Original Paper Acta Haematol 1992;88:67-71
Medical Biochemistry Department, College of Medicine & King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia
Key Words Saudi Arabia Sickel cell disease Sickle cell haemoglobin Thalassaemias
Heterogeneity and Variation of Clinical and Haematological Expression of Haemoglobin S in Saudi Arabs
Abstract Sickle cell haemoglobin (Hb S) occurs at a high frequency in the Eastern (EP), South-Western (SWP) and North-Western (NWP) Provinces of Saudi Arabia and the presentation of the Hb S is believed to exhibit clinical diversity in the different regions. Three areas of Saudi Arabia were screened to determine the frequency of Hb S and a- and p-thalassaemias and glucose-6-phosphate de hydrogenase deficiency genes. Furthermore, in an attempt to investigate and compare the clinical and haematological presentation of sickle cell disease (SCD) in the different regions of Saudi Arabia, the individuals identified as Hb S homozygotes were investigated further. The patients were further classified on the basis of whether there was associated a- or P-thalassaemia. A severity in dex (SI) was calculated for each patient and the clinical presentations and lab oratory findings were compared. The results showed significantly variable severity of SCD in patients from different regions. The patients from the EP generally had a mild clinical presentation, while in the SWP and NWP majority of the patients suffered from a severe disease as judged by the SI. No correla tion could be established between Hb F level and SI, though the WBC level correlated positively with the SI. The lowest SI values were encountered in pa tients with associated a-thalassaemia who also had the lowest WBC count and MCV and the highest RBC count and packed cell volume.
Introduction Red cell genetic disorders including haemoglobin S (Hb S), glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, a- and P-thalassaemia occur at a variable fre quency in different regions of Saudi Arabia [1-5]. In areas with a history of malaria endemicity, where the co-occur rence and interaction of these genes ranks high in the pop
Received: April 4,1991 Accepted: January 27,1992
ulation [6-10] the genetic disorders exist at a high fre quency. The severity and clinical expression of sickle cell dis orders are variable. Several studies have indicated that the sickle cell disease (SCD) in Eastern Saudi Arabia is of a mild nature [11-13]. However, more recent and detailed studies have revealed that two forms of SCD exist in Saudi Arabs, one with a mild course and another with a severe
Prof. Mohsen A.F. El-Hazmi Medical Biochemistry Department (30) College of Medicine & King Khalid Hospital PO Box 2925 Riyadh 11461 (Saudi Arabia)
©1992S. Karger AG, Basel 0001-5792/92/0883-0067 $ 2.75/0
Downloaded by: Kings's College London 137.73.144.138 - 10/22/2017 7:22:39 PM
Mohsen A.F. El-Hazmi
Table 1. SI of SCD (total number of attacks, symp toms and signs/ year)
Total number Hb S a-Thalassaemia -a /a a
- a /- a ß-Thalassacmia G-6-PD deficiency
Eastern Province
North-Western Province
South-Western Province
1,495 0.714
670 0.0082
1,187 0.083
0.367 0.190 0.130 0.282
0.147 0.049 0.049 0.073
0.418 0.124 0.059 0.204
course, similar to that encountered in other populations [14, 15]. Significant clinical diversity has been demon strated in disease severity in the different regions of Saudi Arabia and other Middle-Eastern countries [3,19-21], Sev eral genetic and/or environmental factors have been pro posed to account for the heterogeneity of disease presen tation. Interactions between Hb S and other abnormal genes have been considered as possible modulators of the clinical manifestations of SCD [7, 9,11], In an attempt to investigate and compare the clinical and haematological presentation of SCD in the different regions of Saudi Arabia and the influence of coexisting aand (l°-thalassaemia, this study was conducted on individ uals identified as Hb S homozygotes during screening of three areas of Saudi Arabia.
68
El-Hazmi
Severity of anaemia > 110 g/1 90-110 g/1 70-89 g/1 35 mmol/1) Painful crises (hand-foot syndrome, bone, abdominal)3 Aseptic necrosis (head of femur or humerus) Osteomyelitis Leg ulcers Gallstone Hyposplenism Polyurea/isothinurea Priapism Chest infection Hypoxia (P0j< 70 in room air) Retinopathy Cerebrovascular accidents Deep vein thrombosis Blood transfusion3 Hospitalization3
Score
0 1 2 3 1 1 0-12 1 1 1 1 1 1 1 1 1 1 1 1 0-12 0-12
1 Modified from El-Hazmi (16]. 2 If present, the score indicated was given, while if absent the score was zero. 3 The score depended on the number en countered per year.
Materials and Methods Blood samples (5-10 ml) were collected in EDTA tubes by vene puncture from 3,352 hospital out-patients living in three different re gions of Saudi Arabia (fig. 1). Fresh blood was used to prepare smears for the red cell morphological studies. Haematological analytes and red cell indices were estimated using Coulter Counter ZF6 with a haemoglobinometer attachment. The plasma, red cells and buffy coat were separated by centrifugation of the fresh whole blood. The red cells, washed twice with cold physiological saline, were haemolysed with cold distilled water or 0.02% digitonin. Thereafter, haemolysates were used for the confirmation of haemoglobin types on alka line [22] and acid pH [23], for estimation of Hb A2 using Sickle-Thal Quick Column kit and (f-Thal Quik Column kit and Hb F determina tion by radial immunodiffusion (RID Hb F QUIPlate, Helena Lab oratories) and by alkali denaturation [24]. The G-6-PD level was esti mated in the fresh haemolysate using commercially available kits (Boehringer-Mannheim, GmbH). The DNA was extracted from the buffy coat for diagnosis of a-thalassaemia using the restriction endo nuclease Bam HI [9], p-Thalassaemia was indicated by high Hb A2 level and discriminant factors [25] and confirmed by high a/jl-chain
Heterogeneity and Variation of Expression of Haemoglobin S
Downloaded by: Kings's College London 137.73.144.138 - 10/22/2017 7:22:39 PM
Fig. 1. Sketch map of Saudi Arabia. The shaded areas show the screened regions. The frequency of the abnormal genes is as fol lows:
Findings2
WBC, x 109/l
Fig. 2. The value of haematological parameters, mean cell volume and Hb F in SCD patients from different regions of Saudi Arabia.
Fig. 4. Correlation between SI and white blood cell in SCD
patients.
The significance of the difference in the mean of any two groups was determined using Student’s t test, p < 0.05 was considered statis tically significant. Regression analyses were conducted and correla tion coefficients were determined using an SAS General Linear Model (GLM) Program.
Results
ratio. Biochemical analytes were determined in the plasma using an American Monitor autoanalyzer. The SCD patients (109) were included in this study for further in vestigation. Findings of physical examination and history were re corded during visits. Evaluation of the clinical picture was carried out according to the SI developed from the sum of clinical symptoms and signs, frequency of presentation, hospitalization, and laboratory and radiological findings presented in table 1. The scores/years were add ed and used for obtaining the SI. Out of a total of 109 SCD patients, 54 patients had sickle cell anae mia (SCA) without a-thalassaemia, 32 SCA patients had associated a-thalassaemia and 23 patients were S/p°-thalassaemics. The results of haematological analytes and clinical data were fed into the com puter at King Saud University Computer Centre, Riyadh, and the mean and standard deviation were calculated for each parameter us ing the Statistical Analysis System (SAS).
69
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Fig. 3. Correlation between SI and Hb F level in SCD patients.
The areas screened, the number of individuals and the frequency of the abnormal genes in the three areas are presented in figure 1. The Eastern Province (EP) had the highest frequency of Hb S, a-thalassaemia and G-6-PD deficiency followed by the South-Western Province (SWP) and North-Western Province (NWP). The mean values of haematological parameters in SCD patients from the three regions were obtained and are pre sented in figure 2. A wide range (2-30) was obtained for SI in all groups of patients regardless of the location. However, 90% of pa tients from EP exhibited mild disease with SI < 6, while al most 90% patients from SWP and NWP showed severe disease (SI > 6). Regression analyses between Hb F level and SI were carried out. The correlation coefficient (r) was 0.028 and there was no statistically significant correla tion as shown in figure 3. On the other hand, the SI corre lated positively with the WBC counts (fig. 4). On further classification based on association with a- and fl-thalassaemia, the haematological parameters showed several differences. The results are presented in figure 5. The SI in patients classified as Hb S homozygotes,
and white blood cell count but the mean cell volume was significantly lower, and the Hb F level did not show any significant difference compared to the values in SCA pa tients. The mean value of SI was slightly lower in this group, though it varied widely.
Discussion
Fig. 6. SI in Hb SS, Hb S/ß°-thalassaemia and Hb SS with a-thalas saemia patients.
Hb S homozygotes with a-thalassaemia and Hb S/p°-thalassaemia patients are presented in figure 6. A variable haematological and clinical picture was ob tained depending on whether or not the Hb S homozygous patients had associated a-thalassaemia or were double heterozygous Hb S/ß°-thalassaemia cases. In general, cases with associated a-thalassaemia had improved hae matological parameters and the SI was significantly lower. Compound heterozygotes for Hb S and ß°-thalassaemia had slightly higher total haemoglobin level, red blood cell
70
El-Hazmi
Heterogeneity and Variation of Expression of Haemoglobin S
Downloaded by: Kings's College London 137.73.144.138 - 10/22/2017 7:22:39 PM
Fig. 5. The values of haematological parameters, mean cell vol ume and Hb F in Hb SS, Hb SS with a-thalassaemia and Hb S/p°thalassaemia patients.
The present study shows significant heterogeneity of clinical and haematological expression of sickle cell gene in Saudis. To some extent, this heterogeneity can be ac counted for by coexisting a- and p°-thalassaemias and as shown in a earlier study by the differences in the P-globin gene haplotypes [26], Hb F level does not seem to have a major influence on disease severity in our patients as no correlation could be demonstrated between Hb F level and SI. This is contradictory to several studies that claim a major role of high Hb F levels in SCD amelioration [12, 13]. The genetic factor in patients from the Eastern Prov ince with high Hb F levels appears to be linked to the C -> T mutation at position 158 of the Gy-globin gene [27]. Our earlier studies have confirmed the presence of the C -» T mutation in most patients from the EP of Saudi Arabia and its absence in patients from the SWP, however, we did not find any specific association with Hb F level [28]. In several patients from the EP or SWP without C -* T muta tion at -158, high Hb F levels were frequently encountered [29], In general, SCD patients with associated a-thalassae mia present with a significantly milder disease as assessed by a lower SI and improved haematological parameters compared to the Hb S homozygotes and Hb S/P°-thalassaemia patients. This observation confirms the earlier re ports of a beneficial influence of associated a-thalassae mia [9,11,19]. However, it must be emphasised that since in our group of Hb S homozygous patients some had an equally mild disease despite absence of a-thalassaemia gene there must be some additional factors contributing to the amelioration of the SCD. The coexistence of P°-thalassaemia in the Hb S hetero zygotes produces a clinical and haematological picture very similar to Hb s homozygocity, though some patients, but not all, do have a slightly improved disease. The differ ences in the clinical presentation within the Hb S/[30-thalassaemia patients may be due to the different p-thalassaemia mutations. So far we have encountered the p-thalassaemia mutation IVS-II nt645 in several of our patients. However, more detailed investigations are required to ge netically classify all these patients.
In this study, several patients with high Hb F level had severe disease, while in others with low Hb F levels the dis ease was significantly mild. It thus comes down to the fact that some other, yet unknown, genetic and/or environ mental factors are involved in the heterogeneity of SCD despite the identical single point transversion mutation (A -» T) in all these patients. It seems more and more certain that the clinical and haematological variations are related to differences at the gene level. These include mutations in the various sites of the P-globin gene cluster [26,29,30],
and the locus control region. Further studies involving modulators of the (i-gene, and locus control region are needed to clarify the mechanism involved in sickle cell gene expression and clinical presentation. Acknowledgement This study was supported in part by grant No. AT-4-074 from King Abdulaziz City for Science and Technology (KACST) and in part by King Saud University, Riyadh.
References 11 Weatherall DJ, Clegg JB, Blankson J: A new sickling disorder resulting from interaction of the genes for haemoglobin S and a-thalassae mia. Br J Haematol 1969;17:517-526. 12 Pembrey ME, Wood WG, Weatherall DJ, Perrine RP: Fetal haemoglobin production and the sickle cell gene in the oasis of Eastern Saudi Arabia. Br J Haematol 1978;40:415-429. 13 Perrine RP, Brown MJ, Clegg JB, Weatherall DJ, May A: Benign sickle cell anaemia. Lancet 1972;ii:l 163-1167. 14 Acquaye JK, Omer A, Ganeshaguru K, Sejeny SA, Hoffbrand AV: Non-benign sickle cell ane mia in western Saudi Arabia. Br J Haematol 1985;60:99-108. 15 Babiker MA, Taha SA: Two different patterns of sickle cell disease in children in Saudi Ara bia. Ann Trop Pediatr 1982;2:179-181. 16 El-Hazmi MAF: Studies on sickle cell hetero zygotes in Saudi Arabia - Interaction with a-thalassaemia. Acta Haematol 1986;75:100104. 17 Serjeant GR: Sickle Cell Disease. Oxford, Ox ford University Press, 1985. 18 El-Hazmi MAF, Bahakim HM, Al-Swailem AM, Warsy AS: The features of sickle cell dis ease in Saudi children. J Trop Paediatr 1990; 36:148-155. 19 Padmos MA, Roberts GT, Sackey K, Kulozik A, Bail S, Morris JS, Serjeant BE, Serjeant GR: T\vo different forms of homozygous sickle cell disease occurs in Saudi Arabia. Br J Haematol 1991;79:93-98. 20 Kamel K: Heterogeneity of sickle cell anaemia in Arabs: Review of cases with various amounts of fetal haemoglobin. J Med Genet 1979;16: 428-430.
21 Kamel K, Moafy N: Some aspects of thalassae mia and the world common hemoglobinopa thies in the Middle East; in Bowman JE (ed): Distribution and Evolution of Hemoglobin and Globin Loci. New York, Elsevier, 1983, pp 209-218. 22 Marengo-Rowe AJ : Rapid electrophoresis and quantitation of haemoglobins on cellulose ace tate. J Clin Pathol 1965;18:790-792. 23 Robinson AR, Robson M, Harrison AP, Zuel zer WW: A new technique for differentiation of haemoglobin. J Lab Clin Med 1957;50:745752. 24 Betkc K, Marti HR, Schlicht L: Estimation of small percentages of foetal haemoglobin. Na ture 1959;184:1877. 25 England JM, Fraser PM: Differentiation of iron deficiency from thalassaemia trait by rou tine blood count. Lancet 1973;i:449—452. 26 El-Hazmi MAF: Beta-globin gene haplotype in Saudi sickle cell anaemia patients. Hum Hered 1990;40:177-186. 27 Labié D, Dunda-Belkhodja O, Rouabhi F, Pagnier J, Ragusa A, Nagel RL: The 158 site 5' to the Gy gene and Gy expression. Blood 1985; 66:1463-1465. 28 El-Hazmi MAF: XmnI polymorphism in the gamma-globin gene region among Saudis. Hum Hered 1989;39:12-19. 29 El-Hazmi MAF, Bahakim H, Warsy AS: DNA polymorphism in the p-globin gene cluster in Saudis. 1SH/ASH Meeting, Boston, 1990, Post er No. 1573. 30 El-Hazmi MAF, Bahakim HM, Warsy AS, AlMomen AK, Al-Wazzan A, Al-Fawwaz I, Huraib S: Does Gy/Ay ratio and Hb F level influ ence the severity of sickle cell anaemia. ISH/ ASH Meeting, Boston, 1990, Poster No. 1574.
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1 El-Hazmi MAF: Haemoglobin Disorders: A Pattern for thalassaemia and haemoglobinopathies in Arabia. Acta Haematol 1982;68: 43-51. 2 Gelpi AP: Sickle cell disease in Saudi Arabia. Acta Haematol 1970;43:89-99. 3 Roberts GT, El-Badawi SB, Padmos MA, Sackey K: Regional variations in sickle cell ane mia in Saudi Arabia. Ann Saudi Med 1988;8: 320-328. 4 El-Hazmi MAF: Abnormal haemoglobins and allied disorders in the Middle East-Saudi Ara bia; in Bowman JE (ed): Distribution and Evo lution of Hemoglobin and Globin Loci. New York, Elsevier, 1983, pp 239-249. 5 El-Hazmi MAF: Haemoglobinopathies, thalassaemias and enzymopathies in Saudi Arabia: The present status. Acta Haematol 1987;78: 130-134. 6 El-Hazmi MAF, Warsy AS: The effect of glucose-6-phosphate dehydrogenase deficiency on the haematological parameters and clinical manifestations in patients with sickle cell anae mia. Trop Geogr Med 1989;41:52-56. 7 El-Hazmi MAF, Al-Swailem AR: Sickle cellP°-thalassaemia in Saudi Arabia. Hum Hered 1987;37:211-216. 8 El-Hazmi MAF, Warsy AS: Interaction be tween glucose-6-phosphate dehydrogenase de ficiency and sickle cell gene in Saudi Arabia. Trop Geogr Med 1987;39:32-35. 9 El-Hazmi MAF: Clinical manifestation and laboratory findings of sickle cell anaemia in as sociation with a-thalassaemia in Saudi Arabia. Acta Haematol 1985;74:155-160. 10 El-Hazmi MAF, Warsy AS: Aspects of sick le cell gene in Saudi Arabia - Interaction with glucose-6-phosphate dehydrogenase defi ciency. Hum Genet 1984;68:320-323.
Medical Biochemistry Department, College of Medicine & King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia
Key Words Saudi Arabia Sickel cell disease Sickle cell haemoglobin Thalassaemias
Heterogeneity and Variation of Clinical and Haematological Expression of Haemoglobin S in Saudi Arabs
Abstract Sickle cell haemoglobin (Hb S) occurs at a high frequency in the Eastern (EP), South-Western (SWP) and North-Western (NWP) Provinces of Saudi Arabia and the presentation of the Hb S is believed to exhibit clinical diversity in the different regions. Three areas of Saudi Arabia were screened to determine the frequency of Hb S and a- and p-thalassaemias and glucose-6-phosphate de hydrogenase deficiency genes. Furthermore, in an attempt to investigate and compare the clinical and haematological presentation of sickle cell disease (SCD) in the different regions of Saudi Arabia, the individuals identified as Hb S homozygotes were investigated further. The patients were further classified on the basis of whether there was associated a- or P-thalassaemia. A severity in dex (SI) was calculated for each patient and the clinical presentations and lab oratory findings were compared. The results showed significantly variable severity of SCD in patients from different regions. The patients from the EP generally had a mild clinical presentation, while in the SWP and NWP majority of the patients suffered from a severe disease as judged by the SI. No correla tion could be established between Hb F level and SI, though the WBC level correlated positively with the SI. The lowest SI values were encountered in pa tients with associated a-thalassaemia who also had the lowest WBC count and MCV and the highest RBC count and packed cell volume.
Introduction Red cell genetic disorders including haemoglobin S (Hb S), glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, a- and P-thalassaemia occur at a variable fre quency in different regions of Saudi Arabia [1-5]. In areas with a history of malaria endemicity, where the co-occur rence and interaction of these genes ranks high in the pop
Received: April 4,1991 Accepted: January 27,1992
ulation [6-10] the genetic disorders exist at a high fre quency. The severity and clinical expression of sickle cell dis orders are variable. Several studies have indicated that the sickle cell disease (SCD) in Eastern Saudi Arabia is of a mild nature [11-13]. However, more recent and detailed studies have revealed that two forms of SCD exist in Saudi Arabs, one with a mild course and another with a severe
Prof. Mohsen A.F. El-Hazmi Medical Biochemistry Department (30) College of Medicine & King Khalid Hospital PO Box 2925 Riyadh 11461 (Saudi Arabia)
©1992S. Karger AG, Basel 0001-5792/92/0883-0067 $ 2.75/0
Downloaded by: Kings's College London 137.73.144.138 - 10/22/2017 7:22:39 PM
Mohsen A.F. El-Hazmi
Table 1. SI of SCD (total number of attacks, symp toms and signs/ year)
Total number Hb S a-Thalassaemia -a /a a
- a /- a ß-Thalassacmia G-6-PD deficiency
Eastern Province
North-Western Province
South-Western Province
1,495 0.714
670 0.0082
1,187 0.083
0.367 0.190 0.130 0.282
0.147 0.049 0.049 0.073
0.418 0.124 0.059 0.204
course, similar to that encountered in other populations [14, 15]. Significant clinical diversity has been demon strated in disease severity in the different regions of Saudi Arabia and other Middle-Eastern countries [3,19-21], Sev eral genetic and/or environmental factors have been pro posed to account for the heterogeneity of disease presen tation. Interactions between Hb S and other abnormal genes have been considered as possible modulators of the clinical manifestations of SCD [7, 9,11], In an attempt to investigate and compare the clinical and haematological presentation of SCD in the different regions of Saudi Arabia and the influence of coexisting aand (l°-thalassaemia, this study was conducted on individ uals identified as Hb S homozygotes during screening of three areas of Saudi Arabia.
68
El-Hazmi
Severity of anaemia > 110 g/1 90-110 g/1 70-89 g/1 35 mmol/1) Painful crises (hand-foot syndrome, bone, abdominal)3 Aseptic necrosis (head of femur or humerus) Osteomyelitis Leg ulcers Gallstone Hyposplenism Polyurea/isothinurea Priapism Chest infection Hypoxia (P0j< 70 in room air) Retinopathy Cerebrovascular accidents Deep vein thrombosis Blood transfusion3 Hospitalization3
Score
0 1 2 3 1 1 0-12 1 1 1 1 1 1 1 1 1 1 1 1 0-12 0-12
1 Modified from El-Hazmi (16]. 2 If present, the score indicated was given, while if absent the score was zero. 3 The score depended on the number en countered per year.
Materials and Methods Blood samples (5-10 ml) were collected in EDTA tubes by vene puncture from 3,352 hospital out-patients living in three different re gions of Saudi Arabia (fig. 1). Fresh blood was used to prepare smears for the red cell morphological studies. Haematological analytes and red cell indices were estimated using Coulter Counter ZF6 with a haemoglobinometer attachment. The plasma, red cells and buffy coat were separated by centrifugation of the fresh whole blood. The red cells, washed twice with cold physiological saline, were haemolysed with cold distilled water or 0.02% digitonin. Thereafter, haemolysates were used for the confirmation of haemoglobin types on alka line [22] and acid pH [23], for estimation of Hb A2 using Sickle-Thal Quick Column kit and (f-Thal Quik Column kit and Hb F determina tion by radial immunodiffusion (RID Hb F QUIPlate, Helena Lab oratories) and by alkali denaturation [24]. The G-6-PD level was esti mated in the fresh haemolysate using commercially available kits (Boehringer-Mannheim, GmbH). The DNA was extracted from the buffy coat for diagnosis of a-thalassaemia using the restriction endo nuclease Bam HI [9], p-Thalassaemia was indicated by high Hb A2 level and discriminant factors [25] and confirmed by high a/jl-chain
Heterogeneity and Variation of Expression of Haemoglobin S
Downloaded by: Kings's College London 137.73.144.138 - 10/22/2017 7:22:39 PM
Fig. 1. Sketch map of Saudi Arabia. The shaded areas show the screened regions. The frequency of the abnormal genes is as fol lows:
Findings2
WBC, x 109/l
Fig. 2. The value of haematological parameters, mean cell volume and Hb F in SCD patients from different regions of Saudi Arabia.
Fig. 4. Correlation between SI and white blood cell in SCD
patients.
The significance of the difference in the mean of any two groups was determined using Student’s t test, p < 0.05 was considered statis tically significant. Regression analyses were conducted and correla tion coefficients were determined using an SAS General Linear Model (GLM) Program.
Results
ratio. Biochemical analytes were determined in the plasma using an American Monitor autoanalyzer. The SCD patients (109) were included in this study for further in vestigation. Findings of physical examination and history were re corded during visits. Evaluation of the clinical picture was carried out according to the SI developed from the sum of clinical symptoms and signs, frequency of presentation, hospitalization, and laboratory and radiological findings presented in table 1. The scores/years were add ed and used for obtaining the SI. Out of a total of 109 SCD patients, 54 patients had sickle cell anae mia (SCA) without a-thalassaemia, 32 SCA patients had associated a-thalassaemia and 23 patients were S/p°-thalassaemics. The results of haematological analytes and clinical data were fed into the com puter at King Saud University Computer Centre, Riyadh, and the mean and standard deviation were calculated for each parameter us ing the Statistical Analysis System (SAS).
69
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Fig. 3. Correlation between SI and Hb F level in SCD patients.
The areas screened, the number of individuals and the frequency of the abnormal genes in the three areas are presented in figure 1. The Eastern Province (EP) had the highest frequency of Hb S, a-thalassaemia and G-6-PD deficiency followed by the South-Western Province (SWP) and North-Western Province (NWP). The mean values of haematological parameters in SCD patients from the three regions were obtained and are pre sented in figure 2. A wide range (2-30) was obtained for SI in all groups of patients regardless of the location. However, 90% of pa tients from EP exhibited mild disease with SI < 6, while al most 90% patients from SWP and NWP showed severe disease (SI > 6). Regression analyses between Hb F level and SI were carried out. The correlation coefficient (r) was 0.028 and there was no statistically significant correla tion as shown in figure 3. On the other hand, the SI corre lated positively with the WBC counts (fig. 4). On further classification based on association with a- and fl-thalassaemia, the haematological parameters showed several differences. The results are presented in figure 5. The SI in patients classified as Hb S homozygotes,
and white blood cell count but the mean cell volume was significantly lower, and the Hb F level did not show any significant difference compared to the values in SCA pa tients. The mean value of SI was slightly lower in this group, though it varied widely.
Discussion
Fig. 6. SI in Hb SS, Hb S/ß°-thalassaemia and Hb SS with a-thalas saemia patients.
Hb S homozygotes with a-thalassaemia and Hb S/p°-thalassaemia patients are presented in figure 6. A variable haematological and clinical picture was ob tained depending on whether or not the Hb S homozygous patients had associated a-thalassaemia or were double heterozygous Hb S/ß°-thalassaemia cases. In general, cases with associated a-thalassaemia had improved hae matological parameters and the SI was significantly lower. Compound heterozygotes for Hb S and ß°-thalassaemia had slightly higher total haemoglobin level, red blood cell
70
El-Hazmi
Heterogeneity and Variation of Expression of Haemoglobin S
Downloaded by: Kings's College London 137.73.144.138 - 10/22/2017 7:22:39 PM
Fig. 5. The values of haematological parameters, mean cell vol ume and Hb F in Hb SS, Hb SS with a-thalassaemia and Hb S/p°thalassaemia patients.
The present study shows significant heterogeneity of clinical and haematological expression of sickle cell gene in Saudis. To some extent, this heterogeneity can be ac counted for by coexisting a- and p°-thalassaemias and as shown in a earlier study by the differences in the P-globin gene haplotypes [26], Hb F level does not seem to have a major influence on disease severity in our patients as no correlation could be demonstrated between Hb F level and SI. This is contradictory to several studies that claim a major role of high Hb F levels in SCD amelioration [12, 13]. The genetic factor in patients from the Eastern Prov ince with high Hb F levels appears to be linked to the C -> T mutation at position 158 of the Gy-globin gene [27]. Our earlier studies have confirmed the presence of the C -» T mutation in most patients from the EP of Saudi Arabia and its absence in patients from the SWP, however, we did not find any specific association with Hb F level [28]. In several patients from the EP or SWP without C -* T muta tion at -158, high Hb F levels were frequently encountered [29], In general, SCD patients with associated a-thalassae mia present with a significantly milder disease as assessed by a lower SI and improved haematological parameters compared to the Hb S homozygotes and Hb S/P°-thalassaemia patients. This observation confirms the earlier re ports of a beneficial influence of associated a-thalassae mia [9,11,19]. However, it must be emphasised that since in our group of Hb S homozygous patients some had an equally mild disease despite absence of a-thalassaemia gene there must be some additional factors contributing to the amelioration of the SCD. The coexistence of P°-thalassaemia in the Hb S hetero zygotes produces a clinical and haematological picture very similar to Hb s homozygocity, though some patients, but not all, do have a slightly improved disease. The differ ences in the clinical presentation within the Hb S/[30-thalassaemia patients may be due to the different p-thalassaemia mutations. So far we have encountered the p-thalassaemia mutation IVS-II nt645 in several of our patients. However, more detailed investigations are required to ge netically classify all these patients.
In this study, several patients with high Hb F level had severe disease, while in others with low Hb F levels the dis ease was significantly mild. It thus comes down to the fact that some other, yet unknown, genetic and/or environ mental factors are involved in the heterogeneity of SCD despite the identical single point transversion mutation (A -» T) in all these patients. It seems more and more certain that the clinical and haematological variations are related to differences at the gene level. These include mutations in the various sites of the P-globin gene cluster [26,29,30],
and the locus control region. Further studies involving modulators of the (i-gene, and locus control region are needed to clarify the mechanism involved in sickle cell gene expression and clinical presentation. Acknowledgement This study was supported in part by grant No. AT-4-074 from King Abdulaziz City for Science and Technology (KACST) and in part by King Saud University, Riyadh.
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