METHOD TO DETECT POLYMORPHISM IN THE S182 GENE

Abstract: A method of detecting polymorphisms in the S182 gene by detecting mutations in the S182 gene using selected mismatched PCR primers comprising sequences derived from intronic sequences of the S182 gene is provided. A method of identifying an individual susceptible to late onset Alzheimer's Disease is also provided.

Description: METHOD TO DETECT POLYMORPHISM IN THE S182 GENE BACKGROUND OF THE INVENTION Alzheimer's disease (AD) is a progressive degenerative disease of the central nervous system characterized clinically by dementia and neuropathologically by the presence of numerous senile plaques and neurofibrillary tangles. AD is typically a late onset disease of the elderly. However, a small number of pedigrees have been described wherein an early onset form of 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 old. Genetic factors have been implicated in both early and late onset AD. Mutations in at least four different genetic loci are associated with an inherited susceptibility to AD. The e4 allele of the apolipoprotein E (ApoE) gene on chromosome 19 is associated with late onset AD (Strittmatter et al. Proc. Natl. Acad. Sci. USA 1993, 90:1977-1981; Saunders et al. Neurology 1993, 43: 1467-1472); Corder et al. Science 1993, 261:921-923). Mutations in the β-amyloid precursor protein (βAPP) gene on chromosome 21 have been found in a small number of families with early onset AD (Goate et al. Nature 1991, 349:704-706; Chartier-Harlin et al. Nature 1991, 353:844-846; Murrell et al. Science 1991, 254:97-99; Karlinsky et al. Neurology 1992, 42: 1445-1453). Most recently, a novel AD locus in a gene referred to as STM2 was identified on chromosome 1 (lq31-41) from genetic linkage analysis of "Volga German" kindred (Ephrat Levy-Lahad et al. Science 1995, 269:970-973; ibid. 973- 977). The STM2 gene bears a remarkable similarity to the AD-associated gene, SI 82. The fourth locus (AD3) has been mapped by genetic linkage studies to chromosome 14q24.3 and may account for up to 70% of early-onset autosomal dominant AD. Schellenberg et al. Science 1992, 258:668-670; George-Hyslop et al. Nature Genet. 1992, 2:330-334; Van Boreckhoven et al. Nature Genet. 1992, 2:335- 339). The AD3 locus is associated with the most aggressive form of this disease (onset between 30 and 60 years of age) and it has been suggested that mutations at this locus put into effect a biologically fundamental process leading to AD. Recently, a novel gene with five missense mutations in seven pedigrees segregating early-onset autosomal dominant AD at the AD3 locus, the SI 82 gene, was cloned and described by Sherrington et al. Nature 1995, 375:754-760. Analysis of the nucleotide sequence of the SI 82 transcript revealed heterozygous nucleotide substitutions in the reverse transcriptase-polymerase chain reaction products from affected members of six large pedigrees. The putative open reading frame (ORF) of SI 82 encodes a protein predicted to be a classical seven-transmembrane protein and the pedigree-associated nucleotide substitutions change the encoded amino acids in transmembrane (TM) helices II(L146M), VI(E246A), and VTJ(Y410C) and in loops between TMπ-TMTU(R163H) and TMVI-TMVTI(V286L). No transmembrane ion channel function has been demonstrated for SI 82 to date. A number of other mutants of the SI 82 gene have now been identified. Genomic analysis of the SI 82 gene has defined the intron-exon boundaries of the primary transcription unit. This has led to the development of a method for identification of intronic polymoφhisms which are predictive of disease as well as elucidation of several splicing variants and proximal coding mutations. These intronic sequences are useful in the early detection of mutant forms of the SI 82 gene, a gene which is usually associated with aggressive early-onset AD but may also be involved in the late onset of this disease as well. SUMMARY OF THE INVENTION An object of the present invention is to provide the sequences of the intron- exon boundaries of the S 182 gene. Another object of the present invention is to provide a method for the 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 SI 82 gene through use of intronic sequences. Yet another object of the present invention is to provide the sequence of novel SI 82 mutations and to provide a method for their detection and diagnosis. BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates the sequence of the intron exon boundaries of the SI 82 gene. Coding sequences are in uppercase letters, intronic sequences in lowercase letters. (Seq. LD. Nos. 23-42) Figure 2 provides diagrams of the location of the F74 (and possible F184) intronic mutations. Figure 2 A shows the normal splicing of exon 9. Figure 2B shows the splicing of mutant PS-1 allele in F74 resulting in the loss of exon 9. Figure 2C provides a comparison of wild type and F74 mRNA and amino acid sequences. (Seq. LD. Nos. 43-52) DETAILED DESCRIPTION OF THE INVENTION Genetic linkage strategies placed a gene causing early onset familial Alzheimer's disease (FAD) on the long arm of chromosome 14 between D14S289 and D14S61. Five mutations within the SI 82 gene, which map to this region were recently reported in several families multiply affected by early onset AD (between 30-50 years) (Sherrington et al. Nature 1995, 375:754-760). While conventionally this gene has been thought to only be involved in the rare, familial early onset form of the disease, some evidence for allele sharing between affected family members with late onset disease has also been observed (Schellenberg et al. Am. J. Hum. Genet. 1993, 53:619- 628). This allele sharing between affected family members was not found when standard maximum likelihood methods were used, but was found when the affected pedigree member method of genetic analysis was used, suggesting that the locus was not behaving as an autosomal dominant in the late onset form of the disease. During sequence analysis of early onset Alzheimer disease cases, a common polymoφhism within the intron 3' to exon 9 of the SI 82 gene was identified. The most common allele has an A at nucleotide 16 (allele 1) in the intron while the variant allele has a C at this position (allele 2). The present invention provides a method of detecting this polymoφhism utilizing mismatch PCR primers which introduce a BamHI site when the variant C is at nucleotide 16, but not when the A is present. This allows for the rapid analysis of a large number of samples using PCR followed by digestion with the restriction enzyme for BamHI and agarose gel electrophoresis. Digestion refers to catalytic cleavage of a nucleic acid sequence with a restriction enzyme that acts only at certain sites in the sequence. Restriction enzymes such as that for BamHI are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment. Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res. 1980, 8:4057. The method of the present invention was used to screen for the presence of this polymoφhism in a clinical series of AD cases and age matched controls. This polymoφhism shows a strong association with the occurrence of typical late onset Alzheimer's disease, with homozygosity for the more common of the two alleles causing an apparent doubling of the risk for this form of the disease (Table 1). Table 1 illustrates the association of the presence of an intronic polymoφhism in homozygous form with the occurrence of typical late onset AD. TABLE 1 Alleles 11 12/22 control 51(0.27) 134(0.73) AD 89(0.43) 119(0.57) a familial 14(0.44) 18(0.56) b a cases v. controls chi squares = 9.89, ldf, p = 0.0012, OR = 1.97, CI = 1.29-3.00 b familial cases v. controls chi squares = 3.40, ldf.p = 0.065, OR = 2.04, CI = 0.95-4.41 This polymoφhism does not change the coding sequences of the gene and in the clinical cases examined does not appear to alter the protein. It does, however, indicate that the SI 82 gene is involved in the pathogenesis of at least a proportion of late onset AD cases. Furthermore, if the polymoφhism itself is of biological relevance, it may act by alternative splicing in this area. Intronic sequences in other areas of the SI 82 are believed to be important for similar reasons. A full-length clone of SI 82 was isolated from human cerebellar cDNA libraries. The entire intron-exon structure of this SI 82 gene was determined by comparison of cDNA and genomic DNAs (Figure 1). The SI 82 intronic sequences and their reverse complements near the intron-exon boundaries for the 12 exons were determined and are shown in Table 2. TABLE 2 Region Sequence (5' -> 3') Exon 2-3 gtttgtttctgcttaatgta (Sequence ID NO: 1) gttttttctttcccttttcag (Sequence TD NO: 2) Exon 3-4 gtacagtgt (Sequence ID NO: 3) tgtttttcttgtgcttatag (Sequence ID NO: 4) Exon 4-5 acgtatgagatttgtttt (Sequence ID NO: 5) ttgtgtttgttttattgtag (Sequence TD NO: 6) Exon 5-6 gtgagcatgagacacagatc (Sequence ID NO: 7) tgaaatgctttcttttctag (Sequence ID NO: 8) Exon 7-8 gtaaaacccaagactgataa (Sequence ID NO: 9) ttatgtttttctttttctag (Sequence ID NO: 10) Exon 9-10 gtatgtgcatttctctatgt (Sequence ID NO: 11) ttgtaacctttcctttttag (Sequence ID NO: 12) Exon 10-11 gtatgtgcatttctctatgt (Sequence ID NO: 13) ttgtaacctttcctttttag (Sequence ID NO: 14) Exon 11-12 gtaagtatacactaataaga (Sequence ID NO: 15) ctttcccatcttctccacag (Sequence ID NO: 16) Analysis of the intron-exon boundaries of the novel SI 82 gene indicated that a novel alternative splicing variant of the SI 82 gene had been expressed. Furthermore, using these primers, a mutation in the last nucleotide of the intron between exons 8 and 9 was found in at least one early onset AD family (F74) (Figure 2). A novel mutation was identified in exon 5 which would have been impossible to detect without 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. The following examples are provided for illustrative puφoses only and are not intended to limit the invention. EXAMPLES Example 1: Screening protocol for the T-> G polymorphism 3' of exon 9 A mismatch primer was designed which contained two mismatched base pairs four and five base pairs away from the 3' end of the primer and five and six base pairs from the polymoφhism. When incoφorated in a PCR product, this primer produces a BamHI cut when G is present and no cut when T is present at the polymoφhism site. This enables the two alleles to be distinguished by digestion of a PCR product with BamHI. Forward primer is 5' CACCCATTTACAAGTTTAGC 3' (SEQ ID NO: 17) and the reverse primer is 5' CACTGATTACTAATTCAGGATC 3' (SEQ JD NO: 18). This produces a PCR product of 200 bp which is cleaved by BamHI to produce fragments of 182 and 18 bp. DNA (50-100 ng) was used as a template in 25 μl reactions. The reaction mix consisted of the following concentrations: 0.2 mM dNTP's, 30 pM primer, IX TNK50 buffer, 0.5 U Taq DNA polymerase. PCR was carried out under the following conditions: 94°C for 5 minutes; 94°C for 30 seconds; 45°C for 30 seconds; 72°C for 30 seconds; x 35 cycles; 72°C for 3 minutes. 5 U of BamHI enzyme was added to the PCR products and digestion carried out at 37°C for 3 hours. The digested product was run on a 3% agarose gel which is sufficient to separate the digested products so that 200 bp and 182 bp bands can be distinguished. Example 2: Elucidation of a novel splice variant Expression of an additional form of the SI 82 protein containing an insertion of four amino acids at codons 26-27 (VRSQ) was found. The VRSQ insertion arises from alternative use of a 5' exon donor site in the exon 3/intron 3 (-52 to 75 nt) boundary. (See Figure 1). The ...CAG/gta... boundary of the final Gin codon of exon 3 of the VRSQ motif provides the ideal 5' exon AG donor site and GT intron consensus 5' boundary and use of this splice site results in the insertion of the 12-nts encoding the VRSQ motif. The upstream ...ACT/GTA... boundary of the Thr- Val codons provides the less preferred CT (AG preferred) 5' exonic boundary to the consensus GT 5' intronic boundary and splicing at this site would remove the VRSQ motif. Sherrington et al. Nature 1995, 375:754-760, reports the expression of only the VRSQ minus form. However, screening of fibroblasts with probes covering this region have shown that both splice variants are represented. Primers from the intronic region upstream (forward) and downstream exonic region of this alternate splicing sites allowed for their sequencing and elucidation. Example 3: Elucidation of a novel splicing between exon 8 and 9 A mutation has been found in an early onset AD family designated F74 (it may also occur in another family, F184) in the last nucleotide of the intron between exons 8 and 9, G -> T. This mutation spoils the acceptor site in the middle of Serine 857 and is expected to alter the splicing of this region. This mutation does not change the cDNA sequence and could, therefore, only be identified by sequencing of primers derived from intronic sequences. Example 4: Identification of E120K mutation (G -> A) Additionally, a novel mutation was identified in exon 5 of SI 82 at codon 120 (lys -> glu) arising from a G to A transition (GAA -> AAA). This mutation is near the second putative transmembrane domain (TM2). This mutation is virtually impossible to detect without intronic sequence primers, as it is within 20 bp of the intron-exon boundary in genomic clones. E120K was found by PCR. A 100 μl reaction mix contained water, 10X buffer, 10 mM dNTP's, taq polymerase and 20 μM primer (5'- CCCAACCATAAGAAGAACAG-3' (SEQ ID NO: 19) and 5'- GTGGTAATGTGGTTGGTGAT-3' (SEQ ID NO: 20)). The PCR conditions were 94°C for 5 minutes, (94°C for 30 seconds, 50°C for 30 seconds, 72°C for 45 seconds) x 35, 72°C for 10 minutes. The PCR products were electrophoresed on a 3% agarose gel and visualized by ethidium bromide. The biotinylated primer allows single stranded DNA to be derived using streptavidin-coated magnetic beads and sequenced on an ALF sequencer (Pharmacia) using Autoread kits (Pharmacia). Example 5: Identification of L250S mutation (T -> C) Family 184 was also screened for mutations by PCR. A 100 μl reaction mix contained water, 10X buffer, 5 mM dNTP's, taq polymerase and 20 μM primer (5'- AACAATGGTGTGGTTGGTGA-3' (SEQ ID NO: 21) and 5- AAGTTTTGACATTAAGAGCT-3' (SEQ TD NO: 22)). The PCR conditions were 94°C for 5 minutes, (94°C for 30 seconds, 50°C for 30 seconds, 72°C for 45 seconds) x 35, 72°C for 10 minutes. The PCR products were electrophoresed on a 3% agarose gel and visualized by ethidium bromide. The biotynilated primer allows single stranded DNA to be derived using streptavidin-coated magnetic beads and sequenced on an ALF sequencer (Pharmacia) using Autoread kits (Pharmacia). The base change T to C was found at codon 250 changing leucine to serine, by comparison with the normal PS 1 sequence. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Washington University School of Medicine and University of South Florida (ii) TITLE OF INVENTION: Method for Elucidation and Detection of Polymorphisms, Splice Variants and Proximal Coding Using Intronic Sequences of the Mutations Alzheimer's S182 Gene (iii) NUMBER OF SEQUENCES: 52 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: SmithKline Beecham Corporation (B) STREET: 709 Swedeland Road, P.O. Box 1539 (C) CITY: King of Prussia (D) STATE: PA (E) COUNTRY:USA (F) ZIP: 19406-0939 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: DISKETTE, 3.5 INCH, 1.44 Mb STORAGE (B) COMPUTER: IBM 486 (C) OPERATING SYSTEM: WINDOWS FOR WORKGROUPS (D) SOFTWARE: WORDPERFECT 5.1 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: not yet assigned (B) FILING DATE: Herewith (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 60/007,048 (B) FILING DATE: October 25, 1995 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: William T. Han (B) REGISTRATION NUMBER: 34,344 ( C ) REFERENCE /DOCKET NUMBER : P50388 ( 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: GTTTGTTTCT GCTTAATGTA 20 (2) INFORMATION FOR SEQ ID NO: 2: (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: 2: GTTTTTTCTT TCCCTTTTCA G 21 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTACAGTGT 9 (2) INFORMATION FOR SEQ ID NO: 4: (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: 4: TGTTTTTCTT GTGCTTATAG 20 (2) INFORMATION FOR SEQ ID NO: 5: (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: 5: ACGTATGAGA TTTGTTTT 18 (2) INFORMATION FOR SEQ ID NO: 6: (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: 6: TTGTGTTTGT TTTATTGTAG 20 (2) INFORMATION FOR SEQ ID NO: 7: (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: 7: GTGAGCATGA GACACAGATC 20 ( 2 ) INFORMATION FOR SEQ ID NO : 8 : ( 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: 8: TGAAATGCTT TCTTTTCTAG 20 (2) INFORMATION FOR SEQ ID NO: 9: (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: 9: GTAAAACCCA AGACTGATAA 20 (2) INFORMATION FOR SEQ ID NO: 10: (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: 10: TTATGTTTTT CTTTTTCTAG 20 (2) INFORMATION FOR SEQ ID NO: 11: (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: 11: GTATGTGCAT TTCTCTATGT 20 (2) INFORMATION FOR SEQ ID NO: 12: (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: 12: TTGTAACCTT TCCTTTTTAG 20 (2) INFORMATION FOR SEQ ID NO: 13: (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: 13: GTATGTGCAT TTCTCTATGT 20 (2) INFORMATION FOR SEQ ID NO: 14: (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: 14: TTGTAACCTT TCCTTTTTAG 20 (2) INFORMATION FOR SEQ ID NO: 15: (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: 15: GTAAGTATAC ACTAATAAGA 20 (2) INFORMATION FOR SEQ ID NO: 16: (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: 16: CTTTCCCATC TTCTCCACAG 20 (2) INFORMATION FOR SEQ ID NO: 17: (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: 17: CACCCATTTA CAAGTTTAGC 20 (2) INFORMATION FOR SEQ ID NO: 18: (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: 18: CACTGATTAC TAATTCAGGA TC 22 (2) INFORMATION FOR SEQ ID NO: 19: (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: 19: CCCAACCATA AGAAGAACAG 20 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Sing1e (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GTGGTAATGT GGTTGGTGAT 20 (2) INFORMATION FOR SEQ ID NO: 21: (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: 21: AACAATGGTG TGGTTGGTGA 20 (2) INFORMATION FOR SEQ ID NO: 22: (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: 22: AAGTTTTGAC ATTAAGAGCT 20 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: ATAAAGAAAG GTTTGTTTCT GCTTAATGTA 30 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: GTTTTTTCTT TCCCTTTTCA GAACCTCAAG A 31 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: AGCAATACTG TACGTAGCCA GGTACAGTGT 30 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: TGTTTTTCTT GTGCTTATAG AATGACAAT 29 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GGGCAGCTGT ACGTATGAGA TTTGTTTT 28 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: TTGTGTTTGT TTTATTGTAG AATCTATAC 29 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: TGCTATAAGG TGAGCATGAG ACACAGATC 29 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: TGAAATGCTT TCTTTTCTAG GTCATCCAT 29 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: TACTTGGGGT AAGTTGTGAA ATTTTTGG 28 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: TCTGTGTAAT TTTTTTTCAG GGAACTGTT 29 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: TCAGTATATG GTAAAACCCA AGACTGATAA 30 (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: TTATGTTTTT CTTTTTCTAG ATTTAGTGG 29 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: ATTTACTCCT GTAAGTATTT GAGAAGGATA 30 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: AATTTNGTCT TTCCCAACAG CAACAATG 28 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: AATGCAGAAA GTAGGTAACT TTTATTAGAT 30 (2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: ACTTCCACTT TCTCTTGAAG CACAGAAG 28 (2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39 CCAGAGGAAA GTATGTGCAT TTCTCTATGT 30 (2) INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: TTGTAACCTT TCCTTTTTAG GGGGAGTA 28 ( 2 ) INFORMATION FOR SEQ ID NO : 41 : ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH: 29 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: ATATTAATTG TAAGTATACA CTAATAAGA 29 (2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: CTTTCCCATC TTCTCCACAG GGTTTGTGC 29 (2) INFORMATION FOR SEQ ID NO: 43: (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: 43: ATTTACTCCT CAACAATG 18 (2) INFORMATION FOR SEQ ID NO: 44: (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: 44: AATGCAGAAA GCACAGAA 18 (2) INFORMATION FOR SEQ ID NO: 45: (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: 45: ATTTACTCCT GCACAGAA 18 (2) INFORMATION FOR SEQ ID NO: 46: (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: 46: CCAGAGGAAA GGGGAGTA 18 (2) INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: CAACAGCAAC AA 12 (2) INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: CAACATCAAC AA 12 (2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: ILE TYR SER SER THR MET 1 5 (2) INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: ASN ALA GLU SER THR GLU 1 5 (2) INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: ILE TYR SER CYS THR GLU 1 5 (2) INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS: (A ) LENGTH : 6 (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: PRO GLU GLU ARG GLY VAL 1 5

Claims: What is claimed; 1. A method of detecting polymoφhisms in the SI 82 gene comprising detecting mutations in the SI 82 gene using selected mismatched PCR primers comprising sequences derived from intronic sequences of the SI 82 gene. 2. The method of claim 1 wherein the mismatched PCR primers comprise SEQ ID NOS: 21 and 22. 3. The method of claim 1 wherein the mismatched PCR primers comprise SEQ TD NOS: 19 and 20. 4. The method of claim 1 wherein the mismatched PCR primers comprise SEQ TD NOS: 17 and 18. 5. A method of identifying an individual susceptible to late onset Alzheimer's Disease comprising: providing a sample of genetic material from an individual susceptible to late onset Alzheimer's Disease; and detecting mutations in the SI 82 gene in the sample of genetic material using selected mismatched PCR primers comprising sequences derived from intronic sequences of the S 182 gene. 6. The method of claim 5 wherein the mismatched PCR primers comprise SEQ ID NOS: 21 and 22. 7. The method of claim 5 wherein the mismatched PCR primers comprise SEQ ID NOS: 19 and 20. 8. The method of claim 5 wherein the mismatched PCR primers comprise SEQ ID NOS: 17 and 18.

Inventors: GOATE ALISON M, HARDY JOHN A, GOATE, ALISON, M., HARDY, JOHN, A.
Applicants: GOATE ALISON M, HARDY JOHN A, UNIV SOUTH FLORIDA, UNIV WASHINGTON, GOATE, ALISON, M., HARDY, JOHN, A., UNIVERSITY OF SOUTH FLORIDA, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE
Application year: 1996
Main classification: C12Q1
IPC codes: C12N15, C12Q1