This study aims to define the prevalence and the molecular basis of G6PD deficiency in Negrito tribe of the Malaysian Orang Asli. Four hundred and eighty seven consented Negrito volunteers were screened for G6PD deficiency by using florescent spot test. DNA from deficient individuals underwent PCR-RFLP analysis using thirteen recognized G6PD mutations which are prevalent either in Malaysia or Southeast Asia. When the mutation could not be identified by PCR-RFLP, the entire coding region of the G6PD gene and flanking introns were amplified and subjected to DNA sequencing. In total, 9% (44/486) were found to be G6PD deficient. However, only twenty five out of 45 samples were subjected to PCR-RFLP and DNA sequencing. Of these, three samples were found to carry Viangchan , one Coimbra and 16 with combination of C1311T in exon 11 and IVS11 T93C . Mutation(s) for the five samples remained unknown. The mean G6PD enzyme activity ranged from 0.86 - 5.7 IU/gHb in deficient individuals. Our results show that the frequency of G6PD deficiency in Negrito Orang Asli is higher than among other Malaysian races (almost double) and that the most common molecular variant of G6PD found in Malays and Southeast Asians are less common in Orang Asli. In addition, the dual presence of C1311T in exon 11 and IVS11 T93C in 64% of deficient individuals (16/44), which is the highest reported rate to date, might be a result of genetic drift in this isolated group.
Keywords: G6PD; Mutation; Negrito; Orang Asli; Prevalence
Introduction
The Orang Asli (OA), otherwise referred to as aboriginal people, have been living in different regions of the Malay Peninsula for a long time. The OA are of great interest to anthropologists and population geneticists due to the fact that they are considered to be the likely descendents of a widespread ancient Southeast Asia population 1. The OA are divided into three main tribal groups: Negrito, Senoi, and Proto-Malay. The Negrito is the smallest group of OA with a population of 4,851. Negrito comprise of 6 sub-ethnic groups; Kensiu, Kintak, Lanoh, Jahai, Bateq and Mendriq. Nevertheless, OA have several health problems, besides malnutrition and hygienic issues, including malaria, tuberculosis, leprosy, filariasis, upper respiratory infections and skin problem 2. Specific health needs are catered by JHEOA (OA department) according to these problems through preventive, treatment and promotional action. On the other hand, a few genetic association health problems have been studied. Therefore, very little information is available on the genetic markers or background of the Malaysia OA and their origins.
G6PD deficiency is one of the most common human hereditary diseases 3. Over 400 million people around the world are affected by some 140 different G6PD variants 4. Since each population has their own prevalence and molecular variants for the G6PD gene, it is therefore a suitable marker for anthropologist to study the human origin and migration of these ethnic groups. However, there is a large amount of published data available on G6PD variants in different Asian populations 5-14. According to these publications, Viangchan, Mediterranean, Mahidol, Canton, Gaohe, Coimbra, Andalus, Orissa, Union, Chatham, Kaiping, and Vanua Lava are the most common variants among the aforementioned populations.
Malaysia is a multi-racial country, with the three major ethnic groups being Malay, Chinese and Indian. The only aboriginal people in Malay Peninsula are OA. Comprehensive studies on the G6PD deficiency have predominantly focused on the Malay and Chinese 5..
Malaria was proven to be high in OA inhabitans 15 and the complications associated with the drug treatment of malaria can be avoided if G6PD status was known 16 by the local health provider. It was stated that incidence rate of malaria in OA area is 20 folds higher than urban area 17. Subsequently, we expected the recurrent rate of malaria could also be high. Tishkoff et al 18 has reported that malaria protection is shown by individuals with recurrent history of malaria. Therefore, a simple G6PD screening offered to this population is vital in order to prevent future G6PD associated death by increasing their awareness level.
There is very few information regarding OA neonatal jaundice status because majority of them are delivered at home. However, it shown that from a total of 65 OA admissions into the neonatal unit in one hospital, more than half (33/65) were jaundice whereby 22 of them had G6PD deficiency 19. In addition, OA community recorded the highest death rate for children under five years old in Malaysia 20. The rate was more than 2 times higher than the national rate of 0.6 and more than half of it (59%) did not occur in hospitals.
Consequently, given the lack of information on the G6PD deficiency among the OA, the aim of the present study was to determine the prevalence, and the molecular basis of G6PD deficiency, which will be useful in improving the healthcare delivery among Negrito tribe of OA.
Subjects and Methods
This study was approved by the University Kebangsaan Malaysia (UKM) hospital's ethics committee. All subjects gave their written informed consent.
Subjects: A population screening was performed on 487 Negritos in three Malaysian states, namely, Kedah, Kelantan and Perak. Most of the OA villages were visited using four wheel drive vehicle due to their location in the remote jungle of Peninsula Malaysia. This took place between November 2004 and February 2008. The sub-ethnic group "Mendriq" was excluded from this study as they practiced intermarriages for over a decade ago (as a result of infertility). The age of the participants ranged from 1 to 77 years old.
G6PD screening and activity assay: Florescent spot test was used to detect G6PD deficiency as this method is the most rapid and suitable method for collecting samples in geographically isolated area. G6PD quantification test was done using the G6PD Kit from RANDOX Laboratory LTD (United Kingdom) according to the manufacture instruction. G6PD quantification was done for 25 deficient individuals who then underwent molecular study.
DNA amplification and restriction enzyme analysis: DNA was extracted using the Salting Out method. However, we were only able to collect blood samples from 25 individuals for the further molecular study (including two brothers). These comprise of 9 females and 16 males (12 Lanoh, 11 Kintak, 2 Jahai). PCR-RFLP was undertaken for 13 mutations, namely Viangchan, Mediterranean, Mahidol, Canton, Gaohe, Coimbra, Andalus, Orissa, Union, Chatham, Kaiping, Vanua Lava and A- as described 21-23. These mutations were selected based on their high frequency in three most prevalent ethnic group in Malaysia, namely Malay, Chinese and Indian or other Southeast Asia populations. Assuming their African origin, the African A- mutation was selected for molecular mutational screening.
DNA sequencing: Samples which were negative for the aforementioned mutations were subjected to direct PCR sequencing for 12 exons of G6PD gene and flanking introns. The G6PD gene sequence was obtained from NCBI (reference sequence NC_000023.9). Sequence of each exon was obtained from ENSEMBL (Transcript ENST00000393562). Primers were either design by using free online primer-BLAST program or obtained from published data 8. .PCR was performed according to protocol described elsewhere8. The amplified PCR products was purified by using QiaGene PCR Purification Kit and sent for DNA automated sequencing for both strands on the ABI PRISM 377 DNA Sequencer, with the sequencing service provided by 1st Base Laboratories, Malaysia.
Results
Four hundred and eighty seven Negritos (245 males and 242 females) were screened for the G6PD deficiency. Out of these, 29 males (including two brothers) and 16 females were found to be G6PD deficient. These results show that the overall prevalence of G6PD in Negrito was 9% (44/486) (Table 1).
Table 2 shows the frequency of G6PD deficiency for each sub-tribe separately. The highest incidence was found among Lanoh (28%) whilst the lowest among Kensiu (0%).
The mean G6PD enzyme activity ranged from 0.86 - 5.7 IU/gHb in deficient individuals.
Through the use of the PCR-RFLP method for detecting the 13 known G6PD mutations, the mutation types of 4 males were identified. Three of the males from Lanoh, two of whom were sibling, carried the Viangchan mutation and one male from Jahai was found to carry the Coimbra mutation (Table 2).
By using DNA sequencing, for all coding exons (12 exons) and flanking introns, 16 cases with the combined mutations of C1311T in exon 11 and IVS11 nt T93C were found. Three of the females were heterozygote for C1311T and heterozygote for IVS11 T93C; two others were heterozygote for C1311T and homozygote for IVS11 T93C; and lastly two females were homozygote for both mutations. No mutation was found in the DNA of 5 deficient individuals.
Table 1 prevalence of G6PD in each tribe of Negrito
Tribe % G6PD (Deficient/Total)
Jahai 3% (5/170)
Kintak 18% (11/63)
Lanoh 28% (18/68)
Kensiu 0% (0/118)
Bateq 15% (10/67)
Total 9% (44/487)
Table 2 Classification of molecular analysis of 25 G6PD deficient Negrito
G6PD Variants Number of G6PD activity
deficient (%)
Viangchan 3 (12%) 1.2
Coimbra 1 (4%) 0.86
Combined C1311T 16 (64%) 1.8 - 4.8
& IVS11 T93C
Unknown 5 (20%) 1.2- 5.7
Discussion
Malaysia has implemented nationwide neonatal screening for G6PD deficiency since 1980 but, to date, no data relating to its incidence among Malaysian Orang Asli (OA) has existed. The reason for this is because OA children are predominantly delivered at home. However, the incident rates of G6PD deficiency among Malaysia males is cited as 5.3% with a racial breakdown of 7%, 5% and 3% for Chinese, Malays and Indians males respectively 5. The current study has shown that the incidence rate of G6PD deficiency among Negrito is 11% for male, and 7% for female . Nevertheless, due to the weakness of florescent spot test to detect all the heterozygotes female, higher frequency of G6PDdeficiency are expected in females Negrito. While the frequency of G6PD deficiency in OA Negritos was higher than among other Malaysian races, this finding was expected as we think it is a result of the high epidemic of Malaria in the OA settlement area. Since individuals with an inherited G6PD deficiency are at risk of developing anemia if they are exposed to certain components such as antimalaria drugs, our results strongly suggest the necessity for an appropriate screening method to detect G6PD deficient OAs if any health programs are to be successful. As malnutrition and iron deficiency are prevalent among the OA 24 the ideal screening method should be able to discriminate between hereditary and non-hereditary forms of G6PD deficiency.
Both genetic and geographical factors contribute to the increasing occurrence of different forms of G6PD deficiency in each population 25. Bearing this in mind, the present study is the first attempt to define the molecular variant of the OA G6PD deficiency. Previous research has been undertaken on a very small number of OA; 26. Ainoon et al.27 have reported that 11 mutations are responsible for the G6PD deficiency in Malay and that 89% of them carry Viangchan, Meditarranean and Mahidol. However, an absence of these mutations in 87% of study samples proves that the Negrito was an isolated group for a period of time. On the other hand, the Viangchan variant, which was observed in three males from Lanoh, was found to be the most common mutation in Malay, Thai, Bajo (Indonesian aboriginal group), and Cambodian. The Coimbra variant was observed in Malay, Sikka and Bajo (an Indonesian aboriginal group), Cambodia and Taiwan aboriginals, at a frequency less than 10%. The occurrence of these two variants in OA could be a result of interbreeding with other groups in the distant past.
Unlike C1311T and IVS11 T93C, majority of the known G6PD variants are single missense mutation. A few of them are double or triple missense mutations and small in frame deletion. However, all, except the combination of C1311T and IVS11 T93C, makes change in protein structure by substitution of amino acid(s). Individual who carried this combination is deficient even though the G6PD protein was unchanged. Significant reduction of enzyme activity and consequently its clinical implications have been reported for this combined mutation 28. We assumed there are other still to be identified mutation(s) or SNP(s) causing this low enzyme activity. The presence of combined mutation of C1311T and T93C in a large portion of our study samples most probably is due to the genetic drift in this small isolated population over time. This result is in line with Hill et al.29 who analyzed mitochondrial DNA control-region and coding-region markers in OA and concluded that all of the OA groups have undergone high levels of genetic drift.
No A- variant have been observed in OA which may agree with other report that assumed A- mutation is 3840 to 11760 years old 18 and Negrito was said to be the earliest inhabitant of Malay Peninsula who migrated from Africa more than 50,000 years ago 29.
In summary, we conclude that the prevalence of G6PD is high in the Negrito and we postulate the molecular homogeneity of G6PD mutation in this group may be a result of genetic drift. Further studies are needed to uncover specific mechanism(s) involved with the role of C1311T and IVS11 T93C combination in G6PD deficiency.
Acknowledgments
We would like to thank the JHEOA, all "Penghulu" and "Tok Batin" and the many village folks for their most wonderful and much appreciated cooperation. Also the Malaysian Ministry of Science Technology and Innovation, Sciencefund 02-01-02-SF0316, which has made this study possible. Thank also to staffs and colleagues at the Faculty of Science and Technology for their unwavering support and donations.
