WO2003104489A2 - Mchr1 variant associated with human obesity - Google Patents

Abstract

Described is a novel marker associated with human obesity, a variant of the melanin-concentrating hormone receptor (MCHR1) as well as polynucleotides encoding said polypeptide. Furthermore, diagnostic and therapeutic uses are described which are based on the finding that this MCHR1 variant is associated with human obesity.

Description

MCHRl variant associated with human obesity

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.

Overweight and obesity affect approximately one half of the German population. Obesity is associated with type 2 diabetes mellitus, cardiovascular disorders and other disorders which impose a substantial socio-economic burden. Both environmental and genetic factors are involved in the etiology of obesity. Currently, only a limited number of gene mutations have been implicated in the predisposition to obesity. A molecular genetic diagnostic evaluation will allow the subdifferentiation of different types of obesity which in turn could be associated with different risk profiles for associated disorders. Moreover, identification of gene variants will enable the determination of the relative risk of the individual to develop overweight, obesity or extreme obesity.

Thus, the technical problem underlying the present invention is to provide means for diagnosis and therapy of obesity based on genetic factors.

The solution to said technical problem . is achieved by providing the embodiments characterized in the claims.

The melanin-concentrating hormone (MCH) system is involved in regulation of energy intake and expenditure. In rodents, both intracerebroventricular MCH injection and overexpression of the pro-MCH gene ( Pmch) result in increased food intake and body weight. Fasting induces an upregulation of orexigenic MCH and of the MCH receptor 1 (MCHRl) , one of the two G protein-coupled receptors that bind MCH. MCHRl (also known as GPR24 and SLC1 ) is expressed peripherally and in hypothalamic nuclei and hippocampal regions, which are involved in olfaction and regulation of feeding behavior and body weight. Both Pmch^'/~ and Mchrl^{' '} mice are lean, have a reduced fat mass and an increased metabolic rate. However, whereas Pmch^~/' mice are hypophagic and display normal locomotor activity, Mchrl^~/~ mice are hyperactive and hyperphagic. Association studies with 719 obese individuals and non-obese individuals reveal that obesity is associated with SNP rsl33072 (G/A; Asp-32-Asn) in the MCHRl gene. The transmission disequilibrium test (n=525 trios) revealed preferential transmission of the A-allele (58.5%; p=0.00016) to obese index patients. In vi tro, Asn³²-MCHR1 shows partial loss- of-function. Accordingly, this SNP provides a diagnostic marker for heritable obesity.

Brief description of the drawings

Figure 1 : Nucleotide sequence and determined amino acid sequence of a region of SNP rs!33072 comprising an amino acid exchange

(a) Exon 1 (only translated part with nucleotide and amino acid)

(b) The underlined base and amino acid residue, respectively, indicate the exchanges and correspond to position +94 (bp) and +32 (aa) , respectively, of the MHCRl gene.

Figure 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. Thus, we defined a revised MCHR 1 gene structure with a first exon of 502 bp and a second exon of 1867 bp. 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.

Figure 3 : Functional expression of MCHRl variants in COS-7 cells COS-7 cells were transiently transfected with cDNAs encoding Asp³²-MCHR1 (■) or Asn³²-MCHR1 (A) . Cells were prelabeled with 2 μCi/ l myo-³H-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. After lysis of cells with 0.1 M NaOH and subsequent neutralization of samples with 0.2 M formic acid, inositol phosphate (IP) accumulation was determined by anion exchange chromatography. Half maximal effective agonist concentrations (EC₅0) are indicated. Data are presented as mean ± SEM of three independent experiments, each performed in triplicate.

Thus, the present invention relates to a diagnostic composition containing a polynucleotide being selected from the group consisting of:

(a) a polynucleotide comprising the nucleic acid sequence shown in Figure 1 (b) ;

(b) a polynucleotide encoding a polypeptide comprising the amino acid sequence shown in Figure 1 (b) ;

(c) a polynucleotide having the nucleic acid sequence shown in Figure 1 (a) ;

(d) a polynucleotide encoding a polypeptide having the amino acid sequence shown in Figure 1 (a) ;

(e) 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; (f) 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;

(g) a polynucleotide encoding an MHRCl polypeptide or fragment thereof, wherein said 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; and

(h) a polynucleotide encoding an MHRCl polypeptide or fragment thereof, wherein said polypeptide comprises an amino acid substitution of Asp to Asn at a position corresponding to position +32 of the amino acid sequence of the MCHRl polypeptide.

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 .

The term "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). The position numbers of the amino acid sequences are given relative to the first Met of exon 1 (= position +1) .

The above mentioned 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. For example, where such 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. Similarly, 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. Such polynucleotides may therefore be used to assess the existence of certain SNPs in genomic or mRNA material of a patient.

Particular preferred 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. In particular, 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, whereas 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.

As used herein, the term „hybridizing" 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. However, in certain cases, 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. For example, lower stringency conditions include an overnight incubation at 37°C in a solution comprising 6X SSPE (20X SSPE = 3M NaCl; 9.2M NaH₂P0₄; 0.02M EDTA, pH7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA, following by washes at 50°C with 1 X SSPE, 0.1% SDS. In addition, to achieve even lower stringency, 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 „fragments" 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 .-

In a preferred embodiment of the diagnostic composition of the present invention the polynucleotide is associated with obesity or a predisposition for said disease.

For the manipulation in prokaryotic cells by means of genetic engineering or for purposes of gene therapy the 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 . By means of conventional methods (cf. Sambrook et al . , supra) bases can be exchanged and natural or synthetic sequences can be added. In order to link the DNA fragments with each other adapters or linkers can be added to the fragments. Furthermore, 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. As analysis method usually sequence analysis, restriction analysis and other biochemical or molecular biological methods are used.

Thus, the present invention also relates to a diagnostic composition containing a vector comprising the polynucleotides of the invention. Preferably, 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. Preferably, 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. Preferably, 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. Such 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, however, 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. Preferably, 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 „antibody", 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., Kδhler et al . , Nature 256 (1975), 495). As used herein, the term „antibody" (Ab) or „monoclonal antibody" (Mab) 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. (Wahl et al . , J. Nucl. Med. 24: 316-325 (1983)). Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimerical, single chain, and humanized antibodies.

For certain purposes, e.g. diagnostic methods, 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. This animal has numerous utilities, including as a research model for studying the effects of an altered metabolism and therefore, presents a novel and valuable animal in the development of therapies, treatment, etc. for obesity caused by particular MCHRl variants. Accordingly, in this instance, the non-human mammal is preferably a laboratory animal such as a mouse or rat .

Preferably, 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. This can be achieved by using, for example, 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.

Furthermore, 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. In a preferred embodiment, 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, e.g., 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.

Finally, 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. in situ hybridization, in vitro amplification methods (PCR, LCR, QRNA replicase or RNA- transcription/amplification (TAS, 3SR),- reverse dot blot disclosed in EP-B1 0 237 362) ) , immunoassays, Western Blot and other detection assays that are known to those skilled in the art.

The probe (e.g. a specific antibody or specific oligonucleotide) to be used in the diagnostic method can be detectably labeled. In a preferred embodiment, 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. Alternatively, said method is based on a RIA and said antibody is marked with a radioactive isotope. In a preferred embodiment of the diagnostic method of the invention 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 (¹²⁵I, ¹²¹I) , carbon (¹⁴C) , sulfur (³⁵S) , tritium (³H) , indium (¹¹²In) , and technetium rhodamine, and biotin. In addition to assaying MCHRl variant protein levels in a biological sample, the protein can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X- radiography, NMR or ESR. For X-radiography, 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. A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, ¹³¹I, ¹¹²In, ⁹⁹mTc) , a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal . It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, 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., „Immunopharmacokinetics 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)).

Finally, the present invention also relates to 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.

Examples of 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. As is well known in the medical arts, 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. The skilled person is in a position to select such 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) . In the present invention 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. Even more preferably, the retroviral vector is a derivative of a murine or avian retrovirus . Examples of such 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) . Most preferably, 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 . Since recombinant retroviruses are defective, assistance is required in order to produce infectious particles. 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. Moreover, 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. 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.

In order to achieve expression only in a desired target organ, the functional MCHRl encoding nucleic acid sequences can be linked to a tissue specific promoter and used for gene therapy. Such 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.

The following examples illustrate the invention.

Example 1: General methods

(Λ) Study subjects

A total of four study groups were genotyped:

1) 620 (359 females) extremely obese children and adolescents ascertained via a body mass index (BMI) > 90^th percentile (Hebebrand et al., Int. J. of eating disorders, 19 (4) (1996), 359-369) (mean BMI 33.4 ± 6.6 kg/m²; mean BMI percentile 99.3; mean age 14.0 ± 2.7 years). For the initial mutation screen we chose a subgroup of 215 obese children and adolescents. For transmission disequilibrium tests (TDT) (Spielmann et al., Americ. J. of human genetics, 52(3) (1993), 506-516) both parents of 525 of these children and adolescents were genotyped for four SNPs, TDTs for these and 9 other SNPs were also based on a subgroup of 61 trios (Table 2) . The respective index patients included the 9 patients homozygous for the C- allele of rsl33073, whose DNA was sequenced.

2) 230 (110 females) healthy underweight students (BMI < 15^th percentile; mean BMI 18.3 ± 1.1 kg/m²; mean age 25.2 ± 3.7 years) .

3) 96 (49 females) healthy normal weight students (BMI > 40^th and < 60^th percentile; mean BMI 21.9 ± 1.1 kg/m²; mean age 24.7 ± 2.6 years) . 4) 99 (51 females) healthy overweight students (BMI > 90^th percentile; mean BMI 29.1 ± 3.4 kg/m²; mean age 25.3 ± 3.7 years) .

Written informed consent was given by all participants and in the case of minors, their parents. The Ethics Committee of the

University of Marburg approved this study.

(B) Single strand conformation polymorphism analysis (SSCP) SSCP was performed as described previously (Hinney et al., J. of Clin. Endocrinol. and Metabolism, 84(4) (1999), 1483-1486). PCR products of individuals showing specific aberrant SSCP patterns were sequenced bi-directionally as described previously (Hinney et al. , 1999) .

(C) Sequence analysis of MCHRl locus

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, Weiterstadt, Germany) . Reactions were electrophoresed on ABI 377 automated sequencers.

(D) Expression analyses of MCHRl transcripts

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.

(E) Genotyping of SNPs

All SNPs (Fig. 2) identified by sequencing, were genotyped by restriction fragment length polymorphism analyses (RFLP) or tetra-primer amplification refractory mutation system (ARMS) . (F) Primers and PCR protocols (PCR-RFLP and ARMS-PCR)

SNP rsl33062 (G/A)

Primer Forward SNP rsl33062-F ACT TCA CCC ATG AGG ACC AG Primer Reverse SNP rsl33062-R TGC ATC ATC TAG CAC CCT CA Product-size: 393 bp

1. PCR:

Thermocycler :

Denaturation: 5 min ,94°C

Denaturation: 30 sec,94°C

Annealing: 30 sec,61°C

Elongation: 30 sec,72°C

Elongation: 5 min,72°C

Cooling: 4°C

No. of cycles: 33

Reaction-Mix:

DNA 2 Ml (50 ng) Buffer 2 μl (1 x) (SIGMA) MgCl₂ 1.5 μl (1.5 mM) (SIGMA) Primer F 0.25 μl (6.25 pmol) Primer R 0.25 μl (6.25 pmol) dNTP 0.25 μl (0.25 mM) Taq 0.2 μl (1 U)

(SIGMA) bidest 17.55 μl total volume 25.00 μl

2. RFLP:

Digestion-Mix: Enzyme Cfrl3I (lOU/μl) 0.30 μl (3 U) Buffer: Y⁺Tango 3.00 μl (1 x) bidest 11.7 μl PCR: 15 μl

Water-bath: 37 °C; 1 h

Lengths of fragments: G-Allele: 138 bp, 255 bp A-Allele: 393 bp SNP rsl33063 (T/C)

Primer Forward SNP rsl33063-F ATG AGT AGG CCA GGT GTG GT Primer Reverse SNP rsl33063-RGGA CTG GCT CCA GCT ACA TC Product-size: 384 bp l.PCR:

Thermocycler:

Denaturation: 5 min, 94°C

Denaturation: 30 sec, 94°C

Annealing: 30 sec, 63°C

Elongation: 30 sec, 72°C

Elongation : 5 min , 72°C

Cooling: 4 °C

No. of cycles: 30

Reaction-Mix:

DNA 2.5 μl (50 ng) Buffer 2.5 μl (1 x) (SIGMA) MgCl₂ 1.0 μl (1.0 mM) (SIGMA) Primer F 0 25 μl (6.25 pmol) Primer R 0 4 μl (10 pmol) dNTP 0.25 μl (0.25 mM) Taq 0.2 μl (1 U)

(SIGMA) bidest 17.9 μl total volume 25.00 μl

2.RFLP:

Digestion-Mix: Enzyme: HpyCH4 (5U/μl) 0.30 μl (1,5 U) Buffer: NEB4 3.00 μl (1 x) bidest: 11.7 μl

PCR: 15 μl

Water-bath: 37 °C; 17 h

Lengths of fragments : T-Allele: 384 bp C-Allele: 55 bp, 329 bp

SNP rs!33068 (C/G) Primer Forward SNP 133068-F CCT CCA CCT CTG CTG GTA TT Primer Reverse SNP 133068-R GTG GGG GAT AAA GTC CCT GT Product-size: 150 bp

1. PCR:

Thermocycler:

Denaturation: 5 min, 94°C

Denaturation: 30 sec, 94°C

Annealing: 30 sec, 64°C

Elongation : 30 sec, 72°C

Elongation : 5i min , 72°C

Cooling: 4 °C

No. of cycles: 30

Reaction-: Mix:

DNA 2.5 μl (50 ng)

Buffer 2.5 μl (1 x)

(SIGMA)

MgCl₂ 1.5 μl (1.5 mM)

(SIGMA)

Primer F 0.3 μl (7.5 pmol)

Primer R 0.3 μl (7.5 pmol) dNTP 0.25 μl (0.25 mM)

Taq 0.3 μl (1.5 U)

(SIGMA) bidest 17.35 μl total volume 25.00 μl

2. RFLP:

Digestion- -Mix:

Enzyme : Faul (2U/μl) 0.30 μl (0.6 U) Buffer: SEB 3.00 μl (1 x) bidest 11.7 μl PCR: 15 μl

Water-bath 55 °C; 1 h

Lenghts of fragments : C-Allele: 150 bp G-Allele: 94 bp, 106 bp

SNP rsl33069 (C/A)

Primer Forward inner (A-allele) GGACTTTATCCCCCACCCCACCCTCA Primer Reverse inner (C-allele) TTTTGCAGTAAAAAAAAAAGAAAAAAAGGG Primer Forward outer

GGGGGGGGGGGGGGGGGGGGCCAATACCATGAATTGTCTTTTGAGGGGT

Primer Reverse outer TTGGGTTCATCCAACAAACATTCATTGA Product-size: 115 bp (A-allele); 147 (C-allele) ; 206 (of two outer primers)

1. PCR:

Thermocycler:

Denaturation: 5 min, 94°C

Denaturation: 30 sec, 94°C

Annealing: 30 sec, 60°C

Elongation: 30 sec, 72°C

Elongation: 5 min, 72°C

Cooling: 4 °C

No. of cycles: 30

Reaction-Mix (A-allele) :

DNA 2.5 μl (50 ng) Buffer (PE) 2.5 μl (1 x) MgCl₂ (PE) 3.0 μl (3 mM) Primer F 0.25 μl (6.25 pmol) Primer R 0.25 μl (6.25 pmol) dNTP 0.25 μl (0.25 mM) Taq (PE) 0.25 μl (1.3 U) bidest 16.0 μl total volume 25.00 μl

Reaction-Mix (C-allele) :

DNA 2.5 μl (50 ng) Buffer (PE) 2.5 μl (1 x) MgCl₂ (PE) 3.0 μl (3 mM) Primer F 0.25 μl (6.25 pmol) Primer R 0.25 μl (6.25 pmol) dNTP 0.25 μl (0.25 mM) Taq (PE) 0.3 μl (1.5 U) bidest 15.95 μl total volume 25.00 μl

A alleles and C alleles are to be amplified separately.

SNP rsl33072 (G/A; Asp-32-Asn)

Primer Forward SNP 133072-F gca ggc att cag aag tgg a Primer Reverse SNP 133072-R agg tec ate cag cca gtg Product-size: 302 bp

1. PCR:

Thermocycler:

Denaturation: 5 min, 94°C

Denaturation: 30 sec, 94°C

Annealing: 30 sec, 59°C Elongation: 30 sec, 72°C

Elongation: ! 5 min , 72°C

Cooling: 4 °C

No. of cycles: 30

Reaction- -Mix:

DNA 2.5 μl (50 ng)

Buffer 2.5 μl (1 x)

(SIGMA)

MgCl₂ 1.5 μl (1.5 mM)

(SIGMA)

Primer F 0.3 μl (7.5 pmol)

Primer R 0.6 μl (15 pmol) dNTP 0.5 μl (0.5 mM)

Taq 0.2 μl (1 U)

(SIGMA) bidest 16.90 μl total volume 25. 00 μl

2. RFLP:

Digestion-Mix:

Enzyme : Hpyl88lII (5 U/μl) 0.30 μl (1,5 U)

Buffer: NEB4 3.00 μl (1 x)

BSA 0.35 μl bidest 11.35 μl

PCR: 15 μl

Water-bath 37 °C; 2 h

Lengths of fragments:

A-Allel: 302 bp G-Allel: 178 bp, 124 bp

SNP rsl33073 (T/C)

Primer Forward inner (T-allele) CTGCTGCCCACTGGTCCCCAT

Primer Reverse inner (C-allele) GCCATCAGAGGTGTTGCTGTCG Primer Forward outer GAAGGGAGTGGGGAGGGCAGTT Primer Reverse outer GCCCCTCAGAGCAAAGCAGACC Product-size: 202 bp (T-allele) ; 247 (C-allele) ; 406 (of two outer primers)

1. PCR:

Thermocycler:

Denaturation: 5 min, 94°C Denaturation: 30 sec, 94°C Annealing: 1 min, 65°C Elongation: 1 min, 72°C

Elongation: 5 min, 72°C

Cooling: 4 °C

No. of cycles: 30

Reaction-Mix:

DNA 2.5 μl (50 ng)

Buffer 2.5 μl (1 x)

(PE)

MgCl₂ 2 2..55 μμll ((22..55 mmMW)

(PE)

Primer F inner 0 0..2255 μμll (6.25 pmol)

Primer R inner 00..2255 μμll (6.25 pmol)

Primer F outer 0 0.,1 1 μμll (2 pmol)

Primer R outer 00.,1 1 μμll (2 pmol) dNTP 0 0..2255 μμll ((00..225 mM)

Taq 00..22 μμll ((11 UU))

(PE) bidest 16.35 μl total volume 25.00 μl

SNP rsl33074 (C/T)

Primer Forward SNP 133074-F TCC CAA GCT GGT GGA TAA TG Primer Reverse SNP 133074-R ACC CCA GGT CTC CTT GTT TT Product-size: 181 bp

1. PCR:

Thermocycler :

Denaturation: 5 min, 94°C

Denaturation: 30 sec, 94°C

Annealing: 30 sec, 62°C

Elongation: 30 sec, 72°C

Elongation: 5 min, 72°C

Cooling: 4 °C

No. of cycles: 30

Reaction-Mix:

DNA 2.5 μl (50 ng) Buffer 2.5 μl (1 x)

(SIGMA) MgCl₂ 1.5 μl (1.5 mM)

(SIGMA) Primer F 0.25 μl (6.25 pmol) Primer R 0.25 μl (6.25 pmol) dNTP 0.25 μl (0.25 mM) Taq 0.2 μl (1 U)

(SIGMA) bidest 17.55 μl total volume 25.00 μl 2 . RFLP :

Digestion-Mix:

Enzyme : Tsp509I (lOU/μl) 0.30 μl (3 U)

Buffer: NEB1 3.00 Ml (1 x) bidest 11.7 μl

PCR: 15 μl

Water-bath 65 °C; 1 h

Lengths of fragments : C-Allel: 181 bp T-Allel: 62 bp, 119 bp

SNP rs2032512 (C/A)

Primer Forward SNP rs2032512-F GCC AGA AGT GGA TCT TGA GG Primer Reverse SNP rs2032512-R CCA CAC CTG GCC TAC TCA TT Product-size: 377 bp

2. PCR:

Thermocycler:

Denaturation: 5 min , 94°C

Denaturation: 30 sec, 94°C

Annealing: 30 sec, 62°C

Elongation: 30 sec, 72°C

Elongation: 5 min , 72°C

Cooling: 4 °C

No. of cycles: 30

Reaction-Mix:

DNA 2.5 μl (50 ng) Buffer 2. .5 μl (1 X) (SIGMA) MgCl₂ 3. .0 μl (3.0 mM) (SIGMA) Primer F 0. .25 ^• μl (6.25 pmol) Primer R 0. .25 ^■ μl (6.25 pmol) dNTP 0. .25 ^■ μl (0.25 mM) Taq 0, .2 μl (1 U)

(SIGMA) bidest 16.05 μl total volume 25.00 μl

2. RFLP:

Digestion-Mix: Enzyme : BamHl (lOU/μl) 0.30 μl (3 U)

Buffer: BamHl 3.00 μl (1 x) bidest: 11.7 μl

PCR: 15 μl

Water-bath 37 °C; 1.5 h

Lengths of fragments :

C-Allele: 120 bp, 257 bp A-Allele: 377 bp

SNP rs rsxxxxxx4 (A/G)

Primer Forward SNP rsxxxxxx4-F ACC TGG CCA GCT ACA CAC TT Primer Reverse SNP rsxxxxxx4-R AGG GGA AGC TTT TTG CAG TA Product-size: 190 bp

. PCR:

Thermocycler :

Denaturation: 5 min, 94°C

Denaturation: 30 sec, 94°C

Annealing: 30 sec, 58°C

Elongation: 30 sec, 72°C

Elongation: 5 min , 72°C

Cooling: 4 °C

No. of cycles: 30

Reaction-Mix:

DNA 2.5 μl (50 ng)

Buffer 1.5 μl ( x)

(SIGMA)

MgCl₂ 2.0 μl (2 mM)

(SIGMA)

Primer F 0.25 μl (6.25 pmol)

Primer R 0.25 μl (6.25 pmol) dNTP 0.25 μl (0.25 mM)

Taq 0.2 μl (1 U)

(SIGMA) bidest 17.55 μl total volume 25. 00 μl

2. RFLP:

Digestion-Mix: Enzyme : Bglll (10 U/μl) 0.30 μl (3 U) Buffer: 0⁺ 3.00 μl (1 x) bidest : 11.7 μl

PCR: 15 μl

Water.bath 37 °C; 2 h

Lengths of fragments : A-Allele: 31 bp, 159 bp G-Allele: 31bp, 45 bp, 114 bp

SNP rsxxxxxx5 (C/T)

Primer Forward SNP rsxxxxxx5-F AGA ATC TGC CCT TCC TGC TC Primer Reverse SNP rsxxxxxx5-R CTG AAG GAA GTG AGG AAG CA Product-size: 498 bp

2. PCR:

Thermocycler :

Denaturation: 5 min , 94°C

Denaturation: 30 sec, 94°C

Annealing: 30 sec, 61°C

Elongation: 30 sec, 72°C

Elongation: 5 min, 72°C

Cooling: 4 °C

No. of cycles: 35

Reaction-Mix:

DNA 2.5 μl (50 ng) Buffer 2.5 μl (1 x) (SIGMA) MgCl₂ 3.5 μl (3.5 mM) (SIGMA) Primer F 0.25 μl (6.25 pmol) Primer R 0.25 μl (6.25 pmol) dNTP 0.25 μl (0.25 mM) Taq 0.2 μl (1 U)

(SIGMA) bidest 15.55 μl total volume 25.00 μl

2. RFLP:

Digestion-Mix: Enzyme : PfiFI (lOU/μl) 0.30 μl (3 U) Buffer: NEB4 3.00 μl (1 x) BSA: 0.35 μl bidest: 11.35 μl

PCR: 15 μl

Water-bath 37 °C; 17 h

Lengths of fragments: C-Allele: 183 bp, 315 bp T-Allele: 498 bp

SNP rsxxxxxx6 (T/C)

Primer Forward SNP rsxxxxxx6-F CTC CTG AGC TCA AGC AAT CC Primer Reverse SNP rsxxxxxx6-R GTG GGG GAT AAA GTC CCT GT Product-size: 415 bp

2. PCR:

Thermocycler:

Denaturation: 5 min, 94°C

Denaturation: 30 sec, 94°C

Annealing: 30 sec, 61°C

Elongation: 30 sec, 72°C

Elongation: 5 min, 72°C

Cooling: 4 °C

No. of cycles: 30

Reaction-Mix:

DNA 2.5 μl (50 ng) Buffer 2.5 μl (1 x) (SIGMA) MgCl 1.0 μl (1 mM) (SIGMA) Primer F 0.25 μl (6.25 pmol) Primer R 0.25 μl (6.25 pmol) dNTP 0.25 μl (0.25 mM) Taq 0.1 μl (0.5 U)

(SIGMA) bidest 18.15 μl total volume 25.00 μl

2. RFLP:

Digestion-Mix: Enzyme : Bsrl (lOU/μl) 0.30 μl (3 U) Buffer: NEB3 3.00 μl (1 x) bidest: 11.7 μl PCR: 15 μl

Water-bath 65 °C; 2 h

Lengths of fragments: T-Allele: 415 bp C-Allele: 135 bp, 280 bp

SNP rsxxxxxx7 (T/G)

Primer Forward SNP rsxxxxxx7-F CTT ACT TTT GTG TCC TTC TGG

CTA

Primer Reverse SNP rsxxxxxx7-R GAG CCA TCT GTC TTG GAA GG

Product-size: 146 bp

2. PCR:

Thermocycler :

Denaturation: 5 min, 94°C

Denaturation: 30 sec, 94°C

Annealing: 30 sec, 65°C

Elongation: 30 sec, 72°C

Elongation: 5 min, 72°C

Cooling: 4 °C

No. of cycles : 30

Reaction-Mix:

DNA 2.5 μl (50 ng) Buffer 2.5 μl (1 x) (SIGMA)

(SIGMA) Primer F 0 25 μl (6, .25 pmol) Primer R 0 25 μl (6, .25 pmol) dNTP 0 25 μl (0, .25 mM) Taq 0 2 μl (1 U)

(SIGMA) bidest 16.05 μl total volume 25.00 μl

2. RFLP:

Digestion-Mix: Enzyme: Tfil (10 U/μl) 0.6 μl (6 U) Buffer: NEB3 3.00 μl (1 x) bidest: 11.4 μl PCR: 15 μl

Waterbath 65°C; 17 h

Lengths of fragments : T-Allele: 146 bp G-Allele: 65 bp, 81 bp

SNP rsxxxxxxβ (C/T)

Primer Forward SNP rsxxxxxx8-F AGC CCA GTT TGC TAG GAG GT Primer Reverse SNP rsxxxxxx8-R ACA CAC GGA CAC TCA AGC TG Product-size: 173 bp

2. PCR:

Thermocycler :

Denaturation: 5 min, 94°C

Denaturation: 30 sec, 94°C

Annealing: 30 sec, 61°C

Elongation: 30 sec, 72°C

Elongation: 5 min, 72°C

Cooling: 4 °C

No. of cycles: 30

Reaction-Mix:

DNA 2.5 μl (50 ng) Buffer 2.5 μl (1 x)

(SIGMA) MgCl₂ 2.0 μl (2 mM)

(SIGMA) Primer F 0.25 μl (6.25 pmol) Primer R 0.25 μl (6.25 pmol) dNTP 0.25 μl (0.25 mM) Taq 0.2 μl (1 U)

(SIGMA) bidest 17.05 μl total volume 25.00 μl

2. RFLP:

Digestion-Mix: Enzyme : NLAIII (lOU/μl) 0.2 μl (2 U) Buffer: Puffer 5 3.00 μl (1 x) BSA: 0 . 35 μl bidest: 11.45 μl

PCR: 15 μl

Water-bath 37 °C; 17 h

Lengths of fragments : C-Allele: 173 bp T-Allele: 45 bp, 108 bp

(G) Association studies

For initial, confirmatory and post hoc comparisons of allele and genotype frequencies a hierarchical test procedure was followed: If the allele test is significant using the asymptotic Pearson Chi-square test, differences between genotypes were additionally investigated with the Cochran-Armitage trend test for association.

TDT. All transmission disequilibrium tests (TDT) were carried out using the program Genehunter, version 2.0 beta (Kruglyak et al., Am. J. Hum. Genet . 58, 1347-1363 (1996). The program performs haplotype TDTs with up to four markers for all trios, where phase can be determined unambiguously. Haplotype frequencies were estimated and χ² -statistics for testing association between SNPs were calculated with the program EH, version 1.11 (Xie and Ott, Am. J. Hum. Genet. 53, 1107 (abstract) (1993) .

(H) Genotype relative risks

For the SNP rsl33072, 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.

(I) Accession numbers

Previously known human MCHRl mRNAs, AB063174, NM_005297; ESTs extending the published 5' end of human MCHRl mRNA, BE312542, BG519797, BF313837, BI818110; revised human MCHRl mRNA, AF490537; rat MCHRl mRNA, AF008650; human BAC containing MCHRl , Z86090.

Example 2: Major contribution of a common non-conservative cSNP in the MCHRl to human obesity

Presently, there are two MCHRl mRNA entries in human databases. 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). A shorter CDS starting at codon 70 (Met⁷⁰) downstream of the first methionine codon (Met¹) 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) . Screening of the MCHRl CDS beginning at Met⁷⁰ by single strand conformation polymorphism analysis (SSCP) in 215 extremely obese children and adolescents and 230 underweight students revealed heterozygotes for five silent and five missense variations (allele frequencies < 1 %; data not shown) in addition to the frequent silent SNP rsl33073 in exon 1. Comparison of allele and genotype- frequencies of rsl33073 indicated association of the C-allele with obesity (Table 1) , association was subsequently confirmed with independent study groups of obese and normal weight students .

Table 1: Association analyses of SNP rsl33073 with different study groups

Study group Genotype frequencies* Allele frequencies

CC CT TT C-allele T-allele

1. Extremely obese children 33 115 67 181 249 and adolescents (n = 215) (15.3 ) (53.5 %) (31.2 %) (42.1 %) (57.9 %)

2. Underweight students 23 113 94 159 301 (n = 230) (10.0 %) (49.1 %) (40.9 %) (34.6 %) (65.4 %) initial analysis p = 0.016^x p = 0.021*

3. Overweight students 15 53 31 83 115 (n = 99) (15.2 ) (53.5 %) (31.3 %) (41.9 %) (58.1 %)

4. Normal weight students 7 46 43 60 132 (n = 96) (7.3 %) (47.9 %) (44.8 %) (31.2 %) (68.8 %)

Confirmatory analysis p O.Oir p = 0.011^xx

5. All obese individuals* 114 361 244 589 849 (n = 719) (15.9 ) (50.2 %) (33.9 %) (41.0 %) (59.0 %)

6. All non-obese individuals** 30 159 137 219 433 (n = 326) (9.2 %) (48.8 %) (42.0 %) (33.6 %) (66.4 %)

Post hoc analysis p = 0.001*^* p = 0.001*^* Genotype-frequencies are in Hardy-Weinberg equilibrium, includes study groups 1 + 3 and 405 obese children and adolescents **includes study groups 2 + 4 * two-sided; ** one-sided Finally, a post hoc analysis based on 719 obese and 326 non- obese individuals underscored the association finding (Table 1) . Based on the confirmed association, transmission disequilibrium tests (TDT) performed in an initial sample, a confirmatory sample and in an extended post hoc analysis encompassing 525 trios all revealed a preferential transmission of the C-allele of rsl33073 (Table 2a) .

Table 2: TDTs for initial SNP rsl33073 (a), adjacent SNPs (b) and haplotypes including SNP rs133072 (c)

a) initial TDT confirmatory TDT

108 trios 226 independent^"trio£r -

SNP trans. trans/ trans. two-sided trans/ trans. one-sided allele non-trans rate [%] p-value non-trans rate [%] p-value rsl33073 T/C^* C 61/31 66.3 0.0018 126/84 60.0 0.0019 b) 61 trios 525 trios single marker single marker

SNPs trans. trans/ trans. 2-sided trans/ trans. 2-sided allele^§ non-trans rate [%] p-value non-trans rate [%] p-value rsl33062 G/A A 39/17 69.6 0.0033 rs2032512 C/A A 38/18 67.9 0.0075 rsl33063 T/C C 34/19 64.1 0.0394 rsxxxxxx4 A/G G 14/9 60.9 0.2971 rsxxxχxx5 C/T T 25/16 61.0 0.1599 rsxxxxxxό T/C C 28/15 65.1 0.0474 rsl33068 C/G G 39/18 68.4 0.0054 289/205 58.5 0.00016 rsl33069 C/A A 38/18 67.9 0.0075 rs 133072 G/A^** A 37/18 67.3 0.0104 290/206 58.5 0.00016 rs 133073 T/C^* C 38/17 69.1 0.0046 285/204 58.3 0.00025 rsxxxxxx7 T/G G 14/13 51.9 0.8474 rsl33074 C/T T 27/18 60.0 0.1797 269/229 54.0 0.07306 rsxxxxxxδ C/T T 6/3 66.7 0.3173 Haplotypes formed by two*, three^b and four⁰ SNPs, respectively rsl33068 j rsl33072 j rsl33073 j rsl33074 "229/140 6 i % δooόθ4^r ;

"228/140 62.0 % 0.000004

"190/117 61.9 % 0.000031 ^c186/117 61.4 % 0.000074

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

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

Novel SNPs from database dbSNP Allele-exchanges SNP Contig Pos. AA-exchange

SNP's rsl33062* G/A 95538 rs2032512* C/A 95767

Rsxxxxxxl T/A 95948

Rsxxxxxx2 G/A 96226 rsl33063* T/C 96329 rsl33064 G/A 96401

Rsxxxxxx3 A/G 96796

Rsxxxxxx4* A/G 97003

Rsxxxxxx5* C/T 97723

Rsxxxxxxό* T/C 98838 rsl33068* C/G 99077 rsl33069* C/A 99132 rsl33070 A/G 99443

* genotyped SNPs; grey background= coding SNPs; AA = Aminoacid; Asp = Aspartat; Asn Asparagin

One previously known SNP rsl33072 within the coding region starting with Met¹ could be confirmed. This SNP results in a non- conservative aa exchange (Asp32Asn) and revealed preferential transmission of the A-allele in 525 trios (Table 2c) . TDTs for other identified SNPs were performed in a subgroup (n = 61) and/or in the total of 525 trios. Strong linkage disequilibrium between the four SNPs genotyped for 525 trios was detected (Table 2b) . Haplotypes based on these SNPs revealed that transmission rates in the 525 trios only slightly exceeded the transmission rate of rsl33072 alone.

Due to the location of cSNP rsl33072 in exon 1 of MCHRl the expression of the alternative transcripts was studied. In dbEST, 30 independent cDNA clones were identified. 24 are derived either from brain or germ cells. 11 ESTs are spliced and confirm mRNA 1. No EST extends from exon 2 into the mRNA 2 specific 5' region. In agreement therewith, only mRNA 1 could be amplified but not mRNA 2 from brain specific cDNA. These results support a CDS of exon 1 with methionine codons at positions 1, 6 and 70, which all represent "weak" start codons (Kozak, Gene 234(2) (1999), 187-208). Since the EST assembly does not indicate an alternative transcription start within exon 1, translation most likely starts at Met¹. However, context- dependent leaky scanning cannot be excluded. The structure of proteins translated from Met¹ or Met⁶ start codons will be affected by allelic occurrence of cSNP rsl33072.

Under the assumption that cSNP rsl33072 is functionally relevant, 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. Based on the estimated population frequency of the A-allele of 31.0 % (95 % CI 28.2 - 33.7), the attributable risk estimate is 23.7 % (95 % CI 13.1 % - 34.3 %) . This implies that 24 % of the cases can be attributed to heterozygosity or homozygosity for the A-allele of rsl33072 in the population of obese children and adolescents from which our index patients were "sampled.- Similar- risks apply- to the other SNPs in strong linkage disequilibrium with rsl33072.

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 Asp³²-MCHRl/Asn³²-MCHRl: the A-allele of rsl33072 is associated with loss of function of the MCHRl protein

The MCHRl couples to G_q/u and Gι₀ proteins to activate several intracellular signalling pathways (Hawes et al . , Endocrinology 14 (2000), 4524-4532). Upon functional expression in COS-7 cells, Asn³²-MCHR1 responded to MCH challenge with a decreased maximal inositol phosphate production (4,7 +/- 0.7-fold over basal, n=3) when compared to Asp³²-MCHR1 (12 +/- 0.4-fold over basal; Fig. 3) indicative of a partial loss-of-function mutation. Particular aspects of the complex phenotype of the rodent Mc.hr ^{~ _} (Marsh et al . , Proc. Natl. Acad. Sci. USA 99 (2002), 3240-3245) suggest an explanation of how this in-vitro partial loss-of-function may represent the in-vivo correlate of the observed association of obesity with the A-allele of rsl33072. Based on the unexpected detection of hyperphagia in the knockout model, which is not readily compatible with the orexigenic role of MCH, it is tempting to speculate that in humans the merely partial loss of function leads to overeating without concomitantly inducing the other phenotypical effects apparent in MCHR^"/-mice. 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.

Example 4

Methods

Study subjects

The association and linkage disequilibrium studies were based on four (1-4) study groups ascertained by investigators from Marburg:

5) 620 (359 females) unrelated obese children and adolescents ascertained via a body mass index (BMI) > 90^th percentile (Hebebrand et al. 1996; mean BMI 33.4 ± 6.6 kg/m²; mean

BMI percentile 99.3; mean age 14.0 ± 2.7 years): For the initial mutation screen we chose a

subgroup of 215 extremely obese children and adolescents (127 females; mean BMI 39.8 ±

5.3 kg/m²; mean age 15.3 ± 2.4 years). For transmission disequilibrium tests (TDT; Spielman et al. 1993) both parents of 525 of the children and adolescents (304 females; mean BMI 32.2

± 6.0 kg/m²; mean age 13.7 ± 2.8 years) were genotyped for four SNPs (Table 9a); TDTs for these and 12 other SNPs were also based on a smaller subgroup of 61 trios (Table 9a). For 164 of the 525 trios one (or more) additional obese sib(s) (BMI > 90^th percentile) had also been ascertained; these were also genotyped to allow linkage analyses.

6) 230 (110 females) healthy underweight students (BMI < 15^th percentile; mean BMI

18.3 ± 1.1 kg/m²; mean age 25.2 ± 3.7 years).

7) 96 (49 females) healthy normal weight students (BMI > 40^th and < 60^th percentile; mean BMI

21.9 ± 1.1 kg/m²; mean age 24.7 ± 2.6 years).

8) 99 (51 females) healthy overweight students (BMI > 90^th percentile; mean BMI

29.1 ± 3.4 kg/m²; mean age 25.3 ± 3.7 years).

Written informed consent was given by all participants and in the case of minors, their parents. The Ethics Committee of the University of Marburg and the local ethics committees of the participating investigators approved genotyping of obese subjects, their parents and controls. Mutation screen, initial and confirmatory association and transmission disequilibrium tests and linkage analyses

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.

Table 11: Primers PCR in the initial mutation screen of the CDS of MCHRl transcript defined by Shimomura et al. (1999 ; AB063174) by SSCP

Exonl MCHR-l long-F/ MCHR-1 long-R 100062 - 100302 241 65 AGCCTGGGACTGAAGAAGTT GCTCAGCTCGGTTGTGG

Exonl MCHR-1-F/MCHR-l-R 100286 - 100484 199 63

GCTCAGCTCGGTTGTGG

GCAGTTTGGCTCAGGGG

Exon2 MCHR-2a-F/MCHR-2a-R 101582 - 102169 588 62

GCCCATGTCAAACAGCCAAC

AGGGTGAACCAGTAGAGGTC

Exon2 MCHR-2b-F/MCHR-2b-R 102083 - 102632 550 60

TGCCAGACTCATCCCCT

TTGGAGGTGTGCAGGGT

Association studies: For initial, confirmatory andjrast hoc comparisons of allele and genotype frequencies we followed a hierarchical test procedure: If the allele test is significant using the asymptotic Pearson Chi-square test, we additionally investigate differences between genotypes with the Cochran-Aπnitage trend test for association. In light of hyperactivity, decreased body fat mass and reduced susceptibility to obesity induced by a high-fat diet (Chen et al. 2002) particularly in male MCHRl knockout mice (Marsh et al. 2002) we gender dependently assessed genotypes in relationship to a) maternally recalled motor activity levels of their obese offspring at different ages, b) body composition and c) percent energy consumed as fat in post hoc tests. Mothers of 399 of the 525 obese children and adolescents from family-based studies had been asked to recall age-dependent (age groups: < 1, 1-6, 7-10, 11-14 and > 15 years) motor activity levels of their children as elevated, similar or less than other children of the same age using a structured interview. Body composition (including percent body fat mass) was determined via body impedance analysis (Data Input 2000-S) in 491 of the obese children. The percentage of energy intake consumed as fat was assessed using the Leeds Food Frequency Questionnaire (L- FFQ; Cooling and Blundell 1998) in 140 of the obese children and adolescents.

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

Mutation screen by sequencing: 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.

Table 12: Primers for genomic sequence analysis of 13378 bp of the MCHRl locus

Region*

Alf: GCTTCCACCCTACATTGGTC A2f: AGCATTAACTCTCCCTGCAC Air: AGGTCAATCACGAGGTTAGG A2r: CCATAATGCCAGCTACACAG

A3f: GTCCAGTAGACATGCTCACC A3r: TACCAAAGTGACTGAGCCTG

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

B2r: GGCTGAGGCAGAAGAATCGC

B3f: GTCTCACTCTGTCGCTCAGG B3r: ATGGAGTTGTGGGGGAGGTC B4f: CCCTGACTCACTCTGTTCCC B4Γ: TTCTGCCAGGATCTGCCCAC

B5f: AGGACTATGGAGTTAGCTGG B5Γ: CTTTCTTGCACCTCTGCAAC

B5f2: AGTCTCCCTCTGTCACCC B6f: CCAAAGCAACAATGAGAGACCC B6r: GACCCACCTTGTCATGTACC B6r2: GTCCCTTGTACTTGCGAG

Clf: GGCAGAGTCAGATAGCCAGC C2f: TCCTGACTCTTAACACTCGC Clr: CTAGCACCCTCAAGATCCAC C2r: CAGTGACATCAAGAGGATGG

C3f: GTCACTGTCAAAGTCAGAGG C3r: GCTGGGACTTACTTAGATGG

C4f : TAGTCTTCTAGGTGGCACTG C4π TCACCAGTGGAAATCCCATC

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

Elf: TCAGCTCACTGCAACCCTC E2f : TAGCTGGGACTACAGGCAC Elr: GGAAATTGTCAGTGCAGAGG E2r: TGGATTGACTGAGGTCAGG

E3f: GAGGCAAGAGTCACCACACC E4f : AGTAGCTGGGATTACAGG E3r: TGAGGTCCCTGAGGTTTGGC E4r: GGAAATTGTCAGTGCAGAGG

E5f: TGACCTCAGTCAATCCACC E5r: TCACGCCTGAAATCCCAGC

E6f: CTCACTGCAATTTCCACCTC E6r: CATCCTGGCTAACATGGTG

E7f: CTCTCTTGGTATTCCTGG E7r: GAAGTGAGCTGAGATTGG

E8f: GGTATTACAGGTGTGAGC E8r: GGGTGATTCTGAAGAACAGG

E9f: GCTGGTCTTGAACTCCTG E9r: TTACTACCTGCCAAGCAC

Flf: GCTTCTCAGTGAGGTCTTCC F2f: ACCTCTGCTGGTATTCAGGG Fir: CCCTCAGAGCAAAGCAGACC E3r: TGAGGTCCCTGAGGTTTGGC

C3f: CΓTAACGCCTTTGCCTCTGC

C3r: GAGTCCAGAGGAAACTGTGC

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

Genotyping of SNPs: Thirteen SNPs (Table 9a) were genotyped by restriction fragment length

polymorphism analyses (RFLP; primers Table 13) or tetra-primer amplification refractory

mutation system (ARMS; Ye et al. 2001; primers Table 14).

Table 13: Primers, amplicon length, annealing temperatures and restriction endonucleases for PCR-RFLP

rs MCHR133062-F/MCHR133062-R 95538 393 61 Cfrl3 I

133062 ACTTCACCCATGAGGACCAG TGCATCATCTAGCACCCTCA rs MCHR2032512-F/MCHR2032512-R 95767 377 62 BamH l

2031512 GCCAGAAGTGGATCTTGAGG CCACACCTGGCCTACTCATT rs MCHR133063-F/MCHR133063-R 96329 384 63 HpyCH4 HI

133063 ATGAGTAGGCCAGGTGTGGT GGACTGGCTCCAGCTACATC SNP MCHR1745616-F/MCHR1745616-R 97003 190 58 Bgi π

1745616 ACCTGGCCAGCTACACACTT AGGGGAAGCTTTTTGCAGTA

SNP MCHR1745617-F/MCHR1745617-R 97723 498 61 PfϊF I 1745617 AGAATCTGCCCTTCCTGCTC

CTGAAGGAAGTGAGGAAGCA

SNP MCHR1745618-F/MCHR1745618-R 98838 415 61 Bsrl 1745618 CTCCTGAGCTCAAGCAATCC

GTGGGGGATAAAGTCCCTGT rs MCHR133068-F/MCHR133068-R 99077 150 64 Fau l 133068 CCTCCACCTCTGCTGGTATT

GTGGGGGATAAAGTCCCTGT rs MCHR133072-F/MCHR133072-R 100213 302 59 Hpyl88 iπ 133072 GCAGGCATTCAGAAGTGGA

AGGTCCATCCAGCCAGTG

SNP MCHR1745619-F/MCHR1745619-R 101341 146 65 Tfi l 1745619 CΓTACTTTTGTGTCCTTCTGGCTA

GAGCCATCTGTCTTGGAAGG rs MCHR133074-F MCHR133074-R 103143 181 62 Tsp5091 133074 TCCCAAGCTGGTGGATAATG

ACCCCAGKJTCTCCTTGTTTT rs MCHR3087592-F/MCHR3087592-R 103335 173 61 Nla m 3087592 AGCCCAGTTTGCTAGGAGGT

ACACACGGACACTCAAGCTG

Table 14: Primers, melting and annealing temperatures and amplicon length for tetra- primer ARMS-PCR

rs 133073 100365 Forward inner primer (T-allele) 70 65 202 (T-allele) T>C CTGCTGCCCACTGGTCCCCAT

Reverse inner primer (C-allele) 67 65 247 (C-allele) GCCATCAGAGGTGTTGCTGTCG

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

CA

Reverse inner primer (C-allele) 65 60 147 (C-allele)

TTTTGCAGTAAAAAAAAAAGAAA AAAAGGG

Forward outer primer '6 60 206 (of two

GGGGGGGGGGGGGGGGGGGGCC outer primers)

AATACCATGAATTGTCTTTTGAG GGGT

Reverse outer primer "^ 60

TTGGGTTCATCCAACAAACATTC

ATTGA

Four SNPs (Table 9a) were genotyped by genomic sequencing as described in mutation screen by sequencing (primers Table 12).

Haplotype frequencies were estimated and χ²-statistics for testing association between SNPs

were calculated with the program EH, version 1.11 (Xie and Ott 1993). Haplotype frequencies were estimated for four SNPs (Table 10) in 525 trios (study group 1). Expression analyses

For in silico analysis, ESTs found by BLAST (GenBank release date 01/12, version 127.0; Altschul et al. 1997) were assembled using gap4.5 (Bonfield et al. 1995) and compared to GenBank entries Z86090 and AB063174. For nested and semi-nested RT-PCR, human cDNA library panels MTC Panel I and II (Clontech Laboratories) were used (primers in Table 15). PCR products were sequenced, assembled and edited as described above. Table 15: Primers for expression analyses of the MCHRl transcript

MRNA rtlf/rtlr mchr.4if rt2r

AB063174 TGCAGGCATTCAGAAGTGG CCAGGCTACGGAGGAAGAC

ATGCTGATGAAGGAGAGGG CTGGTGAACTGACTATTGGC rtlf/rt2r

TGCAGGCATTCAGAAGTGG

CTGGTGAACTGACTATTGGC

Functional in vitro studies

Promoter studies. Programs FirstEF ('first exon finder', http://rulai.cshl.org/tools/FirstEF; Davuluri et al. 2001), NNPP ("neural network promotor prediction", http:www.fruitfly.org; Waibel et al. 1989; Reese and Eeckman 1995), Promoterlnspector and ELDorado (http://www.genomatix.de) were used for in silico promoter prediction. For luciferase assay, genomic fragments of 1,181 bp (pos. 98943-100122 in GenBank entry Z86090, i.e. upstream of the putative translation start Met¹) 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 μg reporter gene plasmid and 3 x 10⁶ cells/0.15 ml in 0.4 cm electroporation cuvettes with a gene pulser (Gene zapper 450/2500, IBI, Cambridge, England) at 1200 μFD, 100 Ohm and 300 V. After electroporation 1 x 10⁶ cells/ml/well were cultured in the absence or presence of 500 μM 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). Specific luciferase expression or promoter activity was given in arbitrary units (AU) and assessed normalizing relative light units (raw data) to the protein content of the lysate measured by Bradford protein assay (Sigma). Statistical analysis was performed with the Mann-Whitney-U-test for two independent samples and given as bilateral asymptotic significance p.

Table 16: Primers for promoter studies of the MCHRl -Gens

SW *^A

PCR 1-F: GGAGATCCCTTTCCTGATGG PCR 1-R: GCCTCTCACTGGTGCGATGG PCR 1-F: TGCAGGCATTCAGAAGTGG PCR 1-R: GAGCAGGATGAGACATTTG

Xhol.f TGAGGTCTTCCTCGAGGATCAA Ncol.r GGCTCCCACTGCCATGGCCTAG Results

Mutation screen, initial and confirmatory association and transmission disequilibrium tests

Screening of the MCHRl CDS beginning at Met by SSCP in 215 extremely obese children and

adolescents (subgroup of study group 1) and 230 underweight students (study group 2) revealed heterozygotes for four silent and seven missense variants (allele frequencies < 1 %; Table 4) in addition to two previously known common SNPs in exon 1: rs 133073 (T/C) is silent and rsl33072 (G/A) introduces a non-conservative amino acid exchange in the N-terminus of the receptor (D32N). Three obese children and adolescent were compound heterozygous for two of the seven missense variants (D28V and T411M) as revealed by genotyping of the respective parents.

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

Study group Base position^"1" Exon Effect on amino Position within Frequency of (E) acid sequence^"1-1" the MCHRl^* heterozygotes*

Extremely 100193 C>T E l T25M Ν-ter ED 0.005 obese children 100202 A>T E l D28V Ν-ter ED 0.014 and 101966 C>T E 2 Silent 1L 2 0.005 adolescents 102218 C>T E 2 Silent TM 5 0.009

(n = 215) 102247 C>T E 2 T305M IL 3 0.005

102283 G>A E 2 R317Q H.3 0.005

102491 G>A E 2 Silent C-ter 0.005

102565 C>T E 2 T411M C-ter 0.009

Healthy 100202 A>T E l D28N Ν-ter ED 0.009 underweight 101962 G>A E 2 R21C )H IL 2 0.004 students 102402 A>C E 2 T357P EL 3 0.004

⁺ human BAC containing MCHRl , Z86090

** human MCHRl JC7080

According to http://www.ensembl.org/Homo sapiens/protview?peptide=ENSP00000249016

^# Genotype-frequencies are not different from Hardy-Weinberg equilibrium.

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).

In light of the confirmed association a transmission disequilibrium test (TDT) based on 525 obese children (subgroup of study group 1) and their 1,050 parents was 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 (A-allele of rsl33072 and the C-allele of rsl33073) 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. Gender specific TDTs revealed a non-significantly (p = 0.2) higher transmission of the A-allele of cSNP 133072 to male (transmission rate 62.2 %) than to female obese index patients (transmission rate 56.0 %). Table 5: Association and transmission disequilibrium of SNPs rs 133072 and rs 133073 in obesity rs 133072 (G/A) rs 133073 (T/C)

Association Genotype frequencies* Allele frequencies Genotype frequencies* Allele frequencies

GG GA AA G-allele A-allele TT TC CC T-allele C-allele

All obese individuals 261 354 104 876 562 244 361 114 849 589

(n = 719) (36.3 %) (49.2 %) (14.5 %) (60.9 %) (39.1 %) (33.9 %) (50.2 %) (15.9 %) (59.0 %) (41.0 %)

All non-obese 144 153 29 441 211 137 159 30 433 219 individuals^**

(n = 326) (44.2 %) (46.9 %) (8.9 %) (67.6 %) (32.4 %) (42.0 %) (48.8 %) (9.20 %) (66.4 %) (33.6 %) two sided p value 0.0026* 0.0016" 0.0010" 0.0013"

Transmission transmitted transmitted/ transmission two-sided transmitted transmitted/ transmission two-sided disequilibrium allele non-transm. rate [%] p-value allele non-transm. rate [%] p-value

1 . TRIOS^*" (N = A 290/206 58.5 0.00016 C 285/204 58.3 0.00025

525)

Genotype-frequencies are in Hardy-Weinberg equilibrium.

BMI > 90^th centile; includes 620 (359 females) unrelated obese children and adolescents (mean BMI 33.4 ± 6.6 kg/m²; mean age 14.0 ± years) and 99 (51 females) obese adults (mean BMI 29.1 ± 3.4 kg/m²; 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/m²; mean age 24.7 ± 2.6 years) and 2

(110 females) underweight students (BMI < 15^th percentile; mean BMI 18.3 ± 1.1 kg m²; mean age 25.2 ± 3.7 years) asymptotic Cochran-Armitage-trend test asymptotic Pearson Chi-square test trios based on subgroup of the 620 obese children and both of their parents

Individual and epidemiological risk estimation

Under the assumption that cSNP rs 133072 is functionally relevant, we calculated the genotype

relative risk to develop obesity (defined by BMI > 90^th percentile) 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. Based on the estimated population frequency of the A-allele of 31.0 % (95 % CI 28.2 - 33.7), the attributable risk estimate is 23.7 % (95 % CI 13.1 % - 34.3 %). This implies that about 24 % of the cases can be attributed to heterozygosity or homozygosity for the A-allele of rs 133072 in our study group. Similar risks apply to the other SNPs in strong linkage disequilibrium with rs 133072. Phenotype orientated post hoc tests

In light of hyperactivity in lean Mchrl '^' mice (Chen et al. 2002; Marsh et al. 2002) we explored the post hoc hypothesis, that the MCHRl GG genotype of rs 133072, which is not associated with obesity, is correlated with elevated activity levels. For 399 obese children and adolescents (subgroup of study group 1) mothers had retrospectively rated activity levels of their children at different age periods. Indeed, a trend towards elevated activity levels was detected in those offspring with the genotype GG of the cSNP rs 133072; this effect relied on the male children only (Table 6). Additional post hoc analyses based on 491 male and female obese children and adolescents (subgroup of study group 1) revealed no genotype specific differences for rs 133072 in percent body fat (data not shown); percent body fat was somewhat higher in females with the AA-genotype (n = 55; 36.7 %) than in those with the GG-genotype (n = 184; 34.1 %; p-value 0.07). A final post hoc analysis revealed a higher percent energy intake from fat in obese individuals with the rs 133072 GG-genotype (n = 61; 40%) versus those with the AA- genotype (n = 14; 34.7% p = 0.030). This effect was largely attributable to the difference in percent fat intake in females (39.4%; p = 0.024). Table 6: Descriptive data pertaining to motor restlessness of index patients in relationship to genotype for rsl33072

Age (years) Total Boys Girls

AA AG GG AA AG GG AA AG GG

< 1 (n = 399) 5.3 6.1 8.8 3.85 10.0 12.7 6.9 4.6 6.3

1-6 (n = 399) 8.8 12.3 15.0 11.5 14.8 25.4** 6.5 10.5 7.1

7-10 (n = 399) 10.7 11.0 14.5 1.1 11.4 16.1 13.3 10.7 13.3

11-14 (n = 327) 12.5 10.5 11.0 8.3 12.7 15.8 16.7 9.0 7.1

> 15 (n = 105) 0 4.2 8.9* 0 0 15.8** 0 6.1 3.9 one sided nominal p-values < 0.1* and < 0.05**

Identification of haplotypes and additional SNP and haplotype based TDTs

In order to identify additional SNPs at the MCHRl locus we sequenced the MCHRl CDS as well as 9.808 bp of its 5' region in nine obese children and adolescents who were homozygous for C at sSNP rs 133072 and who had received at least one A-allele from a heterozygous parent in the initial positive TDT (247 trios), as well as in ten obese children and adolescents homozygous for G. 22 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

SNPs ofSNPs ofthe SNPs new SNPs in clone inHGV in Z86090

1 91268 new A/C to submit

2 91276 new A/G to submit

91497 rsl33060 T/A^*

3 94443 rs86708 A/G

95368 rsl33061 indel T^*

4 95538 rsl33062 A/G

5 95767 rs2032512A/C

6 95949 new T/A 1745613

7 96227 new AG 1745614

8 96330 rsl33063 C/T

9 96402 rsl33064A/G

10 96797 new G/A 1745615

11 97004 new A/G 1745616

97718 rs3044564 indel T^*

12 97723 new C/T 1745617

98484 rs2105909 G/A*

13 98848 neuC/T 1745618

14 99803 rsl33068 G/C

15 99142 rsl33069 A/C

16 99450 rsl33070 G/A

99909 rsl33071 T/C^*

17 100220 rsl33072 A/G

18 100373 rsl33073 C/T

19 101352 new T/G 1745619

102660 rs2071827 CT^*

20 103156 rsl33074T/C

21 103270 new A/G 1745620

22 103351 rs3087592 C/T not confirmed in the subgroups of study group 1 All nine patients who were selected for homozygosity of the A allele of rs 133072, were also homozygous at 16 of the 22 SNPs (homozygosity not detected for A-91268-C, A-91276-G, SNP001745617, SNP001745618, SNP001745619 and rs3087592). The 10 obese individuals selected for homozygosity of allele T were homozygousTfor all SNPs apart from SNP001745613, SNP001745616 and SNP001745620 (Table 8). This indicates the existence of two ancestral haplotypes differing in at least 13 sites spanning the sequenced region of 13,378 bp.

Table 8: Genotypes of 22 SNPs in 9 and 10 obese children and adolescents homozygous for C or T of rsl33073, respectively

*Single letters indicate homozygosity.

TDTs for sixteen other identified SNPs (in addition to SNPs rsl33072 and rsl33073) were performed in a subgroup (n = 61 including the 19 individuals selected homozygosity at rsl33072) and/or in the total of 525 trios (subgroups of study group 1). Strong transmission disequilibrium for eleven of these SNPs was detected.

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); ⁸more frequently transmitted allele for all single SNP and haplotype based TDTs; ^#numbers: transmitted/non-transmitted, transmission rate (%), two-sided p-value. Method: S = sequencing; R = RFLP; A = ARMS-RFLP; SS = SSCP

Based on parental genotypes 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)

Allele frequencies marker allele 1 allele 2 rsl33068 0.3600 0.6400 rsl33072 0.3495 0.6505 rsl33073 0.3662 0.6338 rsl33074 0.4552 0.5448

rsl33068-rsl33072- expected (no LD)* estimated (LD)* rsl33073-rsl33074

GGCT 2.10% 31.34% GGCC 2.51% 2.84% GGTT 3.63% 0.04% GGTC 4.34% 0.06% GACT 3.90% 1.49% GACC 4.67% 0.00% GATT 6.76% 0.06% GATC 8.09% 0.18% CGCT 3.73% 0.24% CGCC 4.46% 0.10% CGTT 6.45% 0.09% CGTC 7.72% 0.25% CACT 6.94% 0.32% CACC 8.30% 0.29% CATT 12.01% 11.95% CATC 14.37% 50.75%

* exp./est. freq > 1%

Table 9b: TDTs for haplotypes 525 trios including cSNPs rsl33072 and rsl33073, for both genders and males and females, respectively

525 trios (both genders) 525 trios (males) 525 trios (females) haplotypes haplotypes haplotypes Formed by two^a, three^b and four' SNPs, formed by two⁸, three⁰ and four⁰ SNPs, formed by t wo^a, three^b and four^c SNPs, respectively respectively respectively

Rsl33068 j rsl33072 rsl33073 | rsl33074 rsl33068 j rsl33072 rsl33073 rsl33074 rsl33068 ; rsl33072 rsl33073 j rsl33074 ;

^a 229/140 62.1 % ^a 104/53 66.2 % ^a i^~25/87 59.0 % 0.000004* 0.000047* 0.0091*

^a 230/142 61.8 % j ^a 105/55 65.6 % ^a 125/87 59.0 % 0.000005* i 0.000077* 0.0091*

^a 188/123 60.4 % ^a 84/48 63.6 % ^a 104/75 58.1 % i 0.00023* 0.0017* 0.030* i

^b 228/140 62.0 % 105/53 66.5 % * 123/87 58.6 % 0.000004* 0.000035* 0.013*

^b 190/117 61.9 % i ^b 88/42 67.7 % ^b 102/75 57.6 % j 0.000031* j 0.000055* 0.042* j

^c 186/117 61.4 % ^c 88/42 67.7 % ^c 98/75 56.6 % i j 0.000074* 0.000055* 0.080* j

numbers: transmitted/non-transmitted, transmission rate (%), two-sided p-value.

Discussion

We have detected both association and transmission disequilibrium of a haplotype starting 9.8 kb upstream of MCHRl and extending into its 3' end in obese German children and adolescents. The identification of this haplotype was initially based on detection of association and transmission disequilibrium with SNPs rsl33072 and rsl33073, which were both confirmed in independent study groups and trios, respectively. The transmission rates in our post hoc TDT based on 525 trios were 58.5 % and 58.3 %, respectively (Table 5).

We also sequenced the MCHRl eds and 9.8 kb upstream in probands enriched for relevant variants. We identified two ancestral haplotypes, which within the resequenced segment share almost completely 13 SNPs (Table 8). Each of the ancestral haplotypes harbors additional 9 SNPs, those 5 who were analysed by TDT in 61 trios all led to lower transmission rates (Table 9a). We concluded that this implicates a functional role of either the whole haplotype comprising the A-allele of SNP rsl33072 and the C-allele of SNP rsl33073 or alternatively of one or more of its distinct SNPs. TDTs based on all 525 trios revealed that the haplotypes formed by up to four SNPs led to transmission rates slightly in excess (Tables 6a and b) of those observed upon analysis of any single SNP.

Our original and confirmed association and TDT studies are bolstered by evidence obtained by in silico and in vitro functional analyses pertaining to the detected genomic variations: (a) Within the preferentially transmitted haplotype rs 133072 is the only cSNP; (b) the non-conservative exchange of Asp³² to Asn³² was associated with a change in function of MCHRa; (c) promoter studies based on the comparison of the two genomic fragments encompassing 1,181 bp upstream of the translation initiation of MCHRl comprising either (a) the haplotype associated with obesity (rsl33068: G; rsl33069: A; rsl33070 G) or the haplotype not associated with obesity (rsl33068: C; rsl33069: C; rsl33070: A) also suggest a change in activity: Both basal and cAMP-induced luciferase expression were enhanced by the regulatory region from the haplotype associated with obesity.

We conclude, that in accordance with criteria for a solid association study in common disease (Dahlmann et al. 2002; Hirschhorn and Altshuler 2002; Campbell and Rudan 2002) we have detected association for a highly plausible candidate gene in population-based and family-based studies. This has been confirmed in independent and large study groups from Marburg. A potential functional implication of the cSNP rs 133072 has been detected

Electronic-Database Information

Accession numbers and URLs for data in this article are as follows:

- known human MCHRl mRNAs, AB063174, NM .005297

- ESTs extending the published 5' end of human MCHRl mRNA, BE312542, BG519797, BF313837, BI818110

- revised human MCHRl mRNA, AF490537

- rat MCHRl mRNA, AF008650

- human BAC containing MCHRl, Z86090 -http://www.ncbi.nlm.nih.gov/SNP/ -(http://www.phrap.org/phrap.docs/phrap.html

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Claims

Claims

1. A diagnostic composition containing a polynucleotide being selected from the group consisting of:

(a) a polynucleotide comprising the nucleic acid sequence shown in Figure 1 (b) ;

(b) a polynucleotide encoding a polypeptide comprising the amino acid sequence shown in Figure 1 (b) ;

(c) a polynucleotide having the nucleic acid sequence shown in Figure 1 (a) ;

(d) a polynucleotide encoding a polypeptide having the amino acid sequence shown in Figure 1 (a) ;

(e) 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^V or 3' of said position an insertion of at least one nucleotide;

(f) 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;

(g) a polynucleotide encoding an MCHRl polypeptide or fragment thereof, wherein said polypeptide comprises an amino acid substitution at a position corresponding to position +32 of the amino acid sequence of the MCHRl polypeptide;

(h) a polynucleotide encoding an MCHRl polypeptide or fragment thereof, wherein said polypeptide comprises an amino acid substitution of Asp to Asn at a position corresponding to position +32 of the amino acid sequence of the MCHRl polypeptide;

(i) at least one polynucleotide comprising one or more of the nucleotide exchanges (SNP's) listed in Table 7 and at least 8 bases of sourrounding sequence of the MCHRl gene, whereby the sourrounding sequence may be at the 5' end of the SNP, the 3' end, or both.

2. The diagnostic composition of claim l(c)-(i), wherein the polynucleotide has a length of at least 13 nucleotides .

3. The diagnostic composition of claim 1 or 2, wherein said polynucleotide is associated with obesity or a predisposition for obesity.

4. A diagnostic composition containing a vector comprising the polynucleotide as defined in any one of claims 1 to 3.

5. A diagnostic composition containing a polypeptide or fragment thereof encoded by the polynucleotide as defined in any one of claims 1 to 3.

6. A diagnostic composition containing an antibody specifically binding to the polypeptide as defined in claim 5.

7. The diagnostic composition of claim 6, wherein the antibody specifically recognizes an epitope containing one or more amino acid substitutions resulting from a nucleotide variation as defined in claim 1.

8. The diagnostic composition of any one of claims 1 to 7, wherein the polynucleotide, polypeptide or the antibody is detectably labeled.

9. The diagnostic composition of claim 8, wherein the label is selected from the group consisting of a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, or an enzyme.

10. A diagnostic composition containing one or more primers flanking the nucleic acid variation within the polynucleotide as defined in claim 1.

11. A transgenic non-human animal comprising at least one polynucleotide as defined in claims 1 to 3 or the vector as defined in claim 4.

12. The transgenic non-human animal of claim 11 which is a mouse or a rat .

13. 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 expression of the MHCRl gene or aberrant activity of the MCHRl protein is determined.

14. The method of claim 13, wherein the aberrant expression is a reduced or eliminated expression and the aberrant activity is a partial or complete loss of activity.

15. A pharmaceutical composition comprising a functional MCHRl protein or a polynucleotide encoding said MCHRl protein and a pharmaceutically acceptable excipient, diluent or carrier.

16. Use of an MCHRl protein or polynucleotide as defined in claim 15 for the preparation of a medicament for prophylaxis or treatment of obesity related to the presence of a molecular variant of the MCHRl gene.