JP2002515740A - Variants of the presenilin-2 gene - Google Patents

Abstract

  (57) [Summary] The present invention provides variants of the presenilin-2 gene. Methods of using these genes in the diagnosis of Alzheimer's disease are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION                       Variants of the presenilin-2 gene   Foreword   The present invention was completed during research sponsored by the National Institute of Health. It is. The United States Government has certain rights in the invention.   Background of the Invention   Alzheimer's disease (AD) is an advanced disease characterized by memory loss and dementia It is an active neurodegenerative disease. The main pathological features of AD are neurofibrillary tangles and And senile plaques mainly composed of amyloid protein. These spots are larger 39-43 variants derived from the amyloid precursor protein (APP) Β-amyloid, a peptide of amino acid length (Goate et al., Nature 1991, 3 49, 704-6; Masters et al., PNAS 1985, 82, 4245-4249; Kang et al., Nature 1987, 325, 73. 3-736). Various studies have shown that the increased occurrence of the 42 amino acid peptide has been identified by APP. It shows that it leads to AD in the disease being treated.   AD is typically a senile disease, with up to 6% of them 65 years old and 2 Up to 0% is reflected by being 80 years old. This type of AD is called late-onset It is called. Also, in a small number of families, autosomal chromosomes with age-dependent penetrance It is described as a dominantly unique disease. Most commonly, this disease The age of onset is below 60 years in these families (maturity). That is, this Type AD is termed premature. In both early-onset and late-onset AD, Genetic factors are involved.   Some cases where precocious AD has been isolated as a fully penetrated autosomal dominant trait Have been identified in a large family. Linkage studies in the elderly AD family showed that chromosomes 1, 1 Four genes on 4, 19 and 21 are mutated, causing presenile AD Was identified. The first identified geriatric AD gene is located on chromosome 21. Encodes the βA4-amyloid protein precursor (APP) (Goate et al., Natur e 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 affect about 5% of families Climb. These diseases due to mutations can be transfected or Amyloid, potentially neurotoxic Ab fragment modeled on subcultured cells Has been shown to lead to modified proteolytic processing of APP in a direction that favors cell production. Was. Transgenic overexpression of one mutant APP is shown in a mouse model of AD For the first time, where age-related brain deposition of Ab fragments is nervous With astrocytic and microglial pathologies (Games et al., Nature 1995, 373) , 523-527).   Genetic variability at the apolipoprotein E locus on chromosome 19 is also a cause of AD Theory (Strittmater et al., PNAS 1993, 90, 197). -Arg) Allelic inheritance also reduces age of onset in a dose-dependent manner It also appears to reduce the danger of D (Corder et al., Science 1993, 261, 921-923; C order et al., Nature Genet. 1994, 7, 180-184).   Presenilin-1 (PS-1) is located on chromosome 14 and accounts for an estimated 70% of disease. Harbors the causative mutation, which is responsible for the main inheritance of familial 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-Hy slop et al., Nature Genet. 1992, 2, 330-334; Schellenberg et al., Science 1992, 258. , 668-670). This gene, termed S182 or PS-1, has up to 10 Reading exons (Nos. 3 to 12). PS-1 has at least 7 Presumed to be one integral membrane protein in the transmembrane domain Is imagined. At the time of its isolation, five different missense mutations Identified in eight families of the ream (Sherington et al., Nature 1995, 375, 754-760). varied Of 22 different variants identified in 40 families of ethnic origin (Van Broeckhoven et al., Nature Genet. 1995, 11, 230-233). Two different mutations Was identified at each of codons 139, 146, 163 and 280. But, Most mutations are associated with 5 and 6 hydrophilicities of the 7 putative transmembrane domains. It is dispersed throughout the protein, with mutations found in three of the loops. 10 Mutations were found in six of the coding exons, with 65% of the mutations in exon 5. And 8. The same mutation, but several AD families of different ethnic origins Occurred in the tribe, suggesting that there is an independent mutation event in the PS-1 gene.   A third gene (PS-2) for elderly AD is one in which AD is a founder effect. Chromosome 1 is located in the Volga-German AD family, a group member ( Levy-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 the 7 Volga-German AD family, Recently, a second missense mutation was found in an Italian AD family (Levy-Lahad Et al., Science 1995, 269, 970-973; Rogeav et al., Nature 1995, 376, 775-778; Barin aga, Science 1995, 269, 917-918).   Both PS-1 and PS-2 have about 450 amino acids and have overall homology With 67% similarity and the highest similarity in the TM domain (Levy-Lahad et al., Sc. ience 1995, 269, 973-977; Rogeav et al., Nature 1995, 376, 775-778). This homology Sex degrees indicate similar biological functions. Putative 7 trans of presenilin The membrane domain structure can be a receptor molecule, ion channel or protein It is compatible with the membrane structure. Mutations in PS-1 and PS-2 are Ab₄₂Occurrence of Due to the increase, biological interactions with amyloid have been suggested.   Exon structure and differential splicing in the PS-1 gene were determined. Was. The use of a different splice donor at the 3 'end of exon 3 is Results in clones with or without the VRSQ motif at 29 ( Alzheimer's Disease Collaborative Group. Nature Genet. 1995, 11, 219-222 ). Alternative splicing also occurs at exon 8 in PS-1 and in PS-2. Homologous exon 8 was found in both.   Many other variants have been identified that are separately spliced PS-2 genes . These genes are useful for diagnosing early-onset AD and for drugs useful for treating the disease. Useful for evaluation.   Summary of the Invention   One object of the present invention is to provide novel variant PS-2 sequences.   Another object of the present invention is to provide these novel PS-2 sequences or To provide a method for diagnosing Alzheimer's disease using exon or intron sequences It is.   A further object of the present invention is to provide an Alzheimer's disease model comprising a PS-2 gene variant. To provide Dell system.   BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 is a schematic comparison of the configuration of PS-1 and PS-2. Labeled arrow Indicates a known mutation site, and an unlabeled arrow indicates an intron / exon boundary. Show. The shaded areas in PS-2 indicate different splicing sites. US F # 15 contains exons 3, 4 and 8. WU # 2 is exons 3, 4 and 8 Lacks. WU # 15 only lacks exon 8.   FIG. 2 shows the gene sequence of the PS-2 gene (SEQ ID NO: 31). Detailed description of the invention   The APP gene on chromosome 21, the ApoE gene on chromosome 19 and the Mutations in the S182 or PS-1 gene are identical to those in early Alzheimer's disease. It is a major cause when defined (Strittmater et al., PNAS, 90, 1977-1981 (199 3); Sherrington et al., Nature, 375, 754-760 (1995)). However, Volgaji The Volga-German family and AD are inherited as autosomal dominant traits In other families that are known to be You.   The Volga-German family is a culturally distinct futuristic group of Russia, They did not marry the Russian people. Except for the relative prevalence of AD in this group Very early, in the range of 50 to 70 years. However, clinically and pathologically, AD in these families is indistinguishable from typical AD. Volga-German Autosomal dominant locus involved in AD is restricted to the localization of chromosomes lg31-42 (Levy-Lahad et al., Science, 269, 970-973 (1995)). At this locus Candidate genes located were also identified. Levy-Lahad et al. Found that the deduced amino acid sequence STM2 homologous to the amino acid sequence of S182 (PS-1) was isolated. Ass Point suddenness in STM2 resulting from using isoleucine instead of paragine Mutation (N₁₄₁I) was identified in affected patients. This N₁₄₁The I mutation is Occurs in the amino acid sequence conserved in S182 and a mouse S182 homolog. Ro gaev et al. cloned E5-1 on chromosome 1, which is also homologous to S182. (Nature, 376, 775-778 (1995)). E5-1 (STM2) By analyzing the nucleotide sequence of the pun reading frame, Volga- German (N₁₄₁I) and Italian (M₂₃₉V) For patients affected by pedigree Two missense substitutions were found at conserved amino acid residues in the gene.   Clarify the structure of the PS-2 gene and determine alternative splicing sites The gene was cloned and sequenced, and the altered sp Rice product (variant) and exon / intron boundaries were determined. Intron / To elucidate the sequence of exon boundaries, PS-2 was coded by 10 coding exons. Was revealed. The PS-2 gene sequence corresponds to the EST sequence T03796. After sequencing, the gene was sequenced using the GeneTrapper kit (Gibco BRL, Gaithersburg, MD). It was determined by isolating the S-2 cDNA.   Table 1 shows the intron / exon structure of the PS-2 gene. PS-2 cDNA Indicates the position of the intron that divides. Exon sequence in uppercase, intron sequence In lower case. Exons are counted from the 5 'end of the cDNA sequence.   cDNA sequencing reveals multiple positions where sequence mutations are observed Four occurred at exon boundaries. Key to this splicing data The results are shown in Table 2.   The splicing events in Table 2 correspond to the individual cDNAs from which they were identified as well as the corresponding And the amino acid residues are listed. Splice detected by RT-PCR The marking event is indicated by “+” in the PCR column. All events are designated USF # 15 For full-length cDNA clones. These four cDNA clones FIG. 1 shows the structure of the blade.   Three of the mutations identified contain altered splicing events. These are P Plasmids outside exons 3 and 4 of the S-2 gene, inside or outside exon 8 Include in Ising. Relevant boundaries are amplified by PCR and these clones are tested It was found to occur in all cDNAs of the isolated tissues. Other changed splices Thing was not detected in these tissues. The other two mutations are Levy-Lahad et al. , A small change that differs from the sequence published by Science, 269, 973-977 (1995). Including. These changes are due to the deletion of a glutamic acid residue at codon 324 and codon 3 Changing the amino acid sequence encoded by 57-359 from ERGV to ESQGG 6 base pair insertions.   Conversely, evidence for splicing spontaneously altered in the PS-1 gene Few were recognized. Slight changes previously reported for PS-1 Splicing is added to exon 8 with or without the VRSQ motif It was found at the 3 'end of exon 3 obtained in the clone. These splices The logging event is also observed in PS-2, but has no equivalent in PS-1. Another altered splicing event was observed in PS-2. These events are Causes significant changes in protein structure.   The most shocking altered transcripts are those that lack exons 3 and 4. Exon Variants lacking 3 and 4 have lost the normal starting methionine and For example, it is thought to start with methionine at codon 145. This start site is Transmembrane domain after transmembrane domain 1 2 in the middle. This protein is a Volga-German AD mutant N₁₄₁I And different.   The identification of these variants indicates that certain mutations in the PS-2 gene were "Man" type AD. Therefore, these variants are AD Or in the development of models of AD.   Identification of intron sequences also provides an early test for variant forms of the PS-2 gene Useful for Genomic analysis of the PS-2 gene (see FIG. 1) A method to identify intronic polymorphisms, which is a precursor, will be developed. Splice Intron sequences associated with the DNA mutation and those introns of the PS-2 gene. Mutations in both open reading frames adjacent to the exon boundary Elucidation, detection and diagnosis can be performed by using an intron sequence. Mutations caused by mutations in splice donor or acceptor sites Identification and analysis of variants or variants depends on elucidating these intron sequences. It becomes possible. In addition, a complete analysis of the intron-exon boundary provides The first or last 10 or more of the coding exon near the end of the cDNA Screening primer that enables accurate sequencing of 20 nucleotides Can be manufactured.   Currently, there is no known method of effectively treating various forms of AD. But, Progressive, treatable, very similar to dementia associated with Alzheimer's disease There are several other forms of dementia that cause intellectual decline. Therefore, about AD Diagnostic tests can eliminate early-stage Alzheimer's disease. Would provide useful tools in the diagnosis and treatment of other conditions. Appropriate It may also be useful where treatments for AD are available. Alzheimer Sets that can be used to detect and identify genetic mutations that cause disease There are several methodologies available over recombinant DNA techniques. These are direct Bing, ligase chain reaction (LCR) and polymerase chain reaction (PCR) methods Includes, but is not limited to, jurisprudence.   Detection of variants or mutants using direct probing can be synthetic or Use of oligonucleotide probes produced by nick translation. You. In a preferred embodiment, the probe is a PS-2 gene identified herein. Complementary to at least some of the variants of the gene. The DNA probe is, for example, For subsequent visualization in the Zan blot hybridization procedure, for example, Uses radioactive, enzyme, fluorescent or biotin-avidin labels And may be appropriately labeled. Transfer the labeled probe to nitrocellulose. Of DNA from a patient suspected of having AD bound to a nylon or nylon 66 substrate React with pull. A region carrying a DNA sequence complementary to the labeled DNA Is itself labeled as a result of the rear annealing reaction. By the way, Visualize the area of the filter showing the carousel, for example, by autoradiography I do.   Other probe methods, such as ligase chain reaction (LCR), include mismatched probes, That is, a probe that has complete complementarity with the target except for mutations or changes. Use of The target sequence is then replaced with an Oligonucleotides, ie, oligonucleotides complementary to the PS-2 variants of the invention And oligonucleotides with mismatches and the conditions that distinguish the two To form a hybrid. By manipulating the reaction conditions, complete complementarity It is also possible to achieve hybrid formation only if If there is a mismatch , In which case hybridization is significantly reduced.   Polymerase chain reaction (PCR) is a technique for amplifying specific DNA sequences. Denaturation, primer annealing and using thermostable enzyme Taq polymerase The extension cycle is repeated to increase the concentration of the desired DNA sequence exponentially.   If the nucleotide sequence encoding the PS-2 gene is known, it will be adjacent to the DNA in question. Synthetic oligonucleotides complementary to the flanking sequence can be produced. Oligo The nucleotides are each complementary to one of the two strands. Then, the DNA was Denature at room temperature (eg, 95 ° C.) and then a substantial molar excess of oligonucleotide Annealing in the presence of Oligonucleotides oriented towards each other at the 3 'end Leotide is hybridized to the opposite strand of the target sequence and deoxyribonucleic acid triphosphate is Prime enzyme extension along the nucleic acid template in the presence of the nucleotide. Then the final The product is denatured again for another cycle. Repeat these three steps 7 times However, the DNA segment can be amplified one million times or more. Then got DNA may be sequenced directly to locate genetic changes. Also book The identified PS-2 variants of the invention show that, if there is a mutation, PCR increases DNA. Oligonucleotides that bind only to altered DNA so that they only result in multiplication It is possible to produce tide. According to PCR, allele-specific orientation AD point mutations may be detected using the oligonucleotide hybridisation method.   Alternatively, PCR called amplification of a specific allele ication of specific alleles (PASA) method can also be used; Makes quick and reliable distinctions between different alleles at one base pair To use differential amplification. Newton et al., Nucleic Acid Res., 17, 2503 (1989); N. ichols et al., Genomics, 5, 535 (1989); Okayama et al., J. Lab. Clin. Med., 1214, 10. 5 (1989); Sarkar et al., Anal. Biochem., 186: 64 (1990); Sommer et al., Mayo Clin. Proc., 64, 1361 (1989); Wu, Proc. Nat'l Aca. Sci. USA, 86, 2757 (1989) And Dutton et al., Biotechniques, 11, 700 (1991). PASA is With two oligonucleotide primers, one of which is allele specific Amplifying. The desired allele is effectively amplified while the other Allele (s) are not significantly amplified. Because allele features This is because of a mismatch with the base at or near the 3 'end of the different primer. You. Thus, one or more PSs using PASA or modified PAMSA Mutants of the -2 allele can be specifically amplified. Such amplification If performed on genetic material obtained from the patient, one or more in the patient Can be used as a method to detect the presence of a mutant of the PS-2 allele. Wear. PCR-induced mutation restriction analysis, often referred to as IMPA, It can also be used for body detection.   It is also important to develop experimental models of Alzheimer's disease. Such a mo Dell can be used to screen for reagents that alter the decay process of AD. Early Specific Mutations in the PS-2 Gene as a Cause of Family Alzheimer's Disease If a difference is identified, the genetically modified PS-2 gene or its Developing transgenic model systems and / or whole cell systems with some Both are possible. Drugs are screened using the model system and Alzheimer's The efficacy of a drug in the treatment of a disease can also be evaluated. In addition, these models The system is a means of limiting the biochemistry underlying PS-2 and its relationship to AD And thereby provide a basis for rational drug design.   One type of cell line that can be used in the present invention is naturally induced. Can be. To this end, blood samples from affected individuals were obtained and For example, using the Epstein-Barr virus, the lymphostroid cell line Transform. Once established, such cell lines can be continuously cultivated in suspension culture. Can be grown and used in various in vitro experiments to express and express PS-2. You can study sexing. Another cell line used for these studies is Consists of skin fibroblasts from the patient.   An alternative to constructing cell lines is the PS-2 The mutated gene, or a portion thereof, is transferred to an established cell line of choice, as described above. Operate Such methods are described in Sisodia, Science, 248, 492 (1990) and Olte. rsdork et al. Biol. Chem. , 265, 4492 (1990). Is well known to the public. There, the amyloid precursor peptide gene is found in mammalian cells. Has been transfected.   Baculovirus expression systems also provide for the level of expression of heterologous genes in eukaryotic cells. Has been found to be effective in raising the load.   In addition, the mutated gene is taken out, and the trans It can be used to make transgenic mice. For example, the PS-2 of the present invention The gene can be cloned and placed in a cloning vector. Available black For example, 1Charon35, cosmid or yeast artificial dye Although a color body is mentioned, it is not limited to this. Then, the PS-2 gene Can be transferred to a host animal (non-human animal), for example, a mouse. Transfer As a result, one or more transgenic animals (non-human animals) were obtained. It is preferred to express a variant of the above PS-2 polypeptide.   In addition, a minigen encoding the PS-2 gene can be designed. Take The minigen is a cDNA sequence encoding a variant of the PS-2 polypeptide, preferably A full-length, PS-2 exon combination or downstream polyadenylation system Combination of signal sequence and upstream promoter (and preferably enhancer) Have a match. Such minigen constructs are suitable transgenic hosts, e.g. For example, when introduced into a mouse or rat, the construct is transformed into a PS-2 polypeptide. Will express the variant.   One way to create transgenic animals is to use eukaryotic cell stem (ES) cells. Mutating the desired gene by homologous combination in vitro, The modified ES cell line is then microinjected into host fibroblasts. , Followed by incubation in the foster mother. Frohman and Martin, Cell , 56, 145 (1989). In addition, mutated genes or parts thereof Microinjection into one cell embryo, followed by incubation with foster mother Can be used. Another method of producing transgenic mice Are well known to those skilled in the art.   Transgenic mice are illustrated in US Pat. No. 5,223,610. Used in the evaluation of novel therapeutic compositions and in carcinogenicity studies . These animals are also specifically described in US Pat. No. 5,221,778. As such, it is used to develop predictive animal models for human disease states. This time, Tran Sgenic animals include Alzheimer's disease (US Pat. No. 7,776,626), anti-cancer Multidrug resistance to drugs (US Pat. No. 7,260,827) and carcinogens (US No. 4,736,866). Therefore, chromosome 1 The present invention, which is thought to be the cause of early Alzheimer's disease in a 4-connected family PS-2 gene has been used to develop transgenic animals and evaluate this disease. It provides a useful means for   Using site-directed mutagenesis and / or gene conversion, Alternatively, the allele of the non-human PS-2 gene is changed by transfection. Alternatively, the mutant gene may encode a polypeptide having an altered amino acid as described in the present application. It can be done.   Further, for use in testing the function of the truncated transcript of the PS-2 gene, Antibodies to S-2 and variants thereof can be obtained. These antibodies, for example, For example, it may be a polyclonal antibody or a monoclonal antibody. Also, the present invention , Chimeric, single chain, and humanized antibodies, and Fab fragments, or Also encompasses the production of Fab expression libraries. Various procedures known in the art May be used for the production of such antibodies and fragments.   By direct injection into an animal or administration of a gene to an animal (preferably a non-human). Alternatively, an antibody generated against the PS-2 gene of the present invention may be obtained. Then like that The antibody thus obtained is bound to the PS-2 gene or itself. in this way Thus, these antibodies can also be obtained by using gene fragments.   For preparation of monoclonal antibodies, provide antibodies produced by continuous cell culture. The method provided can be used. Examples include the hybridoma method (Kohler and  Milstein, Nature 1975, 256, 495-497), trioma method, human / B cell hybrid Dorma method (Kozbor et al., Immunology Today 1983, 4, 72), EBV-Hybridoma Method for Production of Lonal Antibodies (Cole et al., Monoclonal  Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp. 77-96) It is.   The present invention was applied by applying a method for producing a single-chain antibody (US Pat. No. 4,946,778). A single-chain antibody against the bright PS-2 gene can be obtained. Also, transje Using a nick mouse to express a humanized antibody against the PS-2 gene of the present invention. Good.   The following non-limiting examples serve to further illustrate the invention. Example Example 1: Cloning of PS-2 gene   PS-2cD using Gene Trapper kit (Gibco BRL) according to the manufacturer's instructions. NA was isolated. Primer 5'-CATTCACTGAGGACACACCC PCMVSP using -3 '(SEQ ID NO: 1) (derived from EST sequence T03796) Library of human / brain transcripts in ORT (Gibco BRL, Gaithersburg, MD) Robe. However, the use of this sequence (three times) always results in different clones. Were isolated and contained 5 'and 3' untranslated gene sequences, but The gene region around the putative codon was missing. Ministry in the above clones Primer 5'-CAAATACGGGAGCGAAGACAG derived from a short region -3 '(SEQ ID NO: 2) and also using the Gene Trapper kit Were rescreened. As a result, clones containing the missing region Can be isolated. Example 2: Preparation of cDNA   Human RNA derived from brain, heart, liver, lung, placenta and skeletal muscle was analyzed by Clontech (Palo Alto, CA). Superscript Preamplification System for First Strand First strand cDNA was synthesized using cDNA Synthesis (Gibco BRL). 0.5 ml In a tube, 2 milligrams of total RNA was combined with 1 mg of oligonucleotide (dT)_{12 ~ 18} Primer, 100 ng random hexamer primer and DEPC treatment Mix with water. The samples were incubated at 70 ° C. for 10 minutes, then placed on ice. Was. Kit components were added according to the manufacturer's protocol and samples were incubated at 37 ° C for 2 hours. Incubation. Superscript II Reverse Transcriptase (RT; 2 00U) was added and the samples were then incubated at 37 ° C. for 1 hour. Next The sample was then incubated at 70 ° C. for 15 minutes, cooled on ice, and then Nase H was added. All samples were stored at -20 <0> C. Further PCR For each reaction, 1 ml of the final product was used for each reaction. Example 3: PCR to determine altered splicing products   Primers are designed throughout the cDNA sequence to vary in various tissues. Splicing can be evaluated. Exon primers and their Table 3 shows the conditions of use. Shows intron primers and their usage conditions It is shown in FIG.   A partial sample of the tissue cDNA was prepared using a Perkin Elmer DNA Thermal Cycler 480 (Perkin Elmer mer, Norwalk, CT). Each PCR reaction system produces 1 ml final cDNA Product, 25 pmol of each primer (forward and reverse), 12.5 nmol dNTP (Pharmacia, Columbus, OH), 1.25 U of Taq polymerase (Promeg a, Madison, WI), a total reaction volume of 50 ml, mineral oil (Fisher, Pittsburgh, PA) was overlaid with 60 ml. At least two putative intros of the PS-2 gene The primers used were designed to span the exon / exon boundaries. For example, LP3 For 13F (forward, exon 2) and LP676R (reverse, exon 4) Straddling the intron exon boundaries of exons 2/3 and 3/4 You. For certain reactions, samples were denatured at 94 ° C. for 5 minutes. Then the sample , At 94 ° C for 0.5 minutes, annealing temperature suitable for the primer pair used For 0.5 minutes and then at 75 ° C. for 0.75 minutes for 35 cycles. After doing this The final extension reaction was performed at 72 ° C. for 10 minutes. The product is purified using ethidium bromide staining. Visualized on a 2% agarose gel (Promega). Cut out the product band, W Purification was performed using izard PCR Preps DNA Purification System (Promega). Final Five microliters of 50 ml of the purified product were used for sequencing. Example 4: Sequencing for Determination of Altered Splicing Products   Exonuclease I and shrimp alkaline phosphatase (PCR sequenci ng kit-USB, Clevland, OH). 5 microphones Of PCR product with exonuclease I at 37 ° C. for 15 minutes It was cubified. The sample is then maintained at 80 ° C. for 15 minutes, after which 2 U of air Bi-alkaline phosphatase was added. The sample is kept at 37 ° C. for 15 minutes, then At 80 ° C. for 15 minutes. 7 ml of this final product is Used in the sequencing protocol described in the ct Sequencing Kit. Original P The forward primer used for the CR reaction was used for manual sequencing. Example 5: PAC isolation   PCR generation amplified using primers R05822F and R05822R Screen the grid library (Genome Systems, Inc.) Thus, an artificial chromosome (PAC) derived from P1 was isolated. 3 containing PS-2 gene Seed PACs were digested with NotI and pulsed-field gel electrophoresis (PFGE) was performed. Size. 200V, 21 hours, 14 ° C, switch time 5-20 seconds PAC DNA was run. The size was 90 kb to 110 kb. 5 'The primer used for detection of the untranslated sequence was 5'-GCTTCTGTCTCAGGGT. TTCTTC-3 '(SEQ ID NO: 3) and 5'-CGGTGTTTGGCTGT TTTATCA-3 ′ (SEQ ID NO: 4). Using intron primer Exons 4 and 7 were detected. Primers R05822F and R0 Exon 12 was detected using 5822R. The PCR reaction conditions are as follows: Denaturation for 5 minutes, then 0.5 minutes at 94 ° C, 0.5 minutes at the appropriate annealing temperature, 7 minutes 35 cycles of 0.5 minutes at 2 ° C. Each reaction was completed by an extension reaction at 72 ° C for 10 minutes. Was. Table 2 shows the annealing temperature of each primer. Example 6: Identification of exon / intron boundaries   Exon / intron boundaries were obtained by PCR / ligation. Purified PAC DNA Was digested with enzymes leaving various blunt ends and ligated to individually designed linkers. The linker-derived primers and PS-2-derived primers for border sequencing were then The sequence was specifically amplified by PCR using the immer.³²P and labeled primer Dideoxy using temperature- and Taq DNA polymerase temperature cycling reaction By using nucleotide sequencing, the product can be distributed in low melting point agarose. Column determinations were made directly. Example 7: Detection of small sequence changes   Small sequence changes using the Pharmacia ALF Fragment Manager (standard Agarose gel / ethidium bromide visualization). 5 ' -Amplify the corresponding cDNA sequence by PCR using fluorescein sticky primer did.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG), EA (AM, AZ, BY, KG, KZ , MD, RU, TJ, TM), AL, AM, AU, BB , BG, BR, CA, CN, CZ, EE, GE, GH, HU, IL, IS, JP, KG, KP, KR, LK, L R, LT, LV, MD, MG, MK, MN, MX, NO , NZ, PL, RO, SG, SI, SK, TR, TT, UA, US, UZ, VN, YU (72) Inventor Hardy, John             United States 32084 Florida Sen             Augustine, Water Street             No. 48 (72) Inventor Goat, Allison M             United States 63117 Missouri Rich             Mondo Heights, Claytonia Terrace             1177th (72) Inventors Full donor, Rebecca A             United States 20838 Maryland Bas             Johnsville, Box 391, Beer Zubi             Le Road No. 21910

Claims (1)

[Claims]   1. A variant of the presenilin-2 gene.   2. Splice exons 3 and 4 outside the presenilin-2 gene A variant of the presenilin 2-gene according to claim 1, comprising:   3. From splicing exon 8 outside the presenilin-2 gene A variant of the presenilin 2-gene according to claim 1.   4. Detect mutants of presenilin-2 gene in DNA samples from patients A method for diagnosing Alzheimer's disease in a patient, comprising:   5. The presenilin-2 donor or acceptor site is replaced with the presenilin-2. Splice with intron primer for gene, analyze the sequence and mutate An identification method comprising identifying a body.