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.
OBJECTIVE:
To evaluate the G6PD(C563T) Mediterranean mutation among Jordanian females who were admitted to Princess Rahma Teaching Hospital (PRTH) with/or previous history of favism.
STUDY DESIGN:
A descriptive study.
PLACE AND DURATION OF STUDY:
Jordanian University of Science and Technology and PRTH, from October 2003 to October 2004.
METHODOLOGY:
After obtaining approval from the Ethics Committee of Jordanian University of Science and Technology, a total of 32 females were included in this study. Samples from 15 healthy individual females were used as a negative control. Blood samples from these patients were collected and analyzed by allele-specific polymerase chain reaction (AS-PCR) to determine the G6PD(C563T) mutation.
RESULTS:
Twenty one out of 32 patients were found to be G6PD(C563T) Mediterranean mutation (65.6%) positive. Three out of 21 patients were homozygous and remaining 18 were heterozygous for G6PD(C563T) Mediterranean mutation. Eleven (34.4%) out of 32 patients were found to be negative for G6PD(C563T) mutation indicating the presence of other G6PD mutations in the study sample.
CONCLUSION:
G6PD(C563T) Mediterranean mutation accounted for 65.6% of the study sample with favism in the North of Jordan. There is likely to be another G6PD deficiency variant implicated in acute hemolytic crisis (favism).
The role of hemolysis in the pathophysiology of neonatal jaundice (NNJ) in patients with glucose 6-phosphate dehydrogenase (G6PD) deficiency has been questioned recently. The aim of the present study was to determine the contribution of hemolysis to the pathophysiology of jaundice in Malay neonates with G6PD deficiency and NNJ.
G6PD deficiency is the most common human enzymopathy and affects 200 million people worldwide. To date more than 400 biochemical variants and at least 60 different point mutations in the G6PD locus have been discovered. In Malaysia the overall incidence of G6PD deficiency among males is 3.1%, being more prevalent among the Chinese and Malays and less common among the Indians. As part of our initial effort to characterise G6PD deficiency in the Malaysian population, we investigated 18 G6PD deficient Chinese male neonates for the G6PD mutation G-->T at nt 1376, a common mutation seen among the Chinese in Taiwan and mainland China. The mutation was detected by a PCR-based technique using primers that artificially create a site for restriction enzyme Xho I. We found 61% (11 out of 18) of the Chinese G6PD deficient male neonates positive for this mutation. Study of enzyme electrophoretic mobility in 7 of the cases positive for this mutation revealed three different patterns of mobility. 107% (5 out of 7), 103% (1 out of 7) and 100% (1 out of 7). This study shows that mutation G-->T at nt 1376 is a common allele causing G6PD deficiency in Malaysians of Chinese origin. The finding of different patterns of electrophoretic mobility among the 7 cases positive for 1376 G-->T mutation supports the notion that diverse biochemical variants may share the same mutation.
Biochemical characteristics of one non-deficient fast G6PD variant (GdSingapore) and six different deficient variants (three new, two Mahidol, one each of Indonesian and Mediterranean) were studied among the Malays of Singapore. The GdSingapore variant had normal enzyme activity (82%) and fast electrophoretic mobilities (140% in TEB buffer, 160% in phosphate and 140% in Tris-HCl buffer systems respectively). This variant is further characterized by normal Km for G6P; utilization of analogues (Gal6P, 2dG6P; dAmNADP), heat stability and pH optimum. The other six deficient G6PD variants had normal electrophoretic mobility in TEB buffer with enzyme activities ranging from 1 to 12% of GdB+. The biochemical characteristics identity them to be 2 Mahidol, 1 Indonesian and 1 Mediterranean variants and three new deficient variants.
This prospective study was performed to quantify glucose-6-phosphate dehydrogenase (G6PD) enzyme activity in deficient males and female heterozygotes. The methods used in the study were the fluorescent spot test, G6PD enzyme electrophoresis on cellulose acetate and quantitative assays. Forty-seven children who had been detected as spot screen deficient at birth were rescreened. Their first degree relatives were also included in the study. The mean enzyme activity of deficient males was 0.74 iu/g Hb (s.d. +/- 0.8), of female heterozygotes was 6.5 iu/g Hb (s.d. +/- 3.2) and of normal males was 12.1 iu/g Hb (s.d. +/- 3.5). The mean activity in deficient males was 6.1% of normal males. Most (35 of 47) of these fell into class 2 in Beutler's classification of G6PD variants. This indicates a population which may be susceptible to favism. Female heterozygotes had an intermediate enzyme activity with a wide scatter. Using a cut off point of enzyme activity of below 9.0 iu/g Hb gave sensitivity and specificity of 87% and 84% in detecting female heterozygotes. This group could be defined more accurately by combining quantitative assays with family studies.
Cord blood from 8,975 babies delivered in Hospital Sultanah Aminah Johor Bahru over a period of eight months (1st August 1985 to 31st March 1986) were screened for G6PD deficiency. The overall incidence was 4.5% in Chinese, 3.5% in Malays and 1.5% in Indian babies. One hundred of these babies were observed in the nursery for seven days and their daily serum bilirubin recorded. The serum bilirubin peaked at 96 hours to a value of 12mg%. None of the babies in the nursery developed a serum bilirubin level of more than 15mg%. Six of the babies with G6PD deficiency that were sent home were readmitted with hyperbilirubinaemia that needed exchange transfusion.
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.
Emerging epidemiological evidence suggests that patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency may have a higher risk of developing diabetes. The aim of the review was to synthesise the evidence on the association between G6PD deficiency and diabetes. A systematic search on Medline, EMBASE, AMED and CENTRAL databases for studies published between January 1966 and September 2016 that assessed the association between G6PD deficiency and diabetes was conducted. This was supplemented by a review of the reference list of retrieved articles. We extracted data on study characteristics, outcomes and performed an assessment on the methodological quality of the studies. A random-effects model was used to compute the summary risk estimates. Fifteen relevant publications involving 949,260 participants were identified, from which seven studies contributed to the meta-analysis. G6PD deficiency was associated with a higher odd of diabetes (odds ratio 2.37, 95% confidence interval 1.50-3.73). The odds ratio of diabetes among men was higher (2.22, 1.31-3.75) compared to women (1.87, 1.12-3.12). This association was broadly consistent in the sensitivity analysis. Current evidence suggests that G6PD deficiency may be a risk factor for diabetes, with higher odds among men compared to women. Further research is needed to determine how G6PD deficiency moderates diabetes.
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
We screened 38 G6PD-deficient male Chinese neonates for known G6PD mutations using established PCR-based techniques. We found 50.0% (19 of 38) were mutation 1376G>T, 34.2% (13 of 38) were mutation 1388G>A, 5.2% (2 of 38 ) were mutation 95A>G and 2.2% (1 of 38) was mutation 1024C>T. In 7% (3 of 38) of the cases the mutations remained uncharacterised. Sixty three percent (24 of 38) of the G6PD deficient neonates had neonatal jaundice with 28.9 % (11 of 38) developing moderate to severe hyperbilirubinemia. The group of neonates with 1388 mutation showed the highest incidence of moderate to severe hyperbilirubinemia requiring phototherapy and/or exchange transfusion respectively. Majority (70%) of the G6PD deficient neonates showed severe enzyme deficiency. However, there was no meaningful association between the level of enzyme activity and the severity of neonatal jaundice. In summary, four mutations account for more than 90% of the G6PD deficiency cases among the Chinese in Malaysia and the pattern of distribution of the molecular variants is similar to those found among the Chinese in Taiwan and southern mainland China. Our findings also suggest the possible association of nt 1388 mutation with severe neonatal jaundice.
The Orang Asli are the indigenous population in peninsular Malaysia and are in fact a diverse sub-ethnic group with different languages. Our aim was to collect data on Orang Asli newborns, from western and central Pahang, that were admitted to a general hospital with paediatric specialist services.
ABO incompatibility and glucose-6-phosphate dehydrogenase deficiency G6PD are common haematological problems affecting the newborn. The resulting haemolytic disease of foetus and newborn (HDFN) caused by either of these pathologies generally follows a benign course. It is typically characterized by mild jaundice without significant anaemia. ABO incompatibility alone as a cause of foetal hydrops is extremely rare. We report a case of a newborn baby girl with an anti-B isoimmunisation and G6PD deficiency manifesting with hydrops foetalis, anaemia and hyperbilirubinaemia, born to a mother with blood group O.
We performed DNA analysis on cord blood samples of 128 Chinese male neonates diagnosed as G6PD deficiency in Hospital Universiti Kebangsaan Malaysia by a combination PCR-restriction enzyme digest technique, Single Stranded Conformation Polymorphism analysis and DNA sequencing. We found 10 different G6PD-deficient mutations exist. The two commonest alleles were G6PD Canton 1376 G>T (42.3%) and Kaiping 1388 G>A (39.4%) followed by G6PD Gaohe 592 G>A (7.0%), Chinese-5 1024 C>T, Nankang 517 T>C (1.5%), Mahidol 487 G>A (1.6%), Chatham 1003 G>T (0.8%), Union 1360 C>T (0.8%), Viangchan 871 G>A (0.8%) and Quing Yang 392 G>T (0.8%). Sixty eight percent (88/125) neonates in this study had neonatal jaundice and 29.7% developed hyperbilirubinemia >250 micromol/l. The incidence of hyperbilirubinemia >250 micromol/l was higher in G6PD Kaiping (43.8%) than G6PD Canton (22%) (p< 0.05). There was no significant difference in the incidence of neonatal jaundice, mean serum bilirubin, mean age for peak serum bilirubin, percentage of babies requiring phototherapy and mean duration of phototherapy between the two major variants. None of the 88 neonates required exchange transfusion. In conclusion we have completely characterized the molecular defects of a group of Chinese G6PD deficiency in Malaysia. The mutation distribution reflects the original genetic pool and limited ethnic admixture with indigenous Malays.
The combination of two silent mutations, c.1311C>T in exon 11 and IVS11 T93C (glucose-6-phosphate dehydrogenase (G6PD) 1311T/93C), with unknown mechanism, have been reported in G6PD-deficient individuals in Asian populations including Malaysian aboriginal group, Negrito. Here, we report the screening of G6PD gene in 103 Negrito volunteers using denaturing high-performance liquid chromatography (dHPLC) and direct sequencing. A total of 48 individuals (46.6%) were G6PD deficient, 83.3% of these carried G6PD 1311T/93C with enzyme activity ranging from 1.8 to 4.8 U gHb(-1). Three novel single-nucleotide polymorphisms (SNPs), rs112950723, rs111485003 and rs1050757, were found in the G6PD 3'-untranslated region (UTR). Strong association was observed between haplotype 1311T/93C and rs1050757G, which is located inside the 35 bp AG-rich region. In silico analysis revealed that the transition of A to G at position rs1050757 makes significant changes in the G6PD mRNA secondary structure. Moreover, putative micro (mi)RNA target sites were identified in 3'-UTR of G6PD gene, two of these in the region encompassing rs1050757. It could be speculated that rs1050757 have a potential functional effect on the downregulation of mRNA and consequently G6PD deficiency either by affecting mRNA stability and translation or mirRNA regulation process. This is the first report of biochemical association of an SNP in 3'-UTR of G6PD gene and the possible role of mRNA secondary structure.
The only currently available drug that effectively removes malaria hypnozoites from the human host is primaquine. The use of 8-aminoquinolines is hampered by haemolytic side effects in glucose-6-phosphate dehydrogenase (G6PD) deficient individuals. Recently a number of qualitative and a quantitative rapid diagnostic test (RDT) format have been developed that provide an alternative to the current standard G6PD activity assays. The WHO has recently recommended routine testing of G6PD status prior to primaquine radical cure whenever possible. A workshop was held in the Philippines in early 2015 to discuss key challenges and knowledge gaps that hinder the introduction of routine G6PD testing. Two point-of-care (PoC) test formats for the measurement of G6PD activity are currently available: qualitative tests comparable to malaria RDT as well as biosensors that provide a quantitative reading. Qualitative G6PD PoC tests provide a binomial test result, are easy to use and some products are comparable in price to the widely used fluorescent spot test. Qualitative test results can accurately classify hemizygous males, heterozygous females, but may misclassify females with intermediate G6PD activity. Biosensors provide a more complex quantitative readout and are better suited to identify heterozygous females. While associated with higher costs per sample tested biosensors have the potential for broader use in other scenarios where knowledge of G6PD activity is relevant as well. The introduction of routine G6PD testing is associated with additional costs on top of routine treatment that will vary by setting and will need to be assessed prior to test introduction. Reliable G6PD PoC tests have the potential to play an essential role in future malaria elimination programmes, however require an improved understanding on how to best integrate routine G6PD testing into different health settings.
Multiplex polymerase chain reaction (PCR) using multiple tandem forward primers and a common reverse primer (MPTP) was recently established as a comprehensive screening method for mutations in X-linked recessive diseases. In the work reported here, MPTP was used to scan for mutations of the glucose-6-phosphate dehydrogenase (G6PD) gene. Mutations in exons 3,4,5,6,7,9, 11, and 12 of the G6PD gene were screened by MPTP in 93 unrelated Malaysian patients with G6PD deficiency. Of the 93 patients, 80 (86%) had identified mutations. Although all of these were missense mutations, identified nucleotide changes were heterogeneous, with 9 mutations involving various parts of the exons. These 9 mutations were G-to-A nucleotide changes at nucleotide 871 of the G6PD gene (G871A), corresponding to G6PD Viangchan, G6PD Mediterranean (C563T), G6PD Vanua Lava (T383C), G6PD Coimbra (C592T), G6PD Kaiping (G1388A), G6PD Orissa (C131G), G6PD Mahidol (G487A), G6PD Canton (G1376T), and G6PD Chatham (G1003A). Our results document heterogeneous mutations of the G6PD gene in the Malaysian population.