Single nucleotide variant counts computed from RNA sequencing and cellular traffic into human kidney allografts

Abstract

Advances in bioinformatics allow identification of single nucleotide polymorphisms (variants) from RNA sequence data. In an allograft biopsy, 2 genomes contribute to the RNA pool, 1 from the donor organ and the other from the infiltrating recipient's cells. We hypothesize that imbalances in genetic variants of RNA sequence data of kidney allograft biopsies provide an objective measure of cellular infiltration of the allograft. We performed mRNA sequencing of 40 kidney allograft biopsies, selected to represent a comprehensive range of diagnostic categories. We analyzed the sequencing reads of these biopsies and of 462 lymphoblastoid cell lines from the 1000 Genomes Project, for RNA variants. The ratio of heterozygous to nonreference genome homozygous variants (Het/Hom ratio) on all autosomes was determined for each sample, and the estimation of stromal and immune cells in malignant tumors using expression data (ESTIMATE) score was computed as a complementary estimate of the degree of cellular infiltration into biopsies. The Het/Hom ratios (P = .02) and the ESTIMATE scores (P < .001) were associated with the biopsy diagnosis. Both measures correlated significantly (r = .67, P < .0001), even though the Het/Hom ratio is based on mRNA sequence variation, while the ESTIMATE score uses mRNA expression. Het/Hom ratio and the ESTIMATE score may offer unbiased and quantitative parameters for characterizing cellular traffic into human kidney allografts.

Landscape of innate immune system transcriptome and acute T cell–mediated rejection of human kidney allografts

“…The role of APCs is also played by macrophages (8, 11). Our finding of increased macrophages in TCMR is consistent with immunohistochemical data demonstrating increased CD68 + macrophages in allograft biopsies of kidney recipients with TCMR (4,55). We found increased M1 macrophages in TCMR, which are activated by IFN-γ, are proinflammatory, and act as an APC (56).…”

“…Kidney transplantation is a life restorative procedure (1,2), but multiple challenges still exist (3). Transplantation is a unique situation from an immunological perspective in that 2 diploid genomes are brought together at the time of transplant surgery (4). Immunological injury to the allograft results from the interactions between the donor and recipient genomes.…”

Technical considerations when designing a gene expression panel for renal transplant diagnosis

“…Gene expression signatures of rejection were first discovered using microarray analysis 2 . However a number of research groups have now assessed gene expression signatures in transplant biopsies placed in an RNA preservative, using a variety of methods, not only microarray analysis [3][4][5][6][7][8][9][10] , but also quantitative real-time polymerase chain reaction (qRT-PCR) [11][12][13] and RNA sequencing 9,14,15 . Further validation of gene signatures for diagnostic use has not proceeded however, partly because a renal transplant biopsy is an invasive procedure that yields a limited amount of tissue.…”

Deep sequencing of DNA from urine of kidney allograft recipients to estimate the donor-specific DNA fraction

“…The presence of donor-specific DNA in urine of kidney allograft recipients has been reported [25]. We have recently conducted a study based on RNA sequencing of tissue biopsies from kidney allograft transplants and found a correlation between the ratio of heterozygous to homozygous SNVs with the rejection phenotype [26]. Moreover, we have shown in another study that DNA methylation could be used to accurately estimate the tissue type composition in recipient urine samples.…”