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WO2003035691A1 - Mutation d'apolipoproteine a-i utilisee dans le diagnostic et le traitement de maladies cardio-vasculaires - Google Patents

Mutation d'apolipoproteine a-i utilisee dans le diagnostic et le traitement de maladies cardio-vasculaires Download PDF

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Publication number
WO2003035691A1
WO2003035691A1 PCT/CA2002/001615 CA0201615W WO03035691A1 WO 2003035691 A1 WO2003035691 A1 WO 2003035691A1 CA 0201615 W CA0201615 W CA 0201615W WO 03035691 A1 WO03035691 A1 WO 03035691A1
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Prior art keywords
mutation
disease
seq
apoa
patient
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PCT/CA2002/001615
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English (en)
Inventor
Alison J. Brownlie
Marie-Pierre Dube
Mark Samuels
Michael R. Hayden
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Xenon Genetics, Inc.
University Of British Columbia
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Publication of WO2003035691A1 publication Critical patent/WO2003035691A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • 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 the field of disease diagnosis and treatment, especially diagnosis and treatment of cardiovascular diseases, such as low HDL diseases, including hypoalphalipoproteinemia, using nucleotide sequences and probes derived from the polynucleotide encoding ApoA-1 polypeptide.
  • OMIM 107680 Online Mendelian Inheritance in Man. It is associated with premature coronary artery disease and stroke (Vergani and Bettale, Clin. Chim. Acta 114: 45-52, 1981 ; Third et al., Metabolism 33:136- 146, 1984; Daniels et al., Stroke 13: 360-365, 1982; Ordovas et al., J. Biol. Chem. 264: 16339-16342, 1986).
  • Apolipoprotein A-I (apoA-l) is the major constituent of the high density lipoproteins (HDL), which is a macromolecular complex composed of cholesterol, phospholipids and triglycerides.
  • HDL high density lipoproteins
  • Epidemiological studies have demonstrated an inverse correlation between the concentrations of HDL cholesterol and apoA-l and the incidence of cardiovascular diseases (E. G. Miller et al., Lancet, 1977:965-968).
  • Other HDL deficiency states are Tangier disease (OMIM 205400) and LCAT deficiency (OMIM 245900) .
  • the gene coding for apoA-l has been cloned and sequenced (Sharpe et al., Nucleic Acids Res. 12(9) (1984) 3917). This gene was initially described to be 1863 bp in length, with 4 exons and 3 introns. The complete genomic sequence has been disclosed in GenBank Accession No. J00098.
  • the cDNA coding for apoA-l has been cloned by Breslow et al. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6861 -5) and the mRNA comprises 897 bp (GenBank Accession No. NMJD0039, see SEQ ID No. 1).
  • Apo-A1 in its mature form is a 243 amino acid sequence following post-translational modification (GenBank Accession No. NP_000030, SEQ ID NO:2).
  • the present invention relates to an isolated polynucleotide comprising a polynucleotide at least 95% identical to a member selected from the group consisting of:
  • the member is (a).
  • the polynucleotide of (a) is a DNA or an RNA, most preferably a cDNA.
  • the present invention relates to an isolated polynucleotide comprising a polynucleotide that encodes the amino acid sequence of SEQ ID NO: 4.
  • the present invention also relates to vectors comprising the isolated polynucleotides of the invention as well as cells, such as recombinant mammalian cells, transfected with these vectors.
  • the present invention relates to an isolated polypeptide comprising an amino acid sequence at least 95% identical to the sequence of SEQ ID NO:4 wherein said amino acid sequence has the proline at residue 178 and wherein any difference with respect to SEQ ID NO: 4 is due solely to conservative amino acid substitutions, as well as to antibodies that selectively, preferably specifically, bind to these polypeptides.
  • the present invention further relates to a method for detecting the presence of a polypeptide of the invention in a sample comprising: (a) contacting the sample with a compound that selectively binds to the polypeptide; and (b) determining said selective binding thereby detecting the presence of the polypeptide.
  • the compound is an antibody.
  • the present invention also relates to probes, such as small oligonucleotides, that hybridize with the nucleotide sequences of the polynucleotides of the invention, especially SEQ ID NO:3, but do not hybridize with the nucleotide sequence of a wild-type ApoA-1 (i.e., SEQ ID NO:1 ) under the same conditions.
  • probes such as small oligonucleotides, that hybridize with the nucleotide sequences of the polynucleotides of the invention, especially SEQ ID NO:3, but do not hybridize with the nucleotide sequence of a wild-type ApoA-1 (i.e., SEQ ID NO:1 ) under the same conditions.
  • Such conditions include stringent conditions.
  • the present invention also relates to a method for detecting the presence of a nucleic acid molecule as disclosed herein according to the invention in a sample, comprising: a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to a complement of the nucleic acid molecule; and b) determining binding of said nucleic acid probe or primer to the nucleic acid molecule in the sample thereby detecting the presence of the nucleic acid molecule of the invention in the sample.
  • the present invention relates to a method for detecting an ApoA-1 mutation comprising amplifying the region of a polynucleotide of the invention that contains the mutation.
  • a preferred embodiment relates to a method for detecting hypoalphalipoproteinemia in a patient comprising the detection of Apolipoprotein A-1 mutation in an isolated nucleic acid molecule of the invention, most preferably where the mutation is a nucleic acid base change from T to C at position 643 of SEQ ID NO:1.
  • the present invention relates to a method for detecting hypoalphalipoprotenemia in a patient by detection of a mutation in Apolipoprotein A-1 polypeptide, wherein the mutation is an amino acid change from Leu to Pro at position 178 of SEQ ID NO:2.
  • kits comprising materials useful in carrying out the methods of the invention.
  • the kit comprises a compound that selectively binds to the mutant polypeptide of the invention, and appropriate instructions for use.
  • said kit comprises a compound that selectively hybridizes to a complement of a polynucleotide of the invention, preferably one comprising SEQ ID NO: 3.
  • the kit of the invention is a kit for detection of a mutation in an isolated nucleic acid molecule (preferably SEQ ID NO:3) from a biological sample, comprising: a) oligonucleotides specific for SEQ ID NO:3; b) a suitably labeled probe(s); and c) instructions for use of the kit in the method of invention.
  • an isolated nucleic acid molecule preferably SEQ ID NO:3
  • Figures 1 is a multipoint linkage analysis of the BC-11 P family on chromosome 11q23.
  • Figure 2 shows haplotype analysis that demonstrates linkage in BC- 11 P to a 13 cM region on chromosome 11q23 containing the Apolipoprotein A-1 (apoA-1) gene.
  • Figure 3 identifies an apoA-1 mutation.
  • Figure 3a shows the genomic DNA sequence trace of two affected individuals and two unaffected individuals.
  • Figure 3b shows that sequence variance in the apoA-1 gene causes an amino acid change from leucine at position 178 to proline (SEQ ID NO:4) in the two affected individuals BC-11 :111-24 and BC-11 :IV-69 in comparison to two unaffected individuals BC-11 :lll-29 and BC-11 :IV-109.
  • Figure 4 is a comparison of ApoA1 polypeptide sequence across species showing Leucine residue at position 178 is highly conserved in all species analyzed with the exception of the pig.
  • Figure 5 shows the structural analysis of the ApoA1 based on the introduction of a proline residue at position 178, which would be expected to disrupt an alpha helical structure.
  • the present invention came to light when families with low HDL were observed to have an apoA-1 mutation not previously observed.
  • L178P leu178Pro
  • the apoA-l mutant according to the invention is useful for diagnosing a subpopulation of low HDL patients with high likelihood of CAD.
  • the nucleic acid and protein according to the invention affords a new way of diagnosing a patient with high susceptibility to various cardiovascular pathologies which then would allow appropriate preventive and/or treatment regimes.
  • Tissue expression cDNA sources, prostate, brain, colon, gall bladder, germ cell, heart, liver, lung, muscle (skeletal), ovary, pool, pooled, spleen, testis, thymus, pooled, whole embryo.
  • L178P mutation was seen in a total of 30 individuals. All of these individuals have HDL levels ⁇ 10 th percentile for age and sex, with the exception of one individual at the 11 th percentile. This mutation has not been observed in 91
  • the present invention features an assay, preferably a DNA based assay, to identify the presence of a low HDL phenotype in a sample, such as a sample derived from a mammal, especially a human.
  • a sample such as a sample derived from a mammal, especially a human.
  • This assay even succeeds in detecting the presence of this low HDL phenotype in several Dutch families described herein.
  • the outline of the assay is described in Example 2 and is based on the fact that the introduction of the mutation creates an additional restriction site that can be used to indicate the presence of the mutation by use of the specified restriction enzyme.
  • such mutation could be detected by other methods, such as rolling circle amplification, such as where primer extension is utilized, and by mismatch ligation, both techniques being well known in the art.
  • the specific mutation may be easily detected by means of such techniques as, but not limited to, direct detection of mutation by various DNA, RNA and protein based methods.
  • the method may include an amplification step, wherein one or more portions of the mRNA corresponding to the ApoAI gene or fragments thereof containing the mutation, is amplified. Methods for assaying the translation products are well known in the art.
  • Test samples which may be assayed by the methods provided herein preferably include blood but may also include saliva, semen, urine, spinal fluid and other tissues.
  • the present invention also provides reagents such as oligonucleotide primers and polypeptides which are useful in performing these methods.
  • similarity means the exact amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so-termed “percent similarity” then can be determined between the compared polypeptide sequences.
  • Techniques for determining nucleic acid and amino acid sequence identity also are well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded thereby, and comparing this to a second amino acid sequence.
  • identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • Two or more polynucleotide sequences can be compared by determining their "percent identity.”
  • Two or more amino acid sequences likewise can be compared by determining their "percent identity.”
  • the programs available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.), for example, the GAP program are capable of calculating both the identity between two polynucleotides and the identity and similarity between two polypeptide sequences, respectively. Other programs for calculating identity or similarity between sequences are known in the art.
  • Detection methods include, for example, but not limited to, probe assays which utilize the nucleic acid molecule(s) provided herein and which also may utilize nucleic acid amplification methods such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), cycling probe technology, third wave technology and hybridization.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Any of the nucleic acid amplification protocols can be used in the method of the invention including the polymerase chain reaction, QB replicase and ligase chain reaction.
  • NASBA nucleic acid sequence based amplification
  • 3SR can be used as described in Compton (1991) Nature 350, 91-92 and AIDS (1993), Vol. 7 (Suppl 2), S108 or SDA (strand displacement amplification) can be used as described in Walker et al (1992) Nucleic Acid Res. 20, 1691 -1696.
  • percent identity when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the "Compared Sequence") with the described or claimed sequence (the “Reference Sequence”).
  • the Percent Identity is then determined according to the following formula:
  • DNA segment refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector.
  • segments are provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Sequences of non-translated DNA may be present downstream from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.
  • isolated in the context of the present invention with respect to polypeptides (or polynucleotides) means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living organism is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • coding region refers to that portion of a gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene.
  • the coding region can be from a normal, mutated or altered gene, or can even be from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.
  • nucleotide sequence refers to a heteropolymer of deoxyribonucleotides.
  • DNA segments encoding the proteins disclosed herein are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.
  • expression product means that polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s).
  • fragment when referring to a coding sequence, means a portion of DNA comprising less than the complete coding region whose expression product retains essentially the same biological function or activity as the expression product of the complete coding region.
  • primer means a short nucleic acid sequence that is paired with one strand of DNA and provides a free 3'OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.
  • promoter means a region of DNA involved in binding of RNA polymerase to initiate transcription.
  • ORF open reading frame
  • reference to a DNA sequence includes both single stranded and double stranded DNA.
  • the specific sequence unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded DNA) and the complement of such sequence.
  • the present invention also relates to probes, comprising oligonucleotide sequences, that specifically hybridize to the mutated form of the ApoA-1 sequence disclosed herein as SEQ ID NO: 3. Such probes will commonly discriminate between the mutated and non-mutated forms of the ApoA-1 gene under stringent conditions, such as where there is a minimum of 95% sequence identity between the probe and a segment of equal length derived from the ApoA-1 gene to be tested.
  • Such probes may be of varying lengths but will commonly be at least about 15 contiguous nucleotides in length, possibly 20 or 25. Longer oligonucleotides may also be used but they must, of course, contain the mutated nucleotide or be complementary to it. Most conveniently, such probes will contain a G residue that is complementary to the C residue of the mutation disclosed herein (as opposed to an A that would be complementary to the T residue of the wild-type) so that hybridization will occur only with the mutated form of the gene. In some embodiments, such probes may act as primers for amplification procedures to detect the mutation. The probes may also be used in pairs that are complementary to contiguous segments that include the mutant nucleotide.
  • the probes upon hybridizing to the ApoA-1 derived sequence, are situated adjacent to each other and can then be ligated enzymatically using a ligase sensitive to a single nucleotide mismatch.
  • one of the probes contains a terminal residue complementary to the mutated nucleotide and so ligation produces a longer oligonucleotide whereas the wild-type sequence results in a nucleotide mismatch at the point where the probes are adjacent so that subsequent ligation fails, thereby detecting a mutation in the ApoA-1 sequence.
  • Non-amplification methods include, but are not limited to, RFLP mutation.
  • SNP detection methods may also be useful, especially where these involve rolling circle amplification using primer extension.
  • Detection methods include, for example, but not limited to, protein assays which utilize the amino acid sequence(s), various protein separation methods, such as electrophoretic gel separation, mass spec, and HPLC. Other methods such immunological methods combined with non-isotopic or and isotopically labeled or use of ligands and reporters are well known in the art.
  • Monoclonal and polyclonal antibodies directed against the mutation carrying epitope contained within these polypeptide sequences are useful for diagnostic tests and for screening for diseases or conditions associated with the variant, especially hypoalphalipoproteinemia.
  • Methods for preparing antibodies and for determining immunological reactivity include, but are not limited to, for example, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA), fluorescence polarization immunoassay (FPIA), chemiluminescent immunoassay (CLIA) and others.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • HA hemagglutination
  • FPIA fluorescence polarization immunoassay
  • CLIA chemiluminescent immunoassay
  • compositions and methods described herein will enable the identification of certain markers as indicative of hypoalphalipoproteinemia.
  • the present invention can be performed on the general population to assess the presence or risk of disease.
  • target patients may be tested to detect a particular type of disease, for example familial hypoalphaproteinemia.
  • subgroups of those patients who already are thought to be at some increased risk such as e.g., a weak family history, may be tested.
  • the present methods of detecting the presence of a disease, assessing the risk of contracting a disease, and detecting the risk may suitably be carried out on any subject selected from the population as a whole. However, it may be preferred to carry out this method on certain selected groups of the general population when screening for the presence of particular types of hypoalphalipoproteinemia. Preferably, the present method is used to screen selected groups which are already known to have an increased risk of contracting the particular type of CAD in question.
  • the mutated polypeptides disclosed herein can be detected also by use of antibodies specific for such polypeptides.
  • antibodies specific for such polypeptides With the advent of modern methods of molecular biology and recombinant technology, it is now possible to produce antibody molecules by recombinant means and thereby generate gene sequences that code for specific amino acid sequences found in the polypeptide structure of the antibodies.
  • Such antibodies can be produced by either cloning the gene sequences encoding the polypeptide chains of said antibodies or by direct synthesis of said polypeptide chains, with in vitro assembly of the synthesized chains to form active tetrameric (H 2 L 2 ) structures with affinity for specific epitopes and antigenic determinants.
  • all antibodies have a similar overall 3 dimensional structure.
  • This structure is often given as H 2 L 2 and refers to the fact that antibodies commonly comprise 2 light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions capable of interacting with a structurally complementary antigenic target.
  • variable regions interacting with the target are referred to as “variable” or “V” regions and are characterized by differences in amino acid sequence from antibodies of different antigenic specificity.
  • the variable regions of either H or L chains contains the amino acid sequences capable of specifically binding to antigenic targets. Within these sequences are smaller sequences dubbed “hypervariable” because of their extreme variability between antibodies of differing specificity. Such hypervariable regions are also referred to as “complementarity determining regions” or "CDR" regions. These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure.
  • the CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains.
  • the variable heavy and light chains of all antibodies each have 3 CDR regions, each non-contiguous with the others (termed L1 , L2, L3, H1 , H2, H3) for the respective light (L) and heavy (H) chains.
  • the accepted CDR regions have been described by Kabat et al, J. Biol. Chem. 252:6609-6616 (1977). The numbering scheme is shown in the figures, where the CDRs are underlined and the numbers follow the Kabat scheme.
  • antibody polypeptides contain constant (i.e., highly conserved) and variable regions, and, within the latter, there are the CDRs and the so-called "framework regions” made up of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs.
  • the antibodies disclosed according to the invention may also be wholly synthetic, wherein the polypeptide chains of the antibodies are synthesized and, possibly, optimized for binding to the polypeptides disclosed herein as being receptors.
  • Such antibodies may be chimeric or humanized antibodies and may be fully tetrameric in structure, or may be dimeric and comprise only a single heavy and a single light chain.
  • Such antibodies may also include fragments, such as Fab and F(ab )' fragments, capable of reacting with and binding to any of the polypeptides disclosed herein as being receptors.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques.
  • the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coll lac or trp, the phage lambda P ⁇ _ promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, • may be employed to transform an appropriate host to permit the host to express the protein.
  • bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS or Bowes melanoma
  • adenoviruses plant cells, etc.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1 , pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are pKK232-8 and pCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P and tip.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill ,in the art.
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae Trp1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ⁇ - factor, acid phosphatase, or heat shock proteins, among others.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wl, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • appropriate means e.g., temperature shift or chemical induction
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required non- transcribed genetic elements.
  • the polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • substitutions may be of a conservative nature, for example, where one amino acid is replaced by an amino acid of similar structure and characteristics, such as where a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be replacement of amino acids of the same or similar size and chemical nature, such as where leucine is replaced by isoleucine.
  • certain amino acid substitutions are more often tolerated than others, and these are often show correlation with similarities in size, charge, polarity, and hydrophobicity between the original amino acid and its replacement, and such is the basis for defining "conservative substitutions.”
  • amino acids may be described chemically as hydrophobic, polar, acidic, or basic. Less conservative substitutions might involve the replacement of one amino acid by another that has similar characteristics but is somewhat different in size, such as replacement of an ala ' nine by an isoleucine residue. Highly non-conservative replacements might involve substituting an acidic amino acid for one that is polar, or even for one that is basic in character.
  • radical substitutions cannot be dismissed as potentially ineffective since chemical effects are not totally predictable and radical substitutions might well give rise to serendipitous effects not otherwise predictable from simple chemical principles.
  • the present invention also relates to a process for diagnosing the presence of an aPOa-1 -linked disease in a patient suspected of being afflicted therewith comprising detecting a mutation in the ApoA-1 gene in the genome of said patient.
  • said mutation is detected in a sample of DNA taken from said patient, most preferably the mutation of SEQ ID NO: 3.
  • said detecting is accomplished by determining the ability of a nucleic acid probe comprising at least 15 contiguous nucleotides that are complementary to a mutated portion of the sequence of SEQ ID NO: 1 , most preferably wherein said probe comprises at least 30 contiguous nucleotides. Longer probes may be employed so long as they specifically detect the mutation.
  • the disease to be detected is a cardiovascular disease, including a disease characterized by low plasma HDL levels, most preferably hypoalphalipoproteinemia.
  • the present invention also relates to a process for determining a patient's risk of developing an ApoA-1 -linked disease where said patient is suspected to be at risk thereof, comprising detecting a mutation in the ApoA-1 gene in the genome of said patient.
  • said mutation is detected in a sample of DNA taken from said patient. Said detecting may also be accomplished in situ.
  • said mutation is a mutation in the nucleotide sequence of SEQ ID NO: 1, especially the mutation of SEQ ID NO: 3.
  • said disease is a cardiovascular disease or a disease characterized by low HDL levels, especially hypoalphalipoproteinemia.
  • the present invention also relates to a process for identifying an agent having therapeutic activity in ameliorating the symptoms of an ApoA-1 -linked disease comprising : (a) contacting a compound with a source of ApoA-1 biological activity,
  • said agent is administered to a patient afflicted with said disease and determining a decrease in one or more symptoms of said disease, thereby identifying an agent having said therapeutic activity.
  • said disease is a cardiovascular disease, or a disease characterized by low HDL levels, especially hypoalphalipoproteinemia.
  • the present invention still further relates to a process for treating an
  • ApoA-1 -linked disease in a patient afflicted therewith comprising administering to said patient an effective amount of an agent that modulates the biological activity of ApoA-1 , wherein such agent was first identified as having such activity by the processes of the invention.
  • said disease is a cardiovascular disease, or a disease characterized by low HDL levels, especially hypoalphalipoproteinemia.
  • said agent is an antibody that binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 3.
  • the present invention also relates to a process that comprises a method for producing a product comprising identifying an agent according to one of the disclosed processes for identifying such an agent (i.e., the therapeutic agents identified according to the assay procedures disclosed herein) wherein said product is the data collected with respect to said agent as a result of said identification process, or assay, and wherein said data is sufficient to convey the chemical character and/or structure and/or properties of said agent.
  • identifying an agent i.e., the therapeutic agents identified according to the assay procedures disclosed herein
  • said product is the data collected with respect to said agent as a result of said identification process, or assay, and wherein said data is sufficient to convey the chemical character and/or structure and/or properties of said agent.
  • the present invention specifically contemplates a situation whereby a user of an assay of the invention may use the assay to screen for compounds having the desired enzyme modulating activity and, having identified the compound, then conveys that information (i.e., information as to structure, dosage, etc) to another user who then utilizes the information to reproduce the agent and administer it for therapeutic or research purposes according to the invention.
  • information i.e., information as to structure, dosage, etc
  • the user of the assay may screen a number of test compounds without knowing the structure or identity of the compounds (such as where a number of code numbers are used the first user is simply given samples labeled with said code numbers) and, after performing the screening process, using one or more assay processes of the present invention, then imparts to a second user (user 2), verbally or in writing or some equivalent fashion, sufficient information to identify the compounds having a particular modulating activity (for example, the code number with the corresponding results).
  • This transmission of information from user 1 to user 2 is specifically contemplated by the present invention.
  • the present invention includes kits which are useful for the detection of the hypoalphalipoproteinemia.
  • the kits contain those components, ingredients, and/or means for carrying out the present methods.
  • the data for the ApoAI gene is shown in row 4.
  • the dark gray area shows the maximum shared haplotypes between families.
  • Light gray shows the minimal shared haplotypes between families.
  • On the far right is a minimal segment.
  • ApoAI Assay The mutation discovered in, exon 4 of apoAI changed one of the wild-type amino acids Leucine into Proline, a T to C substitution.
  • the restriction enzyme Bsu36l (New England Biolabs) was found to predict this mutation and thus can be used as an indicator for the low HDL phenotype in affected individuals.
  • the presence of the 198 basepair fragment means that the digestion worked, absence of the 198 basepair fragment means the digestion has failed.
  • the presence of the 289 and 378 basepair fragments will mean that the ApoAI Mutation is present in the individuals whereas the absence of both or one of these fragments will indicate the absence of the ApoAI mutation.
  • the presence of the 667 basepair fragment is another quality control of a good digestion whereas the presence of the 865 base-pair fragment will mean a partial or unsuccessful digestion. Table 6
  • the Leu178 >Pro mutation was seen in a total of 30 individuals. All of these individuals have HDL levels ⁇ 10 ,h percentile for age and sex, with the exception of one individual at the 11 th percentile.
  • SEQ ID NO. 1 is a wild type ApoAI cDNA sequence with the nucleotide at position 643 showing the mutation and GenBank designation >gi
  • Homo sapiens apolipoprotein A-I (APOA1), (nt 897 bp). The mutation changes T at 643 to C. Start and stop codons are underlined.
  • GenBank designation is >gi
  • the N-terminal underlined sequence indicates the preform.
  • SEQ ID NO. 3 shows a cDNA with the mutation at nucleotide position 643 (from T to C). Start and stop codons are underlined.
  • SEQ ID NO. 4 shows the amino acid sequence with the mutation from Leu at position 178 to Proline (underlined in bold) in the mature form of ApoA- 1.
  • the N-terminal underlined sequence indicates the preform.

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Abstract

L'invention concerne une mutation de l'apolipoprotéine A-I humaine qui résulte du remplacement de la leucine par une proline en position 178. L'invention concerne les acides nucléiques, vecteurs, séquences d'acides aminés et fragments correspondants. L'invention concerne en outre des méthodes de diagnostic et/ou de pronostic de maladies cardio-vasculaires, en particulier l'hypoalphalipoprotéinémie, qui se fondent sur l'identification de la présence ou l'absence de ladite mutation Leu178∊Pro. L'invention concerne également des méthodes d'utilisation de cette mutation dans le traitement de maladies liées à l'ApoA-1.
PCT/CA2002/001615 2001-10-26 2002-10-25 Mutation d'apolipoproteine a-i utilisee dans le diagnostic et le traitement de maladies cardio-vasculaires WO2003035691A1 (fr)

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WO2008102038A3 (fr) * 2007-02-23 2008-10-09 Progenika Biopharma Sa Procédé et produit de génotypage 'in vitro' présentant des applications en médecine anti-âge
WO2009050275A1 (fr) 2007-10-19 2009-04-23 Pronota N.V. Site sensible aux protéases dans l'apolipoprotéine a1 et ses implications thérapeutiques et diagnostiques
CN102559850A (zh) * 2010-12-16 2012-07-11 广州益善生物技术有限公司 一种apoA5基因突变检测特异性引物和液相芯片
WO2017011275A1 (fr) * 2015-07-10 2017-01-19 Nersissian Aram M Compositions de protéine de facteur viii et méthodes de traitement de l'hémophilie a

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008102038A3 (fr) * 2007-02-23 2008-10-09 Progenika Biopharma Sa Procédé et produit de génotypage 'in vitro' présentant des applications en médecine anti-âge
WO2009050275A1 (fr) 2007-10-19 2009-04-23 Pronota N.V. Site sensible aux protéases dans l'apolipoprotéine a1 et ses implications thérapeutiques et diagnostiques
EP2207800A1 (fr) * 2007-10-19 2010-07-21 Pronota NV Site sensible aux protéases dans l'apolipoprotéine a1 et ses implications thérapeutiques et diagnostiques
US8343932B2 (en) 2007-10-19 2013-01-01 Pronota N.V. Protease-sensitive site in apolipoprotein A1, therapeutic and diagnostic implications
CN102559850A (zh) * 2010-12-16 2012-07-11 广州益善生物技术有限公司 一种apoA5基因突变检测特异性引物和液相芯片
WO2017011275A1 (fr) * 2015-07-10 2017-01-19 Nersissian Aram M Compositions de protéine de facteur viii et méthodes de traitement de l'hémophilie a
US10287338B2 (en) 2015-07-10 2019-05-14 Miran NERSISSIAN Factor VIII protein compositions and methods of treating of hemophilia A

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