US 2003.017O678A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0170678A1 Tanzi et al. (43) Pub. Date: Sep. 11, 2003
(54) GENETIC MARKERS FOR ALZHEIMER'S Related U.S. Application Data DISEASE AND METHODS USING THE SAME (60) Provisional application No. 60/348,065, filed on Oct. (75) Inventors: Rudolph E. Tanzi, Hull, MA (US); 25, 2001. Provisional application No. 60/336,983, Kenneth David Becker, San Diego, CA filed on Nov. 2, 2001. (US); Gonul Velicelebi, San Diego, CA (US); Kathryn J. Elliott, San Diego, Publication Classification CA (US); Xin Wang, San Diego, CA (US); Lars Bertram, Brighton, MA (51) Int. C.7 ------C12O 1/68 (US); Aleister J. Saunders, (52) U.S. Cl...... 435/6 Philadelphia, PA (US); Deborah Lynne Blacker, Newton, MA (US) (57) ABSTRACT Correspondence Address: HELLER EHRMAN WHITE & MCAULIFFE LLP Genetic markers associated with Alzheimer's disease are 4350 LA JOLLAVILLAGE DRIVE provided. Also provided are methods of determining the 7TH FLOOR presence or absence in a Subject of one or more polymor SAN DIEGO, CA 92122-1246 (US) phisms associated with Alzheimer's disease and methods of determining the level of risk for Alzheimer's disease in a (73) Assignee: Neurogenetics, Inc. Subject. Further provided are nucleic acid compositions and kits for use in determining the presence or absence in a (21) Appl. No.: 10/281,456 Subject of one or more polymorphisms associated with Alzheimer's disease and kits for determining the level of (22) Filed: Oct. 25, 2002 risk for Alzheimer's disease in a Subject. Patent Application Publication Sep. 11, 2003 Sheet 1 of 34 US 2003/0170678A1
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co o rt wr l Mr. Mr.
Patent Application Publication US 2003/0170678A1
Patent Application Publication Sep. 11, 2003 Sheet 14 of 34 US 2003/0170678A1 Genes on Chromosome 1 O Cytogenetic
O CMOAT CANALICULAR MULTISPECIFIC ORGANIC
ANION TRANSPORTER . . " O ATP-BINDING CASSETTE, SUB-FAMILY C ABCC2 (CFTR/MRP), MEMBER 2 GDB:6089489 10q24-10q24 • ABC
O MRP2
o CMRP o DJS o ATP-BINDIN SUB-FAMILY D ABCDIP2 o ALD1OP1E.I.S.S." GDB99641383,3'- LIMAB1 ABLIM LIMACT IN BINDING PROTEIN 1 GDB.7173461 10q25-10q25 o IMATIN ACADSB o ACYL-COENZYME A DEHYDROGENASE, GDB-388699 F5. SHORT/BRANCHED CHAIN
ACTA2 e ACTSA GDB:1251.97 10q22 ce o ACTIN, ALPHA 2, SMOOTH MUSCLE, AORTA - - - 10q24.
ARP1 (ACTIN-RELATED PROTEIN 1, YEASI) 10q24.2- ACTR1A\ HOMOLOG A (CENTRACTIN ALPHA) CDB:9958719 10q24.3UQ4. o ARP1 o A DISINTEGRIN AND METAL LOPROTEINASE DOMAIN 12 (MELTRIN ALPHA) GDB:9958807 10q26.3- ADAM12 O MLTN 10q26.3 o MCMP o MTNA O A DISINTEGRIN AND METAL LOPROTEASE GDB:4573992 10O26.3-q
o ADENOSINE DEAMINASE, RNA-SPECIFIC, B2 ADARB2 (HOMOLOG OF RAT BLUE) GDB:5929209 10p15-10p15 o RED2
o HRED2
a ADDL 10q24.2 ADD •. ADDUCINA55 N. 3 (GAMMA) -GDB:6O45066 - 10q24.3(4.4 s HS.81 10 ADK to HS.94.382 GDB:1 18979 3. N 8 ADENOSINE KINASE -- o ADORA2L CDB. 128572 10q25.5- ADOR2- . ADENOSINE A2 RECEPTOR-LIKE 1 10q26.5+.
oO ADRARADRA2 GDB:120538:1 10q25qZo ADRA2A o ADRA2R - 1025. ADRENERGIC, ALPHA-2A-, RECEPTOR Patent Application Publication Sep. 11, 2003 Sheet 15 of 34 US 2003/0170678A1
Cytogenetic
O HS.999.13 10 25 ADRB1 a ADRB1R GDB: 19654 :55, ADRENERGIC, BETA-1-, RECEPTOR q AF10 ALL1 FUSED GENE FROM CHROMOSOME 10 GDB:9954451 10p12-10p12
o DDH a DIHYDRODIOL DEHYDROGENASE 1 (TRANS 1,2-DIHYDROBENZENE-1,2-DIOL 1 OD14 AKR1C1 ACIDDEHYDROGENASE, BINDING) HIGH AFFINITY BILE GDB.202890 10p14.p14 o HS.78183 o DDH 0 MBAB
DDH2
DIHYDRODIOL DEHYDROGENASE 2 (TRANS- GDB:385038 10p15 AKR1C2 1,2-DIHYDROBENZENE-1,2-DIOL - || 10p14.
DEHYDROGENASE) O ALDO-KETO REDUCTASE FAMILY 1. MEMBER C3 (3-ALPHA HYDROXYSTEROID GDB:9864779 | 10p15-10p15 AKR1C3 DEHYDROGENASE, TYPE II) a O KAAO 119 O CHDR 10p15 AKR1C4 O CHLORDECONE REDUCTASE GDB:127896 15:3. HS.76790 ALDOAP2 o ALDOLASEPSEUDOGENE A, FRUCTOSE-BISPHOSPHATE2 GDB11895GDB:118995 || 10qter.10pter ALOX5 e ARACHIDONATE 5-LIPOXYGENASE GDB:132455 10q11.2- o HS.894.99 - 10q11.2. o ANKYRIN-LIKE REPEAT PROTEIN o CARP ALRP o C-193 GDB:9993042
o CYTOKINE INDUCIBLE NUCLEAR PROTEIN
o CARDIAC ANKYRIN REPEAT PROTEIN O HS.96029 rrd. -
ANK3 o ANKRIS3,HS.75893 NODE OF RANVIER (ANKYRING) SPB-29. 3. ANKRD2 O ANKYRIN REPEAT DOMAIN 2 (STRETCH GDB:10795364 10q23.1 RESPONSIVE MUSCLE) - 10q25.1 O ANX11 ANXA11 o ANNEXIN XI (56KD AUTOANTIGEN) GDB:313076 10q22-10q25
HS.75510 Ya O ANX2P3 ANXA2P3 : Fic GDB:120553 10q21 o ANX2L3 - 10q22. ... ANNEXIN II (LIPOCORTIN II) PSEUDOGENE 3 ass, GDB:369042 3. 3. ANNEXIN VII (SYNEXIN qZ.Z. FIG. 2B Patent Application Publication Sep. 11, 2003 Sheet 16 of 34 US 2003/0170678A1 Cytogenetic
O ANX8 10q11.2- ANXA8 O ANNEXIN VIII GDB. 128069 5:3. o HS.87268 AP3M1 ADAPTOR-RELATED PROTEIN COMPLEX 3, GDB: 0013490 10q22.1- MU 1 SUBUNIT --- 100.22.5 o ADIPOSE SPECIFIC 2 GOB:9958.670 10-10 - 10q25.1- | ARPP o ANKYRIN REPEAT PROTEIN GDB:11499581 10q25.1
o ATP5C1
o ATP SYNTHASE, H+TRANSPORTING, MITOCHONDRIAL F1 COMPLEX, GAMMA 10q22-10q23
ATP5C1 POLYPEPIDE 1 CD8:137187 14q22.2-
o ATP SYNTHASE, H+TRANSPORTING, 14q22.3 MITOCHONDRIAL F1 COMPLEX, GAMMA
POLYPEPTIOE-LIKE 1
ATP5
e ATP SYNTHASE, H+ TRANSPORTING,
MITOCHONDRIAL FO COMPLEX, SUBUNIT F6
• ATP SYNTHASE, H+ TRANSPORTING, 10pter-1 Oqter
MITOCHONORIAL ATPM
e ATP5A HS,73851
HS.451 - 10p15 o MURINE LEUKEMIA VIRAL (BMI-1) GDB:249556 10p13.
ONCOGENE HOMOLOG
ACVRLK3
BONE MORPHOGENETIC PROTEIN BMPR1A REPOR, TYPE 1A GDB:23.0245 o ACTIVIN A RECEPTOR, TYPE II-LIKE KINASE 3. o HBRAG BRAC 0 KIAAO598 GDB: 10013998 10q26-10q26
o B-CELL RAG ASSOCIATED PROTEIN - m BS69 o ADENOVIRUS 5 E1A BINDING PROTEIN GDB9958263 10-10 BTRC o BETA-TRANSDUCIN REPEAT CONTAINING GDB:995,5268 10q24-10q25 e BUB3 (BUDDING UNINHIBITED BY N BENZIMIDAZOLES 5, YEAST) HOMOLOG ur-ma
o CACNLB2
o MYSB m - CACNB2 o CALCIUM CHANNEL VOLTAGE-DEPENDENT, GDB. 132014 10p12-10p12 BETA 2 SUBUNIT • MYASTHENIC (LAMBERT-EATON) SYNDROME ANTIGEN B
FIG. 2C Patent Application Publication Sep. 11, 2003 Sheet 17 of 34 US 2003/0170678A1
Symbol Alioses Cytogenetic Location callsCALML5 ao CALMODULIN-IKECLP 3 GDB.p 138290 10pter103. CAMKG CAMK2G o CALCIUM/CALMODULIN-DEPENDENT GDB: 138469 10q22
PROTEIN KINASE (CAM KINASE) II GAMMA - 10q22. o CASPASE 7, APOPTOSIS-RELATED CYSTEINE
PRO EASE CASP7 o MCH3 GDB:5750011 10q25-10q25
o CMH-1 o ICE-AP3
CORNEAL DYSTROPHY OF BOWMAN'S LAYER TYPE II (THIEL-BEHNKE)
CDTB GDB:69 19860 10q23-10q24
CDC2 O Fl DMISION CYCLE 2, G1 TO S AND G2 TO GDB:119052 10q21.1 - O. HS.58,393 10q21.1
CADHERIN RELATED 23 - o DEAFNESS, AUTOSOMAL RECESSIVE 12 GDB. 1591799 10q21-10q22
• USHER SYNDROME 1 D (AUTOSOMAL : FSTEROL 25-HYDROXASE GDB9954776. 10q25-10q25
CHAT CHOLINE ACETYLTRANSFERASE GDB.119775 ER3.
o UBIQUTOUSCONSERVED HELIX-LOOP-HELIXKINASE s o IKK1 10q24 o NFKBIKA GDB:46241 O KBKA 10q25. o IKK-ALPHA
• TCF16 CMD1 C SEGi to 1C GDB:9140455 10q21-10q25
• COLLAGEN, TYPE XIII, ALPHA 1 GDB:1 19789 3.
a BPAG2
COL17A COLLAGEN, TYPE XVII, ALPHA 1
o BULLOUS PEMPHIGOID ANTIGEN 2 (18OKD) e BP18O n 9 BCD1
e CORE PROMOTER ELEMENT BENDING COPEB PROTEIN GDB:9120625 10p15-10p15 9 B-CELL, DERVED 1 - . e CPBP e CYTOCHORME C OXIDASE SUBUNIT 5 COX15 COX115 (YEAST) HOMOLOG, CYTOCHROME C GDB9862902 10q24-10q24 OXIDASE ASSEMBLY PROTEIN FIG. 2D Patent Application Publication Sep. 11, 2003 Sheet 18 of 34 US 2003/0170678A1
Cytogenetic
CARBOXYPEPTIDASE N, POLYPEPTIDE 1, CPN1 5OKD GDB,9835951 10pter-10qter 0 CPNE1 .
o CAMP RESPONSIVE ELEMENT MODULATOR
GOB:269801 o HS.79 106
O CHONOROTIN SULFATE PROTEOCLYCAN 6 (BAMACAN) BAM GDB:9958904 10q25-10q25- CSPG6 o HCAP CHONDROITIN SULFATE PROTEOGLYCAN 6 (BAMACAN) o CTSL 10q11.2- CSLL1 o CATHEPSIN L-LIKE 1 saan-n-GDB-250757. 1021, 10q22 CSLL2 O CATHEPSIN L-LIKE 2 GDB250758 1022, d d. 10q22.5- CTSLL3 CATHEPSIN L-LIKE 3 GDB,250760 6,231. O MGA1 CUBN o CUBILINRECEPTOR) (INTRINSIC FACTOR-COBALAMIN GDB,6360496049 O MEGALOBLASTIC ANEMA 1
• CUG TRIPLET REPEAT, RNA-BINDING PROTEIN 2 CUGBP2 O ELAV-TYPE RNA-BINDING PROTEIN GDB:9958105 | 10p15-10p13
O ETR-3 o NAPOR-2 CUL4B O CULLIN 4B GDB:9955.179 10pter-10qter
o HS. 1363 o CYTOCHROME P450, SUBFAMILY XVII GDB: 119829
CYP17 (STEROID 17-ALPHA-HYDROXYLASE), ADRENAL HYPERPLASIA
ge:Episo, SUB FAMILY WA
• P450RAI -
o CYP2C17
CYTOCHROME P450, SUBFAMILY IIC
N (MEPHENYTOIN 4-HYDROXYLASE), pOLYPEPTIDE 17 CYP2C8 o HS.702 y GDB:128983 10q24-10q24 o P450IIC17 o CYTOCHROME P450, SUBFAMILY IIC
(MEPHENYTOIN 4-HYDROXYLASE), POLYPEPTIDE 17 FIG. 2E Patent Application Publication Sep. 11, 2003 Sheet 19 of 34 US 2003/0170678A1
- r Cytogenetic
o CYP2C
a CYTOCHROME P450, SUBFAMILY IIC (MEPHENYTOIN 4-HYDROXYLASE), CYP2C19 EPIDE 19 GDB:1198.31
o CYTOCHROME P450, SUBFAMILY IIC (MEPHENYTOIN 4-HYDROXYLASE) o P450IIC 19 o CYTOCHROME P450, SUBFAMILY IIC 10q24.1- CYP2C8 (MEPHENYTOIN 4-HYDROXYLASE), GDB:127450 10241, W POLYPEPTIDE 8
CYP2C10 o CYTOCHROME P450, SUBFAMILY IIC CYP2C (MEPHENYTOIN 4-HYDROXYLASE), GDB. 131455. YP2C9 POLYPEPTIDE 9 HS.9669 P450IIC9
ETHANOL-INDUCIBLEISSOs P450, suru. IE GDB:119833 10q24.5-
CYP2E o HS.751 83 - 10qter CYP2E1
DNA SEGMENT, SINGLE COPY, PROBE PH4
(TRANSFORMING SEQUENCE, THYROID-1) D1 OS 170 Oa H4PH4 - GDB:GDB:107.96151 10/965 10q21-10q21U9 q e TST1 a PTC1 DFNB25 a DEAFNESS, AUTOSOMAL RECESSIVE 23 GDB-10013912|32 DGS2 PSERS SYNDROME CHROMOSOME GDB:99544.47 10p14-10p15
o DICKKOPF (XENOPUS LAEVIS) HOMOLOG1 10q11.2- o DKK-1 --- DLG5 o P-DLGDISCS, LARGE (DROSOPHILA) HOMOLOG 5 GDB:9955500 10q23-10q25 o KIAAO585
OMBT O DELETED IN MALIGNANT BRAINTUMORS 1 GDB:6943888 1933
DNA2 o DNA2 (DNA REPLICATION HELICASE, YEAST, GDB 313702 10q21.3- HOMOLOG)-LIKE - 10q22.1 DNMT2 N (CYTOSINE-5-)-METHYLTRANSFERASE GDB9836671 10p14-10p12 o TDT GDB: 119 100 10q23 DNTT DEOXYNUCLEOTIDYLTRANSFERASE, 10q24. TERMINAL DOCK1 EASE OF CYTO-KINESIS 1 CDB:9836007 10q26.15 a HS.82295 10q26.3 Patent Application Publication Sep. 11, 2003 Sheet 20 of 34 US 2003/0170678A1
Symbol E" o HVH3 so. 10q25 DUSP5 o DUAL SPECIFICTY PHOSPHATASE 5 GDB-3854.47 625, DUAL SPECIFICITY PHOSPHATASE 8 GDB-6065013 ||38.2 DUSP8P PSEUDOGENE 10q11.2 O DUAL SPECIFICITY PHOSPHATASE 10q11 DUSPP PSEUDOGENE - GDB:3854.49 10q11. a HVH4 o ENOYL COENZYME A HYDRATASE, SHORT 10q26,2- ECHS1 CHAIN, 1, MITOCHONDRIAL GDB520581; e SCEH EARLY GROWTH RESPONSE 2 (KROX-20 (DROSOPHILA) HOMOLOG) GDB:120611 EGR2 o HS.73872 - o KROX20 e EIF5
. • EUKARYOTIC TRANSLATION INITIATION FACTOR 3, SUBUNIT 10 (THETA, 17OKD) O EUKARYOTIC TRANSLATION INITIATION FACTOR 3 EIF3S10 •O HUMANEIF-3 P167 (CF.U58046) GDB;9864.835--ma | 10pter-10qter KIAAO 139 O HS.89996 o EIF3-P170 o EIF3-THEA EIF4EBP2 o EUKARYOTICFACTOR 4BE TRANSLATIONBINDING PROTEIN INITIATION 2 GDB.457248:4372.48 10q21-10q22- -EIF5AP1 o EUKARYOTICFACTOR 5A PSEUDOGENETRANSLATION 1INITIATION GDB:378209GDB3,829 E.10q23.5- EMX2 EMPTY SPIRACLES (DROSOPHILA) GDB:277886 10q26, 1 HOMOLOG 2 - 10q26.1 o CDS9 0 ECTONUCLEOSIDE TRIPHOSPHATE ENTPD1 DIPHOSPHOHYDROLASE 1 GDB:42217.3 CD39 ANTIGEN o HS.31205
0 NTPDASE-1 - 10q24.1
CKN2
o HS.99924 10q11
O EXCISION REPAIR CROSS-COMPLEMENTING 10q21. RODENT REPAIR DEFICIENCY, - GDB: 119882 10q11-10q11
COMPLEMENTATION GROUP 6 10q21.1-
O CSB 10q21.1: RAD26 (YEAST) HOMOLOG • COCKAYNE SYNDROME 2 (LATE ONSET FIG. 2G Patent Application Publication Sep. 11, 2003 Sheet 21 of 34 US 2003/0170678A1
In Cytogenetic
o ERP72
ERP70 O PROTEIN DISULFIDE ISOMERASE RELATED 10pter-10qter PROTEIN (CALCIUM-BINDING PROTEIN, 7pter-7Qter
INTESTINAL-RELATED) o HE TEROGENEOUS NUCLEAR PROTEIN FBRNP SIMILAR TO RA HELIX DESTABILIZING . GDB:995551 0 1 0-10 PROTEIN
• FER-1 (C.ELEGANS)-LIKE 3 (MYOFERLIN) 10q24-10q24 FER13 is MYOF GDB9989905 10q23.32 O KAA 1207 10q25.52
to MYOFERLIN o FIBROBLAST GROWTH FACTOR 8 (ANDROGEN-INDUCED) to HS.57710 o AGF o fibroblast growth factor receptor 2 (bacterio expressed kinase, keratinocyte growth factor receptor, Craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome
HS.82775 BEK
10q25.3- JWS . 10q26.
O CEKS
10q26-10q26
O KGFR o TK14 o TK25 o ECT1
o CFD O K-SAM
FIP2 tumor necrosis foctor alpho-inducible cellular GDB,9955295 10-10 protein containing leucine zipper domains -aximit to . . . w 10q11.2-
FRA1 OG o fragile site, ophidicolin type, common, GDB:125.177 fra (10)(q11.2) - 10q11.2: FRA1 e frequently rearranged in advanced T-cell GDB9954.895 18:02 lymphomas 1623. FUSE o polykaryocytosis promoter GDB 11924 1S. Opter FZD8s s frizzled (Drosophilo) homolog 8 GDB9864883 13:3:.22
o HS.89662
GAD2 o glutamate decarboxylase 2 (pancreatic islets GDB:128595 10p11.23 and brain, 65kD) - 10p11.23. o HS. 1668 e Hs.78048 10p15- - GATA3 e GATA-binding protein 5 GDB.152140 Patent Application Publication Sep. 11, 2003 Sheet 22 of 34 US 2003/0170678A1
Symbol Aliases AccessionID LocationCytogenetic
CBF1 o golgi-specific brefeldin A resistance factor 1 0 KIAAO248 GDB:9785357 10q24-10q24 • Similar to Yeast SEC7 protein GDF10 o growthBMP3B differentiation factor 10 - GDB:7009849 10pter-10qterO
GDP dissociotion inhibitor 2 GD 12 o RABGDIB GDB. 1347 101 10p15-10p15 rob GDP-dissociation inhibitor, beta
CDNFRAGDNF family receptor alpha 1 GDB:566: 1237 10q25-10q25qZ3- UQ
GFRA : stromy receptor olp 10q26-10q26 TRNR1 GDB:43.3257 10p11.2- CKP5 o glycerol kinase pseudogene 5 10p11.2 GLC1E o glaucoma 1, open angle, E (adult-onset) GDB9485054 10p15-10p14
, o HS. 77508 GLUD1 O GLUD GDB:119994 glutomote dehydrogenase 1
GLUDP2 • glutamate dehydrogenase pseudogene 2 GDB:137195 33. CLUDP3 o glutamate dehydrogenase pseudogene 3 GDB: 1571.96 1833. GLUDP5 e glutamate dehydrogenase pseudogene 5 CD8:1w 38.559 10p11.2-10p11.2
o HS.597 O glutamic-oxaloacetic transaminose 1, soluble GDB:120008 GOT1 Rorate aminotransferase 1) GPR10 G protein-coupled receptor 10 GDB:459157 18:-
G-protein-coupled receptor 26 10q26.2-
O in- 10q11.2-
GPR7 G protein-coupled receptor 7 GDB:371714 10q21.14
o GRK5 . 10q24– GPRK5 G protein-coupled receptor kinase 5 GDB250805 10qter HB58 H betO 58 homolod 10q21.1- helicase, lymphoid-specific 10q25.3-
HEP10 a hepatic protein 10 GDB:1 20039 3. F.G. 2 Patent Application Publication Sep. 11, 2003 Sheet 23 of 34 US 2003/0170678A1
Cytogenetic Location
o PRHX o hematopoietically expressed homeobox o HOX11 L-PEN
HHEX eO PRHhomeo box 11-like- - GDB: 138781 o proline-rich homeodomain-containing
transcription factor (symbol provisional)
O HEX o HS.75276 - 10q22 hexokinase 1 GDB.120044 10q22. HMX2 homeo box (H6 family)2. GDB:434.309s 10q25.2-10q26.5
heterogeneous nuclear ribonucleoprotein F o 10q11.21 o hnRNPF GDB5429144 6:23 heterogeneous nuclear ribonucleoprotein H3 HNRPH3 (2H9) GDB.984650 10q22-10q22 O 2H9
o HS.95366 O TOL3 a homeo box 11 (T-cell lymphoma 3 GDB:119607 10q24– HOX11 associated breakpoint). 10q24. a HS. 1096 . -- HS.89583 Hermonsky-Pudlok syndrome 10q25.1- HPS a HS.83951 GDB. 127359 1533. HPS1 human popillomavirus (type 6a) integration 0. 10q24 HPV6A1 site 1 GDB:32.1942 10q24. HSD17B5 hydroxysteroid (17-beta) dehydrogenase 5 GDB:9957758 10p15-10p14 HSD17B7 systeroid (17-beta) dehydrogenase 7 cDB 10796.801.10p11-1011
Hs.73739 o 10q21 HTR7 5-hydroxytryptomine (serotonin) receptor 7 GDB:269830. 10q24. (adenylate cyclose-coupled) HUG1 o HUG1 gene GDB:9957518, 10q24-10q24 IDDM10 insulin-dependent diabetes mellitus 10 GDB:450788, 10p11-10q11 IIDM 17 insulin-dependent diabetes mellitus 17 GDB:9956319 10q25-10q25.
insulin-degrading enzyme GDB: 118817
FIG. 2J Patent Application Publication Sep. 11, 2003 Sheet 24 of 34 US 2003/0170678A1
Location
0 IFI56 o interferon-induced protein with
tetratricopeptide repeats 1 o HS.851.21 a GOP GDB:120620 e IFNAI1 e IFI-56 o interferon-induced protein 56 O GARG-16
8 IF54
interferon-induced protein with tetratricopeptide repeats 2 e G1OP2 GDB:1 19959 8 IFI-54
IFTT2
interferon-induced protein 54
e ciga 2
ISG-54K GARG-39
e interferon-induced protein with tetratricopeptide repeats 4
IF4 59. GDB9864366 10q24–10q24 IFI60 CG-49
IL15RA o HS.interleukin 12505 15 receptor, alpha - GDB:622256 10p15-10p14
o Hs. 1724 IL2RA Af arm---am-argueGDB:119,545 3:...k interleukin 2 receptor, alpha p 10p15 IL9RP2 e interleukin 9 receptor pseudogene 2 GDB:681234 3-5. INPP5A inositol trisphosphate-5-phosphatase, 40kD GDB2281.32 10q26.5- e inositol polyphosphate-5-phosphatase, 40kD ------10q26.3
a SCA8
4-10q24 IOSCA e infantile onset spinocerebellar ataxio GDB-581849 10q23.5-33.9% (autosomal recessive) 10q24.1 k a spinocerebellor ataxia 8 s integrin, beta 1 (fibronectin receptor, beta
polypeptide, antiqen CD29 includes
MDF2, MSK12) a HS.74487 e FNRB GDB. 118732 0 MDF2 e CD29
8 MSK12 GPILA inter-alpha (globulin) inhibitor,. . . . H2 polypeptideGDB. 1201.08 10p15S.S. Patent Application Publication Sep. 11, 2003 Sheet 25 of 34 US 2003/0170678A1
Cytogenetic Locotion
O SLO o potassium lorge conductance calcium KCNMA activated channel, subfamily M, alpha GDB:386031 member 1 - . . o HS. 62679 a KNS1
10p11.23 KIF5B E. SE 5B GDB.155352 10p11.25 e KNS 18cen-18qter 1 kinesin-like 1 GDB: 132856 10q24.1-Q KNSL :gn as 10q24.1+ transcription factor simular to D. melanogaster on LBX1 homeodomain protein lady bird early GDB9958 106 10q24-10q24 o LIM domain binding 1 LDB1 o NL1 GDB;995.7446 10q24–10q25 O CLIM2 LDHAL5 O loctage dehydrogenase A-like-l 5 GDB: 2014.6 10qter10pter o HS. 85226 10q24-10q25 LIPA o lipose A, lysosomal acid, cholesterol esterose GDB:120155 10q25.2- (Wolman disease) 10q23.3%
LMNL2. lamin A/C-like 2 GDB.- 132149 10pterE. 10q21.1 -
| LOAD o late-onset Alzheimer disease susceptibility CDB: 1045O269 Sí. - 10q25.1
LPHB lipophilinprostatein-like B (uteroglobin family member), -aw-no-ma-emCDB:9958087 10q23-10q25 Ooter M150 o membrane external protein (130kD) GDB. 119569. ISE. o COT a ESTF
mitogen-activated protein kinase kinase kinose 8 MAP3K8 a HS.248 GDB. 134538. cot (concer Osaka thyroid) oncogene
o EST • Ewing sarcoma transformant
O PRKM8
o mitogen-activated protein kinase 8
protein kinose motogen-activated 8
(MAP kinase) 10q11.25 o HS.859 GDB9246350 10q11.23 e STRESS-ACTIVATED PROTEIN KINASE JNK1 e JNK O JNK1 O SAPK1 F.G. 2 Patent Application Publication Sep. 11, 2003 Sheet 26 of 34 US 2003/0170678A1
Symbol Alioses AccessionID CytogeneticLocotion e MAT o methionine adenosyltransferase I, alpha MAT1A O - GDB: 129077 10q-10q E. . . - - 10q22-10q22 O SAMS1 o mannose-binding lectin (protein A) 1, O m MBL1P1 pseudogene 1 GDB:995,5523 3: o COLEC3P Q4Z. o MBL • mannose-binding lectin (protein C) 2, Soluble CDB:120167 MB (opsonic defect) O MBP1 MBS3 O MoebiusAdraki e syndrome 3 GDB:9957145 10q22.110q21.3- MGEA5 sO MEA5meningioma expressed antigen 5 GDB:9958216 8: -- (hyaluronidase) qZ4. MGMT o 0-6-methylguanine-DNA methyltransferase GD B:125264. 19928 o HS. 1384 rests - 10q26 a MIPP MINPP1 O MINPP2 GDB9957350 10q25-10q25 o multiple inositol polyphosphate phosphotose 1 o HS.80976 - MKI67 o antigen identified by monoclonal antibody GDB:120185 3.E. Ki-67 Q MPP-1 o M-phase phosphoprotein 1 GDB:9957431 10-10 MRC1 mannose receptor, C type 1 GDB.133759 8:3. o HS.73487 a PSP-94 GDB:128042 o microseminoprotein, beta o PSP94 10q25 10q24-10q25 o myozenin 10q22.1- MYOZ E. w GDB:10797.024 10q22.1 a nuclear receptor coactivator 4 o RFG GDB:9954453 o ELE1 eamm-mm o ARA70 O glucosaminy)ity'? 2 stationers (heparan 5. a NST2 GDB:9958929 10q22-10q22 o HSST2N-deacetylase/N-sulfotransferase (heparan - glucosaminy) 2 FIG. 2M Patent Application Publication Sep. 11, 2003 Sheet 27 of 34 US 2003/0170678A1
w 8 Cytogenetic NEBL O nebulette GBD:995.7579 10p12-10p12 NET-PEN NET neuro epithelioma transforming gene-1 GDB:5885848 10p15-10p15 9 NET1A ranga H-NEU 10q25.1 -
NEURL neuralized (Drosophila)-like GDB9955109 365;
e HS.75090 NFKB2 O LYT-10 GDB:128804
e nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (E85 o nebulin-related anchoring protein GDB6055861 10q24-10q26
neuropilin NRP1 e VEGF165R GDB.9339 107 10p12-10p12 o YSAH NUDI5 o nudixSAH. (nucleoside . . diphosphate. . . linkedw moiety GDB:9956997T 10p14-10p13 x)-type motif 5 o HS.75485 - -- 10q26 Ornithine aminotransferase (gyrate atrophy) GDB, 120246 10q26. OATL3 ornithine aminotransferase-like 3 GDB:2158O3 1 O G 2 6 26x e OR7E73P
o OR7E71P o OR7E72P o OR7E73P o OR7E74P
o OR7E75P olfactory receptor, family 7, subfamily E, GDB-107956340p14-10p13
member 68 pseudogene olfactory receptor, family 7, subfamily E,
member 75 pseudogene o hg674 o hg523 o OR912-46 o OR912-109 e OR912-108 O OR912-110 o P4HA
o HS.89513
procollagen-proline, 2-oxoglutorate 4 GDB:120257 dioxygenase (proline 4-hydroxylase),
alpho polypeptide
0 HS.76768
to SK2 o ATPSK2 PAPSS2 3-prime-phosphoadenosine 5-prime GDB:9954820 10q23-10q24
phosphosulfate synthose 2 F.G. 2N Patent Application Publication Sep. 11, 2003 Sheet 28 of 34 US 2003/0170678A1
Locotion PARG o poly (ADP-ribose) glycohydrolase GDB:9955508 1833;
• Hs.73945 GDB. 138771 3925 PAX2 o paired box homeotic gene 2 --ro 10q24.
o HS.970 - 10q25-10q25
o HS.3192 O PCD o DCOH GDB: 138478
PCBD o 6-pyruvoyl-tetrahydropterin synthase/ dimerization cofactor of hepatocyte nuclear factor 1 alpha (TCF1) - pterin-4-olpho corbinolamine dehydratose protocadherin 15 CDB:11498.174 PCDHis Usher syndrome type IF
a phosphodiesterose 60, c0MP-specific, cone, 10q24 PDE6C alpha prime GDB:596.267 10q24. o CLIM1 o PDZ and LIM domain 1 (elfin) - 10q25.1-
PDLIM1 o carboxy terminal LIM domain protein 1 GDB:9806543. 10q25.1 e CLP-56 e hCLIM1 o PEO a progressive external ophthalmoplegia 1 10q24-10q24 e TWINKLE GDB:632784 10q25.3- PEO1 o C10orf2 --a-as 10q24.3 O FLJ21832 progressive external opthalamoplegio PFKFB3 6-phosphofructo-2-kinase/fructose-2,6- GDB-5772859 10p15-10p14 biphosphotose 3 -
o HS.999 10 10D15.5- PFKP a phosphofructokinase, platelet GDB: 1194.80 10p15.2p o HS.75363 PGAM1 o HS.74575PGAMA GDB-120530 E.- - - N. o phosphoglycerate mutase 1 (brain) C43.
- o phytonoyl-CoA hydroxylose (Refsum disease) 10p15.3-
Refsun disease PIP5K2A o typephosphatidylinositol-4-phosphate II, alpha 5-kinase, GDB-638 1309 10p1310p12.31 - PTX3 U Bired-like homeodomain transcription factor GDB-9858976 10q25-10q25 F.G. 20 Patent Application Publication Sep. 11, 2003 Sheet 29 of 34 US 2003/0170678A1
S -- Cytogenetic ymbol -- Aliases AccessionID Locotion
o PKD2L. a PKOL 10q24-10q24 PKD2L1 o polycystic kidney disease 2-like 1 GDB995.4789 10q25-10q25 o polycystin-like a Hs.77274 w 10q24 plasminogen activator, urokinase GDB. 1 19497 10q24.
PNIA1 ow protein spot in 2-D gels (40kD;pI 5.25) GDB, 127815 10q11.2-site, o NIAAA5 : 1280 - PNIA3 o protein spot in 2-D gels (30kD;pI 5.6) GDB. 128002 10q-10q.
o HS.99950 835, PN IP poncreatic lipase GDB:127916 q
o HS. 108 0q26.1- 10q26.1 POLL o polymerase (DNA-directed), lambda GDB: 10796049.10pter-10qter pe o pyrophosphatase (inorganic) - GDB:1 19505 3.
PPP1R5 proteinsubunit phosphatase5 1,. regulatory (inhibitor) GDB:6262o 11 10q25-10q24-1002
o protein phosphotose 3 (formerly 2B), catalytic 10pter subunit, beta isoform (calcineurin A beta) 10qter PPP3CB o Hs 1335 GDB:151562 10q21-10q22
CALNB pancreatic polypeptide receptor 1 PPYR o Y4NPY4R GDB: 1220228 PP
o AOP1 PRDX3 o peroxiredoxinantioxidant protein 3 1. GDB:9958884 10q25-10q26- o MER5 o PFP o 10q22- . PRF1 o perforin 1 (preforming protein) GDB; 18855 10q22. O HS. 1908 --- PRG1 a PRG GDB: 120312 3. o proteoglycon 1, secretory granule
Pico o protein kinose C, theta 10p15.
o PRKG1B o PRKBR1B
o protein kinose, cGMP-dependent, type I GOB:127918 a PGK a CGKI a protein kinase, cGMP-dependent, regulatory, type I, beta Patent Application Publication Sep. 11, 2003 Sheet 30 of 34 US 2003/0170678A1
Symbol Aliases AccessionID Cytogenetic Locotion PROA Proline(-) auxotroph, complementation of GDB:9954461 10pter-10qter
10q25.5-
PRSS11 protease, serine, 11 (IGF binding) GDB:6392572 365; o HS. 78575 O SAP1 o GLBA GOB: 2O366 a prosaposin (variant Gaucher disease and variant metachromatic leukodystrophy) o pleckstrin and Sec7 domain protein GDB:6542,505 10q24-10q24
o BZS o phosphatose the tensin homolog (mutated in 10q23-10q23 multiple advanced concers 1) GDB:6022948 10q25.5- MHAM
o Bannoyan-Zonana syndrome 10q25.5
multiple homartoma (Cowden syndrome) to MMAC PTER o phosphotriesterose-relatedRPR-1 GDB,9955992 10p12-10p12w
PTPRE • HPTPEprotein tyrosine phosphatase, receptor type, IGDB. 131585 10q26qZb epsilon polypetide - 10q26. o pyrroline-5-carboxylate synthetase (glutamate ommo-semialdehyde Synthetose PYCS .E. yde sy ) GDB,5929054 SS 10q24.5 PYGBL phosphorylase, glycogen; brain-like GDB. 120327 E. PYHG1 protein spot in 2-D gels (218kD) GDB. 119526 SE. 10pter PYHG 18 o protein spot in 2-D gels (31kD) GDB. 119534 10qter
O DOS64 e D10S65 GD8: 120341
o HS.89590 o retinol-binding protein 5, interstitial o HS.857
m RBP4. o retinol-bindingretinol-hind protein- 4, interstitiala a GOB:12O342r 10q24.10q23 ROPA o Refsum disease with increased GDB:9954445 10pter
pipecolicacidemio - 10p11.2 FIG. 20 Patent Application Publication Sep. 11, 2003 Sheet 31 of 34 US 2003/0170678A1
Symbol Aliases AccessionID Cytogenetic Location
o ret proto-oncogene (multiple endocrine neoplasia MEN2A, MEN2B and medullary
thyroid carcinoma 1, Hirschsprung disease) o HS.6253 o PTC GDB: 120346 o MTC1 . O MEN2A
O HSCR1 O MEN2B retinal G protein coupled receptor GDB:439338 10q23-10q23 o RHEB RHEB1 o Ros homolog enriched in brain 1 GDB:373435 10q11-10q11 RPP38 a ribonuclease P (58kD) GDB:995.7654 10-10 o ribosomal protein S24 10q22-10q23 RPS24 o HS.23623 GDB. 128662 623-1625 O RSP-1 - s 10pter RSU1 Ras suppressor protein 1 GDB:203962 8, 1013 o Severe combined immunodeficiency, type a SCIDA (Athabascon) GDB98.56806 || 10pter-10pter 9 A-SCID
o SCYB12 SDF1 :599 GDB:43.3267 e SDFB a strong Cell-derived factor 1 - a stimulator of Fe transport GDB,9956017 10q21-10q21
e SFTP1 a surfactant, pulmonary-associated protein A1 10q22 SFTPA1 b SP-A GDB: 119593 10q25. to SP-A1
e SFTPP1 SFTPAP1 a surfactant, pulmonory-associated protein A GDB:134719 pseudogene 1 arm-an-a-- surfactant-associated protein pseudogene 1 • SFTP4 10q22.2- SFTPD surfactont, pulmonory-associated protein D GDB:132674 10q25.1+ a SP-D SGPL1 ae sphingosine-1-phosphateSPL lyase 1 AlGDB:995.751 A 0 - 1 0021-1Q OG21Q
SHFM3 o splittype hand/foot3 malformation (ectrodactyly) GDB:386050 10q24-10q25- e SUR-8 SHOC2 o KIAAO862 - . . GDB9954459 10q25-10q25 • Suppressor of clear, C. elegans, homolog of Patent Application Publication Sep. 11, 2003 Sheet 32 of 34 US 2003/0170678A1
Symbol Aliases AccessionID CytogeneticLocation a solute carrier family 18 (vesicular SLC18A2 O Reggine) member 2 GDB: 138749
o SVMT a VMAT2 solute carrier family 18 (vesicular SLC18A3 acetylcholine), member 3 GDB:386396 o Hs.459 VACHT a solute carrier family 25 (mitochondrial carrier; Groves disease autoantigen), member 16 OD1 OS 105E SLC25A16 o GT GDB:9954455 e GDA o ML7 o HGT. 1 Graves disease autoantigen SC9ASP soluteexchanger), carrier isoform family 39 pseudoqene(sodium/hydrogen GDB. 1220158 10pter-10qter a SLIL1 -
SLT1 slit(Drosophilo) homolog. 1 -GDB9836963 35'IUC e MEGF4 SNCC O SVUCenyellom Otto DreOS. ConCer-SDeCITIC- p GDB9836247 10q23-10q23
o E o sorbin and SH3 domain containing 1 a SH3D5 o PONSIN GDB:114987.17
SORBS1 SH3P12 e CAP o PF16 10p12
SPAG6 O REPRO-SA-1 GDB,995.7396 10p11.2 o sperm associated antigen 6 a SMNR SPF30 o splicing factor 30, survival of motor neuron- GDB:9955863 10q23-10q23 reloted SPG9 • spastic paraplegia 9 (autosomal dominant) GDB,995,5204 E. ST12 ::gression of tumorigenicity 12 (prostate) GDB9 40457 10pter-10qter SAM Oo STAM1signal tranducing. adaptor molecule (SH3 GDB9954449w 10p14-10p13 domain ond ITAM motif) 1 TACC2N. • transforming,protein 2 acidic coiled-coil containing GDB:9957682anomam 10q26-10q26 o TAC2R
stachykinin receptor 2 o NK-2 receptor GDB:126367 o SKR o NKNAR 10q11 tachykinin 2 receptor (substance K receptor, 10q21k neurokinin 2 receptor FIG.2s Patent Application Publication Sep. 11, 2003 Sheet 33 of 34 US 2003/0170678A1
Symbol Senetic
o TATA box binding protein (TBP)-associated TAF2D factor, RNA polymerase II, D, 100kD GDB:595.018 SE, o TAFII1 OO CZ9.
o BZP o ZEB 8 AREB6 TCF8 a NIL-2-A GDB:128997 o transcription factor 8 (represses interleukin2 expression)
• TCF6 w 10q21 TFAM transcription factor A, mitochondrial GDB:127555 10q21k o TCF6L2 arm-ram- Q 10p12 THC2 thrombocytopenio 2 (autosomal dominant) GDB-10794.765 210p11.1
translocation of mitochondrial precursor GDB:9956986 10q11.21
o tolloid-like 2 LL2 o KIAA0932 protein GDB:9120556 10q25-10q24 KIAAO932
o APT1
- tumor necrosis factor receptor superfomily, o apoptosismember 6 (APO-1) antigen 1 GDB. 152671. TNFRSF6 o HS.82359 a FAS - CD95 O APOP-1
o FAS 1 - TPL2 e tumor progression locus 2 GDB:5875355 10p11-10p11 o thyroid stimulating hormone receptor-like 3 GDB 120470 1 Oqter
Ochoourofacial syndrome syndrome GDB:6380714 10q23-10q24m Hs.75593 10q25.2- UROS uroporphyrinogen III synthase (congenitol GOB: 128112 1653,
erythropoietic porphyria) ------Q4b.
N. Usher syndrome 1F (outosomal recessive, GDB:9571241
severe)
UTF1 O Serentiated embryonic cell tronscription GDB:9955143 10q26-10q26 foctor --- o HS.75550 10q22.1- Patent Application Publication Sep. 11, 2003 Sheet 34 of 34 US 2003/0170678A1
VDAC2 o voltage-dependent anion channel 2 GDB: 138281 E;3. o Hs.78902 sus- 215ter-21ater
o HS2O64 . 10p15
KIAA1351 WDR11 o WD40 repeat domain 11 GDB:11499.060 10q26-10q26 o FLJ10506 WN8B o wingless-type MMTV integration site family, GD8:681241 10q24–10q24 member 8B - - o KIAAO613 10q22.3- ZASP Z-bond alternatively spliced PDZ-motif GDB:9956973 10,235 . ZNFIA zinc finger protein 11a (KOX 2) GDB.125318 1612.
ZNF22 o zinc finger protein 22 (KOX 15) GDB: 125329 1843.
ZNF25 o zinc finger protein 25 (KOX 19) -a-meu-GDB:125,532 3:43.p k ZNF32 o zinc finger protein 52 (KOX 50) looB-125339 3.
.1 e ZNF33 10p11.2- F33A or zinc finger protein 33a (KOX 31) GDB.125340 10.112. ZAF33B e Zincinc fifinger proteintein 33b (KOX 31 ) GDB. 1284.92 10q11.2-62.
o Hs.54488 --
ZNF37A o ZNF37 GDB: 126686 3:43.
s zinc finger protein 37a (KOX 21)
N - - 10q11.2- ZNF37B... y o zinc finger protein 37b (KOX 21) GDB. 128495. 10q11.2
ZWINT 0 ZWO interoctor GOB:995688 1833
FIG. 2U US 2003/017O678A1 Sep. 11, 2003
GENETIC MARKERS FOR ALZHEIMER'S of interfering with the ability of the person to function DISEASE AND METHODS USING THE SAME independently. Other Symptoms, which are variable, include myoclonus and seizures. Duration of AD from the first RELATED APPLICATIONS Symptoms of memory loSS until death is 10 years on average. 0001) Benefit of priority under S119(e) is claimed to U.S. 0005 AD is accompanied by a constellation of neuro provisional application Serial No. 60/348,065, filed Oct. 25, pathological features. The AD brain is characterized by the 2001, entitled “Genetic Markers for Alzheimer's Disease presence of amyloid-containing plaques and neurofibrillary and Methods of Using the Same,” and to U.S. provisional tangles (NFT). A major molecular component of amyloid is application Serial No. 60/336,983, filed Nov. 2, 2001, a highly hydrophobic peptide called AB peptide. A? is an entitled “Genetic Markers for Alzheimer's Disease and ~4.0 kDa protein, about 39-43 amino acids long, that is Methods of Using the Same'. This application is also related derived from a C-terminal region of amyloid precursor to U.S. provisional application Serial No. 60/339,525, filed protein (APP). APP is a membrane-spanning glycoprotein Oct. 25, 2001, entitled “Genes and Polymorphisms on that, in one processing pathway, is cleaved inside the AB Chromosome 10 ASSociated with Alzheimer's Disease and domain to produce C-SAPP, a nonamyloidogenic Secreted Other Neurodegenerative Diseases,” to U.S. provisional form of APP. A? aggregates into antiparallel filaments in a application Serial No. 60/338,010, filed Nov. 8, 2001, B-pleated sheet Structure resulting in the birefringent nature entitled “Genes and Polymorphisms on Chromosome 10 of the AD amyloid. Although AB is the major component of ASSociated with Alzheimer's Disease and Other Neurode AD amyloid, other proteins have also been found associated generative Diseases,” to U.S. provisional application Serial with amyloid plaques, e.g., alpha-1-anti-chymotrypsin No. 60/368,919, filed Mar. 28, 2002, entitled “Genes and Abraham et al. (1988) Cell 52:487-501), cathepsin D Cat Polymorphisms on Chromosome 10 Associated with Alzhe aldo (1990) et al. Brain Res. 513:181-192), non-amyloid imer's Disease and Other Neurodegenerative Diseases,” to component protein Ueda et al. (1993) Proc. Natl. Acad. Sci. U.S. provisional application Serial No. 60/336,929, filed U.S.A. 90:11282-11286), apolipoprotein E (apoE) Namba Nov. 8, 2001 entitled “POLYMORPHIC UROKINASE et al. (1991) Brain Res. 541:163-166; Wisniewski and PLASMINOGEN ACTIVATOR GENES', to U.S. provi Frangione (1992) Neurosci. Lett. 135:235-238; Strittmatter sional application Serial No. 60/338,363, filed Nov. 9, 2001, et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:1977-1981), entitled “POLYMORPHIC UROKINASE PLASMINO apolipoprotein JChoi-Mura (1992) et al. Acta Neuropathol. GEN ACTIVATOR GENES', to U.S. provisional applica 83:260-264; McGeer (1992) et al. Brain Res. 579:337-341), tion Serial No. 60/337052, filed Dec. 4, 2001, entitled heat shock protein 70 Hamos et al. (1991) Neurology “POLYMORPHICUROKINASE PLASMINOGEN ACTI 41:345-350), complement components McGeer and Rogers VATOR GENES', and to Intl. PCT application No. (docket (1992) Neurology 43:447-449), alpha2-macroglobin no. 37481-3308PC), filed the same day herewith, entitled Strauss et al. (1992) Lab. Invest. 66:223-230), interleukin-6 “Genes and Polymorphisms on Chromosome 10 Associated Strauss et al. (1992) Lab. Invest. 66:223-230), proteogly with Alzheimer's Disease and Other Neurodegenerative cans Snow et al. (1987) Lab. Invest. 58:454-458), and Diseases.” The Subject matter of each of these applications serum amyloid P Coria et al. (1988) Lab. Invest. 58:454 is incorporated herein in its entirety. 458). 0002. Subject matter of this application was made in part 0006 Plaques are often surrounded by astrocytes and with support from the United States Government under activated microglial cells expressing immune-related pro Grant Nos. 1 R01 MH60009 (NIMH) and 5P5OAG05134 teins, such as the MHC class II glycoproteins HLA-DR, (NIA). Thus, the U.S. Government may retain certain rights HLA-DP and HLA-DQ, as well as MHC class I glycopro in Such Subject matter. teins, interleukin-2 (IL-2) receptors and IL-1. Also Surround ing many plaques are dystrophic neurites, which are nerve FIELD OF THE INVENTION endings containing abnormal filamentous Structures. 0003. The present invention relates to genetic markers 0007. The characteristic Alzheimer's NFTS contain asSociated with Alzheimer's disease. The invention also abnormal filaments bundled together in neurons and occu relates to methods of determining the presence or absence in pying much of the perinuclear cytoplasm. These filaments a Subject of one or more polymorphisms associated with contain the microtubule-associated protein tau in a hyper Alzheimer's disease and methods of determining the level of phosphorylated form. “Ghost” NFTs are also observed in risk for Alzheimer's disease in a subject. The invention AD brains, which presumably mark the location of dead further relates to nucleic acid compositions and kits for use neurons. Other neuropathological features include granu in determining the presence or absence in a Subject of one or lovascular changes, neuronal loSS, gliosis and the variable more polymorphisms associated with Alzheimer's disease presence of Lewy bodies. and kits for determining the level of risk for Alzheimer's disease in a Subject. 0008. The destructive process of the disease is evident on a groSS level in the AD brain to the extent that in late-stage BACKGROUND OF THE INVENTION AD, Ventricular enlargement and Shrinkage of the brain can be observed by magnetic resonance imaging. The cells 0004 Alzheimer's disease (AD) is a devastating progres remaining at autopsy, however, are grossly different from Sive neurodegenerative disorder, which is the predominant those of a normal brain, characterized by extensive gliosis cause of dementia in people over 65 years of age. Clinical and neuronal loss. Neurons which were possibly involved in Symptoms of the disease typically begin with Subtle short initiating events, are absent, and other cell types, Such as the term memory problems. AS the disease progresses, difficulty activated microglial cells and astrocytes, have gene expres with memory, language and orientation worsen to the point Sion patterns not observed in the normal brain. Thus, the US 2003/017O678A1 Sep. 11, 2003
amyloid plaque Structures and NFTS observed at autopsy are Duijn et al. (1994) Am. J. Hum. Genet. 55: 714-727; Schel most likely the end-products of a lengthy disease process, far lenberg et al. (1988) Science 241:1507; Schellenberg et al. removed from the initiating events of AD. (1991) Am. J. Hum. Genet. 48:563; Schellenberg et al. (1991) Am. J. Hum. Genet. 49:511-517; Kamino et al. (1992) 0009. Accordingly, attempts to use biochemical methods Am. J. Hum. Genet. 51:998; Schellenberg et al. (1993) Am. to identify key proteins and genes in the initiating Steps of J. Hum. Genet. 53:619; Schellenberg et al. (1992) Ann. the disease are hampered by the fact that it is not possible to Neurol. 31:223; Yu et al. (1994) Am. Hum. Genet. 54:631). actually observe these critical initiating events. Biochemical Furthermore, not all people who do carry the APOE4 allele dissection of the postmortem AD brain is an analysis of develop AD, even if they live to old age. products that occur late in the disease long after the early pathogenic processes associated with AD had begun. Recon 0013 There is evidence that genetic factors other than Struction of the pathogenic pathways of AD through bio APOE4 contribute to the risk for late onset AD. A study chemical analysis of lesions of the postmortem AD brain has modeling AD as a quantitative trait estimated that there are its limitations and has been referred to as "molecular archae at least four additional genetic Susceptibility loci for the ology"Schellenberg (1995) Proc. Natl. Acad. Sci. U.S.A. disease Daw et al. (2000) Am. J. Hum. Genet. 66:196). 92:8552-8559). Thus, there is a need to identify genetic markers linked to AD genes, the locations of AD genes and the particular 0.010 The diagnosis of AD is confounded with other disease genes and mutations therein. Useful genetic markers dementing diseases and conditions common in the elderly, and mutations will greatly facilitate detection of a predis including dementia-causing conditions Such as Strokes, position for AD and the development of therapeutics that microvascular disease, brain tumors, thyroid dysfunction, target AD gene-related alterations. drug reactions, Severe depression and a host of other con ditions that can cause intellectual deficits in the elderly. SUMMARY OF THE INVENTION Furthermore, many of the pathological features of AD are not unique to the disease and also occur in the brains of 0014 Provided herein are genetic markers associated normal aged individuals and are associated with diseases with Alzheimer's disease (AD). Genetic markers associated Such as Guam Parkinson disease, dementia pugilistica and with AD are located on chromosome 10. Particular markers progressive Supranuclear palsy. For example, the twisted asSociated with AD are located on chromosome 10q. filaments that form NFTS also occur in certain tangles Included among the genetic markers associated with AD asSociated with other diseases Such as Pick's Disease. provided herein are markers located on chromosome 10q22, 10q23, 10q24, 10q25 and 10q26. In particular embodiments, 0.011 AD is a genetically complex and heterogeneous the markers are located on human chromosome 10. Genetic disorder. Because AD is relatively common in the elderly, markers provided herein are associated with AD Such that clustering of cases in a family may occur by chance, they provide Scores or results indicative of linkage disequi representing possible confounding non-allelic genetic het librium with an AD DNA segment (e.g., an AD gene) or erogeneity, or etiologic heterogeneity with genetic and non asSociation with an allele that conferS protection against AD, genetic cases co-existing in the same kindred. Despite these or of association with AD when tested in linkage disequi problems, it has been found that roughly 40% of early-onset librium or association assessment methods described herein AD (less than 65 years of age) is attributable to missense or known to those of skill in the art for association with an mutations in three genes (APP, PS1 and PS2). In contrast, allele that conferS risk for or protection against AD. Genetic the genetic basis of late onset AD has proven more difficult markers are provided that are in linkage disequilibrium with to disentangle Blacker and Tanzi (1998) Arch. Neurol. an allele that conferS risk for or protection against AD. The 55:294). Substantial evidence Suggests that inherited genetic genetic markers are associated with AD as individual mark defects are involved in late-onset AD. Families with mul erS and/or in combinations, Such as haplotypes, that are tiple late-onset AD cases have been described Bird et al. associated with AD. (1989) Ann. Neurol. 25:12-25; Heston and White (1978) 0015. In further embodiments of the genetic markers Behavior Genet. 8:315-331; Pericak-Vance et al. (1988) asSociated with AD provided herein, the markers are located Exp. Neurol. 102:271-279). on chromosome 10q22, 10q23 or 10q24 or on chromosome 0012 To date, only one genetic risk factor, a common 10q23, 10q24 or 10q25. The genetic markers may, for polymorphism in the apolipoprotein E (APOE) gene, example, be located in a region of chromosome 10 about 30 referred to as the e4 allele (APOE4), has been replicated in cM around and including D10S583, or about 20 cM around independent samples in late-onset ADFarrer et al. (1997).J. and including D10S583, or about 15 cM around and includ Am. Med. Assoc. 278:1349-1356; Blacker et al. (1997) ing D10S583, or about 12 cM around and including Neurology 48:139). However, approximately half of the D10S583, or about 10 cM around and including D10S583. people who develop AD do not carry the APOE e4 allele In particular embodiments, the genetic markers may be found in several other families with high incidence of AD, located in a region of chromosome 10 about 5 cM around including the Volga German (VG) kindreds Brousseau et al. and including D10S583, or about 4 cMaround and including (1994) Neurology 44:342-344; Kuusisto et al. (1994) Brit. D10S583, or about 3 cM around and including D10S583, or Med. J. 309:363; Tsai et al. (1994) Am. J. Hum. Genet. about 2 cM around D10S583, or about 1 cM around and 54:643; Liddelet al. (1994).J. Med. Genet. 31: 197; Cook et including D10S583, or about 0.5 cM around and including al. (1979) Neurology 29: 1402-1412; Bird et al. (1988) Ann. D10S583, or about 0.25 cMaround and including D10S583, Neurol. 23:25-31; Bird et al. (1989) Ann. Neurol. 25:12-25). or about 0.1 cM around and including D10S583. The genetic The known AD loci have been excluded as possible causes markers may also be located in a region of chromosome 10 of the discrepancy Schellenberg et al. (1992) Science about 30 Mb around and including D10S583, or about 28 258:668; Lannfelt et al. (1993) Nat. Genet. 4:218-219; van Mb around and including D10S583, or about 20 Mb around US 2003/017O678A1 Sep. 11, 2003 and including D10S583, or about 15 Mb around and includ 0.025 (i) the region between D10S583 and ing D10S583, or about 10 Mb around and including D10S566, inclusive, D10S583. In further embodiments, the genetic markers may be located in a region of chromosome 10 about 5 Mbaround 0026 (i) the region between D10S583 and and including D10S583, or about 2.5 Mb around and includ D10S1671, inclusive and ing D10S583, or about 1 Mbaround and including D10S583 0027 (k) the region between D10S583 and or about 500 kb around and including D10S583, or about D10S1741, inclusive. 200 kb around and including D10S583, or about 100 kb around and including D10S583, or about 50 kb around and 0028. In particular embodiments of the genetic markers including D10S583, or about 40 kb around and including asSociated with AD provided herein, the markers are located D10S583, or about 20 kb around and including D10S583, or in one or more of the following regions of chromosome 10: about 10 kb around and including D10S583, or about 5 kb 0029 (a) the region extending about 5 cM, or about around and including D10S583, or about 1 kb around and 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 including D10S583. cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, 0016 Genetic markers associated with AD provided or about 0.1 cM proximal (centromeric) from and herein may also be located in one or more of the following including marker D10S583, regions of chromosome 10: 0030) (b) the region extending about 5 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 0017 (a) the region extending about 50 cM, or about cM distal (telomeric) from and including marker 45 cM, or about 33 cM, or about 30 cM, or about 25 D10S583, cM, or about 21 cM, or about 15 cM, or about 10 cM, or about 5 cM, or about 4 cM, or about 3 cM, or 0031 (c) the region extending about 10 Mb, or about 2.7 cM, or about 2.5 cM, or about 2 cM, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about about 1.5 cM, or about 1 cM, or about 0.5 cM, or 500 kb, or about 300 kb, or about 200 kb, or about about 0.1 cM proximal (centromeric) from and 100 kb, or about 80 kb, or about 50 kb, or about 10 including marker D10S583, kb, or about 5 kb, or about 1 kb proximal from and including marker D10S583, and 0018 (b) the region extending about 60 cM, or about 55 cM, or about 50 cM, or about 45 cM, or 0032 (d) the region extending about 10 Mb, or about 40 cM, or about 35 cM, or about 30 cM, or about 8 Mb, or about 5 Mb, or about 4 Mb, or about about 25 cM, or about 20 cM, or about 15 cM, or 3 Mb, or about 2 Mb, or about 1 Mb, or about 500 about 13 cM, or about 12 cM, or about 9 cM, or kb, or about 300 kb, or about 200 kb, or about 100 about 5 cM, or about 2.5 cM, or about 1 cM, or about kb, or about 50 kb, or about 10 kb, or about 5 kb, or 0.5 cM or about 0.1 cM distal (telomeric) from and about 1 kb distal from and including marker including marker D10S583, D1OS583. 0019 (c) the region extending from and including 0033 Also provided herein are combinations or haplo marker D10S583 to the terminus of the q arm of types of genetic markers which are associated with AD. In chromosome 10, one embodiment, each genetic marker in a combination is associated with AD. In other embodiments, Some of the 0020 (d) the region extending from and including genetic markers in the combination are associated with AD marker D10S583 to the centromere of chromosome and Some of the genetic markers are not or none of the 10, genetic markers is associated with AD. In Such embodi 0021 (e) the region extending about 62 Mb, about ments, the combination of markers as a whole is associated 55 Mb, or about 50 Mb, or about 32 Mb, or about 28 with AD, Such as in the case of a haplotype. Mb, or about 25 Mb, or about 20 Mb, or about 15 0034 Genetic markers contained within the combina Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, tions of genetic markers associated with AD provided herein or about 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or are located on chromosome 10. In particular embodiments, about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or the genetic markers are located on human chromosome 10. about 0.01 Mb, or about 1 kb proximal from and In further embodiments of the combinations of genetic including marker D10S583, markers associated with AD, the genetic markers are located on chromosome 10q. For example, each of the genetic 0022 (f) the region extending about 45 Mb, or about markers in the combination may be located on chromosome 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 10q22, 10q23, 10q24, 10q25 or 10q26. In particular embodi Mb, or about 20 Mb, or about 16 Mb, or about 14 ments, each of the genetic markers may be located on Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, chromosome 10q22, 10q23 or 10q24 or on chromosome or about 4 Mb, or about 3 Mb, or about 2 Mb, or 10q23, 10q24 or 10q25. In further embodiments of the about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or combinations of genetic markers associated with AD, each about 0.05 Mb, or about 0.01 Mb, or about 1kb distal of the genetic markers may be located in a region of from and including marker D10S583, chromosome 10 about 30 cM around and including 0023) (g) the region between D10S564 and D10S583, or about 20 cM around and including D10S583, D10S583, inclusive, or about 15 cM around and including D10S583, or about 12 cM around and including D10S583, or about 10 cM around 0024 (h) the region between D10S583 and and including D10S583. Each of the genetic markers may D10S1710, inclusive, also be located in a region of chromosome 10 about 5 cM US 2003/017O678A1 Sep. 11, 2003
around and including D10S583, or about 4 cM around and Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, including D10S583, or about 3 cM around and including or about 4 Mb, or about 3 Mb, or about 2 Mb, or D10S583, or about 2 cM around D10S583, or about 1 cM about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or around and including D10S583, or about 0.5 cM around and about 0.05 Mb, or about 0.01 Mb, or about 1kb distal including D10S583, or about 0.25 cM around and including from and including marker D10S583, D10S583, or about 0.1 cM around and including D10S583. In yet further embodiments, each of the genetic markers may 0.042 (g) the region between D10S564 and be located in a region of chromosome 10 about 30 Mb D10S583, inclusive, around and including D10S583, or about 28 Mb around and 0.043 (h) the region between D10S583 and including D10S583, or about 20 Mb around and including D10S1710, inclusive, D10S583, or about 15 Mb around and including D10S583, or about 10 Mb around and including D10S583. Each of the 0044) (i) the region between D10S583 and genetic markers of the combinations of genetic markers D10S566, inclusive, asSociated with AD may be located in a region of chromo 0.045 () the region between D10S583 and Some 10 about 5 Mb around and including D10S583, or D10S1671, inclusive and about 2.5 Mbaround and including D10S583, or about 1 Mb around and including D10S583 or about 500 kb around and 0046) (k) the region between D10S583 and including D10S583, or about 200 kb around and including D10S1741, inclusive. D10S583, or about 100 kb around and including D10S583, 0047. In particular embodiments of the combinations of or about 50 kb around and including D10S583, or about 40 genetic markers provided herein, each of the genetic markers kb around and including D10S583, or about 20 kb around may be located in one or more of the following regions of and including D10S583, or about 10 kb around and includ chromosome 10: ing D10S583, or about 5 kb around and including D10S583, 0048 (a) the region extending about 5 cM, or about or about 1 kb around and including D10S583. 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 0035) In other embodiments of the combinations of cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, genetic markers associated with AD provided herein, each of or about 0.1 cM proximal (centromeric) from and the genetic markers may be located in one or more of the including marker D10S583, following regions of chromosome 10: 0049 (b) the region extending about 5 cM, or about 0036 (a) the region extending about 50 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 45 cM, or about 33 cM, or about 30 cM, or about 25 cM distal (telomeric) from and including marker cM, or about 21 cM, or about 15 cM, or about 10 cM, D10S583, or about 5 cM, or about 4 cM, or about 3 cM, or 0050 (c) the region extending about 10 Mb, or about 2.7 cM, or about 2.5 cM, or about 2 cM, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about about 1.5 cM, or about 1 cM, or about 0.5 cM, or 500 kb, or about 300 kb, or about 200 kb, or about about 0.1 cM proximal (centromeric) from and 100 kb, or about 80 kb, or about 50 kb, or about 10 including marker D10S583, kb, or about 5 kb, or about 1 kb proximal from and 0037 (b) the region extending about 60 cM, or including marker D10S583; and about 55 cM, or about 50 cM, or about 45 cM, or 0051 (d) the region extending about 10 Mb, or about 40 cM, or about 35 cM, or about 30 cM, or about 8 Mb, or about 5 Mb, or about 4 Mb, or about about 25 cM, or about 20 cM, or about 15 cM, or 3 Mb, or about 2 Mb, or about 1 Mb, or about 500 about 13 cM, or about 12 cM, or about 9 cM, or kb, or about 300 kb, or about 200 kb, or about 100 about 5 cM, or about 2.5 cM, or about 1 cM, or about kb, or about 50 kb, or about 10 kb, or about 5 kb, or 0.5 cM or about 0.1 cM distal (telomeric) from and about 1 kb distal from and including marker including marker D10S583, D1OS583. 0038 (c) the region extending from and including 0052 Genetic markers provided herein may be used in a marker D10S583 to the terminus of the q arm of variety of methods. Provided herein are methods involving chromosome 10, polymorphisms, Such as genetic markers, asSociated with 0039 (d) the region extending from and including AD. In particular embodiments of each of the methods marker D10S583 to the centromere of chromosome provided herein, the genetic markers or polymorphisms or 10, alleles associated with AD are located on human chromo Some 10. Such methods include a method for detecting the 0040 (e) the region extending about 62 Mb, about presence or absence in a Subject of a polymorphism asso 55 Mb, or about 50 Mb, or about 32 Mb, or about 28 ciated with Alzheimer's disease in which chromosome 10 of Mb, or about 25 Mb, or about 20 Mb, or about 15 the Subject is analyzed for a polymorphism, or combination Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, or haplotype of polymorphisms, asSociated with Alzhe or about 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or imer's disease. The polymorphism, or each of the polymor about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or phisms of the combination, may be located on chromosome about 0.01 Mb, or about 1 kb proximal from and 10q, and in particular, on chromosome 10q22, 10q23, including marker D10S583, 10q24, 10q25 or 10q26. In a further embodiment, the 0041 (f) the region extending about 45 Mb, or about polymorphism, or each of the polymorphisms in the com 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 bination, may be located on chromosome 10q22, 10q23 or Mb, or about 20 Mb, or about 16 Mb, or about 14 10q24 or on chromosome 10q23, 10q24 or 10q25. The US 2003/017O678A1 Sep. 11, 2003
polymorphism, or each of the polymorphsims in the com about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or bination, may be located in a region of chromosome 10 about 0.01 Mb, or about 1 kb proximal from and about 30 cMaround and including D10S583, or about 20 cM including marker D10S583, around and including D10S583, or about 15 cM around and including D10S583, or about 12 cM around and including 0059) (f) the region extending about 45 Mb, or about D10S583, or about 10 cM around and including D10S583. 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 The polymorphism, or each of the polymorphisms in the Mb, or about 20 Mb, or about 16 Mb, or about 14 combination, may be located about 5 cM around and includ Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, ing D10S583, or about 4 cM around and including or about 4 Mb, or about 3 Mb, or about 2 Mb, or D10S583, or about 3 cM around and including D10S583, or about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or about 2 cM around D10S583, or about 1 cM around and about 0.05 Mb, or about 0.01 Mb, or about 1kb distal including D10S583, or about 0.5 cM around and including from and including marker D10S583, D10S583, or about 0.25 cMaround and including D10S583, 0060 (g) the region between D10S564 and or about 0.1 cM around and including D10S583. These D10S583, inclusive, methods may also be practiced wherein the polymorphism, or each of the polymorphisms in the combination, is located 0061 (h) the region between D10S583 and about 30 Mb around and including D10S583, or about 28 D10S1710, inclusive, Mb around and including D10S583, or about 20 Mb around 0062 (i) the region between D10S583 and and including D10S583, or about 15 Mb around and includ D10S566, inclusive, ing D10S583, or about 10 Mb around and including D10S583, or about 5 Mb around and including D10S583, or 0063 () the region between D10S583 and about 2.5 Mbaround and including D10S583, or about 1 Mb D10S1671, inclusive and around and including D10S583 or about 500 kb around and 0064) (k) the region between D10S583 and including D10S583, or about 200 kb around and including D10S1741, inclusive. D10S583, or about 100 kb around and including D10S583, or about 50 kb around and including D10S583, or about 40 0065. In a particular embodiment of the method, the kb around and including D10S583, or about 20 kb around polymorphism, or each of the polymorphisms in the com and including D10S583, or about 10 kb around and includ bination, is located in one or more of the following regions ing D10S583, or about 5 kb around and including D10S583, of chromosome 10: or about 1 kb around and including D10S583. 0066 (a) the region extending about 5 cM, or about 0053. In a further embodiment of the method, the poly 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 morphism, or each of the polymorphisms in the combina cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, tion, is located in one or more of the following regions of or about 0.1 cM proximal (centromeric) from and chromosome 10: including marker D10S583, 0067 (b) the region extending about 5 cM, or about 0054 (a) the region extending about 50 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 45 cM, or about 33 cM, or about 30 cM, or about 25 cM distal (telomeric) from and including marker cM, or about 21 cM, or about 15 cM, or about 10 cM, D10S583, or about 5 cM, or about 4 cM, or about 3 cM, or about 2.7 cM, or about 2.5 cM, or about 2 cM, or 0068 (c) the region extending about 10 Mb, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about about 0.1 cM proximal (centromeric) from and 500 kb, or about 300 kb, or about 200 kb, or about including marker D10S583, 100 kb, or about 80 kb, or about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb proximal from and 0.055 (b) the region extending about 60 cM, or including marker D10S583; and about 55 cM, or about 50 cM, or about 45 cM, or about 40 cM, or about 35 cM, or about 30 cM, or 0069 (d) the region extending about 10 Mb, or about 25 cM, or about 20 cM, or about 15 cM, or about 8 Mb, or about 5 Mb, or about 4 Mb, or about about 13 cM, or about 12 cM, or about 9 cM, or 3 Mb, or about 2 Mb, or about 1 Mb, or about 500 about 5 cM, or about 2.5 cM, or about 1 cM, or about kb, or about 300 kb, or about 200 kb, or about 100 0.5 cM or about 0.1 cM distal (telomeric) from and kb, or about 50 kb, or about 10 kb, or about 5 kb, or including marker D10S583, about 1 kb distal from and including marker D1OS583. 0056 (c) the region extending from and including marker D10S583 to the terminus of the q arm of 0070 Also provided are embodiments of the method chromosome 10, wherein the polymorphism is an allele of D10S583. 0057 (d) the region extending from and including 0071. Further provided are embodiments of any of the marker D10S583 to the centromere of chromosome methods for detecting the presence or absence in a Subject of 10, a polymorphism, or combination or haplotype of polymor phisms, associated with Alzheimer's disease, wherein the 0.058 (e) the region extending about 62 Mb, about polymorphism, or combination or haplotype of polymor 55 Mb, or about 50 Mb, or about 32 Mb, or about 28 phisms, may be associated with AD with onset ages of Mb, or about 25 Mb, or about 20 Mb, or about 15 greater than or equal to about 50 years, or greater than or Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, equal to about 60 years, or greater than or equal to about 65 or about 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or years. The method may also be Such that the polymorphism, US 2003/017O678A1 Sep. 11, 2003
or combination or haplotype of polymorphisms, is associ and including D10S583. In further embodiments of the ated with an AD DNA segment that has an effect size method, each of the two or more polymorphisms may be comparable to or greater than the effect size of APOE. The located in a region of chromosome 10 about 5 cM around method may further be Such that the polymorphism, or each and including D10S583, or about 4 cMaround and including or Some of the polymorphisms of the combination, is located D10S583, or about 3 cM around and including D10S583, or within an AD DNA segment. about 2 cM around D10S583, or about 1 cM around and including D10S583, or about 0.5 cM around and including 0.072 Also provided are embodiments of any of the D10S583, or about 0.25 cMaround and including D10S583, methods for detecting the presence or absence in a Subject of or about 0.1 cM around and including D10S583. In yet a polymorphism, or combination or haplotype of polymor further embodiments of the method, each of the two or more phisms, associated with Alzheimer's disease, in which the polymorphisms may be located in a region of chromosome asSociation between the polymorphism, or combination or 10 about 30 Mbaround and including D10S583, or about 28 haplotype, and Alzheimer's disease is Such that it yields a Mb around and including D10S583, or about 20 Mb around positive result (i.e., a result indicative of association or and including D10S583, or about 15 Mb around and includ linkage disequilibrium) in a family-based test for associa ing D10S583, or about 10 Mb around and including tion. The positive result may be a P value less than or equal D10S583. Each of the two or more polymorphisms may be to 0.05 or a value less than 0.05. located in a region of chromosome 10 about 5 Mb around 0073. Further provided are embodiments of any of the and including D10S583, or about 2.5 Mbaround and includ methods for detecting the presence or absence in a Subject of ing D10S583, or about 1 Mbaround and including D10S583 a polymorphism, or combination or haplotype of polymor or about 500 kb around and including D10S583, or about phisms, associated with Alzheimer's disease, in which the 200 kb around and including D10S583, or about 100 kb polymorphism, or combination or haplotype, can be asso around and including D10S583, or about 50 kb around and ciated with an AD gene that accounts for greater than 1% of including D10S583, or about 40 kb around and including the attributtable risk of AD; or the polymorphism may be D10S583, or about 20 kb around and including D10S583, or asSociated with an AD gene that accounts for greater than about 10 kb around and including D10S583, or about 5 kb 2% of the attributtable risk for AD; or the the polymorphism around and including D10S583, or about 1 kb around and may be associated with an AD gene that accounts for greater including D10S583. than 5% of the attributtable risk for AD; or the polymor phism may be associated with an AD gene that accounts for 0077. In other embodiments of the method for detecting greater than 10% of the attributtable risk for AD; or the the presence or absence in a Subject of polymorphisms polymorphism may be associated with an AD gene that asSociated with Alzheimer's disease, each of the two or more accounts for greater than 25% of the attributtable risk for polymorphisms may be located in one or more of the AD. following regions of chromosome 10: 0.074 Also provided are embodiments of any of the 0078 (a) the region extending about 50 cM, or about methods for detecting the presence or absence in a Subject of 45 cM, or about 33 cM, or about 30 cM, or about 25 a polymorphism, or combination or haplotype of polymor cM, or about 21 cM, or about 15 cM, or about 10 cM, phisms, associated with Alzheimer's disease, in which the or about 5 cM, or about 4 cM, or about 3 cM, or polymorphism, or at least one of the polymorphisms of the about 2.7 cM, or about 2.5 cM, or about 2 cM, or combination, is located in one of the following genes: about 1.5 cM, or about 1 cM, or about 0.5 cM, or PLAU, TLL2, PSAP, PSD, KIAAO904, NFKB2, PPP3CB, about 0.1 cM proximal (centromeric) from and CH25H and FERIL3. including marker D10S583, 0079 (b) the region extending about 60 cM, or 0075 Also provided is a method for detecting the pres about 55 cM, or about 50 cM, or about 45 cM, or ence or absence in a Subject of polymorphisms associated about 40 cM, or about 35 cM, or about 30 cM, or with Alzheimer's disease that includes a step of analyzing about 25 cM, or about 20 cM, or about 15 cM, or chromosome 10 of the subject for two or more polymor about 13 cM, or about 12 cM, or about 9 cM, or phisms associated with Alzheimer's disease, wherein at least about 5 cM, or about 2.5 cM, or about 1 cM, or about two of the polymorphisms are associated with different AD 0.5 cM or about 0.1 cM distal (telomeric) from and DNA segments or wherein at least one polymorphism is including marker D10S583, D10S583. In particular embodiments of these methods, the two or more polymorphisms may be located on chromosome 0080 (c) the region extending from and including 10q. For example, each of the two or more polymorphisms marker D10S583 to the terminus of the q arm of may be located on chromosome 10q22, 10q23, 10q24, chromosome 10, 10q25 or 10q26. Each of the two or more polymorphisms 0081 (d) the region extending from and including may be located on chromosome 10q22, 10q23 or 10q24 or marker D10S583 to the centromere of chromosome on chromosome 10q23, 10q24 or 10q25. 10, 0.076. In particular embodiments of the method for 0082 (e) the region extending about 62 Mb, about detecting the presence or absence in a Subject of polymor 55 Mb, or about 50 Mb, or about 32 Mb, or about 28 phisms associated with Alzheimer's disease, each of the two Mb, or about 25 Mb, or about 20 Mb, or about 15 or more polymorphisms may be located in a region of Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, chromosome 10 about 30 cM around and including or about 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or D10S583, or about 20 cM around and including D10S583, about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or or about 15 cM around and including D10S583, or about 12 about 0.01 Mb, or about 1 kb proximal from and cM around and including D10S583, or about 10 cM around including marker D10S583, US 2003/017O678A1 Sep. 11, 2003
0083 (f) the region extending about 45 Mb, or about 0095 Also provided are embodiments of any of the 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 methods for detecting the presence or absence in a Subject of Mb, or about 20 Mb, or about 16 Mb, or about 14 polymorphisms associated with Alzheimer's disease, in Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, which the association between each polymorphism and or about 4 Mb, or about 3 Mb, or about 2 Mb, or Alzheimer's disease is Such that it yields a positive result about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or (i.e., a result indicative of association or linkage disequilib about 0.05 Mb, or about 0.01 Mb, or about 1kb distal rium) in a family-based test for association. The positive from and including marker D10S583, result may be a P value less than or equal to 0.05 or a value less than 0.05. 0084) (g) the region between D10S564 and D10S583, inclusive, 0096. Further provided are embodiments of any of the methods for detecting the presence or absence in a Subject of 0085 (h) the region between D1OS583 and polymorphisms associated with Alzheimer's disease, in D10S1710, inclusive, which at least one of the polymorphisms can be associated 0.086 (i) the region between D1OS583 and with an AD gene that accounts for greater than 1% of the D10S566, inclusive, attributtable risk of AD; or the polymorphism may be asSociated with an AD gene that accounts for greater than 0.087 (i) the region between D1OS583 and 2% of the attributtable risk for AD; or the the polymorphism D10S1671, inclusive and may be associated with an AD gene that accounts for greater than 5% of the attributtable risk for AD; or the polymor 0088 (k) the region between D1OS583 and phism may be associated with an AD gene that accounts for D10S1741, inclusive. greater than 10% of the attributtable risk for AD; or the 0089. In yet further embodiments of the method for polymorphism may be associated with an AD gene that detecting the presence or absence in a Subject of polymor accounts for greater than 25% of the attributtable risk for phisms associated with Alzheimer's disease, each of the two AD. or more polymorphisms may be located in one or more of 0097 Also provided are embodiments of any of the the following regions of chromosome 10: methods for detecting the presence or absence in a Subject of polymorphisms associated with Alzheimer's disease, in 0090 (a) the region extending about 5 cM, or about which at least one of the polymorphisms is located in one of 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 the following genes: PLAU, TLL2, PSAP, PSD, KIAA0904, cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, NFKB2, PPP3CB, CH25H and FERIL3. In such methods, or about 0.1 cM proximal (centromeric) from and one of the polymorphisms may be located in one of the including marker D10S583, identified genes and another of the polymorphisms may be 0091 (b) the region extending about 5 cM, or about located in another of the identified genes. 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 0098. A method of determining the level of risk for cM distal (telomeric) from and including marker Alzheimer's disease in a Subject is also provided. Such D10S583, methods include a step of analyzing chromosome 10 of the 0092 (c) the region extending about 10 Mb, or Subject for the presence or absence of one or more poly about 5 Mb, or about 2 Mb, or about 1 Mb, or about morphisms associated with Alzheimer's disease. Methods of 500 kb, or about 300 kb, or about 200 kb, or about determining the level of risk for Alzheimer's disease in a 100 kb, or about 80 kb, or about 50 kb, or about 10 Subject may include a step of analyzing chromosome 10 of kb, or about 5 kb, or about 1 kb proximal from and a Subject for the presence or absence of a combination or including marker D10S583; and haplotype of polymorphisms associated with AD. In any of these methods, the one or more polymorphisms or the 0.093 (d) the region extending about 10 Mb, or combination of polymorphisms can be Such that they are about 8 Mb, or about 5 Mb, or about 4 Mb, or about indicative of an increased or decreased risk for Alzheimer's 3 Mb, or about 2 Mb, or about 1 Mb, or about 500 disease. In particular methods of determining the level of kb, or about 300 kb, or about 200 kb, or about 100 risk for AD in a Subject, the association between the poly kb, or about 50 kb, or about 10 kb, or about 5 kb, or morphism or combination of polymorphisms and Alzhe about 1 kb distal from and including marker imer's disease yields a result in a family-based test for D1OS583. asSociation that is indicative of linkage disequilibrium between the polymorphism(s) and an allele associated with 0094) Further provided are embodiments of any of the Alzheimer's disease. The association between the polymor methods for detecting the presence or absence in a Subject of phism or combination of polymorphisms and Alzheimer's polymorphisms associated with Alzheimer's disease, disease may yield a positive result in a family-based test for wherein at least one of the polymorphisms may be associ asSociation. The positive result may be a P value less than or ated with AD with onset ages of greater than or equal to equal to 0.05. In particular embodiments, the positive result about 50 years, or greater than or equal to about 60 years, or is a P value less than 0.05. greater than or equal to about 65 years. The method may also be Such that at least one of the polymorphisms is associated 0099. In further embodiments of these methods, the poly with an AD DNA segment that has an effect size comparable morphism or combination of polymorphisms may be asso to or greater than the effect size of APOE. The method may ciated with unaffected members of a family having members further be Such that at least one of the polymorphisms is affected with Alzheimer's disease. The polymorphism or located within an AD DNA segment. combination or polymorphisms may be indicative of a US 2003/017O678A1 Sep. 11, 2003
decreased risk for Alzheimer's disease. The association 0105. In yet further embodiments of the methods, each of between the polymorphism or combination and unaffected the one or more polymorphisms, or each of the polymor members of a family having members affected with Alzhe phisms in the combination of polymorphisms associated imer's disease yields a positive result in a family-based test with AD, may be located in a region of chromosome 10 for association. The positive result may be a P value less than about 30 Mb around and including D10S583, or about 28 or equal to 0.05. In particular embodiments, the positive Mb around and including D10S583, or about 20 Mb around result is a P value less than 0.05. and including D10S583, or about 15 Mb around and includ ing D10S583, or about 10 Mb around and including 0100. In yet further embodiments of the method, the D10S583. In particular embodiments of the methods, each polymorphism or combination of polymorphisms may be of the one or more polymorphisms, or each of the polymor under-represented in cases of a case-control Study. In par phisms in the combination of polymorphisms associated ticular embodiments of the method of determining the level with AD, may be located in a region of chromosome 10 of risk for Alzheimer's disease in a Subject, the polymor about 5 Mb around and including D10S583, or about 2.5 Mb phism is an allele of D10S583 and the presence of the allele around and including D10S583, or about 1 Mb around and of D10S583 is indicative of a decreased risk for AD. The including D10S583 or about 500 kb around and including allele of D10S583 may be about 210 bp. The allele of D10S583, or about 200 kb around and including D10S583, D10S583 may be 209 bp or 211 bp. or about 100 kb around and including D10S583, or about 50 0101. In other embodiments of the method of determin kb around and including D10S583, or about 40 kb around ing the level of risk for Alzheimer's disease in a Subject, the and including D10S583, or about 20 kb around and includ polymorphism or combination of polymorphisms may be ing D10S583, or about 10 kb around and including asSociated with affected members of a family having mem D10S583, or about 5 kb around and including D10S583, or bers affected with Alzheimer's disease and is indicative of an about 1 kb around and including D10S583. increased risk for Alzheimer's disease. The association between the polymorphism or combination of polymor 0106. In particular embodiments of the method of deter phisms and affected members of a family having members mining the level of risk for AD in a Subject, the one or more affected with Alzheimer's disease may yield a positive result polymorphisms, or each of the polymorphisms in the com in a family-based test for association. The positive result bination of polymorphisms associated with AD, may be may be a P value less than or equal to 0.05. In particular, the located in one or more of the following regions of chromo positive result is a P value less than 0.05. Some 10: 0107 (a) the region extending about 50 cM, or about 0102) In yet further embodiments of the method, the 45 cM, or about 33 cM, or about 30 cM, or about 25 polymorphism or combination of polymorphisms is over cM, or about 21 cM, or about 15 cM, or about 10 cM, represented in cases of a case-control Study. or about 5 cM, or about 4 cM, or about 3 cM, or 0103) In particular embodiments of the methods of deter about 2.7 cM, or about 2.5 cM, or about 2 cM, or mining the level of risk for AD in a Subject, the one or more about 1.5 cM, or about 1 cM, or about 0.5 cM, or polymorphisms or combination of polymorphisms associ about 0.1 cM proximal (centromeric) from and ated with AD is located on chromosome 10q. In further including marker D10S583, embodiments, each of the one or more polymorphisms, or 0108 (b) the region extending about 60 cM, or each of the polymorphisms of the combination of polymor about 55 cM, or about 50 cM, or about 45 cM, or phisms, is located on chromosome 10q22, 10q23, 10q24, about 40 cM, or about 35 cM, or about 30 cM, or 10q25 or 10q26. In yet further embodiments of the methods, about 25 cM, or about 20 cM, or about 15 cM, or each of the one or more polymorphisms, or each of the about 13 cM, or about 12 cM, or about 9 cM, or polymorphisms of the combination of polymorphisms, is about 5 cM, or about 2.5 cM, or about 1 cM, or about located on chromosome 10q22, 10q23 or 10q24 or on 0.5 cM or about 0.1 cM distal (telomeric) from and chromosome 10q23, 10q24 or 10q25. including marker D10S583, 0104. In particular embodiments of the method of deter 0109 (c) the region extending from and including mining the level of risk for AD in a Subject, the one or more polymorphisms, or each of the polymorphisms in the com marker D10S583 to the terminus of the q arm of bination of polymorphisms associated with AD, may be chromosome 10, located in a region of chromosome 10 about 30 cM around 0110 (d) the region extending from and including and including D10S583, or about 20 cM around and includ marker D10S583 to the centromere of chromosome ing D10S583, or about 15 cM around and including 10, D10S583, or about 12 cM around and including D10S583, or about 10 cM around and including D10S583. In further 0111 (e) the region extending about 62 Mb, about embodiments of the methods, each of the one or more 55 Mb, or about 50 Mb, or about 32 Mb, or about 28 polymorphisms, or each of the polymorphisms in the com Mb, or about 25 Mb, or about 20 Mb, or about 15 bination of polymorphisms associated with AD, may be Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, located in a region of chromosome 10 about 5 cM around or about 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or and including D10S583, or about 4 cMaround and including about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or D10S583, or about 3 cM around and including D10S583, or about 0.01 Mb, or about 1 kb proximal from and about 2 cM around D10S583, or about 1 cM around and including marker D10S583, including D10S583, or about 0.5 cM around and including 0112 (f) the region extending about 45 Mb, or about D10S583, or about 0.25 cMaround and including D10S583, 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 or about 0.1 cM around and including D10S583. Mb, or about 20 Mb, or about 16 Mb, or about 14 US 2003/017O678A1 Sep. 11, 2003
Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, in which at least one of the polymorphisms, or the combi or about 4 Mb, or about 3 Mb, or about 2 Mb, or nation of polymorphisms, can be associated with an AD about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or gene that accounts for greater than 1%, greater than 2%, about 0.05 Mb, or about 0.01 Mb, or about 1kb distal greater than 5%, greater than 10% or greater than 25% of the from and including marker D10S583, attributtable risk for AD. 0113 (g) the region between D10S564 and 0.125 Also provided are embodiments of any of the D10S583, inclusive, methods for determining the level of risk for AD in a subject in which at least one of the polymorphisms, or at least one 0114 (h) the region between D1OS583 and of polymorphisms in the combination of polymorphisms, is D10S1710, inclusive, located in one of the following genes: PLAU, TLL2, PSAP, 0115 (i) the region between D1OS583 and PSD, KIAAO904, NFKB2, PPP3CB, CH25H and FERIL3. D10S566, inclusive, In Such methods, one of the polymorphisms may be located in one of the identified genes and another of the polymor 0116 (i) the region between D1OS583 and phisms may be located in another of the identified genes. D10S1671, inclusive and 0.126 Further provided is a method for indicating a 0117 (k) the region between D1OS583 and predisposition to or the occurrence of Alzheimer's disease in D10S1741, inclusive. a Subject. The method includes a step of detecting in nucleic 0118. In further particular embodiments of the method of acid obtained from the Subject the presence or absence of a determining the level of risk for AD in a subject, the one or polymorphism on chromosome 10 associated with Alzhe more polymorphisms, or each of the polymorphisms in the imer's disease. Methods for indicating a predisposition to or combination of polymorphisms associated with AD, may be the occurrence of Alzheimer's disease in a Subject may located in one or more of the following regions of chromo include a step of analyzing chromosome 10 of a Subject for Some 10: the presence or absence of a combination or haplotype of polymorphisms associated with AD. In any of these meth 0119 (a) the region extending about 5 cM, or about ods, the presence of the polymorphism or the combination of 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 polymorphisms can be indicative of a predisposition to cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, Alzheimer's disease. In particular embodiments of these or about 0.1 cM proximal (centromeric) from and methods, the polymorphism or combination of polymor including marker D10S583, phisms is on human chromosome 10. 0120 (b) the region extending about 5 cM, or about 0127. Also provided is a method for confirming a phe 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 notypic diagnosis of Alzheimer's disease in a Subject. The cM distal (telomeric) from and including marker method includes a step of detecting in nucleic acid obtained D10S583, from the Subject the presence or absence of a polymorphism on chromosome 10 associated with Alzheimer's disease, 0121 (c) the region extending about 10 Mb, or wherein the presence of the polymorphism confirms a phe about 5 Mb, or about 2 Mb, or about 1 Mb, or about notypic diagnosis of Alzheimer's disease. Methods for con 500 kb, or about 300 kb, or about 200 kb, or about firming a phenotypic diagnosis of Alzheimer's disease in a 100 kb, or about 80 kb, or about 50 kb, or about 10 Subject may include a step of detecting in nucleic acid kb, or about 5 kb, or about 1 kb proximal from and obtained from the Subject the presence or absence of a including marker D10S583; and combination or haplotype of polymorphisms on chromo 0122) (d) the region extending about 10 Mb, or Some 10 associated with AD. In particular embodiments of about 8 Mb, or about 5 Mb, or about 4 Mb, or about these methods, the polymorphism or combination of poly 3 Mb, or about 2 Mb, or about 1 Mb, or about 500 morphisms is on human chromosome 10. kb, or about 300 kb, or about 200 kb, or about 100 0128. A method for predicting a response of a subject to kb, or about 50 kb, or about 10 kb, or about 5 kb, or a drug used to treat Alzheimer's disease is also provided. about 1 kb distal from and including marker The method is performed by detecting the presence or D1OS583. absence of at least one polymorphism on chromosome 10 0123. Further provided are embodiments of any of the asSociated with Alzheimer's disease, or a combination or methods for determining the level of risk for AD in a subject haplotype of polymorphisms on chromosome 10 associated wherein at least one of the polymorphisms, or the combi with AD, wherein the presence of the polymorphism or nation of polymorphisms, is associated with AD with onset combination is indicative of an increased or decreased ages of greater than or equal to about 50 years, or greater likelihood that the drug treatment for Alzheimer's disease than or equal to about 60 years, or greater than or equal to will be effective. In particular embodiments of these meth about 65 years. The method may also be such that at least ods, the polymorphism or combination of polymorphisms is one of the polymorphisms, or the combination of polymor on human chromosome 10. phisms, is associated with an AD DNA segment that has an 0129. Also provided is a method of treating a subject effect size comparable to or greater than the effect size of manifesting an Alzheimer's disease phenotype. The method APOE. The method may further be such that at least one of is performed by detecting in nucleic acid obtained from the the polymorphisms, or the combination of polymorphisms, Subject the presence of a polymorphism on chromosome 10 is located within an AD DNA segment. asSociated with Alzheimer's disease, or a combination or 0.124. Also provided are embodiments of any of the haplotype of polymorphisms on chromosome 10 associated methods for determining the level of risk for AD in a subject with AD, wherein the presence of the polymorphism or US 2003/017O678A1 Sep. 11, 2003
combination of polymorphisms is indicative of the occur about 0.05 Mb, or about 0.01 Mb, or about 1kb distal rence of Alzheimer's disease in a Subject; and Selecting and from and including marker D10S583, administering a treatment that is effective for treating Alzhe 0138 (g) the region between D10S564 and imer's disease. D10S583, inclusive, 0130. In any of the methods of (1) indicating a predis 0139) (h) the region between D10S583 and position to or the occurrence of Alzheimer's disease in a Subject, (2) confirming a phenotypic diagnosis of Alzhe D10S1710, inclusive, imer's disease in a Subject, (3) predicting a response of a 0140) (i) the region between D10S583 and Subject to a drug used to treat Alzheimer's disease and (4) D10S566, inclusive, treating a Subject manifesting an Alzheimer's disease phe 0141 (j) the region between D10S583 and notype, the polymorphism or combination or haplotype of D10S1671, inclusive and polymorphisms may be located on chromosome 10q. In particular embodiments, the polymorphism or combination 0142) (k) the region between D10S583 and or haplotype of polymorphisms may be located on chromo D10S1741, inclusive. Some 10q22, 10q23, 10q24, 10q25 or 10q26. In further 0143. In any of these methods, the polymorphism, or embodiments the polymorphism, or each polymorphism in each polymorphism in the combination or haplotype of the combination or haplotype of polymorphisms, may be polymorphisms, may be located in one or more of the located on chromosome 10q22, 10q23 or 10q24 or on following regions of chromosome 10: chromosome 10q23, 10q24 or 10q25. 0144 (a) the region extending about 5 cM, or about 0131. In any of these methods, the polymorphism, or 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 each polymorphism in the combination or haplotype of cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, polymorphisms, may be located in one or more of the or about 0.1 cM proximal (centromeric) from and following regions of chromosome 10: including marker D10S583, 0132 (a) the region extending about 50 cM, or about 0145 (b) the region extending about 5 cM, or about 45 cM, or about 33 cM, or about 30 cM, or about 25 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 cM, or about 21 cM, or about 15 cM, or about 10 cM, cM distal (telomeric) from and including marker or about 5 cM, or about 4 cM, or about 3 cM, or D10S583, about 2.7 cM, or about 2.5 cM, or about 2 cM, or 0146) (c) the region extending about 10 Mb, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about about 0.1 cM proximal (centromeric) from and 500 kb, or about 300 kb, or about 200 kb, or about including marker D10S583, 100 kb, or about 80 kb, or about 50 kb, or about 10 0133) (b) the region extending about 60 cM, or kb, or about 5 kb, or about 1 kb proximal from and about 55 cM, or about 50 cM, or about 45 cM, or including marker D10S583; and about 40 cM, or about 35 cM, or about 30 cM, or 0147 (d) the region extending about 10 Mb, or about 25 cM, or about 20 cM, or about 15 cM, or about 8 Mb, or about 5 Mb, or about 4 Mb, or about about 13 cM, or about 12 cM, or about 9 cM, or 3 Mb, or about 2 Mb, or about 1 Mb, or about 500 about 5 cM, or about 2.5 cM, or about 1 cM, or about kb, or about 300 kb, or about 200 kb, or about 100 0.5 cM or about 0.1 cM distal (telomeric) from and kb, or about 50 kb, or about 10 kb, or about 5 kb, or including marker D10S583, about 1 kb distal from and including marker 0134) (c) the region extending from and including D1OS583. marker D10S583 to the terminus of the q arm of 0.148. These methods can also be practiced where the chromosome 10, polymorphism, or each polymorphism in the combination of polymorphisms, may be located in a region of chromosome 0135 (d) the region extending from and including 10 about 30 cMaround and including D10S583, or about 20 marker D10S583 to the centromere of chromosome cM around and including D10S583, or about 15 cM around 10, and including D10S583, or about 12 cM around and includ 0136 (e) the region extending about 62 Mb, about ing D10S583, or about 10 cM around and including 55 Mb, or about 50 Mb, or about 32 Mb, or about 28 D1OS583. Mb, or about 25 Mb, or about 20 Mb, or about 15 0149. In further embodiments of any of these methods, Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, the polymorphism, or each polymorphism in the combina or about 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or tion of polymorphisms, may be located in a region of about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or chromosome 10 about 5 cM around and including D10S583, about 0.01 Mb, or about 1 kb proximal from and or about 4 cM around and including D10S583, or about 3 cM including marker D10S583, around and including D10S583, or about 2 cM around 0137 (f) the region extending about 45 Mb, or about D10S583, or about 1 cM around and including D10S583, or 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 about 0.5 cM around and including D10S583, or about 0.25 Mb, or about 20 Mb, or about 16 Mb, or about 14 cM around and including D10S583, or about 0.1 cMaround Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, and including D10S583. or about 4 Mb, or about 3 Mb, or about 2 Mb, or 0150. In yet further embodiments of any of these meth about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or ods, the polymorphism, or each polymorphism in the com US 2003/017O678A1 Sep. 11, 2003 bination of polymorphisms, may be located in a region of including D10S583, or about 4 cM around and including chromosome 10 about 30 Mb around and including D10S583, or about 3 cM around and including D10S583, or D10S583, or about 28 Mb around and including D10S583, about 2 cM around D10S583, or about 1 cM around and or about 20 Mb around and including D10S583, or about 15 including D10S583, or about 0.5 cM around and including Mb around and including D10S583, or about 10 Mb around D10S583, or about 0.25 cMaround and including D10S583, and including D10S583. or about 0.1 cM around and including D10S583. 0151. The polymorphism, or each polymorphism in the O157. In other embodiments of these methods, the gene is combination of polymorphisms, may also be located in a located in a region of chromosome 10 about 30 Mb around region of chromosome 10 about 5 Mb around and including and including D10S583, or about 28 Mb around and includ D10S583, or about 2.5 Mb around and including D10S583, ing D10S583, or about 20 Mb around and including or about 1 Mb around and including D10S583 or about 500 D10S583, or about 15 Mb around and including D10S583, kb around and including D10S583, or about 200 kb around or about 10 Mb around and including D10S583. In further and including D10S583, or about 100 kb around and includ embodiments of the method, the gene is located in a region ing D10S583, or about 50 kb around and including of chromosome 10 about 5 Mb around and including D10S583, or about 40 kb around and including D10S583, or D10S583, or about 2.5 Mb around and including D10S583, about 20 kb around and including D10S583, or about 10 kb or about 1 Mb around and including D10S583 or about 500 around and including D10S583, or about 5 kb around and kb around and including D10S583, or about 200 kb around including D10S583, or about 1 kb around and including and including D10S583, or about 100 kb around and includ D1OS583. ing D10S583, or about 50 kb around and including D10S583, or about 40 kb around and including D10S583, or 0152 Also provided are embodiments of any of the about 20 kb around and including D10S583, or about 10 kb methods in which the association between the polymor around and including D10S583, or about 5 kb around and phism, or a combination of polymorphisms, and Alzhe including D10S583, or about 1 kb around and including imer's disease is Such that it yields a positive result (i.e., a D1OS583. result indicative of association or linkage disequilibrium) in a family-based test for association. The positive result may 0158. In particular embodiments of the method, the gene be a P value less than or equal to 0.05 or a value less than is located in one or more of the following regions of O.05. chromosome 10: (a) the region extending about 50 cM, or about 45 cM, or about 33 cM, or about 30 cM, or about 25 0153. Also provided are embodiments of any of the cM, or about 21 cM, or about 15 cM, or about 10 cM, or methods in which at the polymorphism, or the combination about 5 cM, or about 4 cM, or about 3 cM, or about 2.7 cM, of polymorphisms, can be associated with an AD gene that or about 2.5 cM, or about 2 cM, or about 1.5 cM, or about accounts for greater than 1%, greater than 2%, greater than 1 cM, or about 0.5 cM, or about 0.1 cM proximal (centro 5%, greater than 10% or greater than 25% of the attributtable meric) from and including marker D10S583, risk for AD. 0159) (b) the region extending about 60 cM, or 0154 Also provided are embodiments of any of the about 55 cM, or about 50 cM, or about 45 cM, or methods in which at least one polymorphism, or at least one about 40 cM, or about 35 cM, or about 30 cM, or of the polymorphisms in a combination of polymorphisms, about 25 cM, or about 20 cM, or about 15 cM, or is located in one of the following genes: PLAU, TLL2, about 13 cM, or about 12 cM, or about 9 cM, or PSAP, PSD, KIAAO904, NFKB2, PPP3CB, CH25H and about 5 cM, or about 2.5 cM, or about 1 cM, or about FERIL3. In such methods, one of the polymorphisms may 0.5 cM or about 0.1 cM distal (telomeric) from and be located in one of the identified genes and another of the including marker D10S583, polymorphisms may be located in another of the identified geneS. 0160 (c) the region extending from and including marker D10S583 to the terminus of the q arm of 0.155. A method for identifying a gene as a candidate chromosome 10, Alzheimer's disease gene is also provided. The method is performed by Selecting a gene on chromosome 10 that is or 0161 (d) the region extending from and including encodes a product that has one or more properties which marker D10S583 to the centromere of chromosome relate to one or more phenomena in neurodegenerative 10, disease and thereby identifying a candidate Alzheimer's 0162 (e) the region extending about 62 Mb, about disease gene. In particular embodiments, the gene is on 55 Mb, or about 50 Mb, or about 32 Mb, or about 28 human chromosome 10. Mb, or about 25 Mb, or about 20 Mb, or about 15 0156 The gene can be located, for example, on chromo Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, Some 10q. In particular embodiments, the gene can be or about 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or located on chromosome 10q22, 10q23, 10q24, 10q25 or about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or 10q26. In further embodiments of the method, the gene may about 0.01 Mb, or about 1 kb proximal from and be located on chromosome 10q22, 10q23 or 10q24, or on including marker D10S583, chromosome 10q23, 10q24 or 10q25, or located in a region 0163 (f) the region extending about 45 Mb, or about of chromosome 10 about 30 cM around and including 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 D10S583, or about 20 cM around and including D10S583, Mb, or about 20 Mb, or about 16 Mb, or about 14 or about 15 cM around and including D10S583, or about 12 Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, cM around and including D10S583, or about 10 cM around or about 4 Mb, or about 3 Mb, or about 2 Mb, or and including D10S583, located about 5 cM around and about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or US 2003/017O678A1 Sep. 11, 2003 12
about 0.05 Mb, or about 0.01 Mb, or about 1kb distal 10, or a combination of polymorphisms on chromosome 10, from and including marker D10S583, for association with Alzheimer's disease. In particular embodiments of the method, the polymorphism is on human 0164 (g) the region between D10S564 and chromosome 10. D10S583, inclusive, 0176). In further embodiments, the polymorphism, or 0.165 (h) the region between D10S583 and each of the polymorphisms of the combination of polymor D10S1710, inclusive, phisms, may be located on chromosome 10q22, 10q23, 0166 (i) the region between D10S583 and 10q24, 10q25 or 10q26. In yet further embodiments of the D10S566, inclusive, methods, the polymorphism, or each of the polymorphisms of the combination of polymorphisms, may be located on 0167 (i) the region between D10S583 and chromosome 10q22, 10q23 or 10q24 or on chromosome D10S1671, inclusive and 10q23, 10q24 or 10q25. 0168 (k) the region between D10S583 and 0177. In particular embodiments of the method for iden D10S1741, inclusive. tifying a polymorphism, or combination or haplotype of 0169. In yet further embodiments of the methods, the polymorphisms, associated with Alzheimer's disease, the gene is located in one or more of the following regions of polymorphism, or each of the polymorphisms in the com chromosome 10: bination of polymorphisms, may be located in a region of chromosome 10 about 30 cM around and including 0170 (a) the region extending about 5 cM, or about D10S583, or about 20 cM around and including D10S583, 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 or about 15 cM around and including D10S583, or about cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, 12cM around and including D10S583, or about 10 cM or about 0.1 cM proximal (centromeric) from and around and including D10S583. In further embodiments of including marker D10S583, the methods, the polymorphism, or each of the polymor 0171 (b) the region extending about 5 cM, or about phisms in the combination of polymorphisms, may be 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 located in a region of chromosome 10 about 5 cM around cM distal (telomeric) from and including marker and including D10S583, or about 4 cMaround and including D10S583, D10S583, or about 3 cM around and including D10S583, or about 2 cM around D10S583, or about 1 cM around and 0172 (c) the region extending about 10 Mb, or including D10S583, or about 0.5 cM around and including about 5 Mb, or about 2 Mb, or about 1 Mb, or about D10S583, or about 0.25 cMaround and including D10S583, 500 kb, or about 300 kb, or about 200 kb, or about or about 0.1 cM around and including D10S583. 100 kb, or about 80 kb, or about 50 kb, or about 10 0.178 In yet further embodiments of the methods, the kb, or about 5 kb, or about 1 kb proximal from and polymorphism, or each of the polymorphisms in the com including marker D10S583; and bination of polymorphisms, may be located in a region of 0173 (d) the region extending about 10 Mb, or chromosome 10 about 30 Mb around and including about 8 Mb, or about 5 Mb, or about 4 Mb, or about D10S583, or about 28 Mb around and including D10S583, 3 Mb, or about 2 Mb, or about 1 Mb, or about 500 or about 20 Mb around and including D10S583, or about 15 kb, or about 300 kb, or about 200 kb, or about 100 Mb around and including D10S583, or about 10 Mb around kb, or about 50 kb, or about 10 kb, or about 5 kb, or and including D10S583. In particular embodiments of the about 1 kb distal from and including marker methods, the polymorphism, or each of the polymorphisms D1OS583. in the combination of polymorphisms, may be located in a 0.174. In these methods the one or more phenomena in region of chromosome 10 about 5 Mb around and including neurodegenerative disease can be selected from the group D10S583, or about 2.5 Mb around and including D10S583, consisting of Senile plaques and components thereof, neu or about 1 Mb around and including D10S583 or about 500 ritic plaques, and components thereof, neurofibrillary kb around and including D10S583, or about 200 kb around tangles, tau protein, abnormally phosphorylated tau protein, and including D10S583, or about 100 kb around and includ amyloid precursor protein (APP), processing of APP, AB42 ing D10S583, or about 50 kb around and including protein, Cl-, 3- and Y-Secretases, presenilin proteins, amyloid D10S583, or about 40 kb around and including D10S583, or deposition, Lewy bodies, prions, apoptosis, caspases, about 20 kb around and including D10S583, or about 10 kb inflammation, excitotoxicity, excitotoxins, excessive nitric around and including D10S583, or about 5 kb around and oxide production, oxidative StreSS, proteases, protease including D10S583, or about 1 kb around and including inhibitors, neurotrophic factors, cytokines, calcium-depen D1OS583. dent processes, Signal transduction, altered ionic homeosta 0179. In particular embodiments of the method for iden sis, altered calcium homeostasis, Synaptic molecules, adhe tifying a polymorphism, or combination or haplotype of Sion molecules, molecules involved in membrane turnover, polymorphisms, associated with Alzheimer's disease, the cholesterol and lipid metabolism and transport, cytoskeletal polymorphism, or each of the polymorphisms in the com molecules, neuronal proteins, brain proteins, and cell necro bination of polymorphisms, may be located in one or more SS. of the following regions of chromosome 10: 0175 Also provided is a method for identifying a poly 0180 (a) the region extending about 50 cM, or about morphism, or combination or haplotype of polymorphisms, 45 cM, or about 33 cM, or about 30 cM, or about 25 associated with Alzheimer's disease. This method may be cM, or about 21 cM, or about 15 cM, or about 10 cM, performed by analyzing a polymorphism on chromosome or about 5 cM, or about 4 cM, or about 3 cM, or US 2003/017O678A1 Sep. 11, 2003
about 2.7 cM, or about 2.5 cM, or about 2 cM, or 0194 (c) the region extending about 10 Mb, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about about 0.1 cM proximal (centromeric) from and 500 kb, or about 300 kb, or about 200 kb, or about including marker D10S583, 100 kb, or about 80 kb, or about 50 kb, or about 10 0181 (b) the region extending about 60 cM, or kb, or about 5 kb, or about 1 kb proximal from and about 55 cM, or about 50 cM, or about 45 cM, or including marker D10S583; and about 40 cM, or about 35 cM, or about 30 cM, or 0195 (d) the region extending about 10 Mb, or about 25 cM, or about 20 cM, or about 15 cM, or about 8 Mb, or about 5 Mb, or about 4 Mb, or about about 13 cM, or about 12 cM, or about 9 cM, or 3 Mb, or about 2 Mb, or about 1 Mb, or about 500 about 5 cM, or about 2.5 cM, or about 1 cM, or about kb, or about 300 kb, or about 200 kb, or about 100 0.5 cM or about 0.1 cM distal (telomeric) from and kb, or about 50 kb, or about 10 kb, or about 5 kb, or including marker D10S583, about 1 kb distal from and including marker 0182 (c) the region extending from and including D1OS583. marker D10S583 to the terminus of the q arm of 0196. Further provided are embodiments of any of the chromosome 10, methods for identifying a polymorphism, or combination or 0183 (d) the region extending from and including haplotype of polymorphisms, associated with Alzheimer's marker D10S583 to the centromere of chromosome disease, wherein the polymorphism or combination of poly 10, morphisms on chromosome 10 are analyzed for association with Alzheimer's disease with with onset ages of greater 0184 (e) the region extending about 62 Mb, about than or equal to about 50 years, or greater than or equal to 55 Mb, or about 50 Mb, or about 32 Mb, or about 28 about 60 years, or greater than or equal to about 65 years. Mb, or about 25 Mb, or about 20 Mb, or about 15 Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, 0.197 Also provided are embodiments of any of the or about 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or methods for identifying a polymorphism, or combination or about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or haplotype of polymorphisms, associated with Alzheimer's about 0.01 Mb, or about 1 kb proximal from and disease in which the polymorphism, or at least one of including marker D10S583, polymorphisms in the combination of polymorphisms, is located in one of the following genes: PLAU, TLL2, PSAP, 0185 (f) the region extending about 45 Mb, or about PSD, KIAAO904, NFKB2, PPP3CB, CH25H and FERIL3. 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 In Such methods, one of the polymorphisms may be located Mb, or about 20 Mb, or about 16 Mb, or about 14 in one of the identified genes and another of the polymor Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, phisms may be located in another of the identified genes. or about 4 Mb, or about 3 Mb, or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or 0198 Also provided are the above methods where the about 0.05 Mb, or about 0.01 Mb, or about 1kb distal asSociation between the polymorphism, or the combination from and including marker D10S583, of polymorphsims, and Alzheimer's disease yields a positive 0186 (g) the region between D10S564 and result in a family-based test for association. D10S583, inclusive, 0199 For example, the positive result is a P value less 0187 (h) the region between D1OS583 and than or equal to 0.05, or is a P value less than 0.05. D10S1710, inclusive, 0200 Also provided is a combination containing two or D1OS583 and more oligonucleotides that hybridize to, adjacent to, or to 0188 (i) the region between DNA flanking a DNA segment on chromosome 10, wherein D10S566, inclusive, the DNA segment comprises a polymorphism that is asso 0189 (j) the region between D1OS583 and ciated with AD and at least two of the oligonucleotides D10S1671, inclusive and hybridize to, adjacent to, or to DNA flanking different DNA Segments. In a particular embodiment of the combination, 0.190) (k) the region between D1OS583 and the DNA segment is on chromosome 10q. In further embodi D10S1741, inclusive. ments of the combination, the DNA segment is on chromo 0191 In further particular embodiments of the method, Some 10q22, 10q23, 10q24, 10q25 or 10q26. In yet further the polymorphism, or each of the polymorphisms in the embodiments of the combination, the DNA segment is on combination of polymorphisms, may be located in one or chromosome 10q22, 10q23 or 10q24 or on chromosome more of the following regions of chromosome 10: 10q23, 10q24 or 10q25. 0192 (a) the region extending about 5 cM, or about 0201 In particular embodiments of the combinations, the 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 DNA segment can be located in any of the following regions cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, of chromosome 10: or about 0.1 cM proximal (centromeric) from and including marker D10S583, 0202) (a) the region extending about 50 cM, or about 45 cM, or about 33 cM, or about 30 cM, or about 25 0193 (b) the region extending about 5 cM, or about cM, or about 21 cM, or about 15 cM, or about 10 cM, 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 or about 5 cM, or about 4 cM, or about 3 cM, or cM distal (telomeric) from and including marker about 2.7 cM, or about 2.5 cM, or about 2 cM, or D10S583, about 1.5 cM, or about 1 cM, or about 0.5 cM, or US 2003/017O678A1 Sep. 11, 2003
about 0.1 cM proximal (centromeric) from and 100 kb, or about 80 kb, or about 50 kb, or about 10 including marker D10S583, kb, or about 5 kb, or about 1 kb proximal from and 0203 (b) the region extending about 60 cM, or including marker D10S583; and about 55 cM, or about 50 cM, or about 45 cM, or 0217 (d) the region extending about 10 Mb, or about 40 cM, or about 35 cM, or about 30 cM, or about 8 Mb, or about 5 Mb, or about 4 Mb, or about about 25 cM, or about 20 cM, or about 15 cM, or 3 Mb, or about 2 Mb, or about 1 Mb, or about 500 about 13 cM, or about 12 cM, or about 9 cM, or kb, or about 300 kb, or about 200 kb, or about 100 about 5 cM, or about 2.5 cM, or about 1 cM, or about kb, or about 50 kb, or about 10 kb, or about 5 kb, or 0.5 cM or about 0.1 cM distal (telomeric) from and about 1 kb distal from and including marker including marker D10S583, D1OS583. 0204 (c) the region extending from and including 0218. In the combination, the polymorphism can be, for marker D10S583 to the terminus of the q arm of example, D10S583. chromosome 10, 0219. Also provided is a kit containing two or more 0205 (d) the region extending from and including oligonucleotides that hybridize to, adjacent to, or to DNA marker D10S583 to the centromere of chromosome flanking a DNA segment on chromosome 10 and instruc 10, tions describing procedures for using the oligonucleotide(s) 0206 (e) the region extending about 62 Mb, about in the detection of one or more polymorphisms on chromo 55 Mb, or about 50 Mb, or about 32 Mb, or about 28 Some 10 associated with AD, wherein the DNA segment Mb, or about 25 Mb, or about 20 Mb, or about 15 comprises a polymorphic Site at which a polymorphism that Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, is associated with AD occurs, and at least two of the or about 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or oligonucleotides hybridize to, adjacent to, or to DNA flank about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or ing different DNA segments. about 0.01 Mb, or about 1 kb proximal from and 0220) Further provided is a kit containing two or more including marker D10S583, oligonucleotides that hybridize to, adjacent to, or to DNA flanking a DNA segment on chromosome 10 and instruc 0207 (f) the region extending about 45 Mb, or about tions describing procedures for using the oligonucleotide(s) 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 in methods of detecting the presence or absence of one or Mb, or about 20 Mb, or about 16 Mb, or about 14 more polymorphisms on chormosome 10 associated with Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, AD, wherein the DNA segment comprises a polymorphic or about 4 Mb, or about 3 Mb, or about 2 Mb, or Site at which a polymorphism that is associated with AD about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or occurs, at least two of the oligonucleotides hybridize to, about 0.05 Mb, or about 0.01 Mb, or about 1kb distal adjacent to, or to DNA flanking different DNA segments, from and including marker D10S583, and the procedures provide results on which to base a 0208 (g) the region between D10S564 and determination of a predisposition to or occurrence of AD in D10S583, inclusive, a Subject. 0209 (h) the region between D1OS583 and 0221) Also provided is a kit containing two or more D10S1710, inclusive, oligonucleotides that hybridize to, adjacent to, or to DNA flanking a DNA segment on chromosome 10 and instruc 0210 (i) the region between D1OS583 and tions describing procedures for using the oligonucleotide(s) D10S566, inclusive, in methods of detecting the presence or absence of one or 0211 (i) the region between D1OS583 and more polymorphisms on chormosome 10 associated with D10S1671, inclusive and AD, wherein the DNA segment comprises a polymorphic Site at which a polymorphism that is associated with AD 0212 (k) the region between D1OS583 and occurs, at least two of the oligonucleotides hybridize to, D10S1741, inclusive. adjacent to, or to DNA flanking different DNA segments, 0213. In further embodiments of the combinations, the and the procedures provide results on which to base a DNA segment can be located in any of the following regions prediction of a Subject's response to a treatment for AD. of chromosome 10:: 0222 Further provided is a kit containing two or more oligonucleotides that hybridize to, adjacent to, or to DNA 0214 (a) the region extending about 5 cM, or about flanking a DNA segment on chromosome 10 and instruc 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 tions describing procedures for using the oligonucleotide(s) cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, in methods of detecting the presence or absence of one or or about 0.1 cM proximal (centromeric) from and more polymorphisms on chromosome 10 associated with including marker D10S583, AD, wherein the DNA segment comprises a polymorphic 0215 (b) the region extending about 5 cM, or about Site at which a polymorphism that is associated with AD 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 occurs, at least two of the oligonucleotides hybridize to, cM distal (telomeric) from and including marker adjacent to, or to DNA flanking different DNA segments, D10S583, and the procedures provide results on which to base a 0216 (c) the region extending about 10 Mb, or treatment of AD in a subject. about 5 Mb, or about 2 Mb, or about 1 Mb, or about 0223) Also provided is a kit containing one or more 500 kb, or about 300 kb, or about 200 kb, or about oligonucleotides that hybridize to, adjacent to, or to DNA US 2003/017O678A1 Sep. 11, 2003
flanking a DNA segment on chromosome 10 and one or about 5 cM, or about 2.5 cM, or about 1 cM, or about more control Samples, wherein the DNA segment comprises 0.5 cM or about 0.1 cM distal (telomeric) from and a polymorphic Site at which a polymorphism that is associ including marker D10S583, ated with AD occurs, and the one or more control Samples comprise the DNA segment in which the polymorphic Site 0230 (c) the region extending from and including does not contain a polymorphism associated with AD and/or marker D10S583 to the terminus of the q arm of the DNA segment in which the polymorphic site does chromosome 10, contain a polymorphism associated with AD. 0231 (d) the region extending from and including 0224. In particular embodiments of any of the kits, the marker D10S583 to the centromere of chromosome polymorphism or polymorphisms are located on chromo 10, Some 10q. In further embodiments of any of the kits, the 0232 (e) the region extending about 62 Mb, about polymorphism or polymorphisms are located on chromo 55 Mb, or about 50 Mb, or about 32 Mb, or about 28 Some 10q22, 10q23, 10q24, 10q25 and/or 10q26. In par Mb, or about 25 Mb, or about 20 Mb, or about 15 ticular embodiments of the kits, the polymorphism or poly Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, morphisms are located on chromosome 10q22, 10q23 and/or or about 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or 10q24 or on chromosome 10q23, 10q24 and/or 10q25. about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or 0225. In further embodiments of the kits, the polymor about 0.01 Mb, or about 1 kb proximal from and phism may be located in a region of chromosome 10 about including marker D10S583, 30 cM around and including D10S583, or about 20 cM 0233 (f) the region extending about 45 Mb, or about around and including D10S583, or about 15 cM around and 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 including D10S583, or about 12 cM around and including Mb, or about 20 Mb, or about 16 Mb, or about 14 D10S583, or about 10 cM around and including D10S583. Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, The polymorphism may be located about 5 cM around and or about 4 Mb, or about 3 Mb, or about 2 Mb, or including D10S583, or about 4 cM around and including about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or D10S583, or about 3 cM around and including D10S583, or about 0.05 Mb, or about 0.01 Mb, or about 1kb distal about 2 cM around D10S583, or about 1 cM around and from and including marker D10S583, including D10S583, or about 0.5 cM around and including D10S583, or about 0.25 cMaround and including D10S583, 0234 (g) the region between D10S564 and or about 0.1 cM around and including D10S583. D10S583, inclusive, 0226. In particular embodiments of the kits, the polymor 0235 (h) the region between D10S583 and phism is located about 30 Mb around and including D10S1710, inclusive, D10S583, or about 28 Mb around and including D10S583, 0236 (i) the region between D10S583 and or about 20 Mb around and including D10S583, or about 15 D10S566, inclusive, Mb around and including D10S583, or about 10 Mb around and including D10S583, or about 5 Mb around and including 0237) () the region between D10S583 and D10S583, or about 2.5 Mb around and including D10S583, D10S1671, inclusive and or about 1 Mb around and including D10S583 or about 500 0238 (k) the region between D10S583 and kb around and including D10S583, or about 200 kb around D10S1741, inclusive. and including D10S583, or about 100 kb around and includ ing D10S583, or about 50 kb around and including 0239). In a particular embodiment of the kits, the poly D10S583, or about 40 kb around and including D10S583, or morphism is located in one or more of the following regions about 20 kb around and including D10S583, or about 10 kb of chromosome 10: around and including D10S583, or about 5 kb around and 0240 (a) the region extending about 5 cM, or about including D10S583, or about 1 kb around and including 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 D1OS583. cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, 0227. In a further embodiments of the kits, the polymor or about 0.1 cM proximal (centromeric) from and phism is located in one or more of the following regions of including marker D10S583, chromosome 10: 0241 (b) the region extending about 5 cM, or about 0228) (a) the region extending about 50 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 45 cM, or about 33 cM, or about 30 cM, or about 25 cM distal (telomeric) from and including marker cM, or about 21 cM, or about 15 cM, or about 10 cM, D10S583, or about 5 cM, or about 4 cM, or about 3 cM, or 0242 (c) the region extending about 10 Mb, or about 2.7 cM, or about 2.5 cM, or about 2 cM, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about about 1.5 cM, or about 1 cM, or about 0.5 cM, or 500 kb, or about 300 kb, or about 200 kb, or about about 0.1 cM proximal (centromeric) from and 100 kb, or about 80 kb, or about 50 kb, or about 10 including marker D10S583, kb, or about 5 kb, or about 1 kb proximal from and 0229 (b) the region extending about 60 cM, or including marker D10S583; and about 55 cM, or about 50 cM, or about 45 cM, or 0243 (d) the region extending about 10 Mb, or about 40 cM, or about 35 cM, or about 30 cM, or about 8 Mb, or about 5 Mb, or about 4 Mb, or about about 25 cM, or about 20 cM, or about 15 cM, or 3 Mb, or about 2 Mb, or about 1 Mb, or about 500 about 13 cM, or about 12 cM, or about 9 cM, or kb, or about 300 kb, or about 200 kb, or about 100 US 2003/017O678A1 Sep. 11, 2003 16
kb, or about 50 kb, or about 10 kb, or about 5 kb, or including the Mini Mental State Exam (MMSE) Folstein about 1 kb distal from and including marker and McHugh (1975).J. Psychiatr. Res. 12:196-198; Cockrell D1OS583. and Folstein (1988) Psychopharm. Bull. 24:689-692), the Blessed Test Blessed et al. (1968) Br. J. Psychiatry DESCRIPTION OF THE DRAWINGS 114:797-811 and the Alzheimer's Disease Assessment Scale-Cognitive (ADAS-COG) Test Rosen et al. (1984) 0244 FIG. 1 is a list of short tandem repeat polymor Am. J. Psychiatry 141: 1356–1364; Weyer et al. (1997) Int. phism genetic markers located on human chromosome 10. Psychogeriatr. 9:123-138; and Ihl et al. (2000) Neuropsy 0245 FIG. 2 is a list of genes that have been mapped to chobiol. 4:102-107). human chromosome 10. The list includes gene Symbols, 0249. As used herein, a “property relating to one or more aliases, approximate cytogenetic locations and accession phenomena in neurodegenerative disease' with reference to numbers for locating information, including gene nucleotide a gene, gene product or protein refers to a property of the Sequences, as provided, for example, by the GDB Human gene or gene product that relates to phenomena which have Genome Database available on the internet (http://www.gd been widely described and are known to those of skill in the b.org). art, including morphological, Structural, biological and bio chemical occurrences which can be pathophysiological DETAILED DESCRIPTION aspects of neurodegenerative diseases. The property may be 0246 A. Definitions any aspect or feature of the gene or gene product, including but not limited to its physical composition (e.g., nucleic 0247. Unless defined otherwise, all technical and scien acids, amino acids, peptides and proteins), functional tific terms used herein have the same meaning as is com attributes (e.g., enzymatic capabilities, Such as an enzyme monly understood by one of skill in the art to which the catalyst, inhibitory functions, Such as enzyme inhibition, invention(s) belong. All patents, patent applications, pub antigenic properties, and binding capabilities, Such as a lished applications and publications, Genbank Sequences, receptor or ligand), cellular location(s), expression pattern websites and other published materials referred to through (e.g., expression in the central nervous System) and/or out the entire disclosure herein, unless noted otherwise, are interactions with other compositions. For example, a prop incorporated by reference in their entirety. In the event that erty of a gene or gene product relating to one or more there are a plurality of definitions for terms herein, those in phenomena in neurodegenerative disease may be involve this section prevail. Where reference is made to a URL or ment of the gene and/or product thereof in pathways other Such identifier or address, it understood that Such involved in APP metabolism, A? protein generation, aggre identifiers can change and particular information on the gation and/or degradation, apoptosis, calcium homeostasis, internet can come and go, but equivalent information is inflammation, oxidative StreSS, free radical generation, known and can be readilly accessed, Such as by Searching modification of tau protein (e.g., phosphorylation), axonal the internet and/or appropriate databases. Reference thereto transport, neuroprotection and neurotrophism. evidences the availability and public dissemination of Such information. 0250) As used herein, “late-onset' refers to a type of AD in which AD-associated Symptoms manifest at an age of 0248 AS used herein, an “Alzheimer's disease (AD) sabout 50 years. In late-onset AD, Such Symptoms may phenotype” is used interchangeably with “AD trait” and manifest at any age of about 50 years or older and typically refers to any visible, detectable or otherwise measurable may manifest at sabout 60 years or sabout 65 years. property of an individual diagnosed with AD. Such proper ties include, but are not limited to, dementia, aphasia (lan 0251 AS used herein, “pedigree' refers to a family for guage problems), apraxia (complex movement problems), which information concerning the ancestral relationships agnosia (problems in identifying objects), progressive and transmission of genetic traits over Several generations is memory impairment, disordered cognitive function, altered known. behavior, including paranoia, delusions and loSS of Social 0252) As used herein, “genetic marker” refers to a seg appropriateneSS, progressive decline in language function, ment of DNA with an identifiable location on a chromo Slowing of motor functions Such as gait and coordination in Some. The DNA segment may contain one or more than one later Stages of AD, amyloid-containing plaques, which are nucleotide. The inheritance of a genetic marker may be foci of extracellular amyloid-fi (A?8) protein deposition with followed. Typically, genetic markers useful in genetic analy dystrophic neurites and associated axonal and dendritic SeS are polymorphic Such that two or more alternative forms injury and microglia expressing Surface antigens associated or Sequences or alleles exist in a population. with activation (e.g., CD45 and HLA-DR), diffuse (“preamyloid”) plaques and neuronal cytoplasmic inclusions 0253) As used herein, “allele” refers to variants of a Such as neurofibrillary tangles containing hyperphosphory nucleotide Sequence. An allele can be one of the variant lated tau protein or Lewy bodies (containing C-Synuclein). forms of a gene at a particular locus. Typically, the first Standardized clinical criteria for the diagnosis of AD have identified allele is referred to as the original allele whereas been established by NINCDS/ADRDA (National Institute of all other alleles are referred to as alternative alleles. Differ Neurological and Communicative Disorders and Stroke/ ent alleles can produce variation in inherited characteristics. Alzheimer's Disease and Related Disorders Association) Diploid organisms may be homozygous or heterozygous for McKhann et al. (1984) Neurology 34:939-944). The clinical an allelic form. In an individual, one form of an allele (the manifestations of AD as Set forth in these criteria are dominant one) may be expressed more than another form included within the definition of AD phenotype. For (the recessive one). example, dementia may be established by clinical exam and 0254. As used herein, “genotype” refers to the identity of documented by any of Several neuropsychological tests, the alleles present in an individual or Sample. The term US 2003/017O678A1 Sep. 11, 2003
“genotyping a Sample or individual refers to determining a asSociation between an allele and a trait or trait-causing Specific allele or Specific nucleotide carried by an individual allele. The occurrence of a disease-causing allele may be at a marker. presumed by the occurrence of the disease trait. In Such Studies, it may turn out that a significant association is 0255 As used herein, “haplotype” refers to a collection obtained between an allele and a trait-negative population. of genetic markers. A haplotype can be a combination of Such an association may be indicative of linkage disequi alleles present in an individual or Sample. A haplotype can librium between that allele and a protective allele that be the alleles of different genes received by an individual decreases Susceptibility to disease. ASSociation Studies focus from one parent, or an array of polymorphisms on a chro on population frequencies and explore the relationships OSOC. among frequencies for Sets of alleles between loci. ASSocia 0256 AS used herein, “linkage disequilibrium” with ref tion may be determined using a number of analytical meth erence to the relationship between alleles refers to the ods, including but not limited to case-control Studies, fam deviation from the random occurrence of the alleles in a ily-based asSociation techniques and haplotype analyses. haplotype in populations. Alleles observed together on a ASSociation determinations utilizing alleles that are not chromosome more often than expected from their frequen transmitted from parents to affected individuals as controls cies in the population may be referred to as in linkage and/or related disease family members (e.g., Sib pairs) as disequilibrium. Alleles that are physically close are more affected individuals are particularly useful in accurate deter likely to be inherited together than are alleles that are farther mination of association. Such determinations are in contrast apart. Therefore, variations of Several markers that are close to association Studies using unrelated populations of Subjects to, or within, a particular gene Variant on a chromosome are and matched controls (e.g., case-control Studies), which likely to be inherited together with that gene variant when have the advantage of being relatively simple in terms of the they are in linkage disequilibrium. Thus, genetic markers, Sample Sets and Statistical analyses involved but may be e.g., microSatellite markers and SNP variations, that are in more Susceptible to false positive (type I) and false negative linkage disequilibrium and associated with a disease phe (type I) errors. Thus, in case-control Studies, it is possible in notype can mark the position on the chromosome in which Some instances (e.g., population Stratification, insufficient a Susceptibility gene is located. Sample size and/or poorly matched control groups) to 0257 Generally, linkage disequilibrium spans chromo observe association in the absence of linkage disequilibrium. Some Segments ranging in Size from about SS kb to about 0260 AS used herein, an “Alzheimer's disease (AD) 500 kb or less, such as distances of s80 kb or s50 kb Risch DNA segment” or “ Alzheimer's disease (AD) gene” is a (2000) Nature 405:847-856; Abecasis et al. (2001) Am. J. gene or other DNA segment that either directly causes AD Hum. Genet. 68:191-197; Reich et al. (2001) Nature 41:199 or confers an increased or decreased Susceptibility to AD. A 204). It is common, however, to find Some degree of linkage gene or DNA segment which causes AD may, for example, disequilibrium between alleles that are up to about 1-2 cM have an allele that contains an alteration, e.g., a mutation, apart. Significant linkage disequilibrium between microSat relative to another allele(s) of the gene or DNA segment, ellite loci has been reported to extend to 24 cM Huttley et wherein the alteration can cause or give rise to a defect al. (1999) Genetics 152:1711-1722 and as great as s-21 involved in the manifestation of an AD phenotype. cM Wilson and Goldstein (2000) Am. J. Hum. Genet. 0261) A gene or DNA segment that confers increased 67:926-935). The degree of linkage disequilibrium between Susceptibility to AD may have an allele that predisposes an two alleles can vary based on location within the genome, individual to AD but is not an invariant cause of AD. Thus, population distribution, population frequency and demo an allele that conferS increased Susceptibility to AD graphic history Reich et al. (2001) Nature 41:199-204; increases the likelihood of developing AD but is not suffi Stephens et al. (2001) Science 293:489-493; Wilson and cient alone to cause AD. Such an allele may be referred to Goldstein (2000) Am. J. Hum. Genet. 67:926-935). as a genetic risk ractor for AD and may be one of Several 0258 When a disease-causing allele is in linkage dis genetic risk factors, which in turn may be one type of Several equilibrium with another allele, the frequency of the other types of risk factors. For example, other possible risk factors allele will be increased in a disease population as compared could include environmental risk factors. An allele of a gene to a trait-negative population. This increased frequency is or DNA segment that confers increased susceptibility to AD referred to as "genetic association' or “allelic association' can be over-represented in cases in case control Studies between the other allele and the disease. Thus, association and/or can be associated with affected individuals in a between a disease trait and a marker allele can be indicative family-based association analysis. of linkage disequilibrium between the disease-causing allele 0262. A gene or DNA segment that confers decreased and the marker allele. Similarly, when an allele that confers Susceptibility to AD can be under-represented in cases in protection against a disease is in linkage disequilibrium with case control Studies and/or can be associated with unaffected another allele, the frequency of the other allele may be individuals in a family-based association analysis. increased in a trait-negative population relative to a disease 0263. As used herein, a “DNA segment associated with population. This increased frequency is referred to as genetic Alzheimer's disease (AD)” refers to an allele that either is an asSociation between the other allele and the protective allele. AD gene or DNA segment or is in linkage disequilibrium 0259 Studies of genetic association are commonly used with an AD gene or AD DNA segment. An allele that is an to identify genes involved in complex traits. Genetic asso AD risk factor or AD Susceptibility locus may be in linkage ciation Studies assess correlations between genetic variants disequilibrium with an allele of an AD gene or DNA and trait differences on a population Scale. In association Segment and thus may be a DNA segment associated with based methods of mapping genes that increase Susceptibility AD. DNA segments associated with AD include genes as to disease, evidence is Sought for a Statistically significant well as intergenic regions of DNA. US 2003/017O678A1 Sep. 11, 2003
0264. As used herein, the term “protective” with refer linkage is Statistical evidence that would be expected to ence to an allele refers to an allele that is indicative of a occur one time at random in a genome Scan, Significant decreased risk relative to the general population for a genetic linkage is Statistical evidence expected to occur 0.05 times disease, e.g., AD. The decreased risk associated with a in a genome Scan (that is with probability 5%), highly protective allele may be identified as under-representation of Significant linkage is Statistical evidence expected to occur the allele in caseS relative to controls, and/or as a significant 0.001 times in a genome Scan and confirmed linkage is asSociation between the allele and unaffected members of a Significant linkage from one or a combination of initial family that contains affected members. A protective allele Studies that has Subsequently been confirmed in a further may be a variant of a DNA segment, Such as a gene, that has Sample. In the case of Sibling pair-based linkage analysis, for a risk factor or disease allele. A protective allele may be a example, Suggestive, Significant and highly significant link variant that is “functional” in that it participates in counter age may correspond to LOD Scores of 2.2, 3.6, and 5.4, acting a defect that occurs in a genetic disease, e.g., AD, or respectively. may confer apparent "protection' against a disease by not conferring risk for the disease. 0270. The relationship between two linked loci may also be defined in terms of the physical or genetic distance 0265 AS used herein, the term “penetrance” refers to the between the loci. Thus, two loci may be referred to as linked ratio between the number of trait positive carriers of a when they are located relatively close together on the same particular allele and the total number of carriers of the allele chromosome. For example, in the case of a polymorphic or in the population. Thus, a highly penetrant gene or allele will marker Site on a chromosome linked with a DNA segment have a greater penetrance ratio than a weakly or moderately asSociated with AD, the marker may be located a particular penetrant gene. Penetrance may also be considered as the number of base pairs (bp) or centiMorgans (cM) from the percent probability that a carrier of a particular allele will DNA segment. The particular distance, in bp or cM, between express the corresponding phenotype. two linked loci can vary, but is Small enough So that the 0266. As used herein, “prevalence” refers to the percent linkage Score, e.g., the LOD Score, obtained in linkage age of trait positive individuals that carry a particular allele. analysis of the two loci (e.g., a marker and a trait Such as a disease) is at least indicative of linkage (i.e., the loci are 0267 The terms “recombination fraction” and “recombi “relatively close' to each other) if not at least Suggestive, nation frequency’ are used herein interchangeably and refer Significant or even highly significant linkage. A linked to the probability of a recombination event between two loci marker may be within the DNA segment associated with a in a genome. trait (e.g., AD) and, further, may be a causative polymor 0268 AS used herein, “linked” refers to a relationship phism in a disease (e.g., AD) gene, Such as, for example, a between two loci in a genome. For example, it may refer to polymorphism in an AD gene that is responsible for a defect the relationship between a polymorphic or marker Site on a in an AD gene. When the marker is located within an AD chromosome and a gene, Such as, for example, a gene gene, it is referred to as coincident with the gene. associated with AD. The relationship may be defined in a 0271 AS used herein, the term “effect size” with refer number of ways. For example, the relationship may be ence to a disease gene refers to the degree to which muta defined in terms of the extent to which recombination tions or polymorphisms in a DNA segment, e.g., a gene, between the loci occurs. Typically, the transmission of confer Susceptibility to the disease taking into account the alleles located on different chromosomes occurs in a random magnitude of prevalence and penetrance of the polymor fashion through independent assortment. Loci representative phism. of two Such alleles are considered to be unlinked. If two loci are situated on the same chromosome, the transmission of 0272. As used herein, “indicative of a predisposition to alleles of one locus may be affected by the presence of the Alzheimer's disease (AD)” with reference to an allele means other locus Such that the ratios of alleles are no longer that an individual who possesses the allele without detect independent, and the loci are referred to as “linked.” Two able symptoms of AD is more likely to develop or have AD loci are completely linked when there is no recombination than someone who does not have the allele. The allele may between them; the same alleles or phenotypes are always be over-represented in frequency in individuals with AD as transmitted together from generation to generation within a compared to individuals who do not have AD. Thus, the family. An intermediate State of linkage, referred to as allele can be used to predict disease even in pre-symptom "incomplete linkage” occurs when the transmission of alle atic or pre-diseased individuals. les of two loci deviates consistently and measurably from 0273. As used herein, “indicative of the occurrence of independent assortment but a consistent recombination frac Alzheimer's disease (AD)” with reference to an allele means tion nonetheless exists for the loci. that an individual who possesses the allele and manifests one 0269 Linkage is commonly assessed by the LOD (loga or more symptoms of AD is more likely to have AD than rithm of an odds ratio) score method or other acceptable Someone who does not have the allele and either does or Statistical linkage determination. Positive LOD scores can does not manifest one or more Symptoms of AD. Thus, the be considered as evidence of linkage between two loci. The allele may be used to diagnose AD and, in particular, greater the LOD score, the greater the possibility that the differentially diagnose AD. For example, the allele may be loci are linked. LOD scores >1 are particularly indicative of used to distinguish an individual with AD-associated linkage. Classification of linkage has been proposed see, dementia from an individual with dementia resulting from a e.g., Lander and Kruglyak (1995) Nature Genet. 11:241 condition unrelated to AD. This is particularly of use in 247 based on the number of times it would be expected to diagnosis of AD in individuals about age 50 or greater, about See a result at random in a dense, complete genome Scan for age 60 or greater or about age 65 or greater. In methods of linkage. Under Such a classification Scheme, Suggestive using an allele to diagnose AD, determination of the pres US 2003/017O678A1 Sep. 11, 2003
ence or absence of the allele in an individual may be 0280 At least half of the people who develop late onset conducted in conjunction with other diagnostic tests for AD, AD do not carry an APOEe4 allele. As much as 70% or more including a variety of neuropsychological tests known to of the genetic variance for AD remains unaccounted for by those of skill in the art and referred to herein. APOEe4 and the three known early onset AD genes. Seg 0274. As used herein, “information content” refers to the regation analysis predicts a Susceptibility locus accounting usefulness of a family to be informative for linkage. For a for more than 50% of onset age variation in AD, a consid family to be informative for linkage, at least one of the erably greater fraction than APOEDaw et al. (2000) Am. J. parents must be doubly heterozygous. Thus, the informa Hum. Genet. 66:196-204). Thus, the APOEe4 allele is nei tiveness of a marker is given by its heterozygosity (H), the ther necessary nor Sufficient for AD; instead, it modulates fraction of individuals likely to be heterozygous at that the risk for development of AD Corder et al. (1993) Science locus, or the polymorphism information content (PIC), the 261:921-923; Corder et al. (1994) Nature Genet. 7:180-184). fraction of matings in which a particular parent is expected Additional genetic factors are therefore indicated for AD. to be fully informative. 0281. A number of other genes have been proposed to be 0275 AS used herein, attributable risk refers to that risk factors for late-onset AD or to modify the association percentage of a genetic disease, Such as AD, that would with APOEe4. These include the genes encoding butyrocho disappear Should an alteration a disease gene not exist in a linesterase Lehmann et al. (1997) Human Mol. Genet. population, for example, in humans. 6:1933-1936), low-density lipoprotein receptor-related pro 0276 AS used herein, a combination refers to any asso tein (LRP) Kang et al. (1997) Neurology 49:56-61), very ciation between or among two or more items. The combi low-density lipoprotein receptor (VLDL) Okuizumi et al. nation can be two or more Separate items, Such as two (1995) Nature Genet. 11:207-209), cystatin C(CST3) see compositions or two collections, can be a mixture thereof, Nitsch et al. (1999) Soc. Neuroscience 25 (Part 1):432.11, Such as a Single mixture of the two or more items, or any Abstracts for the 29th Annual Meeting, Oct. 23-28, 1999, variation thereof. Miami Beach, Fla. and Crawford et al. (2000) Neurobiol. Aging 21 (Suppl.):S204, Abstract 927, Abstracts for the 0277 B. Genetics of Alzheimer's Disease (AD) World Alzheimer Congress 2000, Jul. 9-18, 2000, Washing 0278 Alzheimer's disease (AD) is most commonly a late ton, D.C. and C.1-antichymotrypsin Kamboch et al. (1995) onset disease of the elderly occurring at about 50 years of Nature Genet. 10:486-488; Yoshiiwa et al. (1997) Ann. age or older, and most typically at about 60 years of age or Neurol. 42:115-117). None of these genes is located on older. However, a small number of pedigrees have been chromosome 10. described wherein an early onset form of the disease is inherited as an autosomal dominant trait with age-dependent 0282. Several full genome screens for AD susceptibility penetrance. Early onset of AD occurs at ages less than 60 loci have been performed with varying results. For example, years of age, typically at less than about 50 years of age and possible loci which have been proposed in linkage Studies most commonly between the years of about 30 to about 50 include one near the centromere of chromosome 12 (with years of age. Genetic factors have been implicated in early chromosomes 4, 6 and 20 proposed as regions of interest for and late onset AD. follow-up studies) Pericak-Vance et al. (1997) JAMA 278:1237-1241 and Pericak-Vance et al. (1998) Neurobiol. 0279 The genetics of Alzheimer's disease is complex. Aging 19:S39-S42) and one on the proximal long arm of Mutations in at least four genetic loci are associated with chromosome 19 Pericak-Vance et al. (1991) Am. J. Hum. inherited susceptibility to AD (i.e., familial AD). Three Genet. 48:1034-1050). The results of linkage analyses have genes have been associated with early onset AD: APP been reported to Suggest linkage of AD on chromosomes 1, f3-amyloid precursor protein on chromosome 21; Goate et 5, 9, 10, 12, 14, 19 and 21 Kehoe et al. (1999) Hum. Mol. al. (1991) Nature 349:704–706; Chartier-Harlin et al. (1991) Genet. 8:237-245). In the study reported by Kehoe et al. Nature 353:844-846; Murrell et al. (1991) Science 254:97 (1999) Hum. Mol. Genet. 8:237-245), 292 affected sibling 99; Karlinsky et al. (1992) Neurology 42:1445-1453), PS1 pairs with AD and onset age of 265 years were genotyped (presenilin 1) and PS2 (presenilin 2). Only one gene, the e4 using 237 microSatellite markerS Separated by an average allele of the apolipoprotein E (APOE) gene on chromosome distance of 16.3 cM, and the data were analyzed by SPLINK 19, has been associated with late onset AD. This gene was Holman and Clayton (1995) Am. J. Hum. Genet. 57: 1221 identified using both linkage analysis Pericak-Vance et al. 1232 and MAPMAKER/SIBS Lander and Krugylak (1991) Am. J. Human Genet. 48:1034-1050 and association (1995) Nature Genet. 11:241-247 for the whole sample as analysis Corder et al. (1993) Science 261:921-923; Saun well as for samples stratified on the basis of whether both or ders et al. (1993) Neurology 43:1467-1472; Strittmatter et al. neither members of an affected Sibling pair possessed at least (1993) Proc. Natl. Acad. Sci. U.S.A. 90:1977-1981). The one APOE e4 allele. Although multipoint LOD scores 21 magnitude of risk for AD conferred by the APOEe4 allele is were reported for markers on 12 different chromosomes in age dependent, appearing maximal between 60 and 80 years either the Stratified or whole Samples, only markers on of age Tang et al. (1996) Am. J. Hum. Genet. 58:574-584; chromosomes 1, 5, 9, 10 and 19 yielded multipoint LOD Frisoni et al. (1998) J. Neurol. NeuroSurg. Psychiatry Scores > 1 in the whole sample. The linkage peak (LOD score 65:103-106; Bickeboller et al. (1997) Am. J. Hum. Genet. of -2.27) on chromosome 10 observed in the analysis of the 60:439-446). The association of APOEe4 with AD appears whole Sample appears to have been located Somewhere to be strongest in individuals with an onset prior to 70 years within the region of 50-70 cM. A maximum LOD score of of age Farrer et al. (1997) J. Am. Med. Assoc. 278:1349 1.17 was reported for chromosome 10 in analysis of the 1356; Blacker et al. (1997) Neurology 48:139-147) and Stratified Samples (i.e., the APOE e4-negative sample) and weakens with advanced age. appears to be provided by a marker located at about 100 cM. US 2003/017O678A1 Sep. 11, 2003 20
0283) In a follow-up of the study of Kehoe et al. (1999) and e4 alleles are considered variants which affect genetic Hum. Mol. Genet. 8:237-245), additional markers were susceptibility to AD. The e4 allele is associated with an genotyped within each peak region to reduce the interval increased risk and earlier age-at-onset whereas the e2 allele between each pair of markers to <5 cMMyers et al. (2000) confers a decreased risk and older age-at-onset Corder et al. Neurobiol. Aging 21 (suppl.):S103). The peak on chromo (1994) Nat. Genet. 7:180-184). One or more AD genes on some 10 (reported to have yielded a LOD score of 1.87 in chromosome 10, and in particular chromosome 10q, may contrast to the LOD score of 2.27 reported by Kehoe et al. have multiple alleles, one of which is protective against AD Supra) reportedly increased to 3.11 with additional markers and another which confers increased Suceptibility to AD. and Samples and shifted in location towards the pter from 0289. In addition, D10S583, as well as other markers on D10S1211 (70.35 cM) to between D10S1227 and D10S1225 chromosome 10q, particularly markers on chromosome (peak at 60.42 cM). Significantly, this follow-up study 10q22-q26, are linked to AD (see EXAMPLE 2). The peak reports that the initial peak previously found as set forth in linkage occurs on the distal approximately 70-85 cM of the Kehoe et al. (Supra) in the APOE e4-negative sample q arm of chromosome 10, from about 85 cM extending disappeared in this follow-up study using additional affected distally to qter. In terms of the cytogenetic map of chromo Sibling pairs. Another genome Scan for linkage of AD Some 10, the peak linkage extends from 10q22 to qter. The Rogaeva et al. (2000) Neurobiol. Aging 21 (Suppl.):S103 Strongest linkage is on 10q23-q25. Linkage analysis reveals reports that markers on chromosome 10q23 (D10S1423-71 coSegregation of a marker with the disease trait within cM-D10S571) generated negative evidence for linkage. individual families, and thus provides evidence that within 0284. The results of a genome-wide survey for AD risk each family, a particular allele of a marker, Such as an allele loci using a case-control design were reported to indicate of D10S583, is relatively close to at least one DNA segment allelic association with AD at a marker (D10S1423) on that causes AD or confers an increased Susceptibility to AD. chromosome 10p12-14 Zubenko et al. (1998) Genomics The discovery described herein of association between the 50:121-128), approximately 40 cM from the telomere. In a AD-linked marker D10S583 and a population within fami Subsequent case-control Study of marker D10S1423, a Sig lies having affected members, be it association with affected nificant association of marker D10S1423 with AD was family members or with unaffected members, reveals that reported Majores et al. (2000) Neurosci. Lett. 289:224 there is at least one AD DNA segment or AD gene within 226). linkage disequilibrium distance of D10S583 and that there are AD-associated marker alleles in the thus-defined region 0285 C. Genetic Association with Alzheimer's Disease of chromosome 10 that may be used in determining a (AD) on Chromosome 10 predisposition to or the occurence of AD in an individual. 0286 Genetic analysis described herein led to the dis covery of genetic association with AD on chromosome 10. 0290 Although the markers linked to AD as described in The association identifies chromosome 10 as the location of EXAMPLE 2, including D10S583, did not reveal a signifi one or more DNA segments or genes associated with AD and cant association with risk for AD, asSociation analyses of of considerable effect size. In particular, chromosome 10 is multiple alleles of these markers revealed a trend toward identified as containing at least one DNA segment or gene risk. A disease gene, Such as an AD gene, Very likely may asSociated with AD and of comparable or greater impact have Several variant forms that place a perSon at risk for the disease, as well as variant forms that decrease the risk for than APOE, wherein the effect size of the gene is compa AD and forms that are neutral (i.e., have a relative risk of rable to or greater than that of APOE. 1.0). The data from association analyses of the linked 0287. As described herein (see EXAMPLE 3), a diallelic markers described in EXAMPLE 2 are consistent with the test of marker D10S583, located on chromosome 10q23.33, possibility of multiple risk alleles of an AD gene on chro for association with AD using a family-based test method for mosome 10. Thus, the association of an allele of the AD asSociation revealed significant association of an approxi linked marker D10S583 with unaffected AD family mem mately 210-bp allele with protection against AD (nominal bers is consistent with the existence of at least one DNA p=0.004, Bonferronicorrected p=0.04). Thus, D10S583 is in Segment that causes AD or conferS increased Susceptibility linkage disequilibrium with an allele that conferS protection to AD on chromosome 10, and, in particular, chromosome against AD. This finding indicates that there is an allele on 10q22-q26, Such as the region of chromosome 10q23-q25, chromosome 10 that confers in those who carry the allele as well as being indicative of the presence of an allele on protection against AD relative to those who do not carry the chromosome 10 that is protective against AD. allele. 0291 Any other marker found to be in linkage disequi 0288 Furthermore, the finding of association of librium with D10S583 will be associated with an allele D10S583 with AD indicates that there is one or more AD protective against AD and thus will also be evidence of the DNA segments or AD genes on chromosome 10, and in presence of at least one DNA segment that causes AD or particular chromosome 10q, that either directly cause or confers increased Susceptibility to AD on chromosome 10. confer an increased Susceptibility to AD (e.g., a "risk” or Therefore, based on the discovery of association between “disease” allele). A protective allele, such as the allele with D10S583 and AD, additional markers associated with AD or which D10S583 is in linkage disequilibrium, generally has a protective allele may now be identified using methods as a counterpart disease risk allele. For example, the APOE described herein and known in the art. The availabiltiy of gene, located in a peak linkage region on chromosome 19 additional markers is of particular interest in that it will identified in a genetic linkage analysis of late-onset AD increase the density of markers for this chromosomal region families Pericak-Vance et al. (1991) Am. J. Hum. Genet. and can provide a basis for identification of an AD DNA 48:1034-1050), has three common alleles designated e2, e3 Segment or gene in the region of chromosome 10q, and in and e4. The e3 allele is the most common allele, and the e2 particular, chromosome 10q22-q26. An AD DNA segment US 2003/017O678A1 Sep. 11, 2003
or gene may be found in the vicinity of the marker or Set of with AD. In Such embodiments, the combination of markers markers showing the highest correlation with AD. Further as a whole is associated with AD, Such as in the case of a more, the availability of markers associated with AD makes haplotype. possible genetic analysis-based methods of determining a 0296 1. Genetic Markers on Human Chromosome 10 predisposition to or the occurrence of AD in an individual by detection of a particular allele. 0297) a. Genetic Markers 0298. A genetic marker is a DNA segment with an 0292. The discovery of one or more AD-associated genes identifiable location in a chromosome. Genetic markers may on chromosome 10, as evidenced by Significant association be used in a variety of genetic Studies Such as, for example, with AD on chromosome 10, thus provides the basis for locating the chromosomal position or locus of a DNA genetic analysis methods described herein which include: Sequence of interest, and determining if a Subject is predis methods for identifying polymorphisms linked to a DNA posed to or has a particular disease. Segment associated with AD, methods for detecting poly 0299 Because DNA sequences that are relatively close morphisms linked to a DNA segment associated with AD; together on a chromosome tend to be inherited together, methods for identifying polymorphisms associated with AD; tracking of a genetic marker through generations in a family methods for detecting polymorphisms associated with AD; and comparing its inheritance to the inheritance of another methods for detecting the presence of a DNA segment DNA sequence of interest can provide information useful in asSociated with AD in a Subject; methods for determining determining the relative position of the DNA sequence of the level of risk for AD in a subject; methods for determining interest on a chromosome. Genetic markers particularly a predisposition to and/or the occurrence of AD in a Subject; useful in Such genetic Studies are polymorphic. Such mark methods for identifying a region or regions of the human erS also may have an adequate level of heterozygosity to genome containing an AD DNA segment or AD gene; allow a reasonable probability that a randomly Selected methods for predicting response to treatment for AD, meth person will be heterozygous. ods for treating AD, methods for identifying a candidate AD DNA segment or AD gene; and methods for identifying an 0300. The occurrence of variant forms of a particular AD gene. Also provided herein are compositions that may be DNA sequence, e.g., a gene, is referred to as polymorphism. used in methods described herein, Such as nucleic acids that A region of a DNA segment in which variation occurs may may be used as probes or primerS for detection of polymor be referred to as a polymorphic region or site. A polymor phisms linked to AD-associated genes and combinations, phic region can be a single nucleotide (single nucleotide kits and articles of manufacture containing the nucleic acids. polymorphism or SNP), the identity of which differs, e.g., in Such compositions may also be used in methods of deter different alleles, or can be two or more nucleotides in length. mining a predisposition to and/or the occurrence of AD in a For example, variant forms of a DNA sequence may differ Subject. by an insertion or deletion of one or more nucleotides, insertion of a Sequence that was duplicated, inversion of a 0293 D. Genetic Markers Associated with AD Sequence or conversion of a single nucleotide to a different nucleotide. Each individual can carry two different forms of 0294 Genetic markers associated with AD are provided the Specific Sequence or two identical forms of the Sequence. herein. The markers are located on human chromosome 10. More than two forms of a polymorphism may exist for a In particular embodiments of the genetic markers provided Specific DNA marker in the population, but in one family herein, the marker is located located on chromosome 10q22, just four forms are possible: two from each parent. Each 10q23, 10q24, 10q25 or 10q26. In further embodiments, the child inherits one form of the polymorphism from each marker is located on chromosome 10q22, 10q23 or 10q24. parent. Thus, the origin of each chromosome can be traced In yet further embodiments, the marker is located on chro (maternal or paternal origin). mosome 10q23, 10q24 or 10q25. The markers can be over-represented in cases in case-control Studies and/or can 0301 Differences between polymorphic forms of a spe be associated with affected individuals in a family-based cific DNA sequence may be detected in a variety of ways. asSociation analysis. Alternatively, the markers can be For example, if the polymorphism is Such that it creates or under-represented in cases in case-control Studies and/or deletes a restriction enzyme site, Such differences may be asSociated with unaffected individuals in a family-based traced by using restriction enzymes that recognize specific asSociation analysis. The markers can be identified through DNA sequences. Restriction enzymes cut (digest) DNA at linkage disequilibrium or association assessment methods Sites in their specific recognized Sequence, resulting in a described herein or known to those of skill in the art, and collection of fragments of the DNA. When a change exists provide Scores or results indicative of linkage disequilibrium in a DNA sequence that alters a Sequence recognized by a with an AD DNA segment or gene or of association with AD restriction enzyme to one not recognized, the fragments of when tested by Such assessment methods. The genetic DNA produced by restriction enzyme digestion of the region markers are associated with AD as individual markers and/or will be of different sizes. The various possible fragment in combinations, Such as haplotypes, that are associated with sizes from a given region therefore depend on the precise AD. Sequence of DNA in the region. Variation in the fragments produced is termed “restriction fragment length polymor 0295 Also provided herein are combinations of genetic phism” (RFLP). The different sized-fragments reflecting markers which are associated with AD. In one embodiment, variant DNA sequences can be visualized by Separating the each genetic marker in a combination is associated with AD. digested DNA according to its size on an agarose gel and In other embodiments, Some of the genetic markers in the Visualizing the individual fragments by annealing to a combination are associated with AD and Some of the genetic labeled, e.g., radioactively or otherwise labeled, DNA markers are not or none of the genetic markers is associated “probe'. RFLPs occur on average every 10 kb. US 2003/017O678A1 Sep. 11, 2003 22
0302 RFLPs may be somewhat limiting in genetic analy large number of alleles may become a consideration when SeS in that they usually give only two alleles at a locus and using haplotype-based methods, they are not usually not all parents are heterozygous for these alleles and thus intragenic, and they may have relatively high and variable informative for linkage see, e.g., Botstein et al. (1980) Am. mutation rates which may affect linkage disequilibrium J. Hum. Genet. 32:314-331). Newer analytic methods take between a marker and disease mutation. advantage of the presence of DNA sequences that are 0306 SNP markers may also be used in fine mapping and repeated in tandem, for a variable number of repeats, and asSociation analysis, as well as linkage analysis See, e.g., that are Scattered throughout the human genome. The first of Kruglyak (1997) Nature Genetics 17:21-24). Although an these described were variable number tandem repeats of SNP may have limited information content, combinations of core sequences (VNTRs) Jeffreys et al. (1985) Nature SNPs (which individually occur about every 100-300 bases) 314:67-73; Nakamura et al. (1987) Science 235:1616-1622; may yield informative haplotypes. SNP databases are avail Weber (1989) Am. J. Hum. Genet. 44:388-396). VNTRs may able (see, e.g., http://www.ibc.wustl.edu/SNP/; http://ww be detected using unique Sequences of DNA adjacent to the w.ncbi.nlm.nih.gov/SNP/; http://www.genome.wi.mit.edu/ tandem repeat as marker probes, and digesting the DNA with SNP/human/index.html). Assay systems for determining restriction enzymes that do not recognize Sites within the SNPs include synthetic nucleotide arrays to which labeled, core Sequence. VNTRS may also be detected using nucleic amplified DNA is hybridized see, e.g., Lipshutz et al. acid amplification methods. Highly informative VNTR loci (1999) Nature Genet. 21:2-24; single base primer extension have not been found on all chromosome arms, and those methods Pastinen et al. (1997) Genome Res. 7:606–614), which have been identified are often situated near telomeres mass spectroscopy on tagged beads, and Solution assays in Royle et al. (1998) Genomics 3:352-360), leaving regions which allele-Specific oligonucleotides are cleaved or joined of the genome out of reach of these multiallelic marker loci. at the position of the SNP allele, resulting in activation of a 0303 Eukaryotic DNA has tandem repeats of very short fluorescent reporter System see, e.g., Landegren et al. Simple Sequences termed SSRS (simple Sequence repeat (1998) Genome Res. 8:769-776). polymorphisms) Such as, for example, (dC-dA), or (dG-dT), where n=10-60 (termed GT repeat). These are also referred 0307 b. Chromosome 10 to as short tandem repeat polymorphisms (STRPs) and 0308 The estimated physical length of chromosome 10 is microsatellite markers. The (dG-dT) repeats occur every about 144 Mb. The genetic or linkage map of chromosome 30-60kb along the genome Weber et al. (1989) Am. J. Hum. 10, generated based upon frequency of recombination Genet. 44:388-396; Litt et al. (1989) Am. J. Hum. Genet. between genomic Segments, estimates that chromosome 10 44:397-401), and Alu 3' (A)n repeats occur approximately is about 175 centiMorgans (cM) in length. Cytogenetic maps every 5 kb Economou (1990) Proc. Natl. Acad. Sci. U.S.A. of chromosome 10 distinguish regions of the chromosome as 87:2951-4). Repeat polymorphisms include dinucleotide, p or q bands. The bands refer to bands observed on the trinucleotide and tetranucleotide repeats. Dinucleotide Giemsa-Stained chromosome. Approximate locations for the repeats are informative and fairly prevalent in the genome. bands have been determined. For example, band 10q21.3, The small size of the repeat brings about diversity of its the most distal of the 10q21 bands, begins at about 65 Mb allele sizes and thus there is a greater chance that any one and ends at about 70 Mb on chromosome 10. Band 10q22.1, person will be heterozygous for the marker. Trinucleotide the most proximal of the 10q22 bands, begins at about 70 and tetranucleotide repeats are repeats of three and four Mb (or about 86-92 cM) and ends at about 77 Mb on nucleotides. chromosome 10. Band 10q23.1, the most proximal of the 0304 Oligonucleotides corresponding to flanking 10q23 bands, begins at about 85 Mb and ends at about 90 regions of these repeats may be used as primers for the Mb. Band 10q23.33, the most distal of the 10q23 bands, polymerase chain reaction (PCR) Saiki (1988) Science begins at about 99 Mb and ends at about 103 Mb. Band 239:484-491 on a small sample of DNA. By amplifying the 10q25.1, the most proximal of the 10q25 bands, begins at DNA with labeled, e.g., radioactive or fluorescent, nucle about 112 Mb and ends at about 117 Mb. Band 10q25.3, the otides, the Sample may be quickly resolved on a Sequencing most distal of the 10q25 bands, begins at about 119 Mb and gel and Visualized by known methods, e.g., autoradiography ends at about 123 Mb. Band 10q26.11, the most proximal of or fluorescence detection. Because these polymorphisms are the 10q26 bands, begins at about 123 Mb and ends at about comprised of alleles that may differ in length by only a few 125 Mb. base pairs, they generally are not detectable by conventional 0309 More than 400 short tandem repeat polymorphisms Southern blotting as used in traditional RFLP analysis. The which Serve as genetic markers have been mapped on use of PCR to characterize SSRS Such as GT polymorphic chromosome 10. FIG. 1 lists many of these markers and markers enables the use of less DNA, typically only ten provides the Sex-averaged genetic map distance for each nanograms of genomic DNA is needed, and is faster than marker as reported by the Marshfield Center for Medical standard RFLP analysis, because it essentially only involves Genetics see Yu et al. (2001) Nature 409:951-953 and amplification and electrophoresis. (http://research.marshfieldclinic.org/genetics/). Also pro 0305 Microsatellites have been used extensively in link vided in FIG. 1 are references to GenBank, Human Genome age analysis (see, e.g., http://carbon.wi.mit.edu:8000/cgi Draft Sequence and Contig Sequence accession numbers for bin/contig/phys map; http://www.chlc.org/; http://gdb.info many of these markers. biogen.fr/gdb/contact.html#baltimore). They have many 0310 Included among the polymorphic markers in FIG. alleles and therefore are highly informative. Although mic 1 is marker D10S583 discovered, as described herein, to be rosatellites may be used in fine mapping and association asSociated with protection against AD. Also included among analysis, they may have one or more features that should be the markers listed in FIG. 1 are markers linked to AD, considered in connection with Such use. For example, the including D10S583. As described in the EXAMPLES, US 2003/017O678A1 Sep. 11, 2003
markers D10S583, D10S1710, D10S566, D10S1671 and causing a particular trait) or is extremely close to the disease D10S1741 each yielded LOD scores >1 for the whole gene on a chromosome. When a marker analyzed for asso Sample in at least one of Several AD linkage analyses. These ciation with a disease is a functional variant, association is markers are dinucleotide repeat Sequences. the result of the direct effect of the genotype on the pheno typic outcome. When a marker being analyzed for associa 0311) 2. Genetic Association tion is an anonymous marker, the occurrence of association 0312. When two loci are extremely close together, is the result of linkage disequilibrium between the marker recombination between them is very rare, and the rate at and a functional variant. which the two neighboring loci recombine can be So Slow as to be unobservable except over many generations. The 0316 There are a number of methods typically used in resulting allelic association is generally referred to as link assessing genetic association as an indication of linkage age disequilibrium. Linkage disequilibrium can be defined disequilibrium, including the epidemiological case-control as Specific alleles at two or more loci that are observed Study of unrelated Subjects and methods using family-based together on a chromosome more often than expected from controls. Although the case-control design is relatively their frequencies in the population. As a consequence of Simple, it is the most prone to identifying DNA variants that linkage disequilibrium, the frequency of all other alleles prove to be spuriously associated (i.e., association without present in a haplotype carrying a trait-causing allele will also linkage) with disease Cardon and Bell (2001) Nature Rev. be increased (just as the trait-causing allele is increased in an Genet. 2:91-99). Spurious association can be due to the affected, or trait-positive, population) compared to the fre Structure of the population Studied rather than to linkage quency in a trait-negative or random control population. disequilibrium. Thus, for example, if cases and controls are Therefore, association between the trait and any allele in not ethincally comparable, then differences in allele fre linkage disequilibrium with the trait-causing allele will quency can emerge at loci that differentiate the groups Suffice to Suggest the presence of a trait-related DNA seg whether the alleles are causally related to disease or not (a ment in that particular region of a chromosome. On this phenomenon referred to as population Stratification). Link basis, association Studies are used in methods of locating and age analysis of Such spuriously associated allelic variants, discovering disease-Susceptibility genes. however, would not detect evidence of Significant linkage because there would be no familial Segregation of the 0313 A marker locus must be tightly linked to the disease variants. Therefore, putative association between a marker locus in order for linkage disequilibrium to exist between the allele and a disease trait identified in a case-control Study loci. In particular, loci must be very close in order to have should be tested for evidence of linkage between the marker appreciable linkage disequilibrium that may be useful for and the disease before a conclusion of probable linkage asSociation Studies. ASSociation Studies rely on the retention disequilibrium is made. ASSociation tests that avoid Some of of adjacent DNA variants over many generations in historic the problems of the Standard case-control Study utilize ancestries, and, thus, disease-associated regions are theo family-based controls in which parental alleles or haplotypes retically Small in outbred random mating populations. In not transmitted to affected offspring are used as controls. practice, however, it is common to find Some degree of linkage disequilibrium between alleles that are up to about 0317. Although genetic linkage between a marker and a 1 to 2 cM apart, or even 3 to 4 cM apart, and this can be used disease, Such as AD, demonstrates a true genetic basis for the for disease gene mapping Jorde (1995) Am. J. Hum. Genet. disease and indicates proximity between the marker and a 56:11-14; Xiong and Guo (1997) Am. J. Hum. Genet. disease locus which has use in mapping the location of a 60:1513-1531). In contrast, linkage studies, by relying on disease locus on a chromosome, it is determined through identification of haplotypes that are inherited intact over analysis of transmission of marker alleles through individual Several generations (such as in families or pedigrees of families. The Specific alleles Segregating together in one known ancestry) focus on recent, usually observable ances family in a linkage study may well differ from alleles at the try in which there have been relatively few opportunities for Same loci Segregating together in another family. The iden recombination to occur. Thus, disease gene regions identi tities of the differing alleles of a marker that may coSegre fied by linkage will often be large, encompassing many tens gate with a disease trait in different families are not the relevant results in the analysis of linkage. Instead, the result of megabases of DNA. of import in linkage analysis is the finding that any allele of 0314. The power of genetic association analysis to detect a marker coSegregates with a disease trait in Separate genetic contributions to complex disease can be much affected families. Linkage is a property of the relative greater than that of linkage Studies. Linkage analysis can be position loci, not their alleles, and the observed coSegrega limited by a lack of power to exclude regions or to detect loci tion within a given family can involve any allele at the with modest effects. ASSociation tests can be capable of marker locus. detecting loci with Smaller effects Risch and Merikangas (1996) Science 273:1516-1517) which may not be detectable 0318 Linked markers that are not in linkage disequilib by linkage analysis. rium with a DNA segment associated with a disease may be used in determining a predisposition to or the occurrence of 0315. The aim of association studies when used to dis a genetic disease in an individual having an affected relative cover disease-Susceptibility genes is to identify particular of known genotype with respect to the marker. Thus, the genetic variants that correlate with the disease phenotype at allele of the polymorphic marker detected in Such methods the population level. ASSociation at the population level may of determining a predisposition to or the occurrence of a be used in the process of identifying a disease-Susceptibility disease must be in phase with a disease allele. Phase can be gene or DNA segment because it provides an indication that established by determining the presence or absence of an a particular marker is either a functional variant underlying individual's allele for a marker in two relatives (such as the disease (i.e., a polymorphism that is directly involved in relatives of the first or Second degree, and in particular, US 2003/017O678A1 Sep. 11, 2003 24 parents) of the individual who are of known phenotype with clinical definition of a given trait or phenotype. A genetic respect to the disease. For an individual's allele to be in trait may be analyzed by association methods by carefully phase, at least one of the relatives should have the disease Selecting the individuals to be included in the trait-positive and should also be heterozygous for the allele. Strategies for and trait-negative phenotypic groupS. Several criteria are Setting phase in different families are known see, e.g., often useful: clinical phenotype, age at onset, family history Lazarou (1993) Clin. Genet. 43:150-156). Within an affected and Severity. The Selection procedure for continuous or family, the allele of a marker that is in phase with a disease quantitative traits (Such as blood pressure, for example) allele is usually the same in each affected member of the involves Selecting individuals at opposite ends of the phe family because there is a low probability of recombination notype distribution of the trait under Study, So as to include between the two loci. However, in general, the phase rela in these trait-positive and trait-negative populations indi tionship between an allele of a polymorphic marker and viduals with non-overlapping phenotypes. Preferably, case disease allele is different in each family. If a particular allele control populations contain phenotypically homogeneous of a linked marker happens to be in linkage disequilibrium populations. Trait-positive and trait-negative populations with the disease allele, that same allele is likely to be in contain phenotypically uniform populations of individuals phase with the disease allele in any family. Methods for representing each between 1 and 98%, preferably between 1 determining a predisposition to or the occurrence of a and 80%, more preferably between 1 and 50%, and more disease using a linked marker rely on the availability of preferably between 1 and 30%, most preferably between 1 phenotype and genotype information of relatives, at least and 20% of the total population under study, and preferably one of whom has the disease. For late-onset diseases, Such Selected among individuals exhibiting non-overlapping phe information may not always be available. notypes. The clearer the difference between the two trait phenotypes, the greater the probability of detecting an 03.19. In contrast to genetic linkage, which is a property asSociation with markers. The Selection of those drastically of loci, genetic association is a property of alleles. ASSocia different but relatively uniform phenotypes enables efficient tion analysis involves a determiniation of a correlation comparisons in association Studies and the possible detec between a Single, Specific allele and a trait acroSS a popu tion of marked differences at the genetic level, provided that lation, not only within individual families. Thus, a particular the Sample sizes of the populations under Study are signifi allele found through an association Study to be in linkage cant enough. disequilibrium with a disease allele can form the basis of a method of determining a predisposition to or the occurrence 0325 Allelic frequencies of markers in populations can of the disease in any individual. Such methods would not be determined by genotyping pooled DNA samples or involve a determination of phase of an allele and thus would individual Samples. When each individual is genotyped not be limited in terms of the individuals who may be Separately, Simple gene counting may be applied to deter Screened in the method. mine the frequency of an allele or of a genotype in a given population. The proportional representation of the allele for 0320 3. Analysis of Genetic Association the population is then determined. 0321 Association studies explore the relationships 0326. The allelic frequencies of the marker in case and among frequencies for Sets of alleles between loci. ASSocia control populations are analyzed to determine whether a tion Studies may be conducted within the general population Statistically significant association exists between the geno and are not limited to Studies performed on related individu type and phenotype. The Statistical Significance of a corre als in affected families. There are Several methods for testing lation between phenotype and genotype may be determined for association. by any Statistical test known in the art and with any accepted threshold of Statistical significance being required. The 0322 a. Case-Control Studies application of particular methods and thresholds of Signifi 0323 The simplest form of association analysis is the cance are well within the level of skill of one skilled in the case-control Study in which unrelated populations of art. A commonly used Statistical test is a chi-square test with affected (or trait-positive) Subjects (i.e., case individuals) one degree of freedom. A P-value is calculated, which is the and unrelated control (unaffected, trait-negative or random) probability that a Statistic as large or larger than the observed individuals are analyzed. Such population-based association one would occur by chance. If a Statistically significant Studies do not concern familial inheritance but compare the asSociation with a trait is identified for at least one or more prevalence of a particular genetic marker or Set of markers of the analyzed markers, it may be assumed that either the in case-control populations. Marker allele frequencies in asSociated allele is directly responsible for causing the trait each population may be compared using a chi-squared or (i.e., the associated allele is the trait-causing allele), or more Fisher's exact test (see, e.g., linkage.rockefeller.edu/Soft likely the associated allele is in linkage disequilibrium with ware/utilities). the trait-causing allele. 0324. The control group is typically “matched” as much 0327 In testing the association of a particular allele as possible to the case population, particularly to avoid against the disease phenotype, it may be useful to correct the problems of population Stratification. Thus, the control results. One Such correction method is referred to as the group may be ethnically matched to the case population and “Bonferroni” correction in which the probability value matched for the main known confusion factor for the trait required to give Significance is divided by the number of under Study (e.g., age-matched for an age-dependent trait). tests conducted. For example, if five markers are tested, each An important Step in the dissection of complex traits using with five alleles, a probability value of 0.002 would be asSociation Studies is the choice of case-control populations required to declare significance at the 5% level. see Lander and Schork (1994) Science 265:2037-2048). A 0328 Case-control studies can be susceptible to false major Step in the choice of case-control populations is the positive (type I) and false negative (type II) errors. Thus, a US 2003/017O678A1 Sep. 11, 2003
negative result may mean a lack of association or a false Depending on the data structure, TDTs are tests of either negative due to insufficient power to detect association. A linkage or linkage and association. The proposed test Sta positive result may mean an allelic association with disease, tistic is a McNemar's chi-Square and tests the null hypoth the presence of an unknown factor Such as population esis that the putative disease-associated alleles transmitted Stratification between cases and controls or a false positive 50% of the time from heterozygous parents; under the due to an insufficient Sample size for the tests being con alternative hypothesis, the disease-associated allele will be ducted. Calculators (see, e.g., http://www.stat.ucla.edu/cal transmitted more often. culators/powercalc/binomial/case-control/b-case-control 0333. The TDT has been extended to take into account Samp.html) are available to estimate required Sample size for multiallelic marker loci Spielman and Ewens (1996) Am. J. a given marker frequency, relative risk of interest, power and Hum. Genet. 59:983-989; Sham and Curtis (1995) Ann. Significance level (corrected if necessary for multiple tests). Hum. Genet. 59:323-336; Bickeboeller and Clerget-Dar 0329. Typical association studies based on candidate poux (1995) Genet. Epidemiol. 12:865-870; and Rice et al. genes, and in particular, case-control Studies, may have a (1995) Genet. Epidemiol. 12:659-664), the availability of limited ability to discern true medium-sized signals from only one parent Sun et al. (1999) Am. J. Epidemiol. 150:97 false positives see, e.g., Emahazion et al. (2001) Trends 104), analysis of affected sibs or trios Martin et al. (1997) Genet. 17:407-413). Thus, reports of positive association Am. J. Hum. Genet. 61:439-448), multiple analysis of linked findings frequently cannot be replicated. For example, an alleles in haplotypes Clayton and Jones (1999) Am. J. Hum. initial report of a positive association of the GG-genotype of Genet. 65:1161-1169 and Clayton (1999) Am. J. Hum. the TNFRSF6 gene with early-onset AD obtained in an Genet. 65:1170-1177, pooled genotyping of affected chil unrelated case (Scottish sporadic early-onset AD Sample)- dren Risch and Teng (1998) Genome Res. 8:1273-1288) and control study Feuket al. (2000) Hum. Genet. 107:391-396) transmission from parents homozygous at a tightly linked was not replicated in a Subsequent case (Dutch population locus Lie et al. (1999) Am. J. Hum. Genet. 64.793-800). early-onset AD sample)-control study Theuns et al. (2001) Family-based tests, Such as TDT, have largely required Hum. Genet. 108:552-553). A case (Swedish patients with knowledge of parental marker genotypes; however, for sporadic late-onset AD)-control study of 15 polymorphisms late-onset diseases, parental data are often not available. in candidate genes for sporadic AD that previously had been There are tests of linkage and association that use unaffected reported to have significant association with AD found no Siblings as Surrogates for untyped parents from which prob Strong evidence of association for any of the loci in the able parental genotypes may be derived Spielman and Swedish population Prince et al. (2001) Eur: J. Hum. Genet. Ewens (1998) Am. J. Hum. Genet. 62:450-458 (also referred 9:437-444). to as the sib-TDT or S-TDT); Horvath and Laird (1998) Am. J. Hum. Genet. 63:1886-1897; Boehnke and Langefeld 0330 b. Case-Control Studies Using Family-Based Con (1998) Am. J. Hum. Genet. 62:950-961). The discordant trols unaffected Sibling provides information on the alleles not 0331 Case-control studies using family-based controls Segregating to affected individuals. have been developed to address possible errorS relating to 0334. The FBAT is a unified approach to family-based inadequate matching of unrelated cases and controls. Unlike asSociation testing that is similar in design to the TDT but case-control tests, family-based tests are not affected by can accomodate variations in pedigree Structures, arbitrary population Stratification, which can lead to Spurious asso missing genotype information and various different disease ciations of a marker allele with disease Susceptibility. Such models Rabinowitz and Laird (2000) Hum. Hered. 50:227 analytical techniques include the transmission disequilib 233; Laird et al. (2000) Genet. Epi. 19 (Suppl. 1):S36-S42). rium test (TDT) Spielman et al. (1993) Am. J. Hum. Genet. To account for the presence of linkage when using multiple 52:506-516), affected family based control test (AFBAC) affected siblings per nuclear family, the FBAT allows robust Thomson (1995) Am. J. Hum. Genet. 57:487-498 and variance estimation (referred to as EV-FBAT) based on the Schaid and Sommer (1994) Am. J. Hum. Genet. 55:402-409 empirical variance-covariance matrix of the contributions of and the haplotype relative risk test (HRR) Falk and Rubin each family to the score statistic Lake et al. (2000) Am. J. stein (1987) Ann. Hum. Genet. 51:227-233; Terwilliger and Hum. Genet. 67:1515-1525). Ott (1992) Hum. Hered. 42:337-346). In these methods, family members (usually unaffected) can be used as internal 0335) A number of computer software programs are controls. In the HRR and AFBAC tests, an affected indi available for Statistical analysis of genotyping data in fam vidual and two parents are typed for a marker hypothesized ily-based association tests, including the FBAT program to have an allele associated with the disease. The number of Rabinowitz and Laird (2000) Hum. Hered. 50:211-223; see control alleles are derived from the parental alleles not also http://www.biostat.harvard.edu/fbat/default.html), the transmitted to the affected child, and these are compared to GASSOC program of Statistical methods including an exten the number of alleles transmitted to the affected child by a sion of the TDT for multiple marker alleles Schaid (1996) chi-squared test. In the TDT test, one of the parents must be Genet. Epidemiol. 13:423-449; see also http://www.mayo.e- heterozygous for the marker concerned, and the comparison du.statgen/gas.Soc., the Quantitative (Trait) Transmission/ is made between the alleles that are transmitted to the Disequilibrium Test (QTDT) which includes support for the affected child and those that are not. Deviation from the methods of Abecasis et al. (2000) Am. J. Hum. Genet. expected Mendelian 50% transmission is tested by a chi 66:279-292), Fulker et al. (1999) Am. J. Hum. Genet. Squared or Fisher's exact test. 64:259-267), Monks et al. (1998) Am. J. Hum. Genet. 63:1507-1516), Allison (1997) Am. J. Hum. Genet. 60:676 0332 The TDT focuses on alleles transmitted to affected 690; TDTO5) and Rabinowitz (1997) Hum. Hered 47:342 offspring, but is formulated to take account of both the 350See also http://www.well.ox.ac.uk/asthma/OTDT), the linkage and the disequilibrium that underlie the association. Transmission Disequilibrium Test and SIB Transmission US 2003/017O678A1 Sep. 11, 2003 26
Disequilibrium Test (TDT/S-TDT) Spielman et al. (1993) cM, or about 40 cM, or about 35 cM, or about 30 cM, or Am. J. Hum. Genet. 52:506-516 and Spielman and Ewens about 25 cM, or about 20 cM, or about 15 cM, or about 13 (1998) Am. J. Hum. Genet. 62:450-458; see also http:// cM, or about 12 cM, or about 9 cM, or about 5 cM, or about spielman07.med.upenn.edu/TDT.htm, the ASSOC program 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 cM in the Statistical Analysis for Genetic Epidemiology (SAGE) distal (telomeric) from and including marker D10S583, (3) program uses the method of George and Elston (1987) the region extending from and including marker D10S583 to Genet. Epidemiol. 4:193-201; see also http://darwin.cwr the terminus of the q arm of chromosome 10, (4) the region u.edu/pub/sage.html and TRANSMIT see http://www.mrc extending from and including marker D10S583 to the cen bSu.cam.ac.uk/pub/methodology/genetics/. tromere of chromosome 10, (5) the region extending about 62 Mb, about 55 Mb, or about 50 Mb, or about 32 Mb, or 0336 4. Genetic Markers Associated with AD on Chro about 28 Mb, or about 25 Mb, or about Mb, or about 15 Mb, mosome 10 or about 10 Mb, or about 5 Mb, or about 2 Mb, or about 1 0337 As shown by the results of association analyses of Mb, or about 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, or genotyping data with respect to marker D10S583 located on about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or about chromosome 10 (see EXAMPLE 3), provided herein are 1 kb proximal from and including marker D10S583, (6) the genetic markers on chromosome 10 which have an allele region extending about 45 Mb, or about 40 Mb, or about 35 that is associated with AD. Included within the markers Mb, or about 30 Mb, or about Mb, or about 20 Mb, or about provided herein are markers located in the region of 10q22, 16 Mb, or about 14 Mb, or about 13 Mb, or about 8 Mb, or 10q23, 10q24, 10q25 and/or 10q26. In particular, markers about 5 Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, located in the region of 10q22, 10q23 and/or 10q24 or in the or about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or about region of 10q23, 10q24 and/or 10q25 are provided. In 0.05 Mb, or about 0.01 Mb, or about 1 kb distal from and particular embodiments of the markers associated with AD, including marker D10S583, (7) the region between the markers are located in a region of chromosome 10 about D10S564 and D10S583, inclusive, (8) the region between 30 cM around and including D10S583, or about 20 cM D10S583 and D10S1710, inclusive, (9) the region between around and including D10S583, or about 15 cM around and D10S583 and D10S566, inclusive, (10) the region between including D10S583, or about 12 cM around and including D10S583 and D10S1671, inclusive and (11) the region D10S583, or about 10 cM around and including D10S583. between D10S583 and D10S1741, inclusive. A particular In other particular embodiments of the markers associated marker associated with AD provided herein is D10S583. with AD, the markers are located in a region of chromosome Particular regions of chromosome 10 in which markers 10 about 5 cM around and including D10S583, or about 4 asSociated with AD are located can depend on Several cM around and including D10S583, or about 3 cM around factors, including, for example, population. and including D10S583, or about 2 cM around D10S583, or 0339. In other embodiments of the markers provided about 1 cMaround and including D10S583, or about 0.5 cM herein, the markers are located in the following regions of around and including D10S583, or about 0.25 cM around chromosome 10: (1) the region extending about 5 cM, or and including D10S583, or about 0.1 cM around and includ about 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 ing D10S583. In further embodiments of the markers asso cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or ciated with AD, the markers are located in a region of about 0.1 cM proximal (centromeric) from and including chromosome 10 about 30 Mb around and including marker D10S583 (2) the region extending about 5 cM, or D10S583, or about 28 Mb around and including D10S583, about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 or about 20 Mb around and including D10S583, or about 15 cM distal (telomeric) from and including marker D10S583, Mb around and including D10S583, or about 10 Mb around (3) the region extending about 10 Mb, or about 5 Mb, or and including D10S583. In yet further embodiments of the about 2 Mb, or about 1 Mb, or about 500 kb, or about 300 markers associated with AD, the markers are located in a kb, or about 200 kb, or about 100 kb, or about 80 kb, or region of chromosome 10 about 5 Mb around and including about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb D10S583, or about 2.5 Mb around and including D10S583, proximal from and including marker D10S583, and (4) the or about 1 Mb around and including D10S583 or about 500 region extending about 10 Mb, or about 8 Mb, or about 5 kb around and including D10S583, or about 200 kb around Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about and including D10S583, or about 100 kb around and includ 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or ing D110S583, or about 50 kb around and including about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, D10S583, or about 40 kb around and including D10S583, or or about 1 kb distal from and including marker D10S583. about 20 kb around and including D10S583, or about 10 kb 0340 Genetic markers on chromosome 10 associated around and including D10S583, or about 5 kb around and with AD yield scores or results indicative of association with including D10S583, or about 1 kb around and including AD or linkage disequilibrium with an AD DNA segment D1OS583. when tested in association or linkage disequilibrium assess 0338 Further particular regions of chromosome 10 in ment methods. Such methods include but are not limited to which markers associated with AD are located include: (1) case-control Studies and family-based association tech the region extending about 50 cM, or about 45 cM, or about niques. In addition, Such methods can involve separate or 33 cM, or about 30 cM, or about 25 cM, or about 21 cM, or combined tests for linkage of the marker to AD. Preferred about 20 cM, or about 15 cM, or about 10 cM, or about 5 methods are family-based association techniques. Particular cM, or about 4 cM, or about 3 cM, or about 2.7 cM, or about markers associated with AD provided herein yield Scores 2.5 cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or that may be considered Significant evidence of association about 0.5 cM, or about 0.1 cM proximal (centromeric) from with AD or linkage disequilibrium with an AD DNA seg and including marker D10S583 (2) the region extending ment. For example, Such a Score would include a P value about 60 cM, or about 55 cM, or about 50 cM, or about 45 <0.5 and, in particular, a P value <0.5. US 2003/017O678A1 Sep. 11, 2003 27
0341 Particular markers associated with AD further D10S583, or about 0.25 cMaround and including D10S583, include any genetic markers located on chromosome 10, or about 0.1 cM around and including D10S583. In further including but not limited to the markers listed in FIG. 1, that embodiments of the combinations of markers associated yield Scores or results indicative of association with AD or with AD, the markers are located in a region of chromosome linkage disequilibrium with an AD DNA segment when 10 about 30 Mbaround and including D10S583, or about 28 tested in association assessment methods described herein Mb around and including D10S583, or about 20 Mb around and known to those of skill in the art. Included within these and including D10S583, or about 15 Mb around and includ particular markers are any of the following markers that ing D10S583, or about 10 Mb around and including yield Scores or results indicative of association with AD or D10S583. In yet further embodiments of the combinations linkage disequilibrium with an AD DNA segment when of markers associated with AD, the markers are located in a tested in association assessment methods: D10S564, region of chromosome 10 about 5 Mb around and including D10S2470, D10S1755, D10S536, D10S185, D10S1171, D10S583, or about 2.5 Mb around and including D10S583, D10S1173, D10S200, D10S1680, D10S520, D10S1736, or about 1 Mb around and including D10S583 or about 500 D10S574, D10S571, D1OS 1690, ATA105C09, kb around and including D10S583, or about 200 kb around GATA30F07a, D10S677, D10S91, D10S1758, D10S577, and including D10S583, or about 100 kb around and includ D10S1709, D10S184, D10S1726, D10S198, D10S1123, ing D10S583, or about 50 kb around and including D10S1147, D10S603, D10S1266, D10S1265, D10S1264, D10S583, or about 40 kb around and including D10S583, or D10S1778, D10S192, FB7F11, D10S1267, D10S1692, about 20 kb around and including D10S583, or about 10 kb D10S1738, D10S205, D10S1668, D1OS1268, D1OS222, around and including D10S583, or about 5 kb around and D10S1697, GATA64B01, D10S540, D10S467, including D10S583, or about 1 kb around and including GATA114HO9, D1OS254, D10S1663, D10S530, D10S534, D1OS583. D10S521, D1OS108, D10S1741, D10S1760, D10S1121, D10S1795, D10S1750, D10S1246, D10S597, D10S543, 0343. In further embodiments of the combinations of D1OS88, D1OS1682, D10S554, GATA83D05, D10S168, genetic markers associated with AD, the markers are located D10S1269, D10S1681, D10S1776, D10S562, D10S1748, in one or more of the following regions of chromosome 10: D10S1773, D10S1731, ATA103C06, D10S468, D10S1237, (1) the region extending about 50 cM, or about 45 cM, or D10S1158, D10S528, D10S1683, D10S544, D10S531, about 33 cM, or about 30 cM, or about 25 cM, or about 21 D10S1657, D10S545, D10S187, D10S1165, D10S1156, cM, or about 20 cM, or about 15 cM, or about 10 cM, or D10S221, D10S1425, D10S 1693, D10S 1722, D10S1141, about 5 cM, or about 4 cM, or about 3 cM, or about 2.7 cM, D10S1236, D10S190, D10S503, GATA127H01, D10S1785, or about 2.5 cM, or about 2 cM, or about 1.5 cM, or about D10S542, D10S1701, D10S1792, D10S1740, D10S1757, 1 cM, or about 0.5 cM, or about 0.1 cM proximal (centro D10S209, D10S1230, D10S1483, D10S1679, D10S587, meric) from and including marker D10S583 (2) the region D10S1708, D10S1723, D10S1213, D10S2322, D10S216, extending about 60 cM, or about 55 cM, or about 50 cM, or D10S1656, D10S12, D10S575, D10S214, D10S1703, about 45 cM, or about 40 cM, or about 35 cM, or about 30 D10S186, D10S1782, D10S1222, D10S1727, D10S217, cM, or about 25 cM, or about 20 cM, or about 15 cM, or D10S1676, D10S1134, D1OS1655, GGAST14G01, about 13 cM, or about 12 cM, or about 9 cM, or about 5 cM, D10S505, D10S1248, D10S1770, D10S1651, D10S590, or about 2.5 cM, or about 1 cM, or about 0.5 cM or about D10S1675, 1OTEL23, D10S212, D10S555, D10S1711, 0.1 cM distal (telomeric) from and including marker D10S1700, D10S169, D10S 1221, D10S 1223, ATA25D02, D10S583, (3) the region extending from and including marker D10S583 to the terminus of the q arm of chromo D10S1229, D1OS1232, ATA42E11, D10S1239, D10S1423, Some 10, (4) the region extending from and including D10S1435, D1OS110, D1OS466, D10S525, D10S1148, marker D10S583 to the centromere of chromosome 10, (5) D10S1149, D10S1150 and D10S1163. the region extending about 62 Mb, about 55 Mb, or about 50 0342. Also provided herein are combinations of genetic Mb, or about 32 Mb, or about 28 Mb, or about 25 Mb, or markers on human chromosome 10 which are associated about Mb, or about 15 Mb, or about 10 Mb, or about 5 Mb, with AD. In particular embodiments, the markers in the or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.3 combinations are located on chromosome 10q. Such com Mb, or about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or binations include markers located in the region of 10q22, about 0.01 Mb, or about 1 kb proximal from and including 10q23, 10q24, 10q25 and/or 10q26. In further embodiments marker D10S583, (6) the region extending about 45 Mb, or of the combinations of genetic markers, the markers are about 40 Mb, or about 35 Mb, or about 30 Mb, or about Mb, located in the region of 10q22, 10q23 and/or 10q24 or in the or about 20 Mb, or about 16 Mb, or about 14 Mb, or about region of 10q23, 10q24 and/or 10q25. In particular embodi 13 Mb, or about 8 Mb, or about 5 Mb, or about 4 Mb, or ments, the markers contained in a combination of markers about 3 Mb, or about 2 Mb, or about 1 Mb, or about 0.5 Mb, asSociated with AD are located in a region of chromosome or about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or 10 about 30 cMaround and including D10S583, or about 20 about 1 kb distal from and including marker D10S583, (7) cM around and including D10S583, or about 15 cM around the region between D10S564 and D10S583, inclusive, (8) and including D10S583, or about 12 cM around and includ the region between D10S583 and D10S1710, inclusive, (9) ing D10S583, or about 10 cM around and including the region between D10S583 and D10S566, inclusive, (10) D10S583. In other particular embodiments of the combina the region between D10S583 and D10S1671, inclusive and tions of markers associated with AD, the markers are located (11) the region between D10S583 and D10S1741, inclusive. in a region of chromosome 10 about 5 cM around and A particular marker associated with AD provided herein is including D10S583, or about 4 cM around and including D10S583. Particular regions of chromosome 10 in which D10S583, or about 3 cM around and including D10S583, or markers that may be contained within the combinations of about 2 cM around D10S583, or about 1 cM around and genetic markers are located can depend on Several factors, including D10S583, or about 0.5 cM around and including including, for example, population. US 2003/017O678A1 Sep. 11, 2003 28
0344) In other embodiments of the combinations of the region extending about 50 cM, or about 45 cM, or about genetic markers associated with AD provided herein, the 33 cM, or about 30 cM, or about 25 cM, or about 21 cM, or markers are located in one or more of the following regions about 20 cM, or about 15 cM, or about 10 cM, or about 5 of chromosome 10: (1) the region extending about 5 cM, or cM, or about 4 cM, or about 3 cM, or about 2.7 cM, or about about 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 2.5 cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or about 0.5 cM, or about 0.1 cM proximal (centromeric) from about 0.1 cM proximal (centromeric) from and including and including marker D10S583 (2) the region extending marker D10S583 (2) the region extending about 5 cM, or about 60 cM, or about 55 cM, or about 50 cM, or about 45 about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 cM, or about 40 cM, or about 35 cM, or about 30 cM, or cM distal (telomeric) from and including marker D10S583, about 25 cM, or about 20 cM, or about 15 cM, or about 13 (3) the region extending about 10 Mb, or about 5 Mb, or cM, or about 12 cM, or about 9 cM, or about 5 cM, or about about 2 Mb, or about 1 Mb, or about 500 kb, or about 300 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 cM kb, or about 200 kb, or about 100 kb, or about 80 kb, or distal (telomeric) from and including marker D10S583, (3) about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb the region extending from and including marker D10S583 to proximal from and including marker D10S583, and (4) the the terminus of the q arm of chromosome 10, (4) the region region extending about 10 Mb, or about 8 Mb, or about 5 extending from and including marker D10S583 to the cen Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about tromere of chromosome 10, (5) the region extending about 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or 62 Mb, about 55 Mb, or about 50 Mb, or about 32 Mb, or about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, about 28 Mb, or about 25 Mb, or about 20 Mb, or about 15 or about 1 kb distal from and including marker D10S583. Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, 0345 Combinations of genetic markers associated with or about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or AD have a variety of uses. For example, haplotypes asso about 1 kb proximal from and including marker D10S583, ciated with AD may be used in all the methods described (6) the region extending about 45 Mb, or about 40 Mb, or herein that utilize one or more genetic markers associated about 35 Mb, or about 30 Mb, or about 25 Mb, or about 20 with AD. Such haplotypes associated with AD thus may be Mb, or about 16 Mb, or about 14 Mb, or about 13 Mb, or used in methods of identifying the presence or absence in a about 8 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, Subject of a combination of polymorphic markers associated or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.1 with AD by analyzing chromosome 10, and in particular Mb, or about 0.05 Mb, or about 0.01 Mb, or about 1 kb distal chromosome 10q, of the Subject for Such a combination, in methods of indicating a predisposition to or the occurrence from and including marker D10S583, (7) the region between of AD in a Subject, methods of confirming a phenotypic D10S564 and D10S583, inclusive, (8) the region between diagnosis of AD in a Subject, methods of predicting a D10S583 and D10S1710, inclusive, (9) the region between response of a Subject to a drug used to treat AD, methods of D10S583 and D10S566, inclusive, (10) the region between treating a Subject manifesting an AD phenotype, and meth D10S583 and D10S1671, inclusive and (11) the region ods of identifying an AD DNA segment or gene as described between D10S583 and D10S1741, inclusive. herein. 0349 Further particular regions of chromosome 10 in which the polymorphic marker is located include: (1) the 0346 5. Methods for Identifying Genetic Markers Asso region extending about 5 cM, or about 4 cM, or about 2.7 ciated with AD cM, or about 2.5 cM, or about 2 cM, or about 1.5 cM, or 0347 Also provided herein are methods of identifying about 1 cM, or about 0.5 cM, or about 0.1 cM proximal genetic markers associated with AD. The methods include a (centromeric) from and including marker D10S583 (2) the Step of testing a polymorphic marker on chromosome 10 for region extending about 5 cM, or about 2.5 cM, or about 1 asSociation with AD. The testing may involve genotyping cM, or about 0.5 cM or about 0.1 cM distal (telomeric) from DNA from individuals affected with AD, and possibly also and including marker D10S583, (3) the region extending from related or unrelated individuals, with respect to the about 10 Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, polymorphic marker and analyzing the genotyping data for or about 500 kb, or about 300 kb, or about 200 kb, or about asSociation with AD using methods described herein and/or 100 kb, or about 80 kb, or about 50 kb, or about 10 kb, or known to those of skill in the art. For example, Statistical about 5 kb, or about 1 kb proximal from and including analysis of the data may involve a chi-squared or Fisher's marker D10S583, and (4) the region extending about 10 Mb, exact test and may be conducted in conjunction with a or about 8 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, number of programs, Such as the transmission disequilib or about 2 Mb, or about 1 Mb, or about 500 kb, or about 300 rium test (TDT), affected family based control test (AFBAC) kb, or about 200 kb, or about 100 kb, or about 50 kb, or and the haplotype relative risk test (HRR). Case-control about 10 kb, or about 5 kb, or about 1 kb distal from and Strategies can be applied to the testing, as can, for example, including marker D10S583. TDT approaches. 0350 E. Linkage of AD on chromosome 10 0348. In particular embodiments of the methods of iden 0351 Genetic markers linked to a DNA segment associ tifying genetic markers associated with AD, the polymor ated with AD have been identified and are described herein. phic marker is located in the region of 10q22, 10q23, 10q24, The markers are located on human chromosome 10q. Par 10q25 or 10q26. In further embodiments, the marker is ticular markers are located on chromosome 10q22, 10q23, located on chromosome 10q22, 10q23 or 10q24. In yet 10q24, 10q25 or 10q26. The markers tend to coSegregate further embodiments, the marker is located on chromosome with a DNA segment associated with AD, such as an AD 10q23, 10q24 or 10q25. Particular regions of chromosome gene, in families affected with AD. The markers can be 10 in which the polymorphic marker is located include: (1) identified through any linkage assessment methods US 2003/017O678A1 Sep. 11, 2003 29 described herein or known to those of skill in the art, and about 1.5 cM from the DNA segment, or less than about 1.0 provide Scores or results indicative of linkage to AD when cM from the DNA segment, or less than about 0.75 cM from tested by Such linkage determination methods. The markers the DNA segment, or less than about 0.5 cM from the DNA may be used in a variety of methods. For example, a method segment or less than about 0.25 cM from the DNA segment, for detecting the presence or absence in a Subject of a or less than about 0.2 cM from the DNA segment or less than polymorphism linked to a DNA segment associated with about 0.15 cM from the DNA segment or less than about 0.1 Alzheimer's disease includes a Step of analyzing chromo cM from the DNA segment. The particular distance depends Some 10 of the subject for a polymorphism linked to a DNA on the particular marker. The linked markers on chromo Segment associated with Alzheimer's disease. A method for Some 10 may be located within a DNA segment associated identifying a polymorphism linked to a DNA segment with AD and may be a polymorphism in an AD gene, Such asSociated with Alzheimer's disease, includes a step of analyzing a genetic marker on chromosome 10 for linkage to as, for example, a polymorphism in an AD gene that is Alzheimer's disease characterized by a significant or highly responsible for a defect in an AD gene. When the marker is Significant LOD score. Another method for identifying a located within an AD gene, it is referred to as coincident polymorphism linked to a DNA segment associated with with the gene. Alzheimer's disease, includes a step of analyzing a genetic 0355. If two loci are situated on different chromosomes, marker on chromosome 10 for linkage to Alzheimer's dis the transmission of alleles from generation to generation of ease, wherein the marker is located in the region of 10q22, each locus will be random and they are Said to be 10q23, 10q24, 10q25 or 10q26. “unlinked.” If two loci are situated on the same chromo 0352 1. Genetic Linkage Some, the transmission of alleles of one locus will be affected by the presence of the other locus such that the 0353. The closer together two sequences are on a chro ratioS of alleles are no longer independent, and the loci are moSome, the less likely that a recombination event will referred to as “linked.” Thus, two loci may be said to be occur between them, and the more closely linked they are. linked when they are located relatively close together on the Thus, the recombination frequency, i.e., the probability that Same chromosome. Genetic markers provided herein are there is a recombination event between two loci (also located Sufficiently close to a DNA segment associated with referred to as the recombination fraction), can be used as a AD on chromosome 10 Such that the marker and DNA measure of the genetic distance between two gene loci. A Segment are linked. recombination frequency of 1% is equivalent to 1 map unit, or 1 centimorgan (cM), which is roughly equivalent to 1,000 0356. For example, in terms of the physical distance kb of DNA. Loci that segregate independently within a between the linked markers and a DNA segment associated family are unlinked and have a recombination fraction of with AD on chromosome 10, the markers may be less than 50%, whereas linked loci coSegregate within a family and about 72 Mb from the DNA segment, or less than about 65 have a recombination fraction of less than about 50%. For Mb from the DNA segment, or less than about 63 Mb from example, genetic markers linked to a DNA segment associ the DNA segment, or less than about 59 Mb from the DNA ated with AD on chromosome 10 may have a recombination segment, or less than about 55 Mb from the DNA segment, fraction of less than about 50%, or about 45% or less, or or less than about 50Mb from the DNA segment, or less than about 40% or less, or about 35% or less, or about 30% or about 45 Mb from the DNA segment, or less than about 40 less, or about 25% or less, or about 20% or less, or about Mb from the DNA segment, or less than about 35 Mb from 15% or less, or about 10% or less, or about 5% or less or the DNA segment, or less than about 30 Mb from the DNA about 2.5% or less, or about 2% or less, or about 1.5% or less segment, or less than about 25 Mb from the DNA segment, or about 1% or less or about 0.5% or less, or about 0.1% or or less than about 20 Mb from the DNA segment, or less than less, or about 0. The particular recombination fraction about 15 Mb from the DNA segment, or less than about 10 depends on the particular marker. Mb from the DNA segment, or less than about 5 Mb from the DNA segment, or less than about 2.5 Mb from the DNA 0354 For example, in terms of the genetic distance segment, or less than about 1 Mb from the DNA segment, or between the linked markers on chromosome 10 and a DNA less than about 0.5 Mb from the DNA segment, or less than Segment associated with AD on chromosome 10, the mark about 0.1 Mb from the DNA segment, or less than about 0.05 ers may be less than about 85 cM from the DNA segment, Mb from the DNA segment, or less than about 0.01 Mb from or less than about 80 cM from the DNA segment, or less than the DNA segment, or less than about 0.005 Mb from the about 75 cM from the DNA segment, or less than about 70 DNA segment, or less than about 0.001 Mb from the DNA cM from the DNA segment, or less than about 65 cM from Segment. The particular distance depends on the particular the DNA segment, or less than about 60 cM from the DNA marker. segment, or less than about 55 cM from the DNA segment, or less than about 50 cM from the DNA segment, or less than 0357 Two loci are completely linked when there is no about 45 cM from the DNA segment, or less than about 40 recombination between them; the same alleles or phenotypes cM from the DNA segment, or less than about 35 cM from are always transmitted together from generation to genera the DNA segment, or less than about 30 cM from the DNA tion within a family. An intermediate State of linkage, segment, or less than about 25 cM from the DNA segment, referred to as “incomplete linkage” occurs when the trans or less than about 20 cM from the DNA segment, or less than mission of alleles of two loci deviates consistently and about 15 cM from the DNA segment, or less than about 10 measurably from independent assortment (e.g., random cM from the DNA segment, or less than about 5 cM from the transmission of alleles located on different chromosomes) DNA segment, or less than about 4 cM from the DNA but a consistent recombination fraction nonetheless exists segment, or less than about 3 cM from the DNA segment, or for the loci see, e.g., March (1999) Mol. Biotechnol. less than about 2 cM from the DNA segment, or less than 13:113-122). US 2003/017O678A1 Sep. 11, 2003 30
0358 2. Analysis of Genetic Linkage Hum. Genet. 61:1179), ASPEX see, e.g., Badner et al. 0359 Linkage analysis is based upon establishing a cor (1998) Am. J. Hum. Genet. 63:880-888; Hauser et al. (1996) relation between the transmission of genetic markers and Genet. Epidemiol 13:117-137; Davis and Weeks (1997) Am. that of a Specific trait or trait gene throughout generations J. Hum. Genet. 61:1431-1444) and LINKAGE see Lathrop within a family. Thus the aim of linkage analysis is to detect et al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81:3443-3446). marker loci that show coSegregation with a trait of interest 0364 b. Linkage Measurement in a pedigree. 0365 Linkage may be assessed by the LOD (logarithm of 0360) a. Procedures an odds ratio) score method Morton (1955) Am. J. Hum. 0361. In conducting linkage analysis, two positions on a Genet. 7:277-318; Rice et al. (2001) Adv. Genet. 42:99-113) chromosome are followed from one generation to the next or other acceptable statistical linkage determination See also within a family to determine the frequency of recombination Ott (1991) Analysis of Human Genetic Linkage, Baltimore, between them. This can be accomplished by genotyping London, John Hopkins; Terwilliger and Ott (1994) Hand DNA from fully informative individuals within pedigrees book of Human Genetic Linkage, Baltimore, John Hopkins and counting recombinants and nonrecombinants. In a Study University Press; Strachan and Read (1996) Human Molecu of an inherited disease, Such as AD, one chromosomal lar Genetics, Oxford. BIOS Scientific Publishers Ltd.; Sud position, or locus, is marked by the disease gene and the bery (1998) Human Molecular Genetics, Harlow, Addison other position is marked by a DNA sequence (referred to as Wesley Longman; Lander and Schork (1994) Science genetic marker) that shows natural variation in the popula 265:2037-2048). In linkage analysis, a series of likelihood tion, e.g., variable number of tandem repeats (VNTRs), such ratios (relative odds) at various possible values of 0, ranging as minisatellites and microSatellites, Single nucleotide poly from 0 (no recombination) to 0.50 (random assortment) are morphisms (SNPs) and restriction fragment length polymor calculated. The computed likelihoods are usually expressed phisms (RFLPs). RFLPs are variations that modify the as the logarithm of the likelihood ratio (LOD). The use of length of a restriction fragment. Minisatellites are tandemly logarithms allows data collected from different families to repeated DNA sequences present in units of about 5-50 or be combined by Simple addition. Computer programs are more repeats which are distributed along regions of human available that run the analyses involved in Statistical linkage chromosomes ranging from 0.1-20 kb in length. MicroSat determination see, e.g., LIPED; MLINK; Lathrop et al. ellites are tandemly repeated DNA sequences typically (1984) Proc. Natl. Acad. Sci. U.S.A. 81:3443-3446; Terwil present in repeats of lesser units, e.g., up to 4 repeats, than liger and Ott (1994) Handbook of Human Genetic Linkage, those of minisatellites. Because microSatellites and minisat Baltimore, John Hopkins University Press and http://link elliteS present many possible alleles, their informative con age.rockefeller.edu/soft/list.html). A LOD score is the loga tent is very high. SNPs are densely spaced in the human rithm of the ratio of the likelihood that two loci are linked genome and represent the most frequent type of variation. at a given distance (or recombination fraction 0) to the likelihood that they are not linked (recombination fraction 0362 Inheritance of a marker can be determined by 0=0.5; greater than 50 cM apart). The value of 0 at which analyzing DNA from each individual for the presence or the LOD score is the highest is considered to be the best absence of the marker whereas inheritance of the disease estimate of the recombination fraction, the "maximum like gene can be determined by examining whether the indi lihood estimate.” vidual displayS Symptoms of the disease or is a parent of an affected individual or not. In every family, the inheritance of 0366 Positive LOD scores can be considered as evidence the genetic marker is compared to the inheritance of the of linkage. Genetic markers on chromosome 10 linked to a disease State. DNA segment associated with AD yield positive LOD scores when analyzed for linkage to AD by a LOD score 0363 Linkage analysis may be two-point, i.e., comparing method. The positive LOD score may be greater than or the Segregation of a marker and a disease, or multipoint, i.e., equal to about 1.0, or greater than or equal to about 1.5, or Simultaneous analysis of linkage between the disease and greater than or equal to about 1.9, or greater than or equal to Several genetic markers. Multipoint analysis can be advan 2.0, or greater than or equal to about 2.2, or greater than or tageous in mapping a disease gene. For example, the infor equal to about 2.6, or greater than or equal to about 2.7, or mativeness of the pedigree is usually increased in multipoint greater than or equal to about 2.8, or greater than or equal to analysis. Each pedigree has a certain amount of potential about 3.0, or greater than or equal to about 3.12, or greater information, dependent on the number of parents heterozy than or equal to about 3.2, or greater than or equal to about 3.5, or greater than or equal to about 4.0, or greater than or gous for the marker loci and the number of affected indi equal to about 4.5, or greater than or equal to about 5.0, or viduals in the family. However, not all markers are suffi greater than or equal to about 5.4, or greater than or equal to ciently polymorphic as to be informative in all those about 5.5, or greater than or equal to about 6.0, or greater individuals. If multiple markers are considered Simulta than or equal to about 6.5, or greater than or equal to about neously, then the probability of an individual being het 7.0, or greater than or equal to about 7.5, or greater than or erozygous for at least one of the markers is greatly equal to about 8.0, or greater than or equal to about 8.5, or increased. In addition, an indication of the position of the greater than or equal to about 9.0, or greater than or equal to disease gene among the markers may be determined in about 9.5, or greater than or equal to about 10.0 or greater multipoint analysis. This may allow identification of flank than or equal to about 10.5, or greater than or equal to about ing markers, and thus eventually allows isolation of a Small 11.0, or greater than or equal to about 11.5, or greater than region in which the disease gene resides. Examples of or equal to about 12.0, or greater than or equal to about 12.5, computer Software which may be used for multipoint analy or greater than or equal to about 13.0 or greater than or equal sis include GENEHUNTER-PLUS Kruglyak et al. (1996) to about 13.5, or greater than or equal to about 14.0 or Am. J. Hum. Genet. 58:1347; Kong and Cox (1997) Am. J. greater than or equal to about 14.5, or greater than or equal US 2003/017O678A1 Sep. 11, 2003
to about 15.0, or greater than or equal to about 15.5, or sity Press applied to large pedigrees with many affected greater than or equal to about 16.0, or greater than or equal individuals can be useful in the identification of highly to about 16.5, or greater than or equal to about 17.0. The penetrant genes. A number of computer Software programs particular LOD score depends on the particular marker. are available to conduct parametric linkage analysis see, 0367 Criteria have been proposed for use in categorizing e.g., FASTLINK; Lathrop et al. (1984) Proc. Natl. Acad. Sci. linkage analysis results in terms of the extent to which the U.S.A. 81:3443-3446; Cottingham et al. (1993) Am. J. Hum. results may serve as evidence of linkage between loci. For Genet. 53:252-263; Schaffer et al. (1994) Hum. Heredity example, by some criteria Morton (1955) Am. J. Hum. 44:225-237). Genet. 7:277-318), a LOD score of 1.5 or greater is consid 0373 Parametric linkage analysis can be limited due to ered to be “Suggestive' of linkage whereas a LOD Score of its reliance on the choice of a genetic model Suitable for a 3 is considered as Statistically significant evidence for link particular trait, and may be difficult when applied to the age. The Significance level, C., is that which is associated analysis of complex genetic traits Such as those due to the with a likelihood ratio test computed to the base e-X = combined action of multiple genes and/or environmental lod(2ln10). For a LOD score of 3, C-0.0001; for a LOD factors. In the mapping of diseases lacking a clear Mende score of 1.5, O.<0.004. It has also been proposed that a lian inheritance pattern or caused by Several genes of low to multipoint LOD score of s5.4 may be considered as “highly moderate penetrance, it may be more Suitable to utilize Significant' evidence of linkage, whereas “significant evi nonparametric analysis applied to Small Sets of affected dence of linkage may be viewed as a multipoint LOD score relatives, Such as affected Sib pairs. s3.6 or a two-point LOD score s3.3, and “suggestive” 0374 (2) Nonparametric Linkage Analysis evidence of linkage is provided by multipoint LOD scores s2.2 or two-point LOD scores is 1.9 see, e.g., Lander and 0375 Nonparametric linkage analysis involves determin Kruglyak (1995) Nat. Genet. 11:241). ing whether the inheritance pattern of a chromosomal region is not consistent with random Mendelian Segregation by 0368 Linkage analysis methods can be used to screen the showing that affected relatives inherit identical copies of the entire human genome for one or more chromosomal regions region (i.e., allele sharing) more often than expected by containing loci linked to a disease gene. In genome Screen chance. Distortions from expected ratioS of allele Sharing ing procedures, DNA from individual members of families among relatives usually Sibs; See, e.g., Risch (1990) Am. J. in which one or more family members are trait positive is Hum. Genet. 46:229-241 who share a disease phenotype are typed with respect to a Set of genetic markers that includes tested. This form of analysis is independent of the mode of multiple markers from each human chromosome. The reso inheritance of the disease and thus is well-Suited for cases in lution of the Screen depends on the number of markers that which there is not an absolute correlation between pheno are typed and the distance between the markers on each type and genotype, Such as in many multifactorial traits in chromosome. Generally, the higher the density of the col which multiple genes may contribute to observed pheno lection of markers, the higher the resolution of the mapping type. results. Typically, markerS Separated by an average distance of 10 cM or less are considered to provide for a fairly high 0376 Typically, nonparametric linkage analysis is based resolution genome Screen. In particular, an average marker in the analysis of the proportion of alleles shared identical by separation of 9 cM or less is used for high-resolution descent (IBD) between two sibs affected with a disease mapping. The results of the typing are compared to the (affected Sib pairs). The degree of agreement at a marker disease Status of each individual, and these data are Statis locus in two individuals can also be measured by the number tically evaluated using one or more of a variety of linkage of alleles identical by state (IBS). Nonparametric linkage analysis computer Software programs See, e.g., O'Connell analysis can be used in genome wide Scans of multifactorial and Weeks (1995) Nat. Genet. 11:402-408 describing the diseases using linkage maps of genetic markers, e.g., mic VITESSE algorithm). Traditional LOD score analysis is a rosatellite markers. A number of computer Software pro Strong method for evaluating linkage in forms of a disorder grams are available to conduct nonparametric linkage analy showing obvious Mendelian inheritance, but weakens when sis see, e.g., MAPMAKER/SIBS, Lander and Kruglyak the mode of transmission is complex and genetic parameters (1995) Am. J. Hum. Genet. 57:439-454; GENEHUNTER cannot be accurately Specified. In Such cases, Statistical PLUS, Kruglyak et al. (1996) Am. J. Hum. Genet. 58:1347; evaluation of genotyping data may be strengthened through SIMIBD, Davis et al. (1996) Am. J. Hum. Genet. 58:867 use of allele Sharing linkage methods in pairs of affected 880; ASPEX (MLS), Risch (1990) Am. J. Hum. Genet. Siblings or other relative pairs and association Studies. Such 46:222-253). methods are described herein. 0377 3. Analysis of Genetic Linkage of AD on Chromo 0369 C. Statistical Methods in Linkage Analysis Some 10 0378 Genotyping data with respect to multiple markers 0370 Methods of analyzing genetic linkage are termed on human chromosome 10 in AD families were analyzed parametric (i.e., "model-based') if gene frequency and pen using Several different analytic strategies and models (see etrance must be estimated, and nonparametric otherwise. EXAMPLE 2, i.e., using both autosomal dominant and There are many models within each class. recessive parametric two-point analyses and nonparametric 0371 (1) Parametric Linkage Analysis two-point and multipoint analyses) and using a Family Based Association Test (FBAT) for association with AD (see 0372 Parametric linkage analysis see, e.g., Ott (1991) EXAMPLE 3). As described in detail herein, LOD scores Analysis of Human Genetic Linkage, Baltimore, London, indicative of linkage of AD obtained for markers on chro John Hopkins and Terwilliger and Ott (1994) Handbook of moSome 10 were consistently achieved in each of these Human Genetic Linkage, Baltimore, John Hopkins Univer analytical tests. US 2003/017O678A1 Sep. 11, 2003 32
0379 Chromosome 10 markers D10S583, D10S1710, Sci. U.S.A. 94:5344-5348. One class of alleles, referred to D10S566, D10S1671 and D10S1741 each yielded LOD as major genes, can be characterized by Segregation analy scores >1 for the whole sample in at least one of the AD Sis. These genes tend to be rare and have megaphenic effects linkage analyses that were conducted. Analysis of markers (which can be measured as displacement between homozy D10S583 and D10S1671 revealed significant evidence of gotes) that are large relative to the Standard deviation of linkage to AD. The greatest linkage Scores obtained in each liability. A major gene is Sufficient to cause affection against of the analyses of the whole sample were as follows: almost any genetic background, and therefore interaction is parametric two-point LOD score (Z) using the dominant negligible except for modifiers of expression. Another class model=3.3 for D10S583; parametric two-point LOD score of alleles, polygenes, tend to be common and have (Z) using the recessive model=2.9 for D10S1671; non microphenic effects too Small to be characterized. These parametric two-point linkage score (Z)=3.3 for D10S1671; alleles may possibly be detected, however, through allelic nonparametric multipoint LOD score=1.9 for D10S 1710. asSociation at candidate loci. Between these two extreme The greatest linkage Scores obtained in each of the analyses classes of alleles are oligogenes, also called leading factors of the late-onset Sample were as follows: parametric two Wright (1968) Evolution and the Genetics of Populations point LOD score (Z) using the dominant model=3.4 for 1:411-417). These alleles are common and have mesophenic D10S1671; parametric two-point LOD score (Z) using effects too small to be reliably characterized by Segregation the recessive model=3.8 for D10S1671; nonparametric two analysis, but in large Samples they can be detected by point linkage score (Z)=3.8 for D10S1671; nonparametric nonparametric methods and elucidated by combined Segre multipoint LOD score=2.1 for D10S1710. gation and linkage analysis, including allelic association as 0380 Although for each of the specified analyses, one of coupling frequencies MacLean et al. (1984) Comput. markers D10S583, D10S1671 and D10S1710 yielded the Biomed. Res. 17:471-480; Shields et al. (1994) Am. J. Hum. greatest linkage Score, the Second and Sometimes third Genet. 55:540-554). Small numbers of oligogenes interact to highest linkage Scores in each analysis, were, for the most produce affection. One locus may have all three allelic part, also indicative of linkage to AD. Thus, the region of classes; thus, Small effects may be detected through allelic chromosome 10q around and between these markers is asSociation at loci recognized as candidates through larger referred to as a region of peak linkage which may include effects. one or more Specific peaks. Markers flanking this region of 0385 4. Genetic Markers Linked to an AD-Associated peak linkage include the terminus of the q arm of chromo DNA Segment on Chromosome 10 Some 10 and D10S564. 0386 The results of linkage analyses of genotyping data 0381 Stratification of the sample into APOE e4/4-posi with respect to markers located on chromosome 10 in AD tive and -negative Subsets resulted in a lowering of linkage families revealed genetic markers on chromosome 10 that Scores obtained for each marker when analyzed in paramet are linked to one or more DNA segments, e.g., one or more ric two-point linkage analyses using a dominant disease genes, associated with AD. Included within the linked model. The linkage Scores generally were, however, more markers are markers located in the region of 10q22, 10q23, pronounced in families without the APOE e414 genotype. 10q24, 10q25 or 10q26. Particular regions of chromosome 10 in which markers linked to one or more DNA segments 0382 Linkage-based genetic analysis tends to localize associated with AD are located include: (1) the region disease-Susceptiblity genes that have a more Substantial extending about 45 cM, or about 42 cM, or about 33 cM, or effect size. The effect Size of a disease gene is the degree to about 30 cM, or about 25 cM, or about 20 cM, or about 15 which mutations or polymorphisms in a gene confer SuS cM, or about 10 cM, or about 5 cM, or about 3 cM, or about ceptibility to the disease taking into account the magnitude 2.7 cM, or about 2.5 cM, or about 2 cM, or about 1.5 cM, of prevalence and penetrance of the polymorphism. The or about 1 cM, or about 0.5 cM, or about 0.1 cM proximal prevalence of a polymorphic allele refers to the percentage (centromeric) from and including marker D10S583, of affected individuals who carry the polymorphism. Pen D10S1710 and/or D10S1671, (2) the region extending about etrance of a polymorphic allele refers to the percent chance 62 cM, or about 59 cM, or about 50 cM, or about 47 cM, or that a carrier of the allele will express the phenotype. The about 32 cM, or about 30 cM, or about 28 cM, or about 25 concept of penetrance establishes the connection between cM, or about 20 cM, or about 17 cM, or about 15 cM, or genotypes and phenotypes and thus is of central importance about 10 cM, or about 5 cM, or about 2.5 cM, or about 1 cM, in linkage analysis. or about 0.5 cM or about 0.1 cM distal (telomeric) from and 0383. The relative effect size of a gene may be estimated including marker D10S564, D10S583, D10S1710 and/or based on the magnitude of LOD Scores obtained in linkage D10S1671, (3) the region extending from and including any analyses relating to the gene. Maximum LOD Scores of markers D10S564, D10S583, D10S 1710, D10S1671 or obtained in analyses of linkage of AD on chromosome 10 D10S1741 to the terminus of the q arm (4) the region described herein are similar to the maximum LOD scores extending about 46 Mb, or about 40 Mb, or about 32 Mb, or reported in linkage analyses that lead to the discovery of about 25 Mb, or about 20 Mb, or about 16 Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about APOE as an AD-associated gene Pericak-Vance et al. 0.5 Mb, or about 0.3 Mb, or about 0.19 Mb, or about 0.1 Mb, (1991) Am. J. Human Genet. 48:1034-1050). The significant or about 0.05 Mb, or about 0.01 Mb, or about 1 kb proximal linkage of AD on chromosome 10 indicates that the one or from and including marker D10S583, D10S1710 and/or more AD DNA segments or AD genes located on chromo D10S1671, (5) the region extending about 50 Mb, or about Some 10 have a considerable effect size which is comparable 48 Mb, or about 39 Mb, or about 35 Mb, or about 30 Mb, to or greater than that of APOE. or about 25 Mb, or about 20 Mb, or about 15 Mb, or about 0384 Alleles can be considered based on phenotypic 10 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, or classessee, e.g., Lio and Morton (1997) Proc. Natl. Acad. about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.1 US 2003/017O678A1 Sep. 11, 2003
Mb, or about 0.05 Mb, or about 0.01 Mb, or about 1 kb distal D10S1700, D10S169, D10S1221, D10S 1223, ATA25D02, from and including marker D10S564, D10S583, D10S1710 D10S 1229, D1OS1232, ATA42E11, D10S1239, D10S1423, and/or D10S1671, (6) the region between D10S564 and D10S1435, D1OS110, D1OS466, D10S525, D10S1148, D10S583, inclusive, (7) the region between D10S564 and D10S1149, D10S1150 and D10S1163. D10S1710, inclusive, (8) the region between D10S564 and D10S566, inclusive, (9) the region between D10S564 and 0389) 5. Methods for Identifying Genetic Markers Linked D10S1671, inclusive, (10) the region between D10S564 and to an AD-Associated DNA Segment D10S1741, inclusive, (11) the region between the terminus 0390 Methods of identifying genetic markers linked to a of the q arm of chromosome 10 and D10S1741, inclusive, DNA segment associated with AD can include a Step of (12) the region between markers D10S1741 and D10S1671, testing a polymorphic marker on chromosome 10 for linkage inclusive, (13) the region between markers D10S1741 and to AD. The testing may involve genotyping DNA from D10S566, inclusive, (14) the region between markers individuals in AD families with respect to the polymorphic D10S1741 and D10S1710, inclusive, (15) the region marker and analyzing the genotyping data for linkage to AD between markers D10S1741 and D10S583, inclusive and using methods described herein and/or known to those of (16) the region between markers D10S1741 and D10S564, skill in the art. For example, the data may be analyzed in a inclusive. Particular markers linked to a DNA segment LOD score determination method. associated with AD include D10S583, D10S1710 and D1OS1671. 0391) In particular methods of identifying genetic mark ers linked to a DNA segment associated with AD, the 0387 Genetic markers on chromosome 10 linked to a polymorphic marker is located in the region of 10q22, DNA segment associated with AD yield scores or results 10q23, 10q24, 10q25 or 10q26. Particular regions of chro indicative of linkage to AD when tested in linkage assess moSome 10 in which the polymorphic marker is located ment methods. Such methods include but are not limited to include: (1) the region extending about 45 cM, or about 42 LOD score methods, including LOD score methods involv cM, or about 33 cM, or about 30 cM, or about 25 cM, or ing parametric or nonparametric linkage analysis and/or about 20 cM, or about 15 cM, or about 10 cM, or about 5 two-point or multipoint analyses. Particular chromosome 10 cM, or about 3 cM, or about 2.7 cM, or about 2.5 cM, or markers linked to a DNA segment associated with AD yield about 2 cM, or about 1.5 cM, or about 1 cM, or about 0.5 LOD scores that may be considered highly significant, cM, or about 0.1 cM proximal (centromeric) from and Significant, Suggestive or indicative of evidence of linkage. including marker D10S583, D10S1710 and/or D10S1671, 0388 Particular markers linked to a DNA segment asso (2) the region extending about 62 cM, or about 59 cM, or ciated with AD further include any genetic markers located about 50 cM, or about 47 cM, or about 32 cM, or about 30 on chromosome 10, including but not limited to the markers cM, or about 28 cM, or about 25 cM, or about 20 cM, or listed in FIG. 1, that yield scores or results indicative of about 17 cM, or about 15 cM, or about 10 cM, or about 5 linkage to AD when tested in linkage assessment methods cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or described herein and known to those of skill in the art. about 0.1 cM distal (telomeric) from and including marker Included within these particular markers are any of the D10S564, D10S583, D10S1710 and/or D10S1671, (3) the following markers that yield Scores or results indicative of region extending from and including any of markers linkage to AD when tested in linkage assessment methods: D10S564, D10S583, D10S1710, D10S1671 or D10S1741 to D10S564, D10S2470, D10S1755, D10S536, D10S185, the terminus of the q arm (4) the region extending about 46 D10S1171, D10S1173, D10S200, D10S1680, D10S520, Mb, or about 40 Mb, or about 32 Mb, or about 25 Mb, or D10S1736, D10S574, D10S571, D10S1690, ATA105C09, about 20 Mb, or about 16 Mb, or about 10 Mb, or about 5 GATA30F07a, D10S677, D10S91, D10S1758, D10S577, Mb, or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about D10S1709, D10S184, D10S1726, D10S198, D10S1123, 0.3 Mb, or about 0.19 Mb, or about 0.1 Mb, or about 0.05 D10S1147, D10S603, D10S1266, D10S1265, D10S1264, Mb, or about 0.01 Mb, or about 1 kb proximal from and D10S1778, D10S192, FB7F11, D10S1267, D10S1692, including marker D10S583, D10S1710 and/or D10S1671, D10S1738, D10S205, D10S1668, D1OS1268, D1OS222, (5) the region extending about 50 Mb, or about 48 Mb, or D10S1697, GATA64B01, D10S540, D10S467, about 39 Mb, or about 35 Mb, or about 30 Mb, or about 25 GATA114HO9, D1OS254, D10S1663, D10S530, D10S534, Mb, or about 20 Mb, or about 15 Mb, or about 10 Mb, or D10S521, D1OS108, D10S1741, D10S1760, D10S1121, about 5 Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, D10S1795, D10S1750, D10S1246, D10S597, D10S543, or about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or about D1OS88, D1OS1682, D10S554, GATA83D05, D10S168, 0.05 Mb, or about 0.01 Mb, or about 1 kb distal from and D10S1269, D10S1681, D10S1776, D10S562, D10S1748, including marker D10S564, D10S583, D10S1710 and/or D10S1773, D10S1731, ATA103C06, D10S468, D10S1237, D10S1671, (6) the region between D10S564 and D10S583, D10S1158, D10S528, D10S1683, D10S544, D10S531, inclusive, (7) the region between D10S564 and D10S1710, D10S1657, D10S545, D10S187, D10S1165, D10S1156, inclusive, (8) the region between D10S564 and D10S566, D10S221, D10S1425, D10S 1693, D10S 1722, D10S1141, inclusive, (9) the region between D10S564 and D10S1671, D10S1236, D10S190, D10S503, GATA127H01, D10S1785, inclusive, (10) the region between D10S564 and D10S1741, D10S542, D10S1701, D10S1792, D10S1740, D10S1757, inclusive, (11) the region between the terminus of the q arm D10S209, D10S1230, D10S1483, D10S1679, D10S587, of chromosome 10 and D10S1741, inclusive, (12) the region D10S1708, D10S1723, D10S1213, D10S2322, D10S216, between markers D10S1741 and D10S1671, inclusive, (13) D10S1656, D10S12, D10S575, D10S214, D10S1703, the region between markers D10S1741 and D10S566, inclu D10S186, D10S1782, D10S1222, D10S1727, D10S217, sive, (14) the region between markers D10S1741 and D10S1676, D10S1134, D1OS1655, GGAST14G01, D10S1710, inclusive, (15) the region between markers D10S505, D10S1248, D10S1770, D10S1651, D10S590, D10S1741 and D10S583, inclusive and (16) the region D10S1675, 1OTEL23, D10S212, D10S555, D10S1711, between markers D10S1741 and D10S564, inclusive. Par US 2003/017O678A1 Sep. 11, 2003 34 ticular markers linked to a DNA segment associated with Mb, or about 16 Mb, or about 14 Mb, or about 13 Mb, or AD include D10S583, D10S 1710 and D10S1671. about 8 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.1 0392 F. Location of One or More DNA Segments Asso Mb, or about 0.05 Mb, or about 0.01 Mb, or about 1 kb distal ciated with AD on Chromosome 10 from and including marker D10S583, (7) the region between 0393. The discovery of genetic association with AD on D10S564 and D10S583, inclusive, (8) the region between human chromosome 10 as described herein identifies chro D10S583 and D10S1710, inclusive, (9) the region between moSome 10 as the location of one or more DNA segments D10S583 and D10S566, inclusive, (10) the region between associated with AD. The type of AD may be late-onset AD. D10S583 and D10S1671, inclusive and (11) the region The DNA segment or segments may be genes. The DNA between D10S583 and D10S1741, inclusive. Segment or Segments may be an AD DNA segment or AD 0396 Further particular regions of chromosome 10 in gene and may be causative of AD. In particular, chromo which a DNA segment associated with AD may be located Some 10 is identified as containing at least one DNA include: (1) the region extending about 5 cM, or about 4 cM, Segment or gene associated with AD and of comparable or or about 2.7 cM, or about 2.5 cM, or about 2 cM, or about greater impact than APOE, wherein the effect size of the 1.5 cM, or about 1 cM, or about 0.5 cM, or about 0.1 cM gene is comparable to or greater than that of APOE. proximal (centromeric) from and including marker 0394. In one embodiment of a DNA segment associated D10S583 (2) the region extending about 5 cM, or about 2.5 with AD on chromosome 10, the DNA segment is an allele cM, or about 1 cM, or about 0.5 cM or about 0.1 cM distal on chromosome 10 that confers in those who carry the allele (telomeric) from and including marker D10S583, (3) the protection against AD relative to those who do not carry the region extending about 10 Mb, or about 5 Mb, or about 2 allele. In another embodiment of a DNA segment associated Mb, or about 1 Mb, or about 500 kb, or about 300 kb, or with AD on chromosome 10, the DNA segment either about 200 kb, or about 100 kb, or about 80 kb, or about 50 directly causes or confers an increased Susceptibility to AD kb, or about 10 kb, or about 5 kb, or about 1 kb proximal (e.g., a "risk” or “disease' allele). In particular embodi from and including marker D10S583, and (4) the region ments, the DNA segment is an allele on chromosome 10q extending about 10 Mb, or about 8 Mb, or about 5 Mb, or that confers in those who carry the allele protection against about 4 Mb, or about 3 Mb, or about 2 Mb, or about 1 Mb, AD relative to those who do not carry the allele, or either or about 500 kb, or about 300 kb, or about 200 kb, or about directly causes or confers an increased Susceptibility to AD 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, or (e.g., a "risk” or “disease' allele). about 1 kb distal from and including marker D10S583. 0397) G. Methods for Identifying an AD DNA Segment 0395 At least one, or two or more, DNA segment(s) or AD Gene asSociated with AD may be located in the regions of 10q22, 10q23, 10q24, 10q25 or 10q26. In particular, at least one, or 0398 Methods for identifying an AD DNA segment or an two or more, DNA segment(s) associated with AD may be AD gene are provided herein. The methods are based in the located in the regions of 10q22, 10q23 or 10q24. At least genetic analysis of chromosome 10 of individuals affected one, or two or more, DNA segment(s) associated with AD with AD. In particular embodiments, the methods are based may be located in the regions of 10q23, 10q24 or 10q25. in the genetic analysis of chromosome 10q. In further Particular regions of chromosome 10 in which a DNA embodiments, the methods may be based in genetic analysis Segment associated with AD may be located include: (1) the of chromosome 10q22, 10q23, 10q24, 10q25 or 10q26. In region extending about 50 cM, or about 45 cM, or about 33 particular embodiments, the methods may be based in cM, or about 30 cM, or about 25 cM, or about 21 cM, or genetic analysis of chromosome 10 within a region identi about 20 cM, or about 15 cM, or about 10 cM, or about 5 fied herein as containing or near markers associated with cM, or about 4 cM, or about 3 cM, or about 2.7 cM, or about AD. In yet further embodiments, the methods may be based 2.5 cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or in genetic analysis of chromosome 10q22, 10q23 or 10q24. about 0.5 cM, or about 0.1 cM proximal (centromeric) from In particular embodiments, the methods may be based in and including marker D10S583 (2) the region extending genetic analysis of chromosome 10q23, 10q24 or 10q25. about 60 cM, or about 55 cM, or about 50 cM, or about 45 0399 Genetic analysis of chromosome 10 may involve cM, or about 40 cM, or about 35 cM, or about 30 cM, or methods which include, but are not limited to, any one or about 25 cM, or about 20 cM, or about 15 cM, or about 13 more of the following: analysis of one or more genetic cM, or about 12 cM, or about 9 cM, or about 5 cM, or about markers on chromosome 10 for linkage to AD or a DNA 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 cM Segment associated with AD; analysis of association distal (telomeric) from and including marker D10S583, (3) between one or more genetic markers on chromosome 10 the region extending from and including marker D10S583 to and AD; analysis of association between a chromosome 10 the terminus of the q arm of chromosome 10, (4) the region haplotype and AD, calculation of linkage disequilibrium extending from and including marker D10S583 to the cen values from chromosome 10 haplotype data; Sequencing of tromere of chromosome 10, (5) the region extending about DNA in chromosome 10 for polymorphic markers, in par 62 Mb, about 55 Mb, or about 50 Mb, or about 32 Mb, or ticular, SNPs, as genetic markers in further genetic analyses about 28 Mb, or about 25 Mb, or about 20 Mb, or about 15 as described above or as possible AD DNA segments or AD Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, or about gene Variants underlying AD. 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or 04.00) 1. Linkage Analysis in the Identification of AD about 1 kb proximal from and including marker D10S583, DNA Segments or Genes (6) the region extending about 45 Mb, or about 40 Mb, or 04.01 The search for disease-susceptibility genes gener about 35 Mb, or about 30 Mb, or about 25 Mb, or about 20 ally may be conducted using two main analytical methods: US 2003/017O678A1 Sep. 11, 2003
linkage analysis, in which evidence is Sought for coSegre ing from and including marker D10S583 to the terminus of gation between a locus and a putative trait locus within the q arm of chromosome 10, (4) the region extending from families, and association analysis, in which evidence is and including marker D10S583 to the centromere of chro Sought for a Statistically significant association between an mosome 10, (5) the region extending about 62 Mb, about 55 allele and a trait or a trait-causing allele Khoury et al. Mb, or about 50 Mb, or about 32 Mb, or about 28 Mb, or (1993) Fundamentals of Genetic Epidemiology, Oxford Uni about 25 Mb, or about 20 Mb, or about 15 Mb, or about 10 versity Press, N.Y.). These methods can be viewed as tools Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about which may be applied in any of Several approaches to 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, or about 0.1 Mb, disease gene discovery. Two primary approaches to disease or about 0.05 Mb, or about 0.01 Mb, or about 1 kb proximal gene discovery are genetic localization and candidate gene from and including marker D10S583, (6) the region extend Studies. ing about 45 Mb, or about 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 Mb, or about 20 Mb, or about 16 Mb, 0402 a. Candidate Gene Approach or about 14 Mb, or about 13 Mb, or about 8 Mb, or about 5 0403. The candidate gene approach typically takes into Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about account knowledge of biological processes of a disease as a 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or about 0.05 Mb, basis for Selecting genes that encode proteins that could be or about 0.01 Mb, or about 1 kb distal from and including envisioned to be involved in the biological processes. For marker D10S583, (7) the region between D10S564 and example, reasonable candidate genes for blood pressure D10S583, inclusive, (8) the region between D10S583 and disorders could be proteins and enzymes involved in the D10S1710, inclusive, (9) the region between D10S583 and renin-angiotensin System. Candidate genes can be evaluated D10S566, inclusive, (10) the region between D10S583 and genetically as possible disease genes by linkage and/or D10S1671, inclusive and (11) the region between D10S583 asSociation Studies of markers in the candidate gene region. and D10S1741, inclusive. 04.04 (1) Methods of Identifying a Candidate AD Gene 0407. In further particular embodiments of these meth 04.05 The methods of identifying a candidate AD gene ods, the gene is located in a region of chromosome 10 as include a step of Selecting a gene on chromosome 10 that is follows: (1) the region extending about 5 cM, or about 4 cM, or encodes a product that has one or more properties relating or about 2.7 cM, or about 2.5 cM, or about 2 cM, or about to one or more phenomena in neurodegenerative disease. 1.5 cM, or about 1 cM, or about 0.5 cM, or about 0.1 cM Human chromosome 10 contains at least 600 genes. FIG. 2 proximal (centromeric) from and including marker provides a list of many of the genes that are located on D10S583 (2) the region extending about 5 cM, or about 2.5 chromosome 10. Additional genes that have been mapped to cM, or about 1 cM, or about 0.5 cM or about 0.1 cM distal chromosome 10 are also known. Thus, genes on chromo (telomeric) from and including marker D10S583, (3) the Some 10 may be evaluated as possible candidate AD genes region extending about 10 Mb, or about 5 Mb, or about 2 on the basis of, for example, knowledge of the functions of Mb, or about 1 Mb, or about 500 kb, or about 300 kb, or the genes or products thereof and/or their occurrence or about 200 kb, or about 100 kb, or about 80 kb, or about 50 alteration in neurodegenerative diseases, and, in particular, kb, or about 10 kb, or about 5 kb, or about 1 kb proximal AD. from and including marker D10S583, and (4) the region extending about 10 Mb, or about 8 Mb, or about 5 Mb, or 0406 A particular method of identifying a candidate AD about 4 Mb, or about 3 Mb, or about 2 Mb, or about 1 Mb, gene provided herein includes a Step of Selecting a gene on or about 500 kb, or about 300 kb, or about 200 kb, or about chromosome 10q that is or encodes a product that has 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, or properties relating to one or more phenomena in neurode about 1 kb distal from and including marker D10S583. generative disease. In further embodiments of these meth ods, the gene is on chromosome 10q22, 10q23, 10q24, 0408 (2) Properties Relating to Phenomena in Neurode 10q25 or 10q26. In particular embodiments of these meth generative Disease ods, the gene is located within a region identified herein as 04.09. In the methods of identifying a candidate AD gene containing or near markers associated with AD. In further provided herein, a gene on chromosome 10, and, in particu embodiments, the gene is on chromosome 10q22, 10q23 or lar embodiments, on particular regions of chromosome 10 as 10q24. In yet further embodiments, the gene is on chromo described above, is Selected that is or encodes a product that Some 10q23, 10q24 or 10q25. The Step of Selecting a gene has properties relating to one or more phenomena in neu may also be a step of Selecting a gene located in one of the rodegenerative disease. The properties may be any aspect or following regions of chromosome 10: the region extending feature of the gene or gene product, including but not limited about 50 cM, or about 45 cM, or about 33 cM, or about 30 to its physical composition (e.g., nucleic acids, amino acids, cM, or about 25 cM, or about 21 cM, or about 20 cM, or peptides and proteins), functional attributes (e.g., enzymatic about 15 cM, or about 10 cM, or about 5 cM, or about 4 cM, capabilities, Such as an enzyme catalyst, inhibitory func or about 3 cM, or about 2.7 cM, or about 2.5 cM, or about tions, Such as enzyme inhibition, antigenic properties, and 2 cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or binding capabilities, Such as a receptor or ligand), cellular about 0.1 cM proximal (centromeric) from and including location(s), expression pattern (e.g., expression in the central marker D10S583 (2) the region extending about 60 cM, or nervous System and cells and tissues associated therewith) about 55 cM, or about 50 cM, or about 45 cM, or about 40 and/or interactions with other compositions. cM, or about 35 cM, or about 30 cM, or about 25 cM, or about 20 cM, or about 15 cM, or about 13 cM, or about 12 0410 The properties of the gene or gene product that are cM, or about 9 cM, or about 5 cM, or about 2.5 cM, or about selected for in the methods of identifying a candidate AD 1 cM, or about 0.5 cM or about 0.1 cM distal (telomeric) gene are those that relate to one or more phenomena in from and including marker D10S583, (3) the region extend neurodegenerative disease. Such phenomena, which have US 2003/017O678A1 Sep. 11, 2003 36 been widely described and are known to those of skill in the In particular embodiments, the candidate AD gene is located art, are numerous and include morphological, Structural, on chromosome 10q. In further embodiments, the candidate biological and biochemical occurrences which can be patho AD gene is located on chromosome 10q22, 10q23, 10q24, physiological aspects of neurodegenerative diseases. Such 10q25 or 10q26. Candidate genes of particular embodi phenomena include, but are not limited to, Senile plaques, ments, are located within a region identified herein as neuritic plaques, and components of each, neurofibrillary containing or near markers associated with AD. In further tangles, tau protein and abnormal phosphorylation of tau embodiments, the candidate AD gene is on chromosome protein, amyloid precursor protein (APP) and processing 10q22, 10q23 or 10q24. In yet further embodiments, the thereof, AB42 protein, Cl-, 3- and Y-Secretases, presenilin candidate AD gene is on chromosome 10q23, 10q24 or proteins, amyloid deposition, Lewy bodies, prions, apopto 10q25. Candidate AD genes include genes located in the sis see, e.g., Behl (2000) J. Neural Transm. 107:1325 following regions of human chromosome 10: (1) the region 1344), caspases, inflammation see, e.g., McGeer amd extending about 50 cM, or about 45 cM, or about 33 cM, or McGeer (1998) Exp. Gerontol. 33:371-378), excitotoxicity about 30 cM, or about 25 cM, or about 21 cM, or about 20 and excitotoxins, excessive nitric oxide production, oxida cM, or about 15 cM, or about 10 cM, or about 5 cM, or about tive stress see, e.g., Beal (1998) Biochim. Biophys. Acta 4 cM, or about 3 cM, or about 2.7 cM, or about 2.5 cM, or Mol. Cell Res. 1366:211-223 and Wallace et al. (1998) about 2 cM, or about 1.5 cM, or about 1 cM, or about 0.5 Biofactors 7:187-190), proteases, protease inhibitors, neu cM, or about 0.1 cM proximal (centromeric) from and rotrophic factors, cytokines, calcium-dependent processes, including marker D10S583, (2) the region extending about Signal transduction, altered ionic homeostasis, particularly 60 cM, or about 55 cM, or about 50 cM, or about 45 cM, or calcium homeostasis, Synaptic molecules, adhesion mol about 40 cM, or about 35 cM, or about 30 cM, or about 25 ecules, molecules involved in membrane turnover, choles cM, or about 20 cM, or about 15 cM, or about 13 cM, or terol and lipid metabolism and transport, cytoskeletal mol about 12 cM, or about 9 cM, or about 5 cM, or about 2.5 cM, ecules, neuronal and brain proteins, and cell necrosis. For or about 1 cM, or about 0.5 cM or about 0.1 cM distal example, a property of a gene or gene product relating to one (telomeric) from and including marker D10S583, (3) the or more phenomena in neurodegenerative disease may be region extending from and including marker D10S583 to the involvement of the gene and/or product thereof in a pathway terminus of the q arm of chromosome 10, (4) the region involved in APP metabolism, AB protein generation, aggre extending from and including marker D10S583 to the cen gation and/or degradation, apoptosis, calcium homeostasis, tromere of chromosome 10, (5) the region extending about inflammation, oxidative StreSS, free radical generation, 62 Mb, about 55 Mb, or about 50 Mb, or about 32 Mb, or modification of tau protein (e.g., phosphorylation), axonal about 28 Mb, or about 25 Mb, or about 20 Mb, or about 15 transport, neuroprotection and neurotrophism. In particular Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, or about embodiments of the methods of identifying a candidate AD 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, gene provided herein, a gene on chromosome 10 is Selected or about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or that is or encodes a product that has properties relating to about 1 kb proximal from and including marker D10S583, one or more phenomena in Alzheimer's disease See, e.g., (6) the region extending about 45 Mb, or about 40 Mb, or Chapman et al (2001) Trends Genet. 17:254-261 and Selkoe about 35 Mb, or about 30 Mb, or about 25 Mb, or about 20 (2001) Physiol. Rev. 81:741-766 for exemplary phenomena Mb, or about 16 Mb, or about 14 Mb, or about 13 Mb, or in AD). about 8 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, 0411 Properties of the selected gene can relate to phe or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.1 nomena in neurodegenerative disease in many different Mb, or about 0.05 Mb, or about 0.01 Mb, or about 1 kb distal ways. For example, a gene product may be involved in from and including marker D10S583, (7) the region between biological or biochemical processes that have been charac D10S564 and D10S583, inclusive, (8) the region between terized as occurring in AD and/or other neurodegenerative or D10S583 and D10S1710, inclusive, (9) the region between neurological diseases or disorders. Thus, for instance, a gene D10S583 and D10S566, inclusive, (10) the region between product may be an enzyme, inhibitor or regulatory Subunit D10S583 and D10S1671, inclusive and (11) the region in a pathway leading to the generation of a component of an between D10S583 and D10S1741, inclusive. amyloid plaque, e.g., AB, alpha-1-anti-chymotrypsin, cathe 0414 Candidate AD genes of particular interest are psin D, non-amyloid component protein, apolipoprotein E located in a region of chromosome 10 as follows: (1) the (APOE), apolipoprotein J, heat shock protein 70, comple region extending about 5 cM, or about 4 cM, or about 2.7 ment components, alpha2-macroglobin, interleukin-6, pro cM, or about 2.5 cM, or about 2 cM, or about 1.5 cM, or teoglycans and Serum amyloid P. A gene product may also about 1 cM, or about 0.5 cM, or about 0.1 cM proximal be a molecule, e.g., an enzyme, involved in protein phoS (centromeric) from and including marker D10S583, (2) the phorylation leading to hyperphosphorylation. A gene prod region extending about 5 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 cM distal (telomeric) from uct may be a protein, Such as a protein found in the brain, and including marker D10S583, (3) the region extending that is increased, decreased or altered in Some other way in about 10 Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, neurodegenerative diseases, Such as, for example, AD. Thus, or about 500 kb, or about 300 kb, or about 200 kb, or about a property of a gene or gene product can relate to a 100 kb, or about 80 kb, or about 50 kb, or about 10 kb, or phenomenon in neurodegenerative disease if it is directly or about 5 kb, or about 1 kb proximal from and including indirectly involved in, for example, a process or aspect marker D10S583, and (4) the region extending about 10 Mb, characteristic of a neurodegenerative disease. or about 8 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about 1 Mb, or about 500 kb, or about 300 0412 (3) Candidate AD Genes kb, or about 200 kb, or about 100 kb, or about 50 kb, or 0413 Candidate AD genes are also provided herein. The about 10 kb, or about 5 kb, or about 1 kb distal from and candidate AD genes are located on human chromosome 10. including marker D10S583. US 2003/017O678A1 Sep. 11, 2003 37
0415. In particular embodiments of the candidate AD 0416 Particular candidate AD genes include genes listed genes provided herein, the gene is located within a region in TABLE 1. Also listed in TABLE 1 are citations of identified herein as containing or near markers associated publications which may be concerned with the gene and/or with AD. a product of the gene.
TABLE 1. Approximate Location on GenBank Approximate Chromosome Accession Cytogenetic Gene 10 (bp) No. Location Reference SGPL1 77105083- NT O24089a 10q22 Van Veldhoven et 77166645 XM 005931 al., (2000) Biochim XM 046325 Biophys Acta, XM 046326 1487(2–3):128–34 PCBD 77172724- NT O24089a 10q22 Milatovich et al., 77174993 XM 046338 (1993) Genomics XM 046339 16(1):292-5 Hauer et al., (1993) J. Biol. Chem. 268(7):4828–31 FLJ11160 77611041- NT OO8849 10q22.1 77651484 XM 005936 XM O38317 PSAP 77761345- NT 028289a 10q22.1 Morimoto et al., 77796272 XM 045137 (1989) Proc. Natl. XM 045139 Acad. Sci. USA XM 045140 86(9):3389-93 Rorman et. al., (1989) Genomics 5(3):486-92 MYOZ 7842.1968- NT O24037 10q22.1 Takada et. al., 78432O65 XM OO5876 (2001) Proc. Natl. Acad. Sci. USA 98(4):1595-600 FLJ12921 78456597- NT 024037a 10q22.1 7846.2094 XM O15246 LOCS1008 78603718- AF132952 10q22.1 Lai et al., (2000) 78668872 Genome Res. 10(5):7103-13 KIAAO275 78676384- NT O28289 10q22.1 Nagase et. al., 78706358 XM OO5823 (1996) DNA Res. XM 045160 3(5):321-9 CHST3 78756110- NT 028289a 10q22.1 Tsutsumi et. al., 78759658 XM 011902 (1998) FEBS Lett. XM 045142 44.1(2):235–41 Fukuta et. al., (1998) Biochim. Biophys Acta. 1399(1):57-61 CBARA1 78889122- NT O24037 10q22.2 Wiemann et. al., 79102604 XM 011886 (2001) Genome XM 032486 Res. 11(3):422–35 XM 032487 Natter et. at. XM 032489 (1998) FASEB J. 12(14):1559–69 P4HA1 79554896- NT O24037 Oq22.1 Paiunen et. al., 79611864 XM OO5728 (1989) Am. J. Hum. XM 0325.11b Genet. 45(6):829– 34 HSGT1 797O4559- NT O24037 Oq22.1 Sato et. al., (1999) 79741363 XM 032513 Mol. Gen. Genet. 260(6):535–40 ANXAF 79927670- NT O24037 Oq22.2 Shirvan et. al., 7996.1935 XM OO5691 (1994) Biochemistry XM 032526 33(22):6888–901 XM 032527 PPP3CB 79995O85- NT O24037 Oq22.2 Giri et. al., (1991) 80O881.87 XM 011860 Biochem. Biophys XM 032530 Res Commun. 181(1):252–8 PLAU 8O34.1334- NT O24037 Oq22.2 Nagai et al., (1985) 80347653° XM 044353 Gene 36(1-2):183-8 XM 053443 80487562- XM 053444 Oq22.2 80493880 XM 053445
US 2003/017O678A1 Sep. 11, 2003 40
TABLE 1-continued Approximate Location on GenBank Approximate Chromosome Accession Cytogenetic Gene O (bp) No. Location Reference ADRA2A 20968570- M18415 10q24-q26 Hoehe et. al., 20969922 M23533a (1995) J. Mol. Med. 73(6):299-306 SLC18A2 278864.58- NT O29396 10q25 Surratt et. al., 27922683 XM OO5978 (1993) FEBS Lett. XM 048899 318(3):325-30 XM 048900 XM 048901 PRDX3 29883719- NT OO8902 10q25-q26 Tsuji et al., (1995) 29894799 XM 005968 Biochem. J. 307(pt2):377-81 EMX2 28.188763- NT O29396 10q26.1 Katsury et. al., 28193445 XM O49756 (1994) Genomics 22(1):41-5 GPRKS 299623.07- NT OO8902 10q24-qter Bullrich et. al., 3021732O XM 005969 (1995) Cytogenet. Cell Genet. 70(3– 4):250–4 DOCK1 37159760- NT 028285a 10q26.13- Hasegawa et al., 37637128 XM 028933 q26.3 (1996) Mol. Cell. Biol. 16(4):1770–6 "GenBank accession number for chromosomal nucleotide sequence containing the gene. GenBank accession number for mRNA sequence encoded by the gene. One of two or more approximate locations on chromosome 10 according to the UCSC Golden Path Genome Browser August 2001 Freeze (http://genome.ucsc.edu/index.html). One of two or more approximate locations on chromosome 10 according to the UCSC Golden Path Genome Browser August 2001 Freeze (http://genome.ucsc.edu/index.html). 0417 Particular candidate AD genes provided herein 0420. In another embodiment of the methods provided include SNCG (encoding Y-synuclein or persyn), TNFRSF6 herein for identifying an AD DNA segment or an AD gene (encoding tumor necrosis factor receptor Superfamily mem utilizing a candidate gene approach, a step of the method ber 6 or FAS), LIPA (encoding lipase A), IDE (encoding may be Selecting a candidate AD gene located within a insulin degrading enzyme), KNSL1 (encoding kinesin-like region of chromosome 10 identified herein as containing or 1), PLAU (urokinase plasminogen activator), TLL2 (tolloid near a genetic marker associated with AD. like 2), PSAP (prosaposin), PSD (pleckstrin and Sec7 domain protein), KIAA0904, NFKB2 (nuclear factor of 0421. The methods provided herein for identifying an AD kappa light polypeptide gene enhancer in B-cells 2, p49/ Segment or an AD gene utilizing a candidate gene approach p100), PPP3CB (protein phosphatase 3, catalytic subunit, may further include a step of analyzing genetic markers on beta isoform, calcineurin A beta), CH25H (cholesterol chromosome 10 located in or around a Selected candidate 25-hydroxylase) and FERIL3 (C. elegans-like 3, myoferlin). gene for linkage and/or association with AD. A possible benefit in utilizing linkage analysis methods for assessment 0418 (4) Methods for Identifying an AD DNA Segment of candidate genes is that only a few polymorphic micro or AD Gene Satellite markers in the candidate gene region may be required for the analysis. Microsatellite markers within the 0419. In a method provided herein for identifying an AD gene region are Selected, and DNA from AD family Samples DNA segment or an AD gene utilizing a candidate gene is genotyped with respect to those markers. Parametric approach, one Step of the method may be Selecting a and/or nonparametric linkage analyses of the genotype data candidate AD gene on chromosome 10 Such as described may be conducted as described herein or using methods above. The candidate AD gene may be one that is or encodes known in the art. If positive results are obtained in the a product that has properties relating to one or more phe linkage analyses (e.g., a positive LOD score), they are nomena in neurodegenerative disease. Particular embodi indicative of linkage of the DNA segment to a DNA segment ments of the methods provided herein for identifying an AD associated with AD. The LOD score values for markers DNA segment or gene include a Step of Selecting a candidate within the candidate gene region can be compared to deter AD gene on chromosome 10q. In further embodiments of mine if they delineate a region in which an AD gene may be these methods, the methods include a step of Selecting a located. Additional markers at Smaller genetic distances candidate AD gene on chromosome 10q22, 10q23, 10q24, within an identified region may then be analyzed for linkage 10q25 or 10q26. In particular embodiments, the methods to AD in order to narrow in on the location of an AD gene. include a step of Selecting a candidate gene on chromosome The markers may also be assessed through association 10q22, 10q23 or 10q24. In yet further embodiments, the analyses to ascertain whether any might lie within linkage methods include a Step of Selecting a candidate gene on disequilibrium range of an underlying AD DNA segment or chromosome 10q23, 10q24 or 10q25. gene. US 2003/017O678A1 Sep. 11, 2003
0422 The effectiveness of the candidate gene approach is Mb, or about 0.05 Mb, or about 0.01 Mb, or about 1 kb distal influenced by the type and density of the markers utilized in from and including marker D10S583, (7) the region between the Study as well as by Study design and Sample size. D10S564 and D10S583, inclusive, (8) the region between Although linkage analysis of candidate genes may utilize a D10S583 and D10S1710, inclusive, (9) the region between relatively Small number of microSatellite markers in the gene D10S583 and D10S566, inclusive, (10) the region between region, association analysis generally requires a fairly dense D10S583 and D10S1671, inclusive and (11) the region (on the order of kilobases) spacing of markers in the gene between D10S583 and D10S1741, inclusive. region. Thus, SNPs may be more suitable for such associa 0426 In further particular embodiments of these methods tion analyses than microSatellite markers. Therefore, candi the markers analyzed for LOD Scores are located in a region date gene approaches can typically involve Sequence analy of chromosome 10 as follows: (1) the region extending sis of candidate genes, particularly of individuals from about 5 cM, or about 4 cM, or about 2.7 cM, or about 2.5 affected families, to identify useful markers in the genes. cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or about 0423 b. Genetic Localization Approach 0.5 cM, or about 0.1 cM proximal (centromeric) from and including marker D10S583, (2) the region extending about 0424 Genetic localization approaches do not require 5 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or knowledge of the biological or biochemical nature of the about 0.1 cM distal (telomeric) from and including marker disease. In contrast to a full candidate gene approach, which D10S583, (3) the region extending about 10 Mb, or about 5 immediately restricts genetic analysis of a chromosome to a Mb, or about 2 Mb, or about 1 Mb, or about 500 kb, or about Specific gene region determined by a hypothesis based on 300 kb, or about 200 kb, or about 100 kb, or about 80 kb, or trait biology, genetic localization approaches first identify a about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb chromosomal region in which a disease gene or DNA proximal from and including marker D10S583, and (4) the Segment is located and then gradually reduce the size of the region extending about 10 Mb, or about 8 Mb, or about 5 region in order to determine the location of the Specific Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about defective DNA segment as precisely as possible. 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or 0425 For example, in these methods, the position of an about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, AD DNA segment or gene may be localized by determining or about 1 kb distal from and including marker D10S583. LOD scores for different markers on chromosome 10. In 0427. The strategy underlying this method is to select particular embodiments of these methods the markers ana Successive markers progressively closer to an AD gene, each lyzed are located in the region of 10q22, 10q23, 10q24, marker being chosen based on the linkage distance estab 10q25 or 10q26. In further embodiments of these methods, lished for the previous marker. Linkage distance is the the markers analyzed are located within a region identified distance from a marker at which the LOD score of the herein as containing or near markers associated with AD. In marker from an AD gene is maximized. For example, if a particular embodiments, the markers analyzed are on chro first marker gives a maximum LOD score at a linkage mosome 10q22, 10q23 or 10q24 or are on chromosome distance of 10 cM, a second marker would be selected within 10q23, 10q24 or 10q25. In further particular embodiments a 20 cM Segment centered about the first marker. The linkage of these methods, the markers analyzed are located in: (1) distance of the Second marker to the gene is then determined. the region extending about 50 cM, or about 45 cM, or about If this linkage distance is less than that of the first polymor 33 cM, or about 30 cM, or about 25 cM, or about 21 cM, or phic marker, a further marker is Selected within the linkage about 20 cM, or about 15 cM, or about 10 cM, or about 5 distance of the Second marker. If the linkage distance of the cM, or about 4 cM, or about 3 cM, or about 2.7 cM, or about Second marker is greater than that of the first marker, then a 2.5 cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or further marker is Selected within the linkage distance of the about 0.5 cM, or about 0.1 cM proximal (centromeric) from first marker, on the Side distal from the Second marker. By and including marker D10S583, (2) the region extending continually identifying markers progressively closer to the about 60 cM, or about 55 cM, or about 50 cM, or about 45 gene, it is possible to localize the position of the gene to a cM, or about 40 cM, or about 35 cM, or about 30 cM, or relatively small segment of DNA for which more detailed about 25 cM, or about 20 cM, or about 15 cM, or about 13 molecular Studies are feasible. cM, or about 12 cM, or about 9 cM, or about 5 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 cM 0428 Linkage tests can be powerful and specific for gene distal (telomeric) from and including marker D10S583, (3) discovery; however, localization of the disease locus can be the region extending from and including marker D10S583 to achieved only to a certain level of precision (i.e., on the the terminus of the q arm of chromosome 10, (4) the region order of many megabases) which represents a region that extending from and including marker D10S583 to the cen potentially can include hundreds of genes. Additionally, tromere of chromosome 10, (5) the region extending about genes with small or subtle effects may not be detectable by 62 Mb, about 55 Mb, or about 50 Mb, or about 32 Mb, or linkage at all. Linkage Studies are resource intensive and about 28 Mb, or about 25 Mb, or about 20 Mb, or about 15 dependent on the availability of large family collections, Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, or about which are not a requirement of most association-based 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, methods of gene discovery. or about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or about 1 kb proximal from and including marker D10S583, 0429 2. Association Analysis in the Identification of AD (6) the region extending about 45 Mb, or about 40 Mb, or DNA Segments or Genes about 35 Mb, or about 30 Mb, or about 25 Mb, or about 20 0430 Studies based on pedigrees in many cases, particu Mb, or about 16 Mb, or about 14 Mb, or about 13 Mb, or larly based on Study design, may serve to only identify a about 8 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, chromosome that harbors a trait-causing allele and may or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.1 possibly also serve to broadly outline an expansive region in US 2003/017O678A1 Sep. 11, 2003 42 which Such an allele may reside within a chromosome. In 0437. In another embodiment of the methods provided contrast, analyses of genetic association can be a powerful herein for identifying an AD DNA segment or an AD gene method for fine-Scale mapping of a chromosome thereby utilizing a candidate gene approach, a step of the method refining the region in which a trait-causing allele is located may be Selecting a candidate AD gene located within regions and even identifying the position of the allele. of chromosome 10 identified herein as containing or near 0431. There are, however, several situations which can genetic markers associated with AD. lead to the detection of allelic association between loci that 0438. The methods provided herein for identifying an AD are in fact unlinked, Such as, for example, population Segment or an AD gene utilizing a candidate gene approach Stratification. For this reason, linkage disequilibrium-based may further include a step of analyzing genetic markers on mapping is often used to fine-map a disease gene when a chromosome 10 located in or around a Selected candidate possible region of interest has already been identified. Thus, linkage disequilibrium-based gene discovery methods are gene for association with AD. Any Such chromosome 10 usually used to define the chromosomal region containing a markers may be used in the methods. In particular embodi disease gene once linkage has been demonstrated for a ments of these methods, the markers analyzed are chromo chromosome. Some 10 markers described herein that are linked to one or more DNA segments, e.g., genes, associated with AD and/or 0432 Generally, when disequilibrium is suspected, are associated with AD. In further particular embodiments of affected individuals are checked for increased frequency of these methods, the markers analyzed are SNPs. The methods Specific alleles for the marker loci. An exceSS frequency of provided herein for identifying an AD Segment or an AD any allele, as measured against general population frequen gene utilizing a candidate gene approach may include a step cies (using the chi-square Statistics) can indicate linkage of conducting nucleotide Sequence analysis of the candidate disequilibrium. gene to identify polymorphic markers, and in particular 0433 b. Candidate Gene Approach SNPs, which are analyzed for association and/or linkage with AD. 0434 Generally, in a candidate gene approach to the identification of a disease gene using association analysis of 0439 Association studies may be conducted, for polymorphic markers, one or a few markers around or within example, in two Successive Steps. In a first phase, the candidate disease genes, particularly those with hypoth frequencies of a reduced number of markers from the eSized functional importance, are genotyped in a few hun candidate gene are determined in the trait-positive and dred case and control individuals. The Specific characteris control populations. In a Second phase of the analysis, the tics of the associated allele with respect to a candidate gene position of the genetic locus responsible for the given trait function usually give further insight into the relationship is further refined using a higher density of markers from the between the associated allele and the trait (causal or in linkage disequilibrium). If the evidence indicates that the relevant region. However, if the candidate gene under Study asSociated allele within the candidate gene is most probably is relatively Small in length, a Single phase may be Sufficient not the trait-causing allele but is in linkage disequilibrium to establish Significant associations. with the real trait-causing allele, then the trait-causing allele 0440 c. Genetic Localization Approach can be found by Sequencing the vicinity of the associated marker, and performing further association Studies with the 0441 ASSociation analyses may be used in genetic local polymorphisms that are revealed in an iterative manner. ization approaches to disease gene discovery. In these meth 0435. In order for association analysis to be useful, a ods, a region of a chromosome is first identified as being in dense map of markers is generally required since associa proximity to a disease Susceptibility gene by Virtue of tions typically are found over shorter distances. It is esti linkage disequilibrium detected as association between a mated that SNPs occur, on average, every 1,000 bp and have highly informative marker in the region and a disease trait. a low mutation rate, both of which are characteristics that In particular embodiments of these methods, the region may have particular advantages for association analysis. identified is both linked and associated with disease. The Tens of thousands of known SNPs are located on chromo identified region is then analyzed in finer detail through Some 10, with an estimated mean intermarker distance of analysis of additional markers in the region for association about 2 kb, and may be used in methods provided herein for with the disease trait. The application of haplotypes to this identifying an AD Segment or an AD gene utilizing a analysis may provide Stronger evidence of a region being candidate gene approach. asSociated with the disease phenotype than does the use of individual markers. 0436 AS described above, in a method provided herein for identifying an AD DNA segment or an AD gene utilizing 0442. For example, in these methods, the position of an a candidate gene approach, one Step of the method may be AD gene may be localized by conducting AD association Selecting a candidate AD gene on chromosome 10. The analyses on different markers on chromosome 10, individu candidate AD gene may be one that is or encodes a product ally or in combinations. In particular embodiments of these that has properties relating to one or more phenomena in methods, the markers analyzed are on chromosome 10q. In neurodegenerative disease. Particular embodiments of the further embodiments of this method, the markers analyzed methods provided herein for identifying an AD DNA seg are located in the region of 10q22, 10q23, 10q24, 10q25 or ment or gene include a step of Selecting a candidate AD gene 10q26. In further embodiments of these methods, the mark on chromosome 10q. In further particular embodiments of erS analyzed are located within a region identified herein as these methods, the Selected candidate AD gene is on chro containing or near markers associated with AD. In particular mosome 10q22, 10q23, 10q24, 10q25 or 10q26. In particular embodiments, the markers analyzed are on chromosome embodiments, the Selected candidate gene is on chromo 10q22, 10q23 or 10q24 or on chromosome 10q23, 10q24 or Some 10q22, 10q23 or 10q24 or on chromosome 10q23, 10q25. In still further embodiments of these methods, the 10q24 or 10q25. markers analyzed for association with AD are located in: (1) US 2003/017O678A1 Sep. 11, 2003 the region extending about 50 cM, or about 45 cM, or about Some and thus on a Single "background” or “ancestral' 33 cM, or about 30 cM, or about 25 cM, or about 21 cM, or haplotype of linked markers. Consequently, there is com about 20 cM, or about 15 cM, or about 10 cM, or about 5 plete disequilibrium between these markers and the disease cM, or about 4 cM, or about 3 cM, or about 2.7 cM, or about mutation: the disease mutation is found only in the presence 2.5 cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or of a Specific Set of marker alleles. Through Subsequent about 0.5 cM, or about 0.1 cM proximal (centromeric) from generations, recombination events occur between the dis and including marker D10S583, (2) the region extending ease mutation and these marker polymorphisms, and the about 60 cM, or about 55 cM, or about 50 cM, or about 45 disequilibrium gradually dissipates. The pace of this dissi cM, or about 40 cM, or about 35 cM, or about 30 cM, or pation is a function of the recombination frequency, So the about 25 cM, or about 20 cM, or about 15 cM, or about 13 markers closest to the disease gene will manifest higher cM, or about 12 cM, or about 9 cM, or about 5 cM, or about levels of disequilibrium than those that are farther away. 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 cM When not broken up by recombination, “ancestral” haplo distal (telomeric) from and including marker D10S583, (3) types and linkage disequilibrium between marker alleles at the region extending from and including marker D10S583 to different loci can be tracked not only through pedigrees but the terminus of the q arm of chromosome 10, (4) the region also through populations. extending from and including marker D10S583 to the cen tromere of chromosome 10, (5) the region extending about 0447. A haplotype can be tracked through populations 62 Mb, about 55 Mb, or about 50 Mb, or about 32 Mb, or and its Statistical association with a given trait can be about 28 Mb, or about 25 Mb, or about 20 Mb, or about 15 analyzed. Complementing single point (allelic) association Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, or about Studies with multi-point association Studies, also called 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, haplotype Studies, increases the Statistical power of associa or about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or tion Studies. Thus, a haplotype association Study allows one about 1 kb proximal from and including marker D10S583, to define the frequency and the type of the ancestral carrier (6) the region extending about 45 Mb, or about 40 Mb, or haplotype. A haplotype analysis is important in that it about 35 Mb, or about 30 Mb, or about 25 Mb, or about 20 increases the Statistical power of an analysis involving Mb, or about 16 Mb, or about 14 Mb, or about 13 Mb, or individual markers. about 8 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, 0448. In a first stage of a haplotype frequency analysis, or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.1 the frequency of the possible haplotypes based on various Mb, or about 0.05 Mb, or about 0.01 Mb, or about 1 kb distal combinations of markers can be determined. The haplotype from and including marker D10S583, (7) the region between frequency is then compared for distinct populations of trait D10S564 and D10S583, inclusive, (8) the region between positive and control individuals. The number of trait positive D10S583 and D10S1710, inclusive, (9) the region between individuals, which should be subjected to this analysis to D10S583 and D10S566, inclusive, (10) the region between obtain Statistically significant results usually ranges between D10S583 and D10S1671, inclusive and (11) the region 30 and 300, with a preferred number of individuals ranging between D10S583 and D10S1741, inclusive. between 50 and 150. The same considerations apply to the 0443 Markers of particular interest are located in a number of unaffected individuals (or random control) used region of chromosome 10 as follows: (1) the region extend in the study. The results of this first analysis provide hap ing about 5 cM, or about 4 cM, or about 2.7 cM, or about 2.5 lotype frequencies in case-control populations, for each cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or about evaluated haplotype frequency a p-value and an odd ratio are 0.5 cM, or about 0.1 cM proximal (centromeric) from and calculated. If a Statistically significant association is found, including marker D10S583, (2) the region extending about the relative risk for an individual carrying the given haplo 5 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or type of being affected with the trait under study can be about 0.1 cM distal (telomeric) from and including marker approximated. D10S583, (3) the region extending about 10 Mb, or about 5 0449 a. Determination of Haplotype Frequencies Mb, or about 2 Mb, or about 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or about 100 kb, or about 80 kb, or 0450 When genotypes are determined, it is often not about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb possible to distinguish heterozygotes So that haplotype fre proximal from and including marker D10S583, and (4) the quencies cannot be easily inferred. When the gametic phase region extending about 10 Mb, or about 8 Mb, or about 5 is not known, Single chromosomes can be studied indepen Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about dently, for example, by asymmetric PCR amplification (see 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or Newton et al. (1989) Nucleic Acids Res. 17:2503-2516; Wu about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:2757), or by or about 1 kb distal from and including marker D10S583. isolation of single chromosome by limit dilution followed by PCR amplification (see Ruano et al. (1990) Proc. Natl. Acad. 0444. In particular embodiments of the association study Sci. U.S.A. 86:9079-9083). Further, a sample may be hap based disease gene localization methods provided herein, lotyped for sufficiently close markers by double PCR ampli markers analyzed for association are located within a region fication of specific alleles (Sarkar, G. and Sommer S. S. identified herein as containing or near markers associated (1991) Biotechniques). These approaches are not entirely with AD. Satisfying either because of their technical complexity, the 0445 3. Haplotype Analysis additional cost they entail, their lack of generalization at a large Scale, or the possible biases they introduce. To over 0446. When a disease mutation is first introduced into a come these difficulties, an algorithm to infer the phase of population (by a new mutation or the immigration of a PCR-amplified DNA genotypes introduced by Clark, A. G. mutation carrier), it necessarily resides on a single chromo (1990) Mol. Biol. Evol 7:111-122 may be used. Briefly, the US 2003/017O678A1 Sep. 11, 2003 44
principle is to Start filling a preliminary list of haplotypes 0456 Provided herein are methods of detecting an asso present in the sample by examining unambiguous individu ciation between a haplotype and AD. The methods include als, that is, the complete homozygotes and the Single-site Steps of: (a) estimating the frequency of at least one haplo heterozygotes. Then other individuals in the same Sample type in a trait positive population, (b) estimating the fre are Screened for the possible occurrence of previously quency of the haplotype in a control population, and (c) recognized haplotypes. For each positive identification, the determining whether a Statistically significant association complementary haplotype is added to the list of recognized exists between the haplotype and AD. haplotypes, until the phase information for all individuals is 0457. The haplotypes used in these methods include either resolved or identified as unresolved. This method combinations of markers provided herein. In particular assigns a single haplotype to each multiheterozygous indi embodiments of these methods, the markers analyzed in vidual, whereas Several haplotypes are possible when there combinations as haplotypes are on chromosome 10q. In are more than one heterozygous site. further embodiments of this method, the markers analyzed 0451 Alternatively, haplotype frequencies can be esti in combinations as haplotypes are located in the region of mated from the multilocus genotypic data. Any method 10q22, 10q23, 10q24, 10q25 or 10q26. In further embodi known to person skilled in the art can be used to estimate ments of these methods, the markers analyzed in combina haplotype frequencies see, e.g., Lange (1997) Mathemati tions as haplotypes are located within a region identified cal and Statistical Methods for Genetic Analysis, Springer, herein as containing or near markers associated with AD. In N.Y.; Weir (1996) Genetic Data Analysis II: Methods for particular embodiments, the markers analyzed in combina Discrete Population Genetic Data, Sinauer ASSoc., Inc., tions as haplotypes are on chromosome 10q22, 10q23 or Sunderland, Mass., U.S.A.). For example, maximum likeli 10q24 or are on chromosome 10q23, 10q24 or 10q25. In still hood haplotype frequencies can be computed using an further embodiments of these methods, the markers ana Expectation-Maximization (EM) algorithm See, e.g., lyzed in combinations as haplotypes for association with AD Dempster et al. (1977) J. R. Stat. Soc. 39B:1-38; Excoffier are located in: (1) the region extending about 50 cM, or and Slatkin (1995) Mol. Biol. Evol. 12:921-927). This pro about 45 cM, or about 33 cM, or about 30 cM, or about 25 cedure is an iterative process aiming at obtaining maximum cM, or about 21 cM, or about 15 cM, or about 10 cM, or likelihood estimates of haplotype frequencies from multi about 5 cM, or about 4 cM, or about 3 cM, or about 2.7 cM, locus genotype data when the gametic phase is unknown. or about 2.5 cM, or about 2 cM, or about 1.5 cM, or about Haplotype estimations are usually performed by applying 1 cM, or about 0.5 cM, or about 0.1 cM proximal (centro the EM algorithm using for example the EM-HAPLO pro meric) from and including marker D10S583, (2) the region gram Hawley et al. (1994) Am. J. Phys. Anthropol. 18:104) extending about 60 cM, or about 55 cM, or about 50 cM, or or the Arlequin program Schneider et al. (1997) Arlequin. about 45 cM, or about 40 cM, or about 35 cM, or about 30 A Software for Population Genetics Data Analysis, Univ. of cM, or about 25 cM, or about 20 cM, or about 15 cM, or Geneva. The EM algorithm is a generalized iterative maxi about 13 cM, or about 12 cM, or about 9 cM, or about 5 cM, mum likelihood approach. or about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0452 To ensure that the estimation finally obtained is the 0.1 cM distal (telomeric) from and including marker maximum-likelihood estimation, Several values of depar D10S583, (3) the region extending from and including marker D10S583 to the terminus of the q arm of chromo tures are required. The estimations obtained are compared, Some 10, (4) the region extending from and including and, if they are different, the estimations leading to the best marker D10S583 to the centromere of chromosome 10, (5) likelihood are kept. the region extending about 62 Mb, about 55 Mb, or about 50 0453 Estimating the frequency of a haplotype for a set of Mb, or about 32 Mb, or about 28 Mb, or about 25 Mb, or polymorphisms in a population, can be carried out by: 1) about 20 Mb, or about Mb, or about 10 Mb, or about 5 Mb, genotyping at least one polymorphism for each individual in or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.3 a population; 2) genotyping a second polymorphism by Mb, or about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or determining the identity of the nucleotides at the location of about 0.01 Mb, or about 1 kb proximal from and including the polymorphism for both copies of the Second polymor marker D10S583, (6) the region extending about 45 Mb, or phism present in the genome of each individual in the about 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 population; and c) applying a haplotype determination Mb, or about Mb, or about 16 Mb, or about 14 Mb, or about method to the identities of the nucleotides determined in 13 Mb, or about 8 Mb, or about 5 Mb, or about 4 Mb, or Steps a) and b) to obtain an estimate of the frequency. about 3 Mb, or about 2 Mb, or about 1 Mb, or about 0.5 Mb, Methods of estimating the frequency of a haplotype encom or about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or pass methods used alone or in any combination and all about 1 kb distal from and including marker D10S583, (7) others methods known to those of skill in the art in addition the region between D10S564 and D10S583, inclusive, (8) to those described herein. the region between D10S583 and D10S1710, inclusive, (9) 0454) b. Haplotype Analysis in the Identification of AD the region between D10S583 and D10S566, inclusive, (10) DNA Segments or Genes the region between D10S583 and D10S1671, inclusive and 04.55 Methods used for analyses of genetic association of (11) the region between D10S583 and D10S1741, inclusive. individual markers with disease in the identification of AD 0458 In further embodiments of these methods, the DNA segments can likewise be applied to the analysis of markers analyzed in combinations as haplotypes for asso genetic association of haplotypes of the markers with dis ciation with AD are located in a region of chromosome 10 ease for the same purpose. Haplotype analysis can be a most as follows: (1) the region extending about 5 cM, or about 4 powerful method for fine-Scale mapping of a chromosome cM, or about 2.7 cM, or about 2.5 cM, or about 2 cM, or thereby refining the region in which a trait-causing allele is about 1.5 cM, or about 1 cM, or about 0.5 cM, or about 0.1 located and even identifying the position of the allele. cM proximal (centromeric) from and including marker US 2003/017O678A1 Sep. 11, 2003
D10S583, (2) the region extending about 5 cM, or about 2.5 resolution achieved through the analysis of linkage disequi cM, or about 1 cM, or about 0.5 cM or about 0.1 cM distal librium is much higher than that of linkage Studies. (telomeric) from and including marker D10S583, (3) the region extending about 10 Mb, or about 5 Mb, or about 2 0465 Direct calculation of linkage disequilibrium Mb, or about 1 Mb, or about 500 kb, or about 300 kb, or requires a comparison of two genetic positions and can be about 200 kb, or about 100 kb, or about 80 kb, or about 50 used to quantify the extent of linkage disequilibrium in a kb, or about 10 kb, or about 5 kb, or about 1 kb proximal chromosomal region once a single- or multi-locus disease from and including marker D10S583, and (4) the region asSociation has been identified. Haplotypes may be used in extending about 10 Mb, or about 8 Mb, or about 5 Mb, or linkage disequilibrium calculations. about 4 Mb, or about 3 Mb, or about 2 Mb, or about 1 Mb, 0466 Several methods can be used to calculate linkage or about 500 kb, or about 300 kb, or about 200 kb, or about disequilibrium. For example the Standardized multiallelic 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, or disequilibrium coefficient D' may be calculated; it has a about 1 kb distal from and including marker D10S583. Simple interpretation, its Scale is independent of allele fre 0459. In particular embodiments of the haplotype asso quency and it is applicable to both SNP and microsatellite ciation Study-based disease gene localization methods pro data Hedrick (1987) Genetics 117:331-341; Devlin and Vided herein, markers analyzed in combinations for asso Risch (1995) Genomics 29:311-322). ciation with AD are located within a region identified herein 0467 6. Cloning and Sequence Analysis of an AD Gene as containing or near markers associated with AD. Chromosomal Region 0460 4. Interaction Analysis 0468 DNA in one or more regions of chromosome 10q, 0461 The polymorphic markers described herein may and in particular, 10q22-q26, that have been narrowly also be used to identify patterns of markers associated with defined as a possible AD DNA segment- or AD gene detectable traits resulting from polygenic interactions. The containing region based on the results of association analy analysis of genetic interaction between unlinked loci (for SeS described herein may be cloned and Sequenced. The example alleles on chromosome 10) and APOE4 on chro Smaller the candidate AD gene region (e.g., s1 Mb or 1 cM mosome 19) requires individual genotyping using the tech and, particularly, s500 kb or s 1 kb) for such analysis, the niques described herein. The analysis of allelic interaction more efficient the analysis will be. This process may gen among a selected Set of markers (polymorphisms) with erate additional markers and probes for more detailed map appropriate level of Statistical Significance can be considered ping of chromosome 10q with respect to the possible loca as a haplotype analysis. Interaction analysis involves Strati tion of one or more AD DNA segments or genes. fying the case-control populations with respect to a given 0469 It may be useful in narrowing in on a candidate AD haplotype for the first locus and performing a haplotype gene region of chromosome 10, and, in particular, chromo analysis with the Second locus with each Subpopulation. Some 10q22-q26, to identify and analyze additional poly 0462 5. Calculation of Linkage Disequilibrium morphic markers. Methods of identifying new markers are known in the art. For example, cloned human genomic DNA 0463 Linkage disequilibrium is the non-random associa e.g., cosmids, may be Screened for the presence of repetitive tion of alleles at two or more loci and represents a powerful DNA stretches (e.g., di, tri, and tetranucleotide repeats). tool for mapping genes involved in disease traits see, e.g., When a Sequence is positive for hybridization to a Synthetic Ajioka et al. (1997) Am. J. Human Genet. 60:1439-1447). oligonucleotide, it may be Subcloned into a plasmid and Any genetic markers may be used in genetic analysis based Sequenced or may be Sequenced after PCR amplification of on linkage disequilibrium. SNPs, because they are densely the region. PCR primers may be developed to amplify the Spaced in the human genome and can be genotyped in repetitive Stretch identified, and the markers may be tested greater numbers than other types of genetic markers (such as for the presence of polymorphism in a panel of control micro- or minisatellites), are particularly useful in genetic DNAS. In Silico procedures using the many human genome analysis based on linkage disequilibrium. When not broken and Sequence databases may also be used in the identifica up by recombination, “ancestral” haplotypes and linkage tion of additional polymorphic markers. disequilibrium between marker alleles at different loci can be tracked not only through pedigrees but also through 0470 A procedure for direct identification of microsat populations. Direct determination of linkage disequilibrium ellites from yeast artificial chromosomes (YACs) provides (as opposed to the obtaining of indirect evidence of linkage Several new markers from a target region. This procedure is disequilibrium as is obtained in association analysis of a based on a Subtractive hybridization Step that permits sepa ration of the target DNA from the vector background. YAC marker, or haplotype, and a trait) is usually seen as an clones containing Sequence from the 10q22-26 region have asSociation between one Specific allele at one locus and been identified by the CEPH/Genethon consortium (see another specific allele at a Second locus. http://www.cephb.fr/). Markers from YACs that have been 0464) The pattern or curve of disequilibrium between mapped to portions of 10q22-q26, and in particular 10q23 disease and marker loci is expected to exhibit a maximum q25, that are not well represented by currently available that occurs at the disease locus. Consequently, the amount of markers can be isolated. The microSatellite identification linkage disequilibrium between a disease allele and closely procedure may be performed by conducting Subtractive linked genetic markers may yield valuable information hybridization using genomic DNA from a target YAC regarding the location of the disease gene. For fine Scale together with an equivalent amount of a control DNA. This mapping of a disease locus, it is useful to have Some procedure separates YAC DNA from that of the yeast vector. knowledge of the patterns of linkage disequilibrium that Following the subtraction procedure, the subtracted YAC exist between markers in the Studied region. The mapping DNA is purified, digested with restriction enzymes and US 2003/017O678A1 Sep. 11, 2003 46 cloned into a plasmid vector. The cloned products of each isolated and used to genotype AD Subject nucleic acid YAC are Screened using a probe for repetitive Sequences. Samples. If maximal linkage disequilibrium is identified in Each positive clone is sequenced to identify primers for PCR the vicinity of one or two cDNAs, the cDNAS may be the to genotype AD Samples. Alternatively, direct thermal cycle first to be used to screen AD subject DNA for mutations. Sequencing, based on use of a Set of degenerate Sequencing 0474 Coding sequences may also be identified by exon primers that anneal directly to the repeat Sequence can also trapping which targets exons in genomic DNA by identify be used. By typing these markers in AD families, it is ing the consensus Splice Sequences that flank exon-intron possible to narrow the candidate region, for example, to a boundaries. Exons are trapped in the process of cloning Size of less than 1-2 cM, thus limiting the Segment in which DNA, e.g., from P1 clones, into an expression vector. The more extensive mapping efforts are applied. Once the can clones may be transfected into host cells, e.g., COS cells, didate region is narrowed, e.g., to a size of less than about and reverse transcriptase polymerase chain reaction (RT 500 to 1000 kb, a contiguous array (contig) of clones with PCR) may be performed on total or cytoplasmic RNA Smaller inserts than YACs, P1 clones and/or BACs can be isolated from the transfected cells using primers comple developed. P1 clones are phage clones designed to accom mentary to the Splicing vector. Once cDNAS are identified, modate inserts of up to 100 kb. the most plausible candidates may be Screened for Sequence 0471 Along with genetic mapping of candidate AD gene variation (mutations or polymorphisms) by direct Sequenc chromosomal regions on chromosome 10, a physical map of ing, Single Strand conformation polymorphism (SSCP) or chromosome 10q, particularly 10q22-q26, including 10q23 using chemical cleavage assayS. Northern blot analysis or q25, may be developed by, for example, assembling contigs reverse transcriptase polymerase chain reaction (RT-PCR) of of large insert clones that span the region of interest. Low autopsy tissues from affected and unaffected individuals, resolution contigs for most of the human genome are avail RNase protection or chemical cleavage, or any other State able using the YACs and BACs developed by CEPH which of-the-art technique may also be used in identifying the AD may provide a framework to start constructing high resolu gene or genes on chromosome 10q, particularly in the region tion contigs. Alternatively, BAC libraries are available or of 10q22-q26. could be developed to cover the region of interest. Contigs may be generated using P1 clones or BACs once the 0475 7. Expression of an Identified ORF and Analysis of candidate region is narrowed to less than about 1 Mb. Once AD Gene Product a region of 500-1000 kb or less is defined, physical mapping 0476 A candidate cDNA, or open reading frame (ORF), and cloning may be accomplished using P1 and/or BAC may be used to produce a gene product. For example, clones rather than YACs, and P1 or BAC contigs over such nucleic acid containing a candidate AD gene coding a region may be constructed. The P1 or BAC clones may be Sequence may be incorporated into an expression vector used to identify additional markers for further positional which typically will include a promoter that is operably cloning steps. linked to the candidate gene coding Sequence. Usually, the 0472. A starting point of contig construction is microSat promoter is a eukaryotic promoter for expression in a ellite Sequences and non-polymorphic STSS that derive from mammalian cell. The transcription regulation Sequences YACS that Surround a genetically determined AD gene may include an enhancer recognized by the host cell. The candidate region. The STSs may be used to screen the P1 Selection of promoters and enhancers depends on the host library. The ends of the P1 clones may be cloned using PCR cell that is used. The vector may also include host-recog and used to order the P1 clones relative to each other. nized replication Systems, amplifiable genes, Selectable Amplification in a new P1 clone may indicate that it overlaps markers, and other elements. with the previous one. The original set of P1 clones serves 0477. An expression vector containing a candidate AD as building blocks of the complete contig, each end clone is gene coding Sequence may be introduced into a host cell used to rescreen the library and in this way P1 clones are using any one or more of a variety of methods known in the added to the map. Restriction enzyme digestion and South art. The particular method(s) used may depend on the ern blot hybridization permit ordering of the majority of the particular vector and host cell. Suitable methods include, but P1 clones. From each P1 clone, additional microsatellite are not limited to, fusion, conjugation, transfection, trans markers may be identified which allows further reduction of duction, electroporation, injection, calcium phosphate pre the candidate region. ciptation and many other methods. Suitable host cells 0473. From a minimal candidate region defined by include, but are not limited to, bacteria, yeast, filamentous genetic and physical mapping analyses, a Segment may fungi, insect cells and mammalian cells, e.g., mouse, chinese remain that is Sufficiently large to contain multiple different hamster ovary and monkey cells. genes. The P1 clones may be used to identify candidate 0478 Any protein produced upon expression of a candi cDNAs of AD genes which may be screened for mutations date AD gene may be isolated using conventional methods in DNA from AD subjects. Coding sequences from the of protein biochemistry and purification known in the art. surrounding DNA may be identified and screened until a The protein may be analyzed using a variety of methods, probable candidate cDNA is found. It may also be possible including in Silico and functional assayS. For in Silico to identify coding Sequences through human genome methods, structural features (e.g., amino acid sequence) of Sequence database Screening. Candidates may also be iden the protein are compared to those known for certain types of tified by Scanning databases of partially Sequenced cDNAS proteins (e.g., enzymes) to determine if there are regions of known as expressed sequence tags (ESTS). The databases Similarity that may be characteristic of Such classes of may be used to identify clNAS that map to a minimal AD proteins. The information obtained in Such Studies can gene candidate region. The cDNAS may be used as probes provide data used in determining functional aspects of the to hybridize to a P1 contig, and new microSatellites may be protein. For functional Studies, analysis of the protein may US 2003/017O678A1 Sep. 11, 2003 47 also be conducted in cell-based assays using cells expressing Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about cDNA encoding the protein and/or a gene or cDNA con 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, or about 0.1 Mb, taining genetic alterations, e.g., mutations, correlated with or about 0.05 Mb, or about 0.01 Mb, or about 1 kb proximal AD. A candidate cDNA, or open reading frame (ORF), may from and including marker D10S583, (6) the region extend also be expressed in a transgenic, particularly nonhuman, ing about 45 Mb, or about 40 Mb, or about 35 Mb, or about animal. For example, a candidate cDNA may be linked to a 30 Mb, or about 25 Mb, or about 20 Mb, or about 16 Mb, promoter, and the resulting construct injected into a Zygote, or about 14 Mb, or about 13 Mb, or about 8 Mb, or about 5 e.g., a mouse Zygote. The candidate cDNA or gene may Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about contain one or more genetic alterations, e.g., mutations, 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or about 0.05 Mb, correlated with AD. The transgenic animal may also be or about 0.01 Mb, or about 1 kb distal from and including developed to have one or both alleles of a corresponding marker D10S583, (7) the region between D10S564 and endogenous gene inactivated, for example, by using a trans D10S583, inclusive, (8) the region between D10S583 and gene in which a cloned candidate gene having a positive D10S1710, inclusive, (9) the region between D10S583 and selection marker-encoding DNA within it such that it no D10S566, inclusive, (10) the region between D10S583 and longer encodes the candidate protein. Transgenic animals D10S1671, inclusive and (11) the region between D10S583 containing active, mutated and/or inactive candidate genes and D10S1741, inclusive. may be used to Study the function and AD-related mecha 0481. In further embodiments, one or more of the fol nisms of the protein and in identifying and evaluating drugs lowing regions of chromosome 10 is (are) analyzed for a for treatment of AD. polymorphism associated with AD: (1) the region extending 0479. H. Methods for Detecting the Presence of a Poly about 5 cM, or about 4 cM, or about 2.7 cM, or about 2.5 morphism ASSociated with Alzheimer's Disease cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or about 0480 Genetic analysis described herein led to the dis 0.5 cM, or about 0.1 cM proximal (centromeric) from and covery of genetic association with AD on chromosome 10. including marker D10S583, (2) the region extending about The association identifies chromosome 10 as the location of 5 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or one or more AD DNA segments or genes. Based on this about 0.1 cM distal (telomeric) from and including marker discovery which identifies within the entire genome a par D10S583, (3) the region extending about 10 Mb, or about 5 ticular chromosomal location of an AD DNA segment, Mb, or about 2 Mb, or about 1 Mb, or about 500 kb, or about methods are provided herein for detecting the presence in a 300 kb, or about 200 kb, or about 100 kb, or about 80 kb, or subject of a polymorphism or allele associated with AD. The about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb methods include a specific step of analyzing chromosome 10 proximal from and including marker D10S583, and (4) the of the subject for a polymorphism associated with AD. In region extending about 10 Mb, or about 8 Mb, or about 5 particular embodiments of these methods, chromosome 10q Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about is analyzed for a polymorphism associated with AD. In 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or further embodiments of these methods, chromosome 10q22, about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, 10q23, 10q24, 10q25 and/or 10q26 is (are) analyzed for a or about 1 kb distal from and including marker D10S583. polymorphism associated with AD. In further embodiments 0482 In further embodiments of the methods for detect of these methods, a region of chromosome 10 identified ing the presence in a Subject of a polymorphism associated herein as containing or near markers associated with AD is with AD, nucleic acid of the Subject is analyzed for the analyzed for a polymorphism associated with AD. In par presence or absence of marker D10S583. In particular ticular embodiments, chromosome 10q22, 10q23 and/or embodiments of the methods for detecting the presence or 10q24 or chromosome 10q23, 10q24 and/or 10q25 is (are) absence in a Subject of a polymorphism associated with AD, analyzed for a polymorphism associated with AD. In yet chromosome 10 of the Subject is analyzed for the presence further embodiments of these methods, one or more of the following regions of chromosome 10 is (are) analyzed for a or absence of a polymorphism which is associated with AD polymorphism associated with AD: (1) the region extending and linked to a DNA segment or gene associated with AD. about 50 cM, or about 45 cM, or about 33 cM, or about 30 0483 Also provided herein are methods for detecting the cM, or about 25 cM, or about 21 cM, or about 20 cM, or presence in a Subject of a combination or haplotype of about 15 cM, or about 10 cM, or about 5 cM, or about 4 cM, polymorphisms associated with AD. In one embodiment, or about 3 cM, or about 2.7 cM, or about 2.5 cM, or about each polymorphism in the combination is associated with 2 cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or AD. In other embodiments, Some of the polymorphisms in about 0.1 cM proximal (centromeric) from and including the combination are associated with AD and Some of the marker D10S583, (2) the region extending about 60 cM, or polymorphisms are not or none of the polymorphisms is about 55 cM, or about 50 cM, or about 45 cM, or about 40 associated with AD. In embodiments in which some or all of cM, or about 35 cM, or about 30 cM, or about 25 cM, or the polymorphisms are not individually associated with AD, about 20 cM, or about 15 cM, or about 13 cM, or about 12 the combination or haplotype of polymorphisms as a whole cM, or about 9 cM, or about 5 cM, or about 2.5 cM, or about may be associated with AD. The methods for detecting the 1 cM, or about 0.5 cM or about 0.1 cM distal (telomeric) presence in a Subject of a combination or haplotype of from and including marker D10S583, (3) the region extend polymorphisms associated with AD include a specific Step of ing from and including marker D10S583 to the terminus of analyzing chromosome 10 of the Subject for a combination the q arm of chromosome 10, (4) the region extending from or haplotype of polymorphisms associated with AD. In and including marker D10S583 to the centromere of chro particular embodiments of these methods, chromosome 10q mosome 10, (5) the region extending about 62 Mb, about 55 is analyzed for a combination or haplotype of polymor Mb, or about 50 Mb, or about 32 Mb, or about 28 Mb, or phisms associated with AD. In further embodiments of these about 25 Mb, or about 20 Mb, or about 15 Mb, or about 10 methods, the polymorphisms in the combination or haplo US 2003/017O678A1 Sep. 11, 2003 48 type associated with AD are located on chromosome 10q22, 5 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or 10q23, 10q24, 10q25 and/or 10q26. In further embodiments about 0.1 cM distal (telomeric) from and including marker of these methods, a region of chromosome 10 identified D10S583, (3) the region extending about 10 Mb, or about 5 herein as containing or near markers associated with AD is Mb, or about 2 Mb, or about 1 Mb, or about 500 kb, or about analyzed for a combination or haplotype of polymorphisms 300 kb, or about 200 kb, or about 100 kb, or about 80 kb, or asSociated with AD. In particular embodiments, the combi about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb nation or haplotype of polymorphisms associated with AD proximal from and including marker D10S583, and (4) the are located on chromosome 10q22, 10q23 and/or 10q24 or region extending about 10 Mb, or about 8 Mb, or about 5 chromosome 10q23, 10q24 and/or 10q25. In each of these embodiments, the individual polymorphisms contained in Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about the combination or haplotype may be located (1) each in 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or different locations of the identified locations of chromosome about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, 10, (2) all in the same location which is one of the identified or about 1 kb distal from and including marker D10S583. locations on chromosome 10, or (3) any number of a variety 0486 Because of the discovery of chromosome 10 as the of arrangements of the individual polymorphisms of the location of one or more AD genes, it is possible to determine combination within the identified locations of chromosome the presence or absence of one or more polymorphic mark 10, e.g., one polymorphism in a first location, two polymor erS associated with AD in a Subject by focussing analysis of phisms in a Second location, two polymorphisms in a third a nucleic acid Sample of a Subject on a particular region or location, etc. regions of the chromosome identified and described herein. 0484. In yet further embodiments of these methods, each In these methods, the detection of the presence or absence of polymorphism of the combination or haplotype is located in the polymorphism can be accomplished without having to one or more of the following regions of chromosome 10: (1) analyze areas of the Subject's genome other than those the region extending about 50 cM, or about 45 cM, or about identified and Specified herein. The methods also include, 33 cM, or about 30 cM, or about 25 cM, or about 21 cM, or however, optional Steps of analyzing other specific areas of about 20 cM, or about 15 cM, or about 10 cM, or about 5 the Subject's genome which are to known contain markers cM, or about 4 cM, or about 3 cM, or about 2.7 cM, or about associated with AD or AD susceptibility or disease alleles. 2.5 cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or about 0.1 cM proximal (centromeric) from 0487. For example, certain further embodiments of the and including marker D10S583, (2) the region extending methods of determining the presence or absence of a poly about 60 cM, or about 55 cM, or about 50 cM, or about 45 morphism asSociated with AD include an additional Step of cM, or about 40 cM, or about 35 cM, or about 30 cM, or analyzing the subject’s nucleic acid sample for the presence about 25 cM, or about 20 cM, or about 15 cM, or about 13 or absence of the APOE e4 allele. Apollipoprotein E (APOE) cM, or about 12 cM, or about 9 cM, or about 5 cM, or about performs various functions as a protein constituent of 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 cM plasma lipoproteins, including a role in cholesterol metabo distal (telomeric) from and including marker D10S583, (3) lism. The APOE e4 allele is a well-established susceptibility the region extending from and including marker D10S583 to gene for late-onset AD. The APOE-4 allele is neither nec the terminus of the q arm of chromosome 10, (4) the region essary or sufficient for AD, but modulates the risk of extending from and including marker D10S583 to the cen developing AD Corder et al. (1993) Science 261:921-923; tromere of chromosome 10, (5) the region extending about Corder et al. (1994) Nature Genet. 7:180-184). The APOE 62 Mb, about 55 Mb, or about 50 Mb, or about 32 Mb, or e4 polymorphism is designated as SNP528 (nucleotide about 28 Mb, or about 25 Mb, or about Mb, or about 15 Mb, 212240) of Genbank Accession No. AF050154 and is a or about 10 Mb, or about 5 Mb, or about 2 Mb, or about 1 Single nucleotide Substitution which results in the replace Mb, or about 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, or ment of the cysteine at position 112 with an arginine. about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or about 0488 Methods of detecting the presence or absence in a 1 kb proximal from and including marker D10S583, (6) the Subject of a polymorphism associated with AD can be used region extending about 45 Mb, or about 40 Mb, or about 35 for Several purposes. For example, detection of a polymor Mb, or about 30 Mb, or about Mb, or about 20 Mb, or about phism or allele associated with AD can be used in the 16 Mb, or about 14 Mb, or about 13 Mb, or about 8 Mb, or identification and isolation of one or more AD DNA seg about 5 Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, ments or genes on chromosome 10 through the genetic or about 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or about and/or physical analysis of Specific, relatively Small chro 0.05 Mb, or about 0.01 Mb, or about 1 kb distal from and mosomal regions, particularly 10q22, 10q23, 10q24, 10q25 including marker D10S583, (7) the region between and/or 10q26, and more particularly, 10q22, 10q23 and/or D10S564 and D10S583, inclusive, (8) the region between 10q24 or 10q23, 10q24 and/or 10q25. Detection of a poly D10S583 and D10S1710, inclusive, (9) the region between morphism or allele associated with AD can also be used in D10S583 and D10S566, inclusive, (10) the region between the molecular identification of carriers of an AD gene and a D10S583 and D10S1671, inclusive and (11) the region determination of the probability of having the disease or of between D10S583 and D10S1741, inclusive. passing the gene to their offspring. In these methods, the 0485. In further embodiments, each polymorphism of the presence of the polymorphism or allele is indicative of combination or haplotype is located in one or more of the carriers of a DNA segment associated with AD. In addition, following regions of chromosome 10: (1) the region extend detection of a polymorphism or allele associated with AD ing about 5 cM, or about 4 cM, or about 2.7 cM, or about 2.5 can be used in methods of generating pharmacogenetic cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or about profiles which correlate drug response with genotype and 0.5 cM, or about 0.1 cM proximal (centromeric) from and thus can also be used in methods of predicting a response of including marker D10S583, (2) the region extending about a Subject to a drug used to treat AD. US 2003/017O678A1 Sep. 11, 2003 49
0489 Analysis of chromosome 10 in accordance with the at a final concentration of 100-300 nM (Invitrogen Corp., methods of detecting the presence or absence in a Subject of Carlsbad, Calif.) along with Taq polymerase (0.25 U/reac a polymorphism associated with AD can be conducted in a tion; Qiagen, Valencia, Calif. and Roche, Indianapolis, Ind.) variety of ways using analytical methods described herein and dNTPs (2.5uM/rxn; Amersham-Pharmacia, Piscataway, and genetic analysis methods known to those of Skill in the N.J.). All PCR reactions are performed from ~10 ng of art. Any methods whereby the identity of nucleotides in an DNA. General PCR thermo-cycling conditions are as fol nucleic acid Sequence may be determined can be used. lows: initial denaturation 3 minutes at 94 C., followed by 30-35 cycles of denaturation at 94 C. for 45 seconds, 0490 Many methods are available for detecting specific primer-specific annealing temperature (see below) for 45 alleles at human polymorphic loci. The preferred method for seconds, and product extension at 72 C. for 1 minute. Final detecting a particular polymorphism, depends on the nature extension at 72 C. for six minutes. PCR products can be of the polymorphism. Several methods of determining the Visualized on 2% agarose-gels to confirm a single product of presence or absence of allelic variants of a human gene are the correct size. PCR primers and unincorporated dNTPs provided below. Methods that are useful are not limited to can be degraded by adding exonuclease I (Exol, 0.1-0.15 those described below, but include all available methods. U/reaction; New England Biolabs, Beverly, Mass.) and 0491 Generally, nucleic acid detection methods are shrimp alkaline phosphatase (SAP, 1U/reaction; Roche, based in Sequence-specific polynucleotides, oligonucle Indianapolis, Ind.) to the PCR reactions and incubating for otides, probes and primers. Any method known to those of 1 hour at 37° C., followed by 15 minutes at 95° C. to skill in the art for detecting a specific nucleotide within a inactivate the enzymes. The Single base extension Step is nucleic acid Sequence or for determining the identity of a performed by directly adding SBE primer (100 nM; Invit Specific nucleotide in a nucleic acid Sequence is applicable rogen Corp., Carlsbad, Calif.), Thermosequenase (0.4 U/re to the methods of determining the presence or absence of an action; Amersham-Pharmacia, Piscataway, N.J.), and the allelic variant on chromosome 10. Such methods include, appropriate mixture of R110-ddNTP, TAMRA-ddNTP (3 but are not limited to, techniques utilizing nucleic acid uM; NEN, Boston, Mass.), and all four unlabeled ddNTPs hybridization of Sequence-specific probes, nucleic acid (22 or 25 uM; Amersham-Pharmacia, Piscataway, N.J.) to Sequencing, Selective amplification, analysis of restriction the Exol/SAP treated PCR product. Acycloprime-FP SNP enzyme digests of the nucleic acid, cleavage of mismatched detection kits (G/A)(Perkin-Elmer, Boston, Mass.) may also heteroduplexes of nucleic acid and probe, alterations of be used for the SBE reaction. Incorporation of the SNP electrophoretic mobility, primer Specific extension, oligo specific fluorescent ddNTP is achieved by subjecting nucleotide ligation assay and Single-Stranded conformation samples to 35 cycles of 94° C. for 15 seconds and 55° C. for polymorphism analysis. In particular, primer extension reac 30 seconds. The length of the SBE primers are designed to tions that specifically terminate by incorporating a dideoxy yield a melting temperature T of 62-64 C. Fluorescent nucleotide are useful for detection. Several Such general ddNTP incorporation is detected using the AnalystTM AD nucleic acid detection assays are known (see, e.g., U.S. Pat. System (Molecular Devices, Sunnyvale, Calif.) and measur No. 6,030,778). ing fluorescent polarization for R110 (excitation at 490 nm, emission at 520 nm) and TAMRA (excitation at 550 nm, 0492 Any cell type or tissue may be utilized to obtain emission at 580 nm). Genotypes are called manually or nucleic acid Samples, e.g., bodily fluid Such as blood or automatically using the manufacturer's Software ( Allele Saliva, dry Samples Such as hair or skin. caller vers. 1.0, Molecular Devices, Sunnyvale, Calif.). In View of the polymorphic regions provided herein, Specific 0493 1. Primer Extension-Based Methods PCR primers (5' to 3' sequences), annealing temperature, 0494. Several primer extension-based methods for deter product length, SBE primer Sequence, marker location and mining the identity of a particular nucleotide in a nucleic reference Sequence position, can readily be determined by acid sequence have been reported (see, e.g., PCT Applica those of skill in the art using well-known methods. tion Nos. PCT/US96/03651 (WO96/29431), PCT/US97/ 0496 2. Polymorphism-Specific Probe Hybridization 20444 (WO 98/20166), PCT/US97/20194 (WO 98/20019), PCT/US91/00046 (WO91/13075), and U.S. Pat. Nos. 5,547, 0497 Another detection method is allele specific hybrid 835, 5,605,798, 5,622,824, 5,691,141, 5,872,003, 5,851,765, ization using probes overlapping the polymorphic Site and 5,856,092, 5,900,481, 6,043,031, 6,133,436 and 6,197.498.) having about 5, 10, 15, 20, 25, or 30 nucleotides around the In general, a primer is prepared that Specifically hybridizes polymorphic region. The probes can contain naturally occur adjacent to a polymorphic Site in a particular nucleic acid ring or modified nucleotides (see U.S. Pat. No. 6,156,501). molecule. The primer is then extended in the presence of one For example, oligonucleotide probes may be prepared in or more dideoxynucleotides, typically with at least one of which the known polymorphic nucleotide is placed centrally the dideoxynucleotides being the complement of the nucle (allele-specific probes) and then hybridized to target DNA otide that is polymorphic at the Site. The primer and/or the under conditions which permit hybridization only if a per dideoxynucleotides may be labeled to facilitate a determi fect match is found (Saiki et al. (1986) Nature 324:163; nation of primer extension and identity of the extended 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 nucleotide. Specific oligonucleotide hybridization techniques may be 0495. A method of genotyping or determining the pres used for the Simultaneous detection of Several nucleotide ence of an allelic variant involves two-dye fluorescence changes in different polymorphic regions. For example, polarization detected single base extension (FP-SBE (12)) oligonucleotides having nucleotide Sequences of Specific on an LJL-Biosystems Criterion Analyst AD (Molecular allelic variants are attached to a hybridizing membrane and Devices, Sunnyvale, Calif.). PCR primers are designed to this membrane is then hybridized with labeled sample yield products between 200-400 bp in length, and are used nucleic acid. Analysis of the hybridization signal will then US 2003/017O678A1 Sep. 11, 2003 50
reveal the identity of the nucleotides of the Sample nucleic Sci 74:5463. It is also contemplated that any of a variety of acid. In a preferred embodiment, Several probes capable of automated Sequencing procedures may be used when per hybridizing Specifically to allelic variants are attached to a forming the subject assays (1995) Biotechniques 19:448), Solid phase Support, e.g., a “chip'. Oligonucleotides can be including Sequencing by mass spectrometry (see, for bound to a Solid Support by a variety of processes, including example, U.S. Pat. Nos. 5,547,835, 5,691,141, and Interna lithography. For example a chip can hold up to 250,000 tional PCT Application No. PCT/US94/00193 (WO oligonucleotides (GeneChip, Affymetrix, Santa Clara, 94/16101), entitled “DNA Sequencing by Mass Spectrom Calif.). Mutation detection analysis using these chips com etry” by H. Koster; U.S. Pat. Nos. 5,547,835, 5,622,824, prising oligonucleotides, also termed "DNA probe arrays” is 5,851,765, 5,872,003, 6,074,823, 6,140,053 and Interna described e.g., in Cronin et al. (1996) Human Mutation tional PCT Application No. PCT/US94/02938 (WO 7:244 and in Kozal et al. (1996) Nature Medicine 2:753. In 94/21822), entitled “DNA Sequencing by Mass Spectrom one embodiment, a chip includes all the allelic variants of at etry Via Exonuclease Degradation” by H. Koster, and U.S. least one polymorphic region of a gene. The Solid phase Pat. Nos. 5,605,798, 6,043,031, 6,197,498, and International Support is then contacted with a test nucleic acid and Patent Application No. PCT/US96/03651 (WO 96/29431) hybridization to the Specific probes is detected. Accordingly, entitled “DNA Diagnostics Based on Mass Spectrometry” the identity of numerous allelic variants of one or more by H. Koster; Cohen et al. (1996) Adv Chromatogra6:127 genes can be identified in a simple hybridization experiment. 162; and Griffin et al. (1993) Appl Biochem Biotechnol 38:147-159). It will be evident to one skilled in the art that, 0498. 3. Nucleic Acid Amplification-Based Methods for certain embodiments, the occurrence of only one, two or 0499. In other detection methods, it is necessary to first three of the nucleic acid bases need be determined in the amplify at least a portion of a gene prior to identifying the Sequencing reaction. For instance, A-track Sequencing or an allelic variant. Amplification can be performed, e.g., by PCR equivalent, e.g., where only one nucleotide is detected, can and/or LCR, according to methods known in the art. In one be carried out. Other Sequencing methods are known (see, embodiment, genomic DNA of a cell is exposed to two PCR e.g., in U.S. Pat. No. 5,580,732 entitled “Method of DNA primerS and amplification is performed for a number of Sequencing employing a mixed DNA-polymer chain probe' cycles Sufficient to produce the required amount of amplified and U.S. Pat. No. 5,571,676 entitled “Method for mismatch DNA. In another embodiment, the primers are located directed in vitro DNA sequencing”). between 150 and 350 base pairs apart. 0504 5. Restriction Enzyme Digest Analysis 0500 Alternative amplification methods include: self Sustained sequence replication (Guatelli, J. C. et al. (1990) 0505. In some cases, the presence of a specific allele in Proc. Natl. Acad. Sci. U.S.A. 87: 1874-1878), transcriptional nucleic acid, particularly DNA, from a Subject can be shown amplification system (Kwoh, D. Y. et al. (1989) Proc. Natl. by restriction enzyme analysis. For example, a specific Acad. Sci. U.S.A. 86: 1173-1177), Q-Beta Replicase (Lizardi, nucleotide polymorphism can result in a nucleotide P. M. et al. (1988) Bio/Technology 6:1197), or any other Sequence containing a restriction site which is absent from nucleic acid amplification method, followed by the detection the nucleotide Sequence of another allelic variant. of the amplified molecules using techniques well known to 0506 6. Mismatch Cleavage those of skill in the art. These detection Schemes are espe cially useful for the detection of nucleic acid molecules if 0507 Protection from cleavage agents, such as, but not Such molecules are present in Very low numbers. limited to, a nuclease, hydroxylamine or oSmium tetroxide and with piperidine, can be used to detect mismatched bases 0501 Alternatively, allele specific amplification technol in RNA/RNA DNA/DNA, or RNA/DNA heteroduplexes ogy, which depends on Selective PCR amplification may be (Myers, et al. (1985) Science 230:1242). In general, the used in conjunction with the alleles provided herein. Oligo technique of "mismatch cleavage' starts by providing het nucleotides used as primers for Specific amplification may eroduplexes formed by hybridizing a control nucleic acid, carry the allelic variant of interest in the center of the which is optionally labeled, e.g., RNA or DNA, comprising 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. 0502 4. Nucleic Acid Sequencing-Based Methods 0508. In other embodiments, either DNA/DNA or RNA/ 0503) Any of a variety of sequencing reactions known in DNA duplexes can be treated with hydroxylamine or the art can be used to directly Sequence at least a portion of oSmium tetroxide and with piperidine in order to digest a gene and to detect allelic variants, e.g., mutations, by mismatched regions. After digestion of the mismatched comparing the Sequence of the Sample Sequence with the regions, the resulting material is then Separated by Size on corresponding wild-type (control) sequence. Exemplary denaturing polyacrylamide gels to determine whether the Sequencing reactions include those based on techniques control and Sample nucleic acids have an identical nucle developed by Maxam and Gilbert (1977) Proc. Natl. Acad. otide sequence or in which nucleotides they differ (See, for Sci. U.S.A. 74:560) or Sanger et al. (1977) Proc. Natl. Acad. example, Cotton et al. (1988) Proc. Natl. AcadSci U.S.A. US 2003/017O678A1 Sep. 11, 2003
85:4397; Saleeba et al. (1992) Methods Enzymod. 217:286 0514 Several techniques based on this OLA method have 295). The control or sample nucleic acid is labeled for been developed and can be used to detect specific allelic detection. variants of a polymorphic region of a gene. For example, U.S. Pat. No. 5,593,826 discloses an OLA using an oligo 0509. 7. Electrophoretic Mobility Alterations nucleotide having 3'-amino group and a 5'-phosphorylated 0510. In other embodiments, alteration in electrophoretic oligonucleotide to form a conjugate having a phosphorami mobility is used to identify the type of allelic variant of a date linkage. In another variation of OLA described in Tobe gene of interest. For example, Single-Strand conformation et al. (1996) Nucl. Acids Res. 24:3728, OLA combined with polymorphism (SSCP) may be used to detect differences in PCR permits typing of two alleles in a single microtiter well. electrophoretic mobility between mutant and wild type By marking each of the allele-Specific primers with a unique nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. hapten, i.e. digoxigenin and fluorescein, each OLA reaction U.S.A. 86:2766, see also Cotton (1993) Mutat Res 285:125 can be detected by using hapten Specific antibodies that are 144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79). labeled with different enzyme reporters, alkaline phos Single-Stranded DNA fragments of Sample and control phatase or horseradish peroxidase. This System permits the nucleic acids are denatured and allowed to renature. The detection of the two alleles using a high throughput format Secondary Structure of Single-stranded nucleic acids varies that leads to the production of two different colors. according to Sequence, the resulting alteration in electro 0515 9. SNP Detection Methods phoretic mobility enables the detection of even a Single base change. The DNA fragments may be labeled or detected 0516 Several methods have been developed to facilitate with labeled probes. The sensitivity of the assay may be the analysis of Single nucleotide polymorphisms. enhanced by using RNA (rather than DNA), in which the 0517. In one embodiment, the single base polymorphism Secondary Structure is more Sensitive to a change in can be detected by using a specialized exonuclease-resistant Sequence. In another embodiment, the Subject method uses nucleotide, as disclosed, e.g., in Mundy, C. R. (U.S. Pat. No. heteroduplex analysis to Separate double Stranded heterodu 4,656,127). According to the method, a primer complemen plex molecules on the basis of changes in electrophoretic tary to the allelic Sequence immediately 3' to the polymor mobility (Keen et al. (1991) Trends Genet 7:5). phic Site is permitted to hybridize to a target molecule 0511 8. Polyacrylamide Gel Electrophoresis obtained from a particular animal or human. If the polymor phic Site on the target molecule contains a nucleotide that is 0512. In yet another embodiment, the identity of an complementary to the particular exonuclease-resistant allelic variant of a polymorphic region of an gene is obtained nucleotide derivative present, then that derivative will be by analyzing the movement of a nucleic acid comprising the incorporated onto the end of the hybridized primer. Such polymorphic region in polyacrylamide gels containing a incorporation renders the primer resistant to exonuclease, gradient of denaturant is assayed using denaturing gradient and thereby permits its detection. Since the identity of the gel electrophoresis (DGGE) (Myers et al. (1985) Nature exonuclease-resistant derivative of the Sample is known, a 313:495). When DGGE is used as the method of analysis, finding that the primer has become resistant to exonucleases DNA will be modified to ensure that it does not completely reveals that the nucleotide present in the polymorphic Site of denature, for example by adding a GC clamp of approxi the target molecule was complementary to that of the mately 40 bp of high-melting GC-rich DNA by PCR. In a nucleotide derivative used in the reaction. This method has further embodiment, a temperature gradient is used in place the advantage that it does not require the determination of of a denaturing agent gradient to identify differences in the large amounts of extraneous Sequence data. mobility of control and sample DNA (Rosenbaum and 0518. In another embodiment, a solution-based method Reissner (1987) Biophys Chem 265:1275). for determining the identity of the nucleotide of a polymor phic site is employed (Cohen, D. et al. (French Patent Oligonucleotide Ligation ASSay (OLA) 2,650,840; PCT Application No. WO91/02087)). As in the 0513. In another embodiment, identification of the allelic Mundy method of U.S. Pat. No. 4,656,127, a primer is variant is carried out using an oligonucleotide ligation assay employed that is complementary to allelic Sequences imme (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 0519) 10. Genetic Bit Analysis found in a target molecule, the oligonucleotides will hybrid 0520. An alternative method, known as Genetic Bit ize Such that their termini abut, and create a ligation Sub Analysis or GBATM is described by Goelet, et al. (U.S. Pat. Strate. Ligation then permits the labeled oligonucleotide to No. 6,004,744, PCT Application No.92/15712). The method be recovered using avidin, or another biotin ligand. Nick of Goelet, et al. uses mixtures of labeled terminators and a erSon, D. A. et al. have described a nucleic acid detection primer that is complementary to the Sequence 3' to a assay that combines attributes of PCR and OLA (Nickerson, polymorphic Site. The labeled terminator that is incorporated D. A. et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:8923 is thus determined by, and complementary to, the nucleotide 8927). In this method, PCR is used to achieve the exponen present in the polymorphic Site of the target molecule being tial amplification of target DNA, which is then detected evaluated. In contrast to the method of Cohen et al. (French using OLA. Patent 2,650,840; PCT Application No. WO91/02087), the US 2003/017O678A1 Sep. 11, 2003 52 method of Goelet, et al. is preferably a heterogeneous phase 0530 A different multiplex detection format is one in assay, in which the primer or the target molecule is immo which differentiation is accomplished by employing differ bilized to a Solid phase. ent Specific capture Sequences which are position-specifi 0521) 11. Other Primer-Guided Nucleotide Incorporation cally immobilized on a flat Surface (e.g., a chip array). Procedures 0531 14. Other Methods 0522. Other primer-guided nucleotide incorporation pro 0532. Additional methods of analyzing nucleic acids cedures for assaying polymorphic Sites in DNA have been include amplification-based methods including polymerase described (Komher, J. S. et al. (1989) Nucl. Acids Res. chain reaction (PCR), ligase chain reaction (LCR), mini 17:7779-7784; Sokolov, B. P. (1990) Nucl. Acids Res. PCR, rolling circle amplification, autocatalytic methods, 18:3671; Syvanen, A. C., et al. (1990) Genomics 8:684-692, Such as those using QJ replicase, TAS, 3SR, and any other Kuppuswamy, M. N. et al. (1991) Proc. Natl. Acad. Sci. Suitable method known to those of skill in the art. (U.S.A.) 88:1143-1147; Prezant, T. R. et al. (1992) Hum. Mutat. 1:159-164; Ugozzoli, L. et al. (1992) GATA 9:107 0533. Other methods for analysis and identification and 112; Nyren, P. et al. (1993) Anal. Biochem. 208:171-175). detection of polymorphisms, include but are not limited to, These methods differ from GBATM in that they all rely on the allele Specific probes, Southern analyses, and other Such incorporation of labeled deoxynucleotides to discriminate analyses. between bases at a polymorphic Site. In Such a format, Since the Signal is proportional to the number of deoxynucleotides 0534 I. Methods for Determining the Level of Risk for incorporated, polymorphisms that occur in runs of the same AD in a Subject nucleotide can result in Signals that are proportional to the 0535 Methods are provided for determining a subject's length of the run (Syvanen, A. C., et al. (1993) Amer. J. Hum. level of risk for developing or having AD. In these methods, Genet. 52:46-59). chromosome 10 of the Subject is analyzed for the presence 0523 For determining the identity of the allelic variant of or absence of one or more of the following types of a polymorphic region located in the coding region of a gene, polymorphisms or alleles: a polymorphism associated with yet other methods than those described above can be used. AD, a polymorphism that is over-represented in cases of a For example, identification of an allelic variant which case-control Study, a polymorphism associated with unaf encodes a mutated protein can be performed by using an fected members of a family having members affected with antibody Specifically recognizing the mutant protein in, e.g., AD and a polymorphism that is under-represented in cases immunohistochemistry or immunoprecipitation. Binding of a case-control Study. The presence of a polymorphism or assays are known in the art and involve, e.g., obtaining cells allele associated with AD may be indicative of an increased from a Subject, and performing binding experiments with a risk of AD in the subject relative to someone who does not labeled lipid, to determine whether binding to the mutated have the polymorphism or allele. The presence of a poly form of the protein differs from binding to the wild-type morphism or allele that is over-represented in cases of a protein. case-control Study may also be indicative of an increased risk for AD in the subject relative to someone who does not 0524 12. Molecular Structure Determination have the polymorphism or allele. The presence of a poly 0525) If a polymorphic region is located in an exon, either morphism or allele that is associated with AD such that it is in a coding or non-coding region of the gene, the identity of asSociated with unaffected members of a family having the allelic variant can be determined by determining the members affected with AD may be indicative of a decreased molecular structure of the mRNA, pre-mRNA, or cDNA. risk for AD in the subject relative to someone who does not The molecular structure can be determined using any of the have the polymorphism or allele. The presence of a poly above described methods for determining the molecular morphism or allele that is under-represented in cases of a Structure of the genomic DNA, e.g., Sequencing and Single case-control Study may also be indicative of a decreased risk Strand conformation polymorphism. for AD in the subject relative to someone who does not have 0526 13. Mass Spectrometric Methods the polymorphism or allele. 0527 Nucleic acids can also be analyzed by detection 0536. In particular embodiments of these methods for methods and protocols, particularly those that rely on mass determining a Subject's level of risk for developing or spectrometry (see, e.g., U.S. Pat. Nos. 5,605,798, 6.043,031, having AD, chromosome 10q of the Subject is analyzed for 6,197,498, and International Patent Application No. WO the presence or absence of one or more of the polymor 96/29431, International PCT Application No. WO phisms or alleles. In further embodiments of these methods, 98/20019). chromosome 10q22, 10q23, 10q24, 10q25 and/or 10q26 is (are) analyzed for the presence or absence of one or more of 0528 Multiplex methods allow for the simultaneous the polymorphisms or alleles. In yet further embodiments of detection of more than one polymorphic region in a particu these methods, a region identified herein as containing or lar gene. This is the preferred method for carrying out near markers associated with AD is analyzed for the pres haplotype analysis of allelic variants of a gene. ence or absence of one or more of the polymorphisms or 0529 Multiplexing can be achieved by several different alleles. In particular embodiments of these methods, chro methodologies. For example, Several mutations can be mosome 10q22, 10q23 and/or 10q24 is (are) analyzed for the Simultaneously detected on one target Sequence by employ presence or absence of one or more polymorphisms or ing corresponding detector (probe) molecules (e.g., oligo alleles. In yet a further embodiment, chromosome 10q23, nucleotides or oligonucleotide mimetics). Variations in addi 10q24 and/or 10q25 is (are) analyzed for the presence or tions to those set forth herein will be apparent to the skilled absence of one or more polymorphisms or alleles. In further artisan. particular embodiments of these methods, one or more of the US 2003/017O678A1 Sep. 11, 2003 following regions of chromosome 10 is (are) analyzed for markers associated with AD is analZyed in a Subject for the the presence or absence of one or more of the Specified presence or absence of one or more of the Specified poly polymorphisms or alleles: (1) the region extending about 50 morphisms or alleles. cM, or about 45 cM, or about 33 cM, or about 30 cM, or 0539. In particular embodiments of these methods for about 25 cM, or about 21 cM, or about 20 cM, or about 15 determining a Subject's level of risk for developing or cM, or about 10 cM, or about 5 cM, or about 4 cM, or about having AD, chromosome 10 of the subject is analyzed for 3 cM, or about 2.7 cM, or about 2.5 cM, or about 2 cM, or the presence or absence of an allele of marker D10S583, about 1.5 cM, or about 1 cM, or about 0.5 cM, or about 0.1 wherein the presence of an allele of D10S583 is indicative cM proximal (centromeric) from and including marker of a decreased risk for AD. In particular embodiments of D10S583, (2) the region extending about 60 cM, or about 55 such methods, the allele of D10S583 is about 210 bp and cM, or about 50 cM, or about 45 cM, or about 40 cM, or may be, for example, 209 bp or 211 bp. about 35 cM, or about 30 cM, or about 25 cM, or about 20 cM, or about 15 cM, or about 13 cM, or about 12 cM, or 0540 Thus, the present methods for determining a sub ject's level of risk for developing or having AD include about 9 cM, or about 5 cM, or about 2.5 cM, or about 1 cM, examination of one or more polymorphic regions of chro or about 0.5 cM or about 0.1 cM distal (telomeric) from and moSome 10, Such as regions of chromosome 10q, and in including marker D10S583, (3) the region extending from particular chromosome 10q22, 10q23, 10q24, 10q25 or and including marker D10S583 to the terminus of the q arm 10q26. Each allelic variant of a polymorphism may be of chromosome 10, (4) the region extending from and assayed individually or Simultaneously using multiplex including marker D10S583 to the centromere of chromo assay methods. Any methods described herein or known in some 10, (5) the region extending about 62 Mb, about 55 the art for determining the identity of nucleotides in a Mb, or about 50 Mb, or about 32 Mb, or about 28 Mb, or nucleic acid Sequence or alterations in a nucleic acid about 25 Mb, or about 20 Mb, or about 15 Mb, or about 10 Sequence may be used in these methods for the examination Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about of polymorphic regions of chromosome 10. Such methods 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, or about 0.1 Mb, include, but are not limited to, hybridization using specific or about 0.05 Mb, or about 0.01 Mb, or about 1 kb proximal oligonucleotides, direct DNA sequencing, restriction from and including marker D10S583, (6) the region extend enzyme digestion, RNase protection, chemical cleavage and ing about 45 Mb, or about 40 Mb, or about 35 Mb, or about ligase-mediated detection. 30 Mb, or about 25 Mb, or about 20 Mb, or about 16 Mb, or about 14 Mb, or about 13 Mb, or about 8 Mb, or about 5 0541 Genomic DNA used in the methods may be Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about obtained from body cells, Such as, for example, those present 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or about 0.05 Mb, in the blood, tissue biopsy, Surgical Specimen or autopsy or about 0.01 Mb, or about 1 kb distal from and including material. The DNA may be isolated and used directly for marker D10S583, (7) the region between D10S564 and detection of a Specific Sequence or may be amplified prior to D10S583, inclusive, (8) the region between D10S583 and analysis. RNA or cDNA may also be used. D10S1710, inclusive, (9) the region between D10S583 and 0542. Also provided are methods for determining a sub D10S566, inclusive, (10) the region between D10S583 and ject's level of risk for AD which include a step of analyzing D10S1671, inclusive and (11) the region between D10S583 chromosome 10 of the Subject for the presence or absence of and D10S1741, inclusive. a combination or haplotype of polymorphisms or alleles as 0537. In further particular embodiments of these meth follows: a combination or haplotype of polymorphisms ods, one or more of the following regions of chromosome 10 asSociated with AD, a combination or haplotype of poly is (are) analyzed for the presence or absence of one or more morphisms that is over-represented in cases of a case-control of the specified polymorphisms or alleles: (1) the region Study, a combination or haplotype of polymorphisms asso extending about 5 cM, or about 4 cM, or about 2.7 cM, or ciated with unaffected members of a family having members about 2.5 cM, or about 2 cM, or about 1.5 cM, or about 1 affected with AD and a combination or haplotype of poly cM, or about 0.5 cM, or about 0.1 cM proximal (centro morphisms that is under-represented in cases of a case meric) from and including marker D10S583, (2) the region control Study. The presence of a combination or haplotype of extending about 5 cM, or about 2.5 cM, or about 1 cM, or polymorphisms or alleles associated with AD may be indica about 0.5 cM or about 0.1 cM distal (telomeric) from and tive of an increased risk of AD in the subject relative to including marker D10S583, (3) the region extending about Someone who does not have the combination or haplotype. 10 Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, or The presence of a combination or haplotype of polymor about 500 kb, or about 300 kb, or about 200 kb, or about 100 phisms or alleles that is over-represented in cases of a kb, or about 80 kb, or about 50 kb, or about 10 kb, or about case-control Study may also be indicative of an increased 5 kb, or about 1 kb proximal from and including marker risk for AD in the subject relative to someone who does not D10S583, and (4) the region extending about 10 Mb, or have the combination or haplotype. The presence of a about 8 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, combination or haplotype of polymorphisms or alleles that or about 2 Mb, or about 1 Mb, or about 500 kb, or about 300 is associated with AD Such that it is associated with unaf kb, or about 200 kb, or about 100 kb, or about 50 kb, or fected members of a family having members affected with about 10 kb, or about 5 kb, or about 1 kb distal from and AD may be indicative of a decreased risk for AD in the including marker D10S583. Subject relative to Someone who does not have the combi nation or haplotype. The presence of a combination or 0538 In particular embodiments of these methods for haplotype of polymorphisms or alleles that is under-repre determining a Subject's level of risk for developing or Sented in cases of a case-control Study may also be indicative having AD, a region identified herein as containing or near of a decreased risk for AD in the Subject relative to Someone US 2003/017O678A1 Sep. 11, 2003 54 who does not have the combination or haplotype. In these from and including marker D10S583, (7) the region between methods, each polymorphism of the combination or haplo D10S564 and D10S583, inclusive, (8) the region between type may be located in any one of the regions of chromo D10S583 and D10S1710, inclusive, (9) the region between Some 10 explicitly set forth above with respect to the D10S583 and D10S566, inclusive, (10) the region between methods for determining a subject's level of risk for AD D10S583 and D10S1671, inclusive and (11) the region which include a Step of analyzing chromosome 10 of the between D10S583 and D10S1741, inclusive. Subject for the presence or absence of one or more poly morphisms associated with AD, that is over-represented in 0546. In yet further embodiments of these methods, the cases of a case-control Study, associated with unaffected polymorphism or allele is located on one or more of the members of a family having members affected with AD or following regions of chromosome 10: (1) the region extend that is under-represented in cases of a case-control Study. ing about 5 cM, or about 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or about 0543. J. Methods for Indicating a Predisposition to or 0.5 cM, or about 0.1 cM proximal (centromeric) from and Occurrence of Alzheimer's Disease including marker D10S583, (2) the region extending about 0544 Methods are provided for indicating a predisposi 5 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or tion to or the occurrence of Alzheimer's disease in a Subject, about 0.1 cM distal (telomeric) from and including marker which include a step of detecting in nucleic acid obtained D10S583, (3) the region extending about 10 Mb, or about 5 from the Subject the presence or absence of a polymorphism Mb, or about 2 Mb, or about 1 Mb, or about 500 kb, or about or allele on chromosome 10 associated with AD. In these 300 kb, or about 200 kb, or about 100 kb, or about 80 kb, or about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb methods, the presence of the polymorphism is indicative of proximal from and including marker D10S583, and (4) the a predisposition to or the occurrence of AD. region extending about 10 Mb, or about 8 Mb, or about 5 0545. In particular embodiments of the methods for indi Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about cating a predisposition to or the occurrence of Alzheimer's 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or disease in a Subject, the polymorphism or allele is on about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, chromosome 10q. In further embodiments of these methods, or about 1 kb distal from and including marker D10S583. the polymorphism is on chromosome 10q22, 10q23, 10q24, 10q25 or 10q26. In further embodiments of these methods, 0547 Thus, the present methods for determining a pre the polymorphism is in a region of chromosome 10 identi disposition to or occurrence of Alzheimer's disease include fied herein as containing or near markers associated with AD examination of one or more polymorphic regions of chro is analyzed for a polymorphism associated with AD. In moSome 10, in particular chromosome 10q, and in particular particular embodiments, the polymorphism is on chromo chromosome 10q22, 10q23, 10q24, 10q25 or 10q26. Each Some 10q22, 10q23 or 10q24 or on chromosome 10q23, allelic variant of a polymorphism may be assayed individu 10q24 or 10q25. In yet further embodiments of these meth ally or simultaneously using multiplex assay methods. Any ods, the polymorphism or allele is located on one or more of methods described herein or known in the art for determin the following regions of chromosome 10: (1) the region ing the identity of nucleotides in a nucleic acid Sequence or extending about 50 cM, or about 45 cM, or about 33 cM, or alterations in a nucleic acid Sequence may be used in these about 30 cM, or about 25 cM, or about 21 cM, or about 20 methods for the examination of polymorphic regions of cM, or about 15 cM, or about 10 cM, or about 5 cM, or about chromosome 10. Such methods include, but are not limited 4 cM, or about 3 cM, or about 2.7 cM, or about 2.5 cM, or to, hybridization using Specific oligonucleotides, direct about 2 cM, or about 1.5 cM, or about 1 cM, or about 0.5 DNA sequencing, restriction enzyme digestion, RNase pro cM, or about 0.1 cM proximal (centromeric) from and tection, chemical cleavage and ligase-mediated detection. including marker D10S583, (2) the region extending about 0548 Genomic DNA used in the methods may be 60 cM, or about 55 cM, or about 50 cM, or about 45 cM, or obtained from body cells, Such as, for example, those present about 40 cM, or about 35 cM, or about 30 cM, or about 25 in the blood, tissue biopsy, Surgical Specimen or autopsy cM, or about 20 cM, or about 15 cM, or about 13 cM, or material. The DNA may be isolated and used directly for about 12 cM, or about 9 cM, or about 5 cM, or about 2.5 cM, detection of a Specific Sequence or may be amplified prior to or about 1 cM, or about 0.5 cM or about 0.1 cM distal analysis. RNA or cDNA may also be used. (telomeric) from and including marker D10S583, (3) the region extending from and including marker D10S583 to the 0549. Also provided are methods for determining a pre terminus of the q arm of chromosome 10, (4) the region disposition to or occurrence of Alzheimer's disease in a extending from and including marker D10S583 to the cen Subject which include a step of detecting in nucleic acid tromere of chromosome 10, (5) the region extending about obtained from the Subject the presence or absence of a 62 Mb, about 55 Mb, or about 50 Mb, or about 32 Mb, or combination or haplotype of polymorphisms or alleles on about 28 Mb, or about 25 Mb, or about 20 Mb, or about 15 chromosome 10 associated with AD. In these methods, the Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, or about presence of the combination or haplotype of polymorphisms 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, is indicative of a predisposition to or the occurrence of AD. or about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or In particular embodiments of the methods for indicating a about 1 kb proximal from and including marker D10S583, predisposition to or the occurrence of Alzheimer's disease in (6) the region extending about 45 Mb, or about 40 Mb, or a Subject, the combination or haplotype of polymorphisms about 35 Mb, or about 30 Mb, or about 25 Mb, or about 20 or alleles is on chromosome 10q. In further embodiments of Mb, or about 16 Mb, or about 14 Mb, or about 13 Mb, or these methods, each of the polymorphisms of the combina about 8 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, tion or haplotype is on chromosome 10q22, 10q23, 10q24, or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.1 10q25 or 10q26. In further embodiments of these methods, Mb, or about 0.05 Mb, or about 0.01 Mb, or about 1 kb distal each of the polymorphisms of the combination or haplotype US 2003/017O678A1 Sep. 11, 2003
is in a region of chromosome 10 identified herein as con 0551 K. Methods for Predicting Response to Treatment taining or near markers associated with AD. In particular for Alzheimer's Disease and for Treating Alzheimer's Dis embodiments, each of the polymorphisms of the combina CSC tion or haplotype is on chromosome 10q22, 10q23 or 10q24 0552. There are a number of approved prescription thera or on chromosome 10q23, 10q24 or 10q25. In yet further pies available for the treatment of AD, including the cho embodiments of these methods, each of the polymorphisms linesterase inhbibitors tacrine, donepezil and rivastigmine. or alleles of the combination or haplotype is located on one The presence or absence of one or more polymorphisms or or more of the following regions of chromosome 10: (1) the alleles on chromosome 10 which are associated with AD region extending about 50 cM, or about 45 cM, or about 33 may correlate with a Subject's response to a specific thera cM, or about 30 cM, or about 25 cM, or about 21 cM, or peutic drug. In determining Such a pharmacogenetic profile, about 20 cM, or about 15 cM, or about 10 cM, or about 5 one or more alleles or polymorphic markers of chromosome cM, or about 4 cM, or about 3 cM, or about 2.7 cM, or about 10 are correlated with drug response by obtaining genotype 2.5 cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or and/or haplotype data from various groups of patients, in about 0.5 cM, or about 0.1 cM proximal (centromeric) from particular, patients diagnosed phenotypically with AD, to and including marker D10S583, (2) the region extending whom the drug has been administered. The genotype and/or about 60 cM, or about 55 cM, or about 50 cM, or about 45 haplotype of the Subject can then allow a clinician to take a cM, or about 40 cM, or about 35 cM, or about 30 cM, or more individualized approach to preventing the onset, about 25 cM, or about 21 cM, or about 20 cM, or about 15 reducing the progression or alleviating Symptoms of AD by cM, or about 13 cM, or about 12 cM, or about 9 cM, or about tailoring the therapy to increase the chance of a favorable 5 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or effect. about 0.1 cM distal (telomeric) from and including marker 0553 Other therapeutic agents that can be profiled D10S583, (3) the region extending from and including include, but are not limited to, ALCAR, Alpha-tocopherol marker D10S583 to the terminus of the q arm of chromo (Vitamin E), Ampalex, AN-1792 (AIP-001), Cerebrolysin, Some 10, (4) the region extending from and including Daposone, Donepezil (Aricept), ENA-713 (Exelon), Estro marker D10S583 to the centromere of chromosome 10, (5) gen replacement therapy, Galanthamine (Reminyl), Ginkgo the region extending about 62 Mb, about 55 Mb, or about 50 Biloba eXtract, Huperzine A, Ibuprofen, Lipitor, Naproxen, Mb, or about 32 Mb, or about 28 Mb, or about 25 Mb, or Nefiracetam, Neotrofin, Memantine, Phenserine, Rofecoxib, about 20 Mb, or about 15 Mb, or about 10 Mb, or about 5 Selegiline (Eldepryl), Tacrine (Cognex), Xanomeline (skin Mb, or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about patch), Resperidone (RisperidolTM), Neuroleptics, Benzodi 0.3 Mb, or about 0.2 Mb, or about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or about 1 kb proximal from and including aZepenes, Valproate, Serotonin reuptake inhibitors (SRIs), marker D10S583, (6) the region extending about 45 Mb, or Beta and Gamma Secretase Inhibitors, CX-516 (Ampalex), about 40 Mb, or about 35 Mb, or about 30 Mb, or about 25 Statins and AF-102B (Evoxac). Mb, or about 20 Mb, or about 16 Mb, or about 14 Mb, or 0554) Other therapeutic agents include those that are about 13 Mb, or about 8 Mb, or about 5 Mb, or about 4 Mb, neuroprotective. Drugs with anti-oxidative properties, e.g., or about 3 Mb, or about 2 Mb, or about 1 Mb, or about 0.5 flupirtine, N-acetylcysteine, idebenone, melatonin, and also Mb, or about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, novel dopamine agonists (ropinirole and pramipexole) have or about 1 kb distal from and including marker D10S583, (7) been shown to protect neuronal cells from apoptosis and the region between D10S564 and D10S583, inclusive, (8) thus have been Suggested for treating neurodegenerative the region between D10S583 and D10S1710, inclusive, (9) disorders like AD or PD. Also, free radical scavengers, the region between D10S583 and D10S566, inclusive, (10) calcium channel blockers and modulators of certain Signal the region between D10S583 and D10S1671, inclusive and transduction pathways that might protect neurons from (11) the region between D10S583 and D10S1741, inclusive. downstream effects of the accumulation of A-Beta intracel lularly and/or extracellularly. Also, other agents like non 0550. In yet further embodiments of these methods, each steroidal anti-inflammatory drugs (NSAIDs) partly inhibit of the polymorphisms or alleles of the combination or cyclooxygenase (COX) expression, as well as having a haplotype is located on one or more of the following regions positive influence on the clinical expression of AD. Distinct of chromosome 10: (1) the region extending about 5 cM, or cytokines, growth factors and related drug candidates, e.g., about 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 nerve growth factor (NGF), or members of the transforming cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or growth factor-beta (TGF-beta) superfamily, like growth and about 0.1 cM proximal (centromeric) from and including differentiation factor 5 (GDF-5), are shown to protect marker D10S583, (2) the region extending about 5 cM, or tyrosine hydroxylase or dopaminergic neurones from apo about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 ptosis. CRIB (cellular replacement by immunoisolatory bio cM distal (telomeric) from and including marker D10S583, capsule) is gene therapeutical approach for human NGF (3) the region extending about 10 Mb, or about 5 Mb, or Secretion, which has been shown to protect cholinergic about 2 Mb, or about 1 Mb, or about 500 kb, or about 300 neurones from cell death when implanted in the brain kb, or about 200 kb, or about 100 kb, or about 80 kb, or ((2000) Expert Opin Investig Drugs 9(4):747-64). about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb proximal from and including marker D10S583, and (4) the 0555 Provided herein are methods for predicting a region extending about 10 Mb, or about 8 Mb, or about 5 response of a Subject to a drug used to treat Alzheimer's Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about disease by detecting the presence or absence of a polymor 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or phism or allele on chromosome 10 which is associated with about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, AD. A collection or combination or haplotype of polymor or about 1 kb distal from and including marker D10S583. phic regions that individually represent allelic variants that US 2003/017O678A1 Sep. 11, 2003 56 are associated with AD or that as a group on the whole are following regions of chromosome 10: (1) the region extend asSociated with AD may be more informative than a single ing about 5 cM, or about 4 cM, or about 2.7 cM, or about 2.5 allelic variant for indicating whether an individual will cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or about positively respond to a given drug for AD. Each allelic 0.5 cM, or about 0.1 cM proximal (centromeric) from and variant may be assayed individually or simultaneously using including marker D10S583, (2) the region extending about multiplex assay methods, Such as multiplex primer exten 5 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or Sion assays or microarrays comprising probes for Specific about 0.1 cM distal (telomeric) from and including marker alleles. D10S583, (3) the region extending about 10 Mb, or about 5 0556. Accordingly, provided herein are methods for pre Mb, or about 2 Mb, or about 1 Mb, or about 500 kb, or about dicting a response of a Subject to a drug used to treat 300 kb, or about 200 kb, or about 100 kb, or about 80 kb, or Alzheimer's disease which include a step of detecting the about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb presence or absence of at least one polymorphism or allele proximal from and including marker D10S583, and (4) the on chromosome 10 that is associated with AD, wherein the region extending about 10 Mb, or about 8 Mb, or about 5 presence of the polymorphism or allele is indicative of an Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about increased or decreased likelihood that the drug treatment for 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or AD will be effective. In particular embodiments of either of about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, these methods, the polymorphism is located on chromosome or about 1 kb distal from and including marker D10S583. 10q. In further embodiments, the polymorphism is located 0558 Also provided are methods for predicting a on chromosome 10q22, 10q23, 10q24, 10q25 or 10q26. In response of a Subject to a drug used to treat Alzheimer's yet further embodiments of these methods, the polymor disease by detecting the presence or absence of a combina phism is in a region of chromosome 10 identified herein as tion or haplotype of polymorphisms or alleles on chromo containing or near markers associated with AD. In particular Some 10 which is associated with AD, wherein the presence embodiments, the polymorphism is on chromosome 10q22, of the combination or haplotype of polymorphisms or alleles 10q23 or 10q24 or on chromosome 10q23, 10q24 or 10q25. is indicative of an increased or decreased likelihood that the In yet further embodiments of these methods, the polymor drug treatment for AD will be effective. In particular phism is located in one or more of the following regions of embodiments of either of these methods, the combination or chromosome 10: (1) the region extending about 50 cM, or haplotype of polymorphisms is located on chromosome 10q. about 45 cM, or about 33 cM, or about 30 cM, or about 25 In further embodiments, each of the polymorphisms of the cM, or about 21 cM, or about 20 cM, or about 15 cM, or combination or haplotype is located on chromosome 10q22, about 10 cM, or about 5 cM, or about 4 cM, or about 3 cM, 10q23, 10q24, 10q25 or 10q26. In yet further embodiments or about 2.7 cM, or about 2.5 cM, or about 2 cM, or about of these methods, each of the polymorphisms of the com 1.5 cM, or about 1 cM, or about 0.5 cM, or about 0.1 cM bination or haplotype is in a region of chromosome 10 proximal (centromeric) from and including marker identified herein as containing or near markers associated D10S583, (2) the region extending about 60 cM, or about 55 with AD. In particular embodiments, each of the polymor cM, or about 50 cM, or about 45 cM, or about 40 cM, or phisms of the combination or haplotype is on chromosome about 35 cM, or about 30 cM, or about 25 cM, or about 20 10q22, 10q23 or 10q24 or on chromosome 10q23, 10q24 or cM, or about 15 cM, or about 13 cM, or about 12 cM, or 10q25. In yet further embodiments of these methods, the about 9 cM, or about 5 cM, or about 2.5 cM, or about 1 cM, each of the polymorphisms of the combination or haplotype or about 0.5 cM or about 0.1 cM distal (telomeric) from and is located in one or more of the following regions of including marker D10S583, (3) the region extending from chromosome 10: (1) the region extending about 50 cM, or and including marker D10S583 to the terminus of the q arm about 45 cM, or about 33 cM, or about 30 cM, or about 25 of chromosome 10, (4) the region extending from and cM, or about 21 cM, or about 20 cM, or about 15 cM, or including marker D10S583 to the centromere of chromo about 10 cM, or about 5 cM, or about 4 cM, or about 3 cM, some 10, (5) the region extending about 62 Mb, about 55 or about 2.7 cM, or about 2.5 cM, or about 2 cM, or about Mb, or about 50 Mb, or about 32 Mb, or about 28 Mb, or 1.5 cM, or about 1 cM, or about 0.5 cM, or about 0.1 cM about 25 Mb, or about 20 Mb, or about Mb, or about 10 Mb, proximal (centromeric) from and including marker or about 5 Mb, or about 2 Mb, or about 1 Mb, or about 0.5 D10S583, (2) the region extending about 60 cM, or about 55 Mb, or about 0.3 Mb, or about 0.2 Mb, or about 0.1 Mb, or cM, or about 50 cM, or about 45 cM, or about 40 cM, or about 0.05 Mb, or about 0.01 Mb, or about 1 kb proximal about 35 cM, or about 30 cM, or about 25 cM, or about 20 from and including marker D10S583, (6) the region extend cM, or about 15 cM, or about 13 cM, or about 12 cM, or ing about 45 Mb, or about 40 Mb, or about 35 Mb, or about about 9 cM, or about 5 cM, or about 2.5 cM, or about 1 cM, 30 Mb, or about 25 Mb, or about Mb, or about 16 Mb, or or about 0.5 cM or about 0.1 cM distal (telomeric) from and about 14 Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, including marker D10S583, (3) the region extending from or about 4 Mb, or about 3 Mb, or about 2 Mb, or about 1 Mb, and including marker D10S583 to the terminus of the q arm or about 0.5 Mb, or about 0.1 Mb, or about 0.05 Mb, or about of chromosome 10, (4) the region extending from and 0.01 Mb, or about 1 kb distal from and including marker including marker D10S583 to the centromere of chromo D10S583, (7) the region between D10S564 and D10S583, some 10, (5) the region extending about 62 Mb, about 55 inclusive, (8) the region between D10S583 and D10S1710, Mb, or about 50 Mb, or about 32 Mb, or about 28 Mb, or inclusive, (9) the region between D10S583 and D10S566, about 25 Mb, or about 20 Mb, or about 15 Mb, or about 10 inclusive, (10) the region between D10S583 and D10S1671, Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about inclusive and (11) the region between D10S583 and 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, or about 0.1 Mb, D10S1741, inclusive. or about 0.05 Mb, or about 0.01 Mb, or about 1 kb proximal 0557. In yet further embodiments of these methods, the from and including marker D10S583, (6) the region extend polymorphism or allele is located in one or more of the ing about 45 Mb, or about 40 Mb, or about 35 Mb, or about US 2003/017O678A1 Sep. 11, 2003 57
30 Mb, or about 25 Mb, or about 20 Mb, or about 16 Mb, methods, the polymorphism or allele is located in one or or about 14 Mb, or about 13 Mb, or about 8 Mb, or about 5 more of the following regions of chromosome 10: (1) the Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about region extending about 50 cM, or about 45 cM, or about 33 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or about 0.05 Mb, cM, or about 30 cM, or about 25 cM, or about 21 cM, or or about 0.01 Mb, or about 1 kb distal from and including about 20 cM, or about 15 cM, or about 10 cM, or about 5 marker D10S583, (7) the region between D10S564 and cM, or about 4 cM, or about 3 cM, or about 2.7 cM, or about D10S583, inclusive, (8) the region between D10S583 and 2.5 cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or D10S1710, inclusive, (9) the region between D10S583 and about 0.5 cM, or about 0.1 cM proximal (centromeric) from D10S566, inclusive, (10) the region between D10S583 and and including marker D10S583, (2) the region extending D10S1671, inclusive and (11) the region between D10S583 about 60 cM, or about 55 cM, or about 50 cM, or about 45 and D10S1741, inclusive. cM, or about 40 cM, or about 35 cM, or about 30 cM, or about 25 cM, or about 20 cM, or about 15 cM, or about 13 0559). In yet further embodiments of these methods, each cM, or about 12 cM, or about 9 cM, or about 5 cM, or about of the polymorphisms or alleles of the combination or 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 cM haplotype is located in one or more of the following regions distal (telomeric) from and including marker D10S583, (3) of chromosome 10: (1) the region extending about 5 cM, or the region extending from and including marker D10S583 to about 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 the terminus of the q arm of chromosome 10, (4) the region cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or extending from and including marker D10S583 to the cen about 0.1 cM proximal (centromeric) from and including tromere of chromosome 10, (5) the region extending about marker D10S583, (2) the region extending about 5 cM, or 62 Mb, about 55 Mb, or about 50 Mb, or about 32 Mb, or about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 about 28 Mb, or about 25 Mb, or about 20 Mb, or about 15 cM distal (telomeric) from and including marker D10S583, Mb, or about 10 Mb, or about 5 Mb, or about 2 Mb, or about (3) the region extending about 10 Mb, or about 5 Mb, or 1 Mb, or about 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, about 2 Mb, or about 1 Mb, or about 500 kb, or about 300 or about 0.1 Mb, or about 0.05 Mb, or about 0.01 Mb, or kb, or about 200 kb, or about 100 kb, or about 80 kb, or about 1 kb proximal from and including marker D10S583, about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb (6) the region extending about 45 Mb, or about 40 Mb, or proximal from and including marker D10S583, and (4) the about 35 Mb, or about 30 Mb, or about 25 Mb, or about 20 region extending about 10 Mb, or about 8 Mb, or about 5 Mb, or about 16 Mb, or about 14 Mb, or about 13 Mb, or Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about about 8 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or or about 2 Mb, or about 1 Mb, or about 0.5 Mb, or about 0.1 about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, Mb, or about 0.05 Mb, or about 0.01 Mb, or about 1 kb distal or about 1 kb distal from and including marker D10S583. from and including marker D10S583, (7) the region between 0560 Further provided are methods of treating a subject D10S564 and D10S583, inclusive, (8) the region between manifesting an Alzheimer's disease phenotype. Certain D10S583 and D10S1710, inclusive, (9) the region between ambiguous phenotypes, e.g., dementia, mainfested in AD D10S583 and D10S566, inclusive, (10) the region between also occur in connection with other diseases and conditions D10S583 and D10S1671, inclusive and (11) the region which may be treated using drugs and other treatments that between D10S583 and D10S1741, inclusive. are different from drugs and methods used to treat AD. 0562. In yet further embodiments of these methods, the Genotyping of chromosome 10 markers described herein, polymorphism or allele is located in one or more of the and optionally other AD-associated markers, in Subjects following regions of chromosome 10: (1) the region extend manifesting Such an AD phenotype(s) permits confirmation ing about 5 cM, or about 4 cM, or about 2.7 cM, or about 2.5 of AD phenotypic diagnoses and assists in distinguishing cM, or about 2 cM, or about 1.5 cM, or about 1 cM, or about between AD and other possible diseases or disorders. Once 0.5 cM, or about 0.1 cM proximal (centromeric) from and an individual is genotyped as having or being predisposed to including marker D10S583, (2) the region extending about AD, he or she may be treated with any known methods 5 cM, or about 2.5 cM, or about 1 cM, or about 0.5 cM or effective in treating AD. about 0.1 cM distal (telomeric) from and including marker 0561. Accordingly, methods of treating a subject mani D10S583, (3) the region extending about 10 Mb, or about 5 festing an Alzheimer's disease phenotype provided herein Mb, or about 2 Mb, or about 1 Mb, or about 500 kb, or about include Steps of (a) detecting in nucleic acid obtained from 300 kb, or about 200 kb, or about 100 kb, or about 80 kb, or the Subject the presence of a polymorphism or allele on about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb chromosome 10 associated with AD, which is indicative of proximal from and including marker D10S583, and (4) the the occurrence of AD, and (b) selecting a treatment plan that region extending about 10 Mb, or about 8 Mb, or about 5 is effective for treatment of Alzheimer's disease. In particu Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about lar embodiments of these methods, the polymorphism or 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or allele is located on chromosome 10q. In further embodi about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, ments of these methods, the polymorphism or allele is or about 1 kb distal from and including marker D10S583. located on chromosome 10q22, 10q23, 10q24, 10q25 or 0563 Also provided are methods of treating a subject 10q26. In further embodiments of these methods, the poly manifesting an Alzheimer's disease phenotype provided morphism or allele is in a region of chromosome 10 iden herein include steps of (a) detecting in nucleic acid obtained tified herein as containing or near markers associated with from the Subject the presence of a combination or haplotype AD is analyzed for a polymorphism associated with AD. In of polymorphisms or alleles on chromosome 10 associated particular embodiments, the polymorphism is located on with AD, which is indicative of the occurrence of AD, and chromosome 10q22, 10q23 or 10q24 or on chromosome (b) Selecting a treatment plan that is effective for treatment 10q23, 10q24 or 10q25. In yet further embodiments of these of Alzheimer's disease. In particular embodiments of these US 2003/017O678A1 Sep. 11, 2003 58 methods, the combination or haplotype of polymorphisms or about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb alleles is located on chromosome 10q. In further embodi proximal from and including marker D10S583, and (4) the ments of these methods, each of the polymorphisms or region extending about 10 Mb, or about 8 Mb, or about 5 alleles of the combination or haplotype is located on chro Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about mosome 10q22, 10q23, 10q24, 10q25 or 10q26. In further 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or embodiments of these methods, each of the polymorphisms about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, or alleles of the combination or haplotype is in a region of or about 1 kb distal from and including marker D10S583. chromosome 10 identified herein as containing or near 0565 L. Nucleic Acid Compositions, Kits, Articles of markers associated with AD is analyzed for a polymorphism Manufacture and Combinations asSociated with AD. In particular embodiments, each of the polymorphisms of the combination or haplotype is located 0566 Provided herein are combinations of two or more on chromosome 10q22, 10q23 or 10q24 or on chromosome or three or more oligonucleotides which hybridize to, or 10q23, 10q24 or 10q25. In yet further embodiments of these adjacent to, a DNA segment within chromosome 10 that is methods, each of the polymorphisms or alleles of the com associated with AD or hybridize to DNA flanking the DNA bination or haplotype is located in one or more of the Segment. In particular embodiments of the combinations, the following regions of chromosome 10: (1) the region extend oligonucleotides hybridize to, or adjacent to, a DNA Seg ing about 50 cM, or about 45 cM, or about 33 cM, or about ment in chromosome 10q or hybridize to DNA flanking the 30 cM, or about 25 cM, or about 21 cM, or about 20 cM, or DNA segment. In further embodiments of the combinations, about 15 cM, or about 10 cM, or about 5 cM, or about 4 cM, the oligonucleotides hybridize to, or adjacent to, a DNA or about 3 cM, or about 2.7 cM, or about 2.5 cM, or about segment in chromosome 10q22, 10q23, 10q24, 10q25 and/or 2 cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or 10q26 or hybridize to DNA flanking the DNA segment. In about 0.1 cM proximal (centromeric) from and including particular embodiments of the combinations, the oligonucle marker D10S583, (2) the region extending about 60 cM, or otides hybridize to, or adjacent to, or to DNA flanking a about 55 cM, or about 50 cM, or about 45 cM, or about 40 DNA segment in a region of chromosome 10 identified cM, or about 35 cM, or about 30 cM, or about 25 cM, or herein as containing or near markers associated with AD. In about 20 cM, or about 15 cM, or about 13 cM, or about 12 further embodiments of the combinations, the oligonucle cM, or about 9 cM, or about 5 cM, or about 2.5 cM, or about otides hybridize to, or adjacent to, or to DNA flanking a 1 cM, or about 0.5 cM or about 0.1 cM distal (telomeric) DNA segment on chromosome 10q22, 10q23 and/or 10q24 from and including marker D10S583, (3) the region extend or on chromosome 10q23, 10q24 and/or 10q25. In yet ing from and including marker D10S583 to the terminus of further embodiments of the combinations, the oligonucle the q arm of chromosome 10, (4) the region extending from otides hybridize to, or adjacent to, or to DNA flanking a and including marker D10S583 to the centromere of chro DNA segment in chromosome 10 located in one or more of mosome 10, (5) the region extending about 62 Mb, about 55 the following regions: (1) the region extending about 50 cM, Mb, or about 50 Mb, or about 32 Mb, or about 28 Mb, or or about 45 cM, or about 33 cM, or about 30 cM, or about about 25 Mb, or about 20 Mb, or about 15 Mb, or about 10 25 cM, or about 21 cM, or about 20 cM, or about 15 cM, or Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about about 10 cM, or about 5 cM, or about 4 cM, or about 3 cM, 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, or about 0.1 Mb, or about 2.7 cM, or about 2.5 cM, or about 2 cM, or about or about 0.05 Mb, or about 0.01 Mb, or about 1 kb proximal 1.5 cM, or about 1 cM, or about 0.5 cM, or about 0.1 cM from and including marker D10S583, (6) the region extend proximal (centromeric) from and including marker ing about 45 Mb, or about 40 Mb, or about 35 Mb, or about D10S583, (2) the region extending about 60 cM, or about 55 30 Mb, or about 25 Mb, or about 20 Mb, or about 16 Mb, cM, or about 50 cM, or about 45 cM, or about 40 cM, or or about 14 Mb, or about 13 Mb, or about 8 Mb, or about 5 about 35 cM, or about 30 cM, or about 25 cM, or about 20 Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about cM, or about 15 cM, or about 13 cM, or about 12 cM, or 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or about 0.05 Mb, about 9 cM, or about 5 cM, or about 2.5 cM, or about 1 cM, or about 0.01 Mb, or about 1 kb distal from and including or about 0.5 cM or about 0.1 cM distal (telomeric) from and marker D10S583, (7) the region between D10S564 and including marker D10S583, (3) the region extending from D10S583, inclusive, (8) the region between D10S583 and and including marker D10S583 to the terminus of the q arm D10S1710, inclusive, (9) the region between D10S583 and of chromosome 10, (4) the region extending from and D10S566, inclusive, (10) the region between D10S583 and including marker D10S583 to the centromere of chromo D10S1671, inclusive and (11) the region between D10S583 some 10, (5) the region extending about 62 Mb, about 55 and D10S1741, inclusive. Mb, or about 50 Mb, or about 32 Mb, or about 28 Mb, or about 25 Mb, or about 20 Mb, or about 15 Mb, or about 10 0564) In yet further embodiments of these methods, each Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, or about of the polymorphisms or alleles of the combination or 0.5 Mb, or about 0.3 Mb, or about 0.2 Mb, or about 0.1 Mb, haplotype is located in one or more of the following regions or about 0.05 Mb, or about 0.01 Mb, or about 1 kb proximal of chromosome 10: (1) the region extending about 5 cM, or from and including marker D10S583, (6) the region extend about 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 ing about 45 Mb, or about 40 Mb, or about 35 Mb, or about cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or 30 Mb, or about 25 Mb, or about 20 Mb, or about 16 Mb, about 0.1 cM proximal (centromeric) from and including or about 14 Mb, or about 13 Mb, or about 8 Mb, or about 5 marker D10S583, (2) the region extending about 5 cM, or Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 1 Mb, or about 0.5 Mb, or about 0.1 Mb, or about 0.05 Mb, cM distal (telomeric) from and including marker D10S583, or about 0.01 Mb, or about 1 kb distal from and including (3) the region extending about 10 Mb, or about 5 Mb, or marker D10S583, (7) the region between D10S564 and about 2 Mb, or about 1 Mb, or about 500 kb, or about 300 D10S583, inclusive, (8) the region between D10S583 and kb, or about 200 kb, or about 100 kb, or about 80 kb, or D10S1710, inclusive, (9) the region between D10S583 and US 2003/017O678A1 Sep. 11, 2003 59
D10S566, inclusive, (10) the region between D10S583 and The kits and articles of manufacture include one or more D10S1671, inclusive and (11) the region between D10S583 containers (e.g., a tube or vial) which contain one or more and D10S1741, inclusive. or two or more oligonucleotides which hybridize to, or 0567. In yet further embodiments of the combinations, adjacent to, a DNA segment within chromosome 10 that is the oligonucleotides hybridize to, or adjacent to, or to DNA associated with AD or hybridize to DNA flanking the DNA flanking a DNA segment in chromosome 10 located in one Segment. Thus, included within the kits and articles of or more of the following regions of chromosome 10: (1) the manufacture are packaged forms of the combinations pro region extending about 5 cM, or about 4 cM, or about 2.7 vided herein. In particular embodiments of the kits and cM, or about 2.5 cM, or about 2 cM, or about 1.5 cM, or articles of manufacture, the oligonucleotides hybridize to, or about 1 cM, or about 0.5 cM, or about 0.1 cM proximal adjacent to, a DNA segment in chromosome 10q. In further (centromeric) from and including marker D10S583, (2) the embodiments of the kits and articles of manufacture, the region extending about 5 cM, or about 2.5 cM, or about 1 oligonucleotides hybridize to, or adjacent to, a DNA Seg cM, or about 0.5 cM or about 0.1 cM distal (telomeric) from ment in chromosome 10q22, 10q23, 10q24, 10q25 and/or and including marker D10S583, (3) the region extending 10q26 or hybridize to DNA flanking the DNA segment. In about 10 Mb, or about 5 Mb, or about 2 Mb, or about 1 Mb, particular embodiments of the kits and articles of manufac or about 500 kb, or about 300 kb, or about 200 kb, or about ture, the oligonucleotides hybridize to, or adjacent to, or to 100 kb, or about 80 kb, or about 50 kb, or about 10 kb, or DNA flanking a DNA segment in a region of chromosome about 5 kb, or about 1 kb proximal from and including 10 identified herein as containing or near markers associated marker D10S583, and (4) the region extending about 10 Mb, with AD. In further embodiments, oligonucleotides hybrid or about 8 Mb, or about 5 Mb, or about 4 Mb, or about 3 Mb, ize to, or adjacent to, or to DNA flanking a DNA segment on or about 2 Mb, or about 1 Mb, or about 500 kb, or about 300 chromosome 10q22, 10q23 and/or 10q24 or on chromosome kb, or about 200 kb, or about 100 kb, or about 50 kb, or 10q23, 10q24 and/or 10q25. In yet further embodiments of about 10 kb, or about 5 kb, or about 1 kb distal from and the kits and articles of manufacture, the oligonucleotides including marker D10S583. hybridize to, or adjacent to, or to DNA flanking a DNA Segment in chromosome 10 located in one or more of the 0568. The oligonucleotides in particular combinations following regions: (1) the region extending about 50 cM, or may contain 15 or more contiguous nucleotides of the about 45 cM, or about 33 cM, or about 30 cM, or about 25 following nucleotide sequences: SEQ ID NOS. 3-14. For cM, or about 21 cM, or about 20 cM, or about 15 cM, or example, a combination provided herein may contain two or about 10 cM, or about 5 cM, or about 4 cM, or about 3 cM, more of any of the following pairs of oligonucleotides (1) 15 or about 2.7 cM, or about 2.5 cM, or about 2 cM, or about or more contiguous nucleotides of SEQ ID NO. 3 and 15 or 1.5 cM, or about 1 cM, or about 0.5 cM, or about 0.1 cM more contiguous nucleotides of SEQ ID NO. 4, (2) 15 or proximal (centromeric) from and including marker more contiguous nucleotides of SEQ ID NO. 5 and 15 or D10S583, (2) the region extending about 60 cM, or about 55 more contiguous nucleotides of SEQ ID NO. 6, (3) 15 or cM, or about 50 cM, or about 45 cM, or about 40 cM, or more contiguous nucleotides of SEQ ID NO. 7 and 15 or about 35 cM, or about 30 cM, or about 28 cM, or about 25 more contiguous nucleotides of SEQ ID NO. 8, (4) 15 or cM, or about 20 cM, or about 15 cM, or about 13 cM, or more contiguous nucleotides of SEQ ID NO. 9 and 15 or about 12 cM, or about 9 cM, or about 5 cM, or about 2.5 cM, more contiguous nucleotides of SEQ ID NO. 10, (5) 15 or or about 1 cM, or about 0.5 cM or about 0.1 cM distal more contiguous nucleotides of SEQ ID NO. 11 and 15 or (telomeric) from and including marker D10S583, (3) the more contiguous nucleotides of SEQ ID NO. 12, and (6) 15 region extending from and including marker D10S583 to the or more contiguous nucleotides of SEQ ID NO. 13 and 15 terminus of the q arm of chromosome 10, (4) the region or more contiguous nucleotides of SEQ ID NO. 14. extending from and including marker D10S583 to the cen 0569. In particular embodiments of the combinations tromere of chromosome 10, (5) the region extending about provided herein, the oligonucleotides hybridize to, or adja 62 Mb, about 55 Mb, or about 50 Mb, or about 32 Mb, or cent to, or to DNA flanking two or more markers, one of about 25 Mb, or about 20 Mb, or about Mb, or about 10 Mb, which is D10S583. For example, a combination provided or about 5 Mb, or about 2 Mb, or about 1 Mb, or about 0.5 herein may contain two or more pairs of oligonucleotides, Mb, or about 0.3 Mb, or about 0.2 Mb, or about 0.1 Mb, or one pair of which includes 15 or more contiguous nucle about 0.05 Mb, or about 0.01 Mb, or about 1 kb proximal otides of SEQ ID NO. 5 and 15 or more contiguous from and including marker D10S583, (6) the region extend nucleotides of SEO ID NO. 6. ing about 45 Mb, or about 40 Mb, or about 35 Mb, or about 0570. In particular combinations that have only two oli 30 Mb, or about 25 Mb, or about Mb, or about 16 Mb, or gonucleotides, the two oligonucleotides hybridize to, or about 14 Mb, or about 13 Mb, or about 8 Mb, or about 5 Mb, adjacent to, or to DNA flanking different DNA segments in or about 4 Mb, or about 3 Mb, or about 2 Mb, or about 1 Mb, chromosome 10, as described herein. That is, the two or about 0.5 Mb, or about 0.1 Mb, or about 0.05 Mb, or about oligonucleotides may be used to detect two different DNA 0.01 Mb, or about 1 kb distal from and including marker Segments and are not a pair of oligonucleotides used to D10S583, (7) the region between D10S564 and D10S583, detect a Single DNA fragment, Such as, for example, a pair inclusive, (8) the region between D10S583 and D10S1710, of PCR primers that amplify a single DNA segment. In inclusive, (9) the region between D10S583 and D10S566, particular combinations that have only three oligonucle inclusive, (10) the region between D10S583 and D10S1671, otides, at least two of the oligonucleotides hybridize to, or inclusive and (11) the region between D10S583 and adjacent to, or to DNA flanking different DNA segments in D10S1741, inclusive. chromosome 10, as described herein. 0572 In yet further embodiments of the kits and articles 0571 Also provided herein are kits and articles of manu of manufacture, the oligonucleotides hybridize to, or adja facture for use in the practice of methods described herein. cent to, or to DNA flanking a DNA segment in chromosome US 2003/017O678A1 Sep. 11, 2003 60
10 located in one or more of the following regions of with AD, determining a predisposition to or occurrence of chromosome 10: (1) the region extending about 5 cM, or AD, predicting a response to a treatment for AD and/or about 4 cM, or about 2.7 cM, or about 2.5 cM, or about 2 treating AD. cM, or about 1.5 cM, or about 1 cM, or about 0.5 cM, or 0576. The combinations, kits and articles of manufacture about 0.1 cM proximal (centromeric) from and including provided herein may be used in a variety of methods. For marker D10S583, (2) the region extending about 5 cM, or example, combinations, kits and articles of manufacture may about 2.5 cM, or about 1 cM, or about 0.5 cM or about 0.1 be used to determine the presence or absence in nucleic acid cM distal (telomeric) from and including marker D10S583, obtained from a Subject of a polymorphism associated with (3) the region extending about 10 Mb, or about 5 Mb, or AD. In a particular embodiment of a kit or article of about 2 Mb, or about 1 Mb, or about 500 kb, or about 300 manufacture that may be used for this purpose, the kit or kb, or about 200 kb, or about 100 kb, or about 80 kb, or article of manufacture may include labeling or instructions about 50 kb, or about 10 kb, or about 5 kb, or about 1 kb describing procedures for using the oligonucleotide(s) con proximal from and including marker D10S583, and (4) the tained in the kit or article of manufacture in the detection of region extending about 10 Mb, or about 8 Mb, or about 5 one or more or two or more polymorphisms on chromosome Mb, or about 4 Mb, or about 3 Mb, or about 2 Mb, or about 10 associated with AD. 1 Mb, or about 500 kb, or about 300 kb, or about 200 kb, or 0577 Similarly, the combinations, kits and articles of about 100 kb, or about 50 kb, or about 10 kb, or about 5 kb, manufacture may be used, for example, to determine a level or about 1 kb distal from and including marker D10S583. of risk for AD, to determine a predisposition to or occur 0573 The oligonucleotides in particular kits and articles rence of AD in a Subject, to predict a Subject's response to of manufacture may contain 15 or more contiguous nucle a treatment for AD and/or to treat AD in a subject. In otides of the following nucleotide sequences: SEQ ID NOS. particular embodiments of a kit or article of manufacture 1-12. For example, a kit or article of manufacture provided that may be used for these purposes, the kit or article of herein may contain one or more or two or more of the manufacture may include labeling or instructions describing following oligonucleotide pairs: (1) 15 or more contiguous procedures for using the oligonucleotide(s) contained in the nucleotides of SEQ ID NO. 3 and 15 or more contiguous kit or article of manufacture in methods of detecting the nucleotides of SEQ ID NO. 4, (2) 15 or more contiguous presence or absence of one or more or two or more poly nucleotides of SEQ ID NO. 5 and 15 or more contiguous morphisms on chormosome 10 associated with AD, wherein nucleotides of SEQ ID NO. 6, (3) 15 or more contiguous the procedures provide results on which to base a determi nucleotides of SEQ ID NO. 7 and 15 or more contiguous nation of the a Subject's level of risk for AD, a predisposition nucleotides of SEQ ID NO. 8, (4) 15 or more contiguous to or occurrence of AD in a Subject, a prediction of a nucleotides of SEQ ID NO. 9 and 15 or more contiguous Subject's response to a treatment for AD and/or a treatment nucleotides of SEQ ID NO. 10, (5) 15 or more contiguous of AD in a subject. nucleotides of SEQ ID NO. 11 and 15 or more contiguous 0578 For any of these or other uses, the kits or articles of nucleotides of SEQ ID NO. 12, and (6) 15 or more contigu manufacture may contain one or more or two or more pairs ous nucleotides of SEQ ID NO. 13 and 15 or more contigu of oligonucleotides and instructions or labelling describing ous nucleotides of SEO ID NO. 14. use of the oligonucleotide pairs to amplify one or more or 0574. In particular embodiments of the kits and articles of two or more regions of chromosome 10 at which polymor manufacture provided herein, the oligonucleotides hybridize phisms associated with AD. The labelling or instructions to, or adjacent to, or to DNA flanking one or more or two or may further specify possible results of the amplification more of markers, one of which is D10S583. For example, a reaction(s) and interpretation thereof as to whether the kit or article of manufacture provided herein may contain results are indicative of the presence or absence of a poly one or more or two or more oligonucleotide pairs, one pair morphism associated with AD or linked to a DNA segment of which includes 15 or more contiguous nucleotides of SEQ asSociated with AD. In another example, the kit or article of ID NO. 5 and 15 or more contiguous nucleotides of SEQ ID manufacture may contain one or more or two or more NO. 6. oligonucleotides and instructions or labelling describing use of the oligonucleotide(s) in hybridization, Sequencing and/or 0575. The kits and articles of manufacture provided primer eXtension-based methods of detecting the presence of herein may optionally include other elements or reagents in a polymorphism associated with AD. The labelling or addition to the one or more or two or more oligonucleotides instructions may further specify possible results of the which hybridize to, or adjacent to, or to DNA flanking a methods and interpretation thereof as to whether the results DNA segment within chromosome 10, and particularly are indicative of the presence or absence of a polymorphism within Specific regions of chromosome 10 described herein, associated with AD. that is associated with AD. For example, the kits and articles of manufacture may also include one or more oligonucle 0579. The following examples are included for illustra otides that hybridize to, or adjacent to, or to DNA flanking tive purposes only and are not intended to limit the Scope of a DNA segment located on a chromosome other than chro the invention. moSome 10 that is associated with AD or linked to a gene asSociated with AD. In a particular embodiment, Such oli EXAMPLE 1. gonucleotides include those that hybridize to, or adjacent to, or to DNA flanking the APOE gene. Another element which 0580 Genotyping of Chromosome 10 Markers in DNA additionally may be included in the kits and articles of from AD Family Members manufacture is labelling or instructions indicating the Suit 0581 Microsatellite markers on human chromosome 10 ability of the kits for detecting a polymorphism asSociated were analyzed for genetic linkage to AD. The analysis was US 2003/017O678A1 Sep. 11, 2003
conducted by genotyping genomic DNA Samples from AD Medical Genetics (http://research.marshfieldclinic.org/ge family members with respect to Seven microSatellite markers netics/) and GDB (http://www.gdb.org/gdb/). Six of the and performing parametric and nonparametric analyses of Seven microSatellite markers on chromosome 10 are located genotyping data. on 10q23-q25 and the remaining marker (D10S1225) is 0582 Genomic DNA Samples located on 10q21.3 according to the UCSC Golden Path Genome Browser (http://genome.ucsc.edu/index.html). The 0583. The genomic DNA utilized in the linkage analyses was from the full National Institute of Mental Health Seven markers, and their positions on the Sex-averaged (NIMH) Genetics Initiative sample of AD family DNA Marshfield map, are: D10S1225 (80.8 cM), D10S564 (112.6 Blacker et al. (1997) Neurology 48:139-147). Through the cM), D10S583 (115.3 cM), D10S1710 (124.3 cM), NIMH Genetics Initiative, a national resource of clinical D10S566 (127.1 cM), D10S1671 (127.1 cM) and D10S1741 data and biomaterials (DNA samples) collected from indi (128.2 cM). TABLE 2 provides additional information con viduals with AD has been established. AD pedigrees have cerning each locus marker. been ascertained by three extramural sites (Massachusetts General Hospital/Harvard Medical School, University of TABLE 2 Alabama and Johns Hopkins University) and data collection has been coordinated among the three Sites by using a GenBank common protocol that includes uniform assessments and Location Accession Hetero medical, neurologic and psychiatric histories. Locus Marker (cM) Type No. Zygosity D1OS1225 ATA24F10 80.77 TRI GO8772 O.78 0584) In generating the NIMH sample, subjects were D1OSS64 AFMO29xh12 112.58 DI Z23295 O.62 collected following a Standardized protocol applying D1OS583 AFM2892h5 115.27 DI Z24.088 0.87 NINCDS/ADRDA (National Institute of Neurological and D1OS1710 AFMbOSSZb9 124.27 DI Z53234 O60 Communicative Disorders and Stroke/Alzheimer's Disease D1OS566 AFM 154xh2 127.11 DI Z23404 O.65 and Related Disorders ASSociation) criteria for the diagnosis D1OS1671 AFMa222Z.c5 127.11 DI Z52570 0.72 of ADBlacker et al. (1997) Neurology 48:139; McKhannet D1OS1741 AFMcOO5xh5 128.19 DI Z5394O O.58 al. (1984) Neurology 34:939-944). The diagnostic process in Kosambi cM the NIMH AD Genetics Initiative includes a systematic and Marker types: DI = dinucleotide repeat: TRI = trinucleotide repeat. comprehensive examination of all available information from autopsy records, family history, medical records, and patient and/or informant interviews. Definite AD according 0588 Marker genotypes were available for, on average, to age-adjusted Khachaturian criteria is diagnosed on 82% of the Sampled Subjects. Genotyping of the microSat autopsy. Operational criteria for the clinical diagnosis of ellite markers was conducted using nucleic acid amplifica probable or possible AD following NINCDS-ADRDA Work tion procedures essentially as follows. Genomic DNA, ~30 Group guidelines have been developed and are implemented ng, in a 10 ul reaction Volume, was amplified in a poly by the three sites. Case Summaries for all Subjects with a merase chain reaction (PCR). The reaction volume con clinical diagnosis of probable or possible AD are reviewed tained 3 pmol of each primer (primer oligonucleotides were by the site principal investigators and a procedure has been synthesized by Life Technologies, Rockville, Md.), 5ul or implemented to establish a consensus diagnosis. Subjects Taq PCR Master Mix (Qiagen, Inc.) and 0.1 ul of C-33P are followed longitudinally to track changes in diagnoses dATP (1 uCi). After amplification, the products were dena and to compare diagnoses by autopsy. tured for 3 min at 94 C. and then separated by polyacry 0585. Only families in which all sample affecteds had lamide gel electrophoresis (6%, National Diagnostics, onset ages 250 years were included (n=435 families; Atlanta, Ga.) for 1-3 hours at 40-60 Watts and exposed to n=1426 Subjects, mean age of onset=72.5-7.7 years, range film for 24-48 hours. PCR conditions were essentially those 50-97 years). The original sample included a total of 1500 specified by GDB. The primers for amplification of each subjects from 449 families with two or more affected Sub locus marker are shown in TABLE 3. jects per family. Families in which any Sampled individual had an onset age less than 50 years (n=14 families and 74 individuals) were excluded, yielding 1426 individuals from TABLE 3 435 families for this analysis, including 993 affected indi Size SEQ viduals, 429 unaffected, and 4 with phenotype unknown. Forward Reverse Range ID Over the 10 years that the NIMH sample has been followed, Tocus Marker Primer Primer (bp)a Nob a clinical diagnosis of AD has been confirmed at autopsy in D1 OS 1225 ATA24F10 TCCTTTGT, GTTACATAG 173- 1 94% of the cases. All DNA samples are stored in a central CAGTCATGC GAAGCCCG 208 ized cell repository at Rutgers University, New Brunswick, TCA N.J. 0586 Genotyping of Genomic DNA D1 OS 564 AFMO29xh12 TGGGAATGT AGCTCTAACAT 252- 3. GTCTTTATC AGAGGCCAGAT 2.62 0587. The DNA samples were genotyped with respect to CA Seven microSatellite markers located on chromosome 10 and APOE. In Selecting the markers for genotyping on chromo D1 OS 583 AFM2892H5 TCTGACCAA AGAGACTCCAG 201- 5 Some 10, the following genetic maps were considered: AATACCAAA. ATGTTTGATGA, 219 NCBI (http://www.ncbi.nim.nih.gov/), LDB (http://cedar AGAAC genetics. Soton.ac.uk/public html), Marshfield Center for US 2003/017O678A1 Sep. 11, 2003 62
0593. Many genotyping methods, although not all, TABLE 3-continued involve amplification of the nucleic acid region carrying the marker of interest. Such methods specifically increase the Size SEQ Forward Reverse Range ID concentration or total number of Sequences that span the Locus Marker Primer Primer (bp)a NOP marker. Some amplification methods allow for detection of a polymorphism and Simultaneous amplification of a target Sequence. Diagnostic assays may also rely on amplification D1 OS 1710 AFMO5529 GTCTCAGTC TTCATCT- 194- 7. TACTC 8 of DNA segments carrying a marker. While the amplification TCCAGGTGA. AAAGTGGGGC 206 of the target Segment is often employed in genotyping CAATG methods, there are ultrasensitive detection methods known D1 OS 566 AFM1.5 4xh2 CTGAGGTAG AGCTGTGAAAA 200- 9, in the art that do not require amplification. GGGGATGGC ATCACATAT- 212 10 0594 Amplification of nucleic acids may be achieved by TGC any method known in the art. Oligonucleotides appropriate TT TA for use as primers in nucleic acid amplification of chromo D1 OS 1671 AFMa2222C5 ATGCAATGA TGATGATGCTC 92- 11 Some 10 markers include those provided herein as well as GTGCTCCCT AAATGATGG 110 12 any others that Serve to specifically amplify Segments of D1 OS 1741 AFMcO O5xh5 GAGTAGTGT AGATGT- 21 O- 13 chromosome 10 containing a marker. The Spacing of primers TCAGTT determines the length of the Segment to be amplified. CATGGCTCC CCTTGGCT 254 14 Amplified Segments carrying markers can range in size from CT about 25 bp to kilobases, and may typically be on the order Size range for the amplified fragments for each of hundreds of bp. Amplification primers may be labeled or marker. immobilized on a Solid Support. SEQ ID NO for the forward and reverse primers, respectively. 0595 Methods known to those skilled in the art which can be used to detect markers in genotyping include methods 0589 APOE was genotyped as follows see also methods Such as conventional dot blot analyses, Single Strand con described in Blacker et al. (1997) Neurology 48:140). formational polymorphism analysis Orita et al. (1989) Genomic DNA was amplified by polymerase chain reaction Proc. Natl. Acad. Sci. U.S.A. 86:2776-2770), denaturing using the following primers: gradient gel electrophoresis, heteroduplex analysis, mis match cleavage detection, and other techniques See, e.g., Sheffield et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 49:699 5'-TCCAAGGAGCTGCAGGCGGCGCA3' SEQ ID NO : 15 706; White et al. (1992) Genomics 12:301-306; Grompe et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:5855-5892; 5'-ACAGAATTCGCCCCGGCCTGGTACACTGCCA-3 SEQ ID NO : 16 Grompe etal. (1993) Nature Genet. 5:111-117). 0590 For each amplification, 20 ng of human genomic 0596) The nucleotide(s) present at a polymorphic site can DNA, 1 ng of each primer, 200 uM of dCTP, dTTP and be determined by Sequencing methods, which may involve dGTP, 25uM dATP, 20 pCi (alpha-P) dATP, 10% DMSO, initial amplification of the Segment to be sequenced. For 0.1 ul of 100xBSA, 1.6 units Taq DNA polymerase (5 example, an amplification product can be Subjected to auto units/ul; Fisher Biotech, Agawan, Mass.), and 1Xreacton mated dideoxy terminator Sequencing reactions using a buffer (supplied by vendor with 15 mM MgCl) were dye-primer cycle Sequencing protocol. combined in a final volume of 10 ul. Reactions were conducted in V-well plates in a PTC-100 Programmable 0597 Primer extension methods may also be used to Thermal Controller under the following conditions: 5 min at determine the nucleotide at a polymorphic Site. These meth 94° C., 34 cycles of 30 sec at 94° C., 30 sec at 69° C., 1.5 ods involve appropriate primers which hybridize just min at 70° C., followed by a final extension step at 70° C. upstream of the polymorphic base of interest in the target for 10 min. APOE isotypes were then determined by cleav nucleic acid. A polymerase is used to Specifically extend the ing with the restriction enzyme Hhal (5 units) added directly 3' end of the primer with one or more ddNTPs (chain to each well and incubated at 37 C. for 6 hours. Ten ul of terminators), one of which is complementary to the nucle 2xstop dye were added to each well and 5 ul of this mix was otide at the polymorphic Site. The identity of the incorpo then loaded into each well of a 6% nondenaturing polyacry rated nucleotide is then determined. Identification methods lamide gel. Following electrophoresis at 45 mA for 1.5 hr, include fluorescence polarization, pyroSequencing and mass the gel was transferred to Whatman 3M chromatography Spectrometry-based methods. paper, dried by vacuum and exposed to Kodak XAR-5 film. EXAMPLE 2 Autoradiography was carried out for 1 to 16 hrs at -70° C. 0598 Parametric and Nonparametric Linkage Analysis of The resulting genotypes on each autoradiograph were read Chromosome 10 Microsatellite Markers independently by two different observers and scoring of the 0599 Parametric two-point analyses of the genotyping alleles was determined, as presented in Hixon and Vernier data with respect to the chromosome 10 markers were (1990).J. Lipid Res. 31:545). conducted using affecteds only and two different disease 0591. Other Genotyping Methods models: an autosomal dominant disease model (disease gene 0592) A variety of methods may be used in genotyping frequency 0.01) and a recessive disease model (disease gene nucleic acids for particular markers. Any Source of nucleic frequency 0.05). Penetrance in affected individuals corre acids, in purified or non-purified form, can be utilized as the sponded to phenocopy rates of 5% for definite AD (n=278), starting nucleic acid. DNA or RNA may be extracted from 10% for probable AD (n=645) and 14% for possible AD cells, tissueS or body fluids. (n=65). The analyses were performed using the FASTLINK US 2003/017O678A1 Sep. 11, 2003 computer program Cottingham et al. (1993) Am. J. Hum. Genet. 53:252; Schaffer et al. (1994) Hum. Hered 44:225). TABLE 6 The genotyping data were analyzed in terms of the total Sample (all families) and for Samples Stratified by age of Nonparametric Two-Point Analyses onset. Families were considered “late-onset'- if all sampled Locus Marker All Families Late-Onset APOE e4/4- affecteds had onset ages 265 years. Families were classified (Location in cM) Z(p) Z(p) negative Z(p)" as APOE e4/4 positive if at least one affected individual had D1OS1225 (80.8 O.9 (O.2 1.6 (OO6 O.71 (0.24 the e4/4 genotype, and as APOE e4/4 negative otherwise. D1OSS64 S. 8.8 ) 8.8 ) oc s ) The results of the analyses using the autosomal dominant D10S583 (115.3) 3.0 (0.0015) 2.8 (0.0025) 2.5 (0.006) disease model are provided in TABLE 4, and the results of D10S1710 (124.3) 2.4 (0.008) 2.8 (0.003) 2.2 (0.014) the analyses for the full dataset using the autosomal reces- D10S566 (127.1) 1.8 (0.035) 1.5 (0.06) 2.1 (0.02) sive disease model are provided in TABLE 5.
TABLE 4 Parametric Analyses - Autosomal Dominant Disease Model APOE e4/4 APOE e4f4 Locus Marker All Families Late-Onset Postive Negative Zimax (Location in cM) Zinax (G) Zinax (G) Zinax (P) (P) D10S1225 (80.77) 0.4 (0.32) 0.9 (0.26) 0.1 (0.32) 0.4 (0.30) D10S564 (112.6) 0 (0.5) 0 (0.5) 0 (0.5) 0 (0.5) D10S583 (115.3) 3.3 (0.22) 2.8 (0.21) 1.2 (0.20) 2.2 (0.22) D10S1710 (124.3) 0.7 (0.26) 0.9 (0.25) 0.1 (0.28) 0.7 (0.26) D10S566 (127.1) 0.8 (0.28) 0.4 (0.3) 0.2 (0.32) 0.7 (0.26) D10S1671 (127.1) 2.3 (0.22) 3.4 (0.16) 0.1 (0.36) 2.9 (0.18) D10S1741 (128.2) 0.4 (0.29) 0.3 (0.32) 0.3 (0.24) 0.2 (0.32) Locus marker locations in Kosambi cM according to the Marshfield map. Maximum two-point LOD scores (Z) and the recombination fraction (G) used in the calculation. Maximum two-point parametric LOD scores (Z) and the recombination fraction (P) used in the calculation.
0600) TABLE 6-continued TABLE 5 Nonparametric Two-Point Analyses Parametric Analyses - Autosomal Recessive Disease Model Locus Marker All Families Late-Onset APOE e4/4- Locus Marker All Families Late-Onset ( Location in cM) Z(p) Z(p)(p) negativeS. Z(p)Z(p)" (Location in cM) Zas (P) Znak (P) D10S1671 (127.1) 3.3 (0.0005) 3.8 3.7 (<0.0001) (<0.0001) D10S1225 (80.77) 0.4 (0.36) 0.9 (0.32) D10S1741 (128.2) 1.3 (0.09) 0.9 (0.2) 0.8 (0.2) D10S564 (112.6) 0 (0.50) 0 (0.50) D10S583 (115.3) 2.7 (0.28) 2.4 (0.28) Locus marker locations in Kosambi cM according to the Marshfield map. D10S1710 (124.3) 0.8 (0.32) 1.1 (0.30) Two-point nonparametric linkage scores (Z = Z scores for the likelihood D10S566 (127.1) 1.1 (0.32) 0.6 (0.34) ratio) and p-values (p). D10S1671 (127.1) 2.9 (0.28) 3.8 (0.24) D10S1741 (128.2) 0.4 (0.35) 0.3 (0.36) 0602)
Locus marker locations in Kosambi cM according to the Marshfield map. TABLE 7 Maximum two-point LOD scores (Zina) and the recombination fraction (P) used in the calculation for parametric two-point analyses using an Nonparametric Multipoint Analyses autosomal recessive disease model. Locus Marker All Families Late-Onset APOE e4/4- 0601 Nonparametric two-point and multipoint analyses (Location in cM) Z(p) Z(p) negative Z(p)" of the genotyping data were conducted using the GENE D10S1225 (80.8) 0.9 (0.2) 1.6 (0.06) 0.9 (0.2) D10S564 (112.6) 0.4 (0.4) 0.5 (0.3) 0.6 (0.3) HUNTER-PLUS Kruglyak et al. (1996) Am. J. Hum. D10S583 (115.3) 1.1 (0.15) 1.3 (0.1) 1.3 (0.1) Genet. 58:1347 and ASM (Version 1.0 applying the expo D10S1710 (124.3) 1.9 (0.029) 2.1 (0.02) 2.15 (0.016) nential model) Kong and Cox (1997) Am. J. Hum. Genet. D10S566 (127.1) 1.6 (0.05) 1.8 (0.03) 2.0 (0.02) 61:1179 computer programs. The results of the two-point D10S1671 (127.1) 1.3 (0.01) 1.6 (0.05) 1.7 (0.05) nonparametric analyses of the full, age-of-onset-Stratified, D10S1741 (128.2) 1.2 (0.1) 1.6 (0.06) 1.4 (0.08) Locus marker locations in Kosambi cM according to the Marshfield map. and APOE e4/4 status-stratified datasets are provided in Maximum multipoint nonparametric linkage scores (Z = Z scores for the TABLE 6. The results of multipoint nonparametric analyses likelihood ratio) and p-values (p). of the same datasets are provided in TABLE 7. US 2003/017O678A1 Sep. 11, 2003
0603 The results of the parametric two-point analyses of software program (FBAT) Rabinowitz and Laird (2000) Seven microsatellite markers on chromosome 10 in 435 AD Hum. Hered. 50:211-223; see also http://www.biostat.har families using a dominant model revealed Significant evi vard.edu/fbat/default.html to determine if it is within link dence of linkage of AD to chromosome 10 around marker age disequilibrium range of an underlying disease gene. The D10S583 (Z=3.3) in the full sample and around marker D10S1671 in the late-onset sample (Z=3.4). The results analyses were based on estimated empirical variances (to of the parametric two-point analyses using a recessive model account for the presence of linkage) Lake et al. (2000) Am. were similar, with a maximium LOD score of 3.8 for marker J. Hum. Genet. 67:1515-1525 as implemented in FBAT D10S1671 in the late-onset sample. Although linkage was (Version 1.0, 1999). Although the multiallelic test on all 11 generally more pronounced in families without the APOE alleles for marker D10S583 was not significant (p=0.15), the e4/4 genotype, none of the markers had LOD scores greater diallelic test revealed significant association of the 211-bp than 3 in this stratum. allele with protection against AD (nominal p=0.004, Bon 0604. The results of two-point nonparametric linkage ferroni corrected p=0.04). The allele frequency for the analyses also revealed linkage of AD on chromosome 10q 211-bp allele is about 0.096 and the number of informative with the highest linkage scores (Z=Z Scores for the likeli families contributing to the test statistic was 30. hood ratio) provided by markers D10S583, D10S1710 and D10S1671 (Z scores of 2.8, 2.8 and 3.8, respectively, for 0607. The results of the linkage and association analyses the late-onset dataset). Multipoint nonparametric analyses of markers on human chromosome 10 indicate the presence generated maximum Z Scores of 1.9 (p=0.029, full sample), of a single locus or multiple, e.g., two, loci underlying AD 2.1 (p=0.02, late onset) and 2.15 (p=0.016, APOE e4-nega on chromosome 10. The number of AD loci on chromosome tive) at marker D10S1710, which is located between the two 10 can be determined by examination of additional markers markers (i.e., D10S583 and D10S1671) with the greatest distal to marker D10S564, and, in particular, markers in the linkage Signals in the two-point analyses. regions of markers D10S583, D10S1710 and D10S1671. Putative AD gene(s) on chromosome 10 can be identified EXAMPLE 3 through further detailed Studies of linkage disequilibrium as 0605 Association Analyses of a Chromosome 10 Mic well as through assessment of candidate genes. rosatellite Marker 0608 Since modifications will be apparent to those of 0606 Marker D10S583 was analyzed for association skill in this art, it is intended that this invention be limited with AD using the Family-Based Association Test computer only by the Scope of the appended claims.
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