EP0898617A1 - ADNc MUTANT CODANT LA SOUS-UNITE p85$g(a) DU PHOSPHATIDYLINOSITOL 3-KINASE - Google Patents

ADNc MUTANT CODANT LA SOUS-UNITE p85$g(a) DU PHOSPHATIDYLINOSITOL 3-KINASE

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Publication number
EP0898617A1
EP0898617A1 EP97920607A EP97920607A EP0898617A1 EP 0898617 A1 EP0898617 A1 EP 0898617A1 EP 97920607 A EP97920607 A EP 97920607A EP 97920607 A EP97920607 A EP 97920607A EP 0898617 A1 EP0898617 A1 EP 0898617A1
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Prior art keywords
mutation
dna
pi3k
subunit
regulatory subunit
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EP97920607A
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German (de)
English (en)
Inventor
Torben Hansen
Carsten Bo Andersen
Oluf Borbye Pedersen
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Novo Nordisk AS
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Novo Nordisk AS
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Publication of EP0898617A1 publication Critical patent/EP0898617A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)

Definitions

  • the present invention relates to a mutant cDNA sequence encoding the regulatory p85 ⁇ subunit of phosphatidylinositol 3-kinase (PI3K), a method of detecting a mutation in the gene encoding the regulatory p85 ⁇ subunit of phosphatidylinositol 3-kinase, as well as a diagnostic composition and a test kit for use in the method.
  • PI3K phosphatidylinositol 3-kinase
  • Phosphatidylinositol 3-kinase is a heterodimeric protein consisting of a catalytic 110 kDa subunit and an ⁇ -85 kDa subunit that is necessary for docking to growth factor plasma membrane receptors including the insulin receptor and to signalling phosphotyrosine motifs in signalling proteins, e.g. insulin receptor substrate-1 (1,2).
  • PI 3-kinases are also implicated in facilitating membrane trafficking processes.
  • PI3-kinase activation 3,4
  • PI3-kinase has been implicated as a key intermediate in the cytoskeletal rearrangements that accompany secretory processes in platelets, basophil and neutrophil cells (5,6).
  • the yeast homologue VPS34 has been shown to be involved in vesicle trafficking and protein sorting (7).
  • PI3-kinase is thus an attractive candidate protein as a mediator of insulin action on glucose transport as it could act as a point of convergence of signalling and trafficking processes.
  • the gene encoding the regulatory p85 ⁇ subunit of phosphatidylinositol 3-kinase has been examined for genetic variations which might be associated with or causing impaired insulin sensitivity, impaired glucose effectiveness, and/or impaired glucose disappearance.
  • the present invention relates to a nucleic acid sequence comprising a DNA sequence encoding the regulatory p85 ⁇ subunit of human phosphatidylinositol 3-kinase (PI3K), the DNA sequence containing a mutation of at least one nucleotide, or comprising a segment of the cDNA sequence including said mutation. It has been found that such mutations may be localized to the p85 ⁇ subunit of PI3K.
  • PI3K human phosphatidylinositol 3-kinase
  • mutation of the regulatory p85 ⁇ subunit of PI3K gene may be indicative of abnormalities significant for the development of changes in whole body glucose metabolism and whole body insulin action.
  • the mutation may give rise to the substitution of an amino acid in the regulatory p85 ⁇ subunit of PI3K which may cause changes in the structure, such as the tertiary structure, or biochemical properties of the regulatory p85 ⁇ subunit of PI3K.
  • Such changes may interfere with the normal binding of the regulatory p85 ⁇ subunit of PI3K to the insulin receptor kinase and insulin receptor substrates, e.g. IRS-1 and IRS-2 leading to altered glucose metabolism and reduced insulin sensitivity, and may lead to the development of insulin resistant conditions, such as NIDDM, cardiovascular diseases, obesity, and hypertension in human beings and other mammals.
  • the present invention relates to a living system containing a nucleic acid sequence of the invention and capable of expressing the regulatory p85 ⁇ subunit of PI3K wherein at least one amino acid is substituted, inserted or deleted, or the living system contains a nucleic acid sequence of the invention carrying at least one silent mutation.
  • the living system which may comprise a cell or a multicellular organism containing the appropriate signal transduction pathway, may be used to screen for substances which have an effect on the regulatory p85 ⁇ subunit of PI3K activity.
  • the present invention relates to a method of detecting the presence of a mutation in the gene encoding the regulatory p85 ⁇ subunit of PI3K, the method comprising obtaining a biological sample from a subject and analysing the sample for a mutation of at least one nucleotide. It has now been shown that homozygous carriers of a the regulatory p85 ⁇ subunit of PI3K mutation G to A in nucleotide # 1020 exhibit a decreased glucose disappearance rate and a decreased glucose effectiveness and a decreased insulin sensitivity.
  • the present method is useful in diagnosing predisposition to insulin resistance and possibly impaired glucose tolerance and related diseases, such as NIDDM, cardiovascular diseases, obesity, and hypertension, in a subject as well as other disorders resulting from an altered glucose metabolism.
  • Biological samples may, for instance, be obtained from blood, serum, plasma or tissue.
  • the invention further relates to a diagnostic composition and a test kit for use in the method. DETAILED DESCRIPTION OF THE INVENTION
  • Examples of specific mutations found in the cDNA sequence encoding the regulatory p85 ⁇ subunit of PI3K are: C to T in nucleotide # 261 ; T to G in nucleotide # 663; A to G in nucleotide # 810; G to A in nucleotide # 1020 according to the numbering of E.Y. Skolnik et al., Cell 65, 1991, pp. 83-90.
  • the present invention relates to a nucleic acid sequence comprising a cDNA sequence encoding the regulatory p85 ⁇ subunit of PI3K and containing a mutation giving rise to at least one amino acid substitution in the regulatory p85 ⁇ subunit of PI3K protein sequence.
  • the mutation may for instance be located at a site where the amino acid substitution interferes with the binding of the regulatory p85 ⁇ subunit of PI3K to insulin receptor kinase or insulin signalling proteins, e.g. IRS-1 and/or IRS-2, as such a mutation is most likely to be involved in changes in insulin signalling and basal and insulin stimulated glucose uptake. Such changes may therefore lead to reductions in whole body insulin sensitivity and whole body glucose metabolism. Martin, B.C. et al. (Lancet 1992, Vol. 340, 925:929) have previously shown that individuals having low or reduced Si (insulin sensitivity) or Sg (glucose effectiveness) have an increased risk of developing NIDDM.
  • such a DNA sequence is one containing a mutation of G to A in the third position of codon 326 of the regulatory p85 ⁇ subunit of PI3K gene such that methionine 326 is substituted by isoleucine.
  • This mutation is at a domain boundary, directly in front of an SH2 domain of p85 ⁇ . The region is located downstream of a hydrophobic region the function of which is not known but which might be membrane-associated, as the subunit apparently acts as an adaptor allowing the kinase to associate with the membrane.
  • Figure 1 is a diagram of the PI3K p85 ⁇ that shows the position of the codon326 mutation relative to an SH2 domain.
  • silent mutations in the form of silent single nucleotide substitutions may have an influence on splicing (if it is located within a few bases of the exon-intron boundary), mRNA stability (if it changes the folding or the accessibility of the mRNA to RNAses), or protein stability (since codons may be changed into rare codons which may be translated more slowly and thereby influence the folding/stability of the protein (cf. Ikemura, T. and Ozeki, H. : Codon usage and transfer RNA contents: organism-specific codon-choice patterns in preference to the isoacceptor contents. Cold Spring Harbor Symp.
  • the length of the nucleic acid sequence of the invention may vary widely depending on the intended use.
  • the cDNA segment may be as short as 17 nucleotides.
  • the cDNA isolate will typically comprise the full-length cDNA sequence encoding the regulatory p85 ⁇ subunit of PI3K.
  • the nucleic acid sequence of the invention comprising the mutation in the cDNA sequence encoding the regulatory p85 ⁇ subunit of PI3K may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the regulatory p85 ⁇ subunit of PI3K by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor, 1989). The probes used should be specific for the mutation.
  • DNA sequence encoding wild-type of the regulatory p85 ⁇ subunit of PI3K may be modified by site-directed mutagenesis using synthetic oligonucleotides containing the mutation for homologous recombination in accordance with well-known procedures.
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202, Saiki et al., Science 239. 1988, pp. 487-491, or PCR Protocols. 1990, Academic Press, San Diego, California, USA.
  • PCR polymerase chain reaction
  • the DNA isolate of the invention may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3_ (1984), 801 - 805.
  • phosphoamidite method oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed and ligated. This procedure may preferably be used to prepare shorter segments of the regulatory p85 ⁇ subunit of PI3K encoding DNA sequence.
  • the recombinant vector into which the DNA isolate is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures.
  • the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated (e.g. a viral vector).
  • the recombinant vector is preferably an expression vector in which the DNA sequence encoding the regulatory p85 ⁇ subunit of PI3K mutant is operably connected to additional segments required for transcription of the DNA.
  • the vector is derived from plasmid or viral DNA or may contain elements of both.
  • the term "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the regulatory p85 ⁇ subunit of PI3K.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • Suitable promoters for directing the transcription of the mutant DNA encoding the regulatory p85 ⁇ subunit of PI3K in mammalian cells are the SV40 promoter (Subramani et al. , Mol. Cell Biol. I (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
  • the mutant DNA sequence encoding the regulatory p85 ⁇ subunit of PI3K may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., OJL . cit.V
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g. the SV40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
  • the recombinant vector may further comprise a DNA sequence enabling the vector to repli ⁇ cate in the host cell in question.
  • a DNA sequence enabling the vector to repli ⁇ cate in the host cell in question.
  • An example of such a sequence is the SV40 origin of replication.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hygromycin or methotrexate.
  • DHFR dihydrofolate reductase
  • the present invention relates to a variant of the regulatory p85 ⁇ subunit of PI3K containing at least one arnino acid substitution, in particular a variant containing at least one a ⁇ iino acid substitution at a site where the substitution interferes with the binding of the regulatory p85 ⁇ subunit of PI3K to insulin receptor kinase or insulin signalling proteins, e.g. IRS-1 and/or IRS-2, or a fragment thereof including said substitution.
  • insulin receptor kinase or insulin signalling proteins e.g. IRS-1 and/or IRS-2
  • examples of such variants are variants in which methionine 326 is substituted by isoleucine.
  • the living system into which the DNA isolate of the invention is introduced may be a cell which is capable of producing the regulatory p85 ⁇ subunit of PI3K and which has the appropriate signal transduction pathways.
  • the cell is preferably a eukaryotic cell, such as a vertebrate cell, e.g. a Xenopus laevis oocyte or mammalian cell, in particular a mammalian cell.
  • suitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines.
  • the mutant DNA sequence encoding the regulatory p85 ⁇ subunit of PI3K may then be expressed by culturing a cell as described above in a suitable nutrient medium under conditions which are conducive to the expression of the regulatory p85 ⁇ subunit of PI3K- coding DNA sequence.
  • the medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the living system according to the invention may also comprise a transgenic animal.
  • a transgenic animal is one in whose genome a heterologous DNA sequence has been introduced.
  • the transgenic animal is a transgenic non-human mammal, mammals being generally provided with appropriate signal transduction pathways.
  • mammals being generally provided with appropriate signal transduction pathways.
  • it is generally preferred to employ smaller mammals, e.g. rabbits or rodents such as mice or rats.
  • a DNA sequence encoding the mutant regulatory p85 ⁇ subunit of PI3K is operably linked to additional DNA sequences required for its expression to produce expression units.
  • Such additional sequences include a promoter as indicated above, as well 5 as sequences providing for te ⁇ nination of transcription and polyadenylation of mRNA.
  • Construction of the expression unit for use in transgenic animals may conveniently be done by inserting a mutant DNA sequence encoding the regulatory p85 ⁇ subunit of PI3K into a vector containing the additional DNA sequences, although the expression unit may be constructed by essentially any sequence of ligations.
  • the expression unit is then introduced into fertilized ova or early-stage embryos of the selected host species.
  • Introduction of heterologous DNA may be carried out in a number of ways, including microinjection (cf. US 4,873, 191), retroviral infection (cf. Jaenisch, Science 240. 1988, pp. 1468-1474) or site-directed integration using embryonic stem cells
  • a biological sample is obtained from a subject, DNA (in particular genomic DNA) is isolated from the sample and digested with a restriction endonuclease which cleaves DNA at the site of the mutation, and cleavage of the DNA within the gene encoding the regulatory p85 ⁇ subunit of PI3K at this site is determined. After digestion, the resulting DNA fragments may be subjected to electrophoresis on an agarose gel. DNA from the gel may then be blotted onto a nitrocellulose filter and hybridised with a radiolabelled probe.
  • the probe may conveniently contain a DNA fragment of the regulatory p85 ⁇ subunit of PI3K gene spanning the mutation (subtantially according to the method of E.M. Southern, J. Mol. Biol. 2S, 1975, pp. 503, e.g. as described by B.J. Conner et al., Proc. Natl. Acad. Sci. USA fiQ, 1983, pp. 278-282).
  • the DNA isolated from the sample may be amplified prior to digestion with the restriction endonuclease.
  • Amplification may suitably be performed by polymerase chain reaction (PCR) using oligonucleotide primers based on the appropriate sequence of the regulatory p85 ⁇ subunit of PI3K spanning the site(s) of mutation, essentially as described by Saiki et al., Science 230. 1985, pp. 1350-1354.
  • the amplified DNA may be digested with the appropriate restriction endonuclease and subjected to agarose gel electrophoresis.
  • the restriction pattern obtained may be analysed, e.g.
  • wild-type DNA encoding the regulatory p85 ⁇ subunit of PI3K may be subjected to the same procedure, and the restriction patterns may be compared.
  • the sample is preferably analysed for a mutation located at a site where amino acid substitution interferes with the binding of the regulatory p85 ⁇ subunit of PI3K to insulin receptor kinase or insulin signalling proteins, e.g. IRS-1 and /or IRS-2.
  • a mutation located at a site where amino acid substitution interferes with the binding of the regulatory p85 ⁇ subunit of PI3K to insulin receptor kinase or insulin signalling proteins, e.g. IRS-1 and /or IRS-2.
  • IRS-1 and /or IRS-2 insulin signalling proteins
  • a further embodiment of the method of the invention is an adaptation of the method 5 described by U. Landegren et al., Science 241. 1988, pp. 1077-1080, which involves the ligation of adjacent oligonucleotides on a complementary target DNA molecule. Ligation will occur at the junction of the two oligonucleotides if the nucleotides are correctly base paired.
  • the DNA isolated from the sample may be amplified using oligonucleotide primers corresponding to segments of the gene coding for the regulatory p85 ⁇ subunit of PI3K.
  • the amplified DNA may then be analysed by hybridisation with a labelled oligonucleotide probe comprising a DNA sequence corresponding to at least part of the gene encoding the regulatory p85 ⁇ subunit of PI3K and
  • the amplified DNA may furthermore be hybridised with a labelled oligonucleotide probe comprising a DNA sequence corresponding to at least part of the wild-type gene encoding the regulatory p85 ⁇ subunit of PI3K. This procedure is, for
  • PCR-based methods which may be used in the present invention are the allele-specific PCR method described by R. Saiki et al., Nature 324. 1986, pp. 163-166, or D.Y. Wu et al. , Proc. Natl. Acad. Sci. USA ⁇ 6, 1989, pp. 2757-2760, which uses primers specific for the mutation in the regulatory p85 ⁇ subunit of PI3K gene; the ligase chain reaction; restriction
  • a currently preferred method of detecting mutations is by single stranded conformation polymorphism (SSCP) analysis substantially as described by Orita M, Ivahana
  • the label substance with which the probe is labelled is preferably selected from the group consisting of enzymes, coloured or fluorescent substances, or radioactive isotopes.
  • enzymes useful as label substances are peroxidases (such as horseradish peroxidase), phosphatases (such as acid or alkaline phosphatase), ⁇ -galactosidase, urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glucoamylase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, ⁇ -glucosidase, proteases, pyruvate de- carboxylase, esterases, luciferase, etc.
  • peroxidases such as horseradish peroxidase
  • phosphatases such as acid or alkaline phosphatase
  • ⁇ -galactosidase urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glucoamylase, lysozyme, malate dehydrogenase, glucose-6
  • Enzymes are not in themselves detectable but must be combined with a substrate to catalyse a reaction the end product of which is detectable.
  • substrates which may be em- ployed in the method according to the invention include hydrogen peroxide/tetramethylbenzidine or chloronaphthole or o-phenylenediamine or 3-(p- hydroxy phenyl) propionic acid or luminol, indoxyl phosphate, p-nitrophenylphosphate, nitrophenyl galactose, 4-methyl umbelliferyl-D-galactopyranoside, or luciferin.
  • the label substance may comprise coloured or fluorescent substances, including gold particles, coloured or fluorescent latex particles, dye particles, fluorescein, phycoerythrin or phycocyanin.
  • the probe is labelled with a radioactive isotope.
  • Radioactive isotopes which may be used for the present purpose may be selected from I- 125, 1-131, In-Ill, H-3, P-32, C-14 or S-35. The radioactivity emitted by these isotopes may be measured in a beta- or gamma-counter or by a scintillation camera in a manner known per se.
  • the invention further relates to a test kit for detecting the presence of a mutation in the gene encoding the regulatory p85 ⁇ subunit of PI3K, the kit comprising (a) a restriction endonuclease which cleaves DNA at the site of the mutation,
  • Said mutation is preferably the codon326 mutation resulting in a methionin to isoleucin substitution.
  • the first DNA sequence may, for instance, be obtained from genomic DNA or cDNA encoding the regulatory p85 ⁇ subunit of PI3K obtained from a healthy subject (a non- mutation carrier).
  • the second DNA sequence may conveniently be a DNA construct according to the invention.
  • the invention further relates to a test kit for detecting the presence of a mutation in the gene encoding the regulatory p85 ⁇ subunit of PI3K, the kit comprising
  • a labelled oligonucleotide probe comprising a DNA sequence corresponding to at least part of the gene encoding the regulatory p85 ⁇ subunit of PI3K and containing a mutation of at least one nucleotide, which mutation corresponds to the mutation the presence of which in the gene encoding the regulatory p85 ⁇ subunit of PI3K is to be detected.
  • the mutation the presence of which is to be detected is the codon 326 methionin to isoleucin mutation of p85 ⁇ .
  • Appropriate means for amplifying DNA include, for instance, oligonucleotide primers, appropriate buffers and a thermostable DNA polymerase.
  • the kit may further comprise a labelled oligonucleotide probe comprising a DNA sequence corresponding to at least part of the wild-type gene encoding the regulatory p85 ⁇ subunit of PI3K.
  • the invention also relates to the use of a nucleic acid sequence of the invention or a segment thereof as a probe, preferably an oligonucleotide probe, for detection of the presence of a mutation in a regulatory subunit, preferably the p85 ⁇ subunit or any isoform or homologous polypeptide thereof, of PI3K, the regulatory subunit being preferably isolated from a biological sample obtained from a subject.
  • a nucleic acid sequence of the invention or a segment thereof as a probe, preferably an oligonucleotide probe, for detection of the presence of a mutation in a regulatory subunit, preferably the p85 ⁇ subunit or any isoform or homologous polypeptide thereof, of PI3K, the regulatory subunit being preferably isolated from a biological sample obtained from a subject.
  • IVGTT insulin-derived pancreatic beta cell response after a 12 h overnight fasting period.
  • Baseline values of serum insulin, serum C- peptide and plasma glucose were taken in duplicate with 5 min intervals.
  • Glucose was injected i.v. in the contralateral antecubital vein over a period of 1 min (0.3 g/kg body weight of 50% glucose).
  • a bolus of 3 mg tolbutamide/kg body weight was injected during 5 sec to elicit a secondary pancreatic beta cell response.
  • Venous blood was sampled at 2, 4, 8, 19, 22, 30, 40, 50, 70, 90, 180 min, timed from the end of the glucose injection for measurements of plasma glucose, serum insulin and serum C-peptide. All the IVGTT's were done by the same investigator. Glucose induced acute serum insulin and C-peptide responses (0-8 min) were calculated by means of the trapezoidal rule as the incremental values (area under the curve when expressed above basal values). Insulin sensitivity indeks (Si) and glucose effectiveness (Sg) were calculated using the Bergman MINMOD computer program developed specifically for the combined intravenous glucose and tolbutamide tolerance test, cf. Clausen et al. op cit.
  • Intravenous glucose disappearance constant was calculated as the slope of the line relating the natural logarithm of the glucose concentration to the time between 8 and 19 minutes after the glucose bolus. Plasma concentrations of glucose, serum specific insulin and serum C-peptide were measured as described.
  • Genomic DNA was isolated from human leucocyte nuclei isolated from whole blood by digestion with proteinase K followed by phenol extraction on an Applied Biosystems 341 Nucleic Acid Purification System (Applied Biosystems Inc., Foster City, California, USA)
  • Muscle biopsies Needle biopsies of the vastus lateralis muscle (500 mg) from subjects were obtained during local anesthesia (1 % Lidocaine, without epinephrine). A modified
  • RNA was isolated using a 5 394 RNA/DNA extractor (Applied Biosystems Inc., Foster City, California, USA). Purity and concentration of the samples were dete ⁇ riined by measuring the 260/280 nm abso ⁇ tion ratio.
  • cDNA synthesis was synthesized in volumes of 25 ⁇ l containing (in final 0 concentrations) 50 mM Tris-HCI (pH 8.3), 75 mM KCl, 3mM MgCl 2 , 10 mM DDT, 0.2 mM dNTP's, 40 U Rnasin (Promega, Madison, WI), 0.625 ⁇ g Oligo (dT), discipline, 400 U Molony murine leukemia virus reverse transcriptase (M-MLV RT) (Life Technologies, Grand Island, NY), and 1.0 ⁇ g of total muscle RNA. The reactions were performed at 37°C for 1 h, followed by enzyme inactivation for 10 min at 95 °C. Samples were diluted to 5 a final volume of 200 ⁇ l and stored at -20°C.
  • Oligonucleotides used for amplification Oligonucleotides, 18-21 mers in length and with one or two G's and/or Cs at the 3' end, were synthesized on an Applied Biosystems 394 DNA/RNA Synthesizer and applied to a NABTM -10 column (Pharmacia P L Biochemicals 0 Inc., Milwaukee, WI) and used without further purification. Biotin labelling of primers was done during synthesis using DMT-Biotin-C6-PA (Genosys Biotechnologies Inc., Great Britain).
  • the human p85 ⁇ has been published by Skolnik (Skolnik et al., 1991) (GenBank, Genetics Computer Group (Madison; WI), accession number M61906).
  • the published amino acid sequence has 97% homology to bovine p85 ⁇ cDNA (M61745), 95% to mouse 5 p85 ⁇ cDNA (M60651) and only 59% to bovine p85 ⁇ cDNA (M61746).
  • Primers for polymerase chain reaction (PCR) amplification of human p85 ⁇ cDNA were designed from areas of the cDNA sequence, with low homology to bovine p85 ⁇ (Fig. 2).
  • PCR amplification of cDNA A primary PCR amplification was carried out with 5 ⁇ l 0 cDNA as template.
  • the assay conditions were: 10 mM Tris-HCI (pH 9.0), 50 mM KCl,
  • SSCP screening for variants in the regulatory p85 ⁇ subunit of PI3K gene.
  • the coding region of PI3K p85 ⁇ subunit was scanned by PCR-SSCP at the nucleotide level.
  • cDNA from 70 NIDDM patients and 12 healthy control subjects without any known family history of NIDDM was included in the primary SSCP screening.
  • SSCP-analysis (according to the method of Orita et al. , Genomics 5_, 1989, pp. 874-879) was performed on DNA- segments of 250-300 bp with an overlap of 60 base pairs between segments. DNA-segments for SSCP analysis were PCR-amplified in a standard PCR-reaction, using specific primers as described above.
  • PCR-reactions were performed as described above and 3 ⁇ l of the PCR-reaction was mixed with 12 ⁇ l of SSCP loading buffer (95 % deionised formamide, 5 % NaOH, xylene blue, bromo-phenol blue), and 2 ⁇ l were analyzed on a 38 x 31 x 0.04 cm, non-denaturing 5 % acrylamide (49: 1 acrylamide :bisacrylamide) gel in adjacent lanes.
  • SSCP loading buffer 95 % deionised formamide, 5 % NaOH, xylene blue, bromo-phenol blue
  • a "cold gel” containing 1 % glycerol, 1 X TBE-buffer (90 mM Tris- Borate, 2.5 mM EDTA), and 5 % acrylamide (49: 1, acrylamide:bisacrylamide) was run at 8 "C in a cold-room for 3 to 5 h with an applied power of 35 watt.
  • a "warm” gel containing 5 % glycerol, 1 X TBE-buffer (90 mM Tris-Borate, 2.5 mM EDTA), and 5 % acrylamide (49: 1, acrylamide :bisacrylamide), was run at 24°C for 3 to 4 h, applying 65 watt.
  • the electrophoresis buffer was x h x TBE (45 mM Tris Base, 1.25 mM EDTA).
  • the power supplies used were equipped with a temperamre controlling device, to assure constant running temperature (Temperature Controller, Stratagene, La Jolla, CA, USA).
  • the gels were transferred to Whatman 3 MM filter paper, covered in plastic wrap, and autoradiografed at -80°C, for 3 h to 3 days using intensifying screens. Autoradi frets were analyzed visually to reveal variations in migration. SSCP screening and subsequent direct sequencing revealed 4 nucleotide variants, shown in Table 1 below.
  • Table 1 Localisation, character and genotype frequency of nucleotide variants in PI3K p85 ⁇ subunit.
  • the base number is calculated from first sequenced base (accession number M69106), the codon number is calculated from first coding methionin (code 1 , base number 43-45, sequence accession number M69106).
  • Table 2 Nucleotide sequences of DNA primers used for PCR amplification to detect the regulatory p85 ⁇ subunit of PI3K amino acid substitution at codon 326 by SSCP gel analysis.
  • Genomic DNA 100 ng was PCR amplified with primers 10 and 11 (Fig. 2) as described above except for addition of 2.0 mM MgCl 2 and 10% DMSO.
  • the samples were subjected to 40 cycles of amplification: denaturation at 10 95 °C for 30 sec, annealing at 54 °C for 30 sec, extension at 72 °C for 30 sec and final extension for 5 min at 72 °C using GeneAmp PCR system 9600 (Perkin Elmer, Norwalk, CT).
  • PCR samples (15 ⁇ l) were denaturated in 85 ⁇ l 0.2 M NaOH, 1 M NaCI and 2 x 45 ⁇ l were transferred to Hybond-N + filters (Amersham, Buckinghamshire, Great Britain).
  • the membranes were crosslinked twice in a UV Stratalink (Stratagene, La Jolla, CA) at 1200 x 100 ⁇ Joule. Membranes were prehybridized for 1 hour in ⁇ xSSPE, 5xDenhardt, 0.1 % SDS and 100 ⁇ g/ml denaturated salmon sperm. Hybridization was perfomed with allele specific probes (wild type: 5' AAC AAT ATG TCC TTA CA; variant 5' AAC AAT AT ⁇ TCC TTA CA) for 3 hours in ⁇ xSSPE, 5xDenhardt, 0.1 % SDS. Probes were 5'-end-
  • SSCP-variants were sequenced using either an Automated Laser Fluorescence Sequencer (LKB Pharmacia Uppsala, Sweden) and using Fluore-dATP (Pharmacia/Boeringer Mannheim) as label in the AutoRead Sequencing Kit (Pharmacia, Uppsala, Sweden), or by standard dideoxynucleotide sequencing using the Sequenase 2.0
  • DNA for sequencing was PCR-amplified using 5 pmol/ 100 ⁇ l reaction of one biotinylated primer, and single-stranded DNA was isolated using streptavidin-coated magnetic beads (Dynabeads, Oslo, Norway). DNA was sequenced as single stranded DNA according to protocols, using internal sequencing primers.

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Abstract

On décrit une séquence d'acide nucléique comprenant une séquence d'ADN codant une sous-unité régulatrice de phosphatidylinositol 3-kinase (P13K), la séquence d'ADN contenant une mutation d'au moins un nucléotide ou comprenant un segment de séquence d'ADN contenant ladite mutation et pouvant être utilisée comme outil de diagnostic, comme marqueur ou comme sonde. Sont repris ci-après des exemples de mutations dans la sous-unité p85α: De C à T dans le nucléotide 261; De T à G dans le nucléotide 663; De A à G dans le nucléotide 810; De G à A dans le nucléotide 1020.
EP97920607A 1996-05-06 1997-05-02 ADNc MUTANT CODANT LA SOUS-UNITE p85$g(a) DU PHOSPHATIDYLINOSITOL 3-KINASE Withdrawn EP0898617A1 (fr)

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DK53996 1996-05-06
DK53996 1996-05-06
PCT/DK1997/000200 WO1997042310A1 (fr) 1996-05-06 1997-05-02 ADNc MUTANT CODANT LA SOUS-UNITE p85α DU PHOSPHATIDYLINOSITOL 3-KINASE

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US6100090A (en) * 1999-06-25 2000-08-08 Isis Pharmaceuticals Inc. Antisense inhibition of PI3K p85 expression
CA2372542A1 (fr) * 2001-02-20 2002-08-20 Pfizer Products Inc. Animaux transgeniques contenant une forme mutante negative dominante de la sous-unite p85 du gene de la p13-kinase

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DE69230433D1 (de) * 1991-01-18 2000-01-20 Univ New York Cdns-klonierungsverfahren für rezeptortyrosinkinasezielprotein und hgrb-proteine
US5885777A (en) * 1994-10-13 1999-03-23 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Cloning, expression and characterization of a novel form of phosphatidylinositol-3-kinase

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See references of WO9742310A1 *

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