You are on page 1of 9

HUMAN MUTATION Mutation in Brief #574 (2002) O nline

MUTATION IN BRIEF

Glucose-6-Phosphate Dehydrogenase (G6PD) Variants in Malaysian Malays
1 Department 2

O. Ainoon1 ∗, Y.H. Yu1 , A.L. Amir Muhriz1 , N.Y. Boo2 , S.K. Cheong 1 , and N.H. Hamidah1
of Pathology, Faculty of Medicine, National University of Malaysia, Kuala Lumpur, Malaysia; Department of Paediatrics, Faculty of Medicine, National University of Malaysia, Kuala Lumpur, Malaysia

*Correspondence to: Dr. Othman Ainoon, Department of Pathology, Faculty of Medicine, National University of Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, Cheras, 56000, Kuala Lumpur, Malaysia; Tel.: 60391703789; Fax: 603-91737340; E-mail: ainoon@mail.hukm.ukm.my Grant sponsor: Ministry of Science, Technology and Environment, Malaysia; Grant number: IRPA GRANT NO. 06-02-02-0072.
Communicated by Mark H. Paalman

We performed DNA analysis using cord blood samples on 86 male Malay neonates diagnosed as G6PD deficiency in the National University of Malaysia Hospital by a combination of rapid PCR-based techniques, single-stranded conformation polymorphism analysis (SSCP) and DNA sequencing. We found 37.2% were 871G>A (G6PD Viangchan), 26.7% were nt 563 C>T (G6PD Mediterranean ) and 15.1% were 487G>A (G6PD Mahidol) followed by 4.7% 1376G>T (G6PD Canton), 3.5% 383T>C (G6PD Vanua Lava), 3.5% 592C>T ( G6PD Coimbra), 2.3% 1388G>A (G6PD Kaiping), 2.3% 1360C>T (G6PD Union), 2.3% 1003G>A (G6PD Chatham ), 1.2% 131C>G (G6PD Orissa) and 1.2% 1361G>A (G6PD Andalus). Seventy-one (82.6%) of the 86 G6PDdeficient neonates had neonatal jaundice. Fifty seven (80%) of the 71 neonates with jaundice required phototherapy with only one neonate progressing to severe hyperbilirubinemia (serum bilirubin >340 µmol/l) requiring exchange transfusion. There was no significant difference in the incidence of neonatal jaundice , mean serum bilirubin level, mean age for peak serum bilirubin, percentage of babies requiring phototherapy and mean number of days of phototherapy between the three common variants. In conclusion, the molecular defects of Malay G6PD deficiency is heterogeneous and G6PD Viangchan, Mahidol and Mediterranean account for at least 80% of the cases. Our findings support the observation that G6PD Viangchan and Mahidol are common Southeast Asian variants. Their presence in the Malays suggests a common ancestral origin with the Cambodians, Laotians and Thais. Our findings together with other preliminary data on the presence of the Mediterranean variant in this region provide evidence of strong Arab influence in the Malay Archipelago. © 2002 Wiley-Liss, Inc.
Key Words: Glucose-6-phosphate dehydrogenase, G6PD, molecular variants, Malays

INTRODUCTION

Glucose-6-phosphate dehydrogenase (G6PD, MIM# 305900) deficiency is the commonest enzymopathy in human estimated to affect 400 million individuals worldwide. G6PD deficient individuals are usually asymptomatic but acute haemolysis may occur with oxidative stress induced by ingestion of drugs, certain type of food, exposure to certain chemical substances or when there is accompanying infection or hypoxia. Rarely, it may cause chronic non-spherocytic haemolytic anemia. One of the most important complications of G6PD deficiency is severe neonatal hyperbilirubinemia and the risk of developing kernicterus, a problem especially seen in G6PD-deficient individuals in the Mediterranean and Asia (Beutler, 1991; Brown and Boon, 1981; Tan, Received 29 April 2002; accepted revised manuscript 29 October 2002.

© 2002 WILEY-LISS, INC. DOI: 10.1002/humu.9103

2 Ainoon et al.

1981; Fok and Lau, 1986) . To date, at least 400 biochemical and 122 molecular variants of the G6PD enzyme have been identified in various populations (Beutler, 1991). The advances in molecular techniques have allowed the molecular characterisation of the G6PD gene in any population to be carried out with ease. Previous studies have established the molecular abnormalities responsible for G6PD deficiency in several ethnic groups in Asia, including the Chinese (Chang et al., 1992; Tang et al, 1992; Chiu et al., 1993; Lo et al., 1994; Xu et al., 1995; Ainoon et al., 1999) the aboriginal tribes of Ami, Yami and Saisiat in Taiwan (Tang et al., 1995), the Javanese in Indonesia (Soemantri et al., 1995) and other Southeast Asian populations (Kuni Iwai et al., 2001). G6PD deficiency is common in Malaysia with an overall incidence of 3.1% among males and was shown to be more prevalent among ethnic Malays and Malaysian Chinese, and less common among the Indians (Singh, 1986; Alex et al., 1989). It has been shown to be an important cause of severe hyperbilirubinemia and kernicterus necessitating a national screening program for G6PD deficiency for newborns in all state hospitals, a program that has been in place for the last twenty years. We have previously reported a study of the G6PD mutations in Malaysian Chinese (Ainoon et al., 1999). To date, there has only been one report on molecular variant in Malay G6PD involving 3 cases (Iwai et al., 2001). We present here the result of a study on the molecular characterisation of G6PD deficiency in the ethnic Malays in Malaysia. Using established PCR-based techniques, SSCP and direct DNA sequencing we determined the molecular abnormalities in a group of Malay male G6PD-deficient neonates born in the National University Hospital, Kuala Lumpur.
MATERIALS AND METHODS Subjects

All male Malay neonates born in the National University of Malaysia Hospital and diagnosed as G6PDdeficient by routine screening between the period April 1999 to May 2000 were consecutively studied. EDTA cord blood samples collected for each neonate from the Labour Room for routine screening of G6PD deficiency by fluorescent spot test were used for the determination of red cell G6PD activity and DNA extraction. As routinely practised in this hospital, results of all G6PD screening tests were sent back to the ward within 24 hours of receiving the specimen and mothers of babies diagnosed as G6PD deficiency were explained by the attending Paediatrician of the problems related to the disorder and advised to have the babies kept in the Hospital for a minimum of five days to be observed for clinical jaundice. For babies who developed jaundice, peripheral blood was taken for estimation of serum bilirubin. Daily monitoring was carried out clinically and by serial estimation of serum bilirubin. Neonates with serum bilirubin level exceeding 180 µmol/l within the first 48 hours of life and exceeding 250 µmol/l were subjected to phototherapy ( Cockington, 1979). Those infants with bilirubin level >340 µmol/l would require exchange transfusion. Neonates were discharged after the fifth day when they showed both clinical and biochemical improvement with a downward trend of serum bilirubin to a level of less than 230 µmol/l. Babies born to mixed parentage, those born prematurely (<36 weeks) and those with infections or haematoma because of birth injury were excluded from the study.
G6PD activity assay

Red cell G6PD activity assays were performed in all the 87 G6PD-deficient and 335 normal neonates. Based on the principle of measurement of rate of absorbance of reduced NADP+ in red cell haemolysate, the assay was carried out at 250 C according to manufacturer’s instruction using the G6PD Kit by RANDOX Laboratories Ltd (United Kingdom). Measurement of absorbance was performed on the Hitachi Autoanalyser. All G6PD activity assays were performed within 24 hours of collection of samples.
DNA extraction.

Total genomic DNA was isolated from peripheral blood leucocytes of G6PD-deficient neonates according to manufacturer’s instruction using High Pure PCR Template Preparation Kit, Roche Diagnostics Corporation (USA).

G6DP Variants in Malaysian Malays

3

DNA amplification and restriction enzyme analysis

In analysing the DNA, we adopted the strategy of firstly, screening all the G6PD-deficient neonates for two mutations i.e nt 1376 G>T (Canton ) and nt 1388 G>A (Kaiping), previously reported to be common in the Malaysian Chinese G6PD-deficient population (Ainoon et al 1999) and nt 487 G>A (Mahidol) a mutation thought to be common in Southeast Asia population (Panich et al., 1972). Samples showing absence for these three mutations were subjected to SSCP analysis followed by direct DNA sequencing. Mutations at nt 1376 G>T, nt 1388 G>A and nt 487 G>A were detected using PCR-based technique involving amplification of target gene with oligonucleotide primers designed to create restriction sites, followed by restriction enzyme digests of PCR amplified products with Xho I, Nde I and Alu I respectively (Chang et al., 1992). The PCR reaction was optimised for each reaction by using the following concentrations: MgCl2 1.5 mmol/l, DNTP 200 umol/l, each primer 10 pmol and Taq polymerase 1.5u. The PCR was performed on the DNA thermal cycler (Gene Amp PCR System 9700, Applied Biosystems ) at denaturation 940 C 30 seconds, annealing temperature calculated according to Tm of primer pairs for 30 seconds and extension at 720 C 45 seconds. The reaction was run for 35 cycles followed by a further 10 minutes extension. The PCR-digested products were electrophoresed on 3.5% Nu-sieve agarose gel.
SSCP and DNA Sequencing

Samples which were negative for the three mutations were subjected to single stranded conformation polymorphism (PCR-SSCP) analysis using primer sequences for G6PD exons as previously described (Poggi et al., 1990). Amplifications were carried out using fluorescence-tagged primers and amplified fragments were electrophoresed on MDE (Mutation Detection Enhancement Ge l Solution with 5 % polyacrylamide; BioWhittaker Application, USA) at 350 C using the ABI PRISM 377 DNA Sequencer. The amplified DNA fragments were analysed using the software Genescan ABI PRISM 3.1. The exons that showed mobility shifts were subjected to direct sequence analysis. For samples that showed absence of mobility shift by SSCP in any of the G6PD exons, DNA sequence analysis for exons 2 of the G6PD gene were performed using primer -13 sequences similar to that used for SSCP (Poggi et al., 1990). All PCR amplifications were carried out using the DNA thermal cycler (Gene Amp PCR System 9700, Applied Biosystems). Purification of PCR product for preparation of the sequencing template were carried out using CONCERT TM Rapid Gel Extraction System (Life Technologies, USA). Cycle sequencing was performed according to the manufacturer’s instruction using ABI Prism Bigdye Terminator Cycle Sequencing Ready Reaction Kit version 2.0 (Applied Bio Systems, USA). Sequenced products were electrophoresed on 4.5% polyacrylamide gel on the automated DNA sequencer (ABI PRISM 377 Sequencer; APPLIED BIOSYSTEMS, USA) and sequence analysis was carried out using the ABI PRISM Sequence Navigator software.
Data Analysis

Data on incidence of jaundice, the age of onset of jaundice, peak serum bilirubin value, age in days when serum bilirubin peaked, birth weight, mode of treatment and days of phototherapy were analysed. Comparisons of clinical data were carried out for the groups of infants with the three most common mutations (G6PD Viangchan, Mediterranean and Mahidol) using the SPSS (Statistical Package for Social Scientist) version 10.0 (Chicago, USA). Kolmogorov-Smirnov test was used to determine normality of distribution. For comparisons of continuous variables with normal distribution (G6PD activity, serum bilirubin, age for peak bilirubin and duration of phototherapy) student’s t test was used. The Fisher’s exact test was used for comparison of discrete variables (incidence of jaundice, onset of jaundice). P value of less than 0.05 is considered significant.
RESULTS Prevalence of G6PD deficiency

A total of five thousand three hundred and sixty two (2992 males, 2370 females) newborn babies were screened for G6PD deficiency using the semiquantitative fluorescent spot test during the period of study. The overall prevalence of G6PD deficiency was 3.4% (184 of 5362). The overall prevalence of G6PD deficiency among males was 5.3% (160 of 2992) and among females was 1.05 % (25 of 2370). Out of 5362 babies 3355 were Malays, 1682 Chinese and 325 Indians. In the Malay group, the prevalence of G6PD deficiency among males was 4.6% (86 /1852) and among females 1.3% (19/1503). In the Chinese group the prevalence among

4 Ainoon et al.

males and females were 7.2% (69/956) and 0.7% (5/726) respectively. Amo ng the Indian neonates, 5/184 (2.7%) males and 1/141 (0.7%) female had G6PD deficiency.
G6PD mutations

The overall results of mutation analysis following restriction enzyme analysis, SSCP and sequence analysis of the 86 Malay subjects showed the existence of 11 different alleles. The results showed that the three most common G6PD variants in the Malays were 37.2% G6PD Viangchan (871 G>A), 26.7% G6PD Mediterranean (563 C>T) and 15.1% G6PD Mahidol (487 G>A). G6PD Canton and G6PD Kaiping, the two commonest Chinese variants seen among Malaysian Chinese were found to exist only at 4.7% and 3.5% respectively. The other variants found were 3.5% G6PD Vanua Lava (383 T>C), 3.5% G6PD Coimbra (592 C>T), 2.3% G6PD Union (1360C>T), 2.3% G6PD Chatham (1003 G>A), 1.2 % G6PD Orissa (131 C>G) and 1.2% G6PD Andalus (1361 G>A). All cases of mutation 871 G>A, mutation 563 C>T and the one case of nt 131 C>G mutation had two additional silent mutations at nt 1311 C>T and intron 11 G>T. SSCP analysis showed amplicon mobility shifts and were useful in locating the site of the following mutations: Coimbra 592 C>T, Union 1360 C>T, Orissa 131 C>G 1.2% G6PD Andalus 1361 G>A and 1.2% G6PD Chatham (1003 G>A). SSCP was not useful for G6PD Vanua Lava 383 T>C where there was no observed mobility shift of the amplified DNA fragment.
G6PD activity

G6PD activity assay showed that 82 (94%) of 86 babies had severe enzyme deficiency (< 10% mean G6PD activity for normal babies). There was no significant difference in the mean G6PD activity between the major mutations (P>0.1, student’s t test). The results of mutation analysis and G6PD assay activity for Malay G6PDdeficient neonates are shown in Table 1. Table 1. G6PD Mutations, G6PD Activity and Serum Bilirubin of G6PD-deficient Male Malay Neonates No G6PD Activity Neonatal Jaundice G6PD G6PD Of IU/gm Hb Variant Mutation Cases No. of Cases Mean peak SB∗ (%) Range Mean ±SD ( µmol/l )

Viangchan c871 G>A 32 (37.2%) 0.45 –1.03 0.74 28 (87.5%) 224.61 ± 39.6 Mediterranean c563 C>T 23 (26.7%) 0.12-0.72 0.42 17 (73.9%) 231.8 ± 63.1 Mahidol c487 G>A 13 (15.1%) 0.44-1.79 0.91 13 (100%) 212.8 ± 58.7 Canton c1376 G>T 4 (4.7%) 0.13-0.83 0.48 3 232;157; 453 Coimbra c592 C>T 3 (3.5%) Undetectable 2 218; 235 Vanua Lava c383 T>C 3 (3.5%) 0.15-0.23 0.19 2 250; 298 Kaiping c1388 G>A 2 (2.3%) 0.46-1.46 0.96 1 266 Union c1360 C>T 2 (2.3%) 0.1; 0.8 2 135 ; 127 Orissa c131 C>G 1 (1.2%) 0.74;1.28 0 Chatham c1003 G>A 2 (2.3%) 0.62; 0.86 2 339; 250 Andalus c1361 G>A 1 (1.2%) 0.98 1 243.0 Total No. 86 (100%) 71 ∗SB: peak serum bilirubin. Ninety six percent ( 83/86) neonates had severe enzyme deficiency (G6PD activity < 10% normal) with range of G6PD activity: 0 - 1.79 IU/gm Hb; (mean G6PD activity for 330 normal male neonates : 14.1 IU/gm Hb). There was no significant difference in mean G6PD activity (P>0.1 , student’s t test), incidence of jaundice (P> 0.5, Fisher’s exact test ) and mean peak serum bilirubin (P>0.5, student’s t test ) between the 3 major mutations. Neonatal jaundice

Out of 86 Malay G6PD-deficient neonates 71 (82.6%) had neonatal jaundice. Fifty seven (80%) of the 71 babies were given phototherapy. Only one neonate developed severe hyperbilirubinemia (serum bilirubin > 340 µmol/l) requiring exchange transfusion. When the neonates among the 3 groups of common variants (Viangchan, Mediterranean and Mahidol) were compared we found no significant difference in the incidence of jaundice (P>0.5, Fisher’s exact test), mean peak serum bilirubin (P>0.5 student’s t test ) (Table 1), mean age when serum bilirubin peaked (P>0.5, student’s t test ) and duration of phototherapy (P>0.3, student’s t

G6DP Variants in Malaysian Malays

5

test) (Table 2). More than 20% of babies showed serum bilirubin >250 µmol/l in all 3 common variant groups (Viangchan : 6/28 or 21.4%, Mediterranean : 4/17 or 23.5% and Mahidol: 6/13 or 46.2%). Out of the 71 babies with neonatal jaundice 26 (37.7%) developed jaundice on the second day of life (24–48 hours) and in 45 (63.3%) babies the age of onset of jaundice was after 48 hours. None was observed to develop jaundice within the first 24 hours of life. It was observed that all babies with the Mahidol mutation developed jaundice after 48 hours of life (Table 2). Table 2. Molecular Variants and Clinical Data of Male Malay G6PD-deficient Neonates G6PD variant No. of neonates Mean age for Neonates with Days of with peak serum phototherapy photoneonatal Onset of Onset of No. (%) therapy bilirubin ± jaundice jaundice jaundice SD (days) (Mean ± SD) 24-48 hrs >48 hrs
Viangchan (n=28) Mediterranean (n=17) Mahidol (n=13) Canton (n=3) 10 8 0 1 18 9 13 2 4.00 ± 1.6 3.87 ± 1.15 3.8 ± 1.15 4,2, 14 25 14 8 2 ;1 with ET ∗ 3.5 ± 2.2 4.0 ± 2.53 3.1 ± 2.6 2, 1, 14 + ET

Vanua Lava 1 1 4,9 2 2,8 (n=2) Coimbra (n=2) 1 1 4,3 2 6,4 Kaiping (n=1) 1 9 1 5 Union (n=2) 2 3,3 0 0,0 Chatham (n=2) 1 1 4,3 2 3,5 Andalus (n=1) 1 3 1 3 Orissa (n=0) Total : (n=71) 26 (37.7%) 45 (63.3%) 57 (80%) Overall mean age for peak serum bilirubin : 3.9 days of life, overall mean duration of phototherapy: 3.8 days. There is a statistically significant difference in the age of onset of jaundice between the Mahidol variants with the Mediterranean and Viangchan variant (P<0.001, P<0.01 respectively, Fisher’s exact test). ∗The only case of severe hyperbilirubinemia requiring exchange transfusion (ET). DISCUSSION Prevalence of G6PD deficiency

The population in Peninsula Malaysia is divided into indigenuous and non-indigenous ethnic groups. The indigenous group is made up of the Malay language-speaking people called the Malays whose religion is Islam and a few other numerically small but historically important groups collectively known as Orang Asli or aboriginal people. The Malays is the predominant ethnic group and makes up 58.3 % of the Malaysian population. Among the non-indigenous populations, the main communities are the Chinese estimated at 29.4% and the Indians (9.5%) and they are descendents of immigrants who arrived in the mid-nineteenth century to work in the colonial economy. G6PD deficiency is common in Malaysia and has been shown previously to be prevalent among the Malays and the Chinese. In this study, we found that the overall prevalence of G6PD deficiency among males is 5.3% with a racial breakdown of 7.2%, 4.6% and 2.7% among the Chinese, Malays and Indians males respectively, a finding comparable to previous reports (WHO 1985, Singh, 1986; Alex 1989).
G6PD mutations and the anthropological significance

We found 11 different mutations exist in the 86 male Malay G6PD-deficient neonates studied. The three most common variants were G6PD Viangchan 871 G>A (39.5%) associated with C>T mutation at nt 1311, G6PD Mediterranean 563 C>T (26.7%) and G6PD Mahidol 487 G>T (15.1%). G6PD Viangchan has been reported to be a common variant in Laos (Iwai et al., 2001) and recent reports showed a frequency of 54% in a group of G6PD-deficient Thais (Nuchprayoon et al., 2002). G6PD Mahidol is a common G6PD variant among

6 Ainoon et al.

the Thais (Panich et al., 1972; Vulliamy et al., 1989). It was reported in Laotian immigrants in Hawaii (Beutler et al., 1992) and subsequently found to be a dominant variant in Myanmar (Iwai et al., 2001). Our findings in this study appear to be the first documented report on G6PD Viangchan in the Malays. However for G6PD Mahidol, the occurence in the Malay archipelago has been reported in G6PD-deficient individuals from Central Java (Soemantri et al., 1995) and two G6PD-deficient Malaysian Malays (Iwai et al., 2001). The presence of the Viangchan variant at a relatively high frequency and the existence of the Mahidol variant in the Malays appear to reflect a common ancestral origin of the Malays with other Southeast Asian populations specifically the Thais, Laotians and Cambodians. This appear to be a significant finding in the study of Malaysian ethno-history since the facts on the origin of the indigenous people of Malaysia before the 14th century have been inconclusive. The cultural, linguistics and biological differences between the comtemporary Malays and the aboriginal people have long been debated. There are evidence of strong connection between the early inhabitants of Malaysia and the neolithic societies in Southern and Central Thailand which is supported by the fact that the aboriginal people of present day Malaysia speak Mon-Khmer related languages of the Austroasiatic linguistic family spoken by people of the Southern Indochinese mainland (Hall, 1984). Some historians believed that the comtemporary Malays and the aboriginal tribes came from the same genetic pool and the cultural, linguistics and biological differences may have developed through adaptations (Andaya et al., 2001). However, the modern Malay language spoken by the comtemporary Malays belong to the Austronesian linguistic family of people who were believed to have migrated from somewhere in the region of southern China to Taiwan, outward to the Phillipines, Borneo, Indonesia and Peninsula Malaysia. Hence, other scholars suggest that it is possible that the Malays may have the originated from these Austronesian-speaking people (Andaya et al., 2001). The presence of G 6PD Viangchan and Mahidol at high frequencies and the low occurrence of the common Chinese mutations among the Malays shown in this study do not appear to support the latter theory. Malaysia does not belong to the Indochina region, but the sharing of G6PD Viangchan and G6PD Mahidol with Laotians, Cambodians and Thais certainly lent support to the theory of genetic drifts through early population movement from the Indochina Peninsula rather than from South China. The presence of the Mediterranean variant at a high frequency in the Malays is a rather unexpected finding. G6PD Mediterranean 563 C>T mutation was first detected in a subject from Southern Italy (Kurdi- Haidar et al., 1990). Subsequently it was shown to be widely distributed among the different populations in the Mediterranean region (Kurdi –Haidar, 1990; Cappelini et al., 1996; Nafa et al., 1994) and Middle Eastern populations (Daar et al., 1996; Bayoumi et al., 1996; Rizk et al., 2000). G6PD Mediterranean 563 C>T has been found in the context of two haplotypes , one with a silent mutation C>T at nt 1311, common among Europeans (Beutler &Kuhl, 1990; De Vita et al, 1989; Kurdi-Haidar et al 1990; Vives-Cocoran et al, 1990) and the other with nt 1311C common among the people of Punjab in India (Kaeda et al, 1995). It was demonstrated that haplotypes found in the majority of Middle Eastern G6PD-deficient Mediterranean variant are the same as those found in the Italians (Kurdi-Haidar et al., 1990). G6PD Mediterranean was first reported to exist in the Malay archipelago in 31.3% of G6PD-deficient individuals in Central Java (Soemantri et al., 1995). In our study, 27% (23/85) of the Malay G6PD deficiency was caused by the Mediterranean mutation and both haplotypes exist i.e 56% (13/23) cases with the 1311T and 46% (20/23) with 1311C haplotype. The presence of the Mediterranean mutation with 1311T haplotype is most likely caused by ethnic assimilation with the Arabs who came to the Malay Archipelago in the early 9th century as traders and at the same time spread the religion Islam. The presence of the Mediterranean mutation with 1311C haplotype would suggest the effect of ethnic integration with the Indians who came to the peninsula of Malaya as migrant workers in rubber plantations in the early 19th century. The low occurrence of common Chinese mutations G6PD Canton nt 1376 G>T and G6PD Kaiping nt 1388 G>C in the Malays not only put the theory of Austronesian origin of the Malays in dispute but also revealed limited ethnic assimilation between the two groups despite the fact that the Chinese have been here for almost two centuries. In addition, six other variants were found but at much lower frequencies: G6PD Vanua Lava 383 T>C (3.4%), G6PD Coimbra 592 C>T (3.4%), G6PD Union 1360 C>T (2.3%), G6PD Chatham 1003 G>A (2.3 %), G6PD Orissa 131 C>T (1.2%) and G6PD Andalus 1361 G>A (1.2%). All these alleles have previously been found in many other populations (Ganczakowski et al., 1995; Corcoran et al., 1992; Iwai et al., 2001; Hsia et al., 1993; Hirono et al., 1995; Vulliamy et al., 1988; Kaeda et al., 1995 and Kotea et al., 1999) and it is difficult to speculate their origin. The data on these mutations which occur at low frequencies in the Malay population may be due to the different ancestral contributions to the present gene pool in multi-ethnic Malaysia or they could have arisen independently, as in various other populations.

G6DP Variants in Malaysian Malays

7

Silent mutations

All cases of G6PD Viangchan, G6PD Mediterranean and the single case of G6PD Orissa with the silent C>T mutation at nt 1311 had additional second silent mutation at nt 93 T>C in intron 11. Similar findings were reported by Dar et al. (1996) where he found the existence of intron 11 mutation in all cases of G6PD Mediterranean in Oman. The presence of these silent mutations in our G6PD population appear to support the notion that nt 1311 and intron 11 are highly polymorphic sites and the occurrence of the mutations at these sites is widespread.
Neonatal jaundice

The association of jaundice and the risk of kernicterus in G6PD-deficient Malaysian babies have been previously established (Singh, 1986, Alex et al., 1989). In this study the overall incidence of neonatal jaundice in Malay G6PD-deficient neonates was 82.6% (71 of 86) and 18 (21%) babies had peak serum bilirubin level >250 µmol/l. Comparison of mean peak serum birubin however, did not show any significant difference between the three major groups of mutations. The peak serum bilirubin value may not be representative of true values since phototherapy of the hyperbilirubinemic neonates most likely prevented the levels from reaching their natural peak. Although there is no significant difference in the incidence of jaundice between the 3 major mutations, at least 46.2% (6/13) of babies with Mahidol variant had serum bilirubin >250 µmol/l compared to 21.4% (6/28) in the Viangchan and 23.5% (4/17) Mediterranean group. However, the number is too small for any statistical comparison and a larger series of clinical study is required to determine the relationship between the different variants and severity of jaundice and whether, the Mahidol variant had a higher tendency towards hyperbilirubinemia. Perhaps we should also be looking into the coexistence of the variant UDPGT1 promoter allele associated with Gilbert’s syndrome that has been shown to be related to increase incidence and relative risk of hyperbilirubinemia in G6PD-deficient neonates (Kaplan et al., 1997). Severe hyperbilirubinemia (serum bilirubin > 340 µmol/l ) despite phototherapy was seen in only 1 out of 86 neonates. This neonate was a case of G6PD Canton, a variant known to be associated with severe hyperbilirubinemia. All the G6PD-deficient jaundiced babies contributed to late-onset hyperbilirubinemia i.e jaundice developing 24 hours after birth. An interesting observation was that all neonates with the Mahidol mutation characteristically developed jaundice after 48 hours. Perhaps this could be related to G6PD Mahidol being a class III variant. Majority of the babies had peak serum bilirubin just before the 4th day of life and the duration of phototherapy ranged from 1.5–5 days with a mean of 3.9 days. These findings strengthen the notion that G6PD-deficient infants should not be discharged before the fourth day as the danger lies in the fact that we may miss the day of peak serum bilirubin i.e when the infant is most susceptible to developing kernicterus.
Conclusion

We have characterised the molecular defects of G6PD deficiency in the Malays in Malaysia. The three most common alleles are G6PD Viangchan, G6PD Mediterranean and Mahidol causing at least 80% of G6PD deficiency in Malays. These findings, apart from reflecting the effect of ethnic assimilation support the observation that G6PD Viangchan and G6PD Mahidol are common G6PD mutations in Southeast Asia and their presence at high frequency in the Malays suggests a common ancestral origin of the Malays with other Southeast Asian populations. The presence of the Mediterranean haplotype 1311T variant provides the evidence of strong Arab influence in the Malays.
ACKNOWLEDGEMENT We thank the Kumpulan Maybank for donating the ABI PRISM 377 Automated DNA Sequencer. REFERENCES Ainoon O, Joyce J, Boo NY, Cheong SK, Zainal ZA, Hamidah NH. 1999. Glucose-6-phosphate dehydrogenase (G6PD) variants in Malaysian Chinese. Hum Mutat (Online)14 (4):352-359. Alex Tang TH, Balakrishnan S, Zamri A. 1989. Hyperbilirubinemia and erythrocyte glucose-6-phosphate dehydrogenase deficiency in Malaysian children. Med J Malaysia 44:30-4.

8 Ainoon et al.

Antonarakis SE and the Nomenclature Working Group. 1998. Recommendations for nomenclature system for the human gene mutations. Hum Mutat 11 : 1-3. Barbara W Andaya and Leonard N Andaya. 2001.A history of Malaysia. Second Edition. Palgrave Publishers Ltd. Bayoumi FA, Nur-E-Kamal, MS and Tadayyon M. 1996. Molecular characterisation of erythrocyte glucose-6-phosphate dehydrogenase deficiency in Al-Ain District, United Arab Emirate. Hum. Hered. 46:136-41. Beutler E 1991. Glucose-6-phosphate dehydrogenase deficiency. N Engl J Med 324: 169-71. Beutler, Westwood B, Kuhl W and Hsia YE. 1992(a). Glucose-6-phosphate dehydrogenase variants in Hawaii. Hum. Hered 42:327-329. Brown WR, Boon WH.1968. Hyperbilirubinemia and kernicterus in glucose-6-phosphate dehydrogenase-deficient infants in Singapore. Paediatrics. 41:1055-1062. Chang EY, Chiou SS, Perng LI, Chen TC, Liu TC, Lee LS, Chen PH & Tang TK. 1992. Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency by natural and amplification created restriction sites: five mutations account for most G6PD deficiency cases in Taiwan. Blood. 80:1079-1082. Chiu DTY, Zou L, Chen E, Chao L, Louie E, Lubin B, Liu TZ, Du CS. 1993. Molecular characterisation of glucose-6phosphate dehydrogenase (G6PD) deficiency in patients of Chinese descent and identification of new base substitutions in human Genet. 6 :60-62. Corcoran CM, Calabro V, Tamagnini G, Town M, Haidar B, Vulliamy TJ, Mason PJ & Luzzatto L.1992. Molecular heterogeneity underlying the G6PD Mediterranean phenotype. Hum Genet. 88:688-690. Daar S, Vulliamy TJ, Kaeda J, Mason PJ & Luzzatto L. 1996. Molecular characterization of G6PD deficiency in Oman. Hum Hered 46: 172-176. De Vita G, Alcalay M, Sanpietro M, Cappalenni MD, Fiorelli G & Toniolo D. 1989. Two point mutations are responsible for G6PD polymorphism in Sardinia. Am J Genet. 44:233-240. Fok T & Lau S. 1986. Glucose-6-phosphate dehydrogenase deficiency: a preventable cause of mental retardation. BMJ 292:829. Ganczkowski M, Town M , Bowden DK, Vulliamy TJ, Kaneko A, Cleg JB, Weatherall DJ & Luzzatto L. 1995. Multiple glucose-6-phosphate dehydrogenase deficient variants correlate with malarial endemicity in Vanuatu Archipelago (Southwestern Pacific) Am J Hum Genet. 56:294-301. Hall DGE.1984. Sejarah Asia Tenggara. Dewan Bahasa dan Pustaka. Kuala Lumpur. Hirono A, Ishii A, Kere N, Fuji H, Hirono K, Miwa S. 1995. Molecular analysis of glucose-6-phosphate dehydrogenase Hsia YE, Miyakawa F, Baltazar J, Ching NSP, Yuen J, Westwood B & Beutler E. 1993. Frequency of glucose-6-phosphate dehydrogenase (G6PD) mutations in Chinese, Filipinos and Laotians from Hawaii. Hum Genet. 92:470-476. Iwai K, Hirono A, Matsuoka H, Kawamoto F, Horie T, Lin K, Tantular IS, Dachlan YP, Notopuro H, Hidayah NI, Salim AMA, Fuji H, Miwa S , Ishii A. 2001. Distribution of glucose-6-phosphate dehydrogenase mutations in Southeast Asia. Hum Genet. 108 :445-449. Kaeda JS, Chotray GP, Ranjit MR, Bautista JM, Reddy PH, Stevens D, Naidu JM, Brit RP, Vulliamy TJ, Luzzatto L & Mason PJ. 1995. A new glucose-6-phosphate dehydrogenase variant, G6PD Orissa (44 ala-gly) is the major polymorphic variant in tribal populations in India. Am J Hum Genet. 57 : 1335-1341. Kotea R, Kaeda JS, Yan SL, Sem Fa N, Beesoon S, Jankee S, Ramasamy R, Vulliamy T, Bradnock RW, Bautista J, Luzzatto L, Krishnamoorthy R, Mason PJ. 1999. Three major G6PD-deficient polymorphic variants identified among the Mauritian population. Br J Haematol. 104(4):849-54. Kurdi-Haidar B, Mason PJ, Berrebi A, Ankra-Badu G, al-Ali A, Oppenheimer A & Luzzatto L. 1990. Origin and spread of glucose-6-phosphate dehydrogenase variant (G6PD Mediterranean ) in the Middle East. Am J Hum Genetic. 47:10131019.

G6DP Variants in Malaysian Malays

9

Lo YS, Lu CC, Chiou SS, Chen BH, Chang TT and Chang JG. 1994. Molecular characterisation of glucose-6-phosphat e dehydrogenase deficiency in Chinese infants with or without severe neonatal hyperbilirubinemia. Br J Haematol 86:858862. Kaplan M, Renbaum P, Levy-Lahad E, Hammerman C, Lahad A and Beutler E. 1997. Gilbert syndrome and glucose-6phosphate dehydrogenase deficiency: A dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia. Proc Natl Acad Sci 94 : 12128-12132. Nuchprayoon I, Sanpavat S, Nuchparyoon S. 2002. Glucose-6-phosphate dehydrogenase mutations in Thailand: G6PD Viangchan is the most common deficient variant in the Thai population. Hum Mutat (Online) 19 (2): 185-190 Panich V, Sungnate T, Wasi P and Na-Nakorn S. 1972. G6PD Mahidol: the most common glucose-6-phosphate dehydrogenase variant in Thailand. J Med. Assoc Thai 55:576-585. Poggi V, Town M, Foulkes NS & Luzzatto L. 1990. Identification of a single base change in a new human mutant glucose-6phosphate dehydrogenase gene by polymerase chain reaction amplification of the entire coding region from genomic DNA. Biochem J 271:157-160. Rizk SH, Aziz M, El-Ghany HMA, Mansour IM, El-Masry M, Zaied SA, El-Beshlawy A, Ibrahim IYM, Kotb T & El-Nabil H. 2000. Frequency of 563C>T mutation among a group of Egyptian paediatric patients. Lab Hematol 6:127-131. Singh H. 1986. Glucose-6-phosp hate dehydrogenase deficiency : a preventable cause of mental retardation. Br Med J 292:397-398. Soemantri AG, Saha S, Saha N & Tay JSH. 1995. Molecular variants of red cell glucose-6-phosphate dehydrogenase deficiency in Central Java, Indonesia. Hum Hered 45:346-350. Tan KL. 1981. Glucose-6-phosphate dehydrogenase status and neonatal jaundice. Arch Dis Child 56:874-7. Tang TK, Huang CS, Huang MJ, Tam KB, Yeh CH, Tang CJ. 1992. Diverse point mutations result in glucose-6-phosphate dehydrogenase (G6PD) polymorphism in Taiwan. Blood 79 :2135-2140. Vives-Corcoran JL, Kuhl W, Pujades MA & Beutler E. 1990. Molecular genetics of the glucose-6-phosphate dehydrogenase (G6PD) Mediterranean variant and description of a new G6PD mutant, G6PD Andalus (1361A) Am J Hum Genet 47:575-579. WHO Working Group. 1989. Glucose-6-phosphate dehydrogenase deficiency. WHO Bulletin OMS 67(6): 601-611.