Exome Sequencing Reveals Genetic Variability and Identifies Chronic Prognostic Loci in Chinese Sarcoidosis Patients

Abstract

Background: Sarcoidosis is an inflammatory disease characterized by non-caseating granuloma formation in various organs, with several recognized genetic and environmental risk factors. Despite substantial progress, the genetic determinants associated with its prognosis remain largely unknown.

Objectives: This study aimed to identify the genetic changes involved in sarcoidosis and evaluate their clinical relevance.

Methods: We performed whole-exome sequencing (WES) in 116 sporadic sarcoidosis patients (acute sarcoidosis patients, n=58; chronic sarcoidosis patients, n=58). In addition, 208 healthy controls were selected from 1000 G East Asian population data. To identify genes enriched in sarcoidosis, Fisher exact tests were performed. The identified genes were included for further pathway analysis using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Additionally, we used the STRING database to construct a protein network of rare variants and Cytoscape to identify hub genes of signaling pathways.

Results: WES and Fisher's exact test identified 1,311 variants in 439 protein-coding genes. A total of 135 single nucleotide polymorphisms (SNPs) on 30 protein-coding genes involved in the immunological process based on the GO and KEGG enrichment analysis. Pathway enrichment analysis showed osteoclast differentiation and cytokine-cytokine receptor interactions. Three missense mutations (rs76740888, rs149664918, and rs78251590) in two genes (PRSS3 and CNN2) of immune-related genes showed significantly different mutation frequencies between the disease group and healthy controls. The correlation of genetic abnormalities with clinical outcomes using multivariate analysis of the clinical features and mutation loci showed that the missense variant (rs76740888, Chr9:33796673 G>A) of PRSS3 [p=0.04, odds ratio (OR) = 2.49] was significantly associated with chronic disease prognosis. Additionally, the top two hub genes were CCL4 and CXCR4 based on protein-protein interaction (PPI) network analysis.

Conclusion: Our study provides new insights into the molecular pathogenesis of sarcoidosis and identifies novel genetic alterations in this disease, especially PRSS3, which may be promising targets for future therapeutic strategies for chronic sarcoidosis.

Keywords: adaptive immune response; chronic sarcoidosis prognosis; non-synonymous mutations; sarcoidosis; whole-exome sequencing.

Figure 1

Analytical strategy workflow for variant filtration and candidate gene selection. A schematic overview of the steps involved in whole-exome sequencing analysis with pathogenesis candidate gene detection is shown. SNPs, single nucleotide polymorphisms; InDels, insertions and deletions; WES, whole-exome sequencing; MAF, minor allele frequency; MAGMA, Multimarker Analysis on GenoMic Annotation.

Figure 2

KEGG pathway analysis and expression characteristics of 30 immune-related candidate genes. (A) Bar plot of the KEGG pathway enrichment (B) Dot plot of the KEGG pathway enrichment (C) Organ and tissue expression characteristics analyzed by GTEx. A heatmap of the tissue-specific gene expression for 30 immune-related candidate genes in different organs and whole blood from the genotype-tissue expression project (GTEx) v8 54 tissue types of dataset. (D) PPI network of the 30 immune-related candidate genes and three modules were clustered by the STRING database. (E) PPI network of the top 10 hub genes of the 30 immune-related candidate genes.

Figure 3

(A) Characteristics of SNPs of 30 immune-related candidate pathogenesis genes. The details of missense mutations, splice site mutations, and multihit mutations in 116 sporadic sarcoidosis patients are presented as percentages. (B) Two missense mutations in PRSS3 of transcript isoforms. (C) and (D) Mutation details of rs76740888 and rs149664918 on nucleotide sequence and amino acid sequence. (E) Missense mutation of rs78251590 in the CNN2 gene. (F) Mutation details of rs78251590 on nucleotide and amino acid sequences.