Background: Early life impairments leading to lower lung function by adulthood are considered as risk factors for chronic obstructive pulmonary disease (COPD). Recently, we compared the lung transcriptomic profile between two mouse strains with extreme total lung capacities to identify plausible pulmonary function determining genes using microarray analysis (GSE80078). Advancement of high-throughput techniques like deep sequencing (eg. RNA-seq) and microarray have resulted in an explosion of genomic data in the online public repositories which however remains under-exploited. Strategic curation of publicly available genomic data with a mouse-human translational approach can effectively implement “3R- Tenet” by reducing screening experiments with animals and performing mechanistic studies using physiologically relevant in vitro model systems. Therefore, we sought to analyze the association of functional variations within human orthologs of mouse lung function candidate genes in a publicly available COPD lung RNA-seq data-set. Methods: Association of missense single nucleotide polymorphisms, insertions, deletions, and splice junction variants were analyzed for susceptibility to COPD using RNA-seq data of a Korean population (GSE57148). Expression of the associated genes were studied using the Gene Paint (mouse embryo) and Human Protein Atlas (normal adult human lung) databases. The genes were also assessed for replication of the associations and expression in COPD −/mouse cigarette smoke exposed lung tissues using other datasets. Results: Significant association (p < 0.05) of variations in 20 genes to higher COPD susceptibility have been detected within the investigated cohort. Association of HJURP, MCRS1 and TLR8 are novel in relation to COPD. The associated ADAM19 and KIT loci have been reported earlier. The remaining 15 genes have also been previously associated to COPD. Differential transcript expression levels of the associated genes in COPD- and/ or mouse emphysematous lung tissues have been detected. Conclusion: Our findings suggest strategic mouse-human datamining approaches can identify novel COPD candidate genes using existing datasets in the online repositories. The candidates can be further evaluated for mechanistic role through in vitro studies using appropriate primary cells/cell lines. Functional studies can be limited to transgenic animal models of only well supported candidate genes. This approach will lead to a significant reduction of animal experimentation in respiratory research. Keywords: 3R, Alternate models, COPD, Asthma, Lung, Gene, Transcriptomics * Correspondence: email@example.com SRM Research Institute, SRM University, Chennai 603203, India Work Environment Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 287, SE-171 77 Stockholm, Sweden Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vishweswaraiah et al. Respiratory Research (2018) 19:92 Page 2 of 11 Background the lung transcript expression profiles of C3H/HeJ and Progress in the genomics technologies continue to tre- JF1/MsJ mice at the completion of: (I) embryonic lung mendously advance our understanding of chronic lung development; (II) bulk alveolar formation and (III) lung diseases like asthma, chronic obstructive pulmonary dis- growth and maturity . The generated microarray ease (COPD), and idiopathic pulmonary fibrosis. COPD data provides a publicly available resource for perform- alone is the 4th leading cause of death globally [http:// ing genetic association studies as well as functional and www.who.int/mediacentre/factsheets/fs310/en/]. Genetic mechanistic investigations to understand pulmonary predisposition is considered to be an important risk fac- function development and chronic lung disease (eg. tor for COPD susceptibility. This is evident from the fact COPD) susceptibility . Lung developmental pathways that only 15–20% of smokers develop COPD [1, 2]. are recollected in genetic subroutines during repair and Thus, candidate gene identification has been a major remodeling processes following lung injury. Therefore, it focus for COPD research. This has also lead to the is plausible that an individual with hindered lung devel- extensive use of inbred mouse strains for screening ex- opment may have an inefficient repair/remodeling periments and also to the development of transgenic process thereby predisposing them to chronic lung dis- mouse models to identify genetic susceptibility, elucida- eases like COPD [23–25]. A study by Lange et al.  tion of molecular patho-mechanisms and toxicity testing showed that forced expiratory volume in 1 s (FEV )in in COPD research. However, a spin-off of the popularity early adulthood is important for the genesis of COPD of transgenic strains to explore gene-function relation- and that accelerated decline in FEV is not an obligate ships is the increased animal usage . Another corre- feature of COPD. Therefore, in this work, we performed sponding concern is the large number of animals bred an in-silico study, testing the association of functional that are genetically unsuited for the experiment. Breed- variations within human orthologs of mouse lung func- ing surplus often counts for 50% of the offspring . tion candidate genes  in a publicly available RNAseq Moreover, the relevance of a mouse with a single gene dataset of a COPD cohort . inserted or knocked out for studying human diseases is also questioned. This is mainly because complex traits Methods are multi-gene controlled that do not follow Mendelian Figure 1 illustrates the overall analysis strategy followed pattern of inheritance. Pulmonary function and COPD in this study. We focused on the missense single nucleo- are classic examples of such phenomenon [4–18]. Yet tide polymorphisms (SNPs), insertions, deletions and we believe, transgenic models may continue to serve as splice site variations for detecting the functional rele- important resources for studying gene-function relation- vance of the associations. Lung transcriptome data ships particularly in the field of respiratory research. (RNA-seq; GSE57148) from a Korean cohort  were However, the strategy to select candidate genes for using analyzed to call the variants and to identify the SNPs transgenic models to study COPD and other chronic with significant (p < 0.05) allelic frequency differences lung diseases is an important issue that warrants between the COPD cases and controls. attention. Practice of the “3R tenet”-replacement, reduction and Selection of mouse genes refinement warrants a scientist to adequately evaluate Mouse lung microarray dataset was retrieved non-animal alternatives prior to performing animal ex- (GSE80078) from our recently completed project con- periments [19, 20]. Strategic genomics data mining using trasting C3H/HeJ (large total lung capacity) and JF1/MsJ the public repositories can put in practice the “3R-tenet” (small total lung capacity) . Genes exhibiting in- more effectively by: i) reducing screening experiments creased/decreased transcript expression levels by ≥2 fold with animals, ii) performing mechanistic studies in in the lungs of JF1/MsJ mice compared to C3H/HeJ physiologically relevant alternate in vitro model systems were selected for performing the association studies. We and using advanced technologies like RNAi or CRISPR- also included the top 20 genes identified in Kim et al. Cas9 for understanding gene-function relationships, and  study and other COPD associated genes by litera- iii) performing in vivo functional testing using transgenic ture survey resulting in a total of 494 genes for screen- animal models limited to well supported candidate ing. Human orthologs of some genes were not found genes. and many were RIKEN or expressed sequence tags. An accelerated decline in lung function is considered Therefore, the final search list constituted of 355 genes to be the earliest indicator for predisposition, onset and (Additional file 1: Table S1). COPD severity assessment. We previously identified mouse strains (C3H/HeJ and JF1/MsJ) with extreme Human lung transcriptome data total lung capacities [5, 21, 22]. Recently, we performed A publicly available RNA-seq dataset from a Korean co- a large-scale microarray study (GSE80078) to compare hort consisting of 98 COPD cases and 91 control Vishweswaraiah et al. Respiratory Research (2018) 19:92 Page 3 of 11 Fig. 1 Strategic workflow to screen mouse lung developmental genes for their association within a human chronic obstructive pulmonary disease (COPD) cohort transcriptomic (RNAseq) data subjects was selected for the analysis . Based on our usingthe Burrows Wheeler alignment (BWA) tool version search term [(COPD RNA seq human) and “Homo 0.7.10 (http://bio-bwa.sourceforge.net/). The whole genome sapiens”] this was the largest available COPD RNA-seq alignment was performed using ‘BWA-MEM’ algorithm dataset at the Gene expression Omnibus (GEO) data- with default parameters . base. The raw FASTQ files of paired end reads repre- The aligned reads in the Sequence Alignment/Map senting the transcriptome of control and cases were (SAM) format were then sorted using ‘SortSam’ retrieved from the GEO database at the National Centre algorithm of Picard tool v.1.118 (https://sourceforge.net/ for Biological Information (NCBI) through accession projects/picard/). The Sorted SAM file was converted to number GSE57148 (http://www.ncbi.nlm.nih.gov/geo/ binary version of a SAM file (BAM file) using the SAM- query/acc.cgi?acc=GSE57148).The quality of the tools (http://samtools.sourceforge.net/). The resulting raw FASTQ files were analyzed using FASTQC (http:// BAM file was then sorted and indexed using SAMtools www.bioinformatics.babraham.ac.uk/projects/fastqc/) for (http://samtools.sourceforge.net/) for variant calling. The the presence of sequencing adapters and low-quality bases ‘mpileup’ algorithm of SAM tools was used for calling (Phred quality score 30). The quality filtered FASTQ files variants from the sorted BAM file using default parame- (Paired end) for each sample were then mapped against the ters. The resulting variant calling file (VCF) containing Human Reference Genome build hg19 (http://hgdownload. SNPs was used for the further downstream analysis. The soe.ucsc.edu/goldenPath/hg19/bigZips/chromFa.tar.gz) VCF files generated from COPD cases and controls were Vishweswaraiah et al. Respiratory Research (2018) 19:92 Page 4 of 11 separately combined using CombineVariants command RNA-seq dataset of the investigated Korean COPD co- in Genome Analysis Tool Kit (GATK) v.2.3.9 (https:// hort (GSE57148). Our study identified significant associ- www.broadinstitute.org/gatk/). The allele frequency in ation of 16 non-synonymous SNPs, 4 splice junction cases and controls were calculated using VCF tools v.0.1. variations and 3 insertions involving 20 genes out of the 12a (http://vcftools.sourceforge.net/). The calculated al- 355 screened genes to higher COPD susceptibility in the lelic frequencies were considered to compare the differ- investigated cohort (Table 1). ences in SNPs frequencies among the COPD cases and the controls. Association of novel and previously reported genes to COPD Statistics The 20 associated genes include: ATP binding cassette The relative odds with the “cross-products” ratio was subfamily A member 10 (ABCA10); a disintegrin and used for calculating statistical significance. Followed by metallopeptidase domain 19 (ADAM19); basic helix- odds ratio estimation, the confidence interval was loop-helix family member e41 (BHLHE41), CD200 mol- calculated. Ninety five percent confidence level was ecule (CD200); cytochrome b-245, beta polypeptide considered for the estimation . The odds ratio and the (CYBB); glycine amidinotransferasec (GATM); guanylate significance of the associations were calculated using a binding protein 1 (GBP1); holliday junction recognition statistical tool MedCalc (https://www.medcalc.org/calc/ protein (HJURP); KIT proto-oncogene receptor tyrosine odds_ratio.php). Single variant analysis was performed kinase (KIT); leptin receptor (LEPR); LIM domain 7 and the raw p < 0.05 was considered as significant. (LMO7); LDL receptor related protein 1 (LRP1); micro- spherule protein 1 (MCRS1); processing of precursor 4, In silico assessment of functional consequence of the ribonuclease P/MRP subunit (POP4); Patched 1 associated variations on protein biochemistry (PTCH1); sodium channel, voltage-gated, type VII, alpha The polymorphisms with the significant allelic frequency subunit (SCN7A); schlafen family member 12 like differences between the COPD cases and controls were (SLFN12L); toll like receptor 8 (TLR8); tetratricopeptide further analyzed using the visualization tool ‘Golden repeat domain 5 (TTC5) and ventricular zone expressed Helix GenomeBrowse’ (http://www.goldenhelix.com)to PH domain homolog 1 (VEPH1). assess the plausible effect of SNPs on protein biochemis- Our analysis, identified HJURP (rs2286430), MCRS1 try or splicing events. Prosite’ tool of ExPASy was (splice junction), and TLR8 (rs3764880) as three novel used to analyze the effect of amino acid changes on the COPD associated genes (Table 1). The variations (mis- functional domains of proteins. sense SNPs/splice junction variations) on ABCA10 (rs496849), BHLHE41 (rs11048413), CD200 (rs1131199), In silico lung expression domain studies of associated CYBB (not reported in dbSNP), GATM (rs1288775), genes GBP1 (rs1048425), LEPR (rs1137101), LMO7 (2 inser- Transcript expression of the significantly associated tions), LRP1 (splice junction), POP4 (splice junction), genes were screened in embryonic mouse lungs using PTCH1 (splice junction), SCN7A (rs7565062, rs6738031, the online database “GenePaint” . “The Human 1 insertion), SLFN12L (rs2304968), TTC5 (rs3742945), Protein Atlas” database  was used to identify the and VEPH1 (rs11918974) are located on genes previ- immuno-positive lung cells for the significantly associ- ously associated to COPD (Table 1). The associated ated genes in normal adult human lung. SNPs on ADAM19 (rs1422795) and KIT (rs3822214) have been previously reported in relation to COPD Lung transcript expression levels of the associated genes (Table 1). in COPD and cigarette smoke exposed mice The associated 20 genes were scanned for differential In silico protein domain and gene/protein expression transcript expression in several COPD and/ or emphyse- analysis matous lung tissues (GSE: 29133, 22,148, 1650, 47,460 In silico protein domain analysis revealed the ADAM19 and 54,837) [33–37] as well as in mouse cigarette smoke (rs1422795) variation at the position of Chr5: T- exposed lungs (GSE: 8790, 7310, 17,737, and 76,205) 156936364-C resulting in an amino acid exchange of [38–40] using microarray/RNA-seq datasets from GEO Ser17Gly (polar to non-polar) to be located within the database. ADAM metalloprotease domain (Additional file 1: Figure S1). None of the other amino acid changes were Results located within functional domains of the proteins. In A stringent cut off ratio of ≥2 fold increased/decreased silico transcript expression domain analysis using the was used to select the mouse lung function developmen- Gene Paint database (Additional file 1: Table S2) re- tal genes (GSE80078) for association studies in the vealed detectable lung expression of Adam19, Cd200, Vishweswaraiah et al. Respiratory Research (2018) 19:92 Page 5 of 11 Table 1 Details of the gene and corresponding single nucleotide polymorphism (SNP) associated to chronic obstructive pulmonary disease (COPD) susceptibility Gene Gene name Entrez ID Associated SNP Chromosomal Location Ref allele/Alt Ref AA/Alt AA Odds ratio 95% CI z-statistic Significance Association to COPD allele level ABCA10 ATP-binding cassette, 10,349 rs4968849 17: 67178316 A/G M/T 4.09 1.11 to 2.125 0.0336 Novel loci, gene associated sub-family A, member 10 15.01 previously (NLGAP) ADAM19 ADAM metallopeptidase 8728 rs1422795 5: 156936364 T/C S/G 6.21 2.26 to 3.555 0.0004 Loci and gene associated domain 19 17.00 previously BHLHE41 Basic helix-loop-helix 79365 rs11048413 12: 26275555 G/A A/V 5.29 2.71 to 4.879 < 0.0001 NLGAP family, member e41 10.35 CD200 CD200 molecule 4345 rs1131199 3: 112059768 C/G S/C 2.3773 1.11 to 2.248 0.0246 NLGAP 5.05 CYBB Cytochrome b-245, 1536 Novel X: 37658269 C/A Q/K 2.9091 1.35 to 2.726 0.0064 NLGAP beta polypeptide 6.26 GATM Glycine amidinotransferase 2628 rs1288775 15: 45661678 T/A Q/H 2.4309 1.35 to 2.974 0.0029 NLGAP 4.36 GBP1 Guanylate binding protein 1, 2633 rs1048425 1: 89522646 G/C T/S 3.2611 1.70 to 3.566 0.0004 NLGAP interferon-inducible 6.24 HJURP Holliday junction recognition 55355 rs2286430 2: 234761225 C/T E/K 3.36 1.42 to 2.768 0.0056 Novel protein 7.94 KIT V-Kit, sarcoma viral oncogene 3815 rs3822214 4: 55593464 A/C M/L 5.05 1.07 to 2.054 0.04 Loci and gene associated homolog 23.74 previously LEPR Leptin receptor 3953 rs1137101 1: 66058513 A/G Q/R 10.39 5.00 to 6.28 < 0.0001 NLGAP 21.58 LMO7 LIM domain 7 4008 Insertion 13: 76383319 A to G – 3.6316 1.99 to 4.209 < 0.0001 Novel insertion, gene Insertion 6.62 associated Previously (NIGAP) LMO7 LIM domain 7 4008 Insertion 13: 76429504 T insertion – 3.5531 1.50 to 2.903 0.0037 NIGAP 8.36 LRP1 Low density lipoprotein 4035 Splice junction 12: 57605134 G/C – 10.22 1.28 to 2.195 0.0282 Novel splice site; gene receptor-related protein 1 81.58 associated Previously (NSSGAP) MCRS1 Microspherule protein 1 10445 Splice junction 12: 49957330 C/T – 3.365 1.57 to 3.127 0.0018 Novel 7.19 POP4 Processing of precursor 4, 10775 Splice junction 19: 30101540 G/A – 2.7669 1.31 to 2.673 0.0075 NSSGAP ribonuclease P/MRP subunit 5.83 (S. cerevisiae) PTCH1 Patched 1 5727 Splice junction 9: 98242373 G/T – 11.41 2.58 to 3.213 0.0013 NSSGAP 50.37 SCN7A Sodium channel, voltage-gated, 6332 rs7565062 2: 167334085 G/T T/N 4.4175 1.97 to 3.606 0.0003 NLGAP type VII, alpha subunit 9.90 SCN7A Sodium channel, voltage-gated, 6332 Insertion 2: 167289263 AG Insertion – 3.3561 1.17 to 2.263 0.0237 NIGAP type VII, alpha subunit 9.57 Vishweswaraiah et al. Respiratory Research (2018) 19:92 Page 6 of 11 Table 1 Details of the gene and corresponding single nucleotide polymorphism (SNP) associated to chronic obstructive pulmonary disease (COPD) susceptibility (Continued) Gene Gene name Entrez ID Associated SNP Chromosomal Location Ref allele/Alt Ref AA/Alt AA Odds ratio 95% CI z-statistic Significance Association to COPD allele level SCN7A Sodium channel, voltage-gated, 6332 rs6738031 2: 167279922 C/A M/I 2.2817 1.09 to 2.198 0.028 NLGAP type VII, alpha subunit 4.76 SLFN12L Schlafen family member 12 like 100,506,736 rs2304968 17: 33805150 T/C Y/C 2.2 1.07 to 2.151 0.0315 NLGAP 4.51 TLR8 Toll-like receptor 8 51,311 rs3764880 X: 12924826 A/G M/V 2.97 1.11 to 2.181 0.0292 Novel 7.91 TTC5 Tetratricopeptide repeat 91875 rs3742945 14: 20770036 T/C Q/R 2.1591 1.14 to 2.375 0.0176 NLGAP domain 5 4.07 VEPH1 Ventricular zone expressed PH 79674 rs11918974 3: 157081324 A/G S/P 1.8668 1.04 to 2.116 0.0343 NLGAP domain homolog 1 (zebrafish) 3.32 AA amino acids, rs reference sequence, Ref reference, Alt altered, CI confidence interval p < 0.05 was considered as significant Vishweswaraiah et al. Respiratory Research (2018) 19:92 Page 7 of 11 Cybb, Mfleg (HJURP), Kit, Lepr, Lmo7, Lrp1, Mcrs1, Pop4 not exhibiting any differential expression in any of the and Ptch1 in mouse embryo (E14.5; at pseudoglandular investigated datasets. A summary of the expression stage of lung development). This further attests the role pattern of the 20 genes in the investigated COPD lung of the mentioned 11 genes in the process of lung devel- tissue datasets (GSE: 29133, 22,148, 1650, 47,460 and opment. Impairment in the regulation and functionality 54,837) is provided in Additional file 1: Table S3. Mouse of lung developmental genes may result in predisposition cigarette smoke exposure experiments are also another to chronic lung diseases like COPD. In silico lung pro- valuable resource to evaluate molecular patho- tein expression domain analysis using the Human Pro- mechanisms as tobacco smoking is the major risk factor tein Atlas revealed detectable immuno-expression of 18 for COPD. We therefore also evaluated the expression associated genes in macrophages and/or pneumocytes of the 20 associated genes in the datasets generated from and/or nasopharynx (respiratory epithelial cells) and/or lungs of mice exposed to cigarette smoke (GSE: 8790, bronchus (respiratory epithelial cells) (Additional file 1: 7310, 17,737, and 76,205) (Additional file 1: Table S4). In Table S2). Immuno-expression of BHLHE41 and GATM case of mouse studies, Gbp1, Mcrs1, Ptch1, Slfn12l, and were not detectable in the normal human lung tissue. Ttc5 were the genes not exhibiting altered expression Detection of expression of the significantly associated following cigarette smoke exposure. A summary of the COPD susceptibility genes within specific cell types of expression pattern of the 20 genes in the cigarette smoke the normal human lung further supports their specific exposed mouse lung tissue datasets are provided in the role in the normal lung physiology. Additional file 1: Additional file 1: Table S4. Amongst the 20 candidate Figures S1-S4 shows the expression of HJURP, MCRS1 COPD genes identified in our study, transcripts of all ex- and TLR8 in mouse embryonic lungs and normal adult cept GBP1, MCRS1, PTCH1, SLFN12L and TTC5 are human lungs. However, human protein atlas does not differentially expressed in both mouse cigarette smoke provide information on the expression of proteins in exposed lungs and human COPD/emphysematous lungs COPD tissues. Therefore, we investigated the transcript within the investigated datasets. expression levels of the associated genes using available datasets on the lungs of COPD patients and mouse ex- Discussion posed to cigarette smoke. All datasets investigated in this study originated from The associated SNP rs2286430 (C/T) located on the lung samples of human and mouse thereby confirm- HJURP results in an amino acid change of glutamic acid ing the tissue specificity (18, 27, 37–40). The dataset (Glu: acidic, polar and negatively charged) to lysine (Lys: GSE57148 from Kim et al. (27) study consisting of 98 basic, polar and positively charged) in HJURP. Low to COPD patients and 91 control subjects from a Korean medium intensity of HJURP immune positive macro- population. This was the largest available lung RNA-seq phages, pneumocytes, respiratory epithelial cells have dataset of a COPD cohort in GEO database at the time been demonstrated in normal human lung tissue of study. However, for association studies this is a small (Additional file 1: Figure S2) (Human Protein Atlas). sample size. It is important to note that most of the as- Hjurp transcripts has been detected in mouse embryonic sociation studies on COPD genetics and genomics of lungs (Additional file 1: Figure S2). Mcrs1 is expressed in pulmonary function originates from populations with the mouse embryonic lungs (Additional file 1: Figure S3) European ancestry. Therefore, the effect of ethnicity on (Gene Paint). Medium to high intensity immune-positive the current findings cannot be ruled out. Additional file MCRS1 macrophages, pneumocytes, respiratory epithelial 1: Table S5 shows the difference in minor allele frequen- cells have been demonstrated in normal human lung cies of the associated SNPs between Korean population tissue (Additional file 1: Figure S3) (Human Protein Atlas). (http://18.104.22.168/KRGDB/browser/mainBrowser.jsp) TLR8 immuno-positive (high intensity) macrophages are and global population (https://www.ncbi.nlm.nih.gov/ reported in normal human lung (Additional file 1:Figure SNP/) justifying the plausible differences in ethnicity. S4). The intensity of TLR8 immuno-positive staining in Apart from lung specific expression of the associated the respiratory epithelial cells is low (Additional file 1: genes, another strength of our study is the focus on mis- Figure S4) whereas in pneumocytes and embryonic mouse sense SNPs (amino acid change), insertions, deletions, lung TLR8/Tlr8 was not detectable (Human Protein Atlas; and splice junction variations thereby increasing the Gene Paint). functional relevance of these associations. A genome- wide analysis of alternative splicing indicated that 40– Lung transcript expression of the associated genes in 60% of human genes undergo alternative splicing, often other COPD cohorts and mouse studies in a tissue specific manner [41–44]. On the other hand, We investigated the transcript expression levels of the since we performed the study using RNAseq data, our associated 20 genes in several COPD and/ or emphyse- investigation is limited only to the exonic sequences and matous lung tissue data sets. SLFN12L is the only gene therefore could not detect any alterations within the Vishweswaraiah et al. Respiratory Research (2018) 19:92 Page 8 of 11 promoter or intronic region. RNAseq data provides in- Based on the hypothesis on the origin of chronic lung formation only of a single strand. Thus, our study lacks diseases like COPD during the early life events [60–70], information on the homozygosity of the identified asso- we could detect three novel (HJURP, MCRS1 and TLR8) ciations. Availability of the genomic sequence of the COPD candidate genes and replicate the findings in 17 same individuals would have overcome this drawback. other studies using a mouse-human translational datamin- We detected association of 20 genes to higher suscepti- ing approach. Gene set enrichment analysis of the 20 bility for COPD. Our findings on the association of SNPs associated genes identified COPD as one of the top located on ADAM19 (rs1422795) and KIT (rs3822214) to enriched diseases (Additional file 1:Figure S5). HJURP is higher COPD susceptibility replicate the previous findings a centromeric protein (chaperone) that plays a central role by other investigators [12, 45–48]. The rs11048413 SNP in the incorporation and maintenance of histone H3-like on BHLHE41 causing an Ala298Val change have been variant CENPA at centromeres [72–74]. MCRS1 have associated to patient survival in lung adenocarcinoma. been implicated in epithelial-mesenchymal transition, me- The Ala/Val or Val/Val genotype was associated to poor tastasis and growth of lung cancer cells [75–77]. TLR8 is survival rate compared to Ala/Ala genotype . The as- also expressed in human monocytes and myeloid dendritic sociated SNP on GATM (rs1288775) has been linked to cells and Th1-type immune response cells. Mucus hyper- lung cancer phenotypes with and without emphysema secretion is induced by dual TLR7/8 agonist [78, 79]. among African-American population but not among Similarly, the murine TLR8 is involved in the activation of white Americans . The SNP rs3764880 on TLR8 has innate immune responses . Stimulation of TLR8 been associated to tuberculosis. The SNP rs3761624 also causes relaxation of airway smooth muscles thereby pre- located on TLR8 which has been associated to allergic venting broncho-constriction . Association of TLR8 rhinites in a Swedish population is in perfect linkage dise- have been also reported for pulmonary tuberculosis quibrium with rs3764880 suggesting their complementary [82, 83], asthma and related atopic disorders . relationship . The genes ABCA10, BHLHE41, CD200, CYBB, GATM, Conclusions GBP1, LEPR, LMO7, LRP1, POP4, PTCH1, SCN7A, Through this study we could demonstrate a candidate SLFN12L, TTC5, and VEPH1 have been previously asso- gene identification strategy for COPD using mouse- ciated to COPD [52–68]. Moreover, we detected altered human translational approach using existing genomic transcript expression of ABCA10, ADAM19, BHLHE41, datasets in the public repositories. The strategy warrants CD200, CYBB, GATM, GBP1, HJURP, KIT, LEPR, validation in larger sample size and in multiple cohorts. LMO7, LRP1, MCRS1, POP4, PTCH1, SCN7A, TLR8, Cigarette smoke exposure studies in mice are routinely TTC5 and VEPH1 in COPD and emphysematous lungs practiced to model emphysema development, a com- compared to control subjects in various datasets (GSE: monly associated COPD phenotype, as it causes in- 29133, 22,148, 1650, 47,460 and 54,837; Additional file 1: creased pulmonary inflammation, protease activity, Table S3) [33–37]. In case of mouse lungs exposed to oxidative stress and apoptosis . However, cigarette cigarette smoke, altered transcript expression was detected smoke exposure in mice does not result in excessive among Abca8a (ABCA10), Adam19, Bhlhe41, Cd200, Cybb, mucus production or mucus cell metaplasia that is char- Gatm,Hjurp,Kit, Lepr,Lmo7,Lrp1,Pop4,Scn7a, Tlr8,and acteristic of COPD pathogenesis . It is plausible that Veph1 (GSE: 8790, 7310, 17,737, and 76,205; Additional file the different response to cigarette smoke exposure in 1:Table S4)[38–40]. Effect of cigarette smoke exposure on human and mouse lungs may be due to their structural COPD development may act as a confounding factor in the differences . The inbred mouse strains also differ sig- analysis of candidate susceptibility genes in this study. nificantly in their resistance or susceptibility to emphy- However, considering the concept of recapitulation of de- sema development following cigarette smoke exposure velopmental pathways as genetic subroutines during lung as measured by airspace enlargement . This variable repair/remodeling processes, altered regulation of the asso- susceptibility among inbred mouse strains to emphyse- ciated genes in both COPD-and cigarette smoke exposed matous change following cigarette smoke exposure may mouse lungs seems to be reasonable. SNPs on ADAM19 be attributed to their genetic constitution and differ- (rs2277027), PTCH1 (rs16909898), LRP1 (rs11172113) ences in lung development. Most of the COPD tran- and hedgehog interacting protein (HHIP; rs12504628, scriptomic profiling studies have been performed using rs1980057) have been associated to FEV /forced vital cap- lung tissue from severely diseased patients requiring lob- acity (FVC) ratio in samples of European ancestry [10, 12]. ectomy. On the contrary, COPD pathogenesis occurs We previously reported decreased lung Hhip transcript over decades. Molecular mechanisms that are active dur- levels in a mouse model lacking secreted phosphoprotein ing initial phase of the pathogenesis may be completely dif- 1(Spp1) with lower total lung capacity and enlarged alveo- ferent compared to the end stage of the disease. Therefore, lar size compared to control . creation of a translational profile between mouse and Vishweswaraiah et al. Respiratory Research (2018) 19:92 Page 9 of 11 human COPD transcriptomic data is challenging. In this re- Funding This study was supported by the Department of Biotechnology, Government spect, we share similar views as other investigators that it is of India: BT/PR12987/INF/22/205/2015, and VINNOVA (2016–01951) (K.G.). important to carefully evaluate the common lung-biology and -pathobiology existing between mice and human prior Availability of data and materials Microarray data used is available at the Genome Expression Omnibus (GEO) to considering cigarette smokeexposureexperiments in database at National Center for Biotechnology Information NCBI (GSE80078) mouse models . Single gene driven spontaneous em- . Human RNAseq data is also available at NCBI (GSE57148) . physema developing mouse models  identified through Authors’ contributions physiological phenotyping (eg. pulmonary function screen- KG, SV, LG and PN designed and conceived the project; PN, SV, and LG ing) may serve an important tool to understand molecular performed the computational experiments and analyzed the data; KG, SV patho-mechanism but this requires exhaustive supportive and LG wrote the manuscript. All authors have read and approved the manuscript. evidence prior to testing the transgenic model. One way of accumulating convincing supportiveevidenceisexplained Ethics approval and consent to participate in the present work. Mechanistic studies to elucidate the Human participants: human data or human tissue: not applicable. Mice: Not applicable. role of the novel candidate genes can be performed using appropriate cell lines, primary cells and physiologically rele- Consent for publication vant in vitro models . This approach would lead to a Not applicable; Microarray data and RNAseq data from public repository have been used. significant reduction of animal screening experiments in re- spiratory research. Competing interests The authors declare that they have no competing interests. Additional file Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional file 1: Table S1. List of the genes screened for association to higher Chronic Obstructive Pulmonary Disease (COPD) susceptibility. Author details Table S2. Summary of the transcript (Gene Paint; mouse embryo) and 1 2 SRM Research Institute, SRM University, Chennai 603203, India. Department protein (Human Protein Atlas) expression domains of the significantly of Genetic Engineering, School of Bioengineering, Faculty of Engineering associated chronic obstructive pulmonary disease (COPD) genes. and Technology, SRM University, Chennai 603203, India. Work Environment Table S3. Analysis of lung transcript expression of the associated 20 Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box genes in chronic obstructive pulmonary disease (COPD) and/ or 287, SE-171 77 Stockholm, Sweden. emphysematous lung tissues using available datasets [GSE: 29133, 22,148, 1650, 47,460 and 54,837] in Genome Expression Omnibus Received: 31 December 2017 Accepted: 27 April 2018 (GEO) database. ↓: Decreased ↑:Increased ✓: significantly altered. Table S4. Analysis of l transcript expression of the associated 20 genes in mouse cigarette smoke exposed lungs using available References datasets [GSE: 8790, 7310, 17,737, and 6205] in Genome Expression 1. Burrows B, Knudson RJ, Cline MG, Lebowitz MD. Quantitative relationships Omnibus (GEO) database. ↓:Decreased ↑:Increased ✓: significantly between cigarette smoking and Ventilatory function 1, 2. Am Rev Respir Dis. altered. Table S5. 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