RNAseq of amniotic fluid cell-free RNA: insights into the pathophysiology of congenital cytomegalovirus infection

Background Congenital cytomegalovirus (cCMV) is the most common perinatal infection and a significant cause of sensorineural hearing loss, cerebral palsy and neurodevelopmental disability. There is a paucity of human gene expression studies examining the pathophysiology of CMV infection. Objectives The aim of this study was to perform a whole transcriptomic assessment of amniotic fluid from pregnancies with live fetuses to identify differentially-expressed genes and enriched Gene Ontology categories associated with cCMV infection. Study design Amniotic fluid supernatant was prospectively collected from pregnant women undergoing amniocentesis for suspected cCMV due to first trimester maternal primary infection or ultrasound features suggestive of fetal infection. Women who had received therapy to prevent fetal infection were excluded. cCMV was diagnosed via viral PCR of amniotic fluid; CMV-infected fetuses were paired with noninfected controls, matched for gestational age and fetal sex. Paired-end RNA sequencing was performed on amniotic fluid cell-free RNA with the Novaseq6000 at a depth of 30 million reads/sample. Following quality control and filtering, reads were mapped to the human genome and counts summarised across genes. Differentially expressed genes were identified using two approaches: voomWithQualityWeights in conjunction with limma and RUVSeq with edgeR. Genes with a false discovery rate (FDR) < 0.05 were considered statistically significant. Differential exon usage was analysed using DEXSeq. Functional analysis was performed using Gene Set Enrichment Analysis and Ingenuity Pathway Analysis. Manual curation of differentially regulated genes was also performed. Results Amniotic fluid samples were collected from 50 women; 16 (32%) had cCMV confirmed by PCR. After excluding 3 samples without matched controls, 13 CMV-infected samples collected at 18-23 weeks and 13 CMV-negative gestation matched controls were submitted for RNA sequencing and analysis (total n=26). Ten of the 13 pregnancies with CMV-infected fetuses had amniocentesis due to serological evidence of maternal primary infection with normal fetal ultrasound, and three had amniocentesis due to ultrasound abnormality suggestive of CMV infection. Four CMV-infected pregnancies ended in termination (n=3) or fetal death (n=1), and 9 resulted in live births. Pregnancy outcomes were available on 11 of the 13 CMV-negative controls; all resulted in live births of clinically-well infants. Differential gene expression analysis revealed 309 up-regulated and 32 down-regulated genes in the CMV-infected group compared with the CMV-negative group. Gene set enrichment analysis showed significant enrichment of multiple Gene Ontology categories involving the innate immune response to viral infection and interferon signalling. Of the 32 significantly down-regulated genes, 8 were known to be involved in neurodevelopment and preferentially expressed by the brain. Six specific cellular restriction factors involved in host defence to CMV infection were upregulated in the CMV-infected group. Ingenuity Pathway Analysis predicted activation of pathways involved in progressive neurological disease, and inflammatory neurological disease. Conclusions In this next generation sequencing study, we reveal new insights into the pathophysiology of cCMV infection. These data on the upregulation of the intraamniotic innate immune response to CMV infection and the dysregulation of neurodevelopmental genes may inform future approaches to developing prognostic markers and assessing fetal responses to in utero therapy.