Efficient identification of inherited chromosomally integrated human herpesvirus 6 using specimen pooling

Abstract

Background: Human herpesvirus 6 (HHV-6) has a unique ability to integrate into chromosomal telomeres. Vertical transmission via germ cell integration results in offspring with inherited chromosomally integrated (ci)HHV-6 in all nucleated cells, affecting ∼1% of the population.

Objectives: Inherited ciHHV-6 may be a direct or indirect mediator of human disease, but efficient identification of affected individuals is a fundamental roadblock to larger studies exploring the clinical importance of this condition.

Study design: A group testing strategy was designed to efficiently identify individuals with inherited ciHHV-6. DNA was extracted from 2496 cellular samples from hematopoietic cell transplant (HCT) donor-recipient pairs. Pools of 12 samples were screened for HHV-6 DNA with quantitative (q)PCR. Individual samples from high positive pools were tested with qPCR, and high positive individual samples were tested for inherited ciHHV-6 using droplet digital (dd)PCR to determine HHV-6 DNA copies/cellular genome.

Results: Thirty-one pools had high positive HHV-6 DNA detection with >10(3) HHV-6 DNA copies/μg. Each pool had one sample with >10(4) copies/μg HHV-6 DNA. Inherited ciHHV-6 was confirmed by ddPCR in every high positive sample (>10(3) HHV-6 DNA copies/μg), yielding a prevalence of 1.5% in HCT recipients and 0.96% in donors. We performed 580 qPCR tests to screen 2496 samples for inherited ciHHV-6, a 77% reduction in testing.

Conclusions: Inherited ciHHV-6 can be efficiently identified by specimen pooling coupled with modern molecular techniques. This algorithm can be used to facilitate cost-effective identification of patients with inherited ciHHV-6, thereby removing a major hurdle for large-scale study of its clinical impact.

Keywords: Chromosomally integrated; Diagnostic; HHV-6; Herpes; PCR.

Fig. 1

Specimen pooling and screening schematic. (a) Plate 1 is an 8 × 12 plate with individual DNA samples. Each row was combined into 1 well in the (b) pooled plate, which was screened for HHV-6 with qPCR (high positive pools are shaded). (c) Individual samples from high positive pools were screened for HHV-6 by qPCR, and inherited ciHHV-6 was confirmed in high positive individual samples (shaded) using ddPCR.

Fig. 2

HHV-6 DNA detection in pooled and individual samples. This box and whisker plot demonstrates values of high versus low positive HHV-6 DNA detection in pooled and individual samples. A threshold of HHV-6 DNA >10³ copies/μg clearly distinguishes high positive pools and individual samples from low positive pools and individual samples. This correlates with detection of pools and samples with and without inherited ciHHV-6, respectively.

Fig. 3

Flow chart of the inherited ciHHV-6 screening algorithm. 31 individuals with inherited ciHHV-6 were identified in a cohort of 2496 patients using only 580 qPCR tests (208 pools + 372 individual samples) through the application of specimen pooling. ^aAll 31 samples were confirmed to have inherited ciHHV-6 by ddPCR; a subset of negative samples were also tested by ddPCR and confirmed to be without inherited ciHHV-6.