A Preliminary Study of Genetic Diversity of MSP-1 Types in Plasmodium falciparum in Southern Province of Sistan Baluchistan of Iran
Razavi Mohammad Reza,
Plasmodium falciparum merozoite surface protein-1 (MSP-1)
shows extensive antigenic diversity. This is due to the presence of seven
variable blocks, five semi-conserved and also five conserved blocks. The
variable blocks in the MSP-1 gene are principally dimorphic, displaying
either K1 or MAD20 type; except for the block 2 region which is represented
by three alleles, an RO33 type in addition to the other two. Allelic diversity
is reported to be generated by intra-genic recombination between the variable
blocks. A study of allelic variation of MSP-1 gene in Plasmodium falciparum
was carried out in the southern province of Sistan Baluchistan in Iran
in 2001-2003. Samples were obtained from 30 febrile patients and DNA was
extracted and association types between blocks 2 and 6 was identified
on each block using specific primers and compared with those from Vietnam,
Brazil and Africa. The association types obtained, were similar though
less in number than the ones from Vietnam, but more than those from Africa
to cite this article:
Zamani Zahra, Razavi Mohammad Reza, Assmar Mehdi, Sadeghi Sedigheh, Pourfallah Fatemeh, Nasoohi Nikoo, Sheibani Ashraf and Raisi Mohammad, 2007. A Preliminary Study of Genetic Diversity of MSP-1 Types in Plasmodium falciparum in Southern Province of Sistan Baluchistan of Iran. Pakistan Journal of Biological Sciences, 10: 368-372.
Malaria causes over 300 million cases and 2 million deaths annually
world wide (WHO, 1993) Plasmodium falciparum is one of the main
causes of cerebral malaria and the most pathogenic protozoan parasite
(Girard et al., 1988). Increased insecticide resistance by the
mosquito and drug resistance by the parasite itself has made vaccine preparation
an urgency, (Cowman et al., 2000). One of the vaccine candidates
for the blood stage parasites is the 190-200 kD major glycoprotein merozoite
surface protein-1 (MSP-1) which after proteolytic processing, leaves only
a 19 kD C-terminal fragment remains anchored on the merozoite surface
during invasion (Blackman et al., 1992). An immune response to
MSP1 is expected to stop merozoite invasion of erythrocytes, the developmental
stage of the parasite that causes clinical malaria (Cooper et al.,
1992; Pirson and Perkin, 1985; Riley et al., 1992; Riley, 1996).
The protein however, shows extensive polymorphism, but is very strongly
associated with immunity to the disease (Diggs et al., 1993).
An alternative approach could be a multivalent vaccine based on polymorphic
antigens whose composition can be changed regularly to match locally prevalent
antigenic variants. This could be an alternative to those made of highly
conserved but poorly immunogenic antigens (Anderson et al., 1997).
Tanabe et al. (1987) compared different sequences and described
7 variable regions in the MSP1 gene interspersed with conserved or semi-conserved
regions. The 19 kD C-terminus region corresponds approximately to thee
conserved block 17 (Fig. 1) which is a major target of
naturally acquired anti-malarial immunity (Tanabe et al., 1987).
There are two basic versions of each block named after the representative
isolates K1 and MAD20 (Certa et al., 1987). The only known exception
to the allelic dimorphism occurs in block 2 which has a third version
named RO33 (Peterson et al., 1988). Most allelic diversity is generated
by recombination near the 5` end of the gene and the variations in the
tripeptide repeats found in the MAD20 and K1 version of block 2. This
block of MSP1 has been defined as a principal target of antibodies associated
with clinical immunity in Amazonian patients (Da Silveira et al., 1999).
Almost all possible combinations of PfMSP1 variable blocks, are found
in clinical isolates (Da Silveira et al., 1999). A similar study has
been carried out in Brazil, Tanzania and Southern Vietnam and it was seen that some combinations and gene
types from different continents (Kaneko et al., 1997) are significantly
more prevalent than expected by random assortment of allelic types (Ferreira et al., 2003).
Primers for determination of allelic types in variable blocks
of the plasmoium falciparum
MSP1 gene. Conserved, semiconserved and
variable blocks of the gene are shown as white, gray and black boxes, respectively.
Block numbers are after Tanabe et al. (1987)
. Location and directions
of primers are indicated by arrows
Using the PCR strategy developed by Kaneko et
al. (1997), we carried out a study of the patterns of allelic diversity
in Iranian patients infected by P. falciparum between the years
2000-2002 from Sistan Blauchistan State.
MATERIALS AND METHODS
Sample preparation: 2 ml of blood was obtained from 30 willing febrile patients confirmed
as being infected with Plasmodium falciparum by microscopic examination
of Gimesa stained thick blood smears. The patients were residing
in nearby cities in the Sistan Baluchistan province in south-east Iran.
Information revealing their personal data, details of drug therapy and
recent travels to neighboring countries were noted in a questionnaire.
DNA extraction: 1.5 mL blood was centrifuged (1200 rpm for 5 min) at 4°C. The
precipitate was washed with RPMI or PBS. Erythrocytes were initially lysed
in 700 μL PBS buffer containing saponin on ice for 5 min. Cells were
then centrifuged at 10,000 g for 5 min and washed twice with 1 mL PBS.
Lysis was carried out in lysis buffer (50 mM tris (pH 7.8), 50 mM EDTA,
10 mM NaCl, 1% triton X-100 ) and 15 μL proteinase K overnight at
60°C. Phenol chloroform extraction was done according to the method
of Sambrook et al. (1989). DNA was dissolved in 120 μL deionized
DW. The entire experiment was carried out on ice.
PCR amplification: 35 PCR cycles (each one 3 min at 94°C,
45 sec at 91°C, 45 sec at 50°C and 45 sec at 70°C and 7 min
at 70°C) in a thermal cycler (Eppendorf). PCR amplification was performed
in a 25 μL reaction mixture containing 1 μL each of forward
and reverse primers designed by Tanabe et al. (1987), 1 μm
of dNTP, 1 unit of DNA polymerase (Roche ), 2.5 μL of PCR buffer
and 1 μL of DNA were prepared from the infected erythrocytes Every
PCR assay included a tube without template as negative control. PCR products
were electophoressed on 0.8 to 2% agarose gel (depending on predicted
size of PCR products), in 0.5X Tris-borate EDTA buffer and documented
with gel doc (Mitsubishi).
Determination of MSP-1 association type by PCR: Sequences of oligonucleotide primers and PCR protocols was used
to type the blocks as described by Tanabe et al. (1989).
The common primer between block 3 and 5 was detected in all samples
(Fig. 2a) as a heavy band of 500 bp. Allelic family typing
of block 2 with one of the 3 allele specific primers (K2F, M2F, R2F) and
a conserved primer (C3R) (Tanabe et al., 1987) yielded specific
bands of predicted sizes (Fig. 2a). Specific amplification
was also observed for typing of block 6 with a conserved primer C5F and
either of the two allele specific primers (K6R and M6R) (Fig.
2c). All the three variable types of block 2, K1, MAD20 and RO33 type
were detected (Fig. 2b). Nested PCR of block 4 once again
demonstrated the presence of both K1 and MAD20 types (Fig.
In total, 98 MSP-1 association type clones were determined from 30 blood
samples. Twelve out of 30 samples exhibited two different association
type clones and more than 3 association type clones were seen in another
12 isolates including those containing as many as 8 clones. Thus, 24/30
isolates (80%) contained multiple association type clones (Fig.
3). The average number of MSP-1 association type clones per patient
was 3.26 in Sistan Baluchistan in 2001-2003.
Frequency of MSP-1 association types: The analysis of 98 MSP-1 association type revealed 19 distinct types.
The frequency distribution of the expected 24 association types was shown
in the histogram (Fig. 4). As frequency is expressed
only for MSP-1 association type clones and not parasite clones, it is
probable that the same MSP-1 association type clone is shared by different
PCR amplified fragment of the Plasmodium falciparum
MSP-1 gene variable blocks, 2, 3-5, 6 and nested PCR
of block 4 using
specific primers analyzed by 2% agarose gel electrophoresis. MW markers
are 100 bp ladders containing a mixture of 15 double stranded DNA
ranging from 100 to 1500 bp containing an additional 2642 bp band
(Roche). (2a) Lane A is without template and lane M contains MW markers.
And the rest show common primers for analysis of blocks 3-5; 500 bp
fragment amplified by common primers (2b) shows variable block 2-3;
Lane A, without template, lane B positive control, lane C RO33 at
270 bp, lane D, K1 at 264- 269 bp and lane E shows MAD 20 at 154 bp
and lane M, molecular weight marker (2c) Nested PCR using amplified
fragment from block 2-5, lane A without template, lane B positive
control, lane M molecular weight markers, lane I MAD20/MAD20 97 bp,
lane H MAD20/K1 at 94 bp, lane G K1/K1at 80 bp and lane F K1/MAD20
at 84 bp (2d) lane A positive control, lane B MAD20 at 111 bp, lane
C K1 at 119 bp, lane D without template and lane M molecular weight
||Percentage of MSP-I genotype clones
Histogram depicting frequency percentage of MSP1 association
types. Frequency distributions of MSP1 association types were compared by
χ2 test on 2 x R contingency tables; 5% was employed as
the level of significance
|Different kinds of association type numbers combinations of K1, MAD20, RO33 in blocks 2, 4a, 4P and 6.
Particular association types were predominant in Sistan
Baluchistan like type number 17 (with MAD20 type in blocks 2 and 4A, K1 type in block 4P and MAD20 type in block
6) and association type 14 (with MAD20 type in block 2 and block 6 and
K1 type in 4A and 4P). Association types having MAD20 type in block 6
existed at a high rate in all isolates (right half of histogram, Fig.
During the period of our study from 2001-2003, the total number
of malaria cases in Iran dropped from 19,303 to 15,558 cases. In 2001
17,145 P. vivax cases were reported, whereas in 2003, it had dropped
to 13,1318 and the P. falciparum cases from 1875 had gone to 2,219
whereas mixed infection dropped to 195 (Communications from the Center
for Prevention of Communicable Diseases, Tehran, Iran, 2004 (unpublished)).
On the whole, malaria cases had reduced and the downward trend is fortunately
Allelic variations arise due to different associations in the variable
blocks which could be either of K1, MAD20 or RO33 types. The total number
of association types reported so far is 24 (Kaneko et al., 1997;
Da Silveira et al., 1999; Ferreira et al., 2003) though it is
assumed to be much more, as the conserved blocks also show sequential
variations. This is demonstrated by 6 different sizes of the K1 allelic
types in block 2 which could be due to size polymorphism of the tripeptide
repeats, (Jonguwities et al., 1992; Miller et al., 1993;
Daubersies et al., 1996; Kaneko et al., 1997), which was
also observed in this study.
Earlier studies in Vietnam have reported 21 (n = 186), Brazil have shown
15 (n = 620) and Tanzania 13 (n = 60), whereas we have observed 19 of
the 24 reported association types. In the different association types
observed in Sistan Baluchistan province, number 14 (MAD20-K1-K1-MAD20)
and number 17 (MAD-20-K1-K1-MAD20) were seen in the highest frequency
(about 13%). The Vietnam study also had number 17 as the highest frequency
(42%) but number 14 was only 12% of total associations observed.
Predominance of MAD20 type alleles have also been reported in Africa as early
as 1991 by Conway et al. (1991) who used monoclonal antibodies recognizing
polymorphic epitopes of MSP1 have shown the prevalence of MAD20 type paraistes
in bock 6/18. As can be seen in our histogram, types 7, 8, 9,12 and 19 were
not detected at all, in the Vietnam study too, these were represented poorly
as 1, 0, 1, 1 and 6% of total frequencies observed.
The number of samples in this preliminary study were statistically significant
though many more would be required to reach a final conclusion as to the
reason for this variation including samples from Kerman province and Bandar
Abbas, the two southern states from which P.falciparum is reported.
This would require a much longer period of time as malaria is fortunately
following a downward curve in our country.
This study was supported by finance from Pasteur Institute of Iran,
Tehran and National Center for Scientific Research, Tehran, Iran. I wish
to thank Dr. Marcello U. Ferreira for his kind support for this study.
My acknowledgment to my colleagues, Ms. Fatemeh Mirkhani, Ms. Marzieh
Tameemi and Ms. Arpi Hovanisian for their practical assistance.
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