CA2222813A1 - Variant presenilin-2 genes - Google Patents

Abstract

Variant presenilin-2 genes are provided. Methods of using these genes in diagnosing Alzheimer's disease are also provided.

Description

VARI~NT PRESENILIN-2 GENES

INTRODUCTION
This invention was made in the course of research sponsored by the National In.~titu~Ps of 5 Health. The U.S. Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION
.AI,IIr; llr.1'5 Disease (AD) is a progressive neurod,,~ ,dli\re disorder cllcu;.. ~ t by memory loss and dP.mPnti~ The major F ' ~'~ I feature of AD is the presence of ~-u-~ uus 10 ncu~url~illary tangles and senile plaques cu~ osed priniarily of the amyloid protein. These plaques contain beta-amyloid, a peptide varying from 39 to 43 amino acids in length, derived from a larger amyloid ~JI~;UI~UI protein (APP) (Goate et al. Nature 19gl, 349, 704-6; Masters et al.
PNAS 1985, 82, 42454249; Kang et al. Nature 1987, 325, 733-736). Studies have shown that ;l~s in the g~ dliull of the 42 amino acid peptide lead to AD in APP encoded disease.
AD is typically a disease of the elderly, S.rn;. ~;"g up to 6% of those aged 65 and up to 20% of 80 year olds. This type of AD is termed late-onset. In addition, a small number of p~i,5.~s have been des~lilxd wherein the disease is inherited as an ~lt~ nm~l tlom~ mt with age ~r~ r~ ce. Most con~ -ly, the age of onset of the disease is below 60 years in these families (presenile). Thus, this type of AD is termed early-onset. Genetic factors have been 20 ;".~ t~i in both early and late onset AD.
Several large families have been il1r"1;r~d in which ~ lliL AD s~ les as fully losu~ dorninant trait. Linkage analysis studies in presenile AD farnilies have j,lr..,~ir,~i four genes, on ~l.l.)ll~s-,"~-~ 1, 14, 19, and 21, that when mutated, cause presenile AD.
The first presenile AD gene i-lrl 11 ir.Pd maps to chromosorne 21 and codes for the beta A4-amyloid protein ~,~;u,~c" (APP) (Goate et al. Nature 1991, 349, 704-706; Murrell et al. Science 1991, 254, 97-99; Chartier-Harlin et aL Nature 1991, 353, 844-846). Mutations in this gene account for a~uAil-~l~ly 5% of the families. These disease causing ~ n~ have been modeled in~I,.".~r~ 1 or primary cultured cells and have been shown to lead to altered proteolytic ~luces~illg of APP in a way that favors p~olu~;lion of its amylc~ and potentially n~;ulul~JAic Ab fragments. Tld--s~,~ ov~.~A~,.~sioll of one mutant APP has resulted in the first mouse model of CA 02222813 1997-ll-28 AD, in which age-linked cerebral deposition of the Ab fragrnent is 7~Ccr~n~ - ' by neuronal, astrocytic, and ., ~,~,lial pathology (Garnes et al. Nature 1995, 373, 523-527).Genetic variability in the apolip.~l"ut~l E locus on chrornosorne 19 have also been shown to be ill4)UI~ in the etiology of AD (St.i~ et al. PNAS 1993, 90, 1977-1981; Saunders et S al. Neurology 1993, 43, 1467-1472). Inl,~liti~ce of the I4 (112 Cys-Arg) allele has been reported to lower the age of onset in a dose ~ r~ r~-l rnanner. Conversely, il~lcliL~Iee of the apoli~lot~l E I2 allele appears to confer a decreased risk of dcv~ g AD (Corder et al.
Science 1993, 261, 921-923; Corder et a1. Nature Genet. 1994, 7, 180-184).
Presenilin-1 (PS-1), located on cl.~ s~ 14 harbors an P,Stinn~ 70% of the disease 10 causing ".,-l~l;. l-s, rnaking it the rnajor gene for farnilial presenile AD (e~l ~' 10% of all AD
cases~ (Sl-r- 1 ill~ lll et al. Nature 1995, 375, 754-760; van Br~.u~ren et al. Nature Genet. 1992.

2, 335-339; St. George-Hyslop et al. Nature Genet. 1992, 2, 330-334; Srh~llPnhP~g et al. Science 1992, 258, 668-670). This gene, ~ S182 or PS-l, is rnade up of 10 coding exons .~} 3 to 12). PS-1 is ~ d to be an integral ~ Ll~u~e protein with at least 7 15 Il,...s..r.,~ e dornains. At the tirne of its i.co~ ion, five different rnissense mnt~ti-~n.c were it1PntifiPd in 8 chromosorne 14 lir~ced families (Sl-r~ I et al. Nature 1995, 375, 754-760). A
total of ~ different ",..l~ .c in 40 families of various ethnic origins has been i~iP.ntifiP~I (van Broeckhoven et al. Nature Genet. 1995, 11, 23~233). Two drfferent ..,..~ nc were i-lPntifiPA at each of the codons 139, 146, 163, and 280. However, rnost ... "; ~ .,.c are scattered over the 20 protein with mnt~tionc found in S of the 7 putative L~ e domains and in 3 of the 6 hydl~ ~ ' loops. Mutations have been found in 6 of the 10 coding exons, with exon 5 and 8 a ~ g for 65% of the l.~ The same mutation occurs in several AD families ofdifferent ethnic origin, su~P,cting there are ;...lryr~..1r..~l mutation events in the PS-l gene.
A third gene for presenile AD (PS-2) rnaps to ~ hllllnl).CI~IIP I in the Volga-German AD
families, a group of families in which AD is the result of a founder effect (Levy-Lahad et al.
Science 1995, 269, 970-973). This gene (STM-2 or E5-1) was irl~.ntifi~ as a direct result of its high homology to PS-l. The same Ill ~Sr.ll!~e mutation was found in 7 Volga-German AD families and more recently, a second l.hss~l.3e mutation has been found in an Italian AD family (Levy-Lahad et al. Science 1995, 269,97~973; Rogaev et al. Nature 1995, 376, 775-778; Barinaga Science 1995, 269,917-918).

Both PS-I and PS-2 have applv~dllldlly 450 amino acids and share an overall homology of 67% with the highest similarity observed in the TM dornains (Levy-Lahad et al. Science 1995, 269,973-977; Rogaev et al. Nature 1995, 376,775-778. This degree of homology is indicative of a similar biological function. The putative seven ~ " ~l ,. dne domain structure of the presenilins S is cull~ 1c with a function as a receptor l~ le, an ion channel or a ~ e structural protein. It has been determined that ~..--~ "~ in PS-I and PS-2 cause an increase in the ;IdtiUII of Ab42 thus ' lg biological interaction with amyloid.
The exonic structure and the ~ t~-~re of alternate splicing in the PS-1 gene have been determined. The use of an alternate splice donor site at the 3' end of exon 3 results in clones with and without a VRSQ ~tif at codons 26-29 (Al7heimPr's Disease Collaborative Group. Nature Genet. 1995, 11, 219-222). Alternate splicing has also been found in both exon 8 in PS- 1 and the exon 8 in PS-2.
A number of other variant, alternatively spliced PS-2 genes have now been i~lr.lli~lP~l These genes are useful in the ~l;~ cic of early-onset AD and in evaluating agents which rnay be useful for the ~ lll of this disease.
SUMMARY OF THE INVENTION
An object of the present invention is to provide novel, variant PS-2 s~l, Ir~ll C'S
Another object of the present invention is to provide a rnethod of ~ o~;~.g Alzheimer's disease using these novel PS-2 .ce l ~ or the exonic or intronic .se~ rl~ of the PS-2 gene.
Yet another object of this invention is to provide a model system for Alzheirner's disease COIl~l~lllg variant PS-2 genes.

BRIEF DESCRIPIION OF THE FIGURES
- Figure 1 provides a s~ ."~ co.. y ~ nn of the Ol~ of the PS-l and PS-2 gene.
25 Labeled arrows indicate sites of known m~lt~ti-n~ Unlabeled arrows indicate intron/exon bul....l,.. ies Hatched areas in PS-2 indicate sites of alternate splicing. USF#15 contains exons 3, 4 and 8. W.U.#2 lacks exons 3, 4 and 8. W.U.#15 lacks only exon 8.
Figure 2 provides the gene s~ -e of the PS-2 gene (SEQ ID NO: 31).

30 DETAILED DESCR~ION OF THE INVENTION
Mutations in the APP gene on ~hl----~ ---~ 21, the ApoE gene on CI1IUIIIOS(JII~ 19, and the S182 or PS-I gene on chromosome 14 account for the majority of j(l. .llirl~l cases of early onset ~17h~imPr's disease (StlitLIllaLl et al. PNAS 1993, 90, 1977-1981; Sh~llillclc,n et al. Nature 1995, 375, 75~760). However, in the Volga-Gerrnan kindreds, and in several other families in which AD appears to be inherited as an ~ n~ ."~ trait, these loci have been r~r~The Volga-German families are a culturally distinct ~ul~ulalion in Russia, whose5 Iwll~ did not rnarry into the Russian population. The relative onset of AD in this group is eYreptinn~lly early, ranging from 50 to 70 years of age. However, clinically and pathologically, AD in these families is ;,.,~ ;"~ hle from typical AD. The a~ltosom~l d locus, ~ onsibh, for AD in the Volga-German kindreds has been loc~li7P~l to ChI~ C~ P~ lq31~2 (Levy-Lahad et al. Science 1995, 269, 970-973). (~nl' ' genes which rnap to this locus have 10 also been i~ ntifi~ Levy-Lahad et al. isolated STM2 whose p~ t~d amino acid se~l"r..,l~e is l c to that of S 182 (PS-l). A point 1 " ~ l, in STM2, resulting in the ~ub~ uliol- of an icr,~ cin~ for an asparagine (N~4~I), was itlPntifi~l in affected individuals. This N,4,I mnt~ti~m occurs at an arnino acid residue that is conserved in hurnan S182 and at the mouse S182 hom~'~g Rogaev et al. reported the cloning of E5-1 on chrornosorne 1 which is also l-~ cc ~c to S182 (Nature 1995, 376, 775-778). Analysis of the mlrl~ti~l~ se~ r~re of the open reading frarne of E5-1 (STM2) led to the discovery of two missense 5.~1~5~ c at conserved amino acid residues in affected II~ of the Volga-German (N,4,I) and Italian (M~39V) p~licl~s.
In order to better el~, ' the structure of the PS-2 gene and to d - possible sites of alternative splicing, the gene was cloned and s~ ~d and PCR was used to d~t; l.lille alternate 20 splice products (variants) and exon/intron b~ s. F~ ir,l, of intron/exon boundary .r..r~s revealed that PS-2 is encoded by 10 coding exons. The PS-2 gene s~ r~.re was deterrnined by both se.ll.r"~ g the EST seq ~~nre T03796 and isolating PS-2 cDNA's using the GeneTrapper kit (Gibco BRL, Gailht;~ul~" MD).
The intron/exon structure of the PS-2 gene is shown in Table 1. Positions of introns that 25 interrupt the PS-2 cDNA are shown. Exonic se~l~nre is ~ ; -L~d in upper case and intronic s~ in lower case letter. Exons are ~-wl~~red from the 5' end of the cDNA s~ ~enre E;XONl (to-195) . CTTTTcccAAGGTcGcccAGgt~rg~t~t~ rcslg EXON2 ~ k-lk~ CGAGGACGTGGGACTTCTCA

(-194 to -21) GCGGCCCCAAGTGTTCGTGGgt5cg~ttrAgArtrtrt EXON3 tc~ cl~ nGTGCTTCCAGAGGCAGGGCT

(-20 tO 140) GAGAGAACACTGCCCAGTGGgt~ cc~JrA~A~rtg EXON4 c~c~At~ c~c~Ay~GAAGCCAGGAGAACGAGGA

(141 to 355) ACAGAGAAGAATGGACAGCT~t~
EXON5 ~A~A~AA~Al~rA~t~ATcTAcAcGAcATTcAcTG

(356 to 497) ACAAGTACCGCTGCTACAA(~lg~ c~ c~ cc 15 EXON6 Ç~ A~ GTTcATccATGGcTGGTTGA

(498 to 565) TCACCTATATCTACCTTGGGtAA~A~tA~g~.A~A.

EXON7 A~cAr~ grAGAAAGTGCTCAAGACCTACA

(566 to 786) GGGCGCCATCTCTGTGTATC.~A~I~.-A~

EXON8 a~atgt~ ~lgl~ l -GATCTCGTGGCTGTGCTGTG

(787 to 885) CCCTGCCCTGATATACTCATgtg~cccccE;I~c EXON9 AA~A-III.~ ~C~CTGCCATGGTGTGGACGGTT

(886to969) CCCCTACGACCCGGAGATGGgt~
EXON10 ~ Arr~rA ~AGAAGAcTccTATGAcAGT

(970 to 1071) GCTGGAGGAAGAGGAGGAAA~A~ ccAt~trAcA

EXONll t~t~rt~ctrAr~AgGTCAAGGGGGCGTGAAGCTT

CA 02222813 1997-ll-28 W O 97/38133 PCTrUS97/04683 (1072to 1190) CTTCGTGGCCATCCTCATTGt~g~t~ g~, S (1191 to3'end) ct(~ k~ ct~gGGCTTGTGTCTGACCCTCCT
cDNA s~u~n~ing revealed multiple positions at which mltlli(ms in sequence were observed; four of which occurred at exon boun~Lies. A surnmary of this splicing data is provided in Table 2.

Splicing Event PCR cDNA Codons exons 3 and 4 + W.U. #2, W.U. #15 1-119 exon 8 + W.U. #15 263-296 3 bp deletion - IB913 324 6 bp deletion - IB913 357-359 Splicing events in Table 2 are listed with respective cDNAs in which they were i-l~ntifi~l as well as with cc)llr~ ldi-g arnino acid residues. Splicing events detected by RT-PCR are denoted by a "+" in the PCR colurnn. All events are relative to the full length cDNA c10ne, referred to as USF#15. Sirnplified ~ cLu,~ of the four cDNA clones are shown in Figure 1.
Three of the v~i~Liu"~ i-lr.-lir~l involve alternate splicing events. These include splicing out of exon 3 and 4, and splicing in or out of exon 8 in the PS-2 gene. Amplifir~tinn by PCR over the relevant b~u~lJ~ ies showed that these clones occurred in cDNA from all of the tested tissues.
No other alternate splicing was detected these tissues. The other two variations involved small changes from the selr-r .~ bli~hed by Levy-Lahad et al. Science 1995, 269,973-977. These changes include a deletion of a ~ residue at codon 324 and insertion of six base pairs which change the encoded amino acid sr~ e from ERGV to ESQGG at codons 357-359.
In contrast, little evidence has been found for naturally oc.. i.. g alternate splicing in the PS-l gene. The only alternate splicing previously reported for PS-I has been in exon 8 and at the 25 3' end of exon 3 resulting in clones with and without a VRSQ motif. While these splicing events were also observed in PS-2, additional alternative splicing events were observed in PS-2 which CA 02222813 1997-ll-28 W O97/38133 PCTrUS97/04683 were without c~,uivalence in PS-I. Some of these events lead to significant alterations in the structure of the protein.
The rnost striking alternate ~ sclipt~ are those which lack exon 3 and 4. Variants lacking exons 3 and 4 lose the normal start rnPJh: ~ - and if translated would be ~,.. ' : ' to begin S at the methionine at codon 145 . This start site occurs after the proposed l~ c~ ~ ~r~ l ll ,l dile domain I
in the middle of L,;....~n,r...ll"d,le dom.ain 2. This protein is different than the Volga German AD
mutant N,4~I.
The i-lPntific-~tic n of these variants ir.dicates that certain ~ c in the PS-2 gene are Ac.cori~tc~1 with the "Volga German" type of AD. Therefore, these variants may be useful in the 10 rl;~;"f~;c of AD or in the dc~. lO~IIr.l~l of models of AD.
The ic~ntifirAti. n of the intronic ~ u ~ c is also useful in the early dPJc-cti. n of variant forms of the PS-2 gene. The genomic anal.ysis of the PS-2 gene (see Figure 1) has led to the dc~el.~lllelll of a rnethod for iclr.,lir,.,.li..., of intronic poly"ul~ "~ which are predictive of disease. F.lllrirlAtir)n, d~tr~tir~n, and .~ c of ....~ iu..c in both intronic se~l..r..rPi ~csori~
15 with splice variation and in the open reading frames proximal to these intron-exon buullJa.ies of the PS-2 gene can be p~r~ ~d through use of intronic s~lllrl~rr.~. Tllr.llir~r~ n and analysis of mutants or variants arising from ...--l ~ -.c in splice donor or acceptor sites are enabled by hlv..l~dge of these intronic se~ e-~ Fullh~ , a . '~~ analysis of the intron-exon buu-l~i~ makes possible ~,r<l~r-.-; ~ primers that would allow accurate s~lllr..-. e of the first or last 10 to 20 1 ll ~ l ;llr~c of coding exons especially near cDNA termini.
At present there is no known effective therapy for the various forms of AD. However, there are several other forms of dr~n~.ntiA for which ll~llllelll is available and which give rise to plugl~si~e ll~hlAl delellulation closely l~sellll,lillg the dr~n~ntiA AC~; ''~ with Alzheimer's disease. A rliagnl-ctir test for AD would therefore provide a useful tool in the rliaE;noci.C and 25 ll~lll~ll of these other c~ ;r~.C, by way of being able to exclude early onset Alzheirner's &seace. It will also be of value when a suitable therapy for AD is available. There are several mth~Ylr l~gies available from leCOIl~ lalll DNA t~hn~lccy which may be used for dele~iillg and identifying genetic ....~l~ ti~ IèS~ ''~ for Al;~h~l~ disease. These include, but are not limited to, direct probing, ligase chain reaction (LCR) and polymerase chain reaction (PCR) 30 mPth~ I~cY
Detection of variants or mutants using direct probing involves the use of olignn~lc!~ti~
probes which may be prepared synth.-Ji~Ally or by nick trAnCl~~inn. In a plerell~J embodiment, the WO 97/38133 PCTrUS97/04683 probes are CO~ nt:~ry to at least a portion of the variant PS-2 genes i~lPnfifiP~ herein. The DNA probes may be suitably labelled using, for exarnple, a ~ hPl enzyrne label, fluorescent label, or biotin-avidin label, for slll.s~ vic~l~li7~ti~n in for exarnple a Southern blot hybridization ~ll~;t:dUI~. The labelled probe is reacted with a sarnple of DNA from a patients S ~u~;t~d of having AD bound to nitroce]llllr~s~P or Nylon 66 substrate. The areas that carry DNA
se l~r~ ec comple~ u y to the labeled DNA probe become labelled themselves as a c..~lg~lur~re of the re~nnP~ling reaction. The areas of the filter that exhibit such labeling rnay then be vic~l~li7~, for example, by autoradiography.
Alternative probe l: ' qllPC, such as ligace chain reaction (LCR) involve the use of a 10 1":~"~ h probe, i.e., probes which have full c ~ ~ ' ~ .d;.. ;Iy with the target except at the point of the mutation or variation. The target sP~IIlP-nrP is then allowed to hybridize both with the nlignnllrl~tirlPc having full c~ l...llr.l~dly, i.e., cti, ~ r~ collq,L.,~ ,y to the PS-2 variants of the present invention, and olignmlrl~oti-lPs containing a ~ "L~ , under cnn~ ionc which will .li n;,~";cl, between the two. By u, ll~ting the reaction conditions, it is possible to 15 obtain hylL, ;.li".~ n only where there is full c~lllq~l~ .llr~ l ;Iy. If a ",:i"L~ h is present, then there is significantly reduced hyl.. ;. li ,.-~ ;""
The polyrnerase chain reaction (PCR) is a 1~' qnP that aln~ s specific DNA
ce~ r~rP$ Repeated cycles of ~LIldluldli~ll, primer ~nnP~ling and ~ n~;on carried out with a heat stable enzyme Taq polyrnerase leads to ~ontllLidl il-~as~ in the ~;~m~lltldli()ll of desired DNA
20 5~P~ "- PS
Given the knowledge of mlrlPotirlP seqllPnrpc encoding the PS-2 gene, it is possible to prepare synthetic r~ omlcl~otit1Pc co ,l ~~t~ry to the selnr..,rPc which flank the DNA of interest. Each oliE;."-~ lP is c~ ~ ' y to one of the two strands. The DNA is then d~ -dul~d at high l~il~ldulcs (e.g., 95~C) and then rP~nnP~h~l in the presence of a large rnolar 25 excess of o!i~o.,.l.~ The oligcn lr!~ti~lec, oriented with their 3' ends pointing towards each other, hybridize to opposite strands of the target sP~lPnrp and prirne enzymatic e;~lr~ I~ ,n along the nucleic acid template in the presence of the four deoxyribnnllr!~tirl~P. l.i"h p~ The end product is then d~..dulcd again for another cycle. After this thre~step cycle has been repeat several times, an~l;r~=nl;on of a DNA segment by rnore than one rnillion fold can be achieved.
30 The resulting DNA may then be directly sr~ P~ ed in order to locate any genetic alterations.
Alternatively, the i-lPntifiP~ PS-2 variants of the present invention rnake it possible to prepare oli~omlr!~tirlps that will only bind to altered DNA, so that PCR ~11 only result in the multiplication of the DNA if the mutation is present. Following PCR, allele-specific t~lig~ml~lel~ti~l~ hybridization may be used to detect the AD point mllt~tirn Alternatively, an ~ pt~ti-~n of PCR called amplification of specific alleles (PASA) can be employed; this method uses dirrelcll~ial ~mrlifi~tirln for rapid and reliable ~ tin~tjon between S alleles that differ at a single base pair. Newton et al. Nucleic Acid Res. 1989, 17, 2503; Nichols et al. Genomics 1989, 5, 535; Okayama et al. J. Lab. Clin. Med. 1989, 1214, 105; Sarkar et al.
Anal. Biochem. 1990, 186:64; Somrner et al. Mayo Clin. Proc. 1989, 64, 1361; Wu Proc. Nat'l Acad. Sci. USA 1989, 86, 2757; and Dutton et al. Biotechniques 1991, 11, 700. PASA involves A."~,];rl~ n with two cli~ m~ oti~ primers such that one is allele specific. The desired allele is ~fr~ tly ~ lirled, while the other allele(s) is poorly ~- .q)l; r.~ because it 1~ .c with a base at or near the 3' end of the allele specific primer. Thus, PASA or the related rnethod PAMSA can be used to specifically amplify one or more mutant PS-2 alleles. Where such AmrlifirAtion is pelrulllr3d on genetic material obtained from a patient, it can serve as a method of detecting the presence of one or ~re mutant PS-2 alleles in a patient. PCR-induced mutation restriction analysis, often referred to as IMR~ can also be used in the ~ t~tinn of mutants.Also ill4/UI~ t iS thedc~ l of r~pf~ models of Al~ disease. Such models can be used to screen for agents that alter the de~,cllel~lh/e course of AD. Having i-l~.ntifi~
specific ~ n~ in the PS-2 gene as a cause of early onset familial Alzheimer's disease, it is possible using genetic manipulation, to develop transgenic model systerns and/or whole cell systerns cf-.~ g a mutated PS-2 gene or a portion thereof. The model systems can be used for ~,lcelfillg drugs and evaluating the efficacy of drugs in treating Al~ disease. In addition, these rnodel systems provide a tool for defining the underlying l,~ "~ of PS-2 and its 1~ IA1;~ ;I' to AD thereby providing a basis for rational drug design.
- One type of cell system which can be used in the present invention can be naturally derived. For this, blood samples from an affected individual are obtained and pclll~nt;lltly rulll~ into a l~llyllul~' ~1 cell line using, for example, Epstein-Barr virus. Once established, such cell lines can be grown c....l;...~oucly in ~ cultures and can be used in a variety of invitroeApelllllel-ls to study PS-2 cAl lessiOll and processing. Another cell line used in these studies c~nl4~ es skin rlbl~l~l~ derived from patients.
Since the FAD mllt~ti--n is dominant, an all~llldlivè rnethod for CUII~IILIelillg a cell line is to gen~ti~Ally engineer a PS-2 mutated gene, or portion thereof, as dcsclibcd herein, into an e~L~hed cell line of choice. Such methods are well known in the art as exemplified by Sisodia g W O 97/38133 PCT~US97104683 Science 1990, 248, 492 and Oltersdork et al. J. Biol. C~en~ 1990, 265, 4492, wherein an amyloid precursor peptide gene was l~a~l~r~;~ed into n~rnm~ n cells.
Baculovirus ~,~yl~ ivll systems have also been found to be useful for high levelC;A~ Ssiull of h~.~l~gous genes in eukaryotic cells.
The mutated gene can also be excised for use in the creation of llall~O~llic anirnals c.",l~;";~g the mutated gene. For example, a PS-2 gene of the present invention can be cloned and placed in a cloning vector. Examples of cloning vectors which can be used include, but are not ~imited to, ICharon35, cosmid, or yeast artificial cl..v"~s(Jll,r The variant PS-2 gene can then be llal~ cd to a host ~ IL~ ~ animal such as a mouse. As a result of the transfer, the resultant 10 transgenic n~l"."~" anirnal will preferably express one or more of the variant PS-2 polypeptides.
Alternatively, minigenes e,~ ;"g variant PS-2 polypeptides can be rlP.ci~nPA Such minigenes may contain a cDNA se~r.r.l,~ e ~ -colil-g a variant PS-2 polypeptide, preferably full-length, a combination of PS-2 exons, or a co.,l, .~inn thereof, linked to a do~ll~ a polyadenylation signal s~u~nce and an u~ lulllvl~l (and preferably ~ hanc~l). Such a rninigene cu~ u~;l will, when illllu~luc~d into an a~>l).u~ le transgenic host, such as a rnouse or rat, express a variant PS-2 polypeptide.
One a~lua~,l- to creating Llal O - animals is to target a m-lt~tinn to the desired gene by hl logollc l~ b laLiull in an embryonic stem (ES) cell in vitro followed by vlnje~lion of the mQAifi~l ES cell line into a host blastocyst and ~b~ellllrlll in~nb~ti-~n in a foster rnother.
Frohrnan and Martin Cell 1989, 56, 145. Alternatively, the ~ e of microinjection of the mutated gene, or portion thereof, into a one-cell embryo followed by ;"~ im~ in a foster mother can be used. .A~itinnql methods for ~-v lu~ g llal~L ~ animals are well known in the art.
Tlansg - anirnals are used in the ~C.~ lnr~l of new Illel~;ulic cull4,o~ilivl,s and in cal~ ..ngr.l ~ y testing, as ~r~ rl~d by U.S. Patent 5,223,610. These animals are also used in 25 the dcv~lopl~ of predictive anirnal models for human disease states, as exe~rlifi~d in U.S.
Patent5,221,778. Tl. sO ~animalshavenowbeendcvelo~dfor ~ r-~ g ~17h~imPr's&sease(U.S. Patent 7,769,626), multi-drug l~i~ ce to a~;r~ r, agents (U.S. Patent 7,260,827), and carcino" ~ rec (U.S. Patent 4,736,866). Therefore, the PS-2 genes of the present invention which are believed to cause early onset .Al7hPi~r's &sease in Cl~ . so"~ 14-linked 30 pe.li~ provide a useful means for developing ll ,, ~ anirnals to assess this disease.

Site directed m l~gPnP.ci~ and/or gene conversion can also be used to a mutate a non human PS-2 gene allele, either e,~ ge"~ 1y or via tlall~r~licJll, such that the mutated gene encodes a polypeptide with an altered amino acid as described in the present invention.
In addition, ~ il)c ' to the PS-2 gene and variants thereof can be raised for use in the 5 examination of the function of the truncated llans~ of the PS-2 gene. These antibodies can be, for P~ !P polyclonal or nnon~lon~l antibodies. The present invention also includes chirneric, single chain, and 1l~ antibodies, as well as Fab fragrnents, or the product of an Fab ~sion library. Various l)r~lul~s known in the art may be used for the production of such o~ and fragnnents.
Antibodies ge~ t~i against the PS-2 genes of the present invention can be obtained by direct injection into an anirnal or by A~' ' ~ ~Pring the gene to an animal, pl~rtlably a nc,-l,.,.l~-,.
The antibody so obtained will then bind the PS-2 gene or itself. In this manner, even a fragment of the gene can be used to generate thee antibodies.
For ~ iUII of lli~ l all~Odi~s, any l~', which provides antibodies produced by c~ l;."l~ cell line cultures can be used. Examples include the hybridorna ~ lle (Kohler and Milstein, Nature 1975, 256, 495497), the trioma t~rl-n;ql-P the hurnan B-cell hybridorna technique (Kozbor et al., Immlmology Today 1983, 4, 72), and the EBV-hybridoma ' l to produce human l-K,~ Al ~tibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp.77-96).
T~ 3IIP~ llr~ for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain alltil,r ' to the PS-2 genes of this invention.
Also, t, ~ mice rnay be used to express l.~ ~ antibodies to the PS-2 genes of this The following l - " py~ are provided to further illustrate the present invention.
EXAMPLES
Example 1: Cloning ~e PS-2 gene PS-2 cDNA's were isolated using the Gene Trapper kit (Gibco BRL) acc~.d;..g to the m~m-f~rtllrer's directions. A human SU~ " brain library (Gibco BRL, Gaithersburg, MD) in 30 pCMV.SPORT was probcd with the primer 5'-CATTCACTGAGGACACACCC-3' (SEQ ID
NO: I) (derived from the EST selllP.nl~e T03796). However, use of this sequence consi~ ly (three times), resulted in the isolation of different clones which c--nt~inPd the 5' and 3' untranslated -CA 022228l3 l997-ll-28 gene s~ll~nrf-c but were missing the region of the gene around the putative start codon. The library was ~ ~l,ed using the prirner 5'-CAAATACGGAGCGAAGACAG-3' (SEQ ID NO: 2),derived from the region omitted in the previous clones, again using the Gene Trapper kit. This enabled the isolation of a clone containing the missing region.
Example 2: cDNA preparation Human RNA from brain, he~t, liver, lung, placenta, and skeletal muscle was obtained from Clontech (Palo Alto, CA). First strand cDNA was Sy~ ~ following the S~
Preamplifir~tinn System for First Strand cDNA Synthesis (Gibco BRL). Two mg of total RNA
was cull~ ed with 1 mg of r~lig~ cl~oti-~P (dT),2 ,8 prirner, 100 ng of random hexamer primer and DEPC treated water in a 0.5 ml tube. Samples were ;..~ l~1 at 70~C for 10 rninutes and placed on ice. Kit coll~llull~ were added in acc~.dance with the rn~mlf~rhlrer's protocol, and samples were i ~ ~'ul ~. ~t~d at 37~C for 2 minutes. Su~~ t lI Reverse Tl~ls~l i~se (RT; 200 U) was added and sarnples were inruh~t~d at 37~C for 1 hour. Samples were then i l.;LL ' at 70~C
for 15 minutes, chilled on ice, and 2 U of RNAse E~ was added. All sarnples were stored at -20~C.
For ~ll.se~ PCR, 1 ml of final product was used for each reaction.

Example 3: PCR to deternnne alternate splice products Primers were designed throughout the cDNA s~ll--nr~ to allow the ~~ rc.~ r-ll of the 20 alternate splicing in a variety of tissues. Exonic pr~mers, along with the c~ ''t r,n,c for their use are provided in Table 3. Intronic primers, along with the conditions for their use are provided in Table 4.

Primer Name SEQ Se~ - (5'~3') 1 or~ff ~ ~ g ID Temp.
NO (~C) LP313F ~ 5 AGCCTGCTGAGAAGAAGAAACCA EXON 3 50 LPSOlF 6 AGGCAGGGCCCAGAGGATGGAGA EXON 4 50 GA

W O 97138133 PCT~US97/04683 Primer Name SEQ Sequence (5'-3') l.o~ Qn ~r~
ID Temp.
NO (~C) LP71lF 8 ACCATCAAGTCTGTGCGCTTCTAC EXON5 50 5;UTR 23 GCTTCTGTCTCAGGTTCCTTC 5'UTR 54 - FORWARD
5'UTR 24 CGGTGTTTGGCTG m TATCA 5'UTR 54 REVERSE

CA 02222813 1997-ll-28 W O 97/38133 PCTrUS97/04683 PnmerN~ne SEQ Sequence(5'-3') T.or~ g nD Temp.
NO (~C) EXON 4 25 AGCCTCGAGGAGCAGTCAG S' EXON 4 49 INRTONIC
EXON4 26 GCAGACGGAGAGAAGGGT 3'EXON 4 49 INTRONIC
EXON7 27 GGGCAGGCTCTTCTTCAGGG 5'EXON7 57 INTRONIC
EXON 7 28 GAAAGCCACGGCCAGGAAG 3'EXON 7 57 INTRONIC
R05822F 29 TCACGGACAGGAAGCACAGC 5' EXON 12 56 R05822R 30 GTAACAAGAACAGGACTCAG 3' EXON 12 56 W o97/38133 PCTAUS97/04683 Primer SE Se~ (5'-3') Size Annealing Name Q (bp) Temp.
ID (~C) NO

52PS2X3 34 AAA AAT CCG TGC ATT ACA T 50~C/30"

TG
SPS2X4 36 AGC CTC GAG GAG CAG TCA G 53~C/30"

SPS2X5 38 GGT ATC AGT CTC AGG ATC ATG GG 60~C/30"

52PS2X6 40 GTA AAG AGG GCC AGG TTG GG 53~C/30"

52PS2X7 42 GGG CAG GCT CTT CTT CAG GG 60~C/30"

SPS2X8 44 TTA GCA CCG CCT GAG ACG T 48~C/30"

SPS2X9 47 CTC TGA CCA GCT GTT GTT TC 57~C/30"

SPS2X10 49 TTC CAT TCT GTG CAC GCC TC 56~C/30"

SPS2X11 51 ACA GCT CCT GTC CAC ACC A 53~C/30"

CA 022228l3 l997-ll-28 W O 97/38133 PCT~US97/04683 Primer SE Sc4- e (5'-3') Si~ ~ ' Name Q (bp) Temp.
ID (~C) NO
3PS2xl 1 52 ACT AGA GTG TAA AAC TAT ACA A 292 Aliquots of tissue cDNAs were amplified in a Perkin Elmer DNA Thermal Cycler 480(Perkin Elrner, Norwalk, CT). Each PCR reaction c~ .Pd I ml of final cDNA product, 25 pmol of each primer (forward and reverse), 12.5 nmol of dNTP (Pl"~ Columbus, OH), 1.25 U of S Taq polymerase (Promega, Madison, WI) for a total reaction volume of 50 ml overlaid with 60 ml mineral oil (Fisher, Pi~ , PA). Primers used were designed to span at least two putative intron/exon bul~ s of the PS-2 gene. For example, LP313F (forward, exon 2) and LP676R
(reverse, exon 4) were used to span exon 2/3 and exon 3/4 intron exon buundalies. For a given reaction, sarnples were d~ tll~ at 94~C for S minutes. Samples then underwent 35 cycles of 0.5 10 minutes at 94~C, 0.5 minutes at the relevant ~nn~lin~ ll~;lalult; for the given primer pair, and 0.75 minutes at 72~C. This was followed by a final t;AL~Ilsiu" for 10 minutes at 72~C. Products were visualiz_d on a 2% agarose gel (Promega) using ethidium brûmide staining. Product bands were excised and purified using the Wizard PCR Preps DNA Purification System (Prornega). Five rnicroliters of the final 50 ml purified product was used for 5e~ -g Example 4: Sequencing for deternnnation of alternate splice p.~
PCR product was treated with EYnnn~lP~ce 1 and Shrimp Alkaline Ph-~rh~t~cP (PCR
lg kit-USB, Cleveland, OH). Five ""~ of PCR product was ;... ~ ~1 with I U ofEx.. ~ ~ 1 for 15 minutes at 37~C. The sample was then held at 80~C for 15 n~nutes after 20 which 2 U of Shrinnp Alkaline Pl.. !~l)h;.~P. was added. As before, the sarnple was held at 37~C
for 15 minutes and then at 80~C for 15 minutes. This final 7 rnl product was used in the se~uencing protocol described in the Se~ r..~e PCR product S~lllr....-;..g Kit. The forward prirner used in the original PCR reaction was used for manual s~ g 25 Example 5: PAC isolation W O 97/38133 PCTrUS97/04683 P1 derived artificial cl..u.l,oscll~s (PACs) were isolated by s~lcc~ lg a gridded library (Genome Systems, Inc.) with PCR products ~--yl~led with primers R05822F and R05822R.
Three PACs cn~ g the PS-2 gene were digested with Notl and sized using pulse field gel d~:~lu~llulc~ia (PFGE). PAC DNA was run at 200 V for 21 hours at 14~C with switch times S varying from 5-20 seconds. Sizes ranged from 90 kb to 110 kb. Primers used in the detection of the 5' untranslated s~ .ce was 5'-GCTT(~'l'~'l'(l'l'CAG(i'l'l l'(~ l-l'C-3'(SEQ ID NO: 3) and 5'-CG(~'l'~i'l'l'l'GG( :'l'(~'l'l'l'l'ATCA-3' (SEQ ID NO: 4). Int ronic prirners were used to detect presence of exons 4 and 7. Prirners R05822F and R05822R were used to detect exon 12. PCR
reaction c.~".l;~in~-~ were as follows: 5 rninutes d~" alu dliull followed by 35 cycles of 94~C for 0.5 rninutcs, the respective ~nnP~l g lal~.dlulc for 0.5 minutes, and 72~C for 0.5 rninutes. A 10 rninute L.AlCllSiUII at 72~C c- ~ d~ each reaction. ~nnP~l;ng lr- ~ q~ e for each prirner pair are provided in Table 2.

Exsnnple 6: T ' ~ i of ~ t,. boundalies Exon/intron buull~ics were obtained by PCR/ligation lri l.";~lllP~ Purified PAC DNA
was digested with a variety of blunt-cutting enzyrnes and ligated to a .c~ifir:~lly designed linker.
S~uPn~Ps were then specifically amplified by PCR using a linker-derived prirner and a PS-2 derived prirner for boundary sPqlPn~ine S~-Pn~';n~ of products was pu roll~d directly in low-rnelting point agarose by using a rnodified dideox~""~ lr. sP~ Pnrillg rnethod with a 32p end labelled prirner and Taq DNA polyrnerase t~ll~ldtUl~ cycled reactions.

Example 7: Del~c~on of small sequence varia~o~
The Ph~ ALF rlaE;~ rnanager was used to detect srnall se~-lr~ e changes not dt~ hlP by standard agarose gellethidium brornide vi.~ li7~inn Relevant cDNA sefl~lr..l~P.
25 were a-l4~1illed by PCR using a s'-nuol~c~l,l tagged primer.

CA 022228l3 l997-ll-28 WO g7/38133 PCT/US97/04683 SEQUENCE LISTING

~1) GENERAL INFORMATION:

(i) APPLICANTS:University of South Florida, Washington University and The Institute of Genomic Research (ii) TITLE OF INVENTION: Variant Presenilin-2 Genes (iii) NUMBER OF SEQUENCES: 52 (iv) CORRESPONDENCE ADDRESS:
~A) ADDRESSEE: SmithKline Beecham Corporation (B) STREET: 709 Swedeland Road (C) CITY: King of Prussia (D) STATE: PA
(E) COUNTRY USA
(F) ZIP: 19406 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE:
(B) COMPUTER:
(C) OPERATING SYSTEM:
~D) SOFTWARE:
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: not yet assigned (B) FILING DATE: Herewith (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:60/014,860 (B) FILING DATE: March 4, 1996 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: William T. Han (B) REGISTRATION NUMBER: 34,344 (C) REFERENCE/DOCKET NUMBER: ATG50001 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (610) 270-5219 CA 022228l3 l997-ll-28 W O97/38133 PCT~US97/04683 (B) TELEFAX: (610) 270-5090 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 (B~ TYPE: Nucleic Acid (C) STRANDEDNESS: Single ! ~D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear ~iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
G~ CAGGTTTCTT C 21 ~2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acld ~C) STRANDEDNESS: Single W O 97/38133 PCT~US97/04683 (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CGGTGTTTGG c~ ATC A 2l (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

(2) INFORMATION FOR SEQ ID NO: 6:
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(A) LENGTH: 25 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7:
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~A) LENGTH: 24 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

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W O97/38133 PCTnUS97/04683 (A) LENGTH: 24 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO: l0:
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(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l0:
TCAGCGTCAT C~lG~llATG ACC 23 (2) INFORMATION FOR SEQ ID NO: ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: ll:

CA 022228l3 l997-ll-28 WO 97/38133 PCTrUS97/04683 (2) INFORMATION FOR SEQ ID NO: 12:
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(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

(2) INFORMATION FOR SEQ ID NO: 13:
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(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

(2) INFORMATION FOR SEQ ID NO: 14:
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(A) LENGTH: 23 ~B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

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(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
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(2) INFORMATION FOR SEQ ID NO: 16:
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(A) LENG?H: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
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(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

(2) INFORMATION FOR SEQ ID NO: l8:
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(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

(2) INFORMATION FOR SEQ ID NO: l9:

W O 97/38133 PCTrUS97/04683 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv~ ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l9:

(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

(2) INFORMATION FOR SEQ ID NO: 2l:
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(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

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(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO

W O97/38133 rCTrUS97/04683 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

~2) INFORMATION FOR SEQ ID NO: 23:
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~A) LENGTH: 2l ~B) TYPE: Nucleic Acid ~C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
G~ ~l~l CAGGTTCCTT C 2l (2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 2l ~B) TYPE: Nucleic Acid ~C) sTR~Nn~nN~s: Single ~D) TOPOLOGY: Linear ~iv) ANTI-SENSE: NO
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
CGG~ llGG ~ ATC A 2l (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single ~D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
AGCCTCGAGG AGCAGTCAG l9 ~2) INFORMATION FOR SEQ ID NO: 26:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 18 (B) TYPE: Nucleic Acid ~C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
GCAGACGGAG AGAAGGGT l8 (2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
GAAAGCCACG GCCAGGAAG l9 (2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

(2) INFORMATION FOR SEQ ID NO: 30:

-~6-W O97/38133 PCT~US97/04683 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 20 (B) TYPE Nucleic Acld (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO 30:

(2) INFORMATION FOR SEQ ID NO: 3l (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 2277 (B) TYPE Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION SEQ ID NO 31 TCCAGCAGTG AGGAGACAGC CAGAAGCAAG CTATTGGAGC TGAAGGAACC l00 TGAGA~.AA GCTAGTCCCC CCTCTGAATT TTACTGATGA AGAAACTGAG 150 ACATTCATGG CCTCTGACAG CGAGGAAGAA ~l~l~lGATG AGCGGACGTC 450 CCTAATGTCG GCCGAGAGCC CCACGCCGCG ~lC~lGCCAG GAGGGCAGGC 500 AGCACGTGAT CATGCTGTTT GTGCCTGTCA ~ GCAT GATCGTGGTG 700 CATCTACACG ACATTCACTG AGr~rArArc CTCGGTGGGC CAGCGCCTCC 800 CA 022228l3 l997-ll-28 W O97/38133 PCT~US97/04683 GAGGAA&AGG AGGAAAGGGG CGTGAAGCTT GGCCTCGGGG ACTTCATCTT 1500 ATACCACGCT GGCCTGCTTC GTGGCCATCC TCATTGGCTT ~~ ~l~ACC 1600 AGTTTTACAC TCTAGTGCCA TATATTTTTA AGA~"l"l"l"l'~"l' TTCCTTAAAA 1850 CAGATTAGGG CG~JGr-~AAG AGCATCCGGC ATGAGGGCTG AGATGCGCAA 2050 GAAAAGCCAG TTCCCTACGA GGAGTGTTCC CAATGCTTTG TCCATGATGT 2~50 CCTTGTTATT TTATTGCCTT TAGAAACTGA GTC~ TGTTACGGCA 2200 (2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
GACTTGTGTC CAAGTCTC l~
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
CTGTAAGGTA CAGTAGCCG l9 (2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
AAAAATCCGT GCATTACAT l9 (2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

(2) INFORMATION FOR SEQ ID NO: 36:

CA 022228l3 l997-ll-28 WO 97138133 I'CTIUS97104683 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 ~B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO

CA 022228l3 l997-ll-28 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3g:

(2) INFORMATION FOR SEQ ID NO: 40:
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(A) LENGTH: 20 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nuclelc Acid (C) STRANDEDNESS: Single (D) TOPO~OGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

(2) INFORMATION FOR SEQ ID NO: 43:
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(A) LENGTH: 19 (B) TYPE: Nucleic Acid W O 97/38133 PCT~US97/04683 ~C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
GAAAGCCACG GCCAGGAAG l9 ~2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
TTAGCACCGC CTGAGACGT l9 (2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l8 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: ~inear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
AGCTGGTCAG AGTGTTAC l8 (2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

(2) INFORMATION FOR SEQ ID NO: 47:

(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single - (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
CTCTGACCAG ~ "l'C 20 (2) INFORMATION FOR SEQ ID NO: 48:
UU~N~ CHARACTERISTICS:
(A) LENGTH: 18 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO

CA 022228l3 l997-ll-28 W O 97/38133 PCTrUS97/04683 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:

(2) INFORMATION FOR SEQ ID NO: 51:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 ~B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:

Claims (5)

What is Claimed is:

1. A variant presenilin-2 gene.

2. The variant presenilin-2 gene of claim 1 comprising splicing of exon and exon 4 out of a presenilin-2 gene.

3. The variant presenilin-2 gene of claim 1 comprising splicing exon 8 out of a presenilin-2 gene.

4. A method of diagnosing Alzheimer's disease in a patient comprising detecting a mutant presenilin-2 gene in a DNA sample from a patient.

5. A method of identifying mutants in splice donor or acceptor sites of a presenilin-2 gene comprising sequencing splice donor or acceptor sites of the presenilin-2 with intronic primers for the presenilin-2 gene and analyzing the sequences to identify any mutants.