WO1999022024A1 - Detection of neurodegenerative diseases - Google Patents

Detection of neurodegenerative diseases Download PDF

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Publication number
WO1999022024A1
WO1999022024A1 PCT/US1998/022502 US9822502W WO9922024A1 WO 1999022024 A1 WO1999022024 A1 WO 1999022024A1 US 9822502 W US9822502 W US 9822502W WO 9922024 A1 WO9922024 A1 WO 9922024A1
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disease
nucleic acid
alzheimer
acid molecule
replaced
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PCT/US1998/022502
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French (fr)
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Kwan-Fu Rex Sheu
John P. Blass
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Cornell Research Foundation, Inc.
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Priority to AU11975/99A priority Critical patent/AU1197599A/en
Publication of WO1999022024A1 publication Critical patent/WO1999022024A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention provides a method of detecting a predisposition to or a reduced risk for a neurodegenerative disease in a human by detecting polymorphisms which are associated with a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
  • Alzheimer's disease is a progressive neurodegenerative disorder affecting 7% of the population over 65 years of age and characterized clinically by progressive loss of intellectual function and pathologically by a continuing loss of neurons from the brain. More specifically, this pathological impairment usually is correlated with numbers of amyloid containing neuritic plaques in the neocortex and with the loss of presynaptic markers of cholinergic neurons. Neuritic plaques are composed of degenerating axons and nerve terminals, as well as possible astrocytic elements, and these plaques often exhibit a central core.
  • a neurofibrillary tangle is an intraneuronal mass composed of paired helical filaments having unusual properties, which twist and form tangles.
  • Neurofibrillary tangles are comprised of several different proteins.
  • Neurochemical studies show neurotransmitter systems are affected by Alzheimer's disease. The most consistent and severely affected system is that of the cholinergic neurons located in the Nucleus Basalis of Meynert. In addition, in Alzheimer's disease a reduction occurs in somatostatin, substance P and corticotropin releasing factor.
  • paired helical filaments the twisted filaments that form the tangles
  • other diseases such as Guam Parkinson Disease, Dementia Pugilistica and Progressive Supranuclear Palsy.
  • paired helical filaments the twisted filaments that form the tangles
  • other diseases such as Pick's Disease.
  • immunologic studies have shown that epitopes of paired helical filaments exist in Pick bodies, the spherical structures found in affected neurons in the temporal cortex of brains affected by Pick's Disease.
  • the densities of neurofibrillary tangles and neuritic plaques within the cerebral cortex of an Alzheimer's disease patient do not necessarily correlate with the stages of the illness.
  • Alzheimer's disease has been extremely difficult.
  • Ante-mortem diagnosis of the disease has been performed primarily by exclusion.
  • the problem in the diagnosis of Alzheimer's disease is that there is no positive test: the clinician has to rule out other causes of dementia such as strokes, microvascular disease, brain tumors, thyroid dysfunction, drug reactions, severe depression and a host of other conditions that can cause intellectual deficits in elderly people (Davies, "The Neurochemistry of Alzheimer's disease and Senile Dementia", Medicinal Res. Rev., 3(3):221-236 (1983)).
  • Post-mortem diagnosis of Alzheimer's disease has been based on determination of the number of neuritic plaques and tangles in brain tissue which has been stained to visualize these plaques and tangles.
  • diagnostic methods based on neurohistopathological studies, require extensive staining and microscopic examination of several brain sections.
  • the plaques and tangles also may occur in the brains of normal, elderly individuals or individuals with other diseases, a more definitive and reliable method for making the diagnosis on brain tissue is desirable.
  • AD Alzheimer's disease
  • low-density lipoprotein receptor-related protein Kang, D.E. et al., "Genetic Association of the Low-Density Lipoprotein Receptor-Related Protein Gene (LRP), an Apolipoprotein Receptor, with Late-Onset Alzheimer's Disease," Neurology. 49:56-61 (1997)).
  • LRP Low-Density Lipoprotein Receptor-Related Protein Gene
  • very low-density lipoprotein receptor Okuizumi, K. et al., “Genetic Association of the Very Low Density Lipoprotein (VLDL) Receptor Gene with Sporadic Alzheimer's Disease," Nature Genet., 1 1 :207-209 (1995)
  • ⁇ i-antichymotrypsin Kamboch, M.I.
  • Alzheimer's Disease Risk is Modified by ⁇ l-Antichymotrypsin Polymo ⁇ hism," Nature Genet., 10:486-488 (1995)) and a Japanese (Yoshiiwa, A. et al., ⁇ i-Antichymotrypsin as a Risk Modifier for Late-Onset Alzheimer's Disease in Japanese Apolipoprotein E e4 Allele Carriers," Ann. Neurol., 42:1 15-1 17 (1997)) cohort, but not in other Caucasian series (Haines, J.L. et al., "No Genetic Effect of Alpha 1 -antichymotrypsin in
  • Alzheimer's Disease Genomics, 33:53-56 (1996); Christie, J. et al., “Determination of the Alpha- 1 Anti-Chymotrypsin Polymo ⁇ hism in Alzheimer's Disease,” Alzheimer's Res., 2:201-204 (1996)). Whether these apparent discrepancies are due to as yet unrecognized differences in ethnicity or age or other factors in the populations studied is not known.
  • mtDNA mitochondrial DNA
  • DLST is located on chromosome 14q24.3 (Sherrington, R. et al., "Cloning of a Gene Bearing Missense Mutations in Early-Onset Familial Alzheimer's Disease,” Nature 375:754-760 (1995); Cruts, M. et al., “Genetic and Physical Characterization of the Early-Onset Alzheimer's Disease AD3 Locus on Chromosome 14q24.3," Hum. Mol. Genet.. 4:1355-1364 (1995)), and encodes the core dihydrolipoamide succinyltransferase enzyme of the ⁇ -ketoglutarate dehydrogenase complex (KGDHC) (Ali, G.
  • DLST was originally proposed as a candidate gene for AD because of extensive and robust evidence of mitochondrial abnormalities in AD (Blass, J.P., "Pathophysiology of the Alzheimer's Syndrome,” Neurology, 43(suppl. 4):S25-S38 (1993); Beal, M.F., “Age, Energy, and Oxidative Stress in Neurogenerative Diseases," Ann. Neurol., 38:357-366 (1995); Blass, J.P., “Energy/Glucose Metabolism in Neurodegenerative Diseases,” in Wasco, eds., Molecular Mechanisms of Dementia, Towana, NJ:Humana Press, pp.
  • DLST Succinyltransferase
  • a method of screening patients early in the development of Alzheimer's disease utilizing a small sample of body fluid or tissue pre mortem is particularly needed.
  • Genetic markers are desired which are associated with Alzheimer's disease, especially markers which can be used to predict Alzheimer's disease in subjects in whom APOE4 is a relatively weak genetic risk factor.
  • the present invention provides a method of detecting a predisposition to a neurodegenerative disease in a human.
  • a biological sample is obtained from the subject.
  • genetic material in the biological sample is tested for the presence of a polymo ⁇ hism in a gene encoding a Krebs tricarboxylic acid cycle component, wherein the presence of said polymo ⁇ hism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
  • the present invention also provides isolated nucleic acid molecules, or fragment thereof, which encodes a Krebs tricarboxylic acid cycle component, or fragment thereof, and has a polymo ⁇ hism, wherein the presence of said polymo ⁇ hism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
  • the invention further provides a kit for identifying individuals with a predisposition to Alzheimer's disease or another neurodegenerative disease.
  • the kit contains nucleic acid fragments which can be used to amplify a gene encoding a Krebs tricarboxylic acid cycle component, or a fragment thereof, which consists of a position where a polymo ⁇ hism may be located, wherein the presence of said polymo ⁇ hism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
  • the invention provides a kit for identifying individuals with a predisposition to Alzheimer's disease or another neurodegenerative disease, having a restriction enzyme which may be used to detect the presence of a polymo ⁇ hism in a gene encoding a Krebs tricarboxylic acid cycle component, wherein the presence of said polymo ⁇ hism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
  • the invention also provides a method of detecting a predisposition to a neurodegenerative disease in a human subject.
  • a biological sample is obtained from the human subject.
  • the biological sample is contacted with an antibody which preferentially recognizes a Fvrebs tricarboxylic acid cycle component protein, which is encoded by a gene having a polymo ⁇ hism that is indicative of a predisposition or reduced risk for a neurodegenerative disease.
  • an antibody which preferentially recognizes a Fvrebs tricarboxylic acid cycle component protein, which is encoded by a gene having a polymo ⁇ hism that is indicative of a predisposition or reduced risk for a neurodegenerative disease.
  • a Krebs tricarboxylic acid cycle component protein Binding of the antibody to the Krebs tricarboxylic acid cycle component indicates that the polymo ⁇ hism is present which is indicative of a predisposition or reduced risk for a neurodegenerative disease.
  • Figure 1 depicts the polymo ⁇ hisms and expected RFLP of DLST A19,l 17G and T19.183C.
  • the DLST polymo ⁇ hisms A19,l 17G and T19.183C confer restriction sites of Ms el and Acil, respectively.
  • DLST haplotypes consisting of these two polymo ⁇ hisms can be differentiated by RFLP by simultaneous digestion with Msel and Acil of the PCR- amplified DLST fragments #18,747-19,272.
  • the expected restriction fragments for each of the four possible alleles are shown, in which the 230 bp fragment (nucleotide 18,747- 18,976) is common for all alleles.
  • DLST The numbering of DLST described here starts from the first base of exon 1. This base corresponds to base #748 of SEQ. ID. No. 2 (Nakano et al., "Isolation, Characterization and Structural Organization of the Gene and Pseudogene for the Dihydrolipoamide Succinyltransferase Component of the Human 2-Oxoglutarate Dehydrogenase Complex," Eur. J. Biochem.. 224(1):179-189 (1994)).
  • Figure 2 shows the RFLP analysis of DLST genotypes consisting of A 19,117G and T19,183C.
  • Figure 2A shows the RFLP of the six common DLST genotypes.
  • Figure 2B shows that this 296 bp, G,C/A,T-associated DNA is a heterodimer consisting of strands from each of the G,C and A,T alleles.
  • This fragment was extracted from the gel, and PCR-amplified with the forward primer corresponding to nucleotides 18,985 - 19,005 (5' TTATACAGATGGGGGTCTCAC 3') (SEQ. ID. No. 1) and the reverse primer, 19,252 - 19,272 (5' ATCCCCAGGATGGCAGACT 3') (SEQ. ID. No. 2).
  • the PCR was run for 15 cycles with an annealing temperature of 55°C, and the product was then double-digested with Ms el and Acil.
  • the resulting RFLP reproduced the typical restriction pattern of G,C/A,T, including the presence of the 288 bp heterodimer.
  • the present invention provides a method of detecting a predisposition to a neurodegenerative disease in a human subject.
  • a biological sample is obtained from the subject.
  • the sample is then tested for the presence of a polymo ⁇ hism in a gene encoding a Krebs tricarboxylic acid cycle component, wherein the presence of said polymo ⁇ hism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
  • Preferred neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, are related neurological diseases.
  • the testing of the genetic material in the biological sample is carried out by amplifying a region of a gene encoding a Krebs tricarboxylic acid cycle component to provide an amplified fragment.
  • the resulting amplified fragment is then used in order to detect the presence of a polymo ⁇ hism.
  • the biological sample is blood, saliva, cheek scrapings, or urine.
  • DLST dihydrolipoamide succinyltransferase
  • SEQ. ID. No. 3 The sequence of the human gene encoding dihydrolipoamide succinyltransferase (“DLST”) follows (SEQ. ID. No. 3) (Nakano,K.,et al., "Isolation, Characterization And Structural Organization Of The Gene And Pseudogene For The Dihydrolipoamide Succinyltransferase Component Of The Human 2-Oxoglutarate Dehydrogenase Complex," Eur. J. Biochem. 224(1): 179- 189 (1994), which is hereby inco ⁇ orated by reference):
  • AAAAATTCAA AAAGTAAAAA GGGTGTCCAT GAGAAGACTT TTTTATCCTT GTCCTCTAGC 6481 CCCCCGAATG CCTCTTGGGT AGTGTGGCCA CCACTCCGGG TTCCTGGATG ACTTGCAGAG
  • the DLST gene sequence includes approximately on kilobase of upstream sequence priro to the first exon.
  • the gene consists of 15 exons and 14 introns.
  • the amino acid sequence for human dihydrolipoamide succinyltransferase encoded by the intronsis as follows (SEQ. ID. No. 4):
  • the present invention provides a number of polymo ⁇ hisms win the DLST gene which are associated with Alzheimer's disease.
  • the sequence and numbering is based on GenBank Accession # D26535 (Nakano.K.
  • Homozygosity of the haplotypic genotype G (19864 in SEQ. ID. No. 3, C (19930 in SEQ. ID. No. 3) enhances the risk of the epsilon-4 allele of the apolipoprotein E gene for Alzheimer's disease in elderly Ashkenazi Jews.
  • homozygosity of the genotype A (19864 in SEQ. ID. No. 3)
  • T (19930 in SEQ. ID. No. 3) reduced the risk of Alzheimer's disease.
  • DLD dihydrolipoamide dehydrogenase
  • SEQ. ID. No. 5 The sequence of the human gene encoding for DLD is provided below as SEQ. ID. No. 5 (Pons, G. et al., "Cloning And cDNA Sequence Of The Dihydrolipoamide Dehydrogenase Component Of Human Alpha- Ketoacid
  • amino acid sequence for dihydrolipoamide dehydrogenase is as follows (SEQ. ID. No. 6):
  • the nucleotide sequence in SEQ. ID. No. 4 is for a cDNA encoding the DLD gene, thus no introns or sequences upstream of the transcriptional start site are included.
  • the present invention provides nucleotide polymo ⁇ hisms which have been identified within the DLD gene.
  • the nucleic acid molecule has a T replaced by A at nucleotide 1598 in SEQ. ID. No. 5.
  • the nucleic acid molecule has a G replaced by T at nucleotide 1608 in SEQ. ID. No. 5.
  • the most obvious method of characterizing a polymo ⁇ hism entails direct DNA sequencing of the genetic locus that flanks and includes the polymo ⁇ hism. Such analysis can be accomplished using either the "dideoxy-mediated chain termination method,” also known as the “Sanger Method” (Sanger, F. et al., “DNA Sequencing with Chain- Terminating Inhibitors,” Pro Natl. Acad. Sci. U.S.A.. 74:5463-5467 (1977), which is hereby inco ⁇ orated by reference) or the “chemical degradation method,” also known as the “Maxam-Gilbert method” (Maxam, A.M. et al., "A New Method for Sequencing
  • Genomic sequence-specific amplification technologies such as the polymerase chain reaction (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51 :263-273 (1986); Erlich, H. et al, European Patent Appln. 50,424; European Patent Appln. 84,796, European Patent Appln. 258,017, and European Patent Appln. 237,362; Mullis, K., European Patent Appln. 201,184; Mullis K. et al., U.S. Pat. No.
  • Amplification of a selected, or target, nucleic acid sequence may be carried out by any suitable means, either to facilitate sequencing or for direct dtection of polymo ⁇ hisms. (See generally Kwoh, D.
  • amplification techniques include, but are not limited to, polymerase chain reaction, ligase chain reaction ("LCR")(see Barany, Proc Nat'l Acad. Sci. U.S.A. 88:189 (1991), which is hereby incorporated by reference), strand displacement amplification (see generally Walker, G. et al., Nucleic Acids Res. 20, 1691 (1992); Walker. G. et al., Proc Nat'l Acad. Sci. U.S.A.
  • LCR ligase chain reaction
  • nucleic acid sequence-based amplification or "NASBA”
  • RCR repair chain reaction
  • BDA boomerang DNA amplification
  • DNA amplification techniques such as the foregoing involve the use of a probe, a pair of probes, or two pairs of probes which specifically bind to DNA encoding the gene of interest, but do not bind to DNA which do not encode the gene, under the same hybridization conditions, and which serve as the primer or primers for the amplification of the gene of interest or a portion thereof in the amplification reaction.
  • Mundy, et al. U.S. Pat. No. 4,656,127, which is hereby inco ⁇ orated by reference
  • McCy's methods employ a specialized exonuclease-resistant nucleotide derivative.
  • a primer complementary to the allelic sequence immediately 3'-to the polymo ⁇ hic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymo ⁇ hic site on the target molecule contains a nucleotide that is complementary to the particular exonucleotide-resistant nucleotide derivative present, then that derivative will be inco ⁇ orated by a polymerase onto the end of the hybridized primer. Such inco ⁇ oration renders the primer resistant to exonuclease, and thereby permits its detection.
  • the Mundy method has the advantage that it does not require the determination of large amounts of extraneous sequence data. It has the disadvantages of destroying the amplified target sequences and unmodified primer and of being extremely sensitive to the rate of polymerase inco ⁇ oration of the specific exonuclease-resistant nucleotide being used.
  • the method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymo ⁇ hic site will become inco ⁇ orated onto the terminus of the primer.
  • Goelet, P. et al. PCT Appln. No. 92/15712, which is hereby inco ⁇ orated by reference.
  • the method of Goelet, P. et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymo ⁇ hic site.
  • the labeled terminator that is inco ⁇ orated is thus determined by, and complementary to, the nucleotide present in the polymo ⁇ hic site of the target molecule being evaluated.
  • Cohen et al. Fernch Patent 2,650,840; PCT Appln. No.
  • the method of Goelet, P. et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase. It is thus easier to perform, and more accurate than the method discussed by Cohen.
  • OLA Oligonucleotide Ligation Assay
  • the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or. another biotin ligand.
  • OLA is capable of detecting point mutations. Nickerson, D.A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D.A. et al., Proc Natl. Acad. Sci. U.S.A.. 87:8923-8927 (1990), which is hereby inco ⁇ orated by reference).
  • PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
  • Assays such as the OLA, require that each candidate dNTP of a polymo ⁇ hism be separately examined, using a separate set of oligonucleotides for each dNTP.
  • the major drawback of OLA is that ligation is not a highly discriminating process and non-specific signals can be a significant problem.
  • DRED differential restriction endonuclease digestion
  • ASOP allele-specific oligonucleotide probing
  • LMGD ligase-mediated gene detection
  • oligonucleotide probes are synthesized that will hybridize, under appropriate annealing conditions, exclusively to a particular amplified cDNA segment that contains a nucleotide sequence that distinguishes one allele from other alleles of a red blood cell or platelet membrane glycoprotein.
  • This specific probe is discernably labeled so that when it hybridizes to the allele distinguishing cDNA segment, it can be detected, and the specific allele is thus identified.
  • LMGD oligonucleotide probes
  • a pair of oligonucleotide probes are synthesized that will hybridize adjacently to each other, i.e., to a cDNA segment under appropriate annealing conditions, at the specific nucleotide that distinguishes one allele from other alleles of a red blood cell or platelet membrane glycoprotein.
  • Each of the pair of specific probes is labeled in a different manner, and when it hybridizes to the allele-distinguishing cDNA segment, both probes can be ligated together by the addition of a ligase.
  • both types of labeling can be observed together, confirming the presence of the allele-specific nucleotide sequence.
  • the probe pair is not ligatable and, after the probes are isolated from the cDNA segments, both types of labeling are observed separately.
  • the invention in addition to methods for determining whether a patient is at risk for Alzheimer's disease or some other neurodegenerative disorder, also provides isolated nucleic acid molecules which encode a gene encoding a Krebs tricarboxylic acid cycle component, or a fragment of the gene, and a polymo ⁇ hism in, wherein the presence of said polymo ⁇ hism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
  • the present invention also provides fragments of the nucleic acid molecules.
  • fragments of the nucleic acid molecules can be used to hybridize to target nucleic acid molecules to detect the presence of a polymo ⁇ hism.
  • the fragments must be long enough to be useful as a primer in a PCR or LCR type reaction.
  • Preferred fragments are at least twelve bases in length.
  • the nucleic acid molecules or fragments need not be identical to the sequences of
  • nucleic acid molecule needs to have the polymo ⁇ hism and sufficient identity to the remainder of sequence of SEQ. ID. No. 3 or 5 so that the nucleic acid molecule or fragment may be used to differentiate between genetic material having the polymo ⁇ hism and genetic material lacking the polymo ⁇ hism.
  • nucleic acid molecules of the present invention may be linked to other nucleic acid molecules such as vectors or tags to facilitate amplification, purification, or identification.
  • the present invention also provides a kit for identifying individuals with a predisposition to Alzheimer ' s disease or another neurodegenerative, disease.
  • the kit contains nucleic acid fragments which can be used to amplify a gene encoding a Krebs tricarboxylic acid cycle component, or a fragment thereof, which consists of a position where a polymo ⁇ hism may be located, wherein the presence of said polymo ⁇ hism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
  • the invention provides a kit for identifying individuals with a predisposition to Alzheimer's disease or another neurodegenerative disease which contains a restriction enzyme which may be used to detect the presence of a polymorphism in a gene encoding a Krebs tricarboxylic acid cycle component, wherein the presence of said polymo ⁇ hism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
  • a kit utilizing a restriction enzyme analysis also contains nucleic acid fragments which can be used to amplify the gene encoding a Krebs tricarboxylic acid cycle component, or a fragment thereof, which consists of a position where a polymo ⁇ hism may be located.
  • the present invention also provides antibodies which preferentially recognizes a Krebs tricarboxylic acid cycle component protein which is encoded by a gene consisting of a polymo ⁇ hism, wherein the presence of said polymo ⁇ hism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
  • polymo ⁇ hisms will generate amino acid substitutions which can be detected by immunochemical methods.
  • one allele has a substitution where amino acids Ala-Pro are replaced by Arg-Ser at residues 14-15. Another substitution results in the replacement of Thr by Arg at residue 312.
  • antibodies may be used to detect changes in the DLD protein which result from polymorphisms in the DLD gene.
  • Monoclonal antibody production may be effected by techniques which are well- known in the art. Basically, the process involves first obtaining immune cells
  • lymphocytes from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro.
  • the antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line.
  • the resulting fused cells, or hybridomas are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned and grown either in vivo or in vitro to produce large quantities of antibody.
  • a description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler et al., Nature, 256:495 (1975). which is hereby inco ⁇ orated by reference.
  • Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.
  • Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol ("PEG") or other fusing agents (See Milstein et al., Eur. J. Immunol.. 6:511 (1976), which is hereby inco ⁇ orated by reference).
  • PEG polyethylene glycol
  • This immortal cell line which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.
  • Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum.
  • the antigens can be injected at a total volume of 100 ml per site at six different sites. Each injected material will contain adjuvants with or without pulverized acrylamide gel containing the protein or polypeptide after SDS- polyacrylamide gel electrophoresis.
  • the rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost.
  • Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody.
  • This and other procedures for raising polyclonal antibodies are disclosed in E. Harlow et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby inco ⁇ orated by reference.
  • the processes of the present invention encompass use of binding portions of such antibodies.
  • binding portions include Fab fragments, F(ab')2 fragments, and Fv fragments.
  • These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118, New York: Academic Press (1983), which is hereby inco ⁇ orated by reference.
  • the antibodies can be used to screen biological samples from the individual for the presence of altered proteins which are associated with a predisposition or reduced risk for Alzheimer's disease or another neurodegenerative disorder.
  • Antibodies which selectively bind a polymo ⁇ hic DLST or DLD isoform may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation.
  • Antibodies which bind a polymorphic DLST or DLD isoform may likewise be conjugated to detectable groups such as radiolabels (e.g., 35S, 1251, 1311), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
  • radiolabels e.g., 35S, 1251, 1311
  • enzyme labels e.g., horseradish peroxidase, alkaline phosphatase
  • fluorescent labels e.g., fluorescein
  • the invention also provides a method of detecting a predisposition to a neurodegenerative disease in a human subject.
  • a biological sample is obtained from the human subject.
  • the biological sample is contacted with an antibody which preferentially recognizes a Krebs tricarboxylic acid cycle component protein, which is encoded by a gene having a polymo ⁇ hism that is indicative of a predisposition or reduced risk for a neurodegenerative disease.
  • an antibody which preferentially recognizes a Krebs tricarboxylic acid cycle component protein, which is encoded by a gene having a polymo ⁇ hism that is indicative of a predisposition or reduced risk for a neurodegenerative disease.
  • Binding of the antibody to the Krebs tricarboxylic acid cycle component indicates that the polymo ⁇ hism is present which is indicative of a predisposition or reduced risk for a neurodegenerative disease.
  • Detecting the presence of a complex between an antibody or binding portion thereof and protein encoded by a gene carrying a polymo ⁇ hism associated with a neuro degenerative disease can be carried out by any conventional method for detecting antigen- antibody reactions, examples of which can be found, e.g., in Klein, Immunology, New York:John Wiley & Sons, pp. 394-407 (1982), which is hereby inco ⁇ orated by reference.
  • a ploymo ⁇ hism the formation of a complex between the antibody and the protein present in the tissue of fluid sample can be detected by enzyme linked assays, such as ELISA assays.
  • the antibody/protein complex is contacted with a second antibody which recognizes a portion of the antibody that is complexed with the protein.
  • the second antibody is labeled so that its presence (and, thus, the presence of an anntibody /protein complex) can be detected.
  • the antibody or binding portion thereof can be bound to a label effective to permit detection of the protein upon binding of the antibody or binding portion thereof to the protein.
  • Suitable labels include, fluorophores, chromophores, radiolabels, and the like.
  • a radiolabeled antibody or binding portion thereof of this invention can be used for in vitro diagnostic tests.
  • the specific activity of a tagged antibody or binding portion thereof depends upon the half-life and isotopic purity of the radioactive label and how the label is inco ⁇ orated into the antibody or its binding portion. In immunoassay tests, the higher the specific activity, in general, the better the sensitivity.
  • the antibodies or binding portions thereof of the present invention can be used and sold together with equipment to detect the particular label as a kit for in vitro detection of polymo ⁇ hisms associated with neurodegenerative diseases.
  • DNA was prepared with the Easy-DNA kit (Invitrogen, Carlsbad, CA).
  • a DLST fragment was PCR amplified using the forward primer 5' CTGTCACACGGGCTAGCG 3' (SEQ. ID. No. 9) (nt 18,747-18,776) and the reverse primer 5' ATCCCCAGGATGGCAGACT 3' (SEQ. ID. No. 2) (nt 19,252-19,272).
  • PCR components were as above.
  • the PCR ran for 30 cycles with an annealing temperature of 58°C.
  • the sequencing reaction used the forward primer end- labeled with the infrared dye IRD41 (Li-Co ⁇ , Lincoln, NE) and a Li-Cor Model 4000 DNA sequencer.
  • Each of the DLST genotypes corresponded to specific patterns of restriction fragments (Figure 2A), including a 296 bp fragment found consistently and exclusively in genotype G,C/A,T.
  • This 296-bp DNA was a heteroduplex consisting of a G,C and an A,T strands, since this fragment was also detected in DNA mixtures form G,C/G,C and A.T/A.T (far left lanes, Fig. 2A), but not in either of the individual homozygotes.
  • this 296-bp fragment was gel-purified, PCR amplified, and digested with Mvel and Acil, a restriction pattern consistent with A,T/G,C was reproduced ( Figure 2B).
  • Genotypes of APOE were determined by PCR-RFLP (Hixon, J.E. et al., "Restriction Isotyping of Human Apolipoprotein E by Gene Amplification and Cleavage with H/z ⁇ rl," J. Lipid Res., 31 :545-548 (1990), which is hereby inco ⁇ orated by reference).
  • DLST Dihydrolipoyl Succinyltransferase
  • the autopsy series included neuropathologic cases from the Caucasian Jewish cohort and an additional 72 AD and 8 non-AD Caucasian subjects who were autopsy diagnosed.
  • the G19,l 17 was found only in the G,C allele, since no G.T allele was found. Therefore, the effect of homozygosity for the G,C allele (Tables 2, 3 & 4) can as well be attributed to homozygosity for G19,l 17. No relationship was found between AD and DLST genotypes in patients who were APOE4 negative, nor in the overall series regardless of APOE4 status.
  • Example 3 A DLST Genotype Associated with a Reduced Risk for Alzheimer's Disease
  • AD Alzheimer's disease
  • the most common genetic risk factor for AD is the APOE4 allele (Farrer, L.A. et al., "Effects of Age, Sex and Ethnicity on the Association Between Apolipoprotein E Genotype and Alzheimer's Disease,” J. Am. Med. Assoc, 278:1349-1356 (1997); Tang, M.X. et al., "The APOE-e4 Allele and the Risk of Alzheimer Disease Among Africans, Whites and Hispanics,” J. Am. Med. Assoc, 279:751-755 (1998), which are hereby incorporated by reference).
  • DLST genotypes are polymo ⁇ hisms consisting of A19,l 17G and T19,183C.
  • Mechanisms linking effects of DLST and APOE4 in the pathogenesis of AD may involve oxidative stress-related reactive oxygen species and other reactive compounds (Beal, M.F., "Age, Energy, and Oxidative Stress in Neurodegenerative Diseases," Ann. Neurol., 38:357-366 (1995); Blass, J.P., "Energy/Glucose Metabolism in
  • the mechanisms by which the two base pair substitutions studied above might affect the expression of the DLST protein are as yet undetermined.
  • the C19,183T substitution in exon 14 is neutral - both forms encode a glycine (codon 366).
  • a direct effect of that base substitution at this site is hard to explain.
  • the A 19,117G substitution in intron 13 occurs at a potential "branch point" that could affect the excision of introns during procession of mRNA. If this base substitution is pathogenetically related to the disease, the question of why G19,l 17 appears deleterious in Caucasian (Jewish) populations and Al 9,117 in Japanese populations will need to be answered.
  • the genetic evidence is consistent with a mutation in either DLST or a neighboring gene.
  • the likelihood of a pathogenic mutation within DLST is increased by the robust biochemical evidence for the deficiency of KGDHC in AD (Gibson, G.E. et al., "Reduced Activities of Thiamine-Dependent Enzymes in the Brains and Peripheral Tissues of Patients with Alzheimer's Disease," Arch. Neurol., 45:836-840 (1998); Butterworth, R.F. et al, "Thiamine-Dependent Enzyme Changes in Temporal Cortex of Patients with Alzheimer's Disease," Metab.

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Abstract

The present invention provides methods for determining whether an individual is predisposed to Alzheimer's disease or other neurodegenerative disease in a human. Nucleic acid molecules are also provided which have a polymorphism which is associated with a predisposition or reduced risk for Alzheimer's disease or another neurodegenerative disease. The present invention further provides kits which can be used to detect the presence of polymorphisms associated with Alzheimer's disease or neurodegenerative disorders. The present invention also provides antibodies which preferentially recognizes a variant form of a Krebs tricarboxylic acid cycle component protein which is encoded by a gene consisting of a polymorphism and methods of using the antibodies, wherein the presence of said variant form is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.

Description

DETECTION OF NEURODEGENERATIVE DISEASES
The subject matter of this application was made with support from the United States Government under Grant No. AG09014 from the National Institutes of Health. The United States Government may retain certain rights.
The present application claims the benefit of U.S. Provisional Patent Application Serial No. 60/063,105, filed October 24, 1997.
FIELD OF THE INVENTION
The present invention provides a method of detecting a predisposition to or a reduced risk for a neurodegenerative disease in a human by detecting polymorphisms which are associated with a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
BACKGROUND OF THE INVENTION
Alzheimer's disease is a progressive neurodegenerative disorder affecting 7% of the population over 65 years of age and characterized clinically by progressive loss of intellectual function and pathologically by a continuing loss of neurons from the brain. More specifically, this pathological impairment usually is correlated with numbers of amyloid containing neuritic plaques in the neocortex and with the loss of presynaptic markers of cholinergic neurons. Neuritic plaques are composed of degenerating axons and nerve terminals, as well as possible astrocytic elements, and these plaques often exhibit a central core.
Another characteristic pathological feature of Alzheimer's disease is development of neurofibrillary tangles. A neurofibrillary tangle is an intraneuronal mass composed of paired helical filaments having unusual properties, which twist and form tangles. Neurofibrillary tangles are comprised of several different proteins.
Neurochemical studies show neurotransmitter systems are affected by Alzheimer's disease. The most consistent and severely affected system is that of the cholinergic neurons located in the Nucleus Basalis of Meynert. In addition, in Alzheimer's disease a reduction occurs in somatostatin, substance P and corticotropin releasing factor.
None of the above-mentioned pathologic structures, neurochemical alterations, neuritic plaques or neurofibrillary tangles are unique to Alzheimer's disease. These impairments also occur in the brains of normal aged individuals and are associated with other diseases such as Guam Parkinson Disease, Dementia Pugilistica and Progressive Supranuclear Palsy. For example, paired helical filaments, the twisted filaments that form the tangles, also occur in certain tangles associated with other diseases such as Pick's Disease. In fact, immunologic studies have shown that epitopes of paired helical filaments exist in Pick bodies, the spherical structures found in affected neurons in the temporal cortex of brains affected by Pick's Disease. In addition, the densities of neurofibrillary tangles and neuritic plaques within the cerebral cortex of an Alzheimer's disease patient do not necessarily correlate with the stages of the illness.
Accordingly, prior hereto, the diagnosis of Alzheimer's disease has been extremely difficult. Ante-mortem diagnosis of the disease has been performed primarily by exclusion. The problem in the diagnosis of Alzheimer's disease is that there is no positive test: the clinician has to rule out other causes of dementia such as strokes, microvascular disease, brain tumors, thyroid dysfunction, drug reactions, severe depression and a host of other conditions that can cause intellectual deficits in elderly people (Davies, "The Neurochemistry of Alzheimer's disease and Senile Dementia", Medicinal Res. Rev., 3(3):221-236 (1983)).
Post-mortem diagnosis of Alzheimer's disease has been based on determination of the number of neuritic plaques and tangles in brain tissue which has been stained to visualize these plaques and tangles. However, such diagnostic methods, based on neurohistopathological studies, require extensive staining and microscopic examination of several brain sections. Moreover, since the plaques and tangles also may occur in the brains of normal, elderly individuals or individuals with other diseases, a more definitive and reliable method for making the diagnosis on brain tissue is desirable.
As disclosed in U.S. Pat. No. 4,666,829 issued to Glenner et al., attempts have been made to identify the presence of an antigen specific for Alzheimer's disease.
However, the antigen described by Glenner et al. is present in adults of advanced age who do not have Alzheimer's disease (see Ghanbari et al., J. Amer. Med. Assoc, 263:2907- 2910 (1990)). j -
The genetics of Alzheimer's disease (AD) is complex (Wasco, W. et al., "E hiologic Clues from Gene Defects Causing Early Onset familial Alzheimer's Disease," Molecular Mechanisms of Dementia, 1-19 (1997); Roses, "Apolipoprotein E Alleles as Risk Factors in Alzheimer's Disease," Annu. Rev. Med., 47:387-400 (1996)). Three genes have been described in the relatively rare, early onset, forms of AD (APP, PS\ and PS2). In the common, late-onset AD, the major known genetic risk factor is the e4 allele of the APOE gene (APOE4) (Saunders, A.M. et al., "Association of Apolipoprotein E Allele e4 with Late-Onset Familial and Sporadic Alzheimer's Disease," Neurology, 43:1467-1472 (1993); Farrer, L.A. et al., "Effects of Age, Sex, and Ethnicity on the Association Between Apolipoprotein E Genotype and Alzheimer's
Disease - a Meta-analysis," J. Am. Med. Assoc, 278:1349-1356 (1997)). However, at least half of the people who develop AD do not carry this allele, and not all people who do carry this allele develop AD even if they live to old age - not even many of those homozygous for ^4POE4. The association of APOE4 with AD appears to be strongest in patients with an onset prior to 70 years of age (Farrer, L.A. et al., "Effects of Age, Sex, and Ethnicity on the Association Between Apolipoprotein E Genotype and Alzheimer's Disease - a Meta-analysis," J. Am. Med. Assoc, 278:1349-1356 (1997); Blacker, D. et al., "ApoE-4 and Age At Onset of Alzheimer's Disease: The NIMH Genetics Initiative," Neurology, 48:139-147 (1997)). The association weakens with advanced age. Ethnic background also influences the association of APOE4 with AD (Farrer, L.A. et al., "Effects of Age, Sex, and Ethnicity on the Association Between Apolipoprotein E Genotype and Alzheimer's Disease - a Meta-analysis," J. Am. Med. Assoc, 278:1349- 1356 (1997); Maestre, G. et al., "Apolipoprotein E and Alzheimer's Disease: Ethnic Variation in Genotypic Risk," Ann. Neurol., 37:254-259 (1995)). These observations suggest that other genes might mediate the penetrance of APOE4 as a risk factor for AD. A number of other genes have been proposed to be risk factors for AD or to modify the association with APOE4. They include genes for butyrocholinesterase (Lehmann. D. J. et al., "Synergy Between the Genes for Butylcholinesterase K Variant and Apolipoprotein E4 in Late-Onset, Confirmed Alzheimer's Disease," Human Mol. Genet.. 6:1933-1936 (1997)), low-density lipoprotein receptor-related protein (Kang, D.E. et al., "Genetic Association of the Low-Density Lipoprotein Receptor-Related Protein Gene (LRP), an Apolipoprotein Receptor, with Late-Onset Alzheimer's Disease," Neurology. 49:56-61 (1997)). very low-density lipoprotein receptor (Okuizumi, K. et al., "Genetic Association of the Very Low Density Lipoprotein (VLDL) Receptor Gene with Sporadic Alzheimer's Disease," Nature Genet., 1 1 :207-209 (1995)), αi-antichymotrypsin (Kamboch, M.I. et al., "APOE*4-associated Alzheimer's Disease Risk is Modified by αl-Antichymotrypsin Polymorphism," Nature Genet., 10:486-488 (1995); Yoshiiwa, A. et al., αi-Antichymotrypsin as a Risk Modifier for Late-Onset Alzheimer's Disease in Japanese Apolipoprotein E e4 Allele Carriers," Ann. Neurol., 42:1 15-1 17 (1997)), and perhaps a polymoφhism within intron 8 of the PS1 gene (Wragg, M. et al., "Genetic Association Between Intronic Polymorphism in Presenilin-1 Gene and Late-Onset Alzheimer's Disease," Lancet, 347:509-512 (1996); Scott, W.K. et al., "No Association or Linkage Between an Intronic Polymorphism of Presenilin-1 and Sporadic or Late- Onset Familial Alzheimer's Disease," Genet. Epidemiol.. 14:307-315 (1997)). These findings, however, have not been consistently replicated in independent series. For instance, an association of polymoφhism of the αi-antichymotrypsin gene (Ala to Thr at position -15 in the signal peptide) with AD, particularly in the APOE4 positive subjects, was found in an American Caucasian (Kamboch, M.I. et al., "APOE*4-associated
Alzheimer's Disease Risk is Modified by αl-Antichymotrypsin Polymoφhism," Nature Genet., 10:486-488 (1995)) and a Japanese (Yoshiiwa, A. et al., αi-Antichymotrypsin as a Risk Modifier for Late-Onset Alzheimer's Disease in Japanese Apolipoprotein E e4 Allele Carriers," Ann. Neurol., 42:1 15-1 17 (1997)) cohort, but not in other Caucasian series (Haines, J.L. et al., "No Genetic Effect of Alpha 1 -antichymotrypsin in
Alzheimer's Disease," Genomics, 33:53-56 (1996); Christie, J. et al., "Determination of the Alpha- 1 Anti-Chymotrypsin Polymoφhism in Alzheimer's Disease," Alzheimer's Res., 2:201-204 (1996)). Whether these apparent discrepancies are due to as yet unrecognized differences in ethnicity or age or other factors in the populations studied is not known. A number of variations in mitochondrial DNA (mtDNA) have been reported to be associated with AD (Schoffner, J.M. et al., "Mitochondrial DNA Variants Observed in Alzheimer Disease and Parkinson Disease Patients," Genomics, 17:171-184 (1993); Davis, R.E. et al., "Mutations in Mitochondrial Cytochrome C Oxidase Genes Segregate With Late-Onset Alzheimer's Disease," Pro Natl. Acad. Sci. U.S.A.. 94:4526-4531 (1997)), but these results are in general still controversial (Wragg, M.A. et al., "No
Association Found Between Alzheimer's Disease and a Mitochondrial tRNA Glutamine Gene Variant," Neurosci. Lett., 201 : 107-110 (1997); Hirano, M. et al., "Apparent mtDNA Heteroplasimy in Alzheimer's Disease Patients and in Normals Due to PCR amplification of Nucleus-Embedded mtDNA Pseudogenes," Proc. Natl. Acad. Sci. U.S.A., 94:14894- 14899 (1977); Wallace, D. C. et al., "Ancient mtDNA Sequences in the Human Nuclear Genome: A Potential Source of Errors in Identifying pathogenic Mutations," Proc .Natl. Acad. Sci. U.S.A., 94:14900-14905 (1997)).
An association of the DLST gene with Alzheimer's disease has been reported (Sheu, K-FR et al., "A Gene Locus of Dihydrolipoyl Succinyltransferase (DLST) is Associated with Alzheimer's Disease," J. Neurochem.. 66 (Suppl 1) S10B (1996); Sheu K-FR et al.. "A Genetic Association of the Dihydrolipoyl Succinyltransferase Gene with Alzheimer's Disease," Soc. Neusci. Abstr.. 22:2124 (1996); Nakano et al., "Alzheimer's Disease and DLST Genotype," Lancet. 350:1367-1368 (1997)). DLST is located on chromosome 14q24.3 (Sherrington, R. et al., "Cloning of a Gene Bearing Missense Mutations in Early-Onset Familial Alzheimer's Disease," Nature 375:754-760 (1995); Cruts, M. et al., "Genetic and Physical Characterization of the Early-Onset Alzheimer's Disease AD3 Locus on Chromosome 14q24.3," Hum. Mol. Genet.. 4:1355-1364 (1995)), and encodes the core dihydrolipoamide succinyltransferase enzyme of the α-ketoglutarate dehydrogenase complex (KGDHC) (Ali, G. et al., "Isolation, Characterization and Mapping of the Gene Coding the Dihydrolipoyl Succinyltransferase (E2k) of the Human α-Ketoglutarate Dehydrogenase Complex," Somat. Cell Mol. Genet., 20:99-105 (1994); Nakano, K. et al., "Isolation, Characterization, and Structural Organization of the Gene and Pseudogene for the Dihydrolipoamide Succinyltransferase Component of the Human 2-Oxoglutarate Dehydrogenase Complex," Eur. J. Biochem., 224:179-189 (1994)). DLST was originally proposed as a candidate gene for AD because of extensive and robust evidence of mitochondrial abnormalities in AD (Blass, J.P., "Pathophysiology of the Alzheimer's Syndrome," Neurology, 43(suppl. 4):S25-S38 (1993); Beal, M.F., "Age, Energy, and Oxidative Stress in Neurogenerative Diseases," Ann. Neurol., 38:357-366 (1995); Blass, J.P., "Energy/Glucose Metabolism in Neurodegenerative Diseases," in Wasco, eds., Molecular Mechanisms of Dementia, Towana, NJ:Humana Press, pp. 91- 101 (1997)) and specifically of KGDHC (Gibson, G.E. et al., "Reduced Activities of Thiamine-Dependent Enzymes in the Brains and Peripheral Tissues of Patients with Alzheimer's Disease," Arch. Neurol., 45:836-840 (1998); Butterworth, R.F. et al., "Thiamine-Dependent Enzyme Changes in Temporal Cortex of Patients with Alzheimer's Disease," Metab. Brain Pis., 5:179-184 (1990); Sheu K-FR et al., "Abnormality of the α- Ketoglutarate Dehydrogenase Complex in Fibroblasts From Familial Alzheimer's Disease," Ann. Neurol., 35:312-318 (1994); Mastrogiacomo, F. et al, "Brain Protein and α-Ketoglutarate Dehydrogenase Complex Activity in Alzheimer's Disease Brain," Ann. Neurol., 39:592-598 (1996); Blass, J.P. et al., "Inherent Abnormalities in Oxidative Metabolism in Alzheimer's Disease: Interactions with Vascular Abnormalities," Ann. N.Y. Acad. Sci.. 826:382-385 (1997)). KGDHC catalyzes an obligatory step in the Krebs tricarboxylic acid cycle and is also an enzyme of glutamate metabolism. Previous evidence associating polymoφhisms of the DLST gene with AD in Caucasian subjects has been presented (Sheu, K-FR et al., "A Gene Locus of Dihydrolipoyl
Succinyltransferase (DLST) is Associated with Alzheimer's Disease," J. Neurochem.. 66 (Suppl 1) S10B (1996); Sheu K-FR et al, "A Genetic Association of the Dihydrolipoyl Succinyltransferase Gene with Alzheimer's Disease," Soc Neusci. Abstr., 22:2124 (1996)). Nakano et al. (Nakano, K. et al., "Alzheimer's Disease and DLST Genotype," Lancet, 350:1367-1368 (1997)) have reported an association between a haplotype of DLST and AD in Japanese subjects. Recent studies in Sweden also implicate an association between DLST genotype and familial AD.
Therefore, a need still exists to develop a method of diagnosing Alzheimer's disease. In particular, a method of screening patients early in the development of Alzheimer's disease utilizing a small sample of body fluid or tissue pre mortem is particularly needed. Genetic markers are desired which are associated with Alzheimer's disease, especially markers which can be used to predict Alzheimer's disease in subjects in whom APOE4 is a relatively weak genetic risk factor.
SUMMARY OF THE INVENTION
The present invention provides a method of detecting a predisposition to a neurodegenerative disease in a human. A biological sample is obtained from the subject. Then genetic material in the biological sample is tested for the presence of a polymoφhism in a gene encoding a Krebs tricarboxylic acid cycle component, wherein the presence of said polymoφhism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases. The present invention also provides isolated nucleic acid molecules, or fragment thereof, which encodes a Krebs tricarboxylic acid cycle component, or fragment thereof, and has a polymoφhism, wherein the presence of said polymoφhism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
The invention further provides a kit for identifying individuals with a predisposition to Alzheimer's disease or another neurodegenerative disease. The kit contains nucleic acid fragments which can be used to amplify a gene encoding a Krebs tricarboxylic acid cycle component, or a fragment thereof, which consists of a position where a polymoφhism may be located, wherein the presence of said polymoφhism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
In an alternative embodiment, the invention provides a kit for identifying individuals with a predisposition to Alzheimer's disease or another neurodegenerative disease, having a restriction enzyme which may be used to detect the presence of a polymoφhism in a gene encoding a Krebs tricarboxylic acid cycle component, wherein the presence of said polymoφhism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
The invention also provides a method of detecting a predisposition to a neurodegenerative disease in a human subject. A biological sample is obtained from the human subject. The biological sample is contacted with an antibody which preferentially recognizes a Fvrebs tricarboxylic acid cycle component protein, which is encoded by a gene having a polymoφhism that is indicative of a predisposition or reduced risk for a neurodegenerative disease. Then one determines whether the antibody binds to a Krebs tricarboxylic acid cycle component protein. Binding of the antibody to the Krebs tricarboxylic acid cycle component indicates that the polymoφhism is present which is indicative of a predisposition or reduced risk for a neurodegenerative disease.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the polymoφhisms and expected RFLP of DLST A19,l 17G and T19.183C. The DLST polymoφhisms A19,l 17G and T19.183C confer restriction sites of Ms el and Acil, respectively. DLST haplotypes consisting of these two polymoφhisms can be differentiated by RFLP by simultaneous digestion with Msel and Acil of the PCR- amplified DLST fragments #18,747-19,272. The expected restriction fragments for each of the four possible alleles are shown, in which the 230 bp fragment (nucleotide 18,747- 18,976) is common for all alleles. The numbering of DLST described here starts from the first base of exon 1. This base corresponds to base #748 of SEQ. ID. No. 2 (Nakano et al., "Isolation, Characterization and Structural Organization of the Gene and Pseudogene for the Dihydrolipoamide Succinyltransferase Component of the Human 2-Oxoglutarate Dehydrogenase Complex," Eur. J. Biochem.. 224(1):179-189 (1994)). Figure 2 shows the RFLP analysis of DLST genotypes consisting of A 19,117G and T19,183C. Figure 2A shows the RFLP of the six common DLST genotypes. Allele G19,l 17 and T 19,183 (allele G,T) was not found. Restriction fragments agree with those depicted in Figure 1 , including the presence of the 296 bp fragment that always accompanies genotype G,C/A,T. This 296 bp fragment is also present when DNA mixtures of G,C/G,C and A,T/A,T are analyzed, as shown in the two far-right lanes.
Figure 2B shows that this 296 bp, G,C/A,T-associated DNA is a heterodimer consisting of strands from each of the G,C and A,T alleles. This fragment was extracted from the gel, and PCR-amplified with the forward primer corresponding to nucleotides 18,985 - 19,005 (5' TTATACAGATGGGGGTCTCAC 3') (SEQ. ID. No. 1) and the reverse primer, 19,252 - 19,272 (5' ATCCCCAGGATGGCAGACT 3') (SEQ. ID. No. 2). The PCR was run for 15 cycles with an annealing temperature of 55°C, and the product was then double-digested with Ms el and Acil. The resulting RFLP reproduced the typical restriction pattern of G,C/A,T, including the presence of the 288 bp heterodimer.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of detecting a predisposition to a neurodegenerative disease in a human subject. A biological sample is obtained from the subject. The sample is then tested for the presence of a polymoφhism in a gene encoding a Krebs tricarboxylic acid cycle component, wherein the presence of said polymoφhism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases. Preferred neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, are related neurological diseases. Both Alzhiemer's disease and Parkinson's have been associated with the DLST gene as well as mitochondrial genes (See below; Kobayashi et al., "Association between the Gene Encoding the E2 Subunit of the Alpha- Ketoglutarate Dehydrogenase Complex and Parkinson's Disease," Ann. Neurol. 43(l):120-23 (1998); Mizuno, et al., "Mitochondrial Dysfunction in Parkinson's Disease," Ann. Neurol. 44:S99-109 (1998), which is hereby incoφorated by reference).
In a preferred embodiment of the invention, the testing of the genetic material in the biological sample is carried out by amplifying a region of a gene encoding a Krebs tricarboxylic acid cycle component to provide an amplified fragment. The resulting amplified fragment is then used in order to detect the presence of a polymoφhism.
In a preferred embodiment, the biological sample is blood, saliva, cheek scrapings, or urine.
Of the genes which encode a Krebs tricarboxylic acid cycle component, a preferred gene encodes dihydrolipoamide succinyltransferase. The sequence of the human gene encoding dihydrolipoamide succinyltransferase ("DLST") follows (SEQ. ID. No. 3) (Nakano,K.,et al., "Isolation, Characterization And Structural Organization Of The Gene And Pseudogene For The Dihydrolipoamide Succinyltransferase Component Of The Human 2-Oxoglutarate Dehydrogenase Complex," Eur. J. Biochem. 224(1): 179- 189 (1994), which is hereby incoφorated by reference):
1 ACCTTGTTTC TTAAAAAAAC AGCTGGGAGT TTATATTCTA GGGATAGGGT GACAAACGAA
61 CAACTTCCAA AATGCACAAG ATGTATGGGG AACAATTAGT TTGGCTGGAA TTTGCGTTGG
121 GGGGAGATCA GAAGCTGTTG AGAAATAAGG CTATAGTTGG TGAAGTATAC CGACTCGTAA 181 AAAGGCAATG GGATTTAGAA ATTCCTGGGA TTCTGTTTCT TTGACTCGGG GTTAGGAAGA
241 CAGGGAAGTA ACAAGTGCCT TTTTTTTTTT TTTTAATAGT TAAGGGACCT GAGACTGGGG
301 GTGGGGGGCA ATGTCATCTG CCCCAGGCCA CACAAGTGTA AGATCAGGCA TGCATGTCTG
361 CCAAGGTTTC AGTTCAATGT CCGTGCCCCT CTTTTGCTCT CTTCCCAAGG TACAGAGGCG
421 GAACGGCGCG GTGCCCTCGG GTCCCAAGAG AGAAGGGCGC CAAGCTCAGA GCCAGTTCTG 481 TGGAAGGGTA GGGGAAGGCC TGACCCCGGG GGCAATGTGG TGGCGGGACA GTCGTATACA
541 CCTTCAGGAG AACCACCTCA GGCTGACAGG CAGTGCGGAT GGCCCCAAGA GCGCCTTAGG
601 GTTGCCTGCT CCAGAGAGCG GGACTCCTGC GCTGCAGCCG GGATACTGGA CGCCGCCGTG
661 CGCCCACCCC CTGCCAGTGG TTCGCTCCGA ACACCCCTCG TCGGCATGGC GGGTACTGCG
721 GCCCGCCCCA CTTCCGGTTG TTGTCCGGCC CTATATCCGG TGTCCGCCCG CCCTCGGCTC 781 CTCCGCCGTG ATGCTGTCCC GATCCCGCTG TGTGTCTCGG GCGTTCAGCG CTCCGCTCTC
841 CGCCTTCCAG AAGGTACGGT CTGGCCGAGC CGGGGCCCCG ACGGGTGAGG AGTCTGTTGG 901 CGGGCAGAGA GGCGGCCTGG AAGCAGGGGC GCGGCGCGGC CGGGCCGGGC ACCAAGGGCA
961 CTGGGACGCG GAGGCCGCGC GGGCTGGGCG GCCCGGGGCC GTGGTTGCCC TCGGGACCGT
1021 TTTGCGGAGC CCAGCGGCCC CCTGCCTTCT CCAGGCAGCC TGCGGGGAAC GTTTCCTGCT
1081 GTACCCCACC CTCCGCGCGG GAGCTGGGGA GCCGCGCCCG TCCAGATACT TGACTCAGAG 1141 TTAGGGAGGA GGCGCGGCGG AGTCCTCCGG CGGGATTCGG CGCCGTCATC GTCTTGCTCT
1201 GTTAAAATCA GTGGGTATTA ATGAGTCCGT TGGGAAGTTG AGATAGTTTG GCAGGCCCAG
1261 CGTGTTGATT GGGTCTGCCA GCACGGTGTG TTGCATTTAC TTCGGAGTTA CGAGATTCAC 1321 CTGTCACCAC CACTGGGACA GCTTTGAGCG TGTACTTAAA AGAAAAAGCT GGGTTTTTTT
1381 TTCATCTTGG GTGACGTCGA TAATGTCTTC TTTCTCAACA GGGGAACTGC CCTCTAGGGA 1441 GACGTTCCCT GCCTGGTAAG TTCTGCCCTT ACCGTCACCT AAAATGCTCT CCTCCTGGGC
1501 AGAGCAGTAT GTGAGGAACT CGGGGGTGCC TGTGTTTCTC ATAATATTTA AAGACAGTTT
1561 GGTTCAGAGA GTAGTGAGAC GCAATGCTAC ATAATCACGA GCTATCGCTT ATTCATTTAG
1621 CTCGTAGTTG AGTTTCTACT GTGTTAGGCA CTGTGCTAGA CGCTAGGGAT ACAGGAGTGG
1681 ATACAGAAGT GTACAGTTCT TGCCCTCGTG GAGCTCCTAG GCTAGTTGGA TAGTGTCAGT 1741 TGCTAAACAA GCCAACAAAG GCAAATTTTT GTAACTGCTG AGAAGGAAAC GAGGGTGGAC
1801 AGAAGAGCCC TTTTTGAGGA GGTGACTTAA AAGTTATGGC CATGGCCGGG CGCGGTGGCT
1861 GACGCTGTAA TCCCAGCACT CTGGGAGGCT GAGGCGGGCG GATCACGAGG TCAGGAGATC
1921 GAGACCATCC TGGTTAACAC GGTGAAACCC CATCTCTACT AAAAAATACA AAAAATTAGC
1981 CGGGCGTGGT GGCAGGCGCC TGTAGTCCCA GCTCCTCGGG AGGCTGAGGC ACGAGAATGG 2041 CGTGAAGCGG GAGCGGAGTT GCAGTGAGCC GAGTTCGCGC CACTGCACTC CAGCCTGGGC
2101 GACAGAGCGA GACTCCATCT CAAAAAAAAA AAAAAAAAAA AGTTATGGCT AGCAGAATGT
2161 GACGGAGCTA ACCCAGTGAA GCCCAGGAGA AAGAGAATGC ATTCACAAAG ACCCTGAGGA
2221 AGAAAAGAGG TTGGCAGAAC TTAAGAGCTG GAAAAAAGGT GATTTGGCCG GAGTATTTTG
2281 GTTAAGGGAG AGAGTGGCAA GAGATAGAAG ATGGTATAGA GACTGAAGGA GTTTAGATTT 2341 TATCCTAGCA GTTAAAAAGA AAAAAGTGCA TCCATAGCTT TTTCTGTTAT ATGCCTAGCC
2401 TAAGTATTTG TTGAATGAGT TGACCTTGTT GTGGGCATTT CTTTGTGCCA AGCATTTATA
2461 TGAGTGCTTT CTGTGAATTT TTTAGTTAAT CATCTCAAAA CAGTAAAAAG TATTTTCTCT
2521 ATTTTACAAA TGCATAACCC AAGACAAGAG AGCTTAAAAG CCTTGGCCAT GGTCACCCAC
2581 CCAGCTGTTG ATAAGCCAGG TCTACCTGAC CCATCCTGAA CTCTTAACCC ACCATGTTCT 2641 CATTTACTCA TGGACTCAAC AGATCTATTT ATTGAGTGCT TACTATATGC CAGCCACTGT
2701 TCTAGGTAAC AGGGGTATAG ATAGACTCTA GCCTCGAGAT TTTATGGGCT AGCTGGAGAG
2761 AGAGAGCAAA TATACATTTA TGCTCCGACA AGGGCATAAA TTTTGGTTAC TACGAGAGAG
2821 GAATTAAAGG GTGTGATGAG AGGGTGGGAT GATGTAGACC AGCTTGGGAA GGTTGGGAAT
2881 GTCAGTAAAG GCTTCCCTGA AGAAGTGACA TTTAACTGAG ACCTGAATAT AAAGAGCCAA 2941 GAAATGTATT CCAGGCAAAG GGAATTGCTT GTGCAAAGGT TTTCAGCAGG AAGGCTTGAC
3001 CAGTGATAGA CATGGGAAGA AGCCTGTCTG GCAGGAAAGA TAGAGTGGGC TGGACGGTAC 3061 AGGATCGCGT AAAGTGTGGA CTTTATCCTG AGAGTTATGA GAACATAAAG CCCCTAGCAC 3121 AATGTCGGCT TGTAGTGGAC TCTCAATAAC AAACATGTTT TTCAAGTGGG AACCCTGAAC 3181 AATGTCAGTA AACATTTTCT CCACTTCATA GGTGGGCAGG ACAATGGAAA GAGCCTTGCA 3241 CTTGGGGATT TGCATTTGGG CTCAGGGATT CACCTTGGGC TAGTCGCTTA ACCTCCAAAA 3301 GTCCTACTTA TATGAAATGG GAATAATGGC AGATGGCGTT CCACTTCAGA GAGTTGTGAG 3361 ACTGTAAAAT CAAAATCGAC GGCATTATGG GAAAGCTTTT TATAAACCCT AAAGCACTGT 3421 ACAAATGCAA ATTTGCTGTA GGATGAACTC TGAATTGGAC TCCAGGTTCC CAAAATCAGT
3481 GTGAGCCGCC ATCTATTCTA GTTCAAAAAC ATACTATGCT TAGAATTTGC ACTGCATAGT
3541 CATTGGCTGC TGGCACATAA AGGTGACTGG CTTACCTATC TTGGGATGCT TGTGAGGAGG
3601 TGGGCATAAG AGAGGGCTAG CTCTCATTTG GTGTGTCCTG AGCCCATGGT TCAGAAAGAG 3661 TTGTTAATCT GAAAGAAAAA AGACATAAGA AAGAATTCAG AAGAGGCAGC TCTAGTTACT
3721 AGTCATCAGT TGGGGAAATG CACTTAACTA TGATAAGGAC AGCTGACTGG GCAGAAACGG
3781 GTCTCTCACC TTGTTGAGAG TGACCAGGGT GGAAGACTTC AGAAAGGCAT GGGAAGTCAA
3841 TAAACTGAAA GATGGGGACA TGATTTTCTC AGCTCTAGGA CAAACTGGAG ACCCCGAGCC
3901 TAAAGTAGCC AAGTCTTTGC TACTAGGATT CAGGTGATAA GATTTCCAAC CCAATCTTTT 3961 CAAACCCAGA CCAAACATCC TCCTTGCTTC TCAAATTGGG TTATGTATAG TCACAGGATA
4021 AGCCATCCCA TGTCTTTCTC AAGCTTCAGT TGTATTTGAA GACTTGGCGA GTAGAGACAT 4081 TTTATTATGA AAACATACAG GAATAATGAA AAGTTCTGGC TTTTTAAGGT AGATGAGATT 4141 ATATAATCAC TTTGGGCTGT GCCATCAAAT TTTTTCTCTT TCCTCCCCAG CCAAACACAT
4201 ACCCACTCGA TCTCACATAC CCATTTGCTC TCTGCCACTA AGGCTACTTT GGAAAAGTTT 4261 GAGAAGCAGG ACTCTATGAT AAAGATTATA TCCGTTGCCG TTGATCCTGC TCTGTCTCAG
4321 GGTTGGGGGT GAGCAGACCT TTTCCATTCC CAGCATGTAT CCTTTGTGAG ATAAGAGTTG
4381 TTGGTTAAGA GTTATTTTGT TTCTTGCAGG GGTCTCCTTA TGCCAGGGAC CAGGTTACCC 4441 TAACAGCAGG AAGGTTGTGT AAGTATCACT GGGGAGTACA GATATGTGGC CACGGGTTAT 4501 TATTTAAAGA CATAAAGTTC TAAATTTGAC CATCTGATTT CTTCACTGGC CTCCAGACCT 4561 AGAAATTGAG GTGATTATGT TGAGTCCCTA GACTAACCTC AATGTTCACT TTTCCCATGT
4621 GATGTTCCTT GAGCATCAGC TCCGTGTCAG GTTTTGGACT GGGAGTGTGC CCACTCTCCA
4681 CAGGTGTTTG GATTATAATG AAAGTTAAGT TGGAATCCTC TTTTTATGGG ATATAGAAGG
4741 GTGCTCCTAG ATATTCTGTT TGGATGTAAG TGCAGCTGAA AGGCTCGAGG TTACATTTAT
4801 TGTGGAGATT GAGAGCATCC TCTCAGTAGC ATAGTTACTC ATTTTTAAAA ACAGTAAAAG 4861 CATAAAACTG GTTGAGTCCT GGGACTGACT GACTAGTTTG GAGGGAATGC TGTTATTGCA
4921 TAATGCAGAG TTTGGAGAAA GGCTACCCAT GTGTGTTTCA GATTAACTTC TGCGTATGAA
4981 TGTTGCTTAA ATGCTGCTTT GCATTCCAAG TGTTTTTGTG GTGGTGCTAA GAGAATGCTC
5041 TCTAGTAACC TGTTCAGCAG CTGTCAGGCT GCTTTACTCT ACAGGGAGAA TCCTCAAAGT 5101 GAAGAGGTCC AGCGGATGAG CTTCTTACTT ACTCTACATC ACAATGAGGG TTTTTGTTGT 5161 TGTTGTTTTG AGACAGGATC TTGCTCTGCC ACTTTGGCTG GAGTGCGGTA GCCTGATCTT
5221 GGCTCACTGC ATCCTCAGCC TCCTGGGCTC AAGAGATCCT TTCACCTCAG CCTCCTGAGT
5281 AGCTGAGACT ACAGGCACAT GCACCATGCC CGGTTGTTTT TTGTATTTTT TGTAGAGATG
5341 GGGTTGTTTT GCCATGATAC CTGGGGCTGG TGTCAAACTC CTGGGCTCAA GCAATCCAGT
5401 CCACTTGCCT TAGCCTCCCA AAGTGGAGGA TACAGGCGTG AGCTATGGTG CCTGGCTGAT 5461 TTTTAAAATT TGAGTAAATG TTGCACTCAG TACTAGAAAT ATGAATACCG GTGTTATTTT
5521 GGAGGGCAGT TTGATAGTCA TAAGTCTTTG AAGTGTGTGT ACCTTTTCTT TTTTTCTTTT
5581 TTTCAAGATG GAGTCTTGCT CTGTCACCCA GGCTGGAGTG CAGTGGCGCG ATCTCGGCTC
5641 ACTGCAGCCT CTGCCTCTCG GGTTCCAGCA ATTCTCCTGC CTCAGCCTCC TGGGTAGCTG
5701 GGATTACAGG CGCACGCTAC CACACCTGGC TGATTTTTGT ATTAGTAGAG ACGGGGTTTC 5761 ATCTTGTTGG CCAGGCTGGT CTCTGAAACT CCTGACATCA GGTGATCCGC CGACTCAGCC
5821 TCCCAAAGTG CTGGGATTAC AAGCATGAGC CACTGCCCGG CCTGTGTGTT CCTTTTCATC
5881 TAGCAATTCC ATCTCTAGGA ATCTATACTG AAGAACTCCT GGTATATTTA TCTCAGATTT 5941 AATATCTGAT AATATAAATA TTATCAGATT TATCTTAAAA TGGCAAAGAG CAAGGGATTG
6001 GCTAAACTAT AGCATGTTCA CATGTTCACA CTATGAAAAG TGGGAAAAAG CAGGTTACAA 6061 AATAATTTCA TTTTCTACTG TGTGGTATGA TCCCATTGTA AGTATTCTTT TTGCTATTTC 6121 TAATATTCTG TAGTGAACAT CTAGTTATTT AGAGATTCAA AATGTATTAC TCATCTGCAA 6181 TCAAATGAAA ATGCGCTAAT CATGACTCTC TACAATAAAT ACACTAAATA TTTGAAAGTA
6241 AATAATGAAG AAAAAGTAAA CTTTGTGTGG GATCACCCCA ATCATGTCAC CCTAATCCAG
6301 TATCTTTGTG TGTGTTGTGC CTTTTTTGTG AATAAGGTAC CCACTTTATT TAATAAATCT
6361 TTTATTGTAA AACAATTTTT GGTTTTATGT TTTTACTAAT AGTATTATAT TTACATGATT
6421 AAAAATTCAA AAAGTAAAAA GGGTGTCCAT GAGAAGACTT TTTTATCCTT GTCCTCTAGC 6481 CCCCCGAATG CCTCTTGGGT AGTGTGGCCA CCACTCCGGG TTCCTGGATG ACTTGCAGAG
6541 CAGCCTCTGC ACTTGCATGT TACCTGTTAG TTCTTGCCTA TAGGCATGTA CTTTACAAGG
6601 TTACAAACTA TATGTGAAAC AATTTGTATT CTTGTAGTTT TTAAATATCT TCATTTTTTC
6661 TTGGTTATAC ATAGACCTTA TAGTTATTTT GACATTATAA AAGTAAAAAT ACCTAAAGAC
6721 AACTTAAATC TCTTTTACTT TTCTATTATA AAAATGCTTG TTGTGGAATA TTCAGACAAT 6781 CCTGAAGTGT AAAAAGTGAA GTCCCCATCC AAATCATTCT CCCAAATTGT GTAAGTGTGC
6841 AACACATAAC CAGAGATACC ATATATGTCT ACATCTCTCA CCCCTCAGAG GTAACCACTG
6901 TTAATGGCTT TGTGCATACA GGGTGAGCTG ATTCTCCAGT TTATATTATT TACTGTAAAG
6961 GCCATATCAG TAAACGCCTT GGTCTTTGAG GTTCGTTTTG AATGAATCCA GAATGTGTTG
7021 ATGTGCTGTT TTCTAGCAAG AGAACCGAGT AGGAGATACC ATATGGGGTT TGTGTGTGGT 7081 TTTTTTGTTT TTGGGTTTTT TTTTTTTTTT TTTGGCATAA CACCTTGTTT TACTTCCGAG
7141 TTAACCTTGT TAATCTTGCT GCCTTATCTG ATCTAAATCT GTTATCTTGA GGCAGAAATT
7201 CCATTATACC TTGCACAATG GCTTGTACCC TGGGCAGGCC GTAAATCTAT CTCGGATTTA
7261 GTTAAGGAAG AGACCTGGCT GGACATGGTG ACTCACTCCT GTAATCCCAG CACTTTGGGT
7321 AGGATCTTTG AGACCCGTCT GGGCAATGAA ATCCATAGTC TACAAAAAGT AATTTAAAAA 7381 AATTATCTGG GTGGCATGTG CCTGTAGTCC CAGCTACCCG GGAGGCAGAG GTGGGAGGAT
7441 CCCTTAAATC CAGGAGTTTG AGGTTACAGT GAACTATTAT CATGCTACTG CACTCTAGCC
7501 TGGGTGACAG AGCAAGACTC TTATCTCTTA AAAAACAAAA AACCAAATCA AGAAGCTAAG
7561 TCATTTCGGT CTGCAAAGGA CCAAGGTGCT TATTTTTTTA TTGAAAGAGC CAAATGTAAG
7621 TCTGTTTTGT GAGAGTTGGC ATTGCATTGC AGCACAGGGG CCTGAGCATA CTTGGGGAGC 7681 TCACCATTTA AGGTACCTGG GTCGTTAGGC TCTACTCCAG ACCTGCTGAG TCAGAATCCC
7741 TGGGGGAGAG GCCTAGACAA GTGAACTTTG AAAAAGTTCC TCCCCAAGTG ACACTGATGG
7801 ACACCCCTGG TCAAGAGTCA CTGTTTAAGG GGAAGGTGAC CCATGGGGCC TTAACTAGAC
7861 TAAAATGTGA GTGGTTCGCC TGTTAAAAGG AGTTAACGTG TGTTTCTTTT GTAGCATTAA
7921 CAACAGTGTC TTCAGTGTTC GCTTTTTCAG AACTACAGCT GTATGCAGTA AGTACCTGCT 7981 TTCTTGGGAA TGGAATTTTA TGGGAAGAGC AACAATTGAT TGAGTTTAAT TAAAGAAAAA
8041 TATTAAAAAG GAAAAATGAA CTACTTATGA TTTTCTTTTT TGCATTTTTC CTAGAGGATG 8101 ACTTGGTTAC AGTCAAAACC CCAGCGTTTG CAGAATCTGT CACAGAGGGA GATGTCAGGT 8161 GGGAGAAAGG TAAGATTTAG TTTCCTATTT TTTTTTTTTT TTTTTTTGAG ACAGAGTCTT 8221 GCTCTGTTCC CAGCCTGGAG TGCAGTGGTA TGATCTCGGC TCA'CTGCAGC CTCTGCCTCC 8281 CAGGTTCAAG TGATTCTCCT GCCTCAGCCT CCCGAATAGC TGGGATTACA GGTGCCCACC
8341 ACCATGCCTG GCTAAGTTTT TTGTATTTTT AGTAGAGACA GGGTTTCACC ATAGTTGGTC 8401 AGGCTGGTCT CAAACTCCTG ACCTCAGGTG ATCTGCCCAC CTCAGCCTGC CACAAAGTGT u -
8461 TGGGATTACA GGCCTGAGCC ACTGCGTGAG CCACTGCGCC TGGCTCCTGT CACTTCTTAA 8521 GTTTAAGTGC TATCTAAAAT TCTCCCCTCC CCTCAGGATA GTTGTAGAAT TTATAGCTCT 8581 GCCGTTTTCT TTACATATGT ACTTTCTGCT GGCCCTGTAG GAAAGGGTTC TTATAACATA 8641 CCTGCCAAAA TGGGTTTTGT GGCTACTGGA GGCTCCCCGC TAACAGGTAG CCTTGTAGCC 8701 TTTGATTGTC TTTTCAGCTG TTGGAGACAC AGTTGCAGAA GATGAAGTGG TTTGTGAGAT
8761 TGAAACTGAC AAGGTAGGCT TATCTTATAT TCGTACCAGC TTTTCATGGG CTTCCCTTAG 8821 TTAACCTAAT GAAAGAAACA GGGACTTAAC CATTGTTTAG AGATAATTTT TAACTAGCTC 8881 TAACTTCAGT TGTAAAATTA TTGGATTGTT CAATTTAAAA AAAAAACAAC TTTTTACATT 8941 TAACTTTTTT TTTTTTTTTT TTTTTTTGAG ACAGACTTGC TCTGTTGCCC AGGCTGGAGT 9001 GCAATGGCCT GATCTCAGCT CACTGCAACC TCCACCTCCT CCTGGGTTCA AGCGATTCTC
9061 CTTGCCTCAG CCTCCCTAGT ATCTGGGCTT ACAGGCGCAT GCCACAACGC CGGGCTAATT 9121 TTTTGTATTT TTTTGTAGAG ACAGGTGTTG CCAGGCTGGT CTCAAACTCT TGACCTCAGG 9181 TGATCTGCCC GCCTTGGCCT CCCAAAGTGT TGGGATTAGA GCTAGAGCCA CGCACCTGAC 9241 CTGCATTTAA CTTTAAATTG TTTGCTGTGT GTGTACTATG TACTTCATTT AGTAGCTTTA 9301 GTCTTGTAGT GTGTGGTGAG GGGGTGGGGA GGGTAAATAA GCTGTTTCCA CTTTTTCCTG
9361 TATCACTTGG AAACATATTA ACTGAATATT GCCTGAATGT TTCTGAGAAA GGACAGAAAG 9421 TGAATCTTCA CAGCTAGCGA TGACTTGTTA TCACCCTTGC TCTGAAAGTT ATGGCCTTGA
9481 GATGTTTGTG TGCAAGGCCT TATATATTGT GTTGCAACTC ATTGTTACTC ACTGGAGATA 9541 CGCTTGTTGT TGTTGTTGTT TTGAGACGTT TCGCTCTATC ACCCAGGCTG GAGTGCAGTG 9601 TCACGATATG GCTCCACCTG CCGGGCTCAA GTGATTCTCG TGCCTCAGCC TCCCAAGTAG
9661 CTGGGATTAC ATTTTTGTAG TTTTTAGTAG AGATGGGGTA TCACCATGTT GGCCAGGCTG
9721 GTCTTGAACT CCTGACCTCA AGTGATCCGC CTGCCTTGGC ATCCCAATAT GCTGGGACTA
9781 CAGTCATGAA GCCACTGTGC CTGGCTTCAT TGGAGATTCT ATACAGCTAA TTTTCTAATG
9841 CTGGATAAAC ATTTGGACTT CCTCCATCTG TCTTCCTCTT CCAGACATCT GTGCAGGTTC 9901 CATCACCAGC AAATGGCGTG ATTGAAGCTC TTTTGGTACC TGATGGGGGA AAAGTCGAAG
9961 GAGGCACTCC ACTTTTCACA CTCAGGAAAA CTGGTGGTAA AGAAGTTCTC CTGGTGGTCA
10021 AGGTCTCCAG TGTTCCCTCT TGGGATTGGG ACTGAGCATA ATGTGCTAAT TCCCCGATAT
10081 GTCAAAGACT CTTGTCATTC CAGCTGCCCT TAGTTGAGGG AATAAGGGAA TTAAATTTAT
10141 ATATAGTGTG AACTTCAAAT GTCAAATTCT AAAAGAAAAG TGTATTTTTT AAAAAATAAA 10201 CCTTATACTC TGTTCTTTCC ATGGGTGTTT CTTAGCAGGA GCTGAGAAAC TGGACCTTTT
10261 CTTATAGATA TACAGATTGA GCATACCTAA TCTGAAAATT CAAAATCTGA AATGCTCCAA
10321 AACTGAAAAT CCAAAATCTG AAATGCTTCA AAACTTTTTG AGCACTGACA GAATGCTCAA
10381 AGGAAATGCT CATTGTCTTT TATGGGGAAT TCAGTGTTGT TTCTTTTCAC ATCTGTTCTC
10441 TCATATCTCT AGGAAGGGTT GGTATTTCTT TTGGGAAGAA ATGAGAAACG AATTTGTACT 10501 GGACCTGATA GGATTAGAGA TTAATAACTT TATCTGACTC TTAGTTAAAT TTTATAACCA
10561 CTAAAAAGTC TATTTCTTTT TCCTAAGCGA GAATCGTACC CGTAGACCAA CGAGTCACAG
10621 AAGTCTATTT CTTTTTCACT GTTACACTAA TAAGGCAGAT AGACCGCATG ATTGAGGTAG
10681 TCAGGCAAGA CCAGAAGCTC AGAGTAGAAA ACATGTATGG TAGAAGGCTG GGAAATAAGG
10741 TAGTCCTCCT GTTGCAGAAG TTTGTCTTAG CACTAACCCT GGATATAATG TCCAGATGGG 10801 TGAAAAACAG AAACAAACAT TTCATTTAAA GTTGTGAGAT TATTACTTGG CCACTTCAGT
10861 GATTTATGAG TCATCCTGGA TGAAGTGCTT TCTTAAGCCT AAGTCAACCC TCGATGACTA
10921 TTTTATTTTT TGAGATGGAG TTTCACTCTG TCGCCAGGCT GGAGTGCAGT GGCCTGATCT 10981 TGATTCACTA CAACCTCCGC CTCCGGAGTT CAAGTGATTC TCCTGCCTCA GCCTGCCGAG
11041 TAGCTGGGAC TATGGGCACA TGCCACCACG GCCAGCTAAT TTTGTATTTT GGTAGGGACG
11101 GGCTTTCACC ATGTTGGCCA GGATGGTTTT GATCTGTTGA CCTTGTGATC CGCCTGCCTT
11161 GGCCTCCCAA GGGGAGTAAT CCCTTGGCCT GTAATCCTGG GATTACAGGC GTGAGCAACT 11221 GCACCCAGCC TATTTTTTAA TAGTATTCTT GCATATATAA TGTTAAAATA GAGGATGAAG
11281 AGTATCAGTA ATTAGAAATA TGTTATGTAT GCATTTCAAT ATTTTAAATT GCCTAGGTGT
11341 TTTTTTTCCT TGGAAGTTTA CATTTTCTTG GGCTTGATAG GTGCTTTGAA AGTACTTGCT
11401 GAATTTATTC AGTGGAAACC TGTGAAATTG GGAAGGGAGC TAAGTTGAGC CAGTTATCTG
11461 ATGGACATTT AGGGACCCTG AGGACTAGAG AATAGTGCCT TATGTCATTG TGTGTGTGTC 11521 TCATAGTGCT TTGCATATTG CTGATACCCA GTAGACATTC GTTACTTGAT TGGAGCTAGA
11581 GTTTTTGCCA CTAAATTTTG CTTGGTTGCT TCTCATTTCA GACAGTGCCA GTGGCATATA 11641 CTTTTCTTGT TTTTCAGCTG CTCCTGCTAA GGCCAAGCCG GCTGAAGCTC CTGCTGCTGC
11701 AGCCCCAAAA GCAGAACCTA CAGCAGCGGC AGTTCCTCCC CCTGCAGCAC CCATACCCAC 11761 TCAGATGCCA CCGGTGCCCT CGCCCTCACA GCCTCCTTCT GGCAAACCTG GTAGGCTTCC 11821 AACTCCCACA TGTCATGTGG GAGAACATCT CGTCTAATAT GTTCCTTCAA AGGGTAAACT
11881 CTAGACTGAT GATGGTTCTG AACAGGCCAG GTTGGCTGCT TTGTAGAAAG GAATTCTAGA 11941 CTTTGTATCC AAAGGCCTGC TTTGCATCTC AGCTTTCTGT TACTAGCTGT GTGTCTTTAG 12001 GCTAGTCACT TAATTTATCT GAACAGATTG TTCATCTGTG AAGTTGGAGT GATACTAATA 12061 TACCTCAGAA TATCTTGAAG ATTAAGTAAC AGTACATATG AAAGCACTGG CACAAACTCA 12121 GCAGATGTTT CTTTCCTTGT CTGATGCAGC TTTATCCTCT TTTCATTTTC AGTGTCTGCA
12181 GTAAAACCCA CTGTTGCCCC ACCACTAGCT GACGCAGGAG CTGGCAAAGG TCTGCGTTCA
12241 GAACATCGGG TAAGCCTCTG AGGACCACTT TCAGGAAAGA GGCAGGGGGC AGTGTTGAGA
12301 TTAGTGGAGT ATGGGTGTGG TGATGCCTAC CTTTGAATGC CTGGCAGCTC ATTCCCTCAA
12361 CCTTGATAAA GGGAGTTTAG GATATAACTT CTTCATAGTC ACATAACCAT CAAATGATAG 12421 GACTAGAACC TGAGGTCTTC TGAACACTTG TTCAGTGGTT TTCCTACTAT ATTATGCTAC
12481 CAGTGAAAGC CTTGGGTAAG TTAAATATTT GTAGGGGTGG TAGCTTAGTA ACTCACCCCG
12541 TCCTCCCACG ATGTCACACG CTTCAGAGAG GTGGTTTAGT GAGCTCTATT CCTATACTTC
12601 TGGGATACTT AGCTGTAGCT AAGGGATAAT ACTCAGTAGC AATAGCTGTC AGGTGCAGAC
12661 ATAAAGCCTC AAGGAAGTAT GGTCAGGATT GTGGTTCCTC TGCCTGAGAA TACTTCCTAA 12721 AAATATGATA GCCTCGAAAG TTTGTTGCTC ATGAGGTTCT GTTCCACAAA TAGCTATTCT
12781 TATGCAAATC TTTATCAACA GAAAGTGCCC TTTTTCTTAT GATTCTTATG TTGGCTAGAC
12841 TACACTACAC TACATTCTTT TATAAATGGG TTTTGGTTTT TAAATCACTG TTTCTGAGCC
12901 TTCATTCTTG GTCAAGCTTT AGGCACATCT GAGTGAGTAG TTTTGGCCTG TGTTTGCATG
12961 TTTTTGCTTG GAGGAGAAAG GGCCACCACA GGAAAGGGAA GCCTGGAGCT CGTGTACTTA 13021 GATACTGTAG TCCTTGGCCA TCTACTCGTG GCAAGCCGTG TTCTTTCTGA CTACACGGGG
13081 AATGCTTGAC CCAGAGAGAT CAGATTGTCA ATGCTTGTTC CACTCTTACT TTCAGGAGAA 13141 AATGAACAGG ATGCGGCAAC GCATTGCTCA GCGTCTGAAG GAGGCCCAGA ATACATGTGC 13201 AATGCTGACA ACTTTTAATG AGATTGACAT GAGGTAGTGT CTCTAGTCCC TCTTATCCCC 13261 TAGGCCCCTT TTTCTTAGAG AACACGAACT TGCCCCACTC CTTATCTAGT GCTGATTCTA 13321 AAACTAACTT GTCAGGGAAG AGGTATTTGT TTATTGTTTT TTGTTTGTTT GTTTTTGTTT 13381 TTGTTTTTTG AGATGGAGTC TCGCTGTCTC CCAGGCTGGA GTGCAGTGGC ATGATCTCGG 13441 CTCACTGCAA GCTCCGCCTC CCGGGTTCAC GCCATTCTCC TGCCTCAGCC TCCCAGAGTA 13501 GCTGGGAATA CAGGCGCCCC CACCACGCCT GGCTAATTTT TTGTATTTTT TTAGTGGAGA
13561 CGGGGTTTCA CCGTGTTAGC CAGGATGGTC TCGATCTCCT GACCTCGTGA TCTGCCTTCT
13621 CGGCCTCCCA AAGTGCTGGG ATTACAGGCG TGAGCCACCG CGCCTGGCCT GTTTATTGTT
13681 AACTTTCACA GAGTGAGCCG CTGGCTTCTT CTCTAATTGG CTTTCCTAAT ACTATGCCTG 13741 CCTCCTCCAT GACCTTAACC TTAGCCTTGG CTAGGTGTAG TGGCTCACAC CTGTAATCCT
13801 GGCACTTTGG GAGGCCGAGG CAGGCGGATT GCTTGAGTCC AGGAGTTCAA GACCAGCTGG
13861 GTAACAAGGC GAAACCCCAC CTCTACAAAA AATACAAAAA TTAGCCAGGT GTGGTGGTGC
13921 ACATCTGTAG CCCCAGCTAC TTGGGGGGCT GAGATGGGAG GATTGTTTGA CCCCGTCACT
13981 TACATATTTT CCTAAGTGAT TCAGAATCGA GTGGATGAAA AGGCAAAGAT AGGCTGGGCT 14041 TGTCTTTCAT TTTACCCAGC TCTGACAGTG AAATGGAAAA CAAGTTGTGT TTTTAAATAA
14101 CCTTAGAATG TTAACCAAAG GTACTAATTC ACTAGGTATG CCCATTTCTG TAGTGAGCTA
14161 ATTGTTGACT TACCTGTTTT CTTCAAGATT TCAATGTGTT CAAGTATCAG CACTGTCAGG
14221 AAGAGAAATT TGACAAACGT GTTTTGAGTG CGTACTGTAT ACAGAGTACT GTACTGTGGA 14281 ATTCAGTGGG GATAGACGCT GGGCACCATG GCTCGTGCCT ATAATCCCAG AGCTTTGGGA 14341 GGGTGAGGAA GGAGGATCGC TTGAGCTTAG GAGTTGGAAA CCAGCCTGGG CAACATAGCA
14401 AGACTGTCTC TACATAAAAT TTAAAAATTA GCCAAGTGAG GGCTGGGTGC GGTGGCTTGC
14461 GCTTGTAATC GCAACACTTT GGGAGGCTGA GGTGGGTGGA TCACATGAGG CCAGGAGTTC
14521 GAGACCAGCC TGGCCAACGT GGTGAAACTC TGCCTCTACT AAAAATACAA AAATTACCCA
14581 GGCATGGTGG CGTATGCTGT AATCCCAGCT ACTCGGAAGG CTGAGGCACA AGAATTGCTT 14641 GAACCCAGGA GGAGGTTGCA GTGGCTGATA TCGCGCCACT GCACTCCAGC CTGGGCAACA
14701 GAGCGAGATT GTGTCTCAAA AAAAAAATTA TGTATGGTGG CATGGACCTA TAGTCTCAGC
14761 TACTTGGGAG GCTCAAGTGA TCTTCCTGCC TCAGCCTCCC AAATGGCTGG GACCACTACA
14821 GGCATGTGCC ACCAGGCTTG GCCAATTTTT TTAGTTTTTG TAGAGACAAG GTCTCGCTGT
14881 GTTGCCTAGG CTGGTCTCCT GACGTCTAGG TCAAGCCATC CTCCTGCTTT GGTCTCCCAC 14941 TGGGATTACA GGCATGAGCC ACCATTCCTG GCCAAAAAAG AATTTTTAAA CAGGTCTTCA
15001 AGAGTGACTA GCATTTTGTC AGCCTTGTGG AGAAAGGTCA TTCCAGATGA TGTTAACAGT
15061 GTAGACCATG AGATGGGAAA GTAGAAGACA CATTTGAGAA ATTGTCAAAA ATCATTTCTG
15121 TTGTGAGGAT TTTGTATAGC AAAGAATGGT AAAGAAGGTT GGATCCTTAT TTCAGAATGT
15181 TTTGAATGCC AGGGTAGGAG TTTATATTTA ATTCAGGAAG CAAGTCAAGT CTCAGGTCAT 15241 CTCTTTCTTT CTTTTTGTGC CTGAGGGTTT AGTCTGCGAA TCTACTTTTC ATTAAGAGAA
15301 ATTTGCTTTT CTTTGGGAAA GGGTTTGAAA AATAGGGAGA AGGGAGTGTT TTCTTGATAG
15361 GTGCGCTGTG GCATTGTTCA GAGTAGGGCT CTGTGACCTG TTTACTAGAT GCATCCATTC
15421 TGTTGGGAAA CTTTCCGAAG AGACTGGATG ACATTCAAGC CAAAGATTTC TCTCTTGTTC
15481 ATAGGATGTA GGGGAAGGAC CTTGACTAAT GAATCACAAA GGCTGCAGCT TATGTTTCAG 15541 TGCATTAGGG CCAAATTGTA CACAAATTGA ATTGGACTTT GAAATTCTAC CCAGTTTCTT
15601 AAGACAAAAC AGTAAATATT TGTGGCCCTG AAAGATGCAG TTGGCCCAAC AGCTTTGATT
15661 GGAAAGGAAG CGTAATAAGC TGGTCTCCTT TCAGCAGCTA ATCAAGGTGT CACTTAACAA
15721 TGGCCTGCGG TCCTTGCTGC AGCCTAATTT GGTTTAGATC GAGTGCCCTT TGGGTCTTGT 15781 GCTGATCAGC TAACCCATAA GGGACCTTTC TGAGACCTTC TCCCTTCCTC TATTGTCCCA 15841 GCCTTTGCCT CCAAGGGCAG AGCTCTGAAA TAGAAGCTAC TTAATGACCA GACAATAACA
15901 TTTGGGCAGG TTTGAGAGCA GAGATGTTTA TCACATCTCT TTCCCTCTTC TGCAGCTCTT
15961 CTGTCTTGGG AACCAGTAAT GTCTTTAGGG TGGTAATCCT AGCACTAAAT AAGGTTAGAA 16021 TCAGGAAAAC AAGCATTTTA AACCCAATCA GTCCCCCTTT GGGAAAAACT TCCTTTTTCA
16081 AAAACAAAAG ATCTGAAGGG GGCTGTTAAA ATTGTACCAC ATCTTACCTT TCATGCTAAT
16141 GTCGAATTAG GCCATTAGAG GGAGAAAGGG AGAACTGAGC ATTTGGGCCT GAGATAAAGT
16201 TAAATTCAAT TTTCATCGTA GAACTGCATT ACTGCATTCC CGATAAATAC TGAGGTTTAT 16261 GCAGCAAAGT GATGGGAAAG CCATTTCTCA CTAACTCTGC AATGTGTCAT TGAAAAAAAC
16321 CAGTCCCAAA AGAATAAAGG CTGCTCATGT TGTAAAGGCT TTTCAGAAGG CTTGCTGCTT
16381 AATCTGAAGG GAACCAGCCT AAACCACAAG TGGTCCACTT TTCTAAATAC CAAGGAAATC
16441 ACAGCCATTA AACTAATGTT AGGGCAAGAA CTATGTCTTA ATTAGCTTTG TATCTTGGTA
16501 CCTGGCCCTG ACCCTCACAT GTACTAAGGT GTTAGTACCT GTTTGGAAGA ATAAGTGGAT 16561 GGAACTGCCA ACCCTGGAAA GATGAAGAAC GGATATGACT TTTCCGTGGG CCTGAAAGAA
16621 CACTGGGTTA TACCCTTCCC TGCAGAGTCA TGACTGCTTT TTCCCGCATG GAGCCTCATG
16681 TACCCATGAG CTGTTTATTT TAGTCTCCAC TAATCAAGAG ATAGGAGGAA TATCACACGG
16741 GAGTTATACT CAGATTTGGT GGGGTTTTTT TTTTTTTTTT GTATTCTAAC AAGAAATAAA
16801 AAGAATATTG CTTATTTTGT AGATTTTGTT TATACTTTAA AAAATTTTTT TCAGGATGGA 16861 TAGCTCAGTT ATTCAGGGCT TGGATGTTGG TGTGCTGTGT GATCTCTTGT TCATATGATG
16921 TGTCACTGAA ATCTGTGACT TCTAGACCAT GAAGGAGCCC TGTAAAGAGA TCAACCCACC 16981 TTAAAGACAG ATTGGGCCTG ATTCCTGTAG ATTTCTTTGT AAAAGAACAG AGTTACTGTG
17041 TTAACCTCAG GTAGACTATT TCAAAACTTG AACTAAGGTA ATGGTCCAGA GTGAGGGAAA 17101 AGCTCTCACC TAACTTGTGT ATATGGATTG AGAAACCTGC TGTGAAAGTC AAAATGCAGG 17161 CTTTGTGGTC AGTTTGTTTT GTAATGTTTC ACAGTAACAT CCAGGAGATG AGGGCTCGGC 17221 ACAAAGAGGC TTTTTTGAAG AAACATAACC TCAAACTAGG CTTCATGTCG GCATTTGTGA 17281 AGGCCTCAGC CTTTGCCTTG CAGGAACAGC CTGTTGTAAA TGCAGGTGAG TTGCTTGTGG 17341 CTGGAATTGG AGAGGTCCTG GGAGGTAGGT GTATGAGCAC AGACCTGCTC CCACATGCAG 17401 AGGATCCAAC AGACCAAGGC TTTTTATTTG CTACCTATAG AAATATCAGT ATCTTCCCCA 17461 GGGATTTCTT GGTTCACCTT CCTGAAGCAG CATCATGATT ACTTTCTACC CGCCAGCTGG 17521 GCAGAACAGT TTCTCTTCCC AGTGTACAGC TCGGCAGCTG CTCAGGCAGC AGGCAGTTCT 17581 GGGATAGCCC AGCTCTCCAG GCTGTCTCCT GTCAAGGTGG AAGCACGTGT TCTGGCTTCC 17641 TCCCCTCCCC CAGCTCCACA GGCCCTGTTA CCTTGGGCCT GACTTCCACA GTTGGTGTAG
17701 GCTCCTGGCT GACCTTTGAT CTTAGGTCGA TGGGCCTCAC TGGGAGCTTC GGCTTGGCCT 17761 CTCATGGACC TGGTGCTTAT TTTCTTTCCC TTTTTACCTT ACCTACGTCA TTGTAGTGTT
17821 GACCTAACAA GCACTTTGTT TTCCTGTTGA CACACTTCTC TGTTCCTTGG GAGATTGGAG
17881 AGGGGTCAGT GCTACTTTTG CCATCCTCAG CAGTGTCAGG GCCCTAAAGG TCTAGAAGTA
17941 GAAGCCCTAG TCCCTCCTGT TGAGTGGTGC TCTAGGGTGG TAGAAGGCCA TCTTTGTTTT
18001 GCCCAAATGT ATACCTCTTC TTTTTCCCAT TCTTCTCATG CCTCCCTGAC TGGCTCTCAT 18061 CTTTTTCAGT GGCCCCCTTC TGCTTGCCCA CACATGTAGC GGCTCTCAAA AGTTCTCGTG
18121 GCCTCTTTTT CACATTACAT GCTTCCTCTG GATGTCTTAT CTAACAATAC AACATGGACT
18181 TTCTGCGGAA ATGATTCCCA AGCCTTTATT TTCCCATTAG TTGCTAGGTG TACATTTTCA
18241 GATGCTGGTT TGCCATCTCT TGAGGTAGCA TATTTCAAAG CCAGATCACC CCCGCTTACT
18301 CTTGGATCTC CCCTCTTCCT CTTGTTGAGA GTTGGAAGCA GAGAATGGGT AGTATCTCTA 18361 TATCCCTTTT CCTTTGCTAA TTTGCCCTTT TTTGGAACAT TTGTTAACCC TAGTCATTCT
18421 CTGCTAAAGG AGGGCTTGAC TAGAGCTACT TAACGAGAGT TTAGTGCATA CCTATCCTAT
18481 GGAAGCCAGT GTGTTGAGCC CTTCTTAGGG CTGCCAAAGA AATGGATGCT CCTATGCCCA 18541 GGCTCAGTCA GGGACCTTGG GAGATACTAT ATTTGGGATG GGAGTAAGTG GAATGCCAGC 18601 TGCCATGCAT AATGCAGTGG TGTTCATGGT GGTTGGTCAG CCCACACTGT ATCAAGCTCT 18661 GCAGATTTTT ACTCTGTTAA TGCACATATT ACCTCATTAG TCTTGGCCTT CCTGGGGAGT 18721 TGTATGTAAC ATGTATTTTC TCTCTCATAG TGATTGACGA CACAACCAAA GAGGTGGTGT 18781 ATACGGATTA TATTGACATC AGTGTTGCAG TGGCCACCCC ACGGGTATGT TGGGGCAGGA 18841 GGTGGGGAAT GTTGGTCTTA GACCCTCACC TTATCTGTGT GAAGGAGATC ACACAAGAGA
18901 AATCATTTCT TTAATTCTGT ATTTTTAGAA GGGAGTTAGT AAAAGTAACC TTTTTTTCTT 18961 TTAAACCATG CCGCATTCTT TTAACACTTT CTGTTAATCA CACTGAGTAA TGAAGGTATT 19021 CTAGGGAGGG CATACCATGG GTTGAATTCA AGGGAGTTGT TAACTATAAA AGGTACTATT 19081 AATTTGCAAC TGAAAACAGC TTTTCACCCC CTTCAGGGTC TGGTGGTTCC AGTCATCAGG
19141 AATGTGGAAG CTATGAATTT TGCAGATATT GAACGGACCA TCACTGAACT GGGAGAGAAG
19201 GTAAAGTAGA AAGATGTATA CAAGCTGCTA AGCAGGCGAG GGAAGAGAGC CTTCAGAAGG
19261 CTGGGCTCAC TAGCAAGCAG TGCTCATGGA AAGTCAAGTG CATAGCCCCT GAGAAAAGCA
19321 GTTGCCCATG AGAGCTAAGT ATTTTATATA TCTGGTGGAG TTTACCATGC TCTGGTAACT 19381 GAAATCTCAT CAGATGAGAC CTGCTAGAAA AGATCATCTT TGAAGTCTTC AACTGTATGA
19441 AATTGTGCCT TTTCTCTGGT GGAGCACTGG TCACAGACAC CTTAGCCGAA CAACTGTCAC
19501 ACGGAAGCTA GCGCTTGTGC CATCGATCTC GATACAAGCT TTCTGGACTT CCTTTGCTTT
19561 TTTGTTTTTA GAGGCAGGGT CTCGCTGTGT TGCCCAGGCT GGAGTAGAGT GGTAGAGTCA
19621 TAGCTTACTG CAGCCTTGCA CTCCTGGGCT CAAGCGATTC TCCTGCCTCA GCCTCCCAAG 19681 TAGCTGGGAC TACAGGTGTG CACCACCATG CCTGGCTAAT TCTTAAGTTT TTTATACAGA
19741 TGGGGGTCTC ACTATGTTGC CAGGTTGGCC TTGAACTCCT GGCCTCAAGC AGTCCTCCTG
19801 CCTCGGCCTC CCAAAGCGCT GGGATTATGG ACTGACTTTA GGTGAAGGAA ACTGGGGTGG
19861 CTTAATATTT CAATTATGAA ATCTGTTTTA GGCCCGAAAG AATGAACTTG CCATTGAAGA
19921 TATGGATGGT GGTACCTTCA CCATTAGCAA TGGAGGCGTT TTTGGCTCGC TCTTTGGAAC 19981 ACCCATTATC AACCCCCCTC AGTCTGCCAT CCTGGGGATG CATGGCATCT TTGACAGGCC
20041 AGTGGCTATA GGAGGCAAGG TAGGAACCGT CACTTCTAAG GTCCTAGTGG CTAGGTCTCG
20101 ATGAAAGGGA AATCCAACTA AATGCTAATT GTAAAACATT AACTATAATT TCAGGCCTAA
20161 GTTCCATTTC TTAGTTTCTA ATAGCTAAGG CACTGATTAT CAAATTGTGG TCTGGATCAG
20221 CATCATCTGG GACCTTATTA GAAATGCATA TTCTTAGACC CCATCCCAGA CTTAAAGAAG 20281 AAGTGCAGAT GATCCTGATG CATATTCAAG TTTGAGAACC ACTGAGCTGA GGAGGCTTGT
20341 TTGCTTCTAT GGAGTGGGGG ATATAGTTGG AAACGTGGTC CCTTGCCCCT GGGAAACAGA
20401 TGACAATTAT GCATGTCAGT CTGTGAGCTG CCAAGTTGTA TGATAAAGCT TGTAAATTTC
20461 CCGGGAGTTC AGAGAATTAA GTATTAAAAG GGGCTTGACT GGCCCTTAGA AGATACGTTG
20521 GTATCAAAAA AGCTTTCTGG GAACTCCTGT GTTTAGAATC CTTTTTGGGT TACTGACTCC 20581 TGTGTGAAAC CACAGTCTGT AGATCTTCCT GGAAAATGCC CATACACCCA ACTACACATA
20641 CAATTCCAGA ATTTGTAGAC CCTGGCTTAA χχχχTχχ Tχ TTTTTTAAAT TCTAACCCCA
20701 GAGAGAAGCA AGTAGAGGCA GCCAGAACTG AAGGGAAAAC TTTCCTTGTA GGCAGCAGAT
20761 GCGTTAGAGG GCAGAGTATG TTTTTAAAAA ATAAAAGGCA GTTGTGAGAA GACAGTTTTC
20821 TTGGCAAACT TTGTTTCTGA GTGGGGAACG TTTGCACTGA GGGTAAGTCC TGGTCTTTGA 20881 AATACTGTAA ATATGCAGAG CGTAACATCA ATAGGAAAGG CTCTGGAATT AGGAACTTTC
20941 TTATGGGCTG TGCTAAATCT CCTTCAGCGA GGCTGGCTGT GGCTTGCTGG AGACAAACCT
21001 ATTTACCTTT CCTCTCTGTA GGTAGAGGTG CGGCCCATGA TGTACGTGGC ACTGACCTAT 21061 GATCACCGGC TGATTGATGG CAGAGAGGCT GTGACTTTCC TCCGCAAAAT CAAGGCAGCG
21121 GTAGAGGATC CCAGAGTCCT CCTCCTGGAT CTTTAGGAGG AACCCACACA CCCTACAAGT
21181 TGATCATGCA GGAACTGAAA ACCAGTCTTC TCCCTGTCCC CTCATGGGTC CCGGGTTAGC
21241 CTGGTGACAG GCAGACACAT GCTGTTGGCC TCAAGCAAGG AAGCAGAGCA CTGTGTAACC 21301 AGCAGTCACA GGTCTTTTCT TGGCGTTCCT GCCAGGCTCT CCCTCTCTGC ACCTGTCTCA
21361 TAGCCTCGAA TATCTTAATT CCTTAGGCTT AAGAGAGAGA GCCTTAATGG ATGCTCATTC
21421 ATATTCCTGC CTTTCTTCCA TCAGCTCTCT GCAAAGATGA TTTTGCTTTT CCCTAGTGCT 21481 GGTATACTAT AGAGAAACCC CTGGGGATCA TGTGATTAAG TTCCTATCTT TTGAAAGTTT 21541 GTTCTGCAGA GACTTCTAGG AGGATGCTGT GCCTCCCAAG CTCAGAGCAG CCTCTGTCCT 21601 GGCTGTGCAC ATTCTCCCTT GATTCCACTT GTGTGGAGGG ATTGAACACA GGCAAAGAGG
21661 TGCTGCTTTG CTTCTTCAAT GGCACCTTCA TTCTCCGTTG TCATTGACTT CAAGATGCCT
21721 CTTCTACCTC TTCCAGGAAG CACAGGCCAG GGGATCTGGG TGTGTGAGTG GGAGGAGAGG
21781 GCAGAGGTCC CCTGAGGTCA TGCATTGTAA TCATCATAGA AGGAGAGCCC AGGCCTGCCC
21841 TCACGCTCTC CATCATAGGC TGACACCAAG AAGACTCGTC TTGGCACAAT CTCACACAGC 21901 TGGGGCTGTA GCAACCCTTT CCAACCCCTT TGCTGGTTGC TGGGCCTCAT TCTAGCACCT
21961 TGTTCTTAGA GCAGATTCTA GCACATCATG GCAGTGGGAC CAAGCGTGGT CCCGAGGAAG
22021 GGCCAGAGCC TGGTAGAGAC TAGGGAAGGG AGGTCTCCTC TAGACTGACT CACATTGCCT
22081 TGAGCTTTTC AGTTAAGTTG CTGTAAGCAC CTGGGCTGAG GAGGCAGTTT TTGTTCCTTC
22141 CTGCGTTATA GCGGGGCCTT GTCTCTTCCT CTGCAGGACA CAGATCTGGA GGACGTGGAC 22201 TGCGGTAGGA AACCACCCTG AGGGTGTTAG TACCTAGTGG TGAAACGGAT GAGGTCATTT
22261 CTAAGGTGTG TTGCCCGTGG AATCTGGGCA CAACTCATTG GAATTCCTTG GAGCCACTGG
22321 GATTCATGGC TTTGTATCCA ACTGCATCCA GGCCTGAGGC TGCTGACGTT TGACACCAGG
22381 GCCAGTAGAG AGTGCCCTTT TGTATCTTAA GCCAAGTAAG TGAGGCCTGG GGGTGGGGGA
22441 GGGGGGAAGG GGTGGGAGCC AATACTGAGT GCCTGCAGCA TCTACTACTC TGTCTTCACT 22501 ATTCAGAACC TTGTAACTAA AGTATTTAAA GAAACTGATT TTAAATGCAA ATTAAAGGGC
22561 AGATATTCTC AAA
The DLST gene sequence includes approximately on kilobase of upstream sequence priro to the first exon. The gene consists of 15 exons and 14 introns. The amino acid sequence for human dihydrolipoamide succinyltransferase encoded by the intronsis as follows (SEQ. ID. No. 4):
1 MLSRSRCVSRAFSAPLSAFQKGNCPLGRRS PGVSLCQGPGYPNSRKWINNSVFSVR
59 FFRTTAVCK3DDLVTVKTPAFAESVTEGDVR EKAVGDTVAEDEWCEIETDKTSVQVP 117 SPANGVIEALLVPDGGKVEGGTPLFTLRKTGAAPAKAKPAEAPAAAAPKAEPTAAAVP
175 PPAAPIPTQMPPVPSPSQPPSGKPVSAVKPTVAPPLADAGAGKGLRSEHREKMNRMRQ
233 RIAQR KEAQNTCAM TTFNEIDMSNIQEMRARHKEAFLKKHNL'KLGFMSAFVKASAF
291 ALQEQPWNAVIDDTTKEλAΛTTDYIDISVAVATPRGL"WPVIRNVEAMNFADIERTIT
349 ELGEKARKNELAIEDMDGGTFTISNGGVFGS FGTPIINPPQSAlLGMHGIFDRPVAI 407 GGKVEVRPM YVALTYDHR IDGREAVTFLRKIKAAVEDPRVL LDL Althought the roles of all of the introns and exons for the DLST are not well determined, the present invention provides a number of polymoφhisms win the DLST gene which are associated with Alzheimer's disease. The sequence and numbering is based on GenBank Accession # D26535 (Nakano.K. et al., "Isolation, Characterization and Structural Organization of the Gene and Pseudogene for the Dihydrolipoamide Succinyltransferase Component of the Human 2-Oxoglutarate Dehydrogenase Complex," Eur. J. Biochem. 224(1): 179-189 (1994), which is hereby incoφorated by reference). In one allele, CGCTCC is replaced by CCGCTC (nt #829-834 in SEQ. ID. No. 3). This change is located in Exon 1 and amino acids Ala-Pro are replaced by Arg-Ser (#14-15). Other alleles are: T replaced by C (nt #7912 in SEQ. ID. No. 3) (Intron 3); G replaced by A (nt #11791 in SEQ. ID. No. 3) (Exon 8, Neutral substitution); C replaced by G (nt #11803 in SEQ. ID. No. 3) (Exon 8, Neutral substitution); A replaced by G (nt #13159 in SEQ. ID. No. 3) (Exon 10, Neutral substitution); A replaced by G (nt #13159 in SEQ. ID. No. 3) (Intron 10); C replaced by G (nt #18784 in SEQ. ID. No. 3) (Exon 12, Amino acid Thr replaced by Arg (#312); C replaced by A (nt #18822 in SEQ. ID. No. 3) (Exon 12, Neutral, substitution); A replaced by G (nt #19864 in SEQ. ID. No. 3) (Intron 13); T replaced by C (nt #19930 in SEQ. ID. No. 3) (Exon 14, Neutral substitution); C replaced by T (nt #19930 in SEQ. ID. No. 3) (Exon 14, Neutral substitution); and T replaced by C (nt #21508 in SEQ. ID. No. 3). (Exon 15, Outside of the open reading frame).
Homozygosity of the haplotypic genotype G (19864 in SEQ. ID. No. 3, C (19930 in SEQ. ID. No. 3) enhances the risk of the epsilon-4 allele of the apolipoprotein E gene for Alzheimer's disease in elderly Ashkenazi Jews. In American Caucasians, homozygosity of the genotype A (19864 in SEQ. ID. No. 3), T (19930 in SEQ. ID. No. 3) reduced the risk of Alzheimer's disease.
In addition to polymoφhisms in the DLST gene, another preferred gene useful in accordance with the present invention is the gene encoding for dihydrolipoamide dehydrogenase ("DLD"). The sequence of the human gene encoding for DLD is provided below as SEQ. ID. No. 5 (Pons, G. et al., "Cloning And cDNA Sequence Of The Dihydrolipoamide Dehydrogenase Component Of Human Alpha- Ketoacid
Dehydrogenase Complexes," Proc. Natl. Acad. Sci. U.S.A. 85:1422-26 (1988), which is hereby incoφorated by reference). 1 GCTCCCAGCG GAGGTGAAAG TATTGGCGGA AAGGAAAATA CAGCGGAAAA ATGCAGAGCT
61 GGAGTCGTGT GTACTGCTCC TTGGCCAAGA GAGGCCATTT CAATCGAATA TCTCATGGCC
121 TACAGGGACT TTCTGCAGTG CCTCTGAGAA CTTACGCAGA TCAGCCGATT GATGCTGATG
181 TAACAGTTAT AGGTTCTGGT CCTGGAGGAT ATGTTGCTGC TATTAAAGCT GCCCAGTTAG 241 GCTTCAAGAC AGTCTGCATT GAGAAAAATG AAACACTTGG TGGAACATGC TTGAATGTTG
301 GTTGTATTCC TTCTAAGGCT TTATTGAACA ACTCTCATTA TTACCATATG GCCCATGGAA
361 AAGATTTTGC ATCTAGAGGA ATTGAAATGT CCGAAGTTCG CTTGAATTTA GACAAGATGA 421 TGGAGCAGAA GAGTACTGCA GTAAAAGCTT TAACAGGTGG AATTGCCCAC TTATTCAAAC
481 AGAATAAGGT TGTTCATGTC AATGGATATA GAAAGATAAC TGGCAAAAAT CAAGTCACTG 541 CTACGAAAGC TGATGGCGGC ACTCAGGTTA TTGATACAAA GAACATTCTT ATAGCCACGG
601 GTTCAGAAGT TACTCCTTTT CCTGGAATCA CGATAGATGA AGATACAATA GTGTCATCTA
661 CAGGTGCTTT ATCTTTAAAA AAAGTTCCAG AAAAGATGGT TGTTATTGGT GCAGGAGTAA
721 TAGGTGTAGA ATTGGGTTCA GTTTGGCAAA GACTTGGTGC AGATGTGACA GCAGTTGAAT
781 TTTTAGGTCA TGTAGGTGGA GTTGGAATTG ATATGGAGAT ATCTAAAAAC TTTCAACGCA 841 TCCTTCAAAA ACAGGGGTTT AAATTTAAAT TGAATACAAA GGTTACTGGT GCTACCAAGA
901 AGTCAGATGG AAAAATTGAT GTTTCTATTG AAGCTGCTTC TGGTGGTAAA GCTGAAGTTA
961 TCACTTGTGA TGTACTCTTG GTTTGCATTG GCCGACGACC CTTTACTAAG AATTTGGGAC
1021 TAGAAGAGCT GGGAATTGAA CTAGATCCCA GAGGTAGAAT TCCAGTCAAT ACCAGATTTC
1081 AAACTAAAAT TCCAAATATC TATGCCATTG GTGATGTAGT TGCTGGTCCA ATGCTGGCTC 1141 ACAAAGCAGA GGATGAAGGC ATTATCTGTG TTGAAGGAAT GGCTGGTGGT GCTGTGCACA
1201 TTGACTACAA TTGTGTGCCA TCAGTGATTT ACACACACCC TGAAGTTGCT TGGGTTGGCA
1261 AATCAGAAGA GCAGTTGAAA GAAGAGGGTA TTGAGTACAA AGTTGGGAAA TTCCCATTTG 1321 CTGCTAACAG CAGAGCTAAG ACAAATGCTG ACACAGATGG CATGGTGAAG ATCCTTGGGC
1381 AGAAATCGAC AGACAGAGTA CTGGGAGCAC ATATTCTTGG ACCAGGTGCT GGAGAAATGG 1441 TAAATGAAGC TGCTCTTGCT TTGGAATATG GAGCATCCTG TGAAGATATA GCTAGAGTCT
1501 GTCATGCACA TCCGACCTTA TCAGAAGCTT TTAGAGAAGC AAATCTTGCT GCGTCATTTG
1561 GCAAATCAAT CAACTTTTGA ATTAGAAGAT TATATATTTT TTTTTCTGAA ATTTCCTGGG 1621 AGCTTTTGTA GAAGTCACAT TCCTGAACAG GATATTCTCA CAGCTCCAAG AATTTCTAGG
1681 ACTGAATTAT GAAACTTTTG GAAGGTATTT AATAGGTTTG GACAAAATGG AATACTCTTA 1741 TATCTATATT TTACATAAAT TTAGTATTTT TGTTTCAGTG CACTAATATG TAAGACAAAA
1801 AGCTACTTAT TGTAGCATCC TGGAATATCT CCGTCAACTC ATATTTTCAT GCTGTTCATG
1861 AAAGATTCAA TGCCCCTGAA TTTAAATAGC TTTTTTCTCT GATACAGAAA AGTTGAATTT
1921 TACATGGCTG GAGCTAGAAT TTGATATGTG AACAGTTGTG TTTGAAGCAC AGTGATCAAG
1981 TTATTTTTAA TTTGGTTTTC ACATTGGAAA CAAGTCAGTC ATTCAGATAT GATTCAAATG 2041 TCTATAAACC GAACTGATGT AAGT
The amino acid sequence for dihydrolipoamide dehydrogenase is as follows (SEQ. ID. No. 6):
1 MQS SRλYCS AKRGHFNRISHGLQG SAVP RTYADQPIDADVTVIGSGPGGYVAAI
59 KAAQ GFKTVCIEKNETLGGTC NVGCIPSKA LNNΞHYYHMAHGKDFASRGIEMSEV 117 RLNLDKr iMEQKSTAVKALTGGIAHLFKQNKλA^HVNGYRKITGKNQVTATKADGGTQVI 175 DTK ILIATGSEVTPFPGITIDEDTIVSSTGALSLKKVPEKMWIGAGVIGVELGSV 233 QRLGADVTAVEF GHVGGVGID EISKNFQRILQKQGFKFKLNTKVTGATKKSDGKID 291 VS IEAASGGKAEVITCDVLLVCIGRRPFTKN GLEELGIELDPRGRIPVNTRFQTKIP 349 NIYAIGDλTVAGPMLAHKAEDEGI ICVEGMAGGAλ HIDYNCVPSVIYTHPEVAVJVGKSE
407 EQLKEEGIEYKVGKFPF7ΛANSRAKTNADTDGMVKI GQKSTDRV GAHI GPGAGEMV 465 NEAALALEYGAS CED I ARVCHAHPT S EAFRE NLAAS FGKS INF
The nucleotide sequence in SEQ. ID. No. 4 is for a cDNA encoding the DLD gene, thus no introns or sequences upstream of the transcriptional start site are included.
Nevertheless, several polymoφhism have been identified within the sequenced region.
The present invention provides nucleotide polymoφhisms which have been identified within the DLD gene. In a preferred embodiment the nucleic acid molecule has a T replaced by A at nucleotide 1598 in SEQ. ID. No. 5. In another preferred embodiment, the nucleic acid molecule has a G replaced by T at nucleotide 1608 in SEQ. ID. No. 5.
Numerous methods for characterizing or detecting polymoφhisms are known in the art and any of those methods are suitable for the present invention.
The most obvious method of characterizing a polymoφhism entails direct DNA sequencing of the genetic locus that flanks and includes the polymoφhism. Such analysis can be accomplished using either the "dideoxy-mediated chain termination method," also known as the "Sanger Method" (Sanger, F. et al., "DNA Sequencing with Chain- Terminating Inhibitors," Pro Natl. Acad. Sci. U.S.A.. 74:5463-5467 (1977), which is hereby incoφorated by reference) or the "chemical degradation method," also known as the "Maxam-Gilbert method" (Maxam, A.M. et al., "A New Method for Sequencing
DNA," Proc Natl. Acad. Sci. U.S.A.. 74:560-564 (1977), which is hereby incoφorated by reference). Genomic sequence-specific amplification technologies, such as the polymerase chain reaction (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51 :263-273 (1986); Erlich, H. et al, European Patent Appln. 50,424; European Patent Appln. 84,796, European Patent Appln. 258,017, and European Patent Appln. 237,362; Mullis, K., European Patent Appln. 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194, which are hereby incorporated by reference), may be employed to facilitate the recovery of the desired polynucleotides. Amplification of a selected, or target, nucleic acid sequence may be carried out by any suitable means, either to facilitate sequencing or for direct dtection of polymoφhisms. (See generally Kwoh, D. and Kwoh, T., Am Biotechnol Lab, 8, 14 (1990) which is hereby incoφorated by reference.) Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction, ligase chain reaction ("LCR")(see Barany, Proc Nat'l Acad. Sci. U.S.A. 88:189 (1991), which is hereby incorporated by reference), strand displacement amplification (see generally Walker, G. et al., Nucleic Acids Res. 20, 1691 (1992); Walker. G. et al., Proc Nat'l Acad. Sci. U.S.A. 89:392 (1992), which are hereby incoφorated by reference), transcription-based amplification (see Kwoh, D. et al., Proc Nat'l Acad. Sci. U.S.A. 86: 1 173 (1989), which is hereby incoφorated by reference), self-sustained sequence replication (or "3SR") (see Guatelli, J. et al., Proc Nat'l Acad. Sci. U.S.A. 87:1874 (1990), which is hereby incoφorated by reference), the Qb replicase system (see Lizardi, P. et al., Biotechnology, 6, 1 197 (1988), which is hereby incoφorated by reference), nucleic acid sequence-based amplification (or "NASBA") (see Lewis, R., Genetic Engineering News, 12(9), 1 (1992), which is hereby incoφorated by reference), the repair chain reaction (or "RCR") (see Lewis, R., Genetic Engineering News, 12(9), 1 (1992), which is hereby incoφorated by reference), and boomerang DNA amplification (or "BDA") (see Lewis, R., Genetic Engineering News, 12(9), 1 (1992), which is hereby incoφorated by reference). Polymerase chain reaction is currently preferred.
In general, DNA amplification techniques such as the foregoing involve the use of a probe, a pair of probes, or two pairs of probes which specifically bind to DNA encoding the gene of interest, but do not bind to DNA which do not encode the gene, under the same hybridization conditions, and which serve as the primer or primers for the amplification of the gene of interest or a portion thereof in the amplification reaction. Mundy, et al. (U.S. Pat. No. 4,656,127, which is hereby incoφorated by reference) discusses alternative methods for determining the identity of the nucleotide present at a particular polymoφhic site. Mundy's methods employ a specialized exonuclease-resistant nucleotide derivative. A primer complementary to the allelic sequence immediately 3'-to the polymoφhic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymoφhic site on the target molecule contains a nucleotide that is complementary to the particular exonucleotide-resistant nucleotide derivative present, then that derivative will be incoφorated by a polymerase onto the end of the hybridized primer. Such incoφoration renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonucleotide-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymoφhic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. The Mundy method has the advantage that it does not require the determination of large amounts of extraneous sequence data. It has the disadvantages of destroying the amplified target sequences and unmodified primer and of being extremely sensitive to the rate of polymerase incoφoration of the specific exonuclease-resistant nucleotide being used.
Recently, several primer-guided nucleotide incoφoration procedures, i.e microsequencing methods, or assaying polymoφhic sites in DNA have been described (Kornher, J.S. et al., "Mutation Detection Using Nucleotide Analogs that Alter Electrophoretic Mobility," Nucl. Acids. Res., 17:7779-7784 (1989); Sokolov, B.P., "Primer Extension Technique for the Detection of Single Nucleotide in Genomic DNA," Nucl. Acids Res.. 18:3671 (1990); Syvanen, A.C. et al., "A Primer-Guided Nucleotide Incorporation Assay in the Genotyping of Apolipoprotein E," Genomics. 8:684-692 (1990); Kuppuswamy, M.N. et al., "Single Nucleotide Primer Extension to Detect Genetic Diseases: Experimental Application to Hemophilia B (Factor IX) and Cystic Fibrosis Genes." Proc Natl. Acad. Sci. U.S.A., 88:1143-1 147 (1991); Prezant, T.R. et al., "Trapped-Oligonucleotide Nucleotide Incoφoration (TONI) Assay, a Simple Method for Screening Point Mutations," Hum. Mutat.. 1 :159-164 (1992); Ugozzoli, L. et al., "Detection of Specific Alleles by Using Allele-specific Primer Extension Followed by Capture on Solid Support," GATA, 9:107-112 (1992); Nyren, P. et al., "Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay," Anal. Biochem., 208: 171-175 (1993), which are hereby incoφorated by reference). These methods differ from Genetic Bit TM Analysis ("GBA TM " discussed extensively below) in that they all rely on the incoφoration of labeled deoxynucleotides to discriminate between bases at a polymoφhic site. In such a format, since the signal is proportional to the number of deoxynucleotides incoφorated, polymoφhisms that occur in runs of the same nucleotide can result in signals that are proportional to the length of the run (Syvanen, A.C. et al., "Identification of Individuals by Analysis of Biallelic DNA Markers, Using PCR and Solid-Phase Minisequencing." Amer. J. Hum. Genet., 52:46-59 (1993), which is hereby incoφorated by reference).
Cohen, D. et al. (French Patent 2,650.840; PCT Appln. No. WO91/02087, which is hereby incoφorated by reference) discuss a solution-based method for determining the identity of the nucleotide of a polymoφhic site. As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3'-to a polymoφhic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymoφhic site will become incoφorated onto the terminus of the primer.
An alternative method, known as Genetic Bit Analysis TM or GBA TM is described by Goelet, P. et al. (PCT Appln. No. 92/15712, which is hereby incoφorated by reference). In a preferred embodiment, the method of Goelet, P. et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymoφhic site. The labeled terminator that is incoφorated is thus determined by, and complementary to, the nucleotide present in the polymoφhic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087. which are hereby incoφorated by reference) the method of Goelet, P. et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase. It is thus easier to perform, and more accurate than the method discussed by Cohen.
Another solid phase method that uses different enzymology is the "Oligonucleotide Ligation Assay" ("OLA") (Landegren, U. et el., "A Ligase-Mediated Gene Detection Technique," Science. 241 : 1077-1080 (1988), which is hereby incoφorated by reference). The OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or. another biotin ligand. OLA is capable of detecting point mutations. Nickerson, D.A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D.A. et al., Proc Natl. Acad. Sci. U.S.A.. 87:8923-8927 (1990), which is hereby incoφorated by reference). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA. Assays, such as the OLA, require that each candidate dNTP of a polymoφhism be separately examined, using a separate set of oligonucleotides for each dNTP. The major drawback of OLA is that ligation is not a highly discriminating process and non-specific signals can be a significant problem.
Other recently developed variations for detecting the presence of polymoφhisms include: differential restriction endonuclease digestion (DRED), allele-specific oligonucleotide probing (ASOP), and ligase-mediated gene detection (LMGD). Additional methods of analysis would also be useful in this context, such as fluorescence resonance energy transfer (FRET) as disclosed by Wolf et al., Proc Nat. Acad. Sci.
U.S.A.. 85: 8790-94 (1988), which is hereby incoφorated by reference. DRED analysis is accomplished in the following manner. If conditions occur including (1) a particular amplified cDNA segment contains a sequence variation that distinguishes an allele of a polymoφhism and (2) this sequence variation is recognized by a restriction endonuclease, then the cleavage by the enzyme of a particular polynucleotide segment can be used to determine the alloantigen phenotype. In accomplishing this determination, amplified cDNA derived from platelet or red blood cell mRNA is digested and the resulting fragments are analyzed by size. The presence or absence of nucleotide fragments, corresponding to the endonuclease-cleaved fragments, determines which phenotype is present.
In ASOP analysis according to conventional methods, oligonucleotide probes are synthesized that will hybridize, under appropriate annealing conditions, exclusively to a particular amplified cDNA segment that contains a nucleotide sequence that distinguishes one allele from other alleles of a red blood cell or platelet membrane glycoprotein. This specific probe is discernably labeled so that when it hybridizes to the allele distinguishing cDNA segment, it can be detected, and the specific allele is thus identified.
In the course of the third method of analysis, LMGD, as disclosed by Landegren et al., Science, 241: 1077-80 (1988), which is hereby incoφorated by reference, a pair of oligonucleotide probes are synthesized that will hybridize adjacently to each other, i.e., to a cDNA segment under appropriate annealing conditions, at the specific nucleotide that distinguishes one allele from other alleles of a red blood cell or platelet membrane glycoprotein. Each of the pair of specific probes is labeled in a different manner, and when it hybridizes to the allele-distinguishing cDNA segment, both probes can be ligated together by the addition of a ligase.
When the ligated probes are isolated from the cDNA segments, both types of labeling can be observed together, confirming the presence of the allele-specific nucleotide sequence. Where the above-described pair of differently labeled probes bind to a nucleotide sequence containing a distinguishing nucleotide of a different allele, the probe pair is not ligatable and, after the probes are isolated from the cDNA segments, both types of labeling are observed separately.
In addition to methods for determining whether a patient is at risk for Alzheimer's disease or some other neurodegenerative disorder, the invention also provides isolated nucleic acid molecules which encode a gene encoding a Krebs tricarboxylic acid cycle component, or a fragment of the gene, and a polymoφhism in, wherein the presence of said polymoφhism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases. The present invention also provides fragments of the nucleic acid molecules.
Fragments of the nucleic acid molecules can be used to hybridize to target nucleic acid molecules to detect the presence of a polymoφhism. The fragments must be long enough to be useful as a primer in a PCR or LCR type reaction. Preferred fragments are at least twelve bases in length. The nucleic acid molecules or fragments need not be identical to the sequences of
SEQ. ID. No. 3 or 5, excluding the polymoφhism. Rather the nucleic acid molecule needs to have the polymoφhism and sufficient identity to the remainder of sequence of SEQ. ID. No. 3 or 5 so that the nucleic acid molecule or fragment may be used to differentiate between genetic material having the polymoφhism and genetic material lacking the polymoφhism.
The nucleic acid molecules of the present invention may be linked to other nucleic acid molecules such as vectors or tags to facilitate amplification, purification, or identification.
The present invention also provides a kit for identifying individuals with a predisposition to Alzheimer's disease or another neurodegenerative, disease. The kit contains nucleic acid fragments which can be used to amplify a gene encoding a Krebs tricarboxylic acid cycle component, or a fragment thereof, which consists of a position where a polymoφhism may be located, wherein the presence of said polymoφhism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
In yet another embodiment, the invention provides a kit for identifying individuals with a predisposition to Alzheimer's disease or another neurodegenerative disease which contains a restriction enzyme which may be used to detect the presence of a polymorphism in a gene encoding a Krebs tricarboxylic acid cycle component, wherein the presence of said polymoφhism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
In a preferred embodiment, a kit utilizing a restriction enzyme analysis also contains nucleic acid fragments which can be used to amplify the gene encoding a Krebs tricarboxylic acid cycle component, or a fragment thereof, which consists of a position where a polymoφhism may be located.
The present invention also provides antibodies which preferentially recognizes a Krebs tricarboxylic acid cycle component protein which is encoded by a gene consisting of a polymoφhism, wherein the presence of said polymoφhism is indicative of a predisposition or reduced risk for Alzheimer's disease or other neurodegenerative diseases.
Some polymoφhisms will generate amino acid substitutions which can be detected by immunochemical methods. For example for the gene which encodes dihydrolipoamide succinyltransferase, one allele has a substitution where amino acids Ala-Pro are replaced by Arg-Ser at residues 14-15. Another substitution results in the replacement of Thr by Arg at residue 312. Alternatively, antibodies may be used to detect changes in the DLD protein which result from polymorphisms in the DLD gene. Monoclonal antibody production may be effected by techniques which are well- known in the art. Basically, the process involves first obtaining immune cells
(lymphocytes) from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler et al., Nature, 256:495 (1975). which is hereby incoφorated by reference.
Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.
Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol ("PEG") or other fusing agents (See Milstein et al., Eur. J. Immunol.. 6:511 (1976), which is hereby incoφorated by reference). This immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.
Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 ml per site at six different sites. Each injected material will contain adjuvants with or without pulverized acrylamide gel containing the protein or polypeptide after SDS- polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. This and other procedures for raising polyclonal antibodies are disclosed in E. Harlow et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incoφorated by reference. In addition to utilizing whole antibodies, the processes of the present invention encompass use of binding portions of such antibodies. Such binding portions include Fab fragments, F(ab')2 fragments, and Fv fragments. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118, New York: Academic Press (1983), which is hereby incoφorated by reference.
The antibodies can be used to screen biological samples from the individual for the presence of altered proteins which are associated with a predisposition or reduced risk for Alzheimer's disease or another neurodegenerative disorder.
Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof which may be useful for carrying out the method disclosed herein. See Skold et al., U.S. Patent No. 4.727,022; Forrest et al., U.S. Patent No. 4,659,678; David et al., U.S. Patent No. 4,376,1 10; Litman et al., U.S. Patent No. 4,275,149; Maggio et al., U.S. Patent No. 4,233,402; and Isaka et al., U.S. Patent No. 4,230,767, which are hereby incorporated by reference.
Antibodies which selectively bind a polymoφhic DLST or DLD isoform may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation. Antibodies which bind a polymorphic DLST or DLD isoform may likewise be conjugated to detectable groups such as radiolabels (e.g., 35S, 1251, 1311), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
The invention also provides a method of detecting a predisposition to a neurodegenerative disease in a human subject. A biological sample is obtained from the human subject. The biological sample is contacted with an antibody which preferentially recognizes a Krebs tricarboxylic acid cycle component protein, which is encoded by a gene having a polymoφhism that is indicative of a predisposition or reduced risk for a neurodegenerative disease. Then one determines whether the antibody binds to a Krebs tricarboxylic acid cycle component protein. Binding of the antibody to the Krebs tricarboxylic acid cycle component indicates that the polymoφhism is present which is indicative of a predisposition or reduced risk for a neurodegenerative disease.
Detecting the presence of a complex between an antibody or binding portion thereof and protein encoded by a gene carrying a polymoφhism associated with a neuro degenerative disease can be carried out by any conventional method for detecting antigen- antibody reactions, examples of which can be found, e.g., in Klein, Immunology, New York:John Wiley & Sons, pp. 394-407 (1982), which is hereby incoφorated by reference. For in vitro detection such a ploymoφhism, the formation of a complex between the antibody and the protein present in the tissue of fluid sample can be detected by enzyme linked assays, such as ELISA assays. Briefly, the antibody/protein complex is contacted with a second antibody which recognizes a portion of the antibody that is complexed with the protein. Generally, the second antibody is labeled so that its presence (and, thus, the presence of an anntibody /protein complex) can be detected. Alternatively, the antibody or binding portion thereof can be bound to a label effective to permit detection of the protein upon binding of the antibody or binding portion thereof to the protein. Suitable labels include, fluorophores, chromophores, radiolabels, and the like.
For example, a radiolabeled antibody or binding portion thereof of this invention can be used for in vitro diagnostic tests. The specific activity of a tagged antibody or binding portion thereof depends upon the half-life and isotopic purity of the radioactive label and how the label is incoφorated into the antibody or its binding portion. In immunoassay tests, the higher the specific activity, in general, the better the sensitivity. The antibodies or binding portions thereof of the present invention can be used and sold together with equipment to detect the particular label as a kit for in vitro detection of polymoφhisms associated with neurodegenerative diseases.
EXAMPLES
Example 1 - Materials and Methods
Subjects
Diagnoses were by standard clinical (McKhann, G. et al., "Clinical Diagnosis of Alzheimer's Disease: Report of NINCDS-ADRDA Work Group Under the Auspices of the Department of Health and Human Services Task Force on Alzheimer's Disease," Neurology, 34:939-944 (1984), which is hereby incoφorated by reference) or neuropathological criteria (Khachaturian, Z. S., "Diagnosis of Alzheimer's Disease," Arch. Neurol., 42:1097-1105 (1985), which is hereby incoφorated by reference) or both. Patients and controls were matched with respect to age and sex (Table 1). The first series consisted of Caucasian residents of Jewish Home and Hospital for tjie Aged, Bronx, N.Y. (Marin, D.B. et al., "Noncognitive Disturbances in Alzheimer's Disease: Frequency, Longitudinal Course, and Relationship to Cognitive Symptoms," J. Am. Geriatr. Soc, 45:1331-1338 (1997), which is hereby incoφorated by reference) and Hebrew Home of - J l
Aged at Riverdale, New York, N.Y. (Nadler, J.D. et al., "Mental Status Testing in the Elderly Nursing Home Population," J. Geriatr. Psychiatr. Neurol, 8:177-183 (1995), which is hereby incorporated by reference). All subjects were followed closely with serial neuropsychological tests. This ethnically defined cohort was selected to reduce difficulties in inteφreting results with a Caucasian population of mixed ethnic background. The second autopsy series included all autopsied subjects in the Caucasian Jewish cohort and additional Caucasians from the Dementia Research Clinic, Burke Medical Research Institute, and the Pathology Institute, Case Western Reserve University (Permkumar, D.R. et al., "Apolipoprotein E-ε4 Alleles in Cerebral Amyloid Angiopathy and Cerebrovascular Pathology Associated With Alzheimer's Disease," Am. J. Path., 148:2083-2095 (1996), which is hereby incoφorated by reference). Since the neuropathological lesions of AD are believed to precede the clinical disability by many years, it is unlikely that the non-AD controls in this group had a predisposition to developing AD. DNA was prepared by proteinase K digestion and pheno/chloroform extraction.
In some blood samples, DNA was prepared with the Easy-DNA kit (Invitrogen, Carlsbad, CA).
Polymorphisms of DLST The sequence of DLST was from GenBank data (accession number D26535)
(Nakano, K. et al., "Isolation. Characterization, and Structural Organization of the Gene and Pseudogene for the Dihydrolipoamide Succinyltransferase Component of the Human 2-Oxoglutarate Dehydrogenase Complex," Eur. J. Biochem., 224:179-189 (1994), which is hereby incoφorated by reference), numbered starting from the first nucleotide of exon 1. Initial screen by single strand conformation polymoφhism (SSCP) analysis indicated a polymoφhism within nucleotide (nt) 19,126-19,331. The PCR forward primer was 5' CTGGGGTGGCTTAATATTTCA 3' (SEQ. ID. No. 7) (nt 19,105-19,125) and the reverse primer was 5' GAGACCTAGCCACTAGGACCT 3' (SEQ. ID. No. 8) (nt 19,332-19,352). The PCR mixture, like all other PCRs in this study, contained (in lOμl volume) lx concentration of the PCR buffer [10 mM Tris(HCI), pH = 8.9, 10 mM dithiothreitol, 50 mM KC1 and 2.5 mM MgCl2] (Boehringer-Mannheim, Indianapolis, IN). 0.2 mM each of dATP, dCTP, dGTP and dTTP, 0.1 μM of each of the primers, 0.5 JZ ? .
units Taq DNA polymerase (BRL-Gibco, Gaithersburg, MD) and 100 μg test DNA. The reaction ran for 30 cycles, with an annealing temperature of 55°C. For SSCP analysis, the PCR mixture also included 0.1 μCi/μl [α32P]dCTP (New England Nuclear, Boston, MA), and the polymoφhisms were displayed on 0.7x concentration MDE gels (FMC, Rockland, ME). Sequencing analysis of the clonal derivatives, cloned in the pCR-TRAP vector (GeneHunter, Nashville, TN), showed a T19,183C substitution.
Sequencing analysis revealed further a G19,l 17A substitution, in intron 13. A DLST fragment was PCR amplified using the forward primer 5' CTGTCACACGGGCTAGCG 3' (SEQ. ID. No. 9) (nt 18,747-18,776) and the reverse primer 5' ATCCCCAGGATGGCAGACT 3' (SEQ. ID. No. 2) (nt 19,252-19,272).
Concentrations of PCR components were as above. The PCR ran for 30 cycles with an annealing temperature of 58°C. The sequencing reaction used the forward primer end- labeled with the infrared dye IRD41 (Li-Coφ, Lincoln, NE) and a Li-Cor Model 4000 DNA sequencer.
Genotyping of DLST
The A19,l 17G substitution abolished a restriction site of Ms el, while the T19,183C conversion conferred an Acil site (Fig. 1). Thus, identification of the haplotypes was achieved by RFLP analysis by simultaneous digestion With Acil and Msel of the DSLT fragment nt 18,747-19,272 (Figs. 1 and 2). The PCR used the primers nt 18,747-18,776 (forward) and nt 19,252-19,272 (reverse). (See above for primer sequences and PCR conditions.) Restriction digestion was performed by addition of 5μl enzyme mixture to each PCR incubation to final concentrations of 0.5 x buffers 2 and 4 (New England Biolabs, Beverly, MA), and 2 units each of Msel and Acil (New England Biolabs). The digestion mixture was incubated at 37°C overnight. The restriction fragments were resolved in 3.5% MetaPhor agarose gels (FMC) containing ethidium bromide, and visualized under ultraviolet light.
Each of the DLST genotypes corresponded to specific patterns of restriction fragments (Figure 2A), including a 296 bp fragment found consistently and exclusively in genotype G,C/A,T. This 296-bp DNA was a heteroduplex consisting of a G,C and an A,T strands, since this fragment was also detected in DNA mixtures form G,C/G,C and A.T/A.T (far left lanes, Fig. 2A), but not in either of the individual homozygotes. Additionally, when this 296-bp fragment was gel-purified, PCR amplified, and digested with Mvel and Acil, a restriction pattern consistent with A,T/G,C was reproduced (Figure 2B).
Genotypes of APOE were determined by PCR-RFLP (Hixon, J.E. et al., "Restriction Isotyping of Human Apolipoprotein E by Gene Amplification and Cleavage with H/zύrl," J. Lipid Res., 31 :545-548 (1990), which is hereby incoφorated by reference).
Example 2 - Modulation by DLST of the Genetic Risk of Alzheimer's Disease in a Very Elderly Population Previous studies of elderly Caucasian populations (Sheu, K-FR et al., "A Gene
Locus of Dihydrolipoyl Succinyltransferase (DLST) is Associated with Alzheimer's Disease," J. Neurochem., 66 (Suppl 1) SI OB (1996), which is hereby incoφorated by reference), suggested that the G19117,C19183 (G,C) allele would enhance the association of the APOE4 allele with AD. Experiments were designed to test whether this effect would hold even in the population above 85 years old, in whom the association of APOFΛ with AD was expected to be weak.
The data confirmed these hypotheses. In the elderly cohort of residents of two Jewish nursing homes (Table 1), the association of APOE4 with probable AD was found only in the individuals who had the G,C/G,C genotype (Table 2).
Table 1. Demographics of the Study Cohorts
Caucasian Jews Autopsy Series
Alzheimer Control Alzheimer Control
Number (total) 179 250 189 36
Neuropathologic 117 28 189 36
(brain)
Clinical (blood) 62 222 0 0
Age (years)
Mean ± SD 89 ± 7 87 ± 8 85 ± 9 81 ± 12
% Female Subjects 78% 73% 63% 64%
The autopsy series included neuropathologic cases from the Caucasian Jewish cohort and an additional 72 AD and 8 non-AD Caucasian subjects who were autopsy diagnosed.
Table 2. DLST Genotype G,C/G,C Increase the Risk of APOE4 for Probable AD in Elderly Caucasian Residents of Jewish Nursing Homes
Subjects with APOE4 allele
DLST Probable AD Control OR (95% CI)* P#
G.C/G.C 19/38 (50%) 13/62 (21%) 3.77 (1.59, 8.95) 0.0051
G,C/x 36/87 (41%) 37/1 14 (33%) 1.47 (0.82, 2.62) 0.2479 non-G,C 13/54 (24%) 14/74 (19%) 1.36 (0.58, 3.19) 0.6265
Total 68/179 (38%) 64/250 (26%) 1.78 (1.18, 2.69) 0.0084
Age of AD = 89 ± 8 years, control = 87 ± 8 years (mean ± SD). *, OR = odds ratio; 95% CI = confidence interval at 95% level. , by χ2 test.
In this elderly cohort of clinical and autopsy-confirmed cases, APOE4 was not a significant risk factor among individuals who are not homozygous for the G,C allele [odds ratio (OR) - 1.43 with 95% confidence interval (95% CI) = 0.89, 2.30; P = 0.127 (χ2 = 1.868)].
In the subgroup of 302 individuals whose ages were > 85 years (Table 3), an association of APOE4 with probable AD was again seen in those who were homozygous for the G,C allele [OR = 3.19 (95% CI = 1.19, 8.54): P = 0.0382 (χ2 = 4.300)].
Table 3. DLST Genotype G,C/G,C Increases the Risk of APOE4 for Probable AD in Very Elderly (> 85 years old) Caucasian Residents of Jewish Nursing Homes
Subjects with APOE4 allele
DLST Probable AD Control OR (95% CI)* P"
G,C/G,C 15/31 (48%) 10/44 (23%) 3.19 (1.19, 8.54) 0.0382
G,C/x 24/63 (38%) 22/74 (30%) 1.45 (0.71, 2.97) 0.3843 non-G,C 7/42 (17%) 9/48 (19%) 0.87 (2.59, 0.29) 0.9553
Total 46/136 (34%) 41/166 (25%) 1.56 (0.95, 2.57) 0.1064
Age (mean ± SD) of AD = 92 ± 4 years, control = 91 ± 4 years. *, OR = odds ratio; 95% CI = confidence interval at 95% level. , by χ test.
No significant association of APOE4 with AD was seen in the other subjects who were not homozygous for this allele [OR = 1.23 (95% CI = 0.69, 2.21); P = 0.586 (χ2 = 0.296)]. In these "very-elderly" subjects, the Ors for the G,C homozygous, and G,C negative subjects showed a trend consistent with a gene dose-response relationship between the dosage of the G,C allele and the likelihood of an association of AD with the APOE4 allele (Table 3). APOE4 by itself was not a significant risk factor for probable AD in this "very-elderly" cohort [OR = 1.56 (95% CI = 0.95, 2.57); P = 0.106 (χ2 = 2.61)].
In the second, autopsy diagnosed series, the same relationships were found between AD, homozygosity for the G,C allele and possession of APOE4 (Table 4). Table 4. DLST Genotype G,C/G,C Increases the Risk ofAPOE4 for AD in the Autopsy Series
Subjects with APOE4 allele
DLST AD Non-AD OR (95% CI)* p*
G,C/G,C 21/43 (49%) 1/13 (8%) 1 1.45 (1.87, 70.12) 0.0194
G,C/x 45/98 (46%) 6/13 (46%) 0.99 (2.66, 0.37) 0.7794 non-G,C 16/48 (33%) 2/10 (20%) 2.00 (0.38, 10.44) 0.7080
Total 82/189 (43%) 9/36 (25%) 2.30 (1.04, 5.09) 0.0608
Age (mean ± SD) of AD = 85 ± 9 years, non-AD = 81 ± 12 years. *, OR = odds ratio; 95% CI = confidence interval at 95% level. #, by χ2 test.
In this elderly series, APOE4 was a risk factor for AD only in the 25% subjects who were G,C/G,C [OR = 1 1.45 (95% CI = 1.87, 70.12); P = 0.0194 (χ2 = 5.46)]. AD did not associate significantly with APOE4 in those with only one copy or none of the G,C allele [OR = 1.35 (95% CI = 0.49, 2.92); P = 0.684 (χ2 = 0.165)]. The odds ratio in the G,C/G,C homozygotes was over 4-fold that in the overall group or in the heterozygotes or in those not carrying the G,C allele. DLST genotyping thus added significantly to the precision of the genetic definition in this unequivocally diagnosed series of elderly subjects.
The G19,l 17 was found only in the G,C allele, since no G.T allele was found. Therefore, the effect of homozygosity for the G,C allele (Tables 2, 3 & 4) can as well be attributed to homozygosity for G19,l 17. No relationship was found between AD and DLST genotypes in patients who were APOE4 negative, nor in the overall series regardless of APOE4 status.
Example 3 - A DLST Genotype Associated with a Reduced Risk for Alzheimer's Disease
A separate series of genetic screening was conducted, using a cohort of Caucasian Americans from Burke Medical Research Institute and Rehabilitation Hospital, or from the Pathology Institute, Case Western Reserve University Medical School. The diagnosis of AD was as described in Example 1. Ages of AD and control groups were 77 ± 8 and 73 ± 1 1 years (mean ± SD), respectively.
Subjects homozygous for the Al 9,117, T19.183 (A,T) allele of DLST were less likely to have AD when compared with other subjects [OR = 0.35; P = 0.018 (Table 5)]. Heterozygosity for A,T was not protective. As in the previous studies (Nakano, K. et al., "Alzheimer's Disease and DLST Genotype," Lancet. 350:1367-1368 (1997), which is hereby incoφorated by reference), no subjects carrying the G,T allele were found. Thus, the effect of A,T homozygosity was in fact due to homozygosity at T 19, 183. The other DLST polymoφhism, A19,l 17G. did not significantly alter the risk of AD.
The most common genetic risk factor for AD is the APOE4 allele (Farrer, L.A. et al., "Effects of Age, Sex and Ethnicity on the Association Between Apolipoprotein E Genotype and Alzheimer's Disease," J. Am. Med. Assoc, 278:1349-1356 (1997); Tang, M.X. et al., "The APOE-e4 Allele and the Risk of Alzheimer Disease Among Africans, Whites and Hispanics," J. Am. Med. Assoc, 279:751-755 (1998), which are hereby incorporated by reference). Possessing at least one copy of APOE4 was strongly associated with an increased risk of AD in the current series [OR = 5.15 (95% CI = 2.983 - 8.875); P = O.00001 (χ2 = 33.3)]. The "protective" effect of the homozygosity of the £> STT19,183 (A,T) allele shown in Table 5 was due to ihe APOE4 negative subjects (OR = 0.16; P = 0.014) (Table 6). In the APOE4 positive cohort, homozygosity for the A,T allele of DLST was not protective [OR = 1.42 (95% CI = 0.267 - 7.526); P = 1.00 (Fisher exact test)].
Table 5. Distribution of DLST Genotypes in Alzheimer's Disease.
DLST AD Control OR (95% CI) P(χ2)
%(N) %(N)
A,T/A,T 7% (8) 19% (26) 0.35(0.154-0.783) 0.018(5.62)
A,T/G,C 38% (41) 27% (37) 1.69(0.983-2.894) 0.078(3.11)
A,T/A,C 9% (10) 5% (7) 1.92(0.716-5.170) 0.295(1.10)
G,C/G,C 20% (22) 24% (34) 0.79(0.430-1.451) 0.542 (0.37)
G,C/A,C 19% (20) 16% (22) 1.21 (0.621-2.354) 0.698 (0.15)
A,C/A,C 6% (7) 9% (13) 0.67(0.259-1.741) 0.558 (0.34)
Total 100% (7øs 700% (139) 1.00
DLST genotypes are polymoφhisms consisting of A19,l 17G and T19,183C. DLST genotype vs. diagnosis (df = 5): P = 0.051, χ2 = 11.01. OR = odds ratio; 95% CI = confidence interval at 95%.
Table 6. DLST Genotype A,T/A,T Protects Against Alzheimer's Disease in APOE4 Negative* Subjects.
DLST AD Control OR (95% CI) P(X2)
%(N) %(N)
A,T/A,T 4% (2) 22% (24) 0.16(0.042-0.611) 0.014(6.04)
A,T/x 40% (19) 32% (36) 1.41 (0.698-2.044) 0.434(0.61) non-A,T 55% (26) 46% (51) 1.46(0.733-2.893) 0.366 (0.82)
Sum 100% (47) 700% (111) 1.00
*, subjects who do not possess the APOE4 allele. Gene dosage of the A,T allele vs. diagnosis (df = 2); P = 0.023, χ2 = 7.52. Discussion
These results indicate an association between AD and a polymoφhism in DLST, which encodes the core component of KGDHC, a key enzyme of energy and glutamate metabolism. Analysis of polymoφhisms in DLST identified a group of very-elderly (> 85 years) subjects who are homozygous for the G,C allele, in whom the APOE4 allele remained an important risk factor for AD. Nakano et al. (Nakano, K. et al., "Alzheimer's Disease and DLST Genotype," Lancet, 350:1367-1368 (1997), which is hereby incorporated by reference), studying a younger Japanese population, found a higher incidence of the A,C allele in AD patients than controls, independent of the APOE4 status. The differences between their result and ours may reflect differences in age, ethnicity or other unrecognized differences between the populations studied.
Mechanisms linking effects of DLST and APOE4 in the pathogenesis of AD may involve oxidative stress-related reactive oxygen species and other reactive compounds (Beal, M.F., "Age, Energy, and Oxidative Stress in Neurodegenerative Diseases," Ann. Neurol., 38:357-366 (1995); Blass, J.P., "Energy/Glucose Metabolism in
Neurodegenerative Diseases," Molecular Mechanisms of Dementia, 91-101 (1997); Benzi, G. et al., "Age- and Peroxidative Stress-Related Modification of the Cerebral Enzymatic Activities Linked to Mitochondria and Glutathione System," Free Rad. Biol. Med., 19:77-101 (1995); Strittmatter, W.J. et al., "Binding of Human Apolipoprotein E to Synthetic Amyloid β-Peptide: Isoform-specific Effects and Implications for Late-Onset Alzheimer's Disease," Proc Natl. Acad. Sci. U.S.A., 90:8098-8102 (1993); Markesbery, W.R., "Oxidative Stress Hypothesis in Alzheimer's Disease," Free Rad. Biol. Med., 23:134-147 (1997), which are hereby incoφorated by reference). However, such an interaction between DLST and APOE genes will need to be studied directly before confident conclusions are possible.
The mechanisms by which the two base pair substitutions studied above might affect the expression of the DLST protein are as yet undetermined. The C19,183T substitution in exon 14 is neutral - both forms encode a glycine (codon 366). Thus, a direct effect of that base substitution at this site is hard to explain. The A 19,117G substitution in intron 13 occurs at a potential "branch point" that could affect the excision of introns during procession of mRNA. If this base substitution is pathogenetically related to the disease, the question of why G19,l 17 appears deleterious in Caucasian (Jewish) populations and Al 9,117 in Japanese populations will need to be answered. The data described above and earlier (Sheu, K-FR et al., "A Gene Locus of Dihydrolipoyl Succinyltransferase (DLST) is Associated with Alzheimer's Disease," J. Neurochem., 66 (Suppl 1) SI OB (1996); Sheu K-FR et al., "A Genetic Association of the Dihydrolipoyl Succinyltransferase Gene with Alzheimer's Disease," Soc Neusci. Abstr., 22:2124 (1996), which are hereby incoφorated by reference) suggest that a mutation in the DLST gene could be an important risk factor for AD, particularly in "very-elderly" populations. The genetic evidence is consistent with a mutation in either DLST or a neighboring gene. However, the likelihood of a pathogenic mutation within DLST is increased by the robust biochemical evidence for the deficiency of KGDHC in AD (Gibson, G.E. et al., "Reduced Activities of Thiamine-Dependent Enzymes in the Brains and Peripheral Tissues of Patients with Alzheimer's Disease," Arch. Neurol., 45:836-840 (1998); Butterworth, R.F. et al, "Thiamine-Dependent Enzyme Changes in Temporal Cortex of Patients with Alzheimer's Disease," Metab. Brain Pis., 5:179-184 (1990); Sheu K-FR et al., "Abnormality of the α-Ketoglutarate Dehydrogenase Complex in Fibroblasts from Familial Alzheimer's Disease," Ann. Neurol., 35:312-318 91994); Mastrogiacomo, F. et al., "Brain Protein and α-Ketoglutarate Dehydrogenase Complex Activity in Alzheimer's Disease Brain," Ann. Neurol.. 39:592-598 (1996); Blass, J.P. et al., "Inherent Abnormalities in Oxidative Metabolism in Alzheimer's Disease: Interactions with Vascular Abnormalities," Ann. N.Y. Acad. Sci.. 826:382-385 (1997), which are hereby incoφorated by reference). The data therefore supports further investigation of the role of DLST and other KGDHC genes in AD.
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED:
1. A method of detecting a predisposition to a neurodegenerative disease in a human subject, comprising: obtaining a biological sample from the subject; and testing the genetic material in the biological sample for the presence of a polymorphism in a gene encoding a Krebs tricarboxylic acid cycle component, wherein the presence of said polymorphism is indicative of a predisposition or reduced risk for neurodegenerative diseases.
2. The method according to claim 1 , wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or a related neurological disease.
3. The method according to claim 2, wherein the neurodegenerative disease is Alzheimer's disease.
4. The method according to claim 1, wherein said testing the genetic material in the biological sample comprises: amplifying a region of a gene encoding a Krebs tricarboxylic acid cycle component to provide an amplified fragment; and detecting the presence of a polymoφhism in the amplified fragment.
5. The method according to claim 4, wherein said detecting the presence of a polymoφhism comprises subjecting the amplified fragment to direct sequence analysis.
6. The method according to claim 4, wherein said detecting the presence of a polymoφhism comprises subjecting the amplified fragment to conditions effective to hybridize fragments with the polymoφhism to a probe specific for the sequence of said polymoφhism.
7. The method according to claim 4, wherein said detecting the presence of a polymoφhism comprises digesting the amplified fragment with a restriction endonuclease.
8. The method according to claim 1, wherein the biological sample is blood, saliva, cheek scrapings, or urine.
9. The method according to claim 1, wherein the gene encodes dihydrolipoamide succinyltransferase.
10. The method according to claim 1, wherein the gene encodes dihydrolipoamide dehydrogenase.
1 1. An isolated nucleic acid molecule comprising: a gene encoding a Krebs tricarboxylic acid cycle component, and a polymoφhism in said gene encoding a Krebs tricarboxylic acid cycle component, wherein the presence of said polymoφhism is indicative of a predisposition or reduced risk for a neurodegenerative diseases.
12. The nucleic acid molecule according to claim 1 1, wherein the nucleic acid molecule is at least twelve bases in length.
13. The nucleic acid molecule according to claim 1 1 , wherein the gene encoding a Krebs tricarboxylic acid cycle component encodes dihydrolipoamide succinyltransferase.
14. The nucleic acid molecule according to claim 13, wherein the gene encoding dihydrolipoamide succinyltransferase has the nucleic acid sequence according to SEQ. ID. No. 3.
15. The nucleic acid molecule according to claim 13, wherein CGCTCC is replaced by CCGCTC at nucleotides 829-834.
16. The nucleic acid molecule according to claim 13, wherein T replaced by C at nucleotide 7912.
17. The nucleic acid molecule according to claim 13, wherein G replaced by A at nucleotide 1 1791.
18. The nucleic acid molecule according to claim 13, wherein C replaced by G at nucleotide 11803.
19. The nucleic acid molecule according to claim 13, wherein A is replaced by G at nucleotide 13159.
20. The nucleic acid molecule according to claim 13, wherein C is replaced by
G at nucleotide 18784.
21. The nucleic acid molecule according to claim 13, wherein C is replaced by A at nucleotide 18822.
22. The nucleic acid molecule according to claim 13, wherein A is replaced by G at nucleotide 19864.
23. The nucleic acid molecule according to claim 13, wherein T is replaced by C at nucleotide 19930.
24. The nucleic acid molecule according to claim 13, wherein C is replaced by T at nucleotide 19930.
25. The nucleic acid molecule according to claim 13, wherein T is replaced by
C at nucleotide 21508.
26. The nucleic acid molecule according to claim 11 , wherein the gene encoding a Krebs tricarboxylic acid cycle component encodes dihydrolipoamide dehydrogenase.
27. The nucleic acid molecule according to claim 26, wherein the gene encoding dihydrolipoamide dehydrogenase has the nucleic acid sequence according to SEQ. ID. No. 5.
28. The nucleic acid molecule according to claim 26, wherein T is replaced by
A at nucleotide 1598.
29. The nucleic acid molecule according to claim 26, wherein G is replaced by T at nucleotide 1608.
30. A kit for identifying individuals with a predisposition to a neurodegenerative disease, comprising: a nucleic acid molecule which can be used to amplify a gene encoding a Krebs tricarboxylic acid cycle component which has a position where a polymoφhism may be located, wherein the presence of said polymoφhism is indicative of a predisposition or reduced risk for the neurodegenerative disease.
31. The kit according to claim 30. wherein the gene encodes dihydrolipoamide succinyltransferase.
32. The kit according to claim 30. wherein the gene encodes dihydrolipoamide dehydrogenase.
33. A kit for identifying individuals with a predisposition to a neurodegenerative disease, comprising: a restriction enzyme which may be used to detect the presence of a polymoφhism in a gene encoding a Krebs tricarboxylic acid cycle component, wherein the presence of the polymoφhism is indicative of a predisposition or reduced risk for the neurodegenerative disease.
34. The kit according to claim 33, further comprising: a nucleic acid molecule which can be used to amplify the gene encoding a Krebs tricarboxylic acid cycle component which has a position where the polymoφhism may be located.
35. The kit according to claim 33, wherein the gene encodes dihydrolipoamide succinyltransferase.
36. The kit according to claim 33, wherein the gene encodes dihydrolipoamide dehydrogenase.
37. An antibody which preferentially recognizes a Krebs tricarboxylic acid cycle component protein which is encoded by a gene having a polymoφhism, wherein the presence of the polymoφhism is indicative of a predisposition or reduced risk for a neurodegenerative disease.
38. The method according to claim 37, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or a related neurological disease.
39. The method according to claim 38, wherein the neurodegenerative disease is Alzheimer's disease.
40. The antibody according to claim 37, wherein the gene encodes dihydrolipoamide succinyltransferase.
41. The antibody according to claim 40, wherein amino acids Ala-Pro are replaced by Arg-Ser at residues 14-15
42. The antibody according to claim 40, wherein amino acid Thr is replaced by Arg at residue 312.
43. The antibody according to claim 37, wherein the gene encodes dihydrolipoamide dehydrogenase.
44. A method of detecting a predisposition to a neurodegenerative disease in a human subject, comprising: obtaining a biological sample from the human subject; contacting the biological sample with an antibody which preferentially recognizes a Krebs tricarboxylic acid cycle component protein which is encoded by a gene having a polymoφhism which is indicative of a predisposition or reduced risk for a neurodegenerative disease; and determining whether the antibody binds to a Krebs tricarboxylic acid cycle component protein, wherein binding of the antibody to the Krebs tricarboxylic acid cycle component indicates that the polymoφhism is present which is indicative of a predisposition or reduced risk for a neurodegenerative disease.
45. The method according to claim 44, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or a related neurological disease.
46. The method according to claim 45, wherein the neurodegenerative disease is Alzheimer's disease.
47. The antibody according to claim 44, wherein the gene encodes dihydrolipoamide succinyltransferase.
48. The antibody according to claim 47, wherein amino acids Ala-Pro are replaced by Arg-Ser at residues 14-15
49. The antibody according to claim 47, wherein amino acid Thr is replaced by Arg at residue 312.
50. The antibody according to claim 44, wherein the gene encodes dihydrolipoamide dehydrogenase.
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