Relationship of DNA methylation and gene expression in idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is an untreatable and often fatal lung disease that is increasing in prevalence and is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control gene expression and are likely to regulate the IPF transcriptome. OBJECTIVES: To identify methylation marks that modify gene expression in IPF lung. METHODS: We assessed DNA methylation (comprehensive high-throughput arrays for relative methylation arrays [CHARM]) and gene expression (Agilent gene expression arrays) in 94 patients with IPF and 67 control subjects, and performed integrative genomic analyses to define methylation-gene expression relationships in IPF lung. We validated methylation changes by a targeted analysis (Epityper), and performed functional validation of one of the genes identified by our analysis. MEASUREMENTS AND MAIN RESULTS: We identified 2,130 differentially methylated regions (DMRs; <5% false discovery rate), of which 738 are associated with significant changes in gene expression and enriched for expected inverse relationship between methylation and expression (P < 2.2 × 10(-16)). We validated 13/15 DMRs by targeted analysis of methylation. Methylation-expression quantitative trait loci (methyl-eQTL) identified methylation marks that control cis and trans gene expression, with an enrichment for cis relationships (P < 2.2 × 10(-16)). We found five trans methyl-eQTLs where a methylation change at a single DMR is associated with transcriptional changes in a substantial number of genes; four of these DMRs are near transcription factors (castor zinc finger 1 [CASZ1], FOXC1, MXD4, and ZDHHC4). We studied the in vitro effects of change in CASZ1 expression and validated its role in regulation of target genes in the methyl-eQTL. CONCLUSIONS: These results suggest that DNA methylation may be involved in the pathogenesis of IPF.

Methacholine challenge testing: improved patient comfort with a 2-tiered protocol.

BACKGROUND: The methacholine challenge test (MCT) is a test of bronchial hyperreactivity used as an aid in the diagnosis of asthma. MCT results are reported as the provocation concentration at which the forced expiratory volume in 1 second (FEV1) decreases 20% (PC20). The requirement for a 20% or greater decrease in FEV1 results in precipitous decreases in FEV1 in some patients. OBJECTIVE: To improve MCT safety without compromising accuracy. METHODS: We performed a retrospective analysis of 879 consecutive MCTs (derivation cohort). A novel protocol for MCT was developed and validated in a cohort of 564 MCTs performed in a second institution. RESULTS: In comparison with a PC20 cutoff of less than 8 mg/mL, a provocation concentration at which the FEV1 decreases 10% (PC10) cutoff of 1 mg/mL or less has a sensitivity of 86%, a specificity of 98%, a positive predictive value (PPV) of 97%, and a negative predictive value (NPV) of 91%. We propose a novel 2-tiered protocol for MCT. If the PC10 is 1 mg/mL or less, bronchial hyperreactivity is present; if the PC10 is greater than 1 mg/mL, the test is continued until the provocative concentration is 8 mg/mL or a 20% decrease in FEV1 is achieved. Compared with the standard protocol, the proposed protocol has a sensitivity, specificity, PPV, NPV, and overall accuracy of 100%, 98%, 97.6%, 100%, and 99%, respectively. The modified protocol would have enabled us to avoid 26 of 42 cases (62%) in which a 40% or greater decrease in FEV1 occurred and would save 0.65 dose for every MCT performed. The 2-tiered protocol performed well in the validation cohort; sensitivity, specificity, PPV, NPV, and overall accuracy were 100%, 98%, 87%, 100%, and 98%, respectively. CONCLUSION: The proposed 2-tiered protocol is accurate, saves time, and avoids precipitous decreases in FEV1.

Profibrotic role of miR-154 in pulmonary fibrosis.

In this study, we explored the regulation and the role of up-regulated microRNAs in idiopathic pulmonary fibrosis (IPF), a progressive interstitial lung disease of unknown origin. We analyzed the expression of microRNAs in IPF lungs and identified 43 significantly up-regulated microRNAs. Twenty-four of the 43 increased microRNAs were localized to the chromosome 14q32 microRNA cluster. We validated the increased expression of miR-154, miR-134, miR-299-5p, miR-410, miR-382, miR-409-3p, miR-487b, miR-31, and miR-127 by quantitative RT-PCR and determined that they were similarly expressed in embryonic lungs. We did not find evidence for differential methylation in this region, but analysis of transcription factor binding sites identified multiple SMAD3-binding elements in the 14q32 microRNA cluster. TGF-ß1 stimulation of normal human lung fibroblasts (NHLF) caused up-regulation of microRNAs on chr14q32 that were also increased in IPF lungs. Chromatin immunoprecipitation confirmed binding of SMAD3 to the putative promoter of miR-154. Mir-154 was increased in IPF fibroblasts, and transfection of NHLF with miR-154 caused significant increases in cell proliferation and migration. The increase in proliferation induced by TGF-ß was not observed when NHLF or IPF fibroblasts were transfected with a mir-154 inhibitor. Transfection with miR-154 caused activation of the WNT pathway in NHLF. ICG-001 and XAV939, inhibitors of the WNT/ß-catenin pathway, reduced the proliferative effect of miR-154. The potential role of miR-154, one of multiple chr14q32 microRNA cluster members up-regulated in IPF and a regulator of fibroblast migration and proliferation, should be further explored in IPF.

A single testing of serum amyloid a levels as a tool for diagnosis and treatment dilemmas in familial Mediterranean fever.

OBJECTIVES: In a significant proportion of patients with familial Mediterranean fever (FMF), serum amyloid A (SAA) remains elevated during attack-free periods, thereby increasing the risk of developing amyloidosis. The aim of the study was to determine various correlates of elevated SAA and evaluate the role of SAA measurement in the diagnosis and management of FMF. METHODS: We reviewed the medical files of all 204 patients from our FMF center in whom SAA measurements were performed. SAA levels and the resulting diagnostic and therapeutic decisions were analyzed in relation to the reasons of SAA testing and to several clinical and genetic parameters. RESULTS: SAA measurements were made for diagnostic purposes in 29% of the patients. In the remainder, SAA measurements were used for adjustment of colchicine dose. Elevated SAA levels are found in a third of FMF patients during an attack-free period. The highest rate of elevated SAA levels was found in patients with proteinuria (60% of this patient group), followed by noncompliant (40%) and genetically positive asymptomatic patients (38%). Elevated SAA levels during remission were associated with family history of FMF, M694V homozygosity, and elevated C-reactive protein (CRP) (P<0.05 for each). Patients homozygous for the M694V mutation had the highest level of SAA. SAA measurement led to a change in colchicine dose in 30% of the patients, predominantly in noncompliant patients and patients with proteinuria or with atypical manifestations. CONCLUSIONS: Measurement of SAA level may help in the diagnosis of FMF and in adjustment of the colchicine dose.

Attempts at suppression of amyloidogenesis in a mouse model by a variety of anti-inflammatory agents.

OBJECTIVE: The mainstay of AA amyloidosis prevention and treatment is suppression of inflammation. In the present study we have tried to determine the efficacy of a variety of anti-inflammatory agents at suppressing AA amyloidosis in a mouse model of the disease. METHODS: AA amyloidosis was induced in Swiss male mice using amyloid enhancing factor and AgNO(3). Suppression of amyloid formation was studied in comparison to saline, using i.p. injections of several non-steroidal anti-inflammatory agents, TNF-α inhibitors, interferon-α, leflunomide and a variety of chemotherapeutic agents, commonly used in the treatment of inflammatory illnesses such as methotrexate, azathioprine, chlorambucil and cyclophosphamide. The degree of splenic amyloid deposition was determined using Congo red staining of smears and a 5 grade scale. RESULTS: The alkylating agents, chlorambucil and cyclophosphamide, each resulted in a significant 88% reduction in amyloid deposition, yielded the most striking effect on amyloidogenesis suppression in the enhanced mouse model (p<0.0002). The non-steroidal anti-inflammatory agents tested varied widely in their ability to suppress amyloid formation in our mouse model, but only diflunisal was significantly effective, inducing a suppression of 57% (p=0.04). Other chemotherapeutic agents tested, methotrexate and azathioprine, yielded 32% and 27% suppression, which fell short of statistical significance. Surprisingly, the immunomodulatory agents etanercept, infliximab, leflunomide and interferon-α had insignificant effects on amyloid formation in this model. CONCLUSION: Our findings suggest that alkylating agents may have a role in the prevention of amyloidogenesis. Further testing of these agents in animal models and in the clinical setting is needed.

Global methylation patterns in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is characterized by profound changes in the lung phenotype including excessive extracellular matrix deposition, myofibroblast foci, alveolar epithelial cell hyperplasia and extensive remodeling. The role of epigenetic changes in determining the lung phenotype in IPF is unknown. In this study we determine whether IPF lungs exhibit an altered global methylation profile. METHODOLOGY/PRINCIPAL FINDINGS: Immunoprecipitated methylated DNA from 12 IPF lungs, 10 lung adenocarcinomas and 10 normal histology lungs was hybridized to Agilent human CpG Islands Microarrays and data analysis was performed using BRB-Array Tools and DAVID Bioinformatics Resources software packages. Array results were validated using the EpiTYPER MassARRAY platform for 3 CpG islands. 625 CpG islands were differentially methylated between IPF and control lungs with an estimated False Discovery Rate less than 5%. The genes associated with the differentially methylated CpG islands are involved in regulation of apoptosis, morphogenesis and cellular biosynthetic processes. The expression of three genes (STK17B, STK3 and HIST1H2AH) with hypomethylated promoters was increased in IPF lungs. Comparison of IPF methylation patterns to lung cancer or control samples, revealed that IPF lungs display an intermediate methylation profile, partly similar to lung cancer and partly similar to control with 402 differentially methylated CpG islands overlapping between IPF and cancer. Despite their similarity to cancer, IPF lungs did not exhibit hypomethylation of long interspersed nuclear element 1 (LINE-1) retrotransposon while lung cancer samples did, suggesting that the global hypomethylation observed in cancer was not typical of IPF. CONCLUSIONS/SIGNIFICANCE: Our results provide evidence that epigenetic changes in IPF are widespread and potentially important. The partial similarity to cancer may signify similar pathogenetic mechanisms while the differences constitute IPF or cancer specific changes. Elucidating the role of these specific changes will potentially allow better understanding of the pathogenesis of IPF.

Interferon-alpha as a treatment modality for colchicine- resistant familial Mediterranean fever.

OBJECTIVE: Previous reports on interferon-alpha (IFN-alpha) were conflicting with respect to its efficacy in familial Mediterranean fever (FMF) refractory to colchicine treatment. We investigated the effect of IFN-alpha in patients with colchicine-resistant FMF. METHODS: In a prospective, patient self-controlled, open-label study evaluating the safety and efficacy of IFN-alpha in patients with FMF with a severe phenotype, refractory to intensified (oral plus intravenous) colchicine therapy, we advised patients to subcutaneously inject IFN-alpha, 3 million international units, at the onset of the FMF attack. Attacks not treated with IFN-alpha of the same patients and in the same sites served as control attacks. Features of each attack were recorded in a questionnaire, eventually used to compare between IFN-alpha-treated and non-treated attacks. RESULTS: Ten patients with a total of 80 attacks were recruited. Compared to 22 untreated attacks, a > 20% and > 50% reduction in the duration of the attacks was noted in 100% and 90% of the 58 IFN-alpha-treated attacks, respectively (p < 0.001 for both). The severity (degree of pain) of the IFN-alpha-treated attacks was attenuated by > 20% and > 50% in 88% and 49% of these attacks, respectively (p < 0.001 for both). The most common drug-related adverse events were chills and fatigue. CONCLUSION: Early intervention with IFN-alpha injections was associated with reduced attack length and/or severity in a substantial number of bouts, with an acceptable cost of adverse events.