Early detection of active Human CytomegaloVirus (hCMV) infection in pregnant women using data generated for noninvasive fetal aneuploidy testing

Background: Prenatal hCMV infections can lead to severe embryopathy and neurological sequelae in neonates. Screening during pregnancy is not recommended by global societies, as there is no effective therapy. Recently, several groups showed that maternal–fetal hCMV transmission can be strongly reduced by administering anti-viral agents early in pregnancy. This calls for a screening method to identify at risk pregnancies at an appropriate gestational age, with the possibility for large-scale enrolment. Non-Invasive Prenatal Testing (NIPT) for fetal aneuploidy screening early in pregnancy is already implemented in many countries and performed on a large-scale basis. We investigated the use of whole genome cell-free DNA (cfDNA) sequencing data, generated for the purpose of NIPT, as (pre-)screening tool to identify women with active hCMV-infections, eligible for therapy. Methods: Coded raw sequencing NIPT data from 204,818 pregnant women from three testing laboratories were analyzed for the presence of hCMV-cfDNA. Samples were stratified by cfDNA-hCMV load. For validation and interpretation, diagnostic hCMV-qPCR and serology testing were performed on a subset of cfDNA-hCMV-positive (n = 112) and -negative (n = 127) samples. Findings: In 1930 samples (0.94%) hCMV fragments were detected. Validation by hCMV-qPCR showed that samples with high cfDNA-hCMV load tested positive and cfDNA-hCMV-negative samples tested negative. In 32/112 cfDNA-hCMV-positive samples (28.6%) the serological profile suggested a recent primary infection: this was more likely in samples with high cfDNA-hCMV load (78.6%) than in samples with low cfDNA-hCMV load (11.0%). In none of the cfDNA-hCMV-negative samples serology was indicative of a recent primary infection.Interpretation: Our study shows that large-scale (pre-)screening for both genetic fetal aberrations and active maternal hCMV infections during pregnancy can be combined in one cfDNA sequencing test, performed on a single blood sample, drawn in the first trimester of pregnancy. Funding: This work was partly funded by the Prenatal Screening Foundation Nijmegen, the Netherlands.</p

Early detection of active Human CytomegaloVirus (hCMV) infection in pregnant women using data generated for noninvasive fetal aneuploidy testing

Background: Prenatal hCMV infections can lead to severe embryopathy and neurological sequelae in neonates. Screening during pregnancy is not recommended by global societies, as there is no effective therapy. Recently, several groups showed that maternal–fetal hCMV transmission can be strongly reduced by administering anti-viral agents early in pregnancy. This calls for a screening method to identify at risk pregnancies at an appropriate gestational age, with the possibility for large-scale enrolment. Non-Invasive Prenatal Testing (NIPT) for fetal aneuploidy screening early in pregnancy is already implemented in many countries and performed on a large-scale basis. We investigated the use of whole genome cell-free DNA (cfDNA) sequencing data, generated for the purpose of NIPT, as (pre-)screening tool to identify women with active hCMV-infections, eligible for therapy. Methods: Coded raw sequencing NIPT data from 204,818 pregnant women from three testing laboratories were analyzed for the presence of hCMV-cfDNA. Samples were stratified by cfDNA-hCMV load. For validation and interpretation, diagnostic hCMV-qPCR and serology testing were performed on a subset of cfDNA-hCMV-positive (n = 112) and -negative (n = 127) samples. Findings: In 1930 samples (0.94%) hCMV fragments were detected. Validation by hCMV-qPCR showed that samples with high cfDNA-hCMV load tested positive and cfDNA-hCMV-negative samples tested negative. In 32/112 cfDNA-hCMV-positive samples (28.6%) the serological profile suggested a recent primary infection: this was more likely in samples with high cfDNA-hCMV load (78.6%) than in samples with low cfDNA-hCMV load (11.0%). In none of the cfDNA-hCMV-negative samples serology was indicative of a recent primary infection.Interpretation: Our study shows that large-scale (pre-)screening for both genetic fetal aberrations and active maternal hCMV infections during pregnancy can be combined in one cfDNA sequencing test, performed on a single blood sample, drawn in the first trimester of pregnancy. Funding: This work was partly funded by the Prenatal Screening Foundation Nijmegen, the Netherlands.</p

Genomic and molecular characterization of preterm birth.

Preterm birth (PTB) complications are the leading cause of long-term morbidity and mortality in children. By using whole blood samples, we integrated whole-genome sequencing (WGS), RNA sequencing (RNA-seq), and DNA methylation data for 270 PTB and 521 control families. We analyzed this combined dataset to identify genomic variants associated with PTB and secondary analyses to identify variants associated with very early PTB (VEPTB) as well as other subcategories of disease that may contribute to PTB. We identified differentially expressed genes (DEGs) and methylated genomic loci and performed expression and methylation quantitative trait loci analyses to link genomic variants to these expression and methylation changes. We performed enrichment tests to identify overlaps between new and known PTB candidate gene systems. We identified 160 significant genomic variants associated with PTB-related phenotypes. The most significant variants, DEGs, and differentially methylated loci were associated with VEPTB. Integration of all data types identified a set of 72 candidate biomarker genes for VEPTB, encompassing genes and those previously associated with PTB. Notably, PTB-associated genes RAB31 and RBPJ were identified by all three data types (WGS, RNA-seq, and methylation). Pathways associated with VEPTB include EGFR and prolactin signaling pathways, inflammation- and immunity-related pathways, chemokine signaling, IFN-γ signaling, and Notch1 signaling. Progress in identifying molecular components of a complex disease is aided by integrated analyses of multiple molecular data types and clinical data. With these data, and by stratifying PTB by subphenotype, we have identified associations between VEPTB and the underlying biology

Computational analyses to characterise hidden information in short and long read sequencing data of human genomes: there’s more than meets the reference

Next generation sequencing (NGS) has enabled us to accurately determine the nucleotide sequence of short fragments of DNA at a massive scale, which has led to various clinical applications of human genome sequencing. To extract information from these NGS experiments, virtually all analyses make use of a reference assembly of the human genome to map sequenced reads. Importantly, in these experiments a large fraction (~12%) of the sequenced DNA fragments are ignored as the origin of these sequences cannot be traced back to a (single) position on the reference assembly. The origin of these ignored or unmapped fragments is dual. On the one hand these fragments originate from sequence that occurs more than once (repeats). On the other hand, these fragments originate from sequence that is absent from the reference assembly. In practice, many of these unmapped fragments originate from so-called structural variations (SVs) where the sequenced genome differs from the reference assembly. In Part 1 of this thesis, we study this source of sequence variation by making use of so-called long-read sequencing technology and introduce methods to do so. In Part 2 of this thesis, we specifically study the DNA fragments that can’t be traced back to the human reference assembly, but instead seem to originate from DNA viruses

Clinically relevant DNA viruses in pregnancy

Infections by DNA viruses during pregnancy are associated with increased health risks to both mother and fetus. Although not all DNA viruses are related to an increased risk of complications during pregnancy, several can directly infect the fetus and/or cause placental dysfunction. During Non-Invasive Prenatal Testing analysis, the presence of viral DNA can be detected, theoretically allowing screening early in pregnancy. Although treatment options are currently limited, this might rapidly change in the near future. It is therefore important to be aware of the potential impact of these viruses on feto-maternal health. In this manuscript we provide a brief introduction into the most commonly detected DNA viruses in human cell-free DNA sequencing experiments and their pathogenic potential during pregnancy

Distinct fragmentation patterns of circulating viral cell-free DNA in 83,552 non-invasive prenatal testing samples

Aim: The fragmentation characteristics of cell-free DNA (cfDNA) are informative biomarkers in liquid biopsies, including non-invasive prenatal testing (NIPT), as they provide insights into the origins of the cfDNA. Viral infections by DNA viruses can contribute to the available cfDNA in these samples. Here, we characterize the fragment size distribution of viral cfDNA fragments obtained from available anonymous NIPT samples.Methods: A viral database of 224 DNA viruses was generated from the NCBI RefSeq viral database. Paired-end cfDNA sequencing reads from 83.522 NIPT samples that did not map to any of the human chromosomes, or mitochondrial DNA of the human reference genome build GRCh38 (excluding alternative and unplaced contigs) were remapped to the generated viral database. Reads mapping to the 14 most abundant DNA viruses were selected, and fragment size distributions were analyzed in detail.Results: Distinct fragmentation patterns were identified for several DNA viruses, most likely due to differences in viral tropism, chromatinization (binding of nucleosomes), and the topology of the viral DNA. In high viral load parvo B19 positive samples, the fragment size distribution differed between samples, potentially reflecting the state of the infection.Conclusion: These findings outline the potential for liquid biopsies to elucidate the dynamics behind the viral infection, which may potentially have various clinical applications. Our data provide preliminary insights on the use of fragmentomics of viral cfDNA to distinguish between reactivation, reinfection, and primary infection and monitoring the state of viral infections

The cell-free DNA virome of 108,349 Dutch pregnant women

Objective: Viral infections during pregnancy are a major health concern to mother and fetus. By repurposing cell-free Non Invasive Prenatal Testing (NIPT) sequencing data, we investigated prevalence and abundance of viral DNA in a cohort of 108,349 pregnant women. Method: Cell-free DNA (cfDNA) sequencing reads that did not map to any of the human chromosomes or mitochondrial DNA of the human reference genome build GRCh38 were aligned to 224 DNA viruses selected from the NCBI refseq viral database. Results: In total 443,665 reads of viral origin were detected across 42,273 samples representing 165 viral species. Several are known to be potentially harmful during pregnancy and/or childbirth, including Cytomegalovirus, Parvovirus B19 and Hepatitis B. Viral sequences were mostly detected at very low abundance. However, several cases had exceptionally high viral loads for Parvovirus B19, Hepatitis B and others. We found statistically significant associations between presence of viral DNA and gestational age, maternal age, fetal fraction, cfDNA concentration and others. Conclusion: We demonstrate the feasibility to detect viral DNA from typical genome-wide NIPT cfDNA sequencing and describe the main characteristics of the viral DNA in our cohort. Our dataset of detected viral sequence reads is made publicly available to guide future clinical implementations