Gene expression analysis of whole blood from preclinical and clinical cattle infected with atypical bovine spongiform encephalopathy

BACKGROUND: Prion diseases, such as bovine spongiform encephalopathies (BSE), are transmissible neurodegenerative disorders affecting humans and a wide variety of mammals. Variant Creutzfeldt-Jakob disease (vCJD), a prion disease in humans, has been linked to exposure to BSE prions. This classical BSE (cBSE) is now rapidly disappearing as a result of appropriate measures to control animal feeding and monitoring. Besides cBSE, two atypical forms (named H- and L-type BSE) have recently been described in Europe, Japan, and North America. Here we describe the first wide-spectrum microarray analysis in whole blood of atypical BSE-infected cattle. Transcriptome changes in infected animals were analyzed prior to and after the onset of clinical signs. Some of the most significant differentially expressed genes (DEGs) were validated by quantitative real time PCR (RT-qPCR). AIM: The aim of this study was to analyze the transcriptome changes in whole blood from atypical BSE-infected animals prior and after the onset of the clinical signs to understand the peripheral mechanisms of prion infection and to line out some candidate genes that could be further investigated as biomarker of the disease. METHODS: Total RNA from whole blood samples from 8 intracranially BSE-challenged cattle (4 with H-type and 4 with L-type BSE) and 2 non-infected age- and sex-matched controls was isolated and subjected to the microarray analysis using the GeneChip® Bovine Genome Array (Affymetrix). In order to increase the animal cohort, RNA samples from four additional sex-matched control cattle were isolated using the same protocol as the original study group and included in the final statistical analysis. Therefore, 24 RNA samples, divided in 8 preclinical (P1, P2, P4, P5, P7, P8, EP9 and P10), 8 clinical (S1, S2, S3, S4, S7, S8, S9 and S10), and 8 control (c2, c3, cP3, c5, cS5, cP6, cS6 and c9) samples, constituted our animal cohort. After the assessment and inspection of microarray quality controls (RNA degradation plot, RLE and NUSE plots) we identified one low quality control sample (cS5) and excluded it from the final statistical analysis. Gene probes with a p value ≤0.05 and fold-change ≥2 were considered to be differentially expressed. To confirm the microarray results, we performed RT-qPCR using SYBR® green assay (Bio-Rad Laboratories, Inc.) for a selected number of target genes. The RT-qPCR analysis was performed on 22 samples (7 control, 8 preclinical and 7 clinical animals). The normalization accuracy was improved by geometric averaging of multiple reference genes (GAPDH, RPL12 and ACTB) and using two inter-run calibrators to reduce inter-run variation. RESULTS: The microarray analysis revealed a total of 101 differentially regulated probe sets (p value lower than 0.05 and changes in expression higher than 2-fold) in infected animals (clinical and preclinical) versus control group. In the clinical stage, a total of 207 probe sets showed significant alteration in expression levels compared to the control group. Interestingly, a pronounced alteration in the gene expression profile was also found in the preclinical stage, with a total number of 113 differentially expressed probe sets. A set of 35 differentially expressed genes was found to be in common between clinical and preclinical stages and showed a very similar expression pattern in the two phases. To further dissect gene expression alterations during the progression of the disease, we performed a statistical analysis to identify specific changes between the clinical and preclinical stages (CvsP). Indeed, we found 235 DEGs, which were significantly enriched in pathways related to immune response. The comparison of all the analysis, revealed a 22-gene signature with an up/down-down/up pattern of expression, being differentially expressed in preclinical stage and then going back to control levels in the symptomatic phase. One gene, SEL1L3, was progressively downregulated during the progression of the disease. The identified genes belong to several pathways, such as immune response and metabolism, that may play an important role in prion pathogenesis. The RT-qPCR analysis confirmed the microarray results for six out of nine genes selected (XIST, CD40L, GNLY, PDK4, HBA2 and SEL1L3). CONCLUSIONS: The present study has led to the identification of several gene expression changes in whole blood from atypical BSE infected cattle prior and after the manifestation of the pathology. Our findings suggest that it might be feasible to use whole blood RNA transcriptional profiles to distinguish between preclinical and clinical stages of prion infection. Overall, our study confirmed the differential expression of 6 genes (XIST, CD40L, GNLY, PDK4, HBA2 and SEL1L3), which may play several roles in atypical BSE pathogenesis and, possibly, in other prion infections. Even though further studies are required to investigate the specific involvement of all the identified genes in prion diseases, our data support the idea of a relationship of complicity and blindness between prion and the host immune system. As concluding remark, our study underlined the importance of utilizing whole blood, without any additional manipulation, as a source tissue for the development of a preclinical diagnostic test.