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WO2003104489A2 - Variante du mchr1 associee a l'obesite humaine - Google Patents

Variante du mchr1 associee a l'obesite humaine Download PDF

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Publication number
WO2003104489A2
WO2003104489A2 PCT/EP2003/005917 EP0305917W WO03104489A2 WO 2003104489 A2 WO2003104489 A2 WO 2003104489A2 EP 0305917 W EP0305917 W EP 0305917W WO 03104489 A2 WO03104489 A2 WO 03104489A2
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Prior art keywords
mchrl
polynucleotide
polypeptide
gene
allele
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WO2003104489A3 (fr
Inventor
Matthias Platzer
Cornelia Platzer
Thomas Gudermann
Johannes Hebebrand
Anke Hinney
Kathrin Reichwald
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Philipps Universitaet Marburg
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Philipps Universitaet Marburg
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Publication of WO2003104489A3 publication Critical patent/WO2003104489A3/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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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

Definitions

  • the present invention relates to a novel marker associated with human obesity, a variant of the melanin-concentrating hormone receptor (MCHRl) as well as polynucleotides encoding said polypeptide. Furthermore, the present invention relates to diagnostic and therapeutic uses based on the finding that this MCHRl variant is associated with human obesity.
  • MCHRl melanin-concentrating hormone receptor
  • the technical problem underlying the present invention is to provide means for diagnosis and therapy of obesity based on genetic factors.
  • MCH melanin-concentrating hormone
  • Figure 1 Nucleotide sequence and determined amino acid sequence of a region of SNP rs!33072 comprising an amino acid exchange
  • FIG. 2 MCHRl locus a, Exon structure and alternative mRNAs 1 and 2.
  • the previously known UTRs were extended by EST analysis .
  • the 3 ' end of exon 2 was elongated by 596 bp containing two canonical poly (A) signals used in 19 cDNAs.
  • Four cDNA clones extend the published 5' UTR of mRNA 1.
  • Rectangle exon, empty: UTR, filled: CDS; triangle: putative translation start methionine; angular line: splice junction, b, SNPs determined by sequencing of 8.2 kb in 19 individuals.
  • Asterisk newly detected, unmarked: previously reported in dbSNP; boxed: SNP leading to a non-conservative aa exchange.
  • FIG. 3 Functional expression of MCHRl variants in COS-7 cells
  • COS-7 cells were transiently transfected with cDNAs encoding Asp 32 -MCHR1 ( ⁇ ) or Asn 32 -MCHR1 (A) .
  • Cells were prelabeled with 2 ⁇ Ci/ l myo- 3 H-inositol for 18 h and subsequently stimulated with various concentrations of melanin-concentrating hormone (MCH) for 60 min in the presence of 10 mM LiCl.
  • MCH melanin-concentrating hormone
  • IP inositol phosphate
  • the present invention relates to a diagnostic composition containing a polynucleotide being selected from the group consisting of:
  • a polynucleotide capable of hybridizing to a MCHRl gene wherein said polynucleotide is having at a position corresponding to position +94 in Exon I of the MCHRl gene a substitution, deletion or 5 or 3" of said position an insertion of at least one nucleotide;
  • a polynucleotide capable of hybridizing to a MCHRl gene wherein said polynucleotide is having at a position corresponding to position +94 in Exon I of the MCHRl gene an A;
  • polypeptide comprises an amino acid substitution at a position corresponding to position +32 of the amino acid sequence of the MCHRl polypeptide, preferably the amino acid substitution is a non-conservative amino acid substitution;
  • the polynucleotides of the invention can be both DNA and RNA molecules. Suitable DNA molecules are, for example, genomic or cDNA molecules. The polynucleotides of the invention can be isolated from natural sources or can be synthesized according to known methods .
  • MCHRl gene relates to the MCHRl gene having a nucleotide sequence of a clone with gene bank accession numbers ABO63174 or Z86090 (corresponding to a human BAC containing MCHRl) .
  • the position numbers of the nucleic acid sequences are given relative to the ATG codon encoding the first Met of Exon 1 (with the A of ATG corresponding to position number +1 according to Shimomura et al . , 1999; see also Figure 2).
  • polynucleotides also include fragments and variants having at least 80%, preferably at least 90% or 95%, more preferably at least 96, 97, 98 or 99% homology thereto.
  • Polynucleotides used as a hybridization, probe can have, for example, basically or, preferably, exactly, the nucleotide sequence of the corresponding part of the MCHRl gene except the particular variation at position +94, or can be parts of the MCHRl sequence (fragments) .
  • the fragments used as hybridization probes can be synthetic fragments that were produced by means of conventional synthetic methods .
  • Polynucleotides of the present invention include such polynucloeitdes that allow for detection of a certain single nucleotide polymorphism.
  • Such polynucleotides comprise gene sequence surrounding the SNP, typically at least about 8, at least about 10, at least about 13, at least about 16 or at least about 20 nucleotides of wild-type sequence surrounding the SNP.
  • polynucleotides comprise a certain nucleotide that coincides with the position of the SNP (e.g., an A) at their 3' end, they will not be suitable for primer extension, e.g., amplification, of genomic DNA or cDNA that comprises a G in that same position.
  • polynucleotide when the SNP is located within the polynucleotide sequence, such polynucleotide will bind with less efficiency to a target sequence that comprises a different nucleotide (e.g., T) in the position of the SNP where the polynucleotide of the invention comprises e.g., an A.
  • T a different nucleotide
  • Such polynucleotides may therefore be used to assess the existence of certain SNPs in genomic or mRNA material of a patient.
  • compositions of the invention include such compositions that allow the detection of one or more of the the SNPs rsl33068; rsl33069; rsl33070 which are located in the 5' of the MCHRl gene.
  • a patient whose genomic material contains one or more of the SNPs rsl33068: G; rsl33069: A; rsl33070 G may be diagnosed as having increased risk of developing adipositas
  • a patient having one or more of the SNPs rsl33068: C; rsl33069: C; rsl33070: A may be diagnosed as having a lower risk of developing adipositas .
  • a patient whose risk of developing adipositats is estimated, based upon the aforementioned test, as high, may be a candidate for treatment with medication that targets the MCHRl " receptor, because in such patients, the promoter activity of the MCHRl receptor may be higher than in normal subjects.
  • the term “discharhybridizing” relates to hybridization under conventional hybridization conditions, preferably under stringent conditions as described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual 2 nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • a hybridizing polynucleotide can also be detected at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency), salt conditions, or temperature.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC) .
  • Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • the demandsfragments" of the polynucleotides mentioned above are understood to be parts of the polynucleotides that are long enough to allow specific hybridization. These fragments can be used, for example, as probes or primers in the diagnostic method described below and, preferably, are oligonucleotides having a length of at least 10, in particular of at least 13 and, particularly preferred, of at least 20 nucleotides .-
  • the polynucleotide is associated with obesity or a predisposition for said disease.
  • polynucleotides of the invention or parts of these polynucleotides can be introduced into vectors, e.g., plasmids, allowing ,e.g., expression, mutagenesis or a modification of a sequence by recombination of DNA sequences .
  • vectors e.g., plasmids
  • bases can be exchanged and natural or synthetic sequences can be added.
  • manipulations can be performed that provide suitable cleavage sites or that remove superfluous DNA or cleavage sites. If insertions, deletions or substitutions are possible, in vitro mutagenesis, primer repair, restriction or ligation can be performed.
  • analysis method usually sequence analysis, restriction analysis and other biochemical or molecular biological methods are used.
  • the present invention also relates to a diagnostic composition containing a vector comprising the polynucleotides of the invention.
  • the vectors are plasmids, cosmids, viruses, bacteriophages and other vectors usually used in the field of genetic engineering.
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in mammalian cells and baculovirus-derived vectors for expression in insect cells.
  • the polynucleotide of the invention or part thereof is operatively linked to the regulatory elements in the recombinant vector of the invention that guarantee the transcription and synthesis of an mRNA in prokryotic and/or eukaryotic cells that can be translated.
  • the nucleotide sequence to be transcribed can be operably linked to a promoter like a T7, metallothionein I or polyhedrin promoter.
  • the present invention also relates to recombinant host cells transiently or stably containing the polynucleotides (or fragments thereof) or vectors of the invention.
  • a host cell is understood to be an organism that is capable to take up in vi tro recombinant DNA and, if the case may be, to synthesize the polypeptides encoded by the polynucleotides of the invention.
  • these cells are prokaryotic or eukaryotic cells, for example mammalian cells, bacterial cells, insect cells or yeast cells.
  • the host cells of the invention are preferably characterized by the fact that the introduced nucleic acid molecule of the invention either is heterologous with regard to the transformed cell, i.e. that it does not naturally occur in these cells, or is localized at a place in the genome different from that of the corresponding naturally occurring sequence.
  • a further embodiment of the invention relates to a diagnostic composition containing a polypeptide or fragment thereof which is encoded by a polynucleotide of the invention.
  • a polypeptide is, e.g., useful as immunogen for raising antibodies useful for diagnostic purposes and has preferably a length of at least eight amino acids, more preferably of at least 15 amino acids and, particularly preferred, of at least 25 amino acids.
  • Such polypeptide can be produced, e.g., by cultivating a host cell of the invention under conditions allowing the synthesis of the polypeptide and the polypeptide is subsequently isolated from the cultivated cells and/or the culture medium.
  • Isolation and purification of the recombinantly produced polypeptide may be carried out by conventional means including preparative chromatography and affinity and immunological separations using or, e.g., can be substantially purified by the one-step method described in Smith and Johnson, Gene 67; 31-40 (1988) .
  • These polypeptides not only comprise recombinantly produced polypeptides but include isolated naturally occurring polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides or related polypeptides are well understood in the art .
  • These polypeptides are preferably in a substantially purified form.
  • the present invention also relates to an antibody that binds specifically to a polypeptide as defined above, i.e. to a polypeptide containing an amino acid variation associated with obesity or a predisposition for said disease.
  • said antibody specifically recognizes an epitope containing one or more amino acid substitutions, insertions or deletions resulting from a nucleotide variation as described above and, preferably, does not recognize an epitope without said variations, i.e. an epitope of an MCHRl polypeptide not associated with obesity.
  • the term tauantibody preferably, relates to antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specifities, as well as distinct monoclonal antibody preparations.
  • Monoclonal antibodies are made from an antigen containing fragments of the polypeptides of the invention by methods well known to those skilled in the art (see, e.g., K ⁇ hler et al . , Nature 256 (1975), 495).
  • the term is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab') 2 fragments) which are capable of specifically binding to protein.
  • Fab and f(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody.
  • antibodies of the present invention include chimerical, single chain, and humanized antibodies.
  • the polynucleotide, polypeptide or antibody of the present invention can be detectably labeled, for example, with a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, or an enzyme.
  • the present invention also provides a diagnostic composition containing one or more primers flanking the nucleic acid variation located within a polynucleotide of the present invention.
  • Suitable primers can be designed on the basis of the known nucleic acid sequence of the MCHRl gene according to well known methods. Determination of the presence of a variation in the nucleic acid sequence of the amplified product can also be carried out by well known methods, e.g., sequencing, hybridization, e.g., dot blot hybridization etc.
  • the invention also relates to a transgenic non-human animal such as transgenic mouse, rats, hamsters, dogs, monkeys, rabbits, pigs, C. elegans and fish such as torpedo fish comprising a polynucleotide or vector of the invention, preferably wherein said nucleic acid molecule or vector is stably integrated into the genome of said non-human animal, preferably such that the presence of the polynucleotide or vector leads to the expression of a MCHRl polypeptide variant associated with obesity.
  • Said animal may have one or several copies of the same or different polynucleotides encoding one or several forms of a MCHRl polypeptide.
  • the non-human mammal is preferably a laboratory animal such as a mouse or rat .
  • the transgenic non-human animal of the invention further comprises at least one inactivated wild type allele of the corresponding MCHRl gene.
  • This embodiment allows for example the study of the interaction of various mutant forms of MCHRl polypeptides on the onset of the clinical symptoms of the disease. All the applications that have been herein before discussed with regard to a transgenic animal also apply to animals carrying two, three or more transgenes. It might be also desirable to inactivate MCHRl protein expression or function at a certain stage of development and/or life of the transgenic animal.
  • tissue specific, developmental and/or cell regulated and/or inducible promoters which drive the expression of, e.g., an antisense or ribozyme directed against an MCHRl RNA transcript.
  • a suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. 89 USA (1992), 5547-5551) and Gossen et al . (Trends Biotech. 12 (1994), 58-62). Similar, the expression of a mutant MCHRl protein may be controlled by such regulatory elements.
  • the invention also relates to a transgenic mammalian cell which contains (preferably stably integrated into its genome) a polynucleotide according to the invention or part thereof, wherein the transcription and/or expression of the nucleic acid molecule or part thereof leads to reduction of the synthesis of a MCHRl protein.
  • the reduction is achieved by an anti-sense, sense, ribozyme, co-suppression and/or dominant mutant effect.
  • Antisense” and “antisense nucleotides” means DNA or RNA constructs which block the expression of the naturally occurring gene product. Methods for the production of a transgenic non-human animal of the present invention, preferably transgenic mouse, are well known to the person skilled in the art.
  • Such methods comprise the introduction of a polynucleotide or vector of the invention into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom.
  • the non-human animal may be a non-transgenic healthy animal, or may have a disorder, preferably a disorder caused by at least one mutation in the MCHRl protein.
  • Such transgenic animals are well suited for, e.g., pharmacological studies of drugs in connection with mutant forms of the above described MCHRl polypeptide. Production of transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press.
  • the DNA of the embryonal membranes of embryos can be analyzed using, e.g., Southern blots with an appropriate probe.
  • the present invention also relates to a method of diagnosing obesity related to the presence of a molecular variant of the MCHRl gene or a susceptibility to said disorder characterized in that in a sample taken from a subject (a) the presence of a nucleic acid variation as defined in claim 1 or (b) an aberrant, preferably reduced or eliminated, expression of the MHCRl gene or aberrant activity of the MCHRl protein, preferably a partial or complete loss of activity, is determined.
  • Obesity or a susceptibility to obesity can be diagnosed based on the activity or amount of expression of the MCHRl polypeptide.
  • the MCHRl variant polypeptide as defined above or the corresponding DNA or mRNA, e.g. in biological fluids or tissues, may be detected directly in situ, e.g. by in situ hybridization (e.g., according to the examples, below) or it may be isolated from other cell components by common methods known to those skilled in the art before contacting with a probe. Detection methods include Northern Blot analysis, RNase protection, in situ methods, e.g.
  • the probe e.g. a specific antibody or specific oligonucleotide
  • said method uses an antibody specifically binding to the MCHRl variant associated with obesity and allows said diagnosis, e.g., by ELISA and the antibody is bound to a solid support, for example, a polystyrene microtiter dish or nitrocellulose paper, using techniques known in the art.
  • said method is based on a RIA and said antibody is marked with a radioactive isotope.
  • the antibody is labeled.
  • Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I) , carbon ( 14 C) , sulfur ( 35 S) , tritium ( 3 H) , indium ( 112 In) , and technetium rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I) , carbon ( 14 C) , sulfur ( 35 S) , tritium ( 3 H) , indium ( 112 In) , and technetium rhodamine, and biotin.
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
  • the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of "mTc.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific MCHRl protein.
  • In vivo tumor imaging is described in S. . Burchiel et al., StammImmunopharmacokinetics of Radiolabeled Antibodies and Their Fragments". (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B.A. Rhodes, eds., Masson Publishing Inc. (1982)).
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a functional MCHRl protein or a polynucleotide encoding said MCHRl protein and a pharmaceutically acceptable excipient, diluent or carrier. Since it has been found that variation rsl33072 leads to a partial loss of function of the MCHRl protein (see Example 3, below) it can be expected that means allowing to restore the function are therapeutically useful.
  • Suitable pharmaceutical carriers etc. are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular, topical or intradermal administration. The route of administration, of course, depends, e.g., on the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors.
  • dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind and stage of obesity, general health and other drugs being administered concurrently.
  • the delivery of the polynucleotides of the invention can be achieved by direct application or, preferably, by using a recombinant expression vector such as a chimeric virus containing these compounds or a colloidal dispersion system.
  • the colloidal dispersion systems which can be used for delivery of the polynucleotides include macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions (mixed) , micelles, liposomes and lipoplexes,
  • the preferred colloidal system is a liposome.
  • the composition of the liposome is usually a combination of phospholipids and steroids, especially cholesterol.
  • Liposomes which are suitable for the delivery of the desired nucleic acid molecule.
  • Organ-specific or cell-specific liposomes can also be used.
  • the targeting of liposomes can be carried out by the person skilled in the art by applying commonly known methods .
  • This targeting includes passive targeting (utilizing the natural tendency of the liposomes to distribute to cells of the RES in organs which contain sinusoidal capillaries) or active targeting (for example by coupling the liposome to a specific ligand, e.g., an antibody, a receptor, sugar, glycolipid, protein etc., by well known methods) .
  • monoclonal antibodies are preferably used to target liposomes to specific tissues via specific cell-surface ligands.
  • Preferred recombinant vectors useful for gene therapy are viral vectors, e.g. adenovirus, herpes virus, vaccinia, or, more preferably, an RNA virus such as a retrovirus.
  • the retroviral vector is a derivative of a murine or avian retrovirus .
  • retroviral vectors which can be used in the present invention are: Moloney murine leukemia virus (MoMuLV) , Harvey murine sarcoma virus (HaMuSV) , murine mammary tumor virus (MuMTV) and Rous sarcoma virus (RSV) .
  • a non-human primate retroviral vector is employed, such as the gibbon ape leukemia virus (GaLV) , providing a broader host range compared to murine vectors .
  • GaLV gibbon ape leukemia virus
  • Such assistance can be provided, e.g., by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. Suitable helper cell lines are well known to those skilled in the art.
  • Said vectors can additionally contain a gene encoding a selectable marker so that the transduced cells can be identified.
  • the retroviral vectors can be modified in such a way that they become target specific. This can be achieved, e.g., by inserting a polynucleotide encoding a sugar, a glycolipid, or a protein, preferably an antibody.
  • a polynucleotide encoding a sugar, a glycolipid, or a protein, preferably an antibody.
  • Those skilled in the art know additional methods for generating target specific vectors.
  • Further suitable vectors and methods for in vitro- or in vivo-gene therapy are described in the literature and are known to the persons skilled in the art; see, e.g., WO 94/29469 or WO 97/00957.
  • the functional MCHRl encoding nucleic acid sequences can be linked to a tissue specific promoter and used for gene therapy.
  • tissue specific promoters are well known to those skilled in the art (see e.g. Zimmermann et al., (1994) Neuron 12, 11-24; Vidal et al.; (1990) EMBO J. 9, 833-840; Mayford et al . , (1995), Cell 81, 891- 904; Pinkert et al . , (1987) Genes & Dev.. 1, 268-76).
  • the present invention also relates to the use of the above compounds of the invention for the preparation of a pharmaceutical composition for prophylaxis or treatment of obesity related to the presence of a molecular variant of the MCHRl gene.
  • a genomic region of 8.2 kb was covered by five overlapping PCR products . Appropriate numbers of nested PCRs were performed for each region. PCR products were sequenced using the same primers as for PCRs and BigDye Terminator Cycle Sequencing v2.0 kit (Applied Biosystems, Rothstadt, Germany) . Reactions were electrophoresed on ABI 377 automated sequencers.
  • ESTs were downloaded from dbEST (GenBank release date 01/12, version 127.0) and Unigene cluster Hs.248122 (http://www.ncbi.nlm.nih.gov/UniGene). Human brain cDNA library was obtained from Clontech Laboratories, Heidelberg, Germany. Nested and seminested PCR as well as sequencing of PCR products was performed as described above.
  • Enzyme Faul (2U/ ⁇ l) 0.30 ⁇ l (0.6 U) Buffer: SEB 3.00 ⁇ l (1 x) bidest 11.7 ⁇ l PCR: 15 ⁇ l
  • a alleles and C alleles are to be amplified separately.
  • T-Allele 415 bp
  • C-Allele 135 bp, 280 bp
  • Digestion-Mix Enzyme : NLAIII (lOU/ ⁇ l) 0.2 ⁇ l (2 U) Buffer: Puffer 5 3.00 ⁇ l (1 x) BSA: 0 . 35 ⁇ l bidest: 11.45 ⁇ l
  • TDT transmission disequilibrium tests
  • the genotype relative risks to develop obesity BMI > 90 th percentile
  • the frequency of the risk A- allele and the attributable risk were estimated from the trios by unconditional maximum likelihood estimation as described by Schaid and Sommer, Am. J. Hum. Genet . 53, 1114-1126 (1993). These estimates are valid under the assumption of random mating and Hardy-Weinberg equilibrium. Since these assumptions may not be valid for extremely obese individuals (Hebebrand et al . , Int . J. Obes . Relat . Metab. Disord. 24, 345-353 (2000), the maximum likelihood genotype relative risk estimates conditional upon the parental genotypes (CPG) were also calculated (Schaid and Sommer, 1993) . Standard errors were obtained from the inverse of Fisher's information matrix and approximate normal theory confidence intervals were calculated for all estimates.
  • Example 2 Major contribution of a common non-conservative cSNP in the MCHRl to human obesity
  • the alignment of the mRNAs to the genomic sequence indicates the expression of two alternative human transcripts (Fig. 2a) .
  • the first mRNA is composed of two exons encompassing a coding sequence (CDS) for 422 amino acids (aa) (Shimomura et al . , Biochemical and Biophysical Research Communications 261 (3) (1999), 622-626).
  • CDS coding sequence
  • a shorter CDS starting at codon 70 (Met 70 ) downstream of the first methionine codon (Met 1 ) has also been reported (Lakaye et al., Biochimica et Biophysica Acta 1401 (2) (1998), 216-220).
  • the second mRNA was deduced from genomic DNA and consists only of exon 2 of mRNA 1 but elongated upstream by 452 bp (Kolakowski et al., FEBS letters 398(2-3) (1996), 253- 258) .
  • Table 2 TDTs for initial SNP rsl33073 (a), adjacent SNPs (b) and haplotypes including SNP rs133072 (c)
  • SNPs are arranged from 5 'to 3 'of MCHRl; * initial SNP; ** cSNP (Asp32Asn) ; ⁇ more frequently transmitted allele for all single SNP and haplotype based TDTs , # numbers : transmitted/non-transmitted, transmission rate (%) , two sided p-value
  • rsl33073 As no functional significance of rsl33073 is evident, it has been sequenced MCHRl as well as 4.6 kb of its upstream region in nine obese children and adolescents who were homozygous for C at rsl33073 and who had contributed to the positive TDT, as well as in ten obese children and adolescents homozygous for T . These two genotype based groups should be most divergent for functionally relevant SNPs . 18 SNPs were identified (Table 3 ) , 12 of which contribute to two ancestral haplotypes .
  • Table 3 18 SNP's identified by sequencing comprising 8 novel SNPs. 10 SNPSs from databases could be confirmed
  • the genotype relative risk to develop obesity (defined by BMI > 90 th percentile) was calculated as 1.51 (95 % CI 1.17 - 1.86) and 1.95 (95 % CI 1.22 - 2.68) for heterozygous and homozygous carriers of the A-allele, respectively.
  • the attributable risk estimate is 23.7 % (95 % CI 13.1 % - 34.3 %) .
  • MCHRl is the only obvious candidate gene for obesity on chromosome 22ql3. So far, linkage of obesity has not been detected in this chromosomal region. This is likely due to the fact that for a relative risk of the respective allele in the magnitude of 1.5 several thousands of sib-pairs would be required (Rich et al . , Science 273 (1996), 1516-1517). It is concluded that in accordance with criteria for a solid association study in common disease association for a highly plausible candidate gene in population-based and family-based studies has been detected. This has been confirmed in independent and large study groups.
  • Example 3 Inositol phosphate accumulation by MCHRl upon challenge with MCH Asp 32 -MCHRl/Asn 32 -MCHRl: the A-allele of rsl33072 is associated with loss of function of the MCHRl protein
  • the MCHRl couples to G q/ u and G ⁇ 0 proteins to activate several intracellular signalling pathways (Hawes et al . , Endocrinology 14 (2000), 4524-4532).
  • Particular aspects of the complex phenotype of the rodent Mc.hr ⁇ _ Marsh et al . , Proc. Natl.
  • MCHRl is expressed peripherally and in hypothalamic, nuclei and hypocampal regions, which are involved in olfaction and regulation of feeding behaviour and body weight (Saito et al., Nature 400 (1999), 265-269; Chambers et al., Nature 400 (1999), 261-265). Because hyperphagia occurred only in regular chow fed MCHR _/" mice, but not upon maitenance on a high fat diet, it was found that the hyperphagia possibly represents altered nutrient or taste preferences.
  • BMI body mass index
  • SSCP was performed as described previously (Hinney et al. 1999). PCR products of primers corresponding to amplicon 1 of exon 2 (primers Table 11) were digested by both Alul and Mspl (Fermentas). For analysis of the second amplicon of exon 2 Crfl3I (Fermentas) was used. All amplicons were electrophoresed on 21% acrylamide gels (37.5:1, Q Biogene). Gels were run at room temperature for 17 h at 400 V and additionally at 4°C for 18 h at 500 V. Gels were silver stained.
  • PCR products of individuals showing aberrant SSCP patterns were sequenced bi- directionally on a LiCor 4200-2 automatic sequencer using the Base ImaglR 4.0 software (MWG Biotech) as described previously (Hinney et al. 1999) and by Sequence Laboratories (Seq Lab, Gottingen, Germany), respectively.
  • TDT The initial, confirmatory and post hoc transmission disequilibrium tests (TDT) in the German and American families were carried out using the program Genehunter, version 2.0 beta (Kruglyak et al. 1996). The program performs haplotype TDTs with up to four markers for all 525 trios (study group 1), where phase can be determined unambiguously.
  • Model free linkage analysis was performed using the maximum likelihood binomial (MLB) statistics (Abel and Muller-Myshok 1998) as implemented in Mlbgh, Version 1.0. Identification of haplotypes and additional transmission disequilibrium tests
  • a genomic region of 13,378 bp was covered by 8 overlapping PCR products referred to as region A-H, respectively (primers Table 12). Four to eight nested/seminested PCRs were performed in each region. PCR products were sequenced using PCR primers and BigDye Terminator Cycle Sequencing v2.0 kit (Applied Biosystems). Reactions were electrophoresed on ABI 377 automated sequencers. Base calling was performed using phred (Ewing and Green 1998; Ewing et al. 1998). Sequence assembly was done using phrap (http://www.phrap.org/phrap.docs/phrap.html). Trace files were inspected visually in gap4.5.
  • A4f CACTCCTACCATTCAAAGGC
  • A4r AGGCTCTGTGGATACAGTGG
  • A5f GCCATGATTTTGTGAGTCCC
  • A5r CTGGATGTGCTTACTTAGGG
  • A6f ACTATGGCATGTGGAAGAGG
  • A6r GGGAGAAGTCATGCCTGAAG
  • A7f CCCTAAGTAAGCACATCCAGTC A7r: GGAAGTAGTGGTTAGCAGTTGG
  • A8f CTCCCAGGTTCAAACGATTC
  • A8f2 AGTCTCACTCTGTCGCAC
  • A8r GGCTAACACGGTGAAACCTC
  • B Blf GCCTGGCTAAAACTTCTGAC
  • B2f GTGCAGTGGTGTGATCTCGG
  • Blr GCATTGCCCTCAACAATGAC
  • B2f2 AGGTAGCTGAGGCATGAG
  • B3f GTCTCACTCTGTCGCTCAGG
  • B3r ATGGAGTTGTGGGGGAGGTC
  • B4f CCCTGACTCACTCTGTTCCC
  • B4 ⁇ TTCTGCCAGGATCTGCCCAC
  • B5f AGGACTATGGAGTTAGCTGG
  • B5 ⁇ CTTTCTTGCACCTCTGCAAC
  • B5f2 AGTCTCCCTCTGTCACCC
  • B6f CCAAAGCAACAATGAGAGACCC
  • B6r GACCCACCTTGTCATGTACC
  • B6r2 GTCCCTTGTACTTGCGAG
  • C5f GTAGTGAAGACACTGGTGGG C5r: AGCTACTTGGGAGTCTGAGG C5r2: CACTCCCACACATAGTCC
  • Dlf GGGACTATGTGTGGGAGTGG
  • D2f GTCACCTATGCTGAAGTGCC
  • Dlr CCTGGGTGACAGAACGAG
  • D2r TAGGCACAAGCCACAACG
  • D3f CAGAAGTGGATCTTGAGGG
  • D3r TCCAGTGATCCACCAGCC
  • D4f AATCTCACCACTGCACCC
  • D4r GCATGTTAGGACTGGCTC
  • D5f GGCAACATAGTGAGACTCC
  • D5r TGCCATTACCCTCCAGCC
  • D6f GCCAGTCCTAACATGCTTC
  • D6r GTTTGGTGGGAGGTGATTGG
  • E3f GAGGCAAGAGTCACCACACC
  • E4f AGTAGCTGGGATTACAGG
  • E3r TGAGGTCCCTGAGGTTTGGC
  • E4r GGAAATTGTCAGTGCAGAGG
  • E6f CTCACTGCAATTTCCACCTC E6r: CATCCTGGCTAACATGGTG
  • E7f CTCTCTTGGTATTCCTGG
  • E7r GAAGTGAGCTGAGATTGG
  • E9f GCTGGTCTTGAACTCCTG
  • E9r TTACTACCTGCCAAGCAC
  • C4f AGCGAGATAGTTGGAAGCCG
  • C4r TAACCTCTTCAGTCCCAGGC
  • C5f GGAGATCCCTTTCCTGATGG
  • C5r CCCAAGCAGTTTGGCTCAGG
  • Glf CACAGTTTCCTCTGGACTC
  • G2f GCTAAGCCAAGCTGCTCTC
  • Glr AACTCGTCAGCATAGCCAG
  • G2r TGCTCCCACAACCGAGCTG
  • G3f CCAGGCTACGGAGGAAGAC
  • G3r CCTCAGAGCAAAGCAGACC
  • G4f CAGGTGAGTTGACTGGGAG
  • G4r GTGGCAGTAAGGATGTCTC
  • G5f GTTCCAAAGATGCTTGGCAG
  • G5r CTATGTGTAGCCCTGAGTG
  • H Hlf CTCTACAAGACAGTCACCC H2f: CAGTCACCCACAGATATGC Hlr: TTCCACAACCAAGTGACCC H2r: GAGAACTGACATCCTGCTG
  • H3f ATGGCTCAGGGCACTCTGG
  • H3r CATTGCCCATGAGCTGGTG
  • H4f ACTCCACGGTCATCTTCGC H4r: AGGTCAGTGTCTGGGTTGG
  • H5f TGTATGCCAGACTCATCCC
  • H5r TTTGCGGAACGTCTCACAG
  • H6f ACCCAGTTGTCCATCAGCC
  • H6r CAAAGGTCTCATCCTGCTC
  • H7f GACCGCTCGGGAAATGCAG H7r: CCATCGCACCAGTGAGAGGC
  • H8f GTGGAAGGGTACTGACTGG H8r: GTGACTGAGCAAATGTGCC
  • H8f GTGGAAGGGTACTGACTGG H9r: CAGAGTGATGTGGGTGGAG
  • Forward outer primer 68 65 406 (of two GAAGGGAGTGGGGAGGGCAGTT outer primers)
  • Reverse outer primer 68 65 GCCCCTCAGAGCAAAGCAGACC rsl33069 99132 Forward inner primer (A-allele) 74 60 115 (A-allele) C/A GGACTTTATCCCCCACCCCACCCT
  • upstream of the putative translation start Met 1 were PCR-amplified with primers introducing Ncol and Xhol restriction sites, respectively (primers Table 16). Fragments were first cloned into vector pCR2.1-TOPO (Invitrogen) and then directionally cloned into pGL3-Basic Luciferase reporter vector (Promega). For transfection, reporter gene plasmid-DNA was purified with EndoFree® Plasmid Maxi Kit (Qiagen). PC12 rat pheochromocytoma cells were purchased from 'Deutsche Sammlung von Mikroorganismen und Zellkulturen' (Braunschweig, Germany) and maintained in RPMI 1640 with 10% horse serum and 5% FCS (Biochrom, Germany).
  • Transfections were performed by electroporation using 10 ⁇ g reporter gene plasmid and 3 x 10 6 cells/0.15 ml in 0.4 cm electroporation cuvettes with a gene pulser (Gene zapper 450/2500, IBI, Cambridge, England) at 1200 ⁇ FD, 100 Ohm and 300 V. After electroporation 1 x 10 6 cells/ml/well were cultured in the absence or presence of 500 ⁇ M dibutyryl-cAMP (dbcAMP, Sigma) for 24 h, harvested and assayed for luciferase expression using the Promega Luciferase Assay and the Autolumat LB 953 (Berthold, Bad Wildbad, Germany).
  • dbcAMP dibutyryl-cAMP
  • PCR 1-F GGAGATCCCTTTCCTGATGG PCR 1-R: GCCTCTCACTGGTGCGATGG PCR 1-F: TGCAGGCATTCAGAAGTGG PCR 1-R: GAGCAGGATGAGACATTTG
  • Table 4 Variations, SNPs rs 133072 and rs 133073 in the MCHRl in 215 extremely obese children and adolescents and 230 healthy underweight students, respectively
  • SNPs rsl33072 (100213 G>A) and rsl33073 (100365 T>C) are shown in shaded boxes.
  • ED extracellular domain
  • N-ter N-terminal
  • TM transmembrane
  • IL intracellular loop
  • EL extracellular loop
  • C-ter C-terminal
  • Comparison of allele and genotype frequencies of both SNPs rs 133072 and rs 133073 revealed initial association (all two-sided p-values ⁇ 0.04), which was independently confirmed (all onesided p-values ⁇ 0.03); the final comparison based on all 719 obese (study groups 1 and 4) and 326 non-obese subjects (study groups 2 and 3) substantiated association of the A-allele of rsl33072 and of the C-allele of rsl33073 with obesity (all two sided p-values ⁇ 0.008; Table 5). Both SNPs are in tight linkage disequilibrium (p ⁇ 0.001; see below).
  • TDT transmission disequilibrium test
  • 525 obese children subgroup of study group 1
  • their 1,050 parents were performed after both an initial and a confirmatory positive TDT as based on subgroups of 108 and 417 trios, respectively.
  • Preferential transmissions of the alleles that were shown to be associated with obesity were observed (two-sided p-values ⁇ 0.0003; Table 5).
  • the transmission rates for the A-allele and the C-allele in the total trio sample were 58.5 % and 58.3 %, respectively.
  • Table 5 Association and transmission disequilibrium of SNPs rs 133072 and rs 133073 in obesity rs 133072 (G/A) rs 133073 (T/C)
  • BMI > 90 th centile includes 620 (359 females) unrelated obese children and adolescents (mean BMI 33.4 ⁇ 6.6 kg/m 2 ; mean age 14.0 ⁇ years) and 99 (51 females) obese adults (mean BMI 29.1 ⁇ 3.4 kg/m 2 ; mean age 25.3 ⁇ 3.7 years) . includes 96 (49 females) normal weight (BMI > 40 th and ⁇ 60 th percentile; mean BMI 21.9 ⁇ 1.1 kg/m 2 ; mean age 24.7 ⁇ 2.6 years) and 2
  • SNPs were identified (Table 7), 12 of which had been reported previously in dbSNP (rs68708, rsl33062, rs2032512, rsl33063, rsl33064, rsl33068, rsl33069, rsl33070, rsl33072, rsl33073, rsl33074, rs3087592).
  • the ten newly detected SNPs were submitted to HGV (A- 91268-C, A-91276-G, SNP001745613, SNP001745614, SNP001745615, SNP001745616, SNP001745617, SNP001745618, SNP001745619, SNP001745620).
  • Table 7 Identified SNPs by SSCP and genomic sequencing of 13.378 bp the MCHRl locus identified position name ID ofthe
  • Table 8 Genotypes of 22 SNPs in 9 and 10 obese children and adolescents homozygous for C or T of rsl33073, respectively
  • Table 9a TDTs for 16 adjacent SNPs in 61 and 525 trios, respectively and for haplotypes including cSNPs rsl33072 and rsl33073
  • SNPs are arranged from 5' to 3'of MCHRl; * cSNP rsl33073; ** cSNP rsl33072 (Asp32Asn); 8 more frequently transmitted allele for all single SNP and haplotype based TDTs; # numbers: transmitted/non-transmitted, transmission rate (%), two-sided p-value.
  • haplotype frequencies were estimated for the four SNPs rsl33068, rsl33072, rsl33073 and rsl33074, which were genotyped in all 525 trios.
  • P-values for testing association between SNPs were below 10 '5 for all combinations of two SNPs and the combination of all four SNPs demonstrating strong linkage disequilibrium between these markers.
  • the expected and observed frequencies of the haplotypes based on all four SNPs are illustrated in Table 10.
  • Haplotype 1 (rsl33068: G; rsl33072: A; rsl33073: C; rsl33074: T) was transmitted in 61.4 %; gender dependent analyses revealed a higher transmission to males (67.7%) than females (56.6%; Table 9b).
  • Table 10 Haplotypes formed by four SNPs in 525 trios (1050 parents)
  • Table 9b TDTs for haplotypes 525 trios including cSNPs rsl33072 and rsl33073, for both genders and males and females, respectively
  • Chaffer CL Morris MJ. The feeding response to melanin-concentrating hormone is attenuated by antagonism of the NPY Y(l)-receptor in the rat. Endocrinology. 2002 Jan;143(l):191-7.
  • Tritos NA Maratos-Flier E. Two important systems in energy homeostasis: melanocortins and melanin-concentrating hormone. Neuropeptides. 1999 Oct;33(5):339-49. Review.

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Abstract

La présente invention concerne un nouveau marqueur associé à l'obésité humaine, une variante du récepteur MCHR1 (Melanin-Concentrating Hormone Receptor), ainsi que des polynucléotides codant ce nucléotide. L'invention concerne également des utilisations en diagnostic et en thérapie reposant sur le fait que cette variante du MCHR1 est associée à l'obésité humaine.
PCT/EP2003/005917 2002-06-05 2003-06-05 Variante du mchr1 associee a l'obesite humaine Ceased WO2003104489A2 (fr)

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* Cited by examiner, † Cited by third party
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EP2305837A1 (fr) * 2005-05-11 2011-04-06 Aarhus Universitet Procédé de diagnostic et traitement d'une maladie mentale

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WO2000075166A1 (fr) * 1999-06-08 2000-12-14 The Regents Of The University Of California Recepteur de l'hormone concentratrice de la melanine
EP1288310A1 (fr) * 2001-08-31 2003-03-05 Aventis Pharma Deutschland GmbH Utilisation de polymorphismes du nucléotide simple (SNP's) du récepteur (MCH-R) d'hormone concentrant la mélanine pour l'identification d'affections génétiques associées au maintien du poids du corps

Cited By (1)

* Cited by examiner, † Cited by third party
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EP2305837A1 (fr) * 2005-05-11 2011-04-06 Aarhus Universitet Procédé de diagnostic et traitement d'une maladie mentale

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