Interchromosomal segmental duplication drives translocation and loss ofP. falciparumhistidine-rich protein 3

Abstract

Background: Significant progress has been made in the fight against P. falciparum malaria due in part to the widespread adoption of rapid diagnostic tests (RDTs) that detect histidine-rich protein 2 (PfHRP2) and its paralog PfHRP3 encoded by the pfhrp2 and pfhrp3 genes, respectively. Parasites without pfhrp2 and pfhrp3 genes are not detected by these RDTs. Pfhrp3 loss appears to be more common in some geographical regions and has been observed in vitro. We sought to gain insight into geographic patterns of pfhrp3 deletion and define the mechanism of gene loss. Methods: Over 9,830 publicly available, whole-genome sequenced (WGS) P. falciparum field samples were analyzed for genotypes and coverage using PathWeaver for local assembly . DNA from two cultured isolates with pfhrp3 deletion, HB3 and SD01, were sequenced with Oxford Nanopore Technologies (ONT) long-read sequencing, assembled with Canu and Flye, and genes annotated with Companion. Results: Two distinct pfhrp3 deletion patterns were detected: 1) segmental deletion of chromosome 13 just centromeric to pfhrp3 extending to the end of the chromosome, with co-occurring segmental duplication of the chromosome 11 subtelomeric region, and 2) segmental deletion of chromosome 13 starting at various locations centromeric to pfhrp3 without chromosome 11 duplication. Pattern 1 was almost exclusively found in samples from Africa and South America, while pattern 2 was observed predominantly in Southeast Asia. The pattern 1 boundary fell within a 15kb nearly identical duplication on both chromosomes containing ribosomal genes. ONT assembly of HB3 and SD01 parasite lines revealed hybrid chromosomes and long-reads spanning the ribosomal duplication, consistent with recombination between non-homologous chromosomes. Conclusion: Our findings demonstrate duplication-mediated non-homologous recombination creating a hybrid 13-11 chromosome that replaces pfhrp3 and telomeric chromosome 13 with a translocated telomeric chromosome 11 sequence--essentially yielding a deletion of chromosome 13 sequence and interchromosomal duplication of chromosome 11 sequence. Given that existing ribosomal duplications likely predispose to the frequent occurrence of this translocation during meiosis, it suggests that subsequent selective forces are driving its presence or absence in different geographical regions. This mechanism appears to explain pfhrp3 deletion in South America and Africa and may explain why pfhrp3 deletions are more prevalent than pfhrp2 deletions in many localities. However, the forces driving emergence of pfhrp3- parasites may be complex and encompass other genes affected by the recombination event. Further studies of the origins of pfhrp2- and pfhrp3-deleted strains and the selective pressures suppressing their occurrence or driving their expansion are needed.

Global risk of selection and spread ofPlasmodium falciparumhistidine-rich protein 2 and 3 gene deletions

“…Fourth, the country-specific estimates of linkage between pfhrp2 and pfhrp3 deletions provided here assume that the dynamics of these two loci are at equilibrium and that no selective forces are acting to pull certain genotypes, such as pfhrp2-/pfhrp3- , to higher levels. However, we have observed a significant relationship between deletions and malaria prevalence that aligns with recent mechanistic explanations of how pfhrp3 deletions arise and may be driven by low malaria prevalence ( 22 ). If malaria prevalence falls in a region, in addition to the increased selection of pfhrp2 deletions that occurs at low prevalence, the frequency of pfhrp3 deletions may also increase furthering the selection of pfhrp2 deletions.…”

“…Secondly, we note that pfhrp3 deletions are frequently found at higher frequencies than pfhrp2 deletions despite pfhrp2 deletions providing a greater advantage than pfhrp3 deletions with regards to the ability to evade diagnosis by HRP2-based RDTs ( 21 ). This observation reflects the mechanistic ( 22 ) and soft selective processes that are hypothesised to result in the emergence of pfhrp3 deletions ( 5 ). This observation is in contrast to the strong selective sweeps associated with pfhrp2 deletions due to RDT-based test-and-treatment that cause pfhrp2 deletions to be selected on both genetic backgrounds, but more strongly on a pfhrp3-deleted background ( 5 ).…”