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Published by Effandi Abu Bakar

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Published by: Effandi Abu Bakar on May 26, 2011
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HUMAN MUTATION Mutation in Brief #574 (2002) Online
WILEY-LISS, INC.DOI: 10.1002/humu.9103
 Received 29 April 2002; accepted revised manuscript 29 October 2002.
Glucose-6-Phosphate Dehydrogenase (G6PD)Variants in Malaysian Malays
O. Ainoon
, Y.H. Yu
, A.L. Amir Muhriz
, N.Y. Boo
, S.K. Cheong
, and N.H. Hamidah
 Department 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 Universityof Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, Cheras, 56000, Kuala Lumpur, Malaysia; Tel.: 603-91703789; Fax: 603-91737340; E-mail: ainoon@mail.hukm.ukm.my
Grant sponsor:
Ministry of Science, Technology and Environment
Malaysia; Grant number: IRPA GRANTNO. 06-02-02-0072.
Communicated by Mark H. Paalman
We performed DNA analysis using cord blood samples on 86 male Malay neonatesdiagnosed as G6PD deficiency in the National University of Malaysia Hospital by acombination of rapid PCR-based techniques, single-stranded conformation polymorphismanalysis (SSCP) and DNA sequencing. We found 37.2% were 871G>A (G6PDViangchan), 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 (G6PDVanua 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 (G6PDOrissa) and 1.2% 1361G>A (G6PD Andalus). Seventy-one (82.6%) of the 86 G6PD-deficient neonates had neonatal jaundice. Fifty seven (80%) of the 71 neonates with jaundice required phototherapy with only one neonate progressing to severehyperbilirubinemia (serum bilirubin >340
mol/l) requiring exchange transfusion. Therewas no significant difference in the incidence of neonatal jaundice , mean serum bilirubinlevel, mean age for peak serum bilirubin, percentage of babies requiring phototherapyand mean number of days of phototherapy between the three common variants. Inconclusion, the molecular defects of Malay G6PD deficiency is heterogeneous and G6PDViangchan, Mahidol and Mediterranean account for at least 80% of the cases. Ourfindings support the observation that G6PD Viangchan and Mahidol are commonSoutheast Asian variants. Their presence in the Malays suggests a common ancestralorigin with the Cambodians, Laotians and Thais. Our findings together with otherpreliminary data on the presence of the Mediterranean variant in this region provideevidence of strong Arab influence in the Malay Archipelago. ©
2002 Wiley-Liss, Inc.
Key Words: Glucose-6-phosphate dehydrogenase, G6PD, molecular variants, Malays
Glucose-6-phosphate dehydrogenase (G6PD, MIM# 305900) deficiency is the commonest enzymopathy inhuman estimated to affect 400 million individuals worldwide. G6PD deficient individuals are usuallyasymptomatic but acute haemolysis may occur with oxidative stress induced by ingestion of drugs, certain typeof food, exposure to certain chemical substances or when there is accompanying infection or hypoxia. Rarely, itmay cause chronic non-spherocytic haemolytic anemia. One of the most important complications of G6PDdeficiency is severe neonatal hyperbilirubinemia and the risk of developing kernicterus, a problem especiallyseen in G6PD-deficient individuals in the Mediterranean and Asia (Beutler, 1991; Brown and Boon, 1981; Tan,
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 invarious populations (Beutler, 1991).
The advances in molecular techniques have allowed the molecularcharacterisation of the G6PD gene in any population to be carried out with ease. Previous studies haveestablished 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 Javanesein 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 tobe 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 andkernicterus necessitating a national screening program for G6PD deficiency for newborns in all state hospitals, aprogram that has been in place for the last twenty years. We have previously reported a study of the G6PDmutations in Malaysian Chinese (Ainoon et al., 1999). To date, there has only been one report on molecularvariant in Malay G6PD involving 3 cases (Iwai et al., 2001). We present here the result of a study on themolecular characterisation of G6PD deficiency in the ethnic Malays in Malaysia. Using established PCR-basedtechniques, SSCP and direct DNA sequencing we determined the molecular abnormalities in a group of Malaymale G6PD-deficient neonates born in the National University Hospital, Kuala Lumpur.
All male Malay neonates born in the National University of Malaysia Hospital and diagnosed as G6PD-deficient by routine screening between the period April 1999 to May 2000 were consecutively studied. EDTAcord blood samples collected for each neonate from the Labour Room for routine screening of G6PDdeficiency by fluorescent spot test were used for the determination of red cell G6PD activity and DNAextraction. As routinely practised in this hospital, results of all G6PD screening tests were sent back to theward within 24 hours of receiving the specimen and mothers of babies diagnosed as G6PD deficiency wereexplained by the attending Paediatrician of the problems related to the disorder and advised to have the babieskept 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 outclinically and by serial estimation of serum bilirubin. Neonates with serum bilirubin level exceeding 180
mol/l within the first 48 hours of life and exceeding 250
mol/l were subjected to phototherapy ( Cockington,1979). Those infants with bilirubin level >340
mol/l would require exchange transfusion. Neonates weredischarged after the fifth day when they showed both clinical and biochemical improvement with a downwardtrend of serum bilirubin to a level of less than 230
mol/l. Babies born to mixed parentage, those bornprematurely (<36 weeks) and those with infections or haematoma because of birth injury were excluded fromthe study.
G6PD activity assay
Red cell G6PD activity assays were performed in all the 87 G6PD-deficient and 335 normal neonates. Basedon the principle of measurement of rate of absorbance of reduced NADP+ in red cell haemolysate, the assay wascarried out at 25
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 activityassays 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 tomanufacturer’s instruction using High Pure PCR Template Preparation Kit, Roche Diagnostics Corporation(USA).
G6DP Variants in Malaysian Malays
 DNA amplification and restriction enzyme analysis
In analysing the DNA, we adopted the strategy of firstly, screening all the G6PD-deficient neonates for twomutations i.e nt 1376 G>T (Canton ) and nt 1388 G>A (Kaiping), previously reported to be common in theMalaysian Chinese G6PD-deficient population (Ainoon et al 1999) and nt 487 G>A (Mahidol) a mutationthought to be common in Southeast Asia population (Panich et al., 1972). Samples showing absence for thesethree mutations were subjected to SSCP analysis followed by direct DNA sequencing. Mutations at nt 1376G>T, nt 1388 G>A and nt 487 G>A were detected using PCR-based technique involving amplification of targetgene 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 wasoptimised for each reaction by using the following concentrations: MgCl
1.5 mmol/l, DNTP 200 umol/l, eachprimer 10 pmol and Taq polymerase 1.5u. The PCR was performed on the DNA thermal cycler (Gene AmpPCR System 9700, Applied Biosystems ) at denaturation 94
C 30 seconds, annealing temperature calculatedaccording to Tm of primer pairs for 30 seconds and extension at 72
C 45 seconds. The reaction was run for 35cycles 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 conformationpolymorphism (PCR-SSCP) analysis using primer sequences for G6PD exons as previously described (Poggi etal., 1990). Amplifications were carried out using fluorescence-tagged primers and amplified fragments wereelectrophoresed on MDE (Mutation Detection Enhancement Gel Solution with 5 % polyacrylamide;BioWhittaker Application, USA) at 35
C using the ABI PRISM 377 DNA Sequencer. The amplified DNAfragments were analysed using the software Genescan ABI PRISM 3.1. The exons that showed mobility shiftswere 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 2-13 of the G6PD gene were performed using primersequences similar to that used for SSCP (Poggi et al., 1990). All PCR amplifications were carried out using theDNA thermal cycler (Gene Amp PCR System 9700, Applied Biosystems). Purification of PCR product forpreparation of the sequencing template were carried out using CONCERT
Rapid Gel Extraction System (LifeTechnologies, USA). Cycle sequencing was performed according to the manufacturer’s instruction using ABIPrism 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 (ABIPRISM 377 Sequencer; APPLIED BIOSYSTEMS, USA) and sequence analysis was carried out using the ABIPRISM Sequence Navigator software.
Data Analysis
Data on incidence of jaundice, the age of onset of jaundice, peak serum bilirubin value, age in days whenserum 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. Forcomparisons 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 forcomparison of discrete variables (incidence of jaundice, onset of jaundice). P value of less than 0.05 isconsidered significant.
RESULTSPrevalence of G6PD deficiency
A total of five thousand three hundred and sixty two (2992 males, 2370 females) newborn babies werescreened for G6PD deficiency using the semiquantitative fluorescent spot test during the period of study. Theoverall prevalence of G6PD deficiency was 3.4% (184 of 5362). The overall prevalence of G6PD deficiencyamong males was 5.3% (160 of 2992) and among females was 1.05 % (25 of 2370). Out of 5362 babies 3355were Malays, 1682 Chinese and 325 Indians. In the Malay group, the prevalence of G6PD deficiency amongmales was 4.6% (86 /1852) and among females 1.3% (19/1503). In the Chinese group the prevalence among

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