Essay: G6PD deficiency

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2.1 Glucose-6- phosphate dehydrogenase (G6PD)
Glucose-6-Phosphate Dehydrogenase (G6PD) is a significant enzyme in the hexosemonophosphate oxidative pathway which important in the production of nicotinamide adenine dinucleotide phosphate oxidase (NADPH) (Ainoon et al., 1999). This enzyme will help in preventing erythrocytes from oxidative damage and to repair the damage which have occurred (Yan et al., 2010). Oxidative damage can be prevented by glutathione (GSH) which is synthesized and present in high concentration in red blood cell. According to WHO Working Group (1989), NADPH is important in regeneration of GSH by glutathione reductase. Thus, if the red blood cell fails to produce NADPH, it will also reduce the production of GSH which may increase oxidative damage. In addition, the amount of active G6PD enzyme is depending on the age of the erythrocytes. The matured erythrocyte will be damaged more easily when expose to oxidative stress when compared to younger cells. This is because mature erythrocytes do not have nucleus, mitochondria or ribosomes. Hence they cannot produce new proteins including the G6PD enzyme (Anna L. P and Cornelis J. F, 2009).
G6PD enzyme catalyzes the oxidation of G6P in the first step in the Pentose Phosphate pathway. In this pathway, NADP+ is converted into NADPH, while G6P is converted into pentose sugar, ribulose-5-phosphate, and precursor of DNA, RNA, and ATP. Via the anaerobic part of this partway, ribulose-5-phosphate can shuttled into glycolysis. NADPH is essential for biosynthesis and deteoxification reactions and it is act as reducing agent in the cytoplasm cell, which include erythrocytes (Peters & Noorden, 2009). The role of G6PD is described in Figure 2.1 and Figure 2.2 explains the G6PD enzyme metabolic pathway in the HMP.
Figure 2.1 : G6PD catalyzes NADP= to its reduce form, NADPH in the pentose phosphate pathway.
Adopted form (Chowdhry, Bisoyi, & Mishra, 2012)
Figure 2.2 : G6PD enzyme metabolic pathway: the glycolysis and the pentose phosphate cycle (hexose monophosphate shunt).
Adopted from (Minucci, Giardina, Zuppi, & Capoluongo, 2009)
2.2 Genetic of G6PD enzyme
The G6PD gene is located at region q28 which is on the long arm of the X-chromosome (Moiz, 1956). In 1989, Martini et al. has reported that the G6PD enzyme occupied approximately 100 kb of the X chromosomes. In addition, the research also stated that the G6PD gene was 18 kb long and consists of 13 exons. The protein-coding region was divided into 12 segments with sizes between 120 to 236 bp. G6PD is made up of dimers with each monomer being 59 Dalton of molecular weight. It also consisted of 515 amino acids (Moiz, 1956). Figure 2.3 shows the three-dimensional structure of active G6PD dimer. This G6PD gene will give instruction for the production of G6PD enzyme. Thus, if mutation occurs in the G6PD gene, it will affect the production of G6PD enzyme. On the other hand, the activation of this enzyme will occur in the present of the dimer or tetramer which contain tightly bound of NADP+ (Peters & Noorden, 2009).
Figure 2.3 : A three-dimensional model of the active G6PD dimer,
Adopted from (Mason, Bautista, & Gilsanz, 2007)
2.3 Glucose-6-Phosphate dehydrogenase (G6PD) deficiency
G6PD deficiency is an enzymatic disorder of erythrocytes whereby the red blood cells do not produce enough G6PD enzymes or the enzyme itself do not function properly. It has been estimate to affect 400 millions individuals worldwide (Ainoon et al., 2004). According to Ainoon et al. (1999), G6PD deficient patient is usually healthy individual who develop acute hemolysis because of ingestion of certain drugs, food and exposure to certain chemicals or in conditions such as hypoxia or infection.
In 2008, Beutler stated that G6PD deficiency can be found in any population. It has been estimated that 7.5% of world population was reported to carry one or two genes for G6PD deficiency. The proportion is 35% in Africa, 0.1% in Japan and Europe. In addition, 2.9% of the world population was reported as genetically G6PD deficient (WHO Working Group, 1989). However, according to Silao et al. (2009), G6PD deficiency incidence in Malaysia is about 3.5%.
The amount of active G6PD enzyme is affected by the aging factor of the erythrocytes, which means that the amount of the active G6PD enzyme will decrease throughout the aging of the erythrocytes. Thus, the aging erythrocytes become easily to exposed to oxidative stress. This condition happens because the mature erythrocytes do not have nucleus, mitochondria, or ribosomes and thus it can’t produce new proteins. Erythrocytes can’t eliminate stress when it exposed to oxidative stress, thus cause hemolysis (Peters & Noorden, 2009).
2.4 Genetic inheritance and X-chromosome inactivation
It is an X-linked recessive disorder which can happen to both female and male individuals. A male with a single X chromosome will have two phenotype either G6PD normal or deficient hemizygotes, depending on whether the gene carried by the person is normal or abnormal. On the other hand, in female, it can be divided into three subgroups of phenotype since female has 2 X-chromosomes. The three subgroups consist of normal homozygote, deficient homozygote and heterozygote (Kaplan & Hammerman, 2011). Figure 2.4 is described the genetic inheritance of G6PD deficiency.
Some female who are G6PD deficient have mosaic genetic with two populations of erythrocytes which contain G6PD normal level and G6PD deficient level. It is caused by mixed population of erythrocytes which one of the two X-chromosome is randomly inactivated, that call lyonization (Peters & Noorden, 2009)
Figure 2.4 : Inheritance of X-linked recessive disorder
Adopted from U.S, National Library of Medicine, 2015

2.5 Classification of G6PD deficiency
G6PD deficiency has their own genetic variants which grouped them into several classes. The different types of variants have different mutations and different classes of enzyme activity levels. According to the WHO Working Group (1989), G6PD deficiency can be classified into five different classes according to their severity. The Class I variant is for severe deficiency which is associated with a chronic non-spherocytichaemolytic anemia (CNSHA). Class II variant is also for severe deficiency which is less than 10% of residual enzyme activity but without CNSHA. It also includes the common Mediterranean and common severe oriental variants. Class III variant is for moderate deficiency which is 10% – 60% residual enzyme activity and includes the common African form. Class IV variant is for non-deficient enzyme activity and Class V for increased enzyme activity. The classification of G6PD deficiency is show in Table 2.1.
Table 2.1 : Classification of G6PD Deficiency based on their enzyme activity
Adopted from (Chowdhry et al., 2012)

2.6 Complication of G6PD deficiency
G6PD deficiency is an asymptomatic disease in most affected peoples. However, G6PD deficient peoples may have clinical expressions which include acute acquired hemolytic anemia, chronic non-spherocytic hemolytic anemia, favism and neonatal hyperbilirubinemia (Buetler, 1994). According to Ainoon et al. (2002), one od the most significant complication in G6PD deficient peoples is severe neonatal hyperbilirubinemia and the possibility of kernicterus development which can result in permanent neurological damaged and death. This problem is particularly noticed in Mediterranean and Asia G6PD deficient peoples.
2.6.1 Neonatal jaundice
Neonatal jaundice is one of the life and health threatening result of G6PD deficiency. Neonatal jaundice is cause by excessive production of bilirubin and accumulation of piqment in the plasma and tissues. Basically, the production of bilirubin occurred when the destruction of erythrocytes happen either at the end of their normal life span or due to hemolysis. As cited in Cappellini & Fiorelli (2008),previous study suggest that G6PD deficiency is less common in female newborns with jaundice, while about third of all male newborns with neonatal jaundice have G6PD deficiency. Thus, it shows the relation between G6PD deficiency with neonatal jaundice.
As cited in Beutler (1996), neonatal jaundice was primarily occur in Oriental and Mediterranean newborns. In addition, one study revealed that G6PD Aures has been associated with high incidence of jaundice. Beside that, report by USA stated that Afro-American newborns did not have significantly high incidence of neonatal jaundice. However, general observation by USA and survey in Jamica and Africa suggested that G6PD A- newborns have high incidence of neonatal icterus.
2.6.2 Hemolytic anemia in G6PD deficiency
According to Buetler (1994), drug induced hemolysius was the first morbid effect of G6PD deficiency. Several investigations on why some peoples were prone to develop hemolytic anemia when ingesting 8’aminoquinoline antimalarial drug premaquine drug has been done and discovered G6PD deficiency as a result. After one or two days administration of the drug, the hemoglobin level were found to drop. In addition, in early stage of administration, particles of denatured protein adherence to erythrocytes newborn known as Heinz bodies were detected and it will disappear when the hemolysis progresses. Figure 2.5 and Figure 2.6 shows the picture of Heinz Bodies.
As cited in Buetler (1994), peoples with class III G6PD deficiency known as G6PD A- have moderate enzyme activity and self-limited of hemolytic anemia. It is due to condition where only older erythrocytes are destroyed, while young erythrocytes have normal or near normal enzyme activity. Beside that, in peoples with more severe forms of enzyme deficiency, which known as Mediterranean, young erythrocytes are severely deficient of enzyme activity, thus it will resulted of continuous hemolysis until the administration of drug is well stopped. When severe hemolysis occurred, patient may experience back pain and urine color turn to dark.
Figure 2.5 : (A) Heinz bodies detected by supravital staining with methyl violet. (B) Heinz bodies visualised by electron microscopy
Adopted from (Cappellini & Fiorelli, 2008)
Figure 2.6 : The Heinz body test for primaquine sensitivity (G6PD deficiency). The cells (right) are from a primaquine-sensitive (G6PD-deficient) donor; the cells in the left panel, from a normal control.
Adopted from (Ernest Beutler, 2008)
2.6.3 Congenital non-spherocytic hemolytic anemia
In some peoples, variants of G6PD deficiency cause chronic hemolysis, which lead to congenital non-spherocytic hemolytic anemia. Based on WHO classification, this variant is group as class I. As cited in Cappellini & Fiorelli (2008), congenital non-spherocytic hemolytic anemia are occasionally cause by G6PD variants but almost of the occurrence are cause from independent variants. This complication can be diagnosed based on the clinical finding. Many peoples with congenital non-spherocytic hemolytic anemia caused by G6PD deficiency have experienced severe neonatal jaundice, chronic anemia which requires blood transfusion, reticulocytosis, gallstones, and splenomegaly.
2.6.4 Favism
Favism is commonly associated with G6PD deficiency. Favism is one condition coming up due to the consumption of fava beans. As cited in Cappellini & Fiorelli (2008), it can be developed after ingestions of beans, either frozen or dried beans. However, it is usually happen after eating fresh beans and most frequently in the harvested period. Favism does not happen at all G6PD deficient peoples who ingest fava bean and unpredictable response will happen even on the same people. The development of this condition can be cause by several factors such as the health and the amount of fava bean ingested by G6PD peoples.
As cited in Allahverdiyev, Bagirova, & Elcicek (2012), this condition usually occur in children between 2-5 years old and it is 2-3 times more common occur in boy rather than girl. The clinical symptoms for favism are include pallor, jaundice and hemoglobinuria. There are different characteristic symptoms for each individual, even though they come from same family. The characteristic symptoms are dependent on fava bean active metabolites and the genetic variants.
2.7 Factors lead to hemolytic anemia in G6PD deficiency
Most G6PD Deficient peoples were asymptomatic throughout their life and unaware about their status. It will generally manifest as acute hemolysis when erythrocytes undergoes oxidative stress, which is triggered by agent such as drug, infection or ingestion of fava beans. However, G6PD deficiency does not affect expectancy, quality or the activity of the affected peoples (WHO Working Group, 1989).
2.7.1 Drugs
Drug is one of the components that can induce hemolytic anemia. According to Beutler (1989), G6PD deficiency was discovered by investigating thee development of hemolysis in people who has received primaquine, which known as 8-amino-6-methoxy quinolone antimalarial drugs. However, acute hemolysis in G6PD deficient peoples also can be cause by several drugs other than primaquine. An agent deemed to be safe for some G6PD deficient peoples is not necessity safe for all parents because pharmacokinetics between peoples can be varied. Table 2.2 show list of drugs and chemicals associated with substantial hemolysis in patients with G6PD deficiency.
Besides that, administered of drugs with potentially oxidant effect to patient with an underlying clinical condition such as infection also can lead to hemolysis. Hemolysis condition in G6PD deficient peoples is a self-limiting process, thus the clinical significant of anemia and reticulocytes does not always occurred (Cappellini & Fiorelli 2008).

Table 2.2 : Drugs and chemicals associated with substantial hemolysis in patients with G6PD deficiency
Define association Possible association Doubtful association
Antimalarials Primaquine
Pamaquine Chloroquine Mepacrine
Sulfonamides Sulfanilamide
Sulfamethoxazole Sulfadimidine
Glibenclamide Aldesulfone
Sulfones Dapsone – –
Nitrofurantoin Nitrofurantoin – –
Other drugs Nalidixic acid
Co-trimoxazole Ciprofl oxacin
Vitamin K analogues
Ascorbic acid
Mesalazine Aminosalicylic acid
Other chemicals Naphthalene
2,4,6-trinitrotoluene Acalypha indica extract –
Adopted from (Cappellini & Fiorelli, 2008)
2.7.2 Fava beans.
Fava bean, Vicia faba (Figure 2.7), also known as bell beans, broad beans, English dwarf beans, fever beans, haba benas, horse beans, pigeon beans, silkworm beans and tick beans. It’s contains glycosides, divicine and isouramil and convicine which known as oxidant (Moiz, 1956). Cappellini & Fiorelli ( 2008) cited that this oxidant are thought to be toxic element of fava beans which can increase the activity of the hexose monophosphate shunt, thus promoting hemolysis in G6PD deficient patients.
The onset of hemolysis in favism is might be more dangerous compared to the effect of drug administration. However, the possibility of the hemolysis occurrence is similar to the occurrence of the hemolysis after ingestion of drug. Besides that, haemoglobinuria may appear for several days after 24 hours of ingestion of beans, while hemolysis does not occur immediately after ingestion of bean (Buetler , 1994). According to Cappellini & Foirelli (2008), acute hemolytic anemia usually occur around 24 hours after eating the beans.
Figure 2.7 : Fava beans (vicia fava)
2.7.3 Infection
Infection is probably one of the most typical causes of hemolysis in G6PD deficient peoples. There are various type of infections that may promote hemolytic episode such as bacterial, viral or parasitic infection. According to Army, Hospital, & Benning (2005), most common infectious agents causing hemolysis are Salmonella, Escherichia coli, beta-hemolytic streptococci, rickettsial infections, viral infections and influenza A. In addition, Cappellini & Foirelli (2008) stated that hepatitis viruses A and B, cytomegally virus, pneumonia and thypoid fever are notable causes of hemolysis in G6PD deficient peoples. However, the mechanism that related between infection and hemolytic anemia is not clear, but there is one suggestion that during phagocytosis, damaged of the erythrocytes cause by leukocytes is occurred by discharging the active oxygen (Beutler, 1996).
2.8 G6PD mutation and molecular variants of G6PD deficiency
As cited in Ainoon et al. (2003), there are 400 biochemical and 122 molecular variants of the G6PD enzyme that have been recognized in various populations. According to Laosombat et al. (2005), the common G6PD mutation variant reported among Southeast Asian were G6PD Mahidol, G6PD Kaiping, G6PD Canton, G6PD Gaohe, G6PD Union, G6PD Quing Yuan and G6PD Viangchan. In Malaysia, characterization of G6PD mutation has been performed by several groups of researchers. In addition, studies have also included the prevalence of G6PD mutations among Malaysians depending on the common races available in Malaysia. In 2008, a study by Wang et al. has revealed the most common G6PD variants in Malays are G6PD Viangchan, G6PD Mahidol, G6PD Mediterr anean subtypes and Indo-Pakistan subtype. The study also revealed that G6PD Mediterranean Indo-Pakistan subtype and G6PD Namoru were detected among Malaysians Indian. This is not surprising as G6PD Namoru was first detected in Vanuatu Archipelago and was found in abundance in south India (Wang et al., 2008).
According to Ainoon et al. (2003), the first three most common G6PD mutations among Malays were G6PD Viangchan (871 G>A), G6PD Mediterranean (563 C>T) and G6PD Mahidol (487 G>A) which at 37.2%, 26.7% and 15.1%, respectively. Viangchan and Mahidol are common in Thailand which is Malaysia’s neighboring country. Thus, marriage between individuals from the two countries resulted in high incidence of these mutations. In addition, according to Wang et al. (2008), G6PD Viangchan is also common in Cambodia while G6PD Mahidol is common in Myanmar. Besides the two mutations, two subtypes of G6PD Mediterranean were found among Malay population which were Mediterranean subtype and Indo-Pakistan subtype. The Indo-Pakistan subtype was common among Indian population in India, while the Mediterranean subtype were common among Mediterranean people. It showed that the Malays Malaysian have been influenced by several gene flows in very old times ago.
Furthermore, the study also revealed that two most common G6PD mutations among Malaysian Chinese were G6PD Canton (1376 G>T) 4.7% and G6PD Kaiping (1388 G>A) 3.5%. These mutations were common in China and also can be found in Singapore and Vietnam. Other mutations which have been found were G6PD Vanua Lava (383 T>C), G6PD Coimbra (592 C>T), G6PD Union (1360 C>T), G6PD Chatham (1003 G>A), G6PD Orissa (131 C>G), and G6PD Andalus (1361 G>A) which at 3.4%, 3.4%, 2.3%, 2.3%, 1.2% and 1.2%, respectively (Ainoon et al., 2003). On the other hand, in 2004, Ainoon et al. has been done a study among Chinese neonate which give a result on 46.1% for G6PD Canton (1376 G.T), 37.5% for G6PD Kaiping (1388 G.A), 7.0% for G6PD Goahe (592 G>A), 3.1% for G6PD Chinese-5 (1024 C>T), 1.5% for Nankang (517 T>A), 1.6% for Mahidol (487 G>A), and 0.8% for G6PD Chatham (1003 G>T), G6PD Union (1360 C>T), G6PD Viangchan (871 G>A) and Quing Yang (392 G>T).
However, the current study only focuses on G6PD-Mediterranean and G6PD-Canton among G6PD deficient newborns from Malay and Chinese ethnicities. G6PD-Mediterranean is the type of mutation that occur when there is substitution of nucleotide from C to T in position 188, while G6PD-Canton is when there is substitution of nucleotide from G to T in position 459. Both types of mutations are classified in the Class II enzyme activity level. As stated in the previous studies by Wang et al. (2008) and Ainoon et al. (2004), G6PD Canton mutation is one of the common mutations among Malaysian Chinese, while G6PD Mediterranean is one of the common mutations among Malaysian Malay. Table 2.3 represents the G6PD variants among ethnics in Malaysian population.
Table 2.3 : G6PD variants among ethic group in Malaysian population
Race G6PD variant G6PD subtype G6PD mutation
Malay Viangchan – 871 G > A
1311 C > T
IVSII nt93 T > C
Mahidol – 487 G > A
Mediterranean Mediterranean subtype 563 C > T
1311 C > T
IVSII nt93 T > C
Indo- Pakistan subtype 563 C > T
1311 C
IVSII nt93 T
Kaiping – 1388 G > A
Canton – 1376 G > T
Gaohe – 95 A > G
Mediterranean Mediterranean subtype 563 C > T
Indian Mediterranean indo-pakistan subtype 563 C > T
1311 C
IVSII nt93 T
Namoru – 208 T > C
Kaiping – 1388 G > A
Orang asli viangchan – 871 G > A
1311 C > T
IVSII nt93 T > C
Adopted from ( Wang et al. , 2008)
2.9 Detection of G6PD deficiency
At present there are many tests available for the detection of G6PD deficiency. According to Ainoon et al. (2003), diagnosis of G6PD deficiency can be performed by using semiqualitative test or red cell enzyme activity quantitation. The semiquantitative fluorescent spot test (FST) which was initially described by Beutler & Halasz, (1966) and subsequently modified by Beutler & Mitchell, (1968) is widely used for diagnosis and population screening. In Malaysia, the G6PD newborn screening programs used the Beutler’s modified fluorescent spot test (FST) in order to detect deficiency in G6PD (Beutler & Halasz, 1966). According to Nadarajan et al. (2011), FST, spectrophotometric assay and cytochemical assay were most frequently used for identifying of mutation by measuring the NADPH production capacity of G6PD enzyme.
Anna L. P and Cornelis J. F (2009), stated that FST and spectrophotometric assay are based on the formation of fluorescent, while cytochemical assay are based on color formation. As cited in Allahverdiyev et al. (2012), fluorescence is a form of luminescence which use physical change of emission of light upon the excitation of molecules. The type of fluorescence is classified depends on the excitation styles which are photo-luminescence (fluorescence, phosphorescence and delayed fluorescence), chemo-luminescence (ending in a chemical reaction), bioluminescence (via a living organism) and others. Mean while, cytochemical staining assay is a technique based on the tetranitro blue tetrazolium (TNBT) intracellular reduction by the G6PD. The intracellular reduction occurs via exogenous electron carrier 1-methoxyphenazine methosulfate. In the process, the TNBT is reduce to dark-colored water-insoluble formazan and it will be determine by light microscopy. In this method, erythrocytes which have little or no G6PD activity will not be stained (Peters & Noorden, 2009). Figure 2.8 shows the erythrocytes in heterozygous G6PD deficient patient by cytochemical staining method.
Fluorescent Spot test is done by using cord blood sample. The result will be read according to the formation of fluorescence which will be interpreted as deficient for no fluorescence intensity, intermediate for diminished fluorescent intensity and normal for bright fluorescent intensity (Nadarajan et al., 2011). Figure 2.9 shows the interpretation of fluorescence spot test result. According to Kaplan & Hammerman (2011), FST is recommended for standardization in Hematology by the International Committee. They further stated that fluorescence formation indicate the G6PD activity which NADPH will fluoresces intensely when activated by ultraviolet light.
Quantitation enzyme assay is a test done to measure the G6PD enzyme activity level by using a spectrophotometer. The upper limit for total G6PD deficiency is 3.2 U/g Hb, while the upper limit for mild G6PD deficiency is 9.5 U/g Hb. Thus, the interpretation is when the activity level is less than 3.2 U/g Hbit indicates severe deficiency and between 3.2 to 9.5 U/g Hbis mild deficiency (Nadarajan et al., 2011). In 2003, Ainoon et al. stated that FST is a qualitative test and only severe G6PD can be detected when the enzyme level is less than 20% of the normal level. Thus, based on the limitation, the G6PD enzyme level assay is applied in some countries as a screening method instead of FST. Even though there are various screening tests for G6PD deficiency, not all of them are reliable in the detection of female heterozygote. As stated by Beutler & Mitchell (1968), FST is a rapid, simple and cheap test for the identification of hemizygous males and homozygous female only. However, over the years, there are many molecular tests that have been developed to characterize mutations.

Figure 2.8 : Erythrocytes of a heterozygously G6PD-deficient patient after cytochemical staining. The erythrocytes marked with arrows contain G6PD activity; the erythrocytes marked with arrowheads are G6PD deficient. (A) Light microscopical image. (B) Fluorescence image.
Adopted from (Peters & Noorden, 2009)
Figure 2.9 : Fluorescent spot test depicting the fluorescence intensity of dried blood spots (intermediate, deficient and normal) on Whatman 1 filter paper.
Adopted from (Nadarajan et al., 2011) ‘
Nowadays, advancement in molecular techniques has made detection of G6PD mutations possible in many countries (Wong F L, Boo N Y, Ainoon O, and Wang M K 2009). As cited in Ainoon et al. (2003), the advancement of this technique may allow molecular characterization of the various mutations in the G6PD gene to be carried out easily. Several groups have tried to characterize the variants of G6PD gene by using several molecular methods especially assays which were based on polymerase chain reaction (PCR). PCR is a technique to amplify the target DNA. The amplification of the DNA consists of five steps, which are initial heat denaturation step, denaturation step, annealing step, extension step and final extension step. Each of these steps has different temperature and time.
There are various molecular methods based on PCR have been use by researcher, which include Real-Time PCR, Amplification Refractory Mutation System (ARMS-PCR), and Restriction Fragment Length Polymorphism (RFLP-PCR). In addition, Multiplex PCR, Restriction Enzyme PCR and Single Stranded Confirmation Polymorphism PCR (PCR-SSCP) are also has been used. Even though all of these methods are based on PCR technique, each of the methods still has differences between them in part of procedure and instrument use. Besides that, all of these methods also still have their own advantages and disadvantages. One research on comparative study for the detection of mutation has been done by El-Gezeiry group in 2005. The research found that Amplification and refractory mutation system (ARMS) technique is less time consuming and cheaper than restriction fragment length polymorphism. The ARMS technique takes 2 hour 30 minutes for process, while restriction fragment length polymorphism takes 5 hours. (El-gezeiry et al., 2005)
In Malaysia, a study on molecular heterogeneity of G6PD deficiency in Malays has been performed by using Multiplex PCR using multiple tandem forward primers and a common reverse primer (MPTP) (Yusoff et al., 2002). In 2003, Samilchuk et al. has used PCR amplification followed by Restriction Fragment Length Polymorphism (RFLP) to study G6PD mutations and UDP-glucuronosyltransferase promoter polymorphism among G6PD deficient Kuwaitis. The use of Real-Time PCR for the detection of G6PD variant has also been done by Wong F L, Boo N Y, Ainoon O and Wang M K (2009). Beside that, PCR-SSCP has been used to characterize G6PD variants among Malaysian Malays (Ainoon et al., 2003). However, even though various types of PCR are available, DNA sequencing is still the gold standard for detection of any type of mutations.
In the present study, detection of G6PD mutation was conducted Real-Time Polymerase Chain Reaction (PCR) with TaqMan Single Nucleotide Polymorphism (SNP) genotyping assay. Real-Time PCR is also a one of technique which based on PCR. Real-Time PCR has a variety of usage such as for the amplification, detection and quantification of the target DNA. It uses various fluorescent detection chemistries that allow the monitoring of the PCR reaction as it progress. By using TaqMan probe method, the oligonucleotide will be labeled with fluorescent reporter dye at the 5’ end and the quencher dye at the 3’end. The oligonucleotide itself has no significant fluorescence but it will fluoresce when the dye is clipped from the oligonucleotide during the extension period.
The advantage of using TaqMan probe is that the fluorescent signal will be generated only when there is specific hybridization of the probe to the target sequence. Hence, no signal will be generated from any non-specific amplification product that may be formed during the reaction. Thus, this makes the method highly specific. In Malaysia, one study has been done by Wong F L, Boo N Y, Ainoon O and Wang M K(2009) that use fluorescence based Single Nucleotide Polymorphism (SNP) in the detection of G6PD variant by using Real-Time PCR. They also stated that by using PCR Restriction Enzyme method, there are chances of getting significant false positive results due to incomplete restriction enzyme activity.
In addition, they concluded that the TaqMan Minor Groove Binder (MGB) SNP method by Real-Time Polymerase Chain Reaction is less time consuming compared to PCR Restriction Enzyme (PCR-RE) method. This is because the TaqMan MGB SNP method does not require post-amplification steps such as gel electrophoresis which is needed to verify the presence of PCR product, restriction endonuclease digestion. Beside that, it also does not require additional steps for visualization of the restriction pattern. Other than that, the elimination of post PCR process will reduce the labor and time required and prevents the exposure to ethidium bromide which is carcinogenic to human.
Apart from being easy to perform especially for high throughput screening it only takes within 2 hours to obtain the genotyping results. Furthermore, DNA probe will be conjugated to a MGB group which will increase the specificity of the mutation detection. The TaqMan MGB assay is also designed to allow other assays to be simultaneously performed. Therefore, there is potential for it to be used in a clinical laboratory setting for high-throughput detection purposes (Wong F L, Boo N Y, Ainoon O, and Wang M K 2009).

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