WO2014121180A1 - Variantes génétiques chez des sujets atteints de maladie pulmonaire interstitielle - Google Patents

Variantes génétiques chez des sujets atteints de maladie pulmonaire interstitielle Download PDF

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WO2014121180A1
WO2014121180A1 PCT/US2014/014395 US2014014395W WO2014121180A1 WO 2014121180 A1 WO2014121180 A1 WO 2014121180A1 US 2014014395 W US2014014395 W US 2014014395W WO 2014121180 A1 WO2014121180 A1 WO 2014121180A1
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genetic variant
lung disease
subject
interstitial lung
nucleic acid
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PCT/US2014/014395
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English (en)
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Imre Noth
Joe Garcia
Naftali Kaminski
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The University Of Chicago
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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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Idiopathic Pulmonary Fibrosis is a low prevalence, devastating disease of unknown etiology characterized by an interstitial fibrotic process and high mortality.
  • the course of disease is heterogeneous with a 2-5 year median survival from diagnosis.
  • lung transplantation remains the only successful treatment option, while immunosuppression regimens were recently demonstrated as harmful. Therefore, identifying genetic variants associated with susceptibility to IPF and alleles involved in the heterogeneity of disease course and mortality remains a major challenge.
  • SNP single nucleotide polymorphism
  • compositions and methods for identifying genetic variants in interstitial lung disease subjects are also provided. Also provided are compositions and methods of determining whether a human subject has, or is at risk of developing, an interstitial lung disease. In certain embodiments, the methods include detecting whether the genome of the subject comprises a genetic variant of at least one of TOLLIP, SPPL2C, and MDGA2, the presence of the genetic variant indicating that the subject has or is at risk of developing the interstitial lung disease. In certain embodiments, more than one genetic variant of TOLLIP and/or SPPL2C and/or MDGA2 is detected.
  • the method in addition to detecting genetic variants of TOLLIP and/or SPPL2C and/or MDGA2, the method includes detecting whether the genome of the subject includes other genetic variants diagnostic or predictive of risk for interstitial lung disease, e.g., a genetic variant of MUC5B, such as rs35705950.
  • a genetic variant of MUC5B such as rs35705950.
  • Fig. 2A is a flowchart showing the approach used in a three-stage association study
  • Fig. 2B is flowchart of mortality analyses by regression.
  • Fig. 3 QQ plot of the genome-wide association study (GWAS) of idiopathic pulmonary fibrosis (IPF).
  • GWAS genome-wide association study
  • IPF idiopathic pulmonary fibrosis
  • Fig. 4 includes regional association plots showing the IPF-associated regions in Ch11p15.5 (Fig. 4A) and Ch17q21.31 (Fig. 4B).
  • Fig. 5 survival probability over time for people with or without H2 and with or without an SPPL2C variant.
  • Fig. 6A is a KM plot for TOLLIP*/MUC5B risk alleles
  • Fig. 6B is KM plot by Risk Index for WPGS using all 3 genes (TOLLIP, SPPL2C & MUC5B) and categorizing into 4 groups.
  • Fig. 7A-7C is a list of top associated loci with susceptibility to IPF.
  • Fig. 8 is a table listing the sample sources and sizes used in a three stage study.
  • Fig. 9 shows the characteristics of IPF patients used in stage 1 discovery GWAS study.
  • Fig. 10 lists the characteristics of IPF patients by stage and availability.
  • Fig. 1 1 A-11 C is a list of 44 SNPs and their association p-values with susceptibility to IPF from stage 1 , stage 2, and overall.
  • Fig. 12 shows characteristics of IPF case series for mortality analysis.
  • Fig. 13 is a table showing association signals with susceptibility to IPF across stages of six SNPs followed up in Stage 3.
  • Fig. 14 is a table listing SNP effects on mortality.
  • Fig. 15 provides summaries of univariate Cox analysis for mortality.
  • Fig. 16 provides summaries of univariate and multivariate Cox analysis for mortality
  • Fig. 17 provides summaries of Kaplan-Meier survival analysis.
  • Fig. 18 lists predictors of survival in IPF patients identified using a univariant Cox model.
  • Fig. 19A-Fig. 19B lists predictors of survival in IPF patients identified using a multivariate analysis of covariance.
  • Fig. 20 lists 30 regions identified showing the value of aggregation and using information in addition to protein coding SNPs, with the six p values represent highest-ranking SNPs in each region in bold.
  • GWAS genome wide association study
  • the results obtained identified three genetic loci and replicated the association of four novel SNPs (rs11 1521887, rs5743894, rs5743890, and rs17690703) in two novel loci (ch11 p15.5/TOLL/P and ch17q21.3MSPPL2C), and the MUC5B promoter SNP (rs35705950) with IPF susceptibility in European- Americans through a three-stage case-control study.
  • the findings reported herein provide, inter alia, for novel compositions and methods for identifying genetic variants in interstitial lung disease subjects and/or determining whether an individual has, or is at risk for developing, interstitial lung disease and/or compositions and methods for predicting prognosis, e.g., survival time or mortality, of an individual with an interstitial lung disease, for example, a fibrotic interstitial lung disease, such as IPF, or familial interstitial pneumonia.
  • a fibrotic interstitial lung disease such as IPF, or familial interstitial pneumonia.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • Nucleic acid or oligonucleotide or polynucleotide or grammatical equivalents used herein means at least two nucleotides covalently linked together. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 0, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length.
  • Nucleic acids and polynucleotides are a polymers of any length, including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc.
  • the term "nucleotide” typically refers to a single unit of a polynucleotide, i.e., a monomer.
  • Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof.
  • a “genetic variant” refers to a mutation, single nucleotide polymorphism (SNP), deletion variant, missense variant, insertion variant, inversion, or copy number variant.
  • 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.
  • PCR primers are generally between 10 and 40 nucleotides in length, while nucleic acid probes for, e.g., a Southern blot, can be more than a hundred nucleotides in length.
  • the probe or primers can be unlabeled or labeled as described below so that its binding to a target sequence can be detected (e.g., with a FRET donor or acceptor label).
  • the probe or primer can be designed based on one or more particular (preselected) portions of a chromosome, e.g., one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products.
  • PCR polymerase chain reaction
  • the length and complexity of the nucleic acid fixed onto the target element is not critical to the invention. One of skill can adjust these factors to provide optimum hybridization and signal production for a given hybridization and detection procedures, and to provide the required resolution among different genes or genomic locations.
  • Probes and primers can also be immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as in an array.
  • a solid surface e.g., nitrocellulose, glass, quartz, fused silica slides
  • Techniques for producing high density arrays can also be used for this purpose (see, e.g., Fodor (1991) Science 767-773; Johnston (1998) Curr. Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; U.S. Patent No. 5,143,854).
  • 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, but retain the ability to specifically bind to (i.e., hybridize specifically to) the same targets from which they were derived.
  • a probe or primer is "capable of detecting" a genetic variant if it is complementary to a region that covers or is adjacent to the genetic variant.
  • primers can be designed on either side of the SNP, and primer extension used to determine the identity of the nucleotide at the position of the SNP.
  • FRET-labeled primers are used (at least one labeled with a FRET donor and at least one labeled with a FRET acceptor) so that FRET signal will be detected only upon hybridization of both primers.
  • a probe is used in conditions such that it hybridizes only to a genetic variant, or only to a dominant sequence.
  • the probe can be designed to hybridize to a junction point of a genetice inversion, but not to a sequence that does not include the inversion.
  • the term “capable of hybridizing to” refers to a polynucleotide sequence that forms non-covalent, Watson-Crick bonds with a complementary sequence.
  • percent complementarity need not be 100% for hybridization to occur, depending on the length of the polynucleotides, length of the complementary region, and stringency of the conditions.
  • a polynucleotide e.g., primer or probe
  • a polynucleotide can be capable of hybrindizing (binding) to a polynucleotide having 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity over the stretch of the complementary region.
  • Stringency can be increased by reducing the length of the complementary region, reducing the G-C content of the complementary region, increasing temperature and/or detergent levels, varying salt levels and pH, etc. as known in the art.
  • a polynucleotide is capable of hybridizing to a complementary sequence in standard PCR annealing conditions. In the context of detecting genetic variants, the tolerated percent complementarity or number of mismatches will vary depending on the technique used for detection (see below).
  • amplification product refers to a polynucleotide that results from an amplification reaction, e.g., PCR and variations thereof, rtPCR, strand displacement reaction (SDR), ligase chain reaction (LCR), transcription mediated amplification (TMA), or Qbeta replication.
  • a thermally stable polymerase e.g., Taq, can be used to avoid repeated addition of polymerase throughout amplification procedures that involve cyclic or extreme temperatures (e.g., PCR and its variants).
  • label refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include fluorescent dyes, luminescent agents, radioisotopes (e.g., 32 P, 3 H), electron- dense reagents, enzymes, biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by affinity. Any method known in the art for conjugating a nucleic acid or other biomolecule to a label may be employed, e.g., using methods described in Hermanson, Bioconiuqate Techniques 1996, Academic Press, Inc., San Diego.
  • tag can be used synonymously with the term “label,” but generally refers to an affinity-based moiety, e.g., a "His tag” for purification, or a “strepavidin tag” that interacts with biotin.
  • a "labeled" molecule e.g., nucleic acid, protein, or antibody
  • FRET F5rster resonance energy transfer
  • FRET donor donor chromophore
  • FRET acceptor acceptor chromophore
  • a "FRET signal” is thus the signal that is generated by the emission of light from the acceptor.
  • R 0 is about 50-60 A for some commonly used dye pairs (e.g., Cy3-Cy5).
  • FRET signal varies as the distance to the 6 th power. If the donor-acceptor pair is positioned around R 0 , a small change in distance ranging from 1 A to 50 A can be measured with the greatest signal to noise. With current technology, 1 ms or faster parallel imaging of many single FRET pairs is achievable.
  • FRET pair refers to a FRET donor and FRET acceptor pair that are capable of FRET detection.
  • fluorophore fluorophore
  • die fluorescent molecule
  • fluorescent dye fluorescent dye
  • FRET dye and like terms are used synonymously herein unless otherwise indicated.
  • Subject “patient,” “individual” and like terms are used interchangeably and refer to, except where indicated, humans and non-human animals. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision.
  • a patient can be an individual that is seeking diagnosis, treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc.
  • sample refers to a biological sample obtained from a subject. Samples include material that is processed prior to carrying out testing, e.g., genomic DNA separated or purified from other cellular and non-cellular debris.
  • the sample includes genomic DNA from the subject, e.g., cheek swab, blood sample, mucosal sample, buccal swab, skin sample, hair, etc.
  • a "control" sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample.
  • a test sample can be taken from a test condition, e.g., a sample from an individual of unknown disease status, and compared to samples from individuals with known conditions, e.g., healthy, or lacking a given genetic variation (negative control), or pulmonary disease or having a given genetic variation (positive control).
  • a control can also represent an average value gathered from a number of tests or results.
  • controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare signal strength in given conditions, e.g., in the presence of a test probe, or primer.
  • Controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.
  • compositions and methods for determining whether a human subject has or is at risk of developing an interstitial lung disease and/or prognosing interstitial lung disease may be used in conjunction with any other diagnostic or prognostic criterion or method, including, but not limited to, currently known criterion or methods.
  • the method for determining whether a human subject has or is at risk of developing an interstitial lung disease includes detecting whether the genome of the subject comprises a genetic variant of at least one of TOLLIP, SPPL2C, and MDGA2, the presence of the genetic variant indicating that the subject has or is at risk of developing the interstitial lung disease.
  • more than one genetic variant of TOLLIP and/or SPPL2C and/or MDGA2 is detected.
  • the method in addition to detecting genetic variants of TOLLIP and/or SPPL2C and/or DGA2, the method includes detecting whether the genome of the subject includes other genetic variants diagnostic or predictive of risk for interstitial lung disease, e.g., a genetic variant of MUC5B, such as rs35705950.
  • the method for determining whether a human subject has or is at risk of developing an interstitial lung disease includes detecting the presence or absence of one or more SNPs selected from rs11 1521887, rs5743894, rs5743890, rs17690703, and rs7144383.
  • the presence or absence of each SNP may be detected alone or in combination with each other, i.e., the methods of the invention may include detection of one, two, three, four, or five of rs11 1521887, rs5743894, rs5743890, rs17690703, and rs7144383 in any possible combination.
  • the method includes detecting the presence or absence of from one to five of rs1 11521887, rs5743894, rs5743890, rs17690703, and rs7144383 in any combination and the presence or absence of any other SNP associated with an interstitial lung disease or its prognosis, including, without limitation, the MUC5B SNP rs35705950.
  • the method for determining whether a human subject has or is at risk of developing an interstitial lung disease includes detecting the presence of rs1 11521887 ⁇ e.g., G or other non-dominant allele). In some embodiments, the method for determining whether a human subject has or is at risk of developing an interstitial lung disease includes detecting the presence of rs5743894 (e.g., G or other non-dominant allele). In some embodiments, the method for determining whether a human subject has or is at risk of developing an interstitial lung disease includes detecting the presence of rs5743890 (e.g., G or other non-dominant allele).
  • the method for determining whether a human subject has or is at risk of developing an interstitial lung disease includes detecting the presence of rs17690703 (e.g., T or other non-dominant allele). In some embodiments, the method for determining whether a human subject has or is at risk of developing an interstitial lung disease includes detecting the presence of rs7144383 (e.g., G or other non-dominant allele).
  • the method for determining whether a human subject has or is at risk of developing an interstitial lung disease includes detecting one or more genetic variants listed in Fig. 7.
  • the one or more genetic variants may be detected alone or in any possible combination of from two to 52 of the listed genetic variants. If the method includes detecting rs35705950, then the method includes detecting at least one additional genetic variant from the remaining 51 genetic variants listed in Fig. 7.
  • the method includes prognosing an interstitial lung disease in a human subject.
  • the method comprises detecting whether the genome of the subject comprises a genetic variant of TOLLIP and/or SPPL2C prognostic of increased or decreased survival.
  • the methods include detecting whether the genome of the subject comprises a genetic variant of MUC5B and whether the genome comprises a genetic variant of a genetic variant of TOLLIP and/or SPPL2C prognostic of increased or decreased survival.
  • the method includes detecting whether the genome comprises rs17690703 and/or rs5743890, each of which is predictive of decreased survival.
  • the method detects whether the genome comprises rs35705950, which is predictive of increased survival, and rs17690703 and/or rs5743890. In some embodiments, the method comprises detecting rs17690703 (e.g., T or other non-dominant allele), and prognosing reduced survival time for the subject, In some embodiments, the method comprises detecting rs5743890 (e.g., G or other non-dominant allele), and prognosing reduced survival time for the subject.
  • rs17690703 e.g., T or other non-dominant allele
  • rs5743890 e.g., G or other non-dominant allele
  • the method for prognosing the interstitial lung disease in a human subject includes detecting one or more genetic variants listed in Fig. 7.
  • the one or more genetic variants may be detected alone or in any possible combination of from two to 52 of the listed genetic variants. If the method includes detecting rs35705950, then the method includes detecting at least one additional genetic variant from the remaining 51 genetic variants listed in Fig. 7.
  • the present invention provides methods for detecting the presence or absence of at least one genetic variant in a human subject. In certain embodiments, the method includes detecting the presence or absence of at least one genetic variant of at least one of TOLLIP, SPPL2C, and MDGA2 in a sample from the subject.
  • more than one genetic variant of TOLLIP and/or SPPL2C and/or MDGA2 is detected.
  • the method in addition to detecting genetic variants of TOLLIP and/or SPPL2C and/or MDGA2, includes detecting a genetic variant of MUC5B, such as rs355950.
  • the method for detecting the presence or absence of at least one genetic variant in a human subject includes detecting the presence or absence of at least one genetic variant of the genetic variants listed in Fig. 7.
  • the one or more genetic variants may be detected alone or in any possible combination of from two to 52 of the genetic variants listed in Fig. 7. If the method includes detecting rs35705950, then the method includes detecting at least one additional genetic variant from the remaining 51 genetic variants listed in Fig. 7.
  • the at least one genetic variant includes one or more of a single nucleotide polymorphism selected from the group consisting of rs111521887, rs5743894, rs5743890, rs17690703, and rs7144383 in any possible combination.
  • the method for detecting the presence or absence of at least one genetic variant in a human subject includes detecting the presence or absence of heterozygosity in least one genetic variant of the genetic variants listed in Fig. 7.
  • the method for detecting the presence or absence of at least one genetic variant in a human subject includes detecting the presence or absence of homozygosity in least one genetic variant of the genetic variants listed in Fig. 7.
  • the heterozygosity or homozygosity of the one or more genetic variants may be detected alone or in any possible combination of from two to 52 of the genetic variants listed in Fig. 7, wherein the genetic variant may be the same or different in the individual chromosomes present in the diploid human subject.
  • the method includes detecting heterozygosity or homozygosity of rs35705950, then the method includes detecting heterozygosity or homozygosity of at least one additional genetic variant from the remaining 51 genetic variants listed in Fig. 7.
  • the heterozygosity or homozygosity of at least one genetic variant includes the heterozygosity or homozygosity of one or more of a single nucleotide polymorphism selected from the group consisting of rs111521887, rs5743894, rs5743890, rs17690703, and rs7144383 in any possible combination.
  • a method for testing for interstitial lung disease in a human subject involves detecting the level of TOLLIP gene expression in a sample from the subject, a low level of TOLLIP gene expression relative to a control being indicative of interstitial lung disease.
  • the level of gene expression may be detected by measuring, directly or indirectly, TOLLIP mRNA or by measuring Tollip protein by any suitable method, several of which are known in the art.
  • the control may include, for example, a sample from a human that does not have interstitial lung disease or a value or set of values, for example, a normal range, derived from several humans that do not have interstitial lung disease.
  • a low level of TOLLIP gene expression relative to a control (standard control) indicative of interstitial lung disease is a level that is less than about 50% of the control.
  • the present invention includes a method of treating a human subject having an interstitial lung disease comprising detecting the level of TOLLIP expression in a sample from the subject, and if the subject has a low level of TOLLIP expression relative to a control (standard control), administering to the subject an amount of a Tollip agonist, Tollip or a genetic construct expressing TOLLIP effective to treat the interstitial lung disease.
  • An amount effective to treat the interstitial lung disease is an amount effective to delay onset, reduce frequency and/or severity of one or more symptoms, ameliorate one or more symptoms, and/or improve comfort and/or some function of the subject, e.g., respiratory function, relative to an untreated second subject or pool of subjects, or relative to, or to the same subject prior to treatment, or after cessation of treatment.
  • the methods of the invention are not limited to any particular way of detecting the presence or absence of a genetic variant (e.g. SNP) and can employ any suitable method to detect the presence or absence of a variant(s), of which numerous detection methods are known in the art.
  • a genetic variant e.g. SNP
  • any suitable method to detect the presence or absence of a variant(s) of which numerous detection methods are known in the art.
  • DASH Dynamic allele-specific hybridization
  • 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 a variant sequence or to the dominant allelic sequence.
  • 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 genetic variant or dominant allelic sequence, depending on probe design), will result in a lower than expected Tm.
  • DASH genotyping relies on a quantifiable change in Tm, and is thus capable of measuring many types of mutations, not just SNPs.
  • Other benefits of DASH include its ability to work with label free probes and its simple design and performance conditions.
  • Molecular beacons can also be used to detect a genetic variant.
  • 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 targeted genomic DNA sequence.
  • the probe sequence of the molecular beacon encounters its target genomic DNA sequence 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 molecular beacon will preferentially stay in its natural hairpin state and no fluorescence will be observed, as the fluorophore remains quenched.
  • the unique design of these molecular beacons allows for a simple diagnostic assay to identify SNPs at a given location. If a molecular beacon is designed to match a wild-type allele and another to match a mutant of the 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 wild type.
  • a microarray can also be used to detect genetic variants. Hundreds of thousands of probes can be arrayed on a small chip, allowing for many genetic variants or 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 can be addressed by using several redundant probes to interrogate each SNP. Probes can be 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.
  • Restriction fragment length polymorphism can be used to detect genetic variants and SNPs.
  • RFLP makes use of the many different restriction endonucleases and their high affinity to unique and specific restriction sites. By performing a digestion on a genomic sample and determining fragment lengths through a gel assay it is possible to ascertain whether or not the enzymes cut the expected restriction sites. A failure to cut the genomic sample results in an identifiably larger than expected fragment implying that there is a mutation at the point of the restriction site which is rendering it protected from nuclease activity.
  • PCR- and amplification-based methods can be used to detect genetic variants.
  • tetra-primer 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 matches perfectly to only one of the possible alleles.
  • the two primer pairs can be designed such that their PCR products are of a significantly different length allowing for easily distinguishable bands by gel electrophoresis, or such that they are differently labeled.
  • Primer extension can also be used to detect genetic variants.
  • 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. Because primer extension is based on the highly accurate DNA polymerase enzyme, the method is generally very reliable. Primer extension is able to genotype most SNPs under very similar reaction conditions making it also highly flexible. The primer extension method is used in a number of assay formats, and can be detected using e.g., fluorescent labels or mass spectrometry.
  • Primer extension can involve incorporation of either fluorescently labeled ddNTP or fluorescently labeled deoxynucleotides (dNTP).
  • ddNTPs probes hybridize to the target DNA immediately upstream of SNP nucleotide, and a single, ddNTP complementary to the SNP allele is added to the 3' end of the probe (the missing 3'-hydroxyl in didioxynucleotide prevents further nucleotides from being added).
  • Each ddNTP is labeled .with a different fluorescent signal allowing for the detection of all four alleles in the same reaction.
  • allele-specific probes have 3' bases which are complementary to each of the SNP alleles being interrogated.
  • the target DNA contains an allele complementary to the 3' base of the probe, the target DNA will completely hybridize to the probe, allowing DNA polymerase to extend from the 3' end of the probe. This is detected by the incorporation of the fluorescently labeled dNTPs onto the end of the probe. If the target DNA does not contain an allele complementary to the probe's 3' base, the target DNA will produce a mismatch at the 3' end of the probe and DNA polymerase will not be able to extend from the 3' end of the probe.
  • the iPLEX® SNP genotyping method takes a slightly different approach, and relies on detection by mass spectrometer. Extension probes are designed in such a way that many different SNP assays can be amplified and analyzed in a PCR cocktail.
  • the extension reaction uses ddNTPs as above, but the detection of the SNP allele is dependent on the actual mass of the extension product and not on a fluorescent molecule. This method is for low to medium high throughput, and is not intended for whole genome scanning.
  • Primer extension methods are, however, amenable to high throughput analysis. Primer extension probes can be arrayed on slides allowing for many SNPs to be genotyped at once. Broadly referred to as arrayed primer extension (APEX), this technology has several benefits over methods based on differential hybridization of probes. Comparatively, APEX methods have greater discriminating power than methods using differential hybridization, as it is often impossible to obtain the optimal hybridization conditions for the thousands of probes on DNA microarrays (usually this is addressed by having highly redundant probes).
  • Oligonucleotide ligation assays can also be used to detect genetic variants.
  • DNA ligase catalyzes the ligation of the 3' end of a DNA fragment to the 5' end of a directly adjacent DNA fragment. This mechanism can be used to interrogate a SNP by hybridizing two probes directly over the SNP polymorphic site, whereby ligation can occur if the probes are identical to the target DNA.
  • two probes can be designed; an allele-specific probe which hybridizes to the target DNA so that its 3' base is situated directly over the SNP nucleotide and a second probe that hybridizes the template upstream (downstream in the complementary strand) of the SNP polymorphic site providing a 5' end for the ligation reaction. If the allele-specific probe matches the target DNA, it will fully hybridize to the target DNA and ligation can occur. Ligation does not generally occur in the presence of a mismatched 3' base. Ligated or unligated products can be detected by gel electrophoresis, MALDI- TOF mass spectrometry or by capillary electrophoresis.
  • the 5'-nuclease activity of Taq DNA polymerase can be used for detecting genetic variants.
  • the assay is performed concurrently with a PCR reaction and the results can be read in real-time.
  • the assay requires forward and reverse PCR primers that will amplify a region that includes the SNP polymorphic site. Allele discrimination is achieved using FRET, and one or two allele-specific probes that hybridize to the SNP polymorphic site.
  • the probes have a fluorophore linked to their 5' end and a quencher molecule linked to their 3' end. While the probe is intact, the quencher will remain in close proximity to the fluorophore, eliminating the fluorophore's signal .
  • the allele-specific probe if the allele-specific probe is perfectly complementary to the SNP allele, it will bind to the target DNA strand and then get degraded by 5'-nuclease activity of the Taq polymerase as it extends the DNA from the PCR primers. The degradation of the probe results in the separation of the fluorophore from the quencher molecule, generating a detectable signal. If the allele-specific probe is not perfectly complementary, it will have lower melting temperature and not bind as efficiently. This prevents the nuclease from acting on the probe.
  • Fluorescence resonance energy transfer (FRET) detection can be used for detection in primer extension and ligation reactions where the two labels are brought into close proximity to each other. It can also be used in the 5'-nuclease reaction, the molecular beacon reaction, and the invasive cleavage reactions where the neighboring donor/acceptor pair is separated by cleavage or disruption of the stem- loop structure that holds them together. FRET occurs when two conditions are met. First, the emission spectrum of the fluorescent donor dye must overlap with the excitation wavelength of the acceptor dye. Second, the two dyes must be in close proximity to each other because energy transfer drops off quickly with distance. The proximity requirement is what makes FRET a good detection method for a number of allelic discrimination mechanisms.
  • a variety of dyes can be used for FRET, and are known in the art. The most common ones are fluorescein, cyanine dyes (Cy3 to Cy7), rhodamine dyes (e.g. rhodamine 6G), the Alexa series of dyes (Alexa 405 to Alexa 730). Some of these dyes have been used in FRET networks (with multiple donors and acceptors). Optics for imaging all of these require detection from UV to near IR (e.g. Alex 405 to Cy7), and the Atto series of dyes (Atto-Tec GmbH). The Alexa series of dyes from Invitrogen cover the whole spectral range. They are very bright and photostable.
  • Example dye pairs for FRET labeling include Alexa-405/Alex-488, Alexa- 488/Alexa-546, Alexa-532/Alexa-594, Alexa-594/Alexa-680, Alexa-594/Alexa-700, Alexa-700/Alexa-790, Cy3/Cy5, Cy3.5/Cy5.5, and Rhodamine-Green/Rhodamine- Red, etc.
  • Fluorescent metal nanoparticles such as silver and gold nanoclusters can also be used (Richards ei al. (2008) J Am Chem Soc 130:5038-39; Vosch et al.
  • the present invention provides a kit for predicting, diagnosing, or prognosing interstitial lung disease in a human subject, the kit including (e.g. consisting essentially of) at least one probe or primer for detecting the presence or absence of at least one genetic variation.
  • the at least one genetic variation includes a genetic variant of at least one of TOLLIP, SPPL2C, and MDGA2.
  • the kit includes at least one primer or probe for detecting more than one genetic variant of TOLLIP and/or SPPL2C and/or MDGA2.
  • the kit includes at least one probe or primer for detecting additional genetic variants diagnostic or predictive of risk for interstitial lung disease, e.g., a genetic variant of MUC5B, such as rs37055950.
  • the kit includes a probe or primer for detecting one or more SNPs selected from rs11 1521887, rs5743894, rs5743890, rs17690703, and rs7144383.
  • the kit may include probes or primers for detecting rs1 11521887, rs5743894, rs5743890, rs17690703, and rs7144383 alone or in any combination.
  • the kit may include additional primers or probes for detecting the presence of detecting the presence or absence of rs37055950 and rs1 11521887, rs5743894, rs5743890, rs17690703, or rs7144383 in any combination.
  • the kit includes at least one probe or primer includes at least one probe or primer for detecting one or more of the genetic variants listed in Fig. 7.
  • the kit may include probes or primers for detecting the one or more genetic variants listed in Fig. 7 alone or in any possible combination of from two to 52 of the listed genetic variants. If the kit includes a probe or primer for detecting rs35705950, the kit also includes a probe or primer for detecting at least one additional genetic variant from the remaining 51 genetic variants listed in Fig. 7.
  • kits for predicting, diagnosing, or prognosing interstitial lung disease in a human subject “consisting essentially of” certain types of probes or primers is intended to capture kits that include probes or primers that are suitable primarily for detecting genetic variants associated with interstitial lung disease in humans, although the kits may also include additional probes or primers used as controls, for example, probes or primers for detecting housekeeping genes such ⁇ - actin, tubulin, or glyceraldehyde-3-phosphate dehydrogenase, for example.
  • the use of the transitional phrase "consisting essentially of” is intended to exclude arrays containing thousands of probes, the vast majority of which are unrelated to interstitial lung disease.
  • the kits may include buffers, enzymes, labels, and the like, for example, for use in isolating DNA or mRNA, generating cDNA, or for amplifying and/or detecting and/or sequencing specific SNPs.
  • the kit includes (or consists essentially of) a nucleic acid primer capable of hybridizing to a genetic variant in the TOLLIP gene (e.g., a TOLLIP nucleic acid), SPPL2C gene (e.g., a SPPL2C nucleic acid), or MDGA2 gene (e.g., MDGA2 nucleic acid).
  • the genetic variant has been extracted from a human subject with an interstitial lung disease, or suspected of having an interstitial lung disease.
  • the genetic variant is an amplification product of DNA extracted from a human subject with an interstitial lung disease, or suspected of having an interstitial lung disease.
  • the interstitial lung disease is a pulmonary fibrotic condition.
  • the kit includes a first nucleic acid probe (e.g. , a labeled probe) capable of hybridizing to an amplification product of a genetic variant in the TOLLIP gene (e.g., a TOLLIP nucleic acid), SPPL2C gene (e.g., a SPPL2C nucleic acid), or MDGA2 gene (e.g. , MDGA2 nucleic acid).
  • a first nucleic acid probe e.g. , a labeled probe
  • a first nucleic acid probe capable of hybridizing to an amplification product of a genetic variant in the TOLLIP gene (e.g., a TOLLIP nucleic acid), SPPL2C gene (e.g., a SPPL2C nucleic acid), or MDGA2 gene (e.g. , MDGA2 nucleic acid).
  • the kit includes a second nucleic acid probe capable of hybridizing to an amplification product of a genetic variant in the TOLLIP gene (e.g., a TOLLIP nucleic acid), SPPL2C gene (e.g., a SPPL2C nucleic acid), or MDGA2 gene (e.g., MDGA2 nucleic acid).
  • the second nucleic acid probe is capable of hybridizing to a different sequence than the first probe.
  • only one of the nucleic acid probes hybridizes to the variant nucleotide(s) (e.g., in the case of a SNP), while the other nucleic acid probe hybridizes to a nearby sequence.
  • the second probe is labeled, e.g., with a different label than the first probe.
  • the first nucleic acid probe is labeled with a first label
  • the second nucleic acid probe is labeled with a second label, wherein the first and second label form a FRET pair (are capable of fluorescence resonance energy transfer) when hybridized to the genetic variant TOLLIP gene (e.g., a TOLLIP nucleic acid), SPPL2C gene (e.g., a SPPL2C nucleic acid), or MDGA2 gene (e.g. , MDGA2 nucleic acid), or amplification product thereof.
  • TOLLIP gene e.g., a TOLLIP nucleic acid
  • SPPL2C gene e.g., a SPPL2C nucleic acid
  • MDGA2 gene e.g. , MDGA2 nucleic acid
  • the kit includes (or consists essentially of) primers or at least one probe capable of detecting a genetic variant, e.g., as described above, depending on the detection method selected.
  • the kit includes primers or at least one probe capable of detecting a genetic variant in a region selected from the group consisting of 11p15.5, 14q21.3, and 17q21.31.
  • the kit includes primers or at least one probe capable of detecting at least one genetic variant in 11p15.5 (e.g., rs111521887, rs5743894, rs5743890, and rs35705950).
  • the kit includes primers or probes capable of detecting more than one (e.g., 2, 3, 4, 5, 5-10, 10-20, or more) genetic variant in 11p15.5 and 14q21.3 (e.g., rs7144383). In some embodiments, the kit includes primers or probes capable of detecting more than one (e.g., 2, 3, 4, 5, 5-10, 10-20, or more) genetic variant in 11 p15.5 and 17q21.31 (e.g., rs17690703, a genetic inversion, or copy number variation). In some embodiments, the kit includes primers or probes capable of detecting more than one (e.g., 2, 3, 4, 5, or more) genetic variant in 14q21.3 and 17q21.31. In some embodiments, the kit includes primers or probes capable of detecting more than one (e.g., 2, 3, 4, 5, 5-10, 10-20, or more) genetic variant in 11p15.5, 14q21.3, and 17q21.31.
  • the primers and/or probes are labeled, e.g., with fluorescent labels or FRET labels. In some embodiments, the primers and/or probes are unlabeled. In some embodiments, the kit includes primers and/or probes that detect both a variant allelic sequence and the dominant allelic sequence at a selected genetic variant site, e.g., with different labels, or designed to generate amplification or primer extension products with different masses.
  • the kit further includes at least one control sample, e.g., sample(s) with dominant allele(s) at the selected genetic variation site(s), or sample(s) with variant allele(s) at the selected genetic variation site(s).
  • the kit includes a polymerase.
  • nucleic acid complexes e.g., formed in in vitro assays to indicate the presence of a genetic variant sequence.
  • a nucleic acid complex can also be formed to detect the presence of a dominant allelic sequence, depending on the design of the probe or primer, e.g., in assays to distinguish homozygous and heterozygous subjects.
  • the complex comprises a first nucleic acid hybridized to a genetic variant nucleic acid, wherein the genetic variant nucleic acid is a genetic variant in a region selected from 11 p15.5, 14q21.3, and 17q21.31.
  • the genetic variant nucleic acid is an amplification product.
  • the genetic variant nucleic acid is on genomic DNA, e.g., from a subject that has or is suspected of having an interstitial lung disease.
  • the first nucleic acid is an amplification product or a primer extension product.
  • the first nucleic acid is labeled.
  • the nucleic acid complex further comprises a second nucleic acid hybridized to the genetic variant nucleic acid.
  • the second nucleic acid is labeled e.g., with a FRET or other fluorescent label.
  • the first and second nucleic acids form a FRET pair when hybridized to a genetic variant sequence.
  • the genetic variant is in the TOLLIP gene (e.g., rs1 11521887, rs5743894, rs5743890). In some embodiments, the genetic variant is in the MDGA2 gene (e.g., rs7144383). In some embodiments, the genetic variant is in the SPPL2C gene (e.g., rs17690703, a genetic inversion, or copy number variation).
  • an in vitro complex comprising a first nucleic acid probe (e.g., a labeled probe) hybridized to a genetic variant nucleic acid, wherein said genetic variant nucleic acid comprises a genetic variant TOLLIP, SPPL2C or MDGA2 gene sequence, wherein said genetic variant nucleic acid is extracted from a human subject with an interstitial lung disease or suspected of having an interstitial lung disease, or is an amplification product thereof.
  • the complex further comprises a second nucleic acid probe (e.g., labeled with a different label) hybridized to said genetic variant nucleic acid.
  • first nucleic acid probe comprises a first label and said second nucleic acid probe comprises a second label, wherein said first and second label are capable of fluorescence resonance energy transfer.
  • the complex further comprises an enzyme, such as a
  • DNA polymerase e.g., standard DNA polymerase or thermally stable polymerase such as Taq
  • ligase e.g., ligase
  • MUC5B and TOLLIP genes reside on the same genetic locus. Based on the analysis performed, the association of TOLLIP genetic variants was found to be independent from association with the previously reported MUC5B promoter SNP, rs35705950, on IPF susceptibility. Notably, the minor allele of TOLLIP SNP, rs5743890_G, was discovered to be a "protective" allele, as it lowered susceptibility to IPF compared with controls. However, mortality analysis demonstrated that individuals who developed IPF despite having the protective rs5743890_G allele had increased mortality in two independent case series and in a meta-analysis. The MUC5B/TOLLIP region on chromosome 11 p15.5 exemplifies the association patterns, disease susceptibility and outcomes.
  • the Toll interacting protein (Tollip), encoded by the TOLLIP gene, is known to be a critical regulator of Toll-like receptor (TLR)-mediated innate immune responses and transforming growth factor- ⁇ (TGF- ⁇ ) signaling pathway.
  • TLR Toll-like receptor
  • TGF- ⁇ transforming growth factor- ⁇
  • Tollip activates Myd88-dependent NF-kB to modulate TLR signaling and membrane trafficking; interacts with Smad7 to modulate intracellular trafficking and negatively regulated TGF- ⁇ signaling pathway by degrading ubiquitinated TGF- ⁇ type 1 receptor; interacts with caveolin-1 interacting protein in monocytes, regulating signaling in antigen-presenting cells to induce antigen specific proliferation of T-cell proliferation, B cells, or both.
  • TOLLIP polymorphisms are involved in regulation of TLR2 and TLR4 and are associated with susceptibility to tuberculosis, atopic dermatitis, sepsis, and TOLLIP is differentially hypomethylated in IPF lungs. Lastly, failure to upregulate TOLLIP expression in inflammatory bowel disease, may lead to chronic inflammation.
  • Chromosome 17q21 region has been associated with Parkinson's, multiple sclerosis, Alzheimer's, androgenic alopecia, and interestingly, with the response to inhaled corticosteroids in asthma and COPD.
  • the minor allele rs17690703_T in the 17q21.31 region was associated with decreased susceptibility for IPF development and also conferred increased mortality in Inter une, UChicago, and in the meta-analysis.
  • H2 a known inversion, referred to as H2
  • H2 in a large region of conserved LD on the chromosome, which is positively selected in Europeans.
  • CNVs copy number variants
  • MDGA2 a novel region, resides on 14q21.23 and showed association with
  • MDGA2 is a paralog for ICAM, which has been recently demonstrated as a potential biomarker of IPF disease activity. The instant findings indicate the importance of this gene in IPF.
  • IPF is a heterogeneous disease and, by definition, is a diagnosis of exclusion. As such, misdiagnoses are possible, which might lead to a reduction in power.
  • all subjects met currently accepted criteria for diagnosis as outlined by ATS/ERS/JRS/ELAT with many having been vetted with core pathology and radiology as in Inter une, ACE-IPF, as well as participation in variety of studies.
  • a three-stage association study was conducted including a discovery GWAS for susceptibility to IPF in Stage 1 , and replicated the findings in two independent case-control association studies (Stage 2 and Stage 3, respectively). Association with mortality was evaluated in three case series. A flowchart illustrating the strategic approach used is shown in Fig. 2.
  • Stage 1 samples consisting of African-Americans (AA) and European-Americans (EA) were collected for the discovery phase of the genome- wide association study (GWAS), while Stages 2 and 3 consisting of only EA samples were collected for two independent replication studies (replication 1 and 2, respectively). All eligible subjects were at least 35 years of age and reported having symptoms of idiopathic interstitial pneumonia for at least 3 months.
  • a high-resolution computed tomographic scan was required to show definite or probable idiopathic interstitial pneumonia in accordance with predefined criteria, 14 and a surgical lung biopsy confirming UIP, was obtained in 37.3% of subjects in the discovery GWAS stage. Subjects with clinically significant exposure to known fibrogenic agents or another cause of interstitial lung disease were excluded.
  • EA European American
  • Stage 1 genotyping was conducted using the Genome-Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA). Stages 2 and 3 genotyping was conducted using the iPLEX GoldTM Platform (Sequenom, San Diego, CA). Genotype imputation was performed with IMPUTE2 using European ancestry panel data from the 1000 Genomes Project as a reference. Association testing was performed using SNPTEST software (v2.3). 7 Fifty-two SNPs selected in 19 loci showing an association with IPF (p ⁇ 10 "4 ) in Stage 1 were carried forward to Stage 2. As the selected SNPs with the lowest p-value in Stage 1 were all a result of imputation, their association was validated by genotyping using the iPLEX GoldTM Platform. Six SNPs in 3 loci achieving an overall p ⁇ 5x10 "8 (i.e. Stage 1 and 2 combined) were carried forward to Stage 3.
  • Genotypes were recalled plate-by-plate in the study, including those downloaded from dbGaP using "crlmm" package, a new implementation of the Corrected Robust Linear Model with Maximum Likelihood Classification (CRLMM) algorithm, available through the Oligo package at Bioconductor. 18, 19
  • Samples were excluded from the analysis if they failed any of several quality metrics: low call rate (below 97% or 93% for production plate with > 35 samples or with ⁇ 35 samples, respectively), incompatibility between reported gender and genetically determined gender, or incompatibility between reported race and genetically determined race. Samples were also checked for unexpected familial relationships using pairwise IBD estimation in PLINK. 20 The total number of European-American IPF case and control samples passing all initial QC tests was 575 and 1 ,427 (1 ,340 of the available 1,442 cases from dbGaP and 87 of the 103 cases from University of Pittsburgh), respectively.
  • Genome-wide SNP imputation was performed for the cleaned dataset to identify additional SNPs possibly showing associations.
  • SHAPEIT 23 software was used to estimate phased haplotypes from the directly observed genotype data. Haplotypes derived from a European ancestry panel, consisting on samples from CEU, FIN, GBR, IBS and TSI from 1000 Genomes Project (February 2012 release), was used as a reference. Imputation was conducted using IMPUTE2. The inflation factor ( ⁇ ) between cases and controls across all SNPs was 1.06.
  • SNPTEST software (v2.3) 24 was used to calculate p-values based on a one degree-of-freedom score test for a logistic regression which assumes that the allele effect on the genotype for each SNP is additive.
  • the score test implemented in SNPTEST allows for genotypic uncertainty via missing data likelihood, therefore it is applicable to both imputed genotypic data (i.e. in Stage 1) and to directly genotyped data (i.e. all stages).
  • P- values were calculated for each stage separately, for Stages 1 and 2 combined, and finally for a joint analysis with all stages combined as one sample.
  • Model parameters were estimated with a random subset of 200 individuals before imputation on the entire dataset. Regions were deemed for follow-up in Stage 2 if they had a SNP with an association p ⁇ 10 "4 in Stage 1. A minimum of 2 SNPs was selected from each region for Stage 2 genotyping.
  • the linkage disequilibrium (LD) of those two SNPs was low (? ⁇ 0.2), where one of them was the variant with direct genotyping data showing the lowest p-value, and the other was the variant with imputed data showing the lowest p-value. Based on these criteria, a total of 40 SNPs for 19 loci were selected (2 SNPs per loci except for chrl VTOLUP, chrl 7/SPPL2C, and chr7/MAD1L1 regions with 3 SNPs; for c rtlS H region with only 1 SNP).
  • tSNPs tagging SNPs
  • haplotype i 2 included in TagIT 3.03 software.
  • CEU, FIN, GBR, IBS and TSI European individuals
  • Linkage disequilibrium (LD) between SNPs in the MUC5B/T0LLIP region was measured using pairwise r 2 measures. 8
  • the mode of inheritance for these SNPs was determined by comparing the odds ratios of the heterozygous and at-risk homozygous genotypes.
  • a regression-based conditional analysis of the interaction between MUC5B and TOLLIP SNPs on IPF susceptibility was implemented in the R statistical package.
  • Fig. 9 and Fig. 10 Demographic and clinical characteristics of IPF patients and controls in each stage are shown in Fig. 9 and Fig. 10.
  • cases in the discovery stage had a wide range of disease severity and age.
  • the Stage 2 patients were a blend of cases with milder (InterMune) and more severe disease undergoing lung transplantation (LTOG), yielding a very similar group to Stage 1 based on the overall physiologic severity as assessed by forced vital capacity (FVC) and diffusing capacity for carbon monoxide (DLCO) (Fig. 10).
  • FVC forced vital capacity
  • DLCO carbon monoxide
  • the Stage 3 patients were more severe, derived from the LTOG and ACE-lPF study. However all IPF cases met diagnostic criteria 16 and were all of similar age and gender. Characteristics of cases with follow-up data for survival analysis are shown in Fig. 12.
  • a total of 19 genomic loci with an association were identified from Stage 1 discovery GWAS. Fifty-two SNPs were compiled from the combination of genotyped, imputed, and tSNPs.
  • Fig. 7 summarizes annotations for these loci, allele frequency in reference populations (CEU, EUR), IPF cases, controls, as well as their association p-values with susceptibility to IPF.
  • Directly genotyped SNPs in Stage 2 nominally replicated many of the associations with IPF susceptibility detected in Stage 1 GWAS.
  • Five imputed SNPs and the previously identified UC5B promoter SNP reached genome-wide significance levels (p-value ⁇ 4.2 x 10 "8 ) in a joint analysis of Stage 1 and 2. These six SNPs were re-genotyped in Stage 1 samples and the association confirmed.
  • chrl 1 highlighted loci of chrl 1 p15.5 containing SNPs of TOLLIP (rs111521887, rs5743894, rs5743890) and MUC5B (rs35705950); chr17q21.31 of SPPL2C (rs17690703) and Chr14q21.3 of MDGA2 (rs7144383).
  • Diamonds and circles represent individual SNP of the GWA screen using genotyped and imputed data, respectively. Colored diamonds indicate SNP data obtained by the analysis of 542 IPF cases and 542 controls. Additional tSNPs selected for better coverage are included. Associations were assessed assuming recessive and additive modes of inheritance for the MUC5B/TOLLIP locus and the SPPL2C locus, respectively. Levels of linkage disequilibria (r 2 ) with the best-associated SNP (red diamonds) are color-coded. Blue lines indicate recombination fractions as estimated from the European panel sample.
  • the r 2 values of MUC5B promoter SNP, rs35705950, and TOLLIP S Ps were 0.07, 0.16, and 0.01, respectively.
  • These low levels of LD indicate that the signals of association for TOLLIP SNPs are independent from MUC5B (Fig. 4A).
  • the mode of effect for the MUC5B SNP (dominant) was different than that for the TOLLIP SNPs (additive or recessive), providing additional evidence that these are independent signals.
  • EA European ancestry
  • H2 status was based on the presence of all 3 SNPs that tag H2 (rs916793, rs2902662, rs17651213). This method allowed H2 assignment to all but 3 patients in this cohort. The addition of a proxy SNP (rs199448) allowed H2 status to be determined for the 3 remaining patients. These data suggest that presence of an H2 haplotype increases susceptibility to IPF.
  • the cohort of 120 EA individuals was then stratified based on H2 (absent vs. present) and SPPL2C (wild-type (WT) vs variant (Var)) status. Inclusion of SPPL2C in the stratification is necessary given the strong correlation between the two variants and potential confounding by SPPL2C.
  • the vast majority of patients belonged to either the H2(-)/ SPPL2CANT or H2(+)/SPPL2C-Var group, making it difficult to draw a conclusion about the two smaller groups. When comparing one group to another the statistical significance was lost.
  • a barrier inherent in the large amount of data generated by next generation sequencing of genetic regions involves methods to evaluate uncommon or rare variants.
  • regions with common variants have a greater number of uncommon or rare variants as well.
  • One approach using the fundamentals of a logistic regression involves an L1 -regularized regression to accommodate large number of variants.
  • the Lasso method is a shrinkage and selection method for linear regression. It minimizes the usual sum of squared errors, with a bound on the sum of the absolute values of the coefficients. It has connections to soft-thresholding of wavelet coefficients, forward stagewise regression, and boosting methods.
  • a method of determining whether a human subject has or is at risk of developing an interstitial lung disease comprising detecting whether the genome of the subject comprises a genetic variant of at least one of TOLLIP, SPPL2C, and MDGA2 and determining whether the subject has or is at risk of developing an interstitial lung disease, the presence of the genetic variant indicating that the subject has or is at risk of developing the interstitial lung disease.
  • interstitial lung disease is a fibrotic interstitial lung disease.
  • interstitial lung disease is idiopathic pulmonary fibrosis or familial interstitial pneumonia.
  • a method of prognosing an interstitial lung disease in a human subject comprising detecting whether the genome of the subject comprises a genetic variant of TOLLIP or SPPL2C and determining a prognosis for the subject, the presence of the genetic variant gene being prognostic of increased or decreased survival.
  • interstitial lung disease is a fibrotic interstitial lung disease.
  • interstitial lung disease is idiopathic pulmonary fibrosis or familial interstitial pneumonia.
  • a method of detecting the presence or absence of at least one genetic variant in a human subject comprising: detecting the presence or absence of at least one genetic variant of at least one of TOLLIP, SPPL2C, and MDGA2 in a sample from the subject.
  • the at least one genetic variant includes one or more of a single nucleotide polymorphism selected from the group consisting of rs1 1521887, rs5743894, rs5743890, rs17690703, and rs7144383.
  • interstitial lung disease is a fibrotic interstitial lung disease or familial interstitial pneumonia.
  • a method of detecting the presence or absence of at least two genetic variants in a human subject having or suspected of being at risk for developing an interstitial lung disease comprising: detecting the presence or absence of at least two of the genetic variants listed in Fig. 7 in a sample from the subject.
  • a method of testing for interstitial lung disease in a human subject comprising: detecting a level of TOLLIP gene expression in a sample from the subject, a low level of TOLLIP gene expression relative to a control being indicative of interstitial lung disease.
  • a method of treating a human subject having an interstitial lung disease comprising: detecting a level of TOLLIP expression according to any one of embodiments 42-44; and if the subject has a low level of TOLLIP expression relative to a control, administering to the subject an amount of a Tollip agonist, Tollip or a genetic construct expressing TOLLIP effective to treat the interstitial lung disease.
  • kits for predicting, diagnosing, or prognosing interstitial lung disease in a human subject consisting essentially of: at least one probe or primer for detecting the presence or absence of at least one genetic variation in at least one of TOLLIP, SPPL2C, and MDGA2.
  • kits of embodiment 46 wherein the at least one probe or primer includes probes or primers for detecting at least one genetic variation in TOLLIP.
  • kits of embodiment 46 or 47, wherein the at least one probe or primer includes probes or primers for detecting at least one genetic variation in SPPL2C.
  • kit of any one of embodiments 46-50, wherein the genetic variation includes at least one of rs111521887, rs5743894, rs5743890, rs17690703, rs7144383, and rs35705950.
  • kits for predicting, diagnosing, or prognosing interstitial lung disease in a human subject comprising: at least one probe or primer for detecting the presence or absence of at least two genetic variations selected from the genetic variations listed in Fig. 7.
  • kit of embodiment 53 wherein the kit comprises probes and/or primers for detecting the presence or absence of from two to 52 of the genetic variations listed in Fig. 7.
  • kit of embodiment 54 wherein the kit comprises probes and/or primers for detecting the presence or absence of from two to 44 of the genetic variations listed in Fig. 11.
  • a method of determining whether a human subject has or is at risk of developing an interstitial lung disease comprising detecting whether the genome of the subject comprises at least two genetic variants selected from the group of variants listed in Fig. 7 and determining whether the subject has or is at risk of developing an interstitial lung disease, the presence of the genetic variant indicating that the subject has or is at risk of developing the interstitial lung disease.
  • interstitial lung disease is a fibrotic interstitial lung disease.
  • interstitial lung disease is idiopathic pulmonary fibrosis or familial interstitial pneumonia.
  • a method of prognosing an interstitial lung disease in a human subject comprising detecting whether the genome of the subject comprises at least two of the genetic variants listed in Fig. 7 and determining a prognosis for the subject, the presence of the genetic variant gene being prognostic of increased or decreased survival.
  • interstitial lung disease is a fibrotic interstitial lung disease.
  • interstitial lung disease is idiopathic pulmonary fibrosis or familial interstitial pneumonia.
  • a method of prognosing an interstitial lung disease in a human subject comprising detecting whether the genome of the subject comprises an inversion in the 17q21.31 chromosomal region and determining a prognosis for the subject, the presence of the inversion being prognostic of increased or decreased survival.
  • a kit comprising a nucleic acid primer capable of hybridizing to a genetic variant TOLLIP nucleic acid, SPPL2C nucleic acid, or MDGA2 nucleic acid.
  • kit of claim 65 wherein said genetic variant has been extracted from a human subject with an interstitial lung disease or is an amplification product of a nucleic acid extracted from a human subject with an interstitial lung disease.
  • kits of claim 65 or 66 wherein said interstitial lung disease is a pulmonary fibrotic condition.
  • kit of one of claims 65-67 further comprising a first labeled nucleic acid probe capable of hybridizing to an amplification product of said genetic variant TOLLIP nucleic acid, SPPL2C nucleic acid, or MDGA2 nucleic acid.
  • kit of claim 68 further comprising a second labeled nucleic acid probe capable of hybridizing to an amplification product of said genetic variant
  • TOLLIP nucleic acid SPPL2C nucleic acid, or MDGA2 nucleic acid.
  • kit of claim 69 wherein said first labeled nucleic acid probe comprises a first label and said additional labeled nucleic acid probe comprises a second label, wherein said first and second label are capable of fluorescence resonance energy transfer when hybridized to said genetic variant TOLLIP nucleic acid, SPPL2C nucleic acid, or MDGA2 nucleic acid.
  • An in vitro complex comprising a first nucleic acid probe hybridized to a genetic variant nucleic acid, said genetic variant nucleic acid comprising a genetic variant TOLLIP, SPPL2C or MDGA2 gene sequence, wherein said genetic variant nucleic acid is extracted from a human subject with an interstitial lung disease or is an amplification product of a nucleic acid extracted from a human subject with an interstitial lung disease.
  • An in vitro complex comprising a thermally stable polymerase bound to a genetic variant nucleic acid, said genetic variant nucleic acid comprising a genetic variant TOLLIP, SPPL2C or MDGA2 gene sequence, wherein said genetic variant nucleic acid is extracted from a human subject with an interstitial lung disease or is an amplification product of a nucleic acid extracted from a human subject with an interstitial lung disease.
  • Carvalho B Bengtsson H, Speed TP, Irizarry RA. Exploration, normalization, and genotype calls of high-density oligonucleotide SNP array data. Biostatistics (Oxford, England) 2007;8(2):485-99. 19. Carvalho BS, Louis TA, Irizarry RA. Quantifying uncertainty in genotype calls. Bioinformatics (Oxford, England) 2010;26(2):242-9.

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Abstract

L'invention concerne des procédés et des coffrets pour diagnostiquer ou prédire un risque de développement de fibrose pulmonaire interstitielle ou prédire la survie d'individus atteints de fibrose pulmonaire interstitielle.
PCT/US2014/014395 2013-02-01 2014-02-03 Variantes génétiques chez des sujets atteints de maladie pulmonaire interstitielle WO2014121180A1 (fr)

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WO2016172150A1 (fr) * 2015-04-22 2016-10-27 The University Of Chicago Méthodes pour le traitement de la fibrose pulmonaire idiopathique
WO2017121769A1 (fr) * 2016-01-12 2017-07-20 bioMérieux Procédé in vitro permettant de prédire un risque de développement d'une pneumonie chez un sujet

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US20110217315A1 (en) * 2010-01-26 2011-09-08 National Jewish Health Methods and compositions for risk prediction, diagnosis, prognosis, and treatment of pulmonary disorders
US20110311512A1 (en) * 2008-11-14 2011-12-22 Hakon Hakonarson Genetic Variants Underlying Human Cognition and Methods of Use Thereof as Diagnostic and Therapeutic Targets

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US20110311512A1 (en) * 2008-11-14 2011-12-22 Hakon Hakonarson Genetic Variants Underlying Human Cognition and Methods of Use Thereof as Diagnostic and Therapeutic Targets
US20110217315A1 (en) * 2010-01-26 2011-09-08 National Jewish Health Methods and compositions for risk prediction, diagnosis, prognosis, and treatment of pulmonary disorders

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MARTIN ET AL.: "Regulated intramembrane proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/SPPL2b", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 283, no. 3, 18 January 2008 (2008-01-18), pages 1644 - 1652 *
ZHU ET AL.: "Tollip, an intracellular trafficking protein, is a novel modulator of the transforming growth factor-beta signaling pathway", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 287, no. 47, 16 November 2012 (2012-11-16), pages 39653 - 39663 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016172150A1 (fr) * 2015-04-22 2016-10-27 The University Of Chicago Méthodes pour le traitement de la fibrose pulmonaire idiopathique
US10543185B2 (en) 2015-04-22 2020-01-28 The University Of Chicago Method for treating idiopathic pulmonary fibrosis
WO2017121769A1 (fr) * 2016-01-12 2017-07-20 bioMérieux Procédé in vitro permettant de prédire un risque de développement d'une pneumonie chez un sujet

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