Research On The Genetic Characteristics And The Effects Of MUC5B Gene In A Pedigree With Diabetes-associated Interstitial Lung Disease

Background:Interstitial lung diseases(ILD)are a group of diffuse lung diseases that mainly involve the interstitium and alveolar cavity,resulting in the loss of alveolar-capillary functional units.Once inflammation occurs in pulmonary blood vessels,peripheral airways and alveoli,the lung tissue will repair itself,gradually forming pulmonary fibrosis in the process of repeated injury and repair,which is the essential feature of ILD.ILD mainly includes four categories:(1)ILD with known etiology;(2)idiopathic interstitial pneumonia(IIP);(3)granulomatous ILD;(4)rare ILD.Diabetes mellitus(DM)is a systemic metabolic disorder syndrome characterized by the long-term elevated blood glucose due to insulin deficiency or insulin resistance.Chronic injuries caused by diabetes include neurological complications,microvascular complications and macrovascular complications.Because of the extensive alveolar-capillary network and abundant connective tissue,the lungs are prone to be the target organ of chronic injury from diabetes.Studies have shown that patients with diabetes have a significantly higher risk of interstitial lung disease than those without diabetes,and diabetes has become an independent risk factor for interstitial lung disease.The underlying pathogenesis of diabetes-associated interstitial lung disease involve oxidative stress response,excessive production of advanced glycosylation end products or the products’ receptors,epithelial-mesenchymal transition,endoplasmic reticulum stress and abnormal serum alveolar surfactant protein.However,there is a lack of studies on the role of genetic factors in the development of diabetes-associated interstitial lung disease.Objective:The study was aimed to identify the underlying pathogenic genes in a pedigree with diabetes-associated interstitial lung disease through clinical investigation and whole-genome resequencing,so as to lay a foundation for further understanding the pathogenesis of diabetes-associated interstitial lung disease and seeking new treatment strategies.Methods:A pedigree with diabetes-associated interstitial lung disease was included in this study.The pedigree consisted of three generations,and the proband was of the second generation,whose mother was a deceased patient with diabetes-associated interstitial lung disease.The proband and her siblings,a total of six,were almost all patients with diabetes-associated interstitial lung disease,and four of them had been deceased including the proband.There were 11 members in the third generation,including one with diabetes-associated interstitial lung disease,and one with diabetes mellitus but not complicated with interstitial lung disease.The remaining members were all healthy.In the first part of this study,the clinical data of 8 subjects in this pedigree were collected.10 ml of their peripheral blood was collected under aseptic conditions and sent to Shanghai Genechem Co.,ltd.for the whole-genome resequencing.Chromosomal variants(SVs),copy number variants(CNVs),single nucleotide polymorphisms(SNPs)and insertions/deletions(In Dels)were obtained from each sample.By comparing with the reference genome,the genes affected by CNVs,SVs,SNPs or In Dels were selected for annotation,and the variant genes that existed in at least 7(≥80%)subjects were further screened out.The co-existing variant genes or their corresponding proteins were uploaded to Fun Rich(Version 3.1.4)for GO classification,including cell component(CC),biological process(BP),molecular function(MF)and biological pathway enrichment analysis.Go-CC,GO-BP,GO-MF and the biological pathways with P < 0.05 were identified as significant.Meanwhile,the selected genes were also uploaded to STRING Database(version 11.5)to construct a protein-protein interaction(PPI)network.Furtherly,all the potential pathogenic genes were analyzed respectively according to the literature.Finally,the further cytological function experiments centering on MUC5 B were determined,to investigate the role of abnormal MUC5 B protein in the inflammatory response of human normal lung epithelial cells.In the second part of this study,the human normal lung epithelial cells(BEAS-2B)were stimulated with medium containing high concentration of glucose.CCK-8 assay was applied to detect the effect of different concentrations of high glucose on BEAS-2B cells,RT-PCR was applied to analyze the effect of high glucose on the transcription level of MUC5 B,ELISA was applied to analyze the effect of high glucose on the release of MUC5 B,IL-1β and IL-6,and western blot was applied to investigate the effect of high glucose on ERK1/2 activation in BEAS-2B cells.Furthermore,RNA interference was applied to silence MUC5 B gene,and the effects of MUC5 B silencing on the release of IL-1β and IL-6 as well as the activation of ERK1/2 in BEAS-2B cells under high glucose environment were also analyzed.Results:1.Results of the first part of the study:(1)This pedigree of diabetes-associated interstitial lung disease involved three generations.The first and second generations were all almost affected by diabetes and interstitial lung disease,while most of the third generation were healthy except for several patients.After genetic testing,the healthy subjects in the third generation were found to own the same potentially pathogenic genovariation as the patients did.(2)A total of 23 genes were found to be affected by CNVs in at least 7 subjects,among which the genes related to nucleus accounted for the most.The function of21.1% genes was unknown,while the remaining genes were related to transcription factors’ activities,receptors’ activities,transferase activity and DNA binding.Genes related to base,nucleoside,nucleotide and nucleic acid metabolism accounted for the most,followed by the genes related to signal transduction and cell communication.The 23 genes were involved in various signaling pathways such as PI3K/m TOR and Erb B signal pathways,however,no significant enrichment of the corresponding proteins was detected.According to the literature,the relevant studies about the 17 genes affected by CNV,including SH3RF3、UGT2B15、TRIM31、MICB、TRIM40、C6orf10、PGBD2、CHAMP1、PDPR、ZSCAN18、ERICH2、SIRPB1、APOBEC3B、RPL23AP82、CYB561D2、EPHA6 and HCG17,were not found in diabetes or interstitial lung diseases.The other 6 genes,including CREM,GCSH,KALRN,ECE2,HCG18 and GPSM1,had a certain role in the pathogenesis of diabetes and its complications.However,their roles in the development of interstitial lung diseases had not been studied yet.(3)A total of 190 genes were found to be affected by SVs in at least 7 subjects,and these genes were all distributed on autosomes,among which the genes related to X extracellular components accounted for the most.The genes related to cell adhesion molecule activity,extracellular matrix composition and growth factor activity were relatively rich.These genes were mainly involved in the biological process of nervous system,epithelial-mesenchymal transition,chemical synaptic transmission and postsynaptic signal transmission.Interaction network of the proteins corresponding to these genes revealed that 15 proteins were associated with extracellular matrix components,42 were involved in cell signal transduction,54 belonged to glycoproteins,and 28 belonged to secretory proteins.The hub nodes and the four-color nodes(the four-color nodes represented the proteins belonging to secretory protein,glycoprotein and extracellular matrix component at the same time,and participating signal transduction)in the PPI,including PLG,MATN1,ANGPT1,MEPE,COL4A2,NID2,IMPG2,GDF10,SFRP1,ABI3 BP,SBSPON,CDH2,SPP1,PTPRD and NRCAM were analyzed.Whether the nodes played a role in the development of diabetes or interstitial lung disease was unclear.In addition,whether the genes corresponding to these nodes would form fusion genes,affect gene expression or change their phenotypes after being affected by SV had not been studied yet.(4)A total of 70 In Dels in the coding regions were found in at least 7 subjects,including 40 frameshift deletions and 30 frameshift insertions distributed on autosomes and X chromosomes.(1)In the 40 genes with frameshift deletion,most were related to cell membrane components.The function of 26.5% of the genes was unknown,while the remaining genes were related to G protein-coupled receptors’ activities and cellular structural moleculars’ activities.The biological processes of 26.5% of the genes were unknown,while the remaining were related to cell signal transduction and cell communication.The 40 genes were involved in various signaling pathways,however,no significant enrichment of corresponding proteins was detected.According to the literature,only the variants of SLC22A1,TBP,ORAI1,SARM1 and COL18A1 were found to play a certain role in the development of diabetes or be the risk factors of diabetes.However,the remaining 35 genes were rarely studied in the field of diabetes or interstitial lung disease.(2)In the 30 genes with frameshift insertion,the genes related to cytoplasm and nucleus accounted for a large proportion.The function of 45.8% of the genes was unknown,while the remaining genes were related to immune proteins’ activities,transcription factors’ activities and G protein-coupled receptors’ activities.33.3% of the genes were involved in unknown biological processes,while the remaining genes related to cell signal transduction and cell communication accounted for a large proportion.The 30 genes were involved in various signaling pathways,however,no significant enrichment of corresponding proteins was detected.According to the limited literature,5 mutated genes including GIGYF2,ATG3,SRA1,WNK1 and CLECL1 were found to be involved in the development of diabetes and its complications,or be the risk factors for diabetes.The remaining 25 genes had not yet been studied in diabetes or interstitial lung disease.(5)According to the specific genes identified as being associated with the pathogenesis of interstitial lung disease(including familial pulmonary fibrosis)in previous studies,38 SNPs of these specific genes in at least 7 subjects were searched out.The 38 SNPs were found in 10 genes including AKAP13,ATP11 A,DSP,FAM13 A,IL1RN,MAPT,TP53,MUC2,MUC5 B,OBFC1 and SPPL2 C.According to the literature,the roles of AKAP13 SNPs rs8110,rs13225,rs3169121,rs2542604 and rs1808339,ATP11 A SNPs rs7985702 and rs1046790,IL-1RN SNP RS315951,TP53 SNP rs2909430,MUC2 SNPs rs41411848,rs41345745 and rs57737240,MAPT SNP rs2258689,OBFC1 SNPs rs10786775、rs2487999、rs4917405 and rs911547,as well as SPPL2 c SNPs rs242944 and rs171443 in the pathogenesis of diabetes mellitus or interstitial lung diseases were unclear;OBFC1 SNP rs4387287 might be closely related to the susceptibility of diabetes,but the role of this variant in interstitial lung disease remained unclear;DSP rs2076295 and FAM13 A rs2609255 had been confirmed to be associated with some types of interstitial lung diseases,but their roles in diabetes remained unclear;MUC5B SNP rs2943512 had been identified to be significantly associated with the susceptibility of diabetes mellitus,and the over-expressed MUC5 B in the distal airway and alveolar cavity had been confirmed to be closely related to the development of pulmonary fibrosis.(6)To sum up,after the analyzing of the above common genetic variants according to the literature,we found:(1)the roles of most variants in the pathogenesis of diabetes or interstitial lung diseases were unclear;(2)the variants of a few gene,including CREM,GCSH,KALRN,ECE2,HCG18,GPSM1,SLC22A1,TBP,ORAI1,XII SARM1,COL18A1,GIGYF2,ATG3,SRA1,WNK1,CLECL1 and OBFC1,might be involved in diabetes mellitus or its complications,however,their roles in interstitial lung disease remained unclear;DSP rs2076295 and FAM13 A rs2609255 had been confirmed to be associated with some types of interstitial lung diseases,but their roles in diabetes remained unclear;(3)MUC5B SNP rs2943512 had been identified to be significantly associated with the susceptibility of diabetes mellitus,and the over-expressed MUC5 B in the distal airway and alveolar cavity had been confirmed to be closely related to the development of pulmonary fibrosis.Therefore,it was of great significance to investigate the role of MUC5 B SNP rs2943512(A > C)or abnormal MUC5 B protein in the development of diabetes-associated interstitial lung disease.2.Results of the second part of the study:(1)The viability of BEAS-2B cells,which were stimulated by the medium containing 25 m M or 30 m M glucose for 72 hours,was decreased(P<0.001),the transcript level of MUC5 B was increased(P<0.001),and the concentration of MUC5 B in the supernatant was increased(P<0.001)compared with the control group(RPMI1640 medium treatment group,the glucose concentration was 11.11 m M).Finally,30 m M glucose for 72 hours were selected as the subsequent experimental conditions,because the results of this experimental group were the most significantly different from those of the control group.(2)The concentrations of IL-1β and IL-6 in the supernatant of BEAS-2B cells,which were stimulated by the medium containing 30 m M glucose for 72 hours,were both significantly increased compared with the control group(P<0.01,P<0.001).(3)Compared with the control group,the levels of phosphorylated-ERK1/2 were both significantly up-regulated in BEAS-2B cells stimulated with 30 m M glucose for15 minutes and 30 minutes(P<0.05,P<0.001);Compared with the control group,the levels of phosphorylated-P38,phosphorylated-JNK and phosphorylated-IκB in BEAS-2B cells stimulated with 30 m M glucose medium for 15 min to 6 h did not change.(4)After silencing MUC5 B gene,BEAS-2B cells were stimulated with 30 m M glucose for 72 hours.Compared with the control group,the concentrations of IL-1βand IL-6 in the supernatant of the experimental group were significantly decreased(P<0.01,P<0.001).(5)After silencing MUC5 B gene,BEAS-2B cells were stimulated with 30 m M high glucose for 30 minutes.Compared with the control group,the level of phosphorylated-ERK1/2 in the experimental group was significantly decreased(P<0.01).Conclusion:1.In this study,a pedigree with diabetes-associated interstitial lung disease including three generations was selected.8 living members of the pedigree were included as subjects.By conducting the whole-genome resequencing of each subject’s blood,it was found that the healthy subjects had the same potential pathogenic genetic variants as the patients,suggesting there are certain genetic characteristics in the three generations of diabetes-associated interstitial lung disease.2.Some genetic variation in the subjects of this family was detected by genome-wide resequencing,and MUC5 B SNP rs2943512(A > C)had been considered to be an potentially pathogenic gene mutation associated with the pathogenesis of diabetes-associated interstitial lung disease.Therefore,it was of great significance to investigate the role of MUC5 B SNP rs2943512(A > C)or abnormal MUC5 B protein in the development of diabetes-associated interstitial lung disease.3.At the cytological level,high glucose stimulation of BEAS-2B cells could cause MUC5 B overexpression,and at the same time,and the over-expressed MUC5 B could promote the activation of ERK1/2 and the production of IL-1β and IL-6,leading to the injury of alveolar epithelial cells and the development of interstitial lung diseases.Therefore,MUC5 B is expected to be a potential therapeutic target for diabetes-associated interstitial lung disease.