Pathway Analysis of Smokers. Tilley et al.
Tina Cole
2026
R Markdown
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1. Load Libraries Needed for Analysis
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##
## The downloaded binary packages are in
## /var/folders/rg/x_7b05fn3sj3v_jq8q367xzm0000gn/T//Rtmp4VxZC7/downloaded_packages================================================================
2. Acquire Dataset & Prepare for DE Analysis
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##
##
## 14
## smoking status: COPD, GOLD-I, 119 pack-years
## 1
## smoking status: COPD, GOLD-I, 14 pack-years
## 1
## smoking status: COPD, GOLD-I, 22 pack-years
## 1
## smoking status: COPD, GOLD-I, 23 pack-years
## 1
## smoking status: COPD, GOLD-I, 24 pack-years
## 1
## smoking status: COPD, GOLD-I, 26 pack-years
## 1
## smoking status: COPD, GOLD-I, 32.5 pack-years
## 1
## smoking status: COPD, GOLD-I, 48 pack-years
## 1
## smoking status: COPD, GOLD-I, 50 pack-years
## 1
## smoking status: COPD, GOLD-II, 15 pack-years
## 1
## smoking status: COPD, GOLD-II, 20 pack-years
## 1
## smoking status: COPD, GOLD-II, 24 pack-years
## 1
## smoking status: COPD, GOLD-II, 27 pack-years
## 1
## smoking status: COPD, GOLD-II, 27.5 pack-years
## 1
## smoking status: COPD, GOLD-II, 29 pack-years
## 1
## smoking status: COPD, GOLD-II, 33 pack-years
## 1
## smoking status: COPD, GOLD-II, 34 pack-years
## 1
## smoking status: COPD, GOLD-II, 35 pack-years
## 1
## smoking status: COPD, GOLD-II, 60 pack-years
## 1
## smoking status: COPD, GOLD-II, 75 pack-years
## 1
## smoking status: COPD, GOLD-III, 110 pack-years
## 1
## smoking status: COPD, GOLD-III, 53 pack-years
## 1
## smoking status: non-smoker
## 63
## smoking status: smoker, 1 pack-years
## 1
## smoking status: smoker, 16 pack-years
## 1
## smoking status: smoker, 23 pack-years
## 1
## smoking status: smoker, 0.5 pack-years
## 1
## smoking status: smoker, 10 pack-years
## 1
## smoking status: smoker, 10.5 pack-years
## 1
## smoking status: smoker, 11 pack-years
## 2
## smoking status: smoker, 12 pack-years
## 1
## smoking status: smoker, 13 pack-years
## 1
## smoking status: smoker, 14 pack-years
## 1
## smoking status: smoker, 15 pack-years
## 2
## smoking status: smoker, 16 pack-years
## 1
## smoking status: smoker, 17.5 pack-years
## 1
## smoking status: smoker, 18 pack-years
## 1
## smoking status: smoker, 19 pack-years
## 2
## smoking status: smoker, 19.5 pack-years
## 1
## smoking status: smoker, 20 pack-years
## 4
## smoking status: smoker, 21 pack-years
## 1
## smoking status: smoker, 22 pack-years
## 2
## smoking status: smoker, 22.5 pack-years
## 1
## smoking status: smoker, 23 pack-years
## 3
## smoking status: smoker, 24 pack-years
## 3
## smoking status: smoker, 26 pack-years
## 3
## smoking status: smoker, 26.5 pack-years
## 1
## smoking status: smoker, 27 pack-years
## 2
## smoking status: smoker, 28 pack-years
## 1
## smoking status: smoker, 29 pack-years
## 2
## smoking status: smoker, 3 pack-years
## 1
## smoking status: smoker, 3.8 pack-years
## 1
## smoking status: smoker, 30 pack-years
## 2
## smoking status: smoker, 32 pack-years
## 1
## smoking status: smoker, 33 pack-years
## 2
## smoking status: smoker, 35 pack-years
## 1
## smoking status: smoker, 36 pack-years
## 1
## smoking status: smoker, 38 pack-years
## 3
## smoking status: smoker, 43 pack-years
## 1
## smoking status: smoker, 44.3 pack-years
## 1
## smoking status: smoker, 45 pack-years
## 4
## smoking status: smoker, 46 pack-years
## 2
## smoking status: smoker, 47 pack-years
## 2
## smoking status: smoker, 5 pack-years
## 1
## smoking status: smoker, 51 pack-years
## 1
## smoking status: smoker, 56.5 pack-years
## 1
## smoking status: smoker, 60 pack-years
## 1
## smoking status: smoker, 7.6 pack-years
## 1
## smoking status: smoker, 71 pack-years
## 1
## smoking status: smoker, 80 pack-years
## 1
## smoking status: smoker, 9.3 pack-years
## 1## ExpressionSet (storageMode: lockedEnvironment)
## assayData: 6 features, 171 samples
## element names: exprs
## protocolData: none
## phenoData
## sampleNames: GSM101096 GSM101097 ... GSM549782 (171 total)
## varLabels: title geo_accession ... smoking status:ch1 (45 total)
## varMetadata: labelDescription
## featureData
## featureNames: 1007_s_at 1053_at ... 1294_at (6 total)
## fvarLabels: ID GB_ACC ... Gene Ontology Molecular Function (16 total)
## fvarMetadata: Column Description labelDescription
## experimentData: use 'experimentData(object)'
## pubMedIds: 21829517
## 32277131
## Annotation: GPL570##
## Non.smoker Smoker
## 63 94## [1] "ID" "GB_ACC"
## [3] "SPOT_ID" "Species Scientific Name"
## [5] "Annotation Date" "Sequence Type"
## [7] "Sequence Source" "Target Description"
## [9] "Representative Public ID" "Gene Title"
## [11] "Gene Symbol" "ENTREZ_GENE_ID"
## [13] "RefSeq Transcript ID" "Gene Ontology Biological Process"
## [15] "Gene Ontology Cellular Component" "Gene Ontology Molecular Function"Here we see that our data as 63 non-smokers and 94 smokers, including the COPD study participants.
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3. Differential Expression (DE Analysis)
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## Symbol Gene_Name
## 210505_at ADH7 alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide
## 205623_at ALDH3A1 aldehyde dehydrogenase 3 family, member A1
## 207469_s_at PIR pirin (iron-binding nuclear protein)
## 210519_s_at NQO1 NAD(P)H dehydrogenase, quinone 1
## 204059_s_at ME1 malic enzyme 1, NADP(+)-dependent, cytosolic
## 201467_s_at NQO1 NAD(P)H dehydrogenase, quinone 1
## Entrez_ID logFC AveExpr t P.Value adj.P.Val
## 210505_at 131 6411.741 5099.084 14.56583 7.018352e-31 3.837284e-26
## 205623_at 218 27204.698 21314.073 14.35497 2.596014e-30 7.096852e-26
## 207469_s_at 8544 3195.521 3347.489 14.13356 1.028288e-29 1.874054e-25
## 210519_s_at 1728 12922.702 14083.440 13.91535 4.004319e-29 5.473404e-25
## 204059_s_at 4199 2574.942 2294.907 13.65817 1.994186e-28 2.180642e-24
## 201467_s_at 1728 4765.171 4708.853 13.02344 1.061018e-26 9.668530e-23
## B
## 210505_at 7.894573
## 205623_at 7.725022
## 207469_s_at 7.543940
## 210519_s_at 7.362392
## 204059_s_at 7.144492
## 201467_s_at 6.588435## Here we see that the top DE genes are: ADH7 (alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide), ALDH3A1 (aldehyde dehydrogenase 3 family, member A1), PIR (pirin (iron-binding nuclear protein)), NQO1 (NAD(P)H dehydrogenase, quinone 1), ME1 (malic enzyme 1, NADP(+)-dependent, cytosolic), NQO1 (NAD(P)H dehydrogenase, quinone 1), AKR1C1 (aldo-keto reductase family 1, member C1), AKR1C2 /// LOC101930400 (aldo-keto reductase family 1, member C2 /// aldo-keto reductase family 1 member C2-like), NQO1 (NAD(P)H dehydrogenase, quinone 1)## [1] "For context, ADH7 can affect how alcohol is metabolized upregulation and/or variants have been associated with alcohol-related disease risk. It's important to note however that this risk can be mitigated/amplified by other factors. Thus, later we will review some system or pathway analysis."================================================================
4. Visualization and Results Interpretation
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Here we can see that most genes are NOT significantly affected by smoking. However, we can evaluate systemic changes or even the compounded effect of multiple “small gene changes”. This is transcriptomics being converted to systems biology. The way to measure this goes beyond basic DE analysis, although DE analysis can be foundation to downstream analysis [3].
Let’s first do some additional analysis and visualization. We will first filter for low occurrence to improve signal/noise, thereby improving analysis and statistical significance. This improves confidence, reduces false-discovery, increases confidence, and concentrations active transcripts.
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5a. Downstream Analysis: Filtering for Higher Confidence
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## [1] "Probes retained after intensity filtering: 54675"## Symbol Gene_Name
## 210505_at ADH7 alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide
## 205623_at ALDH3A1 aldehyde dehydrogenase 3 family, member A1
## 207469_s_at PIR pirin (iron-binding nuclear protein)
## 210519_s_at NQO1 NAD(P)H dehydrogenase, quinone 1
## 204059_s_at ME1 malic enzyme 1, NADP(+)-dependent, cytosolic
## 201467_s_at NQO1 NAD(P)H dehydrogenase, quinone 1
## Entrez_ID logFC AveExpr t P.Value adj.P.Val
## 210505_at 131 6411.741 5099.084 14.56583 7.018352e-31 3.837284e-26
## 205623_at 218 27204.698 21314.073 14.35497 2.596014e-30 7.096852e-26
## 207469_s_at 8544 3195.521 3347.489 14.13356 1.028288e-29 1.874054e-25
## 210519_s_at 1728 12922.702 14083.440 13.91535 4.004319e-29 5.473404e-25
## 204059_s_at 4199 2574.942 2294.907 13.65817 1.994186e-28 2.180642e-24
## 201467_s_at 1728 4765.171 4708.853 13.02344 1.061018e-26 9.668530e-23
## B
## 210505_at 7.894573
## 205623_at 7.725022
## 207469_s_at 7.543940
## 210519_s_at 7.362392
## 204059_s_at 7.144492
## 201467_s_at 6.588435================================================================
5b. Downstream Analysis: New Plots with filtered probes
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6. Pathway Analysis, Reactome
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7. Pathway Analysis, GO and KEGG - Smokers, up-regulated ONLY
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House Keeping
Pathway Analysis Section: GO, Kegg, & Reactome Analysis Type,Function,Best For: GO (BP),enrichGO,“Broad biological mechanisms (e.g.,”“Cell Proliferation”“)” KEGG,enrichKEGG,“Well-defined metabolic/signaling maps (e.g.,”“Glycolysis”“)” Reactome,enrichPathway,Detailed molecular reactions and hierarchies
GO DETAIL: Category,Question it Answers,Level of Detail BP (Biological Process),What is the overall goal?,System-wide / Cellular program MF (Molecular Function),What is the chemical task?,Molecular / Biochemical CC (Cellular Component),Where is this happening?,Structural / Spatial
Results Explained:
The observed transcriptional changes exert a compounded effect at the network level, whereby coordinated modulation of ER stress, proteasomal degradation, and NRF2-dependent antioxidant pathways converges on central hubs, producing a non-linear amplification of stress tolerance and cellular robustness.
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