WO2017121769A1 - Procédé in vitro permettant de prédire un risque de développement d'une pneumonie chez un sujet - Google Patents

Procédé in vitro permettant de prédire un risque de développement d'une pneumonie chez un sujet Download PDF

Info

Publication number
WO2017121769A1
WO2017121769A1 PCT/EP2017/050505 EP2017050505W WO2017121769A1 WO 2017121769 A1 WO2017121769 A1 WO 2017121769A1 EP 2017050505 W EP2017050505 W EP 2017050505W WO 2017121769 A1 WO2017121769 A1 WO 2017121769A1
Authority
WO
WIPO (PCT)
Prior art keywords
allele
pneumonia
risk
snp
subject
Prior art date
Application number
PCT/EP2017/050505
Other languages
English (en)
Inventor
Sophie BLEIN
Jean-Paul MIRA
Alexandre Pachot
Original Assignee
bioMérieux
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by bioMérieux filed Critical bioMérieux
Publication of WO2017121769A1 publication Critical patent/WO2017121769A1/fr

Links

Classifications

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

Definitions

  • the invention relates to the field of in vitro diagnosis or prognosis.
  • the invention provides in vitro methods for predicting the risk of developing pneumonia in a subject. More specifically, the method comprises determining the presence of one or more PNEUMONIA Risk Allele(s) in a sample from said subject, wherein the presence of said one or more PNEUMONIA Risk Allele(s) indicates whether the subject is at increased or decreased risk of developing pneumonia.
  • VAP ventilator-associated pneumonia
  • a first object of the invention relates to an in vitro method for assessing the risk of developing pneumonia in a subject, said method comprising determining the presence of one or more PNEUMONIA Risk Allele(s) in a sample from said subject wherein said one or more PNEUMONIA Risk Allele(s) indicates whether the subject is at increased or decreased risk of developing pneumonia, and wherein said PNEUMONIA Risk Allele is an allele selected at one or more of the single nucleotide polymorphism (SNP) of the following Table 1 :
  • SNP single nucleotide polymorphism
  • the linkage disequilibrium of a related SNP allele is determined within all populations or superpopulations to which the subject is originating.
  • the linkage disequilibrium of a related SNP allele is determined within a reference population or superpopulation to which the subject is the closest from genetic perspective. In some embodiments, the linkage disequilibrium of a related SNP allele is determined within one of the following reference superpopulation to which the subject is the closest from genetic perspective:
  • SAS - South Asian
  • EAS East Asian
  • the linkage disequilibrium of a related SNP allele is determined within one of the following reference population to which the subject is the closest from genetic perspective:
  • Luhya in Webuye, Kenya, Luhya (LWK);
  • JPT Japan Japanese
  • IBS Spain Spanish
  • the subject is from one of the following superpopulation and the linkage disequilibrium is determined within the superpopulation of the subject:
  • SAS - South Asian
  • EAS East Asian
  • the subject is from one of the following population and the linkage disequilibrium is determined within the population of the subject: - Esan in Nigeria, Esan (ESN);
  • Luhya in Webuye, Kenya, Luhya (LWK);
  • JPT Japan Japanese
  • CEPH - Utah residents
  • CEU Northern and Western European ancestry
  • IBS Spain Spanish
  • the subject is the closest to the European superpopulation and the linkage disequilibrium is determined within the European superpopulation.
  • the subject is a European and the linkage disequilibrium is determined within the following reference population to which the subject is the closest from genetic perspective: - Utah residents (CEPH) with Northern and Western European ancestry (CEU);
  • IBS Iberian Populations in Spain Spanish (IBS); and Toscani in Italia Italian (TSI).
  • said PNEUMONIA Risk Allele is selected among one or more of the SNP allele(s) of the following Table 2:
  • the method comprises determining the presence of one or more SNP allele(s) of the following table 3:
  • detecting said one or more SNP allele(s) is associated to an increased risk of developing pneumonia in said subject.
  • detecting at least SNP5 allele or its related SNP allele(s) in high linkage disequilibrium is associated to an increased risk of developing pneumonia in said subject.
  • the method comprises determining the presence of one or more SNP allele(s) of the following Table 4: SNP# Chromosome Position on genome hg38 / Allele
  • SNPIO 20 23921958 G or its related SNP allele(s) in high linkage disequilibrium wherein detecting said one or more SNP allele(s) is associated to a decreased risk of developing pneumonia in said subject.
  • detecting at least SNPl allele or its related SNP allele(s) in high linkage disequilibrium is associated to a decreased risk of developing pneumonia in said subject.
  • said pneumonia is Pseudomonas aeruginosa pneumonia.
  • said subject is a patient who is in need of receiving mechanical ventilation during at least 48 hours.
  • said risk is a risk of developing serious pneumonia, and for example, serious ventilator-associated pneumonia.
  • the presence of a PNEUMONIA Risk Allele in a subject may be detected by any SNP detection method selected from the group consisting of sequencing methods including without limitation Sanger sequencing, next generation sequencing, pyrosequencing, sequencing by ligation; PCR-based methods including without limitation PCR, real-time PCR, quantitative PCR; any SNP genotyping techniques such as amplification refractory mutation system (ARMS), restriction fragment length polymorphism (RFLP) analysis, denaturing gradient gel electrophoresis (DGGE), single- strand conformation polymorphism (SSCP); and/or any allele discrimination methods including allele-specific hybridization, molecular beacons, allele-specific single base primer extension, Flap endonuclease discrimination, 5' nuclease, oligonucleotide ligation and micro-array analysis of genomic DNA.
  • the method further comprises extracting nucleic acids from the sample, wherein the presence of a PNEUMONIA Risk All
  • the method further comprises determining a prognosis of the subject based on the presence of one or more of the PNEUMONIA Risk Allele(s).
  • the method further comprises adapting health care management for said subject based on the presence of one or more of the PNEUMONIA Risk Allele(s).
  • the method further comprises administering an adapted or preventive treatment of pneumonia to the subject based on the presence of one or more of the PNEUMONIA Risk Allele(s).
  • kits for performing the method as defined above comprises:
  • said means for detecting one or more of the SNP(s) include at least a probe that binds specifically to a nucleic acid comprising one SNP allele of the SNPs 1-10 as defined in Table 1 or 2 or its related SNP allele(s) in high linkage disequilibrium, and/or primers that are capable of amplifying a nucleic acid comprising one SNP allele of the SNPs 1-10 as defined in Table 1 or 2 or its related SNP allele(s) in high linkage disequilibrium.
  • said nucleic acid comprising one SNP allele of the SNPs 1-10 defined in Table 1 or 2 or its related SNP allele(s) in high linkage disequilibrium is either
  • a first object of the invention relates to an in vitro method for assessing the risk of developing pneumonia in a subject, said method comprising determining the presence of one or more PNEUMONIA Risk Allele(s) in a sample from said subject, wherein said one or more PNEUMONIA Risk Allele(s) indicates whether the subject is at increased or decreased risk of developing pneumonia.
  • a subject at risk in developing pneumonia is a patient who is in need of receiving mechanical ventilation during at least 48 hours.
  • a subject at risk in developing pneumonia is a patient in need of surgical intervention, more specifically, in need of digestive, thoracic, cardiac or neuro surgery.
  • a subject at risk in developing pneumonia is a transplanted patient or in need of organ transplantation.
  • a subject at risk in developing pneumonia is a patient suffering from chronic inflammatory disorders.
  • a subject at risk in developing pneumonia is a patient who has had or is in need of having an immunosuppressant and/or anti-inflammatory treatment. In still other embodiments, a subject at risk in developing pneumonia is a patient who is immunocompromised.
  • the method further comprises determining a prognosis of the subject based on the presence of one or more of the PNEUMONIA Risk Allele(s).
  • PNEUMONIA Risk Allele refers to all pneumonia, and more preferably to pneumonia which occurs more than 48 hours after patients have been intubated and received mechanical ventilation (referred hereafter as “ventilator-associated pneumonia” or "VAP").
  • pneumonia is Pseudomonas aeruginosa ventilator-associated pneumonia.
  • prognosis refers to a relative probability that a certain future outcome may occur in a patient.
  • prognosis can refer to the likely severity of the pneumonia (e.g., severity of symptoms, rate of functional decline, survival, etc.). The terms are not intended to be absolute, as will be appreciated by any one of skill in the field of medical diagnostics.
  • a "poor prognosis" in the context of the present invention means that a patient is at higher risk of developing serious pneumonia, in particular serious VAP.
  • the term "serious pneumonia” refers to pneumonia, for example VAP, with one or more of the following characteristics: at least one recurrence of pneumonia, for example, at least one recurrence of VAP during the same intensive care unit (ICU) stay,
  • the wording "assessing the risk of developing pneumonia” means assigning an increased or decreased probability of having/developing pneumonia and/or assigning an increased or decreased probability of having a poor prognosis for a subject having developed the disease, as compared to the average risk in a population.
  • an increased probability does not mean that the subject will develop the disease or will have a poor prognosis for the disease.
  • the method may not also give a precise probability for such risk but may give a relative risk assessment as compared to the average risk in a given population.
  • the method of the invention is an in vitro method which can be carried out on any appropriate biological sample obtained from a subject.
  • biological sample refers to a sample that contains nucleic acid materials reflecting the genomic information of cells, tissue or organs of the subject.
  • said biological sample may be obtained from urine, blood including without limitation peripheral blood or plasma or serum, stool, sputum, saliva, bronchoalveolar fluid, endotracheal aspirates, wounds, cerebrospinal fluid, lymph node, exudate and more generally any human biopsy tissue or body fluids, tissues or materials.
  • the method may further comprise the step of extracting nucleic acids from the biological sample, wherein the presence of a PNEUMONIA Risk Allele is detected in the extracted nucleic acids.
  • said biological sample is blood sample or a derivative thereof such as plasma or serum.
  • PNEUMONIA Risk Allele refers to a single nucleotide polymorphism (SNP) allele that is associated with an increased or decreased risk of developing the disease.
  • SNP single nucleotide polymorphism
  • the inventors have indeed identified that certain single nucleotide polymorphism (SNP) alleles are associated to increased or decreased risk of developing pneumonia in a subject, and more particularly an increased or decreased risk of developing ventilator-associated pneumonia.
  • a “single nucleotide polymorphism (SNP)” is a single base (nucleotide) polymorphism in a DNA sequence among individuals in a population.
  • SNP single nucleotide polymorphism
  • a SNP in which both forms lead to the same polypeptide sequence is termed “synonymous” (sometimes called a silent mutation) - if a different polypeptide sequence is produced they are "nonsynonymous”.
  • a nonsynonymous change may either be "missense” or "nonsense", where a missense change results in a different amino acid, while a nonsense change results in a premature stop codon.
  • the exact sequence of a SNP can be determined from the database of SNPs available at the NCBI website (Entrez SNP, dbSNP build 128).
  • the "position" of the nucleotide of interest gives the location of the SNP in a specified version of the genome, referring to the nucleotide position from the p-terminus of the chromosome in the human genome, see the NCBI SNP website (dbSNP), available on the internet.
  • the version of the genome specified in the context of the present invention is hg38, also known as Genome Reference Consortium Human GRCh38.
  • an allele refers to a particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence, or one of the alternative polymorphisms found at a polymorphic site (for example a SNP).
  • PNEUMONIA Risk Alleles for use in the methods and kits of the invention, include any SNP alleles as described in the following table 5:
  • a linkage disequilibrium refers to co-occurrence of two genetic loci (e.g. SNP allele) at a frequency greater than expected for independent loci based on the allele frequencies.
  • Linkage disequilibrium typically occurs when two loci are located close together on the same chromosome.
  • SNP allele in high linkage disequilibrium have an r 2 value with a specific (tag) SNP allele of greater than or equal to 0.8, greater than or equal to 0.85, greater than or equal to 0.9, or greater than or equal to 0.95, as measured in at least one of the reference populations or superpopulations to which the subject is originating or is the closest from genetic perspective.
  • a reference superpopulation is selected from the following superpopulation as defined in The 1000 Genomes Project Consortium (Nature 1 October 2015, 526, 68-74, A Global reference for human genetic variation):
  • AFR African ancestry
  • EAS East Asian ancestry
  • SAS South Asian ancestry
  • the linkage disequilibrium r 2 value may be measured in the European ancestry population as the reference superpopulation.
  • a reference population is selected from one of the population as defined in The 1000 Genomes Project Consortium (Nature 1 October 2015, 526, 68-74, A Global reference for human genetic variation)
  • the reference population for use in determining the high linkage disequilibrium is selected from one of the following reference population as specifically defined in The 1000 Genomes Project Consortium (Nature 1 October 2015, 526, 68-74, A Global reference for human genetic variation, in particular in the supplementary information Table 1 :
  • Luhya in Webuye, Kenya, Luhya (LWK);
  • JPT Japan Japanese
  • CEPH - Utah residents
  • CEU Northern and Western European ancestry
  • IBS Spain Spanish
  • the subject may be of mixed genetic background with one predominant genetic background.
  • a reference population or superpopulation to which the subject is the closest from genetic perspective may be determined and SNPs in high linkage disequilibrium to such population may be used in the method of the present disclosure.
  • the subject is of mixed but equal genetic background, for example 50% from a reference population and 50% from another population.
  • the linkage disequilibrium may be determined within all the populations from which the subject is originating.
  • the reference population to which a subject is originating or is the closest from genetic perspective there are many commercially available kits that can be used.
  • the AncestryDNA kit as commercialized by Ancestry corporate.
  • the subject is within one of the following superpopulation and the linkage disequilibrium is determined within the superpopulation of the subject:
  • SAS - South Asian
  • EAS East Asian
  • the subject is from one of the following population and the linkage disequilibrium is determined within the population of the subject:
  • Luhya in Webuye, Kenya, Luhya (LWK);
  • JPT Japan Japanese
  • CEPH - Utah residents
  • CEU Northern and Western European ancestry
  • IBS Spain Spanish
  • SNP allele(s) for use in the methods and kits of the invention in high LD with a squared correlation coefficient r 2 superior to 0.8 with some of the SNP alleles of the above Table 5, are described in the Examples below for the European superpopulation.
  • Such SNP alleles with their flanking 5' and 3' sequences are also given in SEQ ID NOs 11-92.
  • the inventors have identified specific SNP alleles that are associated to an increased risk of developing pneumonia, in particular, an increased risk of developing ventilator- associated pneumonia, including an increased risk of developing Pseudomonas aeruginosa VAP.
  • the method comprises determining the presence of one or more of the SNP allele(s) described in the following Table 3:
  • SNP8 14 50504728 c or its related SNP allele(s) in high linkage disequilibrium wherein detecting said one or more SNP allele(s) is associated to an increased risk of developing pneumonia in said subject, for example, an increased risk of developing ventilator-associated pneumonia.
  • SNP5 allele or its related SNP allele(s) in high linkage disequilibrium.
  • SNP5 is SNP rs686155 (SEQ ID NO:5), located at position 109907944 in chromosome 10, and is a SNP located in an intron of XPNPEP1 gene (130bp upstream of exon 3).
  • the inventors have also identified specific SNP alleles that are associated to a decreased risk of developing pneumonia, in particular, a decreased risk of developing ventilator- associated pneumonia, including a decreased risk of developing Pseudomonas aeruginosa VAP.
  • the method comprises determining the presence of one or more of SNP allele(s) described in the following Table 4:
  • SNP1 is SNP rs2077344 (SEQ ID NO: l), located at position 118942520 in chromosome 1 , and is a SNP located in an intron of MARCO gene (lOObp downstream of exon 1). Determining the presence or absence of a PNEUMONIA Risk Allele
  • PNEUMONIA Risk Allele may be determined by any appropriate SNP detection methods in the biological sample.
  • the method of the invention includes determining the presence of a PNEUMONIA Risk Allele by detecting a SNP allele at the nucleic acid level, for example, from genomic DNA extracted from a subject.
  • the presence of the PNEUMONIA Risk Allele is determined on either one or both chromosomes, wherein the identification of the PNEUMONIA Risk Allele (e.g. one or more specific SNPs as described above) on at least one chromosome indicates that the subject is at increased or decreased risk for developing pneumonia, for example at increased or decreased risk for developing serious pneumonia, such as serious VAP.
  • the identification of the PNEUMONIA Risk Allele e.g. one or more specific SNPs as described above
  • the method thus includes determining at least one SNP genotype (e.g. one or more of the SNPs described herein, in particular one or more of SNPs 1-10 described in the above Table 5 or their related SNPs in high linkage disequilibrium), such that the presence of the at least one PNEUMONIA Risk Allele determines genetic predisposition to pneumonia (increased or decreased risk of developing pneumonia) in the subject.
  • SNP genotype e.g. one or more of the SNPs described herein, in particular one or more of SNPs 1-10 described in the above Table 5 or their related SNPs in high linkage disequilibrium
  • the method includes determining whether the subject is homozygous for at least one PNEUMONIA Risk Allele or heterozygous for at least one PNEUMONIA Risk Allele and/or do not have such PNEUMONIA Risk Allele at a given locus.
  • the presence in the genome of a subject of two or more SNPs of interest is determined. For example, two or more SNPs described in the above Table 3 or their related SNP allele(s) in high linkage disequilibrium may be used to determine whether a subject is at increased risk of developing pneumonia. Alternatively, two or more of the SNPs described in the above Table 4 or their related SNP allele(s) in high linkage disequilibrium may be used to determine whether a subject is at decreased risk of developing pneumonia.
  • Methods for detecting the presence of a PNEUMONIA Risk Allele for example one or more of the SNPs described in Table 5 or their related SNP allele(s) in high linkage disequilibrium
  • a PNEUMONIA Risk Allele for example one or more of the SNPs described in Table 5 or their related SNP allele(s) in high linkage disequilibrium
  • Methods for detecting the presence of a PNEUMONIA Risk Allele for example one or more of the SNPs described in Table 5 or their related SNP allele(s) in high linkage disequilibrium
  • determining the presence of one or more PNEUMONIA Risk Allele(s) comprises the step of sequencing a genomic DNA fragment including a SNP of interest (i.e. potentially corresponding to a PNEUMONIA Risk Allele) and analysing the sequence for determining the presence or the absence of a PNEUMONIA Risk Allele.
  • Sequencing methods include without limitation, Sanger sequencing, next generation sequencing, pyrosequencing, sequencing by ligation. Prior to sequencing, amplification of said genomic fragment including the SNP of interest may be carried out from the genomic DNA of the biological sample.
  • determining the presence of one or more PNEUMONIA Risk Allele(s) comprises the step of amplifying a nucleic fragment susceptible of containing one of the PNEUMONIA Risk Allele(s) using PCR-based methods.
  • PCR-based methods useful for SNP detection include without limitation real-time PCR, quantification PCR, high-resolution melting analysis and amplification refractory mutation system PCR (ARMS-PCR).
  • the amplified nucleic acid fragment may be a fragment of at least 20, 30, 40, 50, 100, 200, 300 or at least 500 consecutive nucleotides, for example comprised between 20 and 1000 consecutive nucleotides, preferably between 30 and 200 consecutive nucleotides.
  • the one man skilled in the art will adapt the size of the fragments according to the method used for determining the presence of the SNPs.
  • Other available methods for SNPs genotyping may be used in the methods of the invention.
  • the traditional gel-based approach uses standard molecular techniques, such as amplification refractory mutation system (ARMS), restriction digests and various forms of gel electrophoresis (e.g., RFLP), denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP).
  • High throughput methods include allele discrimination methods (Allele-Specific Hybridization, Molecular Beacons, Allele- Specific Single-Base Primer Extension, 5 'nuclease), High-throughput assay chemistry (Flap endonuclease discrimination, Oligonucleotide ligation), microarray analysis of genomic DNA, pyrosequencing and light cycler.
  • DASH dynamic allele specific hybridization method
  • DASH dynamic allele specific hybridization method
  • DASH genotyping takes advantage of the differences in the melting temperature in DNA that results from the instability of mismatched base pairs. The process can be vastly automated and encompasses a few simple principles.
  • the target genomic segment is amplified and separated from non-target sequence, e.g., through use of a biotinylated primer and chromatography.
  • a probe that is specific for the particular allele is added to the amplification product.
  • the probe can be designed to hybridize specifically to the PNEUMONIA Risk Allele or alternative SNP alleles.
  • the probe can be either labeled with or added in the presence of a molecule that fluoresces when bound to double stranded-DNA.
  • the signal intensity is then measured as temperature is increased until the Tm can be determined.
  • a non-matching sequence (either PNEUMONIA Risk Allele or alternative SNP alleles, depending on probe design) will result in a lower than expected Tm.
  • Molecular beacons can also be used to detect the SNPs in the methods of the invention.
  • This method makes use of a specifically engineered single-stranded oligonucleotide probe.
  • the oligonucleotide is designed such that there are complementary regions at each end and a probe sequence located in between. This design allows the probe to take on a hairpin, or stem-loop, structure in its natural, isolated state. Attached to one end of the probe is a fluorophore and to the other end a fluorescence quencher. Because of the stem- loop structure of the probe, the fluorophore is in close proximity to the quencher, thus preventing the molecule from emitting any fluorescence.
  • the molecule is also engineered such that only the probe sequence is complementary to the genomic DNA that will be used in the assay. If the probe sequence of the molecular beacon encounters its target genomic DNA during the assay, it will anneal and hybridize. Because of the length of the probe sequence, the hairpin segment of the probe will be denatured in favor of forming a longer, more stable probe-target hybrid. This conformational change permits the fluorophore and quencher to be free of their tight proximity due to the hairpin association, allowing the molecule to fluoresce.
  • the probe sequence encounters a target sequence with as little as one non-complementary nucleotide, the molecular beacon will preferentially stay in its natural hairpin state and no fluorescence will be observed, as the fluorophore remains quenched.
  • a molecular beacon is designed to match a PNEUMONIA Risk Allele and another to match the alternative allele, the two can be used to identify the genotype of an individual. If only the first probe's fluorophore wavelength is detected during the assay then the individual is homozygous to the PNEUMONIA Risk Allele. If only the second probe's wavelength is detected then the individual is homozygous to the alternative allele. Finally, if both wavelengths are detected, then both molecular beacons must be hybridizing to their complements and thus the individual must contain both alleles and be heterozygous
  • a microarray can also be used to detect a SNP of interest. Hundreds of thousands of probes can be arrayed on a small chip, allowing for many SNPs to be interrogated simultaneously. Because SNP alleles only differ in one nucleotide and because it is difficult to achieve optimal hybridization conditions for all probes on the array, the target DNA has the potential to hybridize to mismatched probes. This is addressed somewhat by using several redundant probes to interrogate each SNP. Probes are designed to have the SNP site in several different locations as well as containing mismatches to the SNP allele. By comparing the differential amount of hybridization of the target DNA to each of these redundant probes, it is possible to determine specific homozygous and heterozygous alleles.
  • PCR and amplification based-methods can be used alternatively.
  • tetra-primer amplification refractory mutation system PCR or ARMS-PCR, employs two pairs of primers to amplify two alleles in one PCR reaction.
  • the primers are designed such that the two primer pairs overlap at a SNP location but each match perfectly to only one of the possible SNPs. As a result, if a given allele is present in the PCR reaction, the primer pair specific to that allele will produce product but not to the alternative allele with a different SNP.
  • the two primer pairs are also designed such that their PCR products are of a significantly different length allowing for easily distinguishable bands by gel electrophoresis or melt temperature analysis.
  • Primer extension is another possible method for detecting SNP of interest.
  • Primer extension first involves the hybridization of a probe to the bases immediately upstream of the SNP nucleotide followed by a 'mini-sequencing' reaction, in which DNA polymerase extends the hybridized primer by adding a base that is complementary to the SNP nucleotide. The incorporated base that is detected determines the presence or absence of the SNP allele.
  • Taqman 5 ' nuclease genotyping method may also be used.
  • oligonucleotide PCR primers are designed that flank the mutation in question and allow PCR amplification of the region.
  • a third oligonucleotide probe is then designed to hybridize to the region containing the base subject to change between different alleles of the gene.
  • This probe is labelled with fluorescent dyes at both the 5' and 3' ends. These dyes are chosen such that while in this proximity to each other the fluorescence of one of them is quenched by the other and cannot be detected.
  • Extension by Taq DNA polymerase from the PCR primer positioned 5' on the template relative to the probe leads to the cleavage of the dye attached to the 5' end of the annealed probe through the 5' nuclease activity of the Taq DNA polymerase. This removes the quenching effect allowing detection of the fluorescence from the dye at the 3' end of the probe.
  • the discrimination between different DNA sequences arises through the fact that if the hybridization of the probe to the template molecule is not complete (there is a mismatch of some form) the cleavage of the dye does not take place. Thus only if the nucleotide sequence of the oligonucleotide probe is completely complimentary to the template molecule to which it is bound will quenching be removed.
  • a reaction mix can contain two different probe sequences each designed against different alleles that might be present thus allowing the detection of both alleles in one reaction.
  • primers and probes which span one or more fragments that comprise the putative location of a PNEUMONIA Risk Allele (e.g specific SNP as described in the above paragraphs) may be used to detect said PNEUMONIA Risk Allele.
  • probe refers to one or more nucleic acid fragments whose specific hybridization to a sample can be detected.
  • a probe or primer can be of any length depending on the particular technique it will be used for.
  • Such probes or primers which may be used in the methods of the invention may typically be short nucleic acid molecules, for instance DNA oligonucleotides of 10 nucleotides or more in length, which can be annealed to the complementary target nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer or probe and the target nucleic acid strand.
  • the probe or primers can be unlabelled or labelled so that its binding to a target sequence can be detected (e.g. with a FRET donor or acceptor label).
  • a primer can be extended along the target nucleic acid molecule by a polymerase enzyme. Therefore, primers can be used to amplify the target nucleic acid molecule, such as fragments including any of the SNPs 1-10 described in the above Tables 1-5, and/or their related SNPs in high linkage disequilibrium, including any of the SNPs in SEQ ID NO: l 1- 92.
  • the specificity of a probe or a primer increases with its length. Thus, for example, a probe or primer that includes 30 consecutive nucleotides will anneal to a target sequence with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, to obtain greater specificity, probes and primers can be selected that include at least 15, 20, 25, 30,
  • a primer is at least 15 nucleotides in length, such as at least 15 contiguous nucleotides complementary to a target nucleic acid molecule.
  • Particular lengths of primers that can be used to practice the methods of the present disclosure include primers having at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least
  • nucleic acid molecule to be amplified such as a primer of 15-70 nucleotides, 15-60 nucleotides, 15-50 nucleotides, or 15-30 nucleotides.
  • An "upstream” or “forward” primer is a primer 5' to a reference point on a nucleic acid sequence.
  • a “downstream” or “reverse” primer is a primer 3' to a reference point on a nucleic acid sequence. In general, at least one forward and one reverse primer are included in an amplification reaction.
  • Nucleic acid probes and primers can be readily prepared based on the nucleic acid sequence flanking the SNPs of interest for use in the methods of the invention, and for example the genomic sequence including any of SEQ ID NO 1-92.
  • PCR primer pairs can be derived from a known sequence by using computer programs intended for that purpose such as Primer 3 (v. 0.4.0 Whitehead Institute for Biomedical Research, Steve Rozen, and Helen Skaletsky).
  • the SNPs of interest are detected by specific hybridization of nucleic acid probes, such as oligonucleotide probes to genomic DNA or RNA transcripts or corresponding cDNA, potentially containing the PNEUMONIA Risk Alleles, for example containing any one of the SNPs 1-10 or their related SNP allele(s) in high linkage disequilibrium as described in the above paragraphs, including any of the SNPs in SEQ ID NO 11-92.
  • nucleic acid probes such as oligonucleotide probes to genomic DNA or RNA transcripts or corresponding cDNA, potentially containing the PNEUMONIA Risk Alleles, for example containing any one of the SNPs 1-10 or their related SNP allele(s) in high linkage disequilibrium as described in the above paragraphs, including any of the SNPs in SEQ ID NO 11-92.
  • Such probes can also be immobilized on a solid surface (such as nitrocellulose, glass, quartz, fused silica slide)
  • probes and primers can be modified from the target sequence to a certain degree to produce probes that are "substantially identical” or “substantially complementary” to a target sequence, while retaining the ability to specifically bind to (i.e. hybridize specifically to) the same targets from which they are derived.
  • the percent complementary need not be 100% for hybridization or specific binding to occur, depending on the length of the polynucleotides, length of the complementary region and stringency of the conditions.
  • a primer or probe is at least 60%, 70%>, 80%), 90%o, 95%), 99%o or 100% complementary over the stretch of the complementary region. Assessing a risk in a subject and health care management
  • the invention further relates to patient stratification methods based on the risk assessment provided by the above-described methods.
  • a patient identified at increased risk of developing pneumonia may have adapted health care management in order to reduce the risk of developing pneumonia and for example, in order to reduce the risk of developing serious pneumonia, such as VAP.
  • the invention further includes a method comprising (i) identifying whether a patient is at increased risk of developing pneumonia according to the above defined methods, and,
  • the patient stratification method is particularly suitable to patients which are already known at risk of developing pneumonia based on their clinical profile.
  • a subject at risk in developing pneumonia may be a patient in need of surgical intervention, more specifically, in need of digestive, thoracic, cardiac or neuro surgery.
  • a subject at risk in developing pneumonia is a transplanted patient or in need of organ transplantation.
  • a subject at risk in developing pneumonia is a patient suffering from chronic inflammatory disorders.
  • a subject at risk in developing pneumonia is a patient who has had or is in need of having an immunosuppressant and/or anti-inflammatory treatment.
  • a subject at risk in developing pneumonia is a patient who is immunocompromised.
  • Such patients already known to be at risk of developing pneumonia will be assayed for the presence of one or more PNEUMONIA Risk Alleles in their genome, and an adapted health care management will be decided by the clinician accordingly.
  • such adapted health care management may include: daily interruption of sedation, daily weaning trials, head of the bed elevation, oral care, usage of appropriate endotracheal tubes, subglotting secretion drainage via continuous or intermittent suction, new devices to remove bio film from the inside of the endotracheal tube, saline instillation prior to suction, and early tracheostomy.
  • the method further comprises administering an adapted or preventive treatment of pneumonia to the subject, based on the presence or absence of one or more of PNEUMONIA Risk Allele(s).
  • patients with an increased risk of developing pneumonia based on the presence or absence of a PNEUMONIA Risk Allele may be treated with immunostimulating treatment, and/or prophylactic antibiotic treatment in order to reduce the risk of developing pneumonia, for example in order to reduce the risk of developing serious pneumonia such as serious VAP.
  • the invention further includes a method comprising (i) identifying whether a patient is at increased risk of developing pneumonia according to the above defined methods, and,
  • step (ii) treating said patient identified at step (i) with a suitable treatment for treating or preventing pneumonia.
  • treating refers to measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder or slow down or relieve one or more of the symptoms of the disorder.
  • Suitable immunostimulating treatment for preventing pneumonia including without limitation treatment with GM-CSF, IL7, IFNy, anti-PDl .
  • Kits may be prepared for performing the above described methods.
  • one object of the invention relates to a kit for assessing the risk of developing pneumonia in a subject (e.g. from a biological sample, more particularly a blood sample), according to the above described method, said kit comprising: (i) means for detecting one or more of the PNEUMONIA Risk Allele(s) as defined in the above table 1 or 2 or its related SNP allele(s) in high linkage disequilibrium, in particular related SNP allele(s) in high linkage disequilibrium as measured in a European superpopulation;
  • said means for detecting one or more of the PNEUMONIA Risk Allele(s) include at least a probe that binds specifically to a nucleic acid comprising one SNP allele of the SNPs 1-10 as defined in the Table 1 or 2 or its related SNP allele(s) in high linkage disequilibrium, and/or primers that are capable of specifically amplifying a nucleic acid comprising one of the SNPs 1-10 as defined in the Table 1 or 2 or its related SNP allele(s) in high linkage disequilibrium.
  • Said means for detecting PNEUMONIA Risk Allele may therefore comprise, specific primers or probes as described above.
  • the kit of the invention may comprise probes that binds specifically to any one of SEQ ID NOs 1 : 10 or their fragments including the SNP allele with corresponding 5' and 3' flanking regions of at least 5 nucleotides; or, to any one of SEQ ID NOs: 11-92 or their fragments including the SNP allele with corresponding 5 ' and 3 ' flanking regions of at least 5 nucleotides.
  • the kit of the invention may comprise primers that are capable of amplifying (i) a genomic DNA of the subject comprising any one of SEQ ID NOs 1 : 10 or their fragments including the SNP allele with corresponding 5' and 3' flanking regions of at least 5 nucleotides; or, (ii) a genomic DNA of the subject comprising any one of SEQ ID NOs: 11-92 or their fragments including the SNP allele with corresponding 5' and 3 ' flanking regions of at least 5 nucleotides.
  • the kit can include one or more isolated primer or primer pairs for amplifying a target nucleic acid containing a region comprising a SNP associated to increased or decreased risk of developing pneumonia as described above.
  • the kit can include primers for amplifying a haplotype including one, two, three, four, five SNPs among SNPs 1-10 as described above or their related SNP allele(s) in high linkage disequilibrium, wherein the amplified sequence includes the SNP associated with pneumonia.
  • the kit can further include one or more of a buffer solution, a conjugating solution for developing the signal of interest, or a detection reagent for detecting the signal of interest, each in separate packaging, such as a container.
  • the kit includes a plurality of size-associated marker target nucleic acid sequences for hybridization with a detection array.
  • the target nucleic acid sequences can include oligonucleotides such as DNA, RNA, and peptide-nucleic acid, or can include PCR fragments.
  • the kit can also include instructions in a tangible form, such as written instructions or in a computer-readable format.
  • said kit comprises
  • kits further comprises means for detecting other relevant genetic information, for example (i) means for detecting other genetic variants, such as SNPs or gene mutations known to be associated to an increased risk of developing respiratory disorders, including without limitation pneumonia, tuberculosis, chronic obstructive pulmonary disorders, and/or (ii) means for detecting genetic variants (e.g.
  • SNPs or gene mutations known to be associated to resistance to antibiotics, and/or (iii) means for detecting the presence of one or more pathogens including for example Pseudomonas spp, and especially Pseudomonas aeruginosa, Acinetobacter spp., Enterobacter spp., Staphylococcus Aureus, Streptococcus pneumoniae, Escherichia coli, Klebsiella spp., Haemophilus influenza, Moraxella catarrhalis, and Stenotrophomonas maltophilia pneumonia.
  • pathogens including for example Pseudomonas spp, and especially Pseudomonas aeruginosa, Acinetobacter spp., Enterobacter spp., Staphylococcus Aureus, Streptococcus pneumoniae, Escherichia coli, Klebsiella spp., Haemophilus influenza,
  • Figure 1 is a representation of cases and controls in the 2 first axes of Principal Component Analysis.
  • the white squares represent the case patient and the black dots represent the control.
  • Figure 2 is the Manhattan plot of corrected p-values. The line indicates significance threshold after correction
  • Example 1 Identification of SNPs associated to increased or decreased risk of developing ventilator-associated pneumonia
  • VAP Ventilator- Associated pneumonia
  • inclusion criteria included occurrence of at least 1 confirmed VAP due to Pa during the same ICU stay, plus other sign of aggravation, i.e. confirmed recurrences to Pa, or severe pulmonary complication among Acute Respiratory Distress Syndrom or septic shock. Cases were negative for germ other than Pa. Control group included patients that had no VAP due to any germ during mechanical ventilation (MV). Cases and controls were matched on values of age, SAPS2, duration of MV, reason for ICU admission, potential risk factors for VAP (coma, ARDS, head trauma) and outcome. Table 7: patient description
  • Exome Capture and Sequencing were targeted and captured using the Exome Capture kit Agilent SureSelect All Exon 50Mb. Extracted DNA was sequenced on an Illumina HiSeq 2000 (paired-end sequencing, 2x100 bp). Exome Capture and sequencing were performed by KnowMe® company.
  • Multiallelic SNPs (number of distinct alleles observed at least once in our study > 2) were removed. Variants located in repeated regions were excluded (from EnsEMBL, v81 using EnsEMBL perl API). Remaining variants were annotated with Ensembl Variant Effect Predictor Tool (VEP 3 ). Annotation includes: for protein coding variants : gene, codon change, amino acid change, functional consequence predicted by SIFT 4 and Polyphen 5 ; for all variants: rsID if available; description of the genomic context (UTR region, intronic region, intergenic region; regulatory region).
  • PCA was performed with EIGENSTAT smartpca 6 on individual genotype data to check for genetic background homogeneity between cases and controls. Association analyses were performed with a Fisher exact test comparing distribution of alleles in cases and controls with Plink 7 . Multiple Testing correction method FDR from Benjamini and Hochberg 8 ' 9 was applied. Corrected p-values were considered significant if lower than 0.05. Manhattan plot representation was used to assess for the presence of significant associated regions at genome-wide scale. When the difference between minor allele frequency in controls and the frequency reported in public databases (1000 Genomes) was > 0.5, variant was excluded. Variants located outside of the target of the exome capture (exons flanked by 200 bp intronic regions) were not further analyzed. Variants located in genome assembly exceptions were also excluded. Results
  • 10 SNPs remain statistically significantly associated (rs2398157, rs686155, rsl216159, rs7146431) or inversely associated (rs2077344, rs6735771, 5:94570535, rs6483579, rs6044883, rsl797041) with the condition of interest (raw p-values ⁇ 10 "6 ; corrected p-values between 0.00153 and 0.0481).
  • SNPs 6 are located into protein coding genes. None of them leads to an amino acid change, since all are intronic. However, they all are located to less than 140 bp from an exon. Exon flanking regions are known to be potential regulatory regions, as they may contain binding sites for regulation factors, such as splicing factors. The 4 others SNPs are respectively located close or in a pseudogene (RP11-16F15, RP11-461G12) and in unprocessed pseudogenes (OR4A11P, CSTP1).
  • pseudogene RP11-16F15, RP11-461G12
  • OR4A11P unprocessed pseudogenes
  • rs686155 XPNPEP1
  • rsl216159 OR4A11P
  • rs7146431 MA4K5
  • rAggr is a web-based software program for finding markers (SNPs and indels) that are in linkage-disequilibrium (LD) with a set of queried markers, using the 1000 Genomes Project and HapMap genotype databases.
  • rAggr uses an expectation-maximization algorithm adapted from the Haploview software (Barrett et al, Bioinformatics. 2005 Jan 15;21(2):263-5) to calculate pairwise r 2 and D'. All calculations are done "on the fly" by the web server.
  • the software was developed at the University of Southern California by Christopher K. Edlund, David V. Conti and David J. Van Den Berg. For more information on the developers and other software, visit the USC Morris Comprehensive Cancer Center Bioinformatics Core website. Copyright (c) 2015 Christopher K Edlund, David V Conti, David J Van Den Berg.
  • the targeted population is CEU (Utah residents with Northern and Western European ancestry)+FIN (Finnish in Finland)+GBR (British in England and Scotland)+IBS (Iberian populations in Spain)+TSI (Toscani in Italy), which corresponds to individual with European ascendance, enriched with specific European ethnies.
  • Query was performed using rs ID of SNPs of interest.
  • SNPs were considered if they were observed with a Minor Allele Frequency (MAF) higher than 0.001 in the reference population.
  • MAF Minor Allele Frequency
  • SNPs were considered in high linkage disequilibrium with SNPs of interest if located in less than 500kb, and if paired r 2 was comprised between 0.8 and 1.
  • SNP of interest SNP in high LP SEQ ID NOs SNPl (rs2077344) rs 11694929 11 rsl 1694102 12 rsl438838 13 rsl438839 14 rsl898703 15 rs4849735 16 rs55770063 17 rs4848527 18 rsl 114724 19 rs2119110 20 rsl0180048 21 rsl 318645 22

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne le domaine du diagnostic ou pronostic in vitro. L'invention concerne des procédés in vitro permettant de prédire le risque de développement d'une pneumonie chez un sujet. Plus spécifiquement, le procédé consiste à déterminer la présence d'au moins un allèle de risque de pneumonie dans un échantillon dudit sujet, la présence dudit ou desdits allèle(s) de risque de pneumonie indiquant si le sujet présente un risque accru ou réduit de développement de la pneumonie.
PCT/EP2017/050505 2016-01-12 2017-01-11 Procédé in vitro permettant de prédire un risque de développement d'une pneumonie chez un sujet WO2017121769A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP16305022.2 2016-01-12
EP16305022 2016-01-12

Publications (1)

Publication Number Publication Date
WO2017121769A1 true WO2017121769A1 (fr) 2017-07-20

Family

ID=55349767

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/050505 WO2017121769A1 (fr) 2016-01-12 2017-01-11 Procédé in vitro permettant de prédire un risque de développement d'une pneumonie chez un sujet

Country Status (1)

Country Link
WO (1) WO2017121769A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002029100A1 (fr) * 2000-09-29 2002-04-11 Genomics Collaborative, Inc. Procedes permettant de diagnostiquer une pneumonie communautaire et la predisposition ou la sensibilite a la pneumonie communautaire et au choc septique associe par la detection des polymorphismes geniques
WO2005085273A1 (fr) * 2004-03-04 2005-09-15 The University Of British Columbia Haplotypes de la thrombomoduline (thbd) permettant d'etablir un pronostic pour des patients
WO2014121180A1 (fr) * 2013-02-01 2014-08-07 The University Of Chicago Variantes génétiques chez des sujets atteints de maladie pulmonaire interstitielle
WO2014127290A2 (fr) * 2013-02-14 2014-08-21 The Regents Of The University Of Colorado Méthodes pour prédire le risque de pneumonie interstitielle

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002029100A1 (fr) * 2000-09-29 2002-04-11 Genomics Collaborative, Inc. Procedes permettant de diagnostiquer une pneumonie communautaire et la predisposition ou la sensibilite a la pneumonie communautaire et au choc septique associe par la detection des polymorphismes geniques
WO2005085273A1 (fr) * 2004-03-04 2005-09-15 The University Of British Columbia Haplotypes de la thrombomoduline (thbd) permettant d'etablir un pronostic pour des patients
WO2014121180A1 (fr) * 2013-02-01 2014-08-07 The University Of Chicago Variantes génétiques chez des sujets atteints de maladie pulmonaire interstitielle
WO2014127290A2 (fr) * 2013-02-14 2014-08-21 The Regents Of The University Of Colorado Méthodes pour prédire le risque de pneumonie interstitielle

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 19 August 2009 (2009-08-19), "Sequence 15914 from Patent WO2005056837.", XP002755569, retrieved from EBI accession no. EM_PAT:HB575442 Database accession no. HB575442 *
DATABASE Geneseq [online] 18 October 2007 (2007-10-18), "Human single nucleotide polymorphism (SNP) probe SEQ ID NO:171185.", XP002755568, retrieved from EBI accession no. GSN:AGF98693 Database accession no. AGF98693 *
FERNANDO A. RIVERA-CHÁVEZ ET AL: "A TREM-1 Polymorphism A/T within the Exon 2 Is Associated with Pneumonia in Burn-Injured Patients", ISRN INFLAMMATION, vol. 15, no. 1, 1 January 2013 (2013-01-01), pages 45 - 6, XP055258914, DOI: 10.1016/S1072-7515(00)00785-7 *

Similar Documents

Publication Publication Date Title
US10662474B2 (en) Identification of polymorphic sequences in mixtures of genomic DNA by whole genome sequencing
US10294529B2 (en) Microsatellite instability markers in detection of cancer
JP5902843B2 (ja) Ｉｇｆ２遺伝子の対立遺伝子特異的な発現を判定するための一塩基多型ならびに新規および公知の多型の組み合わせ
US20150159220A1 (en) Methods for predicting and detecting cancer risk
EP3460073A1 (fr) Matériels et méthode pour l'identification de vecteurs d'amyotrophie spinale
Hutchinson et al. Allele-specific methylation occurs at genetic variants associated with complex disease
Qu et al. Association of ADAM33 polymorphisms with childhood asthma in a northern Chinese population
Saarela et al. PRKCA and multiple sclerosis: association in two independent populations
US20130136720A1 (en) Methods of using fut2 genetic variants to diagnose crohn's disease
US20150292016A1 (en) Novel markers for mental disorders
US20140148344A1 (en) Association markers for beta thalassemia trait
Boonen et al. Two maternal duplications involving the CDKN1C gene are associated with contrasting growth phenotypes
Reboul et al. Mosaic maternal uniparental isodisomy for chromosome 7q21‐qter
WO2017121769A1 (fr) Procédé in vitro permettant de prédire un risque de développement d'une pneumonie chez un sujet
WO2015124080A1 (fr) Utilisation d'un point de variation du zfp28 dans la préparation d'un kit de diagnostic de la schizophrénie
EP2704800B1 (fr) Procédé de prédiction du développement d'une maladie médiée par le complément
US20140045717A1 (en) Single Nucleotide Polymorphism Biomarkers for Diagnosing Autism
US20190316199A1 (en) Test method for evaluating the risk of anti-thyroid drug-induced agranulocytosis, and evaluation kit
JP5416962B2 (ja) 薬剤による光線過敏症の発症の危険性の判定方法
EP2195448B1 (fr) Procédé de prédiction de la couleur de l'iris
US20130084564A1 (en) Assessment of cancer risk based on rnu2 cnv and interplay between rnu2 cnv and brca1
KR20160014083A (ko) Dna 복제수 변이를 이용한 강직성 척추염 발병 위험도 예측용 조성물 및 이를 이용한 예측 방법
KR20120124507A (ko) 만성 골수성 백혈병 위험도를 예측하는 방법 및 이를 이용한 진단키트
KR20150092937A (ko) 한국인의 고혈압 예측용 ｓｎｐ 마커
JP6082693B2 (ja) 喫煙感受性加齢黄斑変性易罹患性の判定方法及び判定キット

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17700925

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17700925

Country of ref document: EP

Kind code of ref document: A1