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WO2003107008A2 - POLYMORPHISME DIAGNOSTIQUE DE LA 11ss-HYDROXYSTÉROÏDE DÉSHYDROGÉNASE UTILE POUR IDENTIFIER LE RISQUE DE DÉVELOPPEMENT DE LA MALADIE D'ALZHEIMER - Google Patents

POLYMORPHISME DIAGNOSTIQUE DE LA 11ss-HYDROXYSTÉROÏDE DÉSHYDROGÉNASE UTILE POUR IDENTIFIER LE RISQUE DE DÉVELOPPEMENT DE LA MALADIE D'ALZHEIMER Download PDF

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WO2003107008A2
WO2003107008A2 PCT/EP2003/006315 EP0306315W WO03107008A2 WO 2003107008 A2 WO2003107008 A2 WO 2003107008A2 EP 0306315 W EP0306315 W EP 0306315W WO 03107008 A2 WO03107008 A2 WO 03107008A2
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disease
subject
gene coding
activity
gene
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WO2003107008A3 (fr
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Dominique De Quervain
Andreas Papassotiropoulos
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Zurich Universitaet Institut fuer Medizinische Virologie
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Zurich Universitaet Institut fuer Medizinische Virologie
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Priority to AU2003246438A priority Critical patent/AU2003246438A1/en
Priority to EP03759962A priority patent/EP1514119A2/fr
Publication of WO2003107008A2 publication Critical patent/WO2003107008A2/fr
Publication of WO2003107008A3 publication Critical patent/WO2003107008A3/fr
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    • 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
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to methods of diagnosing, prognosticating and monitoring neurodegenerative diseases in a subject, based on the identification of the genetic association of HSD11 B1 gene polymorphisms with increased genetic risk for a neurodegenerative disease, in particular Alzheimer's disease.
  • Alzheimer's disease has a severely debilitating impact on a patient's life. Furthermore, these diseases constitute an enormous health, social, and economic burden. Alzheimer's disease is the most common age-related neurodegenerative condition affecting about 10 % of the population over 65 years of age and up to 45 % over age 85 (for a recent review see Vickers et al., Progress in Neurobiology 2000, 60:139-165). Presently, this amounts to an estimated 12 million cases in the US, Europe, and Japan. This situation will inevitably worsen with the demographic increase in the number of elderly persons ("aging of the baby boomers") in developed countries.
  • AD Alzheimer's disease
  • sporadic AD which accounts for the majority of all AD cases, is multifactorial, and to date, the e4 allele of the gene encoding apolipoprotein E (APOE) is the only well established genetic risk factor (Saunders et al., Neurology 1993, 43:1467-72).
  • AD pathology is characterized by large extracellular ⁇ -amyloid (A ⁇ ) plaques and tau-containing intraneuronal neurofibrillary tangles, which induce neuronal death and synaptic loss.
  • a ⁇ ⁇ -amyloid
  • Hippocampal neurons are among the first cells to degenerate in the brain of patients affected by AD (Ball et al., Lancet 1985, 1 :14-6).
  • the hippocampus which is an important brain region for memory (Squire, Psychol Rev 1992, 99:195-231 ), contains a high density of glucocorticoid receptors ( cEwen et al., Psychol Rev 1986, 66:1121-88; Seckl et al., Brain Res 1991 , 561 :332-7).
  • Glucocorticoids i.e. steroid hormones released by the adrenal cortex, are known to influence cognitive functions.
  • Acute elevation of glucocorticoid levels enhances memory consolidation (Buchanan and Lovallo, Psychoneuroendocrinology 2001 , 26:307-17; Roozendaal, Psychoneuro-endocrinology 2000, 25:213-38) but impairs memory retrieval (de Quervain et al., Nature 1998, 394:787-90; de Quervain et al., Nat Neurosci 2000, 3:313-4; Wolf et al., Behav Neurosci 2001 , 1 15:1002-1 1 ).
  • AD Alzheimer's et al.
  • Am J Psychiatry 1986, 143:300-5 Hartmann et al., Neurobiol Aging 1997, 18:285-9; Hatzinger et al., Neurobiol Aging 1995, 16:205-9; O'Brien et al., Pschol Med 1996, 26:7.14; Pascualy et al., Biol Psychiatry 2000, 48:247-54.
  • glucocorticoids enhance the AD-associated oxidative neuronal damage (Behl, Exp Gerontol 1998, 33:689-96).
  • low glucocorticoid levels may lead to increased AD- associated inflammatory processes which are involved in tissue destruction (Aisen et al., Am J Psychiatry 1994, 151 :1 105-11 13).
  • the glucocorticoid system is implicated in the pathophysiology of AD. This possibility led us to hypothesize that polymorphisms in genes involved in the regulation of the glucocorticoid system may influence the risk for AD.
  • SNPs single nucleotide polymorphisms in genes involved in the regulation of the glucocorticoid system: Corticotropin releasing hormone (CRH), corticotropin releasing hormone binding protein (CRHBP), ACTH-receptor (MC2R), 1 1R-hydroxysteroid dehydrogenase type 1 and 2 (HSD1 1 B1 , HSD1 1 B2), glucocorticoid receptor (NR3C1 ), glucocorticoid modulatory element binding protein 1 and 2 (GMEB1 , GMEB2), glucocorticoid receptor DNA binding factor 1 (GRLF1 ), nuclear receptor coactivator 2 (NCOA2).
  • CSH Corticotropin releasing hormone
  • CHBP corticotropin releasing hormone binding protein
  • M2R ACTH-receptor
  • HSD1 1 B2 1 1R-hydroxysteroid dehydrogenase type 1 and 2
  • GMEB1 , GMEB2 glucocorticoid
  • a further objective of the present invention is to provide methods of monitoring the progression of such a disease and of evaluating a treatment for it. This objective is based on the identification of the genetic association of the gene coding for 11 ⁇ - hydroxysteroid dehydrogenase type 1 (HSD1 1 B1 ) with an increased risk for Alzheimer's disease.
  • HSD1 1 B1 11 ⁇ - hydroxysteroid dehydrogenase type 1
  • level as used herein is meant to comprise a gage of, or a measure of the amount of, or a concentration of a transcription product, for instance an mRNA, or a translation product, for instance a protein or polypeptide.
  • activity shall be understood as a measure for the ability of a transcription product or a translation product to produce a biological effect or a measure for a level of biologically active molecules.
  • activity also refers to enzymatic activity.
  • level and/or “activity” as used herein further refer to gene expression levels or gene activity. Gene expression can be defined as the utilization of the information contained in a gene by transcription and translation leading to the production of a gene product.
  • “Dysregulation” shall mean an upregulation or downregulation of gene expression.
  • a gene product comprises either RNA or protein and is the result of expression of a gene. The amount of a gene product can be used to measure how active a gene is.
  • the term "gene” as used in the present specification and in the claims comprises both coding regions (exons) as well as non-coding regions (e.g. non-coding regulatory elements such as promoters or enhancers, introns, leader and trailer sequences).
  • the term “ORF” is an acronym for "open reading frame” and refers to a nucleic acid sequence that does not possess a stop codon in at least one reading frame and therefore can potentially be translated into a sequence of amino acids.
  • regulatory elements shall comprise inducible and non-inducible promoters, enhancers, operators, and other elements that drive and regulate gene expression.
  • fragment as used herein is meant to comprise e.g. an alternatively spliced, or truncated, or otherwise cleaved transcription product or translation product.
  • derivative as used herein refers to a mutant, or an RNA-edited, or a chemically modified, or otherwise altered transcription product, or to a mutant, or chemically modified, or otherwise altered translation product.
  • a “derivative” may be generated by processes such as altered phosphorylation, or glycosylation, or acetylation, or lipidation, or by altered signal peptide cleavage or other types of maturation cleavage. These processes may occur post-translationally.
  • the term "modulator” as used in the present invention and in the claims refers to a molecule capable of changing or altering the level and/or the activity of a gene, or a transcription product of a gene, or a translation product of a gene.
  • a “modulator” is capable of changing or altering the biological activity of a transcription product or a translation product of a gene.
  • Said modulation may be an increase or a decrease in enzyme activity, a change in binding characteristics, or any other change or alteration in the biological, functional, or immunological properties of said translation product of a gene.
  • agent refers to any substance, chemical, composition or extract that have a positive or negative biological effect on a cell, tissue, body fluid, or within the context of any biological system, or any assay system examined. They can be agonists, antagonists, partial agonists or inverse agonists of a target.
  • agents, reagents, or compounds may be nucleic acids, natural or synthetic peptides or protein complexes, or fusion proteins.
  • oligonucleotide primer or “primer” refer to short nucleic acid sequences which can anneal to a given target polynucleotide by hybridization of the complementary base pairs and can be extended by a polymerase. They may be chosen to be specific to a particular sequence or they may be randomly selected, e.g. they will prime all possible sequences in a mix. The length of primers used herein may vary from 10 nucleotides to 80 nucleotides.
  • Probes are short nucleic acid sequences of the nucleic acid sequences described and disclosed herein or sequences complementary therewith. They may comprise full length sequences, or fragments, derivatives, isoforms, or variants of a given sequence. The identification of hybridization complexes between a "probe” and an assayed sample allows the detection of the presence of other similar sequences within that sample.
  • homolog or homology is a term used in the art to describe the relatedness of a nucleotide or peptide sequence to another nucleotide or peptide sequence, which is determined by the degree of identity and/or similarity between said sequences compared.
  • variant refers to any polypeptide or protein, in reference to polypeptides and proteins disclosed in the present invention, in which one or more amino acids are added and/or substituted and/or deleted and/or inserted at the N-terminus, and/or the C-terminus, and/or within the native amino acid sequences of the native polypeptides or proteins of the present invention.
  • variant shall include any shorter or longer version of a polypeptide or protein.
  • “Variants” shall also comprise a sequence that has at least about 80% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity with the amino acid sequences of HSD11 B1.
  • HSD11 B1 include, for example, proteins and molecules with conservative amino acid substitutions in highly conservative regions.
  • Proteins and polypeptides include variants, fragments and chemical derivatives of HSD1 1 B1 . They can include proteins and polypeptides which can be isolated from nature or be produced by recombinant and/or synthetic means. Native proteins or polypeptides refer to naturally-occurring truncated or secreted forms, naturally occurring variant forms (e.g. splice-variants) and naturally occurring allelic variants.
  • isolated as used herein is considered to refer to molecules that are removed from their natural environment, i.e.
  • sequences encoding such molecules can be linked by the hand of man to polynucleotides, to which they are not linked in their natural state, and that such molecules can be produced by recombinant and/or synthetic means. Even if for said purposes those sequences may be introduced into living or non-living organisms by methods known to those skilled in the art, and even if those sequences are still present in said organisms, they are still considered to be isolated.
  • polymorphism refers to the existence of more than one form of a gene or portion of a gene. It refers to a genetic variation in a nucleotide sequence at a given nucleotide position in the genome, within a given population, and a frequency usually exceeding 1 %. Regions harboring polymorphisms may be a given gene region, coding or non-coding portions of the gene, or even intergenic regions, and are designated as "polymorphic regions". They may cause differences in the nucleotide sequences as well as in the polypeptide sequences, in protein modifications, gene and protein expression processes and DNA replication.
  • single nucleotide polymorphism refers to a polymorphic variation in a nucleotide sequence at a given single nucleotide position in the genome.
  • Single nucleotide polymorphisms may include any single base changes such as a deletion, insertion, or a base exchange.
  • a single nucleotide polymorphism may cause a change in the encoded polypeptide sequence as well.
  • a particular SNP may be indicative for a disease state, a specific feature, or for the risk of developing a disease.
  • locus of a gene refers to a unique position on a chromosome at which the genetic information lies and includes coding sequences, intervening sequences, and regulatory elements of the given gene.
  • the distance between the loci of genes is expressed either in physical terms, or in genetic terms (recombination frequency). It is said that a gene maps to a specific locus on a chromosome.
  • the locus at which the polymorphic variation occurs is the "polymorphic site or polymorphic marker”.
  • the term “allele” or “allelic variant” refers to one of several alternative forms of a gene, or a portion thereof, typically having particular features which result in a particular phenotype.
  • the term “allele” includes any inherited variation in the DNA sequence of a gene located at a given position in the genome.
  • “Allele frequency” or “gene frequency” refers to the frequency with which a given allele is present at a given locus in a given population.
  • An individual or a subject is “homozygous” when two alleles of a given gene of a diploid organism are identical in respect to a given variation or polymorphism.
  • heterozygous is meant that the two alleles at a given locus are different.
  • the terms “risk”, “susceptibility”, “propensity”, and “predisposition” are tantamount and are used with respect to the probability of developing a neurodegenerative disease, preferably Alzheimer's disease.
  • a “haplotype” refers to the polymorphisms located on a single DNA strand, it refers to a series of alleles at several closely linked gene loci on a single chromosome.
  • haplotyping refers to the identification of polymorphisms on a single DNA strand.
  • Genetype is the genetic constitution of an individual or a cell, the types of alleles found at a given locus.
  • Linkage in terms of genetics, refers to gene loci on the same chromosome, localized in a certain distance from each other, so that an independent segregation is not necessarily the case. The closer the gene loci lie to each other, the less frequently they are separated by recombination (crossing-over event between homologous chromosomes) and the higher the probability for linkage. Loci that are physically very close to each other, i.e. recombination between them will usually not occur, tend to be inherited together.
  • the object of "linkage analysis” is to determine whether two loci tend to cosegregate more often than they would, if they were not physically close together. It is to estimate the recombination fraction, to test if the value is less than 0.5 (loci located on different chromosomes), and whether an observed deviation from 0.5 is statistically significant.
  • the ratio of the probability that two gene loci are genetically linked, to the probability that they are not genetically linked, is referred to as "odds ratio”. If the odds for linkage exceed or reach a minimal value (ratio of 1000:1 ), it is assumed that two gene loci are linked.
  • the "LOD score” (logarithm of the odds) refers to the logarithm of the calculated odds ratio, and is the likelihood of a given disease, or phenotype to be localized within the genetic region examined.
  • Linkage disequilibrium (LD) refers to alleles which are nonrandomly associated at closely linked gene loci and operates over distances less than 1 cM. This association of alleles is not necessarily a consequence of close linkage. Allelic association will be shown only if the alleles mark conserved ancestral chromosomal segments, which is usually due to founder effects.
  • positional candidate gene refers to a gene which is considered as a possible locus for a given disease. The assumption is based on known properties as function, expression pattern, chromosomal location etc..
  • susceptibility gene refers to a gene locus which is associated with a given disease and which is a major risk factor for developing said given disease.
  • a "genetic association study” is meant to be a test for differences in the distribution of alleles between unrelated affected subjects (patients diagnosed with a given disease) and controls (healthy subjects). It is tested whether a genetic variant (SNP of a gene) increases disease risk, i.e. is associated with a trait. LD may be also tested, whereby an increased prevalence of a characteristic set of SNP alleles in affected subjects will be identified.
  • AD-type neuropathology refers to neuropathological, neurophysiological, histopathological and clinical hallmarks as described in the instant invention and as commonly known from state- of-the-art literature (see: Iqbal, Swaab, Winblad and Wisniewski, Alzheimer ⁇ s Disease and Related Disorders (Etiology, Pathogenesis and Therapeutics), Wiley & Sons, New York, Weinheim, Toronto, 1999; Scinto and Daffner, Early Diagnosis of Alzheimer ' s Disease, Humana Press, Totowa, New Jersey, 2000; Mayeux and Christen, Epidemiology of Alzheimer ' s Disease: From Gene to Prevention, Springer Press, Berlin, Heidelberg, New York, 1999; Younkin, Tanzi and Christen, Presenilins and Alzheimer ⁇ s Disease, Springer Press, Berlin, Heidelberg, New York, 1998).
  • Neurodegenerative diseases or disorders according to the present invention comprise Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Pick's disease, fronto-temporal dementia, progressive nuclear palsy, corticobasal degeneration, cerebro-vascular dementia, multiple system atrophy, argyrophilic grain dementia and other tauopathies, and mild- cognitive impairment.
  • Further conditions involving neurodegenerative processes are, for instance, age-related macular degeneration, narcolepsy, motor neuron diseases, prion diseases, traumatic nerve injury and repair, and multiple sclerosis.
  • the invention features a method for diagnosing or prognosticating a neurodegenerative disease in a subject, or determining the propensity or predisposition of a subject to develop a neurodegenerative disease.
  • the method comprises detecting in a sample obtained from said subject the presence or absence of a variation in the gene coding for 1 1 ⁇ -hydroxysteroid dehydrogenase type 1 (1 1 ⁇ -HSD-1 ; HSD11 B1 ), wherein the presence of a variation in said gene in said subject indicates a diagnosis or prognosis of a neurodegenerative disease, or an increased propensity or predisposition of developing a neurodegenerative disease as compared to a subject who does not carry a variation in said gene.
  • a variation in the gene coding for HSD11 B1 can be understood as any alteration in the naturally occuring nucleic acid sequence of the said gene, i.e. any alteration from the wildtype.
  • said 11 ⁇ -hydroxysteroid dehydrogenase gene is the gene coding for the 11 ⁇ -hydroxysteroid dehydrogenase type 1 , also termed corticosteroid 11-beta-dehydrogenase isozyme 1 (EC1.1.1.146), or 11-beta-HSD-1 , or HSD11 B1 , or HSD11 , or HSD11 L, or 11-DH, mapping to human chromosome 1 chromosomal band q32.2, located in genomic sequence AL031316 and AL022398 (Genbank protein identification number: AAH12593; SwissProt: P28845; Genbank mRNA accession number: BC012593; Tannin et al., J.
  • said 11 ⁇ -hydroxysteroid dehydrogenase type 1 gene or 11 ⁇ -hydroxysteroid dehydrogenase type 1 protein is also generally referred to as the 11 ⁇ -HSD-1 or HSD11 B1 gene, or 11 ⁇ -HSD-1 or HSD11 B1 protein, or just 11 ⁇ -HSD-1 or HSD11 B1.
  • the variation in the gene coding for HSD11 B1 is a single nucleotide polymorphism located on human chromosome 1 , chromosomal position 198633436, located in the promoter region at position -2037 bases relative to the start codon (single nucleotide polymorphism identification number: rs846911).
  • the variation is a C to A transversion, hereinafter also referred to as the A-allele.
  • SNP rs860185 at position -718 bases relative to the start codon may also act independently as a diagnostic polymorphism according to the claims and methods of the present invention.
  • said neurodegenerative disease or disorder is Alzheimer's disease, and said subjects suffer from Alzheimer's disease.
  • the method according to the present invention may be particularly useful for the identification of individuals that are at risk of developing a neurodegenerative disease. Consequently, the method, according to the present invention, may serve as a means for targeting identified individuals for early preventive measures or therapeutic intervention prior to disease onset, before irreversible damage in the course of the disease has been inflicted.
  • Determining the presence or absence of a polymorphism or variation in the gene coding for HSD11 B1 may comprise determining a partial nucleotide sequence of the DNA from said subject, said partial nucleotide sequence indicating the presence or absence of said polymorphism or variation. It may further be preferred to perform a polymerase chain reaction with the DNA from said subject to determine the presence or absence of said polymorphism or variation. Such techniques are known to those skilled in the art (see Lewin B, Genes V, Oxford University Press, 1994).
  • the invention also relates to the construction and the use of primers and probes which are unique to the nucleic acid sequences of the HSD11 B1 gene, or fragments, or variants thereof, as disclosed in the present invention.
  • the oligonucleotide primers and/or probes can be labeled specifically with fluorescent, bioluminescent, magnetic, or radioactive substances.
  • the invention further relates to the detection and the production of said nucleic acid sequences, or fragments and/or variants thereof, using said specific oligonucleotide primers in appropriate combinations.
  • PCR-analysis a method well known to those skilled in the art, can be performed with said primer combinations to amplify said gene specific nucleic acid sequences from a sample containing nucleic acids.
  • Such sample may be derived either from healthy or diseased subjects. Whether an amplification results in a specific nucleic acid product or not, and whether a fragment of different length can be obtained or not, may be indicative for a neurodegenerative disease, in particular Alzheimer's disease.
  • the invention provides nucleic acid sequences, oligonucleotide primers, and probes of at least 10 bases in length up to the entire coding and gene sequences, useful for the detection of gene mutations and single nucleotide polymorphisms in a given sample comprising nucleic acid sequences to be examined, which may be associated with neurodegenerative diseases, in particular Alzheimer's disease.
  • This feature has utility for developing rapid DNA-based diagnostic tests, preferably also in the format of a kit.
  • the sample taken for genetic analysis comprises DNA obtained from body fluids, tissues, or any suitable cells of the body readily available.
  • the sample is a blood sample.
  • the sample may also consist of body fluids such as saliva, urine, serum plasma, mucus, or cerebrospinal fluid.
  • the invention features a method for diagnosing or prognosticating a neurodegenerative disease in a subject, or determining the propensity or predisposition of a subject to develop a neurodegenerative disease, in particular AD, comprising determining a level, or an activity, or both said level and said activity, of at least one substance which is selected from the group consisting of a transcription product of the gene coding for HSD1 1 B1 , and/or a translation product of the gene coding for HSD11 B1 , and/or a fragment, or derivative, or variant thereof in a sample from said subject and comparing said level, and/or said activity, or both said level and said activity, of at least one of said substances to a reference value representing a known disease or health status, thereby diagnosing or prognosticating a neurodegenerative disease in said subject, or determining the propensity or predisposition of said subject to develop a neurodegenerative disease.
  • the present invention provides a method of monitoring the progression of a neurodegenerative disease in a subject, comprising determining a level, or an activity, or both said level and said activity, of at least one substance which is selected from the group consisting of a transcription product of the gene coding for HSD11 B1 , or a translation product of the gene coding for HSD11 B1 , and/or a fragment, or derivative, or variant thereof in a sample from said subject; and comparing said level, or said activity, or both said level and said activity, of at least one of said substances to a reference value representing a known disease or health status, thereby monitoring the progression of a neurodegenerative disease in said subject.
  • the present invention provides a method of evaluating a treatment for a neurodegenerative disease, comprising determining a level, or an activity, or both said level and said activity, of at least one substance which is selected from the group consisting of a transcription product of the gene coding for HSD 1 B1 , or a translation product of the gene coding for HSD11 B1 , and/or a fragment, or derivative, or variant thereof in a sample obtained from a subject being treated for a neurodegenerative disease; and comparing said level, or said activity, or both said level and said activity, of at least one of said substances to a reference value representing a known disease or health status, thereby evaluating said treatment for a neurodegenerative disease.
  • the sample to be analyzed for determining a level, or an activity, or both said level and said activity, of at least one substance which is selected from the group consisting of a transcription product of the gene coding for HSD11 B1 , or a translation product of the gene coding for HSD1 1 B1 , and/or a fragment, or derivative, or variant thereof is taken from the group comprising body fluid, preferably cerebrospinal fluid, saliva, urine, mucus, blood, or serum plasma, or a tissue, or cells like skin fibroblasts. Most preferably, the sample is taken from cerebrospinal fluid.
  • the methods of diagnosis, prognosis, monitoring the progression or evaluating a treatment for a neurodegenerative disease can be practiced ex corpore, and such methods preferably relate to samples, for instance, body fluids or cells, removed, collected, or isolated from a subject or patient.
  • the reference value of a level, or an activity, or both said level and said activity, of a transcription product of the gene coding for HSD1 1 B1 , or a translation product of the gene coding for HSD1 1 B1 , and/or a fragment, or derivative, or variant thereof is that in a sample from a subject not suffering from a neurodegenerative disease.
  • the determination of a level of transcription products of the gene coding for HSD11 B1 can be performed in a sample from a subject using Northern blots with probes specific for said gene. Another preferred method of measuring said level is by quantitative PCR with primer combinations which amplify said gene-specific sequences from cDNA obtained by reverse transcription of RNA extracted from a sample of a subject. It might further be preferred to measure transcription products by means of chip-based microarray technologies. These techniques are known to those of ordinary skill in the art (see Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001 ; Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, MA, 2000). An example of an immunoassay is the detection and measurement of enzyme activity as disclosed and described in the patent application WO 02/14543.
  • a level and/or an activity of a translation product of the gene coding for HSD1 1 B1 and/or of a fragment, or derivative, or variant thereof, and/or a level of activity of said translation product of the HSD11 B1 gene, and/or of a fragment, or derivative, or variant thereof can be detected using a Western blot analysis, an immunoassay, an enzyme activity assay, and/or a binding assay.
  • assays can measure the amount of binding between said translation product and an anfi- polypeptide antibody by the use of enzymatic, chromodynamic, radioactive, magnetic, or luminescent labels which are attached to either the anti-polypeptide antibody or a secondary antibody which binds the anti-polypeptide antibody.
  • Immunoassays which can be used include e.g. ELISAs, Western blots and other techniques known to those of ordinary skill in the art (see Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999 and Edwards R, Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford; England, 1999). All these detection techniques may also be employed in the format of microarrays, protein-arrays, antibody microarrays, tissue microarrays, electronic biochip or protein-chip based technologies (see Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, MA, 2000).
  • Enzymatic activity of HSD1 1 B1 may be measured by in vitro, cell-based, or in vivo assays. Conveniently, HSD11 B1 enzymatic activity can, for instance, be determined using a dehydrogenase and/or reductase activity assay.
  • the provided methods of diagnosing or prognosticating a neurodegenerative disease in a subject, or determining the propensity or predisposition of a subject to develop a neurodegenerative disease, or monitoring a treatment, or evaluating a treatment of a neurodegenerative disease further comprise comparing a level, or an activity, or both said level and said activity, of a transcription product of the gene coding for HSD1 1 B1 , and/or a translation product of the gene coding for HSD1 1 B1 , and/or a fragment, or derivative, or variant thereof in a series of samples taken from said subject over a period of time.
  • said subject receives a treatment prior to one or more sample gatherings. It is a further preferred embodiment to determine said level, or said activity, or both said level and said activity, in said samples before and after said treatment of said subject.
  • the invention features a kit for diagnosing or prognosticating a neurodegenerative disease, in particular AD, in a subject, or determining the propensity or predisposition of a subject to develop a neurodegenerative disease, in particular AD, said kit comprising: at least one reagent which is selected from the group consisting of (i) reagents that selectively detect a transcription product of the gene coding for HSD1 1 B1 , (ii) reagents that selectively detect a translation product of the gene coding for
  • HSD1 1 B1 (iii) reagents that selectively detect the presence or absence of a variation in the gene coding for HSD1 1 B1 ; and an instruction for diagnosing, or prognosticating a neurodegenerative disease, in particular AD, or determining the propensity or predisposition of a subject to develop such a disease by (i) detecting a level, or an activity, or both said level and said activity, of said transcription product and/or said translation product of the gene coding for HSD 1 B1 , in a sample from said subject; and/or detecting the presence or absence of a variation in the gene coding for HSD11 B1 in a sample from said subject; and (ii) diagnosing or prognosticating a neurodegenerative disease, in particular AD, or determining the propensity or predisposition of said subject to develop such a disease, wherein a varied level, or activity, or both said level and said activity, of said transcription product and/or said translation product compared to a reference value
  • the reagents of the kit selectively detect the single nucleotide polymorphism located at chromosomal position 198633436 of human chromosome 1 , located in the promoter region at position -2037 relative to the start codon of the gene coding for HSD11 B1 (single nucleotide polymorphism identification number: rs846911). It is further preferred that said variation is a C to A transversion.
  • the reagents of the kit selectively detect the single nucleotide polymorphism rs860185 located on human chromosome 1 in the promoter region at position -718 bases relative to the start codon of the gene coding for HSD11 B1.
  • the kit according to the present invention may be particularly useful for the identification of individuals that are at risk of developing a neurodegenerative disease, in particular AD. Consequently, the kit, according to the invention, may serve as a means for targeting identified individuals for early preventive measures or therapeutic intervention prior to disease onset, before irreversible damage in the course of the disease has been inflicted. Furthermore, in preferred embodiments, the kit featured in the invention is useful for monitoring a progression of " a neurodegenerative disease, in particular AD, in a subject. It is further useful in monitoring success or failure of therapeutic treatment of said subject.
  • the invention features a method of treating or preventing Alzheimer's disease or related neurodegenerative diseases, in a subject comprising the administration to said subject in a therapeutically or prophylactically effective amount of an agent or agents which directly or indirectly affect a level, or an activity, or both said level and said activity, of (i) the gene coding for HSD1 1 B1 , and/or (ii) a transcription product of the gene coding for HSD1 1 B1 , and/or (iii) a translation product of the gene coding for HSD1 1 B1 , and/or (iv) a fragment, or derivative, or variant of (i) to (iii).
  • an agent or agents which directly or indirectly affect a level, or an activity, or both said level and said activity, of (i) the gene coding for HSD1 1 B1 , and/or (ii) a transcription product of the gene coding for HSD1 1 B1 , and/or (iii) a translation product of the gene coding for HSD
  • Said agent may comprise a small molecule, or it may also comprise a peptide, an oligopeptide, or a polypeptide.
  • Said peptide, oligopeptide, or polypeptide may comprise an amino acid sequence of a translation product of the gene coding for HSD1 1 B1 , or a fragment, or derivative, or a variant thereof.
  • An agent for treating or preventing a neurodegenerative disease, in particular AD, according to the instant invention may also consist of a nucleotide, an oligonucleotide, or a polynucleotide.
  • Said oligonucleotide or polynucleotide may comprise a nucleotide sequence of the HSD1 1 B1 gene either in sense orientation or in antisense orientation.
  • the method comprises the application of per se known methods of gene therapy and/or antisense nucleic acid technology to administer said agent or agents.
  • gene therapy comprises several approaches: molecular replacement of a mutated gene, addition of a new gene resulting in the synthesis of a therapeutic protein, and modulation of endogenous cellular gene expression by recombinant expression methods or by drugs. Gene-transfer techniques are described in detail (see e.g.
  • the invention features a method of treating or preventing a neurodegenerative disease by means of antisense nucleic acid therapy, i.e. the down-regulation of an inappropriately expressed or defective gene by the introduction of antisense nucleic acids or derivatives thereof into certain critical cells (see e.g. Gillespie, DN&P 1992, 5:389-395; Agrawal and Akhtar, Trends Biotechnol 1995, 13: 197-199; Crooke, Biotechnology 1992, 10:882-6).
  • ribozymes i.e. RNA molecules that act as enzymes, destroying RNA that carries the message of disease has also been described (see e.g.
  • the subject to be treated is a human, and therapeutic antisense nucleic acids or derivatives thereof are directed against the human gene coding for HSD1 1 B1. It is preferred that cells of the central nervous system, preferably the brain, of a subject are treated in such a way. Cell penetration can be performed by known strategies such as coupling of antisense nucleic acids and derivatives thereof to carrier particles, or the above described techniques. Strategies for administering targeted therapeutic oligodeoxynucleotides are known to those of skill in the art (see e.g. Wickstrom, Trends Biotechnol, 1992, 10: 281-287). In some cases, delivery can be performed by mere topical application.
  • RNA interference RNA interference
  • the method comprises grafting donor cells into the central nervous system, preferably the brain, of said subject, or donor cells preferably treated so as to minimize or reduce graft rejection, wherein said donor cells are genetically modified by insertion of at least one transgene encoding said agent or agents.
  • Said transgene might be carried by a viral vector, in particular a retroviral vector.
  • the transgene can be inserted into the donor cells by a nonviral physical transfection of DNA encoding a transgene, in particular by microinjection.
  • Insertion of the transgene can also be performed by electroporation, chemically mediated transfection, in particular calcium phosphate transfection or liposomal mediated transfection (see McCelland and Pardee, Expression Genetics: Accelerated and High-Throughput Methods, Eaton Publishing, Natick, MA, 1999).
  • said agent for treating and preventing a neurodegenerative disease is a therapeutic protein which can be administered to said subject, preferably a human, by a process comprising introducing subject cells into said subject, said subject cells having been treated in vitro to insert a DNA segment encoding said therapeutic protein, said subject cells expressing in vivo in said subject a therapeutically effective amount of said therapeutic protein.
  • Said DNA segment can be inserted into said cells in vitro by a viral vector, in particular a retroviral vector.
  • Said agent, particularly a therapeutic protein can further be administered to said subject by a process comprising the injection or the systemic administration of a fusion protein, said fusion protein consisting of a fusion of a protein transduction domain with said agent.
  • Methods of treatment comprise the application of therapeutic cloning, transplantation, and stem cell therapy using embryonic stem cells or embryonic germ ceils and neuronal adult stem cells, combined with any of the previously described cell- and gene therapeutic methods.
  • Stem cells may be totipotent or pluripotent. They may also be organ-specific.
  • Strategies for repairing diseased and/or damaged brain cells or tissue comprise (i) taking donor cells from an adult tissue. Nuclei of those cells are transplanted into unfertilized egg cells from which the genetic material has been removed. Embryonic stem cells are isolated from the blastocyst stage of the cells which underwent somatic cell nuclear transfer.
  • differentiation factors then leads to a directed development of the stem ceils to specialized cell types, preferably neuronal cells (Lanza et al., Nature Medicine 1999, 9: 975-977), or (ii) purifying adult stem cells, isolated from the central nervous system, or from bone marrow (mesenchymal stem cells), for in vitro expansion and subsequent grafting and transplantation, or (iii) directly inducing endogenous neural stem cells to proliferate, migrate, and differentiate into functional neurons (Peterson DA, Curr. Opin. Pharmacol. 2002, 2: 34-42).
  • Adult neural stem cells are of great potential for repairing damaged or diseased brain tissues, as the germinal centers of the adult brain are free of neuronal damage or dysfunction (Colman A, Drug Discovery World 2001 , 7: 66-71 ).
  • the subject for treatment or prevention can be a human, an experimental animal, e.g. a mouse or a rat, a fish, a fly, or a worm; a domestic animal, or a non-human primate.
  • the experimental animal can be an animal model for a neuro-degenerative disorder, e.g. a transgenic mouse and/or a knock-out mouse with an Alzheimer's-type neuropathology.
  • the invention features a modulator of an activity, or a level, or both said activity and said level of at least one substance which is selected from the group consisting of (i) the gene coding for HSD1 1 B1 and/or (ii) a transcription product of the gene coding for HSD1 1 B1 , and/or (iii) a translation product of the gene coding for HSD1 1 B1 , and/or (iv) a fragment, or derivative, or variant of (i) to (iii).
  • the invention features a pharmaceutical composition
  • a pharmaceutical composition comprising said modulator and preferably a pharmaceutical carrier.
  • Said carrier refers to a diluent, adjuvant, excipient, or vehicle with which the modulator is administered.
  • the invention features a modulator of an activity, or a level, or both said activity and said level of at least one substance which is selected from the group consisting of (i) the gene coding for HSD1 1 B1 , and/or (ii) a transcription product of the gene coding for HSD1 1 B1 and/or (iii) a translation product of the gene coding for HSD1 1 B1 , and/or (iv) a fragment, or derivative, or variant of (i) to (iii) for use in a pharmaceutical composition.
  • the invention provides for the use of a modulator of an activity, or a level, or both said activity and said level of at least one substance which is selected from the group consisting of (i) the gene coding for HSD11 B1 , and/or (ii) a transcription product of the gene coding for HSD1 1 B1 , and/or (iii) a translation product of the gene coding for HSD1 1 B1 , and/or (iv) a fragment, or derivative, or variant of (i) to (iii) for a preparation of a medicament for treating or preventing a neurodegenerative disease, in particular Alzheimer's disease.
  • the present invention also provides a kit comprising one or more containers filled with a therapeutically or prophylactically effective amount of said pharmaceutical composition.
  • the invention features a recombinant, non-human animal comprising a non-native gene sequence coding for a translation product of the HSD1 1 B1 gene, or a fragment, or a derivative, or a variant thereof.
  • the generation of said recombinant, non-human animal comprises (i) providing a gene targeting construct containing said gene sequence and a selectable marker sequence, and (ii) introducing said targeting construct into a stem cell of a non-human animal, and (iii) introducing said non-human animal stem cell into a non-human embryo, and (iv) transplanting said embryo into a pseudopregnant non-human animal, and (v) allowing said embryo to develop to term, and (vi) identifying a genetically altered non-human animal whose genome comprises a modification of said gene sequence in both alleles, and (vii) breeding the genetically altered non-human animal of step (vi) to obtain a genetically altered non-human animal whose genome comprises a modification of said endogenous gene, wherein said gene is mis-expressed, or under-expressed, or over-expressed, and wherein said disruption or alteration results in said non-human animal exhibiting a predisposition to developing symptoms of neuropathology similar to a neurodegenerative disease,
  • the invention features an assay for screening for a modulator of neurodegenerative diseases, in particular Alzheimer's disease, or related diseases and disorders of one or more substances selected from the group consisting of (i) the gene coding for HSD11B1 , and/or (ii) a transcription product of the gene coding for HSD11 B1 , and/or (iii) a translation product of the gene coding for HSD11 B1 , and/or (iv) a fragment, or derivative, or variant of (i) to (iii).
  • a modulator of neurodegenerative diseases in particular Alzheimer's disease, or related diseases and disorders of one or more substances selected from the group consisting of (i) the gene coding for HSD11B1 , and/or (ii) a transcription product of the gene coding for HSD11 B1 , and/or (iii) a translation product of the gene coding for HSD11 B1 , and/or (iv) a fragment, or derivative, or variant of (i) to (
  • This screening method comprises (a) contacting a cell with a test compound, and (b) measuring the level, or the activity, or both the level and the activity of one or more substances recited in (i) to (iv), and (c) measuring the level, or the activity, or both the level and the activity of said substances in a control cell not contacted with said test compound, and (d) comparing the levels of the substance in the cells of step (b) and (c), wherein an alteration in the level and/or activity of said substances in the contacted cells indicates that the test compound is a modulator of said diseases and disorders.
  • the invention features a screening assay for a modulator of neurodegenerative diseases, in particular Alzheimer's disease, or related diseases and disorders of one or more substances selected from the group consisting of (i) the gene coding for HSD1 1 B1 , and/or (ii) a transcription product of the gene coding for HSD11 B1 , and/or (iii) a translation product of the gene coding for HSD1 1 B1 , and/or (iv) a fragment, or derivative, or variant of (i) to (iii), comprising (a) administering a test compound to a test animal which is predisposed to developing or has already developed symptoms of a neurodegenerative disease or related diseases or disorders, and (b) measuring the level and/or activity of one or more substances recited in (i) to (iv), and (c) measuring the level and/or activity of said substances in a matched control animal which is equally predisposed to developing or has already developed symptoms of said diseases and to which animal no such test compound has been administered
  • said test animal and/or said control animal is a recombinant, non-human animal which expresses the HSD1 1 B1 gene, or a fragment, or a derivative, or a variant thereof, under the control of a transcriptional regulatory element which is not the native HSD11 B1 gene transcriptional control regulatory element.
  • the present invention provides a method for producing a medicament comprising the steps of (i) identifying a modulator of neurodegenerative diseases by a method of the herein aforementioned screening assays and (ii) admixing the modulator with a pharmaceutical carrier.
  • said modulator may also be identifiable by other types of screening assays.
  • the present invention provides for a method of testing a compound, preferably an assay for screening a plurality of compounds, for inhibition of binding between a ligand and HSD11 B1 , or a fragment, or derivative, or variant thereof.
  • Said method comprises the steps of (i) adding a liquid suspension of HSD1 1 B1 , or a fragment, or derivative, or variant thereof, to a plurality of containers, and (ii) adding a compound, preferably a plurality of compounds, to be screened for said inhibition to said plurality of containers, and (iii) adding a detectable ligand, preferably a fluorescently detectable ligand, to said containers, and (iv) incubating the liquid suspension of HSD1 1 B1 , or said fragment, or derivative, or variant thereof, and said compounds, and said detectable, preferably said fluorescently detectable ligand, and (v) measuring the amounts of detectable ligand or fluorescence associated with HSD1 1 B1 , or with said fragment, or derivative
  • HSD11 B1 translation product or fragment, or derivative, or variant thereof into artificial liposomes to generate the corresponding proteoliposomes to determine the inhibition of binding between a ligand and said HSD1 1 B1 translation product.
  • Methods of reconstitution of HSD11 B1 translation products from detergent into liposomes have been detailed (Schwarz et al., Biochemistry 1999, 38: 9456-9464; Krivosheev and Usanov, Biochemistry-Moscow 1997, 62: 1064-1073).
  • a fluorescently detectable label it might in some aspects be preferred to use any other detectable label known to the person skilled in the art, e.g. radioactive label, and detect it accordingly.
  • Said method may be useful for the identification of novel compounds as well as for evaluating compounds which have been improved or otherwise optimized in their ability to inhibit the binding of a ligand to HSD1 1 B1 , or a fragment, or derivative, or variant thereof.
  • a fluorescent binding assay in this case based on the use of carrier particles, is disclosed and described in patent application WO 00/52451.
  • a further example is the competitive assay method as described in patent WO 02/01226.
  • the present invention provides a method for producing a medicament comprising the steps of (i) identifying a compound as an inhibitor of binding between a ligand and HSD1 1 B1 by the herein aforementioned inhibitory binding assay and (ii) admixing the compound with a pharmaceutical carrier.
  • a compound as an inhibitor of binding between a ligand and HSD1 1 B1 by the herein aforementioned inhibitory binding assay and (ii) admixing the compound with a pharmaceutical carrier.
  • said compound may also be identifiable by other types of screening assays.
  • the invention features a method of testing a compound, preferably an assay for screening a plurality of compounds, to determine the degree of binding of said compound or compounds to HSD11 B1 , or to a fragment, or derivative, or variant thereof.
  • Said method comprises the steps of (i) adding a liquid suspension of HSD1 1 B1 , or a fragment, or derivative, or variant thereof, to a plurality of containers, and (ii) adding a detectable, preferably a fluorescently detectable compound, or adding a plurality of detectable, in particular fluorescently detectable compounds, to be screened for said binding to said plurality of containers, and (iii) incubating the liquid suspension of HSD1 1 B1 , or said fragment, or derivative, or variant thereof, and said detectable compound, preferably said plurality of detectable compounds, and (iv) measuring the amounts of detectable compound or preferably of fluorescence associated with HSD1 1 B1 , or with said fragment, or derivative, or variant thereof, and (v) determining
  • any other type of detectable label might also be employed. Said method may be useful for the identification of novel compounds as well as for evaluating compounds which have been improved or otherwise optimized in their ability to bind to a HSD11 B1 gene product, or a fragment, or derivative, or variant thereof.
  • the present invention provides a method for producing a medicament comprising the steps of (i) identifying a compound as a binder to HSD11 B1 by the herein aforementioned binding assays and (ii) admixing the compound with a pharmaceutical carrier.
  • said compound may also be identifiable by other types of screening assays.
  • the present invention provides for a medicament obtainable by any of the methods according to the herein claimed screening assays.
  • the instant invention provides for a medicament obtained by any of the methods according to the herein claimed screening assays.
  • the present invention features a protein molecule, said protein molecule being a translation product of the gene coding for HSD1 1 B1 , or a fragment , or derivative, or variant thereof, for the use as a diagnostic target for detecting a neurodegenerative disease, preferably Alzheimer's disease.
  • the present invention further features a protein molecule, said protein molecule being a translation product of the gene coding for HSD1 1 B1 , or a fragment, or derivative, or variant thererof, for the use as a screening target for reagents or compounds preventing, or treating, or ameliorating a neurodegenerative disease, preferably Alzheimer's disease.
  • the present invention features an antibody which is specifically immunoreactive with an immunogen, wherein said immunogen is a translation product of the gene coding for HSD1 1 B1 , or a fragment, or derivative, or variant thereof.
  • the immunogen may comprise immunogenic or antigenic epitopes of portions of a translation product of said genes, wherein said immunogenic or antigenic portion of a translation product is a polypeptide, and wherein said polypeptide elicits an antibody response in an animal, and wherein said polypeptide is immunospecifically bound by said antibody.
  • antibody encompasses all forms of antibodies known in the art, such as polyclonal, monoclonal, chimeric, recombinatorial, anti- idiotypic, humanized, or single chain antibodies as well as fragments thereof (see Dubel and Breitling, Recombinant Antibodies, Wiley-Liss, New York, NY, 1999).
  • Antibodies of the present invention are useful, for instance, in a variety of diagnostic and therapeutic methods, based on state-of-the-art techniques (see Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999 and Edwards R., Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford, England, 1999) such as enzyme-immuno assays (e.g. enzyme-linked immunosorbent assay, ELISA), radioimmuno assays, chemoluminescence-immuno assays, Western-blot, immunoprecipitation and antibody microarrays. These methods involve the detection of translation products of the HSD1 1 B1 gene, or fragments, or derivatives, or variants thereof.
  • enzyme-immuno assays e.g. enzyme-linked immunosorbent assay, ELISA
  • radioimmuno assays e.g. enzyme-linked immunosorbent assay, ELISA
  • said antibodies can be used for detecting the pathological state of a cell in a sample from a subject, comprising immunocytochemical staining of said cell with said antibody, wherein an altered degree of staining, or an altered staining pattern in said cell compared to a cell representing a known health status indicates a pathological state of said cell.
  • the pathological state relates to a neurodegenerative disease, in particular to Alzheimer's disease.
  • Immuno-cytochemical staining of a cell can be carried out by a number of different experimental methods well known in the art.
  • SNP rs846911 is located at -2037 relative to the start codon.
  • SNP rs860185 is located at -718.
  • the A allele of rs846911 generates an OCT-1 binding site.
  • the haplotype containing the rare variants of both SNPs shows a significantly reduced luciferase activity (- 20%) relative to the common variants containing haplotype (right bar).
  • Genetic markers Information on polymorphic sites was derived from the database of single nucleotide polymorphisms (dbSNP) established by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/SNP/index.html). Twentyone SNP candidates in 10 genes were selected for genotyping.
  • dbSNP single nucleotide polymorphisms
  • the mean onset age ( ⁇ standard deviation) of AD was 67 ⁇ 9 years, the mean Mini-mental State Examination (MMSE) score was 20 + 6. There were 21 1 (60%) females among the AD patients.
  • MMSE Mini-mental State Examination
  • Genotyping of eleven SNPs in glucocorticoid-related genes and of APOE in 776 individuals revealed that two genes, APOE and HSD11B1, were associated with AD. The level of genome-wide significance reached by these two genes was equal to 0.002 ( Figure 1A). The final corrected significance p min was 0.006. Separate set- association analysis in the central and south European samples revealed similar results, i.e. APOE and HSD11B1 contributed to AD risk ( Figures 1 B,C). In the south European sample (n 423), the gene encoding the adrenocorticotropic hormone receptor (MC2R) was also associated with the disease.
  • M2R adrenocorticotropic hormone receptor
  • the single nucleotide polymorphism rs84691 1 is located in the promoter region of HSD11B1, 2037 bases 5' to the start codon.
  • allelic distribution of rs860185 was identical to that of rs84691 1 , indicating the presence of two haplotypes spanning 1 .3 kb, the rare and disease- associated haplotype A-T and the frequent haplotype C-A.
  • 1 1 ⁇ -hydroxysteroid dehydrogenase type 1 (1 1 ?-HSD-1 , HSD1 1 B1 ) by acting as an intracellular 1 1 ?-reductase, regenerates biologically active hydrocortisone from inactive cortisone in neurons (Rajan et al., J Neurosci 1996, 16:65-70). This reductase activity would be anticipated to increase intraneuronal glucocorticoid levels, thus potentiating neurotoxicity. Indeed, 1 1-dehydrocorticosterone potentiates in vitro kainate-induced neurotoxicity in cultured hippocampal neurons, which is prevented by HSD1 1 B1 inhibitors (Rajan et al., supra).
  • HSD1 1 B1 knockout mice reportedly ameliorates age-related learning impairment (Yau et al., Proc Natl Acad Sci USA 2001 , 98:4716-21 ).
  • the herein disclosed AD-associated SNP in HSD11 B1 is located in the promoter region of the gene.
  • Computer assisted analysis showed that the risk allele A generates a binding site for the octamer binding factor 1 (OCT-1 ).
  • OCT-1 octamer binding factor 1
  • the random expectation value of the OCT-1 binding site is 4.8 matches per 1000 basepairs of DNA sequence.
  • the C allele of rs846911 failed to induce binding sites for transcription factors at this position.
  • Luciferase-containing constructs (pGL3) were co-transfected with phRL-TK synthetic renilla vector (Promega) to control for transfection efficiency.
  • the Dual Luciferase system (Promega) was used according to the manufacturer's protocol, and experiments were repeated in twenty independent wells. Readings were taken in duplicate on a luminometer.
  • the risk-associated haplotype containing the rare variants of both SNPs reduced luciferase activity by -20% relative to the haplotype containing the common variants ( Figure 3). This difference was highly significant. This result suggests that the effects of the haplotype containing SNPs rs846911 and rs860185 on risk for the development of AD are related to differential regulation of HSD11B1 transcription. Reduced transcription activities, as a result of the rare risk haplotype may lead to low intracellular cortisol levels and thus to increased AD- associated inflammatory processes which promote neuronal death.

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Abstract

La présente invention est fondée sur l'association surprenante d'un polymorphisme dans le gène codant pour HSD11B1 avec un risque génétique accru de développement d'une maladie neurodégénérative, en particulier la maladie d'Alzheimer, et concerne une méthode permettant de diagnostiquer et de pronostiquer une telle maladie, ou de déterminer la propension ou la prédisposition d'un sujet à développer une telle maladie. Ladite méthode consiste à détecter la présence ou l'absence d'un polymorphisme de nucléotide simple dans le gène codant pour HSD11B1.
PCT/EP2003/006315 2002-06-17 2003-06-16 POLYMORPHISME DIAGNOSTIQUE DE LA 11ss-HYDROXYSTÉROÏDE DÉSHYDROGÉNASE UTILE POUR IDENTIFIER LE RISQUE DE DÉVELOPPEMENT DE LA MALADIE D'ALZHEIMER Ceased WO2003107008A2 (fr)

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AU2003246438A AU2003246438A1 (en) 2002-06-17 2003-06-16 DIAGNOSTIC POLYMORPHISM OF 11ss-HYDROXYSTEROID DEHYDROGENASE USEFUL FOR IDENTIFYING RISK OF DEVELOPING ALZHEIMER'S DISEASE
EP03759962A EP1514119A2 (fr) 2002-06-17 2003-06-16 Polymorphisme diagnostique de la 11 -hydroxyst ro de d shydrog nase utile pour identifier le risque de d veloppement de la maladie d'alzheimer

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