bioRxiv preprint doi: https://doi.org/10.1101/748434; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Sensitive detection of highly fragmented cytomegalovirus nucleic acid in human cfDNA 2 3 Vikas Peddu1,%, Benjamin T. Bradley1,%, R. Swati Shree2, Brice G. Colbert1, Hong Xie1, Tracy K. 4 Santo1, Meei-Li Huang1, Edith Y. Cheng2, Eric Konnick1, Stephen J. Salipante1, Keith R. 5 Jerome1,3, Christina M. Lockwood1, Alexander L. Greninger1,3,# 6 7 1Department of Laboratory Medicine, University of Washington, Seattle, WA, USA 8 2Department of Obstetrics and Gynecology, University of Washington, Seattle, WA, USA 9 3Fred Hutchinson Cancer Research Center, Seattle, WA, USA 10 %These authors contributed equally to this work. 11 #corresponding author, Alexander L. Greninger, [email protected] 12 13 Keywords: human cytomegalovirus, CMV, cfDNA, NIPT, sequencing 14 Abstract word count: 247 15 Main text word count: 4313 16 17 bioRxiv preprint doi: https://doi.org/10.1101/748434; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 18 Abstract 19 Congenital human cytomegalovirus (CMV) infections are the leading cause of newborn 20 hearing and central nervous system impairments worldwide. Currently, routine prenatal 21 screening for congenital CMV is not performed in the United States and confirmation in 22 suspected perinatal cases requires invasive sampling by amniocentesis. We hypothesized that 23 detection of CMV from maternal cell-free DNA (cfDNA) plasma could provide a non-invasive 24 indicator of congenital CMV infection. We analyzed sequence data from 2,208 individuals 25 undergoing routine non-invasive prenatal aneuploidy screening at the University of Washington. 26 CMV reads were identified in 117 (5.3%) samples. Positive samples were stratified based on 27 CMV reads per million sample reads (RPM), resulting in ten samples being classified as strong 28 positive (RPM > 0.3) and 107 as intermediate positive (0.01<RPM<0.3). Subsequent qPCR 29 testing identified CMV in 9/10 strong positive samples and 2/32 intermediate positive samples. 30 Median cfDNA insert size derived from CMV was significantly shorter than cfDNA derived from 31 human chromosomes (103 vs 172 bp, p<0.0001), corresponding to the 3rd percentile of human 32 cfDNA insert size. In addition, CMV cfDNA fragment lengths were distributed over a wider range 33 than human cfDNA reads. These studies reveal the highly fragmented nature of CMV cfDNA 34 and offer precise measurements of its length: these features likely explain discrepancies in 35 serum CMV viral loads measurements determined by different qPCR assays, despite 36 widespread efforts to standardize results. More work is required to determine how detection of 37 CMV from maternal cfDNA can be best used as tool for congenital CMV screening or diagnosis. bioRxiv preprint doi: https://doi.org/10.1101/748434; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 38 Introduction 39 Human cytomegalovirus (CMV) is the most common cause of congenital defects in the 40 United States and affects roughly 0.5-1.3% of live births worldwide (1, 2), causing more 41 congenital disease than all disorders tested for in newborn screening combined (4). Congenital 42 CMV is associated with a variety of late-onset permanent disabilities such as hearing loss, 43 microcephaly, vision defects, and intellectual deficits (3). Primary maternal CMV infection is 44 associated with a 40% risk of fetal infection, whereas recurrent infection has an approximately 45 1% transmission rate (3). The high prevalence of recurrent infection means that recurrent 46 disease is the cause of nearly three-quarters of congenital CMV cases (3). Despite the 47 incredible burden of congenital disease, existing methods to screen for fetal CMV infection are 48 limiting in multiple important respects, including a lack of sensitivity and delayed identification of 49 affected patients. 50 Due to the significant risks posed by primary congenital CMV, prenatal CMV screening 51 via maternal serology has been proposed as an attractive approach to limit the incidence of 52 disease in this population (5). Yet, a variety issues can arise during interpretation of serology 53 results, making diagnosis challenging (6–9). Additionally, the utility of serologic screening is 54 limited to mothers with primary infections, thus excluding the substantial majority of congenital 55 CMV cases that are associated with maternal reactivation or reinfection. Consequently, the 56 current gold standard for diagnosing congenital CMV infection prenatally is amniocentesis, an 57 invasive procedure best performed after 21 weeks gestation. Because of the risks associated 58 with this procedure, amniocentesis is considered following radiologic and/or serologic evidence 59 for congenital disease (10–12). 60 If not diagnosed antenatally, additional cases of congenital CMV are identified by testing 61 for viral DNA in newborn saliva or urine within the first three weeks following a failed hearing 62 screen or other concerning clinical characteristics (13). Although this is an important strategy to 63 enable early intervention, the underlying developmental abnormalities are already established at bioRxiv preprint doi: https://doi.org/10.1101/748434; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 64 the time of testing and only demonstrate a modest response to antiviral treatment (14). Further 65 limiting the impact of this approach is the observation that over half of congenital CMV cases 66 manifest symptoms months to years after birth (15). In cases where delayed symptom onset is 67 suspected, the diagnosis of congenital CMV infection is limited to retrospective testing of stored 68 heel stick blood spots since PCR testing for CMV viremia after three weeks of life cannot 69 exclude disease secondary to postnatal infection (16). In summary, the current landscape of 70 congenital CMV screening is hindered by insensitive tools and delayed identification of cases. 71 Non-invasive prenatal testing (NIPT) via maternal cell-free DNA (cfDNA) has already 72 revolutionized the ability to screen for fetal aneuploidies, subchromosomal copy number 73 alterations, and other genetic diseases (17, 18). CMV have previously been detected in cfDNA 74 sequencing data, including NIPT (19). At our institution, we have performed clinical screening 75 for fetal aneuploidies by cfDNA since May 2017. As currently available non-invasive screening 76 methods for congenital CMV infection are fraught with error and uncertainty, leveraging prenatal 77 cfDNA sequencing would provide a new non-invasive approach for examining CMV disease 78 burden and predicting clinical outcomes. 79 80 Methods 81 Study population 82 We included all maternal plasma samples collected between May 2017 to November 2018 for 83 clinically-indicated aneuploidy screening performed at the University of Washington Department 84 of Laboratory Medicine. The 2,208 cfDNA samples in our cohort were derived from pregnant 85 women in the University of Washington (UW) Medicine network. Of these, 727 tests were 86 performed during validation and 1,481 tests were performed during the clinical implementation 87 phase. Metadata was available for the 1,325 patients (1,481 tests) screened during the clinical 88 implementation phase (Supplementary Table 1). A minimum gestational age of 10 weeks was 89 required for testing. Following University of Washington Institutional Review Board review and bioRxiv preprint doi: https://doi.org/10.1101/748434; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 90 approval, maternal and neonatal clinical histories were gathered for those samples with a CMV 91 cfDNA reads per million sample reads (RPM) > 0.3 (10 samples, 9 patients) or those with a 92 CMV cfDNA RPM < 0.3 and a positive qPCR result (2 samples, 2 patients). We collected 93 maternal age, gravidity, parity, preexisting comorbidities, and results from ultrasound studies. 94 Neonatal information included gestational age at birth, birth weight, APGAR scores, and mode 95 of delivery. Additional information gathered from the antenatal period included admission to the 96 neonatal intensive care unit (NICU), length of stay, and any infectious disease testing 97 performed. Given the available cfDNA data, we also included maternal and placental cfDNA 98 fractions and the number of total reads. 99 100 Non-invasive cell-free DNA sequencing 101 cfDNA reads from maternal plasma were generated through a validated, laboratory-developed 102 method used to screen for fetal aneuploidies and copy number alterations. For sample 103 preparation, whole blood from Streck (BCT1) tubes was centrifuged and plasma was isolated as 104 per the package insert. cfDNA was extracted from plasma using the QIAsymphony Circulating 105 DNA Kit. Following measurement of the DNA concentration in the eluate, next-generation 106 sequencing library preparation was performed on the BioMek 4000 using the KAPA HyperPrep 107 kit for adapter and index ligation. The library was purified using the Agencourt AMPureXP kit 108 prior to amplification.