View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Okayama University Scientific Achievement Repository Acta Med. Okayama, 2010 Vol. 64, No. 6, pp. 367ﾝ373 CopyrightⒸ 2010 by Okayama University Medical School. Original Article http ://escholarship.lib.okayama-u.ac.jp/amo/ Incidence and Mutation Analysis of Glucose-6-Phosphate Dehydrogenase Deficiency in Eastern Indonesian Populations Indah S. Tantulara,b, Hiroyuki Matsuokac, Yuichi Kasaharac, Suhintam Pusarawatia,b, Toshio Kanbed, Josef S. B. Tudae, Yasutoshi Kidof, Yoes P. Dachlana,b, and Fumihiko Kawamotof＊ aInstitute of Tropical Disease and bDepartment of Parasitology, Faculty of Medicine, Airlangga University, Surabaya, Indonesia, cDivision of Medical Zoology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan, dDivision of Molecular Mycology and Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan, eDepartment of Parasitology, Faculty of Medicine, Sam Ratulangi University, Manado, Indonesia, and fDivision of International Health, Oita University Faculty of Medicine, Yufu, Oita 879-5593, Japan We conducted a field survey of glucose-6-phosphate dehydrogenese (G6PD) deficiency in the eastern Indonesian islands, and analyzed G6PD variants molecularly. The incidence of G6PD deficiency in 5 ethnic groups (Manggarai, Bajawa, Nage-Keo, Larantuka, and Palue) on the Flores and Palue Islands was lower than that of another native group, Sikka, or a nonnative group, Riung. Molecular analysis of G6PD variants indicated that 19 cases in Sikka had a frequency distribution of G6PD variants simi- lar to those in our previous studies, while 8 cases in Riung had a different frequency distribution of G6PD variants. On the other hand, from field surveys in another 8 ethnic groups (Timorese, Sumbanese, Savunese, Kendari, Buton, Muna, Minahasa, and Sangirese) on the islands of West Timor, Sumba, Sulawesi, Muna and Bangka, a total of 49 deficient cases were detected. Thirty-nine of these 49 cases had G6PD Vanua Lava (383T＞C) of Melanesian origin. In our previous studies, many cases of G6PD Vanua Lava were found on other eastern Indonesian islands. Taken together, these findings may indicate that G6PD Vanua Lava is the most common variant in eastern Indonesian populations, except for Sikka. Key words: Glucose-6-phosphate dehydrogenase deficiency, rapid G6PD test, eastern Indonesian population, molecular analysis, G6PD Vanua Lava n malaria-endemic areas in Southeast Asian malarial drugs on-site, including primaquine. I countries, we have been introducing 2 rapid However, if G6PD-deficient individuals take pri- diagnostic methods for malaria [1, 2] and glucose-6- maquine, a hemolytic attack sometimes occurs by phosphate dehydrogenase (G6PD) deficiency [3, 4]. oxidant stress. Thus, primaquine should not be Using these 2 methods, malaria patients can be administered to malaria patients before confirming informed of the diagnostic results within 30 min after their G6PD condition. In order to transfer these the blood examination, and they are prescribed anti- diagnostic techniques to Southeast Asia, we have been conducting field surveys for malaria and G6PD defi- ciency [5-12] using local staffs as part of their train- Received June 3, 2010 ; accepted July 28, 2010. ＊Corresponding author. Phone : ＋81ﾝ97ﾝ586ﾝ6741; Fax : ＋81ﾝ97ﾝ586ﾝ6741 ing. E-mail : [email protected] (F. Kawamoto) G6PD deficiency is one of the most frequent 368 Tantular et al. Acta Med. Okayama Vol. 64, No. 6 hereditary disorders in the world, and is distributed □ 2 ○ 2 △ 1 ■ 2 ☆ 1 ☆ 1 throughout tropical areas in particular. The open reading frame of this gene is 1,545 base pairs in length, encoded in 13 exons. Most G6PD deficiency is caused by a single nucleotide mutation, resulting in □ 1 ■ 1 one amino-acid change among 515 amino acids. To □ 3 △ 9 ■ 1 ▲ 16 ☆ 23 □ 1 ■ 1 ☆ 1 date, more than 400 G6PD biochemical variants have □ 9 ▲ 1 ♢ 2 ☆ 2 been described, and at least 140 mutations of G6PD △ 1 deficiency have been discovered [13, 14]. □ Vanua Lava (383 T＞C) ● Mahidol (487 G＞A) ○ Mediterranean (563 C＞T) △ Coimbra (592 C＞T) ■ Viangchan (871 G＞A) ▲ Chatham (1003 G＞A) In Indonesia, malaria endemic areas have been ♢Chineseﾝ5 (1024 C＞T) ♤Canton (1376 G＞T) ☆Kaiping (1388 G＞A) widely eradicated, but malaria is still a serious infec- Fig. 1 A detailed map showing the major ethnic groups on the tious disease, particularly in eastern Indonesia and Flores and Palue Islands, and the distribution and frequencies of Kalimantan. In our previous surveys on Flores Island G6PD variants in each group. Numerals indicate the numbers of in eastern Indonesia [5, 9], we detected many G6PD- G6PD-deficient cases confirmed by sequence analysis. deficient individuals as well as malaria patients in 3 ethnic groups, the Sikka, Ende (Ende-Lio) and Bajo populations, and it was interesting that these groups □ 3 ■ 1 had their own frequency distributions of G6PD vari- □ 2 ants. In Flores Island (and Palue Island), 6 more □ 5 ethnic groups, i. e., the Manggarai, Bajawa (or Ngada), Riung, Nage-Keo, Palue and Larantuka (or Lamahorot), are present (Fig. 1), and we further surveyed malaria and G6PD deficiency among these □ 2 groups. In addition, we also surveyed malaria and G6PD deficiency in other eastern Indonesian islands, □ 6 such as West Timor Island, Sumba Island, the □ 1 Southeastern Sulawesi and Muna Islands, and the Northern Sulawesi and Bangka Islands (Fig. 2), and compared their G6PD variants molecularly. □ 27 ■ 6 ▲ 2 ☆ 1 Here, we report that G6PD Vanua Lava is the □ 4 most dominant variant among eastern Indonesian popu- □ Vanua Lava (383 T＞C) ● Mahidol (487 G＞A) ○ Mediterranean (563 C＞T) △ Coimbra (592 C＞T) ■ Viangchan (871 G＞A) ▲ Chatham (1003 G＞A) lations, while the Sikka and the Riung groups each ♢Chineseﾝ5 (1024 C＞T) ♤Canton (1376 G＞T) ☆Kaiping (1388 G＞A) possess many G6PD variants not found in the other Fig. 2 A map showing our study sites in eastern Indonesia, and groups. the distribution and frequencies of G6PD variants in various popula- tions. Numerals indicate the numbers of G6PD-deficient cases Materials and Methods confirmed by sequence analysis. This study was approved by the Ethical Committees of the Health Departments of all regencies Nggelek villages near Lembor (West Manggarai and of Airlangga University, Indonesia, and by the Regency), the Reo village in Riung, the Tiworiwu Ethical Committees of the Oita University Faculty of village in Bajawa (Ngada Regency), the Tonggo village Medicine and Jichi Medical University, Japan. The in Nangaroro (Nage-Keo Regency), and the Waimana, molecular epidemiological data for malaria infection Waiklibang and Lewolaga villages in Larantuka (East obtained in this study will be reported elsewhere. Flores Regency), Flores Island, and at the Field surveys and mutation analysis. In the Reruwarere village, Palue Island (Sikka Regency) East Nusa Tenggara Province, we surveyed malaria from June 2005 to April 2006, and compared them and G6PD deficiency at the Longgot and Wae Nakeng with the incidence of malaria and G6PD deficiency in villages near Labuan Bajo, the Ngedukelu and a Sikka village (Pruda), 40km east from Maumere December 2010 G6PD Variants in Eastern Indonesia 369 city. We also surveyed at the Baturutu, Panite and analysis (see Tables 1 and 3). Maiskolen villages near Soe and Oebobo (South When malaria patients or G6PD-deficient individu- Central Timor Regency), West Timor Island, on als were detected, informed consent was obtained once August 2004. In addition, we surveyed at the again, and 0.2～2.0ml of venous blood was taken for Taimanu, Uma Mapu and Protwora villages near further molecular analysis. G6PD activity in collected Waingapu (East Sumba Regency), Sumba Island, on blood samples was re-confirmed at a laboratory in June 2006. On this island, 2 populations (Sumbanese Surabaya by another G6PD test [15]. This method is and Savunese) are distributed. useful for testing hundreds of blood samples per day, In the Southeast Sulawesi Province, we surveyed although it is very difficult to distinguish heterozygous at the Lambuya village near Kendari (Kendari female samples from normal samples. Regency) and Katobu village in Raha (Muna Regency), G6PD mutation was identified by sequencing both Southeastern Sulawesi Island, and at the Lamaeo strands of the G6PD gene: Genomic DNA was village in Mendo-Wuna (Muna Regency), Muna Island, extracted from 0.1ml of G6PD-deficient blood samples on August 2004. In the North Sulawesi Province, we using a DNA purification kit (Amersham Pharmacia also surveyed at the Kaheka, Libas and Kumersot Biotech, Buckinghamshire, UK). Then, DNA was villages, Bangka Island, and at the Apela, Wolaang, amplified by PCR with 10 sets of primers [6, 10], Noongan, Gmim Kal and Kalooran villages in Tonbatu and the DNA sequences were read by an ABI PRISM (Minahasa Regency), Northern Sulawesi Island, on 310 (Applied Biosystems, Foster City, CA, USA). August 2005 and March 2006. Both strands of each exon were sequenced. We also Informed consent was obtained from each volunteer read some introns of G6PD because silent mutations before diagnoses of malaria and G6PD deficiency. had been found on introns of genomic G6PD; e. g. nt Volunteers were first registered by name, age, gen- 175C＞T on intron 7, nt 163C＞T on intron 8 [16] der, and ethnicity, and then, 3 drops of blood were and nt93T＞C on intron 11 [17]. collected from the fingertip [9]: one for malaria diagnosis, one for hemoglobin concentration, and one Results and Discussion for G6PD test. Malaria was diagnosed by the acridine orange staining method [1, 2], and hemoglobin con- Field surveys and mutation analysis. On centration was measured using a battery-powered, Flores and Palue Islands (Fig. 1), we surveyed 8 HemoCue machine (Angelhorm, Sweden). major ethnic groups, i. e., the Manggarai, Bajawa, For screening of G6PD deficiency, we used a Riung, Nage-Keo, Ende (Ende-Lio), Palue, Sikka and modified method in this study by reducing the volume Larantuka populations.