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WO2004001071A2 - Analyse d'une predisposition genetique et traitement correspondant - Google Patents

Analyse d'une predisposition genetique et traitement correspondant Download PDF

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Publication number
WO2004001071A2
WO2004001071A2 PCT/GB2003/002730 GB0302730W WO2004001071A2 WO 2004001071 A2 WO2004001071 A2 WO 2004001071A2 GB 0302730 W GB0302730 W GB 0302730W WO 2004001071 A2 WO2004001071 A2 WO 2004001071A2
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Prior art keywords
diabetes
type
insulin resistance
lpin2
agent
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PCT/GB2003/002730
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English (en)
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WO2004001071A3 (fr
Inventor
Jan Pullen
Jolyon Holdstock
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Oxagen Limited
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Priority to AU2003236918A priority Critical patent/AU2003236918A1/en
Publication of WO2004001071A2 publication Critical patent/WO2004001071A2/fr
Publication of WO2004001071A3 publication Critical patent/WO2004001071A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to methods of determining genetic predisposition to type 2 diabetes and/or insulin resistance and to methods of treating type 2 diabetes and/or insulin resistance and related conditions, such as obesity.
  • a major objective of the current drive to sequence the human genome and characterise common polymo ⁇ hisms is to understand better the molecular basis for disease. Not only will this lead to new and improved targets for drags, but it will also allow better matching of treatment and disease, and provide assays for genetic risk factors that identify those individuals that are at greatest risk of disease. This would allow both screening and treatment to be better targeted, and to increase the cost-effectiveness of health-care provision.
  • Type 2 diabetes and/or insulin resistance can be broadly defined as a disorder caused by resistance to the action of insulin and glucose intolerance.
  • Type 2 diabetes and/or insulin resistance demonstrates a substantial genetic component, but, with few exceptions, e.g., Calpain 10, specific genetic components have not been identified.
  • Other features of this disease include complications due to micro- and macro-vascular complications such as retinopathy, nephropathy and neuropathies. Genetic factors that influence susceptibility to such disorders offer the prospect of providing important new insights, and will yield important new diagnostic and prognostic tests.
  • the establishment and maintenance of a multi-factorial disease such as type 2 diabetes and/or insulin resistance are the result of a complex interaction between pathways for metabolism and signal transduction.
  • This invention is concerned with the identification of polymo ⁇ hisms in the gene for Lipin 2 that affect gene expression or activity, and which therefore influence the susceptibility of individuals carrying those polymo ⁇ hisms to type 2 diabetes and/or insulin resistance and related conditions.
  • Such related conditions include obesity.
  • the present inventors have shown that the human LPIN2 gene is associated with type 2 diabetes and/or insulin resistance. More specifically, the present inventors have identified four polymo ⁇ hisms in the LPLN2 gene that are associated with type 2 diabetes and/or insulin resistance. These polymo ⁇ hisms are shown in Figure 1 and Table 4.
  • polymo ⁇ hisms Three of the polymo ⁇ hisms identified are single nucleotide polymo ⁇ hisms (SNPs).
  • SNPs single nucleotide polymo ⁇ hisms
  • the fourth polymo ⁇ hism is a deletion and substitution mutation.
  • the polymo ⁇ hisms are defined herein as shown in Table 4. Those skilled in the art can determine whether or not a LPIN2 polynucleotide comprises one or more of the polymo ⁇ hisms using standard methods.
  • a first aspect of the invention provides a method of determining whether an individual is predisposed to type 2 diabetes and/or insulin resistance which method comprises typing the LPIN2 gene region or the LPIN2 protein of the individual, thereby determining whether the individual is genetically predisposed to type 2 diabetes and/or insulin resistance.
  • the LPLN2 gene region is any one of the sequences which are set out in Figures 2, 3 or 4.
  • the LPIN2 protein is the protein encoded by the LPIN2 gene region or equivalent (due to the degenerate code). Typing the LPIN2 gene region or the LPLN2 protein can comprise typing one or more particular part of the gene region or protein.
  • Lipin 2 The gene referred to here is sometimes referred to in the art as either Lipin 2 or LPL 2 and may also be referred to here as either Lipin 2 or LPIN2.
  • the method of typing may comprise identifying whether the individual has 1) one or more polymo ⁇ hisms which is selected from the polymo ⁇ hisms defined in Table 4: 2) any other polymo ⁇ hism which is associated with predisposition to type 2 diabetes and/or insulin resistance and/or 3) has one or more polymo ⁇ hisms which is in linkage disequilibrium with a polymo ⁇ hism which is associated with predisposition to type 2 diabetes and/or insulin resistance.
  • the method may comprise contacting a sample from an individual with a specific binding agent for the polymo ⁇ hism and determining whether the agent binds to the polymo ⁇ hism, wherein binding of the agent to the polymo ⁇ hism indicates that the individual is genetically predisposed to type 2 diabetes and/or insulin resistance.
  • This method includes analysis of linkage disequilibrium polymo ⁇ hisms. Examples of such LD SNPs are given in Table 9.
  • the method may comprise typing for the presence of a polymo ⁇ hism in the LPIN2 gene which is for both alleles of the individual.
  • This method includes analysis of polymo ⁇ hisms in linkage disequilibrium with associated polymo ⁇ hisms. Examples of such SNPs are given in Table 9.
  • a second aspect of the invention relates to a method for treating an individual who has been identified as having a genetic predisposition to type 2 diabetes and/or insulin resistance by a method according to the first aspect of the invention, further comprising administering to the individual a therapeutically effective amount of an agent which prevents or treats type 2 diabetes and/or insulin resistance.
  • prevention or treatment includes any ameliation of the effects of type 2 diabetes and/or insulin resistance.
  • a third aspect of the invention provides the use of an agent which prevents or treats type 2 diabetes and/or insulin resistance in the manufacture of a medicament for prevention or treating an individual, which individual has been identified as having a genetic predisposition to type 2 diabetes and/or insulin resistance, according to a method of the first aspect of the invention.
  • a fourth aspect of the invention provides a method for identifying an agent for the prevention or treatment of type 2 diabetes and/or insulin resistance, which method comprises:-
  • test agent determines whether the test agent is capable of binding to the polypeptide or polynucleotide or modulating the activity or expression of the polypeptide or polynucleotide.
  • a fifth aspect of the invention provides a method of formulating a pharmaceutical composition comprising:-
  • a sixth aspect of the invention provides an agent identified by a method according to the fourth aspect of the invention.
  • a seventh aspect of the invention provides a pharmaceutical composition for the prevention or treatment of type 2 diabetes and/or insulin resistance comprising an agent according to the sixth aspect of the invention and a pharmaceutically acceptable carrier or diluent.
  • An eighth aspect of the invention provides the use of an agent according to the sixth aspect of the invention, in the manufacture of a medicament for treating or preventing type 2 diabetes and/or insulin resistance.
  • a ninth aspect of the invention provides a method of treating type 2 diabetes and/or insulin resistance, which method comprises administering an agent according to the sixth aspect of the invention.
  • the agent may be administered in a therapeutically effective amount. It may be administered to a patient in need thereof.
  • a tenth aspect of the invention provides a method of treating type 2 diabetes and/or insulin resistance, which method comprises :-
  • An eleventh aspect of the invention provides the use of a LPIN2 polynucleotide or polypeptide, not having a mutation or polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance, in the manufacture of a medicament for the prevention or treatment of type 2 diabetes and/or insulin resistance.
  • a twelfth aspect of the invention provides a probe, primer or antibody for use in a method according to the first aspect of the invention, which is capable of selectively detecting a polymo ⁇ hism in the LPIN2 gene associated with type 2 diabetes and/or insulin resistance.
  • a thirteenth aspect of the invention provides a method of identifying a polymo ⁇ hism which can be used to determine whether an individual has a genetic predisposition to type 2 diabetes and/or insulin resistance, the method comprising screening the LPIN2 gene region or LPIN2 protein of one or more individuals.
  • a fourteenth aspect of the invention provides a method of determining whether a candidate polymo ⁇ hism in the LPL 2 gene or LPLN2 protein can be typed to determine whether an individual is genetically predisposed to type 2 diabetes and/or insulin resistance, which method comprises detecting whether the candidate polymo ⁇ hism is (i) associated with type 2 diabetes and/or insulin resistance or (ii) is in linkage disequilibrium with a polymo ⁇ hism which is associated with type 2 diabetes and/or insulin resistance, and thereby determining whether the polymo ⁇ hism can be typed to determine whether an individual has a genetic predisposition to type 2 diabetes and/or insulin resistance.
  • a fifteenth aspect of the invention provides a method for treating or preventing type 2 diabetes and/or insulin resistance in an individual comprising: -
  • a sixteenth aspect of the invention provides a cell line comprising a LPIN2 polynucleotide having a polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance.
  • a seventeenth aspect of the invention provides a non-human animal which is transgenic for a LPIN2 polynucleotide having a polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance.
  • An eighteenth aspect of the invention provides the use of a cell line according to the sixteenth aspect of the invention or a non-human animal according to the seventeenth aspect of the invention in screening for an agent for use in diagnosis or treatment of individuals having a genetic predisposition to type 2 diabetes and/or insulin resistance.
  • a nineteenth aspect of the invention provides a method for determining the efficacy of an agent useful in the treatment of type 2 diabetes and/or insulin resistance in a subject having a polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance in the LPIN2 polynucleotide, which method comprises :-
  • a twentieth aspect of the invention provides a method according to the nineteenth aspect of the invention wherein said polynucleotide or protein is in a subject having said polymo ⁇ hism or in a non-human animal according to the seventeenth aspect of the invention and step (iii) comprises monitoring the effect of administering the agent on one or more phenotype of type 2 diabetes and/or insulin resistance.
  • the invention provides methods for determining whether a subject is susceptible to type 2 diabetes and/or insulin resistance.
  • the method comprises determining whether the subject has a polymo ⁇ hism in the LPIN2 gene or a polymo ⁇ hism in linkage disequilibrium, which polymo ⁇ hism is associated with type 2 diabetes and/or insulin resistance.
  • the polymo ⁇ hism may be associated by linkage disequilibrium.
  • the subject will be a human.
  • the subject is preferably asymptomatic for type 2 diabetes and/or insulin resistance.
  • the subject may show clinical symptoms of predisposition to type 2 diabetes and/or insulin resistance, for example obesity.
  • the subject may have relatives who have been diagnosed as having type 2 diabetes and/or insulin resistance or a predisposition thereto.
  • a subject is genetically predisposed, or susceptible, to type 2 diabetes and/or insulin resistance if the LPIN2 gene with one or both alleles thereof comprise a polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance.
  • the polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance may cause a change in the amino acid sequence of the LPIN2 protein and may thus have a functional effect on the LPIN2 protein.
  • the SNP defined herein as LPN2X10.C145T results in a change in the coding sequence of Leu to Phe in a central region of the LPIN2 protein.
  • the polymo ⁇ hism may have no effect on the amino acid sequence of the LPIN2 protein.
  • Such a polymo ⁇ hism may be in inherited with a second polymo ⁇ hism on the same haplotype that causes a change in the amino acid sequence of the LPIN2 protein.
  • a polymo ⁇ hism may also be in a regulatory region of the LPIN2 gene.
  • a polymo ⁇ hism in a regulatory region of the LPDST2 gene may affect expression of the LPLN2 protein.
  • Preferred polymo ⁇ hisms that do not affect the coding region of the LPIN2 gene are the polymo ⁇ hisms LPN2X2.INS TTAATA, LPN2X19.T220A, LPN2X20.C185T as shown in Table 4.
  • a polymo ⁇ hism suitable for use in diagnosing the existence of or susceptibility to type 2 diabetes and/or insulin resistance may be identified by screening the LPIN2 gene of one or more individuals.
  • One skilled in the art can determine whether a candidate polymo ⁇ hism can be typed to determine whether an individual has, or is susceptible to, type 2 diabetes and/or insulin resistance, by determining whether the candidate polymo ⁇ hism is associated with type 2 diabetes and/or insulin resistance or is in linkage disequilibrium with a polymo ⁇ hism which is associated with type 2 diabetes and/or insulin resistance.
  • nucleic acid includes modified and variant forms of nucleic acid, including synthetic bases and locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • Linkage disequilibrium is the tendency for the alleles at two or more polymo ⁇ hic sites to be correlated in populations.
  • SNPs A/a & B/b with an allele frequency of 50% one would expect to see the four possible haplotypes (combinations of alleles on a chromosome - AB, Ab, aB and bb) at equal frequencies of 25%.
  • haplotypes combinations of alleles on a chromosome - AB, Ab, aB and bb
  • the actual haplotype frequencies might be AB 45%, Ab 2.5%, aB 2.5%, bb45%.
  • the presence of an allele at one or more SNPs alone or in combination can be used to infer the presence of a particular allele at the other SNP.
  • Polymo ⁇ hisms which are in linkage disequilibrium with each other in a population tend to be found together on the same chromosome. Typically one is found at least 30% of the times, for example at least 40%, 50%, 70% or 90%, of the time the other is found on a particular chromosome in individuals in the population.
  • polymo ⁇ hisms which are not functional susceptibility polymo ⁇ hisms, but are in linkage disequilibrium with the functional polymo ⁇ hisms may act as a marker indicating the presence of the functional polymo ⁇ hism.
  • Polymo ⁇ hisms which are in linkage disequilibrium with any of the polymo ⁇ hisms mentioned herein are typically within 500kb, preferably within 400kb, 200kb, 100 kb, 50kb, lOkb, 5kb or 1 kb of the polymo ⁇ hism.
  • the term "gene region" generally encompasses any of these distances from 5' to the transcription start site and 3' to the transcription termination site of the gene.
  • the polymo ⁇ hism is typically an insertion, deletion or substitution with a length of at least 1, 2, 5 or more base pairs or amino acids.
  • the polymo ⁇ hism is typically a substitution of 1 base pair, i.e. a single polynucleotide polymo ⁇ hism (SNP).
  • SNP single polynucleotide polymo ⁇ hism
  • polymo ⁇ hic position is used herein to refer to the nucleotide position(s) which differ between polymo ⁇ hic variants of the LPIN2 gene.
  • the polymo ⁇ hism may be 5' to the coding region, in the coding region, in an intron or 3' to the coding region.
  • the polymo ⁇ hism may be a functional mutation which contributes to type 2 diabetes and/or insulin resistance, but may be a polymo ⁇ hism which is inherited together with a functional mutation of the LPIN2 gene on the same chromosome or may be a polymo ⁇ hism in a regulatory region.
  • the polymo ⁇ hism will be associated with one or more phenotype of type 2 diabetes and/or insulin resistance.
  • the polymo ⁇ hism will generally cause, or be in linkage disequilibrium with a polymo ⁇ hism that causes, a change in any of the characteristics of the LPTN2 protein, such as expression, activity, splicing, cellular localisation or the pattern of expression in different tissues.
  • the polymo ⁇ hism typically has an agonistic or antagonistic effect on any of these characteristics of the receptor or pathway. Generally this will lead to a consequent increase or decrease in the activity of the pathway.
  • a polymo ⁇ hism which can be typed to determine susceptibility to type 2 diabetes and/or insulin resistance may be identified by a method of determining whether a candidate polymo ⁇ hism in the LPIN2 gene is associated with type 2 diabetes and/or insulin resistance.
  • a polymo ⁇ hism which can be typed to determine susceptibility to type 2 diabetes and/or insulin resistance may be identified by detecting polymo ⁇ hisms in a LPIN2 protein or in the gene region expressing the protein. Such a polymo ⁇ hism may be detected using any suitable polymo ⁇ hism detection method, such as sequencing. The detection method may be based on the detection of a difference in a characteristic between a LPLN2 polynucleotide or protein that carries the polymo ⁇ hism and one which does not. Typically, mobility of the proteins, such as mobility on a gel, may be detected. The polymo ⁇ hism may be identified in an individual who has or is suspected as having type 2 diabetes and/or insulin resistance.
  • a polymo ⁇ hism may be identified by any suitable method. For example, DNA samples from heterozygous individuals may be analysed to detect heteroduplexes and the sequences of such heteroduplexes may be determined to enable identification of the polymo ⁇ hism as described in more detail in the Examples.
  • a polymo ⁇ hism which can be typed to determine susceptibility to type 2 diabetes and/or insulin resistance may be identified by a method comprising the determination of whether a candidate polymo ⁇ hism in the LPIN2 gene region or LPIN2 protein is (i) associated with susceptibility to type 2 diabetes and/or insulin resistance or (ii) is in linkage disequilibrium with a polymo ⁇ hism which is associated with type 2 diabetes and/or insulin resistance, and thereby determining whether the polymo ⁇ hism can be typed to diagnose the presence of or susceptibility to type 2 diabetes and/or insulin resistance. Any suitable method may be used to determine whether a candidate polymo ⁇ hism is associated with type 2 diabetes and/or insulin resistance or is in linkage disequilibrium with a polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance.
  • Suitable tests include the transmission disequilibrium test (TDT) and the case/control test.
  • TDT transmission disequilibrium test
  • case/control test excess transmission of an allele is detected by comparing the allele frequencies within a disease population (cases) to the frequencies within a matched control population. The further an allele frequency in the cases differs from the corresponding frequency in the controls the smaller the resulting p-value and larger the significance of the association.
  • the identity of the nucleotide at a polymo ⁇ hic position of the LPLN2 gene is determined for at least one of the alleles carried by a subject. Typically the identity of the nucleotide(s) at the polymo ⁇ hic position is determined for both alleles carried by a subject. The identity of that/those nucleotide(s) may be determined for one or both alleles. The identity of the nucleotide at a polymo ⁇ hic position of the LPIN2 gene of the subject may be determined by any convenient method. Clearly, there are a large number of analytical techniques available to those skilled in the art for determining the identity of a nucleotide at a given position.
  • a sample is obtained from the subject.
  • a nucleic acid preparation is prepared from the sample; and then the sequence of the nucleotide at a polymo ⁇ hic position may be determined.
  • the method is typically carried out in vitro on a sample from the individual.
  • the sample typically comprises a body fluid of the individual and may, for example, be obtained using a swab, such as a mouth swab.
  • the sample may be a blood, urine, saliva, cheek cell or hair root sample.
  • the sample is typically processed before the method is carried out, for example, DNA extraction may be carried out.
  • the polynucleotide or protein in the sample may be cleaved either physically or chemically (e.g. using a suitable enzyme).
  • nucleic acid is prepared from the sample according to techniques well-known to those skilled in the art. Typically, a preparation of total nuclear DNA is prepared. The nucleic acid preparation may then be used to determine the sequence at a polymo ⁇ hic position of the LPLN2 gene. Alternatively, it may not be necessary to include a nucleic acid preparation step and a polymo ⁇ hism discrimination technique may be carried out directly on the sample.
  • allelic variation at a polymo ⁇ hic position requires a polymo ⁇ hism discrimination technique and a signal generation system.
  • an optional amplification step is sometimes used.
  • the polymo ⁇ hism is typically detected by directly determining the presence of the polymo ⁇ hism sequence in a polynucleotide or protein of the individual.
  • a polynucleotide is typically genomic DNA or mRNA, or a polynucleotide derived from these polynucleotides, such as in a library made using polynucleotide from the individual (e.g. a cDNA library).
  • a library made using polynucleotide from the individual e.g. a cDNA library.
  • the presence of the polymo ⁇ hism is determined in a method that comprises contacting a polynucleotide or protein of the individual with a specific binding agent for the polymo ⁇ hism and determining whether the agent binds to a polymo ⁇ hism in the polynucleotide or protein, the binding of the agent to the polymo ⁇ hism indicating that the individual has or is susceptible to type 2 diabetes and/or insulin resistance.
  • the agent will also bind to flanking nucleotides and amino acids on one or both sides of the polymo ⁇ hism, for example at least 2, 5, 10, 15 or more flanking nucleotide or amino acids in total or on each side.
  • determination of the binding of the agent to the polymo ⁇ hism can be done by determining the binding of the agent to the polynucleotide or protein.
  • the agent is able to bind the corresponding wild-type sequence by binding the nucleotides or amino acids which flank the polymo ⁇ hism position, although the manner of binding will be different to the binding of a polynucleotide or protein containing the polymo ⁇ hism, and this difference will generally be detectable in the method (for example this may occur in sequence specific PCR as discussed below).
  • the presence of the polymo ⁇ hism is being determined in a polynucleotide it may be detected in the double stranded form, but is typically detected in the single stranded form.
  • the agent may be a polynucleotide (single or double stranded) typically with a length of at least 10 nucleotides, for example at least 15, 20, 30 or more polynucleotides.
  • the agent may be a molecule structurally related to a polynucleotide, which molecule that comprises units (such as purines or pyrimidines) able to participate in Watson-Crick base pairing.
  • the agent may be a polypeptide, typically with a length of at least 10 amino acids, such as at least 20, 30, 50, 100 or more amino acids.
  • the agent may be an antibody (including a fragment of such an antibody which is capable of binding the polymo ⁇ hism).
  • a polynucleotide agent which is used in the method will generally bind to the polymo ⁇ hism, and flanking sequence, of the polynucleotide of the individual in a sequence specific manner (e.g. hybridise in accordance with Watson-Crick base pairing) and thus, typically, has a sequence which is fully or partially complementary to the sequence of the polymo ⁇ hism and flanking region.
  • the partially complementary sequence is homologous to the fully complementary sequence.
  • the agent is a probe.
  • This may be labelled or may be capable of being labelled indirectly.
  • the detection of the label may be used to detect the presence of the probe on (and hence bound to) the polynucleotide or protein of the individual.
  • the binding of the probe to the polynucleotide or protein may be used to immobilise either the probe or the polynucleotide or protein (and thus to separate it from a composition or solution).
  • the polynucleotide or protein of the individual is immobilised on a solid support and then contacted with the probe.
  • the presence of the probe immobilised to the solid support (via its binding to the polymo ⁇ hism) is then detected, either directly by detecting a label on the probe or indirectly by contacting the probe with a moiety that binds the probe.
  • the solid support is generally made of nitrocellulose or nylon.
  • the method may be based on an ELIS A system.
  • the method may be based on an oligonucleotide ligation assay in which two oligonucleotide probes are used. These probes bind to adjacent areas on the polynucleotide which contains the polymo ⁇ hism, allowing (after binding) the two probes to be ligated together by an appropriate ligase enzyme. However the two probes will only bind (in a manner which allows ligation) to a polynucleotide that contains the polymo ⁇ hism, and therefore the detection of the ligated product may be used to determine the presence of the polymo ⁇ hism.
  • the probe is used in a heteroduplex analysis based system to detect polynucleotide polymo ⁇ hisms.
  • a heteroduplex analysis based system to detect polynucleotide polymo ⁇ hisms.
  • the probe when the probe is bound to polynucleotide sequence containing the polymo ⁇ hism, it forms a heteroduplex at the site where the polymo ⁇ hism occurs (i.e. it does not form a double strand structure).
  • a heteroduplex structure can be detected by the use of an enzyme which is single or double strand specific.
  • the probe is an RNA probe and the enzyme used is RNAse H which cleaves the heteroduplex region, thus allowing the polymo ⁇ hism to be detected by means of the detection of the cleavage products.
  • the method may be based on fluorescent chemical cleavage mismatch analysis which is described for example in PCR Methods and Applications 3, 268-71 (1994) and Proc. Natl. Acad. Sci. 85, 4397-4401 (1998).
  • the polynucleotide agent is able to act as a primer for a PCR reaction only if it binds a polynucleotide containing the polymo ⁇ hism (i.e. a sequence-specific or allele-specific PCR system).
  • a PCR product will only be produced if the polymo ⁇ hism is present in the polynucleotide of the individual.
  • the presence of the polymo ⁇ hism may be determined by the detection of the PCR product.
  • the region of the primer which is complementary to the polymo ⁇ hism is at or near the 3' end of the primer.
  • the polynucleotide agent will bind to the wild-type sequence, but will not act as a primer for a PCR reaction.
  • the method may be an RFLP based system. This can be used if the presence of the polymo ⁇ hism in the polynucleotide creates or destroys a restriction site which is recognised by a restriction enzyme. Thus treatment of a polynucleotide with such a polymo ⁇ hism will lead to different products being produced compared to the corresponding wild-type sequence. Thus the detection of the presence of particular restriction digest products can be used to determine the presence of the polymo ⁇ hism.
  • the presence of the polymo ⁇ hism may be determined based on the change which the presence of the polymo ⁇ hism makes to the mobility of the polynucleotide or protein during gel electrophoresis.
  • SSCP single-stranded conformation polymo ⁇ hism
  • Denaturing gradient gel electrophoresis is a similar system where the polynucleotide is electrophoresed through a gel with a denaturing gradient, a difference in mobility compared to the corresponding wild-type polynucleotide indicating the presence of the polymo ⁇ hism.
  • the presence of the polymo ⁇ hism may be determined using a fluorescent dye and quenching agent-based PCR assay such as the TaqmanTM PCR detection system.
  • a fluorescent dye and quenching agent-based PCR assay such as the TaqmanTM PCR detection system.
  • this assay uses an allele specific primer comprising the sequence around, and including, the polymo ⁇ hism.
  • the specific primer is labelled with a fluorescent dye at its 5' end, a quenching agent at its 3' end and a 3' phosphate group preventing the addition of nucleotides to it. Normally the fluorescence of the dye is quenched by the quenching agent present in the same primer.
  • the allele specific primer is used in conjunction with a second primer capable of hybridising to either allele, 5' of the polymo ⁇ hism.
  • Taq DNA polymerase adds nucleotides to the nonspecific primer until it reaches the specific primer. It then releases nucleotides, the fluorescent dye and quenching agent from the specific primer through its endonuclease activity. The fluorescent dye is therefore no longer in proximity to the quenching agent and fluoresces.
  • the mismatch between the specific primer and template inhibits the endonuclease activity of Taq and the fluorescent dye is not released from the quenching agent. Therefore, by measuring the fluorescence emitted, the presence or absence of the polymo ⁇ hism can be determined.
  • a polynucleotide comprising the polymo ⁇ hic region is sequenced across the region which contains the polymo ⁇ hism to determine the presence of the polymo ⁇ hism.
  • the presence of the polymo ⁇ hism may be determined indirectly, for example by measuring a characteristic of the LPIN2 gene region or protein, such as the expression or activity of an expression product of the LPIN2 gene, such as mRNA or protein.
  • Probes and primers for use in a diagnostic method of the invention may be polynucleotides of the invention as described herein.
  • Other preferred probes and primers comprise all or part of a sequence set out in Table 4.
  • a hybridisation probe may be immobilised on a surface.
  • the hybridisation probe may be immobilised on a micoarray which comprises other hybridisation probes for the detection of other nucleotide sequences.
  • the other hybridisation probes may, for example, be designed for the detection of other polymo ⁇ hisms in LPIN2 and/or in one or more other genes, such as calpain 10, which is associated with type 2 diabetes and/or insulin resistance.
  • the invention also provides a method of detecting the presence, in a sample from a subject, of a genetic alteration in a gene sequence coding for a LPL 2 protein.
  • the genetic alteration may be in the coding or non coding sequence of the LPLN2 gene and may be a substitution, insertion or deletion mutation.
  • the invention also provides a polynucleotide comprising the sequence set out in SEQ ID NO: 1, 2, 3 or 4, or a fragment thereof.
  • the polynucleotide encompasses positions 51 to 56 of SEQ ID NO: 1 (LPN2X2.TNS TTAATA), position 101 of SEQ ID NO: 2 [herein referred to as 201] (LPN2X10.C145T), position 101 of SEQ ID NO: 3 [herein referred to as 202] (LPN2X19.T220A ) or position 101 of SEQ ID NO: 4 (LPN2X20.C185T).
  • the nucleotide at position 101 [herein referred to as 203] SEQ ID NO: 3 is not A and is preferably T, and of SEQ ID No: 4 is not C and is preferably T.
  • a polynucleotide which does not encompass any of these preferred positions is preferably a primer which is part of a primer pair wherein the two primers are on complementary strands and which are on opposite sides of one of these preferred positions.
  • the invention also provides polynucleotides complementary to a polynucleotide of the invention.
  • the polynucleotide may be RNA or DNA including genomic DNA, synthetic DNA or cDNA.
  • the nucleotide sequence is a DNA sequence.
  • Nucleotide sequence information is provided in SEQ ID NOS: 1, 2, 3 and 4. Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press.
  • a variant of a polynucleotide comprising the sequence set out in SEQ ID NO: 1, 2, 3 or 4 or a fragment thereof is also provided.
  • a variant polynucleotide of the invention can hydridise to the sequence or complement of the sequence set out in SEQ ID NO: 1, 2, 3 or 4, at a level significantly above background. Background hybridisation may occur, for example, because of other DNAs present in a DNA library.
  • the signal level generated by the interaction between a polynucleotide of the invention and the sequence set out in SEQ ID NO: 1, 2, 3 or 4 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the sequence set out in SEQ ID NO: 1, 2, 3 or 4.
  • the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P.
  • Selective hybridisation may typically be achieved using conditions of medium stringency and preferably high stringency (for example, 2 x SSC [0.15M sodium chloride and
  • 0.015M sodium citrate] at about 50°C to about 60 °C may be carried out under any suitable conditions known in the art (see Sambrook et al., 1989, supra). For example, if high stringency is required suitable conditions include from 0.1 to 0.2 x SSC at about 60 °C to about 65 °C. If lower stringency is required suitable conditions include 2 x SSC at 60 °C.
  • sequence of SEQ ID NO: 1, 2, 3 or 4 may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10 substitutions provided that the modified sequence can still be used to detect a polymo ⁇ hism under highly stringent conditions.
  • the polynucleotide of SEQ ID NO: 1, 2, 3 or 4 may alternatively, or additionally, be modified by one or more insertions and/or deletions and/or by an extension at either or both ends.
  • a nucleotide sequence which is capable of selectively hybridising to the complement of the sequence set out in SEQ ID NO: 1, 2, 3 or 4, will generally have at least any of the following % sequence identities: 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 to that sequence over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 80, 90 or 100 contiguous nucleotides or most preferably over the full length the sequence.
  • the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).
  • BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
  • HSPs high scoring sequence pair
  • Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873- 5787.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • polynucleotides of the invention Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher sequence identity over longer lengths) being preferred.
  • a polynucleotide which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides.
  • Polynucleotides of the invention may be fragments of the sequence set out in SEQ ID NO: 1, 2, 3 or 4, wherein the fragments comprise the sequence of from 51 to 56 in SEQ ID NO: 1 (LPN2X2.LNS TTAATA), the sequence at position 101 of SEQ ID NO: 2 [herein referred to as 201] (LPN2X10.C145T), the sequence at position 101 of SEQ ID NO: 3 [herein referred to as 202] (LPN2X19.T220A ) or the sequence at position 101 of SEQ ID NO: 4 [herein referred to as 203] (LPN2X20.C185T).
  • the nucleotide at position 101 of SEQ ID No: 3 is not A and is preferably T and at position 101 of SEQ ID NO: 4 is not C and is preferably T.
  • Such fragments will preferably be at least 10, preferably at least 25, or at least 50, for example at least 100 nucleotides in length. They will typically be up to 40, 50, 60, 70 or 90 nucleotides in length.
  • a fragment of the invention is preferably a probe or primer suitable for use in a method of diagnosis according to the invention.
  • a probe or primer is thus capable of selectively detecting a polymo ⁇ hism in the LPLN2 gene.
  • a probe or primer may be from 10 to 50 base pairs, preferably from 15 to 30 and more preferably from 20 to 25 base pairs long.
  • a primer is preferably in a region surrounding the polymo ⁇ hism or encompassing the polymo ⁇ hism at 51 to 56 of SEQ ID NO: 1 (LPN2X2.INS TTAATA), the sequence at position 101 of SEQ ID NO: 2 (LPN2X10.C145T), the sequence at position 101 of SEQ ID NO: 3 (LPN2X19.T220A ) or the sequence at position 101 of SEQ ID NO: 4 (LPN2X20.C185T).
  • the nucleotide at one end of the primer is preferably complementary to the nucleotide at that position.
  • a probe is preferably capable of hybridising to a region including the sequence at position 101 of SEQ ID NO: 2 (LPN2X10.C145T), the sequence at position 101 of SEQ ID NO: 3 (LPN2X19.T220A ) or the sequence at position 101 of SEQ ID NO: 4 (LPN2X20.C185T) or a region including at least part of the sequence from 51 to 56 of SEQ ID NO: 1 (LPN2X2.LNS TTAATA).
  • test kit of the invention comprises means for determining the identity of the nucleotide at a polymo ⁇ hic position of the LPIN2 gene of the subject which means is typically a probe or primer of the invention.
  • Preferred test kits of the invention comprise means for determining the identity of the nucleotides at positions 51 to 56 of SEQ ID NO: 1, position 101 of SEQ ID NO: 2, position 101 of SEQ ID NO: 3 or position 101 of SEQ ID NO: 4.
  • a test kit of the invention may optionally comprise, appropriate buffer(s), enzymes, for example a thermostable polymerase such as Taq polymerase and/or control polynucleotides.
  • a kit of the invention may also comprise appropriate packaging and instructions for use in a method for determining the susceptibility of a subject to type 2 diabetes and/or insulin resistance.
  • a test kit of the invention may also comprise an agent which reduces the risk of type 2 diabetes and/or insulin resistance.
  • the invention also provides a cell comprising a LPLN2 polynucleotide having a polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance.
  • the cell typically expresses a LPIN2 polypeptide which may be mutated as a result of the polymo ⁇ hism.
  • the cell may be any suitable cell, for example a cell line in culture such as HEK293 cells, COS cells, HeLa cells or BHK cells.
  • a cell line in culture such as HEK293 cells, COS cells, HeLa cells or BHK cells.
  • the cell is an adipocyte.
  • the invention also provides a non-human animal transgenic for a LPIN2 polynucleotide having a polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance.
  • the animal is transgenic for a polynucleotide with a polymo ⁇ hism as mentioned above.
  • the genome of all or some of the cells of the animal comprises a polynucleotide of the invention.
  • the animal suffers from type 2 diabetes and/or insulin resistance, or is an animal model thereof, and can be therefore used in a method to assess the efficacy of agents in relieving type 2 diabetes and/or insulin resistance.
  • An animal useful as an animal model of type 2 diabetes and/or insulin resistance may be a knockout animal in which a functional form of the endogenous homologue of the human LPF 2 gene is not expressed.
  • the LPLN2 homologue may be knocked out in all tissues or in only specific tissues. Preferably functional LPIN2 expression is abolished in the adipocytes.
  • a transgenic animal of the invention may be generated by introducing into an embryonic stem cell of a host animal a vector comprising a polynucleotide operably linked to a promoter and optionally other control sequences such as an enhancer sequence (Hogan et al, A Laboratory Manual, Cold Spring Harbour).
  • the transgenic non-human animal is generally a mammal.
  • the transgenic non-human animal is typically of a species commonly used in biomedical research and is preferably a laboratory strain. Suitable animals include non-human primates and rodents. It is preferred that an animal of the invention is a rodent, particularly a mouse, rat, guinea pig, ferret, gerbil or hamster.
  • the invention may be used in the development of new drug therapies which selectively target one or more allelic variants of the LPIN2 gene (i.e. which have different polymo ⁇ hisms). Identification of a link between a particular allelic variant and predisposition to disease development or response to drug therapy may have a significant impact on the design of new drugs. Drugs may be designed to regulate the biological activity of the variants implicated in the disease process while minimising effects on other variants.
  • An agent identified by a method of the invention may be used in the manufacture of a medicament for the treatment of type 2 diabetes and/or insulin resistance.
  • a therapeutically effective amount of such an agent may be administered to a subject having a polymo ⁇ hism in the LPIN2 gene associated with type 2 diabetes and/or insulin resistance.
  • a method of identifying an agent which interacts with a LPLN2 protein encoded by a LPIN2 gene having a polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance is also provided.
  • the polymo ⁇ hism may be a polymo ⁇ hism which alters the amino acid sequence of the LPIN2 protein or may be a polymo ⁇ hism which has no effect on the amino acid sequence of the LPIN2 protein but which is co-inherited with a second polymo ⁇ hism, which may be unknown, that does result in a codon change(s).
  • the method typically comprises: (i) providing a protein as defined above;
  • test agent contacting the protein with a test agent; and (iii) monitoring any interaction between the protein and the test agent, thereby determining whether the test agent interacts with a LPIN2 protein encoded by a LPLN2 gene having a polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance.
  • An agent identified by this method may be administered to a subject having the polymo ⁇ hism in a method of treating type 2 diabetes and/or insulin resistance in the subject.
  • the effectiveness of particular anti-diabetic agents may be affected by or dependent on whether the individual has particular polymo ⁇ hisms in the LPIN2 gene.
  • the invention can allow the determination of whether an individual will respond to a particular anti-diabetic agent by determining whether the individual has a polymo ⁇ hism which affects the effectiveness of that agent.
  • the invention includes a method of treating a patient who has been identified as being able to respond to the agent, which method comprises administering the agent to the patient.
  • certain anti-diabetic agents may produce side effects in individuals with particular polymo ⁇ hisms in the LPIN2 gene.
  • the invention can also allow the identification of a patient who is at increased risk of suffering side effects due to such an anti-diabetic agent by identifying whether an individual has such a polymo ⁇ hism.
  • the polymo ⁇ hism may therefore have the greatest effect on the efficacy of drugs designed to modulate the activity of the LPIN2 or other components in its signalling pathway.
  • the invention may therefore be useful both to predict the clinical response to such agents and to determine therapeutic dose.
  • the present invention thus provides a method for determining the efficacy of, or side effects of, an agent useful in the treatment of type 2 diabetes and/or insulin resistance in a subject having a polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance in the LPIN2 gene, which method comprises:
  • the gene or protein is the endogenous gene or protein in a subject having the polymo ⁇ hism.
  • step (iii) comprises monitoring the effect of administering the agent on one or more phenotype of type 2 diabetes and/or insulin resistance.
  • An agent which modulates the expression or activity of the LPIN2 gene identified by a method of the invention may be used in the manufacture of a medicament for the treatment of type 2 diabetes and/or insulin resistance.
  • a therapeutically effective amount of such an agent may be administered to a subject having a polymo ⁇ hism in the LPF 2 gene associated with type 2 diabetes and/or insulin resistance.
  • the invention also provides a method of formulating a pharmaceutical composition, which method comprises:
  • the invention allows diagnosis of subjects at risk from type 2 diabetes and/or insulin resistance before the onset of disease symptoms or before the onset of severe symptoms.
  • the risk of type 2 diabetes and/or insulin resistance can thus be reduced, prevented or delayed by administration of treatment in advance of type 2 diabetes and/or insulin resistance appearance, for example before any symptoms or phenotypes of type 2 diabetes and/or insulin resistance are apparent.
  • Suitable treatments to reduce the risk of type 2 diabetes and/or insulin resistance include a change of lifestyle. Such changes of lifestyle include for example, an increase in exercise and dietary changes.
  • the treatment can be pharmaceutical, in which case any suitable agent can be used which is known to reduce the risk of type 2 diabetes and/or insulin resistance.
  • Agents which reduce the risk of type 2 diabetes and/or insulin resistance may also be used in the manufacture of a medicament for use in a method of treatment of a subject identified as susceptible to type 2 diabetes and/or insulin resistance.
  • the condition of a subject identified as susceptible to type 2 diabetes and/or insulin resistance can be improved by administration of an agent which reduces the risk of type 2 diabetes and/or insulin resistance.
  • a therapeutically effective amount of an agent which reduces the risk of type 2 diabetes and/or insulin resistance may be given to a human patient identified as susceptible to type 2 diabetes and/or insulin resistance.
  • the present invention provides the use of a substance or composition in the manufacture of a medicament for use in the treatment or prevention of type 2 diabetes and/or insulin resistance in a subject, which subject has been identified as being susceptible to type 2 diabetes and/or insulin resistance using a method of the invention.
  • the invention also provides a method of treating or preventing type 2 diabetes and/or insulin resistance in a subject, which method comprises:
  • Products containing means for determining the identity of the nucleotide at a polymo ⁇ hic position of the LPIN2 gene of a subject, and an agent which reduces the risk of type 2 diabetes and/or insulin resistance as a combined preparation for simultaneous, separate or sequential use in a method of treatment of the human or animal body by therapy may comprise both means for diagnosis and means for therapy.
  • the invention further proves a method of treating type 2 diabetes and/or insulin resistance, comprising providing an agent that complements a polymo ⁇ hism in the LPLN2 gene that is associated with type 2 diabetes and/or insulin resistance, such as any of the polymo ⁇ hisms described above, or any candidate polymo ⁇ hisms identifiable by the polymo ⁇ hism identification methods of the invention.
  • An agent which complements such a polymo ⁇ hism may be in a number of forms.
  • an agent may be, or comprise, a DNA such as a DNA which can bind in the region of the polymo ⁇ hism.
  • a LPIN2 polynucleotide which does not comprise a polymo ⁇ hism associated with type 2 diabetes and/or insulin resistance may thus be administered to the mammal in a method of gene therapy to treat or prevent the onset of type 2 diabetes and/or insulin resistance in a subject having a LPLN2 gene having such a polymo ⁇ hism.
  • Nucleic acid such as RNA or DNA, and preferably, DNA, is provided in the form of a vector, such as the polynucleotides described above, which may be expressed in the cells of the mammal.
  • a method for diagnosing and treating type 2 diabetes and/or insulin resistance in an individual typically comprises:
  • the formulation of an agent which reduces the risk of type 2 diabetes and/or insulin resistance for use in preventing or treating type 2 diabetes and/or insulin resistance will depend upon factors such as the nature of the exact agent, whether a pharmaceutical or veterinary use is intended, etc.
  • An agent which reduces the risk of type 2 diabetes and/or insulin resistance is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g.
  • starches gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations.
  • Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.
  • Liquid dispersions for oral administration may be syrups, emulsions or suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • a therapeutically effective amount of an agent which reduces the risk of type 2 diabetes and/or insulin resistance is administered to a patient.
  • a therapeutically effective amount of an agent is typically an amount which prevents or lessens one or more symptom or phenotype of type 2 diabetes and/or insulin resistance.
  • the dose of an agent which reduces the risk of type 2 diabetes and/or insulin resistance may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
  • a typical daily dose is from about 0.01 to 50 mg per kg of body weight, according to the activity of the specific agent, the age, weight and condition of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration.
  • daily dosage levels are from 20 to 40 micrograms per day.
  • An agent suitable for use in the treatment of type 2 diabetes and/or insulin resistance may be administered in a variety of dosage forms.
  • they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules.
  • the agent which reduces the risk of stroke may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.
  • the agent may also be administered as a suppository. A physician will be able to determine the required route of administration for each particular patient.
  • Nucleic acid may be administered by any available technique.
  • the nucleic acid may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly.
  • the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery.
  • the nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.
  • Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents.
  • these agents includes cationic agents, for example, calcium phosphate and DEAE- Dextran and lipofectants, for example, lipofectam and transfectam.
  • the dosage of the nucleic acid to be administered can be altered.
  • the nucleic acid is administered in the range of lpg to lmg, preferably to lpg to lO ⁇ g nucleic acid for particle mediated gene delivery and lO ⁇ g to lmg for other routes.
  • the present invention also relates to apparatus for carrying out a method as claimed in any one of claims 1, 2, 4, 16 or 17.
  • Such an apparatus comprises:-
  • the apparatus may also comprise means for receiving genetic information.
  • the means for receiving genetic information may be an integral part of the apparatus or remote therefrom.
  • the genetic information relating to the LPLN2 gene may be in the form of a database containing polymo ⁇ hism data.
  • Polymo ⁇ hism data is information concerning one or more of the following for a specific gene: location of polymo ⁇ hic sites, sequence variation at those sites; frequency of polymo ⁇ hisms in one or more populations: the different genotypes and/or haplotypes determined for the gene; frequency of one or more of those genotypes and/or haplotypes in one or more populations; any known association between a trait and a genotype or a haplotype for the gene.
  • a polymo ⁇ hism database is a collection of polymo ⁇ hism data arranged in a systematic or methodological way and capable of being individually assessed by electronic or other means.
  • the genetic information relating to the LPIN2 gene may comprise information relating to a polymo ⁇ hism in the LPIN2 gene which polymo ⁇ hism is associated with the individual being genetically predisposed to type 2 diabetes and or insulin resistance.
  • a further aspect of the invention relates to a computer programme comprising a programme code that, when executed on a computer system, instructs the computer system to perform a method according to any one of claims 1, 2, 4, 16 or 17.
  • the computer programme product may comprise programme code means on a carrier wave.
  • the present invention also relates to a computer programme product comprising a computer-readable storage medium having recorded thereon a computer programme according to the immediately above described aspects of the invention.
  • the invention also relates to a method according to any one of claims 1, 2, 4, 16 or 17, which method is carried out on an apparatus according to claim 24.
  • each or all analytical and mathematical operations involved in practising the method or methods of the present invention may be implemented on an apparatus, such as a computer.
  • an apparatus for example a computer
  • the LPF 2 polymo ⁇ hism data described to may be stored as part of a database (for example an ASCII flat file).
  • the polymo ⁇ hism data may be stored on a computer's hard drive, CD-rom or any one or other storage devices accessible by the computer.
  • the computer system for storing and analysing polymo ⁇ hism data for the LPIN2 gene may comprise one or more of the following:-
  • a communication interface a communication interface, a display device, an input device, a central processing unit and a database containing the polymo ⁇ hism data.
  • the present invention also relates to a database comprising information relating to a polymo ⁇ hism in the LPIN2 gene or protein, which polymo ⁇ hism is associated with an individual's genetic predisposition to type 2 diabetes and/or insulin resistance.
  • Figure 1 is a schematic illustration of the LPIN2 gene indicating the polymo ⁇ hisms described in Example 3.
  • Figures 2, 3 and 4 are each individual human LPIN2 genomic sequences from different sources.
  • Example 1 Determination of LPIN2 Gene Structure, Fragment design and PCR amplification
  • cDNA sequence for LPL 2 was retrieved from Genbank (accession number NM_014646.1). Genomic DNA sequence (accession number NT_011036.7 ) was also retrieved and analysed with the Repeatmasker programme to identify repetitive sequence regions. Repeated regions were avoided in positioning oligonucleotides for DNA fragment design. Twenty exons were identified from the cDNA using the proprietory Retrieve exons programme and aligned with the genomic sequence.
  • the retrieve exons programme is a proprietary software pipeline to: align cDNA sequence with the corresponding genomic DNA sequence, identify the exon boundaries within the cDNA sequence, mask repretitive DNA sequences within the genomic sequence, identify the sequences of database SNPs located within and around the gene and retrieve the cDNA, genomic DNA, masked genomic DNA, exons and database.
  • Intronic oligonucleotides were designed to flank all 20 of the exons. Oligonucleotide sequences for amplification of the Lipin2 gene are presented in Table 1. PCR reaction mixtures and conditions are presented in Tables 2 and 3, respectively.
  • the Lipin2 x2 fragment was amplified according to the protocol of the Qiagen PCR core kit (Qiagen, Cat. No. 201205)
  • exons encoding the 5.2 KB transcript were analysed by Denaturing High Performance Liquid Chromatography (DHPLC) using a Transgenomic WAVE (Transgenomic Inc.).
  • DPLC Denaturing High Performance Liquid Chromatography
  • Transgenomic WAVE Transgenomic Inc.
  • This method is based on an ion-pair reverse-phase chromatography, which separates DNA according to the melting properties of the sequence. The DNA is then eluted on an acetonitrile gradient from the column and viewed as a chromatogram peak.
  • DNA homoduplexes and heteroduplexes are resolved and can therefore be distinguished from each other.
  • melting curves were empirically calculated by running each fragment at a series of temperatures. Melting domains were analysed for each fragment and the optimum temperature identified by selecting the temperature at which the DNA fragment is 25% melted. This was carried out in conjunction with the WAVEMAKER Software (Transgenomic Inc.), which predicts the temperature at which the double stranded DNA fragment will melt into separate stands.
  • the mutation detection temperatures selected for each fragment are detailed in Table 1.
  • a microsatellite marker haptotype associated with type 2 diabetes and/or insulin resistance was identified on chromosome 18. Twelve DNA samples from unrelated individuals, and six individuals, with the microsatellite marker haplotype identified on chromosome 18, were amplified by PCR using the oligonucleotides and conditions in Tables 1-3. Individuals were analysed separately by DHPLC to scan for heteroduplex samples indicative of the presence of polymo ⁇ hisms in the DNA fragment. Heteroduplexes were identified based on separation differences when compared to homozygous individuals.
  • PCR products were sequenced according to the Dynamic ET Terminator cycle sequencing kit protocol (Amersham, cat. No. US80890). Each reaction consisted of 200ng template DNA, 0.25 pmol primer, 8 ⁇ l Termination mix and distilled water to 20 ⁇ l. The cycling conditions used were; 96°C for 30 sec, 50°C for 20 sec, 60°C for 1 min, cycled 25 times, then hold at 4°C until ready to purify.
  • Sequencing products were purified by gel filtration (plO gel) followed by ethanol and Sodium Acetate (NaAc) precipitation. Dry pellets were resuspended in 2 ⁇ l deionised formamide and denatured at 96°C for 3 mins prior to loading on a 48cm sequencing gel and running for 10 hours on an ABI automatic 377 sequencer.
  • the sequence chromatogram files were imported into the Sequencher sequence analysis programme (GENE CODES Co ⁇ .) and aligned to search for sequence variations. Products from both heterozygous and wild type individuals were sequenced in forward and reverse orientations to compare traces.
  • Example 3 Analysis of Functional Effects of Polymo ⁇ hisms Four polymo ⁇ hisms were discovered in the LPIN2 gene (Table 4) The location of each SNP within the gene is shown in Figure 1. One insertion/ deletion (indels) was found intron 2, a leucine (Leu) to phenylalanine (Phe) change in exon 10 [Leu (CTT) ⁇ Phe (TTT)], a SNP in intron 19 and a SNP in exon 20 in the 3' untranslated region (UTR).
  • a pair of oligonucleotides for amplification by PCR was designed on either side of each biallelic polymo ⁇ hism to produce a product size between 50bp and 350bp.
  • a sequencing oligonucleotide was designed to end within 30bp either 5' or 3' to each polymo ⁇ hic site. All amplification oligonucleotides used to generate the complemetery strand to the sequencing primer were labeled with a 5' - Biotin. (see Table 5)
  • PCR amplification oligonucleotides For each marker, all samples genotyped were amplified by PCR using the PCR amplification oligonucleotides. Each reaction used: 20ng DNA (dried down), 0.6 units of AmpliTaq GoldTM DNA polymerase, IX PCR Buffer II, 2.5mM MgCl 2 , ImM dNTP and lOpmol of each PCR oligonucleotide in a final volume of lO ⁇ l. The PCR cycling conditions used were: 95°C for 12 min, 45 cycles of: 94°C for 15 sec, T A for 15 sec (Table 5), 72°C for 30 sec and 72°C for 5 min.
  • sequencing oligonucleotide was annealed to the template by denaturing at 80°C for 2min and then cooling to room temperature for 10 min. Each marker/sample combination was then sequenced/genotyped by pyrosequencingTM on a PSQ96TM (Pyrosequencing AB). Genotype results were stored in the PSQ oracle® database ready for statistical analysis.
  • the population used in the Type 2 Diabetes study came from the S W Netherlands, close to Breda. They were founded in the mid eighteenth century from 150 individuals. Rapid expansion of the population occurred between 1848 and the present time, increasing from 700 to 20,000. During this time the population have been socially and genetically isolated and until recently the migration was less than 5%.
  • HAPIPF found, for example, at http://ideas.repec.Org/c/boc/bocode/s425501.html
  • STATA statistical package
  • SNP 203 Association is seen with SNP 203 and with haplotypes involving 203.
  • the two SNP haplotype 203+202 is strongly associated with the disease, with the association being greatest in non-obese females.
  • the SNPs are listed in the physical mapping order in the table (see Table 9).
  • the most 5' marker in the gene is LPN231 and the most 3' marker is LPIN215.
  • LPN202 and LPN203 fall within this block. Because of the high level of linkage disequilibrium in this region, it is possible to use combinations of genotypes or haplotypes at SNPs within the region as surrogates for genotypes or haplotypes at these two SNPs themselves. For example, haplotype analysis across all 15 SNPs within the high LD block demonstrates that the following haplotypes occur at SNPs LPN203, LPN216, and LPN223:
  • Haplotype frequencies were estimated using the EM algorithm.
  • LPN203 allele T only occurs on chromosomes which also carry both allele G at LPN216 and allele T at LPN223.
  • Haplotypes across LPN216 and LPN223 can therefore act as a tag for alleles at LPN203, rendering the direct genotyping of LPN203 unnecessary in this case. That is to say, the combination GA, at SNPs LPN216 and LPN213 respectively, only ever occurs on chromosomes which carry allele T at LPN202. All other combinations of alleles at LPN216 and LPN223 (ie AA, AT, and GT) only ever occur on chromosomes which carry allele C at LPN203
  • LPN203 alleles to predict susceptibility to diabetes can be carried out with equal accuracy by using alleles at LPN216 and LPN223 in combination.
  • Other possible combinations of SNPs to tag alleles at LPN202 or LPN203 can be found, and software to search for these combinations is readily available, for example SNPtagger
  • the example given above predicts an individual allele by using combinations of alleles at other loci carried on the same chromosome. Similar predictions can be carried out using genotypes (rather than haplotypes) and standard statistical techniques such as linear regression and discriminant function analysis. For example, by giving homozygous A A individuals a score of 1, heterozygotes a score of 1.5 and homozygous GG individuals a score of 2 at LPN216, and similarly giving homozygous A A individuals a score of 1, heterozygotes a score of 1.5 and homozygous TT individuals a score of 2 at LPN223, discriminant function analysis shows that the linear function:
  • LPN203 CC homozygotes have a discriminant score between -4.437 and -0.7995
  • LPN203 CT heterozygotes have a discriminant score between +2.1285 and-5.766 2
  • LPN203 TT homozygotes have a discriminant score of 8.694
  • the discriminant function based on genotypes at LPN216 and LPN223 is calculated as:
  • genotype of individuals at LPN203 can be accurately predicted by converting LPN216 and LPN223 genotypes to numeric values, and evaluating the discriminant function given above.
  • loci within this region of high LD can readily be found which can also act as surrogates for genotypes and haplotypes at LPN202 and LPN203.

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Abstract

L'invention concerne des méthodes pour déterminer une prédisposition génétique au diabète de type 2 et/ou à la résistance à l'insuline, ainsi que des méthodes pour traiter le diabète de type 2 et/ou la résistance à l'insuline ainsi que des états associés, par exemple l'obésité. L'invention concerne des méthodes pour déterminer la prédisposition d'un individu au diabète et/ou à la résistance à l'insuline, par typage du gène LPIN2 ou de la protéine de LPIN2. L'invention concerne également des méthodes, utilisations et traitements associés.
PCT/GB2003/002730 2002-06-25 2003-06-25 Analyse d'une predisposition genetique et traitement correspondant WO2004001071A2 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CAO HENIAN ET AL: "Identification of single-nucleotide polymorphisms in the human LPIN1 gene." JOURNAL OF HUMAN GENETICS. JAPAN 2002, vol. 47, no. 7, 2002, pages 370-372, XP001153218 ISSN: 1434-5161 *
HEGELE R A: "Molecular basis of partial lipodystrophy and prospects for therapy." TRENDS IN MOLECULAR MEDICINE. ENGLAND MAR 2001, vol. 7, no. 3, March 2001 (2001-03), pages 121-126, XP002260772 ISSN: 1471-4914 *
PETERFY M ET AL: "LIPODYSTROPHY IN THE FLD MOUSE RESULTS FROM MUTATION OF A NEW GENE ENCODING A NUCLEAR PROTEIN, LIPIN" NATURE GENETICS, NEW YORK, NY, US, vol. 27, no. 1, January 2001 (2001-01), pages 121-124, XP009017504 ISSN: 1061-4036 *
REUE K ET AL: "Adipose tissue deficiency, glucose intolerance, and increased atherosclerosis result from mutation in the mouse fatty liver dystrophy (fld) gene." JOURNAL OF LIPID RESEARCH. UNITED STATES JUL 2000, vol. 41, no. 7, July 2000 (2000-07), pages 1067-1076, XP001156127 ISSN: 0022-2275 *

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