Antibody Mediated Rejection and T-cell Mediated Rejection Molecular Signatures Using Next-Generation Sequencing in Kidney Transplant Biopsies

Abstract

Recently, interest in transcriptomic assessment of kidney biopsies has been growing. This study investigates the use of NGS to identify gene expression changes and analyse the pathways involved in rejection. An Illumina bulk RNA sequencing on the polyadenylated RNA of 770 kidney biopsies was conducted. Differentially-expressed genes (DEGs) were determined for AMR and TCMR using DESeq2. Genes were segregated according to their previous descriptions in known panels (microarray or the Banff Human Organ Transplant (B-HOT) panel) to obtain NGS-specific genes. Pathway enrichment analysis was performed using the Reactome and Kyoto Encyclopaedia of Genes and Genomes (KEGG) public repositories. The differential gene expression using NGS analysis identified 6,141 and 8,478 transcripts associated with AMR and TCMR. While most of the genes identified were included in the microarray and the B-HOT panels, NGS analysis identified 603 (9.8%) and 1,186 (14%) new specific genes. Pathways analysis showed that the B-HOT panel was associated with the main immunological processes involved during AMR and TCMR. The microarrays specifically integrated metabolic functions and cell cycle progression processes. Novel NGS-specific based transcripts associated with AMR and TCMR were discovered, which might represent a novel source of targets for drug designing and repurposing.

Keywords: RNA-seq experiment; allograft rejection; kidney biopsies; kidney transplantation; molecular signature; next generation sequencing.

FIGURE 1

Antibody-mediated rejection molecular signature. Volcano plot of the significant differentially expressed genes associated with antibody-mediated rejection. Dots are related to each gene. The significant transcripts are displayed according to a 0.05 threshold on the adjusted p-value (vertical grey line). NGS-specific transcripts are highlighted in red, B-HOT-related in yellow and microarray-related in blue. X-axis represents the -log₁₀ of the adjusted p-value (the higher, the smaller is the p-value) and the y-axis represents the log₂ fold change. Differences in gene expression between the AMR and non-AMR group are marked with positive (negative) values correspond to up- (down-)regulated transcripts in the AMR group. In total, 6,141 genes were differentially expressed showing the following distribution: 358 included in the B-HOT gene panel, 5,180 included in the microarray gene panel and 603 NGS-specific. Abbreviations: AMR: antibody-mediated rejection; B-HOT: Banff Human Organ Transplant; NGS: next-generation sequencing.

FIGURE 2

Antibody-mediated rejection map of enriched pathways. Enrichment map of pathways involved in the antibody-mediated rejection, developed on the entire list of upregulated genes. The interaction map contextualizes the pathophysiological categories inter-relations. Vertices represent pathways, dots color intensity refers to the significance of the category and sized of the dots is in accordance to the number of genes in the signature. Edges symbolize the overlap between two pathways, powered by the shared transcripts. The closer two vertices are and the thicker is the edge connecting them, the wider is the overlap between the two pathways.

FIGURE 3

T-cell mediated rejection molecular signature. Volcano plot of the significant differentially expressed genes associated with T-cell mediated rejection. Dots are related to each gene. The significant transcripts are displayed according to a 0.05 threshold on the adjusted p-value (vertical grey line). NGS-specific transcripts are highlighted in red, B-HOT-related in yellow and microarray-related in blue. X-axis represents the -log₁₀ of the adjusted p-value (the higher, the smaller is the p-value) and the y-axis represents the log₂ fold change. Differences in gene expression between the TCMR and non-TCMR group are marked with positive (negative) values correspond to up- (down-)regulated transcripts in the TCMR group. In total, 8,478 genes were differentially expressed showing the following distribution: 439 included in the B-HOT, 6,853 included in the microarray and 1,186 NGS-specific. Abbreviations: B-HOT: Banff Human Organ Transplant; NGS: next-generation sequencing; TCMR: T-cell mediated rejection.

FIGURE 4

T-cell mediated rejection map of enriched pathways. Enrichment map of pathways involved in the antibody-mediated rejection, developed on the entire list of upregulated genes. The interaction map contextualizes the pathophysiological categories inter-relations. Vertices represent pathways, dots color intensity refers to the significance of the category and sized of the dots is in accordance to the number of genes in the signature. Edges symbolize the overlap between two pathways, powered by the shared transcripts. The closer two vertices are and the thicker is the edge connecting them, the wider is the overlap between the two pathways. Only the top 100 pathways are displayed to improve readability.