This survey aims to specify the prevalence and the molecular footing of G6PD lack in Negrito folk of the Malaysian Orang Asli. Four hundred and eighty seven consented Negrito voluntaries were screened for G6PD lack by utilizing florescent topographic point trial. Deoxyribonucleic acid from lacking persons underwent PCR-RFLP analysis utilizing 13 recognized G6PD mutants which are prevailing either in Malaysia or Southeast Asia. When the mutant could non be identified by PCR-RFLP, the full cryptography part of the G6PD cistron and flanking noncoding DNAs were amplified and subjected to DNA sequencing. In entire, 9 % ( 44/486 ) were found to be G6PD deficient. However, merely twenty five out of 45 samples were subjected to PCR-RFLP and DNA sequencing. Of these, three samples were found to transport Viangchan, one Coimbra and 16 with combination of C1311T in exon 11 and IVS11 T93C. Mutation ( s ) for the five samples remained unknown. The average G6PD enzyme activity ranged from 0.86 – 5.7 IU/gHb in lacking persons. Our consequences show that the frequence of G6PD lack in Negrito Orang Asli is higher than among other Malayan races ( about double ) and that the most common molecular discrepancy of G6PD found in Malays and Southeast Asians are less common in Orang Asli. In add-on, the double presence of C1311T in exon 11 and IVS11 T93C in 64 % of lacking persons ( 16/44 ) , which is the highest reported rate to day of the month, might be a consequence of familial impetus in this stray group.
Keywords: G6PD ; Mutation ; Negrito ; Orang Asli ; Prevalence
The Orang Asli ( OA ) , otherwise referred to as Aboriginal people, have been populating in different parts of the Malay Peninsula for a long clip. The OA are of great involvement to anthropologists and population geneticists due to the fact that they are considered to be the likely descendants of a widespread antediluvian Southeast Asia population 1. The OA are divided into three chief 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 wellness jobs, besides malnutrition and hygienic issues, including malaria, TB, leprosy, filariasis, upper respiratory infections and tegument job 2. Specific wellness demands are catered by JHEOA ( OA section ) harmonizing to these jobs through preventative, intervention and promotional action. On the other manus, a few familial association wellness jobs have been studied. Therefore, really small information is available on the familial markers or background of the Malaysia OA and their beginnings.
G6PD lack is one of the most common human familial diseases 3. Over 400 million people around the universe are affected by some 140 different G6PD discrepancies 4. Since each population has their ain prevalence and molecular discrepancies for the G6PD cistron, it is hence a suited marker for anthropologist to analyze the human beginning and migration of these cultural groups. However, there is a big sum of published informations available on G6PD discrepancies in different Asiatic populations 5-14. Harmonizing to these publications, Viangchan, Mediterranean, Mahidol, Canton, Gaohe, Coimbra, Andalus, Orissa, Union, Chatham, Kaiping, and Vanua Lava are the most common discrepancies among the aforesaid populations.
Malaysia is a multi-racial state, with the three major cultural groups being Malay, Chinese and Indian. The lone Aboriginal people in Malay Peninsula are OA. Comprehensive surveies on the G6PD lack have preponderantly focused on the Malay and Chinese 5..
Malaria was proven to be high in OA inhabitans 15 and the complications associated with the drug intervention of malaria can be avoided if G6PD position was known 16 by the local wellness supplier. It was stated that incidence rate of malaria in OA country is 20 creases higher than urban country 17. Subsequently, we expected the recurrent rate of malaria could besides be high. Tishkoff et al 18 has reported that malaria protection is shown by persons with perennial history of malaria. Therefore, a simple G6PD showing offered to this population is critical in order to forestall future G6PD associated decease by increasing their consciousness degree.
There is really few information sing OA neonatal icterus position because bulk of them are delivered at place. However, it shown that from a sum of 65 OA admittances into the neonatal unit in one infirmary, more than half ( 33/65 ) were jaundice whereby 22 of them had G6PD lack 19. In add-on, OA community recorded the highest decease rate for kids under five old ages 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 non happen in infirmaries.
Consequently, given the deficiency of information on the G6PD lack among the OA, the purpose of the present survey was to find the prevalence, and the molecular footing of G6PD lack, which will be utile in bettering the healthcare bringing among Negrito folk of OA.
Subjects and Methods
This survey was approved by the University Kebangsaan Malaysia ( UKM ) infirmary ‘s moralss commission. All topics gave their written informed consent.
Subjects: A population showing was performed on 487 Negritos in three Malayan provinces, viz. , Kedah, Kelantan and Perak. Most of the OA small towns were visited utilizing four wheel thrust vehicle due to their location in the distant jungle of Peninsula Malaysia. This took topographic point between November 2004 and February 2008. The sub-ethnic group “ Mendriq ” was excluded from this survey as they practiced exogamies for over a decennary ago ( as a consequence of sterility ) . The age of the participants ranged from 1 to 77 old ages old.
G6PD showing and activity assay: Florescent topographic point trial was used to observe G6PD lack as this method is the most rapid and suited method for roll uping samples in geographically stray country. G6PD quantification trial was done utilizing the G6PD Kit from RANDOX Laboratory LTD ( United Kingdom ) harmonizing to the industry direction. G6PD quantification was done for 25 lacking persons who so underwent molecular survey.
Deoxyribonucleic acid elaboration and limitation enzyme analysis: Deoxyribonucleic acid was extracted utilizing the Salting Out method. However, we were merely able to roll up blood samples from 25 persons for the farther molecular survey ( including two brothers ) . These comprise of 9 females and 16 males ( 12 Lanoh, 11 Kintak, 2 Jahai ) . PCR-RFLP was undertaken for 13 mutants, viz. Viangchan, Mediterranean, Mahidol, Canton, Gaohe, Coimbra, Andalus, Orissa, Union, Chatham, Kaiping, Vanua Lava and A- as described 21-23. These mutants were selected based on their high frequence in three most prevailing cultural group in Malaysia, viz. Malay, Chinese and Indian or other Southeast Asia populations. Assuming their African beginning, the African A- mutant was selected for molecular mutational showing.
Deoxyribonucleic acid sequencing: Samples which were negative for the aforesaid mutants were subjected to direct PCR sequencing for 12 coding DNAs of G6PD cistron and flanking noncoding DNAs. The G6PD cistron sequence was obtained from NCBI ( mention sequence NC_000023.9 ) . Sequence of each coding DNA was obtained from ENSEMBL ( Transcript ENST00000393562 ) . Primers were either design by utilizing free on-line primer-BLAST plan or obtained from published informations 8. .PCR was performed harmonizing to protocol described elsewhere8. The amplified PCR merchandises was purified by utilizing 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.
Four hundred and eighty seven Negritos ( 245 males and 242 females ) were screened for the G6PD lack. Out of these, 29 males ( including two brothers ) and 16 females were found to be G6PD deficient. These consequences show that the overall prevalence of G6PD in Negrito was 9 % ( 44/486 ) ( Table 1 ) .
Table 2 shows the frequence of G6PD lack for each sub-tribe individually. The highest incidence was found among Lanoh ( 28 % ) whilst the lowest among Kensiu ( 0 % ) .
The average G6PD enzyme activity ranged from 0.86 – 5.7 IU/gHb in lacking persons.
Through the usage of the PCR-RFLP method for observing the 13 known G6PD mutants, the mutant types of 4 males were identified. Three of the males from Lanoh, two of whom were sibling, carried the Viangchan mutant and one male from Jahai was found to transport the Coimbra mutant ( Table 2 ) .
By utilizing DNA sequencing, for all cryptography coding DNAs ( 12 coding DNAs ) and flanking noncoding DNAs, 16 instances with the combined mutants 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 in conclusion two females were homozygote for both mutants. No mutant was found in the Deoxyribonucleic acid of 5 lacking persons.
Table 1 prevalence of G6PD in each folk 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 )
Entire 9 % ( 44/487 )
Table 2 Categorization 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
& A ; IVS11 T93C
Unknown 5 ( 20 % ) 1.2- 5.7
Malaysia has implemented countrywide neonatal showing for G6PD lack since 1980 but, to day of the month, no information relating to its incidence among Malayan Orang Asli ( OA ) has existed. The ground for this is because OA kids are preponderantly delivered at place. However, the incident rates of G6PD lack among Malaysia males is cited as 5.3 % with a racial dislocation of 7 % , 5 % and 3 % for Chinese, Malays and Indians males severally 5. The current survey has shown that the incidence rate of G6PD lack among Negrito is 11 % for male, and 7 % for female. Nevertheless, due to the failing of florescent topographic point trial to observe all the heterozygotes female, higher frequence of G6PDdeficiency are expected in females Negrito. While the frequence of G6PD lack in OA Negritos was higher than among other Malayan races, this determination was expected as we think it is a consequence of the high epidemic of Malaria in the OA colony country. Since persons with an familial G6PD lack are at hazard of developing anaemia if they are exposed to certain constituents such as antimalaria drugs, our consequences strongly suggest the necessity for an appropriate showing method to observe G6PD deficient OAs if any wellness plans are to be successful. As malnutrition and Fe lack are prevailing among the OA 24 the ideal testing method should be able to know apart between familial and non-hereditary signifiers of G6PD lack.
Both familial and geographical factors contribute to the increasing happening of different signifiers of G6PD lack in each population 25. Bearing this in head, the present survey is the first effort to specify the molecular discrepancy of the OA G6PD lack. Previous research has been undertaken on a really little figure of OA ; 26. Ainoon et al.27 have reported that 11 mutants are responsible for the G6PD lack in Malay and that 89 % of them carry Viangchan, Meditarranean and Mahidol. However, an absence of these mutants in 87 % of survey samples proves that the Negrito was an stray group for a period of clip. On the other manus, the Viangchan discrepancy, which was observed in three males from Lanoh, was found to be the most common mutant in Malay, Thai, Bajo ( Indonesian Aboriginal group ) , and Cambodian. The Coimbra discrepancy was observed in Malay, Sikka and Bajo ( an Indonesian Aboriginal group ) , Cambodia and Taiwan Aborigines, at a frequence less than 10 % . The happening of these two discrepancies in OA could be a consequence of crossbreeding with other groups in the distant yesteryear.
Unlike C1311T and IVS11 T93C, bulk of the known G6PD discrepancies are individual missense mutant. A few of them are dual or ternary missense mutants and little in frame omission. However, all, except the combination of C1311T and IVS11 T93C, makes alteration in protein construction by permutation of amino acid ( s ) . Individual who carried this combination is lacking even though the G6PD protein was unchanged. Significant decrease of enzyme activity and accordingly its clinical deductions have been reported for this combined mutant 28. We assumed there are other still to be identified mutant ( s ) or SNP ( s ) doing this low enzyme activity. The presence of combined mutant of C1311T and T93C in a big part of our survey samples most likely is due to the familial impetus in this little stray population over clip. This consequence 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 degrees of familial impetus.
No A- discrepancy have been observed in OA which may hold with other study that assumed A- mutant is 3840 to 11760 old ages old 18 and Negrito was said to be the earliest dweller of Malay Peninsula who migrated from Africa more than 50,000 old ages ago 29.
In drumhead, we conclude that the prevalence of G6PD is high in the Negrito and we postulate the molecular homogeneousness of G6PD mutant in this group may be a consequence of familial impetus. Further surveies are needed to bring out specific mechanism ( s ) involved with the function of C1311T and IVS11 T93C combination in G6PD lack.
We would wish to thank the JHEOA, all “ Penghulu ” and “ Tok Batin ” and the many small town folks for their most fantastic and much appreciated cooperation. Besides the Malayan Ministry of Science Technology and Innovation, Sciencefund 02-01-02-SF0316, which has made this survey possible. Thank besides to staffs and co-workers at the Faculty of Science and Technology for their firm support and contributions.