WO1997038133A1

WO1997038133A1 - Variant presenilin-2 genes

Info

Publication number: WO1997038133A1
Application number: PCT/US1997/004683
Authority: WO
Inventors: John Hardy; Alison M. Goate; Rebecca A. Fuldner
Original assignee: University Of South Florida; Washington University; The Institute Of Genomic Research
Priority date: 1996-04-04
Filing date: 1997-03-20
Publication date: 1997-10-16
Also published as: AU2541497A; CA2222813A1; JP2002515740A; EP0833947A4; EP0833947A1

Abstract

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

Description

VARIANT PRESENI IN-2 GENES

INTRODUCTION

This invention was made in the course of research sponsored by the National Institutes of Health. The U.S. Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and dementia. The major pathological feature of AD is the presence of numerous neurofibrillary tangles and senile plaques composed prirήarily 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 precursor protein (APP) (Goate et al. Nature 1991, 349, 704-6; Masters et al. PNAS 1985, 82, 4245-4249; Kang et al. Nature 1987, 325, 733-736). Studies have shown that increases in the generation of the 42 arriino acid peptide lead to AD in APP encoded disease. AD is typically a disease of the elderly, afflicting 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 pedigrees have been described wherein the disease is inherited as an autosomal dominant with age dependent penetrance. Most commonly, 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 implicated in both early and late onset AD.

Several large families have been identified in which presenile AD segregates as fully penetrant autosomal dominant trait. Linkage analysis studies in presenile AD families have identified four genes, on chromosomes 1, 14, 19, and 21, that when mutated, cause presenile AD. The first presenile AD gene identified maps to chromosome 21 and codes for the beta A4-amyloid protein precursor (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 approximately 5% of the families. These disease causing mutations have been modeled in transfected or primary cultured cells and have been shown to lead to altered proteolytic processing of APP in a way that favors production of its a yloidogenic and potentially neurotoxic Ab fragments. Transgenic overexpression of one mutant APP has resulted in the first mouse model of AD, in which age-linked cerebral deposition of the Ab fragment is accompanied by neuronal, astrocytic, and microglial pathology (Games et al. Nature 1995, 373, 523-527).

Genetic variability in the apolipoprotein E locus on chromosome 19 have also been shown to be important in the etiology of AD (Strittmater et al. PNAS 1993, 90, 1977-1981; Saunders et al. Neurology 1993, 43, 1467-1472). Inheritance of the 14 (112 Cys-Arg) allele has been reported to lower the age of onset in a dose dependent manner. Conversely, inheritance of the apolipoprotein E 12 allele appears to confer a decreased risk of developing AD (Corder et al. Science 1993, 261, 921-923; Corder et al. Nature Genet. 1994, 7, 180-184).

Fτesenilin-1 Q°S-1), located on chromosome 14 harbors an estimated 70% of the disease causing mutations, making it the major gene for familial presenile AD (estimated 10% of all AD cases) (Sherrington et al. Nature 1995, 375, 754-760; van Broeckhoven et al. Nature Genet. 1992. 2, 335-339; St. George-Hyslop et al. Nature Genet. 1992, 2, 330-334; Schellenberg et al. Science 1992, 258, 668-670). This gene, designated S182 or PS-1, is made up of 10 coding exons (numbered 3 to 12). PS-1 is predicted to be an integral membrane protein with at least 7 transmembrane domains. At the time of its isolation, five different missense mutations were identified in 8 chromosome 14 linked families (Sherrington et al. Nature 1995, 375, 754-760). A total of 22 different mutations in 40 families of various ethnic origins has been identified (van Broeckhoven et al. Nature Genet. 1995, 11, 230-233). Two different mutations were identified at each of the codons 139, 146, 163, and 280. However, most mutations are scattered over the protein with mutations found in 5 of the 7 putative transmembrane domains and in 3 of the 6 hydrophilic loops. Mutations have been found in 6 of the 10 coding exons, with exon 5 and 8 accounting for 65% of the mutations. The same mutation occurs in several AD families of different ethnic origin, suggesting there are independent mutation events in the PS- 1 gene.

A third gene for presenile AD Q?S-2) maps to chromosome 1 in the Volga-German AD families, a group of farnUies in which AD is the result of a founder effect fl-evy-Lahad et al. Science 1995, 269, 970-973). This gene (STM-2 or E5-1) was identified as a direct result of its high homology to PS-1. The same missense mutation was found in 7 Volga-German AD families and more recently, a second missense mutation has been found in an Italian AD family O^evy- Lahad et al. Science 1995, 269,970-973; Rogaev et al. Nature 1995, 376, 775-778; Barinaga Science 1995, 269,917-918). Both PS-1 and PS-2 have approximately 450 amino acids and share an overall homology of 67% with the highest similarity observed in the TM domains fl-evy-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 transmembrane domain structure of the presenilins is compatible with a function as a receptor molecule, an ion channel or a membrane structural protein. It has been determined that mutations in PS-1 and PS-2 cause an increase in the generation of Ab ₂ thus indicating biological interaction with amyloid.

The exonic structure and the existence 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 motif at codons 26-29 (Alzheimer's Disease Collaborative Group. Nature

Genet. 1995, 1 1, 219-222). Alternate splicing has also been found in both exon 8 in PS-1 and the homologous exon 8 in PS-2.

A number of other variant, alternatively spliced PS-2 genes have now been identified. These genes are useful in the diagnosis of early-onset AD and in evaluating agents which may be useful for the treatment of this disease. SUMMARY OF THE INVENTION

An object of the present invention is to provide novel, variant PS-2 sequences. Another object of the present invention is to provide a method of diagnosing Alzheimer's disease using these novel PS-2 sequences or the exonic or intronic sequences of the PS-2 gene. Yet another object of this invention is to provide a model system for Alzheimer's disease comprising variant PS-2 genes.

BRIEF DESCRIPTION OF THE FIGURES

- Figure 1 provides a schematic comparison of the organization of the PS-1 and PS-2 gene. Labeled arrows indicate sites of known mutations. Unlabeled arrows indicate intron/exon boundaries. 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 sequence of the PS-2 gene (SEQ ID NO: 31 ).

DETAILED DESCRIPTION OF THE INVENTION

Mutations in the APP gene on chromosome 21, the ApoE gene on chromosome 19, and the SI 82 or PS-1 gene on chromosome 14 account for the majority of identified cases of early onset Alzheimer's disease (Strittmater et al. PNAS 1993, 90, 1977-1981; Sherrington et al. Nature 1995, 375, 754-760). However, in the Volga-German kindreds, and in several other families in which AD appears to be inherited as an autosomal dominant trait, these loci have been excluded.

The Volga-German families are a culturally distinct subpopulation in Russia, whose members did not marry into the Russian population. The relative onset of AD in this group is exceptionally early, ranging from 50 to 70 years of age. However, clinically and pathologically, AD in these families is indistinguishable from typical AD. The autosomal dominant locus, responsible for AD in the Volga-German kindreds has been localized to chromosome lq31-42 (Levy-Lahad et al. Science 1995, 269, 970-973). Candidate genes which map to this locus have also been identified. Levy-Lahad et al. isolated STM2 whose predicted amino acid sequence is homologous to that of S 182 (PS-1). A point mutation in STM2, resulting in the substitution of an isoleucine for an asparagine (Nuil), was identified in affected individuals. This N|₄₁I mutation occurs at an amino acid residue that is conserved in human SI 82 and at the mouse SI 82 homolog. Rogaev et al. reported the cloning of E5-1 on chromosome 1 which is also homologous to SI 82 (Nature 1995, 376, 775-778). Analysis of the nucleotide sequence of the open reading frame of E5-1 (STM2) led to the discovery of two missense substitutions at conserved a ino acid residues in affected members of the Volga-German (NwT) and Italian (M₂₃9V) pedigrees.

In order to better elucidate the structure of the PS-2 gene and to determine possible sites of alternative splicing, the gene was cloned and sequenced and PCR was used to deteirnine alternate splice products (variants) and exon/intron boundaries. Elucidation of intron/exon boundary sequences revealed that PS-2 is encoded by 10 coding exons. The PS-2 gene sequence was determined by both sequencing the EST sequence T03796 and isolating PS-2 cDNAs using the GeneTrapper kit (Gibco BRL, Gaithersburg, MD).

The intron/exon structure of the PS-2 gene is shown in Table 1. Positions of introns that interrupt the PS-2 cDNA are shown. Exonic sequence is presented in upper case and intronic sequence in lower case letter. Exons are numbered from the 5' end of the cDNA sequence. TABLE 1

EXON1

(to - 195) ..CTTTTCCCAAGGTCGCCC AGgtacgatatagcagagccag

EXON2 aaactcttccttgttttaagCGAGGACGTGGGACTTCTCA

EXON2 (- 194 to -21 ) GCGGCCCC AAGTGTTCGTGGgtccgggattcagactctct

EXON3 tccttgtgctccttmccaGTGCTTCCAGAGGCAGGGCT EXON3 (-20 to 140) GAGAGAACACTGCCCAGTGGgtaggtcccaccagcagctg

EXON4 cacgatgtggtttcccacagAGAAGCCAGGAGAACGAGGA EXON4 (141 to 355) ACAGAGAAGAATGGACAGCTgtgagttggggggctggggg

EXON5 acagagaagaatggacagctCATCTACACGACATTCACTG EXON5 (356 to 497) ACAAGTACCGCTGCTACAAGgtgaggccctggccctgccc

EXON6 cgtgcaatttctgttgtctaGTTCATCCATGGCTGGTTGA EXON6

(498 to 565) TCACCTATATCTACCTTGGGtaagtgacagataagcagca

EXON7 aggctccacctggtcctgcaGAAAGTGCTCAAGACCTACA EXON7

(566 to 786) GGGCGCCATCTCTGTGTATGgtaggtgggcagcaagctgg

EXON8 aggatgtctctgtcttcctaGATCTCGTGGCTGTGCTGTG EXON8 (787 to 885) CCCTGCCCTGATATACTCATgtgagtgagccccccgtgcc

EXON9 aacattttctcctcccccagCTGCCATGGTGTGGACGGTT

EXON9

(886 to 969) CCCCTACGACCCGGAGATGGgtgagtatcttggggagcta

EXON 10 cttctcttcctggacacccaAAGAAGACTCCTATGAC AGT

EXON 10

(970 to 1071 ) GCTGGAGGAAG AGGAGGAAAgtaaggtgcccatgttcaca

EXON11 tactgtctctcctcacacagGTCAAGGGGGCGTGAAGCTT EXON 1 1

( 1072 to 1 190) CTTCGTGGCC ATCCTCATTGtgagtggctggggatgcgtc

EXON 12

( 1 191 to 3'end) ctcccctccatgtcctgcagGGCTTGTGTCTG ACCCTCCT cDNA sequencing revealed multiple positions at which mutations in sequence were observed; four of which occurred at exon boundaries. A summary of this splicing data is provided in Table 2.

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 . EB913 324

6 bp deletion - IB913 357-359

Splicing events in Table 2 are listed with respective cDNAs in which they were identified as well as with corresponding amino acid residues. Splicing events detected by RT-PCR are denoted by a "+" in the PCR column. All events are relative to the full length cDNA clone, referred to as USF# 15. Simplified structures of the four cDN A clones are shown in Figure 1.

Three of the variations identified 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. Amplification by PCR over the relevant boundaries 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 sequence published by Levy-Lahad et al. Science 1995, 269,973-977 ^'. These changes include a deletion of a glutamate residue at codon 324 and insertion of six base pairs which change the encoded arnino acid sequence from ERGV to ESQGG at codons 357-359.

In contrast, little evidence has been found for naturally occurring alternate splicing in the PS-1 gene. The only alternate splicing previously reported for PS-1 has been in exon 8 and at the 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 were without equivalence in PS-1. Some of these events lead to significant alterations in the structure of the protein.

The most striking alternate transcripts are those which lack exon 3 and 4. Variants lacking exons 3 and 4 lose the normal start methionine and if translated would be predicted to begin at the methionine at codon 145. This start site occurs after the proposed transmembrane domain 1 in the middle of transmembrane domain 2. This protein is different than the Volga German AD mutant N₎₄ιl.

The identification of these variants indicates that certain mutations in the PS-2 gene are associated with the "Volga German" type of AD. Therefore, these variants may be useful in the diagnosis of AD or in the development of models of AD.

The identification of the intronic sequences is also useful in the early detection of variant forms of the PS-2 gene. The genomic analysis of the PS-2 gene (see Figure 1) has led to the development of a method for identification of intronic polymorphisms which are predictive of disease. Elucidation, detection, and diagnosis of mutations in both intronic sequences associated with splice variation and in the open reading frames proximal to these intron-exon boundaries of the PS-2 gene can be performed through use of intronic sequences. Identification and analysis of mutants or variants arising from mutations in splice donor or acceptor sites are enabled by knowledge of these intronic sequences. Furthermore, a complete analysis of the intron-exon boundaries makes possible sequencing primers that would allow accurate sequence determination of the first or last 10 to 20 nucleotides 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 dementia for which treatment is available and which give rise to progressive intellectual deterioration closely resembling the dementia associated with Alzheimer's disease. A diagnostic test for AD would therefore provide a useful tool in the diagnosis and treatment of these other conditions, by way of being able to exclude early onset Alzheimer's disease. It will also be of value when a suitable therapy for AD is available. There are several methodologies available from recombinant DNA technology which may be used for detecting and identifying genetic mutations responsible for Alzheimer's disease. These include, but are not limited to, direct probing, ligase chain reaction (LCR) and polymerase chain reaction (PCR) methodology.

Detection of variants or mutants using direct probing involves the use of oligonucleotide probes which may be prepared synthetically or by nick translation. In a preferred embodiment, the probes are complementary to at least a portion of the variant PS-2 genes identified herein. The DNA probes may be suitably labelled using, for example, a radiolabel, enzyme label, fluorescent label, or biotin-avidin label, for subsequent visualization in for example a Southern blot hybridization procedure. The labelled probe is reacted with a sample of DNA from a patients suspected of having AD bound to nitrocellulose or Nylon 66 substrate. The areas that carry DNA sequences complementary to the labeled DNA probe become labelled themselves as a consequence of the reannealing reaction. The areas of the filter that exhibit such labeling may then be visualized, for example, by autoradiography.

Alternative probe techniques, such as ligase chain reaction (LCR) involve the use of a mismatch probe, i.e., probes which have full complementarity with the target except at the point of the mutation or variation. The target sequence is then allowed to hybridize both with the oligonucleotides having full complementarity, i.e., oUgonucleotides complementary to the PS-2 variants of the present invention, and oligonucleotides containing a mismatch under conditions which will distinguish between the two. By manipulating the reaction conditions, it is possible to obtain hybridization only where there is full complementarity. If a mismatch is present, then there is significantly reduced hybridization.

The polymerase chain reaction (PCR) is a technique that amplifies specific DNA sequences. Repeated cycles of denaturation, primer annealing and extension carried out with a heat stable enzyme Taq polymerase leads to exponential increases in the concentration of desired DNA sequences.

Given the knowledge of nucleotide sequences encoding the PS-2 gene, it is possible to prepare synthetic oligonucleotides complementary to the sequences which flank the DNA of interest. Each oligonucleotide is complementary to one of the two strands. The DNA is then denatured at high temperatures (e.g., 95°C) and then reannealed in the presence of a large molar excess of oUgonucleotides. The oUgonucleotides, oriented with their 3' ends pointing towards each other, hybridize to opposite strands of the target sequence and prime enzymatic extension along the nucleic acid template in the presence of the four deoxyribonucleotide triphosphates. The end product is then denatured again for another cycle. After this three-step cycle has been repeated several times, amplification of a DNA segment by more than one milhon fold can be achieved. The resulting DNA may then be directly sequenced in order to locate any genetic alterations. Alternatively, the identified PS-2 variants of the present invention make it possible to prepare oUgonucleotides that will only bind to altered DNA, so that PCR will only result in the multipUcation of the DNA if the mutation is present. FoUowing PCR, aUele-specific oUgonucleotide hybridization may be used to detect the AD point mutation.

Alternatively, an adaptation of PCR called amplification of specific alleles (PAS A) can be employed; this method uses differential amplification for rapid and reliable distinction between aUeles 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. /. 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. PAS A involves amplification with two oUgonucleotide primers such that one is allele specific. The desired aUele is efficiently amplified, while the other aUele(s) is poorly ampUfied because it mismatches with a base at or near the 3' end of the aUele specific primer. Thus, PASA or the related method PAMSA can be used to specifically amplify one or more mutant PS-2 aUeles. Where such amplification is performed on genetic material obtained from a patient, it can serve as a method of detecting the presence of one or more mutant PS-2 aUeles in a patient. PCR-induced mutation restriction analysis, often referred to as EMRA, can also be used in the detection of mutants.

Also important is the development of experimental models of Alzheimer's disease. Such models can be used to screen for agents that alter the degenerative course of AD. Having identified specific mutations in the PS-2 gene as a cause of early onset famiUal Alzheimer's disease, it is possible using genetic manipulation, to develop transgenic model systems and/or whole ceU systems containing a mutated PS-2 gene or a portion thereof. The model systems can be used for screening drugs and evaluating the efficacy of drugs in treating Alzheimer's disease. In addition, these model systems provide a tool for defining the underlying biochemistry of PS-2 and its relationship to AD thereby providing a basis for rational drug design.

One type of ceU system which can be used in the present invention can be naturaUy derived. For this, blood samples from an affected individual are obtained and permanently transformed into a lymphoblastoid ceU line using, for example, Epstein-Barr virus. Once established, such cell lines can be grown continuously in suspension cultures and can be used in a variety of in vitro experiments to study PS-2 expression and processing. Another ceU line used in these studies comprises skin fibroblasts derived from patients. Since the FAD mutation is dominant, an alternative method for constructing a ceU line is to genetically engineer a PS-2 mutated gene, or portion thereof, as described herein, into an estabUshed ceU line of choice. Such methods are well known in the art as exemplified by Sisodia Science 1990, 248, 492 and Oltersdork et al. J. Biol. Chem, 1990, 265, 4492, wherein an amyloid precursor peptide gene was transfected into mammaUan cells.

Baculovirus expression systems have also been found to be useful for high level expression of heterologous genes in eukaryotic ceUs. The mutated gene can also be excised for use in the creation of transgenic animals containing 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 limited to, lCharon35, cosmid, or yeast artificial chromosome. The variant PS-2 gene can then be transferred to a host nonhuman animal such as a mouse. As a result of the transfer, the resultant transgenic nonhuman animal will preferably express one or more of the variant PS-2 polypeptides.

Alternatively, minigenes encoding variant PS-2 polypeptides can be designed. Such minigenes may contain a cDNA sequence encoding a variant PS-2 polypeptide, preferably fuU- length, a combination of PS-2 exons, or a combination thereof, linked to a downstream polyadenylation signal sequence and an upstream promoter (and preferably enhancer). Such a minigene construct will, when introduced into an appropriate transgenic host, such as a mouse or rat, express a variant PS-2 polypeptide.

One approach to creating transgenic animals is to target a mutation to the desired gene by homologous recombination in an embryonic stem (ES) ceU in vitro foUowed by microinjection of the modified ES ceU line into a host blastocyst and subsequent incubation in a foster mother. Frohman and Martin Cell 1989, 56, 145. Alternatively, the technique of microinjection of the mutated gene, or portion thereof, into a one-ceU embryo followed by incubation in a foster mother can be used. Additional methods for producing transgenic animals are weU known in the art.

Transgenic animals are used in the assessment of new therapeutic compositions and in carcinogenicity testing, as exemplified by U.S. Patent 5,223,610. These animals are also used in the development of predictive animal models for human disease states, as exemplified in U.S. Patent 5,221,778. Transgenic animals have now been developed for assessing Alzheimer's disease (U.S. Patent 7,769,626), multi-drug resistance to anticancer agents (U.S. Patent 7,260,827), and carcinogenic substances (U.S. Patent 4,736,866). Therefore, the PS-2 genes of the present invention which are beUeved to cause early onset Alzheimer's disease in Chromosome 14-Unked pedigrees provide a useful means for developing transgenic animals to assess this disease. Site directed mutagenesis and or gene conversion can also be used to a mutate a non human PS-2 gene aUele, either endogenously or via transfection, such that the mutated gene encodes a polypeptide with an altered amino acid as described in the present invention.

In addition, antibodies to the PS-2 gene and variants thereof can be raised for use in the examination of the function of the truncated transcripts of the PS-2 gene. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as weU as Fab fragments, or the product of an Fab expression Ubrary. Various procedures known in the art may be used for the production of such antibodies and fragments. Antibodies generated against the PS-2 genes of the present invention can be obtained by direct injection into an animal or by administering the gene to an animal, preferably a nonhuman. 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 preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, Nature 1975, 256, 495-497), the trioma technique, the human B-ceU hybridoma technique (Kozbor et al., Immunology Today 1983, 4, 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp. 77-96). Techniques described for the production of single chain antibodies (U.S. Patent

4,946,778) can be adapted to produce single chain antibodies to the PS-2 genes of this invention. Also, transgenic mice may be used to express humanized antibodies to the PS-2 genes of this invention.

The foUowing nonlimiting examples are provided to further illustrate the present invention.

EXAMPLES

Example 1 : Cloning the PS-2 gene

PS-2 cDNA's were isolated using the Gene Trapper kit (Gibco BRL) according to the manufacturer's directions. A human superscript brain library (Gibco BRL, Gaithersburg, MD) in pCMV.SPORT was probed with the primer 5 -CATTCACTGAGGACACACCC-3' (SEQ ID NO: 1) (derived from the EST sequence T03796). However, use of this sequence consistently (three times), resulted in the isolation of different clones which contained the 5' and 3' untranslated gene sequences but were missing the region of the gene around the putative start codon The Ubrary was rescreened using the pπmer 5'-CAAATACGGAGCGAAGACAG-3' (SEQ ID NO 2), deπved 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, heart, hver, lung, placenta, and skeletal muscle was obtained from Clontech 0°alo Alto, CA) First strand cDNA was synthesized foUowing the Superscript Preamplification System for First Strand cDNA Synthesis (Gibco BRL) Two mg of total RNA was combined with 1 mg of oUgonucleotide (dT)i₂ is primer, 100 ng of random hexamer pπmer and DEPC treated water in a 05 ml tube Samples were incubated at 70°C for 10 minutes and placed on ice Kit components were added in accordance with the manufacturer's protocol, and samples were incubated at 37°C for 2 minutes Superscπpt II Reverse Transcπptase (RT, 200 U) was added and samples were incubated at 37°C for 1 hour Samples were then incubated at 70°C for 15 minutes, chilled on ice, and 2 U of RNAse H was added AU samples were stored at -20°C For subsequent PCR, 1 ml of final product was used for each reaction

Example 3: PCR to deteπnine alternate splice products

Pπmers were designed throughout the cDNA sequence to aUow the assessment of the alternate spUcing in a vanety of Ussues Exonic pπmers, along with the conditions for their use are provided in Table 3 Intronic pπmers, along with the conditions for their use are provided in Table

4

TABLE 3

Primer Name SEQ Sequence (5'-3') Location Annealing

ID Temp.

NO (°C)

LP313F 5 AGCCTGCTGAGAAGAAGAAACCA EXON 3 50

LP501F 6 AGGCAGGGCCCAGAGGATGGAGA EXON 4 50 GA Primer Name SEQ Sequence (5'-3') Location Annealing

ED Temp.

NO (°C)

LP676R 7 AGAGTGACAGGCACAAACAGCAT EXON 5 50 G

LP711F 8 ACCATCAAGTCTGTGCGCTTCTAC EXON 5 50

LP861R 9 AGATGGTCATAACCACGATGACG EXON 6 50

LP835F 10 TCAGCGTCATCGTGGTTATGACC EXON 6 50

LP960R 11 AGGTAGATATAGGTGAAGAGGAA EXON 7 50

LP921F 12 TCTTCACTGATGCTGCTGTTCC EXON 7 59

LP1012R 13 AAGAGGGTGGGGTAGTCCATGGC EXON 8 50

LP1081F 14 AGCAGGCCTACCTCATCATGATC EXON 8 50

LP1254R 15 TCATTTCTCTCCTGGGCAGTTTC EXON 9 50

LP1231F 16 TAGAAACTGCCCAGGAGAGAAAT EXON 9 50

LP1290F 17 TGGTGTGGACGGTTGGCATGGCG EXON 10 48

LP1334R 18 GGTCCAGCTTCGCCATGCCAACC EXON 9 59

LP1460R 19 TCTTCCTCCAGCTCCTCCCCTGG EXON 1 1 50

LP1380F 20 ATGACAGTTTTGGGGAGCCTTCA EXQN 1 1 50

LP1535R 21 TGGCAGCCGCCTTGCCCACCAGC EXON 12 48

LP1820R 22 ACTAGAGTGTAAAACTATACAA EXON 12 50

5;UTR 23 GCTTCTGTCTCAGGTTCCTTC 5' UTR 54 FORWARD

5'UTR 24 CGGTG'l Ti'UGC TG IT 1TA TCA 5' UTR 54 REVERSE Primer Name SEQ Sequence (5'-3') Location Annealing

ID Temp.

NO (°C)

EXON 4 25 AGCCTCGAGGAGCAGTCAG 5' EXON 4 49 INRTONIC

EXON 4 26 GCAGACGGAGAGAAGGGT 3'EXON 4 49 INTRONIC

EXON 7 27 GGGCAGGCTCTTCTTCAGGG 5'EXON 7 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

TABLE 4

Primer SE Sequence (5'-3') Size Annealing Name Q (bp) Temp.

ID (°C)

NO

53PS2X2 32 GAC TTG TGT CCA AGT CTC

33PS2X2 33 CTG TAA GGT ACA GTA GCC G 325

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

3PS2X3 35 GCT GGT GTG GAG CTG CAG GTA CAG 405 TG

5PS2X4 36 AGC CTC GAG GAG CAG TCA G 53°C/30"

3PS2X4 37 GCA GAC GGA GAG AAG GGT 240

5PS2X5 38 GGT ATC AGT CTC AGG ATC ATG GG 60°C/30"

3PS2X5 39 TGG GGA AGA CTG GAG CTC GAT G 263

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

3PS2X6 41 GTG CAG CAC TGG GGA CGA TTT 360

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

3PS2X7 43 GAA AGC CAC GGC CAG GAA G 255

5PS2X8 44 TTA GCA CCG CCT GAG ACG T 48°C/30"

3PS2X8 45 AGC TGG TCA GAG TGT TAC 510

32PS2X8 46 CCC CTC CTG AAC TCA TGC CT

5PS2X9 47 CTC TGA CCA GCT GTT GTT TC 57°C/30"

3PS2X9 48 AGC CTC CAC CCT CTG TCT 249

5PS2X10 49 TTC CAT TCT GTG CAC GCC TC 56°C/30"

3PS2X10 50 ACC TGC CCC CAC CAC AAT G 244

5PS2X1 1 51 ACA GCT CCT GTC CAC ACC A 53°C/30" Primer SE Sequence (5'-3') Size Annealing

Name Q (bp) Temp.

ID (°C)

NO

3PS2xl l 52 ACT AGA GTG TAA AAC TAT ACA A 292

AUquots of tissue cDNAs were ampUfied in a Perkin Elmer DNA Thermal Cycler 480 (Perkin Elmer, Norwalk, CT). Each PCR reaction contained 1 ml of final cDNA product, 25 pmol of each primer (forward and reverse), 12.5 nmol of dNTP (Pharmacia, Columbus, OH), 1.25 U of Taq polymerase (Promega, Madison, WI) for a total reaction volume of 50 ml overlaid with 60 ml mineral oil O^isher, Pittsburgh, PA). Primers used were designed to span at least two putative intron/exon boundaries 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 boundaries. For a given reaction, samples were denatured at 94°C for 5 minutes. Samples then underwent 35 cycles of 0.5 minutes at 94°C, 0.5 minutes at the relevant anneaUng temperature for the given primer pair, and 0.75 minutes at 72°C. This was foUowed by a final extension for 10 minutes at 72°C. Products were visualized on a 2% agarose gel 0?romega) using ethidium bromide staining. Product bands were excised and purified using the Wizard PCR Preps DNA Purification System 07τomega). Five microUters of the final 50 ml purified product was used for sequencing.

Example 4: Sequencing for determination of alternate splice products

PCR product was treated with Exonuclease 1 and Shrimp Alkaline Phosphatase 0?CR sequencing kit-USB, Cleveland, OH). Five microUters of PCR product was incubated with 1 U of Exonuclease 1 for 15 minutes at 37°C. The sample was then held at 80°C for 15 minutes after which 2 U of Shrirnp Alkaline Phosphatase was added. As before, the sample was held at 37°C for 15 minutes and then at 80°C for 15 minutes. This final 7 ml product was used in the sequencing protocol described in the Sequenase PCR product Sequencing Kit. The forward primer used in the original PCR reaction was used for manual sequencing.

Example 5: PAC isolation PI derived artificial chromosomes 0?ACs) were isolated by screening a gridded Ubrary (Genome Systems, Inc.) with PCR products ampUfied with primers R05822F and R05822R. Three PACs containing the PS-2 gene were digested with NotI and sized using pulse field gel electrophoresis (PFGE). PAC DNA was run at 200 V for 21 hours at 14°C with switch times varying from 5-20 seconds. Sizes ranged from 90 kb to 1 10 kb. Primers used in the detection of the 5' untranslated sequence was 5'-GCTTCTGTCTCAGGTTTCTTC-3'(SEQ ID NO: 3) and 5'- CC<JTGTTTGGCTGTTTTATCA-3' (SEQ ID NO: 4). Int ronic primers were used to detect presence of exons 4 and 7. Primers R05822F and R05822R were used to detect exon 12. PCR reaction conditions were as foUows: 5 minutes denaturation foUowed by 35 cycles of 94°C for 0.5 minutes, the respective anneaUng temperature for 0.5 minutes, and 72°C for 0.5 minutes. A 10 minute extension at 72°C concluded each reaction. AnneaUng temperature for each primer pair are provided in Table 2.

Example 6: Identification of exon intron boundaries Exon/intron boundaries were obtained by PCR/Ugation techniques. Purified PAC DNA was digested with a variety of blunt-cutting enzymes and ligated to a specificaUy designed linker. Sequences were then specificaUy ampUfied by PCR using a linker-derived primer and a PS-2 derived primer for boundary sequencing. Sequencing of products was performed directly in low- melting point agarose by using a modified dideoxynucleotide sequencing method with a ³²P end labeUed primer and Taq DNA polymerase temperature cycled reactions.

Example 7: Detection of small sequence variations

The Pharmacia ALF fragment manager was used to detect smaU sequence changes not detectable by standard agarose gel/ethidium bromide visualization. Relevant cDNA sequences were ampUfied by PCR using a 5'-fluorescein tagged primer.

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 (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: CATTCACTGA GGACACACCC 20

(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: CAAATACGGA GCGAAGACAG 20

(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: GCTTCTGTCT CAGGTTTCTT C 21

(2) INFORMATION FOR SEQ ID NO: 4:

(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: 4: CGGTGTTTGG CTGTTTTATC A 21

(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: AGCCTGCTGA GAAGAAGAAA CCA 23

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AGGCAGGGCC CAGAGGATGG AGAGA 25

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AGAGTGACAG GCACAAACAG CATG 24

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ACCATCAAGT CTGTGCGCTT CTAC 24

(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: AGATGGTCAT AACCACGATG ACG 23

(2) INFORMATION FOR SEQ ID NO: 10: (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: 10: TCAGCGTCAT CGTGGTTATG ACC 23

(2) INFORMATION FOR SEQ ID NO: 11: (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: 11: AGGTAGATAT AGGTGAAGAG GAA 23

(2) INFORMATION FOR SEQ ID NO: 12: (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: 12: TCTTCACTGA TGCTGCTGTT CC 22

(2) INFORMATION FOR SEQ ID NO: 13: (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: 13: AAGAGGGTGG GGTAGTCCAT GGC 23

(2) INFORMATION FOR SEQ ID NO: 14: (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: 14: AGCAGGCCTA CCTCATCATG ATC 23

(2) INFORMATION FOR SEQ ID NO: 15: (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: 15: TCATTTCTCT CCTGGGCAGT TTC 23

(2) INFORMATION FOR SEQ ID NO: 16: (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: 16:

TAGAAACTGC CCAGGAGAGA AAT 23

(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: TGGTGTGGAC GGTTGGCATG GCG 23

(2) INFORMATION FOR SEQ ID NO: 18: (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: 18: TGGTGTGGAC GGTTGGCATG GCG 23

(2) INFORMATION FOR SEQ ID NO: 19: (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: 19: TCTTCCTCCA GCTCCTCCCC TGG 23

(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: ATGACAGTTT TGGGGAGCCT TCA 23

(2) INFORMATION FOR SEQ ID NO: 21: (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: 21: TGGCAGCCGC CTTGCCCACC AGC 23

(2) INFORMATION FOR SEQ ID NO: 22: (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 : 22 : ACTAGAGTGT AAAACTATAC AA 22

( 2 ) INFORMATION FOR SEQ ID NO : 23 : ( 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: 23: GCTTCTGTCT CAGGTTCCTT C 21 (2) INFORMATION FOR SEQ ID NO: 24: (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: 24: CGGTGTTTGG CTGTTTTATC A 21

(2) INFORMATION FOR SEQ ID NO: 25: (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: 25: AGCCTCGAGG AGCAGTCAG 19

(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 18

(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

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GGGCAGGCTC TTCTTCAGGG 20

(2) INFORMATION FOR SEQ ID NO: 28: (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: 28: GAAAGCCACG GCCAGGAAG 19

(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: TCACGGACAG GAAGCACAGC 20

(2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 30: GTAACAAGAA CAGGACTCAG 20

(2) INFORMATION FOR SEQ ID NO: 31: (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: GAATTCCCGG GTCGACCCAC GCGTCCGCGA GCGGCGGCGG AGCAGGCATT 50 TCCAGCAGTG AGGAGACAGC CAGAAGCAAG CTATTGGAGC TGAAGGAACC 100 TGAGACAGAA GCTAGTCCCC CCTCTGAATT TTACTGATGA AGAAACTGAG 150 GCCACAGAGC TAAAGTGACT TTTCCCAAGG TCGCCCAGCG AGGACGTGGG 200 ACTTCTCAGA CGTCAGGAGA GTGATGTGAG GGAGCTGTGT GACCATAGAA 250 AGTGACGTGT TAAAAACCAG CGCTGCCCTC TTTGAAAGCC AGGGAGCATC 300 ATTCATTTAG CCTGCTGAGA AGAAGAAACC AAGTGTCCGG GATTCAGACC 350 TCTCTGCGGC CCCAAGTGTT CGTGGTGCTT CCAGAGGCAG GGCTATGCTC 400 ACATTCATGG CCTCTGACAG CGAGGAAGAA GTGTGTGATG AGCGGACGTC 450 CCTAATGTCG GCCGAGAGCC CCACGCCGCG CTCCTGCCAG GAGGGCAGGC 500 AGGGCCCAGA GGATGGAGAG AACACTGCCC AGTGGAGAAG CCAGGAGAAC 550 GAGGAGGACG GTGAGGAGGA CCCTGACCGC TATGTCTGTA GTGGGGTTCC 600 CGGGCGGCCG CCAGGCCTGG AGGAAGAGCT GACCCTCAAA TACGGAGCGA 650 AGCACGTGAT CATGCTGTTT GTGCCTGTCA CTCTGTGCAT GATCGTGGTG 700 GTAGCCACCA TCAAGTCTGT GCGCTTCTAC ACAGAGAAGA ATGGACAGCT 750 CATCTACACG ACATTCACTG AGGACACACC CTCGGTGGGC CAGCGCCTCC 800 TCAACTCCGT GCTGAACACC CTCATCATGA TCAGCGTCAT CGTGGTTATG 850 ACCATCTTCT TGGTGGTGCT CTACAAGTAC CGCTGCTACA AGTTCATCCA 900

TGGCTGGTTG ATCATGTCTT CACTGATGCT GCTGTTCCTC TTCACCTATA 950

TCTACCTTGG GGAAGTGCTC AAGACCTACA ATGTGGCCAT GGACTACCCC 1000

ACCCTCTTGC TGACTGTCTG GAACTTCGGG GCAGTGGGCA TGGTGTGCAT 1050

CCACTGGAAG GGCCCTCTGG TGCTGCAGCA GGCCTACCTC ATCATGATCA 1100

GTGCGCTCAT GGCCCTAGTG TTCATCAAGT ACCTCCCAGA GTGGTCCGCG 1150

TGGGTCATCC TGGGCGCCAT CTCTGTG AT GATCTCGTGG CTGTGCTGTG 1200

TCCCAAAGGG CCTCTGAGAA TGCTGGTAGA AACTGCCCAG GAGAGAAATG 1250

AGCCCATATT CCCTGCCCTG ATATACTCAT CTGCCATGGT GTGGACGGTT 1300

GGCATGGCGA AGCTGGACCC CTCCTCTCAG GGTGCCCTCC AGCTCCCCTA 1350

CGACCCGGAG ATGGAAGAAG ACTCCTATGA CAGTTTTGGG GAGCCTTCAT 1400

ACCCCGAAGT CTTTGAGCCT CCCTTGACTG GCTACCCAGG GGAGGAGCTG 1450

GAGGAAGAGG AGGAAAGGGG CGTGAAGCTT GGCCTCGGGG ACTTCATCTT 1500

CTACAGTGTG CTGGTGGGCA AGGCGGCTGC CACGGGCAGC GGGGACTGGA 1550

ATACCACGCT GGCCTGCTTC GTGGCCATCC TCATTGGCTT GTGTCTGACC 1600

CTCCTGCTGC TTGCTGTGTT CAAGAAGGCG CTGCCCGCCC TCCCCATCTC 1650

CATCACGTTC GGGCTCATCT TTTACTTCTC CACGGACAAC CTGGTGCGGC 1700

CGTTCATGGA CACCCTGGCC TCCCATCAGC TCTACATCTG AGGGACATGG 1750

TGTGCCACAG GCTGCAAGCT GCAGGGAATT TTCATTGGAT GCAGTTGTAT 1800

AGTTTTACAC TCTAGTGCCA TATATTTTTA AGACTTTTCT TTCCTTAAAA 1850

AATAAAGTAC GTGTTTACTT GGTGAGGAGG AGGCAGAACC AGCTCTTTGG 1900

TGCCAGCTGT TTCATCACCA GACTTTGGCT CCCGCTTTGG GGAGCGCCTC 1950

GCTTCACGGA CAGGAAGCAC AGCAGGTTTA TCCAGATGAA CTGAGAAGGT 2000

CAGATTAGGG CGGGGAGAAG AGCATCCGGC ATGAGGGCTG AGATGCGCAA 2050

AGAGTGTGCT CGGGAGTGGC CCCTGGCACC TGGGTGCTCT GGCTGGAGAG 2100

GAAAAGCCAG TTCCCTACGA GGAGTGTTCC CAATGCTTTG TCCATGATGT 2150

CCTTGTTATT TTATTGCCTT TAGAAACTGA GTCCTGTTCT TGTTACGGCA 2200

GTCACACTGC TGGGAAGTGG CTTAATAGTA ATATCAATAA ATAGATGAGT 2250

CCTGTTAGAA AAAGCGGCCG CTCTAGA 2277

(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 18

(2) INFORMATION FOR SEQ ID NO: 33: (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: 33: CTGTAAGGTA CAGTAGCCG 19

(2) INFORMATION FOR SEQ ID NO: 34: (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: 34: AAAAATCCGT GCATTACAT 19

(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: GCTGGTGTGG AGCTGCAGGT ACAGTG 26 (2) INFORMATION FOR SEQ ID NO: 36: ( i ) SEQUENCE CHARACTERI STICS :

(A) LENGTH: 19

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: AGCCTCGAGG AGCAGTCAG 19

(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: GCAGACGGAG AGAAGGGT 18

(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: GGTATCAGTC TCAGGATCAT GGG 23

(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 (xi ) SEQUENCE DESCRI PTION : SEQ ID NO : 39 : TGGGGAAGAC TGGAGCTCGA TG 22

( 2 ) INFORMATION FOR SEQ ID NO : 40 : ( 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: 40: GTAAAGAGGG CCAGGTTGGG 20

(2) INFORMATION FOR SEQ ID NO: 41: (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: 41: GTGCAGCACT GGGGACGATT T 21

(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: GGGCAGGCTC TTCTTCAGGG 20

(2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19

(B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 43: GAAAGCCACG GCCAGGAAG 19

(2) INFORMATION FOR SEQ ID NO: 44: (l) 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: 44: TTAGCACCGC CTGAGACGT 19

(2) INFORMATION FOR SEQ ID NO: 45: (l) 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: 45: AGCTGGTCAG AGTGTTAC 18

(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

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 46: CCCCTCCTGA ACTCATGCCT 20

(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

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 47: CTCTGACCAG CTGTTGTTTC 20

(2) INFORMATION FOR SEQ ID NO: 48: (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: 48: AGCCTCCACC CTCTGTCT 18

(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: TTCCATTCTG TGCACGCCTC 20

(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 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: ACCTGCCCCC ACCACAATG 19

(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: ACAGCTCCTG TCCACACCA 19

(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: ACTAGAGTGT AAAACTATAC AA 22

Claims

What is Claimed is:

1. A variant presemlin-2 gene.

2. The variant preseniUn-2 gene of claim 1 comprising splicing of exon 3 and exon 4 out of a presenilin-2 gene.

3. The variant presenihn-2 gene of claim 1 comprising spUcing 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 spUce donor or acceptor sites of a preseniUn-2 gene comprising sequencing spUce donor or acceptor sites of the presenilin-2 with intronic primers for the presenilin-2 gene and analyzing the sequences to identify any mutants.