[go: up one dir, main page]

WO2000079000A1 - Methode de detection de la maladie d'alzheimer a apparition precoce - Google Patents

Methode de detection de la maladie d'alzheimer a apparition precoce Download PDF

Info

Publication number
WO2000079000A1
WO2000079000A1 PCT/EP2000/005942 EP0005942W WO0079000A1 WO 2000079000 A1 WO2000079000 A1 WO 2000079000A1 EP 0005942 W EP0005942 W EP 0005942W WO 0079000 A1 WO0079000 A1 WO 0079000A1
Authority
WO
WIPO (PCT)
Prior art keywords
presenilin
psen1
promoter
polymorphism
upstream
Prior art date
Application number
PCT/EP2000/005942
Other languages
English (en)
Inventor
Jessie Theuns
Marc Cruts
Christine Van Broeckhoven
Original Assignee
Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw filed Critical Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
Priority to AU62656/00A priority Critical patent/AU6265600A/en
Publication of WO2000079000A1 publication Critical patent/WO2000079000A1/fr

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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 relates generally to the field of neurological and physiological dysfunctions associated with Alzheimer's disease (AD). More particularly, the invention concerns the use of a variant in the preseniline-1 (PSEN1 ) regulatory region to determine in a human cell or tissue obtainable from a human being whether said human being has or is at risk for developing early-onset AD or AD.
  • PSEN1 preseniline-1
  • the present invention also relates to methods to screen for molecules that inhibit the reduction of PSEN-1 levels associated with polymorphisms in the PSEN-1 promoter region and molecules obtained via said methods.
  • D14S1028, a simple tandem repeat (STR) polymorphism located upstream of PSEN1 (p 0.04).
  • STR simple tandem repeat
  • the intron 8 association was explained by linkage disequilibrium to the dominant PSEN1 mutations.
  • the regulatory region of PSEN1 and D14S1028 associations remained significant.
  • the association of PSEN1 with AD was independent of the apolipoprotein E (APOE) genotype.
  • the present invention also discloses that the PSEN1 promoter polymorphisms -2154 G/A, -2818 A G and -2823 l/D are associated with an increased risk for AD.
  • the present invention further discloses that, especially with regard to the PSEN1 promoter variation/polymorphism -48 C/T, a neuron-specific twofold decrease in PSEN1 promoter activity for the -48C risk allele can in homozygous individuals lead to a critical decrease in PSEN1 expression.
  • the present invention aims at providing a method for determining whether a human subject has or is at risk for developing early-onset Alzheimer disease or Alzheimer disease comprising the step of detecting the presence or absence of a genetic lesion in the presenilin-1 gene of said subject, wherein said genetic lesion comprises a polymorphism in the presenilin-1 promoter region or regulatory region upstream of presenilin-1 and wherein the presence of said genetic lesion identifies a subject that has or is at risk for developing early-onset Alzheimer disease or Alzheimer disease.
  • the present invention aims at providing a method as described above, wherein said polymorphism in the presenilin-1 promoter region is chosen from the group comprising: -48 C/T; -280 C/G; -2154 G/A, -2818 A/G and -2823 l/D and/or wherein said polymorphism in the regulatory region upstream of presenilin-1 is a simple tandem repeat polymorphism at D14S1028.
  • the present invention also aims at providing a transgenic non-human animal comprising in its genetic material a human presenilin-1 variant/mutation/polymorphism (the latter three terms can be used interchangeably) in the presenilin-1 promoter region and/or in the presenilin-1 regulatory region upstream of presenilin-1 and to provide a usage of said transgenic non-human animal to screen for therapeutic molecules to treat Alzheimer disease.
  • a human presenilin-1 variant/mutation/polymorphism the latter three terms can be used interchangeably
  • the present invention further aims at providing a method to screen for molecules that inhibit the reduction of presenilin-1 levels induced by a polymorphism in the preseniiin-1 promoter region (specifically with regard to the polymorphisms in the presenilin-1 promoter region -48 CT and/or -280 C/G) and/or regulatory region upstream of presenilin-1 comprising:
  • the present invention finally relates to molecules obtainable by using a transgenic animal and/or a method as described above. Table and Figure legends
  • Table 5 Sequence variations between the Neurogen sequence (N) and AF109907.
  • Table 6 Overlapping primer sets of the PSEN1 promoter region
  • Table 7 Polymorphisms detected in the PSEN1 5' upstream region. The nucleotide positions are relative to the uppermost transcription initiation site of exon 1A. Allele frequencies were determined in the 118 control individuals using SSCP.
  • FIG. 1 Map of the chromosome 14q24.3 region containing PSENL The position of the STR polymorphisms and the 5' ⁇ 3' orientation of PSEN1 are as published by Cruts et al. (37). Distances are in centimorgan (cM) according to the Genethon sex averaged genetic map of chromosome 14 (39), or in kilobases (kb) defined by restriction mapping of PSEN1 and YAC fragmentation. (Del-Favero,J et al., Gene,1999;193-201 ).
  • Figure 2 Functional analysis of polymorphisms in the PSEN1 5' upstream region.
  • the positions of the PSEN1 -48C>T and -280C>G promoter polymorphisms are shown by arrows. Restriction enzyme recognition sites used for subcloning into the pGL3 basic vector are represented in bold. The deletion constructs in the pGL vector are represented by horizontal lines. Position of the 5' and 3' end of each construct is given based on the exonlA transcriptional start site t2 reported by Rogaev et al. (12) (L76518). Total length is given in bp. The relative luciferase activity of each construct in N2A and HEK293 cells is presented as a percentage of the SV40 promoter construct (%RLA). Figure 4:.
  • Transcriptional activity of the PSEN1 -48C/T variants in transient transfection experiments Bars represent firefly/reniila luciferase ratios for the different constructs (relative luciferase activity, RLA). Transcriptional activities are presented as percentage of the activity of the wild type construct (-280C, white bars). Values are the mean ⁇ SEM of in duplo determinations of at least 3 experiments of 3 independent DNA preparations each.
  • Figure 5 EMSA analysis of allele-specific effect of the -280C>G mutation, on the interaction of nuclear protein complexes present in HEK293 cells with the (-291/-268) region. The same effect is seen for mouse (N2A) and human neuroblastoma (Kelly) cells (data not shown). 1-5 ng 32 P- labelled double-stranded probes were incubated with 10 ⁇ g nuclear extract from HEK293 cells. Lanes 1-6: include WT -280C construct. Lanes 7-16: include mutant -280G construct. In competition experiments 100 or 200 ng unlabeled probe was added before the labelled probe. In supershift experiments 400ng ⁇ -NF-1 was pre-incubated with the extract prior to incubation with labelled probe. Specific complexes are indicated with filled arrows. Supershifted bands are indicated with open arrows.
  • di-allelic PSEN1 polymorphisms in the promoter region, the 5' untranslated region (UTR) (8), intron 8 (11) and the 3' UTR were analysed. Furthermore linkage disequilibrium with STR polymorphisms flanking PSEN1 was examined. For PSEN2 three polymorphisms in exons 3 and 4 and intron 11 (8) were analysed, since no polymorphisms in the promoter region or UTRs were described. According to the current invention inside the PSEN1 gene, di-allelic polymorphisms were examined (Table 1 and Table 7).
  • EOAD early onset AD
  • additional polymorphisms 2154G>A and -2823I/D
  • the invention concerns a method for determining whether a human subject has or is at risk for developing early-onset Alzheimer disease (AD) or Alzheimer disease comprising the step of detecting the presence or absence of a genetic lesion in the presenilin-1 gene of said subject, wherein said genetic lesion comprises a polymorphism in the presenilin-1 (PSEN-1 ) promoter region or regulatory region upstream of PSEN-1 and wherein the presence of said genetic lesion identifies a subject that has or is at risk for developing early-onset AD or AD.
  • PSEN1 promoter polymorphism as such has recently been identified while performing a systematic mutation analysis of the coding and non-coding exons of PSEN1 in this same series of early-onset AD cases (8).
  • the polymorphism is a C to T nucleotide change at nucleotide position -48, a C to G nucleotide change at nucleotide position -280, a G to A nucleotide change at nucleotide position -2154, a A to G nucleotide change at nucleotide position -2818, and a deletion (D)/insert (I) of 13 bp (5'-GCATGTCCTGGGT-3') at position -2823, upstream of exon 1A (8).
  • PSEN1 has 13 exons of which exons 1A, 1 B and 2 are 5' non-coding exons (33).
  • Differential expression of APOE in brain of AD patients with specific APOE promoter polymorphisms has been associated with an increased risk for late-onset AD independent of the APOE ⁇ 4 genotype (33-35).
  • Recently two early-onset AD cases were identified wherein the first out-frame PSEN1 mutation in intron 4 leads to deleted transcripts and early truncation of the PSEN1 protein (36). In these cases it was hypothesised that AD would result from haplo-insufl ⁇ ciency of wild type PSEN1 protein.
  • Exons 1A and 1B are alternatively used exons and sequence analysis of PSEN1 clones from a hippocampal cDNA library suggested that only exon 1A containing transcripts are present in brain (38). Also, there is no linkage disequilibrium between the exon 1 B polymorphism and the promoter or D14S1028 polymorphism. No association was found with polymorphisms in the 3'UTR or downstream of PSENL
  • the present invention concerns the use of a variant in the PSEN1 regulatory region to determine in a human cell or tissue obtainable from a human being whether said human being has or is at risk for developing early-onset AD or AD.
  • Individuals with a high risk for AD (present in family pedigree) or, individuals not previously known to be at risk, or people in general may be screened routinely using for instance probes to detect the presence of a promoter PSEN 1 polymorphism by a variety of techniques.
  • Genomic DNA used for the diagnosis may be obtained from body cells, such as those present in the blood, tissue biopsy, surgical specimen or autopsy material. The DNA may be isolated and used directly for detection or may be amplified by known techniques prior to analysis.
  • a transgenic mouse model for AD has incorporated in its mouse genome the human promoter region or regulatory regions upstream PSEN1 mutated to manifest the symptoms.
  • the transgenic mouse exhibits symptoms of cognitive memory loss or behavioural disturbances.
  • Said transgenic mouse can be used as starting point for rational drug design to provide therapeutic drugs or other types of small chemical molecules.
  • the current invention discloses a first study that examines several polymorphic markers spanning the whole gene as well as flanking regions and shows association to multiple markers in the region upstream of PSEN1. In contrast to the study of Wragg et al.
  • Plasmid B22 contains the sequence from 6.5 kb upstream of exonlA to 2.6 kb downstream of exon 1 B, and was used as the starting clone in this invention.
  • the present invention also relates to a functional analysis of the PSEN1 promoter region in relation to the EOAD related variations in the PSEN1 proximal promoter: - 280C>G and -48C>T.
  • Deletion analysis of the PSEN1 upstream region showed that the smallest promoter fragment, extending from -47 to +823 though still containing the most upstream TSS (54), confers maximum promoter activity in both N2A and HEK293 cells even exceeding SV40 promoter activity in N2A cells.
  • Matlnspector v2.2 predicted three major elements with a 100% core and at least 95% matrix similarity in this fragment: Ets-1 at position -40, Myb at position -28 and SP1 at position -12. Both the Ets-1 and SP1 , strictly conserved between human and mouse sequences, site were recently reported to be functional PSEN1 promoter elements in SK-N-SH and HepG2 cells (54, 55). All larger fragments show lower promoter activity suggesting that both -3525 ⁇ 48 and +824/+1386 confer an overall inhibitory effect on PSEN1 promoter activity. 3' deletion of the +824/+ 1225 region increases promoter activity more than 4-fold, either due to the presence of negative regulators or to distance effects.
  • promoter activity is not completely abolished.
  • the remaining promoter activity can be due to transcription from the more downstream located TSSs t3, t4, t5 reported by Rogaev et al. (51 ).
  • Another, more likely scenario is that transcription initiates from exon 1 B driven by promoter elements located downstream of the major exon 1A TSSs.
  • transcriptional activity increases with deletion of the +16/+202 region, deleting all reported TSSs of the exon 1A transcripts. This effect is the most pronounced in HEK293 cells with a nearly 3-fold increase, hereby reaching expression levels corresponding to one third of the maximal promoter activity.
  • exon 1B transcripts Another reason for the underrepresentation of exon 1B transcripts in cDNA libraries might be differences in efficiency of reverse transcription of exons 1A versus 1 B. Also the difference in length between the exon 1A and exon 1 B transcripts could lead to preferential cloning of the shorter 1 A transcript.
  • the overall transcription level is on average twice as high in N2A cells as compared to HEK293 cells.
  • Northern blot analysis does not show a significant difference in mRNA levels between human brain and kidney (51 ).
  • a recent analysis of the mouse PSEN1 promoter shows preferential transcriptional activity in neuron-like cells, supporting a cell-type-specific pattern of PSEN1 expression.
  • To determine whether the observed difference in human PSEN1 expression levels is species- or tissue-specific we study PSEN1 promoter activity in human neuroblastoma cells.
  • EMSA revealed that the nuclear extracts obtained from Kelly, HEK293 and N2A cells all contained nuclear proteins specifically recognising this region of the PSEN1 promoter resulting in two specific DNA-protein complexes A and B.
  • For the mutant -280G probe we demonstrated that a major part of the DNA is involved in the formation of an alternative complex A which migrates slower.
  • complex B is competed more efficiently with mutant than wild type probe and vice versa for complex A. Since no third complex was detected, binding sites are most probably partially or completely overlapping, therefore proteins have to compete for binding and can never bind together.
  • binding of these proteins does not seem to be exclusive for either of the alleles. Binding of specific nuclear factors to DNA is a dynamic process influenced by the binding affinities of the DNA sequence. Overlapping binding sites for different TF are known to be important for tissue-specific gene regulation (60-62).
  • Matlnspector v2.2 predicted the creation of a potential NF1 binding site (core similarity 1 / matrix similarity 0.872) for the -280G mutation. Competition with the NF-1 consensus sequence alters the ratio between the two complexes significantly. Since the predicted binding affinities for NF-1 were higher for -280G than for -280C we expected the upper complex A to be competed by the NF-1 consensus probe. However, the presence of unlabeled NF-1 probe decreases the formation of complex B in favour of the formation of complex A. Also, pre-incubation of the nuclear extracts with anti-NF-1 antibody gives rise to a supershift of the lower band, confirming the competition data.
  • NF1 proteins exhibit ubiquitous patterns of expression, and it is therefore unlikely that they play a primary role in determining cell- or tissue-specific transcription.
  • NF1 proteins have been implicated in brain-specific gene expression (66, 67) and more recently in human neuroblastoma-specific expression of the human 3 nAchR subunit gene (58).
  • the obvious lack of restricted expression for the NF1 gene family has led to the hypothesis that NF1 may either activate or silence gene expression in a cell-specific manner by participating in a combinatorial code involving cofactors, best exemplified by liver-specific vitellogenin gene expression (68).
  • This same type of mechanism may account for cell type-specific expression of PSENL
  • the nature of the binding to the PSEN1 NF1 site by these NFI-like proteins may be different and therefore the effect of -280C>G on transcriptional activity may differ between different cell types.
  • the present invention demonstrates that the -48C>T polymorphism, located in the PSEN1 core promoter, even has a larger effect on PSEN1 promoter activity than -280C>G and provides evidence that -48C>T modifies transcriptional activity of the PSEN1 promoter in a cell-type specific manner.
  • the -48C>T polymorphism, associated with EOAD, is located just upstream of the PSEN1 core promoter.
  • the 12 bp fragment ending at -48 contains a strong negative regulatory element involved in cell-type-specific inhibition of PSEN1 transcription.
  • a 50% reduction of PSEN1 levels significantly increased A ⁇ 42 production (70). Since most of our EOAD patients are homozygous for -48C, the corresponding 50% decrease in promoter activity will have a major effect on PSEN1 expression levels, possibly leading to increased A ⁇ 42 production.
  • the present invention relates to a method to screen for molecules which inhibit the reduction of presenilin-1 levels induced by a polymorphism in the presenilin-1 promoter region, in particular induce by the polymorphisms -48 C/T and/or -280 C/G, and/or regulatory region upstream of presenilin-1 comprising 1 ) exposing said polymorphism in the presenilin-1 promoter region or regulatory region upstream of presenilin-1 to said molecules, and 2) monitoring said preseniiin-1 levels.
  • the present invention also relates to molecules obtainable by using a transgenic animal as described above and/or a method as described above.
  • 'drug screening assays' or 'bioassays' typically include the step of screening a candidate/test compound or agent for the ability to interact with (e.g. bind to) the presenilin-1 promoter region and/or regulatory region upstream of presenilin-1 and/or factors interacting with said regions such as NF-1 as indicated above.
  • Candidate compounds or agents, which have this ability can be used as drugs to combat or prevent pathological conditions of AD.
  • Candidate/test compounds such as small molecules, e.g. small organic molecules, and other drug candidates can be obtained, for example, from combinatorial and natural product libraries as described above.
  • the assays are cell-free assays which include the steps of combining the presenilin-1 promoter region and/or regulatory region upstream of presenilin-1 and/or factors interacting with said regions such as NF-1 and a candidate/test compound, e.g., under conditions which allow for interaction of (e.g.
  • binding of) the candidate/test compound with the presenilin-1 promoter region and/or regulatory region upstream of presenilin-1 and/or factors interacting with said regions such as NF-1 to form a complex and detecting the formation of a complex, in which the ability of the candidate compound to interact with ) the presenilin-1 promoter region and/or regulatory region upstream of presenilin-1 and/or factors interacting with said regions such as NF-1 is indicated by the presence of the candidate compound in the complex.
  • Formation of complexes between the presenilin-1 promoter region and/or regulatory region upstream of presenilin-1 and/or factors interacting with said regions such as NF-1 and the candidate compound can be quantitated, for example, using standard immunoassays.
  • the presenilin-1 promoter region and/or regulatory region upstream of presenilin-1 and/or factors interacting with said regions such as NF-1 employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intra-cellularly.
  • Interaction e.g., binding of of presenilin-1 promoter region and/or regulatory region upstream of presenilin-1 and/or factors interacting with said regions such as NF-1 presenilin-1 promoter region and/or regulatory region upstream of presenilin-1 and/or factors interacting with said regions such as NF-1 to a target molecule
  • any vessel suitable for containing the reactants include microtiter plates, test tubes, and microcentrifuge tubes.
  • AD Association of polymorphisms with AD - Patients were derived from a population-based epidemiological study of early-onset AD (27). Within two areas of the Netherlands, the study aimed at a complete ascertainment of all AD patients in whom the disease onset was at or before the age of 65 years. Age at onset was defined as the age at which memory loss or changes in behaviour were first noted. For this study, the clinical diagnosis of AD was independently confirmed by two neurologists using a standardised protocol according to the NINCDS-ADRDA criteria for AD. Of the 201 eligible patients, 198 (99%) participated in the study. Family history of dementia addressed all first, second and third degree relatives of the patients. Patients who were known to be related were excluded from the association studies presented here.
  • Blood samples for DNA extraction were collected of 102 (52%) of the participating patients.
  • the mean age at onset of the patients was 56.7 ⁇ 5.4 years and the mean attained age at the time of the study was 63 ⁇ 4.4 years.
  • PCR polymerase chain reaction
  • the STR polymorphisms at D1S479, D14S1028, D14S77, D14S1004, D14S43 and D14S61 were PCR amplified using published primers of which one was fluorescently labelled.
  • the alleles were separated on a 6% polyacrylamide gel containing 8M urea using an ABI373A automated DNA sequencer (Applied Biosystems, Foster City, USA) and analysed using the GeneScan 672 software (Applied Biosystems). Alleles were numbered as in the CEPH Genotype Database. When additional alleles were observed, new numbers were assigned (Table 2).
  • the Bst ⁇ polymorphism 0.9 kb downstream of the c-fos oncogene (FOS)(29), the BsfXi polymorphism in exon 1 B of PSEN1 (5'UTR), and the Ddel, BbtP ⁇ and PvuW polymorphisms in exons 3 and 4 and intron 11 of PSEN2 (8) were detected by restriction fragment length polymorphism (RFLP) analysis of the respective PCR amplified exons. PCR amplification was performed using published primers (8, 29) and the products were digested overnight with 5 U of the appropriate restriction enzyme.
  • RFLP restriction fragment length polymorphism
  • PCR primers 3UTR7 (5'-AAACAGTACAGCTATTTCTCATC- A-3') and 3UTR8 (5'-GCTTCAACAGCCATTTTACTC-3') were developed to amplify an A to T polymorphism at nucleotide 952 in the 3'UTR of PSEN1 that recently has been identified.
  • the amplification products were digested with 5 U ⁇ //alll resulting in a constant fragment of 37 bp and 2 alleles of 308 bp (T-allele) and 162 + 146 bp (A-allele) respectively.
  • the alleles were separated by agarose gel electrophoresis and visualised on a UV transiliuminator after ethidium bromide staining.
  • the longer restriction fragment was assigned allele number 1
  • the shorter one was assigned allele number 2.
  • the polymorphism in intron 8 of PSEN1 was analysed by PCR-RFLP analysis using a mismatch primer and BamHI digestion as described (11 ). Allele numbering was in accordance with the original publication (11 ).
  • the polymorphism at codon Gly366 in exon 14 of the dihydrolipoyl succinyltransferase (DLST) gene (30) and the PSEN1 promoter polymorphism (8) were scored by single-strand conformational polymorphism (SSCP) analysis. PCR was performed using published primers (8,30). PCR amplification products of DLST exon 14 were loaded on a 1x HydroLink MDE gel (J.T. Baker, Phillipsburg, USA) and separated by electrophoresis for 20 h at 800 V and at room temperature.
  • SSCP single-strand conformational polymorphism
  • the promoter polymorphism of PSEN1 was analysed on precast ExcelGel gels (Pharmacia Biotech, Uppsala, Sweden), electrophoresis was for 3.5 h at 600 V using the MultiPhorll electrophoresis system (Pharmacia Biotech). After electrophoresis, the SSCP bands were visualised by silver staining. The allele corresponding to the higher band in the SSCP analysis was assigned number 1 , while the lower allele was assigned number -Statistical analysis: the significance of the association between early-onset AD and the polymorphisms studied was assessed using the likelihood ratio test or the Fisher exact test when appropriate. For di-allelic markers, genotype and allele frequencies were compared between cases and controls.
  • Plasmid B22 was subcloned into the appropriate restriction sites of the pOCUS-2 vector (Novagen Inc., Madison, WI). The pOCUS-2 constructs were transferred into chemically competent ⁇ donor cells, which carry the ⁇ transposon on an F factor, and one of the resulting colonies was mated with the ⁇ recipient cells according to the manufacturers protocol. For each subclone 96 resulting colonies were randomly selected and stored in a 96 well plate containing LB medium with 20% glycerol.
  • the transposition site of the different clones was mapped by colony PCR combining one of the two vector-specific primers (POCUSUP or POCUSDOWN) with a phosphorylated transposon-specific primer (GDIR) in separate reactions (Strathmann et al., 1991 ). Colonies were selected based on the length of their PCR products and DNA was prepared for sequencing using the Wizard Plus SV Minipreps DNA Purification System. Plasmid sequencing was accomplished using the Thermo SequenaseTM II Dye Terminator Cycle Sequencing Kit (Amersham Life Science, Cleveland, USA) and the supplier's protocol using the published primers GD1 and GD2 (Strathmann et al., 1991 ). The sequences were assembled using the Lasergene software for Windows (DNASTAR inc., Madison, USA). To close the remaining gaps, primers flanking the gaps were designed with the Lasergene software and used in cycle sequencing to complete the sequence.
  • Direct sequencing was also used for linking the first three fragments.
  • 9643 bp In total we sequenced 9643 bp, with 6698 bp upstream of exon 1B with an average coverage higher than 4. 83% of this sequence was sequenced at least twice and on both strands, 13% at least twice but only on one strand, and only 4% was sequenced only once. In the latter case however, the sequencing results were optimal.
  • the percentage of the bases Guanine and Cytosine in the total sequence lies within the normal range.
  • the GC content does not change in the 6.7 kb fragment upstream of exon 1 B nor in the 6.4 kb upstream of exon 1 A.
  • Overlapping primer sets were designed based on the 7kb sequence upstream of exon 1B using the Lasergene software.
  • a polymerase chain reaction (PCR) was performed using 200 ng genomic DNA as template in a 25 I reaction mixture containing 25 pmol of each PCR primer, 1 unit (U) Taq DNA polymerase (Gibco BRL, Gaithersburg, USA), 0.2 mM of each dNTP (Pharmacia Biotech, Uppsala, Sweden), 0.05 % W1 and 1x Taq buffer (GIBCO BRL).
  • the PCR OptimizerTM Kit In order for some primers to work, the PCR OptimizerTM Kit (Invitrogen Corporation, CA, USA) was used. Deviating buffer conditions, additives and the MgCI 2 conditions are described in table 2.
  • the PCR amplification consisted of 30 cycles of 60 sec at 94°C, 90 sec at the empirically defined optimal annealing temperature, and 120 sec at 72°C (table 5).
  • SSCP single-strand conformational polymorphism
  • overlapping primer sets were used to amplify fragments of the PSEN1 5' upstream region.
  • PCR products were analysed on precast ExcelGel gels (Pharmacia Biotech), electrophoresis was for 3.5 h at 600 V using the MultiPhorii electrophoresis system (Pharmacia Biotech). After electrophoresis, the SSCP bands were visualised by silver staining. When aberrant SSCP patterns were observed the sequences of the fragments were determined using cycle sequencing.
  • PCR amplification products were pre-treated with 10 U exonuclease I and 2 U shrimp alkaline phosphatase to remove excess PCR primers and nucleotides.
  • PCR amplification product (5 ⁇ l) was used as template in the cycle sequencing reaction using the ABI PRISM Dye Terminator Cycle Sequencing Core Kit according to the supplier's protocol using the same primers as in the PCR amplification. Comparison of the sequences was done using the Lasergene software. Sequence variations in the PSEN1 5' upstream region were analysed by restriction enzyme digestion of the amplified products when they involved the creation or abolition of a restriction enzyme recognition site (Restriction fragment length polymorphism, RFLP).
  • Genomic PCR amplification products were digested over night (ON) using 10 U of the corresponding restriction enzyme (table 6) at the appropriate reaction temperature.
  • the restriction fragments were separated on a 1.5-3% agarose gel, depending on the allele sizes, and visualised on a UV trans-illuminator after EtBr staining.
  • allelic distribution of the different polymorphisms was compared with the distribution of the alleles of the exon 1A polymorphism reported above.
  • overlapping primer sets were designed to screen the 3.3 kb sequence upstream of and including exon 1A, for polymorphisms (Table 6).
  • PCR-SSCP analysis was performed on a selection of 12 patients and 10 controls of the Rotterdam EOAD sample (van Duijn et al., 1994). Altered SSCP patterns were observed for 5 primer sets (Table 7). Cycle sequencing of the different patterns revealed 7 sequence variations in total (Table 7). The different patterns found for primer set Prom2 could be explained by the C/T variation at 48 bp upstream of exon 1A (-48) reported earlier (Cruts et al., 1998b). For primer set Proml only patient 1093-000 showed an altered SSCP pattern. This patient has an onset-age of 56 years and a familial background. Cycle sequencing revealed a C to G transition at 280 bp upstream of exon 1A (-280), creating an Ncol site.
  • the analysis of this sequence variation was extended to the complete Rotterdam EOAD sample (van Duijn et al., 1994) using SSCP analysis.
  • the SSCP pattern of primer set Proml 3 was extremely complex and five samples, giving different patterns, were sequenced.
  • the complex SSCP patterns were due to two different sequence variations present on the fragment amplified by the respective primer set.
  • the first sequence variation was a G/A variation at position -2154 bp, which can be detected by BsmBI digestion; when A is present the site disappears. This polymorphism is in complete linkage disequilibrium with the exon 1A polymorphism in the samples examined. G is the most frequent allele and is associated with a higher risk for EOAD.
  • the second variation is a variable T-stretch at position -2319, which has only been examined in the five samples that have been sequenced. Thus far, four different alleles have been found, ranging from 12 to 15 T's. All samples examined are heterozygous with a combination of two subsequent alleles e.g. 12 and 13 T's.
  • the fourth primer set, Proml 5 showed four different SSCP patterns in the selection of 22 samples. Cycle sequencing revealed two different sequence variations in the fragment amplified with the respective primers.
  • the first variation is a 13 bp insert or deletion of the sequence 5'-GCATGTCCTGGGT-3' at position -2823.
  • Mouse Neuro2a-neuroblastoma (N2a) cells were routinely propagated in a minimal essential medium with Earie's salt (Life technologies), 10% foetal bovine serum (Life technologies), 2mM L-glutamine (Life technologies), 200IU/ml Penicillin, 200 ⁇ g/ml Streptomycin (Life technologies) and 0.1 mM nonessential amino acids (Life technologies).
  • Human embryonic kidney cells (HEK293) were routinely propagated in Optimem (Life technologies) with 10% foetal bovine serum (Life technologies), 200 lU/ml Penicillin and 200 ⁇ g/ml Streptomycin (Life technologies).
  • N2a and HEK293 cells were seeded in 6-well tissue culture dishes, at 9 X 10 4 and 7 X 10 5 cells/well respectively, and allowed to recover for 24 hours.
  • Cells were co-transfected with 1 ⁇ g of one of the PSEN1 promoter constructs, pGL3 basic vector or pGL3 promoter plasmid (containing an SV40 promoter upstream of the firefly luciferase gene, Promega) and 20 ng of pRL-TK plasmid (which contains a herpes simplex virus thymidine kinase promoter upstream of the renilla luciferase gene, Promega), using the Lipofectamine procedure (Life technologies) as described in the manufacturer's protocol.
  • Plasmid B22 containing exons 1A and AB of PSEN1 and approximately 6.5 kb of upstream sequences, was previously restriction mapped (52) and sequenced (AF205592) (50). Genomic fragments obtained from plasmid B22 by restriction digestion or PCR amplification, were subcloned upstream of the firefly luciferase gene in the promoteriess pGL3-basic vector.
  • Deletion of the -3525/-1219 region had no effect on promoter activity.
  • Deletion of -1218/-515 increased transcriptional activity 3-fold in N2A and 2-fold in HEK293 cells.
  • deleting -514/-321 decreased promoter activity 2-fold in N2A and with 30% in HEK293 cells.
  • Further deletion of the -320/-142 sequence increased activity again, 3-fold in N2A and nearly 2-fold in HEK293.
  • Deletion of -141/-59 had no effect on promoter activity in N2A cells but decreased expression significantly in HEK293 cells.
  • Deletion of the -59/-48 fragment increased promoter activity more than 3-fold in HEK293 cells and with 50% in N2A cells.
  • Complex B showed a higher mobility resulting in the lower band (B) on the gel in figure 5.
  • Complex A migrated slower in the gel, resulting in the upper band (A) ( Figure 5).
  • Both complexes were present for both -280C and -280G alleles, however, the ratio of the amount of the 2 complexes differs significantly.
  • the amount of complex B formed on the -280C probe is higher than the amount of complex A (intensity band B>A, Figure 5 lane 1 ), whereas a significant shift towards formation of complex A was observed for -280G (intensity band A>B, Figure 5 Iane7).
  • the target fragments were extracted from gel and ligated into the corresponding restriction sites of the vector: the 4911 bp Bg/ll/BamHI fragment spanning nucleotides -3525/+1386, the 2443 bp H/noflll fragment spanning -1218/+1225, the 1739 bp Nnel/H/ndlll fragment spanning -514/+1225, the 1545 bp Kpnl/H/n ⁇ lll fragment spanning -320/+1225, the 1366 bp Sacl/H/ndlll fragment spanning -141/+1225 and the 625 bp Xnol/H/ndlll fragment spanning +603/+1225 (numbering according to the exon 1A transcription start site (TSS) t2 reported by Rogaev et al.
  • TSS exon 1A transcription start site
  • Genomic fragments were obtained by PCR amplification of plasmid B22 DNA (52) using the proof-reading Fu polymerase (Promega) and primers designed to our published sequence of the human PSEN1 upstream region (AF205592) (50). Primers were designed to incorporate restriction enzyme sites at the end of the amplified products to facilitate subcloning of the fragments in the pGL3-basic vector.
  • the Quick-change in vitro mutagenesis kit (Stratagene, La Jolla, CA, USA) was used to introduce the T allele at position -48 in the 1545 bp Kpnl/H/ ' ncflll fragment using primer 5'-gtgggccggccgccaacgaTgccagagccggaaatgacg-3' and its reverse complement.
  • -48T clones were selected by Hgal digestion.
  • Mouse Neuro2a-neuroblastoma (N2a) cells were propagated in a minimal essential medium with Earie's salt (Life Technologies, Gaithersburg, USA), 10% foetal bovine serum (Life technologies), 2mM L-glutamine (Life technologies), 200 lU/ml penicillin, 200 g/ml Streptomycin (Life technologies) and 0.1 mM nonessential amino acids (Life technologies).
  • Human embryonic kidney cells (HEK293) were propagated in Optimem (Life technologies) with 10% foetal bovine serum (Life technologies), 200 lU/ml penicillin and 200 g/ml Streptomycin (Life technologies).
  • N2a and HEK293 cells were seeded in 6-well tissue culture dishes, at 9 X 10 4 and 7 X 10 5 cells/well respectively, and allowed to recover for 24 hours.
  • Cells were co-transfected with 20 ng of pRL-TK plasmid containing the herpes simplex virus thymidine kinase promoter upstream of the renilla luciferase gene (Promega) and 1 ⁇ g of either one of the PSEN1 promoter constructs or one of the control plasmids, using the Lipofectamine procedure (Life technologies) as described in the manufacturer's protocol.
  • Transfected cells were cultured for 48 hours, washed with 1ml phosphate-buffered saline (PBS, Life Technologies), and lysed with Passive lysis buffer (Promega).
  • Firefly luciferase activities (LA F ) and renilla luciferase activities (LA R ) were measured sequentially using a Dual-Luciferase reporter assay system (Promega) and a model TD-20E Luminometer (Turner design).
  • RLA relative luciferase activity
  • HEK293, N2A and Kelly human neuroblastoma cells were grown under normal growth conditions to a density of 0.5-1 * 10 6 cells/ml. Approximately 10 9 cells were harvested in PBS (Life Technologies), washed twice in PBS, pelleted by centrifugation at 950 x g and 4°C for 15 min, frozen in liquid nitrogen and stored at -80°C. Nuclear extracts were prepared according to a modified Dignam et al. (14) procedure.
  • Single strand oligonucleotides were designed spanning either the PSEN1 proximal promoter sequence variations or the consensus sequence of the NF-1 binding site: - 48C, 5'-gccgccaacgaCgccagagccgga-3'; -48T, 5'-gccgccaacgaTgccagagccgga-3'; -280C, 5'-aggatggccatCgcttgtatgccg-3'; -280G, 5'-aggatggccatGgcttgtatgccg-3'; NF-1 , 5'-ttttggattgaagccaatatgataa-3', mNF-1 , 5'-ttttggattgaaTAAaatatgataa-3'.
  • Blunt-ended double stranded probes were obtained by annealing of the oligonucleotides with their respective reverse complements and were labelled with [ ⁇ - 32 P]dATP and T4 polynucleotide kinase (Life Technologies). All probes were purified on a non- denaturing 15% polyacrylamide gel in 0.5 x TBE.
  • 0.1-0.5 ng 32 P-labeled double stranded probe (10 4 Cerenkov counts) was added to a total reaction volume of 20 ⁇ l containing 2-10 ⁇ g nuclear extract, 1x Hepes binding buffer [12% glycerol, 20 mM HEPES or TRIS, 50 mM KCI, 1 mM EDTA, 1 mM DTT, 1 mM PMSF] and 0.5 ⁇ g poly (dl-dC) (Boehringer-Mannheim, Germany).
  • unlabelled double stranded probes were added to the reaction mixture prior to addition of the labelled probe.
  • Presenilin-1 intron 8 polymorphism is not associated with autopsy- confirmed late-onset Alzheimer's disease. Neurosci.Lett. 1997; 222: 68-69.
  • Van Broeckhoven C, Backhovens H, Cruts M, et al. APOE genotype does not modulate age of onset in families with chromosome 14 encoded Alzheimer's disease. Neurosci.Lett. 1994; 169: 179-180.
  • NRSF neuron-restrictive silencer factor
  • Sp1 can displace GHF-1 from its distal binding site and stimulate transcription from the growth hormone gene promoter. Mol.Cell Biol., 10, 1811- 1814. 62. Hoch, M., Gerwin, N., Taubert, H. and Jackie, H. (1992) Competition for overlapping sites in the regulatory region of the Drosophila gene Kruppel. Science, 256, 94-97.
  • Genotype 11 74 74 83 72 0.79
  • Prom9F TAGATGGGTTTTAGCCTGTATTTGTT 1,5 mM 60 359
  • Prom9R TTCCTATCGCATCAATCTCCA

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne d'une manière générale le domaine des dysfonctionnements neurologiques et physiologiques associés à la maladie d'Alzheimer (AD). Plus particulièrement, l'invention concerne l'utilisation d'un variant dans la région régulatrice de la préséniline-1 (PSEN1) pour déterminer dans une cellule ou un tissu humain pouvant être prélevé sur un sujet humain si ce dernier présente ou a un risque de développer la maladie d'Alzheimer ou la maladie d'Alzheimer à apparition précoce. La présente invention concerne également des méthodes de criblage de molécules inhibant la réduction de niveaux de PSEN-1 associés à des polymorphismes dans la région promoteur de PSEN-1 ainsi que des molécules obtenues par lesdites méthodes.
PCT/EP2000/005942 1999-06-22 2000-06-22 Methode de detection de la maladie d'alzheimer a apparition precoce WO2000079000A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU62656/00A AU6265600A (en) 1999-06-22 2000-06-22 Method for detection of early-onset alzheimer's disease

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP99201991.9 1999-06-22
EP99201991 1999-06-22

Publications (1)

Publication Number Publication Date
WO2000079000A1 true WO2000079000A1 (fr) 2000-12-28

Family

ID=8240333

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2000/005942 WO2000079000A1 (fr) 1999-06-22 2000-06-22 Methode de detection de la maladie d'alzheimer a apparition precoce

Country Status (2)

Country Link
AU (1) AU6265600A (fr)
WO (1) WO2000079000A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1724588A3 (fr) * 2001-03-12 2007-09-05 Isis Innovation Limited Méthodes de diagnostic de la maladie d'Alzheimer
CN111057758A (zh) * 2019-12-23 2020-04-24 福州艾迪康医学检验所有限公司 检测psen1基因突变的引物、方法及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034099A2 (fr) * 1995-04-28 1996-10-31 Hsc Research And Development Limited Partnership Sequences genetiques et proteines associees a la maladie d'alzheimer et leur utilisation
EP0785282A2 (fr) * 1996-01-19 1997-07-23 Washington University School Of Medicine Diagnostique et prognostique de maladies en utilisant séquences du gène présenilin-1
EP0811695A2 (fr) * 1996-06-06 1997-12-10 Washington University Gènes S182(PS-1) mutés
WO1998051781A1 (fr) * 1997-05-14 1998-11-19 Merck & Co., Inc. Animal transgenique exprimant une proteine preseniline 1 mutante non native de type sauvage de la maladie d'alzheimer familiale dans un contexte depourvu de preseniline 1 native

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034099A2 (fr) * 1995-04-28 1996-10-31 Hsc Research And Development Limited Partnership Sequences genetiques et proteines associees a la maladie d'alzheimer et leur utilisation
EP0785282A2 (fr) * 1996-01-19 1997-07-23 Washington University School Of Medicine Diagnostique et prognostique de maladies en utilisant séquences du gène présenilin-1
EP0811695A2 (fr) * 1996-06-06 1997-12-10 Washington University Gènes S182(PS-1) mutés
WO1998051781A1 (fr) * 1997-05-14 1998-11-19 Merck & Co., Inc. Animal transgenique exprimant une proteine preseniline 1 mutante non native de type sauvage de la maladie d'alzheimer familiale dans un contexte depourvu de preseniline 1 native

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
ALDUDO J ET AL: "DGGE method for the mutational analysis of the coding and proximal promoter regions of the Alzheimer's disease presenilin - gene: two novel mutations.", HUMAN MUTATION, (1999) 14 (5) 433-9., XP000892681 *
ANWAR R ET AL: "Molecular analysis of the presenilin 1 ( S182 ) gene in "sporadic" cases of Alzheimer's disease: identification and characterisation of unusual splice variants.", JOURNAL OF NEUROCHEMISTRY, (1996 APR) 66 (4) 1774-7., XP000891244 *
CRUTS ET AL: "Presenilin mutations in Alzheimer's disease", HUMAN MUTATION, vol. 11, 1998, pages 183 - 190, XP000891200 *
CRUTS M ET AL: "Estimation of the genetic contribution of presenilin -1 and -2 mutations in a population-based study of presenile Alzheimer disease.", HUMAN MOLECULAR GENETICS, (1998 JAN) 7 (1) 43-51., XP002133454 *
CRUTS M ET AL: "MOLECULAR GENETIC ANALYSIS OF FAMILIAL EARLY-ONSET ALZHEIMER'S DISEASE LINKED TO CHROMOSOME 14Q24.3", HUMAN MOLECULAR GENETICS,GB,OXFORD UNIVERSITY PRESS, SURREY, vol. 4, no. 12, 1 December 1995 (1995-12-01), pages 2363 - 2371, XP000604935, ISSN: 0964-6906 *
MITSUDA, N. ET AL: "Transcriptional regulation of the presenilin -1 gene.", SOCIETY FOR NEUROSCIENCE ABSTRACTS, (1997) VOL. 23, NO. 1-2, PP. 823. MEETING INFO.: 27TH ANNUAL MEETING OF THE SOCIETY FOR NEUROSCIENCE, PART NEW ORLEANS, LOUISIANA, USA OCTOBER 25-30, 1997, XP000892532 *
THEUNS J. ET AL: "Genetic variability in the regulatory region of presenilin 1 associated with risk for Alzheimer's disease and variable expression.", HUMAN MOLECULAR GENETICS, (12 FEB 2000) 9/3 (325-331)., XP002150840 *
THEUNS, J. (1) ET AL: "Polymorphisms in the 5' regulatory region of presenilin 1 associated with an increased risk for early-onset Alzheimers disease.", AMERICAN JOURNAL OF HUMAN GENETICS, (OCT., 1999) VOL. 65, NO. 4, PP. A53. MEETING INFO.: 49TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF HUMAN GENETICS SAN FRANCISCO, CALIFORNIA, USA OCTOBER 19-23, 1999 THE AMERICAN SOCIETY OF HUMAN GENETICS., XP000892683 *
VAN DUIJN C M ET AL: "Genetic association of the presenilin -1 regulatory region with early-onset Alzheimer's disease in a population-based sample.", EUROPEAN JOURNAL OF HUMAN GENETICS, (1999 OCT-NOV) 7 (7) 801-6., XP000892690 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1724588A3 (fr) * 2001-03-12 2007-09-05 Isis Innovation Limited Méthodes de diagnostic de la maladie d'Alzheimer
JP2008245654A (ja) * 2001-03-12 2008-10-16 Isis Innovation Ltd アルツハイマー病の診断スクリーニング
JP2010166922A (ja) * 2001-03-12 2010-08-05 Isis Innovation Ltd アルツハイマー病の診断スクリーニング
CN111057758A (zh) * 2019-12-23 2020-04-24 福州艾迪康医学检验所有限公司 检测psen1基因突变的引物、方法及其应用

Also Published As

Publication number Publication date
AU6265600A (en) 2001-01-09

Similar Documents

Publication Publication Date Title
Kaisaki et al. Mutations in the hepatocyte nuclear factor-1α gene in MODY and early-onset NIDDM: evidence for a mutational hotspot in exon 4
US6140473A (en) Antibodies specific for MTS2 Polypeptide
Theuns et al. Genetic variability in the regulatory region of presenilin 1 associated with risk for Alzheimer’s disease and variable expression
Biggs Iii et al. Identification and characterization of members of the FKHR (FOX O) subclass of winged-helix transcription factors in the mouse
AU694502B2 (en) Mts gene, mutations therein, and methods for diagnosing cancer using mts gene sequence
Schaffer et al. Gene structure and alternative splicing of glycogen synthase kinase 3 beta (GSK-3β) in neural and non-neural tissues
Wong et al. Two novel human RAB genes with near identical sequence each map to a telomere-associated region: the subtelomeric region of 22q13. 3 and the ancestral telomere band 2q13
Karges et al. Primary structure, gene expression and chromosomal mapping of rodent homologs of the MEN1 tumor suppressor gene
AU778975B2 (en) Therapeutics and diagnostics based on an IL-1B mutation
Kanters et al. Novel donor splice site mutation in the KVLQT1 gene is associated with long QT syndrome
US20080261231A1 (en) Diabetes gene
US6153386A (en) Method to determine predisposition to hypertension
Parant et al. Organization, expression, and localization of the murine mdmx gene and pseudogene
WO2000079000A1 (fr) Methode de detection de la maladie d'alzheimer a apparition precoce
WO2000071751A1 (fr) Gene du diabete
WO1999002735A2 (fr) Dosages diagnostiques et kits utilises pour traiter les troubles dus a un exces de masse corporelle et les troubles cardiovasculaires
Riggs et al. Characterization of the LIM/homeodomain gene islet-1 and single nucleotide screening in NIDDM
US6083694A (en) Method for elucidation and detection of polymorphisms, splice variants, and proximal coding mutations using intronic sequences of the alzheimer's S182 gene
JP5119430B2 (ja) 多発性嚢胞腎疾患関連遺伝子及びその利用
US6210949B1 (en) Mouse MTS2 gene
US7960117B2 (en) Promoter mutations that increase amyloid precursor protein expression
US6060301A (en) Vector containing MTS1E1β gene
WO1997038133A1 (fr) Variants du gene preseniline 2
US20030033619A1 (en) Kainate receptor subunit GLUR7 polymorphisms for diagnosing predisposition and for therapy of mood disorders
Jain et al. A Haplotype of Angiotensin Receptor Associated with Human Hypertension Increases Blood Pressure in Transgenic Mice

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP