Transcriptome analysis of peripheral blood of Schistosoma mansoni infected children from the Albert Nile region in Uganda reveals genes implicated in fibrosis pathology

Over 290 million people are infected by schistosomes worldwide. Schistosomiasis control efforts focus on mass drug treatment with praziquantel (PZQ), a drug that kills the adult worm of all Schistosoma species. Nonetheless, re-infections have continued to be detected in endemic areas with individuals living in the same area presenting with varying infection intensities. Our objective was to characterize the transcriptome profiles in peripheral blood of children between 10–15 years with varying intensities of Schistosoma mansoni infection living along the Albert Nile in Uganda. RNA extracted from peripheral blood collected from 44 S. mansoni infected (34 high and 10 low by circulating anodic antigen [CAA] level) and 20 uninfected children was sequenced using Illumina NovaSeq S4 and the reads aligned to the GRCh38 human genome. Differential gene expression analysis was done using DESeq2. Principal component analysis revealed clustering of gene expression by gender when S. mansoni infected children were compared with uninfected children. In addition, we identified 14 DEGs between S. mansoni infected and uninfected individuals, 56 DEGs between children with high infection intensity and uninfected individuals, 33 DEGs between those with high infection intensity and low infection intensity and no DEGs between those with low infection and uninfected individuals. We also observed upregulation and downregulation of some DEGs that are associated with fibrosis and its regulation. These data suggest expression of fibrosis associated genes as well as genes that regulate fibrosis in S. mansoni infection. The relatively few significant DEGS observed in children with schistosomiasis suggests that chronic S. mansoni infection is a stealth infection that does not stimulate a strong immune response.


RNA extraction and purification
126 RNA was extracted from blood collected in PAXgene Blood RNA tubes using Trizol 127 (Invitrogen, USA) protocol (11). The RNA was quantified using Qubit (Invitrogen, USA) and 128 samples with concentration >1µg were shipped to the Centre for Genomics Research at the 129 University of Liverpool for sequencing where the quality of the samples was checked using an 130 Agilent Bioanalyser. 151 In addition to high prevalence of schistosomiasis, our previous study found high levels of 152 stunting and under nutrition in this study population (4). To understand the association of BMI 153 and stunting with gene expression, we conducted linear regression analysis with BMI and 154 stunting as dependent variables and sex and age as covariates for each analysis.  Association of DEGs with schistosomiasis or fibrosis 161 We searched Pubmed abstracts and titles using the name of each of the 63 differentially 162 expressed genes and the terms "schistosomiasis" or "schistosoma" for association of the DEGs 163 with schistosomiasis. We further searched using the term "fibrosis" with each of the unique 164 DEGs for association with fibrosis.  Table S7). Functional analysis of the expressed genes showed 234 that stunting was linked to pathways of catabolic process and lipid metabolic processes. The 235 genes upregulated in children with high BMI were linked to pathways involved with 236 biosynthesis, signal transduction, cellular nitrogen compound metabolic processes and cellular 237 protein modification among others (Table S8).  278 polycomb repressive complex 2 subunits (SUZ12) to be linked to pathways in REACTOME.
279 The T cell receptor beta constant 2 (TRBC2) was found to be associated with T cell signalling 280 and regulation pathways.
281 Genes associated with fibrosis.
282 Whilst schistosome infections cause lethargy and other non-specific symptoms, death is mainly 283 caused by the fibrosis accumulating around eggs lodged in the tissues, particularly in the 284 hepatic portal vein. A search through Pubmed abstracts and titles using the term "fibrosis" with 285 each of the 63 genes in turn identified 27 gene names that appeared in articles that also 286 mentioned fibrosis of which 13 had well documented associations with fibrosis (Table S5).
287 Although our study was of whole blood and schistosomiasis associated fibrosis occurs in 288 extracellular matrix, six of the 13 of the well documented DEGS associated with fibrosis had 289 been previously found to be differentially expressed in comparisons of liver fibroses with 290 healthy tissues (28) suggesting that expression data from whole blood could be informative. 291 For 11 genes it was possible to predict a direction of effect on fibrosis that would be caused by 292 a change in gene expression. The expression changes in five genes (OGG1, OGT, ITGA4, PRMT7, 293 SUZ12), were predicted to increase fibrosis in participants with high parasitaemia and the 294 remaining 6 genes (MALAT1, TPT1, A1CF, SSPN, SUV39H1, ZNF217) were predicted to reduce 296 their expression profiles. A prospective study to determine whether these genes could be useful 297 biomarkers of morbidity risk may be more informative. 298 TGFB1 has been described as the master regulator of fibrosis (29). Although it was not 299 differentially expressed in our study, seven of the 13 genes associated with fibrosis were also 300 associated with TGFB1. SUZ12 suppresses p27 (30) and p27 promotes TGFB1      566 Table S6: DEGs associated with TGFB1.
567 Table S7: Expressed genes in stunting and BMI.
568 Table S8: GO terms and pathways associated with differentially expressed genes by stunting 569 and BMI.
570 Table S9: Cell types that differ in relative abundance between S. mansoni infected and 571 uninfected children.