WO2008107377A2 - Combinations of markers for sepsis risk assessment - Google Patents

Combinations of markers for sepsis risk assessment Download PDF

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WO2008107377A2
WO2008107377A2 PCT/EP2008/052474 EP2008052474W WO2008107377A2 WO 2008107377 A2 WO2008107377 A2 WO 2008107377A2 EP 2008052474 W EP2008052474 W EP 2008052474W WO 2008107377 A2 WO2008107377 A2 WO 2008107377A2
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gene
nucleic acid
acid variant
polymorphism
subject
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French (fr)
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WO2008107377A3 (en
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Lieve Nuytinck
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Innogenetics N.V.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to methods and kits for identifying a subject at risk of developing sepsis, severe sepsis or septic shock.
  • Systemic inflammatory conditions represent one such class of diseases. Sepsis is a complex clinical syndrome as a result of a systemic inflammatory response to live bacteria and/or bacterial products. This response is expressed as a compendium of a variety of different clinical signs and symptoms such as fever, increased blood leukocyte counts, unexplained thrombocytopenia, mental confusion, transient hypotension, and organ stress and dysfunction. The individual response is determined by many factors, including the virulence of the organism, the size of the inoculum, and the patient's coexisting conditions.
  • Sepsis develops when the initial, appropriate host response to an infection becomes amplified, and is then deregulated.
  • the most common sites of infection are the lungs, the abdominal cavity, the urinary tract, and primary infections of the bloodstream. If untreated, septic patients may develop acute respiratory or renal failure, multi-organ dysfunction, shock, and death.
  • Sepsis follows a well-described time course, progressing from systemic inflammatory response syndrome ("SIRS")-negative to SIRS-positive to sepsis, which may then progress to severe sepsis, septic shock, multiple organ dysfunction (“MOD”), and ultimately death. Sepsis also may arise in an infected individual when the individual subsequently develops SIRS.
  • SIRS systemic inflammatory response syndrome
  • MOD multiple organ dysfunction
  • Sepsis also may arise in an infected individual when the individual subsequently develops SIRS.
  • the evolution of the stage of sepsis towards severe sepsis, then towards septic shock is not systematic since approximately 64% of the septic patients develop a severe sepsis, and 23% of the patients in sepsis evolve/move in septic shock. Before this ultimate stage of septic shock, treatments must be prescribed with the patient in order to stop and to reverse this process.
  • SIRS Systemic inflammatory response syndrome
  • Sepsis is defined as the presence of SIRS in the setting of infection. Severe sepsis is defined as sepsis with evidence of end-organ dysfunction as a result of hypoperfusion. Septic shock is defined as sepsis with persistent hypotension despite fluid resuscitation and resulting tissue hypoperfusion (American College of Chest Physicians, 1992).
  • sepsis represents a major burden to the health care system in most developed countries since septic patients require admission and aggressive treatment in the intensive care units (ICU) and are generally hospitalized for longer than 3 weeks.
  • ICU intensive care units
  • the difficulty in early detection of sepsis is reflected by the high morbidity and mortality associated with the disease. Sepsis is estimated to affect 18 million people worldwide each year and to kill 1400 people each day.
  • determining the risk would be made by a technique that accurately, rapidly, and simultaneously measures a plurality of markers at a single point in time, thereby minimizing disease progression during the time required for detection. It has been shown that genetic factors of importance for the inflammatory response such as polymorphisms in the promoter region of the tumour necrosis factor alpha (TNF-alpha) and Interleukin-1 alpha (IL-I alpha) gene may be associated with susceptibility and outcome of septic shock.
  • TNF-alpha tumour necrosis factor alpha
  • IL-I alpha Interleukin-1 alpha
  • the present invention presents a new reliable tool for an improved risk assessment for the development of sepsis, severe sepsis and/or septic shock. It has been found that the analysis of specific gene variants is very relevant to discriminate good or bad prognosis for a patient. The identification of alleles/genotypes linked with an adverse, or alternatively, favourable clinical outcome in sepsis, severe sepsis or septic shock can decrease morbidity and mortality through improved risk assessment and the administration of prophylactic or "personalized" medicine.
  • susceptibility of an individual to develop sepsis, severe sepsis or septic shock can be made with improved accuracy, by the methods and tools of the invention.
  • the invention provides combinations of markers with increased reliability for predicting susceptibility to sepsis, severe sepsis or septic shock.
  • the methods of the present invention can improve personalised therapy for sepsis, severe sepsis or septic shock.
  • the improved risk assessment can be performed using one analysis technique, in a time- and cost-efficient way.
  • a first aspect of the invention provides methods of gaining information about the risk of a subject to develop sepsis, severe sepsis or septic shock, comprising:
  • Another aspect of the invention provides methods of identifying the risk of a subject to develop sepsis, severe sepsis or septic shock, comprising detecting at least two nucleic acid variants selected from the group consisting of:
  • - position -502 of the TLR6 gene being a C/T polymorphism (dbSNP number rslO39559)
  • - position 17962 of the TLR7 gene being a T/A polymorphism (dbSNP number rsl79008)
  • - position 9300 or 12979 of the TLR8 gene being an C/T polymorphism (dbSNP number rs5744080), - position 3392 of the Sigirr gene being a G/A polymorphism (dbSNP number rs3210908), and
  • the method can be carried out in vitro or ex vivo.
  • the methods of the invention comprise the step of determining: - the nucleic acid variant rsl 800450 in the MBL2 gene and the nucleic acid variant rs2563298 in de CD14 gene, and/or
  • nucleic acid variant rs5744080 in the TLR8 gene and the nucleic acid variant rs2989727 or rsl 071583 in de FCNl gene, and/or
  • nucleic acid variant rsl 7514136 in the FCN2 gene and the nucleic acid variant rsl898830 in de TLR2 gene or the nucleic acid variant rsl927914 in the TLR4 gene, or the nucleic acid variant rsl 039559 in the TLR6 gene, or the nucleic acid variant rs5744080 in the TLR8 gene, and/or
  • nucleic acid variant rs7851696 in the FCN2 gene and the nucleic acid variant rs3210908 or rs3087588 in de Sigirr gene or the nucleic acid variant rs3775291 in the TLR3 gene, or the nucleic acid variant rsl7549193 or rsl 7514136 in the FCN2 gene, and/or
  • nucleic acid variant rsl 79008 in the TLR7 gene and the nucleic acid variant rslO39559 in de TLR6 gene; wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop sepsis, severe sepsis or septic shock.
  • Specific embodiments of the above-cited methods of the invention comprise methods wherein the step of determining the occurrence of at least two nucleic acid variants in one or more genes in that subject comprises the step of determining the presence or absence of a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • nucleic acid variants in one or a combination of genes involve the detection of two or more nucleic acid variants or SNPs located at position 162 of the MBL2 gene, at position 1336 of the CD14 gene, at position -1981 or 7919 of the FCNl gene, at position -4,-64, 6359 or 6424 of the FCN2 gene, at position 70465 of the MASPl gene, at position -15607 of the TLR2 gene, at position 6301 of the TLR3 gene, at position -2026 of the TLR4 gene, at position -502 of the TLR6 gene, at position 17962 of the TLR7 gene, at position 9300 or 12979 of the TLR8 gene, and/or at position 3392 or 3402 of the Sigirr gene, of the genomic DNA sequence.
  • heterozygous or homozygous presence (or, where appropriate, absence) of the at least one nucleic acid variant is indicative of having or of a risk of having or developing sepsis, severe sepsis or septic shock.
  • nucleic acid variant rs5744080 in the TLR8 gene and the nucleic acid variant rs2989727 or rsl 071583 in de FCNl gene, and/or
  • nucleic acid variant rs7851696 in the FCN2 gene and the nucleic acid variant rs3210908 or rs3087588 in de Sigirr gene or the nucleic acid variant rs3775291 in the TLR3 gene, or the nucleic acid variant rsl7549193 or rsl 7514136 in the FCN2 gene, and/or
  • nucleic acid variant rsl 79008 in the TLR7 gene and the nucleic acid variant rslO39559 in de TLR6 gene, wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop sepsis, severe sepsis or septic shock.
  • the methods comprise detecting at least two nucleic acid variants as given in Table 2.
  • the invention encompasses a method and kit for the identification of the risk of a subject to develop sepsis, severe sepsis or septic shock., said method comprising the step of determining he combined presence of a nucleic acid variant as given in Table 4.
  • nucleic acid variants as presented in Table 4, wherein it is determined if the nucleic acid variant is present in 0, 1 or 2 copies.
  • the present invention provides a method of identifying a subject at risk of developing severe sepsis or septic shock comprising: determining the presence of at least one nucleic acid variant in the MASP2 gene and at least one nucleic acid variant in the FCN2 gene in said subject; and/or determining the presence of at least one nucleic acid variant in the FCN2 gene and at least one nucleic acid variant in the LBP gene or at least one nucleic acid variant in the CIqRl gene, or at least one nucleic acid variant in the TLR6 gene, or at least one nucleic acid variant in the CD 14 gene in said subject; and/or determining the presence of at least one nucleic acid variant in the LBP gene and at least one nucleic acid variant in the TLR6 gene or at least one nucleic acid variant in the TLRl gene in said subject; and/or determining the presence of at least one nucleic acid variant in the LBP gene and at least one nucleic acid variant in the TLR6 gene or at least one nucleic
  • - position 1336 of the CD14 gene being a G/T polymorphism (dbSNP number rs2563298), - position -7202 of the TLRl gene being a A/G polymorphism (dbSNP number rs5743551),
  • TLR2 gene being a T/C polymorphism (dbSNP number rs3804100),
  • TLRlO gene being a T/C polymorphism (dbSNP number rs7694115),
  • TLRlO gene - position -992 of the TLRlO gene being a T/A polymorphism (dbSNP number rsl 1466645), - position 721 of the TLRlO gene being a A/C polymorphism (dbSNP number rsl 1096957),
  • nucleic acid variant rs 12711521 in the MASP2 gene and the nucleic acid variant rsl7514136 or rsl7549193 in de FCN2 gene and/or - the nucleic acid variant rsl 7514136 in the FCN2 gene and the nucleic acid variant rsl780627 in de LBP gene, or the nucleic acid variant rs3746731 in the CIqRl gene, or the nucleic acid variant rslO39559 in the TLR6 gene, or the nucleic acid variant rs2563298 in the CD 14 gene, and/or
  • TLR8 gene and/or
  • nucleic acid variant rs2232613 in the LBP gene and the nucleic acid variant rsl 039559 in de TLR6 gene, and/or
  • nucleic acid variant rs2232618 in the LBP gene and the nucleic acid variant rs5743611 in de TLRl gene, and/or
  • nucleic acid variant rs5743551 in the TLRl gene and the nucleic acid variant rslO39559 or rs3821985 in de TLR6 gene, and/or
  • nucleic acid variant rs4833095 in the TLRl gene and the nucleic acid variant rslO39559 or rs3821985 in de TLR6 gene and/or - the nucleic acid variant rs3804100 in the TLR2 gene and the nucleic acid variant rsl 039559 in de TLR6 gene, and/or
  • nucleic acid variant rsl 039559 in the TLR6 gene and the nucleic acid variant rs2159377 in de TLR8 gene or the nucleic acid variant rsl 1466645, rsl 1096956 or rs4129009 in the TLRlO gene, and/or
  • nucleic acid variant rs3821985 in the TLR6 gene and the nucleic acid variant rs7694115, rsl 1096957, rsl 1466645, rsl 1096956 or rs4129009 in the TLRlO gene; wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop severe sepsis or septic shock.
  • the present invention also encompasses a method of identifying the risk of a subject to develop sepsis, sever sepsis or septic shock comprising determining the presence of the nucleic acid variant at position 745 of the genomic
  • DNA sequence of the TLR6 gene wherein the presence of the nucleic acid variant is indicative of the risk to develop sepsis, severe sepsis or septic shock.
  • said nucleic acid variant is a single nucleotide polymorphism (SNP).
  • SNP is characterized as being a C/T polymorphism (dbSNP number rs5743810) in the TLR6 gene.
  • Specific embodiments of the present invention relate to methods for determining the presence of a marker which is a nucleic acid variant in a gene, whereby the detection is performed by any method known to the skilled person to reveal differences in nucleic acid sequences.
  • Such methods are, but not limited to, one or more methods selected from the group consisting of: DNA or RNA hybridization, sequencing, PCR, primer extension, multiplex ligation-dependent probe amplification (MLPA), oligonucleotide ligation assay (OLA) and restriction site analysis.
  • Preferable detection assays are LiPA, DoPA or a Microarray.
  • the methods of the present invention are performed in vitro on a biological sample of a subject of which the susceptibility to sepsis, and/or severe sepsis or septic shock is to be determined.
  • methods and tools of the present invention are used for the identification of the risk of a subject to develop an increased severity of sepsis, and more specific to determine the risk of developing severe sepsis or septic shock. More particularly, susceptibility to increased severity of severe sepsis or septic shock is determined based on the combined presence of a nucleic acid variant as given in Table 4.
  • kits for the determination of the risk of a subject to develop sepsis, severe sepsis or septic shock, which kits comprise: one or more reagents for detecting the presence of one, or at least two nucleic acid variants in a gene, preferably selected from the genes given in Table 1.
  • kits of the present invention comprise (a) one or more allele specific primers and/or or more oligonucleotide probes for detecting the presence of one, or at least two nucleic acid variants in the genes selected from Table
  • kits of the present invention comprise one or more allele specific primers and/or or more oligonucleotide probes for detecting the presence of at least two nucleic acid variants as given in Table 4.
  • kits of the present invention comprise one or more allele specific primers and/or or more oligonucleotide probes for detecting the presence of the nucleic acid variant at position g.745 in the TLR6 gene, as described herein.
  • methods and tools of the present invention are used for the identification of a subject at risk for developing a modified response to sepsis, severe sepsis or septic shock therapy. More particularly, the likeliness of a subject to have a modified response to sepsis, severe sepsis or septic shock therapy is determined based on the combined presence of a nucleic acid variant in the genes of Table 1.
  • methods for identifying a subject at risk of having or developing a modified response to sepsis, severe sepsis or septic shock therapy, said method comprising: determining the presence of at least two nucleic acid variants in said subject as given in Table 2; wherein the combined presence of the at least two nucleic acid variants identifies whether a subject is at risk of having or developing a modified response to sepsis, severe sepsis or septic shock therapy.
  • Yet another aspect of the present invention provides methods of treating a subject at risk of developing sepsis, severe sepsis or septic shock, said method comprising determining the presence of at least two nucleic acid variants in one or more genes as given in Table 1 or Table 2 in said subject; and/or determining the presence of the nucleic acid variant at position g.745 in the TLR6 gene; and determining the appropriate treatment regiment for the so -identified subject.
  • appropriate medicaments well known to the skilled person, against sepsis, severe sepsis or septic shock will be administered.
  • Figure 1 Fragment of the CIqRl gene comprising a nucleic acid variant.
  • Figure 2 Fragment of the CD 14 gene comprising a nucleic acid variant.
  • Figure 3 Fragments of the FCNl gene comprising a nucleic acid variant.
  • Figure 4 Fragments of the FCN2 gene comprising a nucleic acid variant.
  • Figure 5 Fragments of the LBP gene comprising a nucleic acid variant.
  • Figure 6 Fragment of the MASPl gene comprising a nucleic acid variant.
  • Figure 7 Fragment of the MASP2 gene comprising a nucleic acid variant.
  • Figure 8 Fragment of the MBL2 gene comprising a nucleic acid variant.
  • Figure 9 Fragments of the Sigirr gene comprising a nucleic acid variant.
  • Figure 10 Fragments of the TLRl gene comprising a nucleic acid variant.
  • Figure 11 Fragments of the TLR2 gene comprising a nucleic acid variant.
  • Figure 12 Fragment of the TLR3 gene comprising the nucleic acid variant.
  • Figure 13 Fragment of the TLR4 gene comprising a nucleic acid variant.
  • Figure 14 Fragment of the TLR5 gene comprising a nucleic acid variant.
  • FIG. 15 Fragments of the TLR6 gene comprising the nucleic acid variant.
  • Figure 16 Fragment of the TLR7 gene comprising a nucleic acid variant.
  • Figure 17 Fragments of the TLR8 gene comprising a nucleic acid variant.
  • FIG. 18 Fragments of the TLRlO gene comprising a nucleic acid variant.
  • a “gene” refers not only to the coding sequence, but to all sequences that are part of that gene: the introns and exons, the regulatory regions including the promoter region and possible other regulatory sequences, such as 5'UTR, 3'UTR or sequences further up- or downstream.
  • Table 1 gives for each gene of the present invention the number of exons, the reference gDNA sequence (with the used version between brackets), the reference mRNA sequence and the reference protein sequence, as given by the NCBI.
  • transcript/protein entries can be provided for a single gene entry.
  • wild-type sequence refers to the reference sequence.
  • the reference nucleic acid and protein sequences indicated in the current invention are derived from NCBI (http://www.ncbi.nlm.nih.gov/) and indicated by their respective accession number (Table 1), as is well known to the person skilled in the art.
  • the nomenclature for the 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.
  • nucleotide numbering of the coding DNA and RNA reference sequence is as follows:
  • nucleotide +1 is the A of the ATG-translation initiation codon • there is no nucleotide 0
  • nucleotide number is preceded by "g.” when a genomic or by "c.” when a cDNA reference sequence is used. Substitutions are designated by ">”.
  • 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 about 100 nucleotides or less in length is often also referred to as an oligonucleotide.
  • nucleic acid variant or “polymorphism” as used in the present invention refers to a position comprising one or more nucleotides in the nucleic acid sequence which differs relative to a reference nucleic acid sequence.
  • 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 polymorphism or nucleic acid variant can be an insertion, deletion, substitution, tandem repeat or similar.
  • Single nucleotide polymorphism refers to the variation of a single nucleotide. This includes the replacement of one nucleotide by another and deletion or insertion of a single nucleotide.
  • SNPs are biallelic markers although tri-and tetra-allelic markers also exist.
  • SNP A ⁇ C may comprise allele C or allele A.
  • a nucleic acid variant when referring to the SNP A ⁇ C may include a C or A at the polymorphic position.
  • the SNPs of the present invention are disclosed in the NCBI database dbSNP, and are characterised by an "rs" number. Said rs number can be used to retrieve summary information on the known variation at the locus, a list of the specific reports that characterize a SNP, and links to other NCBI resources.
  • determining 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. In practice, both the absence and the presence of a certain event can function as markers. Accordingly, reference to determining the presence of a nucleic acid variant for sepsis, and/or severe sepsis or septic shock, generally encompasses determining whether the variant is present or absent in a sample. As such, this also includes the possible finding that the marker is not present in the sample, i.e. determining the absence of a nucleic acid variant.
  • 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 (besides the direct detection of the nucleic acid variant) also be done by determining the homozygous presence of the corresponding allele not comprising the nucleic acid variant.
  • allele specific oligonucleotide primers or probes for detecting the presence of a SNP can be specific for the allele where the
  • haplotype 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 means the genetic constitution of an individual, either overall or at a specific locus, and defines the combination of alleles the individual carries.
  • homozygous refers to having two of the same alleles at a locus; the term “heterozygous” refers to having different alleles at a locus.
  • SIRS as used herein is defined as a clinical manifestation secondary to an insult, mostly of infectious origin.
  • SIRS sepsis
  • severe sepsis as used herein is defined as sepsis with evidence of end-organ dysfunction as a result of hypoperfusion.
  • septic shock as used herein is defined as sepsis with persistent hypotension despite fluid resuscitation.
  • tissue 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 hair root, 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, synovial fluid, 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 pre-treatment may be selected appropriately by those skilled in the art depending on the biological sample to be subjected.
  • the present invention provides methods and tools for identifying the risk of a subject to develop sepsis, severe sepsis or septic shock.
  • the "subject" on which the method of the present invention can be carried out can be any vertebrate animal, more particularly any mammalian animal, and is most particularly a human. It is envisaged that the methods of the invention can be applied to a non-human subject such as (but not limited to) a cow, a pig, a sheep, a goat, a horse, especially racing horses, a monkey, a rabbit, a dog, a cat, a mouse, a rat, a hamster or a primate or any laboratory test animal.
  • a non-human subject such as (but not limited to) a cow, a pig, a sheep, a goat, a horse, especially racing horses, a monkey, a rabbit, a dog, a cat, a mouse, a rat, a hamster or a primate or any laboratory test animal.
  • the present invention provides methods for identifying the risk of a subject to develop sepsis, severe sepsis or septic shock based on a combined determination of the presence in that subject of at least two nucleic acid variants in a specific gene or combination of genes.
  • determination of the occurrence of a polymorphism in a gene allows the identification of a subject at risk of developing sepsis, severe sepsis or septic shock with improved accuracy, compared to the determination of a single polymorphism in an individual gene.
  • the SNP rs5743810 in the TLR6 gene can be used on its own to determine the risk of a subject to develop sepsis, severe sepsis or septic shock
  • the determination of the presence of one or more polymorphisms of a gene is ensured at the DNA level.
  • the determination includes determining the occurrence of one variant allele of a gene (heterozygous for the variant allele) or the presence of two variant alleles of a gene (homozygous for the presence the variant allele). Determination of the occurrence of a variant allele at the DNA level is described in more detail below.
  • the determination of the presence of a variant allele can be performed at the protein level, based on quantitative expression of the expressed protein, qualitative assessment of the expressed protein (differences in protein sequence) and/or a functional assessment of the expressed protein (e.g. the ability to bind to GIcNAc).
  • quantitative expression of the expressed protein e.g. the expression of a protein
  • qualitative assessment of the expressed protein e.g. the expression of a protein
  • functional assessment of the expressed protein e.g. the ability to bind to GIcNAc.
  • the methods of the present invention involve the determination of two or more polymorphisms in a gene or a combination of genes, more particularly the determination of two or more polymorphism in a gene or combination of genes selected from the group consisting of: mannose-binding lectin (MBL2), MBL-associated Serine Protease 1 (MASPl), MBL-associated Serine Protease 2 (MASP2), Ficolin 1 (FCNl), Ficolin 2 (FCN2), Complement component Iq Receptor 1 (CIqRl), Lipopolysaccharide-binding protein (LBP), Monocyte Differentiation Antigen CD 14 (CD 14), Single Immunoglobulin domain-containing ILlR-related protein (Sigirr), Toll- like Receptor 1 (TLRl), Toll- like Receptor 2 (TLR2), Toll-like Receptor 3 (TLR3), Toll-like Receptor 4 (TLR4), Toll-like Receptor 5 (TLR5),
  • MBL2
  • the polymorphism in the gene is characterized as indicated in Table 2.
  • the presence of at least two nucleic acid variants in one gene or a combination of genes is associated with the risk to develop sepsis, severe sepsis or septic shock, with an odds ratio of at least 3.25, 3.26, 3.27, 3.28 or 3.29, preferably 3.5 and even more preferably 4.0, or more, or alternatively, with an odds ratio of less than 0.3, 0.29, 0.28, 0.27 or 0.26, and preferably 0.25, or less.
  • the upper and lower limit of the confidence interval for this odds ratio is ⁇ 1 and >1, respectively.
  • Table 2 gives NCBI's accession number for each SNP, the gene, the position of the SNP, the nucleic acid polymorphism and the amino acid change. It is to be understood that the nucleotide variant also encompasses the complement nucleic acids.
  • the relevant part of the genomic DNA of the gene comprising the nucleic acid variant as given in Table 2 is provided in Figures 1-18. Accordingly, in a specific embodiment, the polymorphism or nucleic acid variant is detected in the specified gene comprising said fragment, i.e. comprising the sequence as given by SEQ ID NO 1 to 35, respectively.
  • the method of the present invention comprises a step wherein one or more of the sequences represented by SEQ ID NO 1 to 35 are amplified.
  • Amplification of one gene fragment can be done separately, or simultaneously with one ore more of the other fragments.
  • certain nearby located gene regions represented by SEQ ID NO 1 to 35, respectively, and within a single gene can be amplified in one reaction.
  • the methods of the invention comprise the step of determining whether the one or more nucleic acid variants in the gene are present in 0, 1 or 2 copies, more particularly whether a nucleic acid variant in the gene is present in one or in both alleles.
  • it is determined not only whether an allele carrying the nucleic acid variation is present or absent, but if it is present, it is also determined whether there is only one allele carrying the nucleic acid variant (heterozygous presence) or whether both alleles carry the nucleic acid variant (homozygous presence). Either heterozygous or homozygous presence of the at least one nucleic acid variant is indicative of having or a risk of having or developing sepsis, severe sepsis or septic shock. This is shown in Table 4 (Example 1).
  • the methods of the invention comprise determining the presence of at least two genotypes. More specific, the presence of 0, 1 or 2 alleles in a single gene or in two different genes is linked to sepsis, severe sepsis or septic shock. In a more specific embodiment, the methods of the invention comprise determining the presence of a combination of at least two genotypes as given in Table 4.
  • 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), Gut (2001), and in standard textbooks, for example "Laboratory Protocols for Mutation Detection”, Ed. by U. Landegren, Oxford University Press, 1996 and “PCR”, 2nd Edition” by Newton & Graham, BIOS Scientific Publishers Limited, 1997.
  • the step of determining the presence of a nucleic acid variant in a gene in the methods of the present invention can be carried out in vivo or in vitro. Most typically, however, detection of nucleic acid variants in the genes are performed in vitro in a biological sample obtained from the subject.
  • a nucleic acid comprising a sequence of interest can be obtained from a biological sample, more particularly from a sample comprising 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 DNA 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).
  • nucleic acid of interest 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 methods of the present invention optionally comprise the steps of isolating nucleic acids from the sample and/or an amplification step.
  • the detection of the presence or absence of a nucleic acid variant is determined by DNA or RNA hybridization, sequencing, PCR, primer extension, multiplex ligation-dependent probe amplification (MLPA), oligonucleotide ligation assay (OLA) or restriction site analysis.
  • MLPA multiplex ligation-dependent probe amplification
  • OLA oligonucleotide ligation assay
  • Another aspect of the invention is the use of the present methods to identify a subject at risk of developing a modified response to sepsis, severe sepsis or septic shock therapy. Identifying the most effective treatment by first determining the presence of specific genotypes may lead to a considerable decrease in morbidity. The methods of the invention thus provide a tool for improved risk assessment and may help to determine the most appropriate, "personalized" therapy.
  • kits for use in the methods as described herein More specific the present invention encompasses a kit for identifying the risk of a subject to develop sepsis, severe sepsis or septic shock, comprising one or more reagents for detecting the presence (or, where appropriate absence) of at least two nucleic acid variants in a gene or a combination of genes.
  • kits of the present invention provide the tools for the detection of: - the presence of at least one nucleic acid variant in the MBL2 gene and at least one nucleic acid variant in the CD 14 gene in said subject; and/or the presence of at least two nucleic acid variants in the FCN2 gene, and/or the presence of at least one nucleic acid variant in the TLR8 gene and at least one nucleic acid variant in the FCNl gene in said subject, and/or the presence of at least one nucleic acid variant in the FCN2 gene and at least one nucleic acid variant in the gene selected from the group consisting of: the MASPl gene, the TLR3 gene, the TLR2 gene, the TLR3 gene, the TLR4 gene, the TLR6 gene, the TLR8 gene, and the Sigirr gene in said subject, and/or - the presence of at least one nucleic acid variant in the TLR7 gene and at least one nucleic acid variant in the TLR6 gene in said subject; and wherein the combined presence of at least one
  • the kit of the present invention comprises one or more reagents for detecting the presence of the nucleic acid variant at position g.745 of the TLR6 gene.
  • the nucleic acid variant is the SNP rs5743810.
  • the invention relates to a kit for identifying the risk of a subject to develop sepsis, severe sepsis or septic shock, comprising one or more reagents for detecting the presence (or, where appropriate absence) of at least two nucleic acid variants selected from the group as given in Table 2. More particularly the kits of the present invention provide the tools for the detection of the specific combination of at least two nucleic acid variants as provided in Table 4.
  • kits of the present invention comprise an oligonucleotide probe suitable for detection of a sequence within the gene and/or an oligonucleotide pair suitable for amplification of a sequence within a polynucleic acid.
  • Oligonucleotides for use in the kits or methods of the present invention typically are isolated nucleic acid molecules comprising at least 8 nucleotides and specifically hybridizing with a target nucleic acid sequence, e.g. the wild type or variant sequence of the gene including the position of the nucleic acid variant, or the complementary thereof.
  • the oligonucleotide comprises at least 9, 10, 11, 12, 13, 14 or 15 nucleotides and up to 40, 30, 25, 24, 23, 22, 21, or 20 nucleotides.
  • the oligonucleotides can be used as a primer or probe.
  • primers or probes will comprise nucleotide sequences entirely complementary to the corresponding target sequence, e.g. a wild type or variant locus in the target gene.
  • 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.
  • the primers or probes will amplify or hybridize with the sequence characterized by SEQ ID NOs 1-35.
  • an oligonucleotide primer designed to specifically recognize and amplify either a wild type or variant allele at a locus is referred to as an allele specific primer (or primer pair).
  • an allele specific probe i.e. an oligonucleotide probe that specifically hybridizes to either a wild type or variant allele.
  • the hybridization conditions are to be stringent as known in the art.
  • Stringent refers to conditions under which a nucleotide sequence will no longer bind to unrelated 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 be prevented or dissolved.
  • the primers or probes may carry one or more labels to facilitate detection.
  • the nature of the label is not critical to the invention and may be fluorescent, chemiluminescent, enzymatic, radioactive, chemical or other, provided it doesn't interfere with correct hybridization of the oligonucleotide.
  • kits of the present invention will comprise one or more oligonucleotide primers and/or one or more probes specific for the one or more allele(s) containing the nucleic acid variant(s).
  • the kits for detection according to the methods of the present invention comprise one or more oligonucleotide primers and/or one or more probes specific for the wild type allele, not containing the nucleic acid variant, whereby an indication of the presence of a wild type allele is indicative of the absence of a nucleic acid variant.
  • the kits of the present invention comprise both oligonucleotide primers (and/or probes) specific for the "variant" and the "wild type” allele. The latter embodiment is particularly suited to determine the copy number of the variant alleles.
  • the one or more allele specific primers and/or probes for detecting the nucleic acid variants will typically comprise at least part of the nucleic acid sequence as identified in Table 1, e.g. as characterized by SEQ ID NO 1-35 respectively, or the complementary thereof, wherein the part of the nucleic acid sequence is envisaged to potentially comprise one or more nucleic acid variants. More specifically, kits are envisaged which comprise combinations of at least two primers and/or probes capable of hybridizing to and/or amplifying the region comprising
  • TLR6 gene which is a C/T polymorphism (dbSNP number rs5743810)
  • kits comprising two or more, i.e.
  • nucleic acid variants are selected from the group as given in Table 2. Even more preferably the two or more nucleic acid variants are those combinations as specifically given in Table 4.
  • kits of the invention optionally contain a variety of other reagents, e.g. depending on the detection procedure.
  • an agent for denaturing nucleic acids an enzyme capable of modifying a double stranded or single stranded nucleic acid molecule, a hybridization buffer, or components necessary for producing a hybridization buffer, a wash solution, or components necessary for producing a wash solution, a means for detecting partially or completely denatured polynucleic acids, a means for detecting hybrids formed in the preceding hybridization, a means for detecting enzymatic modifications of nucleic acids, a means for attaching an oligonucleotide to a known location on a solid support, a labelled antibody, and/or a means for attaching an antigen to a known location on a solid support.
  • methods are provided for treating a subject at risk of developing sepsis, severe sepsis or septic shock, which method comprises identifying the subject as being at risk of developing sepsis, severe sepsis or septic shock.
  • methods are provided for treating a subject having an increased risk of developing sepsis, severe sepsis or septic shock, using the methods as described herein.
  • 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 for a protective effect on the risk of developing a disorder or disease (OR ⁇ 1).
  • the 95% confidence interval (95% CI) is the range of numerical values in which we can be confident (to a computed probability, here 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 get narrower and "precision" is increased. To confidently accept a calculated OR as reliable, important or clinically significant, the lower boundary of the confidence interval, or lower confidence limit, should be >1 if the OR>1, or the upper boundary of the confidence interval should be ⁇ 1 if the OR ⁇ 1.
  • DNA extraction of whole blood samples was performed using PUREGENE ® DNA Purification Kit.
  • the relevant areas of the genes listed in Table A, were amplified and hybridized to (i) either LiPA strips for MBL2 according to the protocol described by the manufacturer of the kit (INNO-LiPA MBL2, Innogenetics NV), (ii) or DoPA (Dot Probe Assay) strips for the remaining genes.
  • the protocol for the DoPA strips is similar to the protocol for the MBL2 LiPA strips.
  • the relevant regions were amplified using biotinylated oligonucleotides.
  • the polymorphisms were detected by use of a reverse hybridization method with probes designed to recognize the polymorphisms. After stringent wash 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 OR of the FCN2 promoter SNP rsl7514136 is 2.4 (see Table 3) while this SNP is found in 7 significant combinations having an OR ranging from 3.26 to 6.2. This also applies to all other combinations listed in Table 4.
  • SNP's also proved to be of relevance if used individually. For example, the following SNP's showed an OR that have a significant predictive value in predicting the risk to develop sepsis, severe sepsis or septic shock:

Abstract

The present invention relates to methods for determiningthe risk ofa subject to develop sepsis, severe sepsis or septic shockwhich comprise detecting the presence ofat least twonucleic acid variantsin a singlegeneor a combination of genes. The invention further provides tools and kits for performing these methods and the use of these methods in personalised therapy.

Description

COMBINATIONS OF MARKERS FOR SEPSIS RISK ASSESSMENT
FIELD OF THE INVENTION
The present invention relates to methods and kits for identifying a subject at risk of developing sepsis, severe sepsis or septic shock.
BACKGROUND OF THE INVENTION
Early detection of a disease condition allows for a more effective therapeutic treatment with a correspondingly more favourable clinical outcome. In many cases, however, early detection of disease symptoms is problematic due to the complexity of the disease. Hence, a disease may become relatively advanced before diagnosis is possible. Systemic inflammatory conditions represent one such class of diseases. Sepsis is a complex clinical syndrome as a result of a systemic inflammatory response to live bacteria and/or bacterial products. This response is expressed as a compendium of a variety of different clinical signs and symptoms such as fever, increased blood leukocyte counts, unexplained thrombocytopenia, mental confusion, transient hypotension, and organ stress and dysfunction. The individual response is determined by many factors, including the virulence of the organism, the size of the inoculum, and the patient's coexisting conditions.
Sepsis develops when the initial, appropriate host response to an infection becomes amplified, and is then deregulated. The most common sites of infection are the lungs, the abdominal cavity, the urinary tract, and primary infections of the bloodstream. If untreated, septic patients may develop acute respiratory or renal failure, multi-organ dysfunction, shock, and death.
Sepsis follows a well-described time course, progressing from systemic inflammatory response syndrome ("SIRS")-negative to SIRS-positive to sepsis, which may then progress to severe sepsis, septic shock, multiple organ dysfunction ("MOD"), and ultimately death. Sepsis also may arise in an infected individual when the individual subsequently develops SIRS. The evolution of the stage of sepsis towards severe sepsis, then towards septic shock is not systematic since approximately 64% of the septic patients develop a severe sepsis, and 23% of the patients in sepsis evolve/move in septic shock. Before this ultimate stage of septic shock, treatments must be prescribed with the patient in order to stop and to reverse this process.
The basis of sepsis is the presence of infection and the subsequent physiologic alterations in response to that infection, namely the activation of the inflammatory cascade. Systemic inflammatory response syndrome (SIRS) is a term used to define this clinical condition, and it is considered present if abnormalities in 2 of the following 4 clinical parameters exist: (1) body temperature, (2) heart rate, (3) respiratory rate, and (4) peripheral leukocyte count.
Sepsis is defined as the presence of SIRS in the setting of infection. Severe sepsis is defined as sepsis with evidence of end-organ dysfunction as a result of hypoperfusion. Septic shock is defined as sepsis with persistent hypotension despite fluid resuscitation and resulting tissue hypoperfusion (American College of Chest Physicians, 1992).
From a financial perspective, sepsis represents a major burden to the health care system in most developed countries since septic patients require admission and aggressive treatment in the intensive care units (ICU) and are generally hospitalized for longer than 3 weeks. The difficulty in early detection of sepsis is reflected by the high morbidity and mortality associated with the disease. Sepsis is estimated to affect 18 million people worldwide each year and to kill 1400 people each day.
A need, therefore, exists for a method of determining the risk to develop sepsis sufficiently early to allow effective prevention and intervention. There is a continuing urgent need in the art to diagnose the risk to develop sepsis accurately, without the need for monitoring a patient over time. Ideally, determining the risk would be made by a technique that accurately, rapidly, and simultaneously measures a plurality of markers at a single point in time, thereby minimizing disease progression during the time required for detection. It has been shown that genetic factors of importance for the inflammatory response such as polymorphisms in the promoter region of the tumour necrosis factor alpha (TNF-alpha) and Interleukin-1 alpha (IL-I alpha) gene may be associated with susceptibility and outcome of septic shock. In addition, genetic variation within the fibrinolytic system seems to be of importance for the outcome of sepsis (Menges et al. 2001; Dahmer MK et al. 2005). Despite the different associations of genetic risk factors with sepsis, severe sepsis and/or septic shock, there is a continuous search for more accurate genetic markers that provide a reliable risk assessment for the development of sepsis, severe sepsis and/or septic shock.
The present invention presents a new reliable tool for an improved risk assessment for the development of sepsis, severe sepsis and/or septic shock. It has been found that the analysis of specific gene variants is very relevant to discriminate good or bad prognosis for a patient. The identification of alleles/genotypes linked with an adverse, or alternatively, favourable clinical outcome in sepsis, severe sepsis or septic shock can decrease morbidity and mortality through improved risk assessment and the administration of prophylactic or "personalized" medicine.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide methods and kits for identifying the risk of a subject to develop sepsis, severe sepsis or septic shock.
It is an advantage of the present invention that susceptibility of an individual to develop sepsis, severe sepsis or septic shock can be made with improved accuracy, by the methods and tools of the invention. For example, the invention provides combinations of markers with increased reliability for predicting susceptibility to sepsis, severe sepsis or septic shock.
It is a further advantage of the present invention that patients having been more accurately assessed for the risk to develop sepsis, severe sepsis or septic shock can be treated in a more timely and appropriate manner.
Accordingly, the methods of the present invention can improve personalised therapy for sepsis, severe sepsis or septic shock.
It is a further advantage of the present invention that the risk assessment can be performed by simple tests which can be developed into routine methods and standard kits.
It is a further advantage of particular embodiments of the present invention that the improved risk assessment can be performed using one analysis technique, in a time- and cost-efficient way.
A first aspect of the invention provides methods of gaining information about the risk of a subject to develop sepsis, severe sepsis or septic shock, comprising:
- determining the presence of at least one nucleic acid variant in the MBL2 gene and at least one nucleic acid variant in the CD 14 gene in said subject; and/or determining the presence of at least two nucleic acid variants in the FCN2 gene, and/or determining the presence of at least one nucleic acid variant in the TLR8 gene and at least one nucleic acid variant in the FCNl gene in said subject, and/or - determining the presence of at least one nucleic acid variant in the FCN2 gene and at least one nucleic acid variant in the gene selected from the group consisting of: the MASPl gene, the TLR3 gene, the TLR2 gene, the TLR4 gene, the TLR6 gene, the TLR8 gene, and the Sigirr gene in said subject, and/or determining the presence of at least one nucleic acid variant in the TLR7 gene and at least one nucleic acid variant in the TLR6 gene in said subject; wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop sepsis, severe sepsis or septic shock.
Another aspect of the invention provides methods of identifying the risk of a subject to develop sepsis, severe sepsis or septic shock, comprising detecting at least two nucleic acid variants selected from the group consisting of:
- position 162 of the MBL2 gene being a G/A polymorphism (dbSNP number rsl 800450),
- position 1336 of the CD14 gene being a G/T polymorphism (dbSNP number rs2563298),
- position -1981 of the FCNl gene being a G/A polymorphism (dbSNP number rs2989727), - position 7919 of the FCNl gene being a G/A polymorphism (dbSNP number rslO71583),
- position -64 of the FCN2 gene being a A/C polymorphism (dbSNP number rs7865453),
- position -4 of the FCN2 gene being a G/A polymorphism (dbSNP number rsl7514136),
- position 6359 of the FCN2 gene being a C/T polymorphism (dbSNP number rsl7549193),
- position 6424 of the FCN2 gene being a G/T polymorphism (dbSNP number rs7851696), - position 70465 of the MASPl gene being a G/A polymorphism (dbSNP number rs3733001),
- position -15607 of the TLR2 gene being a G/A polymorphism (dbSNP number rsl898830),
- position 6301 of the TLR3 gene being a C/T polymorphism (dbSNP number rs3775291),
- position -2026 of the TLR4 gene being a G/A polymorphism (dbSNP number rsl 927914),
- position -502 of the TLR6 gene being a C/T polymorphism (dbSNP number rslO39559), - position 17962 of the TLR7 gene being a T/A polymorphism (dbSNP number rsl79008),
- position 9300 or 12979 of the TLR8 gene being an C/T polymorphism (dbSNP number rs5744080), - position 3392 of the Sigirr gene being a G/A polymorphism (dbSNP number rs3210908), and
- position 3402 of the Sigirr gene being a G/T polymorphism (dbSNP number rs3087588); wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop sepsis, severe sepsis or septic shock.
Optionally, the method can be carried out in vitro or ex vivo.
According to a specific embodiment, the methods of the invention comprise the step of determining: - the nucleic acid variant rsl 800450 in the MBL2 gene and the nucleic acid variant rs2563298 in de CD14 gene, and/or
- the nucleic acid variant rs5744080 in the TLR8 gene and the nucleic acid variant rs2989727 or rsl 071583 in de FCNl gene, and/or
- the nucleic acid variant rs7865453 in the FCN2 gene and the nucleic acid variant rs3775291 in de TLR3 gene or the nucleic acid variant rs3733001 in the MASPl gene, and/or
- the nucleic acid variant rsl 7514136 in the FCN2 gene and the nucleic acid variant rsl898830 in de TLR2 gene, or the nucleic acid variant rsl927914 in the TLR4 gene, or the nucleic acid variant rsl 039559 in the TLR6 gene, or the nucleic acid variant rs5744080 in the TLR8 gene, and/or
- the nucleic acid variant rsl 7549193 in the FCN2 gene and the nucleic acid variant rsl 898830 in de TLR2 gene or the nucleic acid variant rs5744080 in the TLR8 gene, and/or
- the nucleic acid variant rs7851696 in the FCN2 gene and the nucleic acid variant rs3210908 or rs3087588 in de Sigirr gene, or the nucleic acid variant rs3775291 in the TLR3 gene, or the nucleic acid variant rsl7549193 or rsl 7514136 in the FCN2 gene, and/or
- the nucleic acid variant rsl 79008 in the TLR7 gene and the nucleic acid variant rslO39559 in de TLR6 gene; wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop sepsis, severe sepsis or septic shock.
Specific embodiments of the above-cited methods of the invention comprise methods wherein the step of determining the occurrence of at least two nucleic acid variants in one or more genes in that subject comprises the step of determining the presence or absence of a single nucleotide polymorphism (SNP). More specific embodiments, whereby the methods of the present invention involve the determination of at least two nucleic acid variants in one or a combination of genes, involve the detection of two or more nucleic acid variants or SNPs located at position 162 of the MBL2 gene, at position 1336 of the CD14 gene, at position -1981 or 7919 of the FCNl gene, at position -4,-64, 6359 or 6424 of the FCN2 gene, at position 70465 of the MASPl gene, at position -15607 of the TLR2 gene, at position 6301 of the TLR3 gene, at position -2026 of the TLR4 gene, at position -502 of the TLR6 gene, at position 17962 of the TLR7 gene, at position 9300 or 12979 of the TLR8 gene, and/or at position 3392 or 3402 of the Sigirr gene, of the genomic DNA sequence.
Further particular embodiments of the methods of the present invention involve determining whether the at least two nucleic acid variants in the gene(s) is present in
0, 1 or 2 copies, wherein the heterozygous or homozygous presence (or, where appropriate, absence) of the at least one nucleic acid variant is indicative of having or of a risk of having or developing sepsis, severe sepsis or septic shock.
Specific embodiments of the methods of the present invention relate to the combined detection of at least two nucleic acid variants as follows:
- the nucleic acid variant rsl 800450 in the MBL2 gene and the nucleic acid variant rs2563298 in de CD14 gene, and/or
- the nucleic acid variant rs5744080 in the TLR8 gene and the nucleic acid variant rs2989727 or rsl 071583 in de FCNl gene, and/or
- the nucleic acid variant rs7865453 in the FCN2 gene and the nucleic acid variant rs3775291 in de TLR3 gene, or the nucleic acid variant rs3733001 in the MASPl gene, and/or - the nucleic acid variant rsl 7514136 in the FCN2 gene and the nucleic acid variant rsl898830 in de TLR2 gene, or the nucleic acid variant rsl927914 in the TLR4 gene, or the nucleic acid variant rsl 039559 in the TLR6 gene, or the nucleic acid variant rs5744080 in the TLR8 gene, and/or - the nucleic acid variant rsl 7549193 in the FCN2 gene and the nucleic acid variant rsl 898830 in de TLR2 gene, or the nucleic acid variant rs5744080 in the TLR8 gene, and/or
- the nucleic acid variant rs7851696 in the FCN2 gene and the nucleic acid variant rs3210908 or rs3087588 in de Sigirr gene, or the nucleic acid variant rs3775291 in the TLR3 gene, or the nucleic acid variant rsl7549193 or rsl 7514136 in the FCN2 gene, and/or
- the nucleic acid variant rsl 79008 in the TLR7 gene and the nucleic acid variant rslO39559 in de TLR6 gene, wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop sepsis, severe sepsis or septic shock.
In further specific embodiments, the methods comprise detecting at least two nucleic acid variants as given in Table 2.
In a further embodiment, the invention encompasses a method and kit for the identification of the risk of a subject to develop sepsis, severe sepsis or septic shock., said method comprising the step of determining he combined presence of a nucleic acid variant as given in Table 4.
Further specific embodiments of the above-described methods comprise determining the combined presence of at least two nucleic acid variants as presented in Table 4, wherein it is determined if the nucleic acid variant is present in 0, 1 or 2 copies.
Certain polymorphisms also influence the severity of sepsis and are useful in predicting those individuals who will develop more severe disease. In an alternative embodiment, the present invention provides a method of identifying a subject at risk of developing severe sepsis or septic shock comprising: determining the presence of at least one nucleic acid variant in the MASP2 gene and at least one nucleic acid variant in the FCN2 gene in said subject; and/or determining the presence of at least one nucleic acid variant in the FCN2 gene and at least one nucleic acid variant in the LBP gene or at least one nucleic acid variant in the CIqRl gene, or at least one nucleic acid variant in the TLR6 gene, or at least one nucleic acid variant in the CD 14 gene in said subject; and/or determining the presence of at least one nucleic acid variant in the LBP gene and at least one nucleic acid variant in the TLR6 gene or at least one nucleic acid variant in the TLRl gene in said subject; and/or determining the presence of at least one nucleic acid variant in the TLRl gene and at least one nucleic acid variant in the TLR6 gene in said subject; and/or determining the presence of at least one nucleic acid variant in the TLR2 gene and at least one nucleic acid variant in the TLR6 gene in said subject; and/or determining the presence of at least one nucleic acid variant in the TLR5 gene and at least one nucleic acid variant in the TLR6 gene in said subject; and/or - determining the presence of at least one nucleic acid variant in the TLR6 gene and at least one nucleic acid variant in the TLR8 gene or at least one nucleic acid variant in the TLRlO in said subject; wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop severe sepsis or septic shock.
A specific embodiment of the above-method relates to the detection of at least two nucleic acid variants selected from the group consisting of:
- position 16349 of the MASP2 gene being a G/T polymorphism (dbSNP number rsl2711521), - position -4 of the FCN2 gene being a G/A polymorphisms (dbSNP number rsl7514136),
- position 6359 of the FCN2 gene being a C/T polymorphism (dbSNP number rsl7549193),
- position 22713 of the LBP gene being a T/C polymorphism (dbSNP number rsl780627),
- position 22736 of the LBP gene being a C/T polymorphism (dbSNP number rs2232613),
- position 26842 of the LBP gene being a T/C polymorphism (dbSNP number rs2232618), - position 1621 of the CIqRl gene being a C/T polymorphism (dbSNP number rs3746731),
- position 1336 of the CD14 gene being a G/T polymorphism (dbSNP number rs2563298), - position -7202 of the TLRl gene being a A/G polymorphism (dbSNP number rs5743551),
- position 239 of the TLRl gene being a G/C polymorphism (dbSNP number rs5743611),
- position 743 of the TLRl gene being a A/G polymorphism (dbSNP number rs4833095),
- position 1350 of the TLR2 gene being a T/C polymorphism (dbSNP number rs3804100),
- position 1775 of the TLR5 gene being a A/G polymorphism (dbSNP number rs2072493), - position -502 of the TLR6 gene being a C/T polymorphism (dbSNP number rslO39559),
- position 1083 of the TLR6 gene being a G/C polymorphism (dbSNP number rs3821985),
- position 9009 or 12688 of the TLR8 gene being an C/T polymorphism (dbSNP number rs2159377),
- position -1883 of the TLRlO gene being a T/C polymorphism (dbSNP number rs7694115),
- position -992 of the TLRlO gene being a T/A polymorphism (dbSNP number rsl 1466645), - position 721 of the TLRlO gene being a A/C polymorphism (dbSNP number rsl 1096957),
- position 1032 of the TLRlO gene being a G/T polymorphism (dbSNP number rsl 1096956), and
- position 2323 of the TLRlO gene being a A/C or G/T polymorphism (dbSNP number rs4129009); wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop severe sepsis or septic shock. Even more specific embodiments of the methods of the present invention relate to the combined detection of at least two nucleic acid variants as follows:
- the nucleic acid variant rs 12711521 in the MASP2 gene and the nucleic acid variant rsl7514136 or rsl7549193 in de FCN2 gene, and/or - the nucleic acid variant rsl 7514136 in the FCN2 gene and the nucleic acid variant rsl780627 in de LBP gene, or the nucleic acid variant rs3746731 in the CIqRl gene, or the nucleic acid variant rslO39559 in the TLR6 gene, or the nucleic acid variant rs2563298 in the CD 14 gene, and/or
- the nucleic acid variant rsl 7549193 in the FCN2 gene and the nucleic acid variant rsl780627 in de LBP gene or the nucleic acid variant rs5744080 in the
TLR8 gene, and/or
- the nucleic acid variant rs2232613 in the LBP gene and the nucleic acid variant rsl 039559 in de TLR6 gene, and/or
- the nucleic acid variant rs2232618 in the LBP gene and the nucleic acid variant rs5743611 in de TLRl gene, and/or
- the nucleic acid variant rs5743551 in the TLRl gene and the nucleic acid variant rslO39559 or rs3821985 in de TLR6 gene, and/or
- the nucleic acid variant rs4833095 in the TLRl gene and the nucleic acid variant rslO39559 or rs3821985 in de TLR6 gene, and/or - the nucleic acid variant rs3804100 in the TLR2 gene and the nucleic acid variant rsl 039559 in de TLR6 gene, and/or
- the nucleic acid variant rs2072493 in the TLR5 gene and the nucleic acid variant rsl 039559 in de TLR6 gene, and/or
- the nucleic acid variant rsl 039559 in the TLR6 gene and the nucleic acid variant rs2159377 in de TLR8 gene, or the nucleic acid variant rsl 1466645, rsl 1096956 or rs4129009 in the TLRlO gene, and/or
- the nucleic acid variant rs3821985 in the TLR6 gene and the nucleic acid variant rs7694115, rsl 1096957, rsl 1466645, rsl 1096956 or rs4129009 in the TLRlO gene; wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop severe sepsis or septic shock.
It has furthermore been found that a specific single nucleic acid variant is indicative for the risk to develop sepsis, severe sepsis or septic shock. As such, the present invention also encompasses a method of identifying the risk of a subject to develop sepsis, sever sepsis or septic shock comprising determining the presence of the nucleic acid variant at position 745 of the genomic
DNA sequence of the TLR6 gene, wherein the presence of the nucleic acid variant is indicative of the risk to develop sepsis, severe sepsis or septic shock.
In a preferred embodiment, said nucleic acid variant is a single nucleotide polymorphism (SNP). The SNP is characterized as being a C/T polymorphism (dbSNP number rs5743810) in the TLR6 gene.
Specific embodiments of the present invention relate to methods for determining the presence of a marker which is a nucleic acid variant in a gene, whereby the detection is performed by any method known to the skilled person to reveal differences in nucleic acid sequences. Such methods are, but not limited to, one or more methods selected from the group consisting of: DNA or RNA hybridization, sequencing, PCR, primer extension, multiplex ligation-dependent probe amplification (MLPA), oligonucleotide ligation assay (OLA) and restriction site analysis. Preferable detection assays are LiPA, DoPA or a Microarray. According to a particular embodiment, the methods of the present invention are performed in vitro on a biological sample of a subject of which the susceptibility to sepsis, and/or severe sepsis or septic shock is to be determined.
In another aspect of the present invention methods and tools of the present invention are used for the identification of the risk of a subject to develop an increased severity of sepsis, and more specific to determine the risk of developing severe sepsis or septic shock. More particularly, susceptibility to increased severity of severe sepsis or septic shock is determined based on the combined presence of a nucleic acid variant as given in Table 4.
Yet another aspect of the present invention provides reagents, combinations of reagents and kits for the determination of the risk of a subject to develop sepsis, severe sepsis or septic shock, which kits comprise: one or more reagents for detecting the presence of one, or at least two nucleic acid variants in a gene, preferably selected from the genes given in Table 1. Specific embodiments of the kits of the present invention comprise (a) one or more allele specific primers and/or or more oligonucleotide probes for detecting the presence of one, or at least two nucleic acid variants in the genes selected from Table
1, more particularly for detecting one, or at least two nucleic acid variants as given in Table 2.
Further specific embodiments of the kits of the present invention comprise one or more allele specific primers and/or or more oligonucleotide probes for detecting the presence of at least two nucleic acid variants as given in Table 4.
Alternatively or additionally, the kits of the present invention comprise one or more allele specific primers and/or or more oligonucleotide probes for detecting the presence of the nucleic acid variant at position g.745 in the TLR6 gene, as described herein.
In yet another aspect, methods and tools of the present invention are used for the identification of a subject at risk for developing a modified response to sepsis, severe sepsis or septic shock therapy. More particularly, the likeliness of a subject to have a modified response to sepsis, severe sepsis or septic shock therapy is determined based on the combined presence of a nucleic acid variant in the genes of Table 1. Accordingly, methods are provided for identifying a subject at risk of having or developing a modified response to sepsis, severe sepsis or septic shock therapy, said method comprising: determining the presence of at least two nucleic acid variants in said subject as given in Table 2; wherein the combined presence of the at least two nucleic acid variants identifies whether a subject is at risk of having or developing a modified response to sepsis, severe sepsis or septic shock therapy.
Yet another aspect of the present invention provides methods of treating a subject at risk of developing sepsis, severe sepsis or septic shock, said method comprising determining the presence of at least two nucleic acid variants in one or more genes as given in Table 1 or Table 2 in said subject; and/or determining the presence of the nucleic acid variant at position g.745 in the TLR6 gene; and determining the appropriate treatment regiment for the so -identified subject. Preferably, appropriate medicaments, well known to the skilled person, against sepsis, severe sepsis or septic shock will be administered.
The present description including the Examples is to be understood in conjunction with the following Figures intended to illustrate the invention without implying any limitation of the invention to the specific embodiments described therein.
FIGURE LEGENDS
Figure 1: Fragment of the CIqRl gene comprising a nucleic acid variant. Figure 2: Fragment of the CD 14 gene comprising a nucleic acid variant.
Figure 3: Fragments of the FCNl gene comprising a nucleic acid variant.
Figure 4: Fragments of the FCN2 gene comprising a nucleic acid variant.
Figure 5: Fragments of the LBP gene comprising a nucleic acid variant.
Figure 6: Fragment of the MASPl gene comprising a nucleic acid variant. Figure 7: Fragment of the MASP2 gene comprising a nucleic acid variant.
Figure 8: Fragment of the MBL2 gene comprising a nucleic acid variant.
Figure 9: Fragments of the Sigirr gene comprising a nucleic acid variant.
Figure 10: Fragments of the TLRl gene comprising a nucleic acid variant.
Figure 11: Fragments of the TLR2 gene comprising a nucleic acid variant. Figure 12: Fragment of the TLR3 gene comprising the nucleic acid variant.
Figure 13: Fragment of the TLR4 gene comprising a nucleic acid variant.
Figure 14: Fragment of the TLR5 gene comprising a nucleic acid variant.
Figure 15: Fragments of the TLR6 gene comprising the nucleic acid variant.
Figure 16: Fragment of the TLR7 gene comprising a nucleic acid variant. Figure 17: Fragments of the TLR8 gene comprising a nucleic acid variant.
Figure 18: Fragments of the TLRlO gene comprising a nucleic acid variant. DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In any case the meaning of a term should not be limited to less than would be commonly understood by one of ordinary skill. The examples are only illustrative and not limiting.
Definitions Unless otherwise specified, a "gene" refers not only to the coding sequence, but to all sequences that are part of that gene: the introns and exons, the regulatory regions including the promoter region and possible other regulatory sequences, such as 5'UTR, 3'UTR or sequences further up- or downstream.
Table 1 gives for each gene of the present invention the number of exons, the reference gDNA sequence (with the used version between brackets), the reference mRNA sequence and the reference protein sequence, as given by the NCBI.
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Due to the presence of alternative splice forms, multiple transcript/protein entries can be provided for a single gene entry.
As used herein, the term "wild-type" sequence refers to the reference sequence. The reference nucleic acid and protein sequences indicated in the current invention are derived from NCBI (http://www.ncbi.nlm.nih.gov/) and indicated by their respective accession number (Table 1), as is well known to the person skilled in the art. The nomenclature for the 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 • there is no nucleotide 0
• the nucleotide 5' of the ATG-translation initiation codon is -1.
To avoid confusion, the nucleotide number is preceded by "g." when a genomic or by "c." when a cDNA reference sequence is used. Substitutions are designated by ">".
The term "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 about 100 nucleotides or less in length is often also referred to as an oligonucleotide.
The term "nucleic acid variant" or "polymorphism" as used in the present invention refers to a position comprising one or more nucleotides in the nucleic acid sequence which differs relative to a reference nucleic acid sequence.
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. The term "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 polymorphism or nucleic acid variant can be an insertion, deletion, substitution, tandem repeat or similar. The term "Single nucleotide polymorphism (SNP)" refers to the variation of a single nucleotide. This includes the replacement of one nucleotide by another and deletion or insertion of a single nucleotide. Typically, SNPs are biallelic markers although tri-and tetra-allelic markers also exist. For example, SNP A\C may comprise allele C or allele A. Thus, a nucleic acid variant when referring to the SNP A\C may include a C or A at the polymorphic position. The SNPs of the present invention are disclosed in the NCBI database dbSNP, and are characterised by an "rs" number. Said rs number can be used to retrieve summary information on the known variation at the locus, a list of the specific reports that characterize a SNP, and links to other NCBI resources.
The phrase "determining the presence", e.g. of a marker or variant as used herein, refers to determining whether or not the relevant genetic, physiological and/or biochemical event, linked with the occurrence of a disease is present. In practice, both the absence and the presence of a certain event can function as markers. Accordingly, reference to determining the presence of a nucleic acid variant for sepsis, and/or severe sepsis or septic shock, generally encompasses determining whether the variant is present or absent in a sample. As such, this also includes the possible finding that the marker is not present in the sample, i.e. determining the absence of a nucleic acid variant. In both cases 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 (besides the direct detection of the nucleic acid variant) also be done by determining the homozygous presence of the corresponding allele not comprising the nucleic acid variant. Similarly, allele specific oligonucleotide primers or probes for detecting the presence of a SNP can be specific for the allele where the
SNP is not present. The term "haplotype" means a particular pattern of sequential polymorphisms found on a single chromosome. As used herein, the term "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. The term "genotype" means the genetic constitution of an individual, either overall or at a specific locus, and defines the combination of alleles the individual carries. The term "homozygous" refers to having two of the same alleles at a locus; the term "heterozygous" refers to having different alleles at a locus. The term "SIRS" as used herein is defined as a clinical manifestation secondary to an insult, mostly of infectious origin.
The term "sepsis" as used herein is defined as the presence of SIRS in the setting of a documented infection.
The term "severe sepsis" as used herein is defined as sepsis with evidence of end-organ dysfunction as a result of hypoperfusion.
The term "septic shock" as used herein is defined as sepsis with persistent hypotension despite fluid resuscitation.
The term "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 hair root, a skin sample or organ sample (e.g. liver). The term "body fluid" refers to all fluids that are present in the body including but not limited to blood, plasma, serum, synovial fluid, 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 pre-treatment may be selected appropriately by those skilled in the art depending on the biological sample to be subjected.
The present invention provides methods and tools for identifying the risk of a subject to develop sepsis, severe sepsis or septic shock.
The "subject" on which the method of the present invention can be carried out can be any vertebrate animal, more particularly any mammalian animal, and is most particularly a human. It is envisaged that the methods of the invention can be applied to a non-human subject such as (but not limited to) a cow, a pig, a sheep, a goat, a horse, especially racing horses, a monkey, a rabbit, a dog, a cat, a mouse, a rat, a hamster or a primate or any laboratory test animal. In a first aspect, the present invention provides methods for identifying the risk of a subject to develop sepsis, severe sepsis or septic shock based on a combined determination of the presence in that subject of at least two nucleic acid variants in a specific gene or combination of genes.
According to the present invention, determination of the occurrence of a polymorphism in a gene, in combination with determination of the presence of another polymorphism in the same or another gene, allows the identification of a subject at risk of developing sepsis, severe sepsis or septic shock with improved accuracy, compared to the determination of a single polymorphism in an individual gene. As an exception however, it has been found that the SNP rs5743810 in the TLR6 gene can be used on its own to determine the risk of a subject to develop sepsis, severe sepsis or septic shock
According to a particular embodiment of the methods of the invention, the determination of the presence of one or more polymorphisms of a gene is ensured at the DNA level. Most particularly, the determination includes determining the occurrence of one variant allele of a gene (heterozygous for the variant allele) or the presence of two variant alleles of a gene (homozygous for the presence the variant allele). Determination of the occurrence of a variant allele at the DNA level is described in more detail below.
Additionally or alternatively, the determination of the presence of a variant allele can be performed at the protein level, based on quantitative expression of the expressed protein, qualitative assessment of the expressed protein (differences in protein sequence) and/or a functional assessment of the expressed protein (e.g. the ability to bind to GIcNAc). Methods for determining serum concentrations and altered function of a protein are described, for instance in Hummelshoj et al. (2005).
In a first embodiment, the methods of the present invention involve the determination of two or more polymorphisms in a gene or a combination of genes, more particularly the determination of two or more polymorphism in a gene or combination of genes selected from the group consisting of: mannose-binding lectin (MBL2), MBL-associated Serine Protease 1 (MASPl), MBL-associated Serine Protease 2 (MASP2), Ficolin 1 (FCNl), Ficolin 2 (FCN2), Complement component Iq Receptor 1 (CIqRl), Lipopolysaccharide-binding protein (LBP), Monocyte Differentiation Antigen CD 14 (CD 14), Single Immunoglobulin domain-containing ILlR-related protein (Sigirr), Toll- like Receptor 1 (TLRl), Toll- like Receptor 2 (TLR2), Toll-like Receptor 3 (TLR3), Toll-like Receptor 4 (TLR4), Toll-like Receptor 5 (TLR5), Toll-like Receptor 6 (TLR6), Toll-like Receptor 7 (TLR7), Toll- like Receptor 8 (TLR8), and Toll- like Receptor 10 (TLRlO). Sequence characteristics of the genes and associated proteins are given in Table 1. In a particular embodiment, the polymorphism can be detected in the corresponding cDNA or RNA sequence.
According to yet a further embodiment, the polymorphism in the gene is characterized as indicated in Table 2. According to a particular embodiment, the presence of at least two nucleic acid variants in one gene or a combination of genes is associated with the risk to develop sepsis, severe sepsis or septic shock, with an odds ratio of at least 3.25, 3.26, 3.27, 3.28 or 3.29, preferably 3.5 and even more preferably 4.0, or more, or alternatively, with an odds ratio of less than 0.3, 0.29, 0.28, 0.27 or 0.26, and preferably 0.25, or less. Most particularly the upper and lower limit of the confidence interval for this odds ratio is <1 and >1, respectively.
Table 2 gives NCBI's accession number for each SNP, the gene, the position of the SNP, the nucleic acid polymorphism and the amino acid change. It is to be understood that the nucleotide variant also encompasses the complement nucleic acids.
Figure imgf000022_0001
Figure imgf000023_0001
vl = variant 1 v2 = variant 2 na: not applicable
Furthermore, the relevant part of the genomic DNA of the gene comprising the nucleic acid variant as given in Table 2 is provided in Figures 1-18. Accordingly, in a specific embodiment, the polymorphism or nucleic acid variant is detected in the specified gene comprising said fragment, i.e. comprising the sequence as given by SEQ ID NO 1 to 35, respectively.
In a particular embodiment, the method of the present invention comprises a step wherein one or more of the sequences represented by SEQ ID NO 1 to 35 are amplified. Amplification of one gene fragment can be done separately, or simultaneously with one ore more of the other fragments. As will be evident for the skilled person, certain nearby located gene regions represented by SEQ ID NO 1 to 35, respectively, and within a single gene can be amplified in one reaction.
In a further embodiment, the methods of the invention comprise the step of determining whether the one or more nucleic acid variants in the gene are present in 0, 1 or 2 copies, more particularly whether a nucleic acid variant in the gene is present in one or in both alleles. Thus, according to this embodiment it is determined not only whether an allele carrying the nucleic acid variation is present or absent, but if it is present, it is also determined whether there is only one allele carrying the nucleic acid variant (heterozygous presence) or whether both alleles carry the nucleic acid variant (homozygous presence). Either heterozygous or homozygous presence of the at least one nucleic acid variant is indicative of having or a risk of having or developing sepsis, severe sepsis or septic shock. This is shown in Table 4 (Example 1).
According to yet a further embodiment, the methods of the invention comprise determining the presence of at least two genotypes. More specific, the presence of 0, 1 or 2 alleles in a single gene or in two different genes is linked to sepsis, severe sepsis or septic shock. In a more specific embodiment, the methods of the invention comprise determining the presence of a combination of at least two genotypes as given in Table 4.
Different analytical procedures suitable for the detection of the presence or absence of the nucleic acid variants mentioned herein are known in the art.
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), Gut (2001), and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford University Press, 1996 and "PCR", 2nd Edition" by Newton & Graham, BIOS Scientific Publishers Limited, 1997.
The step of determining the presence of a nucleic acid variant in a gene in the methods of the present invention can be carried out in vivo or in vitro. Most typically, however, detection of nucleic acid variants in the genes are performed in vitro in a biological sample obtained from the subject.
Typically, a nucleic acid comprising a sequence of interest can be obtained from a biological sample, more particularly from a sample comprising 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 DNA 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., Cold Spring Harbor Laboratory Press 1989, Cold Spring Harbor, NY, USA; Ausubel et al., Current Protocols in Molecular Biology 2003, John Wiley & Sons). If needed, e.g. when the quantity of the nucleic acid is low or insufficient for the assessment, the nucleic acid of interest may be amplified. Such 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).
Accordingly the methods of the present invention optionally comprise the steps of isolating nucleic acids from the sample and/or an amplification step.
Numerous methods for detecting a single nucleotide anomaly in nucleic acid sequences are well-known in the art. 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), GeneChip™ microarrays, dynamic allele-specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, TaqMan™ (5 'nuclease assay), and molecular beacons; allele-specific PCR methods such as intercalating dye, FRET primers, and Alphascreen™; primer extension methods such as ARMS (amplification refractory mutation system), kinetic or real-time PCR, SNPstream™, Genetic Bit Analysis™ (GBA), multiplex minisequencing, SnaPshot™, Pyrosequencing™, MassEXTEND™, MassArray™, GOOD assay, microarray minisequencing, APEX (arrayed primer extension), sequence specific priming (SSP), microarray primer extension, Tag arrays, coded microspheres, template-directed incorporation (TDI), fluorescence polarization; oligonucleotide ligation methods such as colorimetric OLA (oligonucleotide ligation assay), sequence-coded OLA, microarray ligation, ligase chain reaction, padlock probes, and rolling circle amplification; endonuclease cleavage methods such as restriction site analysis (RFLP) and Invader™ assay. More specific assays that can be used are LiPA, DoPA and a microarray.
In a particular embodiment, the detection of the presence or absence of a nucleic acid variant is determined by DNA or RNA hybridization, sequencing, PCR, primer extension, multiplex ligation-dependent probe amplification (MLPA), oligonucleotide ligation assay (OLA) or restriction site analysis.
Another aspect of the invention is the use of the present methods to identify a subject at risk of developing a modified response to sepsis, severe sepsis or septic shock therapy. Identifying the most effective treatment by first determining the presence of specific genotypes may lead to a considerable decrease in morbidity. The methods of the invention thus provide a tool for improved risk assessment and may help to determine the most appropriate, "personalized" therapy.
Another aspect of the invention relates to a kit for use in the methods as described herein. More specific the present invention encompasses a kit for identifying the risk of a subject to develop sepsis, severe sepsis or septic shock, comprising one or more reagents for detecting the presence (or, where appropriate absence) of at least two nucleic acid variants in a gene or a combination of genes. More particularly the kits of the present invention provide the tools for the detection of: - the presence of at least one nucleic acid variant in the MBL2 gene and at least one nucleic acid variant in the CD 14 gene in said subject; and/or the presence of at least two nucleic acid variants in the FCN2 gene, and/or the presence of at least one nucleic acid variant in the TLR8 gene and at least one nucleic acid variant in the FCNl gene in said subject, and/or the presence of at least one nucleic acid variant in the FCN2 gene and at least one nucleic acid variant in the gene selected from the group consisting of: the MASPl gene, the TLR3 gene, the TLR2 gene, the TLR3 gene, the TLR4 gene, the TLR6 gene, the TLR8 gene, and the Sigirr gene in said subject, and/or - the presence of at least one nucleic acid variant in the TLR7 gene and at least one nucleic acid variant in the TLR6 gene in said subject; and wherein the combined presence of at least two nucleic acid variants is indicative of the risk to develop sepsis, severe sepsis or septic shock.
Alternatively, or additionally, the kit of the present invention comprises one or more reagents for detecting the presence of the nucleic acid variant at position g.745 of the TLR6 gene. Specifically, the nucleic acid variant is the SNP rs5743810.
In a specific embodiment, the invention relates to a kit for identifying the risk of a subject to develop sepsis, severe sepsis or septic shock, comprising one or more reagents for detecting the presence (or, where appropriate absence) of at least two nucleic acid variants selected from the group as given in Table 2. More particularly the kits of the present invention provide the tools for the detection of the specific combination of at least two nucleic acid variants as provided in Table 4.
Although different reagents suitable for detecting the presence of a nucleic acid variant in a gene can be envisaged, in a particular embodiment they will include an oligonucleotide probe suitable for detection of a target polynucleic acid and/or an oligonucleotide pair suitable for amplification of a target polynucleic acid. Specific embodiments of the kits of the present invention comprise an oligonucleotide probe suitable for detection of a sequence within the gene and/or an oligonucleotide pair suitable for amplification of a sequence within a polynucleic acid.
Oligonucleotides for use in the kits or methods of the present invention typically are isolated nucleic acid molecules comprising at least 8 nucleotides and specifically hybridizing with a target nucleic acid sequence, e.g. the wild type or variant sequence of the gene including the position of the nucleic acid variant, or the complementary thereof.
More particularly, the oligonucleotide comprises at least 9, 10, 11, 12, 13, 14 or 15 nucleotides and up to 40, 30, 25, 24, 23, 22, 21, or 20 nucleotides. The oligonucleotides can be used as a primer or probe. In general such primers or probes will comprise nucleotide sequences entirely complementary to the corresponding target sequence, e.g. a wild type or variant locus in the target gene. Of course, 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. Preferably, the primers or probes will amplify or hybridize with the sequence characterized by SEQ ID NOs 1-35.
An oligonucleotide primer (or primer pair) designed to specifically recognize and amplify either a wild type or variant allele at a locus is referred to as an allele specific primer (or primer pair). The same applies for an allele specific probe, i.e. an oligonucleotide probe that specifically hybridizes to either a wild type or variant allele.
For the detection of specific alleles, or for the detection of specific nucleic acid variants, the hybridization conditions are to be stringent as known in the art. "Stringent" refers to conditions under which a nucleotide sequence will no longer bind to unrelated 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 be prevented or dissolved.
The primers or probes may carry one or more labels to facilitate detection. The nature of the label is not critical to the invention and may be fluorescent, chemiluminescent, enzymatic, radioactive, chemical or other, provided it doesn't interfere with correct hybridization of the oligonucleotide.
Typically, the kits of the present invention will comprise one or more oligonucleotide primers and/or one or more probes specific for the one or more allele(s) containing the nucleic acid variant(s). However, in another embodiment, it is envisaged that the kits for detection according to the methods of the present invention comprise one or more oligonucleotide primers and/or one or more probes specific for the wild type allele, not containing the nucleic acid variant, whereby an indication of the presence of a wild type allele is indicative of the absence of a nucleic acid variant. In a further embodiment, the kits of the present invention comprise both oligonucleotide primers (and/or probes) specific for the "variant" and the "wild type" allele. The latter embodiment is particularly suited to determine the copy number of the variant alleles.
Accordingly, in specific embodiments, the one or more allele specific primers and/or probes for detecting the nucleic acid variants will typically comprise at least part of the nucleic acid sequence as identified in Table 1, e.g. as characterized by SEQ ID NO 1-35 respectively, or the complementary thereof, wherein the part of the nucleic acid sequence is envisaged to potentially comprise one or more nucleic acid variants. More specifically, kits are envisaged which comprise combinations of at least two primers and/or probes capable of hybridizing to and/or amplifying the region comprising
- position 162 of the MBL2 gene which is a G/A polymorphism (dbSNP number rsl 800450),
- position 1336 of the CD14 gene which is a G/T polymorphisms (dbSNP number rs2563298),
- position -1981 of the FCNl gene which is a G/A polymorphism (dbSNP number rs2989727), - position 7919 of the FCNl gene which is a G/A polymorphism (dbSNP number rslO71583),
- position -64 of the FCN2 gene which is a AJC polymorphism (dbSNP number rs7865453),
- position -4 of the FCN2 gene which is a G/A polymorphism (dbSNP number rsl7514136),
- position 6359 of the FCN2 gene which is a C/T polymorphism (dbSNP number rsl7549193),
- position 6424 of the FCN2 gene which is a G/T polymorphism (dbSNP number rs7851696), - position 70465 of the MASPl gene which is a G/A polymorphism (dbSNP number rs3733001),
- position -15607 of the TLR2 gene which is a G/A polymorphism (dbSNP number rsl 898830), - position 6301 of the TLR3 gene which is a C/T polymorphism (dbSNP number rs3775291),
- position -2026 of the TLR4 gene which is a G/A polymorphism (dbSNP number rs 1927914), - position -502 of the TLR6 gene which is a C/T polymorphism (dbSNP number rslO39559),
- position 745 of the TLR6 gene which is a C/T polymorphism (dbSNP number rs5743810),
- position 17962 of the TLR7 gene which is a T/A polymorphism (dbSNP number rsl79008),
- position 9300 or 12979 of the TLR8 gene which is an C/T polymorphism (dbSNP number rs5744080),
- position 3392 of the Sigirr gene which is a G/A polymorphism (dbSNP number rs3210908), and - position 3402 of the Sigirr gene which is a G/T polymorphism (dbSNP number rs3087588), wherein the designated positions either have the wild type nucleotides or nucleic acid variants thereof.
Accordingly, the present invention also provides kits comprising two or more, i.e.
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more, allele specific primers and/or oligonucleotide probes, for detecting the presence of at least two, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38, nucleic acid variants. Preferably, the two or more nucleic acid variants are selected from the group as given in Table 2. Even more preferably the two or more nucleic acid variants are those combinations as specifically given in Table 4.
Apart from the one or more specific reagents for detecting the presence of markers for sepsis, severe sepsis or septic shock described above, the kits of the invention optionally contain a variety of other reagents, e.g. depending on the detection procedure. The following is a non-exhaustive list of reagents that may be part of the kit's contents, the person skilled in the art will understand that this merely is illustrative of the possibilities: an agent for denaturing nucleic acids, an enzyme capable of modifying a double stranded or single stranded nucleic acid molecule, a hybridization buffer, or components necessary for producing a hybridization buffer, a wash solution, or components necessary for producing a wash solution, a means for detecting partially or completely denatured polynucleic acids, a means for detecting hybrids formed in the preceding hybridization, a means for detecting enzymatic modifications of nucleic acids, a means for attaching an oligonucleotide to a known location on a solid support, a labelled antibody, and/or a means for attaching an antigen to a known location on a solid support.
In yet another aspect of the invention, methods are provided for treating a subject at risk of developing sepsis, severe sepsis or septic shock, which method comprises identifying the subject as being at risk of developing sepsis, severe sepsis or septic shock. Thus, methods are provided for treating a subject having an increased risk of developing sepsis, severe sepsis or septic shock, using the methods as described herein.
Other arrangements of the methods and tools embodying the invention will be obvious for those skilled in the art. It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for the methods and tools according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention.
The present invention is illustrated by the following Examples, which should not be understood to limit the scope of the invention to the specific embodiments therein. EXAMPLES
Statistical analysis
For the present examples, 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). In the present examples, 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 for a protective effect on the risk of developing a disorder or disease (OR<1).
The 95% confidence interval (95% CI) is the range of numerical values in which we can be confident (to a computed probability, here 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 get narrower and "precision" is increased. To confidently accept a calculated OR as reliable, important or clinically significant, the lower boundary of the confidence interval, or lower confidence limit, should be >1 if the OR>1, or the upper boundary of the confidence interval should be <1 if the OR<1.
Patient samples Whole blood samples were collected via a prospective study that involved 169 consecutive ICU (Intensive Care unit) patients (conform the inclusion criteria of the study). Patients were diagnosed according different degrees of sepsis: 87 patients with no sepsis; 17 patients with sepsis; 16 patients with severe sepsis and 49 patients with septic shock. For each patient, informed consent to participate in the study is available.
Materials & Methods
DNA extraction of whole blood samples was performed using PUREGENE® DNA Purification Kit. The relevant areas of the genes listed in Table A, were amplified and hybridized to (i) either LiPA strips for MBL2 according to the protocol described by the manufacturer of the kit (INNO-LiPA MBL2, Innogenetics NV), (ii) or DoPA (Dot Probe Assay) strips for the remaining genes. The protocol for the DoPA strips is similar to the protocol for the MBL2 LiPA strips.
Table A
Figure imgf000033_0001
In general, the relevant regions were amplified using biotinylated oligonucleotides. The polymorphisms were detected by use of a reverse hybridization method with probes designed to recognize the polymorphisms. After stringent wash 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).
SNP analysis and results
From the hybridization results for each locus the genotype was determined and odds ratios for individual SNP (Table 3) and combinations of two SNP's were calculated. From these, the most valuable SNP combinations were selected using the following criteria: OR > 3.25 or OR < 0.3 and the number of patients with that particular SNP combination is > 20 in the study population.
Of the SNP's studied, 40 combinations were found to comply to the selection criteria (see Table 4), involving 38 singular SNP's (see Table 3). From both Tables it is clear that combinations of SNP's are far better predictors for the risk to develop sepsis, severe or septic shock than used individually. In Table 3, showing the OR of the individual SNP's, all but one OR's are between 3.04 (FCN2, rsl7514136) and 0.31 (TLR6, rslO39559). As a consequence of the criteria applied all relevant combinations have an OR of either > 3.25 and < 0.3, and thereby are all better indicators to assess the risk to develop sepsis, severe sepsis and septic shock. For example, the OR of the FCN2 promoter SNP rsl7514136 (AG) is 2.4 (see Table 3) while this SNP is found in 7 significant combinations having an OR ranging from 3.26 to 6.2. This also applies to all other combinations listed in Table 4.
Table 3. Overview OR and 95% confidence interval for single SNP's in sepsis all group (includes patients with sepsis, severe sepsis or septic shock) and severe+shock group (includes patients with severe sepsis or septic shock) sepsis all severe+shock gene SNP genotype OR 95% conf int OR 95% conf int
MBL 2 rs 1800450 AG 0,73 0,37-1,42 0,81 0,40-1,63
MASPl rs3733001 GG 0,9 0,49-1,64 0,89 0,47-1,70
MASP 2 rsl2711521 TT 1,26 0,68-2,33 1,52 0,78-2,97
FCNl rs2989727 AG 0,93 0,51-1,70 0,99 0,52-1,89
FCNl rslO71583 AG 0,95 0,52-1,74 1,02 0,54-1,95
FCN2 rs7865453 AC 0,56 0,26-1,20 0,54 0,23-1,23
FCN2 rsl7514136 AA 0,43 0,23-0,79 0,38 0,19-0,74
FCN2 rsl7514136 AG 2,43 1,31-4,52 3,04 1,56-5,92
FCN2 rsl7549193 CC 0,52 0,28-0,96 0,51 0,26-0,98
FCN2 rsl7549193 CT 1,99 1,08-3,68 2,27 1,18-4,38
FCN2 rs7851696 GT 0,52 0,25-1,12 0,51 0,22-1,16
CIqRl rs3746731 CT 1,11 0,61-2,04 0,96 0,50-1,84
LBP rsl780627 CT 0,67 0,37-1,24 0,55 0,28-1,06
LBP rs2232613 CC 0,83 0,35-2,01 0,89 0,35-2,29
LBP rs2232618 CT 0,47 0,21-1,08 0,48 0,20-1,17
CD14 rs2563298 GT 0,7 0,38-1,29 0,64 0,33-1,23
Sigirr rs3210908 GG 0,73 0,37-1,42 0,84 0,43-1,64
Sigirr rs3087588 GG 0,73 0,37-1,42 0,84 0,43-1,64
TLRl rs5743551 AA 1,01 0,55-1,84 1 0,53-1,90
TLRl rs5743611 GG 0,78 0,35-1,72 0,84 0,36-1,95
TLRl rs4833095 AA 1,01 0,55-1,84 1 0,53-1,90
TLR2 rsl898830 AA 1,85 1,00-3,39 1,91 1,00-3,65
TLR2 rs3804100 TT 1,16 0,53-2,54 1,49 0,62-3,59
TLR3 rs3775291 CC 0,8 0,44-1,46 0,87 0,46-1,66
TLR4 rs 1927914 AA 1,18 0,63-2,20 1,37 0,71-2,66
TLR5 rs2072493 AA 0,68 0,35-1,32 0,6 0,30-1,21
TLR6 rslO39559 CC 0,51 0,24-1,12 0,31 0,12-0,80
TLR6 rslO39559 CT 1,61 0,98-2,96 2,05 1,06-3,95
TLR6 rs5743810 TT 0,51 0,22-1,23 0,27 0,09-0,86
TLR6 rs3821985 CC 0,73 0,40-1,35 0,63 0,33-1,21
TLR7 rsl79008 AA 0,71 0,36-1,41 0,88 0,42-1,85
TLR8 rs2159377 CC 1,44 0,74-2,77 1,37 0,68-2,77
TLR8 rs5744080 CC 1,56 0,84-2,88 1,38 0,72-2,67
TLR8 rs5744080 CT 0,64 0,31-1,41 0,72 0,34-1,53
TLRlO rs7694115 TT 0,99 0,53-1,85 1,01 0,52-1,96
TLRlO rs 1146645 TT 0,92 0,48-1,77 0,89 0,45-1,79
TLRlO rsl 1096957 AA 1,1 0,59-2,05 1,06 0,54-2,06
TLRlO rsl 1096956 GG 1,16 0,63-2,14 1,18 0,61-2,28
TLRlO rs4129009 AA 0,92 0,48-1,77 0,89 0,45-1,79 Table 4. Overview OR and 95% confidence interval for combined SNP's (SNPl + SNP2) in sepsis all group (includes patients with sepsis, severe sepsis or septic shock) and severe+shock group (includes patients with severe sepsis or septic shock)
Cu
Figure imgf000036_0001
Figure imgf000037_0001
35 Use of a single polymorphism
Some SNP's also proved to be of relevance if used individually. For example, the following SNP's showed an OR that have a significant predictive value in predicting the risk to develop sepsis, severe sepsis or septic shock:
SNP GENOTYPE OR 95% CONF. INTERV
- FCN2 rsl7514136 AA 0.38 0.19-0.74
- FCN2 rsl7514136 AG 3.04 1.56-5.92
- TLR6 rsl039559 CC 0.31 0.12-0.80
- TLR6 rs5743810 TT 0.27 0.09-0.86
In this study, for the first three SNP's, combinations with other SNP's were found that revealed higher OR than for each SNP individually. However, this was not the case for the last SNP (TLR6 rs5743810). No combinations with an OR lower than 0.27 were found. Hence, this SNP is a preferred candidate to be used on its own to determine the risk of a subject to develop sepsis, sever sepsis or septic shock.
REFERENCES
American College of Chest Physicians, Crit Care Med, 1992; 20: 864-875.
Dahmer MK, et al, Genetic Polymorphisms in sepsis (2005) Pediatr Crit Care Med 6(3) Suppl: S61-S73.
Den Dunnen JT and Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: A discussion. Hum. Mut. 2000; 15: 7-12.
Gut LG. (2001) Automation in genotyping of single nucleotide polymorphisms. Hum. Mutat. 17: 475-492.
Hummelshoj et al. Hum MoI Genet. 2005;14(12):1651-8.
IFCC. (1987) Approved recommendation on the theory of reference values. Part. 5.
Statistical treatment of collected refereece values. Determination of reference limits. J.
Clin. Chim Acta 1987;170:S13-S32
Menges T, Hermans PW, Little SG, Langefeld T, Boning O, Engel J, Sluijter M, de
Groot R, Hempelmann G. Plasminogen-activator-inhibitor-1 4G/5G promoter polymorphism and prognosis of severely injured patients. Lancet. 2001 Apr
7;357(9262): 1096-7
Nollau P, Wagener C. (1997) Methods for detection of point mutations: performance and quality assessment. IFCC Scientific Division, Committee on Molecular Biology
Techniques. Clin Chem., 43(7): 1114-28.
Sambrook J., Fritsch E. and Maniatis T. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
Stuyver L., Wyseur A., van Arnhem W., Hernandez F., Maertens G. (1996) A second generation line probe assay for hepatitis C virus. J. Clin. Microbiol. 34: 2259-2266. Stuyver L., Wyseur A., Rombout A., Louwagie J., Scarcez T., Verhofstede C, Rimland D., Schinazi R.F., Rossau R. (1997) Line probe assay (LiPA) for the rapid detection of drug-selected mutations in the HIV-I reverse transcriptase gene. Antimicrob. Agents Chemother. 41 : 284-291.
Syvanen A.C. (2001) Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat. Rev. Genet. 2: 930-942.
Van Geyt C, De Gendt S., Rombaut A., Wyseur A., Maertens G., Rossau R., Stuyver L. (1998) A line probe assay for hepatitis B virus genotypes. In: R.F. Schinazi, J.P. Sommadossi, and H. Thomas (eds.). Therapies of viral hepatitis. International Medical Press, London, UK, pp. 139-145.

Claims

1. A method of identifying the risk of a subject to develop sepsis, severe sepsis or septic shock comprising: - determining the presence of a nucleic acid variant at position -4,-64, 6359 and/or 6424 in the FCN2 gene and a nucleic acid variant selected from the group consisting of: a nucleic acid variant at position 70465 in the MASPl gene, a nucleic acid variant at position 6301 in the TLR3 gene, a nucleic acid variant at position -15607 in the TLR2 gene, a nucleic acid variant at position -2026 in the TLR4 gene, a nucleic acid variant at position 9300 and/or 12979 in the TLR8 gene, and a nucleic acid variant at position 3392 and/or 3402 in the Sigirr gene in said subject, and/or determining the presence of a nucleic acid variant at position 9300 and/or 12979 in the TLR8 gene and a nucleic acid variant at position -1981 and/or 7919 in the FCNl gene in said subject, and/or determining the presence of a nucleic acid variant at position 17962 in the TLR7 gene and a nucleic acid variant at position -502 in the TLR6 gene in said subject, and/or determining the presence of a nucleic acid variant at position 162 in the MBL2 gene and a nucleic acid variant at position 1336 in the CD14 gene in said subject; and/or determining the presence of a nucleic acid variant at position -4 and at position 6424 in the FCN2 gene; and/or determining the presence of a nucleic acid variant at position 6359 and at position 6424 in the FCN2 gene; wherein the genomic DNA sequence (gDNA) of said genes is as defined in Table 1, and wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop sepsis, severe sepsis or septic shock.
2. The method of claim 1, wherein one or more of said nucleic acid variant is a single nucleotide polymorphism (SNP).
3. The method of claim 2, wherein the SNP is characterized as follows: - position 162 of the MBL2 gene is a G/A polymorphism (dbSNP number rsl 800450),
- position 1336 of the CD14 gene is a G/T polymorphisms (dbSNP number rs2563298), - position -1981 of the FCNl gene is a G/A polymorphism (dbSNP number rs2989727),
- position 7919 of the FCNl gene is a G/A polymorphism (dbSNP number rslO71583),
- position -64 of the FCN2 gene is a A/C polymorphism (dbSNP number rs7865453),
- position -4 of the FCN2 gene is a G/A polymorphism (dbSNP number rsl7514136),
- position 6359 of the FCN2 gene is a C/T polymorphism (dbSNP number rsl7549193), - position 6424 of the FCN2 gene is a G/T polymorphism (dbSNP number rs7851696),
- position 70465 of the MASPl gene is a G/A polymorphism (dbSNP number rs3733001),
- position -15607 of the TLR2 gene is a G/A polymorphism (dbSNP number rsl898830),
- position 6301 of the TLR3 gene is a C/T polymorphism (dbSNP number rs3775291),
- position -2026 of the TLR4 gene is a G/A polymorphism (dbSNP number rsl 927914), - position 17962 of the TLR7 gene is a T/A polymorphism (dbSNP number rsl79008),
- position 9300 or 12979 of the TLR8 gene is an C/T polymorphism (dbSNP number rs5744080),
- position 3392 of the Sigirr gene is a G/A polymorphism (dbSNP number rs3210908), and
- position 3402 of the Sigirr gene is a G/T polymorphism (dbSNP number rs3087588).
4. A method of identifying the risk of a subject to develop sepsis, severe sepsis or septic shock comprising determining the presence of:
- the nucleic acid variant rs7865453 in the FCN2 gene and the nucleic acid variant rs3775291 in de TLR3 gene, or the nucleic acid variant rs3733001 in the MASPl gene, and/or
- the nucleic acid variant rsl 7514136 in the FCN2 gene and the nucleic acid variant rsl898830 in de TLR2 gene, or the nucleic acid variant rsl927914 in the TLR4 gene, or the nucleic acid variant rs5744080 in the TLR8 gene, and/or
- the nucleic acid variant rsl 7549193 in the FCN2 gene and the nucleic acid variant rsl 898830 in de TLR2 gene, or the nucleic acid variant rs5744080 in the
TLR8 gene, and/or
- the nucleic acid variant rs7851696 in the FCN2 gene and the nucleic acid variant rs3210908 or rs3087588 in de Sigirr gene, or the nucleic acid variant rs3775291 in the TLR3 gene, or the nucleic acid variant rsl7549193 or rs 17514136 in the FCN2 gene, and/or
- the nucleic acid variant rs5744080 in the TLR8 gene and the nucleic acid variant rs2989727 or rsl 071583 in de FCNl gene, and/or
- the nucleic acid variant rsl 79008 in the TLR7 gene and the nucleic acid variant rslO39559 in de TLR6 gene, and/or - the nucleic acid variant rsl 800450 in the MBL2 gene and the nucleic acid variant rs2563298 in de CD 14 gene; wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop sepsis, severe sepsis or septic shock.
5. The method of any one of claims 1 to 4, which comprises determining whether said nucleic acid variant in the gene is present in 0, 1 or 2 copies, wherein the heterozygous or homozygous presence of the combination of the at least two nucleic acid variants is indicative of the risk to develop sepsis, severe sepsis or septic shock.
6. The method of any one of claims 1 to 5, wherein the step of detecting the presence of the nucleic acid variant is performed by DNA or RNA hybridization, sequencing, PCR, primer extension, multiplex ligation-dependent probe amplification (MLPA), oligonucleotide ligation assay (OLA) and/or restriction site analysis.
7. The method of any one of claims 1 to 6, wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop a modified response to therapy.
8. The method of any one of claims 1 to 7, wherein the step of determining the presence of the nucleic acid variant is performed in vitro in a biological sample obtained from said subject.
9. A kit for the determining the risk of a subject to develop sepsis, severe sepsis or septic shock comprising one or more reagents for detecting the presence of: a nucleic acid variant at position -4,-64, 6359 and/or 6424 in the FCN2 gene and a nucleic acid variant selected from the group consisting of: a nucleic acid variant at position 70465 in the MASPl gene, a nucleic acid variant at position 6301 in the TLR3 gene, a nucleic acid variant at position -15607 in the TLR2 gene, a nucleic acid variant at position -2026 in the TLR4 gene, a nucleic acid variant at position 9300 and/or 12979 in the TLR8 gene, and a nucleic acid variant at position 3392 and/or 3402 in the Sigirr gene in said subject, and/or a nucleic acid variant at position 9300 and/or 12979 in the TLR8 gene and a nucleic acid variant at position -1981 and/or 7919 in the FCNl gene in said subject, and/or a nucleic acid variant at position 17962 in the TLR7 gene and a nucleic acid variant at position -502 in the TLR6 gene in said subject, and/or a nucleic acid variant at position 162 in the MBL2 gene and a nucleic acid variant at position 1336 in the CD14 gene in said subject; wherein the genomic DNA sequence (gDNA) of said genes is as defined in Table 1.
10. The kit according to claim 9 wherein the reagents consist of one or more allele specific primers and/or one or more oligonucleotide probes for detecting the presence of the nucleic acid variants.
11. The kit of claim 10, wherein the nucleic acid variant is characterized as follows:
- position 162 of the MBL2 gene is a G/A polymorphism (dbSNP number rsl 800450),
- position 1336 of the CD14 gene is a G/T polymorphisms (dbSNP number rs2563298),
- position -1981 of the FCNl gene is a G/A polymorphism (dbSNP number rs2989727),
- position 7919 of the FCNl gene is a G/A polymorphism (dbSNP number rslO71583), - position -64 of the FCN2 gene is a A/C polymorphism (dbSNP number rs7865453),
- position -4 of the FCN2 gene is a G/A polymorphism (dbSNP number rsl7514136),
- position 6359 of the FCN2 gene is a C/T polymorphism (dbSNP number rsl7549193),
- position 6424 of the FCN2 gene is a G/T polymorphism (dbSNP number rs7851696),
- position 70465 of the MASPl gene is a G/A polymorphism (dbSNP number rs3733001), - position -15607 of the TLR2 gene is a G/A polymorphism (dbSNP number rsl898830),
- position 6301 of the TLR3 gene is a C/T polymorphism (dbSNP number rs3775291),
- position -2026 of the TLR4 gene is a G/A polymorphism (dbSNP number rsl927914),
- position 17962 of the TLR7 gene is a T/A polymorphism (dbSNP number rsl79008),
- position 9300 or 12979 of the TLR8 gene is an C/T polymorphism (dbSNP number rs5744080), - position 3392 of the Sigirr gene is a G/A polymorphism (dbSNP number rs3210908), and
- position 3402 of the Sigirr gene is a G/T polymorphism (dbSNP number rs3087588).
12. A method of treating a subject having an increased risk of developing sepsis, severe sepsis or septic shock comprising: determining the presence of a nucleic acid variant at position -4,-64, 6359 and/or 6424 in the FCN2 gene and a nucleic acid variant selected from the group consisting of: a nucleic acid variant at position 70465 in the MASPl gene, a nucleic acid variant at position 6301 in the TLR3 gene, a nucleic acid variant at position -15607 in the TLR2 gene, a nucleic acid variant at position - 2026 in the TLR4 gene, a nucleic acid variant at position 9300 and/or 12979 in the TLR8 gene, and a nucleic acid variant at position 3392 and/or 3402 in the S igirr gene in said subject, and/or determining the presence of a nucleic acid variant at position 9300 and/or 12979 in the TLR8 gene and a nucleic acid variant at position -1981 and/or 7919 in the FCNl gene in said subject, and/or determining the presence of a nucleic acid variant at position 17962 in the TLR7 gene and a nucleic acid variant at position -502 in the TLR6 gene in said subject, and/or determining the presence of a nucleic acid variant at position 162 in the MBL2 gene and a nucleic acid variant at position 1336 in the CD 14 gene in said subject; and/or - determining the presence of a nucleic acid variant at position -4 and at position
6424 in the FCN2 gene; and/or determining the presence of a nucleic acid variant at position 6359 and at position 6424 in the FCN2 gene; wherein the genomic DNA sequence (gDNA) of said genes is as defined in Table l, and determining the treatment regimen,
13. A method of identifying the risk of a subject to develop severe sepsis or septic shock comprising: - determining the presence of a nucleic acid variant located at position 16349 in the MASP2 gene and a nucleic acid variant located at position -4 and/or 6359 in the FCN2 gene in said subject; and/or determining the presence of a nucleic acid variant at position -4 and/or 6359 in the FCN2 gene and at a nucleic acid variant located at position 22713, 22736 and/or 26842 in the LBP gene in said subject, or a nucleic acid variant located at position 1621 in the CIqRl gene in said subject, or a nucleic acid variant located at position -502 of the TLR6 gene, or a nucleic acid variant located at position 1336 in the CD 14 gene in said subject; and/or - determining the presence of a nucleic acid variant located at position 22713,
22736 and/or 26842 in the LBP gene and a nucleic acid variant at position - 502 and/or 1083 in the TLR6 gene in said subject or a nucleic acid variant located at position -7202 , 239 and/or 743 in the TLRl gene in said subject; and/or - determining the presence of a nucleic acid variant located at position -7202 ,
239 and/or 743 in the TLRl gene and a nucleic acid variant located at position -502 and/or 1083 in the TLR6 gene; and/or determining the presence of a nucleic acid variant located at position 1350 in the TLR2 gene and a nucleic acid variant located at position -502 and/or 1083 in the TLR6 gene; and/or determining the presence of a nucleic acid variant located at position 1775 in the TLR5 gene and a nucleic acid variant located at position -502 and/or 1083 the TLR6 gene; and/or determining the presence of a nucleic acid variant located at position -502 and/or 1083 in the TLR6 gene and a nucleic acid variant located at position
9009 and/or 12688 in the TLR8 gene or a nucleic acid variant located at position -1883, -992, 721, 1032 and/or 2323 in the TLRlO; wherein the genomic DNA sequence (gDNA) of said genes is as defined in Table 1, and wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop severe sepsis or septic shock.
14. The method of claim 13, wherein one or more of said nucleic acid variant is a single nucleotide polymorphism (SNP).
15. The method of any of claims 13 to 14, wherein the nucleic acid variant is characterized as follows:
- position 16349 of the MASP2 gene is a G/T polymorphism (dbSNP number rsl2711521), - position -4 of the FCN2 gene is a G/A polymorphisms (dbSNP number rsl7514136),
- position 6359 of the FCN2 gene is a C/T polymorphism (dbSNP number rsl7549193), - position 22713 of the LBP gene is a T/C polymorphism (dbSNP number rsl780627),
- position 22736 of the LBP gene is a C/T polymorphism (dbSNP number rs2232613),
- position 26842 of the LBP gene is a T/C polymorphism (dbSNP number rs2232618),
- position 1621 of the CIqRl gene is a C/T polymorphism (dbSNP number rs3746731),
- position 1336 of the CD14 gene is a G/T polymorphism (dbSNP number rs2563298), - position -7202 of the TLRl gene is a A/G polymorphism (dbSNP number rs5743551),
- position 239 of the TLRl gene is a G/C polymorphism (dbSNP number rs5743611),
- position 743 of the TLRl gene is a A/G polymorphism (dbSNP number rs4833095),
- position 1350 of the TLR2 gene is a T/C polymorphism (dbSNP number rs3804100),
- position 1775 of the TLR5 gene is a A/G polymorphism (dbSNP number rs2072493), - position -502 of the TLR6 gene is a C/T polymorphism (dbSNP number rslO39559),
- position 1083 of the TLR6 gene is a G/C polymorphism (dbSNP number rs3821985),
- position 9009 or 12688 of the TLR8 gene is an C/T polymorphism (dbSNP number rs2159377),
- position -1883 of the TLRlO gene is a T/C polymorphism (dbSNP number rs7694115),
- position -992 of the TLRlO gene is a T/A polymorphism (dbSNP number rsl 1466645), - position 721 of the TLRlO gene is a A/C polymorphism (dbSNP number rsl 1096957),
- position 1032 of the TLRlO gene is a G/T polymorphism (dbSNP number rsl 1096956), and - position 2323 of the TLRlO gene is a A/C or G/T polymorphism (dbSNP number rs4129009).
16. A method of identifying the risk of a subject to develop severe sepsis or septic shock comprising determining the presence of: - the nucleic acid variant rs 12711521 in the MASP2 gene and the nucleic acid variant rsl7514136 or rsl7549193 in de FCN2 gene, and/or
- the nucleic acid variant rsl 7514136 in the FCN2 gene and the nucleic acid variant rsl780627 in de LBP gene, or the nucleic acid variant rs3746731 in the CIqRl gene, or the nucleic acid variant rslO39559 in the TLR6 gene, or the nucleic acid variant rs2563298 in the CD14 gene, and/or
- the nucleic acid variant rsl 7549193 in the FCN2 gene and the nucleic acid variant rsl780627 in de LBP gene or the nucleic acid variant rs5744080 in the TLR8 gene, and/or
- the nucleic acid variant rs2232613 in the LBP gene and the nucleic acid variant rsl 039559 or rs5743810 in de TLR6 gene, and/or
- the nucleic acid variant rs2232618 in the LBP gene and the nucleic acid variant rs5743611 in de TLRl gene, and/or
- the nucleic acid variant rs5743551 in the TLRl gene and the nucleic acid variant rslO39559 or rs3821985 in de TLR6 gene, and/or - the nucleic acid variant rs4833095 in the TLRl gene and the nucleic acid variant rslO39559 or rs3821985 in de TLR6 gene, and/or
- the nucleic acid variant rs3804100 in the TLR2 gene and the nucleic acid variant rsl 039559 in de TLR6 gene, and/or
- the nucleic acid variant rs2072493 in the TLR5 gene and the nucleic acid variant rsl 039559 in de TLR6 gene, and/or
- the nucleic acid variant rsl 039559 in the TLR6 gene and the nucleic acid variant rs2159377 in de TLR8 gene, or the nucleic acid variant rsl 1466645, rsl 1096956 or rs4129009 in the TLRlO gene, and/or - the nucleic acid variant rs3821985 in the TLR6 gene and the nucleic acid variant rs7694115, rsl 1096957, rsl 1466645, rsl 1096956 or rs4129009 in the TLRlO gene; wherein the combined presence of the at least two nucleic acid variants is indicative of the risk to develop severe sepsis or septic shock.
17. The method of any one of claims 13 to 16, which comprises determining whether the nucleic acid variant in the gene is present in 0, 1 or 2 copies, wherein the heterozygous or homozygous presence of the combination of the at least two nucleic acid variants is indicative of the risk to develop severe sepsis or septic shock.
18. The method of any one of claims 13 to 17, wherein the step of detecting the presence of the nucleic acid variant is performed by DNA or RNA hybridization, sequencing, PCR, primer extension, multiplex ligation-dependent probe amplification (MLPA), oligonucleotide ligation assay (OLA) and/or restriction site analysis.
19. The method according to claim 6 or 18, wherein the detection of the presence of the nucleic acid variant is performed using a LiPA, DoPA or a Microarray.
20. A method of identifying the risk of a subject to develop sepsis, severe sepsis or septic shock comprising determining the presence of the nucleic acid variant at position 745 of the genomic DNA sequence of the TLR6 gene, as defined in Table 1 , wherein the presence of the nucleic acid variant is indicative of the risk to develop sepsis, severe sepsis or septic shock.
21. The method of claim 20, wherein said nucleic acid variant in is a single nucleotide polymorphism (SNP).
22. The method of claim 21, wherein the SNP is characterized as follows: position 745 of the TLR6 gene being a C/T polymorphism (dbSNP number rs5743810).
23. The method of any one of claims 20 to 22, which comprises determining whether said nucleic acid variant in the gene is present in 0, 1 or 2 copies, wherein the heterozygous or homozygous presence of the nucleic acid variant is indicative of the risk to develop sepsis, severe sepsis or septic shock. 5. • . ' ■ ' .. .
24. The method of any one of claims 20 to 23, wherein the step of detecting the presence of the nucleic acid variant is performed by DNA or RNA hybridization, sequencing, PCR, primer extension, multiplex ligation-dependent probe amplification (MLPA), oligonucleotide ligation assay (OLA) and/or restriction0 site analysis. . .
25. The method of any one of claims 20 to 24, wherein the presence of nucleic acid variant is indicative of the risk to develop a modified response to therapy. 5
26. The method of any one of claims 20 to 25, wherein the step of determining of the presence of the nucleic acid variant is performed in vitro in a biological- sample obtained from said subject.
27. A kit for the determining the risk of a subject to develop sepsis, severe sepsis or0 septic shock comprising one or more reagents for detecting the presence of the nucleic acid variant at position 745 of the genomic DNA sequence of the TLRό gene, as defined in Table 1.
28. The kit according to claim 27 comprising one or more allele specific primers5 and/or one or more oligonucleotide probes.
29. A kit for determining the risk of a subject to develop sepsis, severe sepsis, or septic shock, comprising one or more reagents for the detection of at least one of . the SNP combinations as given in Table 4.
30. A kit according to claim 31 wherein the genotypes are detected.
RECTIFIED SHEET (RULE 91) ISA/EP
1. A method for determining the risk of a subject to develop sepsis, severe sepsis, or septic shock, comprising the detection of at least one. of the SNP combinations as given in Table 4. ■
RECTIFIED SHEET (RULE 91) ISA/EP
PCT/EP2008/052474 2007-03-02 2008-02-29 Combinations of markers for sepsis risk assessment WO2008107377A2 (en)

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