Clinical Analysis Of 3620 Cases Of Thyroid-Associated Ophthalmopathy And Its Pathogenesis Based On RNA-Seq

Objective:TAO(Thyroid associated ophthalmopathy)is a specific autoimmune disease and the most common orbital disease in adults.Clinical manifestations include upper eyelid contracture,upper eyelid lag,incomplete eyelid closure,exophthalmos,restricted eye movement,conjunctival congestion and edema.In severe cases,exposure keratitis and compressive optic neuropathy(CON)may occur,even blindness or need to remove the eyeballs,which have a great impact on the patient’s physical and mental health and life.The clinical manifestations of TAO patients can vary widely,and there is no guideline for TAO treatment in China.Clinical understanding of this disease is still limited,which makes misdiagnosis and mistreatment more common.Several studies have reported the incidence,gender differences and risk factors,and clinical characteristics analysis of TAO,but the results are diverse and limited by the small sample size.Clinical analysis of TAO in a large-scale cohort is of great significance to promote the understandings of diagnosis and treatment and improve the prognosis of patients.Therefore,the first part of this study retrospectively collected and analyzed the TAO patients who have visited outpatient clinic of our hospital in the past 10 years,and summarized the demographic characteristics,symptoms and signs,treatment,prognosis of TAO patients in order to provide guidance and reference for the diagnosis and treatment of TAO and provide evidence for preparing guidelines on the diagnosis and treatment of TAO in China in the future.At present,the pathogenesis of TAO is still not fully understood.Studies have shown that its occurrence is related to multiple factors such as heredity,immunity and environment.The judgment assessment of TAO activity and severity is important for choosing the timing and strategy of treatment.Moreover,the severity of the disease has an important impact on the prognosis of patients according to our clinical observation and analysis.In previous studies,more attention has been paied on the pathogenesis differences between TAO patients with active and inactive periods,and studies on the severity of TAO are relatively rare.The current assessment of the severity of TAO is mainly based on the symptoms and signs.There are no effective serological indicators to assist diagnosis and assessment.In view of the above background,the second part of this study used RNA-seq technology to compare the difference in the gene expression levels of peripheral blood mononuclear cells between the NOSPECS grades 0-3 and NOSPECS grades 4-6 patients,the differential genes(DEGs)were analyzed by Gene Ontology Analysis(GO analysis),KEGG analysis,and Protein-protein interaction Network(PPI)analysis to explore the core signal pathways and key proteins related to its pathogenesis,which can be used a molecular marker to provide evidence for clinical diagnosis and prognosis evaluation of the disease.Considering that TAO is an autoimmune disease,the pathological process may involve systemic immune response and local orbital tissue immune microenvironment.In order to further deepen the research on the pathogenesis of TAO,the third part of this study used RNA-seq technology to compare the difference in gene expression levels of orbital connective tissue between TAO NOSPECS grade 4-6 cases and NOSPECS grade 0-3 cases to obtain the differential genes involved in the local immune microenvironment and the core signal pathway and key proteins of local immunity through GO analysis,KEGG analysis,and PPI analysis.Additionally,we conducted a comparative analysis of differential genes in peripheral blood and orbital adipose connective tissue in patients with different severity of TAO,in order to reveal the mechanism of the occurrence and development of TAO.Materials and Methods:(1)Clinical research: All patients diagnosed with TAO and attending the ophthalmic clinic at West China Hospital in a ten-year period,from October 1,2009,to October 1,2019,were included in the study.The medical records of outpatient cases,examination results and patient’s eye photography will be photographed and archived.The eye photography includes extraocular motility in a total of 9 cardinal positions of gaze(front,up,down,left,right,upper left,upper right,lower left,and lower right).In special patients,photos of tear caruncle,conjunctiva,and cornea with fluorescent staining were also recorded.The visit data from each patient were stored in a folder named after the patient’s name and date of visit.Information obtained from the same patient are stored in a folder from the first visit to the last visit.Medical records of all registered patients were collected in an Excel database.The registration content includes general information,symptoms,eye signs,examination results,treatment,prognosis,etc.Statistical analysis of the collated data was conducted by using SPSS v23 software.(2)Peripheral blood mononuclear cell(PBMCs)transcriptome sequencing analysis of TAO patients:(1)Sample collection: 14 TAO patients were collected from the outpatient clinic of our hospital at the first time of visit,of which 7 cases were in the NOSPECS grade 0-3,and 7 cases in the NOSPECS grade 4-6.The two groups of patients had never been treated on the eyes,all patients present with hyperthyroidism,and antithyroid drugs(ATD)were used for hyperthyroidism treatment.Tumors,other autoimmune diseases,diabetes and infections were excluded in these patients.Two milliliters of peripheral blood was drawn through the median cubital venipuncture into the EDTA anticoagulation tube.(2)After extracting PBMCs,total RNA extraction was perform and m RNA quantity and quality were detected.(3)RNA sequencing was performed on the samples that met the requirements to obtain raw data.(4)Bioinformatics analysis of sequencing data: include quality control of raw data,quantification and correction of gene expression,analysis of differentially expressed genes(DEGs)between the two groups.(5)DEGs functional annotation,KEGG signal pathway analysis,and protein-protein interaction network(PPI)analysis of DEGs.(6)RT-PCR verification on key genes of interest obtained after biosynthesis.(3)Transcriptome sequencing analysis of TAO orbital fat connective tissue samples:(1)Sample collection: The orbital connective tissue of TAO patients who underwent orbital fat decompression in our hospital was collected.Among the 6 cases enrolled,3 cases were NOSEPECS grade 0-3 and 3 cases were grade 4-6.The patients were in stable period and the appearance has improved after operation with normal thyroid function for more than half year.There was no glucocorticoid therapy in the past six months,and infection,other autoimmune diseases,and diabetes were ruled out in these patients.During the operation,0.5-1ml of orbital fat connective tissue was taken out and placed into RNA later,and transported to the Ophthalmology Laboratory of West China Hospital of Sichuan University at room temperature for storage at-80°.The samples were shipped in dry ice to Beijing Novogene Company for RNA extraction and sequencing.(2)Total RNA extraction,m RNA quality detection,m RNA concentration measurement after orbital fat connective tissue cells.(3)RNA sequencing was performed on the samples that met the requirements to obtain raw data.(4)Bioinformatics analysis of sequencing data: quality control of raw data,quantification and correction of gene expression,analysis of DEGs between the two groups.(5)GO(gene ontology)analysis,KEGG pathway analysis,and PPI analysis of DEGs.(6)Analysis of the DEGs differences between peripheral blood mononuclear cells(PBMCs)and orbital fat connective tissue cells in TAO with different severity.Results:(1)TAO clinical research:1.A total of 3620 TAO patients were enrolled.Female patients were more common,and the male to female ratio is 1:1.83.The age of onset in males is order than females,with 45-50 years old in males and 40-45 years old in females.2.TAO was identified most frequently after thyroid diseases were diagnosed,accounting for 46.90%(1288 cases),followed by 39.44%(1083 cases)at the same time with thyroid diseases,and 13.66%(375 cases)before thyroid diseases was found.In addition,the thyroid function was related to the onset of one eye or both eyes,showing significantly statistical difference.3.The most common symptom of TAO was upper eyelid lag,followed by ptosis and eyelid retraction.The NOSPECS score and CAS score were significantly different between male and female patients.Eye lesions in male patients were usually more severe than in female patients.4.Exophthalmos accounted for 80.8% of the total number of eyes.Unlike exophthalmos,most eyes(5101,81.67%)had normal intraocular pressure,and only2.32% of eyes had intraocular pressure> 30 mm Hg.5.The most commonly affected extraocular muscle was the inferior rectus muscle.Approximately 11.7% of patients had lacrimal gland enlargement.6.Risk factor analysis in 2170 patients with a clear history of smoking showed that age,time of onset,gender,thyroid function,whether or not smoking are related to the severity of TAO disease and are the independent risk factors of severity of TAO disease.There was no statistical difference between the treatment modalities of hyperthyroidism with the severity of the TAO.7.The average course of TAO from the active phase to the stationary phase is11.35 months,and 75.50% of the TAOs have an active course of less than 12 months.Regarding the prognosis of TAO,only 2.1% of patients showed complete remission.(2)TAO peripheral blood sequencing analysis:1.After performing high-throughput transcriptome sequencing bioinformatics analysis on the peripheral blood of TAO patients,a total of 602 genes were differentially expressed between the 4-6 grade TAO and the 0-3 grade TAO.Among them,243 genes were up-regulatedly expressed in the 4-6 grade TAO group,and 359 genes were down-regulatedly expressed.2.The functional enrichment analysis of differential genes showed that the expression of differential genes up-regulated in PBMCs of patients with grade 4-6TAO was significantly enriched in biological processes including chemotaxis,acute inflammation,bone marrow leukocyte migration,and macrophages cell migration.Significantly enriched molecular functions were mainly sulfur compound binding,glycosaminoglycan binding,and heparin binding.Significantly enriched cell function areas were mainly extracellular matrix,endocytic membrane vesicles,lysosome cavity,and filopodia.3.In the PBMCs of patients with grade 4-6 TAO,the significantly enriched biological functions of down-regulated expression genes included keratinization,and the cell function area that were significantly enriched was the keratinocyte membrane mantle.4.The KEGG pathway analysis of DEGs in PBMCs of patients with grades 0-3TAO and grade 4-6 TAO showed that the significant enrichment signal pathways in up-regulated genes of 4-6 grade TAO include AGE-RAGE signaling pathway in diabetic complications,Cytokine-cytokine receptor interaction.Down-regulated DEGs did not significantly enrich in the signal pathway.5.The network of interactions between differentially expressed genes and protein products was constructed by using STRING database,and 10 hub genes were selected through the Cyto Hubba plug-in of Cytoscape,including CCL2,VCAM1,C3AR1,IL1 A,COL5A1,CXCL2,PTX3,FBN1,NID1 and HP.6.RT-PCR verification was performed on CCL2,C3AR1,FNB1,and PTX3,and the results were consistent with the sequencing results,proving the reliability of the sequencing results and the availability of subsequent analysis results.(3)TAO orbital connective tissue sequencing analysis and the study of its relationship with peripheral blood:1.After the high-throughput transcriptome sequencing bioinformatics analysis of the peripheral blood of TAO patients,a total of 1749 genes showed differential expression between the 4-6 grade TAO and the 0-3 grade TAO patients.Among them,1172 genes showed up-regulated expression in the 4-6 grade TAO,and 577 genes showed down-regulated expression.2.The functional enrichment analysis of differential genes showed that the main biological processes of up-regulated expression DEGs in the orbital fat connective tissue cells of grade 4-6 TAO patients significantly enriched in oxidative phosphorylation,oxygen transport,ATP metabolism process,muscle fibril assembly,muscle cell development,extracellular matrix decomposition,cell killing of other organisms,etc.The DEGs were mainly enriched in the cellular functional areas such as ribosomes,mitochondria,respiratory chain,oxidoreductase complex,extracellular matrix.The main molecular functions during the pathogenesis process included the structural composition of ribosomes,oxidoreductase activity,the structural composition of muscle,oxygen binding,and antioxidant activity.3.As for the down-regulated expression DEGs in the orbital fat connective tissue cells of grade 4-6 TAO patient,the involved biological processes included axon development and kidney development.They were significantly enriched in cell areas such as extracellular matrix,intercellular connections,and synaptic membrane.4.For up-regulated expressed DEGs in orbital adipose connective tissue cells in patients with grade 4-6 TAO,the significantly enriched signal pathways were mainly oxidative phosphorylation,ribosomes,myocardial contraction,non-alcoholic fatty liver.5.Differential genes that were down-regulated expressed in orbital adipose connective tissue cells of patients with grade 4-6 TAO were involved in neuroactive ligand receptor interaction,calcium signaling pathway,serotonergic synapse,c AMP signaling pathway,TGF-β signaling pathway,and other pathways such as glutamatergic synapse and linoleic acid metabolism.6.By comparisons of the DEGs in PBMCs between 0-3 grade TAO and 4-6 grade TAO and the DEGs of orbital fat connective tissue cells between grade 0-3 and 4-6grade TAO patients,we found there were 45 common DEGs in the two types of cells.7.GO function annotation and KEGG pathway enrichment analysis for 45 common differential genes indicated that the AGEs-RAGE signaling pathway,TNF signaling pathway,and human T-cell leukemia virus 1 infection signaling pathways were beyond the significant enrichment threshold.A total of 131 GO terms were enriched including DNA binding transcription activator activity,positive regulation of endothelial cell proliferation,positive chemotaxis,antimicrobial peptide-mediated antimicrobial humoral immune response,adipocyte differentiation,positive regulation of mast cell chemotaxis,vascular endothelial growth factor Receptor binding,response to hypoxia,glucocorticoid receptor binding,etc.Conclusion:1.The first part of this study conducted clinical analysis of 3620 patients with thyroid-related ophthalmopathy and concluded that TAO is most common in women and is closely related to hyperthyroidism.TAO with normal thyroid function usually develops in monocular.CAS score combined with MRI can improve the detection of TAO.NOSPECS scores should be slightly refined regarding the criteria for corneal involvement.The clinical symptoms of TAO are diverse and there are still many mistaken treatment of TAO in domestic.Clinical management of TAO should be individualized according to CAS or NOSPECS assessments and a multidisciplinary approach is warranted.A minority of patients showed complete remission.2.In the second part,through transcriptome sequencing technology,it was found that there were significant differences in the peripheral blood gene expression between TAO patients with NOSPECS grade 0-3 and 4-6.The up-regulation of differential genes in the peripheral blood of patients with grade 4-6 are mainly related to inflammation and control of inflammation is an important means of TAO treatment.Through functional annotation of DEGs and signaling pathways,10 Hub genes related to the pathogenesis of TAO are derived.Among them,C3AR1 gene and AGEs-RAGE signaling pathway may be a new study field of TAO,which may provide new insights for the study of TAO pathogenesis.C3AR1,as an immunological factor that can be tested by serological ELISA,is expected to provide assistance in the assessment of the severity of TAO.Additionally,this study will provide a reference for molecular therapy in the future.3.The third part studied the role of the immune microenvironment of the orbital connective tissue of TAO patients in the pathogenesis of TAO.In the local reaction,oxidative stress is one of the main pathological processes.Comparative analysis of DEGs in PBMCs and orbital adipose connective tissue in TAO patients indicates that there are fewer differential genes in common between the two types of cells.Most of the biological processes of the common DEGs resulting from the significant enrichment of common differential genes are consistent with the known pathological processes of TAO.The AGEs-RAGE signal pathway studied in the second part was once again enriched in the common DEGs of peripheral blood and orbital tissue.