tactgattag atgataattt totttgcagg acatgggctt 51840 aaagg cattc accaactcaa agattcgitaa accoaaaatg tdtccacago ttcaacagta 51900 tgaaatgcat ggacctgaag gtctacgtgt aggtttittat ggtaaacaaa aaattaataa 51960 atatatattg cctaatatat toaccaaatt ttaaattittt taaaagatac aatgttgacaa 52020 aaattaacaa acaaaaagga catgtgagtc. atacatatta aggttattgc tica aggtoat 52080 atagtaatat aaactcaaat totagtaaat ggaggtacat gtgttaggct gaaaggaaag. 52140 agaaagtttc caag.cgtagg attagtgtaa acagaaatag aaatgttcac acaacaacac 52200 tacattctoc atcagtcagg taaaaaagct gttcaactitc cccaaaacat cag coaatat 52260 titatgttgga accacacaca ttcatcaatg acatgagcta cittctittgat gaactgaata 52320 gtaatcaag.c atttatttat agtcc catat tigtoaaatca to attgagga taagttgagt 52380 acagagaaaa gagggtoaaa aatcaaggaa agcatttaag gagaatcagt togctictato 52440 gaattcacta toaa.gc.ctac cqcatattitt attatttata aattatattg tataatcctt 52500 tatcagtaag atatatagta aattitatgca totataggta tatatatata tatatatata 52560 tatatatata tatgcacaat tttitttittcc agagagctgg ttgttgaaata ttgaccagoa 52620 cacccittaat agaaggtogaa tagagtgaag gaalactaact gtaatcttct gacac gatag 52680 agaataaaaa gtctittatta ttattaaact tittcc citcct gtaaactgtt totcaaag.cc 52740 actgtcataa catgtgtgtc. agtttittcct to citcctgca gaggagcaca ccaatggaaa 52800 cittggattcc td.ccccitctt cacggccttg tdtcataaca citctocactt agcacagogg 52860 cago agccac ataggacitcc acagagtcct totgc.cccac agacaatcca agc citctgtc. 52920 tgccaaagtc. tctatagoct citgctitatga cccitcctccc ccagotcact cotag ctitat 52980 tottgtttitt ttttgttgtt tatttgtttgtttgtttgtt tdagacgaag totcactctg. 53040 togcto aggt cqc coaggct ggagtgcagt gg.cgc.cgtot cqgcc toggc citctoaaagt 53100 gctgggatta cagg catgag ccaccgc.gcc cqgcc to tcc togcttgtta agt calaccala 53160 catattitcct cotcaaagaa gotttccttg atgaccoaag cq.cccitctoc toctitccitct 53220 US 2003/0162202 A1 Aug. 28, 2003 66

-continued citatttcatc agacgatttgtttitttgttt ttttitttittg gtggcaacta ttacattcto 53280 toataagctt tatctgtatgtttattgtaa tdtcttctitc citcactcacc atagagtcag 53340 atgtaatggg aagaggcc at gcacgc.ctgg to catgttga agagcct cac acggaga.ccg 53400 tacgaaagta citt.ccctgag acatggat.ct gggatttggt ggtggtaaag taagtaactt 53460 cctgcatatg caatatgcaa caatagaggit coctdactat tittcaactot ttgctagttt 53520 ttctgtttitt attgttittat atgtttatga tigtacaacat gatgttittga tatatatata 53580 tacacataca catagtgaaa tdattactac agtcaa.gcaa attaacacat coattagctc. 53640 acattgttac citattitttgt gtgcatagda agcacaccita aaatctatoc tottagccaa 53700 gtttcagtat coaatacagt attatcaact gcagtccitca tactgtactt tag atttcta 53760 aatttattoa toctacgitaa cittcaactitt gtaccctittg acctgcttct tcc catccct 53820 gcaaccocaa ccacctittct actictotgtt cotatgtatt caacttcttt tag catccac 53880 atacaagtaa gatcacgcag tacttgtcct gtgtctggct tatttcactt agcataatct 539 40 cccc.caggitt catccatgtt gtoacaagtg gcaggatctt cittccactta aggctgaata 54000 atatto catt acgcatagoc acaatttatt tatccattca cocag acact taggttgttt 54060 ttatatottg goaactgtga ataatgctgt aatgaacacc aaaacacata tatctotaca 54.120 aggtgctitat titcatttcct ttgggagtat acccagaaga gggatttctg. g.gacatatgg 54 180 tagtttcc at tittaaaattt ttgaggitatc. tccatactgt tittccataat ggctgtggca 54240 atgtacattt goaccaaggg gagtgaacaa attcc titcca gagagacact ggaaacagag 54.300 ttittatcc.gt gaaacaagcc agtggggcag gtggaga gaa agagc.cggala togccatata 5 4 360 citcc ctittca ggctoctagga gitottgttta cittctoccitt toactcc cag atgcaggctg. 54.420 tittagaagcc caa.cccittag gag acago ca gaaatgg gag attittgcct g ttctotctgt 54 480 attgagccaa goggtggtgg g g g c gogtag ccactgg cat togctoaaagg cct atttaaa 54540 accaccitctg. tattoactgt ggtotaggga gacitcctgat tigcagagctc catctactcc 54 600 tggagctagg tatttagga gccagaccct taggtacgag citgtaaa.cat toggggtgctt 54 660 gatgcataga caaattatto coaggggitat gttcagacct ggttittatcc gtgggg.cgag 54.720 ccaggggaag gaggcatggg aagtgcc.citt actgcttittc agcct tcct g taagttctgtc. 54780 gttitccctg.c toctitctgct tcc cagtgca ggctagttag aag cocaiaca citcagtcago 54840 aactgataaa gtgggtag ac gaag.ccctitc cagggagaaa citgggagctg. ittagctaatt 54900 tittcagtact gaagtacttic tittcaaacgc attgctaatt toaggagatg tittaacataa 54960 atacatcago taagaagttct tactaatcta attgcatcag agctaaaaaa titttgttgcaa 55020 atttaattct aagattitcca gaaaatgggc ataag gacct aatacaacca agg actocac 55 080 agattgcctg taagagatcc citcactggitt agcaatcc to agittaaatac agatticagtic 55 140 aggcc.cacta tacacaatag tagtagaatt taaaattata aag cagocct cagtgaaata 55200 gcattttaga gaagagaact taagaacaat citcaaactg.c atgttaaatt tataactata 55260 ttgttctgtaa aagattatgc tacaattctg atatactaca attaaaaaca gttggaagaa 55320 aagg actitat attcccatct caatccttga ttatacticto cattattggit atttctattg 55380 agtgtttitta agt catggca gtagaatcat toctagg gat totcitcc cta gaaaggatgt 55 440 atttaactgc titactittctg. titcttcactt acacticcitct coagctoagic aggtgtggct 55500 US 2003/0162202 A1 Aug. 28, 2003 67

-continued gagg taggag taa.cagtc.cc tdacaccatc accgagtgga agg caggggc cittctgcct g 55560 totgaagatg citgg acttgg tatctottcc actg.cctcitc. tcc gag cctt coag.ccctitc 55 620 tttgtggagc ticacaatgcc titactctgtg attcgtggag aggccttcac actica aggcc 55 680 acggtoctaa actacctitcc caaatgcatc cqggtaagga totctittcct aaattaaata 55740 caaggtagco atcaagtaaa ttaaaagttg cattcctagg aatactgcaa acticttgitat 55800 gcaaaatatg cittactag at attcatgatt gtaaaagtta caggttittga gatctocaaa 55860 taccaaattig caccataaag caagtttcta gcc cittataa cccttcaaag titagtatttg 55920 tgtgg tatga agaattctga caggggtgac aaaagtcagt actictattot catgacagat 55.980 totacaaggt ttcaacctct acg atctoat atatttaact tittcgtagct cattcattat 56040 attaaaccita attittaaaag togtttgttga gcatcttacc tittgctgaaa ccataactgt 56100 ttataagttct totatoctot gcc.gg gagat cogggggaat ggtoaaagtt coagaccaaa 56160 gaggtagagc agcatgcc at cattacctitt coctitcc tot gotcc catca totgaaagag 56220 caggttgctt coaaaataac toagatttac citgtgitaaat citgatacatt aag atccact 56280 taaatatatt toaggtactt gatcttcatt tatatoatct ttaatgtgag goaacctgaa 56340 atctaaccaa titcctdaaga tactttacitt caaac coatt citcccitctgttctoctitccc 56400 totctactcc ctittctitatg tdtggitttca ggtoagtgttg cagotggaag cctotccc.gc 56.460 cittcctagot gtc.ccagtgg agaaggaaca agcgcct cac togcatctgttg caaacggg.cg 56.520 gcaaactgtg toctgggcag taaccocaaa gtcattaggit gag caaaaaa citgctagaga 56580 taattctota citcaaagatt gtatatggca gtgggaacct tatattgagt gctactitcct 56640 tdaggaaaag accactag at gctg.cgattt tttitcctittg ccttittatto taagatgcct 56700 acaaggatat cotcaacatc. tccaccittga attctdagta toattcacct citcatttgca 56760 tgtttcc.gtt cotgcttctg. tattittaata aaacaaaagt ttacagag cattgaacattt 56820 citaaatcttg agtttggagg catggaggaa gqggaagatg citattoattt citactggcct 5 6880 tttittittcag gaaatgtgaa titt cactgtg agcgcagagg cactagagtic toaa.gagctg. 56940 tgtggg actd aggtgc ctitc agttcctgaa cacggaagga aag acacagt catcaagcct 57 000 citgttggttg aagtaagtaa acctaaataa tatatag toc acaataatat ataatatatg 57060 tgggtaatat aataatatat ggatattitta taatattatt citcatgitatic totctgtcct 57 120 atctotctot to atttacitt totgttttgttgggggittitt tattitttgtt tittgagg cag 57 180 agtc.ttgctc tdtcatccag gotggagtgc agtgg cagga totctgcto a citgcaacct c 57240 cgc.citcctgg gttcaa.gcaa ttctogtgcc toagc citcct gagtagctgg gattacaggit 57300 gtgcaccacc acgcc.cagot agtttttgta tttittagtag agacaggatt toaccacgtt 57 360 ggcc aggctg gtc.tc.gaact cotggcctica agtgatctgc ccaccitcago titc.ccaaagt 57 420 gctgggatta taggtgtgag coaccatgca cagoctocct ttgatttact ttcttaattit 57480 titccttcatt tattoatgcatcgaactacc toctacgitat attgcttata totacagaat 57540 tittcttagat aatacagttcaaatccttct cittcactato caaatatotg tdgtoccitcc 576.00 attaaaacac atgttctgaa ggtoagtcca ttctoactag cittittctittc titttacctaa 57660 agcctgaagg act agagaag gaaacaac at tdaactc.cct actttgtc.ca totaggtaaga 57.720 gtdaac catc ataatttaaa aaacattaaa gtctaacatt taaagttcaa agaacattta 57780 US 2003/0162202 A1 Aug. 28, 2003 68

-continued tatattatto citacacttitc. tctgtgatct aag acctgaa goaccatcaa tag catttgac 57840 aaatgtggaa aatagttctt aggaaggcca agtaatttga totagaat atc cctaggcct g 57900 cattctgagt cittgatctitt togcagdacct gtgcaaacac caaatgacitt totgaccagt 57960 gtatgg tatg ggcataggta gaaagtgggit agaatcaaaa ttaat attac caaaagg gat 58 020 gttitccittaa ataattaata atgcaaacta tagg acggctgaatttagggc attctaacac 58 080 tgagttttac atagocaa.ca gtatttgata acgggattgc tatttcc caa aggaaaagtt 581 40 gtdatggcct ttaccattat tdtcatatta atatotgttt gatgccitatc cc.gtacctaa 58.200 tgcc citatica alacatttgag aaggaact ga agaaacttac aggaaaaatt taatacacta 58260 agaaattitat cagdacaatig cattctoacc ccaaaccaac attgaatcaa catcatacat 58320 aggttcattg cctittctotg actacctaca aatttagtat gtttittcgta citaaatactt 58380 tatctattoa totgttgcca agatgtaaca cataaaatgt accotaaaaa cataactitcc 58440 ttgtcattta gccittattitc tacatttaag tdaactgatt accitatcatt caatccttitt 58500 atcatgacitt citcc.gtttct gagttactica ttittgatgta totcttaagt gitaagggcta 585.60 atcatcaaat agttttacta aattitcattt taattaccaa cataatcaaa tatgcctacc 58620 taattittaca aaaatatatt cittctittaaa aaaaaaaaca gaacatcaca ttaaaggitta 58680 atgtcacccc cct gaacatt tttcagtact ttgccatcca tittatctitta gaaataatgt 58740 gtgtagatgt atatgtttgt ggatgtgttga tttacatata ataaactgta taagtttcat 58800 totataaatc actgtttgtt titt cactcag catcctgttct to gag attta cctatgttaa 58860 attgtag atc taggtotttc cittggaattig cittittaagcc tataatataa atacatcaca 58920 attctgctta gtgttittagt cittcctatat tiggittttittcaactattoac tagctittaaa 58.980 aaattagtta gttaattata ataagagcct cittaatgaac atatgcaagt acago taggg 590 40 tagatccaaa atgttaaatt cotgagtcag agag catatg catagatatg catagittittg 59 100 tittggttggit tattgttgtt gttctittaat tacattgitaa act gaccatt tataattgta 59 160 tatatataca gcatacaaag tdatgttatg atttatgaat aaaatgttgaaataattaaat 59220 caagctaacc tdaaatactt atgttttgttg gtgggaacat ttgaaattct citaag caagt 59280 ttgaaacata aaatacacta ttattaacta tatto accat gctgtgcaat agatcccaaa 59340 aagaaaaaaa totattoctit citgtctgaga citttgttgtcc cittgaacacc acctitcctitt 59 400 tacticcagot to atccitcca taaccaccat tctactcitct gctcctgttga atttgaatgt 59 460 tittagctitcc acatacaaat gagaacatgc aatatttgtt titcctatacct ggcttattt 59520 cacataacat aatctoctoc agatttaatc atgctg.ccat aaatagoaga atgttcttgt 59580 tttittaaaat ggaatggaat tctatgttgta tataccaaat tittctittatc tatto atctg. 59640 ttgatgacac titatgattcc ataactagac atcagtaatt tottagg gat tacattcaat 597.00 atgtag attg ctittgg gtag tdtgg acatt ttaacagtat taattctitcc aatccatgaa 59760 cattgtattt tttittcattt atttgtgttc. tctittgattt citttcatcag tattittataa 59820 ttitt cactgt acatttcacc toctitgatta gatttatttc tacatattgt ttatagotat 59880 tgtaaatggg attgtttitta tittctittcto aatcattcat tigittagtgaa cagaaaatac 59940 tactgattitt tatgttgttaa titttgttatct tdcaactitta ttgcattcat ttataagttc 60000 ttgcag coct ttggtgaagt cittittgagct tccaatatat aagataatgt catcaccaac 60060 US 2003/0162202 A1 Aug. 28, 2003 69

-continued agtgaaaatt ttacttctitc cittgttcaatt taggatattitt to atttcttt titcatgtttg 60 120 attgctcittg citgctacttic cagtgctact ttgaaaataa atggtgg cag tdggitatcct 60 180 tgtcttgttc cag atcttaa aggaaagttct ttcaattittc. cactgttaaa tatgtaagct 60240 ataggitttat catacatgcc ctittattgttg ttgaggaaca ttgcttgitat atctaatttg 60300 gtgagagttt tatcataaaa gag cattgaa ttctgtcaaa tacttitttct coatctaaca 60360 agatgatggit atggitttitta coctitcattctgtaaatgta atgitatcaca tittattgata 60420 tgcatatgtt gaacaattitt to catctoag ggataaatcc cacttgacta ggtagatgat 60480 ccttittact.g. tattgttgga tittagtctg.c tagtttattt cqtgttggttg gttggttggit 60540 ttagttttitt aagtgatgag cittittgctgt gttcc.ccagg citgg acttga acticcitgagt 60600 tdaag caatc ctaccaccitc agccaccoac aggtgitatgc caccatgctt agctgctatg 60660 ctag tattitt attgaggatt tittgcatcta tattoatcaa gaatattggit citgitaattct 60720 ttitttgtaat gtctttittat gacattggta totagg gtaat gcttgccitca taaaatgagc 60780 ttgaaagtat tcc titcctct tccagotttt ggcagagittt gagaaggatt g g tattoaitt 60840 citttittaaat gataagtaga acctagoagt gaagccaaca gttattaggc titttctittaa 60900 tggaaaactt tittattactg attcaatcto tttactcatt atttgtcagt toagattittc. 60960 tatttcttca to attcagtc. ttggtagitat gtatatgtct aggaatgcat tcatttctitc 61020 tagatcatcc aatttattgg tagtatactta to cataatag totctitatga toctittgtat 61080 ttctgtggta totagocataa tittctotttc atttctgatt ttatatattt aaggcctccc 61140 tottttittct tagcta acct agctaaaagt titttgttctgt citttittaaaa aaacaattca 61200 gtttcattga tottttgtat totttittcta gtctotattt gatttatttctgctotaatc. 61260 tittattattt toctittcttctg.ccaattitt gagcttactt tattottctt titcctactitc. 61320 totgag gaat atcattagta totttattgg aaatttittct tctttitttgg tatttattgt 61380 tataagctitt cotcttagaa ttgcttttac tittatgctat gttttgttat gctccatttc. 61440 catgttcatt totcttaaga tatttittgaattitccttitta aatttctitta ttgaccoatt 61500 ggctgttcag gag catgtca tittaattittc atatatttgt gaattitttitc taatticcitcc 61560 tgttacitcat ttctagttitt catag tattg togg to agaaa agatacttga tacgatgaaa 61620 cgattitcagt cittcttaaat ttgctaag ac ttgttttgttg gg.ctaaaata tdatctatot 61680 tggagaatgt ttcttgttgttg cittgagatga aatgttctgt atgitatccat taggtotatt 61740 tgatctaaag tattgg to aa gttcaacgtt ttcttattaa ttittctttct ggataatcta 61800 to catttitta aaagtgagat gttgaaattic cct gatatta citgcattgca acatatotct 61860 cccttcaacc tittaatattt gttittatata tittaggtgct coaatgttgg atatgtatag 6.1920 atttacaatt gttatatoct tittgatgaat tigacccttitt atcattatat aatgttctoc 61980 tttgttctott totacagttt ttgactittaa gtctgttttgttgaatataa gtatagotac 62040 ccctgctotc ttittagtc.cc catttaccta gaacatcttt titccttctot toactittcag 62100 totatgttgttg toctitaaaaa ttaggtgagt citcttgtaat tag catatgt titcggtoctg. 62160 tttittittaaa toc attcagt cactittatgt cittittaaatg g g gaatttag to catttgca 62220 ttcaaggtaa ttattaattit aaaaatgact togg tactacc attttgttgt tittctggttg 62280 ttittgcttct ttgttcctct tittgctgtct tcctttgttgg totgatgttctgtag toggta 62340 US 2003/0162202 A1 Aug. 28, 2003 70

-continued tgatttgaat cittittaaaat titttgttctg tacttctatt aaagatttitt gccittgctgt 62400 tactatgggg tttacagtca atttcaagct gataacaact taactittgcattctttcact 62460 cccccacaca catttitatgt cqttgatgtc agaatttaca tattttgtaa tatgtattta 62520 ttgacaattt atttittagct atgcttgtta ttaatattitt gtcttittaac ccttgtacta 62580 gagataaaat tigctittaaat accatcatta cagtcataga gtattittgaa tatggctcta 62640 tattacittat accattaaat tttgttgcttt tatgtttittg tattattaat tagggg.cctt 62700 ttgtttcago ttaaagaact coctitcagta attcctgaag goaggccitaa tattgacgac 62760 toccittagct tittagtttgt citgggaatgt tittatttcto cotcatttct gaaag acago 62820 tittgctggat gaagaattct td attccatg ttgtttittat ttttgtttitc ctitcagtact 62880 ttgaatatat tattocactc. tcc citcagoc td.ccgggitta citgctaaaaa tocatggata 62940 gttgitattgg aatticcitttg tatgttgatat gtttctittat caccittctgc titttcagaat 63000 ttttittttgt citttgattitt tdatagittta attattgttgt cittagtgagc agttctttca 63060 tittgaattitc actggag acc totgttgccitc ctgtacttgg atgctag cat citatccccta 63120 attagg gaag titttcagocc titactgcttt ttttitttittctgattctata citcttitttitt 63180 toattattgc tittaaatgtg ctittatagtc. tctittcttct tcttctggac tittctittaat 63240 gcaaaggttt gattitcatga tagatgtc.cca taattitccat aggctttctt cattcttittg 63300 totttctgct cittctgcctg gataatticca aatactictat citttgagctc actgattctt 63360 citgcttgatc aagttctgctg. ittgagcttac tittgaattitt taattittagt cattgtatto 63 420 tittattitcca ggatttctat ttggitttctt tittgattgtt totatttatt ttittattitta 63480 caatattagc taagttgcag ataattgttt citatttcttt tttittittata citttaagttc. 63540 tagg gtacat gtgcacaatg togcaggttta ttacatatgt atacatgtgc catgttggtg 63600 tgctgcacco attaactcgt catttacatt agctatatot cotaatgcta toccitcc ccc 63660 atc.cccitcac cocacaacag gocccggtgt gtgatgttcc cctitcctgttg tocaagttgtt 63720 citcattgttcaatticccacc tatgagtgag aacatg.cggit gtttgtttitt citgtc.cttgc 63 780 aatagtttgc tigagaatgat ggtttccago ttcatccato tcc caggaaa caa.caggtoc 63840 tggagaggat gtggagaaat aggaacactt ttacactgtt gotgggacta taalactagtt 63900 caac cactgt ggaagttcaat gtggc gattc citcagggatc tagaactaga aatacatttg 63960 accoagccat cocattact g g gtatatacc caaaggatta taaatcatgc tigctataaag 64020 acacatgcac acatatgttt attgttggcac tattoacaat cqtttctatgtcaaacttct 64080 cagtttgttg gtgitattgtt ttgcaaattt catttaattt totatttata tattottgta 64140 gtocactgaa tittcttcaag aggattatto tgaattctitt gtcagtgatt toatagatct 64200 ttatttctat gaggtoaatt ttittgagctt ggccagtttc titttggaggit gtcatcattc. 64260 cittgattott cataatcctg tdtccttgca ttatttgttgc atttgag gag aaag.ccactt 64320 cittctggittt ttataggitat totttggcag ggataaaggt ttgctattta gtotagocta 64380 taattctgga aagatcagtt gotgacaa.cc ttgag caggc agagtttitca toggttc.cct 64 440 agttggctgg gcc act gcct ttgct cittat gtttgg tagg gcc acto gtt go.gc.cittgct 64500 citctggcaag atcactgttt totcitctgct atctggtgga gctgctggct g g g tactaca 645 60 atggcc totg gtcaggccag to acaagatg tdttgcctgg citggatgatt citgctatttg 64 620 US 2003/0162202 A1 Aug. 28, 2003 71

-continued ggacctgaag ttaggcaggg to acaatc.cg ggctgtgagg ttagg tagag ttgttgcttg 64680 ggatgggcag aaataaatac tatacttctt agatgttgcat aatagaggat tdctaccc.ca 64740 cc cittgttgaa toggagc catg gagtgaggitt ttggctdagt to agctaccc tittagacitcc 64800 caggtoaag.c atatttaacc cctacacttic tatgaaatac acagaggtgg tdtctgctac 64860 citgggtgggg to actogcat aaccitctgaa gotgggctta cag actogcc atctggaaac 64920 tdaagctagg ttgaactitcc caacatgctt citgaaagtga ccagotcagt tittgcagatg 64.980 ggctatocag ttggctggta totctgaatg ggtgc catag citggcagaaa cacagaa.gca 65040 citaccaaaat coacatgctg gtoactgtga gctctgtcct tctttgtttc tacct gacct 65100 cattactitcc totgttcc.cg gtgaaatgag accagagtgg gctitcct gag aagtgtc.ttt 65160 gaatacttga gaatcttgat gtotacccct g gttctottc ccc.gctgtag aaactgtgac 65220 cc cagggaac toctotcitat citggcattgt gctaaccitaa aggagtggga acaatgacat 65280 ggtoaaagtg agaccattct tcc tactcitt citaatttgtc titcactcagt totatgaaca 65.340 atgtaggtgt cotag acttig tittccaagta ttggggittitt caaaatagat tittctgatct 65 400 gtggatagoa gctagttgga citttctgtgg agg gaggaag atcct gagac tittctag to c 65 460 atcatcttgc tittattotga attittaattgaactacaaaa cqaaaatcct cotctittatt 65520 acctaaatgc atttatactt coaccaggat acatttccat agtgttatat ttgccatcat 65580 citgttaccat caaagtttitt aattittaatt tittgccaaaa atttagaaaa aaaaattittg 65640 citgttgttitt aatttatatt ttcttaatta caaggatgac cittatttittg catgtttatt 65700 aattgcctitt ataatctittg gctatttgtc titttgagtag tittttittctg acticagttgt 65760 atgacactaa ttctittatct gttgaatatgttgcagatat tittctittcac ttggtoattt 65820 ttittaactitt gtttatgg catctgttttitt tacaaaagtt ttaac attaa ttittatgaga 65880 aagg gaag at aactgctaca tttitt cattt gtatataatt caccaatact aaaattgtag 65.940 taaatgitatgttcatcagta gcagttattt tatttitcagt gag to aag cattttattittg 66000 cittago catt totcctittaa citatgctitat ggcttitttitt taagaaacat tittaatatga 66060 attittatgag gaagggattc agtatgaaaa taaataccat acttctotca tttitcatttc. 66120 atataattta coaagattaa aatggaagta aatgitatgtt catgagtagt aattattitta 66180 ttittcaatgc atcaaatact gttctgtctitc actitccttac cotcaattitt citaggittitt c 66240 cataaaaata citatctttgt atatgaaatt tdaagaaaga acatagoatt attatagaat 66300 tdagg accitt ttgttgg gtaa titttactitat gtatactitat agg gctttgttgttggtgtt 66360 ttct coatiac aactgttgag taaggaagtt gotggtggga actaaataga to atcttgttg 66 420 ataaccgtct tatgtcagoc atcagatgac agcaactgaa toacaacatc accaggctot 66480 tacaatttgttgttcttattt g g catgcg at tctacataaa ttactgaaaa gatcattgaa 66540 gaagaaattic tdaaaatcac aggaaaccag tag cocattt ttaagatatt tatatattac 66600 tgttgtatta aaggcggaca acttittcagg aggagtttag gtataaggca tagtoctago. 66660 ttctgggtoa tag agctgtt tagaaagata taatgcagaa ataattittca tatgtctgat 66720 ttgcttattt citctaggtgg tdaggtttct gaagaattat coctaaaact gccaccaaat 66780 gtggtagaag aatctgcc.cg agcttctgtc. tcagttittgg gtgagtc.to c agc.ccctagt 66840 ggatcc.gggc attaacagot totattatac tatttittatt toccataaat atttactaaa 66900 US 2003/0162202 A1 Aug. 28, 2003 72

-continued aataatacta taattittaac ttctttctitc. tcttcttctt taggcttgtt tattgctitt c 66960 aatcatactt citatcc ctgg aagaatcatc ctitcctaaaa attctdaatt totaagcto a 67020 actaattatt totgcttaat gactittgata gatgataatc. tccaagctitt atgactitcc c 67080 atctotccca ttctotagga gacatattag gotctg.ccat gcaaaacaca caaaatctitc. 67140 to cagatgcc citatggctgt ggaga.gcaga atatggtoct citttgct colt aac atctato 67200 tactggatta totaaatgaa acacagoagc titacticciaga gatcaagttcc aaggc cattg 67260 gctatotcaa cactggtgag to attacittg agtaagg gala aacttgaatgttatttcaac 67320 tggattitccc agtaggitttc agittactitat gaatattatg atacattagc titagctoact 67380 atgatagotg citatgatagt taatttcaag gaaactatoc actictocaac citccaataaa 67440 atatttaagg citcagaaact cotaatctat gacaacaaaa tittaagaaat gito acaagag 67500 aagccaaggt acttittagta atttctocac cctoag catg cacattaatc cattgttgctg. 67560 tittcgittaat cittcctttcc aggttaccag agacagttga actacaaaca citatgatgg c 67620 toctacagca cctittgggga gcgatatggc aggaaccagg gcaac acct g gtaaggaaag 67 680 aacaatttitt to agcttctt tttgttgttgcc agctotttta catgitattac citcaattata 6.7740 ttcacagdaa cactatoaga tatgtattat cag accgatg gtttgttata citagataaat 67800 ccaccalagat tag caaggta atcagaagaa aacct gatat coaaatacat gttatgttag 67860 gcttgtttcc aaaatggatc ctattaataa totaccaagg ttittctttct gaaatggcta 67920 ttctittctaa agtagctaccataac catga gttittaaaat gatattgcca gtgaacatat 67.980 ataactitcca gataaaccat gttaactitca gcttatattg tdacattcta agtcattcag 68040 cittgacittgg aatgaattica ttaataagag gaaacaattg agaaggaaac agtaatataa 68100 aacatttittt taaatcccita aagtaaag.ca atattaaaat titactgcatg taagagctgc 68160 atgtgagaag attctgtcat citgcagaagg aaatctotaa agataagaga gatttaaagc 68220 cittactcaag taactaacaa aaataagtac attcaaatta cittgaatgta aatttgttca 68280 accattgttgg aagacagtat gg.cgattctt caaggat.cta galaccagaaa taccatttga 68340 cctagtaatc ccattactgg gtatataccc aaaggaatat aaatcattct actataatga 68400 cacatgcaca totatgttta togcgg cact atttacaata acaaagttcat ggaacta acc 68460 caaatgctica totaatgacag actggataaa gaaaatgtgg tacatataca totato gaata 68520 citatgcagoa ataaaaagaa atgaaatcat gtc.ctittgca gggacatgga tigaagctgga 68580 agccatcago citcagoaaac taacacagga acagaaaacc aaacaccaca tttctoactic 68640 ataagtggga gttaa.gcaat gagaacacac ggacacaggg acaggaacaa cacacaccag 68.700 ggcctgttgg gaggtgtggg gtgacgggag g galactaagc ggatgggtoa ataggtgcaa. 68760 gaaaccacca tagg cacacgt atactitatgt aacaaacctg. cacgttctg.c acatgitatct 68820 cggalactaaa ataaaattaa atatactaag acticcictgtg gcaaaga gag agittagcaag 68880 gaaatactac atctagoaga ttaatcaggc agacitaaaga ttaatcaagg agataagctic 68940 totaagtaca caagaattitt gttagctaac toacatcata tdaagcctgttgctgttgaag 69000 tggittataaa accattttga caacataaac atcatgattig cittccitccot g g toaggctc. 69 060 acag cotttgttctgaag acttittgcc.caa gotcgagcct acatcttcat cqatgaag ca 69 120 cacattaccc aagcc.citcat atggctdtcc cagaggcaga agg acaatgg citgtttcagg 69180 US 2003/0162202 A1 Aug. 28, 2003 73

-continued agctctgggt cactgctcaa caatgccata aaggtgaatc attctggagc tagttittgat 69240 ttgtcc atta toatat citgc aag gatgagg ataggaagtg ataatgttgaaaaattictaag 69300 ggaaag.ccitc agaggaaaat aaaacctgga tiggcaccalaa aaagagggga tagaacaaaa 69360 gttgattgtg atactittgcc citatagggat ggatatgggit aag gatgaat to catgacac 69420 agcagaatag aaagaactaa toaatagoat tctdagaagt tdaattatto agatctotct 694.80 citcg tattoa cagg gaggag tagaagatga agtgacccto tcc.gc.ctata toaccatc.gc 695 40 ccttctggag attcctcitca cagtcactgt agg taccacc ccattccitct gctgaag gag 69600 agttctggat gcaatgaaac tactgacctg. citgtctgaaa tactatocta ttaaaag caa 69660 agcatcagot ttctttctat gcaatgccag tactitcc cag atctacagag aatttgg to a 69.720 gcc.cattaag aaaggtttaa atttitcccag taatticccct aggctattta ccaccaccac 69780 toaaaaaaga atcttaaaga tigitatcttitt gaatgtgaga ataacagata aaaataatat 69840 tatatotatt gataagaatg aggaatcgitt ggaaaaatgc gtttgaaaaa cittctgtgct 69900 gtgatcc.gtg tatttgcct g g gaatgctaa tatgcctgtt tacatagott agttccctitc 69960 ttgttctgcc titcacagdac cct gttgtcc gcaatgccct gttittgcct g gag to agcct 70020 ggaaga cago acaagaaggg gaccatggca gcc atgtata taccaaag.ca citgctggcct 7 0080 atgcttittgc cct ggcaggit aaccaggaca agaggaagga agtactcaag to acttaatg 701 40 aggaagctgt gaagaaaggt gagag cacac citgagatcct tct cotggcc catcc totgt 70200 atcaagaact gcatggcaaa aatcc citcac toctacctoc totgatccct gtotccticto 70260 ttcttittcta tatatoatat atattttgtc. catattgcat cittataaaat citaggattitc. 70320 ttaatcaaat cagaaatcag aag acaagag gCogtgcaga tigcttcticaa ttacgatggg 70380 gttatatoct gacaaactica ttgtaaagttc taaaaaatct taagtgggac cattgtaagt 70440 cagg gaccat citctatagta togctggtaag aagag cattc. tctggag act agctocaaaa 70500 tgttgctacct atgtgagctt g g gaaagttca ttaactitcct tatgtttcag titccttcatc. 70560 agtaaaatgg ggataataat agtatttacc to acagagct gttgtaataa atgaattggt 70620 acacgtaaaa cacttagtag agtacatgtc. acatagdaaa toctataaaa gtact agitta 70680 ttacaattaa catato agitt citcaatatat gcc caaccot tacctgg tac attatataac 70740 cittaaacata agaaaataat catggaagta acticcittgaatgaattctgg tattittaa.gc. 70800 ccatttcata agaccaataa tattgaccaa totactcata titcacacagt acttctacat 70860 ataccatggit citatatgagc ggttgaagaa atagaaaata aaatgcaaat cacaagatgt 70920 ccattaaaac agtctacctt tttcctttga cago cattaa ttcttcttaa aatgtattga 70980 gaaatattitt ataatagata tacaaaaggg cataagctat alactagaaaa cactgtacaa 71040 citct citcata gattaagaaa tagaaaatta cc.gacatggg aaaaataaat cocttgttgta 71100 toactaccac citccagaggc aatcattatc ctgaatttgt cqttacaatt coatggattt 71160 ccittatattt ttgctgcata totatoccita actaatattt agaatcttca catgttgtttc. 71220 atcttgagag aaacaagatt tittatttcto tcttattoaa gaaacaagag aaacatttitt 71280 gaatatttca gcagottagt tittttgtttgtctgctttitt attittctaag titcaiacacta 71340 tgttgatgaa gaccoctata caagtatgtg cagagtcago toattaattt toactgctgc 71400 ataatatatt acagtctata aattagtcat aatttacaca togagtttct cotcatggat 71460 US 2003/0162202 A1 Aug. 28, 2003 74

-continued tttittittatgttittgctatt aaaaaaaatg citgcaatgaa tacticatgttg cct gttittct 71520 tgttgcatctg. tatttctoca aattatgctt tdagaag cat aatgactgat tagtgggcta 71580 agcacatctt coccattgct gaatattgcc aaagagcagt togct tcc.ca cagoagtgta 71640 tattggttcc cattgtttca catccatgtc agcctittggit atttcaagga ttact gtatt 71700 tttitttittca atttaatgag tagaaactict actatttcat gtgatctgttg atcctcacaa 71760 gaaactgata aagacacact ttataggaaa totaacaaac to cagotata gttctaatata 71820 acatcataaa taggaaatag coag actoaa tdag acaact gctgtgccat ttctttctoc 71880 caccaa.cccg actatagdaa cqatttgaaa acatagatag goataggctt citgacitccag 71940 catcaatato tdc cittagct gggctaaaac acaccalaatt cagatttaca toga agggaaa 72000 agcatctaca tacagtacag gggattataa togg catgca attct cattt cagdactggc 72060 ttgg gtactt to accttgaa ttaaatataa atatgtaggc acttataaat atctttittct 72120 catctittaag acaactctgt coattgggag cqc cotcaga aaccolaaggc accagtgggg 721.80 catttittacg aaccoc aggc ticc citctgct gaggtggaga tigacatccta totgcto citc. 72240 gcttatctoa cqgcc.ca.gcc agc.cccalacc toggagg acc to accitctgc aaccalacatc. 72300 gtgaagtgga toacgaag ca gcagaatgcc caggg.cggitt totcc to cac ccaggttggt 72360 gatttgccaa aaccttitt at titcaccittca ggtag caaaa gatttgaatgaaaaagaaac 72420 aaacacatcc aagaagaaaa aaatacagat gacagta act togaaatgagg aaaagttitt C 72.480 agitatccaag gataatggaa ataaaagcaa atcaaagttca aagagggcca aaaggaaatg 72540 citcagaatcc cgg caccoca togctgtgtt attatccatc. tcc tattitcc cataacaaca 72600 citgcct tcct caag cagoag toggag cacca gcagaatgaa goagatgtct cotgc cattc. 72660 toctgaaag.c totagg gtct citttcaaact gttcaaagga actictactica aaatccaaca 72720 acctcitccitc gcaaatctot coattcttag gtc.ccctitta ataggcttitt citcaaaacta 72780 cacattttgt gctitcc ccta titcaccttitt tttittitttitt ttttittaaga cagagtcttg 72840 citctgtcacc taggctggaa to cagtggtg caatctoggc ticactgcaac citccatctoc 72900 caggttcaag cq attctagt gccitcago ct cocaagtatc taggattaca gtoatgttgca 72960 atcatgcct g g ctaatttitt gtatttittag tag agacgag gttittgccat gttgcccagg 73020 citgatctoga agticcitgagc ticaggcaatc catcc.gc.citt gocct cacaa agtgctagga 73 080 ttataggtgt gag coactgc gtocago.ccc ctattoacct cittaatacac aaacatttat 73140 toatcaggag cataaagaac tdtctittatt catccaacct cotaaatcta gctatataac 73200 catgitatctgaacaattcat tdatatgtac acagoagaaa gttittatctt cagagaattic 73260 ggatgtttgc titatataccc taaaacggaa aaaatgttgac aaaatgg cat tccatccitat 73320 titccattgta ttaatcttitt atcatatgaatgaaaaaaac taagtaattt tattaaaggit 73380 tatcattcat ttattagaaa catattattt gaaggaggcc aag caggittt aatgttgttg 73440 aggata cata ccagoaga.ca ttcactggga acaggaaatc atccaataaa aagggaaagc 73500 caaataaaaa tdtcattaaa tocagaag at aattataata citcatcttitt atttcttittg 73560 gagaaact ga agcatgactic toc to atggc tigcaaagaac cittgg gttct citcCaggaca 73620 citgaccitcag caactgagca aagtttaata toggaga gag ccagactgaa citttgcttga 73680 gtggtggcag atatgagcat agttgttcaag aaaga catgt tag caaatag citgatgccaa 73740 US 2003/0162202 A1 Aug. 28, 2003 75

-continued taactgattg ccattcacat gtttitccaca titccatgtcc cacatatact tacagagaga 73800 aaaggatcaa ttittctgata aataaaataa acatgtaggg catacagtoc alaggtagata 73860 tgttgaatgtt atggttctitc aactatotaa agattataat caatcttgaa attacagotc. 73920 citat atttaa gtag taggg aagtaggaaa toaaagtccc to acatgggit citttgaaaaa 73980 tatctoagcc citcaaag.cct tataatgccc aatgg gttct citcactcatc tdtctotaac 74040 aggacacagt ggtggctcitc catgctotgt coaaatatgg agcagocaca tttaccagga 74100 citgggaaggc tigcacaggtg act atccagt cittcagg gac attitt.ccago: aaatticcaag 74160 tggacaacaa caa.ccgcc to ttact gcago aggtotcatt gccagagct g cct ggggaat 74220 acagoatgaa agtgacagga gaaggatgtg totacct coa ggtgagactic ttggg caggt 7428O gaggacagga cagatgagga cagoagctgt totcitctgag aagttcctaac to agaaaa.ca 74340 atgggacaga totagagaaag g gttagggiac gtggacagga attctgg gala agggcaaaaa 74 400 actgattittg totttgatgttctatagaca toctitgaaat acaatattot cocagaaaag 74 460 gaag agttcc cctittgctitt aggagtgcag actctgc citc aaacttgtga tigaac coaaa 74520 gcccacacca gctitccaaat citccctaagt gtcaggtaag accittctgac totato acct 74580 aatcctaaga ataaccacca gitottcttitc gggaactcct ctittaagtaa agcagtgcaa 74640 cagtagatat ttgcactatt cacaaaaaat gcaatgtatt citcttaagtt gatataattt 74700 citcaatgatg g g gtttacat tdtccatcca ggatctacta ttgttgcaacc toattgttta 74760 aagggitaata attitcc citca ataacaacta agtaaatatt acccattgcc totgacctga 74.820 attccttgtt atgtaatgaa atcctatatt attcttgctt tattgaagat agagatgaag 74880 aattattgaa aagtttgaat agaaggaagt agtgacticct tagttagaat toc tactggc 74940 aataataaat citcaggittat atatgatata attaatttgg g g g gaagata cacttatatg 75000 catcaatatt taaatagotg cag atctgat aaaaaactct citctocacaa acatattatt 75060 acttggttgg agatactatt caggaaaaaa gttaggacaa aatacatgta acaaataact 75 120 ggcacacatc aaaaagaatg agatcatgac ctittgcagga acatggatgg agatggaggit 751.80 cattatccitt ggcaaactag cacaggaatg gaaaaccaaa cactgcatgttctoatttgt 75240 aagtgg gagc taaatgatga galacatatgg acacaaaaag gagaacaa.ca gacaccagag 75 300 ccitacttgag g gttaaggat gg gaggaggg agaagat cag aaaaaaacaa caatticagtg 75.360 caaaatttag tacccaagtg ataaagtaat citgitacacca aaccoccatg acacgagttt 75420 acctatataa caaacctgca tatgtacgcc tdaacctaaa agittaagtat atatatatat 75480 atttittittca tittaatttgg totatatata toccaaaaaa taaattaagc agtccaaatt 75540 toggatgcaa actictogggg acaagacgct aggtgtttct aagtgttttgttgaaag.cca 75 600 gtgtttaagt aaacattata aattattgtt gttitttgtaa ataatgtaga citgaaattta 75660 ttattoataa tatacatcat tttgtcagot gaaagaaaat aaaagtaaac aaataaataa 75720 aataactggc atagattaga ggtoacaaac agcctgtgcatcacttgtag agctttctta 75780 aaatgcagat cotcago.cgg gcgtggtggc ticacgc.ctgt aatctoag.ca citttgggagg 75840 ccaaggcggg cagattacct taggtoggga gttcaag acc agcct gacca acatggaga a 75900 accocqtctg tactaaaaat acaaaattag toggacgtgg toggtgcatgc citgitaatccc 75960 agctactcgg gaggct gagg caggagaatc acttgaacco aggaggcgga ggttgcggtg 76020 US 2003/0162202 A1 Aug. 28, 2003 76

-continued agcc gaaatc atgccattgc acticcago: ct gggcaagaag agtgaaaaac to catcaaaa 76080 aaaaaaaatg cagatcctica gcc cc catac act agacatt citgattcatc aggtotagag 76140 tagg gcctgg totctgtggc tittaacaggc titcctaaaaa ttctacgcac accacttitta 76200 caaaccactg ggataagata tittgggaaga cittacgtgta ccttittagag citgtggaatg 76260 cittaacatgg acatagaaga agaaaatatt taaaaacaca gaaaaccota atcctttcct 76320 cc ccto gatc. citcagttaca cagggagc.cg citctgccitcc aacatgg.cga togttgatgt 76380 gaagatgg to tctggctt.ca titc.ccctgaa gocaa.ca.gtg aaaatggtag gtttatcata 764-40 acco cagact gcc ctattitt atttaatgat gitatgttatcc ccagoataag acaatactaa 76500 tatcaaaata citattaaagt caatctotat caaag.ccitta toctitttitcc agctdagaaa 76560 tataatcaca totgtttgta togaatgctga ccatgtgcag agc actotgc taggacccat 76 620 gactacaaga aaaagattgt cagoaggttc ctgcttittca atttcttctt agcttagaat 76680 tittgctaaga agataaaaga tatgaacacg aaccagtgaa aaatatgaaa atgactgatt 76740 ggcataaact ataagtatta cagaagttaa aagaaaaata gagcaa.gcaa aac aggaaaa 76800 aaactoctita toaagaaata gaaactgaat tigaactittga aatatgagta actaccaagt 76860 ttaggatact tagctgtctt ttcttcagat aaataactitt acacattagt cqtgttgttat 76920 actaatagta aaccotttat gcctttcatt tittaattgta ttacattata tatttccitta 76.980 caaaaag.cat ttgaagaatt citaccctdag g gttattittg goaatacaaa gatttitttct 77040 citgg atcc cc caggggittitc atctattitat taacatttgt g g tatttcaa ttittctitcag 77100 cittgaaagat citaaccatgt gag coggaca gaagttcagoa gcaac catgt cittgatttac 77160 cittgataagg taagagaact tccagtctat ttgcaaaaaa acgtagataa taatcctcta 77220 agggaacatc toggaaggta aatgcattitt agaaa.catca citt.ccatgct agaaatttga 77280 gaattctaat gttaactcta aaagaatgtt cittctotcct ttatttatat titcaccaggg 77340 attacaggta gaaatggctt attatgatct toggatatga atattoctaa aatcc cataa 77400 gcaagaaatc titcacaaaat gtgtttatta tattgacaag titttittggat accoagtaat 77.460 ataaggaagt agcccttgttg attagtcaat tattagttaa ttatcaacat acticaacaac 77520 aatatgaaag ggaaaaaaaa citgtcagtict coacaaggac ttgaaccata aaataataag 77580 accagttcac cagtaalacca atctgattitt atagatatgt gtggtag gag agtttgttca 77640 tgcataagtt gatgggaatt atagitttaca aattittatga aacttaagcc tdggaag atc. 77700 aaccttittag atgcctctitt gag totacgc aagtattoct gcaag acaga gaagttcaaac 77760 tataccaaat citctggatat taaaaaatga acacagttag to atccaata aaaagtatat 77820 atcatttacc cccatgaaca gagctatgta ttgg cattga cagaggtata td.cgittatgt 77880 tagittattta agaaataatc tdgagaattt atcatcc cct citgagagatt totgcacaat 77940 ttaattaagg accotatagt gtgctgtagg ataataaag.c tttitccc.cca aaaaacaggit 78000 gaatacittaa actaattcaa agagagaaga aagctitcctgaaaggtoatt taattgactt 78060 ttgcttitcca ggtgttcaaat cag acactga gcttgttctt cacggttctg caagatgtcc 78120 cagtaagaga totgaalacca gccatagtga aagtctatoa ttacitacgag acgggtgagt 7818O gag agtgatt ttcacgtaga aatattta at toctogatcac agaaatticag gtttaggaga 78240 tgttgttgggg ttatttatta cattaagtaa ttacattatc actitcattitt gtctocatca 78300 US 2003/0162202 A1 Aug. 28, 2003 77

-continued agtctgatgc cccitctttitt gtotcittata catacattat agaaacaacc tacattataa 78360 atttatcaac tactaataca aaacacctgt gggatattta gttcc ctittt catcagataa 78420 atgg actota togacaatatg agatttaagt aagtagaa.ca totgaagagt cottcaggag 78.480 tittgggataa aagaatatat aaaac act at atttgaaagg agaatataag gtagcaa.gca 78540 acacatcaga tigaatgatgc titatgtttct g g tacaatac tagttctitccc acaacaaact 78600 cctitccittgg cct gitatccc acagatgttt gctttctittc. tcactitcatg taatgattitc. 78660 tggitttitttgttggittttitt ttttittcaga tigagtttgca attgctgagt acaatgctoc 78720 ttgcag caaa ggtaagccac toacactcct coaaaaggca gtcagagctc ctitcagottg 78780 cccc.ccaaac cittctoctitc ataaaacgct g g g taaatat ttgttcaaaaa catcaaatta 78840 citcacactg.c acattattat agaaaaacac atttattgga gaggg.ccgct g actotgtca 78900 aaccitcagag agticcatagg attgctitatg g gtaatgatt toggaatagat ttggtttccc 78960 actgtact ga ttaggtttcc ttggg cacta tyctacccag alactaaggga aagaatactic 79020 totgctcatg gag accoaaa totgtcttaa tttitttittct titccaatgtc. acagatcttg 79 080 gaaatgcttg aagaccacaa goctogaaaag toctittgctd gag to citgtt citcagagctic 79.140 cacagaagac acgtgtttitt gtatctittaa agacittgatg aataaac act titttctgg to 79.200 aatgtcttitc cct gtttcct gttcattcaa taaatatoat tigtacatttc catatgattc 79.260 ccaatagaat accalagatta aacttaaagg aatcaagtgc tigaaggacitt cagaatacaa 79320 aaaaatgata cagtgatgtc. g.gtctgagta ggcttcatgt aaggactgtg g g gaaagaag 79380 aaagtattgg gttatgtact aggaaagtgt aaagtgtgtt togttatggg aatacccitat 79440 gaaaaaccoa aagggtgaat ttt tatgaga aaataaaaga citg acttcac cagaaaagac 795 00 tttittacatt aaaatgaagt agaatgaaat acaacattga acatgtcata ttgag aggca 79560 agataattgg gacittgacct gaattgggag tatgttgtcc tatgttacac caaaatctgc 79 620 cact gatgag agtgat cagt cagittaacct g g g gtttcag attcaataat agatgagctg. 79680 aaaataatga agg gaggatt catgcagaag cacgttittct cagaagaagg aatgttgtatg 79740 actcaaagttc caaataggag tattatattg gatcatctitt cittctggaac tittgagccag 79800 gattaaagga tagctgtaaa gtcaaggaga tattotgatg cagaaatcag ttcto acaac 79860 atctgattga tigtotgatgt citcacaacat citctittagtc tatttittaaa atatataatt 79.920 ttctittgcag taagtattgc g acatatatt to cattctat agaaggggaa goaaaacttic 79.980 aggagtttitt galagtaggaa aggttaaag.c aggaggattg agccaagaga gttctgaggac 80040 aatcgtagga gtcc tactict tcatttggca caaaaatgac aatgcttagt taggcagaag 80 100 gtgagtatgg attgtataaa citaagaactg gaaaagacitt to cagttcaa gaatccitta 80 160 gctctgtc.tc. caggctagac aaaataagaa ataaaagcta toactitctgt gtggtgctta 80220 tagaatagaa tta acatato agcattatgg gatctittagg gtgtc.gctitt cotggcc agt 80280 citagtggcac ctittgcct ga gttittgctict g g gcc cactg. g.gctoctitct gcc cacticgc. 80.340 acttgctacc aacctggatc ccggatccaa gqgagattga gacgggtgga gcagaggggt 80 400 gtgctagggg totgtgagca agcgtggcca citgtgcagto: acacacacaa gotgctg.ccc 80460 aggttgggca gcticcaggtg ccago acagg citcto catga ggtggctgga ccagg cacac 80520 aacaaacago titcc.ccctgg accaggcgca toacaag cag citt.ccaa.cag togg cactggc 80580