Identification of Chromosomally Integrated Human Herpesvirus 6 by Droplet Digital PCR

Sedlak, Ruth Hall; Cook, Linda; Huang, Meei Li; Magaret, Amalia; Zerr, Danielle M.; Boeckh, Michael; Jerome, Keith R.

doi:10.1373/clinchem.2013.217240

Cited by 115 publications

(89 citation statements)

References 30 publications

(39 reference statements)

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“…Three days after infection, cells were transferred into a 25-cm 2 flask and cultured with or without BRACO-19 (100 nM) for 21 days. At that point in time, cells were detached using trypsin and DNA was isolated and tested by ddPCR for the presence of HHV-6 and the cellular reference gene RPP30, as described previously (82). U65-U66-specific primers were validated previously (12,83) and used for the detection of the integrated HHV-6 genome.…”

Section: Methodsmentioning

confidence: 99%

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Gilbert-Girard

Gravel

Artusi

et al. 2017

J Virol

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Human herpesviruses 6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes using a mechanism that remains poorly understood. To achieve a better understanding of the HHV-6A/B integration mechanism, we made use of BRACO-19, a compound that stabilizes G-quadruplex secondary structures and prevents telomere elongation by the telomerase complex. First, we analyzed the folding of telomeric sequences into G-quadruplex structures and their binding to BRACO-19 using G-quadruplex-specific antibodies and surface plasmon resonance. Circular dichroism studies indicate that BRACO-19 modifies the conformation and greatly stabilizes the G-quadruplexes formed in G-rich telomeric DNA. Subsequently we assessed the effects of BRACO-19 on the HHV-6A initial phase of infection. Our results indicate that BRACO-19 does not affect entry of HHV-6A DNA into cells. We next investigated if stabilization of G-quadruplexes by BRACO-19 affected HHV-6A's ability to integrate its genome into host chromosomes. Incubation of telomerase-expressing cells with BRACO-19, such as HeLa and MCF-7, caused a significant reduction in the HHV-6A integration frequency (P Ͻ 0.002); in contrast, BRACO-19 had no effect on HHV-6 integration frequency in U2OS cells that lack telomerase activity and elongate their telomeres through alternative lengthening mechanisms. Our data suggest that the fluidity of telomeres is important for efficient chromosomal integration of HHV-6A and that interference with telomerase activity negatively affects the generation of cellular clones containing integrated HHV-6A.IMPORTANCE HHV-6A/B can integrate their genomes into the telomeres of infected cells. Telomeres consist of repeated hexanucleotides (TTAGGG) of various lengths (up to several kilobases) and end with a single-stranded 3= extension. To avoid recognition and induce a DNA damage response, the single-stranded overhang folds back on itself and forms a telomeric loop (T-loop) or adopts a tertiary structure, referred to as a G-quadruplex. In the current study, we have examined the effects of a G-quadruplex binding and stabilizing agent, BRACO-19, on HHV-6A chromosomal integration. By stabilizing G-quadruplex structures, BRACO-19 affects the ability of the telomerase complex to elongate telomeres. Our results indicate that BRACO-19 reduces the number of clones harboring integrated HHV-6A. This study is the first of its kind and suggests that telomerase activity is essential to restore a functional telomere of adequate length following HHV-6A integration.

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Section: Methodsmentioning

confidence: 99%

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Gilbert-Girard

Gravel

Artusi

et al. 2017

J Virol

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“…13 We used droplet digital (dd)PCR to determine the ratio of HHV-6 DNA to human cells (using human ribonuclease P DNA [RPP30], a reference gene for cell count). 13,16,19 A ratio of 1 6 0.07 was considered positive for iciHHV-6 and distinguished between iciHHV-6 and naturally acquired infection. This assay was validated against a fluorescence in situ hybridizationconfirmed iciHHV-6 cell line.…”

Section: Laboratory Assaysmentioning

confidence: 99%

Outcomes of hematopoietic cell transplantation using donors or recipients with inherited chromosomally integrated HHV-6

Hill

Magaret

Hall-Sedlak³

et al. 2017

Blood

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• Inherited ciHHV-6 was detected in 1.4% of HCT recipients and 0.9% of their donors.• Acute GVHD grades 2-4 and cytomegalovirus viremia were more frequent when recipients or donors had inherited ciHHV-6.Human herpesvirus 6 (HHV-6) species have a unique ability to integrate into chromosomal telomeres. Mendelian inheritance via gametocyte integration results in HHV-6 in every nucleated cell. The epidemiology and clinical effect of inherited chromosomally integrated HHV-6 (iciHHV-6) in hematopoietic cell transplant (HCT) recipients is unclear.We identified 4319 HCT donor-recipient pairs (8638 subjects) who received an allogeneic HCT and had archived pre-HCT peripheral blood mononuclear cell samples. We screened these samples for iciHHV-6 and compared characteristics of HCT recipients and donors with iciHHV-6 with those of recipients and donors without iciHHV-6, respectively. We calculated Kaplan-Meier probability estimates and Cox proportional hazards models for post-HCT outcomes based on recipient and donor iciHHV-6 status. We identified 60 HCT recipients (1.4%) and 40 donors (0.9%) with iciHHV-6; both recipient and donor harbored iciHHV-6 in 13 HCTs. Thus, there were 87 HCTs (2%) in which the recipient, donor, or both harbored iciHHV-6. Acute graft-versus-host disease (GVHD) grades 2-4 was more frequent when recipients or donors had iciHHV-6 (adjusted hazard ratios, 1.7-1.9; P 5 .004-.001). Cytomegalovirus viremia (any and high-level) was more frequent among recipients with iciHHV-6 (adjusted HRs, 1.7-3.1; P 5 .001-.040). Inherited ciHHV-6 status did not significantly affect risk for chronic GVHD, hematopoietic cell engraftment, overall mortality, or nonrelapse mortality. Screening for iciHHV-6 could guide donor selection and post-HCT risk stratification and treatment. Further study is needed to replicate these findings and identify potential mechanisms. (Blood. 2017;130(8):1062-1069

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“…To aid in the recognition of inherited ciHHV-6 in the transplant setting, we recently developed a clinical droplet digital PCR (ddPCR) assay to identify inherited ciHHV-6 in cellular patient specimens (11). A subsequent study demonstrated the utility of ddPCR in distinguishing HHV-6A and -B (12).…”

mentioning

confidence: 99%

Detection of Human Herpesvirus 6B (HHV-6B) Reactivation in Hematopoietic Cell Transplant Recipients with Inherited Chromosomally Integrated HHV-6A by Droplet Digital PCR

et al. 2016

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eThe presence of inherited chromosomally integrated human herpesvirus 6 (ciHHV-6) in hematopoietic cell transplant (HCT) donors or recipients confounds molecular testing for HHV-6 reactivation, which occurs in 30 to 50% of transplants. Here we describe a multiplex droplet digital PCR clinical diagnostic assay that concurrently distinguishes between HHV-6 species (A or B) and identifies inherited ciHHV-6. By applying this assay to recipient post-HCT plasma and serum samples, we demonstrated reactivation of HHV-6B in 25% (4/16 recipients) of HCT recipients with donor-or recipient-derived inherited ciHHV-6A, underscoring the need for diagnostic testing for HHV-6 infection even in the presence of ciHHV-6. Human herpesvirus 6A and -B (HHV-6A and -B) are known to be able to integrate within human chromosomal subtelomeric regions through homologous recombination at HHV-6 direct repeat regions (1). If this integration occurs within germ line cells, the offspring produced from those cells carry a copy of HHV-6A or -B in every nucleated cell; this condition is called inherited chromosomally integrated HHV-6A or -B (ciHHV-6A or -B) (2). The presence of ciHHV-6A or -B in every cell of the body has unknown effects (3), but two recent studies suggested a link between inherited ciHHV-6A or -B and angina pectoris (4, 5). Also, several reports have demonstrated HHV-6 reactivation and associated disease from inherited ciHHV-6 (6, 7).HHV-6B reactivation (but not HHV-6A reactivation) occurs in 30 to 50% of hematopoietic cell transplant (HCT) recipients, typically within the first 2 to 6 weeks posttransplantation. This has been associated with complications, such as central nervous system dysfunction, fever and rash, myelosuppression, graft rejection, and acute graft-versus-host disease (GVHD), some of which have also been reported after solid organ transplantation (8). While further research is needed to determine the clinical significance of inherited ciHHV-6A or -B after transplantation, it is well recognized that inherited ciHHV-6A or -B complicates molecular diagnostic testing for HHV-6B reactivation (9, 10). Specifically, tissue and blood samples from patients with inherited ciHHV-6A or -B will have high levels of HHV-6A or -B detected by routine quantitative PCR (qPCR), obscuring detection of HHV-6B reactivation.To aid in the recognition of inherited ciHHV-6 in the transplant setting, we recently developed a clinical droplet digital PCR (ddPCR) assay to identify inherited ciHHV-6 in cellular patient specimens (11). A subsequent study demonstrated the utility of ddPCR in distinguishing HHV-6A and -B (12). Here we describe an improved assay that identifies inherited ciHHV-6 and determines which species, HHV-6A or HHV-6B, is responsible for the integration by using a single reaction mixture. We also show that this assay can aid in the diagnosis of reactivated HHV-6B in HCT recipients with inherited ciHHV-6A, which further highlights the need for improved diagnostics for HHV-6 reactivation, particularly in immunosuppressed patien...

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Identification of Chromosomally Integrated Human Herpesvirus 6 by Droplet Digital PCR

Cited by 115 publications

References 30 publications

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Outcomes of hematopoietic cell transplantation using donors or recipients with inherited chromosomally integrated HHV-6

Detection of Human Herpesvirus 6B (HHV-6B) Reactivation in Hematopoietic Cell Transplant Recipients with Inherited Chromosomally Integrated HHV-6A by Droplet Digital PCR

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