Epstein-Barr Virus in Burkitt Lymphoma in Africa Reveals a Limited Set of Whole Genome and LMP-1 Sequence Patterns: Analysis of Archival Datasets and Field Samples From Uganda, Tanzania, and Kenya

Abstract

Epstein-Barr virus (EBV) is associated with endemic Burkitt lymphoma (eBL), but the contribution of EBV variants is ill-defined. Studies of EBV whole genome sequences (WGS) have identified phylogroups that appear to be distinct for Asian versus non-Asian EBV, but samples from BL or Africa, where EBV was first discovered, are under-represented. We conducted a phylogenetic analysis of EBV WGS and LMP-1 sequences obtained primarily from BL patients in Africa and representative non-African EBV from other conditions or regions using data from GenBank, Sequence Read Archive, or Genomic Data Commons for the Burkitt Lymphoma Genome Sequencing Project (BLGSP) to generate data to support the use of a simpler biomarker of geographic or phenotypic associations. We also investigated LMP-1 patterns in 414 eBL cases and 414 geographically matched controls in the Epidemiology of Burkitt Lymphoma in East African children and minors (EMBLEM) study using LMP-1 PCR and Sanger sequencing. Phylogenetic analysis revealed distinct genetic patterns of African versus Asian EBV sequences. We identified 281 single nucleotide variations (SNVs) in LMP-1 promoter and coding region, which formed 12 unique patterns (A to L). Nine patterns (A, AB, C, D, F, I, J, K and L) predominated in African EBV, of which four were found in 92% of BL samples (A, AB, D, and H). Predominant patterns were B and G in Asia and H in Europe. EBV positivity in peripheral blood was detected in 95.6% of EMBLEM eBL cases versus 79.2% of the healthy controls (odds ratio [OR] =3.83; 95% confidence interval 2.06-7.14). LMP-1 was successfully sequenced in 66.7% of the EBV DNA positive cases but in 29.6% of the controls (ORs ranging 5-11 for different patterns). Four LMP-1 patterns (A, AB, D, and K) were detected in 63.1% of the cases versus 27.1% controls (ORs ranges: 5.58-11.4). Dual strain EBV infections were identified in WGS and PCR-Sanger data. In conclusion, EBV from Africa is phylogenetically separate from EBV in Asia. Genetic diversity in LMP-1 formed 12 patterns, which showed promising geographic and phenotypic associations. Presence of multiple strain infection should be considered in efforts to refine or improve EBV markers of ancestry or phenotype.Lay SummaryEpstein-Barr virus (EBV) infection, a ubiquitous infection, contributes to the etiology of both Burkitt Lymphoma (BL) and nasopharyngeal carcinoma, yet their global distributions vary geographically with no overlap. Genomic variation in EBV is suspected to play a role in the geographical patterns of these EBV-associated cancers, but relatively few EBV samples from BL have been comprehensively studied. We sought to compare phylogenetic patterns of EBV genomes obtained from BL samples in Africa and from tumor and non-tumor samples from elsewhere. We concluded that EBV obtained from BL in Africa is genetically separate from EBV in Asia. Through comprehensive analysis of nucleotide variations in EBV’s LMP-1 gene, we describe 12 LMP-1 patterns, two of which (B and G) were found mostly in Asia. Four LMP-1 patterns (A, AB, D, and F) accounted for 92% of EBVs sequenced from BL in Africa. Our results identified extensive diversity of EBV, but BL in Africa was associated with a limited number of variants identified, which were different from those identified in Asia. Further research is needed to optimize the use of PCR and sequencing to study LMP-1 diversity for classification of EBV variants and for use in epidemiologic studies to characterize geographic and/or phenotypic associations of EBV variants with EBV-associated malignancies, including eBL.

Burkitt Lymphoma Incidence in Five Continents

“…While underdiagnosis and/or underascertainment of BL are possible explanations for the discrepant low BL rates in Asia, that explanation is inconsistent with the high rates for nasopharyngeal cancer [93] and extra-nodal NK/T-cell lymphomas [94], both associated with EBV, in Asia. The low BL rates could be an epidemiological manifestation indicating that EBV strains circulating in Asia have a lower population attributable risk for BL than EBV in Africa [63]. EBV phylogenetic data have confirmed that EBV in Asia is genetically distinct from EBV in Africa [63,[95][96][97].…”

“…EBV phylogenetic data have confirmed that EBV in Asia is genetically distinct from EBV in Africa [63,[95][96][97]. Moreover, the EBV in US/Europe is phylogenetically closer to that in Africa [63,96], which is intriguing given that BL rates in US/Europe intermediate to those in Africa versus Asia. Molecular analysis of EBV variants in regions with different BL rates could lead to discovery of EBV variants with differential risk for BL.…”

“…The low BL rates could be an epidemiological manifestation indicating that EBV strains circulating in Asia have a lower population attributable risk for BL than EBV in Africa [63]. EBV phylogenetic data have confirmed that EBV in Asia is genetically distinct from EBV in Africa [63,[95][96][97]. Moreover, the EBV in US/Europe is phylogenetically closer to that in Africa [63,96], which is intriguing given that BL rates in US/Europe intermediate to those in Africa versus Asia.…”

“…This pattern suggests that there might be multiple risk factors that influence the within-and between-regional variation of BL rates. Because BL has a multi-factorial etiology, including EBV [63,64], P. falciparum [65], HIV [25], and immune dysregulation, including from genetic or nutritional factors [66], the mosaic pattern observed here provides an epidemiological framework, as did the endemic/sporadic classification, for discovery of etiological factors that vary regionally, leading to pockets of high or deficit BL rates. Second, we confirm that BL predominates in males, including in countries that reported only pediatric cases.…”

“…Although not usually considered, other infections that are spread by mosquitoes could contribute [65]. Finally, the role of EBV variants [63,64], nutritional deficiencies, such as dietary selenium [79] or magnesium [66], are exposures that could modify geographical patterns of BL.…”