EP1192275A2 - Prediction des risques de pathologie interstitielle pulmonaire - Google Patents

Prediction des risques de pathologie interstitielle pulmonaire

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Publication number
EP1192275A2
EP1192275A2 EP00921536A EP00921536A EP1192275A2 EP 1192275 A2 EP1192275 A2 EP 1192275A2 EP 00921536 A EP00921536 A EP 00921536A EP 00921536 A EP00921536 A EP 00921536A EP 1192275 A2 EP1192275 A2 EP 1192275A2
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Prior art keywords
allele
tnf
irn
ild
gene
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Gordon W. Duff
Francesco Saverio Di Giovine
Moria Whyte
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Interleukin Genetics Inc
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Interleukin Genetics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • ILD Interstitial lung disease
  • ILD Interstitial lung disease
  • fnterstitial Lung Disease and Idiopathic Pulmonary Fibrosis is a broad term applied to disorders of both known and unknown etiology characterized by fibrosis and inflammation.
  • Representative known etiologies for ILD include occupational exposures (silicosis, asbestosis, berylliosis, coal miner's pneumoconiosis and hard metal pneumoconiosis), infectious exposures (fungal disease and post- viral syndromes), systemic rheumatoid disorders (rheumatoid arthritis, systemic lupus erythematosis, Sjogren's syndrome, systemic sclerosis, dermatomyositis/polymyositis, mixed connective tissue disease and ankylosing spondilytis) and other miscellaneous causes (drug- induced pneumonitis, oxygen toxicity, radiation exposure, hypersensitivity pneumonitis and
  • IPF acute interstitial pneumonitis
  • IPF interstitial pneumonitis
  • UPF interstitial pneumonitis
  • DIP desquamative interstitial pneumonitis
  • IPF IPF-like sarcoidosis
  • IPF IPF-like sarcoidosis
  • the cause for IPF is, by definition, unknown. Contributory factors have been identified, including organic dust exposure, antecedent viral illnesses, and cigarette smoking. Smoking may also cause pulmonary bronchiolitis, an entity histologically similar to DIP that can also lead to fibrosis.
  • IPF irritable bowel syndrome
  • the disease is usually a progressive one, with early symptoms worsening over time. Upon presentation, the patient may manifest dyspnea at rest, dyspnea worsening with exertion, and a non-productive cough. Constitutional symptoms of malaise and weight loss may also be found. There may be accompanying rheumatological symptoms such as arthralgias even in the absence of discrete rheumatological disease. Findings on physical examination generally include tachypnea, exercise induced cyanosis and bibasilar, fine, late inspiratory crackles. These crackles are thought to be associated with subpleural fibrosis, although vibrations from the airway walls may also be involved.
  • Dyspnea and limitation of physical activity may be quantitated as part of the patient's physical evaluation.
  • Dyspnea is thought to be due to reduced lung compliance and increased elastic work of breathing.
  • the patient may have sufficiently advanced pulmonary disease to have developed pulmonary hypertension or even cor pulmonale.
  • the anatomic changes to the lungs and the pulmonary vasculature may not be reversible by the time the patient presents for medical evaluation.
  • the typical IPF patient is of middle age and is somewhat more likely to be male than female. Sudden onset of symptoms for which no etiology can be identified is suggestive of AIP or Hamman-Rich syndrome. AIP patients are younger, and may include children. Although the disease has a poor prognosis, those who survive may have no pulmonary residua. Another syndrome of acute onset is bronchiolitis obliterans with organizing pneumonia (BOOP). This disorder, involving damage to the small airways and adjacent lung parenchyma, tends to have a good prognosis.
  • BOOP bronchiolitis obliterans with organizing pneumonia
  • IPF is a diagnosis of exclusion.
  • a familial form of IPF has been described that is thought to be autosomal dominant with variable penetrance (Bitterman et al., "Familial IPF: evidence of lung inflammation in unaffected family members," N. Engl. J. Med. 314:1343-1341, 1986). Further evaluation of the symptomatic patient requires laboratory investigation.
  • pulmonary function tests are presently available: pulmonary function tests, radiological studies, bronchioalveolar lavage and lung biopsy. No non-invasive diagnostic modality exists to date that will unequivocally yield the diagnosis of IPF.
  • Radiological studies that are undertaken include plain chest Xrays (CXRs) and CT scans.
  • CXRs plain chest Xrays
  • CT scan is thought to be superior to CXR in the evaluation of IPF.
  • Limitations in the diagnostic accuracy of CT scan in IPF have been noted. For example, the diffuse patchiness of the disease may be missed by CT scan. Further, CT scan may miss early cases of IPF.
  • Other techniques, including gallium scans, MRI and PET scans have been undertaken to diagnose IPF, without notable success.
  • Bronchioalveolar lavage is performed to recover fluid from the bronchioalveolar tree that can be analyzed for cellular elements, pathogens and secreted proteins.
  • This technique involves instilling a saline solution into the bronchioalveolar tree through a flexible bronchoscope that has been wedged in a third or fourth order bronchus. The fluid retrieved is then analyzed. It is still considered by most authorities to be a research tool, rather than a diagnostic method of proven clinical utility. Nonetheless, the identification of inflammatory substances correlates with other indicia of active lung inflammation, even though the technique is not standardized. Serial BALs may be followed in a particular patient to monitor a response to therapy or to predict a prognosis, although its overall diagnostic accuracy is questionable.
  • Lung biopsy may be required to establish the diagnosis of IPF.
  • Lung biopsy usually involves an open procedure through a thoracotomy because it permits sampling of grossly affected areas as well as more proximal areas that may be in the early stage of the disease.
  • Open lung biopsy results in serious complications 11-23% of the time.
  • Thorascopic techniques have lowered this complication rate while still providing diagnostic accuracy equivalent to the open procedures.
  • a surgical intervention with general anesthesia is required, with the morbidity thereby entailed.
  • the decision to proceed with a biopsy must take into consideration the patient's often fragile clinical status and the likelihood of post-operative complications, as well as the clinical utility of the information the biopsy will provide. Patients are not infrequently treated empirically in order to avoid the biopsy that would yield the definitive diagnosis.
  • Pulmonary fibrosis has also been identified as a co-morbid condition in other lung disorders.
  • ARDS adult respiratory distress syndrome
  • pulmonary fibrosis in the adult respiratory distress syndrome (ARDS) patient is correlated with a 57% mortality rate, in contrast to the 0% mortality in those patients without pulmonary fibrosis.
  • ARDS adult respiratory distress syndrome
  • Pulmonary fibrosis is a frequent and serious complication of treating early stage invasive breast cancer with wide excision and radiation (B ⁇ ttner et al., "Local production of interleukin-4 during radiation-induced pneumonitis and pulmonary fibrosis in rats: macrophages as a prominent source of interleukin-4," Am. J. Respir. Cell. Mol. Biol. 17(3): 15- 25, 1997).
  • mice Some genetic variability in susceptibility to radiation induced pulmonary fibrosis has been identified in mice (Johnston, et al., "Differences in correlation of mRNA gene expression in mice sensitive and resistant to radiation-induced pulmonary fibrosis," Radiat. Res. 142 (2): 197-203, 1995).
  • TGF- beta is one of the cytokines that is involved in the development of post-radiation pulmonary fibrosis, though other cytokines have also been implicated (Yi, et al., "Radiation-induced lung injury in vivo: expression of transforming growth factor-beta precedes fibrosis," Inflammation 20(4):339-52, 1996, Zhang, et al, "Cytokines and pulmonary fibrosis," Biol Signals 5(4):232-9, 1996, Johnston, et al., "Early and persistent alterations in the expression of interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor alpha mRNA levels in fibrosis-resistant and sensitive mice after thoracic irradiation," Radiat.
  • IPF is understood to have a 50% five-year survival rate.
  • Planning treatment has been stymied by the inadequacy of diagnostic methods (Sharma, "Idiopathic pulmonary fibrosis,” Curr. Opin. Pulm. Med. 2(5):343-6, 1996).
  • ILD patients may be candidates for more aggressive therapies, including corticosteroids, antimetabolites, cytotoxic drugs, colchicine or combinations thereof (Entzian, et al., "Anti- inflammatory and antifibrotic properties of colchicine: implications for idiopathic pulmonary fibrosis," Lung 175(l):4 ⁇ -5 ⁇ , 1997, Hunninghake, et al., "Approaches to the treatment of pulmonary fibrosis," Am. J. Respir. Crit. Care Med. 151(3 Pt 7):915-8, 1995).
  • Anticytokine therapies have also been proposed for use in IPF, but these agents are sufficiently complex in their pharmacological behavior that precise diagnosis should precede their utilization.
  • heritable diseases have depended on either the identification of abnormal gene products (e.g., sickle cell anemia) or an abnormal phenotype (e.g., mental retardation). These methods are of limited utility for heritable diseases with late onset and no easily identifiable phenotypes such as, for example, Alzheimer's disease. With the development of simple and inexpensive genetic screening methodology it is now possible to identify polymorphisms that indicate a propensity to the development of a disease, even when the disease is of polygenic origin. The number of diseases that can be screened by molecular biological methods continues to grow with increased understanding of the genetic basis of multifactorial disorders.
  • abnormal gene products e.g., sickle cell anemia
  • phenotype e.g., mental retardation
  • Genetic screening can be broadly defined as testing to determine if a patient has mutations (or alleles or polymorphisms) that either cause a disease state or are "linked” to the mutation causing a disease state.
  • Linkage refers to the phenomenon that DNA sequences which are close together in the genome have a tendency to be inherited together. Two sequences may be linked because of some selective advantage of co- inheritance. More typically, however, two polymo ⁇ hic sequences are co-inherited because of the relative infrequency with which meiotic recombination events occur within the region between the two polymo ⁇ hisms.
  • the co-inherited polymo ⁇ hic alleles are said to be in linkage disequilibrium with one another because, in a given human population, they tend to either both occur together or else not occur at all in any particular member of the population. Indeed, where multiple polymo ⁇ hisms in a given chromosomal region are found to be in linkage disequilibrium with one another, they define a quasi-stable genetic "haplotype.” In contrast, recombination events occurring between two polymo ⁇ hic loci cause them to become separated onto distinct homologous chromosomes. If meiotic recombination between two physically linked polymo ⁇ hisms occurs frequently enough, the two polymo ⁇ hisms will appear to segregate independently and are said to be in linkage equilibrium.
  • one or more polymo ⁇ hic alleles of the haplotype can be used as a diagnostic or prognostic indicator of the likelihood of developing the disease.
  • This association between otherwise benign polymo ⁇ hisms and a disease-causing polymo ⁇ hism occurs if the disease mutation arose in the recent past, so that sufficient time has not elapsed for equilibrium to be achieved through recombination events. Therefore identification of a human haplotype which spans or is linked to a disease-causing mutational change, serves as a predictive measure of an individual's likelihood of having inherited that disease-causing mutation.
  • prognostic or diagnostic procedures can be utilized without necessitating the identification and isolation of the actual disease-causing lesion.
  • the statistical correlation between a disorder and a polymo ⁇ hism does not necessarily indicate that the polymo ⁇ hism directly causes the disorder. Rather the correlated polymo ⁇ hism may be a benign allelic variant which is linked to (i.e. in linkage disequilibrium with) a disorder-causing mutation which has occurred in the recent human evolutionary past, so that sufficient time has not elapsed for equilibrium to be achieved through recombination events in the intervening chromosomal segment.
  • detection of a polymo ⁇ hic allele associated with that disease can be utilized without consideration of whether the polymo ⁇ hism is directly involved in the etiology of the disease.
  • a broad-spanning human haplotype (describing the typical pattern of co-inheritance of alleles of a set of linked polymo ⁇ hic markers) can be targeted for diagnostic pu ⁇ oses once an association has been drawn between a particular disease or condition and a corresponding human haplotype.
  • the determination of an individual's likelihood for developing a particular disease of condition can be made by characterizing one or more disease- associated polymo ⁇ hic alleles (or even one or more disease-associated haplotypes) without necessarily determining or characterizing the causative genetic variations.
  • the IL-1 gene cluster is on the long arm of chromosome 2 (2ql3) and contains at least the genes for IL-1 alpha (IL-1 A), IL-1 beta (IL-1B), and the IL-1 receptor antagonist (IL- 1RN), within a region of 430 Kb (Nicklin, et al, Genomics, 19:382-4 (1994)).
  • the agonist molecules, IL-1 alpha and IL-1 beta have potent pro-inflammatory activity and are at the head of many inflammatory cascades. Their actions, often via the induction of other cytokines such as IL-6 and IL-8, lead to activation and recruitment of leukocytes into damaged tissue, local production of vasoactive agents, fever response in the brain and hepatic acute phase response.
  • IL-1 molecules bind to type I and to type II IL-1 receptors, but only the type I receptor transduces a signal to the interior of the cell. In contrast, the type II receptor is shed from the cell membrane and acts as a decoy receptor.
  • the receptor antagonist and the type II receptor therefore, are both anti-inflammatory in their actions.
  • IL-IRN allele 2 has been found to be associated with coronary artery disease (co-owned PCT/US/98/04725, and USSN 08/813456), osteoporosis (co- owned U.S. Patent No. 5,698,399), nephropathy in diabetes mellitus (Blakemore, et al. (1996) Hum. Genet. 97(3): 369-74), alopecia areata (Cork, et al., (1995) J. Invest. Dermatol. 104(5 Supp.): 15S-16S; Cork et al. (1996) Dermatol Clin.
  • IL-1 A allele 2 from marker -889 and IL-1B (Taql) allele 2 from marker +3954 are associated with periodontal disease (co-owned U.S. Patent No. 5,686,246; Kornman and di Giovine (1998) Ann Periodont 3: 327-38: Hart and Kornman (1997) Periodontol 2000 14: 202-15; Newman (1997) Compend Contin Educ Dent 18: 881-4; Kornman et al. (1997) J. Clin Periodontol 24: 72-77).
  • the IL-1A allele 2 from marker -889 has also been found to be associated with juvenile chronic arthritis, particularly chronic iridocyclitis (McDowell, et al.
  • IL-1B (Taql) allele 2 from marker +3954 of IL-1B has also been found to be associated with psoriasis and insulin dependent diabetes in DR3/4 patients (di Giovine, et al. (1995) Cytokine 7: 606; Pociot, et al. (1992) Eur J. Clin. Invest. 22: 396-402). Additionally, the IL-IRN (VNTR) allele 1 is associated with diabetic retinopathy (See Co-owned US ⁇ N 09/037472, and PCT/GB97/02790).
  • the tumor necrosis factor (TNF) locus lies in the class III region of the major histocompatibility complex (MHC) on the short arm of chromosome 6, approximately 250 kilobases (kb) centromeric of the human leukocyte antigen (HLA)-B locus and 850 kb telomeric of the class II region (Carroll et al. (1987) Proc Natl Acad Sci USA 84:8535-9; Dunham et al. (1987) Proc Natl Acad Sci USA 84:7237-41).
  • MHC major histocompatibility complex
  • TNF- ⁇ and lymphotoxin- ⁇ lie within a 7-kb stretch and are separated by 1.1 kb in a tandem arrangement, LT- ⁇ lying telomerically. Both consist of four exons and three introns and encode short 5' untranslated and longer 3' untranslated stretches in the corresponding mRNA (Nedospasov et al. (1986) Cold Spring Harbor Symp Quant Biol 511:611-24; Nedwin et al. (1985) Nucleic Acids Res 13:6361- 73).
  • the MHC is a 4-megabase (Mb) stretch of DNA on the short arm of chromosome 6 (Campbell et al. (1993) Immunol Today 14:349-52), comprising approximately 0.1% of the human genome. It is known to contain 110 genes, most of which code for immunologically relevant proteins (Trowsdale (1993) Trends Genet 9:117-22).
  • a striking feature of the MHC is the high degree of polymo ⁇ hism of the genes in the class I and II regions (Bodmer et al. (1991) Tissue Antigens 37:97-104).
  • haplotypes HLA-A1-B8-DR3-DQ2 and HLA-A2-B44-DR4-DQ8 occur more frequently than the products of their individual allelic frequencies would suggest (Tiwari et al. (1985) New York: Springer-Verlag).
  • Genes in the class III region have also been shown to be polymo ⁇ hic.
  • the complement cluster containing the genes for the two isotypes of C4: C4A and C4B, as well as the genes for C2 and factor B, lies at the centromeric end of this region in close proximity to the two steroid 21-hydroxylase genes (Campbell et al. (1988) Annu Rev Immunol 6:161-95).
  • These genes are also highly polymo ⁇ hic, with large deletions involving several genes associated with particular MHC haplotypes (Schneider et al. (1986) J Clin Invest 78:650-57; Braun et al. (1990) J Exp Med 171:129-40).
  • the 70-kd heat-shock protein which contains a restriction fragment length polymo ⁇ hism (RFLP) (Pugliese et al. (1992) Diabetes 41 : 788-91) and at the telomeric end lies the TNF locus, which is also polymo ⁇ hic (see below).
  • RFLP restriction fragment length polymo ⁇ hism
  • Susceptibility is, however, multifactorial. as shown by studies of concordance rates for disease in monozygotic twins. If a disease is purely genetic then a concordance rate of 100% would be expected. However, the rate varies from 5% for multiple sclerosis to 30% for rheumatoid arthritis. These observations indicate that additional environmental factors, perhaps viral or bacterial, are important in disease susceptibility.
  • TNF ⁇ is mainly secreted by macrophages.
  • the expression of TNF-a is induced by bacterial lipopolysaccharides, mitogens, and viruses, and it is regulated both transcriptionally and postranscriptionally (Golfeld et al. (1990) Proc Natl Acad Sci USA 87: 9769-73; Golfeld et al. (1991) J Exp Med 174: 73-81; Han et al. (1990) J Exp Med 171: 465-75; Han et al. (1991) J Immunol 146: 1843-48).
  • TNFA 5'- and 3'- flanking regions surrounding the coding sequence, and as well as sequence occurring in the introns distributed between the coding exons.
  • There are approximately 1000 base pairs in the TNFA 5'- flanking region which contain elements critical to transcriptional control, including three putative NFkB type consensus sequences, a Y-box of the MHC class II promotors, and a cyclic adenosine 3' 5'- monophosphate (cAMP) response element (CRE) similar to that of the somatostatin promotor.
  • cAMP cyclic adenosine 3' 5'- monophosphate response element
  • the NFkB element nearest the coding sequence is an area of intense study, with overlapping elements involved in transcriptional inhibition by cyclosporin A and activation by the nuclear factor C/EBPB in T cells.
  • the third intron possesses enhancer activity which stems from viral enhancer sequences within this region.
  • the 3' untranslated region (3'UTR) contains evolutionarily conserved TA-rich sequences, also present in the 3'UTR of other inflammation-related genes including granulocyte macrophage colony stimulating factor (GM- CSF) and the human hepatocyte inducible nitric oxide synthase genes.
  • GM- CSF granulocyte macrophage colony stimulating factor
  • NF-kB transcription factor nuclear factor kB
  • IkB 37-kD inhibitor
  • polymo ⁇ hisms in the TNFA promoter may cause polymo ⁇ hism-specific differences in the transcriptional response of TNFA to LPS and other TNFA inducing stimuli, thereby at least partly accounting for interindividual differences in clinical presentations among patients with similar disease processes, as well as fundamental interindividual differences in genetic predisposition to such processes.
  • polymo ⁇ hisms in or near the TNFA promoter have been described in humans (Pociot F., et. al 1993 Gene).
  • C3H/HeJ mice differ from their wild-type congenics only by mutation of the "LPS gene" located on murine chromosome 4. These mice are resistant to challenges with large doses of endotoxin which are uniformly fatal to wild-type animals. Macrophages from C3H/HeJ mice, when stimulated with LPS in vitro, failed to secrete the TNF- ⁇ protein; however, nearly normal increases in TNFA mRNA were noted.
  • TNFA human and mouse IL-1 A human and mouse GM- CSF, human and mouse interferon (IFN) a, human and mouse c-fos, and others.
  • IFN interferon
  • 3'UTR of the human hepatocyte inducible nitric oxide synthase gene was also shown to contain a TTATTTAT consensus sequence and several TA-rich flanking sequences. Thus far, these sequences have been shown to have two important regulatory roles. First, the presence of TA-rich regions confers mRNA instability and therefore a short mRNA half-life. Furthermore, substitution of the 3'UTR of GM-CSF for the 3'UTR of ⁇ -globin caused a decrease in mRNA half-life from several hours to several minutes.
  • TNFA mRNA half-life is indeed short, there is yet no definite evidence to demonstrate that TNFA mRNA half-life is appreciably altered following LPS stimulation.
  • the second regulatory role of TTATTTAT concerns the efficiency of translation of mRNA.
  • TTATTTAT a > 90% inhibition of protein production resulted from inclusion of a single UUAUUUAU sequence in the 3'UTR of a reporter construct (Kruys, V. et. al. (1989) Science 245: 852-55; Kruys et al. (1987) Proc. Natl. Acad Sci USA 84: 6030-34).
  • transgenic mice in which the TNFA 3'UTR has been replaced by the 3'UTR of the globin gene displayed disregulated TNF- ⁇ biosynthesis and developed chronic inflammatory polyarthritis (Keffer J., et al. (1991) EMBO J 10: 4025-31).
  • polymo ⁇ hisms in the 3'UTR of various mouse strains including mutations in the TTATTTAT sequences in New Zealand white and Mus spretus strains indicated that polymo ⁇ hisms in the 3'UTR of the TNFA gene may be associated with TNFA mediated diseases in animals, and potentially in humans as well (Beutler et al. (1993) Gene 129: 279-83).
  • a 190-base pair fragment of the TNFA 3'UTR which included the TTATTTAT motifs was amplified and sequenced from each patient. All patients and control subjects exhibited the normal wild-type sequence, with no deletions, insertions, or substitutions in TTATTTAT; these data imply that mutations in this region occur infrequently, if at all in patients with diseases examined in this study. Nonetheless, it remains possible that 3'UTR polymo ⁇ hisms are responsible for interindividual differences in the response to LPS.
  • TNF- ⁇ is initially synthesized as a prohormone which contains in humans, 76 additional amino acids at the N-terminus. This sequence is cleaved, followed by trimerization and secretion of the mature 157 amino acid sub-units (Ceoh, et al. (1989) J Biol Chem.26: 16256- 80). A 26-kD membrane form of TNF-a is also described which may participate in macrophage killing of target cells (Kriegler et al. (1988) Cell.53: 45-53). There is no information concerning altered regulation of post-translational events following LPS stimulation of macrophages.
  • TNF- ⁇ is a cytokine with a wide variety of functions: it can cause cytolysis of certain tumor cell lines, it is implicated in the induction of cachexia, it is a potent pyrogen causing fever by direct action or by stimulation of interleukin 1 secretion, and it can stimulate cell proliferation and induce cell differentiation under certain conditions.
  • the TNF locus in the class III region of the MHC is also a good candidate gene cluster in autoimmune and inflammatory diseases, but because of the high degree of linkage disequilibrium across the MHC, it is difficult to determine which genes on a haplotype are important in the aetiology of a disease.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • TNF-A gene locus lies in the class III region of the major histocompatibility complex (MHC) and so the association between a particular TNF polymo ⁇ hism and a particular disease or disorder may result from linkage disequilibrium with particular MHC class III alleles.
  • MHC major histocompatibility complex
  • the haplotype HLA-A1-B8-DR3-DQ2 known as the "autoimmune haplotype” has been associated with a number of autoimmune diseases, including insulin dependent diabetes, Graves' disease, myastenia gravis, SLE, dermatitis he ⁇ etiformis and coeliac disease (Svejgaard et al. (1989) Genet Epidemiol 6: 1-14; Welch et al. (1988) Dis Markers 6: 247-55; Ahmed (1993) J Exp Med 178: 2067-75).
  • TNF does have an important role to play in infectious diseases; in a large study of patients with malaria in the Gambia, TNFA allele 2 homozygosity was strongly associated with death from cerebral malaria, and no association with clinical outcome was found with any other marker in the class I and II regions of the MHC (McGuire et al. (1994) Nature 371 : 508-511). Investigations of other infectious diseases will be very interesting in this regard.
  • TNF locus A number of polymo ⁇ hisms have been described in the TNF locus. Three RFLP's have been described in the LT- ⁇ gene.
  • T ⁇ FB1 The association of T ⁇ FB1 with phenotype is not clear; however, one study demonstrating association with high LT- ⁇ production and no association with T ⁇ F- ⁇ production (Messer et al.
  • TNFA consists of a (CA) n sequence and has 12 alleles.
  • TNFB (CT) n sequence has 7 alleles (Jongeneel et al. (1991) Proc Natl Acad Sci USA 88:9717-21).
  • TNFc is a biallelic (CT) n sequence that lies in the first intron of LT- ⁇ (Nedospasov et al.
  • TNFd and TNFe lie 8-10 kb downstream of the TNF- ⁇ gene; both consist of (CT) n sequences and have 7 and 3 alleles, respectively (Udalova et al. (1993) Genomics 16:180-86).
  • CT CT
  • LT- ⁇ Ncol RFLP LT- ⁇ Ncol RFLP
  • TNF production occurs at the transcriptional and post-transcriptional levels (Sariban et al. (1988) J. Clin Invest 81:1506-10). Sequences within the 5' DNA control the rate of transcription (Goldfeld et al. (1991) J Exp Med 174:73-81). This region of the gene was therefore investigated for polymo ⁇ hisms and a biallelic polymo ⁇ hism was discovered at -308 relative to the transcriptional start site involving the substitution of guanine by adenosine in the uncommon (TNF2) allele (Wilson et al. (1992) Hum Mol Genet 1 :353).
  • TNF2 adenosine in the uncommon
  • the TNF2 allele was found to be very strongly associated with HLA-A1-B8-DR3-DQ2 haplotype (Wilson et al. (1993) J Exp Med 177:577-560), raising the possibility that the association of this haplotype with autoimmune diseases and high TNF- ⁇ production may be related to polymo ⁇ hism within the TNF- ⁇ locus.
  • a second polymo ⁇ hism has recently been described in the TNF- ⁇ promoter region at -238, in a putative Y box (D'Alfonso et al. (1994) Immunogenetics 39:150-54), the rare allele of which in linkage disequilibrium with HLA-B18 and -B57.
  • the lung is divided into a multitude of terminal respiratory units or acini at the end of the multiply branched terminal bronchioles.
  • Acini contain minute respiratory bronchioles which in turn give off clusters of alveolar sacs that are formed of multiple alveoli.
  • a cluster of three to five terminal bronchioles, each with its appended acini, is referred to as a pulmonary lobule.
  • the architecture of the lung is vital to the function of gas exchange.
  • the design of the respiratory tree is to permit gas exchange to occur at the alveolar level while protecting the terminal structures from airborne particles.
  • the first line of defense in the respiratory tree is mechanical: cough and glottic closure protect the proximal respiratory tree.
  • the bronchioles are lined with pseudostratified columnar, ciliated epithelial cells admixed with goblet cells adapted for the secretion of mucus. These cells are part of the lung's defense against inhaled particulate matter. Secreted mucus traps particles while the cilia fan the secretions up the tracheobronchial tree to be expelled. Repeated branching of bronchioles render access to the distal alveoli more difficult.
  • the alveoli are intimately associated with a network of anastamosing capillaries that maximize the surface area for gas exchange.
  • the alveolar walls or septae consist of structures derived from both capillary endothelium and respiratory epithelium. Beginning on the blood side, moving towards the air side, these structures include: 1) capillary endothelium; 2) a basement membrane and surrounding interstitial tissue between the vascular system and the respiratory system; 3) the alveolar epithelium; 4) pulmonary surfactant.
  • Gas exchange takes place when the oxygen in the alveolus passes through its wall, to be taken up by the red blood cells in an adjacent capillary, a process that is coincident with the diffusion of dissolved carbon dioxide from the bloodstream into the alveolus.
  • the layers of the alveolar wall are organized to achieve these two gas exchange functions.
  • Alveolar epithelium is made up of two main cell types: Type I pneumocytes, covering about 95% of the alveolar surface, and Type II pneumocytes.
  • Type II pneumocytes have two important functions. These cells are the source of pulmonary surfactant, and they are responsible for the repair of the alveolar epithelium after it is damaged. Loosely attached to the alveolar epithelium or floating free within the alveolus are alveolar macrophages. The alveolar walls are perforated by numerous pores that permit solid and liquid material to pass readily between adjacent alveoli.
  • ILDs are among the conditions affecting the interstitium between the alveolar wall and the capillary endothelium area. ILDs are characterized by a combination of inflammation and fibrosis. Alveolitis is understood to be the initial abnormality in ILD. Infiltrates of lymphocytes and plasma cells are observed early in the course of the disease. Soon after, there follows interstitial edema with the loss of Type I pulmonary epithelial cells and capillary endothelial cells. Desquamation may accompany these processes. Desquamation is defined as the state in which Type II pneumocytes and alveolar macrophages come to fill the alveolar lumen. Desquamation indicates active inflammation.
  • Type II pneumocytes proliferate in areas where the lung damage is less severe and where there is less fibrosis.
  • Cuboidal epithelial cells and metaplastic squamous epithelial cells regenerate the alveolar epithelium in areas that have been more severely damaged. As the damaged areas heal, there is an accumulation of fibroblasts and collagen within the alveolar septae. This results in the dense alveolar septal fibrosis that characterizes more advanced ILD.
  • IPF is understood to be one form of ILD.
  • IPF There are two histologically identifiable forms of IPF: UTP and DIP.
  • DIP tends to be clinically milder, characterized pathologically by mild inflammation of the alveolar interstitium, preservation of the alveolar architecture and the presence of large numbers of macrophages in the alveolar air spaces.
  • UIP the alveolar wall is thickened and the lung parenchyma is undergoing reorganization, with inflammatory cells and fibrosis evident.
  • DIP and UIP may represent different stages of the same disease process. The characteristic changes may be found in different places in the same specimen.
  • Ultrastructural histopathology may help differentiate IPF from those ILD conditions associated with other etiologies, such as those ILDs associated with systemic rheumatologic disorders. Endothelial cell swelling and intracellular tuboreticular structures are seen in patients with systemic rheumatologic disorders and associated ILD; the tuboreticular structures are also seen in patients with viral pneumonia. These findings, however, are not observed in IPF patients.
  • AIP is another form of ILD. Its course tends to be rapid, progressive and often fatal. It can be distinguished from IPF on the basis of routine histology.
  • the histological changes found in AIP include intra-alveolar hyaline membranes, interstitial septal widening, endothelial and epithelial damage and cellular fibroblast proliferation without marked collagen deposition. These changes are also found in adult respiratory distress syndrome, which is known to resolve in many cases without permanent lung damage.
  • AIP frequently progresses to a condition of permanent fibrosis, as does IPF.
  • hype ⁇ lasia of the Type II pneumocytes typically takes place within the first few weeks of the disease.
  • the collapsed alveoli coalesce into a single thickened alveolar septum.
  • Type I pneumocytes proliferate along the basement lamina of these alveolar septae, adding to their thickness.
  • Intra-alveolar exudates further thicken the lung parenchyma.
  • AIP there is a characteristic collapse of entire alveoli with apposition of their walls.
  • the final histology of AIP resembles that seen in PF, although their histopathologies are distinct in the earlier stages of the disorders.
  • alveolitis understood herein to be an accumulation of inflammatory and immune effector cells within the alveolar spaces and walls. Inflammatory and immune effector cells within the lung consist mostly of macrophages, with lymphocytes, neutrophils and eosinophils also present. In alveolitis, macrophages and neutrophils predominate. The accumulation of leukocytes in alveolitis has two consequences: it distorts normal alveolar architecture, and it results in the release of mediators that can injure parenchymal cells and stimulate fibrosis.
  • the initial stimulus for alveolitis in ILD can take a number of forms, including environmental inhalants, drug exposure, radiation and infection; the stimulus can also be unknown, as in IPF.
  • the stimulus may have a direct toxic effect on the alveolar epithelium, the capillary endothelium or both, as is the case with certain chemicals, radiation and oxygen free radicals. Beyond this direct toxicity, though, the key event is the triggering of the sequence of inflammatory processes, including recruitment and activation of inflammatory and immune effector cells.
  • the end stage of these processes in ILD is a fibrotic lung in which the alveoli are replaced by cystic spaces separated by thick bands of connective tissue infiltrated with inflammatory cells.
  • alveolar macrophages have as their primary function the ingestion and elimination of foreign material that has entered the alveolus. They are part of the alveolar immune system. For example, they avidly bind particles opsonized by IgG or complement. Most AMs are derived from monocytes in the bloodstream. Bloodborne monocytes pass into the alveolar wall to become AMs. AMs are normally found in the alveolar tissues, comprising about 2-5%> of the normal lung parenchyma. In contrast, neutrophils (PMNs) are rarely found in the alveoli or interstitium in healthy people.
  • AMs neutrophils
  • AMs In response to inflammatory stimuli or in response to certain cytokines, AMs become activated.
  • macrophages produce a large number of enzymes, cytokines and other inflammatory proteins.
  • activated AMs secrete complement components Clq, C2, C3 and C5 that are essential for clearance of opsonized organisms and immune complexes.
  • Important cytokines released by the activated macrophage include IL-1 and TNF, both of which have autocrine and paracrine effects.
  • TNF provides auto-stimulation to monocytes and macrophages to maintain full activation. TNF further stimulates PMNs to full activation.
  • the activated PMN may act as a primary phagocyte, responsible for ingesting and killing invading organisms.
  • These cells may further release free oxygen radicals and lysosomal enzymes into the tissue fluid, causing extracellular killing of pathogens.
  • Side-effects of the release of these cellular cytotoxic products include tissue necrosis, further inflammation and the activation of the coagulation cascade.
  • More PMNs are attracted from adjacent micro vessels by the release of complement cleavage products and TNF. As these PMNs marginate within the microvascular adjacent to the alveoli, they can cause endothelial damage, increased vascular permeability and subsequent exudation of cells and serum proteins into the tissue space. Furthermore, when activated, AMs secrete IL-1.
  • IL-1 In response to macropfiage-derived IL-1, endothelial cells and fibroblasts secrete additional IL-1, thereby amplifying the inflammatory response.
  • IL-1 induces the expression of adhesion molecules on the endothelial cells and is chemotactic for lymphocytes.
  • IL-1 is further understood to induce angiogenesis and fibrosis.
  • the PMNs effects on the local microvessels increase their permeability, thereby increasing the fluid load in the lung parenchyma.
  • the component proteins and cells for the process of fibrosis are the component proteins and cells for the process of fibrosis.
  • Mesenchymal cells of the interstitium considered to be incompletely differentiated AMs, synthesize type I collagen, type III collagen, fibronectin and other matrix proteins found in fibrotic lungs. These cells are more numerous in IPF, and their synthetic products are altered. They are thought to contribute to the interstitial accumulation of fibrous tissue in IPF and related conditions.
  • the lung damage in ILD and in IPF is produced by both AMs and PMNs. It is thought that interactions among the AMs and PMNs, with the release of their cytokines and other active substances, play an important role both in slowly progressive pulmonary fibrosis and in the more fulminant conditions. The inflammatory effects of these cellular mechanisms combine with the processes of fibroplasia to result in the alveolar damage and architectural distortion that characterizes ILDs in general and IPF in particular.
  • Macrophages directly damage lung parenchyma by the release of their activated products such as free oxygen radicals. Further, activated AMs attract and activate PMNs and other inflammatory cells. They release chemotactic factors for PMNs such as leukotriene B4, growth factors for fibroblasts such as fibronectin, platelet derived growth factor and insulin-like growth factor, and proinflammatory cytokines. Macrophages release both IL-1 beta and its specific inhibitor, IL-lRa. It is understood that the IL-lbeta/TL-lRa ratio is increased in patients with IPF, providing a proinflammatory environment.
  • PMNs directly damage lung tissue in multiple ways. Their presence in bronchioalveolar lavage fluid has been correlated with a poor prognosis in IPF. Once activated, PMNs release several cytotoxic substances, including oxidants, proteinases such as collagenase, and products of lipid peroxidation. The reactive products of respiratory oxygenation (superoxide, hydrogen peroxide, hydroxyl radicals and hypochlorous acid) react with essentially all cellular components, causing denaturation and cross-linkage of proteins, changes in membrane permeability and damage to nucleic acids and cellular organelles. Proteolytic enzymes released by PMNs such as elastase and metalloproteinase can digest all the architectural components of the lung interstitium.
  • Neutrophil oxidants act synergistically with these enzymes, heightening local tissue damage. Hypochlorous acid, for example, inactivates the proteolytic inhibitors like alpha- 1 antitryptase that would otherwise check the action of neutrophil elastase. Oxidation products activate neutrophil collagenase. Lipid peroxidation products cause changes in vascular permeability. These substances, further, are chemotactic for neutrophils and lymphocytes. Thus, the inflammatory cycle controlled by PMNs is auto- amplifying.
  • the present invention provides novel methods for identifying whether a patient has or is predisposed to developing an interstitial lung disorder (ILD), such as, but not limited to, IPF.
  • the method comprises determining whether an ILD associated allele is present in a nucleic acid sample obtained from the subject.
  • the ILD associated allele is IL-IRN (+2018) allele 2, TNFA (-308) allele 2, or alternatively a nucleic acid sequence that is in linkage disequilibrium with IL-IRN (+2018) allele 2 or TNFA (-308).
  • the ILD associated allele can be detected by any of a variety of techniques including: 1) performing a hybridization reaction between a nucleic acid sample and a probe that is capable of hybridizing to an ILD associated allele; 2) sequencing at least a portion of an ILD associated allele; or 3) determining the electrophoretic mobility of an ILD associated allele or fragment thereof (e.g., fragments generated by endonuclease digestion).
  • the allele can optionally be subjected to an amplification step prior to performance of the detection step.
  • Preferred amplification steps are selected from the group consisting of: the polymerase chain reaction (PCR), the ligase chain reaction (LCR), strand displacement amplification (SDA), cloning, and variations of the above (e.g.
  • Primers for amplification may be selected to either flank the marker of interest (as required for PCR amplification) or directly overlap the marker (as in ASO hybridization). Oligonucleotides primers that hybridize to II- 1 and TNFA genes can easily be selected with commercially available primer selection programs.
  • the sample is hybridized with a set of primers, which hybridize 5' and/or 3' in a sense or antisense sequence to the ILD associated allele, and is subjected to a PCR amplification.
  • kits for performing the above-described assays can include nucleic acid sample collection means and a means for determining whether a subject carries an ILD associated allele.
  • the kit may also comprise control samples, either negative or positive, or standards.
  • the kit may also include an algorithmic device for assessing identity match.
  • the algorithmic device may be used in conjunction with controls, or may be used independently of controls.
  • the kits of the invention may also contain a variety of additional components such as a DNA amplification reagent, a polymerase, a nucleic acid purification reagent, a restriction enzyme, a restriction enzyme buffer, a nucleic acid sampling device, deoxynucleotides (dNTPs), and the like.
  • Information obtained using the assays and kits described herein is useful for determining whether a non- symptomatic subject has or is likely to develop ILD, or more generally, a disease or condition that is caused by or contributed to by the allelic pattern detected.
  • the information alone or in conjunction with information on another genetic defect contributing to ILD allows customization of therapy for preventing the onset of symptoms associated with ILD, or for preventing the progression of the disease to end-stage, irreversible fibrosis. For example, this information can enable a clinician to: 1) more effectively prescribe a therapeutic that will address the molecular basis of ILD; and 2) better determine the appropriate dosage of a particular therapeutic for a particular subject
  • the invention features methods for treating or preventing the development of an ILD in a subject, by administering to the subject, a pharmaceutically effective amount of an ILD therapeutic of the invention.
  • the invention provides in vitro and in vivo assays for screening test compounds to identify ILD therapeutics.
  • the screening assay comprises contacting a cell transfected with an ILD causative mutation that is operably linked to an appropriate promoter with a test compound and determining the level of expression of a protein in the cell in the presence and in the absence of the test compound.
  • the ILD causative mutation results in decreased production of IL-1 receptor antagonist, and increased production of the IL-1 receptor antagonist or TNF- ⁇ in the presence of the test compound indicates that the compound is an agonist of IL-1 receptor antagonist or TNF- ⁇ activity.
  • the invention features transgenic non-human animals and their use in identifying antagonists of IL-1 ⁇ , IL-l ⁇ or TNF- ⁇ activity or agonists of IL-lRa activity.
  • FIG. 1 shows the nucleic acid sequence for IL-1 A (GEN X03833; SEQ ID No.
  • FIG. 2 shows the nucleic acid sequence for IL-1B (GEN X04500; SEQ ID No.
  • FIG. 3 shows the nucleic acid sequence for the secreted IL-IRN (GEN X64532; SEQ ID No. 3).
  • FIG.4 shows the nucleic acid sequence for TNF-A (GenBank Accession Nos. X02910, X02159, SEQ ID NO. 4).
  • the position of allelic form 1 of the TNF-A (-308) polymo ⁇ hism is indicated by a lower case “g” in bold at position 308 (allele 2 corresponds to "A” at this position).
  • Sequences complementary to the primers used in TNF-A (-308) polymo ⁇ hism typing experiments are underlined.
  • the position of allelic form 1 of the TNF-A (-238) polymo ⁇ hism is indicated by a lower case "g” in bold at position 378 (allele 2 corresponds to "A” at this position).
  • Sequences complementary to the primers used in TNF-A (-238) polymo ⁇ hism typing experiments correspond to nucleotide residues 190 to 212 (forward primer) and 379 to 399 (reverse primer).
  • allele refers to the different sequence variants found at different polymo ⁇ hic sites in DNA obtained from a subject.
  • IL-IRN VNTR
  • the sequence variants may be single or multiple base changes, including without limitation insertions, deletions, or substitutions, or may be a variable number of sequence repeats.
  • Allelic variants at a certain locus are commonly numbered in decreasing order of frequency. In a biallelic situation the frequent allele is allele 1, the rarer allele will be allele 2.
  • 2/2 - Refers to the homozygous allele 2/allele 2 state.
  • 2/1 - Refers to the heterozygous allele 2/allele 1 state.
  • allelic pattern refers to the identity of an allele or alleles at one or more polymo ⁇ hic sites.
  • an allelic pattern may consist of a single allele at a polymo ⁇ hic site, as for IL-IRN (+2018) allele 1, which is an allelic pattern having at least one copy of IL-IRN allele 1 at position +2018 of the IL-IRN gene loci.
  • an allelic pattern may consist of either a homozygous or heterozygous state at a single polymo ⁇ hic site.
  • IL1-RN (VNTR) allele 2,2 is an allelic pattern in which there are two copies of the second allele at the VNTR marker of IL-IRN and that corresponds to the homozygous IL-RN (VNTR) allele 2 state.
  • an allelic pattern may consist of the identity of alleles at more than one polymo ⁇ hic site.
  • antibody as used herein is intended to refer to a binding agent including a whole antibody or a binding fragment thereof which is specifically reactive with an IL-1 or TNF ⁇ polypeptide.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating an antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.
  • the antibody of the present invention is further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for an IL-1 or TNF ⁇ polypeptide conferred by at least one CDR region of the antibody.
  • Bioactivity or “bioactivity” or “activity” or “biological function”, which are used interchangeably, for the pu ⁇ oses herein means an effector or antigenic function that is directly or indirectly performed by an IL-1 or TNF ⁇ polypeptide (whether in its native or denatured conformation), or by any subsequence thereof.
  • Biological activities include binding to a target peptide, e.g., a receptor.
  • a bioactivity can be modulated by directly affecting the polypeptide.
  • a bioactivity can be modulated by modulating the level of a polypeptide, such as by modulating expression of the gene encoding the polypeptide.
  • bioactive fragment refers to a fragment of a full-length polypeptide, wherein the fragment specifically mimics or antagonizes the activity of a wild-type polypeptide.
  • the bioactive fragment preferably is a fragment capable of interacting with a receptor.
  • an aberrant activity refers to an activity which differs from the activity of the wild-type or native polypeptide or which differs from the activity of the polypeptide in a healthy subject.
  • An activity of a polypeptide can be aberrant because it is stronger than the activity of its native counte ⁇ art.
  • an activity can be aberrant because it is weaker or absent relative to the activity of its native counte ⁇ art.
  • An aberrant activity can also be a change in an activity.
  • an aberrant polypeptide can interact with a different target peptide.
  • Cells Cells
  • host cells or “recombinant host cells” are terms used interchangeably herein to refer not only to the particular subject cell, but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact be identical to the parent cell, but is still included within the scope of the term as used herein.
  • a “chimera,” “mosaic,” “chimeric mammal” and the like, refers to a transgenic animal, which has a knock-out or knock-in construct in at least some of its genome-containing cells.
  • control refers to any sample appropriate to the detection technique employed.
  • the control sample may contain the products of the allele detection technique employed or the material to be tested.
  • the controls may be positive (e.g., IL-IRN (+2018) allele 2 or TNFA (-308) allele 2) or negative (e.g., allele 1 of the described marker) controls.
  • the control sample may comprise DNA fragments of the appropriate size.
  • the control sample may comprise a sample of mutant protein.
  • the control sample may comprise the material to be tested.
  • the controls may be a sample of genomic DNA or a cloned portion of the IL-1 gene cluster.
  • the control sample is preferably a highly purified sample of genomic DNA.
  • disruption of the gene and “targeted disruption” or any similar phrase refers to the site specific interruption of a native DNA sequence so as to prevent expression of that gene in the cell as compared to the wild-type copy of the gene.
  • the interruption may be caused by deletions, insertions or modifications to the gene, or any combination thereof.
  • Genotyping refers to the analysis of an individual's genomic DNA (or a nucleic acid corresponding thereto) to identify a particular disease causing or contributing mutation or polymo ⁇ hism, directly or based on detection of a mutation or polymo ⁇ hism (a marker) that is in linkage disequilibrium with the disease causing or contributing gene.
  • haplotype refers to a set of alleles that are inherited together as a group (are in linkage disequilibrium). As used herein, haplotype is defined to include those haplotypes that occur at statistically significant levels (p corr ⁇ 0.05). As used herein, the phrase an "IL-1 haplotype” refers to a haplotype in the IL-1 loci and a "TNFA haplotype” refers to a haplotype in the TNFA loci.
  • interstitial lung disease refers to that group of lung disorders of both known and unknown etiology that are characterized by parenchymal inflammation and fibrosis.
  • the primary pathological locus of these conditions is the interstitial tissue in the alveolar wall between the alveolar epithelium and the capillary endothelium, although the pathological changes in these disorders are not limited to the interstitium.
  • This term includes, but is not limited to those disorders selected from the group consisting of acute interstitial pneumonitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, usual interstitial pneumonitis, desquamative interstitial pneumonitis, bronchiolitis obliterans organizing pneumonia, mineral exposure pneumonitis and fibrosis (silicosis, asbestosis, berylliosis, coal dust pneumoconiosis, hard metal pneumoconiosis), post-adult respiratory distress syndrome fibrosis, hypersensitivity pneumonitis, drug-related pneumonitis, radiation-exposure pneumonitis, oxygen-exposure pneumonitis, sarcoidosis, Goodpasture's syndrome, idiopathic pulmonary hemosiderosis, eosinophilic pneumonia, histiocytosis X, giant cell pneumonitis, lymphocytic interstitial pneumonitis, and the inflammatory/fibrotic manifestations of systemic rhe
  • the term may also encompass other interstitial lung disorders. These include, but are not limited to individuals who are at risk of developing lung disease which is histo logically similar to IPF, such as patients with connective tissue diseases, (e.g., SLE, systemic sclerosis); patients being considered for treatment with chemotherapeutic agents or the anti-arrhythmic amiodarone; and individuals at risk for occupational exposure (e.g.
  • extrinsic allergic alveolitis - sarcoidosis where high levels of IL-IRN are expressed within granulomas
  • chronic inflammatory lung diseases including adult respiratory distress syndrome (ARDS, low concentrations of IL-IRN in bronchoalveolar lavage samples have been shown to be associated with poor prognosis in patients with ARDS); pulmonary embolic diseases, especially the resolution of repeated pulmonary emboli of any type; infectious lung diseases such as tuberculosis (mycobacterial), mycoplasmal, bacterial, viral, protozoan, helminthic and other lung infections associated with an inflammatory response; and reactivity to lung irritants.
  • ILD associated allele refers to an allele whose presence in a subject indicates that the subject is susceptible to developing interstitial lung disease.
  • ILD associated alleles include allele 2 of the +2018 marker of IL-IRN (contains an Msp 1 site); allele 2 of the -308 marker of TNFA (is not cut by Nco I), allele 2 of the VNTR marker of IL-IRN (240 bp PCR product); allele 4 of the 222/223 marker of IL-IA (132 mobility units (mu) PCR product); allele 4 of the gz5/gz6 marker of IL-IA (91 mu PCR product); allele 1 of the -889 marker of IL-IA (contains an Ncol site); allele 1 of the +3954 marker of IL-1 B contains two Taql sites); allele 2 of the -511 marker of IL-1B (contains a Bsu36I site); allele 3 of the gaat.p33330 marker (
  • an "ILD causative functional mutation” refers to a mutation which causes or contributes to the development of interstitial lung disease in a subject. Preferred mutations occur within the IL-1 complex or TNF-A.
  • An ILD causative functional mutation occurring within an IL-1 gene e.g. IL-IA, IL-1B or IL-IRN
  • a TNA A gene or a gene locus, which is linked thereto may alter, for example, the open reading frame or splicing pattern of the gene, thereby resulting in the formation of an inactive or hypoactive gene product.
  • a mutation which occurs in intron 6 of the IL-IA locus corresponds to a variable number of tandem repeat 46 bp sequences corresponding to from five to 18 repeat units (Bailly, et al. (1993) Eur. J. Immunol. 23: 1240-45).
  • These repeat sequences contain three potential binding sites for transcriptional factors: an SP1 site, a viral enhancer element, and a glucocorticoid-responsive element; therefore individuals carrying IL-IA intron 6 VNTR alleles with large numbers of repeat units may be subject to altered transcriptional regulation of the IL-IA gene and consequent perturbations of inflammatory cytokine production.
  • an IL-1B (-511) mutation occurs near a promoter binding site for a negative glucocorticoid response element (Zhang et al. (1997) DNA Cell Biol 16: 145-52). This element potentiates a four-fold repression of IL-1 B expression by dexamethosone and a deletion of this negative response elements causes a 2.5 -fold increase in IL-1B promoter activity.
  • the IL- 1B (-511) polymo ⁇ hism may thus directly affect cytokine production and inflammatory responses.
  • an "ILD therapeutic” refers to any agent or therapeutic regimen (including pharmaceuticals, nutraceuticals and surgical means) that prevents or postpones the development of or alleviates the symptoms of an interstitial lung disease in a subject.
  • An ILD therapeutic can be a polypeptide, peptidomimetic, nucleic acid or other inorganic or organic molecule, preferably a "small molecule” including vitamins, minerals and other nutrients.
  • an ILD therapeutic can modulate at least one activity of an IL-1 and/or TNF- ⁇ polypeptide, e.g., interaction with a receptor, by mimicking or potentiating (agonizing) or inhibiting (antagonizing) the effects of a naturally-occurring polypeptide.
  • An agonist can be a wild-type protein or derivative thereof having at least one bioactivity of the wild-type, e.g., receptor binding activity.
  • An agonist can also be a compound that upregulates expression of a gene or which increases at least one bioactivity of a protein.
  • An agonist can also be a compound which increases the interaction of a polypeptide with another molecule, e.g., a receptor.
  • An antagonist can be a compound which inhibits or decreases the interaction between a protein and another molecule, e.g., a receptor or an agent that blocks signal transduction or post-translation processing (e.g., IL- 1 converting enzyme (ICE) inhibitors).
  • ICE IL- 1 converting enzyme
  • a preferred antagonist is a compound which inhibits or decreases binding to a receptor and thereby blocks subsequent activation of the receptor.
  • An antagonist can also be a compound that downregulates expression of a gene or which reduces the amount of a protein present.
  • the antagonist can be a dominant negative form of a polypeptide, e.g., a form of a polypeptide which is capable of interacting with a target peptide, e.g., a receptor, but which does not promote the activation of the receptor.
  • the antagonist can also be a nucleic acid encoding a dominant negative form of a polypeptide, an antisense nucleic acid, or a ribozyme capable of interacting specifically with an RNA.
  • antagonists are molecules which bind to a polypeptide and inhibit its action.
  • Such molecules include peptides, e.g., forms of target peptides which do not have biological activity, and which inhibit binding to receptors. Thus, such peptides will bind the active site of a protein and prevent it from interacting with target peptides.
  • Yet other antagonists include antibodies interacting specifically with an epitope of a molecule, such that binding interferes with the biological function of the polypeptide.
  • the antagonist is a small molecule, such as a molecule capable of inhibiting the interaction between a polypeptide and a target receptor. Alternatively, the small molecule can function as an antagonist by interacting with sites other than the receptor binding site.
  • An antagonist can be any class of molecule, including a nucleic acid, protein, carbohydrate, lipid or combination thereof, but for therapeutic pu ⁇ oses is preferably a small molecule.
  • Preferred ILD therapeutics include: corticosteroids (e.g. prednisone and methylprednisone), cyclophosphamide (e.g. cytoxan), colchicine. azathioprine (e.g I uran), methotrexate, penicillamine. cyclosporine and other immunosuppressive agents (e.g. chlorambucil and vincristine sulfate).
  • Idiopathic pulmonary fibrosis refers generally to those pulmonary disorders characterized by diffuse interstitial inflammation and fibrosis for which no underlying causative disease process can be identified. As used herein, this term refers to a discrete syndrome wherein symptoms of respiratory difficulty are observed, accompanied in advanced cases by hypoxemia and cyanosis with secondary pulmonary hypertension. Lung histopathology reveals septal fibrosis, that constitutes a significant physiological alveolocapillary block. Mo ⁇ hological changes in the lung vary according to the stage of the disease.
  • the lungs are grossly firm in consistency with microscopic findings of pulmonary edema, intra- alveolar exudation, hyaline membranes, alveolar septal mononuclear infiltration, and hype ⁇ lasia of Type II pneumocytes which appear as cuboidal or columnar cells lining the alveolar spaces.
  • the intra-alveolar exudate organizes into fibrous tissue, and fibrosis and inflammation lead to a thickening of the intra-alveolar septae.
  • the lungs are solid with alternating areas of fibrosis and normal lung consistency.
  • the lung consists of spaces lined by cuboidal or columnar epithelium separated by inflammatory fibrous tissue.
  • IPF is understood to represent a stereotyped inflammatory response of the alveolar wall to injuries of different types, durations or intensities (Kobzik and Schoen, "The lung,” pp. 673-734 in Robbins' Pathological Basis of Disease, eds. Coltran et al. (Philadelphia: W.B. Saunders, 1994) at 714).
  • the initiating injury results in interstitial edema with the accumulation of inflammatory cells, a condition generally termed alveolitis.
  • the Type I membranous pneumocyte is commonly injured by these processes.
  • the Type II pneumocytes then proliferate in an attempt to reconstitute the alveolar epithelial lining.
  • Fibroblasts enter the area as part of the region's attempt to heal the injured area. Fibroplasia in the interalveolar septae and IN the intra-alveolar exudate results in the obliteration of the normal pulmonary architecture.
  • IL-1 gene cluster and "IL-1 loci” as used herein include all the nucleic acid at or near the 2ql3 region of chromosome 2, including at least the IL-IA, IL-1B and IL-IRN genes and any other linked sequences. (Nicklin et al, Genomics 19: 382-84, 1994).
  • the gene accession number for IL-IA, IL-1B, and IL-IRN are X03833, X04500, and X64532, respectively.
  • IL-1 functional mutation refers to a mutation within the IL-1 gene cluster that results in an altered phenotype (i.e. effects the function of an IL-1 gene or protein). Examples include: IL- lA(+4845) allele 2, IL-1B (+3954) allele 2, IL-1B (+6912) allele 2 and IL-IRN (+2018) allele
  • IL-1X (Z) allele Y refers to a particular allelic form, designated Y, occurring at an IL-1 locus polymo ⁇ hic site in gene X, wherein X is IL-IA, B, or RN or some other gene in the IL-1 gene loci, and positioned at or near nucleotide Z, wherein nucleotide Z is numbered relative to the major transcriptional start site, which is nucleotide +1, of the particular IL-1 gene X.
  • IL-1X allele (Z) refers to all alleles of an IL-1 polymo ⁇ hic site in gene X positioned at or near nucleotide Z.
  • IL-IRN (+2018) allele refers to alternative forms of the IL-IRN gene at marker +2018.
  • IL-IRN (+2018) allele 1 refers to a form of the IL-IRN gene which contains a cytosine (C) at position +2018 of the sense strand.
  • C cytosine
  • IL-IRN (+2018) allele 2 refers to a form of the IL- IRN gene which contains a thymine (T) at position +2018 of the plus strand.
  • IL-IRN (+2018) allele 2 refers to the homozygous IL-1 RN (+2018) allele 2 state.
  • IL-IRN (+2018) allele 1,1 refers to the homozygous IL-1 RN (+2018) allele 1 state.
  • IL-IRN (+2018) allele 1,2 refers to the heterozygous allele 1 and 2 state.
  • IL-1 related as used herein is meant to include all genes related to the human IL- 1 locus genes on human chromosome 2 (2q 12-14). These include IL-1 genes of the human IL-1 gene cluster located at chromosome 2 (2q 13-14) which include: the IL-IA gene which encodes interleukin-1 ⁇ , the IL-1B gene which encodes interleukin-1 ⁇ , and the IL-IRN (or IL-lra) gene which encodes the interleukin-1 receptor antagonist. Furthermore these IL-1 related genes include the type I and type II human IL-1 receptor genes located on human chromosome 2 (2ql2) and their mouse homo logs located on mouse chromosome 1 at position 19.5 cM.
  • Interleukin-1 ⁇ , interleukin-1 ⁇ , and interleukin-1 RN are related in so much as they all bind to IL-1 type I receptors, however only interleukin-1 ⁇ and interleukin-1 ⁇ are agonist ligands which activate IL-1 type I receptors, while interleukin-1 RN is a naturally occurring antagonist ligand.
  • IL-1 is used in reference to a gene product or polypeptide, it is meant to refer to all gene products encoded by the interleukin-1 locus on human chromosome 2 (2q 12-14) and their corresponding homologs from other species or functional variants thereof.
  • the term IL-1 thus includes secreted polypeptides which promote an inflammatory response, such as IL-l ⁇ and IL-l ⁇ . as well as a secreted polypeptide which antagonize inflammatory responses, such as IL-1 receptor antagonist and the IL-1 type II (decoy) receptor.
  • IL-1 receptor refers to various cell membrane bound protein receptors capable of binding to and/or transducing a signal from IL-1 locus-encoded ligand.
  • the term applies to any of the proteins which are capable of binding interleukin-1 (IL-1) molecules and, in their native configuration as mammalian plasma membrane proteins, presumably play a role in transducing the signal provided by IL-1 to a cell.
  • IL-1 interleukin-1
  • the term includes analogs of native proteins with IL-1 -binding or signal transducing activity. Examples include the human and murine IL-1 receptors described in U.S. Patent No. 4,968,607.
  • IL-1 nucleic acid refers to a nucleic acid encoding an IL-1 protein.
  • IL-1 polypeptide and IL-1 protein are intended to encompass polypeptides comprising the amino acid sequence encoded by the IL-1 genomic DNA sequences shown in Figures 1, 2, and 3, or fragments thereof, and homologs thereof and include agonist and antagonist polypeptides.
  • Increased risk refers to a statistically higher frequency of occurrence of the disease or condition in an individual carrying a particular polymo ⁇ hic allele in comparison to the frequency of occurrence of the disease or condition in a member of a population that does not carry the particular polymo ⁇ hic allele.
  • interact as used herein is meant to include detectable relationships or associations (e.g. biochemical interactions) between molecules, such as interactions between protein-protein, protein-nucleic acid, nucleic acid-nucleic acid and protein-small molecule or nucleic acid-small molecule in nature.
  • an isolated nucleic acid encoding one of the subject IL-1 polypeptides preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the IL-1 gene in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequences, and most preferably less than 1.5kb of such naturally occurring flanking sequence.
  • kb kilobases
  • isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • a "knock-in" transgenic animal refers to an animal that has had a modified gene introduced into its genome and the modified gene can be of exogenous or endogenous origin.
  • a “knock-out" transgenic animal refers to an animal in which there is partial or complete suppression of the expression of an endogenous gene (e.g, based on deletion of at least a portion of the gene, replacement of at least a portion of the gene with a second sequence, introduction of stop codons, the mutation of bases encoding critical amino acids, or the removal of an intron junction, etc.).
  • a “knock-out construct” refers to a nucleic acid sequence that can be used to decrease or suppress expression of a protein encoded by endogenous DNA sequences in a cell.
  • the knock-out construct is comprised of a gene, such as the IL-IRN gene, with a deletion in a critical portion of the gene so that active protein cannot be expressed therefrom.
  • a number of termination codons can be added to the native gene to cause early termination of the protein or an intron junction can be inactivated.
  • IL-IRN 5'/neo/ IL-IRN 3' where IL- 1RN5' and IL-IRN 3', refer to genomic or cDNA sequences which are, respectively, upstream and downstream relative to a portion of the IL-IRN gene and where neo refers to a neomycin resistance gene.
  • a second selectable marker is added in a flanking position so that the gene can be represented as: IL-lRN/neo/IL-lRN/TK, where TK is a thymidine kinase gene which can be added to either the IL-1RN5' or the IL-1RN3' sequence of the preceding construct and which further can be selected against (i.e. is a negative selectable marker) in appropriate media.
  • TK is a thymidine kinase gene which can be added to either the IL-1RN5' or the IL-1RN3' sequence of the preceding construct and which further can be selected against (i.e. is a negative selectable marker) in appropriate media.
  • This two-marker construct allows the selection of homologous recombination events, which removes the flanking TK marker, from non-homologous recombination events which typically retain the TK sequences.
  • the gene deletion and/or replacement can be from the exons, introns, especially in
  • Linkage disequilibrium refers to co-inheritance of two alleles at frequencies greater than would be expected from the separate frequencies of occurrence of each allele in a given control population.
  • the expected frequency of occurrence of two alleles that are inherited independently is the frequency of the first allele multiplied by the frequency of the second allele. Alleles that co-occur at expected frequencies are said to be in "linkage equilibrium”.
  • the cause of linkage disequilibrium is often unclear. It can be due to selection for certain allele combinations or to recent admixture of genetically heterogeneous populations.
  • an association of an allele (or group of linked alleles) with the disease gene is expected if the disease mutation occurred in the recent past, so that sufficient time has not elapsed for equilibrium to be achieved through recombination events in the specific chromosomal region.
  • allelic patterns that are comprised of more than one allele a first allelic pattern is in linkage disequilibrium with a second allelic pattern if all the alleles that comprise the first allelic pattern are in linkage disequilibrium with at least one of the alleles of the second allelic pattern.
  • linkage disequilibrium is that which occurs between the alleles at the IL-IRN (+2018) and IL-IRN (VNTR) polymo ⁇ hic sites.
  • the two alleles at IL-IRN (+2018) are 100% in linkage disequilibrium with the two most frequent alleles of IL-IRN (VNTR), which are allele 1 and allele 2.
  • the term "marker” refers to a sequence in the genome that is known to vary among individuals.
  • the IL-IRN gene has a marker that consists of a variable number of tandem repeats (VNTR).
  • VNTR variable number of tandem repeats
  • the marker IL-IRN (+2018) as described herein can be used for identification of propensity to develop ILD.
  • a “mutated gene” or “mutation” or “functional mutation” refers to an allelic form of a gene, which is capable of altering the phenotype of a subject having the mutated gene relative to a subject which does not have the mutated gene.
  • the altered phenotype caused by a mutation can be corrected or compensated for by certain agents. If a subject must be homozygous for this mutation to have an altered phenotype, the mutation is said to be recessive. If one copy of the mutated gene is sufficient to alter the phenotype of the subject, the mutation is said to be dominant. If a subject has one copy of the mutated gene and has a phenotype that is intermediate between that of a homozygous and that of a heterozygous subject (for that gene), the mutation is said to be co-dominant.
  • non-human animal of the invention includes mammals such as rodents, non- human primates, sheep, dogs, cows, goats, etc.
  • Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse, though transgenic amphibians, such as members of the Xenopus genus, and transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation.
  • transgenic amphibians such as members of the Xenopus genus
  • transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation.
  • chimeric animal is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant gene is expressed in some but not all cells of the animal.
  • tissue-specific chimeric animal indicates that one of the recombinant IL-1 genes is present and/or expressed or disrupted in some tissues but not others.
  • non-human mammal refers to any members of the class Mammalia, except for humans.
  • nucleic acid refers to polynucleotides or oligonucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs (e.g. peptide nucleic acids) and as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • polymo ⁇ hism refers to the coexistence of more than one form of a gene or portion (e.g., allelic variant) thereof.
  • a portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a "polymo ⁇ hic region of a gene".
  • a specific genetic sequence at a polymo ⁇ hic region of a gene is an allele.
  • a polymo ⁇ hic region can be a single nucleotide, the identity of which differs in different alleles.
  • a polymo ⁇ hic region can also be several nucleotides long.
  • propensity to disease means that certain alleles are hereby discovered to be associated with or predictive of ILD.
  • the alleles are thus over-represented in frequency in individuals with disease as compared to healthy individuals. Thus, these alleles can be used to predict disease even in pre-symptomatic or pre-diseased individuals.
  • Small molecule as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5kD and most preferably less than about 4kD. Small molecules can be nucleic acids, peptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.
  • the term “specifically hybridizes” or “specifically detects” refers to the ability of a nucleic acid molecule to hybridize to at least approximately 6 consecutive nucleotides of a sample nucleic acid.
  • Systemic rheumatologic disorder refers to a disease selected from the group including at least the following disorders: systemic lupus erythematosis, Sjogren's syndrome, systemic sclerosis, dermatomyositis/polymyositis, mixed connective tissue disease, ankylosing spondylitis and the seronegative spondyloarthropathies.
  • Transcriptional regulatory sequence is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
  • transgene means a nucleic acid sequence (encoding, e.g., one of the IL-1 polypeptides, or an antisense transcript thereto) which has been introduced into a cell.
  • a transgene could be partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can also be present in a cell in the form of an episome.
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
  • a "transgenic animal” refers to any animal, preferably a non-human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical crossbreeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • transgene causes cells to express a recombinant form of one of the IL-1 or TNF ⁇ polypeptides, e.g. either agonistic or antagonistic forms.
  • transgenic animals in which the recombinant gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below.
  • transgenic animal also includes those recombinant animals in which gene disruption of one or more genes is caused by human intervention, including both recombination and antisense techniques. The term is intended to include all progeny generations. Thus, the founder animal and all FI, F2, F3, and so on, progeny thereof are included.
  • treating is intended to encompass curing as well as ameliorating at least one symptom of a condition or disease.
  • vector refers to a nucleic acid molecule, which is capable of transporting another nucleic acid to which it has been linked.
  • One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • plasmid and "vector” are used interchangeably as the plasmid is the most commonly used form of vector.
  • vector is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • wild-type allele refers to an allele of a gene which, when present in two copies in a subject results in a wild-type phenotype. There can be several different wild-type alleles of a specific gene, since certain nucleotide changes in a gene may not affect the phenotype of a subject having two copies of the gene with the nucleotide changes.
  • the present invention is based, at least in part, on the identification of alleles that are associated (to a statistically significant extent) with the development of interstitial lung disease in subjects.
  • alleles that are associated (to a statistically significant extent) with the development of interstitial lung disease in subjects.
  • IL-IRN (+2018) allele 2 and TNFA (-308) allele 2 have been shown to be associated with ILD. Therefore detection of these alleles in a subject indicate that the subject has or is predisposed to the development of an
  • IL-IRN (+2018) allele 2 also referred to as exon 2 (8006)
  • alleles of the 11-1 (44112332) proinflammatory haplotype are known to be in linkage disequilibrium with IL-IRN (+2018): allele 4 of the 222/223 marker of IL-IA (a dinucleotide repeat polymo ⁇ hism (HUGO GDB: 190869); allele 4 of the gz5/gz6 marker of IL-IA (a trinucleotide repeat polymo ⁇ hism (HUGO GDB: 177384; Zuliani et al, Am. J. Hum. Genet. 46:963-69, 1990); allele 1 of the -889 marker of IL-IA (a single base variation marker- HUGO GDB: 210902; McDowell et al, Arthritis and Rheumatism 38:221-28,
  • allele 1 of the +3954 marker of IL-1B (a single base C/T variation; di Giovine et al, Cytokine 7:606 (1995); Pociot et al. Eur J. Clin. Invest. 22:396-402, 1992); allele 2 of the -511 marker of IL-1B; allele 3 of the gaat.p33330 marker; and allele 3 of the Y31 marker.
  • IL-IRN Three other polymo ⁇ hisms in an IL-IRN alternative exon (Exon lie, which produces an intracellular form of the gene product.
  • GEN X77090 are in linkage disequilibrium with IL-IRN (+2018) allele 2. These include: the IL-IRN exon lie (1812) polymo ⁇ hism (GenBank:X77090 at 1812); the IL-IRN exon lie (1868) polymo ⁇ hism (GenBank:X77090 at 1868); and the IL-IRN exon lie (1887) polymo ⁇ hism (GenBank:X77090 at 1887).
  • allelic patterns described above one of skill in the art can readily identify other alleles (including polymo ⁇ hisms and mutations) that are in linkage disequilibrium with IL-IRN (+2018) allele 2, and are thereby associated with ILD.
  • a nucleic acid sample from a first group of subjects without ILD can be collected, as well as DNA from a second group of subjects with ILD.
  • the nucleic acid sample can then be compared to identify those alleles that are over-represented in the second group as compared with the first group, wherein such alleles are presumably associated with ILD.
  • alleles that are in linkage disequilibrium with an ILD associated allele can be identified, for example, by genotyping a large population and performing statistical analyses to determine which alleles appear more commonly together than expected.
  • the group is chosen to be comprised of genetically related individuals. Genetically related individuals include individuals from the same race, the same ethnic group, or even the same family. As the degree of genetic relatedness between a control group and a test group increases, so does the predictive value of polymo ⁇ hic alleles which are ever more distantly linked to a disease-causing allele. This is because less evolutionary time has passed to allow polymo ⁇ hisms which are linked along a chromosome in a founder population to redistribute through genetic cross-over events.
  • race-specific, ethnic-specific, and even family-specific diagnostic genotyping assays can be developed to allow for the detection of disease alleles which arose at ever more recent times in human evolution, e.g., after divergence of the major human races, after the separation of human populations into distinct ethnic groups, and even within the recent history of a particular family line.
  • Linkage disequilibrium between two polymo ⁇ hic markers or between one polymo ⁇ hic marker and a disease-causing mutation is a meta-stable state. Absent selective pressure or the sporadic linked reoccurrence of the underlying mutational events, the polymo ⁇ hisms will eventually become disassociated by chromosomal recombination events and will thereby reach linkage equilibrium through the course of human evolution. Thus, the likelihood of finding a polymo ⁇ hic allele in linkage disequilibrium with a disease or condition may increases with changes in at least two factors: decreasing physical distance between the polymo ⁇ hic marker and the disease-causing mutation, and decreasing number of meiotic generations available for the dissociation of the linked pair.
  • Appropriate probes may be designed to hybridize to a specific gene of the IL-1 locus, such as IL-IA, IL-1B or IL-IRN, TNFA or a related gene.
  • IL-IA, IL-1B or IL-IRN, TNFA or a related gene are shown in Figures 1-4, respectively, and further correspond to formal SEQ ID Nos. 1-4, respectively.
  • these probes may inco ⁇ orate other regions of the relevant genomic locus, including intergenic sequences. Indeed the IL-1 region of human chromosome 2 spans some 400,000 base pairs and, assuming an average of one single nucleotide polymo ⁇ hism every 1,000 base pairs, includes some 400 SNPs loci alone. Yet other polymo ⁇ hisms available for use with the immediate invention are obtainable from various public sources.
  • the human genome database collects intragenic SNPs, is searchable by sequence and currently contains approximately 2,700 entries (http://hgbase.interactiva.de). Also available is a human polymo ⁇ hism database maintained by the Massachusetts Institute of Technology (MIT SNP database (http://www.genome.wi.mit.edu SNP/human/index.html)). From such sources SNPs as well as other human polymo ⁇ hisms may be found.
  • IL-1 locus genes are flanked by a centromere proximal polymo ⁇ hic marker designated microsatellite marker AFM220ze3 at 127.4 cM (centiMorgans) (see GenBank Ace. No. Z17008) and a distal polymo ⁇ hic marker designated microsatellite anchor marker AFM087xal at 127.9 cM (see GenBank Ace. No. Z16545).
  • These human polymo ⁇ hic loci are both CA dinucleotide repeat microsatellite polymo ⁇ hisms, and, as such, show a high degree of heterozygosity in human populations.
  • one allele of AFM220ze3 generates a 211 bp PCR amplification product with a 5' primer of the sequence TGTACCTAAGCCCACCCTT-TAGAGC (SEQ ID No. 5) and a 3' primer of the sequence TGGCCTCCAGAAACCTCCAA (SEQ ID No. 6).
  • one allele of AFM087xal generates a 177 bp PCR amplification product with a 5' primer of the sequence GCTGATATTCTGGTGGGAAA (SEQ ID No.7) and a 3' primer of the sequence GGCAAGAGCAAAACTCTGTC (SEQ ID No. 8).
  • Equivalent primers corresponding to unique sequences occurring 5' and 3' to these human chromosome 2 CA dinucleotide repeat polymo ⁇ hisms will be apparent to one of skill in the art.
  • Reasonable equivalent primers include those which hybridize within about 1 kb of the designated primer, and which further are anywhere from about 17 bp to about 27 bp in length.
  • a number of other human polymo ⁇ hic loci occur between these two CA dinucleotide repeat polymo ⁇ hisms and provide additional targets for determination of an ILD prognostic allele in a family or other group of genetically related individuals.
  • the National Center for Biotechnology Information web site www.ncbi.nlm.nih.gov/genemap/
  • nucleotide segments of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of human chromosome 2 q 12-13 or cDNAs from that region or to provide primers for amplification of DNA or cDNA from this region.
  • the design of appropriate probes for this pu ⁇ ose requires consideration of a number of factors. For example, fragments having a length of between 10, 15, or 18 nucleotides to about 20, or to about 30 nucleotides, will find particular utility. Longer sequences, e.g., 40, 50, 80, 90, 100, even up to full length, are even more preferred for certain embodiments.
  • oligonucleotides of at least about 18 to 20 nucleotides are well accepted by those of skill in the art as sufficient to allow sufficiently specific hybridization so as to be useful as a molecular probe.
  • relatively stringent conditions For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids. For example, relatively low salt and/or high temperature conditions, such as provided by 0.02 M-0.15M NaCl at temperatures of about 50° C to about 70° C. Such selective conditions may tolerate little, if any, mismatch between the probe and the template or target strand.
  • the preferred method for detecting a specific polymo ⁇ hic allele may depend, in part, upon the molecular nature of the polymo ⁇ hism.
  • the preferred method of detection used for a single nucleotide polymo ⁇ hism may differ from that employed for a VNTR polymo ⁇ hism.
  • detection of specific alleles may be nucleic acid techniques based on hybridization, size, or sequence, such as restriction fragment length polymo ⁇ hism (RFLP), nucleic acid sequencing, and allele specific oligonucleotide (ASO) hybridization.
  • the methods comprise detecting in a sample DNA obtained from a woman the existence of an allele associated with ILD.
  • a nucleic acid composition comprising a nucleic acid probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence to an allele associated with ILD can be used as follows: the nucleic acid in a sample is rendered accessible for hybridization, the probe is contacted with the nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected.
  • Such technique can be used to detect alterations or allelic variants at either the genomic or mRNA level as well as to determine mRNA transcript levels, when appropriate.
  • an allele associated with ILD at a VNTR polymo ⁇ hism. such as IL-IRN (VNTR) allele 2 may be determined.
  • the number of tandem repeats of the IL-IRN (VNTR) polymo ⁇ hic site may be determined by amplifying the nucleic acid to be analyzed, and determining the identity of the allele of that site by analyzing the size of said amplification product.
  • a preferred detection method is ASO hybridization using probes overlapping an allele associated with ILD and has about 5, 10, 20, 25, or 30 nucleotides around the mutation or polymo ⁇ hic region.
  • several probes capable of hybridizing specifically to other allelic variants involved in EOM are attached to a solid phase support, e.g., a "chip" (which can hold up to about 250,000 oligonucleotides).
  • Oligonucleotides can be bound to a solid support by a variety of processes, including lithography. Mutation detection analysis using these chips comprising oligonucleotides, also termed "DNA probe arrays" is described e.g., in Cronin et al, Human Mutation 7:244, 1996.
  • a chip comprises all the allelic variants of at least one polymo ⁇ hic region of a gene.
  • the solid phase support is then contacted with a test nucleic acid and hybridization to the specific probes is detected. Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment.
  • Amplification techniques are known to those of skill in the art and include, but are not limited to cloning, polymerase chain reaction (PCR), polymerase chain reaction of specific alleles (ASA), ligase chain reaction (LCR), nested polymerase chain reaction, self sustained sequence replication (Guatelli, J.C. et al, Proc. Natl. Acad. Sci. USA 87:1874-78, 1990), transcriptional amplification system (Kwoh, O.Y. et al, Proc. Natl Acad. Sci. USA 86:1173-77, 1989), and Q- Beta Replicase (Lizardi, P.M. et al, Bio/Technology 6:1197, 1988).
  • Amplification products may be assayed in a variety of ways, including size analysis, restriction digestion followed by size analysis, detecting specific tagged oligonucleotide primers in the reaction products, allele-specific oligonucleotide (ASO) hybridization, allele specific 5' exonuclease detection, sequencing, hybridization, and the like.
  • ASO allele-specific oligonucleotide
  • PCR based detection means can include multiplex amplification of a plurality of markers simultaneously. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously. Alternatively, it is possible to amplify different markers with primers that are differentially labeled and thus can each be differentially detected. Of course, hybridization based detection means allow the differential detection of multiple PCR products in a sample. Other techniques are known in the art to allow multiplex analyses of a plurality of markers.
  • the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic. mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to IL-IRN (+2018) allele 2 or any nucleic acid sequence in linkage disequilibrium with that allele under conditions such that hybridization and amplification of the desired marker occurs, and (iv) identifying the amplification product.
  • nucleic acid e.g., genomic. mRNA or both
  • IL-IRN (+2018) allele 2 or TNFA (-308) allele 2 is identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases. and fragment length sizes are determined by gel electrophoresis.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence IL-IRN (+2018) allele 2 or any nucleic acid sequence in linkage disequilibrium with it.
  • Exemplary sequencing reactions include those based on techniques developed by Maxim and Gilbert (Proc. Natl. Acad. Sci. USA 74:560, 1977) or Sanger (Sanger et al, Proc. Nat. Acad. Sci. USA 74:5463, 1977). It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing the subject assays (Biotechniques 19:448, 1995), including sequencing by mass spectrometry (see, for example PCT publication WO 94/16101; Cohen et al, Adv.
  • protection from cleavage agents can be used to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNA heteroduplexes (Myers et al, Science 230:1242, 1985).
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • mismatched bases in RNA/RNA or RNA/DNA or DNA/DNA heteroduplexes (Myers et al, Science 230:1242, 1985).
  • mismatch cleavage starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type allele with the sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S 1 nuclease to enzymatically digest the mismatched regions.
  • either DNA/DNA or RNA DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. (See. for example, Cotton et al, Proc. Natl. Acad. Sci. USA 85:4397, 1988: Saleeba et al. Methods Enzymol 217:286-95, 1992)
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes).
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et ⁇ /., Carcinogenesis 15:1657-62, 1994).
  • a probe based on IL-IRN (+2018) allele 2 is hybridized to a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. (See, for example, U.S. Patent No. 5,459,039.)
  • alterations in electrophoretic mobility will be used to identify IL-IRN (+2018) allele 2 or any nucleic acid sequence in linkage disequilibrium with it.
  • SSCP single strand conformation polymo ⁇ hism
  • Single-stranded DNA fragments of sample and control IL-IRN (+2018) alleles or alleles of any nucleic acid sequence in linkage disequilibrium with them are denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al, Trends Genet. 7:5, 1991).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner, Biophys. Chem. 265:12753, 1987).
  • oligonucleotide primers may be prepared in which the known mutation or nucleotide difference (e.g., in allelic variants) is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al, Nature 324:163, 1986); Saiki et al, Proc. Natl. Acad. Sci. USA 86:6230, 1989).
  • Such allele specific oligonucleotide hybridization techniques may be used to test one mutation or polymo ⁇ hic region per reaction when oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations or polymo ⁇ hic regions when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation or polymo ⁇ hic region of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al, Nucleic Acids Res. 17:2437-2448, 1989) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner, Tibtech 11:238, 1993.
  • amplification may also be performed using Taq ligase for amplification (Barany, Proc. Natl. Acad. Sci USA 88:189, 1991). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • identification of the allelic variant is carried out using an oligonucleotide ligation assay (OLA), as described, e.g., in U.S. Pat. No. 4,998,617 and in Landegren et al, Science 241:1077-80, 1988.
  • OLA oligonucleotide ligation assay
  • the OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target.
  • One of the oligonucleotides is linked to a separation marker, e.g,. biotinylated, and the other is detectably labeled.
  • the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand.
  • Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson et al, Proc. Natl. Acad. Sci. USA 87:8923-27, 1990. In this method, PCR is used to achieve the exponential amplification of target DNA. which is then detected using OLA.
  • U.S. Patent No. 5,593,826 discloses an OLA using an oligonucleotide having 3'-amino group and a 5'-phosphorylated oligonucleotide to form a conjugate having a phosphoramidate linkage.
  • OLA combined with PCR permits typing of two alleles in a single microtiter well.
  • each OLA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase.
  • This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors.
  • the single base polymo ⁇ hism can be detected by using a specialized exonuclease-resistant nucleotide, as disclosed, e.g., in U.S. Pat. No.4,656,127 (Mundy et al).
  • a primer complementary to the allelic sequence immediately 3' to the polymo ⁇ hic site is permitted to hybridize to a target molecule obtained from a particular animal or human.
  • the polymo ⁇ hic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be inco ⁇ orated onto the end of the hybridized primer. Such inco ⁇ oration renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymo ⁇ hic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of extraneous sequence data.
  • a solution-based method is used for determining the identity of the nucleotide of a polymo ⁇ hic site.
  • a primer is employed that is complementary to allelic sequences immediately 3' to a polymo ⁇ hic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymo ⁇ hic site will become inco ⁇ orated onto the terminus of the primer.
  • GBA TM Genetic Bit Analysis
  • the method of Goelet et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymo ⁇ hic site.
  • the labeled terminator that is inco ⁇ orated is thus determined by, and complementary to, the nucleotide present in the polymo ⁇ hic site of the target molecule being evaluated.
  • the method of Goelet et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.
  • RNA is initially isolated from available tissue and reverse-transcribed, and the segment of interest is amplified by PCR.
  • the products of reverse transcription PCR are then used as a template for nested PCR amplification with a primer that contains an RNA polymerase promoter and a sequence for initiating eukaryotic translation.
  • the unique motifs inco ⁇ orated into the primer permit sequential in vitro transcription and translation of the PCR products.
  • RNA DNA (as opposed to RNA) is used as a PCR template when the target region of interest is derived from a single exon.
  • DASH Dynamic Allele Specific Hybridization
  • a target sequence is amplified by PCR in which one primer is biotinylated.
  • the biotinylated product strand is bound to a streptavidin or avidin coated microtiter plate well, and the non- biotinylated strand is rinsed away with alkali.
  • An oligonucleotide probe specific for one allele, is hybridized to the target at low temperature. This forms a duplex DNA region that interacts with a double strand-specific intercalating dye. Upon excitation, the dye emits fluorescence proportional to the amount of double stranded DNA (probe-target duplex) present. The sample is then steadily heated while fluorescence is continually monitored. A rapid fall in fluorescence indicates the denaturing (or "melting") temperature of the probe-target duplex. When performed under appropriate buffer and dye conditions, a single-base mismatch between the probe and the target results in a dramatic lowering of melting temperature (Tm) that can be easily detected (Howell, W.M. et al., (1999) Nature Biotechnology 17:)87-88.
  • Tm melting temperature
  • the DNA sample is obtained from a bodily fluid, e.g, blood, obtained by known techniques (e.g. venipuncture) or saliva.
  • a bodily fluid e.g. blood
  • nucleic acid tests can be performed on dry samples (e.g. hair or skin).
  • the cells or tissues that may be utilized must express the genes of the IL-1 loci.
  • Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary.
  • Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, PCR in situ Hybridization: Protocols and Applications (Raven Press, NY, 1992)).
  • profiles may also be assessed in such detection schemes.
  • Finge ⁇ rint profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT- PCR.
  • kits for detecting a propensity for ILD in a patient may contain one or more oligonucleotides, including 5' and 3' oligonucleotides that hybridize 5' and 3' to an ILD associated marker (e.g. IL-IRN (+2018) allele 2 or TNFA (-308) allele 2), or any nucleic acid sequence in linkage disequilibrium with that marker, or detection oligonucleotides that hybridize to the ILD associated marker.
  • the kit may also contain one or more oligonucleotides capable of hybridizing near or at other alleles of the TNFA gene or an IL-1 gene.
  • PCR amplification primers should hybridize between 25 and 2500 base pairs apart, preferably between about 100 and about 500 bases apart, in order to produce a PCR product of convenient size for subsequent analysis.
  • oligonucleotides may be any of a variety of natural and/or synthetic compositions such as synthetic oligonucleotides, restriction fragments, cDNAs, synthetic peptide nucleic acids (PNAs), and the like.
  • the assay kit and method may also employ labeled oligonucleotides to allow ease of identification in the assays. Examples of labels which may be employed include radio-labels, enzymes, fluorescent compounds, streptavidin, avidin, biotin, magnetic moities. metal binding moities, antigen or antibody moities, and the like.
  • the kit may, optionally, also include DNA sampling means such as the AmpliCardTM (University of Sheffield, Sheffield, England S10 2JF; Tarlow, et al, J. of Invest. Dermatol 103:387-389, 1994) and the like; DNA purification reagents such as NucleonTM kits, lysis buffers, proteinase solutions and the like; PCR reagents, such as lOx reaction buffers, thermostable polymerase, dNTPs, and the like; and allele detection means such as the Hinfl restriction enzyme, allele specific oligonucleotides, degenerate oligonucleotide primers for nested PCR from dried blood.
  • DNA sampling means such as the AmpliCardTM (University of Sheffield, Sheffield, England S10 2JF; Tarlow, et al, J. of Invest. Dermatol 103:387-389, 1994) and the like
  • DNA purification reagents such as NucleonTM kits,
  • comparison of an individual's IL-1 and/or TNF-A profile to the population profile for the disease permits the selection or design of drugs that are expected to be safe and efficacious for a particular patient or patient population (i.e., a group of patients having the same genetic alteration).
  • the ability to target populations expected to show the highest clinical benefit, based on genetic profile can enable: 1) the repositioning of marketed drugs with disappointing market results; 2) the rescue of drug candidates whose clinical development has been discontinued as a result of safety or efficacy limitations, which are patient subgroup-specific; and 3) an accelerated and less costly development for drug candidates and more optimal drug labeling (e.g. since measuring the effect of various doses of an agent on an ILD causative mutation is useful for optimizing effective dose).
  • the treatment of an individual with a particular therapeutic can be monitored by determining protein (e.g. IL-l ⁇ , IL-l ⁇ , IL-lRa or TNA ⁇ ), mRNA and/or transcriptional level. Depending on the level detected, the therapeutic regimen can then be maintained or adjusted (increased or decreased in dose).
  • protein e.g. IL-l ⁇ , IL-l ⁇ , IL-lRa or TNA ⁇
  • the effectiveness of treating a subject with an agent comprises the steps of: (i) obtaining a preadministration sample from a subject prior to administration of the agent; (ii) detecting the level or amount of a protein, mRNA or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the protein, mRNA or genomic DNA in the post-administration sample; (v) comparing the level of expression or activity of the protein, mRNA or genomic DNA in the preadministration sample with the corresponding protein, mRNA or genomic DNA in the postadministration sample, respectively; and (vi) altering the administration of the agent to the subject accordingly.
  • Cells of a subject may also be obtained before and after administration of a therapeutic to detect the level of expression of genes other than an IL-1 gene or TNFA, to verify that the therapeutic does not increase or decrease the expression of genes which could be deleterious. This can be done, e.g., by using the method of transcriptional profiling.
  • mRNA from cells exposed in vivo to a therapeutic and mRNA from the same type of cells that were not exposed to the therapeutic could be reverse transcribed and hybridized to a chip containing DNA from numerous genes, to thereby compare the expression of genes in cells treated and not treated with the therapeutic.
  • Modulators of IL-1 e.g. IL-l ⁇ , IL-l ⁇ or IL-1 receptor antagonist
  • TNF ⁇ or a protein encoded by a gene that is in linkage disequilibrium with an IL-1 or TNF-A gene can comprise any type of compound, including a protein, peptide, peptidomimetic, small molecule, or nucleic acid.
  • Preferred agonists include nucleic acids (e.g. encoding an IL-1 protein or TNF ⁇ or a gene that is up- or down-regulated by an IL-1 or TNF ⁇ protein), proteins (e.g. IL-1 or TNF ⁇ proteins or a protein that is up- or down-regulated thereby) or a small molecule (e.g.
  • Preferred antagonists which can be identified, for example, using the assays described herein, include nucleic acids (e.g. single (antisense) or double stranded (triplex) DNA or PNA and ribozymes), protein (e.g. antibodies) and small molecules that act to suppress or inhibit IL-1 or TNFA transcription and/or protein activity.
  • nucleic acids e.g. single (antisense) or double stranded (triplex) DNA or PNA and ribozymes
  • protein e.g. antibodies
  • small molecules that act to suppress or inhibit IL-1 or TNFA transcription and/or protein activity.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining The LD50 (the dose lethal to 50% of the population) and the Ed50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50- Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissues in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i- e - > the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • Such information can be used to more accurately determine useful doses in humans.
  • Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, injection, inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the compounds of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences. Meade Publishing Co.. Easton, PA.
  • systemic administration injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous.
  • the compounds of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch or
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable delivery systems include microspheres which offer the possibility of local noninvasive delivery of drugs over an extended period of time. This technology utilizes microspheres of precapillary size which can be injected via a coronary catheter into any selected part of the e.g. heart or other organs without causing inflammation or ischemia. The administered therapeutic is slowly released from these microspheres and taken up by surrounding tissue cells (e.g. endothelial cells).
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through nasal sprays or using suppositories.
  • the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
  • a wash solution can be used locally to treat an injury or inflammation to accelerate healing.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the invention further features cell-based or cell free assays, e.g., for identifying ILD therapeutics.
  • a cell expressing an IL-1 receptor, TNF ⁇ receptor or a receptor for a protein that is encoded by a gene which is in linkage disequilibrium with TNF-A or an IL-1 gene, on the outer surface of its cellular membrane is incubated in the presence of a test compound alone or in the presence of a test compound and a IL-1, TNF- ⁇ or other protein and the interaction between the test compound and the receptor or between the protein (preferably a tagged protein) and the receptor is detected, e.g., by using a microphysiometer (McCormell et al.
  • This assay system thus provides a means of identifying molecular antagonists which, for example, function by interfering with protein- receptor interactions, as well as molecular agonist which, for example, function by activating a receptor.
  • Cellular or cell-free assays can also be used to identify compounds which modulate expression of an IL-1 or TNF-A gene or a gene in linkage disequilibrium therewith, modulate translation of an mRNA, or which modulate the stability of an mRNA or protein. Accordingly, in one embodiment, a cell which is capable of producing an IL-1, TNF- ⁇ or other protein is incubated with a test compound and the amount of protein produced in the cell medium is measured and compared to that produced from a cell which has not been contacted with the test compound. The specificity of the compound vis a vis the protein can be confirmed by various control analysis, e.g., measuring the expression of one or more control genes. In particular, this assay can be used to determine the efficacy of antisense, ribozyme and triplex compounds.
  • Cell-free assays can also be used to identify compounds which are capable of interacting with a protein, to thereby modify the activity of the protein.
  • a compound can, e.g., modify the structure of a protein thereby effecting its ability to bind to a receptor.
  • cell-free assays for identifying such compounds consist essentially in a reaction mixture containing a protein and a test compound or a library of test compounds in the presence or absence of a binding partner.
  • a test compound can be, e.g., a derivative of a binding partner, e.g., a biologically inactive target peptide, or a small molecule.
  • one exemplary screening assay of the present invention includes the steps of contacting a protein or functional fragment thereof with a test compound or library of test compounds and detecting the formation of complexes.
  • the molecule can be labeled with a specific marker and the test compound or library of test compounds labeled with a different marker.
  • Interaction of a test compound with a protein or fragment thereof can then be detected by determining the level of the two labels after an incubation step and a washing step. The presence of two labels after the washing step is indicative of an interaction.
  • An interaction between molecules can also be identified by using real-time BIA (Biomolecular Interaction Analysis, Pharmacia Biosensor AB) which detects surface plasmon resonance (SPR), an optical phenomenon. Detection depends on changes in the mass concentration of macromolecules at the biospecific interface, and does not require any labeling of interactants.
  • a library of test compounds can be immobilized on a sensor surface, e.g., which forms one wall of a micro-flow cell. A solution containing the protein or functional fragment thereof is then flown continuously over the sensor surface. A change in the resonance angle as shown on a signal recording, indicates that an interaction has occurred. This technique is further described, e.g., in BIAtechnology Handbook by Pharmacia.
  • Another exemplary screening assay of the present invention includes the steps of (a) forming a reaction mixture including: (i) an IL-1, TNF- ⁇ or other protein, (ii) an appropriate receptor, and (iii) a test compound; and (b) detecting interaction of the protein and receptor.
  • the compounds of this assay can be contacted simultaneously.
  • a protein can first be contacted with a test compound for an appropriate amount of time, following which the receptor is added to the reaction mixture. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay can also be performed to provide a baseline for comparison.
  • Complex formation between a protein and receptor may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled proteins or receptors, by immunoassay, or by chromatographic detection.
  • detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled proteins or receptors
  • immunoassay or by chromatographic detection.
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the receptor, e.g.
  • an 35s-labeled receptor and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired.
  • the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintilant), or in the supernatant after the complexes are subsequently dissociated.
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of protein or receptor found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples.
  • Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, either protein or receptor can be immobilized utilizing conjugation of biotin and streptavidin.
  • Transgenic animals can also be made to identify agonists and antagonists or to confirm the safety and efficacy of a candidate therapeutic.
  • Transgenic animals of the invention can include non-human animals containing an ILD causative mutation under the control of an appropriate endogenous promoter or under the control of a heterologous promoter.
  • the transgenic animals can also be animals containing a transgene, such as reporter gene, under the control of an appropriate promoter or fragment thereof. These animals are useful, e.g., for identifying drugs that modulate production of an IL-1 or TNF- ⁇ protein, such as by modulating gene expression. Methods for obtaining transgenic non-human animals are well known in the art.
  • the expression of the ILD causative mutation is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis- acting sequences that control expression in the desired pattern.
  • such mosaic expression of a protein can be essential for many forms of lineage analysis and can additionally provide a means to assess the effects of, for example, expression level which might grossly alter development in small patches of tissue within an otherwise normal embryo.
  • tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the mutation in certain spatial patterns.
  • temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.
  • Genetic techniques which allow for the expression of a mutation can be regulated via site-specific genetic manipulation in vivo, are known to those skilled in the art.
  • the transgenic animals of the present invention all include within a plurality of their cells an ILD causative mutation transgene of the present invention, which transgene alters the phenotype of the "host cell".
  • an ILD causative mutation transgene of the present invention which transgene alters the phenotype of the "host cell".
  • either the crelloxP recombinase system of bacteriophage PI Lakso et al. (1992) PNAS 89:6232-6236; Orban et al. (1992) PNAS 89:6861-6865
  • FLP recombinase system of Saccharomyces cerevisiae O'Gorman et al.
  • Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences.
  • lo.xP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination.
  • the orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.
  • genetic recombination of the target sequence is dependent on expression of the Cre recombinase.
  • Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element.
  • the activation of expression of the EOM causative mutation transgene can be regulated via control of recombinase expression.
  • crelloxP recombinase system to regulate expression of an ILD causative mutation transgene requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject protein. Animals containing both the Cre recombinase and the ILD causative mutation transgene can be provided through the construction of "double" transgenic animals. A convenient method for providing such animals is to mate two transgenic animals each containing a transgene. Similar conditional transgenes can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the transgene. Exemplary promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Patent No. 4,833,080.
  • conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the transactivating protein, e.g. a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner.
  • a gene encoding the transactivating protein e.g. a recombinase or a prokaryotic protein
  • the transgene could remain silent into adulthood until "turned on” by the introduction of the transactivator.
  • the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal.
  • Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell.
  • the specific line(s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness.
  • the haplotype is a significant factor. For example, when transgenic mice are to be produced, strains such as C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, ME).
  • Preferred strains are those with H-2 b , H-2 d or H-21 haplotypes such as C57BL/6 or DBA 1.
  • the line(s) used to practice this invention may themselves be transgenics, and/or may be knockouts (i.e., obtained from animals which have one or more genes partially or completely suppressed) .
  • the transgene construct is introduced into a single stage embryo.
  • the zygote is the best target for micro injection.
  • the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1-2 pi of DNA solution.
  • the use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be inco ⁇ orated into the host gene before the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). As a consequence, all cells of the transgenic animal will carry the inco ⁇ orated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.
  • the nucleotide sequence comprising the transgene is introduced into the female or male pronucleus as described below. In some species such as mice, the male pronucleus is preferred. It is most preferred that the exogenous genetic material be added to the male DNA complement of the zygote prior to its being processed by the ovum nucleus or the zygote female pronucleus.
  • ovum nucleus or female pronucleus release molecules which affect the male DNA complement, perhaps by replacing the protamines of the male DNA with histones, thereby facilitating the combination of the female and male DNA complements to form the diploid zygote.
  • the exogenous genetic material be added to the male complement of DNA or any other complement of DNA prior to its being affected by the female pronucleus.
  • the exogenous genetic material is added to the early male pronucleus, as soon as possible after the formation of the male pronucleus, which is when the male and female pronuclei are well separated and both are located close to the cell membrane.
  • the exogenous genetic material could be added to the nucleus of the sperm after it has been induced to undergo decondensation.
  • Sperm containing the exogenous genetic material can then be added to the ovum or the decondensed sperm could be added to the ovum with the transgene constructs being added as soon as possible thereafter.
  • transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
  • the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention.
  • a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism.
  • the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes.
  • the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism.
  • a euploid zygote is preferred. If an aneuploid zygote is obtained, then the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.
  • the biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.
  • the number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. As regards the present invention, there will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences.
  • exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.
  • Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.
  • Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product.
  • DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene.
  • the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
  • Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
  • suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like.
  • Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
  • Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal.
  • the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both.
  • the partner may be a parental line.
  • in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
  • transgenic animals produced in accordance with the present invention will include exogenous genetic material. Further, in such embodiments the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.
  • a transcriptional control element e.g., a promoter
  • Retroviral infection can also be used to introduce the transgene into a non-human animal.
  • the developing non-human embryo can be cultured in vitro to the blastocyst stage.
  • the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).
  • Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).
  • the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al.
  • the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.
  • transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
  • ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) N ⁇ twre 322:445-448).
  • Transgenes can be efficiently introduced into the ES cells by D ⁇ A transfection or by retrovirus- mediated transduction.
  • Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
  • Jaenisch, R. (1988) Science 240: 1468-1474 For review see Jaenisch, R. (1988) Science 240: 1468-1474.
  • Plasma samples are taken by venipuncture and stored uncoagulated at -20°C prior to DNA extraction.
  • Ten milliliters of blood are added to 40 ml of hypotonic red blood cell (RBC) lysis solution (10 mM Tris, 0.32 Sucrose , 4 mM MgCl 2 , 1% Triton X-100) and mixed by inversion for 4 minutes at room temperature (RT).
  • Samples are then centrifuged at 1300 g for 15 minutes, the supernatant aspirated and discarded, and another 30 ml of RBC lysis solution added to the cell pellet.
  • RBC hypotonic red blood cell
  • the pellet is resuspended in 2ml white blood cell (WBC) lysis solution (0.4 M Tris, 60 mM EDTA, 0.15 M NaCl, 10% SDS) and transferred into a fresh 15 ml polypropylene tube.
  • WBC white blood cell
  • Sodium perchlorate is added at a final concentration of 1M and the tubes are first inverted on a rotary mixer for 15 minutes at RT, then incubated at 65°C for 25 minutes, being inverted periodically. After addition of 2 ml of chloroform (stored at-20° C), samples are mixed for 10 minutes at room temperature and then centrifuged at 800 G for 3 minutes.
  • Oligonucleotide primers designed to amplify the relevant region of the gene spanning the polymo ⁇ hic site are synthesized, resuspended in Tris-EDTA buffer (TE), and stored at -20°C as stock solutions of 200 uM. Aliquots of working solutions (1 : 1 mixture of forward and reverse, 20 ⁇ M of each in water) are prepared in advance.
  • PCR reaction mixtures are prepared as detailed below.
  • DNA template is dotted at the bottom of 0.2 ml tubes or microwells. The same volume of water or negative control DNA is also randomly tested.
  • a master-mix (including all reagents except templates) is prepared and added to the wells or tubes, and samples are transferred to the thermocycler for PCR.
  • PCR can be performed in 0.5 ml tubes. 0.2 ml tubes or microwells. according to the thermocycler available. The reaction mixture is overlaid with mineral oil if a heated lid (to prevent evaporation) is not available.
  • a master mix of restriction enzyme buffer and enzyme is prepared and aliquotted in suitable volumes in fresh microwells. Digestion is carried out with an oil overlay or capped microtubes at the appropriate temperature for the enzyme on a dry block.
  • Restriction buffer dilutions are calculated on the whole reaction volume (i.e. ignoring salt concentrations of PCR buffer). Restriction enzymes are used 3-5 times in excess of the recommended concentration to compensate for the unfavorable buffer conditions and to ensure complete digestion.
  • Polyacrylamide-gel electrophoresis (PAGE) of the PCR sample is carried out in Tris-Borate-EDTA buffer and at constant voltage. Depending on the size discrimination need, different PAGE conditions are used (9 to 12% acrylamide, 1.5 mm x 200) and different DNA size marker ( X174-Hae III or X 174-Hinf 1). A 2% agarose horizontal gel can be used for genotyping the IL-IRN (VNTR) marker.
  • VNTR IL-IRN
  • Reaction buffer is 20mM Tris-HCl (pH 8.4), 50mM KC1, 1.75 mM MgCl 2 , 0.2 mM dNTPs, 0.001 mM primers, 0.05% W-l (Gibco-BRL), 100 ng. template, 1.25 Units Taq Polymerase, Cycling is performed at [96 " , 1 min] x 1 ; [94 , 1 min; 57 , 1 min; 70 , 2 min;] x 35; [70 ° , 5 min] x 1; 4 ° C.
  • One part of the PCR products are digested with Alu I, the other with Msp I (37 ' C overnight). Restriction products are sized on PAGE 9%>.
  • Alu I will produce 126 + 28bp fragments for allele 1, while it does not digest allele 2 (154 bp).
  • Msp I will produce 125 + 29bp with allele 2, while allele 1 is uncut (154 bp). The two reactions will give inverted patterns of digestion for homozygote individuals, and identical patterns in heterozygotes.
  • PCR products are digested with Neo I (37 C overnight), electrophoresis by PAGE 6%. Neo I digestion produces 87 + 20 for allele 1, while it does not cut allele 2 (107 bp). Heterozygotes will have 107 + 87 + 29 bp fragments.
  • IL-IRN polymo ⁇ hism (+2018) and the related IL-IRN VNTR confer increased risk of developing IPF and implies that unopposed IL-1 beta biological activities may play a pathophysiological role in this condition.
  • the rare allele of IL- IRN VNTR/IL-IRN (+2018) is associated with lower IL-IRN protein production in vivo (Carter et al., 1978) and in vitro (Tountas et al., 1997). Lower levels of IL-lRa will significantly dampen the anti-inflammatory activity of this cytokine, with a net effect of increased proinflammatory effects of IL-1 alpha and IL-1 beta.
  • Probe 1 5' - C (- FAM) AACCAACTAGTTGCTGGATACTTGCAAG (- TAMRA)
  • Probe 2 5' - C (- TET) AACCAACTAGTTGCCGGATACTTGCAAG (- TAMRA) -
  • VNTR IL-IRN
  • the PCR product sizes are direct indication of number of repeats: the most frequent allele (allele 1) yields a 412 bp product. As the flanking regions extend for 66 bp, the remaining 344 bp imply four 86 bp repeats. Similarly, a 240 bp product indicates 2 repeats (allele 2), 326 is for 3 repeats (allele 3), 498 is 5 (allele 4), 584 is 6 (allele 6). Frequencies in a North British Caucasian population for the four most frequent alleles are 0.734, 0.241, 0.021 and 0.004. Table 8 TNF (-238) Polymorphism Typing
  • MgCE is used at 2 mM final, and PCR primers at 0125 uM. Cycling is performed at [94°, 1 min; 61 °, 1 min, 72°, 1 min;] x35; [72°, 5 min] xl; 4°C. Each PCR reaction is added of 5 Units of Avail in addition to 3ul of the specific 10X restriction buffer. Incubation is at 37°C overnight. Electrophoresis is by PAGE 12%.
  • Genotyping studies were done by investigators at the National Institute of Occupational Safety and Hazards on samples obtained from subjects who worked in mines. The diagnosis of silicosis was based on gross and microscopic analyses of the lungs at autopsy. The controls were miners with no evidence on autopsy of silicosis or other occupational lung disorders.

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Abstract

L'invention concerne des méthodes et des kits nouveaux permettant de déterminer si un sujet est atteint d'une pathologie interstitielle pulmonaire ou s'il est susceptible de développer une telle pathologie, telle que la fibrose pulmonaire. L'invention concerne également des méthodes de traitement d'une pathologie interstitielle pulmonaire et des essais de criblage pour identifier de nouvelles thérapeutiques contre ces pathologies.
EP00921536A 1999-04-02 2000-03-31 Prediction des risques de pathologie interstitielle pulmonaire Withdrawn EP1192275A2 (fr)

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US20030148288A1 (en) * 2002-02-01 2003-08-07 Yi-Wei Tang Colorimetric genetic test for clinically significant TNF polymorphism and methods of use thereof
WO2004035822A1 (fr) * 2002-10-15 2004-04-29 Novartis Ag Methodes pour prevoir des phenomenes d'oedeme comme effets secondaires de traitement medicamenteux
WO2006100446A2 (fr) * 2005-03-23 2006-09-28 Astrazeneca Uk Limited Procede
EP1888779A4 (fr) * 2005-05-20 2009-06-10 Synergenz Bioscience Ltd Méthodes d'analyse de polymorphismes et leurs utilisations
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ES2660113T3 (es) * 2011-12-21 2018-03-20 The Provost, Fellows, Foundation Scholars, & the other members of Board, of the College of the Holy & Undiv. Trinity of Queen Procedimientos relacionados con la fibrosis pulmonar idiopática (FPI)
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