INCIDENCE OF SEVERE GLUCOSE-6-PHOSPHATE DEHYDROGENASE(G6PD) DEFICIENCY IN COUNTRYSIDE VILLAGES OF THE CENTRALCITY OF MANISA, TURKEYErsin Minareci, Selim Uzunoğlu, Orkide MinareciCelal Bayar University, Faculty of Sciences and Arts, Biology Department, Campus ofMuradiye, Manisa, TurkeyAim: The primary objective of this study was to determine the incidence of severe G6PDdeficiency in selected countryside villages of central city of Manisa in Turkey. Secondarily toinform and protect G6PD deficient people from acute hemolytic crisis and neonatal jaundice bydelivery of the updated protective food and drug list prepared in the light of the WHO- G6PDWorking Committe reports.Methods: In this study, the incidence of severe G6PD deficiency were screened by Beutler ’sFluorescence Spot test among 1604 people living in the contryside villages of central city ofManisa in Turkey.Results: Thirty five out of 1604 tested people were found to have severe G6PD deficiency.The incidence of severe G6PD deficiency were 2.2 % in sampled population. There was adifference for the incidence between male (3.2%) and female (1.14%) as expected due toX-linked heritance. There was no significant differences in the prevalence of severe G6PDdeficiency between the countryside villages connected to central city of Manisa.Conclusion: The high incidence of severe G6PD deficiency implies that this inheritedmetabolite disorder is an important health problem in Manisa region and it is necessary to carryout large-scale screening in the whole population since severe- full G6PD deficiency relatedhealth problems are preventable. For this reason it must be included in the pool of geneticscreening tests in regional health policy.Key words: Severe G6PD deficiency, fluorescence spot test, acute hemolytic anemia, Manisa,Turkey.Eur J Gen Med 2006; se(G6PD, EC 22.214.171.124; locus linked to the Xchromosome at the q28 locus) expressed inall tissues, is the first enzyme of pentosephosphate pathway, where 5-carbon sugar,ribose, and NADPH were synthesized bycoupled oxidation/reduction reactions (1).The function of the normal G6PD enzymeis critical to human survival since G6PD isthe only enzyme producing NADPH requiredfor antioxidative repair systems in circulatederythrocytes (2).G6PD deficiency describes all structuraland functional disorders reducing the catalyticfunction of G6PD protein expressed fromG6PD gene. It is the most common humanCorrespondence: Selim UzunoğluBiology Department, Faculty of Sciences and Arts, CelalBayar University, Campus of Muradiye, 45030,Manisa/TurkeyPhone: 902362412151-118, Fax:902362412158E-mail: email@example.com deficiency in the world. It affects anestimated 400 million people and displays thecharacteristics of X-linked inheritance (3,4).Severe G6PD deficiency causes severalmild to severe health problems either directlyor indirectly depending on the conditions.G6PD deficient individuals can expect severalclinical manifestations including hemolyticanemia, neonatal jaundice, abdominal and/orback pain, dizziness, headache and dyspnea.If the update and right combination ofscientific information and technology wasbuilt up and applied, the occurence of healthproblems would be prevented. The fifteenpercentage of normal enzyme activity andits lower values were categorized as severeG6PD deficiency based on the classification
6Minareci et al.Table 1. The list of sampling countryside villages and their living population size and thenumber of tested people (9).ProvinceMANİSACountyCenterThe samplingcountryside villagesTotal Size ofpopulationThe number oftested ı BozköyYukarı KayapınarBelen Yenice 23391604of World Health Organization (WHO). Thissevere form of G6PD deficiency should bescreened in populations where the incidence isone percentage and higher. Protective adviceshould be given to given to severity deficientpeople (5). The 7.5% of world population arecarriers of G6PD deficiency, and 2.9% wereG6PD deficient according to WHO reports(6).G6PDdeficiencyscreeningstudiescarried out in Aegean region indicates thatthe frequency of severe G6PD deficiencyin population conforms to the screeningcriteria of WHO G6PD Deficiency WorkingCommittee.If severe G6PD deficient people consumecertain oxidative foods such as fava beansand drugs, an acute hemolytic anemia can beinduced in affected individuals especially inchildren and adults. If the cause of acutehemolytic anemia developing with jaundicecould not be find in first 12 hours and bloodcould not be supplied, the person would beexposed to death risk between 18 and 72hours. It may also lead to neonatal jaundiceand related health problems in many G6PDdeficient newborns (7, 8). Fortunatelywhen severe G6PD deficient people werediagnosed earlier by screening tests andgiven updated forbidden food and drug list,the health problems related to the G6PDdeficiency could be definitely prevented.Having considering that there is no sign ofthe disease in normal conditions but leadingto the deathly events conditionally, it makesthe G6PD deficiency screening studies veryimportant and beneficial in population level.The primary objective of this study wasto determine the incidence of severe G6PDdeficiency in selected countryside villages ofcentral city of Manisa in Turkey. Secondarilyto inform and protect G6PD deficient peoplefrom acute hemolytic crisis and neonataljaundice by delivery of the updated protectivefood and drug list prepared in the light of theWHO- G6PD Working Committe reports.MATERIAL AND METHODSThe blood samples were collected fromnon-related 1604 people (male 816; female788) living in some countryside villages ofcentral city of Manisa. The size of livingpopulation in the country side villages ofcentral city of Manisa is about 8672 (9). Thesevere G6PD deficiency screening study werecarried out in the villages given in Table 1. Thegeographic locations of the villages where thetested people live were given in Figure 1. Themales and females were randomly screened inthe villages. There was no gender bias in thisstudy. The blood samples were collected onlyfrom healthy people.The diagnosis of severe G6PD deficientcases was made preferentially by usingstandardized home made fluorescence spottest developed by Uzunoglu S instead ofcommercial Beutler ’s fluorescence spot test(10). The preferred and used screening testwere modified and standardized accordingto guidelines of International HematologyStandardization Committee and Sigma G6PDstandards with normal (non-deficient) (9-12 Ugr/Hb) and patient (full-deficient) controls(0.0- 0.4 U gr/Hb).Blood samples from finger tips for eachperson were collected into heparinizedeppendorf tubes and were stored in ice-boxes.They were kept in 4 o C until fluorescencespot test were run.The modified protocol of Beutler ’s
Glucose 6-phosphate dehydrogenase deficiency7Table 2. The incidence of full-severe G6PD deficient cases in the tested people from sampledpopulation.MaleFemaleTotalTested people(n)The number of healthypeople with negative result81678816047907791569Fluorescece spot test was performed (10).Five ml blood from each sample weredelivered into each eppendorf tube containing45 ml spot test reagent. 10 ml samplestaken out from spot test reactions weredropped separately in whatman paper withone size after 10 minutes of incubation atroom temperature. Dried spots containingthe mixture of hemolysate and spot testreagent were evaluated directly by lookingat the spot colour under the ultraviolet light(366 nm wavelength) in dark room. In thistest, the blood samples with both normalG6PD activity and mild deficiency appearin heavy green fluorescence colour basedon the amount of hemoglobin in the bloodsample. This greenish colour was interpretedas negative result in terms of severe G6PDdeficiency and normal healthy person. Ifdried completed test reaction spots were inheavy brownish colour, this was accepted aspositive result in terms of severe-full G6PDdeficiency. Three dilutions of positive fulldeficient blood samples were reassayed intriplicate in order to avoid the possible falsepositive results due to high concentrations ofhemoglobin.RESULTSThirty-five out of 1604 people screenedby spot test were found to be severeG6PD deficient. The prevalence of severeG6PD deficiencywas 2.2% in sampledpopulation(Table 2). The distribution of severedeficient G6PD cases in males and femaleswas as below: twenty-six out of thirty-fivecases was male. Only nine out of thirty-fivecases was female. The percentage of severedeficient patients was 74.3% in males and25.7% in females. There was also markeddifferences in prevalence between males(3.22%) and females (1.14%). The differencewas statistically significant (p 0.001).The distribution of thirty-five severe G6PDdeficient cases in villages were displayed inTable 3. The high numbers in positive caseswere found in Ucpinar (10), Yenikoy (9)The full-severeG6PD deficient casesn(%)263.291.14352.2and Horozkoy (7) villages. There were nopositive cases in the villages of Akgedik andKarakılınçlı withmountainous geographyand high altitude (Table 3).DISCUSSIONFluorescence spot test has the highestvalidity and specificity in the diagnosis ofsevere G6PD deficiency for both homozygotemales and females. Moreover it wasstandardized by International HematologyStandardization Committee, and its usein screening severe G6PD deficient caseswas advised by WHO-G6PD group (6,10).Therefore the Florescence spot testwaspreferred in qualitative screening of severefull G6PD deficient cases in order to determinethe incidence of severe G6PD deficiency inthe villages of central city of Manisa. It wasnot necessary to determine quantitativelythe activity of G6PD enzyme since the aim ofstudy was to find the severe G6PD deficientcases in the sampled population.Before using the spot test, the mixtureof test reagents, prepared manually, werecalibrated by using Sigma G6PD controlnormal (Sigma G–6888) and G6PD control–patient (severe-full deficient) (Sigma G–5888)standards. In the evaluation of fluorescencespot test findings, the colour of spot test madeby Sigma standards were used as a reference.The heavy green colour indicated the G6PDnon-deficient blood sample, while the lightand heavy brown colour was the indicator offull-severe deficient blood samples. In orderto prevent the false positive results among thepositively diagnosed blood samples, dilutiongradient of blood samples were studied andthe differences in colour formation wereoptimized (11,12).Thirty-five out of 1604 people screenedby spot test were found to be severeG6PD deficient. The prevalence of severeG6PD deficiencywas 2.2% in sampledpopulation. The incidence was above the 1%in population based on WHO-G6PD groupcriteria ofinherited disease significance
8Minareci et al.Table 3. The distribution of severe deficient G6PD cases in the sampled villages of centralcity of Manisa.The sampling villages ofcentral city of ManisaAltitude 80m, First groupÜçpınarYeniköyYağcılarHorozköyY. KayapınarAltitude 80m, Second groupSancaklı BozköyBelen YeniceTepecikAkgedikKarakılınçlıThe number oftested peopleThe full-severe G6PDdeficient casesn(% 006,27,16,6-level.The percentage of full-severe deficientpatients was 74.3% in males and 25.7% infemales.There was also marked differencesin prevalence between males (3.22%) andfemales (1.14%) in sampled population. Thisremarkable difference conforms the X linkedinheritance of G6PD deficiency.The high numbers in positive cases werefound in Ucpinar (10), Yenikoy (9) andHorozkoy (7) villages. There was remarkabledifference in the number of positive casescompared the other villages. There were nopositive cases in the villages of Akgedik andKarakılınçlı withmountainous geographyand higher altitude. The locations of villageswith more cases were close to Gediz river anddelta. The high incidence of severe G6PDdeficiency in the sampled Turkish populationmay be due to the geographic region with thehistory of malaria endemic and was surroundedby Gediz river and several waterlands.There were few studies on the incidenceof severe G6PD deficiency in Aegean region.In one study made in Manisa by DemircanK (1999) in newborns and adults (n 601),the activity of G6PD enzyme were measuredquantitatively and the incidence of severeG6PD deficiency were found to be 2.82 %.The incidence in males and females were5.37 % and 0.62 % respectively (13). Theincidence of our sampled population (2.2 %)was quite close to the data(2.82 %) obtainedby Demircan K.In one qualitative screening study(n:1950) by using fluorescence spot test, theprevalence of severe G6PD deficiency werefound to be 1.23% in the province of Denizli(14).In another study (1985) by using 500chord blood samples,the incidence ofsevere G6PD deficiency was revealed tobe 2.4% in Antalya province located inthe mediterranean cost of Turkey (15). Oursevere G6PD prevalence data (2.2%) wassimiliar to the data in the mentioned literatureand confirms that severe G6PD deficiency isquite high in Aegean region. Moreover, theregional people consume the fava beans inseasonal diet composition. There are manydrugsthat may induce oxidative stressleading to hemolytic crisis in severe G6PDdeficient people. Therefore there is a need forscreening for G6PD in the region and informthem about the disease and the list of food anddrugs that may lead to acute hemolytic crisis.In conclusion, this high incidence of severeG6PD deficiency implies that this inheritedmetabolite disorder is an important healthproblem in Manisa region and it is necessaryto carry out large-scale screening in the wholepopulation since severe G6PD deficiencyrelated health problems are preventable. Forthis reason it must be included in the spectrumof genetic screening tests in regional healthpolicy.ACKNOWLEDGEMENT: This study is the summary ofmaster thesis of Ersin Minareci under the supervisorof Selim Uzunoğlu submitted to The Institute of NaturalSciences. Celal Bayar University, Manisa. 2000.
Glucose 6-phosphate dehydrogenase deficiency9Figure 1. The location map of the research area where countryside villages of central cityof Manisa were displayed.REFERENCES1. BeutlerE.Glucose-6-phosphatedehydrogenase deficiency. N Engl J Med1991;324:169–742. Scott MD Luo L, Lubin BH et al.NADPH,NotGlutathione,StatusModulates Oxidant Senstivity in Normaland Glucose-6-Phosphate DehydrogenaseDeficient Erythrocytes. Blood 1991;77(9):2059-643. Mehta A, Mason PJ, Vulliamy TJ. Glucose6-phosphate dehydrogenase deficiency.Bailliere’s Best Pract Res Clin Haematol2000;13(1):21–384. Luzzatto L, Mehta A, Vulliamy TJ. In:C.R. Scriver, A.L. Beaudet, W.S. Slyand D. Valle, Editors, The Metabolic andMolecular Basis of Inherited Disease,McGraw-Hill, New York, 2001: 4517–535. Beutler E. G6PD Deficiency. Blood 1994;84 (11): 3613-366. WHOWorkingGroup.Glucose-6phosphate dehydrogenase deficiency. BullWHO 1989;67:601–117. Valaes T. Severe neonatal nase deficiency, pathogenesisand global epidemiology. Acta PaediatrSuppl 1994;394:58–768. Meloni T, Forteloni G, Meloni GF. Marked126.96.36.199.13.decline of favism after neonatal glucose6-phosphate dehydrogenase screening andhealth education: the northern Sardinianexperience. Acta Haematol 1992;87:29Annual official statistical reports ofManisa city. The Health Ministry RegionalOffice in Manisa 1998Uzunoğlu S. The investigation ofMediterranean mutation causing to severeG6PD deficiency in Aegean region byusing DNA technology. Ph.D thesis.Dept. of Medical Biology. The Institute ofHealth Sciences. Dokuz Eylul University1995:14-46Barbette SS, Versie DJ. Modification ofNeonatal Screening Test For ncy. Clinica Chimica Acta 1976;71:239–44Solem E, Prizer C. Mass screening oved fluorescent spot test.Clinica Chimica Acta 1985;152:135–42Demircan K. The determination of normalactivity values of Glucose 6-phosphatedehydrogenase in newborns and adultsin Manisa region. Phd thesis. Dept ofMedical Biology.The Institute of HealthSciences. Celal Bayar University. Manisa.1999
1014. Keskin N, Ozdes I, Keskin A, Acikbas I,Bagci H. Incidence and molecular analysisof glucose-6-phosphate dehydrogenasedeficiency in the province of Denizli,Turkey. Med Sci Monit 2002;8(6):453Minareci et al.15. Dolunay M. The prevalence of G6PDdeficiency and its biochemical phenotypein the central part of Antalya province.Ms.C thesis, Dept of Biochemistry.Faculty of Medicine. Akdeniz University.1988
G6PD deficient according to WHO reports (6). G6PD deficiency screening studies carried out in Aegean region indicates that the frequency of severe G6PD deficiency in population conforms to the screening criteria of WHO G6PD Deficiency Working Committee. If severe G6PD deficient people consume certain oxidative foods such as fava beans
chromosomes (XX). The gene coding for G6PD enzyme is located on the X chromosome. As males have only one X chromosome and thus one copy of G6PD gene, they would have G6PD deficiency if the G6PD gene carries a mutation. The G6PD activity in the blood will be reduced. A female who has a mutant G6PD gene on one of her X
It is a lifelong condition, however G6PD deficient can have a completely normal life as long as they avoid triggers such as drugs and chemical exposures that can cause oxidant stress. Prevalence of G6PD in Malaysia: Figure 2: An overview of G6PD deficiency prevalence in Malaysia 
foods is crucial to prevent future episodes of hemolysis. In many cases all members of the patient’s family should avoid such precipitating foods. G6PD deficiency is a sex-linked disorder. As result males who inherit a G6PD mutation are hemizygous for the defect all their RBCs are affected. Females who inherit a heterozygous G6PD mutation
G6PD Mahi-dol is the best-characterized Asian variant and is con-sidered predominant across Myanmar and Thailand [40,41]. Enzyme activity in G6PD Mahidol individuals is reduced to 5-32% of normal levels , and its prima-quine sensitivity phenotype lies between that of the G6PD A- and Mediterranean variants [43,44]. As for the
3 PATH A Guide to Fluorescent Spot Testing for G6PD Deflciency I. Introduction Glucose-6-phosphate dehydrogenase (G6PD) deficien
6 Key points to remember G6PD is an inherited condition and cannot be spread from one person to another. Most people with G6PD deficiency have a completely normal life as long as they avoid certain foods and drugs. Some people with the condition will get anaemia or jaundice, especially after taking medicine or eating food
in an enzyme called G6PD, which helps defend red blood cells against stresses. Primaquine damages these cells so that they burst, leading to anaemia. This is a major problem because G6PD deficiency is common in regions where malaria is present: in some areas up
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