SCREENING ELSEVIER Screening 4 (1995) 27-34 Frequencies of glucose 6-phosphate dehydrogenase pyruvate kinase and hexokinase deficiencies in the Basrah population of Iraq Menhel M. Al-Naamaaa, Lamia M. Al-Naama*b, Tagreed A. Al-Saadoonb “Chemistry Department, College of Education, Basrah University, Basrah, Iraq bBiochemistry Department, College of Medicine, Basrah University, Basrah, Iraq Received 3 April 1992; revision received 29 July 1993; accepted 9 January 1995 Abstract Introduction: The frequenciesof enzyme deficienciesthat causehemolytic anemiaamong Iraqi peoplein southernIraq have beenstudied. These hereditary red blood cell enzymesin- clude glucose6-phosphate dehydrogenase (G6PD), pyruvate kinase (PK) and hexokinase (HK). The mostcommon enzyme deficiency is G6PD deficiency, an X-linked recessivetrait. PK deficiency is associatedwith chronic hemolytic anemia, but HK deficiency is a rare autosomalrecessive enzymopathy. Methods: A total of 1497clinically normal neonatesand 1629apparently healthy adults from Iraqi Arab original inhabitantsof Basrahwere studied. Blood samplesobtained from the umbilical cord of the newbornsor by venipuncture from adultswere collected in ACD anticoagulantand storedat 4°C. Erythrocyte G6PD activity was measuredqualitatively by the fluorescentspot test or methemoglobinreduction test and con- firmed quantitatively by spectrophotometricanalysis. PK and HK activities were estimated only quantitatively by spectrophotometry.Results: Only sevenpartially deficient infants out of 506 infants and 343 adults were detectedwith PK deficiency. On this basis,the incidence would be 0.82 with an estimatedgene frequency of about 0.004. Partial HK deficiency was found in only one femaleinfant out of 220 infants and 246 adults in both sexes.No caseof completedeficiency of theseenzymes was identified. G6PD deficiencywas identified in 131 out of 1040male infants and adults. All the affected malesseemed to have severedeficiency (hemizygotes).On this basis,we estimatedthe genefrequency to be about 12.6%.In the female population, 3 1 out of 771 femaleinfants and adultshad severeenzyme deficiency, a frequency of 4.0% (possiblehomozygosity) while 67 femalesshowed intermediate or moderateenzyme activity (heterozygosity),a prevalenceof 8.7%.Discussion.. Among the Iraqi Arab populations that are original inhabitantsof Basrah,erythrocytes G6PD deficiency is by far the mostcom- * Correspondingauthor. Elsevier Science Ireland Ltd. SSDI 0925-6164(95)00103-S 28 M. M. AI-Naamaa et al. /Screening 4 (1995) 27-34 mon enzyme deficiency that causes hemolytic anemia. The gene frequencies for male infants and adults were 11.7% and 13.1%, respectively. The observed female incidences, for both infants and adults, were far from those expected. Factors that might be the causes of such discrepancies were discussed. Keywords: Glucose 6-phosphate dehydrogenase (G6PD); Pyruvate kinase; Hexokinase; Screening; Newborn; Cord blood; Adults; Iraq 1. Introduction We have studied the frequency of certain hereditary enzymopathies that cause he- molytic anemia among Iraqi people in southern Iraq. These enzymes deficiencies in red blood cells include those of glucose 6-phosphate dehydrogenase (G6PD), pyru- vate kinase (PK) and hexokinase (HK). G6PD is the first enzyme of the oxidative hexose monophosphate shunt, PK catalyses the conversion of phosphoenolpyruvate to pyruvate in the Embden-Meyerhof pathway, and HK catalyses the phosphoryla- tion of glucose to its metabolically active form, glucose 6-phosphate. Each of these deficiencies has been responsible for hereditary non-spherocytic congenital hemo- lytic anemia [l-5], and each may also be associated with other effects, both hematological and extra-hematological [2,6,7]. The most common of these three enzyme deficiencies is G6PD deficiency, an X- linked recessive trait which exists in many genetic variants among different popula- tions [8]. Some variants are associated with normal enzyme activity and others with reduced G6PD activity [l]. It has been estimated that more than 200 million people have G6PD deficiency [1,7,8]. PK deficiency, an autosomal recessive disorder, is associated with chronic hemolytic anemia and accounts for over 80% of the cases of hemolytic anemia involving the Embden-Meyerhof pathway [2]. Valentine et al. [2] have documented over 300 patients with hemolytic anemia due to PK deficiency. Although PK deficiency has worldwide distribution, its prevalence in many parts of the world is still unknown. It is mostly found in Caucasians of north European ancestry, but has also been reported from the Mediterranean, Far East and Mexico [2]. HK deficiency is rare but several cases have been reported in different popula- tions [9]. In 14 patients it has been identified as an autosomal recessive enzymopathy causing life-long hemolytic anemia [2]. A high prevalence of G6PD deficiency has been reported in Iraq, particularly in Basrah [ 10,111, but also in other regions and among different ethnic groups [ 121. No observations from Iraq have been published about other enzyme deficiencies such as PK and HK, which play important roles in red blood cell metabolism. With this aim in mind, we conducted a survey to estimate their deficiencies in Iraqi male and female populations from Basrah, in adults and in cord blood samples. 2. Materials and methods A total of 3126 blood samples were collected from apparently healthy unrelated individuals. All were Iraqi Arabs, originally inhabitants of Basrah in southern Iraq M. M. Al-Nuamaa et al. /Screening 4 (1995) 27-34 29 (90% Moslem and 10% Christian). No Kurds or other specific ethnic groups were included. The first group consisted of 1497 clinically normal neonates (871 males and 626 females) delivered at the Basrah maternity and Al-Tahreer hospitals. The second group included 1629 adults (945 males and 684 females) students and employees of the university, blood donors, and people attending the out-patient clinics at the Basrah Teaching Hospital. Their ages ranged from 20 to 45 years. Any subject with a known family history of enzyme deficiency was excluded. Blood samples of 5.0 ml obtained from the umbilical cords of the newborns or by venipuncture from the adults, were collected in ACD anticoagulant and stored at 4°C until assayed within 24 h. Erythrocyte G6PD activity was measured qualitatively by the fluorescent spot test of Beutler and Mitchell [ 131 or methemoglobin reduction test [14] and confirmed quantitatively by spectrophotometric analysis using a SP8- 100 with cell temperature controller (Pye-Unicam, United Kingdom), according to a recommended method [ 151. PK activity was determined by standard methods developed by the International Committee for Standardisation in Haematology [ 161. Red blood cell HK was estimated quantitatively using the method described by Beutler [17]. All enzyme activities were calculated as pmol substrate converted per 10” erythrocytes per min at 37°C. Each sample reading was a mean of triplicate determinations. Normal activity was expressed as the mean of the normal values. 3. Results The frequencies of PK and HK deficiencies among neonates and adults are shown in Table 1. Tanaka [18] had reported that severe PK deficiency was defined as Table 1 Frequencies of pyruvate kinase and hexokinase deficiencies for newborn infants and adults in the Basrah population of Iraq Population PK HK Number Partial deficiency Number Partial deficiency tested tested n o/o n ‘%, Infants Male 336 5 I.5 III 0 0 Female 170 2 1.2 109 I 0.91 Total 506 7 1.4 220 I 0.45 Adults Male 198 0 0 131 0 0 Female 145 0 0 115 0 0 Total 343 0 0 246 0 0 All age groups Male 534 5 0.94 242 0 0 Female 315 2 0.63 224 I 0.45 Total 849 7 0.82 466 I 0.21 Mean normal activity at 37°C: infants PK 5.10 f 0.81 and HK 0.61 f 0.16 U/10” RBC; adults PK 3.61 + 0.55 and HK 0.41 f 0.06 U/1O’o RBC. 30 M.M. Al-Naamaa et al. /Screening 4 (1995) 27-34 Table 2 Frequency of glucose 6-phosphate dehydrogenase deficiency in the neonate population of the Basrah area of Iraq Sex Number Normal Intermediate Severe tested n % n % n % Male 424 374 88.3 0 0 50 11.7 Female 341 307 88.6 29 8.3 II 3.1 Mean normal G6PD activity is 3.29 f 1.07 U/lO’o RBC at 37°C. Deficiency is defined with activity ranges: intermediate, 0.33-1.92; severe O-O.33 U/lO’o RBC. 5%-20% of the mean normal enzyme activity while heterozygotes showed moderate enzyme reduction at less than 60% of mean normal activity. Among the 506 neo- nates, 7 (5 males and 2 females) had moderate PK deficiency, a prevalence of 1.4%. Only one neonate (a female) out of 220 infants (0.45%) was deficient in HK. No adults were deficient in either PK or HK. Thus, the overall frequencies of PK and HK deficiencies were 0.82% and 0.2%, respectively (Table 1). No case of complete deficiency of either of these enzymes was identified. Table 2 shows the frequency of G6PD deficiency in newborn infants. Of the 771 neonates tested, 90 showed G6PD deficiency, a prevalence of 11.6% for both sexes. Those with G6PD deficiency were separated according to whether the activity was less than 10% (severe) or lo%-60% (intermediate) of the mean normal activity [19]. Severe deficiency was detected in 50 males (11.7%) and 11 females (3.1%); intermedi- ate deficiency was demonstrated in 29 females (8.3%). For adults, 139 were found to be G6PD deficient, a prevalence of 13.3%. Severe deficiency was detected in 81 males (13.1%) and 20 females (4.7%) while 38 females (8.9”/,) demonstrated interme- diate G6PD activity (Table 3). For all age groups (infants and adults), severe G6PD deficiency was noted in 12.6% of males (hemizygotes) and 4.0% of females (homozygoses), while intermediate enzyme activity was found in 8.7% of females (heterozygoses) (Table 4). The gene frequency for G6PD deficiency was estimated from the data obtained.