Correction to 'Genotyping of Malaysian G6PD-deficient neonates by reverse dot blot flow-through hybridisation'.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genotyping of Malaysian G6PD-deficient neonates by reverse dot blot flow-through hybridisation.

G6PD deficiency is the commonest enzyme deficiency found in humans. Current diagnostic methods lack sensitivity to detect all cases of G6PD deficiency. We evaluated the reverse dot blot flow-through hybridisation assay designed to detect simultaneously multiple known G6PD mutations in a group of Malaysian neonates. Archival DNA samples from 141 G6PD-deficient neonates were subjected to reverse dot blot flow-through hybridisation assay using the GenoArray Diagnostic Kit (Hybribio Limited, Hong Kong) and DNA sequencing. The method involved PCR amplification of 5 G6PD exons using biotinylated primers, hybridisation of amplicons to a membrane containing oligoprobes designed for G6PD mutations known to occur in the Malaysian population and colour detection by enzyme immunoassay. The assay detected 13 of the 14 G6PD mutations and genotyped 133 (94.3%) out of 141 (102 males, 39 females) cases. Among the 39 female G6PD-deficient neonates, there were 7 homozygous and 6 compound heterozygous cases. The commonest alleles were G6PD Viangchan 871G > A (21%) and G6PD Mahidol 487G > A(20%) followed by G6PD Mediterranean 563C > T, (14%), G6PD Vanua Lava 383T > C (12%), G6PD Canton 1376G > T (10%), G6PD Orissa 131C > G (6.3%) G6PD Coimbra 592C > T (5.6%) plus 6 other mutations. DNA sequencing of remaining cases revealed 6 cases of intron 11 nt 93C > T not previously reported in Malaysia and two novel mutations, one case each of nt 1361G > T and nt 1030G > A. We found the reverse dot blot assay easy to perform, rapid, accurate and reproducible, potentially becoming an improved diagnostic test for G6PD deficiency.

Prenatal diagnosis of aneuploidies in amniotic fluid by multiple ligation-dependent probe amplification (MLPA) analysis.

Prenatal diagnosis is essential in the new era of diagnosis and management of genetic diseases in obstetrics. Multiple ligation-dependent probe amplification (MLPA) is a recent technique for prenatal diagnosis for the relative quantification of 40 different nucleic acid sequences in one single reaction. We had utilized the MLPA technique in detecting aneuploidies in amniotic fluid samples from 25 pregnant women from the Obstetrics and Gynaecology Department UKMMC, versus the quantitative fluorescent polymerase chain reaction (QF-PCR) method. Conclusive results were obtained in 18 cases and all were concordant with that of the QF-PCR. All four cases of trisomies were correctly identified including one case with maternal cell contamination.

Detection of Partial G6PD Deficiency using OSMMR2000-D Kit with Hb Normalization

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide including Malaysia. Screening of cord blood for partial G6PD deficiency is important as they are also prone to develop acute haemolysis. In this study, we determined the prevalence of partial G6PD deficient in paediatric population aged 1 month-12 years and normal term female neonates using OSMMR-D kit with haemoglobin (Hb) normalization and compare it with florescence spot test (FST). A total of 236 children, aged between between 1 month-12 years and 614 normal term female neonates were recruited for this study. Determination of normal means for G6PD activity and; cut-off points for partial and severe deficiency were determined according to WHO Working Group (1989). Determination of prevalence for partial deficiency for both groups (female patient) was done using this enzyme assay kit and findings were compared with FST. In this study, 15.7% (18/115) female children were classified as partial G6PD deficient by quantitative enzyme method (G6PD activity: 4.23-5.26U/gHb). However, FST only detected 0.9% (1/115) with minimal G6PD activity. The prevalence of partial G6PD deficiency in female neonate group was 3.42% (21/614) by enzyme assay versus 0.49% (3/614) by FST. This study concluded that our routine screening method using FST was unable to diagnose female heterozygotes. We recommend using this quantitative enzyme assay method by OSMMR-D kit since it was more sensitive in detecting G6PD deficiency in female neonates compared to FST.

Prevalence of iron deficiency anaemia and thalassaemia trait among undergraduate medical students.

BACKGROUND: Anaemia is a global health problem including Malaysia. In adults, anaemia may affect work productivity. Iron deficiency anaemia and thalassaemia are common causes of anaemia in Malaysia. However, there is scarcity of data on national prevalence of iron deficiency anaemia and thalassaemia, especially in young adults. This cross sectional study was performed to determine the prevalence of iron deficiency anaemia and thalassaemia among medical students of Universiti Kebangsaan Malaysia Medical Centre (UKMMC). MATERIALS AND METHODS: Blood samples collected in EDTA tubes were analyzed for haemoglobin level and red cell parameters such as MCV, MCH and red cell counts. Samples with abnormal red cell indices were sent for analysis of RBC morphology, iron status, haemoglobin analysis and DNA analysis. RESULTS: A total of 400 samples were available for this study. Fifty-eight (14.5%) students had hypochromic microcytic red cell indices in which 44 (11%) showed thalassaemia red cell indices while 14 (3.5%) had iron deficiency red cell indices which were finally confirmed by serum iron/TIBC analysis. Amongst those suspected to have thalassaemia, 12 (27.3%) were confirmed as alpha thalassaemia trait (αα/--(SEA)), 11 (25%) as Haemoglobin-E trait, 8 (18.2%) as beta thalassaemia trait and 2 (4.5%) as Haemoglobin Constant Spring (αα/α(CS)α). However, eleven students (25%) with thalassaemia red cell indices could not be confirmed with the common thalassaemia primers available, thus causes have yet to be established. CONCLUSION: Our prevalence of thalassaemia was high and thus we opine that better screening methods should be adopted.

A study of JAK2 (V617F) gene mutation in patients with chronic myeloproliferative disorders.

BACKGROUND AND AIMS: Chronic myeloproliferative diseases (MPDs) are heterogenous group of haematological malignant disorders. It is now a well recognized fact that the JAK2 (V617F) mutation occurs in majority of the patients with polycythaemia vera (PV) and half of those with myelofibrosis and essential thrombocythaemia. The presence of JAK2 (V617F) mutation is considered an important criterion for the exclusion of secondary-reactive from clonal disorders. In the present uni-institutional study, we analyzed the JAK2 (V617F) mutation status in the ethnic Malay and Chinese patients who were diagnosed as MPDs. MATERIALS AND METHODS: The study was performed on known cases of chronic MPDs either at diagnosis or during the follow-up. A total of 45 cases were studied with informed consent. The allele specific PCR, ARMS-PCR and RQ-PCR methods were used. RESULTS: The frequency of the JAK2 (V617F) mutation varied between the MPD subtypes, with the mutation being most frequent in PV (95.8%) and 39% showed homozygous mutant allele. The mutation was detected in 52.9% cases of ET, of which 36.4% were homozygous for the mutant allele and 1 case of MF was homozygous for the mutant allele. CONCLUSION: Screening for the mutation in all cases suspected of chronic MPD could be beneficial in differentiating patients with reactive erthrocytosis or thrombocytosis from the true clonal MPDs especially polycythaemia vera.

Human mesenchymal stromal cells could deliver erythropoietin and migrate to the basal layer of hair shaft when subcutaneously implanted in a murine model.

Mesenchymal stromal cells (MSC) are an attractive cell-targeting vehicle for gene delivery. MIDGE (an acronym for Minimalistic, Immunologically Defined Gene Expression) construct is relatively safer than the viral or plasmid expression system as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. The objective of this study was to test the ability of the human MSC (hMSC) to deliver the erythropoietin (EPO) gene in a nude mice model following nucleofection using a MIDGE construct. hMSC nucleofected with MIDGE encoding the EPO gene was injected subcutaneously in Matrigel at the dorsal flank of nude mice. Subcutaneous implantation of nucleofected hMSC resulted in increased hemoglobin level with presence of human EPO in the peripheral blood of the injected nude mice in the first two weeks post-implantation compared with the control groups. The basal layer of the hair shaft in the dermal layer was found to be significantly positive for immunohistochemical staining of a human EPO antibody. However, only a few basal layers of the hair shaft were found to be positively stained for CD105. In conclusion, hMSC harboring MIDGE-EPO could deliver and transiently express the EPO gene in the nude mice model. These cells could be localized to the hair follicle and secreted EPO protein might have possible role in hair regeneration.

Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells.

Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary cells and hard-to-transfect cell lines. Herein, we compared the efficiency and viability of nucleofection with cationic lipofection, and used the more efficient transfection method, nucleofection, to deliver a construct of minimalistic, immunologically defined gene expression encoding the erythropoietin (MIDGE-EPO) into hMSC. MIDGE construct is relatively safer than the viral and plasmid expression systems as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. Using a plasmid encoding the luciferase gene, we demonstrated a high transfection efficiency using the U-23 (21.79 ± 1.09%) and C-17 (5.62 ± 1.09%) pulsing program in nucleofection. The cell viabilities were (44.93 ± 10.10)% and (21.93 ± 5.72)%, respectively 24 h post-nucleofection. On the other hand, lipofection treatment only yielded less than 0.6% efficiencies despite showing higher viabilities. Nucleofection did not affect hMSC renewability, immunophenotype and differentiation potentials. Subsequently, we nucleofected MIDGE-EPO using the U-23 pulsing program into hMSC. The results showed that, despite a low nucleofection efficiency with this construct, the EPO protein was stably expressed in the nucleofected cells up to 55 days when determined by ELISA or immunocytochemical staining. In conclusion, nucleofection is an efficient non-viral transfection approach for hMSC, which when used in conjunction with a MIDGE construct, could result in extended and stable transgene expression in hMSC.

Prevalence of uridine glucuronosyl transferase 1A1 (UGT1A1) mutations in Malay neonates with severe jaundice.

A number of genetic risk factors have been implicated in the development of neonatal severe hyperbilirubinaemia. This includes mutations in the uridine glucoronosyl transferase 1A1 (UGT1A1) gene which is responsible for unconjugated hyperbilirubinemia in Gilbert's Syndrome. We studied the prevalence of UGT1A1 gene mutations in a group of Malay neonates to determine whether they are risk factors to severe neonatal jaundice. One hundred and twenty-five Malay neonates with severe hyperbilirubinemia were studied. Ninety-eight infants without severe hyperbilirubinaemia were randomly selected from healthy Malay term infants (controls). DNA from EDTA cord blood samples were examined for UGT1A1 mutations nt211G > A and nt247T > C using established Taqman SNP genotyping assays and the UGT1A1*28 variant was detected by the Agilent 2100 bioanalyzer. All samples were also screened for common Malay G6PD variants using established techniques. The frequency of UGT1A1 211G > A mutation is significantly higher in the severely hyperbilirubinemic group (13%) than the control group (4%; p = 0.015) and all the positive cases were heterozygous for the mutation. There was no significant difference in the frequency of UGT1A1*28 mutation between the severely hyperbilirubinemic (3.5%) and the control group (0.01%; p = 0.09). None of the neonates in both groups carried the nt247 T > C mutation. The prevalence of G6PD mutation was significantly higher in the severely jaundiced group than control (9% vs 4%; p = 0.04). In conclusion, nt 211 G > A alleles constitute at least 12% of UGT1A1 mutations underlying unconjugated hyperbilirubinemia and appears to be a significant independent risk factor associated with severe neonatal hyperbilirubinemia in the Malay newborns.

Real-time quantification for BCR-ABL transcripts in chronic myeloid leukaemia patients in UKMMC, Malaysia.

Molecular pathogenesis of chronic myeloid leukemia (CML) is well established and molecular monitoring for patients with CML has become an important practice in the management of patients on imatinib therapy. In the present study, we report the use of RQ-PCR method for detection of BCR-ABL fusion gene for our CML cases. We performed a two-step RQ-PCR on bone marrow aspirates or peripheral blood of 37 CML patients. Quantitative expression of BCR-ABL fusion gene was carried out relative to the expression of a housekeeping gene as endogenous control to compensate for uneven cell numbers, RNA quality, or variations in reverse transcription efficiencies. Twenty-four of these patients were pre-treated with hydroxyurea or alpha interferon prior to the imatinib therapy. Their BCR-ABL fusion gene levels were monitored for 18 months. All samples processed were evaluable. The PCR amplification efficiency of the ABL gene is 90.5% (0.2158) and the BCR-ABL gene, 93.4% (0.1573).

G6PD enzyme activity in normal term Malaysian neonates and adults using a OSMMR2000-D kit with Hb normalization.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the commonest causes of neonatal jaundice in Malaysia. Screening of cord blood for G6PD deficiency by the semiquantitative fluorescent spot test (FST) is performed in Malaysia but this test can miss cases of partial G6PD deficiency. The OSMMR-D kit assay measures G6PD activity and hemoglobin (Hb) concentration, allowing direct expression of results in U/gHb. We evaluated this method and established the normal range for G6PD activity in normal term neonates and adults. EDTA blood from 94 neonates and 295 adults (age 15-59 years old) with normal Hb and FST were selected. The normal means for G6PD activity for neonates and adults were 12.43 +/- 2.28 U/gHb and 9.21 +/- 2.6 U/gHb, respectively; the reference ranges for normal G6PD activity in neonates and adults were 10.15-14.71 U/gHb and 6.61-11.81 U/gHb respectively. There were no significant differences in mean normal G6PD activity between the Malays and Chinese racial groups or between genders. The upper and lower limit cut-off points for partial deficiency in neonates were 7.4 U/gHb (60% of the normal mean) and 2.5 U/gHb (20% of the normal mean), respectively. For adults, the upper and lower limit cut-off points for partial deficiency in adults were 5.52 U/gHb (60% of the normal mean) and 1.84 U/gHb (20% of the normal mean), respectively. The quantitation of G6PD enzymes using this OSMMR-D kit with Hb normalization was simple since the Hb was analyzed simultaneously and the results were reproducible with a CV of less than 5%.

Comparison of detection of glucose-6-phosphate dehydrogenase deficiency using fluorescent spot test, enzyme assay and molecular method for prediction of severe neonatal hyperbilirubinaemia.

INTRODUCTION: This study aimed to compare the detection rates of glucose-6-phosphate dehydrogenase (G6PD) deficiency in neonates by fluorescent spot test (FST), enzyme assay and molecular methods, and to identify which method was a significant predictor of severe hyperbilirubinaemia. METHODS: 74 term infants of Chinese descent admitted with severe hyperbilirubinaemia (total serum bilirubin equal or greater than 300 micromol/L) and 125 healthy term infants born in the hospital without severe hyperbilirubinaemia were recruited into the study. Specimens of blood were collected from each infant for FST, G6PD enzyme assay and TaqMan minor groove binder single nucleotide polymorphism genotyping assay. RESULTS: 26 (13.1 percent) infants were diagnosed to have G6PD deficiency by FST. They had significantly lower median enzyme levels (0.8 IU/g Hb, interquartile range [IQR] 0.4-4.3) than those diagnosed to be normal (12.0 IU/g Hb, IQR 10.3-15.8) (p-value is less than 0.0001). Based on the enzyme assay, 39 (19.6 percent) infants had G6PD deficiency at an enzyme cut-off level of less than 8.5 IU/g Hb. G6PD mutation was detected in 27 (13.6 percent) infants. Logistic regression analysis showed that the only significant predictors of severe hyperbilirubinaemia were G6PD deficiency based on a cut-off level of less than 8.5 IU/g Hb (adjusted odds ratio [OR] 5.3, 95 percent confidence interval [CI] 2.4-11.4; p-value is less than 0.0001) and exclusive breast-feeding (adjusted OR 11.4, 95 percent CI 3.1-42.4; p-value is less than 0.0001). The gender and birth weight of infants, FST results, G6PD mutation and the actual G6PD enzyme levels were not significant predictors. CONCLUSION: A G6PD enzyme level of less than 8.5 IU/g Hb is a significant predictor of severe hyperbilirubinaemia.

Juvenile myelomonocytic leukaemia: a case series.

Juvenile myelomonocytic leukaemia (JMML), previously known as juvenile chronic myeloid leukaemia (JCML) is a rare, myelodysplastic - myeloproliferative disease typically presenting in early childhood. This disorder is difficult to distinguish from other myeloproliferative syndrome such as chronic myeloid leukaemia (CML) because of the similarities in their clinical and bone marrow findings. However, because of its unique biological characteristics such as absolute monocytosis with dysplasia, absence of Philadelphia chromosome or BCR-ABL fusion protein, hypergammaglobulinaemia and raised fetal haemoglobin level, this disorder does not satisfy the criteria for inclusion in the CML or chronic myelomonocytic leukaemia (CMML) group, as seen in adult patients. We describe a series of three patients with JMML, who had almost similar clinical and laboratory findings, and discuss the difficulty in the classification and treatment of the disease.

Assessment of P-gp and MRP1 activities using MultiDrugQuant Assay Kit: a preliminary study of correlation between protein expressions and its functional activities in newly diagnosed acute leukaemia patients.

Multidrug resistance (MDR) is believed to be responsible for poor response of patients towards chemotherapy particularly patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). The best-characterized resistance mechanism is the one mediated by permeability-glycoprotein (P-gp) encoded by MDR1 gene, which is responsible for drug efflux. We studied P-gp and multidrug resistance-associated protein 1 (MRP1) expression and functional activities in 43 newly diagnosed acute leukemia cases (19 paediatric ALL cases and 24 adult AML cases). The expression and functional activities were examined using flow cytometry and MultiDrugQuant assay kit (involving calcein AM uptake and efflux). P-gp and MRP1 expression and its functional activities were observed in 68.4% of paediatric ALL. In adult AML cases, all cases expressed MRP1 and its functional activities but only 58.3% were positive for P-gp and its functional activities. We were able to show a significant correlation between the expression of the multidrug resistant protein (P-gp and MRP1) and their functional activity in adult AML and paediatric ALL samples.

Expression of multidrug resistance (MDR) proteins and in vitro drug resistance in acute leukemias.

The expression of the multidrug resistance (MDR) proteins may influence the outcome of treatment in patients with acute leukemia. The aim of this study was to determine the IC50 of cytotoxic drugs (cytosine arabinoside, ara-C and daunorubicin, dnr) using the in vitro 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay method. A total of 82 newly diagnosed acute leukemia cases (43 adult myeloid leukaemia, AML cases and 39 acute lymphoblastic leukaemia, ALL cases) and 16 relapsed cases (8 AML cases and 8 ALL cases) were studied. The MTS assay was performed using two cytotoxic drugs, dnr and ara-C. Cells were incubated with different concentrations of drugs for 4 days and the IC50 was extrapolated from the viability curve. In newly diagnosed cases, we found that childhood ALL samples showed higher IC50 values of dnr (0.040 +/- 2.320) compared to adult AML samples (0.021 +/- 0.158). In contrast, newly diagnosed adult AML samples showed higher IC50 values of ara-C (0.157 +/- 0.529) compared to childhood ALL samples (0.100 +/- 2.350). In relapsed cases, two samples of childhood ALL showed IC50 values of dnr (0.910 +/- 1.760) and ara-C (1.310 +/- 2.390), which was higher compared to childhood AML samples (0.129 +/- 0.214 and 0.210 +/- 0.003, respectively). However, there was no correlation between IC50 values of these drugs tested with clinical outcome. In conclusion, we found that MTS assay is an easy, rapid and non laborious method to study in vitro drug resistance in acute leukaemia cases.

G6PD deficiency with hemolytic anemia due to a rare gene deletion--a report of the first case in Malaysia.

A 2-year-old Chinese boy was referred to Hospital UKM for investigation of recurrent episodes of dark-coloured urine and pallor since birth. He was born prematurely at 34 weeks gestation and developed severe early-onset neonatal jaundice requiring exchange blood transfusion. Screening at birth showed Glucose-6-phosphate dehydrogenase (G6PD) deficiency. On admission, physical examination revealed pallor, jaundice and mild hepatomegaly. Results of laboratory investigations showed a hemoglobin level of 11.0 g/dl with a hemolytic blood picture, reticulocytosis of 20% and red cell G6PD activity reported as undetectable. The patient's DNA was analysed for G6PD mutations by PCR-based techniques and DNA sequencing and results showed a 24 bp deletion of nucleotide 953-976 in the exon 9 of the G6PD gene. DNA analysis was also performed on blood samples of the patient's mother and female sibling confirming their heterozygous status, although both showed normal red cell G6PD activity levels. The patient was discharged well and his parents were appropriately advised on the condition and the importance of taking folic acid regularly. This is a first case report in Malaysia of G6PD deficiency causing chronic-hemolytic anemia. The rare 24 bp deletion causes the G6PD Nara variant, previously reported only in two other unrelated males, a Japanese and a Portuguese both with chronic hemolytic anemia.

Assessing donor chimerism using flow cytometry in paroxysmal nocturnal haemoglobinuria after stem cell transplantation--a case report.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemopoietic stem cell disorder arising from somatic mutation of the X-linked PIG-A gene which leads to deficiency of the glycosylphosphatidylinositol (GP1) membrane anchor proteins such as CD 59 (MIRL: membrane inhibitor of reactive lysis) and CD 55 (DAF: decay accelerating factor). Allogeneic peripheral blood stem cell transplant (PBSCT) is a curative mode of treatment in symptomatic PNH patients. Assessment of donor chimerism for PBSCT can be performed by various methods including short tandem repeat loci (STR) and variable number of tandem repeats (VNTR). Flow cytometry, which is much cheaper and faster, also can be used to assess engraftment in patients with PNH. Engrafted patients will show the presence of CD 55 and CD 59 on their red cells and white cells. We describe here the usefulness of flow cytometry in the assessment of donor chimerism following allogeneic PBSCT, in a case of PNH.

Allogeneic haemopoietic stem cell transplantation using non-myeloablative conditioning--a local experience.

Allogeneic haemopoietic stem cell transplantation was initially considered as a means of delivering supralethal doses of chemotherapy with or without total body irradiation for the treatment of malignancy. However, it has become clear that this mode of therapy does not eradicate the malignancy in many patients and its benefit is largely due to the immune mediated graft versus malignancy effect. This has led to development of alternative strategy to utilize a less intensive preparative regimen pre-transplantation that provides sufficient immunosuppression to achieve engraftment of an allogeneic stem cell graft, thus allowing the evolution of a graft versus malignancy effect post-transplantation. Since September 1999, we had carried out 10 cases of allogeneic peripheral blood stem cell transplantation: one case of aplastic anaemia, four cases of acute myeloid leukemia (AML) in first remission, and five cases of chronic myeloid leukemia (CML) in chronic phase. The preparative regimen was non-myeloablative comprising Fludarabine with Cyclophosphamide or Busulphan. Recovery from transplantation was rapid with no or brief period of neutropenia or thrombocytopenia. Engraftment was established by determining donor's short tandem repeats in the recipient's bone marrow at day 30, 60 and 100 post-transplantation. Seven cases (70%) show partial or complete donor's chimerism by day 30 indicating successful engraftment. No treatment mortality was noted at day 100. Graft versus host disease was generally limited. Up to the date of reporting, two patients with CML had graft failure, one was successfully re-transplanted later. Two patients with AML had since relapsed and passed away. The others remain alive and well. The cost of transplantation on average was estimated to be about a quarter of that using a myeloablative regimen. It appears that this treatment strategy is a promising approach for the management of blood disorders.

Semiquantitative screening test for G6PD deficiency detects severe deficiency but misses a substantial proportion of partially-deficient females.

Neonatal screening for G6PD deficiency has long been established in many countries. The aim of the study was to determine whether the routine semiquantitative fluorescent spot test could detect all cases of G6PD deficiency, including those cases with partial deficiency (residual red cell G6PD activity between 20-60% of normal). We compared the results of G6PD screening by the semiquantitative fluorescent spot test and quantitative G6PD activity assay on a group of 976 neonates and 67 known female heterozygotes. The values for mean G6PD activity of G6PD-normal neonates and 293 healthy adult females were determined. There was no significant difference in the mean normal G6PD activity between the two racial groups in the neonates (669 Malays, 307 Chinese) and in the 293 healthy adult females (150 Malays, 143 Chinese) group. The values for the upper limits of total deficiency (20% of normal residual activity) for neonates and adult females were 2.92 U/gHb and 1.54 U/gHb, respectively. The upper limits of partial deficiency (60% of normal residual activity) were 8.7 U/gHb and 4.6 U/gHb respectively. The prevalence of G6PD deficiency among the male neonates was 5.1% (26) by both the fluorescent spot test and the enzyme assay method. The G6PD activity levels of all 26 cases of G6PD-deficient male neonates were < 20% normal (severe enzyme deficiency). In the female neonate group, the frequency of G6PD deficiency was 1.3% (6 of 472) by the fluorescent spot test and 9.35% (44 of 472) by enzyme assay. The 6 cases diagnosed as deficient by the fluorescent spot test showed severe enzyme deficiency (< 2.92 U/gHb). The remaining 38 female neonates had partial enzyme deficiency and all were misdiagnosed as normal by the fluorescent spot test. In the female heterozygote group, G6PD deficiency was diagnosed in 53% (35 of 67) by enzyme assay and in 7.5% (4 of 67) of cases by the fluorescent spot test. The 4 cases detected by fluorescent spot test had severe enzyme deficiency (<1.6 U/gHb). The remaining 31 (46.3%) cases, diagnosed as normal by fluorescent spot test, showed partial G6PD deficiency. In conclusion, we found that the semiquantitative fluorescent spot test could only diagnose cases of total G6PD deficiency and misclassified the partially-deficient cases as normal. In this study, the overall prevalence of G6PD deficiency was 3.28% by the semiquantitative fluorescent spot test and 7.17% by enzyme assay. This means that 3.9% of G6PD-deficient neonates were missed by the routine fluorescent spot test and they were found to be exclusively females. This study demonstrates a need to use a method that can correctly classify female heterozygotes with partial G6PD deficiency. The clinical implication is that these individuals may be at risk of the hemolytic complication of G6PD deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) variants in Malaysian Malays.

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 G6PD-deficient 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 micromol/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.