Interchromosomal segmental duplication drives translocation and loss of P. falciparum histidine-rich protein 3
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eLife assessment
This valuable study investigates likely molecular mechanisms underlying the increasingly common deletions of the hrp2 and hrp3 genes of the human malaria parasite Plasmodium falciparum, that render parasites undetectable by widely used rapid diagnostic tests. The generation of additional long-read data, alongside a new analysis of 19,000 public short-read sequenced genomes, makes this the most detailed investigation currently available on this topic. The authors provide solid evidence for chromosomal breakage with subsequent telomere healing as the mechanism for hrp2 deletion, with more complex patterns for hrp3 deletion, but further methodological details would bolster confidence in the conclusions and enable replication of the results.
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Abstract
Most malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and PfHRP3, but deletions of pfhrp2 and phfrp3 genes make parasites undetectable by RDTs. To better understand these deletions, we analyzed 19,289 public whole-genome-sequenced P. falciparum field samples. Pfhrp2 deletion only occurred by chromosomal breakage with subsequent telomere healing. Pfhrp3 deletions involved loss from pfhrp3 to the telomere and showed 3 patterns: no other associated rearrangement with evidence of telomere healing at breakpoint (Asia; Pattern 13 - ); associated with duplication of a chromosome 5 segment containing multidrug-resistant-1 gene (Asia; Pattern 13 - 5 ++ ); and most commonly, associated with duplication of a chromosome 11 segment (Americas/Africa; Pattern 13 - 11 ++ ). We confirmed a 13-11 hybrid chromosome with long-read sequencing, consistent with a translocation product arising from recombination between large interchromosomal ribosome-containing segmental duplications. Within most 13 - 11 ++ parasites, the duplicated chromosome 11 segments were identical to each other. Across parasites, multiple distinct haplotype groupings were consistent with emergence due to clonal expansion of progeny from intrastrain meiotic recombination. Together, these observations suggest negative selection normally removes 13 - 11 ++ pfhrp3 deletions , and specific conditions are needed for their emergence and spread including low transmission, findings that can help refine surveillance strategies.
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eLife assessment
This valuable study investigates likely molecular mechanisms underlying the increasingly common deletions of the hrp2 and hrp3 genes of the human malaria parasite Plasmodium falciparum, that render parasites undetectable by widely used rapid diagnostic tests. The generation of additional long-read data, alongside a new analysis of 19,000 public short-read sequenced genomes, makes this the most detailed investigation currently available on this topic. The authors provide solid evidence for chromosomal breakage with subsequent telomere healing as the mechanism for hrp2 deletion, with more complex patterns for hrp3 deletion, but further methodological details would bolster confidence in the conclusions and enable replication of the results.
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Reviewer #1 (Public Review):
Summary:
Deletion of the hrp2 and hrp3 loci in P. falciparum poses an immediate public health threat. This manuscript provides a more complete understanding of the dynamic nature with which these deletions are generated. By delving into the likely mechanisms behind their generation, the authors also provide interesting insight into general Plasmodium biology that can inform our broader understanding of the parasite's genomic evolution.Strengths:
The sub-telomeric regions of P. falciparum (where hrp2 and hrp3 are located) are notoriously difficult to study with short-read sequence data. The authors take an appropriate, targeted approach toward studying the loci of interest, which includes read-depth analysis and local haplotype reconstruction. They additionally use both long-read and short-read data to …Reviewer #1 (Public Review):
Summary:
Deletion of the hrp2 and hrp3 loci in P. falciparum poses an immediate public health threat. This manuscript provides a more complete understanding of the dynamic nature with which these deletions are generated. By delving into the likely mechanisms behind their generation, the authors also provide interesting insight into general Plasmodium biology that can inform our broader understanding of the parasite's genomic evolution.Strengths:
The sub-telomeric regions of P. falciparum (where hrp2 and hrp3 are located) are notoriously difficult to study with short-read sequence data. The authors take an appropriate, targeted approach toward studying the loci of interest, which includes read-depth analysis and local haplotype reconstruction. They additionally use both long-read and short-read data to validate their major findings. There is an extensive set of supplementary plots, which helps clarify several aspects of the data.Weaknesses:
In this first version, there are a few factors that hinder a full assessment of the robustness and replicability of the results. First, a number of the analyses lack basic details in the methods; for instance, one must visit the authors' personal website to find some of the tools used. Second, there are several tricky methodological points that are not fully documented. Read depths are treated (and plotted) discretely as 0/1/2 without any discussion of how thresholds were used and determined. For read mapping to standard vs hybrid chromosomes, there is no documentation on how assignments were made if partially ambiguous or how final sample calls were determined when some reads were discordant. There is no mention of how missing data were handled. Without this, it is difficult to know when conclusions were based on analyses that were more quantitative (for instance, using pre-determined read thresholds) or more subjective (with patterns being extracted visually). Third, while a new method is employed for local haplotype reconstruction (PathWeaver), the manuscript does not include details on this approach or benchmarking data with which to evaluate its performance and understand any potential artifacts. -
Reviewer #2 (Public Review):
This work investigates the mechanisms, patterns, and geographical distribution of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum. Rapid diagnostic tests (RDTs) detect P. falciparum histidine-rich protein 2 (PfHRP2) and its paralog PfHRP3 located in subtelomeric regions. However, laboratory and field isolates with deletions of pfhrp2 and pfhrp3 that can escape diagnosis by RDTs are spreading in some regions of Africa. They find that pfhrp2 deletions are less common and likely occur through chromosomal breakage with subsequent telomeric healing. Pfhrp3 deletions are more common and show three distinct patterns: loss of chromosome 13 from pfhrp3 to the telomere with evidence of telomere healing at breakpoint (Asia; Pattern 13-); duplication of a chromosome 5 segment containing pfhrp1 on chromosome 13 …
Reviewer #2 (Public Review):
This work investigates the mechanisms, patterns, and geographical distribution of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum. Rapid diagnostic tests (RDTs) detect P. falciparum histidine-rich protein 2 (PfHRP2) and its paralog PfHRP3 located in subtelomeric regions. However, laboratory and field isolates with deletions of pfhrp2 and pfhrp3 that can escape diagnosis by RDTs are spreading in some regions of Africa. They find that pfhrp2 deletions are less common and likely occur through chromosomal breakage with subsequent telomeric healing. Pfhrp3 deletions are more common and show three distinct patterns: loss of chromosome 13 from pfhrp3 to the telomere with evidence of telomere healing at breakpoint (Asia; Pattern 13-); duplication of a chromosome 5 segment containing pfhrp1 on chromosome 13 through non-allelic homologous recombination (NAHR) (Asia; Pattern 13-5++); and the most common pattern, duplication of a chromosome 11 segment on chromosome 13 through NAHR (Americas/Africa; Pattern 13-11++). The loss of these genes impacts the sensitivity of RDTs, and knowing these patterns and geographic distribution makes it possible to make better decisions for malaria control.
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Reviewer #3 (Public Review):
Summary:
The study provides a detailed analysis of the chromosomal rearrangements related to the deletions of histidine-rich protein 2 (pfhrp2) and pfhrp3 genes in P. falciparum that have clinical significance since malaria rapid diagnostic tests detect these parasite proteins. A large number of publicly available short sequence reads for the whole genome of the parasite were analyzed, and data on coverage and discordant mapping allowed the authors to identify deletions, duplications, and chromosomal rearrangements related to pfhrp3 deletions. Long-read sequences showed support for the presence of a normal chromosome 11 and a hybrid 13-11 chromosome lacking pfhrp3 in some of the pfhrp3-deleted parasites. The findings support that these translocations have repeatedly occurred in natural populations. The …Reviewer #3 (Public Review):
Summary:
The study provides a detailed analysis of the chromosomal rearrangements related to the deletions of histidine-rich protein 2 (pfhrp2) and pfhrp3 genes in P. falciparum that have clinical significance since malaria rapid diagnostic tests detect these parasite proteins. A large number of publicly available short sequence reads for the whole genome of the parasite were analyzed, and data on coverage and discordant mapping allowed the authors to identify deletions, duplications, and chromosomal rearrangements related to pfhrp3 deletions. Long-read sequences showed support for the presence of a normal chromosome 11 and a hybrid 13-11 chromosome lacking pfhrp3 in some of the pfhrp3-deleted parasites. The findings support that these translocations have repeatedly occurred in natural populations. The authors discuss the implications of these findings and how they do or do not support previous hypotheses on the emergence of these deletions and the possible selective pressures involved.Strengths:
The genomic regions where these genes are located are challenging to study since they are highly repetitive and paralogous and the use of long-read sequencing allowed to span the duplicated regions, giving support to the identification of the hybrid 13-11 chromosome.All publicly available whole-genome sequences of the malaria parasite from around the world were analysed which allowed an overview of the worldwide variability, even though this analysis is biased by the availability of sequences, as the authors recognize.
Despite the reduced sample size, the detailed analysis of haplotypes and identification of the location of breakpoints gives support to a single origin event for the 13-5++ parasites.
The analysis of haplotype variation across the duplicated chromosome-11 segment identified breakpoints at varied locations that support multiple translocation events in natural populations. The authors suggest these translocations may be occurring at high frequency in meiosis in natural populations but are strongly selected against in most circumstances, which remains to be tested.
Weaknesses:
Relying on sequence data publicly available, that were collected based on diagnostic test positivity and that are limited by sequencing availability, limits the interpretation of the occurrence and relative frequency of the deletions. In the discussion, caution is needed when identifying the least common and most common mechanisms and their geographical associations. The identification of only one type of deletion pattern for Pfhrp2 may be related to these biases.The specific objectives of the study are not stated clearly, and it is sometimes difficult to know which findings are new to this study. Is it the first study analyzing all the worldwide available sequences? Is it the first one to do long-read sequencing to span the entire duplicated region?
Another aspect that should be explained in the introduction is that there was previous information about the association of the deletions to patterns found in chromosomes 5 and 11. In the short-read sequences results, it is not clear if these chromosomes were analysed because of the associations found in this study (and no associations were found to putative duplications or deletions in other chromosomes), or if they were specifically included in the analysis because of the previous information (and the other chromosomes were not analysed).
An interesting statement in the discussion is that existing pfhrp3 deletions in a low-transmission environment may provide a genetic background on which less frequent pfhrp2 deletion events can occur. Does it mean that the occurrence of pfhrp3 deletions would favor the pfhrp2 deletion events? How, and is there any evidence for that?
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