Screening 4 (1995) 27-34. Frequencies of glucose 6-phosphate dehydrogenase pyruvate kinase and hexokinase deficiencies in the. Basrah population of Iraq.
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 bBiochemistry
Received
Department, Department,
3 April
College College
1992; revision
of Education, of Medicine, received
Basrah Basrah
University,
Basrah,
University,
Basrah,
29 July 1993; accepted
9 January
Iraq Iraq
1995
Abstract Introduction: The frequenciesof enzymedeficienciesthat causehemolytic anemiaamong Iraqi peoplein southernIraq have beenstudied.Thesehereditary red blood cell enzymesinclude glucose6-phosphatedehydrogenase(G6PD), pyruvate kinase(PK) and hexokinase (HK). The mostcommonenzymedeficiencyis G6PD deficiency,an X-linked recessivetrait. PK deficiency is associatedwith chronic hemolytic anemia,but HK deficiency is a rare autosomalrecessiveenzymopathy.Methods: A total of 1497clinically normal neonatesand 1629apparentlyhealthy adultsfrom Iraqi Arab original inhabitantsof Basrahwerestudied. Blood samplesobtainedfrom the umbilicalcord of the newbornsor by venipuncturefrom adultswerecollectedin ACD anticoagulantandstoredat 4°C. Erythrocyte G6PD activity was measuredqualitatively by the fluorescentspot test or methemoglobinreductiontest and confirmed quantitatively by spectrophotometricanalysis.PK and HK activitieswere estimated only quantitatively by spectrophotometry.Results: Only sevenpartially deficient infantsout of 506infantsand 343adultsweredetectedwith PK deficiency.On this basis,the incidence would be 0.82with an estimatedgenefrequencyof about 0.004. Partial HK deficiencywas found in only one femaleinfant out of 220 infants and 246adultsin both sexes.No caseof completedeficiencyof theseenzymeswasidentified. G6PD deficiencywasidentified in 131 out of 1040maleinfants and adults.All the affected malesseemed to have severedeficiency (hemizygotes).On this basis,weestimatedthe genefrequencyto beabout 12.6%.In the female population,31out of 771 femaleinfantsand adultshad severeenzymedeficiency,a frequency of 4.0%(possiblehomozygosity)while 67 femalesshowedintermediateor moderateenzyme activity (heterozygosity),a prevalenceof 8.7%.Discussion.. Among the Iraqi Arab populations that areoriginal inhabitantsof Basrah,erythrocytesG6PD deficiencyisby far the mostcom-
* Corresponding author. Elsevier SSDI
Science Ireland Ltd. 0925-6164(95)00103-S
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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); Screening; Newborn; Cord blood; Adults; Iraq
Pyruvate kinase; Hexokinase;
1. Introduction We have studied the frequency of certain hereditary enzymopathies that cause hemolytic anemia among Iraqi people in southern Iraq. These enzymes deficiencies in red blood cells include those of glucose 6-phosphate dehydrogenase (G6PD), pyruvate 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 phosphorylation of glucose to its metabolically active form, glucose 6-phosphate. Each of these deficiencies has been responsible for hereditary non-spherocytic congenital hemolytic 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 Xlinked recessive trait which exists in many genetic variants among different populations [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 populations [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
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(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 SP8100 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 population
of pyruvate of Iraq
Population
kinase and hexokinase
deficiencies
PK Number tested
for newborn
infants
and adults in the Basrah
HK Partial
deficiency
n
o/o
Number tested
Partial
deficiency
n
‘%,
Infants Male Female Total
336 170 506
5 2 7
I.5 1.2 1.4
III 109 220
0 I I
0 0.91 0.45
Adults Male Female Total
198 145 343
0 0 0
0 0 0
131 115 246
0 0 0
0 0 0
All age groups Male Female Total
534 315 849
5 2 7
0.94 0.63 0.82
242 224 466
0 I I
0 0.45 0.21
Mean normal activity at 37°C: infants 3.61 + 0.55 and HK 0.41 f 0.06 U/1O’o
PK 5.10 f 0.81 and HK 0.61 f 0.16 U/10” RBC.
RBC;
adults
PK
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Table 2 Frequency of glucose 6-phosphate dehydrogenase deficiency in the neonate population of the Basrah area of Iraq Sex
Male Female
Number tested
Normal n
424 341
374 307
Intermediate
Severe
%
n
%
n
%
88.3 88.6
0 29
0 8.3
50 II
11.7 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 neonates, 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%); intermediate 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 intermediate 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. For male infants, it was 11.7%. This means that for females, 20.7% would be expected to be heterozygous and 1.4% to be homozygous. Likewise, the gene frequency Table 3 Frequency of glucose 6-phosphate dehydrogenase deficiency in the adult population of Basrah in Iraq Sex
Male Female
Number tested
Normal n
616 424
535 366
Intermediate
Severe
%
n
%
n
%
86.9 86.3
0 38
0 8.9
81 20
13.1 4.1
Mean normal G6PD activity is 2.14 f 0.40 U/lOt” RBC at 37°C. Deficiency is defined with activity ranges: intermediate, 0.22-1.21; severe, O-O.22 U/10” RBC.
M. M. AI-Naamaa Table 4 Frequency
of G6PD
Sex
deficiency
Number tested
Male Female
1040 771
et al. /Screening
in all age groups
4 (1995)
in the Basrah
Normal
27-34
population
31
of Iraq
Intermediate
Severe
n
u%,
n
%,
n
YU
909 613
81.4 87.3
0 67
0 8.7
131 31
12.6 4.0
estimated from male adults was 13.1%. This means that for adult females, 22.8% would be expected to be heterozygous and 1.7”/0 to be homozygous. The observed female incidences, for both infants and adults, were far from the expected. 4. Discussion Our results showed that G6PD deficiency is by far the most common of the three enzyme deficiencies that cause hemolytic anemia in Iraq (Table 5). The frequency of Table 5 Prevalence of G6PD, PK and HK deficiency nean region and Arabian Peninsula Country
Reference
Populations Adults
Present
study
in adults
and neonates
surveyed Neonates
1497
IlO1 1121
177 563
Iraq Saudi Arabia
1201
261
889 I04
1211 1221 1231 1241 1251
Jordan
426 306 395
Wpt
[‘W
Libya
1271
296 500 200
Turkey Lebanon WHO Iraq Iran Saudi Arabia
1271 1271
105 549
78
‘GBPD
G6PD
PK
HK
13.1 II.7 13.0 8.9 8.4 12.4 24.0 7-22 4-22 15.0 10.1 14.6 26.4 2.5 3.8 I I.4 3.1
0 I.4
0 0.45
3-6
l-2
171
frequencies
7-9.9 10-14.9 15-22 are for male only;
PK and HK
in the Mediterra-
u/u Frequency
1629
Basrah Iraq
of populations’
frequencies
are for both sexes.
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G6PD deficiency varies widely in the Mediterranean region and the Arabian Peninsula (Table 5). The high frequency of G6PD found in the present study is matched by earlier reports from Iraq, Saudi Arabia, Jordan, Turkey, Iran, Libya and Egypt. G6PD deficiency is transmitted as a sex-linked trait with severe enzyme deficiency occurring only in hemizygote males and homozygote females, while heterozygous females often have normal or moderately decreased enzyme levels [ 1,8]. Our observed gene frequency for combined male infants and adults was 12.6%, i.e. 131 deficient out of 1040 tested (Table 4), all of whom had severe G6PD deficiency. Comparable results were found in different parts of Iraq (10,12,20). Not all female heterozygotes for G6PD deficiency have intermediate activity; some may be normal and others may have low activity, depending on X-inactivation. Thus, the best estimate of gene frequency is from data in males, since enumeration of affected males should give the gene frequency [28]. The ‘expected’ frequency of G6PD deficient severe and intermediate females was calculated from the 11.7% and 13.1% hemizygous neonatal and adult males, respectively, This means that for female infants and adults, 22% would be expected to be heterozygous and 1.6% to be homozygous. In this study, the observed combined frequency for heterozygotes was 8.7% and for homozygotes 4.0%, so that less than half the expected heterozygotes and an excess of apparent homozygotes were detected (Table 4). These discrepancies are similar to what others have found and are probably attributable to considerable overlap of heterozygote activities with the normal range [29], and in part to random X-chromosome inactivation [8]. This observation is consistent with earlier studies on the effect of X-chromosome inactivation and X-chromosome mosaicism on the expression of G6PD deficiency in heterozygous women [ 1,8]. The effects of high rate of consanguinity and other forms of inter-marriages cannot be ruled out. The region we studied is known for its high prevalence of sickle cell disease [30]. Consanguineous marriage is common and malaria was endemic, although no cases of malaria have been reported since 1979 [31]. It is known that both G6PD deticiency and sickle cell disease have important and protective actions against malaria [ 1,7,8]. Similar findings have also been reported from countries neighboring on Iraq [21-25,321. Moderate deficiencies of pyruvate kinase and hexokinase were identified in this area, with combined frequencies of 0.82% and 0.2%, respectively, among the newborn infants and adults tested (Table 1). The gene frequency for recessive PK deficiency calculated from the combined heterozygous incidence was 0.004%. No case of complete deficiency was identified. Comparable frequencies have been reported for PK and HK deficiencies in the Saudi Arabia population [21], Jordan [25] and for Chinese infants [33], whereas our frequencies were higher than those reported for the Spanish population [34]. This variation could be attributed to race and geographical distribution. PK deficiency data has also been acquired. However, most of the published reports were either isolated cases or clinical observations and studies, except for the Saudi report [21], not designed to determine the frequency of population deficiencies. Extensive population screening may reveal a higher frequency of these enzyme defects in other populations. In addition, a study of patients with various blood disorders has shown the occurrence of acquired erythrocyte en-
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zymopathies in leukemias, preleukemic states and bone marrow insufficiencies with or without sideroblastosis [6]. In conclusion, we report the frequency of G6PD, PK and HK deficiency in the Iraqi popdation in newborns and adults. More extensive studies are required to correlate the phenotypes and clinical manifestations of these genetic abnormalities with the incidence of acquired red blood cell enzymopathies in the Iraqi population. Acknowledgements We wish to thank Dr. Farkad K. Al-Dorky the cord blood samples.
for her kind assistance in collecting
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