2. 146 D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156
dent folding, similar to some other P. falciparum ment and institutional ethical committees. DNA from
proteins with low primary sequence homology but not part of each blood sample was prepared by Proteinase
apparent 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. DNA
inhibiting parasite fertilisation, and thus it is a candi- was dissolved in sterile nanopure H2O for use as tem-
date for a transmission-blocking vaccine [14]. plate in polymerase chain reaction amplification (PCR)
The small number of known nucleotide polymor- assays. It was considered important to determine the
phisms in Pfs48 /45 of P. falciparum are all nonsynony- haplotypes of Pfs48 /45 alleles, which is possible in
mous, causing amino acid changes, and are located blood samples containing only one detectable haploid
between 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 [23], were used to
gether with the sequence in the most closely related identify samples with apparently only one P. falciparum
species P. reichenowi (a parasite of chimpanzees) [16] clone. Thus, 255 of such isolates were studied here: 57
reveals an excess of nonsynonymous polymorphisms from Nigeria, 56 from Sudan, 52 from South Africa, 55
within P. falciparum, compared to a slight excess of from Brazil, and 35 from Malaysia.
synonymous substitutions between the species [7]. This
indicates that positive selection may be operating on 2.2. Molecular genotyping of Pfs48 /45 sequences
alleles of Pfs48 /45 in P. falciparum. A survey of two
adjacent polymorphic codons in the gene in natural Five previously identified polymorphic sites in the
populations showed that different alleles were very gene [15] were studied, in codons 253, 254, 304, 314,
common or at fixation in different continents [17]. This and 322. Each of these polymorphic amino acids is
is in strong contrast to the more evenly distributed predicted to be on exposed loops in the secondary
alleles of asexual antigen genes [6,18], but no compari- structure of Pfs48/45 [12]. From each isolate, a central
son has yet been made with a set of putatively neutral portion of the gene, containing codons 235–346 was
loci. Here, 255 P. falciparum isolates were sampled amplified by PCR in a 20 ml volume in wells of a
from 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), 100
the Pfs48 /45 gene (on Chromosome 13), and at 11 mM of each dNTP, 1 × Bioline Taq polymerase buffer
microsatellite loci considered to be evolving in a neutral including 1.5 mM MgCl2, and 1.2 units of Bioline Taq
manner 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 for
2. Materials and methods PCR from each isolate, which yielded an abundant
PCR product for most isolates. For some isolates with
2.1. 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 first
identified in studies on malaria in five geographical round reaction of 30 cycles with the above conditions
locations (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 to
Malaysia in 1997; Porto Velho in Rondonia state, give a GC clamp and an EcoR1 site, for earlier work
western Amazonian Brazil in 1997. The Malaysian and [17]. Two microlitres of first round PCR product was
Brazilian populations (similar to ones previously de- used as a template for the second round PCR which
scribed [21,22]) had generally lower P. falciparum en- was performed exactly as described above for the single
demicity than the African populations (previously round PCR. Final PCR products were denatured and
studied for other polymorphisms [23]). 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 of
agulant or spotted and dried on filter paper, with allele-specific probes to the DNA arrayed on replicate
permission and under guidelines of the relevant govern- membranes was performed. The Pfs48 /45 allele-specific
3. D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156 147
probes were 18-mer oligonucleotides: 5%-TATCAT- allele at each locus). Allelic diversity at each locus (each
AAAAACTTAACT-3% (253-4 KN), 5%-TATCATG- of the Pfs48 /45 SNPs and composite haplotypes, and
AAAAGTTAACT-3% (253-4 EK), 5%-TATCATAAAA- each of the microsatellite loci) was estimated as the
AGTTAACT-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-
i
3% (304 D), 5%-ACAGATAGTTTAGATATT-3% (314 L), lates sampled and pi is the frequency of each different
5%-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 [26], with the FSTAT version 1.2 program [27].
with a replicate membrane in a separate tube in 5 ml For each locus, FST indices were calculated among the
TMAC 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 was
55°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 variation
sual 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 to
codon SNP in each of the isolates a single allele was differences in copy numbers (n) of tri-nucleotide TAA
determined, and composite haplotypes were thus (n) repeats [25], potentially conforming to a stepwise
identified. 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 using
microsatellite loci the RSTCALC 2.2 program [28]. 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 of
consisting of locus-specific sequences flanking polymor- discrete alleles.
phic tri-nucleotide repeats [20,25]. The names (and
chromosomal locations) [1] of the loci are: Polya
(Chr4), TA42 (Chr5), TA81 (Chr5), TA1 (Chr6), TA87
(Chr6), TA109 (Chr6), ARA2 (Chr11), TA102 (Chr12), 3. Results
PfPK2 (Chr12), Pfg377 (Chr12), TA60 (Chr13). Alleles
at these loci (except locus TA102 ) were typed using a
semi-nested PCR method previously described [19], 3.1. Pfs48 /45 allele and haplotype frequencies
with some modifications made for convenience in the
choice 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 each
TA109, 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 greater
Locus TA102 was amplified under the same cycling haplotype diversity is seen in the three African popula-
conditions as the other loci [19], 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 populations
TACGATAGGTT-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 exist
second round primer TA102-F 5%-GCTCCAAGAT- among the populations in allele frequencies at each
GATTAAAGA-3% together with TA102-R (with kind individual codon and in frequencies of haplotypes, the
advice from T.J.C. Anderson). Alleles at all loci were most pronounced differences being among populations
identified by sizing of PCR products electrophoresed on in different continents. Different alleles are at complete
an ABI 377, using GENESCAN and GENOTYPER or near fixation in different populations. Codons 253-4
software (Applied Biosystems, UK). show a geographical allele frequency distribution con-
sistent with that reported for five other populations in
2.4. Statistical analyses which these codons were typed using a different method
[17]. 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 have
derived by direct counting (as single-clone isolates were the EK allele, all isolates from Southeast Asia have the
chosen for the study, each isolate contained only one KK allele, whereas in Africa the KN allele is most
4. 148
D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156
Fig. 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. [17]: Amapa in northeastern Amazonian Brazil, n= 40; The Gambia, n =57;
Cameroon, n = 29; Tanzania, n= 31; Thailand, n= 27).
5. D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156 149
common, but all three alleles are present in all African was statistically analysed and shown to be much higher
populations 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 polymorphic
3.2. 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 of
numbers 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 [25]. These were therefore also
alleles at the microsatellite loci is higher in the African studied using the RST index of inter-population variance
populations (in Nigeria, Sudan, and South Africa, re- (which takes into account the variance in allele size due
spectively, mean number of alleles per locus is 9.9, 7.9 to gain or loss of repeat copies by SMM). Table 4
and 9.1) than in the populations from Brazil (4.4) or shows the RST estimates for these loci, alongside the FST
Malaysia (5.2). Similarly, the allelic diversity (H) is estimates for comparison. Among populations in the
higher in the African populations (for Nigeria, Sudan, three African countries, the average RST value over all
and South Africa, respectively, over all loci mean H= six loci was similar to the average FST value, but among
0.80, 0.71 and 0.78) than in Brazil (mean H =0.54) and populations in the different continents the average RST
Malaysia (mean H= 0.66). This trend is similar to that value (0.256) was somewhat higher than the average
noted for the Pfs48 /45 gene, but much less pronounced. FST value (0.183). It should be noted that the values
A large proportion of microsatellite alleles are shared vary widely among the loci for the variance among the
among 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 of
frequencies. inter-continental variation shown for Pfs48 /45. Overall,
the analysis suggests that divergence at microsatellite
3.3. Quantification of 6ariance in allele frequencies loci could be somewhat greater than the FST values
among 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 1
Pfs48 /45 allele and haplotype frequencies in five geographical populations of P. falciparum
Codon 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 – 1.00
254 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 1.00
322 S 1.00 1.00 1.00 1.00 0.03
N – – – – 0.97
5-codon haplotypes
K-K-D-L-N – – – – 0.97
K-K-D-L-S – 0.05 – – 0.03
K-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 –
6. 150
D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156
Table 2
Numbers of alleles and diversity index (H) at 11 microsatellite loci in five geographical populations of P. falciparum
Locus 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 0.79
TA42 9 4 7 4 4 0.48 0.20 0.64 0.30 0.58
TA60 8 11 7 4 6 0.82 0.80 0.72 0.64 0.79
TA81 8 5 7 3 6 0.82 0.76 0.81 0.46 0.78
TA87 9 8 8 6 5 0.86 0.73 0.83 0.64 0.69
TA102 9 7 9 3 7 0.85 0.85 0.83 0.63 0.79
TA109 12 9 10 5 2 0.83 0.73 0.76 0.41 0.28
ARA2 10 9 9 3 5 0.86 0.83 0.85 0.47 0.79
Pfg377 6 5 5 2 3 0.63 0.49 0.55 0.51 0.37
PFPK2 10 7 15 6 3 0.86 0.67 0.85 0.66 0.55
POLYA 13 12 14 5 8 0.86 0.90 0.96 0.69 0.81
Mean 9.9 7.9 9.1 4.4 5.2 0.80 0.71 0.78 0.54 0.66
9. D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156 153
Table 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 [30], 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 some
frequencies in the Pfs48 /45 gamete surface protein extent [31]. This could have affected the microsatellite
gene. In a quantitative analysis, the inter-population FST estimates here, and in another study [29], particu-
fixation indices for Pfs48 /45 alleles and haplotypes are larly at the level of comparisons among continents. To
consistently much higher than those for the microsatel- explore this, it would be useful if fixation indices among
lite alleles, whether the analysis considers different pop- P. falciparum populations were studied with a broad
ulations within Africa, or different continents. The random sample of neutral SNPs in the genome, and
geographical 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 genes
of several other populations [29]. 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 processes
therefore hypothesised that the exceptionally skewed alone. Future large scale SNP discovery throughout the
frequencies of Pfs48 /45 alleles may be due to divergent P. falciparum genome would therefore yield resources
selection operating on the protein in different for addressing this question, as recognised for the hu-
populations. man genome [32].
An important critical question is whether fixation To focus on Pfs48/45, it is reasonable to suggest a
indices based on microsatellite loci may underestimate role in gamete recognition and fertilisation, as it is
the 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 selection
mutational changes in lengths of repeats (which may can operate in mating type genes [33] and other genes
make ancestrally distinct alleles appear the same) [28]. determining aspects of sexual reproduction [34,35]. The
To investigate this, an additional analysis of variation importance of understanding the parameters affecting
at 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 of
gence 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
10. 154 D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156
alleles were inherited from each parent [37– 40], al- Table 4
Comparison of RST and FST estimates of inter-population variance at
though these clones had different Pfs48/45 types [15].
six microsatellite loci considered to evolve according to a SMM
However, the maternally-inherited mitochondrial DNA
of the HB3 parent was severely under-represented in Locus Variance among populations
progeny of both crosses [41,42]. It is plausible that
Three African countries Three different continents
directional incompatibility occurs in the case of particu-
lar male-female Pfs48/45 interactions, but interpreting RST FST RST FST
the 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 [28].
defect in male gametocyte production or viability in the
Dd2 parent [43].
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 [44]. 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 [48], or by an artefact due
to null alleles in laboratory typing [49]. 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 allelic
Fig. 2. Inter-population variance (FST) in allele frequencies at five replacement of Pfs48 /45 and other candidate genes for
SNPs 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 the
type, and the 11 microsatellite loci are plotted separately. Mean Pfs48 /45 gene (and its homologue Pbs48 /45 in the
values for the Pfs48/45 codons and the microsatellite loci are shown rodent malaria parasite P. berghei ) impairs the ability
with white bars on the right. All values are significantly 0 (at the of male gametes to attach to and fertilise female
P B 0.01 level) except for four microsatellite loci (TA60, TA102,
gametes [50]. This male-specific effect, although un-
TA109, Pfg377 ) among the three African populations. (Asterisks
indicate where FST indices for two codons could not be determined as known until completion of the present paper, strongly
the same allele was at or near fixation in all the three African supports the plausibility of the gamete compatibility
populations). hypothesis.
11. D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156 155
Acknowledgements [15] 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 Parasitol
Cox-Singh, D.E. Arnot, and M.U. Ferreira for help 1995;69:115 – 8.
with sample collection, T.J.C. Anderson for advice and [16] 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 and
manuscript, and R. Carter, G.A.T. Targett, D.A. Baker
comparison with its homologue in Plasmodium falciparum. Exp
and C.J. Drakeley for discussions on Pfs48/45 function. Parasitol 1997;87:150 – 2.
The research was supported by the Wellcome Trust [17] Drakeley CJ, Duraisingh MT, Povoa M, Conway DJ, Targett
(Grant Ref. 055487/Z/98/Z) and the Medical Research GAT, Baker DA. Geographical distribution of a variant epitope
Council (Grant Ref. G9803180). of Pfs48/45, a Plasmodium falciparum transmission-blocking vac-
cine candidate. Mol Biochem Parasitol 1996;81:253 – 7.
[18] Anderson TJC, Day KP. Geographical structure and sequence
evolution as inferred from the Plasmodium falciparum S-antigen
locus. Mol Biochem Parasitol 2000;106:321 – 6.
References [19] Anderson TJC, Su X-Z, Bockaire M, Lagog M, Day KP. Twelve
microsatellite markers for characterisation of Plasmodium falci-
parum from finger prick blood samples. Parasitology
[1] Su X-Z, Ferdig MT, Huang Y, Huynh CQ, Liu A, You J, et al.
1999;119:113 – 25.
A genetic map and recombination parameters of the human
[20] Su X-Z, Wellems TE. Towards a high resolution Plasmodium
malaria parasite P. falciparum. Science 1999;286:1351 –3.
falciparum linkage map: polymorphic markers from hundreds of
[2] Ferdig MT, Su X-Z. Microsatellite markers and genetic mapping
simple sequence repeats. Genomics 1996;33:430 – 44.
in Plasmodium falciparum. Parasitol Today 2000;16:307 – 12.
[21] Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS,
[3] Beaumont MA, Nichols RA. Evaluating loci for use in the
Rahman HA. A genus- and species-specific nested polymerase
genetic analysis of population structure. Proc R Soc Lond B
chain reaction malaria detection assay for epidemiologic studies.
1996;263:1619 – 26.
Am J Trop Med Hyg 1999;60:687 – 92.
[4] McDonald JH. Detecting natural selection by comparing geo-
[22] Camargo LMA, dal Colletto GMD, Ferreira MU, Gurgel SDM,
graphic variation in protein and DNA polymorphisms. In: Gold-
Escobar AL, Marques A, et al. Hypoendemic malaria in Rondo-
ing B, editor. Non-Neutral Evolution: Theories and Molecular
nia (Brazil, Western Amazon Region): seasonal variation and
Data. London: Chapman and Hall, 1994:88 –100.
risk groups in an urban locality. Am J Trop Med Hyg
[5] Hughes MK, Hughes AL. Natural selection on Plasmodium
1996;55:32 – 8.
surface proteins. Mol Biochem Parasitol 1995;71:99 –115.
[23] Conway DJ, Roper C, Oduola AMJ, Arnot DE, Kremsner PG,
[6] Conway DJ. Natural selection on polymorphic malaria antigens Grobusch MP, et al. High recombination rate in natural popula-
and the search for a vaccine. Parasitol Today 1997;13:26 –9. tions of Plasmodium falciparum. Proc Natl Acad Sci USA
[7] Escalante AA, Lal AA, Ayala FJ. Genetic polymorphism and 1999;96:4506 – 11.
natural selection in the malaria parasite Plasmodium falciparum. [24] Conway DJ, Fanello C, Lloyd JM, Al-Joubori BMA-S, Baloch
Genetics 1998;149:189 –202. AH, Somanath SD, et al. Origin of Plasmodium falciparum
[8] Verra F, Hughes AL. Evidence for ancient balanced polymor- malaria is traced by mitochondrial DNA. Mol Biochem Parasitol
phism at the apical membrane antigen-1 (AMA-1) locus of 2000;111:137 – 45.
Plasmodium falciparum. Mol Biochem Parasitol 2000;105:149 – [25] Anderson TJC, Su X-Z, Roddam A, Day KP. Complex muta-
53. tions in a high proportion of microsatellite loci in the protozoan
[9] Kocken CHM, Narum DL, Massougbodji A, Ayivi B, Dubbeld malaria parasite Plasmodium falciparum. Mol Ecol 2000;9:1599 –
MA, van der Wel A, et al. Molecular characterisation of Plas- 608.
modium reichenowi apical membrane antigen-1 (AMA-1), com- [26] Weir BS, Cockerham CC. Estimating F statistics for the analysis
parison with P. falciparum AMA-1, and antibody-mediated of population structure. Evolution 1984;38:1358 – 70.
inhibition of red cell invasion. Mol Biochem Parasitol [27] Goudet J. FSTAT (Version 1.2): a computer program to calcu-
2000;109:147 – 56. late F statistics. J Hered 1995;86:485 – 6.
[10] Conway DJ, Cavanagh DR, Tanabe K, Roper C, Mikes ZS, [28] Goodman SJ. RST Calc: a collection of computer programs for
Sakihama N, et al. A principal target of human immunity to calculating estimates of genetic differentiation from microsatel-
malaria identified by molecular population genetic and immuno- lite data and determining their significance. Mol Ecol
logical analyses. Nature Med 2000;6:689 –92. 1997;6:881 –5.
[11] Kocken CHM, Jansen J, Kaan AM, Beckers PJA, Ponnudurai [29] Anderson TJC, Haubold B, Williams JT, Estrada-Franco JG,
T, Kaslow DC, et al. Cloning and expression of the gene coding Richardson L, Mollinedo R, et al. Microsatellite markers reveal
for the transmission-blocking target antigen Pfs48/45 of Plas- a spectrum of population structures in the malaria parasite
modium falciparum. Mol Biochem Parasitol 1993;61:59 –68. Plasmodium falciparum. Mol Biol Evol 2000;17:1467 – 82.
[12] Carter R, Coulson A, Bhatti S, Taylor BJ, Elliott JF. Predicted [30] Balloux F, Brunner H, Lugon-Moulin N, Hausser J, Goudet J.
disulphide-bonded structures for three uniquely related proteins Microsatellites can be misleading: an empirical and simulation
of Plasmodium falciparum, Pfs230, Pfs48/45 and Pf12. Mol study. Evolution 2000;54:1414 – 22.
Biochem Parasitol 1995;71:203 –10. [31] Hedrick PW. Highly variable loci and their interpretation in
[13] Templeton TJ, Kaslow DC. Identification of additional members evolution and conservation. Evolution 1999;53:313 – 8.
define a Plasmodium falciparum gene superfamily which includes [32] The International SNP Map Working Group. A map of human
Pfs48/45 and Pfs230. Mol Biochem Parasitol 1999;101:223 – 7. genome sequence variation containing 1.42 million single nucle-
[14] Roeffen W, Mulder B, Teelen K, Bolmer M, Eling W, Targett otide polymorphisms. Nature 2001;409:928 –33.
GAT, et al. Association between anti-Pfs48/45 reactivity and P. [33] Palumbi SR. All males are not created equal: fertility differences
falciparum transmission-blocking activity in sera from depend on gamete recognition polymorphisms in sea urchins.
Cameroon. Parasite Immunol 1996;18:103 –9. Proc Natl Acad Sci USA 1999;96:12632 – 7.
12. 156 D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156
[34] Tsaur S, Ting CT, Wu C-I. Positive selection driving the evolu- genetic crosses of Plasmodium falciparum. Mol Cell Biol
tion of a gene of male reproduction. Mol Biol Evol 1993;13:7349 – 57.
1998;15:1040 – 6. [43] Guinet F, Dvorak JA, Fujioka H, Keister DB, Muratova O,
[35] Wyckoff GJ, Wang W, Wu C-I. Rapid evolution of male repro- Kaslow DC, et al. A developmental defect in Plasmodium falci-
ductive genes in the descent of man. Nature 2000;403:304 –9. parum male gametocytogenesis. J Cell Biol 1996;135:269 – 78.
[36] Rich SM, Ayala FJ. Population structure and recent evolution of [44] Swanson WJ, Vacquier VD. Concerted evolution in an egg
Plasmodium falciparum. Proc Natl Acad Sci USA 2000;97:6994 – receptor for a rapidly evolving abalone sperm protein. Science
7001. 1998;281:710 –2.
[37] Walliker D, Quakyi IA, Wellems TE, McCutchan TF, Szarfman [45] Kumar N. Target antigens of malaria transmission blocking
A, London WT, et al. Genetic analysis of the human malaria immunity exist as a stable membrane bound complex. Parasite
parasite Plasmodium falciparum. Science 1987;236:1661 –6. Immunol 1987;9:321 – 35.
[38] Wellems TE, Panton LJ, Gluzman IY, do Rosario VE, Gwadz [46] Babiker HA, Ranford-Cartwright LC, Currie D, Charlwood JD,
RW, Walker-Jonah A, Krogstad DJ. Chloroquine resistance not Billingsley P, Teuscher T, Walliker D. Random mating in a
natural population of the malaria parasite Plasmodium falci-
linked to mdr-like genes in a Plasmodium falciparum cross.
parum. Parasitology 1994;109:413 – 21.
Nature 1990;345:253 –5.
[47] Paul REL, Packer MJ, Walmsley M, Lagog M, Ranford-
[39] Walker-Jonah A, Dolan SA, Gwadz RW, Panton LJ, Wellems
Cartwright LC, Paru R, Day KP. Mating patterns in malaria
TE. An RFLP map of the Plasmodium falciparum genome,
parasite populations of Papua New Guinea. Science
recombination rates and favoured linkage groups in a genetic
1995;269:1709 – 11.
cross. Mol Biochem Parasitol 1992;51:313 –20. [48] Hill WG, Babiker HA, Ranford-Cartwright LC, Walliker D.
[40] Ranford-Cartwright LC, Balfe P, Carter R, Walliker D. Fre- Estimation of inbreeding coefficients from genotypic data on
quency of cross-fertilization in the human malaria parasite Plas- multiple alleles, and application to estimation of clonality in
modium falciparum. Parasitology 1993;107:11 –8. malaria parasites. Genet Res 1995;65:53 –61.
[41] Creasey AM, Ranford-Cartwright LC, Moore DJ, Williamson [49] Anderson TJC, Paul REL, Donnelly CA, Day KP. Do malaria
DH, Wilson RJM, Walliker D, Carter R. Uniparental inheri- parasites mate non-randomly in the mosquito midgut? Genet
tance of the mitochondrial gene cytochrome b in Plasmodium Res 2000;75:285 – 96.
falciparum. Curr Genet 1993;23:360 –4. [50] van Dijk MR, Janse CJ, Thompson J, Waters AP, Braks A,
[42] Vaidya AB, Morrisey J, Plowe CV, Kaslow DC, Wellems TE. Dodemont H, et al. A central role for P48/45 in malaria parasite
Unidirectional dominance of cytoplasmic inheritance in two male gamete fertility. Cell 2001;104:153 – 64.
.
![Molecular & Biochemical Parasitology 115 (2001) 145– 156
www.parasitology-online.com.
Extreme geographical fixation of variation in the Plasmodium
falciparum gamete surface protein gene Pfs48 /45 compared with
microsatellite loci
David J. Conway a,*, Ricardo L.D. Machado b, Balbir Singh c, Patricia Dessert a,
Zsuzsanna S. Mikes a, Marinete M. Povoa b, Ayoade M.J. Oduola d, Cally Roper a
a
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
b
Ser6ico de Parasitologia, Instituto E6andro Chagas, Belem, PA, Brazil
¸
c
Faculty of Medicine and Health Sciences, Uni6ersiti Malaysia Sarawak, Kota Samarahan 94300, Sarawak, Malaysia
d
Postgraduate Institute of Medical Research and Training, College of Medicine, Uni6ersity of Ibadan, Ibadan, Nigeria
Received 27 October 2000; received in revised form 15 March 2001; accepted 20 March 2001
Abstract
Comparing patterns of genetic variation at multiple loci in the genome of a species can potentially identify loci which are under
selection. The large number of polymorphic microsatellites in the malaria parasite Plasmodium falciparum are available markers
to screen for selectively important loci. The Pfs48 /45 gene on Chromosome 13 encodes an antigenic protein located on the surface
of parasite gametes, which is a candidate for a transmission blocking vaccine. Here, genotypic data from 255 P. falciparum isolates
are presented, which show that alleles and haplotypes of five single nucleotide polymorphisms (SNPs) in the Pfs48 /45 gene are
exceptionally skewed in frequency among different P. falciparum populations, compared with alleles at 11 microsatellite loci
sampled widely from the parasite genome. Fixation indices measuring inter-population variance in allele frequencies (FST) were in
the order of four to seven times higher for Pfs48 /45 than for the microsatellites, whether considered (i) among populations within
Africa, or (ii) among different continents. Differing mutational processes at microsatellite and SNP loci could generally affect the
population structure at these different types of loci, to an unknown extent which deserves further investigation. The highly
contrasting population structure may also suggest divergent selection on the amino acid sequence of Pfs48/45 in different
populations, which plausibly indicates a role for the protein in determining gamete recognition and compatibility. © 2001 Elsevier
Science B.V. All rights reserved.
Keywords: Plasmodium falciparum; Gametes; Pfs48/45; Genetics; Microsatellites; Selection
1. Introduction caused by directional selection for favoured alleles,
whereas unusually even frequencies may be due to
A high density of informative microsatellite markers balancing selection maintaining different alleles [3,4].
on all 14 chromosomes of the protozoan malaria para- Several asexual blood stage antigens of P. falciparum
site Plasmodium falciparum [1] should help studies to are apparently under selection which maintains allelic
identify functionally important loci in the genome [2]. variation [5–9], and a detailed study of polymorphisms
in the merozoite surface protein 1 gene (msp1 ) has
One approach is to identify loci with extreme statistical
prospectively identified a domain encoding a target of
distributions of allele frequencies among populations.
allele-specific protective immune responses [10].
Exceptionally skewed frequency distributions may be
In contrast to many antigens of the P. falciparum
asexual blood stages, there is a relatively low level of
Abbre6iations: SMM, stepwise mutation model; SNP, single nucle- sequence polymorphism in antigens of sexual gameto-
otide polymorphism.
* Corresponding author. Tel.: +44-20-79272331; fax: + 44-20-
cyte and gamete stages of the parasite. A major surface
76368739. antigen of these stages is the protein Pfs48/45 [11],
E-mail address: david.conway@lshtm.ac.uk (D.J. Conway). which has an unusual predicted disulphide bond-depen-
0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 6 - 6 8 5 1 ( 0 1 ) 0 0 2 7 8 - X](https://image.slidesharecdn.com/2001extremegeographicalfixation-121204072613-phpapp02/85/2001-extreme-geographical-fixation-1-320.jpg)
![146 D.J. Conway et al. / Molecular & Biochemical Parasitology 115 (2001) 145–156
dent folding, similar to some other P. falciparum ment and institutional ethical committees. DNA from
proteins with low primary sequence homology but not part of each blood sample was prepared by Proteinase
apparent 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. DNA
inhibiting parasite fertilisation, and thus it is a candi- was dissolved in sterile nanopure H2O for use as tem-
date for a transmission-blocking vaccine [14]. plate in polymerase chain reaction amplification (PCR)
The small number of known nucleotide polymor- assays. It was considered important to determine the
phisms in Pfs48 /45 of P. falciparum are all nonsynony- haplotypes of Pfs48 /45 alleles, which is possible in
mous, causing amino acid changes, and are located blood samples containing only one detectable haploid
between 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 [23], were used to
gether with the sequence in the most closely related identify samples with apparently only one P. falciparum
species P. reichenowi (a parasite of chimpanzees) [16] clone. Thus, 255 of such isolates were studied here: 57
reveals an excess of nonsynonymous polymorphisms from Nigeria, 56 from Sudan, 52 from South Africa, 55
within P. falciparum, compared to a slight excess of from Brazil, and 35 from Malaysia.
synonymous substitutions between the species [7]. This
indicates that positive selection may be operating on 2.2. Molecular genotyping of Pfs48 /45 sequences
alleles of Pfs48 /45 in P. falciparum. A survey of two
adjacent polymorphic codons in the gene in natural Five previously identified polymorphic sites in the
populations showed that different alleles were very gene [15] were studied, in codons 253, 254, 304, 314,
common or at fixation in different continents [17]. This and 322. Each of these polymorphic amino acids is
is in strong contrast to the more evenly distributed predicted to be on exposed loops in the secondary
alleles of asexual antigen genes [6,18], but no compari- structure of Pfs48/45 [12]. From each isolate, a central
son has yet been made with a set of putatively neutral portion of the gene, containing codons 235–346 was
loci. Here, 255 P. falciparum isolates were sampled amplified by PCR in a 20 ml volume in wells of a
from 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), 100
the Pfs48 /45 gene (on Chromosome 13), and at 11 mM of each dNTP, 1 × Bioline Taq polymerase buffer
microsatellite loci considered to be evolving in a neutral including 1.5 mM MgCl2, and 1.2 units of Bioline Taq
manner 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 for
2. Materials and methods PCR from each isolate, which yielded an abundant
PCR product for most isolates. For some isolates with
2.1. 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 first
identified in studies on malaria in five geographical round reaction of 30 cycles with the above conditions
locations (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 to
Malaysia in 1997; Porto Velho in Rondonia state, give a GC clamp and an EcoR1 site, for earlier work
western Amazonian Brazil in 1997. The Malaysian and [17]. Two microlitres of first round PCR product was
Brazilian populations (similar to ones previously de- used as a template for the second round PCR which
scribed [21,22]) had generally lower P. falciparum en- was performed exactly as described above for the single
demicity than the African populations (previously round PCR. Final PCR products were denatured and
studied for other polymorphisms [23]). 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 of
agulant or spotted and dried on filter paper, with allele-specific probes to the DNA arrayed on replicate
permission and under guidelines of the relevant govern- membranes was performed. The Pfs48 /45 allele-specific](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)