Pre-eclampsia (typically characterized by new-onset hypertension and proteinuria in the second half of pregnancy) represents a major determinant of the global burden of disease1,2. Its pathophysiology involves placental dysfunction, but the mechanism is unclear. Viral infection can cause organ dysfunction, but its role in placentally related disorders of human pregnancy is unknown3. We addressed this using RNA sequencing metagenomics4,5,6 of placental samples from normal and complicated pregnancies. Here, we show that human herpesvirus 6 (HHV-6, A or B) RNA was detected in 6.1% of cases of pre-eclampsia and 2.2% of other pregnancies. Fetal genotyping demonstrated that 70% of samples with HHV-6 RNA in the placenta exhibited inherited, chromosomally integrated HHV-6 (iciHHV-6). We genotyped 467 pre-eclampsia cases and 3,854 controls and found an excess of iciHHV-6 in the cases (odds ratio of 2.8, 95% confidence intervals of 1.4–5.6, P = 0.008). We validated this finding by comparing iciHHV-6 in a further 740 cases with controls from large-scale population studies (odds ratio of 2.5, 95% confidence intervals of 1.4–4.4, P = 0.0013). We conclude that iciHHV-6 results in the transcription of viral RNA in the human placenta and predisposes the mother to pre-eclampsia.
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The sequencing data have been deposited in the European Genome-phenome Archive (EGA) with the following accession numbers: EGAD00001003457 (RNA-seq controls), EGAD00001003507 (RNA-seq FGR cases), EGAD00001003508 (RNA-seq pre-eclampsia cases), EGAD00001004197 (metagenomics) and EGAD00001004592 (SureSelect).
Custom scripts for the bioinformatics analyses are available at https://github.com/sung/HHV6-Nat-Micr-2020.
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We are grateful to the following: the participants of the POP, case–control and GOPEC studies; L. Bibby, S. Ranawaka, K. Holmes, J. Gill, C. L. Soh and R. Millar for technical assistance; the UCL Pathogen Genomics Unit; the Sanger Institute core sequencing facility and the Pathogen Informatics team. This work was supported by the Women’s Health theme of the NIHR Cambridge Biomedical Research Centre, the Medical Research Council (MR/K021133/1 to G.C.S.S., D.S.C.-J., J.P. and S.J.P. and G1100221 to G.C.S.S. and D.S.C.-J.) and the UCL/UCLH NIHR Biomedical Research Centre (to J.B. and C.V.). S.L., M.C.d.G., J.D., S.J.P., J.P., D.S.C.-J. and G.C.S.S. received grants from the Medical Research Council (UK); F.G., U.S., S.G., E.C., D.S.C.-J. and G.C.S.S. received grants from the National Institute for Health Research (UK); U.S. received grants from the Stillbirth & Neonatal Death Society (Sands); and J.P. received grants from the Wellcome Trust and grants from Pfizer.
J.D. reports being an employee of GlaxoSmithKline and A.S. reports being an employee of Robinson College (Cambridge, UK). J.P. reports personal fees from Next Gen Diagnostics Llc., outside the submitted work; S.J.P. reports personal fees from Specific and personal fees from Next Gen Diagnostics, outside the submitted work; D.S.C.-J. reports grants from GlaxoSmithKline Research and Development Limited, outside the submitted work; G.C.S.S. reports grants and personal fees from GlaxoSmithKline Research and Development Limited, personal fees and non-financial support from Roche Diagnostics Ltd, outside the submitted work; D.S.C.-J. and G.C.S.S. report grants from Sera Prognostics Inc. and non-financial support from Illumina Inc., outside the submitted work. A.M., W.K.L., C.D., C.V. and J.B. have nothing to disclose.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Characteristics of the study groups in the Pregnancy Outcome Prediction (POP) study.
Data are expressed as median (IQR) or n (%) as appropriate. The overall rate of pre-eclampsia in these participants was 6.4%. For fields where there is no category labelled ‘missing’, data were 100% complete. Maternal age was defined as age at recruitment. All other maternal characteristics were defined by self-report at the 20 weeks questionnaire, from examination of the clinical case record, or linkage to the hospital’s electronic databases. Socio-economic status was quantified using the Index of Multiple Deprivation (IMD) 2007, which is based on census data from the area of the mother’s postcode. Stillbirths (n=8) and spontaneous preterm deliveries (n=100) were included in the analysis, while miscarriages (n=7) and terminations of pregnancy (n=11) were excluded. Abbreviations: non-cases denote patients without pre-eclampsia; FTE denotes full time education; BMI denotes body mass index; DM denotes diabetes mellitus.
Placental HHV-6 positive samples identified by RNA-seq. Reads were identified by Kraken as aligning to the HHV-6 genomes and mapped with BWA to the HHV-6A or HHV-6B reference genomes (Supplementary Methods); note that the total number of reads/sample recognized by the two software is not always identical. CON denotes a healthy pregnancy without FGR or pre-eclampsia (see Methods); FGR denotes fetal growth restriction; PE denotes a patient with pre-eclampsia; HHV-6A denotes human herpesvirus 6, variant A; HHV-6B denotes human herpesvirus 6, variant B; DRL: direct repeat left; DRR: direct repeat right. Repetitive regions are in Italic. HHV-6A and HHV-6B genomes have been described by Dominguez G et al53.
HHV-6A and HHV-6B representative signals in cord (a) and parental (b) DNA samples detected using a multiplex qPCR approach. These analyses were performed in 5,061 and 86 samples, respectively, and each sample was analyzed in triplicate. qPCR amplification curves for the HHV-6A and HHV-6B 9 U67/68 genes are represented in green and red, respectively; RNase P curves are in blue and confirmed presence of DNA in the wells. ciHHV-6 corresponds to a high HHV-6 DNA signal in the sample measured by qPCR, that is within 4 cycles of the RNase P signal. HHV-6 non-integrated corresponds to a HHV-6 DNA signal in the sample detected at more than 4 Ct higher compared to the RNase P signal. Negative samples lack HHV-6A or HHV-6B DNA signal. c) RT-qPCR amplification plot of placental RNA samples showing detection of the HHV-6 U100 gene. Eight representative samples are shown, two with viral transcript amplification (total n=48 samples, each analyzed in triplicate). Five negative controls (samples without reverse transcriptase enzyme in the RT reaction) lacked U100 amplification (not shown). U100 curves are in black and RNase P curves are in blue. Rn (normalized reporter value) represents the fluorescence of the reporter dye normalized to the signal of the passive reference dye for a given reaction. The ΔRn is the Rn value of an experimental reaction minus the Rn value of the baseline signal generated by the instrument. This parameter indicates the magnitude of the fluorescent signal generated in the qPCR assay. ciHHV-6 denotes chromosomally integrated human herpesvirus 6; HHV-6A denotes human herpesvirus 6, variant A; HHV-6B denotes human herpesvirus 6, variant B; RNase P denotes the human positive control gene RPPH1.
DNA-seq reads of 2 randomly selected samples were compared to the HHV-6B reference genome. *Informative SNP sites, that is SNPs present in just one of the two analyzed samples (gapped vertical red lines). **SNPs present in both analyzed samples, that is sites concordantly different from the reference genome, were considered not informative (continuous vertical red lines). Throughout the 162kb HHV-6B genome 187 SNPs were classified as informative.
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Gaccioli, F., Lager, S., de Goffau, M.C. et al. Fetal inheritance of chromosomally integrated human herpesvirus 6 predisposes the mother to pre-eclampsia. Nat Microbiol 5, 901–908 (2020). https://doi.org/10.1038/s41564-020-0711-3
Mucosal Immunology (2021)