A novel gene-screening approach reveals QSOX1/IL1RAP as promising biomarkers for the severity of non-alcoholic fatty liver disease

Background and Aims Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease that ranges from simple steatosis to inflammation, fibrosis, and cirrhosis. To address the unmet need for new NAFLD biomarkers, we aimed to identify candidate biomarkers using publicly available RNA sequencing (RNA-seq) and proteomics data. Methods A novel approach involving unsupervised gene clustering was performed using homogeneously processed and integrated RNA-seq data of 625 liver specimens to screen for NAFLD biomarkers in combination with public proteomics data from healthy controls and NAFLD patients. Additionally, we validated the results in the NAFLD and healthy cohorts using enzyme-linked immunosorbent assay (ELISA) of plasma and immunohistochemical staining (IHC) of liver samples. Results We generated a database () for exploring gene expression changes along NAFLD progression to facilitate the identification of genes and pathways involved in the disease’s progression. Through cross-analysis of the gene and protein clusters, we identified 38 genes as potential biomarkers for NAFLD severity. Up-regulation of Quiescin sulfhydryl oxidase 1 ( QSOX1 ) and down-regulation of Interleukin-1 receptor accessory protein ( IL1RAP ) were associated with increasing NAFLD severity in RNA-seq and proteomics data. Remarkably, the QSOX1/IL1RAP ratio in plasma demonstrated effectiveness in diagnosing NAFLD, with an area under the receiver operating characteristic (AUROC) of up to 0.95 as quantified by proteomics profiling and an AUROC of 0.82 with ELISA. Conclusions We discovered a significant association between the levels of QSOX1/IL1RAP and NAFLD severity. Furthermore, the QSOX1/IL1RAP ratio shows promise as a non-invasive biomarker for diagnosing NAFLD and assessing its severity. (ChiCTR2300073940). Lay Summary This study aimed to find non-invasive biomarkers for non-alcoholic fatty liver disease (NAFLD). Researchers utilized a new gene clustering method to analyze RNA-seq data from 625 liver samples. The identified biomarkers were further validated using plasma proteomics profiling, enzyme-linked immunosorbent assay (ELISA), and liver immunohistochemical staining (IHC) in three separate groups of healthy controls and NAFLD patients. The study revealed that the levels of QSOX1 were elevated while IL1RAP levels were reduced with increasing severity of NAFLD. Notably, the ratio of QSOX1 to IL1RAP expression in plasma showed promise as a non-invasive diagnostic tool for assessing the severity of NAFLD, eliminating the reliance on liver biopsy. Highlights ![Figure][1] ### Competing Interest Statement Henning Gronbaek has received research grants from Abbvie, Intercept, ARLA Food for Health, ADS AIPHIA Development Services AG. Consulting Fees from Ipsen, NOVO, Pfizer. Lecturer for AstraZeneca and EISAI; and on Data Monitoring Committee at CAMURUS AB. All other authors have no conflicts of interest to declare. ### Funding Statement This research was funded by the Shenzhen Science and Technology Project and Sanming Project of Medicine in Shenzhen, China (grant nos. SZSM201612074, JCYJ20180302173542393 and JCYJ20170817094901026). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Shenzhen Traditional Chinese Medicine Hospital, China gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. * AUROC : area under the receiver operating characteristic avg_log2FC : log fold-change of the average expression between the two groups BMI : body mass index CK18 : circulating keratin 18 fragments ECM : extracellular matrix ELISA : enzyme-linked immunosorbent assay F : Fibrosis score FIB-4 : Fibrosis-4 GEO : Gene Expression Omnibus GO : Gene Ontology GRCh37 : Genome Reference Consortium Human Build 37 HCC : hepatocellular carcinoma IHC : immunohistochemistry staining IL1RAP : Interleukin-1 receptor accessory protein lg : log10 PCA : Principal components analysis QSOX1 : Quiescin sulfhydryl oxidase 1 RNA-seq : RNA sequencing N : NAS score NAFL : Non-alcoholic Fatty Liver NAFLD : Non-alcoholic fatty liver disease NAFLD-DB : NAFLD gene expression database NAFLD_ngt : NAFLD with normal glucose tolerance NAFLD_T2D : NAFLD with type 2 diabetes NAS : NAFLD activity scores NASH : Non-alcoholic Steatohepatitis ncRNAs : non-coding RNAs scRNA-seq : single-cell RNA sequencing snRNA-seq : Single-nuclei RNA sequencing SZTCMH : Shenzhen Traditional Chinese Medicine Hospital, China THBS2 : thrombospondin 2 TMM : Trimmed Mean of M-values TPM : Transcript Per Million [1]: pending:yes