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European Journal of Medical Genetics 57 (2014) 603e606

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European Journal of Medical Genetics
journal homepage: http://www.elsevier.com/locate/ejmg

Clinical research

Sickle cell anemia and a-thalassemia: A modulating factor in
homozygous HbS/S patients in Oman
S.M. Hassan a, c, *, M. Al Muslahi b, M. Al Riyami b, E. Bakker c, C.L. Harteveld c, P.C. Giordano c
a

Molecular Genetic Laboratory, Genetic Centre, Muscat, Oman
Royal Hospital, Muscat, Oman
c
The Reference Hemoglobinopathies Laboratory, Dept. of Human and Clinical Genetics, Leiden University Medical Centre, Leiden, The Netherlands
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 23 May 2014
Accepted 21 September 2014
Available online 28 September 2014

We report the general phenotype severity and the hematological presentation in a cohort of 125 sickle
cell anemia (SCA) patients with identical homozygous HbS/S genotype and categorized by identical bS
haplotype, both with and without alpha thalassemia. No clear general phenotype correlation was found
when patients were compared regardless of the haplotype but overall, patients with homozygous alpha
thalassemia (a/a) had the highest Hb, HCT, RBC and the lowest MCV, MCH and MCHC levels. When
patients with identical haplotype were compared, the mildest hematological and clinical conditions were
observed in patients of the Asian/Asian haplotype, also known as Arab-Indian haplotype, and carriers of
a-thalassemia, suggesting an additional ameliorating effect of alpha thalassemia. In conclusion, our results show that alpha thalassemia improves the hematological conditions but amelioration of the general
disease severity is only noticed when compared in cohorts of the same haplotype.
Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords:
a-thalassemia
bS haplotype
Sickle cell anemia

1. Introduction
Sickle cell anemia (SCA) is in general a severe condition caused
by different genotype combinations of which HbS homozygosis is
the most common. The pathophysiology of SCA is complex and
involves HbS polymerization in hypoxic conditions in the postcapillary veins, erythrocyte sickling, chronic and acute vasoocclusive events, hemolysis and progressive organ and tissue
damage at variable levels [Ware and Aygun, 2009]. Although it is
known that both environmental and genetic factors may
contribute to this variability [Steinberg, 2005], patients with the
same HbS/S genotype often display very different phenotypes in
which the clinical manifestations may range from very severe to
milder or can even in some cases be almost asymptomatic and be
diagnosed accidentally [Serjeant, 1989; Giordano et al., 2011]. The
clinical and hematological severity of SCA can be influenced by a
number of factors among which the main one is the level of fetal
hemoglobin (HbF) in postnatal life which is related to the beta
globin gene haplotype [Kulozik et al., 1986]. The role of coinherited alpha thalassemia influencing or not the phenotype of
SCA has been long debated [Steinberg, 2009].

* Corresponding author. Hemoglobinopathies Laboratory, Molecular Genetics,
Genetic Centre, Ministry of Health, Muscat, Oman.
E-mail address: suha_85@hotmail.com (S.M. Hassan).
http://dx.doi.org/10.1016/j.ejmg.2014.09.005
1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.

The alpha-globin chains that are needed to form sufficient Hb
tetramers are coded by two alpha genes located on the short arm of
chromosome 16. The two a-globin genes, alpha 2 and alpha 1, are
separated by less than 4 kb and code for identical alpha-globin
chains.
Alpha thalassemia occurs at a high prevalence in Oman [Alkindi
et al., 2010] with a3.7 kb deletion being the commonest [Hassan
et al., 2010].
The influence of alpha thalassemia on SCA has been reported to
ameliorate the hematological and clinical manifestation of the
disease in several populations [El-Hazmi, 1985]. Alpha thalassemia
lowers the mean cell volume (MCV) and the mean cell hemoglobin
(MCH), and both these changes might be expected to be beneficial
to patients with sickle cell disease improving rheology and
reducing the concentration of the Hb molecules in the red cells
[Steinberg, 2005]. However, to study the effect of alpha
thalassemia on the severity of the disease one needs to compare
cohorts of patients with identical genotype (HbS/S mutation) and
haplotype (beta globin cluster).
Both alpha thalassemia and SCA are frequent in Oman [Alkindi
et al., 2010] and the HbS mutation is present on severe and mild
haplotypes. Therefore, in order to establish if alpha thalassemia
has any effect on the severity of the disease, we have studied the
hematological and general clinical phenotype of 125 patients, all
HbS/S homozygous, with or without alpha thalassemia and

3% e e e 75% 57. 2. To assess if presence or absence of alpha thalassemia has an effect on disease expression. based upon their disease history. hematocrit (HCT). clinical and hematological comparison of the patient’s history was made between genetically equivalent cohorts with and without alpha thalassemia.5% 33.3% 33. 2012] and genotyping errors were ruled out by random sample sequencing. Materials and methods Blood samples were collected in EDTA at the Ministry of Health Hospitals in Oman. the milder condition was present in 75% of the patients with (a/a) while none had the mild condition in absence of alpha thalassemia and none of them were severe (Table 1b).7/a3.3. comparing the general clinical phenotype with the genetic background of the patients. different b-cluster haplotypes and clinical severity.4% 87. 2000] for all the samples while the alpha-globin genes were sequenced for selected samples. was found in 55 patients (44%). Bio-Rad Laboratories.1.4% e e 33. Hercules. Effect of a-thalassemia on the hematological parameters of HbS/S patients The presence of alpha-thalassemia homozygosis (a/a) resulted in significantly higher mean hemoglobin (Hb) levels. Alpha-globin genotype was established by GAP-PCR for the most common 7 alpha thalassemia deletion defects [Liu et al. We subdivided the phenotypes as mild.5% 66.7% 17. Five patients were not included in the haplotype grouping analysis because each had a single unique haplotype with no comparable cases. 2010]..1% e 25% 42. Genotype (a) Asian/Asian haplotype a/a (n ¼ 20) a/aa (n ¼ 9) aa/aa (n ¼ 18) (b) Asian/Oman haplotype a/a (n ¼ 5) a/aa (n ¼ 7) aa/aa (n ¼ 2) (c) Asian/CAR haplotype a/a (n ¼ 3) a/aa (n ¼ 3) aa/aa (n ¼ 2) (d) Benin/Benin haplotype a/a (n ¼ 16) a/aa (n ¼ 8) aa/aa (n ¼ 1) (e) CAR/Oman haplotype a/a (n ¼ 5) a/aa (n ¼ 6) aa/aa (n ¼ 1) (f) CAR/CAR haplotype a/a (n ¼ 4) a/aa (n ¼ 5) aa/aa (n ¼ 4) Mild Intermediate Severe 82. 2009] and molecular characterization of the genotypes and haplotypes was performed at Leiden University Hemoglobinopathies center. The cohort consisted of individuals that had been previously diagnosed with SCA. We found a Central African Republic (CAR) derivative haplotype in some patients and named it the Oman haplotype. position rs968857 5260458) [Phylipsen et al.6%) had the heterozygous genotypes (a3. CA.4%) had a normal alpha-globin genotypes (aa/aa). In spite of the limited number of cases found on other haplotypes.2) combination while 42 patients (33.6% 12. No alpha deletions or point mutations were found in the studied samples.604 S.7% e 60% 83. 3.7/aa) and 28 individuals (22. 2012]. Hematological and clinical data were obtained from routine hematology and from patient’s medical history documented by the treating physician. was twice more frequent in absence of alpha thalassemia. Similarly.M.3% 66. 4/year ¼ severe). Patients with Table 1 Association between alpha thalassemia. Among the severe haplotype. the same pattern seems to appear in the Benin/Benin and in the CAR/Oman cohorts (Table 1d and e) while no association seems to be present in the severe CAR/CAR cohort (Table 1f). Effect of a-thalassemia in grouped HbS/S patients of mild and severe haplotypes HbS/S patients with Asian/Asian and Asian/Oman were grouped under mild haplotype while those with Benin/Benin.. Homozygosis or compound heterozygosis (a/a). In the smaller cohorts of HbS/S patients with Asian/Oman haplotype. red blood cells counts (RBC) but lower levels of fetal hemoglobin (HbF).9% 100% e e e 33. / European Journal of Medical Genetics 57 (2014) 603e606 categorized in specific and different haplotypes. Effect of a-thalassemia in HbS/S patients of specific haplotypes HbS/S patients with Asian/Asian haplotype (also known as ArabIndian) had a mild presentation in 82% and 87% of the cases with (a/a) and (a/aa) thalassemia respectively. When HbS/S patients classified as mild. number of painful crisis per year (3 ¼ mild. Asian/CAR was not included in the 2 groups as it had an intermediate phenotype. all patients without alpha thalassemia were presented with a severer phenotype (Table 2b). DNA extraction was done using the commercial Qiagen kit as per the manufacturer instruction [Hassan et al.7/a4. no clear correlation was found.3% 100% e e e e 20% e 100% 80% 100% . In the few HbS/S patients with Asian/CAR haplotype and (a/a) all three conditions (mild. The diagnosis was confirmed on HPLC (Variant II. None of the Asian/Asian presented with a severe condition (Table 1a). The median age was 24 years. 6 ¼ severe) and blood transfusion (2/year ¼ mild. a-thalassemia’s frequency in the different cohorts The gene frequency of a-thalassemia among HbS/S patient was confirmed to be very high. A group of 125 individuals confirmed homozygous HbS/S were enrolled in this study. were compared with the presence or absence of a-thalassemia regardless the haplotype. intermediate and severe by the occurrence of symptoms such as: acute chest syndrome. Specifically 54 had the (a3. Our goal is to build a bridge between the clinician and the geneticists... Results 3. CAR/Oman and CAR/CAR under severe haplotype. Hassan et al.1% e 75% 42. intermediate and severe) were observed but the severe phenotype was 3 time higher in absence of alpha thalassemia (Table 1c). 3. USA) [Van Delft et al.. mean cell volume (MCV). 3. mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) than the group with a normal alpha genotype (aa/aa). stroke and the presence of avascular necrosis (absence ¼ mild.2. Patients grouped as mild haplotype had a mild presentation in cases with (a/a) and (a/aa) thalassemia than cases without alpha thalassemia (Table 2a).4. We premise therefore that our study is not meant to go deeply into clinical details. This in contrast with 66% of the cases without alpha thalassemia. This haplotype differs from CAR by a single variation at HcII RFLP G>A (SNP F5. The haplotype of the b-globin gene cluster was determined by melting curve analysis as previously reported [Phylipsen et al. presence ¼ severe). an intermediate state.9% 100% e e e 40% 16. The beta genotype was confirmed by direct Sanger DNA sequencing.7) and 1 had the (a3.7% e 33. 3.3% 100% e e e 25% 57. intermediate and severe.

7 86.1 30.4 68.3 aa/aa (n ¼ 1) 5.8 7. Hassan et al.2%).6 a/a (n ¼ 20) 18.0 32.7 25.4 32.0 3.3% 73. our results also show that alpha thalassemia.5 (38. 2010] and by Alkindi et al.4 4.9 2.8 23. intermediate or severe conditions.0 29. The overall distributions of hematologic parameters in Hb S/S patients with three different alphaglobin genotypes are summarized in Table 3.1 69.2 35. This is probably due to a bias deriving by the selection of homozygous HbS/S patients with a much higher chance of being the progeny of consanguineous parents.3 29.8 2.9 83.5 7.6 9.9 9.5%) [Alkindi et al.1 34.9 84.7% 26.3%) measured by our self in a previous study [Hassan et al.2 76.6% e 76% 68.8 26. 1983].2 11.9 26.9 74.5 aa/aa (n ¼ 4) 7.3 26. Hematological data and HbF Alpha thalassemia is believed to improve the survival of the erythrocytes in SCA resulting in a milder form of anemia due to decreased hemolysis [de Ceulaer et al. 1996]. 4.1 9. RBC and lowered HbF.9 72 84..1 2. our study is not meant to be a detailed clinical report but rather a correlation study based upon the occurrence of general symptoms that may indicate mild.1 25.1 a/aa (n ¼ 3) 14. Genotype (a) Mild haplotype a/a (n ¼ 25) a/aa (n ¼ 16) aa/aa (n ¼ 20) (b) Severe haplotype a/a (n ¼ 25) a/aa (n ¼ 19) aa/aa (n ¼ 6) Mild Intermediate Severe 77.9 87.7% 40% e e e e e e 24% 31. In our study the reduction of HbF expression was particularly relevant in patients with the mild “high HbF” (Asian) haplotypes and (a/a) alpha thalassemia.5 32. Data are summarized in Table 4.8 15.4 9. 1998.1 27.0 3. / European Journal of Medical Genetics 57 (2014) 603e606 Table 2 Association between alpha thalassemia.2 26.1 3.8 66.M.5.5 2.6% intermediate while no severe cases were present.7 aa/aa (n ¼ 1) 4..2 26.3 8. Mukherjee et al. In patients with Asian/Oman and Asian/CAR haplotypes the HbF level was however found increased (Table 4b and c). As mentioned in the introduction.1 22.1 8.7 28.6 29.4% were mild and 17.8 33. Previous and present studies In a similar study reported by Mukherjee et al.1 605 Table 4 Effects of various a-thalassemia genotypes on the average hematological parameters in HbS/S patients based on their haplotype. 82.5 9.3 10.1 aa/aa (n ¼ 2) 10.8%) and to severe (40%) cases. 3.7 34.2 (b) Asian/Oman haplotype a/a (n ¼ 5) 9.9 23. 2001].1 69.2 89.2 80.7 11.2 33.S.7 90.6 8. Therefore we have selected groups with identical genotypes and haplotypes with and without alpha thalassemia.7 29.7 3. the presence of alpha thalassemia in Hb S/S homozygotes resulted in significantly higher mean Hb and HCT levels as well as higher RBC counts and these three parameters indicate a better RBC survival as a consequence of lower hemolysis. Mukherjee et al.9 33.3 69. MCH and MCHC in patients with Asian/Asian.2 3.. Influence of a-thalassemia on hematological parameters in HbS/S patients of different haplotypes Alpha thalassemia increased the mean Hb. Conversely.6 4. patients with homozygous alpha thalassemia had on average lower levels of fetal Hb (HbF) and a lower MCHC than patients with a normal alpha-globin genotype (aa/aa).1 30.2 4.6 22.9 34. The clinical presentation of our total cohort with homozygous alpha thalassemia ranged from mild Table 3 The effects of the various a-thalassemia genotypes on the mean hematological parameters in HbS/S patients regardless of the haplotype.7 9. in patients with severe “low HbF” haplotypes (CAR and Benin) the HbF remained either unchanged or increased in the presence of alpha thalassemia while .3 79.4% 100% heterozygous alpha complement (a/aa) showed intermediate mean hematological values. 1998].9 9. to intermediate (21.3 23.1 34.3 29.3 a/aa (n ¼ 8) 3.4 9. Although external and accidental factors cannot be avoided.7 9.7 33. The same correlation was also observed in patients with Asian/Oman haplotype and (a/a) in which 75% had a mild disease and 25% were intermediate.5 31. In HbS/S patients of the severe Benin/Benin haplotype only a decreased MCV and MCH were observed.2 34.3 35. studied patients of the Asian/Asian haplotype with high HbF levels [Mukherjee et al.5 5. 2010].7 29.6 aa/aa (n ¼ 2) 9. In addition.7 25. 5.4 9.7 10. although ameliorating the hematological parameters.9 74.1 22. CAR/Oman and CAR/CAR haplotypes (Table 4a.9 2. e and f).4 33.8 4.4 21.8 3.6 HCT (%) RBC (1012/L) MCV (fl) MCH (pg) MCHC (g/dl) 29.1 3.7 25. 6.8 33.0 9.. we do believe that our cohorts are as comparable as possible.. a/a (n ¼ 55) a/aa (n ¼ 42) aa/aa (n ¼ 28) Hb F (%) Hb (g/dl) HCT (%) RBC (1012/L) MCV (fl) MCH (pg) MCHC (g/dl) 10. when compared with Asian haplotypes without a-thalassemia [Adekile and Haider.1 3. all SCA patients from Western India with homozygous a-thalassemia had a mild phenotype [Mukherjee et al.6 35.2 a/aa (n ¼ 5) 5. In our study.8 25.0 28.0 35.5 26.6 34.9 23.4 33.6 34. Discussion When performing correlation studies between genotype and phenotype it is important to compare cohorts that are not only phenotypically similar but genotypically identical in order to reduce genetic variables to a minimum. The frequency of alpha thalassemia in the present cohort (72%) was higher than the (58. How relative can be the effect of alpha thalassemia in SCA is shown by many contrasting reports [Ballas.6 32. mild and severe b-cluster haplotypes and clinical severity.6 79.3 3.9 2. On the other hand. HCT. Considering the variability of the definition “intermediate” our results are quite compatible with the observations of the Indian study and with the conclusions of other authors reporting milder conditions among Saudis and Kuwaitis carrying the Asian haplotype with a-thalassemia.0 (e) CAR/Oman haplotype a/a (n ¼ 5) 4.3 27.9 27.2 a/aa (n ¼ 7) 17.4 10.8 82.4 (c) Asian/CAR haplotype a/a (n ¼ 3) 12.9 23.5 23..2 8. Hb F (%) Hb (g/dl) (a) Asian/Asian haplotype 13.1 (f) CAR/CAR haplotype a/a (n ¼ 4) 4.5 77.8 22.3% 60% 22.5 25.6 33. MCV. 1997] and our data on the 47 patients with Asian/Asian haplotype shows that among cases with (a/a). is of little effect in reducing the symptoms of the HbS/S homozygous with the severe CAR and Benin haplotypes.3 a/aa (n ¼ 6) 5.6 a/aa (n ¼ 9) aa/aa (n ¼ 18) 20. (48.5 (d) Benin/Benin haplotype a/a (n ¼ 16) 5.3 31..6 4.5 35.

differences in the mild and severe forms became more evident. Al Zadjali S. Miles K. Jaffer Mohammed A. Advances in the use of hydroxyurea. Shelton JB. Al Abri Q. Serjeant GR. Sci World J 2009. et al.106:75. Al-Awamy B. Mukherjee MB. Schroeder WA. Surve R.M.18(9):46e67. Ducrocq R. N Engl J Med 1982. Phylipsen M. and S-b-thalassemia patients in Southern California. Huynh V. Hassan et al. Lamb J. Baylan U.. References Adekile AD.. Glader BE. et al.96:150e4.20(2):107e21. Higgs DR. Jaffer Al-Lawatiya F. . Colah RB. Harteveld CL. The interaction of alpha-thalassaemia and homozygous sickle cell disease.55:104e9. Kanhai WA. In addition. Hassan SM. Am Soc Hematol Educ Program 2009. a-thalasemia reduces the haemolytic rate in homozygous sickle cell disease. Rapid detection of a-thalassaemia deletions and a-globin gene triplication by multiplex polymerase chain reactions. 7. Old JM.32:578e87. 2000]. Higgs et al. Van Delft P. N Engl J Med 1983. Kulozik AE. [1982] also observed that (a/a) individuals had the lowest cell volume and hemoglobin content per cell (MCHC) which reduce the polymerization risk of the Hb molecules in the smaller and less dense cells. Serjeant GR. Fisher CA. Monovalent cation changes in sickle erythrocytes: a direct reflection of a-globin gene number.39: 239e44. Clegg JB. Steinberg MH. Ghosh K. but also by environmental factors and life style. Yamsri S.74: 155e60. Am J Hum Genet 1986. Hemoglobin 2010.108:295e9. Powars DR. Powars DR.309:189. Br J Haematol 2000. 1985]. However. Bakker-Verweij M. Weatherall DJ.34(2):127e34. providing a larger reserve of internal volume per given amount of polymer and thus giving protection against the deleterious consequences of membrane stretching during deoxygenation [Embury et al. Evaluating five dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnic populations. Harteveld CL. Serjeant BE. Essan GJ. Effect of alpha-globin genotype on the pathophysiology of sickle cell disease. Huisman W. Gangakhedkar RR. Hemoglobin 2010. Aldridge BE. Hayes RJ.and alpha-thalassemia in Oman. a-gene status and hematologic data from SS. Noninvasive prenatal diagnosis of beta-thalassemia and sickle-cell disease using pyrophosphorolysis-activated polymerization and melting curve analysis. et al. SC. Predicting clinical severity in sickle cell anaemia. An overview. Indian J Med Res 1998.107:178e81.129(4):465e81. Diagn.. The influence of fetal hemoglobin on the clinical expression of sickle cell anemia. 1985]. However. Forecasting hemoglobinopathy burden through neonatal screening in Omani neonates. Beta-cluster haplotypes. Other studies have shown that coexistence of a-thalassemia enhances the levels of HbF associated with a specific haplotype such as the Senegal [Schroeder et al. bs haplotype and a-globin gene patterns among sickle cell anemia patients in Kuwait. et al. MCH and MCHC associated with alpha thalassemia should diminish the amount of intravascular sickling while the decreased intra-erythrocytic hemoglobin S concentration associated with a-thalassemia should diminish polymerization (sickling) and herewith the degree of chronic hemolytic anemia typical of SCA [Ballas. variability in clinical outcomes of sickle cell disease is not modulated by genetic factors alone. Br J Haematol 2005. Lu CY.306:1441e6. low MCV in (a/a) generates a relatively larger cellular surface compared to (aa/ aa) which might give the cells the property of increased membrane redundancy. The influence of alpha-thalassaemia on the haematological & clinical expression of sickle cell disease in western India. Ware RE. 1989]. Al Haddabi H. 2001]. Ann N Y Acad Sci 1989.565:109e19. Am J Hematol 1997. Liu YT. Schroeder WA. Effect of a-thalassemia on sickle-cell anemia linked to the Arab-Indian haplotype in India. Morbidity. Rajab A. it seems evident from the present study that alpha-thalassemia can modulate the hematological picture of HbS/S but not clearly the overall severity manifestation in patients of all haplotypes. We do believe that smaller cells (low MCV) might just be faster. El-Hazmi MAF. Giordano PC.. Lenters E. [1985] reported an increase in HbF levels [El-Hazmi. Genetic etiologies for phenotypic diversity in sickle cell anemia. passing the risk area of the post-capillary veins as this seems to be the same rheological advantage observed in mild SCA phenotypes with microcytic hypochromic parameters due to iron deficiency [Liu et al. In addition. Daar S. Iron depletion: an ameliorating factor for sickle cell disease? ISRN Hematol 2011. Extended molecular spectrum of beta. et al. Kay LM. de Korte M. Wainscoat JS. et al. / European Journal of Medical Genetics 57 (2014) 603e606 little changes were observed in these patients also at the RBC level. Kar BC. Steinberg MH. Chan L.34(2):135e44. Lu CY. Higgs et al. Clegg JB. other factors (both genetic and environmental) are involved making the interpretation of phenotype/genotype association studies more complex. Prenat. Chan LS. Geographical survey of beta S-globin gene haplotypes: evidence for an independent Asian origin of the sickle-cell mutation. Ann N Y Acad Sci 1989. Al Madhani A. Conclusion Although some of the haplotype cohorts are small. Weatherall DJ. Serjeant GR. Pediatr Pathol Mol Med 2001. Hamza N. Boon EMJ.2011:473152. Ballas SK. Higgs DR. Tamankar A. et al. Aygun B. Weatherall DJ. Hayes RJ.565:262e78. Harteveld CL. Haider MZ. Mukherjee MB. Gangakhedkar RR. Alkindi S. Treffers EE. et al. 1989] and Benin haplotypes [Powars et al. Embury SH. Acta Haematol 1996. 2012. Clinical manifestation and laboratory findings of sickle cell anaemia in association with aethalassaemia in Saudi Arabia.606 S. Int J Lab Hematol 2009. Geography and the clinical picture of sickle cell disease. [1982] reported a decrease in the level of HbF in SCA patients with homozygous alpha thalassemia while Embury et al. when the cohort was subdivided into larger groups of similar haplotypes. Jansen DTSL.(1):62e9. Kadam MD. In conclusion the lower MCV.31(5):484e95. Hemoglobin 1989. J Lab Clin Med 1985. de Ceulaer D. Acta Haematol 1985. et al. Backer K.13:325e53.