WO2007028795A1 - Variants d'acide nucleique dans le gene masp-2, gene associe a une immunite innee alteree - Google Patents

Variants d'acide nucleique dans le gene masp-2, gene associe a une immunite innee alteree Download PDF

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WO2007028795A1
WO2007028795A1 PCT/EP2006/066035 EP2006066035W WO2007028795A1 WO 2007028795 A1 WO2007028795 A1 WO 2007028795A1 EP 2006066035 W EP2006066035 W EP 2006066035W WO 2007028795 A1 WO2007028795 A1 WO 2007028795A1
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masp
nucleic acid
disease
gene
risk
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Lieve Nuytinck
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Innogenetics N.V.
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
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    • 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
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a method and kit for identifying a subject at risk of, or having, an indication associated with altered innate immunity.
  • the present invention is based on the determination of the mannan-binding lectin-associated serine protease 2 (MASP-2) genotype and/or serum level.
  • MASP-2 mannan-binding lectin-associated serine protease 2
  • Immunity to infection is mediated by two systems, the acquired (or adaptive) immune system and the innate (or natural) immune system.
  • the innate immunity system is an evolutionary ancient form of immunity and offers the main resistance to microbial pathogens within the first minutes, hours or days of an infection (Fujita et al., 2002).
  • Innate immunity recognition is mediated by germ-line-encoded receptors, which means that the specificity of each receptor is genetically predetermined.
  • the strategy of the innate immune response may not be to recognize every possible antigen, but rather to focus on a few, highly conserved structures (patterns) present in large groups of microorganisms. These structures are referred to as pathogen-associated molecular patterns (PAMPs), and the receptors of the innate immune system that evolved to recognize them are called pattern-recognition receptors (PRRs) or pattern-recognition molecules (PRMs).
  • PAMPs can be protein, lipid, nucleic acid, and carbohydrate (Lu et al., 2002).
  • PRRs As soon as the PRRs identify the corresponding predetermined carbohydrate pattern on a pathogen, they immediately trigger effector cells to destroy the invading microorganism, rather than after having to undergo a proliferative cycle, as is the case for the time-delayed adaptive immune response.
  • PRRs can be divided into three classes: signaling, endocytic, and secreted (Medzhitov R. et al., 2004).
  • Innate immunity refers to antigen-nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to an antigen. This is the immunity one is born with and is the initial response by the body to eliminate microbes and prevent infection.
  • the complement system is essential for the operation of the innate and adaptive immune defense.
  • This pathway is triggered by oligomeric lectins that recognize arrays of neutral carbohydrates on the surface of pathogens and share the ability to associate with enzymes called mannan-binding lectin-associated serine proteases (MASPs).
  • MASPs mannan-binding lectin-associated serine proteases
  • MASP-2 MASP-I and MASP-3 have also been described.
  • Alternative splicing of the MASP-2 gene generates MBL-associated protein 19 (MAp 19), a nonenzymic protein comprising the N-terminal CUBl -EGF segment of MASP-2 prolonged by four unique residues.
  • MAp 19 MBL-associated protein 19
  • MASP-3 is a splice variant of the MASP-I gene.
  • MASP-2 Thiel S. et al. (1997) identified MASP-2 and its role in complement activation.
  • the MASP-2 polypeptide and nucleic acid sequence are also reported by Stover et al.(1999) and in WO02/06460.
  • Structurally the MASP-2 polypeptide is composed of an N-terminal CUBl domain, followed by an epidermal growth factor (EGF) domain, a second CUB domain (CUB 2), two complement control domains(CCPl and CCP2), and serine protease domain.
  • EGF epidermal growth factor
  • CCP2 complement control domain
  • serine protease domain serine protease domain.
  • the domains of MASP-2 are involved in functional interactions with different proteins of the complement system.
  • the CUBl and EGF domains have been suggested to play a major role in association of MASP-2 with MBL in the MBL-MASP complexes.
  • MASP-2 is the enzyme of the MBL/MASP complex needed for activation of complement factor C4 (Thiel S., 2006). Only few reports are published on the role of the MASP-2 protein in innate immunity. Ytting H. et al. (2005) disclose that MASP-2 levels correlate significantly with recurrence and poor survival of colorectal cancer.
  • MASP2 deficiency missense mutation D 105 G
  • a MASP2 deficiency was at least partly responsible for the manifestations of disease (i.e. frequent infections and chronic inflammatory disease, including pulmonary fibrosis) in a patient.
  • said new genetic markers provide a reliable diagnosis or prediction of the risk to develop a disease or disorder influenced by innate immunity.
  • Identification of such allotypes and haplotypes may not only provide insight as to why the response to treatment varies amongst individuals, but also may potentially decrease morbidity and mortality through improved risk assessment and the administration of prophylactic or "personalized” medicine.
  • the present invention provides a method and kit for identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, based on the MASP-2 genotype, concentration or functionality.
  • the present invention provides a method and kit for identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, comprising detecting in a sample the presence or absence of at least one nucleic acid variant in the MASP-2 gene.
  • the present invention provides a method and kit for identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, comprising detecting in a sample the presence or absence of at least one nucleic acid variant in the MASP-2 gene, or part thereof, whereby the presence of at least one nucleic acid variant identifies whether a subject is at risk or has an indication associated with an altered innate immunity.
  • a specific region of interest in the MASP-2 gene is the C-terminal region, and more specific the region encoding the CCP2 (Sushi 2) domain of the MASP-2 protein. Of particular interest is intron 7, exon 9 and exon 11.
  • the methods and kits of the present invention can also be carried out in combination with other methods for identifying a subject at risk of, or having, an indication associated with altered innate immunity.
  • the methods and kits are carried out in combination with a method for the detection of the presence or absence of a nucleic acid variant, or other markers, in any other gene.
  • Any detection method is part of the present invention.
  • Preferred methods and means for the detection of the presence or absence of the nucleic acid variants of the present invention are hybridization, sequencing, PCR, primer extension, MLPA, OLA and restriction site analysis, or a combination thereof.
  • the method and kits of the present invention identify a subject at risk of, or having, an indication associated with altered innate immunity, and comprises measuring the concentration or functionality of the MASP-2 protein in a biological sample, wherein an increased or decreased MASP-2 concentration or altered functionality compared to a reference value representing a known health status indicates that said subject is at risk or suffers from a disorder associated with altered innate immunity.
  • a further embodiment of the present invention relates to a method for selecting an appropriate treatment or therapeutic agent for a subject at risk of, or having, an indication associated with altered innate immunity, comprising determining the status or activity of the innate immunity by the methods of the present invention and selecting an appropriate treatment or therapeutic agent.
  • FIGURE LEGENDS
  • Figure 1 human MASP-2 wt gDNA sequence (SEQ ID NO: 1): exon sequences and possible polymorphism positions are indicated in respectively grey and bold/boxed. Nucleotide +1 is the A of the ATG-translation initiation codon which is bold and underlined.
  • CDS (var2) join(3370..3374,3458..3686,3844..4021,5038..5169,
  • transcript variant 2 is encoded by transcript variant 2; MBL-associated plasma protein of 19 kD; small
  • Figure 2 human MASP-2 wt protein sequence including signal peptide (SEQ ID NO:2): positions of possible amino acid changes are indicated in bold/boxed.
  • the determination of the nucleic acid sequence and/or the MASP-2 concentration or functionality makes it possible to estimate or identify whether a subject is at risk of, or has, an indication associated with altered innate immunity.
  • the method of the present invention determines the presence of both variant and normal nucleic acids of the MASP-2 gene, or part thereof, in a sample.
  • MASP-2 gene refers to the gene encoding the mannan-binding lectin-associated serine protease 2 protein, and also to analogous or derivatives thereof. "Part thereof refers to the region of interest or the target region, i.e. the region of the MASP-2 gene comprising a nucleic acid variant.
  • a part thereof refers to the 5 'UTR, the promotor region, exon 1 , intron 1 , exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10 and/or exon 11.
  • the current invention relates to a method of identifying a subject at risk of, at risk of having, or having, an indication associated with altered innate immunity, comprising detecting the presence or absence of at least one nucleic acid variant in the MASP-2 gene, or part thereof, whereby the presence of at least one nucleic acid variant identifies whether a subject is at risk of, or has, an indication associated with an altered innate immunity.
  • nucleic acid refers to a single stranded or double stranded nucleic acid sequence and may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides, or may have been adapted for therapeutic purposes. There is no limitation in length. A nucleic acid that is up to about 100 nucleotides in length, is often also referred to as an oligonucleotide.
  • the reference nucleic acid and protein sequences indicated in the current invention are derived from GeneBank (NCBI) and indicated by their respective accession number, as is well known to the person skilled in the art.
  • NCBI GeneBank
  • the nomenclature for the MASP-2 nucleotide and amino acid changes as used herein is generally accepted and recommended by den Dunnen and Antonarakis (2000). Frequent updates of the nomenclature for the description of sequence variations are provided on the web-site of the Human Genome Variation Society. Accordingly, the nucleotide numbering of the coding DNA and RNA reference sequence is as follows:
  • nucleotide +1 is the A of the ATG-translation initiation codon
  • nucleotide 5' of the ATG-translation initiation codon is -1.
  • nucleotide number is preceded by "g.” when a genomic or by "c.” when a cDNA reference sequence is used. Substitutions are designated by ">”.
  • the MASP-2 gene (mannan-binding lectin serine protease 2) is located at chromosome 1 and comprises 11 exons.
  • the reference nucleic acid sequence for MASP-2 is NT 021937 (SEQ ID NO:1; gDNA; Version: NT_021937.17 GI:51458380) and NM_ 006610 (mRNA varl) and NM 139208.
  • the reference MASP-2 protein sequence is NP_ 006601 (SEQ ID NO:2; Version NP 006601.2 GL21264363).
  • the reference MApl9 protein sequence is NP_631947.
  • nucleic acid variant or “polymorphism” or “variant” as used in the present invention, means that the nucleic acid sequence at a certain position in the MASP-2 gene differs relative to one or more reference nucleic acid sequences.
  • nucleic acid polymorphism The most simple nucleic acid polymorphism is a polymorphism affecting a single nucleotide, i.e. a single nucleotide polymorphism or SNP. Nucleic acid polymorphisms further include any number of contiguous and/or non-contiguous differences in the primary nucleotide sequence of the nucleic acid under investigation relative to the primary nucleotide sequence of one or more reference nucleic acids.
  • polymorphic position or "position” refers to the nucleic acid position at which a nucleic acid polymorphism arises.
  • Nucleic acid sequences comprising at least one such polymorphism are referred to as "polymorphic nucleic acid sequences", “polymorphic polynucleotides”, “polymorphic sequences” or the like.
  • the polymorhism or nucleic acid variant can be an insertion, deletion, substitution, tandem repeat or similar.
  • detecting the presence refers to determining whether or not the relevant genetic, physiological and/or biochemical event, linked with the occurrence of a disease is present.
  • both the absence and the presence of a certain event can function as markers.
  • reference to detecting the presence of a nucleic acid variant or a biochemical marker generally encompasses determining whether the marker is present, either based on the absence or the presence of the variant or biochemical marker in a sample. Moreover, this also includes the possible finding that the marker is not present in the sample, i.e. determining the absence (or presence) of a nucleic acid variant or biochemical marker.
  • determining the presence of the marker can also be done indirectly, e.g. where the presence of a nucleic acid variant is linked to disease, the occurrence of this marker can also be done by determining the homozygous presence of the corresponding allele not comprising the nucleic acid variant.
  • allele specific oligonucleotide primers and probes for detecting the presence of a SNP can be specific for the allele where the SNP is not present.
  • hap Io type means a particular pattern of sequential polymorphisms found on a single chromosome.
  • allele is one of several alternative forms of a gene or DNA sequence at a specific chromosomal location (locus). At each autosomal locus an individual possesses two alleles, one inherited from the father and one from the mother.
  • gene “genotype” means the genetic constitution of an individual, either overall or at a specific locus.
  • allotype refers to any of the genetically determined variants in the constant region of a given subclass of an immunoglobulin that is detectable as an antigen by members of the same species having a different constant region.
  • the present invention relates to a method according to the present invention, wherein the MASP-2 genotype has at least one variant allele of the MASP-2 gene (heterozygous).
  • the method of the invention relates to a method according to the present invention, wherein the MASP-2 genotype has two variant alleles of the MASP-2 gene (homozygous).
  • homozygous refers to having two of the same alleles at a locus.
  • heterozygous refers to having different alleles at a locus.
  • the method of the invention comprises the step of determining whether one or more nucleic acid variants in the MASP-2 gene are present in 0, 1 or 2 copies, more particularly whether a nucleic acid variant in the MASP-2 gene is present in one or both alleles.
  • the MASP-2 gene has 11 exons.
  • the invention relates in particular to any polymorphism located within the MASP-2 gene as identified in SEQ ID NO:1, or in the corresponding cDNA or mRNA sequence.
  • the polymorphism is located in the C-terminal region, or more specific in intron 7, exon 11 and in the region encoding the CCP2 domain of the MASP-2 protein.
  • This CCP2 encoding region starts at position g.16328 and ends at position g.17031 of the gDNA sequence of the MASP-2 gene (SEQ ID NO:1).
  • SEQ ID NO:17031 of the gDNA sequence of the MASP-2 gene
  • Of particular interest is intron 7, exon 9 and exon 11 of the MASP-2 gene.
  • intron 7 may contain a nucleic acid variant at position 12226 of the gDNA sequence. More specific, the nucleic acid variant is a G>A substitution/mutation and is identified as rs6695096 (dbSNP NCBI).
  • exon 9 may contain a nucleic acid variant at position 16349 of the gDNA sequence. More specific, the nucleic acid variant is a G>T substitution/mutation, is identified as rs 12711521 (dbSNP NCBI), and results in the amino acid change D371Y. Additionally, exon 9 may contain a nucleic acid variant at position 16368 of the gDNA sequence. More specific, the nucleic acid variant is a T>C substitution/mutation, is identified as rs223346 (dbSNP NCBI), and results in the amino acid change V377A. Also exon 11 is polymorphic and contains a nucleic acid variant at position 19741 of the gDNA sequence. More specific, the nucleic acid variant is a C>T substitution/mutation and is identified as rsl782455 (dbSNP NCBI).
  • wild-type sequence is analogous to the reference sequence.
  • the nucleic acid sequence of the wild type human MASP-2 gene is identified by SEQ ID NO:1.
  • the allele may be normal as in the reference sequence, or it may be a variant, such as a structural or a non- structural variant.
  • the amino acid sequence of the wild type human MASP-2 protein is identified by SEQ ID NO:2.
  • the present invention also covers analogues of MASP-2.
  • An "analogue” is a compound (or molecule) that is a (chemical) structural derivative of MASP-2. It is also used to describe a molecule which may be structurally similar (but not identical) to another, and which exhibits many or some of the same biological functions of MASP-2.
  • An analogue is to be understood as being any peptide sequence capable of the same biological functions as wild-type MASP-2, including recombinant MASP-2.
  • innate immunity refers to the natural ability of an organism to defend itself against invasions by pathogens. "Pathogens” as used herein, may include, but are not limited to bacteria, fungi, parasites, viruses and algae. In addition, innate immunity includes immune responses that affect other diseases, such as cancer, inflammatory diseases, neurological diseases, autoimmune diseases and various infections.
  • an "indication or condition associated with aberrant, modified or altered innate immunity” refers to any indication or disease resulting from a decreased or increased defense mechanism.
  • a decreased defense can increase the susceptibility for infection or inflammation or can increase the risk for acquiring of a particular disease.
  • An increased defense might result in neurological disease, autoimmune disease or inflammatory diseases.
  • MASP-2 deficiencies are associated with an increased risk for infections, inflammation, neurological disease and autoimmune conditions, and influence the severity and/or course of several diseases. Accordingly, MASP-2 deficiencies can be linked with increased susceptibility for disease and/or prognosis for more severe or more frequent disease, or worse outcomes due to complication. Alternatively, it is shown in the present invention that MASP-2 deficiencies can also be associated with a decreased (i.e. lower or no) risk for infections, inflammation, cancer or autoimmune conditions, indicating the protective effect of the MASP-2 deficiency. In general, MASP-2 deficiencies can be linked with altered activity of innate immunity. Furthermore, treatment options can be considered and include eventual MASP-2 replacement therapy. With the methods of the present invention, the risk for developing a disorder associated with an altered activity of innate immunity can be determined.
  • the present invention relates to a method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising detecting the presence or absence of at least one nucleic acid variant in the MASP-2 gene.
  • the present invention relates to a method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising detecting the presence or absence of at least one nucleic acid variant in the MASP-2 gene, whereby the presence of at least one nucleic acid variant identifies a subject at risk of, or having an increased or decreased (reduced) susceptibility for disease.
  • the presence of at least one nucleic acid variant has a protective effect on the development of a disease.
  • the presence of at least one MASP-2 nucleic acid variant identifies a subject at risk of, or having an increased severity of disease.
  • the presence of at least one MASP-2 nucleic acid variant identifies a subject at risk of, or having a modified response to therapy for a disease. Furthermore, the presence of at least one MASP-2 nucleic acid variant identifies a subject at risk of, or having increased risk of transplant rejection.
  • the altered innate immunity is associated with an increased or reduced susceptibility for or severity of infection, an autoimmune disease, cystic fibrosis, a cardiovasular disease, a neurological disease or cancer.
  • infection encompasses bacterial, viral, fungal, parasitic or algae infection.
  • diseases caused by infections are sepsis, severe sepsis, septic shock, otitis media, and recurrent infections especially in children.
  • Sepsis is defined as presence of infection and several of other parameters of general clinical nature, inflammatory, hemodynamic and tissue perfusion parameters. Severe sepsis is the presence of sepsis complicated by organ dysfunction.
  • Septic shock is defined as the presence of severe sepsis accompanied by acute circulatory failure.
  • Otitis media is an infection of the middle ear.
  • the altered innate immunity is associated with one or more of the following autoimmune diseases: rheumatoid arthritis (RA), spondyloarthropathy, systemic lupus erythematosus (SLE), Sjogren's disease, multiple sclerosis (MS), Crohn's disease, coeliac disease, Type 1 diabetes, Kawasaki disease, asthma, atopic dermatitis, dermatomyositis or Behcet's disease.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • MS multiple sclerosis
  • coeliac disease Type 1 diabetes
  • Kawasaki disease asthma
  • atopic dermatitis dermatomyositis or Behcet's disease.
  • the altered innate immunity is associated with one or more of the following cancers: (1) solid tumors such as colon cancer, colorectal cancer, gastric cancer, cervical cancer, lung cancer, liver cancer, kidney cancer or brain cancer, and (2) haematological malignancies such as a) Leukemias: acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML) and chronic lymphoid leukemia (CLL), b) Lymphomas: Hodgkins and non-Hodgkin's lymphomas and c) Myelomas.
  • AML acute myeloid leukemia
  • ALL acute lymphoid leukemia
  • CML chronic myeloid leukemia
  • CLL chronic lymphoid leukemia
  • Lymphomas Hodgkins and non-Hodgkin's lymphomas
  • Myelomas Myelomas.
  • the altered innate immunity is associated with one or more of the following cardiovascular diseases: bypass failure, atherosclerosis, myocardial reperfusion injury, coronary artery disease or heart disease.
  • the altered innate immunity is associated with one or more of the following neurological diseases: Alzheimer's disease, myasthenia gravis, multiple sclerosis, microbial infections, head trauma and stroke, Pick's disease, Parkinson's disease, dementia with Lewy bodies, Huntington disease, chromosome 13 dementias, Down's syndrome, cerebrovascular disease, Rasmussen's encephalitis, viral meningitis, NPSLE, amyotrophic lateral sclerosis, Creutzfeldt- Jacob disease, Gerstmann-Straussler-Scheinker disease, transmissible spongiform encephalopathies or ischemic reperfusion damage.
  • the altered innate immunity is associated with an increased severity of disease influencing the course of a disease.
  • the disease status can be aggravated leading to a higher mortality.
  • autoimmune disease for example RA
  • the severity or damage to the joints can be more pronounced, as measured by radiology.
  • the altered innate immunity is associated with a modified response to therapy for a specific disease resulting in adverse effects. This can for example by seen in vaccinations or in NSAID therapy.
  • the present invention relates to a method of identifying a subject at risk of, or having, an increased or decreased susceptibility for (recurrent) otitis media, rheumatoid arthritis, recurrent infections, sepsis, severe sepsis, septic shock, or cancer, more specific a solid tumor or a haematological malignancy, comprising detecting the presence or absence of at least one nucleic acid variant in the MASP-2 gene. Accordingly, the presence of the nucleic acid variant is associated with an increased or decreased (reduced) risk for developing a disease. In the latter case, the nucleic acid variant has a protective effect.
  • the present invention thus also relates to a method for determining a protective effect on the development of (recurrent) otitis media, rheumatoid arthritis, recurrent infections, sepsis, severe sepsis, septic shock or cancer, more specific a solid tumor or a haematological malignancy, comprising detecting the presence or absence of at least one nucleic acid variant in the MASP-2 gene.
  • the nucleic acid variant is located in the C-terminal region of the MASP-2 gene, or more specific in intron 7, exon 11 and the region encoding the CCP2 domain of the MASP-2 protein. Even more specific, the nucleic acid variant is located in intron 7, exon 9 or exon 11 of the MASP-2 gDNA sequence. More particular, the nucleic acid variant is located at position 12226, 16349, 16368 and/or 19741 of the MASP-2 gDNA sequence.
  • defect refers to an alteration in the genomic DNA sequence when compared to the wild type sequence. This may result in altered (enhanced or decreased) expression levels or may result in changes (increase or decrease) in the functionality of the encoded protein, or even may result in a change of function of the protein ("gain of function").
  • the "subject" on which the method of the present invention is carried out can be any subject of which the risk of an altered innate immunity needs to be determined.
  • the subject may be a non-human subject such as (but not limited to) a cow, a pig, a sheep, a goat, a horse, a monkey, a rabbit, a dog, a cat, a mouse, a rat, a hamster, a zebrafish, a pufferfish (Fugu), a fly, a worm or C. elegans. More preferably, the subject is a primate. Even more preferably, the subject is a human.
  • nucleic acid from any nucleated cell can be used as the starting point for such assay techniques and may be isolated according to standard nucleic acid preparation procedures well known to those of skill in the art.
  • Many current methods for the detection of allelic variation are reviewed by Nollau et al. (1997), and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford University Press, 1996 and “PCR", 2 nd Edition” by Newton & Graham, BIOS Scientific Publishers Limited, 1997 (incorporated herein by reference).
  • the method of the present invention can be carried out in vivo or in vitro. Preferred, however, is in vitro detection of nucleic acid variants in the MASP-2 gene in a biological sample obtained from the subject.
  • biological sample means a tissue sample or a body fluid sample.
  • a tissue sample includes (but is not limited to) buccal cells, a brain sample, a skin sample or organ sample (e.g. liver).
  • body fluid refers to all fluids that are present in the body including but not limited to blood, plasma, serum, lymph, urine, saliva or cerebrospinal fluid.
  • the biological sample may also be obtained by subjecting it to a pre treatment if necessary, for example, by homogenizing or extracting. Such a pretreatment may be selected appropriately by those skilled in the art depending on the biological sample to be subjected.
  • a nucleic acid comprising an intended sequence prepared from a biological sample may be prepared from DNA (e.g. gDNA or cDNA) or RNA (e.g. mRNA). Release, concentration and isolation of the nucleic acids from the sample can be done by any method known in the art. Currently, various commercial kits are available such as the QIAamp Blood Kit from Qiagen (Hilden, Germany) for the isolation of nucleic acids from blood samples, or the 'High pure PCR Template Preparation Kit' (Roche Diagnostics, Basel, Switzerland) or the DNA purification kits (PureGene, Gentra, Minneapolis, US). Other, well-known procedures for the isolation of DNA or RNA from a biological sample are also available (Sambrook et al., 1989; Ausubel et al., 2003).
  • the nucleic acid may be amplified.
  • amplification procedures can be accomplished by those methods known in the art, including, for example, the polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification, rolling circle amplification, T7- polymerase amplification, and reverse transcription polymerase reaction (RT-PCR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • strand displacement amplification strand displacement amplification
  • rolling circle amplification rolling circle amplification
  • T7- polymerase amplification T7- polymerase amplification
  • RT-PCR reverse transcription polymerase reaction
  • the present invention is not limited by any particular method used to detect the target sequences disclosed herein. Examples of such methods are described by Gut (2001) and Syvanen (2001), and include, but are not limited to, hybridization methods such as reverse dot blot, Line Probe Assay (LiPA), geneChipTM microarrays, dynamic allel-specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, TaqManTM (5 'nuclease assay) and molecular beacons; allele-specific PCR methods such as intercalating dye, FRET primers and AlphascreenTM; primer extension methods such as ARMS, kinetic PCR, SNPstreamTM, Genetic Bit AnalysisTM (GBA), multiplex minisequencing, SNaPshot, pyrosequencing, MassExtend, MassArray, Goodassay, microarray miniseq, APEX (arrayed primer extension), sequence specific priming (SSP), microarray primer extension, Tag arrays
  • the detection of the presence or absence of a nucleic acid variant is determined by DNA or RNA hybridization, sequencing, PCR, primer extension, MLPA, oligonucleotide ligation assay (OLA) or restriction site analysis, or a combination thereof.
  • the detection of the presence or absence of a nucleic acid variant is determined by the Line Probe Assay (LiPA), or by the 4MATTM assay.
  • the method of the present invention optionally comprises the steps of isolating nucleic acids from the sample and/or an amplification step.
  • the present invention also provides isolated oligonucleotides, i.e. primers and probes, in order to amplify and/or detect nucleic acid variants and/or the wild type sequence of the MASP-2 gene.
  • the wild type sequence of the MASP-2 gene is identified by SEQ ID NO: 1.
  • Such primers or probes, specifically hybridizing to the target nucleic acid are of any convenient length such as to consist of at least 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides and up to 40 nucleotides, up to 35 nucleotides, up to 30 nucleotides or more conveniently up to 25 nucleotides in length.
  • a preferred length of the primers or probes is thus 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.
  • such primers or probes will comprise nucleotide sequences entirely complementary to the corresponding wild type or variant locus in the MASP-2 gene.
  • one or more nucleotides may be added or one or more mismatches may be introduced, provided that the discriminatory power of the oligonucleotide primer or probe is not unduly affected.
  • Specific length and sequence of the probes and primers will depend on the complexity of the required nucleic acid target, as well as on the reaction conditions such as temperature and ionic strength. In general, the hybridization conditions are to be stringent as known in the art.
  • “Stringent” refers to the condition under which a nucleotide sequence can bind to related or non-specific sequences. For example, high temperature and lower salt increases stringency such that non-specific binding or binding with low melting temperature will dissolve.
  • the primers or probes of the invention may carry one or more labels to facilitate detection.
  • the primer or probe consists of 10 to 30 nucleotides, preferably 15 to 30 nucleotides, and is capable of specifically forming a hybrid with a part of the MASP-2 gene and is at least one or more selected from the group consisting of:
  • an oligonucleotide capable of hybridizing under a stringent condition with the sequence as represented by SEQ ID NO:1, or the complement thereof;
  • the present invention relates to an isolated oligonucleotide consisting of 10 to 30 nucleotides, optionally 15 to 30 nucleotides, for detecting the presence of one or more nucleic acid variants in SEQ ID NO:1, or the complementary strand.
  • the nucleic acid variant is located at position 12226, 16349, 16368 and/or 19741 of SEQ ID NO:1.
  • the polymorphism located in the MASP-2 gene may also be detected in vitro by determining in the isolated MASP-2 protein, as identified in the present invention, the presence or absence of an amino acid change by sequencing said protein.
  • the amino acid change may also be detected by any conventional method known in the art, for example by mass-spectroscopy, gel electrophoresis, MALDI-TOF mass spectroscopy, ELISA, protein arrays, determination of the molecular weight, or by iso electrofocusing .
  • ficolin genes i.e.
  • the present invention also relates to a method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising the step of detecting the presence or absence of a nucleic acid variant in the MASP-2 gene and detecting the presence or absence of one or more nucleic acid variants in any other gene.
  • the "other" gene is selected from the group consisting of: the ficolin genes (i.e.
  • other risk factors can be determined in combination with the method of the method
  • the present invention also encompasses a method for determining whether a subject has a risk of developing a disease wherein the nucleic acid variants in the MASP-2 gene are detected by their protein phenotype.
  • the invention discloses how decreased or increased levels of MASP-2 and lack of functional MASP-2 is crucial in the innate immunity defense.
  • the method encompasses the measurement of one or more proteins.
  • the present invention relates to a method for identifying a subject at risk of, or having, an indication associated with altered innate immunity comprising the steps of: a) determining the concentration or functionality of the MASP-2 protein in a sample, b) identifying if said subject is at risk of, or has, an indication associated with altered innate immunity.
  • the current invention provides a method of identifying a subject at risk of, or having, an indication associated with altered innate immunity, comprising measuring the concentration or functionality of the MASP-2 protein in a biological sample, wherein an increased or decreased MASP-2 concentration or altered functionality compared to a reference value representing a known health status indicates that said subject is at risk of or suffers from a disorder associated with altered innate immunity.
  • a "MASP-2 protein” is a protein encoded by the MASP-2 gene as described in the present invention, or a variant thereof.
  • the human wild type protein is encoded by SEQ ID NO:1 and is identified by SEQ ID NO:2.
  • concentration or “level”, as used in the present invention, refers to the presence or absence and/or amount of a certain protein.
  • a change in the concentration of a protein refers to a measurable increase or decrease, including total absence or presence, in the protein concentration when compared to a control subject.
  • "A known health status" or "control subject”, as defined in the present invention is a subject of the same species as the subject under examination which is free from, or not at direct risk of developing a disease associated with altered innate immunity.
  • the healthy subject can be of the same weight, age, and gender as the subject who is being diagnosed or prognosed for an altered innate immunity. In some cases, it might be preferred to use a reference value from the subject which is diagnosed.
  • concentration obtained upon analyzing the subject under examination relative to the concentration obtained upon analyzing a control subject will depend on the particular analytical protocol and detection technique that is used. Accordingly, those skilled in the art will understand that, based on the present description, any laboratory can establish, for the MASP-2 protein, a suitable "reference range”, “reference level range”, “concentration range” or “range of levels” (those terms are used interchangeable) characteristic for control subjects according to the analytical protocol and detection technique in use.
  • the concentration obtained for the subject under examination can then be compared with this reference range and based on this comparison, a conclusion can be drawn as to whether the subject has a risk of developing a disease as described herein.
  • a cut-off value suitable for determining whether a subject is at risk of, or has, an altered innate immunity.
  • Methods for defining cut-off values include (but are not limited to) the methods described by IFCC (1987).
  • the reference value can be that of a level or concentration of the MASP-2 protein in a sample, preferably a body fluid, from a subject not suffering from a disease associated with altered innate immunity.
  • the healthy subject can be of the same weight, age, and gender as the subject who is being diagnosed or prognosed for an altered innate immunity. In some cases, it might be preferred to use a reference value from the subject which is diagnosed.
  • the MASP-2 protein that is detected in the method of the present invention may be detected by any method known to those skilled in the art. They can be identified by their structure, by partial amino acid sequence determination, by functional assay, by enzyme assay, by various immunological methods, or by biochemical methods, known to those skilled in the art.
  • Biochemical methods include (but are not limited to) capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, two-dimensional liquid phase electrophoresis (2-D- LPE; Davidsson et al. 1999) or detection of the migration pattern in gel electrophoreses.
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) is a widely used approach for separating proteins from complex mixtures (Patterson and Aebersold, 1995). It can be performed in one- or two-dimensional (2- D) configuration. For less complicated protein preparation, one-dimensional SDS- PAGE is preferred over 2-D gels, because it is simpler.
  • 2-D gel electrophoresis incorporates isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension, leading to a separation by charge and size (O'Farrell, 1975).
  • IEF isoelectric focusing
  • 2-D PAGE is a powerful technique for separating very complex protein preparations, resolving up to 10 000 proteins from mammalian tissues and other complex proteins (Klose and Kobalz, 1995; Celis et al., 1996; Yan et al., 1997).
  • the MASP-2 proteins of the present invention can be identified by their isoelectric focusing point (pi) and their molecular weight (MW) in kilodaltons (kD).
  • the level of MASP-2 protein can also be detected by an immunoassay.
  • an "immunoassay” is an assay that utilizes an antibody to specifically bind to the antigen (i.e. the MASP-2 protein). The immunoassay is thus characterized by detection of specific binding of the MASP-2 protein to an antibody.
  • Immunoassays for detecting MASP-2 proteins may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte (i.e. the MASP-2 protein) is directly measured. In competitive assays, the amount of analyte (i.e.
  • the MASP-2 protein) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent (i.e. the antibody) by the analyte (i.e. the MASP-2 protein) present in the sample.
  • a competition assay a known amount of the (exogenous) MASP-2 protein is added to the sample and the sample is then contacted with the antibody.
  • the amount of added (exogenous) MASP-2 protein bound to the antibody is inversely proportional to the concentration of the MASP-2 protein in the sample before the exogenous MASP-2 protein is added.
  • the antibodies can be bound directly to a solid substrate where they are immobilized.
  • immunological methods include but are not limited to fluid or gel precipitation reactions, immunodiffusion (single or double), agglutination assays, immunoblotting , immunospotting (such as line immunoassays or LIA), Immunoelectrophoresis, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), TRIFMA (Christiansen et al, 1999), Western blots, liposome immunoassays (Monroe et al., 1986), complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays or immunoPCR.
  • An overview of different immunoassays is given in Wild (2001), Ghindilis et al. (2002) and Kilpatrick (2002).
  • the method of the present invention may also be used in determining whether and which therapeutic agent might be suitable for a patient being at risk of, or having an indication associated with altered innate immunity.
  • the therapeutic agent may be used to prevent or treat the indication or disease.
  • preventing a disease means inhibiting or reversing the onset of the disease, inhibiting or reversing the initial signs of the disease, inhibiting the appearance of clinical symptoms of the disease.
  • treating a disease includes substantially inhibiting the disease, substantially slowing or reversing the progression of the disease, substantially ameliorating clinical symptoms of the disease or substantially preventing the appearance of clinical symptoms of the disease.
  • kits for use in the method as described herein encompasses a kit for identifying a subject at risk of, at risk of having, at risk of having, or having, an indication associated with altered innate immunity.
  • This kit can be based on the detection of nucleic acid variants in the MASP-2 gene of said subject or it can be based on the detection of MASP-2 proteins.
  • the kit of the present invention comprises reagents that selectively detect a nucleic acid variant in the MASP-2 gene or that selectively detect a MASP-2 protein.
  • a kit based on the detection of nucleic acid variants in the MASP-2 gene may comprise: (a) a means or reagent for detecting the presence or absence of one or more nucleic acid variants in the MASP-2 gene of said subject; and
  • step (b) optionally, a means for determining, from the nucleic acid variants detected with the means of step (a), whether the subject is at risk of, or has, an indication associated with altered innate immunity.
  • the kit comprises a means for detecting the presence or absence of one or more nucleic acid variants in the C-terminal region, more specific in the region encoding the CCP2 domain of the MASP-2 protein, or even more specific in intron 7, exon 9 and/or exon 11 of the MASP-2 gene.
  • the kit comprises:
  • step (b) optionally, a means for determining, from the nucleic acid variants detected with the means of step (a), whether the subject is at risk of, or has, an indication associated with altered innate immunity.
  • the means or reagents in step (a) of said kit may comprise:
  • the means or reagent in step (a) of said kit comprises at least one oligonucleotide probe suitable for detection of a target MASP-2 nucleic acid. Said probe specifically hybridizes to the target MASP-2 nucleic acid.
  • the target MASP-2 nucleic acid is located in the C-terminal region of the MASP-2 gene, and more specific in the region encoding the CCP2 domain of the MASP-2 protein.
  • the target MASP-2 nucleic acid is located in intron 7, exon 9 or exon 11, or part thereof. Even more specific, the target MASP-2 nucleic acid is located at position 12226, 16349, 16368 and/or 19741 of the MASP-2 gene. The designated positions either have the wild type nucleotides or nucleic acid variants thereof.
  • the oligonucleotide suitable for detection of a target MASP-2 nucleic acid is a SSP (sequence specific primer).
  • the oligonucleotide suitable for detection of a target MASP-2 nucleic acid is a probe configured to hybridize to said MASP-2 polynucleic acid to form an invase cleavage structure.
  • the cleavage structure can be detected by a cleavage agent or enzyme such as a structure-specific nuclease, a 5 'nuclease, a FEN-I endonuc lease or a polymerase.
  • the means or reagent in step (a) also includes at least one pair of primers suitable for amplification of a target MASP-2 polynucleic acid. Said primers specifically hybridize to the MASP-2 nucleic acid.
  • the target nucleic acid is the C-terminal region of the MASP-2 gene, and more specific the region encoding the CCP2 domain. More particular, the target polynucleic acid is intron 7, exon 9 or exon 11 of the MASP-2 gene, or part thereof. Even more specific, the target MASP-2 polynucleic acid comprises or consists of position 12226, 16349, 16368 and/or 19741 of the MASP-2 gene. The designated positions either have the wild type nucleotides or nucleic acid variants thereof.
  • hybridization buffer means a buffer allowing a hybridization reaction between the oligonucleotides and the polynucleic acids present in the sample, or the amplified products, under the appropriate stringency conditions.
  • wash solution means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.
  • the means for detecting the presence or absence of nucleic acid variants in the MASP-2 gene is an INVADER assay (see e.g. WO97/27214, incorporated herein by reference), or the 4MATTM assay (Innogenetics N. V., Ghent, Belgium).
  • the means for detecting the presence or absence of nucleic acid variants in the MASP-2 gene is a line probe assay (LiPA; Stuyver et al, 1996; Stuyver et al, 1997; Van Geyt et al, 1998).
  • the selected set of probes is immobilized to a membrane strip in a line fashion.
  • An alternative is the immobilization of the probes in a "dotted fashion" (dot spots; DoPA). Said probes may be immobilized individually or as mixtures to the delineated locations.
  • the amplified MASP-2 polynucleic acids can be labelled with biotine, and the hybrid can then, via a biotine-streptavidine coupling, be detected with a non- radioactive colour developing system.
  • oligonucleotides may be coupled to microspheres or chips.
  • An example of an assay that provides for simultaneous detection includes (but is not limited to) the xMAPTM technology (Luminex 100 IS, Austin, Texas, USA) and the PamGene technology (PamGene, 's-Hertogenbosch, The Netherlands).
  • the means in step (b) of said kit for determining, from the nucleic acid variants in the MASP-2 gene detected with the means of step (a), whether the subject is at risk of, or has, an indication associated with altered innate immunity include a table, a chart, or similar, generally referred to as "a predisposition risk algorithm", indicating the MASP-2 nucleic acid variants or haplotypes that confer a risk (increased or decreased) for or the existence of an indication associated with altered innate immunity.
  • a predisposition risk algorithm indicating the MASP-2 nucleic acid variants or haplotypes that confer a risk (increased or decreased) for or the existence of an indication associated with altered innate immunity.
  • a predisposition risk algorithm indicating the MASP-2 nucleic acid variants or haplotypes that confer a risk (increased or decreased) for or the existence of an indication associated with altered innate immunity.
  • a predisposition risk algorithm indicating the MASP-2 nucleic acid variants or
  • the kit of the present invention may include, in additions to the means or reagent for detecting the presence or absence of a nucleic acid variant in the MASP-2 gene, a means for detection other risk factors, e.g. nucleic acid variants in a gene, for an indication associated with an altered innate immunity.
  • the kit additionally includes a means, preferably probes, for detecting the genotype of or a nucleic acid variant in at least one of the genes selected from the group consisting of: the ficolin genes (FCNl, FCN2 and FCN3), ClQRl, LBP, BPI (Bacterial/permeability- increasing protein), CD 14 (CD 14 antigen precursor), beta- catenin (CTNNBI, Cadherin Associated Protein beta), ILlO (Interleukin 10), RP 105 (LY64, lymphocyte antigen 64 homolog radioprotective), MBL2 (Mannose Binding Protein), MD-I (RP105-associated), MD-2 (MD2 Protein, Lymphocyte antigen 96), MYD88 (Myeloid differentiation primary response gene 88), NODl (Caspase recruitment domain 4, CARD4), NOD2 (Caspase recruitment domain family, member 15, CARD 15) and the Toll Like Receptor genes, i.e. TLRl, TLR2, TLR
  • a kit based on the detection of MASP-2 proteins may comprise an antibody that specifically recognizes the MASP-2 protein that is detected.
  • a preferred kit for carrying out the method of the invention comprises: an antibody (primary antibody) which forms an immunological complex with the MASP-2 protein to be detected; - a monoclonal antibody (secondary antibody) which specifically recognizes the MASP-2 protein to be detected; a marker either for specific tagging or coupling with said secondary antibody; appropriate buffer solutions for carrying out the immunological reaction between the primary antibody and the MASP-2 protein, between the secondary antibody and the primary antibody-MASP-2 protein complex and/or between the bound secondary antibody and the marker; or - possibly, for standardization purposes, a purified MASP-2 protein.
  • the statistical analysis of the data is based on the determination of odds ratios (OR) using standard procedures.
  • An odds ratio is calculated by dividing the odds in the treated or exposed (case) group by the odds in the control group.
  • the odds of an event are calculated as the number of events divided by the number of non-events. If the odds of an event are greater than one the event is more likely to happen than not (the odds of an event that is certain to happen are infinite); if the odds are less than one the chances are that the event won't happen (the odds of an impossible event are zero).
  • the strength of association was reported as odds ratios (OR) (with 95% lower (LCL) and upper (UCL) confidence limit), indicating the factor by which the risk of developing a disorder or disease is increased (OR>1), or indicating the factor by which the risk of developing a disorder or disease is decreased (OR ⁇ 1) (protective effect).
  • the 95% confidence interval (95% CI) is the range of numerical values in which we can be confident (to a computed probability, such as 90 or 95%) that the population value being estimated will be found. Confidence intervals indicate the strength of evidence; where confidence intervals are wide, they indicate less precise estimates of effect. The larger the trial's sample size, the larger the number of outcome events and the greater becomes the confidence that the true relative risk reduction is close to the value stated. Thus the confidence intervals narrow and "precision" is increased. In a "positive finding" study the lower boundary of the confidence interval, or lower confidence limit, should still remain important or clinically significant if the results are to be accepted. In a "negative finding" study, the upper boundary of the confidence interval should not be clinically significant if you are to confidently accept this result.
  • Table 1 identifies the MASP-2 nucleic acid and protein variants of the present invention
  • Example 1 Detection of nucleic acid variants in the MASP2 gene from patients with Rheumatoid arthritis and from control subjects
  • the relevant regions were amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 1. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time.
  • Example 2 Detection of nucleic acid variants in the MASP2 gene from children with recurrent infections and from control subjects
  • the control group (C) consisted of 205 healthy individuals. From each patient, informed consent to participate in the study is available.
  • nucleic acid variants in the MASP2 gene were amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Dot Probe
  • hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al.
  • the strength of association was reported as odds ratios (OR).
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time. Frequencies of MASP2 genotypes in children with recurrent infections and control individuals
  • Example 3 Detection of nucleic acid variants in the MASP2 gene from children with a haematological malignancy or solid tumor and from control subjects
  • the relevant regions were amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 1. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996), Stuyver et al. (1997) and Van Geyt et al. (1998).
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, if the study repeated multiple times, would contain the true effect 95% of the time.
  • M major allele (wt)
  • m minor allele (variant)
  • Example 4 Detection of nucleic acid variants in the MASP-2 gene from patients newly diagnosed with rheumatoid arthritis (RA) and from control subjects
  • RA was included in the differential diagnosis. After 1 year, diagnosis established 149 RA patients and 402 non-RA patients (control population).
  • nucleic acid polymorphisms To determine the presence or absence of nucleic acid variants in the MASP-2 gene, the relevant regions (see Table 1) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 1. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996, J. Clin. Microbiol.
  • the p-value represents the calculated probability, well known to the skilled person.
  • the above data show the value of said SNPs in the differential diagnosis of RA versus other diseases such as osteoarthritis, abarticular rheumatic symptoms, spondyloarthropathy, connective tissue diseases including polymyalgia rheumatica, psoriatic arthritis, crystal induced arthritis, fibromyalgia and aspecific arthralgias, being other and undifferentiated diseases including infections, malignancies, and neurological disorders.
  • diseases such as osteoarthritis, abarticular rheumatic symptoms, spondyloarthropathy, connective tissue diseases including polymyalgia rheumatica, psoriatic arthritis, crystal induced arthritis, fibromyalgia and aspecific arthralgias, being other and undifferentiated diseases including infections, malignancies, and neurological disorders.
  • Example 5 Detection of nucleic acid variants in the MASP-2 gene from patients admitted to the ICU who develop sepsis/severe sepsis or septic shock and those who do NOT develop sepsis/severe sepsis or septic shock.
  • Consecutive cohorts of 200 patients presented at an ICU were incorporated in the study. At discharge of ICU, patients were evaluated whether or not sepsis/severe sepsis or septic shock has been developed using clinical criteria well known by ICU practitioners. From this study, 88 patients were diagnosed as NOT having sepsis/severe sepsis or septic shock, while 82 of the patients indeed developed sepsis/severe sepsis or septic shock.
  • nucleic acid variants in the MASP2 gene were amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 1. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996, J. Clin. Microbiol. 34:2259-2266), Stuyver et al. (1997, Antimicrob. Agents Chemoter. 41:284-291) and Van Geyt et al. (1998, in: Therapies of viral Hepatitis. International Medical Press, London, UK pp. 139-145).
  • Example 6 Detection of nucleic acid variants in the MASP2 gene from patients admitted to the ICU who develop septic shock and those who do
  • nucleic acid polymorphisms To determine the presence or absence of nucleic acid variants in the MASP2 gene, the relevant regions (see Table 1) were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method (Dot Probe Assay) with probes designed to recognize the polymorphisms as given in Table 1. After stringent washing at 56°C, hybridized probes were incubated with a streptavidine-alkaline phosphatase conjugate. The presence of a hybridized probe was revealed using NBIT/BCIP color development. Details on the reverse hybridization are described in Stuyver et al. (1996, J. Clin. Microbiol.
  • the 95% confidence interval (95% CI) is the interval computed from the sample data which, were the study repeated multiple times, would contain the true effect 95% of the time.
  • Barber RC O'Keefe GE. Characterization of a single nucleotide polymorphism in the lipopolysaccharide binding protein and its association with sepsis. Am J Respir Crit Care Med. 2003 May 15;167(10):1316-20.
  • Bochkov VN Kadi A, Huber J, Gruber F, Binder BR, Leitinger N. Protective role of phospholipid oxidation products in endotoxin- induced tissue damage. Nature. 2002 Sep 5;419(6902):77-81
  • Bochkov VN Mechtcheriakova D, Lucerna M, Huber J, Malli R, Graier WF, Hofer E, Binder BR, Leitinger N.
  • Oxidized phospholipids stimulate tissue factor expression in human endothelial cells via activation of ERK/EGR-1 and Ca(++)/NFAT. Blood. 2002 Jan l;99(l):199-206.
  • Pavcnik-Arnol M Hojker S, Derganc M. Lipopolysaccharide-binding protein in critically ill neonates and children with suspected infection: comparison with procalcitonin, interleukin-6, and C-reactive protein. Intensive Care Med. 2004 Jul;30(7): 1454-60.

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Abstract

La présente invention porte sur une méthode et sur un kit d'identification d'un sujet à risque ou présentant une indication associée à une immunité innée altérée. Cette invention est basée sur la détermination des génotypes MASP-2 et/ou des taux sériques.
PCT/EP2006/066035 2005-09-09 2006-09-05 Variants d'acide nucleique dans le gene masp-2, gene associe a une immunite innee alteree WO2007028795A1 (fr)

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WO2010078411A1 (fr) 2008-12-30 2010-07-08 Children's Medical Center Corporation Procédé de prédiction d'une appendicite aiguë

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009103992A1 (fr) * 2008-02-19 2009-08-27 Queen Mary & Westfield College Variation génétique associée à la maladie coeliaque
WO2010078411A1 (fr) 2008-12-30 2010-07-08 Children's Medical Center Corporation Procédé de prédiction d'une appendicite aiguë
EP3032258A1 (fr) 2008-12-30 2016-06-15 Children's Medical Center Corporation Procédé de prédiction d'une appendicite aiguë
EP3913367A1 (fr) 2008-12-30 2021-11-24 Children's Medical Center Corporation Procédé de prédiction d'une appendicite aiguë

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