Polyomavirus BK Nephropathy-Associated Transcriptomic Signatures: A Critical Reevaluation

Abstract

Background: Recent work using DNA microarrays has suggested that genes related to DNA replication, RNA polymerase assembly, and pathogen recognition receptors can serve as surrogate tissue biomarkers for polyomavirus BK nephropathy (BKPyVN).

Methods: We have examined this premise by looking for differential regulation of these genes using a different technology platform (RNA-seq) and an independent set 25 biopsies covering a wide spectrum of diagnoses.

Results: RNA-seq could discriminate T cell-mediated rejection from other common lesions seen in formalin fixed biopsy material. However, overlapping RNA-seq signatures were found among all disease processes investigated. Specifically, genes previously reported as being specific for the diagnosis of BKPyVN were found to be significantly upregulated in T cell-mediated rejection, inflamed areas of fibrosis/tubular atrophy, as well as acute tubular injury.

Conclusions: In conclusion, the search for virus specific molecular signatures is confounded by substantial overlap in pathogenetic mechanisms between BKPyVN and nonviral forms of allograft injury. Clinical heterogeneity, overlapping exposures, and different morphologic patterns and stage of disease are a source of substantial variability in "Omics" experiments. These variables should be better controlled in future biomarker studies on BKPyVN, T cell-mediated rejection, and other forms of allograft injury, before widespread implementation of these tests in the transplant clinic.

FIGURE 1

Quality control data from a representative sequencing chip. After filtering for bases with a quality value of AQ20 the most frequent sequences had a read length of 100 to 125 nucleotides (left panel). Alignment of Reads to hg19 Ampliseq Transcriptome ERCC v1 showed 90% base alignment with an average depth of coverage of 136.6×, and mean raw accuracy of 97.6% (middle and right panels).

FIGURE 2

Log ratio (M) versus mean average (A) plot for RNA-seq data derived from 5 biopsies each from the TCMR and STA groups (left panel). The same data is also illustrated in the form of a volcano plot (right panel). Genes differentially expressed at an FDR less than 0.05 are coded blue. FDR, false discovery rate.

FIGURE 3

IPA showing the top 10 biologic pathways corresponding to genes that were found to be significantly (-log p > 1.31) up-regulated in biopsies with TCMR versus STA. The largest proportion of upregulated genes (18/44, 40.9%) is seen in the “Primary immunodeficiency signaling” pathway, whereas the next largest proportion (22/82, 26.8%) belongs to the “Communication between innate and adaptive immune cells” pathway.

FIGURE 4

Expression of 4 previously reported ‘BKPyVN specific’ genes (LTF, CFD, RPS15 and NOSIP) in 5 biopsies each with ATI, BKPyVN, STA, and TCMR. Data are expressed as RNA-seq counts for the individual samples (circles) after normalization for library counts to correct for differences in library size.