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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Patel, S. S. Articles by Zimmerman, P. A. Search for Related Content PubMed PubMed Citation Articles by Patel, S. S. Articles by Zimmerman, P. A. Related Collections Red Cells Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 December 2001, Vol. 98, No. 12, pp. 3489-3491 BRIEF REPORT The association of the glycophorin C exon 3 deletion with ovalocytosis and malaria susceptibility in the Wosera, Papua New Guinea Sheral S. Patel, Rajeev K. Mehlotra, William Kastens, Charles S. Mgone, James W. Kazura, and Peter A. Zimmerman From the Division of Geographic Medicine, Case Western Reserve University, and University Hospitals of Cleveland, Cleveland, OH; and Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea.     Abstract Top Abstract Introduction Study design Results and discussion References Erythrocyte polymorphisms, including ovalocytosis, have been associated with protection against malaria. This study in the Wosera, a malaria holoendemic region of Papua New Guinea, examined the genetic basis of ovalocytosis and its influence on susceptibility to malaria infection. Whereas previous studies showed significant associations between Southeast Asian ovalocytosis (caused by a 27- base pair deletion in the anion exchanger 1 protein gene) and protection from cerebral malaria, this mutation was observed in only 1 of 1019 individuals in the Wosera. Polymerase chain reaction strategies were developed to genotype individuals for the glycophorin C exon 3 deletion associated with Melanesian Gerbich negativity (GPCex3). This polymorphism was commonly observed in the study population (GPCex3 frequency = 0.465, n = 742). Although GPCex3 was significantly associated with increased ovalocytosis, it was not associated with differences in either Plasmodium falciparum or P vivax infection measured over the 7-month study period. Future case-control studies will determine if GPCex3 reduces susceptibility to malaria morbidity. (Blood. 2001;98:3489-3491) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References The geographic overlap between malaria and red blood cell (RBC) disorders led Haldane to hypothesize that many polymorphisms in the human genome have arisen by natural selection to protect from severe malaria infection and thereby increase reproductive fitness of populations living in malaria endemic regions.1 In Papua New Guinea, Southeast Asian ovalocytosis, caused by a 27-base pair (bp) deletion in the anion exchanger 1 protein gene (AE127), is observed in many coastal malaria holoendemic areas and is associated with protection from cerebral malaria.2,3 While AE127 has not been reported in residents of the malaria holoendemic Wosera region of Papua New Guinea,2 ovalocytic RBCs are common. Additional polymorphisms characterizing the human population in the Wosera include an exon 3 deletion of the integral membrane sialoglycoprotein glycophorin C (GPC).4-6 This deletion (GPCex3) changes serologic phenotypes of the Gerbich (Ge) blood group system in Melanesians.7 Because GPC is involved in maintaining the integral RBC membrane lattice, the association between GPCex3 and ovalocytosis was tested. Furthermore, because GPCex3 is distributed within a malaria holoendemic region, we tested the association between GPCex3 and susceptibility to malaria infection.     Study design Top Abstract Introduction Study design Results and discussion References Study population and malaria The study was conducted in the Wosera region of Papua New Guinea, where all 4 human Plasmodium species are transmitted year-round.8 Blood samples were collected monthly from permanent residents (median age, 17 years; range, 1-86 years) of 6 villages within the Wosera from July 1998 to January 1999. The human investigations institutional review boards of Case Western Reserve University, University Hospitals of Cleveland, and the Papua New Guinea Medical Research Advisory Committee approved all protocols. Malaria and red blood cell morphology Thick and thin films stained with 4% Giemsa were prepared at the time of blood collection (Figure 1A-D).8 Malaria parasites were identified by light microscopy. Parasite densities were recorded as the number of parasites per 200 leukocytes (average 8000 leukocytes per microliter).8 Thin smears were examined by light microscopy for the proportion of ovalocytes (erythrocytes with length-width ratio more than 1:1) without knowledge of genotypic results.2 Because previous studies have shown that elliptocytes are rare in the Wosera, the distinction between ovalocytes and elliptocytes (RBC with length:width more than 2:1) was not made.9 Blood smears from North Americans were prepared using a modified Wright stain (Diff-Quick Stain Set, Dade-Behring, Newark, DE). One reader reviewed all of the blood smears. Two additional observers independently reviewed a subset of smears. Genotyping for AE1 and GPC polymorphisms Blood was collected in ethylenediaminetetraacetic acid vacutainer tubes and stored at 70°C until DNA extraction was performed with the QIAmp96 DNA blood kit (Qiagen, Valencia, CA). Genotyping of band 3 was performed as previously described.10 New polymerase chain reaction (PCR) genotyping strategies for GPC are described in Figure 1E-G. View larger version (62K): [in this window] [in a new window]   Figure 1. Representative blood smears and GPC genotyping results. (A) North American control. Arrows indicate examples of ovalocytes: (B) Melanesian, Southeast Asian ovalocytosis (heterozygous for AE127); (C) Melanesian homozygous for GPCex3; and (D) Melanesian heterozygous for GPCex3. GPC genotyping; lane 1: homozygous wt (wt/wt); lane 2: GPCex3 heterozygote (wt/); lane 3: GPCex3 homozygote (/); lane 4: North American, wt/wt genotype. Because exon 2 and exon 3 of the GPC gene are highly homologous, 2 reactions are needed to delineate all 3 genotypes. All PCR amplifications were performed as previously described.12 (E) To assess the presence of exons 2 and 3, primers (GPCup 5'-CAGATTCTTGTCCTCTGTTCACAG-3' and GPCdn 5'-TCAAAACCACCTCTGAGGGAGAG-3') annealing to conserved sequence around exons 2 and 3 were used (thermocycling program: 94°C for 30 seconds, 68°C for 30 seconds, and 72°C for 30 seconds [× 40]). PCR products were subjected to electrophoresis on a 4% 5:1 GTG NuSieve:LE agarose (FMC Bioproducts, Rockland, ME) gel. A 264-bp band (exon 2) is detected in wt/wt, wt/, and /. A 240 bp band (exon 3) is detected in only wt/wt and wt/. The heteroduplex product is created by hybridization between complementary strands of exon 2 and 3 amplicons. (F) To identify individuals with the wt allele, primers annealing to a 51-bp intron 2 insert (GenBank AF342984) and downstream intron 2 polymorphism (GenBank M24627)5 (GPC349up 5'-GGAAACTGCCGTGACTTCAGA-3' and 1679dn 5'-CATGGTCCTTGCAACAGTTGC-3') were used to amplify a 1376-bp product (thermocycling program: 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 90 seconds [× 40]). PCR products were subjected to electrophoresis on a 1.5% 5:1 GTG NuSieve:LE agarose gel. (G) To identify the GPCex3 allele and differentiate between wt/wt and wt/, primers annealing to a 51-bp intron 2 insert and downstream intron 3 polymorphism (GenBank M24628)5 (GPC349up and GPC1680dn 5'-AGGTTAGAATCATACCCCAGG-3') were used. Because the 3.4-kb deletion of the GPC gene includes exon 3, this PCR produces a 1377-bp band. To ensure that absence of the 1376-bp (D) and 1377-bp (E) bands was not caused by heterogeneity in PCR master mix, an additional primer, GPCdn, along with GPC349up, amplified conserved regions downstream of exon 2. The 155-bp product produced in all genotypes by GPC349up and GPCdn is not shown. All gels were stained with a 1:10 000 dilution of SYBR Gold (Molecular Probes, Eugene, OR), and products were visualized using a Storm 860 (Molecular Dynamics, Sunnyvale, CA). Statistical analysis Categorical variables were analyzed by the 2 test and continuous variables by the Wilcoxon or Kruskal-Wallis test. Statistical Analysis Systems version 8.1 software package (Cary, NC) was used.     Results and discussion Top Abstract Introduction Study design Results and discussion References Ovalocytes in the Wosera To assess the relationship between ovalocytosis and Melanesian ancestry, 13 North Americans and 199 individuals from the Wosera were compared. The frequency of ovalocytes per 1000 RBCs was significantly higher in residents of the Wosera than North Americans (median, 292 ± 131.4; median, 24 ± 22.6, respectively, Wilcoxon, P < .0001). Representative blood smears from a North American Caucasian and 3 Melanesians with different GPC and band 3 genotypes are illustrated in Figure 1A-D. AE127 Previous studies reported an absence of AE127 in the Wosera (n = 216).2 Consistent with these findings, only 1 of 1019 residents from the Wosera had this mutation (Figure 1B). The PCR product from this individual was cloned and sequenced, verifying that this individual carried AE127 (not shown).10 GPC genotype in the Wosera GPC genotyping was performed on 742 individuals (Figure 1E-G). The first reaction, screening for the presence or absence of the wild-type (wt) GPC allele where exons 2 and 3 are both present (Figure 1E), identified homozygous GPCex3 individuals (lane 3). The second (Figure 1F) and third reactions (Figure 1G) amplified GPC sequence within the wt or GPCex3 alleles, respectively, and allowed homozygous wt individuals (Figure 1F-G, lanes 1 and 4) to be distinguished from heterozygous individuals (Figure 1F-G, lane 2). Allele frequencies for GPC wt and GPCex3 were 0.535 and 0.465, respectively. Genotyping showed 211 (28.4%) of 742 individuals as homozygous wt, 372 (50.1%) of 742 as heterozygotes, and 159 (21.4%) of 742 as GPCex3 homozygotes. This distribution is in Hardy-Weinberg equilibrium, indicating that GPCex3 does not confer a selective disadvantage. This is in contrast to AE127, a balanced polymorphism, where the disadvantage of lethality in the homozygous form is outweighed by the selective advantage against severe malaria for heterozygotes.2 GPC genotype and ovalocytosis The association between ovalocytosis and GPC genotype was evaluated in 134 individuals who did not carry AE127. The wt individuals (n = 32) had the lowest proportion of ovalocytes per 1000 RBCs (median, 238 ± 115.1). Heterozygous individuals (n = 52) had a higher proportion of ovalocytes (median, 297 ± 103.8), while homozygotes (n = 49) had the highest of all 3 genotypes (median, 312 ± 145.9). In a comparison of all 3 genotypes, the proportion of ovalocytes was significantly associated with GPCex3 (Kruskal-Wallis, P = .021). Individual comparisons among the 3 genotypic groups showed significant differences by a one-sided Wilcoxon test (wt/wt vs wt/GPCex3, P = .0392; wt/wt vs GPCex3/GPCex3, P = .0045). These results suggest that GPCex3 contributes to ovalocytosis in the Wosera. When erythrocyte morphology was compared between homozygous wt individuals from the Wosera and North Americans, the former had a significantly increased ovalocyte frequency (Wilcoxon, P < .0001). This suggests that altered RBC morphology in the Wosera is a heterogenous condition caused by additional unknown mutations in RBC membrane proteins, such as protein 4.1 and spectrin as well as environmental or nutritional factors. GPC genotype and infection status The prevalence of infection with P falciparum or P vivax determined by blood smear has been examined in relation to serologic Ge antigen status in one published study of 266 people.11 This study observed a lower combined smear positive rate for P falciparum and/or P vivax infection in Ge-negative individuals, suggesting that Ge antigen negativity protects against infection.11 To examine the relationship between GPC genotype and susceptibility to malaria infection more rigorously, we studied a larger population over 7 months. Results for genotype and infection status were available for 325 to 696 individuals at each of 7 monthly intervals. This analysis showed that the prevalence or density of P falciparum and P vivax infection was not significantly different for individuals in the 3 GPC genotypic groups at any time (Table 1). These results parallel findings of other RBC polymorphisms, such as AE127, where genotypic differences are associated with reduced susceptibility to severe malaria morbidity with no effect on susceptibility to infection.3 The relationship of GPCex3 to malaria morbidity in young children, the age group most susceptible to the clinical phenotype, requires further study.                                View this table: [in this window] [in a new window]   Table 1. Glycophorin C genotype and infections over time     Acknowledgments We thank the residents of the Wosera for participating in this study. We thank Chloe Hill for reviewing blood smears.     Footnotes Submitted January 30, 2001; accepted July 30, 2001. Supported by National Institutes of Health grants AI-36478-07 and AI-07024. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Sheral S. Patel, Pediatric Infectious Diseases, Div of Geographic Medicine, School of Medicine W123, 2109 Adelbert Rd, Cleveland, OH 44106; e-mail: ssp6{at}po.cwru.edu.     References Top Abstract Introduction Study design Results and discussion References 1. Miller LH, Good MF, Milon G. Malaria pathogenesis. Science. 1994;264:1878-1883[Abstract/Free Full Text]. 2. Mgone CS, Koki G, Paniu MM, et al. Occurrence of the erythrocyte band 3 (AE1) gene deletion in relation to malaria endemicity in Papua New Guinea. Trans R Soc Trop Med Hyg. 1996;90:228-231[CrossRef][Medline] [Order article via Infotrieve]. 3. Allen SJ, O'Donnell A, Alexander NDE, et al. Prevention of cerebral malaria in children in Papua New Guinea by Southeast Asian Ovalocytosis band 3. Am J Trop Med Hyg. 1999;60:1056-1060[Abstract]. 4. Booth PB, McLoughlin K. The Gerbich blood group system, especially in Melanesians. Vox Sang. 1972;22:73-84[Medline] [Order article via Infotrieve]. 5. Colin Y, Le Van Kim C, Tsapis A, et al. Human erythrocyte glycophorin C gene structure and rearrangements in genetic variants. J Biol Chem. 1989;264:3773-3780[Abstract/Free Full Text]. 6. High S, Tanner MJA, MacDonald EB, Anstee DJ. Rearrangements of the red-cell membrane glycophorin C (sialoglycoprotein beta) gene. Biochem J. 1989;262:47-54[Medline] [Order article via Infotrieve]. 7. Serjeantson SW, White BS, Bhatia K, Trent RJ. A 3.5 kb deletion in the glycophorin C gene accounts for the Gerbich-negative blood group in Melanesians. Immunol Cell Biol. 1994;72:23-27[Medline] [Order article via Infotrieve]. 8. Genton B, Al-Yaman F, Beck HP, et al. The epidemiology of malaria in the Wosera area, East Sepik Province, Papua New Guinea, in preparation for vaccine trials, I: malariometric indices and immunity. Ann Trop Med Parasitol. 1995;89:359-376[Medline] [Order article via Infotrieve]. 9. Sapak P, Sleigh A, Williams G, Peter W, Ginny M, Waranduo M. Measurement of ovalocyte frequency in peripheral blood smears in defining ovalocytosis in Papua New Guinea. Trop Med Int Health 1998;10:809-817[CrossRef]. 10. Jarolim P, Palek J, Amato D, et al. Deletion in erythrocyte band 3 gene in malaria-resistant Southeast Asian ovalocytosis. Proc Natl Acad Sci U S A. 1991;88:11022-11026[Abstract/Free Full Text]. 11. Serjeantson SW. A selective advantage for the Gerbich-negative phenotype in malarious areas of Papua New Guinea. P N G Med J. 1989;32:5-9[Medline] [Order article via Infotrieve]. 12. Zimmerman PA, Woolley I, Masinde GL, et al. Emergence of the FY*(null) in a Plasmodium vivax-endemic region of Papua New Guinea. 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	Copyright © 2001 by American Society of Hematology         Online ISSN: 1528-0020