BR112015019502B1 - IN VITRO AND/OR EX VIVO METHOD FOR DETERMINING WHETHER A HUMAN SUBJECT PRESENTS OR IS AT RISK OF DEVELOPING INTERSTITIAL LUNG DISEASE - Google Patents

Abstract

METHOD FOR DETERMINING WHETHER A HUMAN SUBJECT PRESENTS OR IS AT RISK OF DEVELOPING INTERSTITIAL LUNG DISEASE; METHOD FOR MONITORING THE PROGRESSION OF INTERSTITIAL LUNG DISEASE IN A HUMAN SUBJECT; METHOD FOR EVALUATING THE EFFECTIVENESS OF TREATMENT AGAINST INTERSTITIAL LUNG DISEASE IN A HUMAN SUBJECT; ASSAY SYSTEM FOR PREDICTING RESPONSE TO THERAPY AGAINST INTERSTITIAL LUNG DISEASE IN A HUMAN SUBJECT; KIT TO PREDICT, DIAGNOSE OR PROGNOSE INTERSTITIAL LUNG DISEASE; AND COMPLEX IN VITRO. Biomarkers, methods, and assay systems for identifying poor prognosis of interstitial pneumonia (pulmonary fibrosis) in an individual diagnosed with suspected interstitial pneumonia are disclosed.

Description

CROSS REFERENCES TO RELATED ORDERS

[0001] This application claims priority to Provisional Application No. US61/764986, filed on February 14, 2013, the disclosure of which is incorporated herein in its entirety.

GOVERNMENT DECLARATION OF SUPPORT

[0002] This invention was made with government support under grant numbers R01-HL095393, R01-HL097163, P01-HL092870, RC2-HL101715, U01-HL089897, U01-HL089856, U01-HL108642 and P50-HL0894932 awarded by the National Heart, Lung and Blood Institute, and grant number 1I01BX001534 awarded by the Veterans Administration. The government has certain rights to this invention.

FIELD OF INVENTION

[0003] The present disclosure generally relates to biomarkers, methods and assay kits for identifying and evaluating the prognosis of individuals with or suspected of having interstitial lung disease. Petition 870230035817, of 04/28/2023, p. 12/32

BASICS OF THE INVENTION

[0004] Idiopathic interstitial pneumonias (IIPs) represent a group of lung diseases commonly characterized by pulmonary fibrosis or progressive scarring of the alveolar interstitium, which can lead to significant morbidity and mortality due to hypoxemic respiratory failure. Although some forms of pulmonary fibrosis are associated with known environmental exposures (e.g., asbestos), drug toxicity, radiation exposures, or collagen vascular diseases (e.g., scleroderma), IIPs have no known etiology. The most common and serious IIP is idiopathic pulmonary fibrosis (IPF), which has a median survival of 2-3 years after diagnosis. There is no pharmacological therapy for IPF approved for use in the United States, and lung transplantation is the only life-prolonging intervention. Although all IIPs have a variable clinical course, they frequently progress to end-stage lung disease and death. Although it appears that the risk of IIP is likely determined by multiple genetic variants and environmental toxins, the causes of IIP are only beginning to emerge.

[0005] It is necessary to identify genetic variants, which act independently or in combination, which are indicative of different histological types of interstitial lung diseases, as well as methods of identifying these genetic variants in an individual, diagnosed with or suspected of being predisposed to the development of interstitial lung disease. Solutions to these and other problems in the art are provided in this document.

BRIEF SUMMARY OF THE INVENTION

[0006] Provided herein are methods and materials for determining whether a subject (i.e., an individual) has or is at risk of developing an interstitial lung disease, such as interstitial pneumonia (e.g., FIP, IPF, or IIP). Also provided are methods for determining the prognosis of an individual diagnosed with or suspected of having an interstitial lung disease (e.g., an individual with a family history of interstitial pneumonia). In some embodiments, the interstitial lung disease is a fibrotic interstitial pneumonia, such as idiopathic pulmonary fibrosis or familial interstitial pneumonia. In some embodiments, the individual is a human.

[0007] Also provided herein are methods for detecting a genetic variant (e.g., single nucleotide polymorphism) of a human subject with an interstitial lung disease. The method includes detecting a polymorphism described below in a biological sample from the human subject. In some embodiments, the method includes obtaining and/or testing the biological sample. As described below, in some embodiments, the polymorphism is rs868903, rs7934606, rs6421972, rs7480563, rs7942850, rs4077759, rs2334659, rs7122936, rs2301160, rs3829223, or rs285747 6. In some embodiments, the genetic variant is selected from any of the SNPs listed in Tables 1 and 2.

[0008] Also provided herein are methods for treating an interstitial lung disease in a human subject in need of such treatment, for example, in a subject diagnosed as having or likely to have an interstitial lung disease using the methods described herein. The method includes detecting a genetic variant, as described below, in a biological sample from the human subject and administering an effective amount of an interstitial lung disease treatment. In some embodiments, the method includes obtaining and/or testing the biological sample. As described below, in some embodiments, the genetic variant is the rs868903, rs7934606, rs6421972, rs7480563, rs7942850, rs4077759, rs2334659, rs7122936, rs2301160, rs3829223, or rs285 polymorphism 7476. In some embodiments, the genetic variant is selected from any of the SNPs listed in Tables 1 and 2.

[0009] One embodiment of the disclosure refers to a method that includes detecting one or more genetic variants (e.g., a polymorphism in a marker gene or plurality of marker genes) in a biological sample from an individual. Polymorphisms are selected from rs2736100, rs2076295, rs3778337, rs4727443, rs868903, rs7934606, rs6421972, rs7480563, rs7942850, rs4077759, rs2334659, rs 7122936, rs2034650, rs1992272, rs1981997, rs17563986, rs8070723, rs12610495, rs2109069, rs1379326, rs1881984, rs10936599 ,rs1997392,rs6793295,rs2609255,rs2853676,rs10484326,rs10748858,rs2067832,rs11191865,rs2301160,rs3829223,rs2857476,rs12787 69, rs1007177, rs10518693, rs393152, rs12373139, rs17690703, rs2532274, rs2532269, rs2668692, rs169201, rs199533 and rs415430.

[0010] In a related embodiment, the polymorphism is selected from the group consisting of rs2736100, rs2076295, rs3778337, rs4727443, rs868903, rs7934606, rs6421972, rs7480563, rs7942850, rs407775 9, rs2334659, rs7122936, rs2034650, rs1992272, rs1981997, rs17563986, rs8070723, rs12610495 and rs2109069. In some embodiments, detection comprises detecting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 such polymorphisms in any combination.

[0011] In related embodiments, the polymorphism is selected from the group consisting of rs868903, rs7934606, rs6421972, rs7480563, rs7942850, rs4077759, rs2334659, rs7122936, rs2301160, rs382922 3, and rs2857476. In some embodiments, detection comprises detecting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 such polymorphisms in any combination.

[0012] In related embodiments, the polymorphism is selected from the group consisting of rs2736100, rs868903, rs1881984 and rs2853676. In some embodiments, detection comprises detecting at least 1, 2, 3, or 4 such polymorphisms in any combination.

[0013] In related embodiments, the polymorphism is rs868903.

[0014] In a related embodiment, the polymorphism is selected from the group consisting of rs1379326, rs1881984, rs10936599, rs1997392, rs6793295, rs2609255, rs2853676, rs10484326, rs10748858, rs20 67832, rs11191865, rs2301160, rs3829223, rs2857476, rs1278769, rs1007177, rs10518693, rs393152, rs12373139, rs17690703, rs2532274, rs2532269, rs2668692, rs169201, rs199533 and rs415430. In some embodiments, detection comprises detecting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 of these polymorphisms in any combination.

[0015] In related embodiments, the method includes detecting one or more additional polymorphisms in the biological sample of the individual in which the polymorphism is rs35705950.

[0016] In related embodiments, the individual may be homozygous for one or more of the polymorphisms mentioned above. In other related embodiments, the individual may be heterozygous for one or more of the polymorphisms cited above.

[0017] In each of these embodiments, the detection of at least one of the polymorphisms is indicative of an individual who has a modified risk of developing interstitial lung disease (e.g., the individual has an increased or reduced risk of developing interstitial lung disease) .

[0018] In some modalities, the individual is at high risk of developing sporadic interstitial lung disease. In some modalities, the individual is at high risk of developing familial interstitial lung disease. In some modalities, the individual is at high risk of developing idiopathic pulmonary fibrosis (IPF). In other modalities, the individual has a reduced risk of developing sporadic IIP. In some embodiments, the individual has a reduced risk of developing familial IPI. In some embodiments, the individual has a reduced risk of developing idiopathic pulmonary fibrosis (IPF).

[0019] In these modalities, the detection of at least one of the polymorphisms may be indicative of the progression of the individual's interstitial lung disease. In some embodiments, detection of at least one of the polymorphisms may be indicative of a lack of progression of interstitial lung disease, or a slow progression of interstitial lung disease in the individual. In some embodiments, detection of at least one of the polymorphisms may be indicative of rapid progression of interstitial lung disease in the individual.

[0020] In each of these modalities, the presence of one or more of the polymorphisms can be compared to a control, such as a defined standard or reference group of polymorphisms that have been associated with the risk of developing interstitial lung disease, a diagnosis of a specific interstitial lung disease, a progression of interstitial lung disease, a clinical outcome of interstitial lung disease in an individual, or responsiveness to treatment of interstitial lung disease, as determined in accordance with a statistical procedure for predicting risk .

[0021] In one embodiment of this method, the presence of polymorphisms can be detected by obtaining a sample of the individual's genomic DNA and determining the presence or absence of the polymorphism at the specific locus. In some embodiments, the presence or absence of the polymorphism is determined by at least one method selected from multiplexed locus-specific PCR amplification, multiplexed single-based extension (SBE). ) of locus-specific amplicons and multiplexed resolution of SBE products using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

[0022] In another embodiment of this method, the presence of the marker is determined by obtaining RNA from the biological sample (e.g., tissue sample); generation of cDNA from RNA; optional amplification of the cDNA with probes or primers for genetic sites containing the polymorphisms; determination of the presence or absence of at least one of the polymorphisms in the biological sample.

[0023] These methods may include comparing the presence of one or more of the polymorphisms in the biological sample to a standard set of one or more polymorphism(s) that have been correlated with the development of an interstitial lung disease or the progression of the disease in a diagnosed individual (e.g., someone with stable IIP disease or slowly, severely, or rapidly progressing IIP disease), or a control or standard set of one or more polymorphism(s) that has been correlated with not developing interstitial lung disease or the non-development of pathological symptoms of the disease, such as lung scarring (fibrosis). In this modality, the individual is identified as at modified risk (e.g., at increased or reduced risk) of developing or progressing (e.g., progressing rapidly, slowly, or not progressing) with the development of interstitial lung disease or pathologic manifestations of lung disease. interstitial (lung scars (fibrosis)) if the presence of the one or more polymorphisms coincides with the standard set of one or more polymorphism(s) that has been correlated with the risk of developing interstitial lung disease or the severity or extent of disease progression interstitial lung. Alternatively, the individual may be predicted to have a reduced risk of or not develop interstitial lung disease or clinically not progress with pathologic manifestations of interstitial lung disease if the presence of the one or more polymorphisms does not match the standard set of one or more polymorphisms. (s).

[0024] One embodiment of such methods for determining whether an individual is at high or low risk of developing interstitial lung disease, or at high or low risk of rapidly progressing with the development of lung scarring (fibrosis), includes detecting the presence of at least one polymorphism selected from the polymorphism(s) listed above, such as one or more of the SNPs listed in Tables 1 and 2, e.g., rs35705950, rs868903, rs2736100, rs2853676, rs1881984, rs2736100, rs2609255, rs1048432 6, rs2076295, rs10748858, rs2067832, rs11191865, rs1278769, rs12610495 and rs2109069. The presence of at least one of the polymorphisms is indicative of the possibility of an individual developing or progressing (e.g., progressing rapidly) with the development of lung scarring (fibrosis) and interstitial lung disease.

[0025] These modalities may include performing a follow-up step with the individual, such as a clinical assessment, a chest computed tomography (CT scan) and analysis by a radiologist.

[0026] Another embodiment of the present disclosure is a testing system for predicting the need for treatment (e.g., palliative therapy or lung transplantation) in an individual diagnosed with interstitial lung disease. The assay system includes a means for detecting the presence of at least one polymorphism selected from the group consisting of rs35705950, rs868903, rs2736100, rs2853676, rs1881984, rs2736100, rs2609255, rs10484326, rs2076295, rs10 748858, rs2067832, rs11191865, rs1278769, rs12610495 and rs2109069. In one embodiment of the assay system, the means for detecting the polymorphisms includes a nucleic acid probe with at least 10 to 50 contiguous nucleic acids of the nucleic acid sequence comprising the polymorphism. The nucleic acid probes are preferably disposed on an assay surface which may include a chip, array, or fluidity card. The assay system may include a control selected from information containing a predetermined polymorphism or set of polymorphisms that has been correlated with the risk of developing interstitial lung disease, or the progression of interstitial lung disease, or increased or decreased life expectancy in patients with interstitial lung disease.

[0027] In any of the embodiments of the present disclosure, the detection step may include, but is not limited to, using a nucleotide probe that hybridizes to at least one genetic site comprising the polymorphism. In one aspect, the probe may be a chimeric probe (e.g., one that hybridizes to more than one of the polymorphism sites). In another aspect, the detection step may include detecting the number of copies of the polymorphism in one or more cells in the biological sample (i.e., determining whether the individual is heterozygous or homozygous for the polymorphism).

[0028] In one aspect of this embodiment, the comparison step comprises comparing the presence of one or more of the polymorphisms in the biological sample to a control set of polymorphisms from patients with rapidly progressing interstitial lung disease, or a control set of polymorphisms of patients with slow or no progression of interstitial lung disease.

[0029] In any of the embodiments of the disclosure, an individual may be selected by their risk of developing interstitial lung disease or by diagnosis or prognosis (e.g., predicted to not progress or to progress slowly or rapidly with pathological features of interstitial lung disease, such as lung scars) through assessment of a clinical covariate, including histological appearance and/or marker(s) in the individual's lung tissue.

[0030] Also provided herein are methods for detecting an expression level of one or more marker genes (e.g., biomarkers) in a human subject with an interstitial lung disease. The method includes detecting a level of one or more marker genes described below in a biological sample from the human subject. In some embodiments, the method includes obtaining and/or testing the biological sample. As described below, in some embodiments, the marker gene is TERT, MUC2, TOLLIP, DSP, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69, or homologues or variants thereof. In some embodiments, the marker gene is selected from TERT, MUC2, TOLLIP, homologs and variants thereof.

[0031] Also provided herein are methods for treating interstitial lung disease in a subject in need of such treatment. The method includes detecting a level of one or more marker genes described below in a biological sample from the human subject and administering an effective amount of an interstitial lung disease treatment. In some embodiments, the method includes obtaining and/or testing the biological sample. As described below, in some embodiments, the marker gene is TERT, MUC2, TOLLIP, DSP, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69, or homologues or variants thereof.

[0032] One embodiment of the disclosure relates to a method that includes detecting a gene expression level (e.g., RNA or protein expression) of a marker gene or plurality of marker genes in a biological sample from an individual. The marker gene(s) are selected from a marker gene with at least 95% sequence identity with a sequence selected from: TERT (telomerase reverse transcriptase; NC_000005.9; AY407349) ; TOLLIP (toll-interacting protein; NC_000011.9; AY419805), MUC2 (mucin 2, mucus/gel-forming oligomeric; NC_000011.9; DQ036653), DSP (desmoplakin; NC_000006.11; DQ030635), DISP2 (expedited homolog 2 ; NC_000015.9), MAPT (microtubule-associated tau protein; NC_000017.10; AY413628), DPP9 (dipeptidyl peptidase 9; NC_000019.9; DQ053109), CSMD1 (CUB and Sushi 1 multiple domains; NC_000008.10; DQ037810) , MYNN (myoneurin; NC_000003.11; AY407169), LRRC34 (leucine-rich repeat containing 34; NC_000003.11), FAM13A (sequence similarity family 13, member A; NC_000004.11), OBFC1 (oligonucleotide-binding fold /oligosaccharide containing 1; NC_000010.10), ATP11A (ATPase, class VI, type 11A; NC_000013.10), IVD (isovaleryl-CoA dehydrogenase; NC_000015.9; AY418331), CRHR1 (corticotropin-releasing hormone receptor 1; NC_000017 .10; AY414327), IMP5 (importin 5; NC_000013.10), LOC100128977 (MAPT antisense RNA 1; NC_000017.10), KIAA1267 (KAT8 regulatory NSL complex subunit 1; NC_000017.10; NG_032784), NSF (N-ethylmaleimide sensitive factor; NC_000017.10), WNT3 (wingless-type MMTV integration site family, member 3; NC_000017.10; AY413892), C17orf69 (CRHR1 intronic transcript 1 (non-protein coding ; NC_000017.10). In some embodiments, the marker gene has at least 95% sequence identity over a range of at least 10, 15, 20, 25, 30, 50, 70, 80, 100, 200 or more nucleotides contiguous areas of the selected gene. In some embodiments, the marker gene is a homologue or variant of at least one of the above that, although distinct from the selected marker gene, includes the same genetic variation.

[0033] In a related embodiment, the marker gene(s) are selected from a marker gene with at least 95% sequence identity with a sequence selected from a plurality of marker genes comprising MUC5B and at least one marker gene with at least 95% sequence identity (e.g., at least 96, 97, 98, 99, or 100% identity over a range of at least 10, 15, 20, 25, 30, 50, 70, 80, 100, 200 or more contiguous nucleotides) with a sequence selected from the group consisting of TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, TOLLIP, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69. Again, the marker gene may be a homologue or variant of the selected marker gene that includes the same genetic variant.

[0034] In a related embodiment, the marker gene(s) are selected from a marker gene with at least 95% sequence identity (e.g., at least 96, 97, 98, 99 or 100% identity) over a range of at least 10, 15, 20, 25, 30, 50, 70, 80, 100, 200 or more contiguous nucleotides with a sequence selected from a plurality of marker genes comprising the gene set of TERC, DSP, MUC2, DISP2, MAPT, DPP9, or homologs or variants thereof. In a related embodiment, the marker gene(s) are selected from a marker gene with at least 95% sequence identity to a sequence selected from a plurality of marker genes comprising the set of TERT, MUC2, TOLLIP genes, or homologs or variants thereof.

[0035] In a related embodiment, the marker gene(s) are selected from a marker gene with at least 95% sequence identity (e.g., at least 96, 97, 98, 99 or 100% identity) with a sequence selected from a plurality of marker genes comprising the gene pool of TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, TOLLIP, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69, or homologues or variants thereof. In related embodiments, the methods may further include detecting a gene expression level (e.g., RNA or protein expression) of one or more additional marker genes in the subject's biological sample. The additional marker gene(s) are selected from a marker gene with at least 95% sequence identity (e.g., at least 96, 97, 98, 99, or 100% identity) with a sequence selected from MUC5B and TERC, SFTPC and SFTPA2. In related embodiments, the additional marker gene is MUC5B.

[0036] In a related embodiment, detection of the expression level of the marker gene(s) can be conducted by detecting polypeptides encoded by the marker gene(s) and/or polypeptide fragments of the marker genes, and/or a polynucleotide (e.g., mRNA) that is fully complementary to at least a portion of the marker genes.

[0037] In some embodiments, the detection of elevated gene expression of the markers is indicative of an individual who has an elevated risk of developing interstitial lung disease. In some modalities, the individual is at risk of developing sporadic IIP. In some modalities, the individual is at risk of developing familial IPI. In some modalities, the individual is at risk of developing idiopathic pulmonary fibrosis (IPF).

[0038] In some embodiments, the genes detected in these methods share 100% sequence identity with the corresponding marker genes.

[0039] In each of these embodiments, the levels of at least one of the plurality of markers can be determined and compared to a standard level or reference range of gene expression that can be determined in accordance with a statistical procedure for predicting risk. .

[0040] In one embodiment of this method, the presence of polypeptides can be detected using a reagent that specifically binds to the polypeptide, or a fragment thereof. In one embodiment, the reagent is selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment.

[0041] In another embodiment of this method, the presence of the marker is determined by obtaining RNA from a tissue sample from a subject; generation of cDNA from RNA; cDNA amplification with probes or primers for marker genes; obtaining, from the amplified cDNA, the expression levels of the genes or gene expression products in the sample.

[0042] These methods may include comparing the expression level of the marker gene or plurality of marker genes in the biological sample to a control level of the marker gene(s) including: a control level of the marker gene that has been correlated with the diagnosis or development of, or progression of, interstitial lung disease. In these embodiments, the individual is predicted to develop or progress with the pathological manifestations of interstitial lung disease (such as lung scarring (fibrosis)) if the expression level of the marker gene in the individual's biological sample is statistically similar or higher. the level of control of marker gene expression that has been correlated with the incidence of interstitial lung disease or with the development of interstitial lung disease, or progressive interstitial lung disease. Alternatively, the individual is predicted not to develop or may be predicted not to progress or to progress slowly with the development of interstitial lung disease if the level of the marker gene in the individual's biological sample is statistically lower than the gene control level. marker that has been correlated with the incidence of interstitial lung disease or the development of interstitial lung disease, or progressive interstitial lung disease.

[0043] Additionally, or alternatively, these modalities may include comparing the level of expression of the marker gene or plurality of marker genes in the biological sample to a level of the marker gene(s) in a second individual who has developed or have progressive interstitial lung disease. In this modality, the individual is predicted to develop or have progressive interstitial lung disease if the level of expression of the marker gene in the individual's biological sample is statistically similar to, or greater than, the level of expression of the marker gene(s) (s) in the second individual. Alternatively, the individual is predicted not to develop or have progressive interstitial lung disease if the level of the marker gene in the individual's biological sample is lower than the level of expression of the marker gene(s) in the individual. second individual.

[0044] One embodiment of these methods for determining whether an individual will develop or rapidly progress with the development of lung scarring (fibrosis) and interstitial lung disease includes detecting a gene expression level of a gene with at least 95% sequence identity with each one of MUC5B, DSP and DPP9, or homologues or variants thereof, in a biological sample from an individual. In some embodiments, the detected genes preferentially share 100% sequence identity with the corresponding marker genes. The method can also be conducted by detecting a level of polypeptides encoded by the genes, and/or polypeptide fragments, and/or a polynucleotide that is fully complementary to the genes. In this modality, a high level of expression of the plurality of markers is indicative of the possibility of an individual developing or rapidly progressing with the development of lung scarring (fibrosis) and interstitial lung disease.

[0045] Another embodiment of the disclosure is a method for monitoring the progression of interstitial lung disease in a subject by measuring the expression level of one or more (e.g., a plurality) of the marker genes set forth above in a first biological sample obtained of the subject and comparison of the expression level to a control. In related embodiments, a method is provided for monitoring the progression of interstitial lung disease in a subject by measuring the level of expression of a plurality of marker genes in a first biological sample obtained from the subject, measuring the level of the plurality of markers in a second biological sample obtained from the subject, and comparing the level of the marker measured in the first sample with the level of the marker measured in the second sample. In this embodiment, the plurality of marker gene(s) is selected from a marker gene with at least 95% sequence identity with a sequence selected from a marker gene as set forth above. Alternatively, in this embodiment, the plurality of marker gene(s) is selected from a marker gene with at least 95% sequence identity to a sequence selected from MUC5B, DSP and DPP9 or homologues or variants thereof. same. Preferably, the second biological sample is obtained from the subject at a time subsequent to obtaining the first biological sample. Alternatively, the first biological sample and the second biological sample are obtained from the subject more than once, over a series of times.

[0046] In a related embodiment, detection of the expression level of the marker gene(s) can be conducted by detecting polypeptides encoded by the marker gene(s), and/or or polypeptide fragments of the marker genes, and/or a polynucleotide that is fully complementary to at least a portion of the marker genes. In some embodiments, the genes detected in these methods share 100% sequence identity with the corresponding marker genes.

[0047] These modalities may include performing a follow-up step, such as a computed tomography of the chest (computed tomography of the chest) and analysis by a radiologist.

[0048] Another embodiment of the disclosure is a method of evaluating the effectiveness of a treatment for interstitial lung disease in a subject by comparing the expression level of a gene marker measured in a first sample obtained from the subject with a control value associated with the development or progression of interstitial lung disease. Another embodiment of the disclosure is a method of evaluating the effectiveness of a treatment for interstitial lung disease in a subject by comparing the level of expression of a gene marker measured in a first sample obtained from the subject with the level of expression of the gene marker in a second sample obtained from the subject at a later time, and perform a follow-up step, such as computed tomography of the chest (computed tomography of the chest) or analysis of a lung sample by a radiologist. In this embodiment, a decrease in the level of the marker in the second sample relative to the first sample is an indication that the treatment is effective in treating interstitial lung disease in the subject. In some embodiments, the first sample is collected before a treatment has been administered to the subject, and the second sample is obtained after the treatment has been administered to the subject. In another embodiment, samples are obtained and the comparison is repeated over a series of times. In this embodiment, the plurality of marker gene(s) is selected from a marker gene with at least 95% sequence identity with a sequence selected from a marker gene described above. Alternatively, in this embodiment, the plurality of marker gene(s) is selected from a marker gene with at least 95% sequence identity to a sequence selected from MUC5B, DSP and DPP9 or homologues or variants thereof. same.

[0049] In a related embodiment, detection of the expression level of the marker gene(s) can be conducted by detecting polypeptides encoded by the marker gene(s), and/or or polypeptide fragments of the marker genes, and/or a polynucleotide that is fully complementary to at least a portion of the marker genes. In some embodiments, the genes detected in these methods share 100% sequence identity with the corresponding marker genes.

[0050] Another embodiment of the present disclosure is a testing system for predicting the need for lung transplantation in an individual diagnosed with interstitial lung disease. The assay system includes a means for detecting expression of a marker gene or plurality of marker genes with at least 95% sequence identity to a sequence selected from MUC5B, DSP and DPP9, or homologues or variants thereof. In some embodiments, the genes detected in these methods share 100% sequence identity with the corresponding marker gene.

[0051] In one embodiment of the assay system, the means for detecting includes a nucleic acid probe with at least 10 to 50 (e.g., 10, 15, 20, 25, 30, 10-50, 20-40, 10 -100, 50-100, etc.) contiguous nucleic acids of the marker gene(s) or complementary nucleic acid sequences thereof. In another embodiment of the assay system, the means for detecting includes binding ligands that specifically detect polypeptides encoded by the marker genes. Such binding ligands may include antibodies, derivatives of antigen-binding antibodies, or fragments of antigen-binding antibodies. The nucleic acid probes and/or binding ligands can be arranged on an assay surface, such as a bead, microfluidic surface, chip, array, or fluidity card.

[0052] The assay system may include a control selected from information containing a predetermined control level of the marker gene that has been correlated with progression or life expectancy in patients with interstitial lung disease.

[0053] In any of the embodiments of the present disclosure, the detection step may include, but is not limited to, using a nucleotide probe that hybridizes to at least one of the marker gene(s). In one aspect, the probe may be a chimeric probe (e.g., one that hybridizes to more than one of the biomarker genes). In another aspect, the detection step may include detecting the number of copies of the biomarker genes per cell in one or more cells in the biological sample, and/or detecting amplification of the marker gene per cell in one or more cells in the biological sample. In embodiments, the gene expression detection step is performed by TaqMan® Gene Signature Array, as described in Patent Nos. US6,514,750 and 6,942,837, and 7,211,443 and 7,235,406, each of which is incorporated by reference in its entirety.

[0054] In one aspect of this embodiment, the comparison step comprises comparing the biomarker level in the biological sample to a control level of the biomarker in one or more control samples from patients with rapidly progressing interstitial lung disease. In one aspect, the level of biomarker control is the level that has been correlated with slow or no progression of interstitial lung disease.

[0055] In any of the embodiments of the disclosure, selection of an individual predicted to develop or have a progressive interstitial lung disease may include assessment of a clinical covariate including histological appearance and/or marker(s) in the individual's lung tissue.

[0056] Methods are further provided for determining whether a human subject has or is at risk of developing interstitial lung disease comprising: detecting, in a biological sample from the subject, at least one of: a) the presence of a genetic variant selected from the group consisting of: rs2736100, rs2076295, rs3778337, rs4727443, rs868903, rs7934606, rs6421972, rs7480563, rs7942850, rs4077759, rs2334659, rs712293 6, rs2034650, rs1992272, rs1981997, rs17563986, rs8070723, rs12610495, rs2109069, rs1379326, rs1881984, rs10936599, rs1997392, rs6793295, rs2609255, rs2853676, rs10484326, rs10748858, rs2067832, rs11191865, rs2301160, rs3829223, rs2857476, rs1278769, rs1007177 , rs10518693, rs393152, rs12373139, rs17690703, rs2532274, rs2532269, rs2668692, rs169201, rs199533, and rs415430; b) gene expression level of a marker gene or plurality of marker genes selected from the group consisting of: a marker gene with at least 95% sequence identity with a sequence selected from the group consisting of TERT, DSP, MUC2 , DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, TOLLIP, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69, or homologs or variants thereof; c) polypeptides encoded by the marker genes of b); d) polypeptide fragments of c); and e) a polynucleotide that is fully complementary to at least a portion of a marker gene of b); wherein the presence of at least one genetic variant, polypeptide, fragment, and/or complementary polynucleotides, and/or increased or reduced gene expression of the marker gene indicates that the subject has or is at risk of developing interstitial lung disease. In some embodiments, the presence of a genetic variant is determined by PCR. In some embodiments, the presence of the genetic variant is determined by detecting a Forster resonance energy transfer (FRET). In some embodiments, the presence of the genetic variant is determined by detecting the presence or level of expression of a polypeptide, for example, using an antibody, an antigen-binding antibody derivative, and a specific antigen-binding antibody fragment. for the polypeptide. In some embodiments, interstitial lung disease is a fibrotic lung disease, idiopathic pulmonary fibrosis (IPF), familial interstitial pneumonia (FIP), or idiopathic interstitial pneumonia (IIP).

[0057] Also provided are methods for monitoring the progression of interstitial lung disease in a human subject, comprising i) measuring expression levels of a plurality of gene markers in a first biological sample obtained from the subject, wherein the plurality of markers comprises a plurality of markers selected from the group consisting of: a) a marker gene with at least 95% sequence identity with a sequence selected from the group consisting of TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN , LRRC34, FAM13A, OBFC1, TOLLIP, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69, or homologs or variants thereof; b) polypeptides encoded by the marker genes of a); c) polypeptide fragments from b); and d) a polynucleotide that is fully complementary to at least a portion of a marker gene of a); ii) measuring expression levels of the plurality of markers in a second biological sample obtained from the subject; and iii) compare the marker expression level measured in the first sample with the marker level measured in the second sample. In some embodiments, the method further comprises measuring the expression level of the plurality of markers in at least one additional biological sample obtained from the subject at least once more, and comparing the expression level of the markers measured in the first and second samples to the level of the marker measured in at least one additional sample. In some embodiments, the method further comprises recommending treatment for interstitial lung disease when the expression level of the marker in the second sample is higher than that in the first sample. In some embodiments, the interstitial lung disease is fibrotic lung disease, idiopathic pulmonary fibrosis (IPF), familial interstitial pneumonia (FIP), or idiopathic interstitial pneumonia.

[0058] Also provided are methods for evaluating the effectiveness of treatment for interstitial lung disease in a human subject, the method comprising: determining the expression level of a marker measured in a first sample obtained from the subject at a time t0, wherein the marker is selected from the group consisting of a) a marker gene with at least 95% sequence identity with a sequence selected from the group consisting of TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34 , FAM13A, OBFC1, TOLLIP, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69, or homologs or variants thereof; b) polypeptides encoded by the marker genes of a); c) polypeptide fragments from b); and d) a polynucleotide that is fully complementary to at least a portion of a marker gene of a); ii) determine the expression level of the marker in a second sample obtained from the subject at a later time t1; and iii) perform a follow-up step selected from the performance of a computed tomography of the chest and perform a pathological examination of the subject's lung tissues; wherein a decrease in the expression level of the marker in the second sample relative to the first sample is an indication that the treatment is effective in treating interstitial lung disease in the subject. In some embodiments, time t0 is before the treatment has been administered to the subject, and time t1 is after the treatment has been administered to the subject. In some embodiments, time t0 is after the treatment has been administered to the subject and time t1 is later than time t0 after the treatment has been administered to the subject. In some embodiments, the treatment is administered multiple times. In some embodiments, the comparison is repeated for biological samples obtained from the subject over a series of times. Additionally provided are assay systems for predicting response to therapy for interstitial lung disease in a human subject comprising a means for detecting at least one of: a) presence of a genetic variant selected from the group consisting of: rs2736100, rs2076295, rs3778337, rs4727443, rs868903, rs7934606, rs6421972, rs7480563, rs7942850, rs4077759, rs2334659, rs7122936, rs2034650, rs1992272, rs1981997, rs17 563986, rs8070723, rs12610495, rs2109069, rs1379326, rs1881984, rs10936599, rs1997392, rs6793295, rs2609255, rs2853676, rs10484326, rs10 748858, rs2067832,rs11191865, rs2301160, rs3829223, rs2857476, rs1278769, rs1007177, rs10518693, rs393152, rs12373139, rs17690703, rs2532274, rs2532269, rs2668692, rs169201, rs199533, and rs415430; and b) gene expression level of a marker gene or plurality of marker genes selected from the group consisting of: a marker gene with at least 95% sequence identity with a sequence selected from the group consisting of TERT, DSP, MUC2 , DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, TOLLIP, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69, or homologs or variants thereof; c) polypeptides encoded by the marker genes of b); d) polypeptide fragments of c); and e) a polynucleotide that is fully complementary to at least a portion of a marker gene of b); In some embodiments, the means for detecting comprises nucleic acid probes comprising at least 10 to 50 contiguous nucleic acids of the polymorphisms or marker gene(s), or nucleic acid sequences complementary thereto. In some embodiments, the means for detecting comprises nucleic acid primers or probes that hybridize to a sequence adjacent to or comprising the genetic variant(s) of (a). In some embodiments, at least one of the primers or probes is labeled with a Forster resonance energy transfer (FRET) acceptor, and at least one of the primers or probes is labeled with a FRET donor. In some embodiments, the means for detecting comprises binding ligands that specifically detect polypeptides encoded by the marker genes (e.g., an antibody, antigen-binding antibody derivative or antigen-binding antibody fragment). In some embodiments, the means for detecting comprises at least one of the nucleic acid probes and/or binding ligands disposed on an assay surface (e.g., chip, array, bead, microfluidic surface, or fluidity card). In some embodiments, the probes comprise nucleic acid sequences complementary to at least 10 to 50 contiguous nucleic acids of the marker genes.

[0059] Kits are additionally provided for predicting, diagnosing or prognosing interstitial lung disease. In some embodiments, the kit comprises at least one nucleic acid probe or primer for detecting a genetic variant in a gene selected from the group consisting of: TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, TOLLIP, MUC5B, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, C17orf69, and WNT3. In some embodiments, the kit includes reagents for amplifying the selected genetic variant(s), e.g., primers that amplify a nucleic acid in the selected gene, polymerase (e.g., a thermostable polymerase, such such as Taq or other DNA or RNA polymerase), buffers, etc. In some embodiments, the at least one probe or primer is complementary to a nucleotide variant (e.g., the recessive SNP) of the genetic variant. In some embodiments, the at least one probe or primer is complementary to (hybridizes to) the selected genetic variant polynucleotide sequence or an amplification product thereof. In some embodiments, at least one probe or primer is labeled. In some embodiments, the label is a fluorescent label, or a FRET acceptor or donor. In some embodiments, the kit comprises at least one probe or primer labeled with a Forster resonance energy transfer (FRET) acceptor, and at least one probe or primer labeled with a FRET donor. In some embodiments, the kit includes at least one probe or primer each to detect a genetic variant in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 of the above genes in any combination. In some embodiments, the at least one nucleic acid probe or primer is included in an array, bead, microfluidic surface, or chip. In some embodiments, the kit includes at least one control sample, for example, comprising a nucleic acid having the dominant allele of the at least one selected genetic variant, or comprising a nucleic acid having the polymorphic allele of the at least one selected genetic variant.

[0060] Kits are further provided for predicting, diagnosing or prognosticating interstitial lung disease comprising at least one nucleic acid probe or primer for detecting a genetic variant selected from the group consisting of: rs2736100, rs2076295, rs3778337, rs4727443, rs868903, rs7934606 , rs6421972, rs7480563, rs7942850, rs4077759, rs2334659, rs7122936, rs2034650, rs1992272, rs1981997, rs17563986, rs8070723, rs1261049 5 rs2109069, rs1379326, rs1881984, rs10936599, rs1997392 rs6793295, rs2609255, rs2853676, rs10484326, rs10748858 , rs2067832 , rs111918 65, rs2301160, rs3829223 ,rs2857476,rs1278769,rs1007177,rs10518693,rs393152,rs12373139,rs17690703,rs2532274,rs2532269,rs2668692,rs169201,rs199533 and rs415430. In some embodiments, the kit includes reagents for amplifying the nucleic acid comprising the genetic variant (e.g., PCR primers on both sides of the polymorphic nucleotide, polymerase, buffer, etc.). In some embodiments, the at least one probe or primer is complementary to a variant nucleotide (e.g., SNP) of the genetic variant. In some embodiments, the at least one probe or primer is complementary to (hybridizes to) the selected genetic variant polynucleotide sequence or an amplification product thereof. In some embodiments, at least one probe or primer is labeled. In some embodiments, the label is a fluorescent label, or a FRET acceptor or donor. In some embodiments, the kit comprises at least one probe or primer labeled with a Forster resonance energy transfer (FRET) acceptor, and at least one probe or primer labeled with a FRET donor. In some embodiments, the kit includes at least one probe or primer each to detect a genetic variant in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 of the above genes in any combination. In some embodiments, the at least one nucleic acid probe or primer is included in an array, bead, microfluidic surface, or chip. In some embodiments, the kit includes at least one control sample, for example, comprising a nucleic acid having the dominant allele of the at least one selected genetic variant, or comprising a nucleic acid having the polymorphic allele of the at least one selected genetic variant.

[0061] In vitro-formed complexes are additionally provided for detecting a biomarker (e.g., genetic variant) associated with interstitial lung disease (e.g., fibrotic lung disease, idiopathic pulmonary fibrosis (IPF), familial interstitial pneumonia (FIP), or pneumonia idiopathic interstitial disease (IIP)). Interstitial lung disease can be fibrotic lung disease. Interstitial lung disease can be IPF. Interstitial lung disease can be FIP. Interstitial lung disease can be IIP. In some embodiments, the complex comprises a first nucleic acid probe hybridized to a genetic variant nucleic acid, wherein the genetic variant nucleic acid comprises a TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, gene sequence. FAM13A, OBFC1, TOLLIP, MUC5B, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3 or C17orf69 genetic variant, wherein said genetic variant nucleic acid is extracted from a human subject with or suspected of having a disease interstitial lung disease or is an amplification product of a nucleic acid extracted from a human subject with or suspected of having interstitial lung disease. In some embodiments, the complex further comprises a second labeled nucleic acid probe hybridized to said genetic variant nucleic acid. In some embodiments, said first labeled nucleic acid probe comprises a first label and said second labeled nucleic acid probe comprises a second label, wherein said first and second labels are capable of Forster resonance energy transfer (FRET). In some embodiments, the complex further comprises a polymerase (e.g., a thermostable polymerase or other DNA or RNA polymerase) or ligase. In some embodiments, the complex further comprises a nucleic acid primer hybridized to the genetic variant nucleic acid.

[0062] Other features and advantages of the disclosure will become apparent to one skilled in the art from the following detailed description.

BRIEF DESCRIPTION OF FIGURES

[0063] Figure 1 shows the results of GWAS on 439,828 SNPs with 1616 cases and 4683 controls in the additive model. SNPs above the red line were significant across the entire genome at P < 5 x 10-8. These SNPs and SNPs between the red and blue lines, corresponding to 5 x 10-8 < P-value < 0.0001 were selected for follow-up in 876 cases and 1890 controls.

[0064] Figure 2 shows locus-specific plots corresponding to discovery GWAS results for all loci that achieved genome-wide significance in the discovery GWAS analysis and meta-analysis of discovery and replication results.

[0065] Figure 3 shows locus-specific plots corresponding to discovery GWAS results for four additional loci that achieved genome-wide significance following meta-analysis of discovery and replication results.

[0066] Figure 4 shows relative expression of DSP in lung tissue from 100 cases and 94 controls a) relative expression by case/control status b) relative expression by genotype at rs2076295 in DSP.

[0067] Figure 5 shows a Quantile-Quantile (Q-Q) plot of observed vs. observed p-value distribution. expected for GWAS across 439,828 high-quality SNPs.

[0068] Figure 6 shows the chromosomal locations, SNPs and genes for significant loci throughout the genome.

[0069] Figure 7 shows the Linkage Disequilibrium between the significant SNPs throughout the genome at 11p15 and rs35705950. Color indicates D'estimated = 1; white, an estimated D' = 0. Numbers in squares correspond to r2*100. Estimates based on joint case and control genotypes as used in the analyzes for Table 2 and Table 6.

[0070] Figure 8 depicts a genome-wide linkage scan in families with interstitial lung disease, where the rs3570950 polymorphism was found to be predictive.

[0071] Figure 9 shows odds ratios of SNPs in MUC2, MUC5AC and MUC5B being associated with interstitial lung disease.

[0072] Figure 10 shows confirmation of the relevance of the MUC5B promoter SNP rs3570950 in several study groups.

[0073] Figure 11 shows the longer duration of survival associated with patients with interstitial lung disease who carry the rs3570950 SNP.

[0074] Figure 12 shows the longer duration of survival associated with patients with interstitial lung disease who carry the rs3570950 SNP in different study groups.

[0075] Figure 13 compares different study groups for longer duration of survival associated with patients with interstitial lung disease who carry the rs3570950 SNP.

[0076] Figure 14 shows the structure of the MUC5B gene and the effect of the rs3570950 SNP.

[0077] Figure 15 compares MUC5B expression in normal vs. normal lung tissue. with IPF.

[0078] Figure 16 shows the expression of MUC5B in normal vs. normal lung tissue. with IPF in individuals carrying wild-type (GG) vs. wild-type (GG) genes. MUC5B variant (GT or TT).

[0079] Figure 17 shows that the expression of MUC5B and surfactant protein C (SPC) is increased (upregulated) in lung tissue with IPF.

[0080] Figure 18 depicts effects associated with the rs3570950 MUC5B SNP.

[0081] Figure 19 compares effects of genetics for genes associated with pulmonary fibrosis.

[0082] Figure 20 shows fibrotic lung tissue in patients carrying the rs3570950 SNP.

[0083] Figure 21 shows the increased probability of interstitial lung disease in patients carrying at least one variant rs3570950 allele.

[0084] Figure 22 compares effects of genetics for genes associated with pulmonary fibrosis.

[0085] Figure 23 depicts the genome-wide association study (GWAS) to associate genetic markers with various interstitial lung diseases.

[0086] Figure 24 shows the geographic origin of individuals considered in the study.

[0087] Figure 25 shows an overview of the GWAS results.

[0088] Figure 26 shows the genetic location of SNPs associated with interstitial lung disease.

[0089] Figure 27 shows the relative frequency of fibrotic conditions in genotypic and replication populations.

[0090] Figure 28 shows the genetic location of SNPs associated with interstitial lung disease in the replication population.

[0091] Figure 29 shows the combined results of the GWAS studies and the locations of SNPs associated with interstitial lung disease.

[0092] Figure 30 shows the effect of ancestry on SNPs on chromosome 17q21.

[0093] Figure 31 shows the odds ratios (ORs) and P-values for the effect of ancestry at various SNPs on chromosome 17q21.

[0094] Figure 32 shows the ORs and P-value for the association of the MUC5B promoter SNP with interstitial lung disease.

[0095] Figure 33 summarizes the GWAS findings of interstitial lung disease in terms of SNP location.

[0096] Figure 34 summarizes the GWAS findings of interstitial lung disease in terms of SNP location.

[0097] Figure 35 summarizes the GWAS findings of interstitial lung disease in terms of gene function.

DETAILED DESCRIPTION

[0098] Unless otherwise defined, technical and scientific terms used in this document have the same meaning as commonly understood by a person ordinarily skilled in the art. See, for example, Lackie, Dictionary of Cell and Molecular Biology, Elsevier (4th ed. 2007); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). The term "one" or "an" is intended to mean "one or more". The term "comprise" and variations thereof, such as "comprises" and "comprising", when preceding the citation of a step or an element, is intended to mean that the addition of additional steps or elements is optional and not excluded. The following definitions are provided to facilitate the understanding of certain terms frequently used in this document and are not intended to limit the scope of this disclosure.

[0099] The terms "subject", "patient", "individual", etc. They are not intended to be limiting and can generally be interchanged. That is, an individual described as a "patient" does not necessarily have a given illness, but may merely be seeking medical advice.

[0100] A "control", "control sample", "standard control", or "control value" refers to a sample that serves as a reference, generally a known reference, for comparison with a test sample. For example, a test sample may be taken from a patient suspected of having a given lung disease and compared with samples from a patient with known lung disease, a known polymorphism carrier, or a known normal individual (without disease). A control may also represent an average value collected from a population of similar individuals, for example, patients with lung disease or healthy individuals with a similar medical background, age, weight, etc. A control value can also be obtained from the same individual, for example, from a sample obtained previously, before the disease, or before treatment. One skilled in the art will recognize that controls can be designed to evaluate any number of parameters.

[0101] Someone skilled in the art will understand which controls are valuable in a given situation and will be able to analyze data based on comparisons to control values. Controls are also valuable in determining the significance of data. For example, if values for a given parameter vary widely in controls, the variation in test samples will not be considered significant.

[0102] The term "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 mean at least two nucleotides covalently linked together. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length to about 100 nucleotides in length. Nucleic acids and polynucleotides are polymers of any length, including longer lengths, for example, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. The term "nucleotide" typically refers to a single unit of a polynucleotide, that is, a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides or modified versions thereof.

[0103] As used herein, a "genetic variant" refers to a mutation, single nucleotide polymorphism (SNP), deletion variant, missense variant, insertion variant, inversion, or copy number variant. A genetic variant can be used as a biomarker and can result in increased or decreased expression levels, or differential modification.

[0104] The term "biomarker" refers to a biometric that can be detected in a biological sample (or sample derived from or processed from a biological sample) and compared to a control sample as indicative of a particular condition. . Examples of biomarkers include genetic variants, increased or decreased expression levels (determined by detection of chromatin opening, transcription product, or translation product), and differential modification (e.g., methylation of nucleic acids, or phosphorylation, glycosylation, or multimerization of proteins). A "marker gene" is a gene affected by a biomarker. That is, a marker gene may include a genetic variation in its genomic form, be expressed at a higher or lower level, or be differentially modified as indicative of a particular condition, for example, interstitial lung disease.

[0105] The terms "probe" or "primer" refer 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. For example, PCR primers are generally between 10 and 40 nucleotides in length, while nucleic acid probes, for example a Southern blot, can be over a hundred nucleotides in length. The probe or primers may or may not be labeled, as described below, so that their binding to a target sequence can be detected (e.g., with a FRET donor or acceptor tag). The probe or primer can be designed based on one or more particular (pre-selected) portions of a chromosome, for example, one or more clones, an isolated entire chromosome or chromosome fragment, or a collection of amplification products from polymerase chain reaction (PCR). The length and complexity of the nucleic acid fixed to the target element are not critical to the invention. One skilled in the art can adjust these factors to provide optimized hybridization and signal production for a given hybridization and detection procedure and to provide the necessary resolution between different genes or genomic locations.

[0106] Probes and primers can also be immobilized on a solid surface (e.g., nitrocellulose slides, glass, quartz, fused silica), such as in a matrix. Techniques for producing high-density matrices can also be used for this purpose (see, for example, Fodor (1991) Science 767-773; Johnston (1998) Curr. Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 10871092; Kern (1997) Biotechniques 23: 120-124; Patent No. US5,143,854). One skilled in the art will recognize that the exact sequence of probes and primers in particular 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 be specifically bind (i.e., specifically hybridize to) the same targets from which they were derived.

[0107] A probe or primer is "capable of detecting" a genetic variant if it is complementary to a region that encompasses or is adjacent to the genetic variant. For example, to detect a SNP, primers can be designed on both sides of the SNP, and primer extension used to determine the identity of the nucleotide at the position of the SNP. In some embodiments, FRET-labeled primers are used (at least one labeled with a FRET donor and at least one labeled with a FRET acceptor), so that this FRET signal will be detected only upon hybridization of both primers. In some embodiments, a probe is used under conditions such that it hybridizes only to a genetic variant, or only to a dominant sequence.

[0108] Again, in the context of nucleic acids, the term "capable of hybridizing to" refers to a polynucleotide sequence that forms Watson-Crick bonds with a complementary sequence. One skilled in the art will understand that the percentage of complementarity does not need to be 100% for hybridization to occur, depending on the length of the polynucleotides, the length of the complementary region (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more bases in length), and the rigor of the conditions. For example, a polynucleotide (e.g. primer or probe) may be able to bind to a polynucleotide with 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity across the size of the complementary region. In the context of detecting genetic variants, the percentage of tolerated complementarity or number of incompatibilities will vary depending on the technique used for detection (see below).

[0109] In the context of nucleic acids, the term "amplification product" refers to a nucleic acid (e.g., 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 during amplification procedures involving extreme or cyclic temperatures (e.g., PCR and its variants).

[0110] The terms "marker", "detectable fraction," "detectable agent" and similar terms refer to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels include fluorescent dyes, luminescent agents, radioisotopes (e.g., 32P,3H), electron-dense reagents, enzymes, biotin, digoxigenin, or haptens, and proteins or other entities that may become detectable, e.g. by affinity. Any method known in the art for conjugating a nucleic acid or other biomolecule to a marker can be employed, for example, using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego. The term "tag" can be used synonymously with the term "tag", but generally refers to an affinity-based moiety, for example, a "His tag" for purification, or "strepavidin tag" that interacts with biotin.

[0111] A "tagged" molecule (e.g., nucleic acid, protein, or antibody) is one that is linked either covalently, through a ligand or chemical bond, or non-covalently, through ionic, van der bonds. Waals, electrostatic, or hydrogen to a tag, so that the presence of the molecule can be detected by detecting the presence of the tag attached to the molecule.

[0112] Forster resonance energy transfer (abbreviated FRET), also known as fluorescence resonance energy transfer, is a mechanism that describes the transfer of energy between two chromophores. A donor chromophore (FRET donor), initially in its excited electronic state, can transfer energy to an acceptor chromophore (FRET acceptor), which is typically less than 10 nm away, through nonradiative dipole-dipole coupling. The energy transferred to the FRET acceptor is detected as an emission of light (energy) when the FRET donor and acceptor are in close proximity. A "FRET" signal is therefore the signal that is generated by the emission of light from the acceptor. The efficiency of Forster resonance energy transfer between a donor dye and an acceptor dye separated by a distance of R is given by E = 1/[1+(R/R0)6], with R0 being the Forster radius of the donor-acceptor pair where E = ^. Ro is about 50-60 Å for some commonly used dye pairs (e.g. Cy3-Cy5). The FRET signal varies as the distance to the 6th power. If the donor-acceptor pair is positioned around R0, a small change in distance ranging from 1 Â to 50 Â can be measured with the highest signal to noise. With current technology, 1 ms or faster parallel imaging of many single FRET pairs is achievable.

[0113] A "FRET pair" refers to a FRET donor and FRET acceptor pair that is capable of detecting FRET.

[0114] The terms "fluorophore", "dye", "fluorescent molecule", "fluorescent dye", "FRET dye" and similar terms are used synonymously in this document unless otherwise indicated.

[0115] As used herein, the terms "treat" and "prevent" are not intended to be absolute terms. Treatment may refer to any delay in onset, reduction in frequency or severity of symptoms, improvement in symptoms, improvement in patient comfort and/or respiratory function, etc. The treatment effect can be compared to an individual or a pool of individuals who are not receiving a given treatment, or to the same patient before or after stopping treatment.

[0116] The term "prevent" refers to a decrease in the occurrence of symptoms of lung disease in a patient. As indicated above, prevention can be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur without treatment.

[0117] The term "therapeutically effective amount", as used herein, refers to the amount of the therapeutic agent sufficient to improve the disorder, as described above. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least 100%. Therapeutic efficacy can also be expressed as an "n-fold" increase or decrease. For example, a therapeutically effective amount may have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold or more effect on a control.

[0118] The term "diagnosis" refers to a relative probability that a lung disease is present in the subject. Likewise, the term "prognosis" refers to a relative probability that a certain future outcome may occur in the subject. For example, in the context of the present invention, prognosis may refer to the probability that an individual will develop a lung disease, or the likely severity of the disease (e.g., severity of symptoms, rate of functional decline, survival, etc.). The terms are not intended to be absolute, as will be appreciated by anyone skilled in the field of medical diagnosis.

[0119] The terms "correlated" and "associated", in reference to determining a lung disease risk factor, refer to comparing the presence or amount of the risk factor (e.g., dysregulation or genetic variation in a mucin gene) in an individual to its presence or amount in people known to be suffering from, or at risk of, a lung disease, or in people known to be free from lung disease, and to assign an increased or decreased probability of having/developing the disease to an individual based on the assay result(s).

[0120] The present inventors have discovered polymorphisms and gene expression profiles that are important contributors to the risk of IIP. These findings include eight new genetic risk loci (4q22, 6p24, 7q22, 10q24, 13q34, 15q14-15, 17q21, and19p13) and the role of risk variants in three previously reported genes/loci (TERC[3q26], TERT[ 5p15] and MUC5B [11p15]) in IIP. Prior to this discovery, the only two genes with a common variant reproducibly associated with IIP were TERT and MUC5B. Altogether, the common risk variants associated with IIP suggest that this disease is primarily mediated by defects in host defense, cell-cell adhesion, and premature cellular senescence. These findings can be used to guide intervention and treatment trials in this complex disease.

[0121] According to one definition, a biological marker is "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions." NIH Biomarker Definitions Working Group (1998). Biological markers may also include patterns or sets of characteristics indicative of particular biological processes ("marker panel"). The marker measurement can be increased or decreased to indicate a particular biological event or process. Furthermore, if a marker measurement typically changes in the absence of a particular biological process, a constant measurement may indicate the occurrence of that process.

[0122] Marker measurements can be absolute values (e.g., the molar concentration of a molecule in a biological sample or the presence or absence of a polymorphism) or relative values (e.g., the relative concentration of two molecules in a sample biological). The quotient or product of two or more measurements can also be used as a marker. For example, some doctors use total blood cholesterol as a marker of the risk of developing coronary artery disease, while others use the ratio of total cholesterol to HDL cholesterol.

[0123] In the disclosure, markers are primarily used for diagnostic and prognostic purposes. However, they may also be used for therapy, drug screening, and individual stratification purposes (e.g., to group individuals into a number of "subsets" for assessment), as well as other purposes described herein, including assessing the efficacy of an interstitial lung disease therapy.

[0124] The practice of disclosure employs, unless otherwise indicated, conventional methods of generally known techniques of analytical biochemistry, microbiology, molecular biology and recombinant DNA within the skill of the art. Such techniques are fully explained in the literature. (See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2000; DNA Cloning: A Practical Approach, Vol. I & II (Glover, ed.); Oligonucleotide Synthesis (Gait, ed., Current Edition); Nucleic Acid Hybridization (Hames & Higgins, eds., Current Edition); Transcription and Translation (Hames & Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, Vol. I & II (Tijessen, ed.); Fundamental Virology, 2nd Edition, Vol. I & II (Fields and Knipe, eds.)).

[0125] The terminology used in this document is to describe particular embodiments and is not intended to be limiting. As used herein, the singular forms "a," "and," and "the" include plural referents unless the content and context clearly dictate otherwise. Thus, for example, a reference to "a marker" includes a combination of two or more such markers. Unless otherwise defined, all scientific and technical terms are to be understood as having the same meaning commonly used in the art to which they pertain. For the purposes of this disclosure, the following terms are defined below.

[0126] As used herein, the term "tag" includes polypeptide tags and polynucleotide tags. For clarity of the disclosure, aspects of the disclosure will be described in relation to "polypeptide tags" and "polynucleotide tags". However, phrases made herein regarding "polypeptide tags" are intended to apply to other polypeptides of the disclosure. Likewise, phrases made herein regarding "polynucleotide" tags are intended to apply to other polynucleotides of the disclosure, respectively. Thus, for example, a polynucleotide described as encoding a "polypeptide tag" is intended to include a polynucleotide that encodes: a polypeptide tag, a polypeptide that has substantial sequence identity to a polypeptide tag, modified polypeptide tags, fragments of a polypeptide tag, precursors of a polypeptide tag and successors of a polypeptide tag, and molecules comprising a polypeptide tag, homologous polypeptide, a tag or a modified polypeptide fragment, precursor or successor of a polypeptide tag (e.g. a fusion protein).

[0127] As used herein, the term "polypeptide" refers to a polymer of amino acid residues that has at least 5 contiguous amino acid residues, e.g., 5, 6, 7, 8, 9, 10, 11 or 12 or more amino acids in length, including each whole number up to the total length of the polypeptide. A polypeptide can be composed of two or more polypeptide chains. A polypeptide includes a protein, a peptide, an oligopeptide, and an amino acid. A polypeptide can be linear or branched. A polypeptide may comprise modified amino acid residues, amino acid analogues or non-naturally occurring amino acid residues, and may be interrupted by residues other than amino acids. Included within the definition are amino acid polymers that have been modified, whether naturally or by intervention, for example, formation of a disulfide bond, glycosylation, lipidation, methylation, acetylation, phosphorylation, or by manipulation, such as conjugation with a labeling component. . Also included are antibodies produced by a subject in response to overexpressed polypeptide tags.

[0128] As used herein, a "fragment" of a polypeptide refers to a plurality of amino acid residues that is smaller than the complete polypeptide. For example, a fragment of a given polypeptide may comprise at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 20 contiguous amino acid residues, or at least 30 contiguous amino acid residues of the complete polypeptide. As used herein, a polynucleotide "fragment" refers to a polymer of nucleic acid residues comprising a nucleic acid sequence that has at least 5, 10, or 15 contiguous nucleic acid residues, at least 30 contiguous nucleic acid residues nucleic acids, at least 60 contiguous nucleic acid residues, or at least 90% of a polynucleotide sequence. In some embodiments, the fragment represents a domain (e.g., a functional domain) of the complete polypeptide. In some embodiments, the fragment represents the complete polypeptide minus a given domain. In some embodiments, the fragment is an antigenic fragment and the size of the fragment will depend on factors such as whether the epitope recognized by an antibody is a linear epitope or a conformational epitope. Thus, some antigenic fragments will consist of longer segments, while others will consist of shorter segments (e.g., 5, 6, 7, 8, 9, 10, 11, or 12 or more amino acids in length, including every whole number up to the length total polypeptide). Those skilled in the art are well versed in methods for selecting antigenic fragments bound by antigen-binding antibodies, antibody derivatives, and antibody fragments.

[0129] In some embodiments, a polypeptide tag is a member of a biological pathway. As used herein, the term "precursor" or "successor" refers to molecules that precede or follow the polypeptide tag or polynucleotide tag in the biological pathway. Thus, once a polypeptide tag or polynucleotide tag is identified as a member of one or more biological pathways, the present disclosure may include additional precursor or successor members of the biological pathway. Such identification of biological pathways and their members is within the skill of one in the art.

[0130] As used herein, the term "polynucleotide" refers to a single nucleotide or a polymer of nucleic acid residues of any length. The polynucleotide may contain deoxyribonucleotides, ribonucleotides and/or their analogues and may be single or double stranded. A polynucleotide may comprise modified (e.g., methylated) nucleic acids, nucleic acid analogues, or non-naturally occurring nucleic acids, and may be interrupted by residues other than nucleic acids. For example a polynucleotide includes a gene, a gene fragment, cDNA, isolated DNA, mRNA, tRNA, rRNA, isolated RNA of any sequence, recombinant polynucleotides, primers, probes, plasmids and vectors. Included in the definition are nucleic acid polymers that have been modified, either naturally or by intervention.

[0131] As used herein, a component (e.g., a marker) is referred to as "differentially expressed" in one sample compared to another sample when the method used to detect the component provides a different level or activity when applied to the two samples. A component is referred to as "increased" in the first sample if the method for detecting the component indicates that the level or activity of the component is greater in the first sample than in the second sample (or if the component is detectable in the first sample but not in the second sample). Conversely, a component is referred to as "decreased" in the first sample if the method for detecting the component indicates that the level or activity of the component is lower in the first sample than in the second sample (or if the component is detectable in the second sample , but not in the first sample). In particular, marker is referred to as "increased" or "decreased" in a sample (or set of samples) obtained from a subject with interstitial lung disease (or a subject suspected of having interstitial lung disease, or who is at risk for developing interstitial lung disease). interstitial lung disease) if the level or activity of the marker is higher or lower, respectively, compared to the level of the marker in a sample (or set of samples) obtained from a subject without interstitial lung disease, or a reference value or range.

[0132] The markers identified as being expressed in interstitial lung disease are of significant biological interest. A genome-wide association cas control study (GWAS; 1616 cases and 4683 controls) and replication study (876 cases and 1890 controls) of IIP was conducted. All types of fibrotic IIP were included in the case group since: a) distinguishing between IIP diagnoses is often problematic due to substantial clinical, pathological and radiological overlap; and b) there is strong evidence for shared genetic susceptibility. Both familial and sporadic IIPs were also included in the case group because the MUC5B, TERT, TERC, and SFTPC variants provide evidence that sporadic IIP is genetically similar to the familial form of this disease. The results indicate that IIPs are caused by multiple genetic variants, acting independently or in combination, and that the same genetic variants can lead to different histological types of IIP.

[0133] As explained in detail below, when polymorphism and gene expression profiles were compared with clinical parameters and the common risk variants associated with IIP, the results indicate that this disease is primarily mediated by defects in host defense, cell-cell adhesion, cell and early cellular senescence. These findings can be used to guide intervention trials in this complex disease.

[0134] In addition to the discovery of biomarkers that can be used individually or in any combination in assays and kits for the diagnosis of, prognosis of, or other evaluation or study of interstitial lung disease, biomarkers not previously recognized to play a role in the process infectious disease of interstitial lung disease can now be studied in more detail and/or be used as targets for the discovery of other disease modulators or therapeutic agents. Disclosure markers include the polymorphisms: rs1379326, rs1881984, rs10936599, rs1997392, rs6793295, rs2609255, rs2853676, rs10484326, rs10748858, rs2067832, rs11191 865, rs2301160, rs3829223, rs2857476, rs1278769, rs1007177, rs10518693, rs393152, rs12373139, rs17690703, rs2532274, rs2532269, rs2668692, rs169201, rs199533 and rs415430. The disclosure markers also include elevated gene expression in the following genes: TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, TOLLIP, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, and C17orf69.

[0135] Given the name of the gene, the protein (also referred to herein as the "complete protein", denoted as "Protein") and other peptide fragments of such measured proteins may be obtained (by any means), and such other peptide fragments are included within the scope of the disclosure. The methods of the present disclosure can be used to evaluate fragments of the expression products of the listed genes, as well as molecules that contain an entire listed molecule, or at least a significant portion thereof (e.g., single epitope measured), and modified versions of the tags . Accordingly, such fragments, larger molecules and modified versions are included within the scope of the disclosure.

[0136] Homologs and alleles of disclosure markers can be identified by conventional techniques. As used herein, a homologue of a gene or polypeptide, for example, from a human or other animal, has a high degree of structural and functional similarity to the identified gene or polypeptide. Identification of homologues of human polypeptide tags and other organisms identified herein will be familiar to those skilled in the art. In general, nucleic acid hybridization is a suitable method for identifying homologous sequences from another species (e.g., human, cow, sheep) that correspond to a known sequence. Standard nucleic acid hybridization procedures can be used to identify related nucleic acid sequences of selected percent identity. For example, one could construct a library of cDNAs reverse transcribed from the mRNA of a selected tissue (e.g., colon) and use the nucleic acids encoding polypeptides identified herein to scan the library for related nucleotide sequences. The scan is preferably performed using high stringency conditions (described elsewhere in this document) to identify sequences that are closely related by sequence identity. Nucleic acids identified in this way can be translated into polypeptides and the polypeptides can be tested for activity.

[0137] Furthermore, the present disclosure includes polynucleotides and polypeptides that have sequence identity substantially similar to the markers of the present disclosure. As used herein, two polynucleotides or polypeptides have "substantial sequence identity" when there is at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 99% sequence identity, or 100% sequence identity between their amino acid sequences, or when polynucleotides (e.g., polynucleotides encoding polypeptides) are capable of form a stable duplex with each other under stringent hybridization conditions. In the context of the present disclosure, a genetic variant can be detected in a marker gene, even if the marker gene has more than one site of genetic variation. That is, a selected genetic variant can be detected in the test sample, for example, from an individual suspected of having interstitial lung disease, by determining the sequence of a marker gene comprising the genetic variant, and comparing it with the sequence of the marker gene. of a control or control population. The full-length marker gene test and control sequences may include more than one genetic variant and thus may differ from each other, i.e., may not be 100% identical. One skilled in the art will recognize that the genetic variant can be detected in a sequence that is shorter than the length of the marker gene sequence, for example, by using PCR to amplify a fragment of the marker gene that includes the genetic variant site or a probe that is complementary to a sequence that includes the genetic variant site. Where aspects or embodiments refer to sequence identity, such sequence identity may be with respect to a portion of the sequence as disclosed herein (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45 , 50, 60, 70, 80, 90, 100, or more nucleic acid or amino acid bases in length).

[0138] Conservative amino acid substitutions can be made in polypeptides to provide functionally equivalent variants of the above polypeptides, i.e., the variants maintain the functional capabilities of the polypeptides. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequences known to one skilled in the art. For example, by determining that a peptide is a polypeptide associated with interstitial lung disease, one can make conservative amino acid substitutions in the amino acid sequence of the peptide, and still cause the polypeptide to retain its specific antibody binding characteristics. Furthermore, one skilled in the art will appreciate that allelic variants and SNPs will give rise to substantially similar polypeptides and the same or similar polypeptide fragments.

[0139] Numerous comparison studies have been performed to identify markers using various groups of individuals with interstitial lung disease and without interstitial lung disease (e.g., "control"). The tables list markers that were found to be present or differentially expressed with statistical significance. Therefore, these biomarkers are indicators of interstitial lung disease and disease progression. Where a polypeptide marker was found to be statistically significant in a plurality of studies, the data associated with the observations of greatest statistical significance are presented. Accordingly, in one aspect, the disclosure provides polypeptide biomarkers of interstitial lung disease. In another embodiment, the disclosure provides a polypeptide with substantial sequence identity to a polypeptide tag. In another embodiment, the disclosure provides a molecule comprising an above polypeptide or polynucleotide. As used herein, a compound is referred to as "isolated" when it has been separated from at least one component with which it is naturally associated. For example, a polypeptide can be considered isolated if it is separated from contaminants, including metabolites, polynucleotides, and other polypeptides. Isolated molecules can be prepared synthetically or purified from their natural environment. Standard quantification methodologies known in the art can be employed to obtain and isolate the molecules of the disclosure.

[0140] Some variation is inherent in measurements of the physical and chemical characteristics of markers. The magnitude of the variation depends, to some extent, on the reproducibility of the separation medium and the specificity and sensitivity of the detection medium used to make the measurement. Preferably, the method and technique used to measure the markers is sensitive and reproducible.

[0141] The dataset set out in the Tables reflects the method that was used to detect the markers. When a sample is processed and analyzed as described in the Example, the retention time of the marker is about the stated value for the marker; that is, within about 10% of the declared value, within about 5% of the declared value, or within about 1% of the declared value, and the marker has a mass-to-charge ratio of about the declared value for the marker; that is, within about 10% of the declared value, within about 5% of the declared value, or within about 1% of the declared value.

[0142] Another embodiment of the present disclosure relates to an assay system including a plurality of antibodies, or antigen-binding fragments thereof, or aptamers for detecting the expression of differentially expressed biomarkers in individuals with interstitial lung disease. The plurality of antibodies, or antigen-binding fragments thereof, or aptamers, consists of antibodies, or antigen-binding fragments thereof, or aptamers that selectively bind to proteins differentially expressed in individuals with interstitial lung disease, and which may be detected as protein products using antibodies or aptamers. Furthermore, the plurality of antibodies, or antigen-binding fragments thereof, or aptamers, comprise antibodies, or antigen-binding fragments thereof, or aptamers that selectively bind to proteins or portions thereof (peptides) encoded by any of the genes in the tables provided in this document.

[0143] Certain embodiments of the present disclosure utilize a plurality of biomarkers that have been identified herein as being present or differentially expressed in subjects with interstitial lung disease. As used herein, the terms "patient" "a subject who has interstitial lung disease", "subject with interstitial pneumonia", "patient with interstitial lung disease", "subject with interstitial pneumonia", etc. are intended to refer to subjects who have been diagnosed with interstitial lung disease (e.g., IIP, IPF, FIP). The terms "non-subject", "normal individual", "a subject who does not have interstitial lung disease", etc. are intended to refer to a subject who has not been diagnosed with interstitial lung disease. A subject without interstitial lung disease may be healthy and have no other disease, or he may have a disease other than interstitial lung disease.

[0144] The plurality of biomarkers within the above limitation includes at least two or more biomarkers (e.g., at least 2, 3, 4, 5, 6, and so on, in whole number increments, up to all disclosed biomarkers) , and includes any combination of such biomarkers. Such biomarkers are selected from any of the polymorphisms or polypeptides listed in the tables provided herein and polypeptides encoded by any of the genes listed in the Tables. In some embodiments, the plurality of biomarkers used in the present disclosure includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or all biomarkers that have been demonstrated to be being predictive of the development or progression of or clinical outcome of an individual diagnosed with or suspected of having interstitial lung disease, such as interstitial pneumonia.

[0145] The polypeptide and polynucleotide markers of the disclosure are useful in methods for diagnosing interstitial lung disease, determining the extent and/or severity of disease, monitoring disease progression, response to therapy, and/or the need for a lung transplant . The markers are also useful in methods for treating interstitial lung disease and for evaluating the effectiveness of treatment for the disease. Such methods can be performed on human and non-human subjects. Markers can also be used as pharmaceutical compositions or in kits. Tags can also be used to screen candidate compounds that modulate expression. The markers can also be used to screen candidate drugs for treating interstitial lung disease. Such screening methods can be performed on human and non-human subjects.

[0146] Polypeptide tags can be isolated by any suitable method known in the art. Natural polypeptide tags can be purified from natural sources by standard methods known in the art (e.g., chromatography, centrifugation, differential solubility, immunoassay). In one embodiment, polypeptide tags can be isolated from a serum sample using the chromatographic methods disclosed herein. In another embodiment, polypeptide tags can be isolated from a sample by contacting the sample with substrate-bound antibodies or aptamers that specifically bind to the tag.

[0147] Polynucleotide tags can be found in mRNA, cDNA or genomic DNA transcripts and can include polynucleotides that encode the polypeptides of the disclosure. In one embodiment, the disclosure provides polynucleotides that encode a polypeptide tag, or a molecule comprising such a polypeptide. In another embodiment, the disclosure provides polynucleotides that encode a polypeptide with substantial sequence identity to a polypeptide tag, or a molecule comprising such a polypeptide.

[0148] In another embodiment, the disclosure provides polynucleotides that encode a polypeptide that is a fragment, precursor, successor or modified version of a marker, or a molecule that comprises such a polypeptide.

[0149] In another embodiment, the disclosure provides polynucleotides that have substantial sequence similarity to a polynucleotide that encodes a polypeptide that is a fragment, precursor, successor or modified version of a marker, or a molecule comprising such a polypeptide. Two polynucleotides have "substantial sequence identity" when there is at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, at least about 95% of sequence identity, or at least 99% sequence identity between their amino acid sequences, or when polynucleotides are capable of forming a stable duplex with each other under stringent hybridization conditions. Such conditions are described elsewhere in this document. As described above in relation to polypeptides, the disclosure includes polynucleotides that are allelic variants, the result of SNPs, or those in alternative codons to those present in natural materials as inherent in the degeneracy of the genetic code.

[0150] In some embodiments, the described polynucleotides can be used as surrogate markers of interstitial lung disease. Thus, for example, if the level of a polypeptide marker is increased in subjects with interstitial lung disease, an increase in the mRNA encoding the polypeptide marker may be interrogated instead of the polypeptide marker (e.g., to diagnose interstitial lung disease). in a subject).

[0151] Polynucleotide markers can be isolated by any suitable method known in the art. Natural polynucleotide tags can be purified from natural sources by standard methods known in the art. In one embodiment, a polynucleotide tag can be isolated from a mixture by contacting the mixture with substrate-bound probes that are complementary to the polynucleotide tag under hybridization conditions.

[0152] Alternatively, polynucleotide tags can be synthesized by any suitable chemical or recombinant method known in the art. In one embodiment, for example, the markers can be synthesized using the methods and techniques of organic chemistry. In another embodiment, a polynucleotide tag can be produced by polymerase chain reaction (PCR).

[0153] The present disclosure also encompasses molecules that specifically bind to the polypeptide or polynucleotide tags of the present disclosure. In one aspect, the disclosure provides molecules that specifically bind to a polypeptide tag or a polynucleotide tag. As used herein, the term "specifically binding" refers to the interaction between binding pairs (e.g., an antibody and an antigen or aptamer and its target). In some embodiments, the interaction has an affinity constant of at most 10-6 mol/liter, at most 10-7 mol/liter, or at most 10-8 mol/liter. In other embodiments, the phrase "specifically binds" refers to the specific binding of one protein to another (e.g., an antibody, fragment thereof, or binding partner to an antigen), wherein the level of binding, such as measured by any standard assay (e.g., an immunoassay), is statistically significantly greater than the background control for the assay. For example, when performing an immunoassay, controls typically include a reaction well/tube that contains the antibody or antigen-binding fragment alone (i.e., in the absence of antigen), in which an amount of reactivity (e.g., binding is not specific to the well) by the antibody or antigen-binding fragment thereof in the absence of antigen is considered as background. Binding can be measured using a variety of methods standard in the art, including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc.).

[0154] Binding molecules include antibodies, aptamers and antibody fragments. As used herein, the term "antibody" refers to an immunoglobulin molecule capable of binding to an epitope present on an antigen. The term is intended to encompass not only intact immunoglobulin molecules, such as monoclonal and polyclonal antibodies, but also bispecific antibodies, humanized antibodies, chimeric antibodies, anti-idiopathic (anti-ID) antibodies, single chain antibodies, Fab fragments, F(ab' fragments), fusion proteins and any modifications of the foregoing that comprise an antigen recognition site of the required specificity. As used herein, an aptamer is a non-naturally occurring nucleic acid with a desirable action on a target. A desirable action includes, but is not limited to, binding to the target, catalytically changing the target, reacting with the target in a way that modifies/alters the target or the functional activity of the target, covalently attaching to the target as in a suicide inhibitor, facilitate the reaction between the target and another molecule. In some embodiments, the action is specific binding affinity for a target molecule, such as the target molecule being a three-dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism that relies predominantly on Watson/Watson base pairing. Crick or triple helix ligation, in which the nucleic acid ligand is not a nucleic acid with the known physiological function of being bound by the target molecule.

[0155] In one aspect, the disclosure provides antibodies or aptamers that specifically bind to a SNP marker, or to a molecule comprising an above component (e.g., a protein comprising a polypeptide encoded by a marker gene).

[0156] In another embodiment, the disclosure provides antibodies or aptamers that specifically bind to a polypeptide with substantial sequence identity to a marker gene, or to a molecule comprising a polypeptide above.

[0157] In another embodiment, the disclosure provides antibodies or aptamers that specifically bind to a polypeptide tag or a polynucleotide tag that is structurally different from a tag specifically identified in the tables provided herein, but has the same (or nearly the same same) function or properties, or to a molecule comprising an above component.

[0158] Another embodiment of the present disclosure relates to a plurality of aptamers, antibodies, or antigen-binding fragments thereof, for detecting the expression of differentially expressed biomarkers in individuals with interstitial pneumonia. The plurality of aptamers, antibodies, or antigen-binding fragments thereof, consists of antibodies, or antigen-binding fragments thereof, that selectively bind to proteins differentially expressed in individuals with interstitial lung disease, and which can be detected as protein products using antibodies. Furthermore, the plurality of aptamers, antibodies, or antigen-binding fragments thereof, comprise antibodies, or antigen-binding fragments thereof, that selectively bind to proteins or portions thereof (peptides) encoded by any of the genes of the tables provided in this document.

[0159] In accordance with the present disclosure, a plurality of aptamers, antibodies, or antigen-binding fragments thereof, refers to at least 2, and more preferably at least 3, and most preferably at least 4, and more preferably at least 5, and more preferably at least 6, and more preferably at least 7, and more preferably at least 8, and most preferably at least 9, and most preferably at least 10, and so on, in increments from one to any suitable number of antibodies, or antigen-binding fragments thereof, including, in some embodiments, antibodies representing all of the biomarkers described herein, or antigen-binding fragments thereof.

[0160] Certain antibodies that specifically bind to polypeptide tags and polynucleotide tags of the disclosure may already be known and/or available for purchase from commercial sources. In any case, the antibodies of the disclosure may be prepared by any appropriate means known in the art. For example, antibodies can be prepared by immunizing an animal host with a marker or an immunogenic fragment thereof (conjugated to a carrier, if necessary). Adjuvants (e.g., Freund's adjuvant) may optionally be used to enhance the immune response. Serum containing polyclonal antibodies with high affinity for the antigenic determinant can then be isolated from the immunized animal and purified.

[0161] Alternatively, antibody-producing tissue from the immunized host can be collected and a cellular homogenate prepared from the organ can be fused to cultured cancer cells. Hybrid cells that produce monoclonal antibodies specific to a marker can be selected. Alternatively, the antibodies of the disclosure may be produced by chemical synthesis or recombinant expression. For example, a polynucleotide encoding the antibody can be used to construct an expression vector for producing the antibody. The antibodies of the present disclosure can also be generated using various phage display methods known in the art.

[0162] Antibodies or aptamers that specifically bind to markers of the disclosure can be used, for example, in methods for detecting biomarkers of this disclosure using methods and techniques known in the art. In some embodiments, for example, antibodies are conjugated to a detection molecule or moiety (e.g., a dye and enzyme) and may be used in ELISA or sandwich assays to detect markers of the disclosure.

[0163] In another embodiment, antibodies or aptamers against a polypeptide tag or polynucleotide tag of the disclosure can be used to analyze a tissue sample (e.g., a thin cortical slice) for the tag. Antibodies or aptamers can specifically bind to the marker, if any, present in the tissue sections and allow localization of the marker in the tissue. Similarly, antibodies or aptamers labeled with a radioisotope can be used for image capture or in vivo treatment applications.

[0164] Another aspect of the disclosure provides compositions comprising a polypeptide or polynucleotide tag of the disclosure, a binding molecule that is specific for a polypeptide or polynucleotide tag (e.g., an antibody or an aptamer), an inhibitor of a tag of polypeptide or polynucleotide, or other molecule that can increase or decrease the level or activity of a polypeptide tag or polynucleotide tag. Such compositions may be pharmaceutical compositions formulated for use as a therapy.

[0165] Alternatively, the disclosure provides a composition comprising a component that is a fragment, modification, precursor or successor of a marker of the invention, or a molecule comprising an above component.

[0166] In another embodiment, the disclosure provides a composition comprising a polynucleotide that binds to a polypeptide or a molecule comprising a polynucleotide above.

[0167] In another embodiment, the disclosure provides a composition comprising an antibody or aptamer that specifically binds to a polypeptide or a molecule comprising an antibody or aptamer above.

Methods for detecting a genetic variant

[0168] The present disclosure also provides methods for detecting biomarkers of the present disclosure. The practice of the present disclosure employs, unless otherwise indicated, conventional methods of analytical biochemistry, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are fully explained in the literature. (See, for example, Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2000; DNA Cloning: A Practical Approach, Vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, Vol. I & II (P. Tijessen, ed.); Fundamental Virology, 2nd Edition, Vol. I & II (B. N. Fields and D. M. Knipe, eds.)).

[0169] 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 may employ any suitable method for detecting the presence or absence of a variant(s), of the which numerous detection methods are known in the art. Dynamic allele-specific hybridization (DASH) can be used to detect a genetic variant. DASH genotyping takes advantage of differences in melting temperature in DNA that result from the instability of mismatched base pairs. The process can be largely automated and encompasses a few simple principles. Thus, the aspects and modalities described in this document provide methods for evaluating the presence or absence of SNPs in a sample (e.g., biological sample) from a subject suspected of having or developing an interstitial lung disease (e.g., due to family history ). In certain embodiments, one or more SNPs are screened in one or more samples from a subject. SNPs may be associated with one or more genes, for example, one or more genes or other genes associated with mucous secretions, as disclosed herein.

[0170] Typically, the target genomic segment is amplified and separated from the non-target sequence, for example, through the 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 specifically hybridize to a variant sequence or dominant allele sequence. The probe can be 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 the temperature is increased until the Tm can be determined. A mismatched sequence (genetic variant or dominant allelic sequence, depending on the probe design) will result in a lower Tm than expected.

[0171] DASH genotyping is based 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.

[0172] Molecular flags 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 between them. This design allows the probe to incorporate a hairpin, or hairpin-loop, structure in its natural isolated state. A fluorophore is attached to one end of the probe, and a fluorescence quencher is attached to the other end. Due to the hairpinloop 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 designed in such a way that only the probe sequence is complementary to the targeted genomic DNA sequence.

[0173] If the molecular beacon probe sequence encounters its target genomic DNA sequence during the assay, it will pair and hybridize. Due to the length of the probe sequence, the hairpin segment of the probe will be denatured in favor of forming a longer and more stable probe-target hybrid. This conformational change allows the fluorophore and quencher to be freed from their close proximity due to hairpin association, allowing the molecule to fluoresce.

[0174] If, on the other hand, the probe sequence encounters a target sequence with only one non-complementary nucleotide, the molecular beacon preferentially remains in its natural hairpin state and no fluorescence will be observed, since the fluorophore remains deactivated. The unique design of these molecular beacons allows a simple diagnostic assay to identify SNPs at a given location. If one 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 an individual's genotype. If only the wavelength of the fluorophore of the first probe is detected during the assay, then the individual is homozygous for wild type. If only the wavelength of the second probe is detected, then the individual is homozygous for the mutant allele. Finally, if both wavelengths are detected, then both molecular beacons must be hybridized to their complements and thus the individual must contain both alleles and be heterozygous.

[0175] A microarray can also be used to detect genetic variants. Hundreds of thousands of probes can be arrayed on a small chip, allowing many genetic variants or SNPs to be interrogated simultaneously. Since SNP alleles differ by only one nucleotide and since it is difficult to achieve optimal hybridization conditions for all probes on the array, target DNA has the potential to hybridize to incompatible probes. This can be resolved by using multiple redundant probes to interrogate each SNP. Probes can be designed to have the SNP site in several different locations, as well as contain incompatibilities with the SNP allele. By comparing the differential amount of hybridization of target DNA to each of these redundant probes, it is possible to determine specific homozygous and heterozygous alleles.

[0176] Restriction fragment length polymorphism (RFLP) can be used to detect genetic variants and SNPs. RFLP makes use of the many different restriction endonucleases and their high affinity for unique and specific restriction sites. By performing a digestion on a genomic sample and determining fragment lengths through an in-gel assay, it is possible to determine whether the enzymes cut the expected restriction sites or not. A failure to cut the genomic sample results in a fragment that is identifiably larger than expected, implying that there is a point mutation at the restriction site that is protecting it against nuclease activity.

[0177] PCR and amplification-based methods can be used to detect genetic variants. For example, 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 one SNP site, but each perfectly matches only one of the possible alleles. 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 the alternative allele with a different allele sequence. 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 marked differently.

[0178] Primer extension can also be used to detect genetic variants. Primer extension involves first hybridizing a probe to bases immediately upstream of the SNP nucleotide, followed by a 'minisequencing' 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. Since primer extension is based on the highly accurate DNA polymerase enzyme, the method is generally very reliable. Primer extension is capable of genotyping 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, for example, fluorescent markers or mass spectrometry.

[0179] Primer extension may involve the incorporation of fluorescently labeled ddNTP or fluorescently labeled deoxynucleotides (dNTP). With ddNTPs, probes hybridize to target DNA immediately upstream of the 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 more nucleotides from being added). Each ddNTP is labeled with a different fluorescent signal, allowing detection of all four alleles in the same reaction. With dNTPs, allele-specific probes have 3' bases that are complementary to each of the SNP alleles being interrogated. If the target DNA contains an allele complementary to the 3' base of the probe, the target DNA will hybridize completely to the probe, allowing DNA polymerase to extend from the 3' end of the probe. This is detected by the incorporation of fluorescently labeled dNTPs at the end of the probe. If the target DNA does not contain an allele complementary to the 3' base of the probe, the target DNA will produce a mismatch at the 3' end of the probe, and the DNA polymerase will not be able to extend from the 3' end of the probe.

[0180] The iPLEX® SNP genotyping method takes a slightly different approach, and is based on mass spectrometer detection. Extension probes are designed such that many different SNP assays can be amplified and analyzed in one PCR cocktail. The extension reaction uses ddNTPs as above, but 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.

[0181] Primer extension methods are, however, amenable to high-throughput analysis. Primer extension probes can be arranged on slides, allowing many SNPs to be genotyped at the same time. Widely known as arrayed primer extension (APEX), this technology has several advantages over methods based on differential probe hybridization. Comparatively, APEX methods have greater discriminatory power than methods that use differential hybridization, since it is often impossible to obtain optimal hybridization conditions for the thousands of probes on DNA microarrays (this is often solved by having highly redundant probes). .

[0182] Oligonucleotide binding 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 to the polymorphic SNP site, whereby binding can occur if the probes are identical to the target DNA. For example, two probes can be designed: an allele-specific probe that hybridizes to the target DNA so that its 3' base lies directly over the SNP nucleotide; and a second probe that hybridizes to the template upstream (downstream on the complementary strand) of the polymorphic SNP 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 binding can occur. Binding generally does not occur in the presence of an incompatible 3' base. Bound or unbound products can be detected by gel electrophoresis, MALDI-TOF mass spectrometry, or by capillary electrophoresis.

[0183] The 5'-nuclease activity of Taq DNA polymerase can be used to detect genetic variants. The assay is performed simultaneously with a PCR reaction and results can be read in real time. The assay requires sense 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 attached to their 5' end and a deactivator molecule attached to their 3' end. As long as the probe is intact, the deactivator will remain in close proximity to the fluorophore, eliminating the fluorophore signal. During the PCR amplification step. If the allele-specific probe is perfectly complementary to the SNP allele it will bind to the target DNA strand and then be degraded by the 5'-nuclease activity of Taq polymerase as it extends the DNA from the PCR primers. Degradation of the probe results in separation of the fluorophore from the deactivating molecule, generating a detectable signal. If the allele-specific probe is not perfectly complementary, it will have a lower melting temperature and will not bind as efficiently. This prevents the nuclease from acting on the probe.

[0184] Forster resonance energy transfer (FRET) detection can be used for detection in primer extension and ligation reactions where the two labels are placed in close proximity to each other. It can also be used in the 5'-nuclease reaction, the molecular beacon reaction, and invasive cleavage reactions where the adjacent donor/acceptor pair is separated by cleavage or disruption of the hairpin-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, since energy transfer drops off rapidly with distance. The proximity requirement is what makes FRET a good detection method for a number of allelic discrimination mechanisms.

[0185] A variety of dyes can be used for FRET and are known in the art. The most common 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). The optics for imaging all of them require UV to near-IR detection (e.g. Alex 405 to Cy7) and the Atto series of dyes (Atto-Tec GmbH). Invitrogen's Alexa series of dyes covers the entire spectral range. They are very bright and photostable.

[0186] Exemplary 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 et al. (2008) J Am Chem Soc 130:5038-39; Vosch et al. (2007) Proc Natl Acad Sci USA 104:12616- 21; Petty and Dickson (2003) J Am Chem Soc 125:7780- 81. Available filters, dichroics, multichroic mirrors and lasers can affect the choice of dye.

Methods for detecting markers, including polynucleotide and polypeptide expression levels

[0187] The markers of the disclosure can be detected by any method known to those skilled in the art, including, without limitation, LC-MS, GC-MS, immunoassays, hybridization and enzymatic assays. Detection can be quantitative or qualitative. A wide variety of conventional techniques are available, including mass spectrometry, chromatographic separations, 2D gel separations, binding assays (e.g., immunoassays), competitive inhibition assays, and so on. Any method effective in the art for measuring the presence/absence, level, or activity of a polypeptide or polynucleotide is included in the disclosure. It is within the ability of one skilled in the art to determine which method would be most appropriate for measuring a specific marker. So, for example, an ELISA assay may be better suited for use in a doctor's office, while a measurement that requires more sophisticated instrumentation may be better suited for use in a clinical laboratory. Regardless of the method selected, it is important that measurements are reproducible.

[0188] The disclosure markers can be measured by mass spectrometry, which allows direct measurements of analytes with high sensitivity and reproducibility. Numerous mass spectrometry methods are available. Electrospray ionization (ESI), for example, allows the quantification of differences in the relative concentration of various species in one sample versus another; Absolute quantification is possible through normalization techniques (e.g. using an internal standard). Matrix-assisted laser desorption ionization (MALDI) or related SELDI® (Ciphergen, Inc.) technology can also be used to make a determination of whether a tracer was present and the relative or absolute level of the tracer. Mass spectrometers that allow time-of-flight (TOF) measurements have high accuracy and resolution and are capable of measuring low-abundance species, even in complex matrices such as serum or CSF.

[0189] For protein tags, quantification can be based on derivatization in combination with isotopic labeling, referred to as isotope-coded affinity tags ("ICAT"). In this and related methods, a specific amino acid in two samples is differentially and isotopicly labeled and subsequently separated from the peptide background by solid phase capture, washing, and release. The intensities of molecules from the two sources with different isotopic labels can be accurately quantified relative to each other. Quantification can also be based on the isotope dilution method by introducing an isotopically labeled peptide or protein analogous to those being measured. Furthermore, quantification can also be determined without isotopic standards using the direct intensity of the analyte compared to another measurement of a standard in a similar matrix.

[0190] Additionally, one- and two-dimensional gels have been used to separate proteins and quantify gel spots by silver staining, fluorescence or radioactive labeling. These differently colored dots were detected using mass spectrometry and identified by tandem mass spectrometry techniques.

[0191] In one embodiment, the markers are measured using mass spectrometry in connection with a separation technology, such as liquid chromatography/mass spectrometry or gas chromatography/mass spectrometry. In particular, high-resolution reversed-phase coupling liquid chromatography, time-of-flight mass spectrometry (TOF) and high mass accuracy ESI allow measurement of the spectral intensity of a large number of biomolecules from a relatively small amount of any complex biological material. Analyzing a sample in this manner allows the marker (characterized by a specific RT and m/z) to be determined and quantified.

[0192] As will be appreciated by one skilled in the art, many other separation technologies can be used in conjunction with mass spectrometry. For example, a wide selection of separation columns is commercially available. Furthermore, separations can be performed using custom chromatographic surfaces (e.g., a bead onto which a marker-specific reagent has been immobilized). Molecules subsequently retained in the medium can be eluted for analysis by mass spectrometry.

[0193] Liquid chromatography-mass spectrometry analysis produces a mass intensity spectrum, the peaks of which represent various components of the sample, each component having a characteristic mass-to-charge ratio (m/z) and retention time (RT). . The presence of a peak with the m/z and RT of a marker indicates that the marker is present. The peak representing a marker can be compared to a corresponding peak from another spectrum (e.g., from a control sample) to obtain a relative measurement. Any normalization technique in the art (e.g., an internal standard) can be used when a quantitative measurement is desired. "Deconvolution" software is available to separate overlapping peaks. Retention time depends, to some extent, on the conditions employed in the performance of liquid chromatography separation. Suitable conditions, those used to obtain the retention times appearing in the Tables, are defined in the Example. The mass spectrometer preferably provides high mass accuracy and high mass resolution. The mass accuracy of a well-calibrated Micromass TOF instrument, for example, is reported to be approximately 5 mDa, with m/Δm resolution greater than 5000.

[0194] In some embodiments, the level of the markers can be determined using a standard immunoassay, such as sandwich ELISA using corresponding antibody pairs and chemiluminescent detection. Commercially available or custom monoclonal or polyclonal antibodies are typically used. However, the assay can be adapted for use with other reagents that specifically bind to the marker. Standard protocols and data analysis are used to determine tracer concentrations from assay data.

[0195] A number of the assays discussed above employ a reagent that specifically binds to the marker. Any molecule that is capable of specifically binding to a marker is included within the disclosure. In some embodiments, the binding molecules are antibodies or antibody fragments. In other embodiments, the binding molecules are different species of antibodies, such as aptamers. Thus, for example, the binding molecule may be an enzyme for which the label is a substrate. Binding molecules can recognize any epitope of the targeted markers.

[0196] As described above, binding molecules can be identified and produced by any method accepted in the art. Methods for identifying and producing antibodies and antibody fragments specific to an analyte are well known. Examples of other methods used to identify binding molecules include binding assays with random peptide libraries (e.g., phage display) and design methods based on an analysis of the tag structure.

[0197] The markers of the disclosure may also be detected or measured using a number of chemical derivatization or reaction techniques known in the art. Reagents for use in such techniques are known in the art and are commercially available for certain classes of target molecules.

[0198] Finally, the chromatographic separation techniques described above can also be coupled to an analytical technique other than mass spectrometry, such as fluorescence detection of labeled molecules, NMR, capillary UV, evaporative light scattering or electrochemical detection.

[0199] Measuring the relative amount of an RNA or protein marker of the disclosure may be by any method known in the art (see, for example, Sambrook, J., Fritsh, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Typical methodologies for RNA detection include extraction of RNA from a cell or tissue sample, followed by hybridization of a labeled probe (e.g., a complementary polynucleotide) specific for the target RNA to the extracted RNA, and detection of the probe (e.g., example, Northern blotting). Typical methodologies for protein detection include protein extraction from a cell or tissue sample, followed by hybridization of a labeled probe (e.g., an antibody) specific for the target protein to the protein sample, and detection of the probe. The labeling group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Detection of specific proteins and polynucleotides can also be assessed by gel electrophoresis, column chromatography, direct sequencing, or quantitative PCR (in the case of polynucleotides) among many other techniques well known to those skilled in the art.

[0200] Detection of the presence or number of copies of all or part of a marker gene of the disclosure can be performed using any method known in the art. Typically, it is convenient to assess the presence and/or quantity of a DNA or cDNA by Southern analysis, in which total DNA from a cell or tissue sample is extracted, hybridized with a labeled probe (e.g., a complementary DNA molecule ), and the probe is detected. The labeling group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Other useful methods of detecting and/or quantifying DNA include direct sequencing, gel electrophoresis, column chromatography, and quantitative PCR, as known to one skilled in the art.

[0201] The similarity of polynucleotides can be assessed by hybridizing single-stranded nucleic acids with complementary or partially complementary sequences. Such experiments are well known in the art. High stringency hybridization and washing conditions, as referred to herein, refer to conditions that permit the isolation of nucleic acid molecules with at least about 80% nucleic acid sequence identity to the nucleic acid molecule being used. as a probe in the hybridization reaction (i.e., conditions that allow about 20% or less nucleotide mismatch).

[0202] Very high stringency hybridization and washing conditions, as referred to herein, refer to conditions that allow the isolation of nucleic acid molecules with at least about 90% nucleic acid sequence identity to the nucleic acid molecule. nucleic acid being used as a probe in the hybridization reaction (i.e., conditions that allow about 10% or less nucleotide mismatch). As discussed above, one skilled in the art can use the formulas in Meinkoth et al., ibid. to calculate the appropriate hybridization and washing conditions to achieve these particular levels of nucleotide mismatch. Such conditions will vary depending on the formation of DNA:RNA or DNA:DNA hybrids. Calculated melting temperatures for DNA:DNA hybrids are 10 °C lower than for DNA:RNA hybrids. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na+) at a temperature between about 20°C and about 35°C (lower stringency), more preferably, between about 28°C and about 40°C (more stringent) and even more preferably, between about 35°C and about 45°C (further stringent), with appropriate washing conditions. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na+) at a temperature between about 30°C and about 45°C, more preferably, between about 38°C. °C and 50 °C and even more preferably between about 45 °C and about 55 °C, with similarly stringent washing conditions. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide, and a G+C content of about 40%. Alternatively, Tm can be calculated empirically as set out in Sambrook et al., supra, pages 9.31 to 9.62. In general, washing conditions should be as stringent as possible and should be appropriate for the chosen hybridization conditions. For example, hybridization conditions may include a combination of temperature and salt conditions that are approximately 20-25°C below the calculated Tm of a particular hybrid, and washing conditions typically include a combination of temperature and salt conditions that are about 12-20°C below the calculated Tm of the particular hybrid. An example of suitable hybridization conditions for use with DNA:DNA hybrids includes a 2-24 hour hybridization in 6X SSC (50% formamide) at about 42°C, followed by wash steps that include one or more washes at room temperature in about 2X SSC, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash at about 37°C in about 0.1X-0.5X SSC, followed by at least a wash at about 68°C in about 0.1X-0.5X SSC). Other hybridization conditions, and, for example, those most useful with nucleic acid arrays, will be known to those skilled in the art.

Diagnosis, monitoring and treatment of interstitial lung disease

[0203] The present disclosure includes methods for diagnosing interstitial lung diseases, such as interstitial pneumonia, idiopathic interstitial pneumonia, familial interstitial pneumonia, idiopathic pulmonary fibrosis, etc., stratifying patients between different types of interstitial lung disease, and/or excluding other types of lung disease that cause similar symptoms and show similar abnormalities on chest x-rays, and related methods. In general, it is expected that the biomarkers described in this document will be measured in combination with other signs, symptoms, and clinical tests of interstitial lung disease, such as radiographs, pathological evaluation of lung tissue, or biomarkers of interstitial lung disease reported in the literature. Likewise, more than one of the biomarkers of the present disclosure can be measured in combination. Measuring the biomarkers of the disclosure along with any other markers known in the art, including those not specifically listed herein, falls within the scope of the present disclosure. Appropriate markers for this modality include those that have been identified as present or increased in samples obtained from biological samples, and especially from lung, compared to normal or control samples. Other suitable tags for this embodiment include fragments, precursors, successors and modified versions of such tags, polypeptides with substantial sequence identity to such tags. Other appropriate markers for this embodiment will be apparent to one skilled in the art in light of the disclosure herein.

[0204] The term "interstitial lung disease" or "ILD" is used herein in accordance with its simple and common meaning in the art. Interstitial lung diseases are lung diseases that affect the interstitium. ILDs can be characterized by shortness of breath, chronic cough, fatigue and weakness, and/or rapid loss of appetite. Whenever an aspect or modality in this document refers to ILD, the ILD may be IIP. Whenever an aspect or modality in this document refers to ILD, the ILD may be FIP. Whenever an aspect or modality in this document refers to ILD, the ILD may be IPF. Whenever an aspect or modality in this document refers to ILD, the ILD may be IIP. Additional fibrotic lung diseases include Acute Interstitial Pneumonia (AlP), Interstitial Lung Disease associated with Respiratory Bronchiolitis (RBILD), Desquamative Interstitial Pneumonia (DIP), Nonspecific Interstitial Pneumonia (NSIP), Bronchiolitis Obliterans with Organizing Pneumonia (BOOP). AIP is a rapidly progressing and histologically distinct form of interstitial pneumonia. The pathological pattern is an organized form of diffuse alveolar damage (DAD) that is also found in acute respiratory distress syndrome (ARDS) and other acute interstitial pneumonias of known causes (see Clinical Atlas of Interstitial Lung Disease (2006 ed.). pp 61-63).

[0205] RBILD is characterized by inflammatory lesions of the respiratory bronchioles in smokers. The histological appearance of RBILD is characterized by the accumulation of pigmented macrophages within the respiratory bronchioles and adjacent air spaces, variably peribronchial fibrotic alveolar septal thickening, and minimal associated mural inflammation (see Wells et al. (2003) No Breath. Crit. Care Med. vol. 24).

[0206] PID is a rare interstitial lung disease characterized by the accumulation of macrophages in large numbers in alveolar spaces associated with interstitial inflammation and/or fibrosis. Macrophages often contain light brown pigment. Lymphoid nodules are common, as are scattered but distinct eosinophilic infiltrates. DIP is more common in smokers (see Tazelaar et al. (Sep. 21, 2010) Histopathology).

[0207] NSIP is characterized pathologically by uniform interstitial inflammation and the appearance of fibrosis over a short period of time. NSIP differs from other interstitial lung diseases in that it has a generally good prognosis. Furthermore, the temporal uniformity of parenchymal changes seen in NSIP contrasts sharply with the temporal heterogeneity of usual interstitial pneumonia (see Coche et al. (2001) Brit J Radiol74:189).

[0208] BOOP, unlike NSIP, can be fatal within days of the first acute symptoms. It is characterized by rapid onset of acute respiratory distress syndrome; therefore, clinically, rapidly progressing BOOP may be indistinguishable from acute interstitial pneumonia. Histologic features include clusters of mononuclear inflammatory cells that form granulation tissue and obstruct the distal airways and alveolar spaces. These granulation tissue obstructions may form polyps that migrate within the alveolar ducts or may be focally attached to the wall. (see White & Ruth-Saad (2007) Crit. Care Nurse 27:53).

[0209] More details about the characteristics and therapies available for these diseases can be found, for example, on the American Lung Association website at lungusa.org/lung-disease/pulmonary-fibrosis. Diagnostic indicators of lung disorders include biopsy (e.g., VATS or surgical lung biopsy), high-resolution computed tomography (HRTC), or respiration metrics such as forced expiratory volume (FEV1), vital capacity (VC), forced vital capacity (FVC) and FEV1/FVC.

[0210] Idiopathic interstitial pneumonias (IIP) may include idiopathic pulmonary fibrosis and familial interstitial pneumonia (FIP). Idiopathic interstitial pneumonias (IIP) are a subset of diffuse interstitial lung diseases of unknown etiology (the term "idiopathic" indicates unknown origin). IIPs are characterized by expansion of the interstitial compartment (i.e., that portion of the lung parenchyma sandwiched between the epithelial and endothelial basement membranes) with an infiltration. The infiltrate may be accompanied by fibrosis, either in the form of abnormal collagen deposition or proliferation of fibroblasts capable of synthesizing collagen.

[0211] Idiopathic Pulmonary Fibrosis (IPF) occurs in thousands of people around the world with a doubling of prevalence in the last 10 years. The onset of IPF occurs around 50 to 70 years of age and begins with progressive shortness of breath and hypoxemia. Median survival from IPF is about 3-5 years. The etiology and pathogenesis of the condition is not well understood. About 5-20 percent of all cases of IPF have a family history and inheritance appears to be autosomal dominant.

[0212] Methods for determining whether a subject has interstitial lung disease are provided herein. In another aspect, the disclosure provides methods for diagnosing interstitial lung disease in a subject. These methods comprise obtaining a biological sample from a subject suspected of having interstitial pneumonia, or at risk of developing interstitial lung disease, detecting the presence or level or activity of one or more biomarkers in the sample, and comparing the result to the level or activity present of the marker(s) in a sample obtained from a control or normal subject, or at a reference range or value. As used herein, the term "biological sample" includes a sample of any bodily fluid or tissue (e.g., mucus, whole blood, peripheral blood mononuclear cells (PBMC), serum, plasma, blood, cerebrospinal fluid, urine, saliva, pulmonary).

[0213] One skilled in the art will understand that a blood sample or a buccal swab would be expected to contain the same genetic sequence information as a lung cell. For detection of a given expression level, samples of lung tissue and other biological fluids are typically used. Biological samples may include a sample of lung mucosa or biological fluid, such as blood or blood components (plasma, serum), sputum, mucus, urine, saliva, etc. A sample of the lung mucosa can be obtained using methods known in the art, for example, a bronchial epithelial brush or exhaled air condensate. Additional methods include bronchial biopsy, bronchial lavage, bronchoalveolar lavage, whole lung lavage, transendoscopic biopsy, translaryngoscopic catheter, and transtracheal lavage. A review of commonly used techniques, including comparisons and safety concerns, is provided in Busse et al. (2005) Am J Respir Crit Care Med 172:807–816. For lavage techniques, a bronchoscope can be inserted to the desired level of the airway. A small volume of physiologically acceptable sterile fluid (e.g., buffered saline) is released and immediately aspirated. The lavage material contains mucosal cells and upper epithelia (Riise et al. (1996) Eur Resp J9:1665). For use of a bronchial epithelial brush, a sterile non-irritating cytology brush (e.g., nylon) can be used. Multiple strokes may be taken to ensure representative sampling. The brush is then agitated in physiologically acceptable fluid and the cells and debris are separated using routine methods (Riise et al. (1992) Eur Resp J 5:382). Cellular components can be isolated using methods known in the art, for example, centrifugation. Similarly, subcellular components (e.g., exosomes or vesicles) can be isolated using known methods or commercial separation products (available from BioCat, System Bio, Bioscientific, etc.). An exemplary method is described, for example, by Thery et al. (2006) Current Prot. Cell Biol.

[0214] Typically, the standard biomarker level or reference range is obtained by measuring the same marker or markers in a set of normal controls. Measurement of the standard biomarker level or reference range does not need to be done simultaneously; it may be a historic measure. Preferably, the normal control matches the individual with respect to some attribute(s) (e.g., age). Depending on the difference between the measured level and the standard level or reference range, the individual may be diagnosed as having interstitial lung disease or as not having interstitial lung disease. In some embodiments, interstitial lung disease is diagnosed in the individual if the level of expression of the biomarker or biomarkers in the interstitial sample is statistically more similar to the level of expression of the biomarker or biomarkers that have been associated with interstitial lung disease than the level of expression of the biomarker or biomarkers that were associated with normal controls.

[0215] What is currently known as interstitial lung disease includes a number of related but distinct conditions. Classifications can be made, and these types can be further distinguished into subtypes. Any and all various forms of interstitial lung disease are intended to be within the scope of the present disclosure. In effect, by providing a method for forming subsets of individuals based on the level of biomarker measurement, the compositions and methods of the present disclosure can be used to discover and define various forms of the disease.

[0216] The methods of the present disclosure can be used to make the diagnosis of interstitial pneumonia regardless of other information, such as the individual's symptoms or the results of other clinical or paraclinical tests. However, the methods of the present disclosure may be used in conjunction with such other data points.

[0217] Since a diagnosis is rarely based solely on the results of a single test, the method can be used to determine whether or not a subject is more likely to have interstitial lung disease, or is more likely to have interstitial lung disease than than having another disease, based on the difference between the measured reference level or range and biomarker standard. Thus, for example, an individual with a putative diagnosis of interstitial lung disease may be diagnosed as being "more likely" or "less likely" to have interstitial lung disease in light of information provided by a method of the present disclosure. If a plurality of biomarkers are measured, at least one and even all of the measured biomarkers must differ, in the appropriate direction, for the subject to be diagnosed as having (or being more likely to have) interstitial lung disease. In some embodiments, such a difference is statistically significant.

[0218] The biological sample can be any tissue or fluid, including a serum or tissue sample, but other biological fluids or tissues can be used. Possible biological fluids include, but are not limited to, mucus, whole blood, peripheral blood mononuclear cells (PBMC), plasma, urine, saliva, and lung tissue. In some embodiments, the level of a marker may be compared to the level of another marker or some other component in a different tissue, fluid, or biological "compartment." Thus, a differential comparison can be made of a marker in tissue and serum. It is also within the scope of the disclosure to compare the level of a marker with the level of another marker or some other component within the same compartment.

[0219] As will be apparent to those ordinarily skilled in the art, the above description is not limited to making an initial diagnosis of interstitial lung disease, but is also applicable to confirming a provisional diagnosis of interstitial lung disease or "ruling out" such a diagnosis. Additionally, an increased or decreased level or activity of the marker(s) in a sample obtained from a subject suspected of having interstitial lung disease, or at risk of developing interstitial lung disease (e.g., with a genetic predisposition), is indicative that the subject has or is at risk of developing interstitial lung disease.

[0220] Based on the diagnosis, a practitioner can further determine a course of treatment for interstitial lung disease. Therapy options are limited, but may include palliative measures, decongestants, analgesics, immunosuppression, lung transplantation. Furthermore, based on the present disclosure, treatment may include targeted gene or antibody therapy to reduce or correct the expression of the disclosed biomarkers to more normal levels. Treatment may be adjusted over time depending on ongoing monitoring of the subject, e.g., measurement of expression levels of currently reported biomarkers, or other measures such as radiology, oxygen capacity, comfort levels, etc.

[0221] The disclosure also provides a method for determining a subject's risk of developing interstitial lung disease, the method comprising obtaining a biological sample from a subject, detecting the presence, level or activity of a marker in the sample, and comparing the result to the presence, level, or activity of the marker in a sample obtained from a subject without interstitial lung disease, or to a range or reference value in which the presence or an increase or decrease of the marker correlates with the risk of developing interstitial lung disease .

[0222] The disclosure also provides methods for determining the stage or severity of interstitial lung disease, the method comprising obtaining a biological sample from a subject, detecting the presence, level or activity of a marker in the sample, and comparing the result to the presence, level or activity of the marker in a sample obtained from a normal subject or control, or to a range or reference value in which the presence or an increase or decrease of the marker is correlated with the stage or severity of the disease.

[0223] In another aspect, the disclosure provides methods for monitoring disease progression in a subject who has interstitial lung disease, the method comprising obtaining a first biological sample from a subject, detecting the level or activity of a marker in the sample, and compare the result with the level or activity of the marker in a second sample obtained from the subject at a later time, or with a range or reference value in which an increase in the marker is correlated with disease progression.

[0224] A significant difference in the elevation of the measured value of one or more of the gene markers indicates that the individual has (or is more likely to have, or is at risk of having, or is at risk of developing, or has an increased risk of of developing the progression of) interstitial lung disease. If only one biomarker is measured, then this value must increase to indicate interstitial lung disease. If more than one biomarker is measured, then a diagnosis of interstitial pneumonia may be indicated by a change in only one biomarker, all biomarkers, or any number in between. In some embodiments, multiple markers are measured, and a diagnosis of interstitial lung disease is indicated by changes in multiple markers. For example, a panel of markers may include markers that are increased in level or activity in samples from a subject with interstitial lung disease compared to samples from a subject without interstitial lung disease. The measurements may be of (i) a biomarker of the present disclosure, (ii) a biomarker of the present disclosure and another factor known to be associated with interstitial lung disease (e.g., computed tomography); (iii) a plurality of biomarkers of the present disclosure, (iv) a plurality of biomarkers comprising at least one biomarker of the present disclosure and at least one biomarker reported in the literature; (v) a biomarker or plurality of biomarkers of the present disclosure and at least one clinical covariate that may include the subject's age, pathological evaluation results, and (vi) any combination of the foregoing. Furthermore, the amount of change in a biomarker level can be an indication of the relative probability of disease progression.

[0225] The marker(s) may be detected in any biological sample obtained from the subject, by any suitable method known in the art (e.g., immunoassays, hybridization assay). Preferably, the marker(s) is detected in a whole blood sample obtained from the individual.

[0226] In an alternative embodiment of the disclosure, a method is provided for monitoring interstitial lung disease in an individual over time to determine whether the disease is progressing. The specific techniques used in implementing this embodiment are similar to those used in the embodiments described above. The method is performed by obtaining a biological sample, such as serum or lung tissue, from the subject at a certain time (t1); measuring the level of at least one of the biomarkers in the biological sample; and comparing the measured level with the level measured relative to a biological sample obtained from the subject at a previous time point (t0). Depending on the difference between the measured levels, one can see whether the marker level increased, decreased, or remained constant over the interval (t1-t0). A further shift of a marker in the direction indicating interstitial pneumonia, or the additional measurement of increased interstitial lung disease markers, would suggest disease progression during the interval. Subsequent sample acquisitions and measurements can be performed as many times as desired over a series of times t2 to tn.

[0227] The ability to monitor an individual by making serial marker level determinations would represent a valuable clinical tool. Rather than the limited "instant status" provided by a single test, such monitoring would reveal trends in tracer levels over time. In addition to indicating disease progression, monitoring marker levels in an individual could be used to predict exacerbations or indicate the clinical course of the disease. For example, as will be apparent to one skilled in the art, the biomarkers of the present disclosure could be further investigated to distinguish between any or all known forms of interstitial lung disease or any types or subtypes of the disease described later. Furthermore, the sensitivity and specificity of any method of the present disclosure could be further investigated in connection with distinguishing interstitial lung disease from other diseases or for predicting relapse or remission.

[0228] In an analogous manner, the administration of a drug or combination of drugs can be evaluated or reevaluated in light of the test results of the present disclosure. For example, the drug(s) may be administered differently to different subject populations, and measurements corresponding to administration may be analyzed to determine whether differences in the biomarker signature of the invention before and after after administration of the drug are significant. Results from different drug regimens can also be compared with each other directly. Alternatively, the results of the trial may indicate the preference of one drug regimen over another, or indicate that a specific drug regimen should or should not be administered to an individual with interstitial pneumonia. In one embodiment, the finding of elevated levels of the marker genes of the present disclosure in an individual with interstitial lung disease is indicative of a poor prognosis. In another embodiment, the absence of elevated levels of the marker genes of the present disclosure in an individual with interstitial lung disease is indicative of a good prognosis.

[0229] In another aspect, the disclosure provides methods for screening candidate compounds for use as therapeutic compounds in the treatment of interstitial lung disease. In one embodiment, the method comprises screening candidate compounds for those that provide clinical progress after administration to a patient with interstitial lung disease for whom a lung sample has been shown to have elevated levels of the markers of the present disclosure.

[0230] In an analogous manner, the markers of the present disclosure can be used to evaluate the effectiveness of a therapeutic intervention in a subject. The same approach described above would be used, except if an appropriate treatment was initiated, or an in-progress treatment was changed, before the second measurement (i.e. after t0 and before t1). Treatment may be any therapeutic intervention, such as drug administration, dietary restriction, or surgery, and may follow any suitable schedule over any period of time as appropriate for the intervention. The before and after measurements could then be compared to determine whether the treatment had an effect or not. As will be appreciated by one skilled in the art, the determination may be confounded by other overlapping processes (e.g., an exacerbation of the disease during the same period).

[0231] In an additional embodiment, the markers can be used to screen candidate drugs, for example, in a clinical trial, to determine whether a candidate drug is effective in treating interstitial lung disease. At time t0, a biological sample is obtained from each subject in the population of subjects diagnosed with interstitial pneumonia. Then, assays are performed on each subject's sample to measure levels of a biological marker. In some embodiments, only a single marker is monitored, while in other embodiments a combination of markers, until the total number of markers provided herein, is monitored. Next, a predetermined dose of a candidate drug is administered to a portion or subpopulation of the same subject population. Administration of the drug may follow any suitable schedule over any period of time. In some cases, varying doses are administered to different subjects within the subpopulation, or the drug is administered by different routes. At time t1, after drug administration, a biological sample is acquired from the subpopulation and the same assays that were performed previously are performed on the biological samples to obtain measurement values. As before, subsequent sample acquisitions and measurements can be performed as many times as desired over a series of times t2 to tn. In a study, a different subpopulation of the subject population serves as a control group, which is administered a placebo. The same procedure is then followed for the control group: obtain the biological sample, process the sample, and measure the biological markers to obtain a measurement chart.

[0232] Specific doses and routes of delivery can also be examined. The method is performed by administering the candidate drug at specific doses or routes of delivery to subjects with interstitial lung disease; obtaining biological samples, such as serum or tissue, from subjects; measuring the level of at least one of the biomarkers in each of the biological samples; and, comparing the level measured for each sample with other samples and/or a standard level. Typically, the standard level is obtained by measuring the same marker or markers in the subject before administration of the drug. Depending on the difference between measured and standard levels, the drug may be considered to have an effect on interstitial lung disease. If multiple biomarkers are measured, at least one and even all of the biomarkers must change, in the expected direction, for the drug to be considered effective. Preferably, multiple markers must change for the drug to be considered effective, and preferably such a change is statistically significant.

[0233] As will be apparent to those ordinarily skilled in the art, the above description is not limited to a candidate drug, but is applicable to determining whether any therapeutic intervention is effective in treating interstitial lung disease.

[0234] In a typical embodiment, a population of subjects with interstitial lung disease is selected for the study. The population is typically selected using standard protocols for selecting subjects for clinical trials. For example, the subjects are generally healthy, are not taking other medications, and are evenly distributed in age and sex. The subject population can also be divided into multiple groups; for example, different subpopulations may be suffering from different types or different degrees of the disorder for which the drug candidate is intended. Stratification of the population of individuals can be done based on the biomarker levels of the present disclosure.

[0235] In general, a number of statistical considerations must be made when designing the assay to ensure that statistically significant changes in biomarker measurements can be detected after drug administration. The amount of change in a biomarker depends on a number of factors, including drug strength, drug dose, and treatment program. It will be apparent to someone skilled in statistics how to determine appropriate subject population sizes. Preferably, the study is designed to detect relatively small effect sizes.

[0236] Subjects may optionally be "detoxified" from any prior drug use for an appropriate period of time. Detox removes effects of any previous medications so that an accurate baseline measurement can be taken. At time t0, a biological sample is obtained from each subject in the population. Then, an assay or variety of assays is performed on each subject's sample to measure levels of the particular biomarker of disclosure. Assays may use conventional methods and reagents as described above. If the sample is blood, then assays are typically performed on serum or plasma. For other fluids or tissues, additional sample preparation steps are included as necessary before the assays are performed. The assays measure values of at least one of the biological markers described in this document. In some embodiments, only a single marker is monitored, while in other embodiments a combination of factors, up to the total number of markers, is monitored. Markers can also be monitored in conjunction with other measures and factors associated with interstitial lung disease (e.g., MRI imaging). The number of biological markers whose values are measured depends, for example, on the availability of assay reagents, biological fluids, and other resources.

[0237] Next, a predetermined dose of a candidate drug is administered to a portion or subpopulation of the same subject population. Administration of the drug may follow any suitable schedule over any period of time, and the subpopulation may include some or all of the subjects in the population. In some cases, varying doses are administered to different subjects within the subpopulation, or the drug is administered by different routes. Appropriate doses and routes of administration depend on specific characteristics of the drug. At time t1, after drug administration, another biological sample (the "t1 sample") is acquired from the subpopulation. Typically, the sample is the same type of sample and processed in the same manner as the sample acquired from the subject population prior to administration of the drug (the "sample"). The same tests are performed on sample t1 as on sample t0 to obtain measurement values. Subsequent sample acquisitions and measurements can be performed as many times as desired over a series of times t2 to tn.

[0238] Typically, a different subpopulation of the subject population is used as a control group, to which a placebo is administered. The same procedure is then followed for the control group: obtain the biological sample, process the sample, and measure the biological markers to obtain measurement values. Furthermore, different drugs can be administered to any number of different subpopulations to compare the effects of multiple drugs. As will be apparent to those ordinarily skilled in the art, the above description is a very simplified description of a method involving a clinical trial. Clinical trials have many more procedural requirements, and it must be understood that the method is typically implemented following all of these requirements.

[0239] Paired measurements of the various biomarkers are now available for each subject. The different measurement values are compared and analyzed to determine whether the biological markers changed in the expected direction for the drug group but not for the placebo group, indicating that the drug candidate is effective in treating the disease. In some embodiments, such a change is statistically significant. The measurement values at time t1 for the group receiving the candidate drug are compared with standard measurement values, preferably the values measured before the drug was given to the group, i.e. at time t0. Typically, the comparison takes the form of statistical analysis of the measured values of the entire population before and after administration of the drug or placebo. Any conventional statistical method can be used to determine whether changes in biological marker values are statistically significant. For example, pairwise comparisons can be made for each biomarker using a parametric paired t-test or a non-parametric signal or signal rank test, depending on the distribution of the data.

[0240] Additionally, tests can be performed to ensure that statistically significant changes found in the drug group are not also found in the placebo group. Without such testing, it cannot be determined whether the observed changes occur in all individuals and, therefore, are not a result of candidate drug administration.

[0241] Gene marker expression values are higher in samples collected from individuals with interstitial lung disease. A significant decrease in the measured value of one or more of the gene expression markers indicates that the drug is effective. If only one biomarker is measured, then this value must decrease to indicate the effectiveness of the drug. If more than one biomarker is measured, then drug efficacy can be indicated by changes in just one biomarker, all biomarkers, or any number in between. In some embodiments, multiple markers are measured, and drug efficacy is indicated by changes in multiple markers. Measurements may be of both the biomarkers of the present disclosure and other measurements and factors associated with interstitial lung disease. Furthermore, the amount of decrease in a gene biomarker level may be an indication of the relative efficacy of the drug.

[0242] In addition to determining whether a particular drug is effective in treating interstitial lung disease, disclosure biomarkers can also be used to examine dose effects of a candidate drug. There are a number of different ways in which varying doses can be examined. For example, different doses of a drug may be administered to different subject populations, and measurements corresponding to each dose may be analyzed to determine whether differences in the biomarkers of the invention before and after administration of the drug are significant. In this way, a minimum dose needed to effect a change can be estimated. Additionally, results from different doses can be compared with each other to determine how each biomarker behaves as a function of dose. Based on the results of drug screenings, disclosure markers can be used as teragnostics; that is, they can be used to individualize medical treatment.

Kits

[0243] In another aspect, the disclosure provides a kit for detecting polynucleotide or polypeptide marker(s) of the present disclosure. The kit may be prepared as an assay system including any of assay reagents, assay controls, protocols, exemplary assay results, or combinations of these components designed to provide the user with a means of evaluating the level of expression of the marker(s). es) of this disclosure.

[0244] In another aspect, the disclosure provides a kit for diagnosing interstitial lung disease in a subject including reagents for detecting at least one polypeptide or polynucleotide marker in a biological sample from a subject.

[0245] The kits of the disclosure may comprise one or more of the following: an antibody, wherein the antibody specifically binds to a polypeptide tag, a labeled binding partner for the antibody, a solid phase on which the antibody is immobilized or its binding partner, a polynucleotide probe that can hybridize to a polynucleotide tag, primer pairs that, under appropriate reaction conditions can initiate amplification of at least a portion of a polynucleotide tag or a polynucleotide that encodes a polypeptide marker (e.g., by PCR), instructions on how to use the kit, and a label or insert indicating regulatory approval for diagnostic or therapeutic use.

[0246] The disclosure further includes polynucleotide or polypeptide microarrays comprising polypeptides of the disclosure, polynucleotides of the disclosure, or molecules, such as antibodies, which specifically bind to the polypeptides or polynucleotides of the present disclosure. In this aspect of the disclosure, standard microarray technology techniques are used to evaluate the expression of polypeptide biomarkers and/or identify biological components that bind to such polypeptides. Protein microarray technology is well known to those ordinarily skilled in the art and is based on, but not limited to, obtaining an array of identified peptides or proteins on a fixed substrate, target binding molecules or biological constituents for the peptides, and evaluation of such connection. Polynucleotide arrays, particularly arrays that bind to polypeptides of the disclosure, may also be used for diagnostic applications, such as to identify subjects who have a condition characterized by the expression of polypeptide biomarkers, e.g., interstitial lung disease.

[0247] The assay systems of the present disclosure may include a means of detecting, in a sample of lung cells, a level of amplification of the marker gene(s) and/or a level of polysomy of the marker gene(s). ) marker gene(s). The assay system preferably also includes one or more controls. Controls may include: (i) a control sample to detect interstitial lung disease in an individual; (ii) a control sample to detect the absence of interstitial lung disease; and (iii) information that contains a predetermined level of control of gene markers to be measured in relation to the diagnosis of or progression of interstitial lung disease.

[0248] In another embodiment, a means for detecting the level of expression of the marker(s) of the disclosure generally may be any type of reagent which may include, but is not limited to, antibodies and antigen-binding fragments of the themselves, peptides, binding partners, aptamers, enzymes and small molecules. Additional reagents useful for performing an assay using such a detection means may also be included, such as reagents for performing immunohistochemistry or other binding assay.

[0249] The means for detecting the assay system of the present disclosure may be coupled with a detectable label or detectable marker. Such a label may be any suitable label that permits detection of the reagents used to detect the gene or protein of interest and includes, but is not limited to, any composition or marker detectable by spectroscopic, photochemical, electrical, optical or chemical means. Labels useful in the present disclosure include: biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g. , 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in an ELISA), and colorimetric markers such as colloidal gold beads or colored glass or plastic (e.g. example, polystyrene, polypropylene, latex, etc.).

[0250] Furthermore, the means for detecting the assay system of the present disclosure may be immobilized on a substrate. Such substrate may include any substrate suitable for immobilizing a detection reagent as would be used in any of the detection methods described above. Briefly, a suitable substrate for immobilizing a detection medium includes any solid support, such as any organic solid, biopolymer or inorganic support that can form a bond with the detection medium without significantly affecting the activity and/or capacity of the detection medium. detection to detect the desired target molecule. Exemplary organic solid supports include polymers such as polystyrene, nylon, phenol-formaldehyde resins, and acrylic copolymers (e.g., polyacrylamide). The kit may also include reagents suitable for detecting the reagent and/or for labeling positive or negative controls, wash solutions, dilution buffers, and the like. The assay system may also include a set of written instructions for using the system and interpreting the results.

[0251] The assay system may also include a means for detecting a control marker that is characteristic of the cell type being sampled, generally it may be any type of reagent that can be used in a method for detecting the presence of a known marker ( at the protein or nucleic acid level) in a sample, as by a method for detecting the presence of a biomarker of this disclosure. Specifically, the medium is characterized in that it identifies a marker specific to the cell type being analyzed that positively identifies the cell type. For example, in an interstitial lung disease assay, it is desirable to screen lung cells for the level of biomarker expression and/or biological activity. Therefore, the means for detecting a control marker identifies a marker that is characteristic of a lung cell such that the cell is distinguished from other cell types. Such a means increases the precision and specificity of the assay of the present disclosure. Such means for detecting a control marker includes, but is not limited to: a probe that hybridizes, under stringent hybridization conditions, to a nucleic acid molecule encoding a protein marker; PCR primers that amplify such nucleic acid molecule; an aptamer that specifically binds to a conformationally distinct site on a target molecule; and/or an antibody, antigen-binding fragment thereof, or antigen-binding peptide that selectively binds to the control marker in the sample. Nucleic acid and amino acid sequences for many cellular markers are known in the art and can be used to produce such reagents for detection.

[0252] In some embodiments, the kit includes (or essentially consists of) primers or at least one probe capable of detecting a genetic variant, for example, as described above, depending on the detection method selected. In some embodiments, the kit includes primers or at least one probe capable of detecting a genetic variant in a region selected from the group consisting of 5p15, 6p24, 7q22, 11p15, 15q14-15, 17q21, 19p13, and 8p23. In some embodiments, the kit includes primers or at least one probe capable of detecting at least one genetic variant in 6p24 (e.g., rs2076295 or rs3778337). In some embodiments, the kit includes primers or at least one probe capable of detecting at least one genetic variant at 7q22 (e.g., rs4727443). In some embodiments, the kit includes primers or at least one probe capable of detecting at least one genetic variant in 11p15 (e.g., rs868903, rs7934606, rs6421972, rs7480563, rs7942850, rs4077759, rs2334659, rs2334659, rs7122936 ). In some embodiments, the kit includes primers or at least one probe capable of detecting at least one genetic variant in 5p15 (e.g., rs2736100). In some embodiments, the kit includes primers or at least one probe capable of detecting at least one genetic variant in 15q14-15 (e.g., rs2034650, rs1992272). In some embodiments, the kit includes primers or at least one probe capable of detecting at least one genetic variant at 17q21 (e.g., rs1981997, rs17563986, rs8070723). In some embodiments, the kit includes primers or at least one probe capable of detecting at least one genetic variant in 19p13 (e.g., rs12610495, rs2109069). In some embodiments, the kit includes primers or at least one probe capable of detecting at least one genetic variant in 8p23 (e.g., rs1379326). 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 at 5p15, 6p24, 7q22, 11p15, 15q14 - 15.17q21, 19p13, and 8p23, in any combination.

[0253] In some embodiments, the primers and/or probes are labeled, for example, with fluorescent or FRET labels. In some embodiments, the primers and/or probes are labelless. In some embodiments, the kit includes primers and/or probes that detect both a variant sequence and a dominant allelic sequence at a selected genetic variant site, e.g., with different markers, or designed to generate amplification or extension products. primer with different masses.

[0254] In some embodiments, the kit additionally includes at least one control sample, e.g., the sample(s) with dominant allele(s) at the selected site(s) of genetic variation (s), or sample(s) with variant allele(s) at the selected genetic variation site(s).

In vitro complexes

[0255] Nucleic acid complexes are provided herein, for example, formed in in vitro assays to indicate the presence of a genetic variant sequence. One skilled in the art will understand that a nucleic acid complex can also be formed to detect the presence of a dominant allelic sequence, depending on the probe or primer design, for example, in assays to distinguish homozygous from heterozygous subjects.

[0256] In some embodiments, the complex comprises a first nucleic acid first hybridized to a genetic variant nucleic acid, wherein the genetic variant nucleic acid is in a region selected from 5p15, 6p24, 7q22, 11p15, 15q14-15, 17q21, 19p13, and 8p23, or in a gene selected from TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, TOLLIP, MUC5B, ATP11A, IVD, CRHR1, IMP5, LOC100128977 , KIAA1267, NSF, and WNT3. In some embodiments, the genetic variant nucleic acid is an amplification product. In some embodiments, the genetic variant nucleic acid is in genomic DNA, for example, from a subject who has or is suspected of having an interstitial lung disease. In some embodiments, the first nucleic acid is an amplification product or a primer extension product. In some embodiments, the first nucleic acid is labeled. In some embodiments, the nucleic acid complex further comprises a second nucleic acid hybridized to the genetic variant nucleic acid. In some embodiments, the second nucleic acid is labeled, for example, with a FRET or other fluorescent label. In some embodiments, the first and second nucleic acids form a FRET pair when hybridized to a genetic variant sequence.

[0257] In some embodiments, the nucleic acid complex further comprises an enzyme, such as a DNA polymerase (e.g., standard DNA polymerase or thermostable polymerase, such as Taq) or ligase.

[0258] The present disclosure includes, but is not limited to, the following modalities:

[0259] 1. A method for determining whether an individual is expected to develop and/or rapidly progress with interstitial pneumonia comprising: detecting, in a biological sample from the individual, at least one of: a) the presence of a polymorphism marker selected from the group consisting of: rs2736100, rs2076295, rs3778337, rs4727443, rs868903, rs7934606, rs6421972, rs7480563, rs7942850, rs4077759, rs2334659, rs71 22936, rs2034650, rs1992272, rs1981997, rs17563986, rs8070723, rs12610495, rs2109069, rs1379326, rs1881984, rs10936599, rs1997392, rs6793295, rs2609255, rs2853676, rs10484326, rs10748858, rs2067832, rs11191865, rs2301160, rs3829223, rs285747 6, rs1278769, rs1007177, rs10518693, rs393152, rs12373139, rs17690703, rs2532274, rs2532269, rs2668692, rs169201, rs199533, and rs415430; and, b) a gene expression level of a marker gene or plurality of marker genes selected from the group consisting of: a marker gene with at least 95% sequence identity with at least one sequence selected from the group consisting of MUC5B, TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, TOLLIP, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69, or homologues or variants thereof ; c) polypeptides encoded by the marker genes of b) d) polypeptide fragments of c); and e) a polynucleotide that is fully complementary to at least a portion of a marker gene of b); wherein the presence of the plurality of markers is indicative of the possibility of an individual developing interstitial pneumonia or developing progressive IIP disease.

[0260] 2. The method of modality 1, in which the detected genes share 100% sequence identity with the corresponding marker gene in b).

[0261] 3. The method of embodiment 1, wherein the presence or level of at least one of the plurality of markers is determined and compared to a standard level or reference set.

[0262] 4. The method of embodiment 1, wherein the standard level or reference set is determined in accordance with a statistical procedure for predicting risk.

[0263] 5. The method of modality 4, wherein the statistical procedure for risk prediction comprises using the sum of the gene expression of the marker or markers or the presence or absence of a set of markers, weighted by a Risk coefficient Proportional.

[0264] 6. The method of embodiment 1, wherein the presence of the at least one marker is determined by detecting the presence or absence or level of expression of a polypeptide.

[0265] 7. The method of embodiment 6, wherein the method further comprises detecting the presence of the polypeptide using a reagent that specifically binds to the polypeptide or a fragment thereof.

[0266] 8. The method of embodiment 7, wherein the reagent is selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment.

[0267] 9. The method of modality 1, in which the presence of the marker is determined by obtaining the genomic DNA sequence at the locus of the polymorphism.

[0268] 10. The method of modality 1, in which the presence of the marker is determined by obtaining RNA from the biological sample; generation of cDNA from RNA; cDNA amplification with probes or primers for marker genes; obtaining, from the amplified cDNA, the expression levels of the genes or gene expression products in the sample.

[0269] 11. The method of any of modality 1, wherein the individual is a human.

[0270] 12. The method of any of embodiment 1, further comprising: a) comparing the expression level of the marker gene or plurality of marker genes in the biological sample to a control level of the marker gene(s) ) selected from the group consisting of: a marker gene control level that has been correlated with interstitial lung disease, the risk of developing IIP or having progressive interstitial pneumonia; and a level of marker control that has been correlated with slow or no progression of interstitial lung disease or interstitial pneumonia, or low risk of developing an IIP; and b) select the individual as predicted to progress rapidly in the development of interstitial pneumonia if the level of marker gene expression in the individual's biological sample is statistically similar to, or greater than, the control level of marker gene expression that has been correlated with interstitial lung disease or rapid progression of interstitial pneumonia, or c) select the individual as predicted not to develop interstitial pneumonia, or to progress slowly, if the level of the marker gene in the individual's biological sample is statistically lower than the control level of the marker gene that has been correlated with interstitial lung disease or rapid progression of interstitial pneumonia.

[0271] 13. The method of modality 1, further comprising: comparing the presence of a polymorphism, in the biological sample, with a set of genetic variants or polymorphic markers from an individual or control group that has developed interstitial pneumonia, and selecting the individual as predicted to develop or progress with interstitial pneumonia if the polymorphic markers present in the biological sample are identical or statistically similar to a set of polymorphic markers from the individual or control group or, select the individual as predicted to develop or rapidly progress with interstitial pneumonia if the polymorphic markers present in the biological sample are not identical or statistically similar to the set of genetic variants or polymorphic markers of the individual or control group.

[0272] 14. A method for monitoring the progression of interstitial lung disease or interstitial pneumonia in a subject, comprising: i) measuring expression levels of a plurality of gene markers in a first biological sample obtained from the subject, wherein the plurality of markers comprise a plurality of markers selected from the group consisting of: a marker gene with at least 95% sequence identity with a sequence selected from the group consisting of MUC5B, TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, TOLLIP, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69, or homologs or variants of the same complementary markers; b) polypeptides encoded by genes a) c) polypeptide fragments of d); and e) a polynucleotide that is entirely at least a portion of a marker gene of b); ii) measuring expression levels of the plurality of markers in a second biological sample obtained from the subject; and iii) compare the marker expression level measured in the first sample with the marker level measured in the second sample.

[0273] 15. The method of embodiment 14, wherein the detected marker genes share 100% sequence identity with the corresponding marker gene in a).

[0274] 16. The method of modality 14, further comprising performing a follow-up step selected from the group consisting of computed tomography of the chest and pathological examination of the subject's lung tissues.

[0275] 17. The method of embodiment 14, wherein the first biological sample from the subject is obtained at a time t0, and the second biological sample from the subject is obtained at a later time t1.

[0276] 18. The method of embodiment 14, wherein the first biological sample and the second biological sample are obtained from the subject and are obtained more than once over a series of times.

[0277] 19. A method for evaluating the effectiveness of a treatment for interstitial lung disease or interstitial pneumonia in a subject, the method comprising comparing: i) the level of expression of a marker measured in a first sample obtained from the subject at a time t0, wherein the marker is selected from the group consisting of a) a marker gene with at least 95% sequence identity with a sequence selected from the group consisting of TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, TOLLIP, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69, or homologs or variants thereof; b) polypeptides encoded by the marker genes of a) c) polypeptide fragments of b); and d) a polynucleotide that is fully complementary to at least a portion of a marker gene of a); ii) the level of the marker in a second sample obtained from the subject at time t1; and iii) perform a follow-up step selected from chest computed tomography and pathological examination of the subject's lung tissues; wherein a decrease in the level of the marker in the second sample relative to the first sample is an indication that the treatment is effective in treating interstitial pneumonia in the subject.

[0278] 20. The method of embodiment 19, wherein the detected genes share 100% sequence identity with the corresponding marker gene in a).

[0279] 21. The method of embodiment 19, wherein the time t0 is before the treatment has been administered to the subject, and the time t1 is after the treatment has been administered to the subject.

[0280] 22. The method of embodiment 19, wherein the comparison is repeated over a series of times.

[0281] 23. An assay system for predicting subject prognosis therapy for interstitial pneumonia comprising a means for detecting at least one of: a) the presence of a polymorphism marker selected from the group consisting of: rs2736100, rs2076295, rs3778337, rs4727443, rs868903, rs7934606, rs6421972, rs7480563, rs7942850, rs4077759, rs2334659, rs7122936, rs2034650, rs1992272, rs1 981997, rs17563986, rs8070723, rs12610495, rs2109069, rs1379326, rs1881984, rs10936599, rs1997392, rs6793295, rs2609255, rs2853676, rs1 0484326, rs10748858, rs2067832, rs11191865, rs2301160, rs3829223, rs2857476, rs1278769, rs1007177, rs10518693, rs393152, rs12373139, rs1769070 3, rs2532274, rs2532269, rs2668692, rs169201, rs199533, and rs415430; and, b) a gene expression level of a marker gene or plurality of marker genes selected from the group consisting of: a marker gene with at least 95% sequence identity with a sequence selected from the group consisting of TERT, DSP, MUC2, DISP2, MAPT, DPP9, CSMD1, MYNN, LRRC34, FAM13A, OBFC1, TOLLIP, ATP11A, IVD, CRHR1, IMP5, LOC100128977, KIAA1267, NSF, WNT3, C17orf69, or homologs or variants thereof; c) polypeptides encoded by the marker genes of b) d) polypeptide fragments of c); and e) a polynucleotide that is fully complementary to at least a portion of a marker gene of b).

[0282] 24. The assay system of embodiment 23, wherein the means for detecting comprises nucleic acid probes comprising at least 10 to 50 contiguous nucleic acids of the polymorphisms or marker gene(s), or nucleic acid sequences complementary to them.

[0283] 25. The assay system of embodiment 23, wherein the means for detecting comprises binding ligands that specifically detect polypeptides encoded by the marker genes.

[0284] 26. The assay system of embodiment 23, wherein the detected genes share 100% sequence identity with the corresponding marker gene in b).

[0285] 27. The assay system of embodiment 23, wherein the means for detecting comprises at least one of nucleic acid probe and binding ligands disposed on an assay surface.

[0286] 28. The test system of embodiment 27, wherein the test surface comprises a fluidity chip, matrix or card.

[0287] 29. The assay system of embodiment 28, wherein the probes comprise nucleic acid sequences complementary to at least 10 to 50 nucleic acid sequences of the marker genes.

[0288] 30. The assay system of embodiment 28, wherein the binding ligands comprise antibodies or binding fragments thereof.

[0289] 31. The assay system of embodiment 23, further comprising: a control selected from information containing a predetermined level of control or set of genetic variants or polymorphic markers that has been correlated with the diagnosis, development, progression or expectation of life in interstitial lung disease or patients with IIP.

[0290] 32. A method for detecting a gene expression level of one or more marker genes in a human subject with interstitial pneumonia, comprising: obtaining a biological sample from a human subject with interstitial pneumonia; detect the expression level of a gene selected from TERT, MUC2, TOLLIP, MUC5B, DPP9, DSP and homologs or variants thereof, in one or more cells of the individual's biological sample.

[0291] 33. The method of modality 32, further comprising detecting the expression level of a gene selected from TERT, MUC2, TOLLIP, MUC5B, DPP9, DSP and homologues or variants thereof, in one or more cells of the sample biological status of the individual.

[0292] 34. The method of modality 32, further comprising detecting the expression level of a gene selected from MUC5B, TERC, SFTPC, SFTPA2 and homologues or variants thereof in one or more cells of the individual's biological sample.

[0293] 35. A method for treating interstitial pneumonia in a subject in need of such treatment, comprising: detecting a level of one or more marker genes selected from TERT, MUC2, TOLLIP, MUC5B, DPP9, DSP or homologues or variants thereof, in a biological sample obtained from the human subject; and administering an effective amount of an interstitial pneumonia treatment.

[0294] 36. The method of modality 35, further comprising detecting the expression level of a gene selected from TERT, MUC2, TOLLIP, MUC5B, DPP9, DSP and homologues or variants thereof, in one or more cells of the sample biological status of the individual.

[0295] 37. The method of modality 35, further comprising detecting the expression level of a gene selected from MUC5B, TERC, SFTPC, SFTPA2 and homologues or variants thereof in one or more cells of the individual's biological sample.

[0296] The Examples that follow are illustrative of specific disclosure modalities and various uses thereof. They are described for explanatory purposes only and should not be taken as limiting the disclosure.

EXAMPLES

[0297] Provided herein is a genome-wide case-control association study (GWAS; 1616 cases and 4683 controls) and replication study (876 cases and 1890 controls) of individuals with IIP, including all types of IIP. fibrotic. Different types of IIP were included in the study because: a) distinguishing between IIP diagnoses is often problematic due to substantial clinical, pathological and radiological overlap; and b) there is strong evidence for shared genetic susceptibility (e.g., more than 40% of families with FIP have more than one type of IIP among affected family members). Samples from individuals with both familial and sporadic IIP were also included in this GWAS study because the MUC5B, TERT, TERC, and SFTPC variants provide evidence that sporadic IIP is genetically similar to the familial form of this disease.

[0298] With the goal of identifying additional genetic risk factors that collectively increase our understanding of IIP, the present inventors have completed a genome-wide case-control association study (GWAS; 1616 cases and 4683 controls) and replication study (876 cases and 1890 controls) of IIP. All types of fibrotic IIP were included in the case group. The inventors also included both familial and sporadic IIPs.

Study populations

[0299] Case definition. We used standard criteria established by the American Thoracic Society/European Respiratory Society to determine the diagnostic classification of all patients in the discovery and replication phases. We excluded cases with known explanations for the development of fibrotic IIP, including infections, systemic disorders, or relevant exposures (e.g., asbestos). To maximize power and minimize potential confounding by ancestry, we included only non-Hispanic white (NHW) participants in the GWAS and replication. All subjects gave written consent as part of the IRB-approved protocols for their recruitment, and the GWAS study was approved by the National Jewish Health IRB and Colorado Combined Institutional Review Board (COMIRB).

[0300] Discovery GWAS. We genotyped 1914 patients with IIP from 7 cohorts (familial interstitial pneumonia [n = 566], National Jewish Health IIP population [n = 238], InterMune IPF trials [n = 720], UCSF [n = 66], population with IIP from Vanderbilt University [n = 105], and the National Heart Lung and Blood Institute Lung Tissue Research Consortium [n = 219]) and compared them with genotypes from 4683 off-study controls. After genotype quality control, we included 1616 cases in analyses.

[0301] A family with familial interstitial pneumonia (FIP) is defined by the presence of at least 2 cases of definite or probable IIP in individuals genetically related within 3 degrees. Recruitment of families based at three large referral centers (Vanderbilt University, Duke University, and National Jewish Health) has occurred since 1999. We included only 1 case of IIP among first-degree relatives. The National Jewish Health IIP cohort consists of patients with sporadic IIP who have been clinically evaluated and enrolled at National Jewish Health as part of ongoing research protocols associated with medical care. Details of the recruitment criteria for the Intermune IPF Y-Interferon Intervention Trial cases have been described in detail. Briefly, eligible patients who had IPF were 40 to 79 years of age with clinical symptoms for at least 3 months and evidence of disease progression in the previous 12 months. We included all available cases regardless of treatment assignment. The National Heart Lung and Blood Institute Lung Tissue Research Consortium (NHLBI LTRC) was established to provide lung tissue and DNA to the research community. We included DNA from these subjects with a diagnosis of IIP.

[0302] We use de-identified control genotypes generated at the Center d’Étude du Polymorphisme Humain (CEPH) as part of other studies. Potential controls were those who were NHW, had been genotyped on the same platform as our cases, and were appropriately approved by us for use as controls in other studies. We selected a subset of controls, corresponding to approximately 3 controls for 1 case, based on genetic similarity to cases that passed our genotyping quality control thresholds (see Statistical Analyzes below).

[0303] Replication. We genotyped a total of 1027 NHW IIP cases and 2138 NHW controls for replication of the top SNPs from GWAS. Replication controls were from individual replication groups (n = 138) and a subset (n = 2000) of controls from the Chronic Obstructive Pulmonary Disease (COPD) Gene Study. We selected controls to be frequency matched to replication cases based on age and sex. After quality control, we included 876 cases and 1890 controls in analyses.

[0304] Expression. We measured gene expression in a subset of IIP cases from the Lung Tissue Research Consortium and National Jewish Health GWAS (n = 100) and controls from National Jewish Health (n = 94). Whole lung samples were obtained from the International Institute for the Advancement of Medicine (Edison, NJ). Eligible controls and cases had sufficient RNA from lung tissue biopsy available for assay; cases with IPF were chosen preferentially over other IIP diagnoses. National Jewish Health controls also had genome-wide SNP data available.

DNA preparation, storage and quality control

[0305] Genomic DNA was isolated from both whole blood and lung tissue collected for biopsy on the Autopure LS (Qiagen) or Qiacube (Qiagen) automation platform, respectively. Prior to extraction on the Qiacube using the DNAeasy kit, fibrotic lung tissues were first homogenized using Lysing Matrix D tubes and a FastPrep-24 benchtop homogenizer (MPBiomedicals). After isolation, all DNA was analyzed for concentration and purity on the NanoDrop ND-1000 Spectrophotometer. Samples were excluded if DNA was <50 ng/μl or had an A260/A280 ratio outside the range of 1.7-2.0.

[0306] Prior to submission to the CNG, all samples were requantified using the QuantiT PicoGreen dsDNA Assay Kit (Invitrogen), normalized with 1xTE, and had aliquots deposited in screw cap tubes with individual barcodes. Due to volume limitations with liquid handling robots, an absolute minimum quantity for submission to CNG was 30 μl to 50 ng/μl. If samples did not meet this minimum quantity, an alternative extraction was performed or the sample was withdrawn from the study.

[0307] Upon receipt of replicate samples, they were transferred to robotics-compatible 96-well plates, quantified with PicoGreen, and normalized with 1xTE. In accordance with BMGC submission guidelines, 400ng of DNA was submitted for each member of the GWAS and replication cohorts. In an effort to minimize confounding by batch effects, samples were aliquoted into 96-well plates in a randomized fashion across all cohorts with two duplicates per plate using the Tecan Evo200 liquid handling robot.

Genome-wide genotyping

[0308] Barcoded DNA samples were received in standard tubes along with sample information and were subjected to strict quality control (QC). Concentration, fragmentation and PCR response were determined. Samples from cases and controls were randomly distributed into loops of 96 wells. Processing was performed under full LIMS control in a fully automated Illumina BeadLab equipped with 8 Tecan liquid handling robots, 6 Illumina BeadArray readers, and 2 Illumina iScans. Genotyping was performed using the Illumina Human610 quad array. Replication genotyping

[0309] We genotyped 198 SNPs with P-values less than 0.0001 (see Statistical Analyzes) in 1027 laces ial cases and 2000 COPDgene controls. We also genotyped the MUC5B laces i SNP rs35705950, which is not on the Illumina 660 Quad beadchip, to allow adjustment of the chromosome 11p15 replication SNPs to rs35705950. Furthermore, to allow for statistical testing on the follow-up set (using raw genotypes from both GWAS cases and replication cases and controls) with adjustments for covariates that were not available in the out-of-study controls, we also genotyped a subset of cases. of GWAS. Details of the validation assays are described below. After genotyping quality control, we included 876 cases and 1890 controls in the replication analyzes and 859 of the GWAS cases in the pooled analyses.

[0310] Prior to genotyping, all samples underwent quality control by real-time Q-PCR quantification (“QC1”) and uniplex genotyping using Taqman (“QC2”). Samples that failed QC1 or QC2, although continued with genotyping, were subsequently removed from analysis.

[0311] Validation genotyping was performed with a combination of multiplexed (Sequenom iPLEX) and uniplexed (Taqman) assays. First, the Sequenom iPLEX multiplexed genotyping assay design was performed on an input set of 198 SNPs (Table 3), using a combination of web-based (AssayDesigner Suite, available at sequenom.com) and desktop software tools. (AssayDesigner) (Sequenom, San Diego). Of 198 input SNPs, 193 were efficiently placed into a set of 6 assays of the following plexities: 35, 35, 35, 35, 31 and 22 SNPs. Sequenom iPLEX genotyping is based on multiplexed locus-specific PCR amplification, multiplexed single-based extension (SBE) of locus-specific amplicons, and multiplexed base assignment resolution ( base calling) of SBE products using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

[0312] Primers for the Sequenom assay were purchased from IDT (Coralville, Iowa), and all steps of the iPLEX procedure were performed using laces i and methods from Sequenom (San Diego, CA) according to the manufacturer's instructions. Reactions were performed in 384-well loops and analyzed using the Sequenom MassARRAY Analyzer 4 system with iPLEX Gold loops and SpectroCHIP arrays. Results were analyzed using a combination of laces ial software (Typer 4, Sequenom) and custom data management tools. Of 193 assays across 6 multiplexes, 179 were successful in generating usable genotyping data.

[0313] The remaining 5 SNPs that were not successfully included in the original Sequenom iPLEX projects (rs2736100, rs35705950, rs13225346, rs10822856, rs10139381, rs10751635), as well as a laces SNP (rs35705950) published in previous studies, were genotyped using Taqman assays commercial (Life Technologies, San Diego, CA). The dbSNP rs#s of these SNPs, as well as the commercial product IDs of the assays employed, are shown in Table 3. Reactions were performed in 384-well loops and fluorescence reading using an Applied Biosystems ABI7900HT Sequence Detection System (Applied Biosystems, Foster City, CA).

Gene expression

[0314] Total RNA was isolated from approximately 30 mg of lung tissue preserved by snap-freezing or RNA-later using the Ambion mirVana kit (Life Technologies). RNA concentration was determined by Nanodrop ND-1000 (Thermo Scientific) and RNA integrity was determined using the 2100 Bioanalyzer (Agilent). Single-stranded cDNA conversions were performed using the Superscript III First-Strand Synthesis System (Invitrogen) and expression analysis was performed using pre-designed Taqman assays run on the Viia7 Real-Time PCR instrument (Life Technologies). (DPP9: Hs00373589; DSP: Hs00189422 and variant 1 assay DSP is Hs00950584; FAM13A: Hs00208453; IVD: Hs01064832; MUC5B: Hs00861588; MUC2: Hs00149374; OBFC1: Hs009 98588; WNT3: Hs00902257; WNT9B: Hs00916642; GAPDH: 4333764F) . All assays were performed in triplicate with GAPDH used as an endogenous control. As an additional control, one sample per plate was run in duplicate from the cDNA conversion step.

STATISTICAL ANALYSIS

[0315] Selection of off-study controls for discovery GWAS. An ancestry analysis was performed using EIGENSTRAT3.0 software. HapMap data and reference European samples were used as proxies for European, West African, and East Asian populations to infer the key informative components of ancestry that were projected onto the case and control samples. Putative non-European samples were flagged as outliers and eliminated from subsequent analyses. We obtained controls with close genetic matching to cases from a large database of anonymized genotypes of Europeans. From this database, we selected a subset of the control genotype data in order to obtain three matched controls per case through the use of a cluster-based approach with the support vector machine (R package "e1071"), followed by application of a pairwise matching algorithm (R package "optmatch"). With this selection, the genomic inflation factor (evaluated with adjustment for population structure with the GEMMA software) was 0.99.

[0316] Removal of first-degree relatives. We included only one individual among first-degree relatives based on an estimated relatedness coefficient >0.45. To estimate the percentage variation in disease risk explained by GWAS SNPs which is sensitive to hidden relatedness, we additionally removed only one individual among those with estimated relatedness coefficient > 0.025.

[0317] Exclusion of individuals and prioritization of SNPs for discovery GWAS. In addition to laboratory-excluded individuals, we excluded individuals with 1) evidence to be a genetic outlier based on a pairwise estimate of identity-by-state (IBS) with the 5th nearest neighbor that was >4 standard deviations of the mean IBS pairwise estimate across all pairs, 2) unresolved sex mixing and matching between clinical and genomic data, 3) heterozygosity along SNPs greater or less than 4 standard deviations from mean heterozygosity across all individuals, and 4) genotype assignments to less than 98% of SNPs that pass laboratory quality control. Based on this quality control, we excluded 298 cases and 165 controls. In addition to laboratory quality control measures, we prioritize signals of association for follow-up based on other criteria. We tested differential missingness through a chi-square test of missingness proportions between cases and controls and HWE deviations through a 1-df goodness-of-fit test. We prioritized SNPs with 1) MAF > 0.05, 2) HWE p-value > 0.0001 in cases and controls evaluated separately, 3) p-value for differential absence between cases and controls > 0.001, if less than 2% of absence, and > 0.05, if between 2% and 5% of absence.

[0318] GWAS association test. We tested the association between each SNP and IIP using an exact mixed model approach to take into account both subtle relatedness and population stratification between our cases and controls that is implemented in the genome-wide efficient mixed model association software package. (GEMMA). We tested the association under an additive model for our preliminary analysis and subsequently took the minimum of the recessive and dominant model p-values if there was significant lack of fit relative to the additive model (p < 0.05). from a linear regression that assumed independence between samples (such a test is not currently implementable in the GEMMA software). We adjust for gender in all models. We compare the distribution of p-values obtained under the additive model to those expected under the null hypothesis of no genome-wide association and report the quantile-quantile (Q-Q plot) and genomic inflation factor (À) to verify the absence of systematic trends due to experimental factors or other confounding factors such as population stratification. We selected all SNPs with a p-value < 0.0001 for follow-up in replication populations and visually inspected genotype spectra for all 198 selected SNPs to ensure the quality of genotype assignment. We calculated odds ratios and 95% confidence intervals (CIs) from a sex-adjusted logistic regression model that assumed independence between cases and controls, since the linear model in GEMMA uses the link function of identity instead of the logarithm of the odds link function. As such, the CIs may be slightly narrower than those based on the full mixed models.

[0319] Replication association. We tested the association between each SNP and replication IIP in replication cases and controls using the freely available SNPGWA software (see URLs). We tested the association under the genetic model from the GWAS that gave the minimum p-value (143 under an additive model, 24 under a dominant model and 31 under a recessive model). A p-value < 0.0025 was considered statistically significant replication for the 20 genome-wide significant GWAS SNPs. The p-values for the other 178 SNPs were used in the meta-analysis of the GWAS and replication cohorts.

[0320] Meta-analysis. To obtain a joint measure of association between each of the 181 SNPs successfully genotyped in the replication and IIP set, we performed a meta-analysis of the GWAS and replication results. We use the weighted inverse normal method. Let Zi (i = GWAS or replication) be the test statistic of the association test in the ith study and let vi (i = GWAS or replication) be the corresponding weight. Here, we took the weight to be the square root of the total sample size in the ith study, since effect estimates from GWAS and replication were not on the same scale. Note that this method explicitly takes into account the directionality of the association. Thus, highly significant associations with conflicting directions do not exhibit strong statistical association. We used METAL (available at sph.umich.edu/csg/abecasis/metal/) to perform meta-analysis. SNPs with PJoint < 5 x 10-8 were considered statistically significant across the entire genome. We created locus-specific plots of discovery GWAS results for all loci that were genome-wide significant in the meta-analysis.

[0321] Multi-SNP models. To assess the independence of the effects of genome-wide significant SNPs from the meta-analysis, we used logistic regression models within each locus using the combined case group (GWAS and replication) and replication controls. Specifically, within each locus with a genome-wide significant SNP, we tested the association between IIP and each of the other validation panel SNPs within that locus after adjusting for the most significantly associated SNP at that locus (on chromosome 11p15, we set it to rs35705950). To assess the robustness of each SNP association to the effects of age in addition to sex, we tested the association between IIP and each SNP adjusted for age and sex.

[0322] Expression analysis. We tested differential gene expression in the lung between 100 cases and 94 controls using a two-sample t-test. We also tested differential expression by genotype using the combined case and control group via ANOVA across the three genotype groups unless there were <5 individuals in a genotype group; in this case, we group together the rare homozygous and heterozygous groups. A p-value < 0.05 was considered statistically significant.

Results Genome-wide Discovery

[0323] We genotyped 1914 cases of fibrotic IIP that self-reported non-Hispanic white on the Illumina 660 Quad beadchip. Of these, 14, 126, 8, and 150 were excluded due to being a genetic outlier, evidence of being a first-degree relative of another case, high heterozygosity, or absence of >2% of genotypes across all SNPs, respectively (see Statistical methods); 1616 cases were included in the analyses. Among 15,352 off-study controls also genotyped on the Illumina 660 Quad beadchip, we used 4683 controls most genetically similar to our cases, based on identity-by-state comparisons across the entire genome.

[0324] We compared IIP cases and controls on 439,828 SNPs with 1) MAF > 0.05, 2) HWE p-value > 0.0001 in cases and controls evaluated separately, 3) p-value for differential absence between cases and controls > 0.001, if less than 2% absence, and > 0.05, if between 2% and 5% absence. Neither the QQ plot of p-values (Figure 5) nor the estimated genomic inflation factor of 0.99 suggested any systematic trends, such as those related to population stratification. Under an additive model for the minor allele at each SNP, we identified 19 SNPs, representing 7 chromosomal locations, with significant associations across the entire genome (P < 5 x 10-8) (Figure 1 and Table 1). We identified another genome-wide significant SNP (rs1379326) that represents a single locus, under a recessive model (Table 1).

REPLICATION AND META-ANALYSIS

[0325] We selected the 20 significant SNPs across the entire genome and 178 additional SNPs with 5 x 10-8 < p-value < 0.0001 (SNPs between the top and bottom lines in Figure 1; see Tables 3 and 4 for SNP location, genotype, and HWE information, and Table 5 for association information for all 198 SNPs) for genotyping in a replication cohort of 1027 IIP cases and 2138 controls. After genotype quality control, we included 876 cases and 1890 controls successfully genotyped at 181 of the SNPs. 13 of the 20 genome-wide significant SNPs were associated with IIP in the replication cohort at P < 0.0025, corresponding to the conservative Bonferroni correction for 20 tests (Table 1, middle columns). 18 of the 20 genome-wide significant SNPs, representing 7 loci, from GWAS (Figure 2) were genome-wide significant in the meta-analysis (Table 1, last column). An additional 25 SNPs representing 9 chromosomal locations (5 overlapping GWAS loci and 4 additional loci (Figure 3)) were significant across the entire genome in the meta-analysis (Table 1).

[0326] The SNP most strongly associated in the GWAS discovery, rs868903 (PGWAS = 1.3 x 10-22; PMeta = 9.2 x 10-26), is in the promoter of the MUC5B gene on chromosome 11p15, which we report to be associated with IPF and FIP and has been confirmed in other studies. Ten additional SNPs in the MUC5B region, including SNPs in the MUC2 and TOLLIP genes, were also significant across the entire genome in the joint analysis and not in strong LD with rs868903 (Figure 2d). The SNPs rs2736100 (PMeta = 1.7 x 10-19) and rs2853676 (PMeta = 3.3 x 10-8) on chromosome 5p15 are in the TERT gene (Figure 2a) and rs1881984 (PMeta = 4.5 x 10-8 ) is close to the TERC gene (Figure 3a); Rare mutations in TERT and TERC have been reported to be associated with FIP and IPF, and rs2736100 was previously reported in the TERT gene. The remaining 8 genome-wide significant loci are novel IIP loci (Figure 6). Five of the association signals on chromosomes 4q22, 6p24, 10q24, 13q34, and 19p13 appear located in single genes. SNP rs2609255 (PMeta = 2.2 x 10-11) is in the FAM13A gene (sequence similarity family 13, member A) on chromosome 4q22 (Figure 3b). SNPs rs10484326 (PMeta = 5.5 x 10-9) and rs2076295 (PMeta = 1.1 x 10-19) are in the DSP (desmoplakin) gene on chromosome 6p24 (Figure 2b). SNPs rs10748858 (PMeta = 2.7 x 10-8), rs2067832 (PMeta = 3.7 x 108) and rs11191865 (PMeta = 2.4 x 10-8) are in the OBFC1 gene (possessing oligonucleotide binding fold 1 - oligonucleotide-binding fold containing 1) on chromosome 10q24 (Figure 3c). SNP rs1278769 (PMeta = 6.7 x 10-9) is in the ATP11A gene (ATPase, class VI, type 11A) on chromosome 13q34 (Figure 3d). SNPs rs12610495 (PMeta = 1.7 x 10-12) and rs2109069 (PMeta = 2.4 x 10-11) are in the DPP9 (dipeptidyl-peptidase 9) gene on chromosome 19p13 (Figure 2g). The other three chromosomal regions (7q22, 15q14-15 and 17q21) have no significant SNPs in any gene or SNPs with significant associations in multiple genes (Table 1 and Figure 2c, 2e, 2f). The estimated odds ratios (OR) for all significant SNPs across the entire genome range from ~1.1 to ~1.6 (Table 1; ORs for MAF that are less than 1 correspond to ORs for major allele in same track).

Investigating fitted models for significant SNPs across the entire genome

[0327] To adjust for the previously discovered MUC5B promoter SNP (rs35705950; not on the Illumina 660 Quad beadchip), we genotyped a subset of the GWAS discovery cases on the same platform and at the same time as the replication cases for the SNPs in the Table 1. We combined the raw genotypes of these cases (n = 859) with the replication cases and controls for pooled analyses.

[0328] To assess evidence of multiple signals of independent association within each region, we tested the association with each SNP in a given region after adjusting for the most significant SNP in that region based on meta-analysis. For the chromosome 11p15 region, we adjusted for rs35705950 due to our previous findings and the strength of the association we observed between rs35705950 and IIP in our current study population (OR [95% CI]: 4.51 [3.91, 5 .21] PJoint = 7.21 x 10-95). After adjusting for rs35705950, only one of the SNPs at 11p15 (rs4077759) remained nominally associated with IIP (P = 0.03; Table 2), while rs35705950 remained highly significant in all models, suggesting that the associations we observed with other SNPs were due to weak LD with rs35705950 (see Figure 6 for LD between SNPs). The reductions in significance of SNPs in the other regions after adjusting for the top SNP were consistent with LD between SNPs (Table 6) and did not provide evidence for multiple signals of association. It is noteworthy that the SNP rs1881984 near the TERC gene is no longer significant after adjusting for the SNP rs6793295 in the LRRC34 gene.

[0329] Finally, we adjust for age in addition to sex for all significant SNPs across the entire genome; with the exception of rs7942850 on chromosome 11 (adjusted to Page = 0.06), all SNPs remained significant after adjustment (Table 6).

EXPRESSION OF KEY GENES IN LUNG TISSUE

[0330] We measured the expression of DPP9, DSP, FAM13A, IVD, MUC5B, MUC2, DISP2, OBFC1, WNT3 and WNT9B in lung tissue from 100 IPF cases and 94 controls using validated Taqman and quantitative PCR Genotyping Assays (Applied Biosystems , Foster City, CA) to test for differences between cases and controls and to test the association between genotypes at the SNPs most strongly associated in each gene with expression of that gene. We confirm our results from a smaller study in which MUC5B is more highly expressed in lung tissue of cases compared to controls (P = 5.6 x 10-11), but consistent with our previous findings for rs35705950 among IPF cases, rs868903 was not associated with MUC5B expression. DSP was more highly expressed in cases compared to controls (P = 0.0002), and expression differed by genotype at rs2076295 (P = 0.002); relative DSP expression increased with the copy number of the putative allele at risk (Figure 4). There are two isoforms of desmoplakin generated by alternative splicing. rs2076295 is contained in a binding site for the transcription factor PU.1, which has been implicated in alternative splicing of target genes; however, we saw no evidence for a differential effect of the rs2076295 genotype upon expression of the primary isoform compared to the alternative isoform. There was nominal evidence for higher expression of DPP9 in cases compared to controls (P = 0.03), but neither rs12610495 (P = 0.46) nor rs2109069 (P = 0.72) were associated with DPP9 expression. Neither FAM13A, IVD, or OBFC1 differed in expression between cases and controls or by genotype (all P > 0.12); MUC2, DISP2, WNT3, and WNT9B showed little or no expression in these lung samples.

PERCENTAGE VARIATION IN DISEASE RISK EXPLAINED BY SNPS GWAS

[0331] We estimated the percentage of disease risk explained by all 439,828 GWAS SNPs tested for association using a variance components model over a range of prevalence estimates for IIP (50 per 100,000 to 100 per 100,000). We found that GWAS SNPs accounted for approximately 30% (s.e. 2%) to 33% (s.e. 3%) of IIP risk. Since we did not include the MUC5B promoter SNP (rs35705950) in this analysis, this is a conservative estimate of the contribution of common SNPs to IIP risk.

DISCUSSION

[0332] These findings provide compelling evidence that common genetic variation is an important contributor to the risk of interstitial lung diseases, such as IIP. We identified 8 novel genetic risk loci (4q22, 6p24, 7q22, 10q24, 13q34, 15q14-15, 17q21, and19p13) and confirmed the role of risk variants in three previously reported genes/loci (TERC[3q26], TERT[ 5p15] and MUC5B [11p15]) in IIP. Prior to this report, the only two genes with a common variant reproducibly associated with IIP were TERT and MUC5B. Altogether, the common risk variants associated with IIP suggest that this disease is primarily mediated by defects in host defense, cell-cell adhesion, and premature cellular senescence. Furthermore, these findings can be used to guide intervention trials in this complex disease.

[0333] Secreted mucins (MUC5B) in the distal small airways appear to play a role in the development of IIP. The data do not suggest a strong effect of SNPs in other genes (MUC2 or TOLLIP) in the 11p15 region after accounting for the effect of the MUC5B promoter SNP rs35705950 that we previously identified as a key risk factor for IIP. The rs868903 SNP in the MUC5B gene promoter that was one of the most strongly associated SNPs in GWAS, replication and meta-analysis is not in strong LD (r2 = 0.13) with rs35705950 and is closer to the transcription start site for MUC5B than rs35705950 (3 kb vs, 1.5 kb respectively). Although lung tissue from patients with IIP has higher MUC5B concentrations than controls, none of these MUC5B promoter variants appear to be entirely responsible for the increased expression of MUC5B in patients with IIP, suggesting that other genetic variants or environmental toxins play a role. role in this disease. Dysregulated lung mucins likely initiate or exacerbate fibroproliferation through one of the following mechanisms: 1) altered host mucosal defense; 2) interference with alveolar repair; or 3) direct cellular toxicity (endoplasmic reticulum stress or apoptosis), stimulating a fibroproliferative response initiated by excess production of lung mucins.

[0334] Genes that maintain telomere length appear to play a role in the development of IIP. Prior to this report, associations between pulmonary fibrosis and TERT and TERC involved rare variants of TERT and TERC and a common variant of TERT. Mutations in these genes are associated with shortened telomeres in alveolar epithelial cells, suggesting that these genetic variants may increase the risk of pulmonary fibrosis through potentiated apoptosis or necrosis of the alveolar epithelium. Furthermore, dyskeratosis congenita, a congenital disorder that resembles premature aging and often involves pulmonary fibrosis, has been associated with mutations in TERT and TERC. This GWAS identified common variants in TERT and near TERC, and in another gene that influences telomere length, OBFC1 (Pmeta = 2.4 x 10-08). A common variant in OBFC1 has been associated with telomere length in two GWAS studies of human leukocyte telomere length in the general population. It appears that the risk associated with these genes is not limited to rare variants, but represents variation in common risk. Altogether, these findings emphasize the importance of telomere length and early cellular senescence in the pathogenesis of pulmonary fibrosis.

[0335] The results implicate changes in cell-cell adhesion in the risk of developing IIP. Variants in the DSP gene have been strongly associated with IIP and with DSP expression in lung tissue from patients with IIP. The DSP gene encodes the protein desmoplakin, a component of the desmosome, an adhesive intercellular molecule that firmly binds adjacent cells and forms a dynamic structure with other proteins (plakogobin and plakophilins) that tether the cytoskeleton to the cell membrane. Desmosomes are particularly important for maintaining the integrity of tissues that experience mechanical stress (such as the peripheral portions of the lung), and there is strong evidence that desmosome disruption disrupts epithelial homeostasis. Mutations in DSP have been associated with arrhythmogenic right ventricular dysplasia, keratodermas and alopecia, directly implicating desmoplakin in diseases with loss of tissue integrity. More specifically, mutations in DSP have been associated with cardiac interstitial fibrosis, based on overexpression in mouse cardiac tissue. An additional potential mechanism for DSP involvement is through alterations in the wnt/β-catenin signaling pathway, which has been consistently observed in pulmonary fibrosis. It has been shown that desmoplakin influences the wnt/β-catenin signaling pathway through the regulation of another desmosome component, γ-catenin. These studies and the finding that overexpression of DSP in IIP is associated with the variant allele of rs2076295 provide a strong biomechanical or biological rationale for the role of genetic variation in DSP in contributing to pulmonary fibrosis.

[0336] The results implicate other cell adhesion molecules in the risk of developing IIP. The DPP9 gene is a member of the same protein family as the fibroblast activation protein, which has been shown to be expressed in fibroblastic foci but not in adjacent healthy lung in IPF. DPP9 is expressed in epithelia and has been shown to alter cell adherence in human embryonic kidney cells. Furthermore, the catenin-cadherin-associated protein alpha 3 (CTNNA3) gene was nearly significant in the pooled analysis (Pmeta = 9.8 x 10-07), is located at 10q22, and is a cell adhesion molecule that physically interacts with β-catenin and mediates cell adhesion. Altogether, these findings suggest that pulmonary fibrosis is caused by defects in cell-cell adhesion or defects in the cytoskeleton that are unable to accommodate the stress associated with mechanical stretching of the lung.

[0337] FAM13A is a signal transduction gene that is sensitive to hypoxia and a SNP (rs7671167) in this gene was recently found to be protective in chronic obstructive pulmonary disease. The other significant loci across the entire genome are not as tightly localized to a single gene, although interesting candidates emerge. There are several markers associated with IIP that are all in strong LD on chromosome 17q21, spanning 1.14 Mb. An obvious candidate among these genes is the WNT3 gene, given the changes in wnt signaling observed in IIP; however, we found no evidence for WNT3 expression in the lung. 17q21 is a structurally complex genetic region, with a large (>1 Mb) inversion polymorphism and smaller copy number variants (CNVs) associated with the disease. Interestingly, the LRRC37A and LRRC37A2 genes in this region are from the same family as the LRRC34 gene on chromosome 3, adjacent to the TERC gene, which had one of the strongest association signals in the replication samples. In both the 17q21 region of chromosome and the complex mucin region on chromosome 11p15, it is likely that deep sequencing and array-based copy number measurement followed by functional testing of putative genes/alleles/CNVs will be required to further characterize the architecture genetics of these associations with IIP observed. Although it has been proposed that pulmonary fibrosis results from activation of developmental pathways or aberrant lung repair, findings suggest that these mechanisms are secondary to a primary defect in host defense or cell-cell adhesion. Since genes involved in lung epithelial integrity (DSP, DPP9 and CTNNA3) and lung mucins (MUC5B) are genetic risk variants, defects in these mechanisms are likely primary contributors to the development of pulmonary fibrosis. Given the importance of environmental exposures (e.g., exposure to cigarette smoke, asbestos, and silica) in the development of other forms of interstitial lung disease, it stands to reason that common inhaled particles, such as those associated with cigarette smoke or air pollution , over the years cause exaggerated interstitial damage in people who have defects in pulmonary host defense or cell-cell adhesion. Shortened telomeres and subsequent premature cellular senescence likely alter host defense or may enhance 'host defense provocation' to the lung, in a manner analogous to asbestos or cigarette smoke. Thus, excessive lung injury through enhanced environmental exposure, endogenous defects in critical homeostatic mechanisms, or subtle defects in host defense can, over the years, lead to the development of pulmonary fibrosis. More attention should be directed to host defense and cell-cell adhesion when considering targets amenable to drug treatment for this complex disease.

[0338] The present findings should substantially influence future pharmacological, genetic and diagnostic studies of IIP. The cumulative GWAS SNPs reported here explain approximately one-third of the variability in risk of developing IIP, suggesting that further examination of common variation with larger cohorts is warranted, in addition to studies of rare variation, epigenetic features, and gene-environment interactions. Although the clinical manifestations of these diseases have been well defined, it is becoming increasingly clear that each type of IIP is caused by multiple genetic variants that likely have distinct prognoses and may respond differently to pharmacological intervention. Consequently, subjects with IIP for genotyping in future therapeutic trials can inform drug development by identifying agents that are effective in selective patients. Indeed, the lack of attention to pharmacogenetic approaches in IIP trials may explain why few agents have been found to alter the course of these diseases. Furthermore, the genetic heterogeneity of IIP suggests that characterizing genetic variants is useful in redefining IIP types so that we can provide more accurate prognostic information for patients and their families.

[0339] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to persons skilled in the art and should be included within the spirit and scope of this application and scope of the attached claims. All publications, patents, patent applications, websites and databases cited herein are incorporated by reference in their entirety for all purposes. TABLE 1: SIGNIFICANT LOCI ACROSS THE ENTIRE GENOME IN DISCOVERY GWAS (GWAS P -VALUE < 5 X 10-8) aBased on NCBI Construct 36, bGene name if SNP falls in the coding region of the gene, cAdjusted for sex MAF: Minor allele frequency; secondary allele defined as secondary allele in combined case and control group; OR: Odds ratio for the secondary allele; CI: Confidence interval TABLE 2: SIGNIFICANT LOCIS ACROSS THE ENTIRE GENOME FROM META-ANALYSIS (GWAS 5X10-8 < P-VALUE <.0001 AND META-ANALYSIS P-VALUE < 5X10-8) aBased on NCBI Construct 36, bGene name if SNP falls in gene coding region, cAdjusted for sex MAF: Minor Allele Frequency; Minor allele defined as Minor allele in combined case and control group; OR: Odds ratio for the secondary Allele; CI: Confidence Interval TABLE 3: GENOTYPE COUNTS AND HARDY-WEINBERG EQUILIBRIUM (HWE) P-VALUES BETWEEN CASES AND CONTROLS IN THE DISCOVERY SET FOR ALL 198 SNPS TAKEN IN THE REPLICATION SET. aGenomic position based on NCBI Construct 36 bSecondary allele in cases listed first. cSecondary: Count of subjects with secondary allele dHet: Count of heterozygous subjects eMain: Count of homozygous subjects with main allele (most frequent allele) fP-value for goodness-of-fit test HWE TABLE 4: GENOTYPE COUNTS AND P-VALUES OF HARDY-WEINBERG EQUILIBRIUM (HWE) BETWEEN CASES AND CONTROLS IN THE REPLICATION SET FOR ALL REPLICATION SUCCESSFULLY GENOTYPED SNPS. aGenomic position based on NCBI Construct 36 bSecondary allele in cases listed first. cSecondary: Count of subjects with secondary allele dHet: Count of heterozygous subjects eMain: Count of homozygous subjects with main allele (most frequent allele) fP-value for HWE goodness-of-fit test Table 5: Association information for all 198 SNPs chosen for replication. Blank and pooled replication columns correspond to SNPs not successfully genotyped in MAF replication: Minor allele frequency; secondary allele defined as secondary allele in combined case and control groups; OR: Odds ratio for the secondary allele; CI: Confidence Interval aBased on pooled analysis of GWAS and replication cases compared to controls to allow adjustment for rs35705950, which is not in the GWAS panel, and age; GWAS cases were regenotyped for the Table 1 SNPs and rs35705950 using the same platform and at the same time as replication cases and controls. bEach SNP was tested for association in a logistic regression model that also included the most strongly associated SNP from the meta-analysis at that locus. The exception is chromosome 11p15, where each SNP was tested for association in a logistic regression model that also included rs35705950.

Claims (8)

1. IN VITRO AND/OR EX VIVO METHOD TO DETERMINE WHETHER A HUMAN SUBJECT PRESENTS OR IS AT RISK OF DEVELOPING INTERSTITIAL LUNG DISEASE, characterized by comprising: the detection, in a biological sample of the subject, of the presence of a genetic variant rs2076295, as per established in the sequence SEQ. ID. NO: 1; and wherein the presence of said genetic variant indicates that the subject has or is at risk of developing interstitial lung disease. 2. METHOD, according to claim 1, characterized by the presence of the genetic variant rs2076295, as established in the sequence SEQ. ID. NO: 1 be determined and compared to a reference set. 3. METHOD, according to claim 2, characterized in that the reference set is determined according to a statistical procedure for risk prediction. 4. METHOD, according to claim 1, characterized by the presence of the genetic variant rs2076295, as established in the sequence SEQ. ID. NO: 1 be determined by PCR. 5. METHOD, according to claim 1, characterized by the presence of the genetic variant rs2076295, as established in the sequence SEQ. ID. NO: 1 be determined by detecting a Forster resonance energy transfer (FRET). 6. METHOD, according to claim 1, characterized in that interstitial lung disease is a fibrotic lung disease. 7. METHOD, according to claim 1, characterized in that interstitial lung disease is idiopathic pulmonary fibrosis (IPF), familial interstitial pneumonia (FIP) or idiopathic interstitial pneumonia (IIP). 8. METHOD, according to claim 1, characterized by further comprising: a) comparing the presence of the genetic variant rs2076295, as established in the sequence SEQ. ID. NO: 1 in the biological sample with a set of genetic variants from a subject or control group presenting interstitial lung disease, and b1) the selection of the individual as presenting or being at risk of developing interstitial lung disease if the genetic variant rs2076295, as established in the sequence SEQ. ID. NO: 1 present in the biological sample is the same as the genetic variants of the subject or control group, or b2) the selection of the individual as not presenting or at risk of developing interstitial lung disease if the genetic variant present in the biological sample is not the same than the genetic variants of the subject or control group.