Hereditary elliptocytosis

MASAKO KIMURA,1 YUKO SHIMIZU,1 WANNAPA SETTHEETHAM-ISHIDA,2 AUGUSTINUS SOEMANTRI,3 DANAI TIWAWECH,4 AMORNRAT ROMPHRUK,5 PHAIBOOL DUANGCHAN,6 AND TAKAFUMI ISHIDA1

Abstract Screening for a 27-bp deletion in the band 3 protein gene that causes Southeast Asian/Melanesian ovalocytosis (SAO) was carried out using the PCR method among 15 Southeast Asian populations of Thailand (Akha, Hmong, Isaan, Red Karen, White Karen, Black Lahu, Lisu, Manni, Shan, and central Thais) and Indonesia (Bugis, Dayak, Javanese, Madurian, and Toraja). Individuals with the 27-bp deletion were identified only in the Bugis of southern Sulawesi, the Dayak of southern Borneo, and Javanese of central Java. The gene frequency of the 27-bp deletion in the general population was rather low: 0.012 and 0.013 in the Dayak and the Bugis, respectively. This restricted ethnic and geographic distribution of the 27-bp deletion suggests (1) local differentiation in the prevalence of this deletion in a given ethnic group and (2) the presence of molecular heterogeneity of SAO.

Southeast Asian ovalocytosis (SAO), a hereditary trait characterized by oval-- shaped red blood cells, is inherited through an autosomal dominant mode. This condition is widespread among Southeast Asian and Oceanian populations (Lie-Injo 1976). Microscopic observation of red blood cell morphology has shown the prevalence of SAO carriers in several populations, such as in Malaysia, Borneo, Sumatra, Sulawesi, and Papua New Guinea (Amato and Booth 1977; Lie-Injo 1965,1976, Ganesan et al.1975; Sofro 1986). However, there are no standardized or objective criteria for the diagnosis of SAO. Unlike other types of hereditary elliptocytosis, carriers of SAO demonstrate little hemolysis and are basically asymptomatic (Lie-Injo 1965). Serjeantson et al. (1977) hypothesized that SAO is resistant against malaria because the distribution of SAO coincides with malaria endemic areas. Higher frequencies of SAO in adults than in the young of the same population in malaria endemic areas (Baer et al. 1976) support this hypothesis and suggest a selective advantage of SAO.

Recent studies have revealed a molecular basis of SAO (Jarolim et al. 1991; Mohandas et al. 1992; Schofield et al. 1992). SAO was first attributed to an elongated form of a major erythrocyte membrane protein, band 3 protein, because of the slower mobility of a band 3 protein fragment detected by SDS polyacrylamide gel electrophoresis (Liu et al. 1990). This fragment later turned out to be due to a point mutation called Memphis (Mueller and Morrison 1977; Jarolim et al. 1992). The Memphis mutation, which is found in human populations all over the world, does not give rise to red blood cell deformity, but it coincides with SAO (Jarolim et al. 1991).

Heterozygous presence of a 27-bp deletion was found in exon 11 of the anion exchanger 1 gene, which encodes erythrocyte band 3 protein among SAO carriers in Malaysia, the Philippines, and Papua New Guinea (Jarolim et al. 1991). The 27-bp deletion results in 9 amino acid residues deleted at the border between the transmembrane domain and the cytoplasmic domain of band 3 protein. This deletion is responsible for the partial dysfunction of band 3 protein to serve as an anion transporter and cytoskeletal protein (Moriyama et al. 1992). The 27-bp deletion in the band 3 protein gene was subsequently reported in Indonesia (Takeshima et al. 1994; Alimsardjono et al. 1997), Mauritius (Tanner et al. 1991), and South Africa (Ravindranath et al. 1994).

An interesting feature of the molecular basis of SAO is that no homozygotes for the 27-bp deletion have been found. It is conceivable that homozygous status is lethal during fetal development, because band 3 protein operates in the kidney for ion transport (Liu et al. 1994). If SAO caused by the 27-bp deletion shows resistance to malaria, heterozygotes of the 27-bp deletion are more fit than either type of homozygote in malaria endemic areas. Thus SAO provides an example of a heterozygote advantage and a balanced polymorphism.

No surveys for the prevalence of the 27-bp deletion have been done among any ethnic groups in Southeast Asia, except Papua New Guinea (Mgone et al. 1996), from the population genetic and anthropological points of view. To shed light on the distribution and possible origin of this deletion, we conducted molecular screening for this deletion mutation in several ethnic groups in Southeast Asia.

Materials and Methods

Subjects. One thousand three hundred fifty-nine individuals from 15 different groups in Southeast Asia were the subjects of this study. To represent the population, we sampled unrelated individuals, except for the Manni, whose total population size was less than 100 at the time of sampling. Blood specimens were collected after subjects gave informed consent.

In Thailand individuals from 10 different groups were studied (Table 1 and Figure 1): 169 Akha, 62 Lisu, 54 Black Lahu, 74 Shan, 112 Red Karen, 102 White Karen, 103 Hmong, 77 Isaan, 43 Manni, and 173 central Thais. Central Thais were the residents of Bangkok who had mixed ethnic origins, mainly Thai and Chinese. Isaan is the Thai name of the Lao, who speak Laotian and live in northeastern Thailand. The Manni are one of the nomadic groups who have maintained a hunting and gathering mode of life in southern Thailand (Ishida et al. 1998) and have been classified as Negritos (Martin 1905). Their language is a branch of Mon-Khumer and they have been called Sakai. The rest of the populations are so-called hill tribes in northern Thailand; they are classified into Sino-Tibetan language speaking groups, except for the Hmong, whose language belongs to the Miao-Yao branch of the ThaiAustro linguistic group (Anderson 1993).

In Indonesia individuals from five different groups were studied (Table 1 and Figure 1): 120 Bugis in southern Sulawesi, 97 Toraja in central Sulawesi, 78 Madurians from Madura Island, 42 Dayak (Kahayan group) in central Kalimantan Province of Indonesian Borneo, and 53 members of a family affected with hereditary optic neuropathy in central Java.

Preparation of Genomic DNA and Amplification of DNA by PCR. Genomic DNAs were isolated from peripheral blood lymphocytes or from Epstein-Barr virus immortalized cells by using the NaI method (Wang et al. 1994).

Amplification of genomic DNA was performed by using specific primers (5'-GGGCCCAGATGACCCTCTGC-3' for bases 1098-1117 and 5'GCCGAAGGTGATGGCGGGTG-3' for bases 1272-1253) that span the 27bp deletion (Jarolim et al. 1991). An initial denaturation at 95C for 5 min was followed by 30 cycles of denaturation at 94C for 1 min and annealing and extension at 70C for 1 min with a final extension at 70C for 5 min. Polymerase chain reaction (PCR) amplified products were size-fractionated in 2.5% agarose gel and visualized by ethidium bromide staining. The expected sizes of the PCR products are 175 bp and 148 bp for the normal and the mutant gene, respectively.

Results and Discussion

Out of IS different groups a 27-bp deletion in the band 3 gene was detected among 3 groups in Indonesia (Table 1 and Figure 1). The gene frequency of the deletion ranged between 0.0 and 0.013, with the highest frequencies occurring in the Bugis of southern Sulawesi (0.013) and in the Dayak of southern Borneo (0.012). Among the 53 individuals of the family with hereditary optic neuropathy in central Java, 6 were heterozygous for the 27-bp deletion, representing an inheritance of this deletion through 3 generations (Figure 2). There was no association between presence of the 27-bp deletion and development of optic neuropathy among them. No deletion was found among the Thailand populations, the Toraja of central Sulawesi, or the Madurians.

It was not until 1991 that the molecular basis of SAO was revealed as a 27-bp deletion in the band 3 protein gene (Jarolim et al. 1991). A number of studies on the presence of ovalocytosis in Southeast Asia and Melanesia (Malay Peninsula, Borneo, Sulawesi, and Papua New Guinea) have been reported since the 1930s. In 1939 one report on SAO in central Celebes (Sulawesi) stated that nearly half the residents, who were Toradja (Toraja), carried ovalocytosis (determined by microscopic observation) (Bonne and Sandground 1939). Provided that all these carriers of ovalocytosis were classified as having SAO, the gene frequency of the 27-bp deletion in this population would be approximately 0.25, because there were no homozygotes for this deletion. In our survey none of the 97 Toraja in central Sulawesi had the 27-bp deletion. The individuals of Toraja tested in these two studies were not identical, and the sampling sites were 120 km apart. This suggests that (1) a great difference in the gene frequency of the 27-bp deletion, even in a given ethnic group, is present and (2) not a single molecular basis contributed to the microscopically diagnosed cases of SAO in the 1930s.

We may encounter a similar phenomenon in the Dayak of Borneo. More than 10% (12.7% for Land Dayak and 9.0% for Sea Dayak) of the natives of Sarawak were diagnosed as having hereditary ovalocytosis (SAO) (microscope observation) (Ganesan et al. 1975). The frequency of SAO in Land and Sea Dayak corresponds to gene frequencies of the 27-bp deletion of 0.064 and 0.045, respectively. However, our study of the 27-bp deletion in 42 Kahayan Dayaks of central Kalimantan Province of Indonesian Borneo showed that the gene frequency is as low as 0.012.

In an earlier report Bonne and Sandground (1939) pointed out the importance of genetic studies of ovalocytosis, whereas we now emphasize the significance of the molecular basis of morphologically defined SAO.

Resistance to malaria in individuals with SAO has been documented since the 1970s by population-based statistical analyses (Baer et al. 1976; Cattani et al. 1987) and recent molecular studies (Mgone et al. 1996; Genton et al. 1995). However, inconsistent arguments, such as on parasite density and species (Serjeantson et al. 1977; Cattani et al. 1987: Foo et al. 1992), require further studies. If there are subclasses of morphologically defined SAO and if they are caused by different molecular bases, data obtained in population studies must be reconsidered based on the molecular criteria; otherwise, the conclusions are indeterminate. Diagnosis of SAO morphologically without confirming the 27-bp deletion may have affected evaluations of resistance to malaria in SAO individuals. In fact, among Indonesian islanders we found no significant difference in the parasite species between malaria-- infected individuals with and without the 27-bp deletion (data not shown).

During our study, we found some cases of red blood cell deformity without the 27-bp deletion that were indistinguishable from SAO under the microscope. On the other hand, individuals with the 27-bp deletion had oval-- ocytic erythrocytes. As mentioned, the presence of molecular heterogeneity of SAO is indicated, and the geographic range of molecularly defined SAO may fall within the range of morphologically defined SAO. The 27-bp deletion in the erythrocyte band 3 gene has thus far been reported in Indonesia, the Philippines, Malaysia, Papua New Guinea, Mauritius, and South Africa (Jarolim et al. 1991; Takeshima et al. 1994; Alimsardjono et al. 1997; Ravindranath et al. 1994; Mgone et al. 1996). In addition to these reports, the presence of the deletion in southern Thailand (C. Nopparatana, personal communication, 1996; Kimura et al., unpublished data, 1998) and the absence of the deletion in central, northern, and northeastern Thailand and in Taiwan (unpublished data) indicate the northern limits of the ethnogeographic distribution of this deletion: one between southern and central Thailand and the other between the Philippines and Taiwan.

The absence and the low frequencies of the 27-bp deletion in Thai and Indonesian populations suggest the presence of molecular heterogeneity of SAO (morphologically defined) and local heterogeneity in gene frequency of the 27-bp deletion in a given ethnic group. In some populations studied here the number of individuals may be too small to exclude the possible presence of the deletion among them. Further molecular epidemiologic surveys for band 3 mutations in other parts of Southeast Asia would contribute to the understanding of not only the origin and the distribution of the 27-bp deletion but also the role of this mutation in the genetic adaptation to malaria, other than Duffy blood groups, G6PD deficiency, and hemoglobinopathies.

Acknowledgments We are grateful to Prathom and Patra Nuankum, Mae Hong Sorn Health Centre, and Hiroki Oota, University of Tokyo, for their assistance with the sampling. We also thank Yupa Urwijitaroon, Chanvit Leelayuwat, Suporto, Susanto, Marbaniati, and Yvonne Sukri for their generous arrangements for this study. This research was supported by the Japan Society for Promotion of Science, Monbusho, and the University of Tokyo.

Received 20 January 1998; revision received 13 April 1998.

Literature Cited

Alimsardjono, H., I.S. Mukono, Y.P. Dachlan et al.1997. Deletion of twenty-seven nucleotides within exon 11 of the band 3 gene identified in ovalocytosis in Lombok Island, Indonesia. Jpn. J. Hum. Genet. 42:233-236.

Amato, D., and P.B. Booth. 1977. Hereditary ovalocytosis in Melanesians. Papua New Guinea Med. J. 20:2632.

Anderson, E.F. 1993. Plants and People of the Golden Triangle. Bangkok, Thailand: Silkworm Books.

Baer, A., L.E. Lie-Injo, Q.B. Welch et al. 1976. Genetic factors and malaria in the Temuan. Am. J. Hum. Genet. 28:179-188.

Bonne, C., and J.H. Sandground. 1939. Echinostomiasis in Celebes veroorzaakt door het eten van zoetwatermosselen. Geneeskd. Tijdschr. Ned. Indie 79:3016-3034. Cattani, J.A., F.D. Gibson, M.P. Alpers et al. 1987. Hereditary ovalocytosis and reduced susceptibility to malaria in Papua New Guinea. Trans. R. Soc. Trop. Med. Hyg. 81:705709.

Foo, L.C., V. Rekhraj, G.L. Chiang et al. 1992. Ovalocytosis protects against severe malaria parasitemia in the Malayan aborigines. Am. J. Trop. Med. Hyg. 47:271-275. Ganesan, J., L.E. Lie-Injo, and O.B. Poon. 1975. Abnormal hemoglobins, glucose-6-phosphate dehydrogenase deficiency, and hereditary ovalocytosis in the Dayaks of Sarawak. Hum. Hered. 25:258-262.

Genton, B., F. Al-Yaman, C.S. Mgone et al. 1995. Ovalocytosis and cerebral malaria. Nature

378:564-565.

Ishida, T., J. Suzuki, P. Duangchan et al. 1998. Preliminary report on the short stature of Southeast Asian forest dwellers, the Manni, in southern Thailand: Lack of an adolescent

spurt in plasma IGF-I concentration. Southeast Asian J. Trop. Med. Public Health 29 (in press).

Jarolim, P., J. Palek, D. Amato et al. 1991. Deletion in erythrocyte band 3 gene in malaria-- resistant Southeast Asian ovalocytosis. Proc. Natl. Acad. Sci. USA 88:11,022-11,026. Jarolim, P., H.L. Rubin, S. Zhai et al. 1992. Band 3 Memphis: A widespread polymorphism with abnormal electrophoretic mobility of erythrocyte band 3 protein caused by substitution AAG - GAG (Lys -> Glu) in codon 56. Blood 80:1592-1598. Lie-Injo, L.E. 1965. Hereditary ovalocytosis and hemoglobin E ovalocytosis in Malayan aborigines. Nature 208:1329.

Lie-Injo, L.E. 1976. Genetic relationships of several aboriginal groups in Southeast Asia. In The Origin of the Australians, R.L. Kirk and A.G. Thorne, eds. Human Biology Series 6, Australian Institute of Aboriginal Studies. Canberra, Australia: Humanities Press, 277306.

Liu, S.-C., P. Jarolim, H. Rubin et al. 1994. The homozygous state for the band 3 protein mutation in Southeast Asian ovalocytosis may be lethal. Blood 84:3590-3591. Liu, S.-C., S. Zhai, J. Palek et al. 1990. Molecular defect of the band 3 protein in the human

erythrocyte membrane. New Engl. J. Med. 323:1530-1538. Martin, R. 1905. Die Inlandstamme der Malayischen Halbinsel. Jena, Germany: Verlag von Gustav Fischer.

Mgone, C.S., G. Koki, M.M. Paniu et al. 1996. Occurrence of the erythrocyte band 3 (AEI) gene deletion in relation to malaria endemicity in Papua New Guinea. Trans. R. Soc. Trop. Med. Hyg. 90:228-231.

Mohandas, N., R. Winardi, D. Knowles et al. 1992. Molecular basis for membrane rigidity of

hereditary ovalocytosis. J. Clin. Invest. 89:686-692.

Moriyama, R., H. Ideguchi, C.R. Lombordo et al. 1992. Structural and functional characterization of band 3 from Southeast Asian ovalocytosis. J. Biol. Chem. 267:25,792-25,797. Mueller, T.J., and M. Morrison. 1977. Detection of a variant of protein 3, the major transmem

brane protein of the human erythrocyte. J. Biol. Chem. 252:6573-6576. Ravindranath, Y., G. Goyette Jr., and R.M. Johnson. 1994. Southeast Asian ovalocytosis in an African American family. Blood 84:2823-2824.

Schofield, A.E., M.J.A. Tanner, J.C. Pinder et al. 1992. Basis of unique red cell membrane

properties in hereditary ovalocytosis. J. Molec. Biol. 223:949-958. Serjeantson, S., K. Bryson, D. Amato et al. 1977. Malaria and hereditary ovalocytosis. Hum. Genet. 37:161-167.

Sofro, A.S. 1986. Ovalocytosis in Indonesia. Medika 10:954958. Takeshima, Y., A.S. Sofro, P. Suryantoro et al. 1994. Twenty-seven nucleotide deletion within exon I I of the erythrocyte band 3 gene in Indonesian ovalocytosis. Jpn. J. Hum. Genet. 39:181-185.

Tanner, M.J.A., L. Bruce, P.G. Martin et al. 1991. Melanesian hereditary ovalocytes have a

deletion in red cell band 3. Blood 78:2785-2786.

Wang, L., K. Hirayasu, M. Ishizawa et al. 1994. Purification of genomic DNA from human whole blood by isopropanol-fractionation with concentrated Nal and SDS. Nucleic Acids Res. 22:1774-1775.

1 Human Genetics Unit, Department of Biological Sciences, School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.

2 Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.

3 Department of Child Health, Faculty of Medicine, Diponegoro University, Semarang, Indonesia. 4 Research Division, National Cancer Institute, Bangkok, Thailand.

5 Blood Transfusion Centre, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. 6 Srinakharinwirot University, Bangkok, Thailand.

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