In this study we show that diversity in Block 2 is underestimated by only genotyping three allele families as there is a fourth recombinant allele family that is distributed worldwide. We also demonstrate that the distribution of fragment size polymorphism in MSP-1 Block 2 cannot be explained by neutrality. Finally, we observe that within allele families, amino acid motifs are not conserved within alleles of the same fragment size. These results suggest a higher level of complexity that may hamper our ability to elucidate allele family specific immune responses elicited by this vaccine target and its overall use as genetic marker in other types of epidemiologic investigations.
In order to test whether the fragment size polymorphism in Block 2 is under selection, we genotyped DNA from 362 samples collected from parasitemic Kenyan children. The number of observed alleles is higher than those reported for other populations such as Tanzania (Babiker et al., 1994
) with 10 K1 and five Mad20 alleles detected, Sudan (Babiker et al., 1997
) with four K1 and four Mad20 alleles detected, Papua New Guinea (Paul et al., 1995
) with four K1 and four Mad20 alleles detected, and Thailand (Paul et al., 1998
) with three K1 and four Mad 20 alleles detected. Although this overall pattern is consistent with other malarial antigens (Escalante et al., 2001
), it is important to note that several of these studies used PCR followed by hybridization with allele-specific probes to genotype Block 2 (Babiker et al., 1994
; Paul et al., 1995
). This technique yields products that range in size from 400 to 600 bp in length. As a result, it is possible that some unique alleles were not recognized because it is difficult to differentiate tripeptide size differences in products this large. We also observed more alleles than studies that used a similar nested PCR technique detecting smaller product sizes. Jelinek et al. (1999)
detected six K1 alleles and one Mad20 allele in Uganda; Snounou et al. (2000)
detected four K1 alleles and five Mad20 alleles in Thailand; and Robert et al. (1996)
detected 10 K1 alleles and 5 Mad20 alleles in Senegal. These data support the idea that it is difficult to make comparisons between populations whose genotyping was performed in different laboratories, using different protocols with varying sensitivities. This fact was noted by Farnert et al. (2001)
, who conducted a study of the comparability of genotyping results from different laboratories and emphasized the need for standardization of genotyping methods. The inability to compare results across different populations and laboratories poses and additional problem with using Block 2 as a reliable molecular marker.
The role of genetic recombination in generating the observed polymorphisms in P. falciparum
has been a focus of controversy for more than 10 years (Tibayrenc et al., 1991
; Babiker et al., 1997
; Rich et al., 1997
; Conway et al., 1999
; Escalante et al., 2001
). Studies examining this issue have focused mostly on antigens that are targets of potential antimalarial vaccines. Unfortunately, because these genes are under positive natural selection, it is difficult to differentiate the effect of selection from recombination using point mutations (McCutchan et al., 1992
; Escalante et al., 1998
). Nevertheless, intragenic recombination has been implicated as a major factor to explain the observed polymorphisms in MSP-1 (Conway and McBride, 1991
; Jongwutiwes et al., 1991
; Hughes, 1992
; Qari et al., 1998
; Conway et al., 1999
; Sakihama et al., 1999
). Polymorphisms in tandem repeat regions, such as Block 2, have often been considered evidence of recombination (McCutchan et al., 1988
); however, alternative hypotheses such as mitotic intragenic recombination have been raised for MSP-2 (Irion et al., 1997
) and CSP (Rich et al., 1997
). Indeed, other investigations suggest that the number of repeats appears to be independent of the number of recombination events and that the number of repeats is relatively high compared with the number of documented recombination events in the surrounding area (Sakihama et al., 2004
). Nevertheless, our finding of a recombinant in Block 2 of MSP-1 between the Mad20 and RO33 allele families supports the notion that sexual intragenic recombination is an important factor in the evolution of genetic diversity in this repetitive region. This kind of event cannot be clouded by the action of natural selection and cannot be explained by mitotic intragenic changes.
In a previous study, we have described the possible origins of the MR allele family, and postulated that this family arose from a single recombination event (Takala et al., 2002
). In this study, we have shown that the MR family is found worldwide and that the recombination site occurs at the same position in MR alleles from all the localities tested. These data support the hypothesis that the MR family derives from a single recombination event. The alternative hypothesis that this recombinant appears de novo at different locations exactly in the same place is a less parsimonious explanation. The worldwide distribution of the MR allele family as well as the observed allelic polymorphisms in this family suggest that this recombination event could be as old as the population expansion of P. falciparum
outside of Africa. However, our data do not allow us to quantitate how often these recombination events take place in a given population. Such measures demand different kinds of studies (Conway et al., 1999
; Su et al., 1999
); however, we are providing evidence that this process has generated a new allele family in Block 2 of MSP1. Further investigation is also needed to understand the processes responsible for the expansion and spread of the MR family in the population.
Our analysis has also shown that the number of tandem repeats in Block 2 appears to be under positive natural selection. The EW and EWS tests are capable of detecting departure from neutrality due to the action of balancing selection or advantageous alleles. We found evidence that the fragment size polymorphisms in the K1 and MAD20 allele families are not neutral. The tests failed to detect departure from neutrality in the case of MR, even though the distribution of fragment sizes had a similar shape to that of K1 and M20. A plausible explanation for this result is reduced statistical power due to the low frequency of MR alleles. A larger study would be required to apply the tests under conditions comparable to the other allele families.
Our results provide new insight about genetic polymorphisms that complement those of Conway (Conway et al., 2000
), who used Wright’s Fst test to demonstrate that the frequency of the Block 2 allele families is under selection. Although clear evidence was provided that natural selection acts on the frequency of the allele families themselves (MAD20-like and K1-like, respectively), no evidence has been reported on how the fragment size polymorphism is maintained within each allele family. Immune responses are usually considered the driving selective force acting on antigens; however, it is not clear how the number of repeats could modulate the quality of the immune response. If there is not a clear phenotype that can be recognized by the putative selective force then we cannot convincingly argue for positive natural selection (Escalante et al., 2004
). An alternative explanation is that the number of repeats could affect the stability of the protein itself. Such an alternative has been explored in CSP where simulations demonstrated that structure could explain the observed frequency of fragment sizes (Escalante et al., 2002
Previous studies have made the observation that fragment size polymorphisms of antigens are not good markers for certain types of molecular epidemiological studies (e.g. studies of evolutionary history) since they may have their own dynamics due to the action of selection (Anderson et al., 2000
). We have shown that at least in the Kenyan population included in this study, the fragment size polymorphisms in Block 2 are not neutral. These results support the use of other markers, such as microsatellites, to conduct such studies. The role played by natural selection, if any, in the fixation and worldwide distribution of the MR allele family demands further investigation.
We have also demonstrated that within allele families, alleles of the same size may have different amino acid motifs. The frequency of this phenomenon cannot be accurately determined from this study as the number of alleles examined is small. However, this result has significant implications for studies using Block 2 fragment length polymorphism as a marker of diversity. For example, studies examining the association between Block 2 alleles and immunity may not observe a correlation between fragment size and immune response because correlations may be masked by one fragment size containing a mixture of repeats. In addition, studies that rely on allele frequencies (e.g. studies of selection, linkage disequilibrium, or population structure) may also be affected by grouping together alleles based on fragment size that are actually different from each other.
In summary, we have shown that the new allele family, MR, is present worldwide, and that it appears to be the product of a single recombination event. This evidence suggests that sexual intragenic recombination is a mechanism that can generate new genetic variants of malarial antigens, and that it should be considered in the study of genes encoding vaccine and drug targets. We have also shown that the fragment size polymorphisms in Block 2 of MSP-1 are under positive natural selection. Finally, we demonstrate the amino acid sequences in alleles of the same size and allele family are not conserved. These results suggest that fragment size may not be an accurate marker for genetic diversity within MSP1 Block 2 and support the use of other markers such as microsatellites to conduct molecular epidemiological studies.