146 D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156dent folding, similar to some other P. falciparum ment and institutional ethical committees. DNA fromproteins with low primary sequence homology but not part of each blood sample was prepared by Proteinaseapparent in any non-Plasmodium proteins [12,13]. Anti- K digestion, followed by two extractions in phe-bodies against Pfs48/45 can inhibit infection and devel- nol:chloroform:isoamyl alcohol (25:24:1), one extrac-opment in mosquitoes at an early stage, presumably by tion in chloroform, and ethanol precipitation. DNAinhibiting parasite fertilisation, and thus it is a candi- was dissolved in sterile nanopure H2O for use as tem-date for a transmission-blocking vaccine . plate in polymerase chain reaction ampliﬁcation (PCR) The small number of known nucleotide polymor- assays. It was considered important to determine thephisms in Pfs48 /45 of P. falciparum are all nonsynony- haplotypes of Pfs48 /45 alleles, which is possible inmous, causing amino acid changes, and are located blood samples containing only one detectable haploidbetween codons 253– 322 in a central part of the gene genotype of P. falciparum, so genotypic data on other[7,15]. Analysis of P. falciparum allele sequences to- polymorphic loci, including msp1 , were used together with the sequence in the most closely related identify samples with apparently only one P. falciparumspecies P. reichenowi (a parasite of chimpanzees)  clone. Thus, 255 of such isolates were studied here: 57reveals an excess of nonsynonymous polymorphisms from Nigeria, 56 from Sudan, 52 from South Africa, 55within P. falciparum, compared to a slight excess of from Brazil, and 35 from Malaysia.synonymous substitutions between the species . Thisindicates that positive selection may be operating on 2.2. Molecular genotyping of Pfs48 /45 sequencesalleles of Pfs48 /45 in P. falciparum. A survey of twoadjacent polymorphic codons in the gene in natural Five previously identiﬁed polymorphic sites in thepopulations showed that different alleles were very gene  were studied, in codons 253, 254, 304, 314,common or at ﬁxation in different continents . This and 322. Each of these polymorphic amino acids isis in strong contrast to the more evenly distributed predicted to be on exposed loops in the secondaryalleles of asexual antigen genes [6,18], but no compari- structure of Pfs48/45 . From each isolate, a centralson has yet been made with a set of putatively neutral portion of the gene, containing codons 235–346 wasloci. Here, 255 P. falciparum isolates were sampled ampliﬁed by PCR in a 20 ml volume in wells of afrom ﬁve newly surveyed geographical populations, and 96-well plate using 100 nM primers 5%-TTTTCAA-the distribution of allele frequencies was studied at ﬁve GAAGGAAAAGAAAAAGCC-3% (fwd) and 5%-single nucleotide polymorphisms (SNPs) in codons of CACCAGGACAATTTAAACCTACC-3% (rev), 100the Pfs48 /45 gene (on Chromosome 13), and at 11 mM of each dNTP, 1 × Bioline Taq polymerase buffermicrosatellite loci considered to be evolving in a neutral including 1.5 mM MgCl2, and 1.2 units of Bioline Taqmanner and representing a random sample of such loci Polymerase. Ampliﬁcation employed an initial denatu-from the parasite genome [1,19,20]. ration step of 94°C for 4 min, followed by 45 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. One or 2 ml of dissolved DNA was routinely used for2. Materials and methods PCR from each isolate, which yielded an abundant PCR product for most isolates. For some isolates with2.1. Study populations and samples a low DNA concentration the product of this single PCR was undetectable, so DNA from these isolates was Human subjects with P. falciparum infections were ampliﬁed by a nested PCR with an additional ﬁrstidentiﬁed in studies on malaria in ﬁve geographical round reaction of 30 cycles with the above conditionslocations (three in Africa, one in Southeast Asia, one in using an outer pair of primers 5%-GCGC-South America): Ibadan in Oyo state, south-western GAATTCTTCCCATTTAGTCCAAAAGAC-3% (fwd)Nigeria in 1996; Daraweesh in Gedaref state, eastern and 5%-GCGCGAATTCGTTACATCCGTGTATGA-Sudan in 1992– 1995; KwaZulu-Natal, northern South CTTT-3% (rev), amplifying codons 145–432. Ten nucle-Africa in 1996; Malinsau in Sabah state in Borneo, East otides at the 5%-end of each primer had been added toMalaysia in 1997; Porto Velho in Rondonia state, give a GC clamp and an EcoR1 site, for earlier workwestern Amazonian Brazil in 1997. The Malaysian and . Two microlitres of ﬁrst round PCR product wasBrazilian populations (similar to ones previously de- used as a template for the second round PCR whichscribed [21,22]) had generally lower P. falciparum en- was performed exactly as described above for the singledemicity than the African populations (previously round PCR. Final PCR products were denatured andstudied for other polymorphisms ). Peripheral blood 1.5 ml aliquots were dotted on replicate nylon mem-samples were obtained by venepuncture (10 ml) or branes, left to dry and cross-linked with 1200 mJ ultra-ﬁngerprick (500 ml), collected in sodium heparin antico- violet light. High stringency hybridisation ofagulant or spotted and dried on ﬁlter paper, with allele-speciﬁc probes to the DNA arrayed on replicatepermission and under guidelines of the relevant govern- membranes was performed. The Pfs48 /45 allele-speciﬁc
D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156 147probes were 18-mer oligonucleotides: 5%-TATCAT- allele at each locus). Allelic diversity at each locus (eachAAAAACTTAACT-3% (253-4 KN), 5%-TATCATG- of the Pfs48 /45 SNPs and composite haplotypes, andAAAAGTTAACT-3% (253-4 EK), 5%-TATCATAAAA- each of the microsatellite loci) was estimated as theAGTTAACT-3% (253-4 KK), 5%-TCAAATGTTAGTT- expected heterozygosity index for each population,CTAAA-3% (304 V), 5%-TCAAATGATAGTTCTAAA- H= [n/(n − 1)][1− p 2], where n is the number of iso- i3% (304 D), 5%-ACAGATAGTTTAGATATT-3% (314 L), lates sampled and pi is the frequency of each different5%-ACAGATAGTATAGATATT-3% (314 I), 5%-TT- allele at the locus.GATGATAGTGCACATA-3% (322 S), 5%-TTGAT- The among-population variance in allele frequencies,GATAATGCACATA-3% (322N). Oligonucleotides were FST, was calculated using Weir & Cockerham’s q esti-3%-end labelled with digoxigenin, and each hybridised mator , with the FSTAT version 1.2 program .with a replicate membrane in a separate tube in 5 ml For each locus, FST indices were calculated among theTMAC buffer (3 M tetramethylammonium chloride/50 three populations in Africa, and among the three differ-mM Tris, pH 8.0/2 mM EDTA, pH 8.0/0.1% SDS) at ent continents. Testing whether each FST value was55°C. Full details of reagents and procedures for allele- signiﬁcantly greater than zero was performed by per-speciﬁc hybridisation, detection, development and vi- mutation with 1000 runs on FSTAT. Allelic variationsual scoring of results were as described previously in at six of the microsatellite loci (TA60, TA81, TA87,studies of other P. falciparum SNPs [23,24]. For each ARA2, PfPK2, and Pfg377 ) appears to be due solely tocodon SNP in each of the isolates a single allele was differences in copy numbers (n) of tri-nucleotide TAAdetermined, and composite haplotypes were thus (n) repeats , potentially conforming to a stepwiseidentiﬁed. mutation model (SMM). For these six loci, an addi- tional (SMM-based) ﬁxation index, an unbiased estima-2.3. Molecular genotyping of P. falciparum tor of Slatkin’s RST, was calculated for these loci usingmicrosatellite loci the RSTCALC 2.2 program . This latter estimate takes into account the variation in the number of Eleven microsatellite loci were studied, each of which repeats between alleles, within and among populations,is a single locus in the haploid P. falciparum genome, rather than being based solely on the frequencies ofconsisting of locus-speciﬁc sequences ﬂanking polymor- discrete alleles.phic tri-nucleotide repeats [20,25]. The names (andchromosomal locations)  of the loci are: Polya(Chr4), TA42 (Chr5), TA81 (Chr5), TA1 (Chr6), TA87(Chr6), TA109 (Chr6), ARA2 (Chr11), TA102 (Chr12), 3. ResultsPfPK2 (Chr12), Pfg377 (Chr12), TA60 (Chr13). Allelesat these loci (except locus TA102 ) were typed using asemi-nested PCR method previously described , 3.1. Pfs48 /45 allele and haplotype frequencieswith some modiﬁcations made for convenience in thechoice of ﬂuorescent dye used to label the internal The allele frequencies of each of the Pfs48 /45 poly-primer for some of the loci (FAM used for Pfg377, morphic codons, and the composite haplotypes in eachTA109, and TA42 ; HEX used for TA1, TA60, and population, are given in Table 1. Nine different ﬁve-TA81 ; NED used for TA87, PfPK2, ARAII and Polya). codon haplotypes were present in total. Much greaterLocus TA102 was ampliﬁed under the same cycling haplotype diversity is seen in the three African popula-conditions as the other loci , using ﬁrst round tions (for Nigeria, Sudan and South Africa, respec-ampliﬁcation primers TA102-3(F) 5%-GAAGCTAG- tively, H=0.59, 0.70 and 0.62) than in populationsTACGATAGGTT-3% and TA102-R 5%-CAAATAAAT- sampled in South America (Brazil, H= 0.00) or South-TTGCATCCTGGTC-3% and a FAM-labelled nested east Asia (Malaysia, H= 0.06). Major differences existsecond round primer TA102-F 5%-GCTCCAAGAT- among the populations in allele frequencies at eachGATTAAAGA-3% together with TA102-R (with kind individual codon and in frequencies of haplotypes, theadvice from T.J.C. Anderson). Alleles at all loci were most pronounced differences being among populationsidentiﬁed by sizing of PCR products electrophoresed on in different continents. Different alleles are at completean ABI 377, using GENESCAN and GENOTYPER or near ﬁxation in different populations. Codons 253-4software (Applied Biosystems, UK). show a geographical allele frequency distribution con- sistent with that reported for ﬁve other populations in2.4. Statistical analyses which these codons were typed using a different method . The combined data from all the ten populations Allele frequencies in each population sample were are shown in Fig. 1. All isolates typed from Brazil havederived by direct counting (as single-clone isolates were the EK allele, all isolates from Southeast Asia have thechosen for the study, each isolate contained only one KK allele, whereas in Africa the KN allele is most
148 D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156Fig. 1. Geographical distribution of allele frequencies at codons 253-4 of Pfs48 /45 in P. falciparum. The ten populations include the ﬁve in the present study (Rondonia in western Brazil, n=55;Nigeria, n = 57; Sudan n= 56; South Africa, n= 52; East Malaysia, n =35) and ﬁve in a previous study (Ref. : Amapa in northeastern Amazonian Brazil, n= 40; The Gambia, n =57;Cameroon, n = 29; Tanzania, n= 31; Thailand, n= 27).
D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156 149common, but all three alleles are present in all African was statistically analysed and shown to be much higherpopulations and the EK allele is common in the east of for the Pfs48 /45 alleles than for the microsatellites. Fig.the continent. 2 shows the variance (FST) among the three populations within Africa (mean FST for Pfs48 /45 polymorphic3.2. Microsatellite allele frequencies and di6ersity codons= 0.29, mean FST for the microsatellite loci= 0.04) and among the three continents (mean FST for The 11 microsatellite loci were highly polymorphic, Pfs48 /45 polymorphic codons=0.69, mean FST for mi-with total numbers of alleles per locus ranging from 7 crosatellite loci= 0.17). None of the microsatellite loci(locus Pfg377 ) to 19 (locus TA1 ). Table 2 shows the individually had an FST index as high as that of any ofnumbers of alleles at each of these loci, and the allelic the Pfs48 /45 codons.diversity (expected heterozygosity, H), in each of the Six of the microsatellite loci may conform to a SMM,ﬁve populations. Full tabulation of all allele frequencies variation being entirely due to gain or loss of tri-nucle-in each population is given in Table 3. The number of otide TAA repeat copies . These were therefore alsoalleles at the microsatellite loci is higher in the African studied using the RST index of inter-population variancepopulations (in Nigeria, Sudan, and South Africa, re- (which takes into account the variance in allele size duespectively, mean number of alleles per locus is 9.9, 7.9 to gain or loss of repeat copies by SMM). Table 4and 9.1) than in the populations from Brazil (4.4) or shows the RST estimates for these loci, alongside the FSTMalaysia (5.2). Similarly, the allelic diversity (H) is estimates for comparison. Among populations in thehigher in the African populations (for Nigeria, Sudan, three African countries, the average RST value over alland South Africa, respectively, over all loci mean H= six loci was similar to the average FST value, but among0.80, 0.71 and 0.78) than in Brazil (mean H =0.54) and populations in the different continents the average RSTMalaysia (mean H= 0.66). This trend is similar to that value (0.256) was somewhat higher than the averagenoted for the Pfs48 /45 gene, but much less pronounced. FST value (0.183). It should be noted that the valuesA large proportion of microsatellite alleles are shared vary widely among the loci for the variance among theamong populations, and the alleles which are ‘private’ three different continents, and a high RST value (0.548)to any one population are generally at quite low for locus Pfg377 nearly approaches the FST value offrequencies. inter-continental variation shown for Pfs48 /45. Overall, the analysis suggests that divergence at microsatellite3.3. Quantiﬁcation of 6ariance in allele frequencies loci could be somewhat greater than the FST valuesamong populations would indicate (particularly among populations in dif- ferent continents), but not to the extent seen at the The among-population variance in allele frequencies Pfs48 /45 locus.Table 1Pfs48 /45 allele and haplotype frequencies in ﬁve geographical populations of P. falciparumCodon Allele Nigeria (n= 57) Sudan (n =56) South Africa (n= 52) Brazil (n = 55) Malaysia (n =35)253 E 0.05 0.27 0.56 1.00 – K 0.95 0.73 0.44 – 1.00254 K 0.12 0.36 0.62 1.00 1.00 N 0.88 0.64 0.38 – –304 D 0.02 0.05 – – 1.00 V 0.98 0.95 1.00 1.00 –314 I 0.68 0.64 0.10 – – L 0.32 0.36 0.90 1.00 1.00322 S 1.00 1.00 1.00 1.00 0.03 N – – – – 0.975-codon haplotypesK-K-D-L-N – – – – 0.97K-K-D-L-S – 0.05 – – 0.03K-K-V-I-S 0.05 0.04 0.02 – –K-K-V-L-S 0.02 – 0.04 – –K-N-D-L-S 0.02 – – – –K-N-V-I-S 0.58 0.50 0.04 – –K-N-V-L-S 0.28 0.14 0.35 – –E-K-V-I-S 0.05 0.11 0.04 – –E-K-V-L-S – 0.16 0.52 1.00 –
150 D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156Table 2Numbers of alleles and diversity index (H) at 11 microsatellite loci in ﬁve geographical populations of P. falciparumLocus Numbers of different alleles within each population Allelic diversity (H) within each population Nigeria (n=57) Sudan (n= 56) South Africa Brazil (n =55) Malaysia Nigeria (n = 57) Sudan (n= 56) South Africa Brazil (n=55) Malaysia (n= 52) (n =35) (n=52) (n= 35)TA1 15 10 9 7 8 0.92 0.85 0.76 0.54 0.79TA42 9 4 7 4 4 0.48 0.20 0.64 0.30 0.58TA60 8 11 7 4 6 0.82 0.80 0.72 0.64 0.79TA81 8 5 7 3 6 0.82 0.76 0.81 0.46 0.78TA87 9 8 8 6 5 0.86 0.73 0.83 0.64 0.69TA102 9 7 9 3 7 0.85 0.85 0.83 0.63 0.79TA109 12 9 10 5 2 0.83 0.73 0.76 0.41 0.28ARA2 10 9 9 3 5 0.86 0.83 0.85 0.47 0.79Pfg377 6 5 5 2 3 0.63 0.49 0.55 0.51 0.37PFPK2 10 7 15 6 3 0.86 0.67 0.85 0.66 0.55POLYA 13 12 14 5 8 0.86 0.90 0.96 0.69 0.81Mean 9.9 7.9 9.1 4.4 5.2 0.80 0.71 0.78 0.54 0.66
D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156 153Table 3 (Continued)Locus Allele Nigeria (n= 57) Sudan (n = 56) South Africa (n =52) Brazil (n =55) Malaysia (n=35) 184 0.02 – 0.02 – – 187 – – 0.02 – – 190 0.02 0.02 0.04 – – 193 – – 0.04 – – 196 – 0.04 0.02 – – 199 0.04 – – – – 202 – 0.02 – – –POLYA 138 0.02 0.02 0.02 – – 141 0.02 – 0.12 – – 144 – 0.04 0.10 – – 147 0.02 0.06 0.06 – – 150 0.04 0.13 0.06 – – 153 0.19 0.07 0.12 0.17 – 156 0.28 0.04 0.12 – 0.03 159 0.12 – 0.10 – – 162 0.07 0.11 0.02 – 0.15 165 0.05 0.04 0.04 0.06 0.12 168 0.02 0.24 0.06 – 0.38 171 0.05 0.06 0.04 – – 174 0.02 0.17 0.08 – 0.12 177 0.11 0.04 0.06 0.07 0.03 180 – – – – 0.15 183 – – – 0.50 0.03 189 – – – 0.21 –4. Discussion ability to resolve high levels of inter-population diver- gence , and a large amount of existing allelic diver- There is extreme geographical divergence of allele sity at any locus can also restrict the estimates to somefrequencies in the Pfs48 /45 gamete surface protein extent . This could have affected the microsatellitegene. In a quantitative analysis, the inter-population FST estimates here, and in another study , particu-ﬁxation indices for Pfs48 /45 alleles and haplotypes are larly at the level of comparisons among continents. Toconsistently much higher than those for the microsatel- explore this, it would be useful if ﬁxation indices amonglite alleles, whether the analysis considers different pop- P. falciparum populations were studied with a broadulations within Africa, or different continents. The random sample of neutral SNPs in the genome, andgeographical variance in microsatellite allele frequencies compared to the indices derived from microsatellites.determined here is similar to that seen in a large study Most known P. falciparum SNPs are within genesof several other populations . Asexual blood stage which have been studied individually for their func-antigen genes have shown similar or even lower geo- tional and immunological interest, and their allele fre-graphical variance in allele frequencies [6,10,18]. It is quencies may not be determined by neutral processestherefore hypothesised that the exceptionally skewed alone. Future large scale SNP discovery throughout thefrequencies of Pfs48 /45 alleles may be due to divergent P. falciparum genome would therefore yield resourcesselection operating on the protein in different for addressing this question, as recognised for the hu-populations. man genome . An important critical question is whether ﬁxation To focus on Pfs48/45, it is reasonable to suggest aindices based on microsatellite loci may underestimate role in gamete recognition and fertilisation, as it isthe actual genetic distance between populations. This located on the surface of gametes. In other eukaryotes,could potentially result from the reversible nature of there is evidence that very strong directional selectionmutational changes in lengths of repeats (which may can operate in mating type genes  and other genesmake ancestrally distinct alleles appear the same) . determining aspects of sexual reproduction [34,35]. TheTo investigate this, an additional analysis of variation importance of understanding the parameters affectingat six of the microsatellite loci was performed here sexual reproduction in different populations of P. falci-assuming such a SMM (yielding the RST index), but this parum is recognised [23,29,36]. In the laboratory, mat-did not greatly increase the estimate of genetic diver- ing experiments have been performed between clones ofgence at these loci. However, it has been noted that a P. falciparum (crosses involving parental clones HB3×high mutation rate at microsatellite loci may lower the 3D7, and HB3×Dd2). In each cross, nuclear DNA
154 D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156alleles were inherited from each parent [37– 40], al- Table 4 Comparison of RST and FST estimates of inter-population variance atthough these clones had different Pfs48/45 types . six microsatellite loci considered to evolve according to a SMMHowever, the maternally-inherited mitochondrial DNAof the HB3 parent was severely under-represented in Locus Variance among populationsprogeny of both crosses [41,42]. It is plausible that Three African countries Three different continentsdirectional incompatibility occurs in the case of particu-lar male-female Pfs48/45 interactions, but interpreting RST FST RST FSTthe results of the HB3× Dd2 cross is confounded by a TA60 0.058 0.029 0.148 0.076 TA81 0.059 0.053 0.154 0.153 TA87 0.033 0.069 0.047 0.160 ARA2 0.079 0.027 0.425 0.199 Pfg377 0.013 0.000 0.548 0.368 PfPK2 0.016 0.064 0.152 0.143 Averagea 0.034 0.040 0.256 0.183 a Average values are calculated as follows: for FST, the arithmetic mean of the value for the six loci; for RST, averaging (over loci) of variance in repeat copy numbers prior to calculation . defect in male gametocyte production or viability in the Dd2 parent . It is unlikely that a single protein such as Pfs48/45 would alone determine gamete compatibility, and it may have a recognition function on only one gametic sex, and interact with a different protein on the oppo- site sex. Rapid evolution of sperm proteins and egg receptors may both be involved in reproductive isola- tion of some sexual species . It is notable that Pfs48/45 has been shown to bind strongly to another major protein of the gamete surface (Pfs230) with simi- larity in secondary structure [12,13,45], and it will be important to consider this interaction (within and be- tween gametes) in determining compatibility. The hypothesis that Pfs48/45 alleles affect gamete recognition may be tested in the ﬁeld and laboratory. Natural populations generally show a deﬁcit in the proportion of heterozygotes at the diploid stage [46,47], explained primarily by the fact that mosquitoes acquire parasite gametocytes from only one or a few clones of P. falciparum per blood feed , or by an artefact due to null alleles in laboratory typing . If the het- erozygote deﬁcit at the Pfs48 /45 locus were observed to be more extreme than that for many other marker loci analysed in diploid stages, it would suggest a particular effect in determining gamete compatibility. Laboratory mating experiments could potentially use genetically modiﬁed parasites to determine the effects of allelicFig. 2. Inter-population variance (FST) in allele frequencies at ﬁve replacement of Pfs48 /45 and other candidate genes forSNPs in codons in the Pfs48 /45 gene and at 11 microsatellite loci: A,Among the three African populations; B, among the three continents. gamete compatibility mechanisms. Of particular signiﬁ-Pfs48 /45 codons are numbered and plotted after the 5-codon haplo- cance, a recent study has shown that disruption of thetype, and the 11 microsatellite loci are plotted separately. Mean Pfs48 /45 gene (and its homologue Pbs48 /45 in thevalues for the Pfs48/45 codons and the microsatellite loci are shown rodent malaria parasite P. berghei ) impairs the abilitywith white bars on the right. All values are signiﬁcantly 0 (at the of male gametes to attach to and fertilise femaleP B 0.01 level) except for four microsatellite loci (TA60, TA102, gametes . This male-speciﬁc effect, although un-TA109, Pfg377 ) among the three African populations. (Asterisksindicate where FST indices for two codons could not be determined as known until completion of the present paper, stronglythe same allele was at or near ﬁxation in all the three African supports the plausibility of the gamete compatibilitypopulations). hypothesis.
D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156 155Acknowledgements  Kocken CHM, Milek RLB, Lensen THW, Kaslow DC, Schoen- makers JGG, Konings RNH. Minimal variation in the transmis- sion-blocking vaccine candidate Pfs48/45 of the human malaria We are very grateful to O.A.T. Ogundahunsi, J. parasite Plasmodium falciparum. Mol Biochem ParasitolCox-Singh, D.E. Arnot, and M.U. Ferreira for help 1995;69:115 – 8.with sample collection, T.J.C. Anderson for advice and  Milek RLB, Kocken CHM, Kaan AM, Jansen J, Meijers H,discussion on microsatellite analysis and the Konings RNH. Plasmodium reichenowi: deduced amino acid sequence of sexual stage-speciﬁc surface antigen Prs48/45 andmanuscript, and R. Carter, G.A.T. Targett, D.A. Baker comparison with its homologue in Plasmodium falciparum. Expand C.J. Drakeley for discussions on Pfs48/45 function. 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