Inflammation and transcriptional responses of peripheral blood mononuclear cells in classic ataxia telangiectasia

Abstract

Introduction: Classic ataxia telangiectasia (A-T) is an autosomal recessive disease characterized by early onset ataxia, immune deficiency, sino-pulmonary disease, lymphoid/solid malignancies and telangiectasias. Prior studies have suggested that chronic inflammation and premature aging may contribute to the development of malignancy and pulmonary disease in people with A-T. To further examine the link between A-T and inflammation, we hypothesized that subjects with classic A-T would have greater enrichment of inflammatory pathways in peripheral blood mononuclear cells (PBMCs) compared to non A-T age-matched controls. To test this hypothesis we used RNAseq as an unsupervised approach to identify biological processes altered in people with classic A-T.

Methods: PBMCs were isolated from subjects with classic A-T and compared to non-A-T age-matched healthy controls. RNAseq with differential gene expression analyses was then performed. Selected genes were validated by RT-qPCR using cohorts of subjects consisting of classic A-T, mild A-T or non-A-T controls. Subjects with mild A-T were characterized by later onset/mild neurologic features and normal/near normal immune status.

Results: RNAseq revealed 310 differentially expressed genes (DEGs) including genes involved in inflammation, immune regulation, and cancer. Using gene set enrichment analysis, A-T subjects were found to have biological processes enriched for inflammatory and malignancy pathways. In examining a cohort of A-T subjects in which baseline serum IL8 and IL6 levels were measured previously, an association was found between higher serum IL8 levels and higher likelihood of developing malignancy and/or death in a subsequent 4-6 year period.

Conclusion: RNAseq using PBMCs from subjects with classic A-T uncovered differential expression of immune response genes and biological processes associated with inflammation, immune regulation, and cancer. Follow-up of A-T subjects over a 4-6 year period revealed an association between higher baseline serum IL8 levels and malignancy/death. These findings support a role for inflammation as a contributing factor in A-T phenotypes.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Principal Component analysis (PCA) illustrating variance across 13,511 genes in the data set.

Variance is primarily due to classic A-T phenotype (PC1). Red- Classic A-T phenotype (n = 3) and Blue- non-A-T healthy age-matched controls (n = 3). Ellipses represent normal contour lines with one standard deviation probability.

Fig 2

A. Principal Component analysis using 310 DEGs identified in the RNAseq dataset. A PC1 variance of 86.7% indicates that A-T phenotype accounts for the majority of variance between the two groups. B. Heat map of the 310 differentially expressed genes. AT1, AT2, AT3 represents individual samples in the classic A-T group and Ctr1, Ctr2 and Ctr3 represent individual samples in the non-A-T healthy age-matched control group. The rows and columns are grouped using hierarchical clustering. Red- represents increased gene expression and blue represents decreased gene expression. DEGs were identified using edgeR analysis, with a FDR q-value cutoff of 0.05.

Fig 3

A. Hallmark processes negatively enriched in controls (positively enriched in A-T). Attached bar graph includes hallmark processes negatively enriched in non A-T age-matched healthy controls (positively enriched in A-T) with a FDR q-value < = 0.01. Processes highlighted in red are processes involved in inflammation, cancer and cell proliferation. B. Example gene set enrichment plot for TNF-α signaling via NFκB.

Fig 4. Volcano plot of 310 DEGs depicting genes downregulated in the classic A-T group (right side) and upregulated in the classic A-T group (left side) compared to non-A-T healthy age-matched controls.

Fig 5

Left pair of box plots demonstrate median and interquartile ranges for log transformed serum IL-8 levels for subjects who developed and did not develop malignancy following specimen collection; subjects who had developed malignancies prior to specimen collection were excluded from this pair of plots. The right pair of box plots depicts similar data for subjects alive at the end of the follow-up period compared to those who died. *Regression p values adjusted for age, sex, and IL6 were 0.009 for any malignancy and 0.034 for death.

Fig 6. Four differentially expressed genes (DEGs) from RNAseq data validated by RT-PCR in subjects recruited prospectively.

Box plots of selected DEGs by clinical group (non-A-T healthy controls, Mild A-T phenotype and Classic A-T phenotype). *Adjusted p<0.05 for Mild A-T phenotype compared to non-A-T healthy controls. **Adjusted p<0.05 for Classic A-T phenotype compared to non-A-T healthy controls. RNA was isolated from PBMCs obtained from Classic A-T (n = 7), Mild A-T (n = 8) and non-A-T healthy controls (n = 6). P values were derived through linear regression and adjusted for age.