Background
Congenital cytomegalovirus infection 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 cytomegalovirus infection.
Objective
This study aimed 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 congenital cytomegalovirus infection.
Study Design
Amniotic fluid supernatant was prospectively collected from pregnant women undergoing
amniocentesis for suspected congenital cytomegalovirus infection because of first-trimester
maternal primary infection or ultrasound features suggestive of fetal infection. Women
who had received therapy to prevent fetal infection were excluded. Congenital cytomegalovirus
infection was diagnosed via viral polymerase chain reaction of amniotic fluid; cytomegalovirus-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 Novaseq 6000 at a depth of 30 million reads per sample. Following quality control
and filtering, reads were mapped to the human genome and counts summarized across
genes. Differentially expressed genes were identified using 2 approaches: voomWithQualityWeights
in conjunction with limma and RUVSeq with edgeR. Genes with a false discovery rate
<0.05 were considered statistically significant. Differential exon use was analyzed
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 congenital cytomegalovirus
infection confirmed by polymerase chain reaction. After excluding 3 samples without
matched controls, 13 cytomegalovirus-infected samples collected at 18 to 23 weeks
and 13 cytomegalovirus-negative gestation-matched controls were submitted for RNA
sequencing and analysis (N=26). Ten of the 13 pregnancies with cytomegalovirus-infected
fetuses had amniocentesis because of serologic evidence of maternal primary infection
with normal fetal ultrasound, and 3 had amniocentesis because of ultrasound abnormality
suggestive of cytomegalovirus infection. Four cytomegalovirus-infected pregnancies
ended in termination (n=3) or fetal death (n=1), and 9 resulted in live births. Pregnancy
outcomes were available for 11 of the 13 cytomegalovirus-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 cytomegalovirus-infected group compared with the cytomegalovirus-negative
group. Gene set enrichment analysis showed significant enrichment of multiple Gene
Ontology categories involving the innate immune response to viral infection and interferon
signaling. 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 defense to cytomegalovirus infection were up-regulated
in the cytomegalovirus-infected group. Ingenuity Pathway Analysis predicted the activation
of pathways involved in progressive neurologic disease and inflammatory neurologic
disease.
Conclusion
In this next-generation sequencing study, we revealed new insights into the pathophysiology
of congenital cytomegalovirus infection. These data on the up-regulation of the intraamniotic
innate immune response to cytomegalovirus infection and the dysregulation of neurodevelopmental
genes may inform future approaches to developing prognostic markers and assessing
fetal responses to in utero therapy.
Key words
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Article Info
Publication History
Published online: May 21, 2022
Accepted:
May 13,
2022
Received in revised form:
May 9,
2022
Received:
December 24,
2021
Publication stage
In Press Journal Pre-ProofFootnotes
The authors report no conflict of interest.
This study was funded by a Sylvia and Charles Viertel Clinical Investigator grant and the National Health and Medical Research Council (grant numbers 1196010 and 1105603 to L.H. and grant number 1146128 to N.J.H.). The funding sources had no involvement in the study design, collection, analysis, data interpretation, or writing of the report.
Data sharing: All analysis code presented in this manuscript can be found at https://jovmaksimovic.github.io/amnio-cell-free-RNA/index.html. The analysis website was created using the workflowr (1.6.2) R package. The GitHub repository associated with the analysis website is at: https://github.com/JovMaksimovic/amnio-cell-free-RNA.
Ethical approval precluded deposition of the sequencing datasets in a public genomic data repository; however the de-identified, processed, unnormalized read counts may be available to researchers on reasonable request to [email protected] , subject to Mercy Health ethics committee approval.
Cite this article as: Hui L, De Catte L, Beard S, et al. RNA-Seq of amniotic fluid cell-free RNA: a discovery phase study of the pathophysiology of congenital cytomegalovirus infection. Am J Obstet Gynecol 2022;XX:x.ex–x.ex.
Identification
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