LETTER
Inherited chromosomally integrated human herpesvirus 6 (iciHHV-6), present in 1 to 2% of the population, is a condition in which the HHV-6 genome is integrated near one telomere on a single chromosome in every nucleated cell (1, 2). Telomeric shortening at sites adjacent to HHV-6 integration suggests that iciHHV-6 may disrupt local telomere stability, a mechanism by which the integrated virus could promote oncogenesis (3). This mechanism of oncogenicity occurs in fowl infected with Marek’s disease virus, an alphaherpesvirus that leads to fatal T-cell lymphomas following integration into a host’s chromosome (4). The specific chromosome harboring the integrated genome varies among individuals with iciHHV-6, and studies of associations between iciHHV-6 and hematologic conditions have not considered the location of HHV-6 integration (5–7). We aimed to identify chromosome-specific HHV-6 integration sites and compare their distributions among (i) hematopoietic cell transplant (HCT) recipients with hematologic malignancies versus healthy HCT donors and (ii) HCT recipients with myeloid versus lymphoid neoplasms.
We screened cryopreserved peripheral blood mononuclear cells (PBMCs) from 4,319 HCT donor-recipient pairs (8,638 individuals) for iciHHV-6 using pooled droplet digital PCR as previously reported (8) and identified 41 HCT donors and 61 HCT recipients (102 individuals) with iciHHV-6. The specific chromosome arm carrying iciHHV-6 was defined in 30 HCT donors and 47 recipients (77 individuals; 9 related donor/recipient pairs) with available samples using sequencing and maximum likelihood phylogenetic reconstruction (n = 59), fluorescent in situ hybridization (FISH) (n = 3), optical mapping (n = 1), sequencing and FISH (n = 7), or sequencing, FISH, and optical mapping (n = 3), as previously described (9, 10). Integration sites were inferred from a related donor or recipient site for four individuals.
We identified 10 distinct subtelomeric regions of HHV-6 integration. The most common sites were 9q and 17p. The distributions of integration loci were not statistically significantly different between HCT donors and recipients (Table 1), nor were they statistically significantly different between recipients with myeloid (n = 33) and those with lymphoid (n = 14) neoplasms (Table 2). The distributions of integration loci among donors and recipients did not change when related pairs were excluded. Notably, integration at 9q was twice as frequent in HCT recipients (17/47; 36%) compared to healthy donors (5/30; 17%) and among individuals with myeloid (14/33; 42%) versus lymphoid (3/14; 21%) malignancies.
TABLE 1.
Characteristics and integration loci among hematopoietic cell transplant recipients and healthy donors
| Parameter | Value for group |
|||||
|---|---|---|---|---|---|---|
| All recipients and donors (n = 77) |
Recipients and donors, excluding related pairs (n = 59) |
|||||
| Recipients (n = 47) | Donors (n = 30) | P valuea | Recipients (n = 38) | Donors (n = 21) | P valuea | |
| Median age (yrs) (IQR)b | 47 (34–59) | 48 (30–58) | 0.86 | 45 (32–59) | 39 (28–51) | 0.38 |
| No. (%) of subjects assigned female at birth | 17 (37.0) | 15 (50.0) | 0.23 | |||
| No. (%) of subjects of race | ||||||
| White | 46 (98.0) | 17 (56.7) | 37 (97.4) | 9 (42.9) | ||
| Unknown | 1 (2.0) | 13 (43.4) | 1 (2.6) | 13 (57.1) | ||
| No. (%) of subjects with disease typec | ||||||
| Myeloid neoplasm | 33 (70.2) | 26 (68.4) | ||||
| Lymphoid neoplasm | 14 (29.8) | 12 (31.6) | ||||
| No. (%) of subjects with integration sited | 0.45 | 0.42 | ||||
| 9p | 2 (4.3) | 0 (0.0) | 2 (5.3) | 0 (0.0) | ||
| 9q | 17 (36.2) | 5 (16.7) | 16 (42.1) | 4 (19.1) | ||
| 11p | 2 (4.3) | 2 (6.7) | 1 (2.6) | 1 (4.8) | ||
| 15p | 1 (2.1) | 0 (0.0) | 1 (2.6) | 0 (0.0) | ||
| 17p | 14 (29.8) | 12 (40.0) | 10 (26.3) | 8 (38.1) | ||
| 17q | 0 (0.0) | 1 (3.3) | 0 (0.0) | 1 (4.8) | ||
| 18q | 5 (10.6) | 4 (13.3) | 3 (7.9) | 2 (9.5) | ||
| 19p | 2 (4.3) | 1 (3.3) | 2 (5.3) | 1 (4.8) | ||
| 19q | 4 (8.5) | 4 (13.3) | 3 (7.9) | 3 (14.3) | ||
| Xp | 0 (0.0) | 1 (3.3) | 0 (0.0) | 1 (4.8) | ||
| No. (%) of subjects with HHV-6 speciese | 0.52 | 0.54 | ||||
| A | 11 (23.4) | 9 (30.0) | 8 (21.1) | 6 (28.6) | ||
| B | 36 (76.0) | 21 (70.0) | 30 (78.9) | 15 (71.4) | ||
Wilcoxon’s rank sum test was used to evaluate continuous variables, and chi-squared or Fisher’s exact tests were used to evaluate categorical variables.
The age at the time of hematopoietic cell donation was available for 23 donors, 9 of whom donated to a related recipient with iciHHV-6. IQR, interquartile range.
Myeloid neoplasms/conditions included aplastic anemia (n = 1), acute myelogenous leukemia (n = 18), myelodysplastic syndrome/refractory anemia with excess blasts/chronic myelomonocytic leukemia (n = 5), and chronic myelogenous leukemia (n = 9). Lymphoid neoplasms included acute lymphoblastic leukemia (n = 8), chronic lymphocytic leukemia (n = 1), non-Hodgkin’s lymphoma (n = 3), and multiple myeloma (n = 2). Neoplasms among recipients with related donors included acute myelogenous leukemia (n = 2), chronic myelogenous leukemia (n = 2), myelodysplastic syndrome (n = 3), and non-Hodgkin’s lymphoma (n = 2).
iciHHV-6 was located at the same site among the nine related donor/recipient pairs: 11p (1 pair), 17p (4 pairs), 18q (2 pairs), 19q (1 pair), and 9q (1 pair).
Three recipients with species A had related donors with species A; 6 recipients with species B had related donors with species B. Among the 20 individuals with HHV-6 species A, the sites of integration were 17p (n = 7; 35.0%), 17q (n = 1; 5.0%), 18q (n = 9; 45.0%), and 19p (n = 3; 15.0%). Among the 57 individuals with HHV-6 species B, the sites of integration were 9p (n = 2; 3.5%), 9q (n = 22; 38.6%), 11p (n = 4; 7.0%), 15p (n = 1; 1.8%), 17p (n = 19; 33.3%), 19q (n = 8; 14.0%), and Xp (n = 1; 1.8%).
TABLE 2.
HHV-6 species and integration sites among hematopoietic cell transplant recipients with myeloid or lymphoid neoplasms (n = 47)
| Parameter | Value for group |
||
|---|---|---|---|
| Myeloid neoplasm (n = 33) | Lymphoid neoplasm (n = 14) | P valuea | |
| No. (%) of subjects with integration site | 0.17 | ||
| 9p | 2 (6.1) | 0 (0.0) | |
| 9q | 14 (42.4) | 3 (21.4) | |
| 11p | 1 (3.0) | 1 (7.1) | |
| 15p | 0 (0.0) | 1 (7.1) | |
| 17p | 7 (21.2) | 7 (50.0) | |
| 18q | 3 (9.1) | 2 (14.3) | |
| 19p | 2 (6.1) | 0 (0.0) | |
| 19q | 4 (12.2) | 0 (0.0) | |
| No. (%) of subjects with HHV-6 speciesb | 0.71 | ||
| A | 7 (21.2) | 4 (28.6) | |
| B | 26 (78.8) | 10 (71.4) | |
P values were calculated using chi-squared or Fisher’s exact tests.
Among the 11 individuals with HHV-6 species A, the sites of integration were 17p (n = 4; 36.4%), 18q (n = 5; 45.5%), and 19p (n = 2; 18.2%). Among the 36 individuals with HHV-6 species B, the sites of integration were 9p (n = 2; 5.6%), 9q (n = 4; 7.2%), 11p (n = 2; 5.6%), 15p (n = 1; 2.8%), 17p (n = 10; 27.8%), and 19q (n = 4; 11.1%).
This is the largest investigation of its kind identifying iciHHV-6 integration sites. Among individuals with hematologic malignancies and healthy controls, we identified 9q and 17p as the most prevalent sites of HHV-6 integration. These integration sites are also the ones most commonly reported in the literature, particularly among people of European descent (7, 11–13). Although we found no clear association between iciHHV-6 integration sites and malignancies, the study was underpowered to conclude significant differences. We did observe a trend toward more frequent carriage of iciHHV-6 on 9q in HCT recipients than in healthy donors, but the study’s relatively small sample size restricted more definitive comparisons of iciHHV-6 loci and specific malignancies. Larger investigations are needed to confirm these findings and elucidate the potential mechanisms of iciHHV-6-related oncogenicity.
Data availability.
Data are available from the authors upon reasonable request. Stata version 16.1 (StataCorp, College Station, TX) was used to perform statistical analyses.
ACKNOWLEDGMENTS
We are grateful to Joyce Maalouf and Jessica Hirianto for their assistance with data organization.
This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (T32AI118690 to M.R.H.) and the HHV-6 Foundation’s Dharam Ablashi Research Fund pilot grant. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
M.R.H. received speaking honoraria from Cigna LifeSource and Thermo Fisher Scientific. D.M.Z. received consulting fees from Allovir and research support from Merck. A.L.G. reports contract testing from Abbott, Cepheid, Novavax, Pfizer, Janssen, and Hologic and research support from Gilead and Merck, outside the described work. M.B. received consulting fees from Symbio, Gilead Sciences, and Allovir and research support from Gilead Sciences. J.A.H. received consulting fees from Gilead Sciences, Amplyx, Allovir, Allogene, Takeda, CRISPR, Karius, CSL Behring, Octapharma, OptumHealth, and Symbio and research support from Takeda, Allovir, Karius, Gilead, Merck, Deverra, and Oxford Immunotec.
Contributor Information
Madeleine R. Heldman, Email: madeleine.heldman@gmail.com.
Joshua A. Hill, Email: jahill3@fredhutch.org.
Felicia Goodrum, University of Arizona.
REFERENCES
- 1.Arbuckle JH, Pantry SN, Medveczky MM, Prichett J, Loomis KS, Ablashi D, Medveczky PG. 2013. Mapping the telomere integrated genome of human herpesvirus 6A and 6B. Virology 442:3–11. doi: 10.1016/j.virol.2013.03.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Arbuckle JH, Medveczky MM, Luka J, Hadley SH, Luegmayr A, Ablashi D, Lund TC, Tolar J, De Meirleir K, Montoya JG, Komaroff AL, Ambros PF, Medveczky PG. 2010. The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro. Proc Natl Acad Sci USA 107:5563–5568. doi: 10.1073/pnas.0913586107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Huang Y, Hidalgo-Bravo A, Zhang E, Cotton VE, Mendez-Bermudez A, Wig G, Medina-Calzada Z, Neumann R, Jeffreys AJ, Winney B, Wilson JF, Clark DA, Dyer MJ, Royle NJ. 2014. Human telomeres that carry an integrated copy of human herpesvirus 6 are often short and unstable, facilitating release of the viral genome from the chromosome. Nucleic Acids Res 42:315–327. doi: 10.1093/nar/gkt840. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Greco A, Fester N, Engel AT, Kaufer BB. 2014. Role of the short telomeric repeat region in Marek’s disease virus replication, genomic integration, and lymphomagenesis. J Virol 88:14138–14147. doi: 10.1128/JVI.02437-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Hill JA, Magaret AS, Hall-Sedlak R, Mikhaylova A, Huang ML, Sandmaier BM, Hansen JA, Jerome KR, Zerr DM, Boeckh M. 2017. Outcomes of hematopoietic cell transplantation using donors or recipients with inherited chromosomally integrated HHV-6. Blood 130:1062–1069. doi: 10.1182/blood-2017-03-775759. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Hubacek P, Muzikova K, Hrdlickova A, Cinek O, Hyncicova K, Hrstkova H, Sedlacek P, Stary J. 2009. Prevalence of HHV-6 integrated chromosomally among children treated for acute lymphoblastic or myeloid leukemia in the Czech Republic. J Med Virol 81:258–263. doi: 10.1002/jmv.21371. [DOI] [PubMed] [Google Scholar]
- 7.Nacheva EP, Ward KN, Brazma D, Virgili A, Howard J, Leong HN, Clark DA. 2008. Human herpesvirus 6 integrates within telomeric regions as evidenced by five different chromosomal sites. J Med Virol 80:1952–1958. doi: 10.1002/jmv.21299. [DOI] [PubMed] [Google Scholar]
- 8.Hill JA, HallSedlak R, Magaret A, Huang M-L, Zerr DM, Jerome KR, Boeckh M. 2016. Efficient identification of inherited chromosomally integrated human herpesvirus 6 using specimen pooling. J Clin Virol 77:71–76. doi: 10.1016/j.jcv.2016.02.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Wight DJ, Aimola G, Aswad A, Lai C-YJ, Bahamon C, Hong K, Hill JA, Kaufer BB. 2020. Unbiased optical mapping of telomere-integrated endogenous human herpesvirus 6. Proc Natl Acad Sci USA 117:31410–31416. doi: 10.1073/pnas.2011872117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Aswad A, Aimola G, Wight D, Roychoudhury P, Zimmermann C, Hill J, Lassner D, Xie H, Huang M-L, Parrish NF, Schultheiss H-P, Venturini C, Lager S, Smith GCS, Charnock-Jones DS, Breuer J, Greninger AL, Kaufer BB. 2021. Evolutionary history of endogenous human herpesvirus 6 reflects human migration out of Africa. Mol Biol Evol 38:96–107. doi: 10.1093/molbev/msaa190. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Ward KN, Leong HN, Nacheva EP, Howard J, Atkinson CE, Davies NW, Griffiths PD, Clark DA. 2006. Human herpesvirus 6 chromosomal integration in immunocompetent patients results in high levels of viral DNA in blood, sera, and hair follicles. J Clin Microbiol 44:1571–1574. doi: 10.1128/JCM.44.4.1571-1574.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Clark DA, Nacheva EP, Leong HN, Brazma D, Li YT, Tsao EH, Buyck HC, Atkinson CE, Lawson HM, Potter MN, Griffiths PD. 2006. Transmission of integrated human herpesvirus 6 through stem cell transplantation: implications for laboratory diagnosis. J Infect Dis 193:912–916. doi: 10.1086/500838. [DOI] [PubMed] [Google Scholar]
- 13.Luppi M, Marasca R, Barozzi P, Ferrari S, Ceccherini-Nelli L, Batoni G, Merelli E, Torelli G. 1993. Three cases of human herpesvirus-6 latent infection: integration of viral genome in peripheral blood mononuclear cell DNA. J Med Virol 40:44–52. doi: 10.1002/jmv.1890400110. [DOI] [PubMed] [Google Scholar]
Associated Data
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Data Availability Statement
Data are available from the authors upon reasonable request. Stata version 16.1 (StataCorp, College Station, TX) was used to perform statistical analyses.