WHOLE1.indd ABSTRACT Background: Haemoglobinopathies are a major cause of morbidity in the Sultanate of Oman and premarital screening is being encouraged in order to reduce the number of affected births. The identification of β-thalassaemia carrier status is an essential prerequisite of any screening programme. However, the level of Haemoglobin (Hb) A2, which is used to detect β-thalassaemia carriers, can be affected by other factors including iron deficiency, concurrent α thalassaemia and the type of DNA mutation present. Objec- tives: The following study was undertaken to ascertain if the Hb A2 level is an appropriate tool for the identification of β-thalassaemia carriers in the Omani population. Method: Hb A2 was measured by high performance liquid chromatography (HPLC) in 60 obligate carriers of β-thalassaemia. 58 subjects had Hb A2 levels above 3.5% indicating β-thalassaemia trait. Two subjects had slightly lower levels and were found to be iron deficient. After therapy both these subjects’ Hb A2 levels increased to above 3.5%. Conclusion: In the absence of iron deficiency, Hb A2 is an accurate marker for the presence of β-thalassaemia trait in the Sultanate of Oman. Key words: Hb A2 , β-thalassaemia trait, Sultanate of Oman Diagnosis of Beta-Thalassaemia Carriers in the Sultanate of Oman *Shahina Daar1, David Gravell2 SULTAN QABOOS UNIVERSITY MEDICAL JOURNAL JUNE 2006 VOL 6, NO. 1 SULTAN QABOOS UNIVERSITY© IN THE NORMAL POPULATION HAEMOGLOBIN A2 (α2δ2) is physiologically unimportant as it is present in very small quantities (<3% of total haemo- globin). However, the carrier state for β-thalassaemia is characterised by an increased level of Hb A2 above 3.5%, low mean cell volume (MCV) and low mean cell haemoglobin (MCH).1 Exceptions to this are a group of heterozygotes that may have normal Hb A2 levels; these include the ‘silent’ thalassaemias and coinheritance of δ-thalassaemia.2 The mechanism underlying the increase in Hb A2 appears to be through increased αδ-dimer formation. In a normal red cell, α chains will preferentially com- bine with β chains rather than δ chains which are more positively charged; however, in the presence of a re- duced quantity of β chains and a relative excess of α chains as found in β-thalassaemia, more Hb A2 will be formed.3 Beta-thalassaemia heterozygotes are usually clini- cally asymptomatic. Although there is a decrease in β globin gene output, there is very little ineffective eryth- ropoiesis, with the proteolytic mechanisms within the red cell having the capacity to deal with this degree of chain imbalance. Thus, in presence of the normal 1 Department of Haematology, Sultan Qaboos University, College of Medicine and Health Sciences, P.O.Box 35, Al-Khod, Muscat 123, Sultanate of Oman, 2 Department of Haematology, Sultan Qaboos University Hospital, P.O.Box 35, Al-Khod, Muscat 123, Sultanate of Oman. *To whom correspondence should be addressed. Email: asdoc@omantel.net.om O R I G I N A L S T U D Y سلطنة (β-Thalassaemia) في ثاالسيميا البيتا حاملي تشخيص عمان قبل ما (غربلة) تقصي تشجيع مت وقد عمان، سلطنة في للمراضة االسباب الرئيسية من (الهيموجلوبني) املقدمة: امراض خضاب الدم امللخص: حال، أية على للتقصي. ألي برنامج جوهري مطلب هو البيتا ثاالسيميا حاملي على التعرف أن بهذه األمراض. املصابني األطفال والدة لتقليل الزواج α) ، ووجود الفا ثاالسيميا احلديد كعوز اخرى بعوامل يتأثر أن ميكن ثاالسيميا ، البيتا حاملي لكشف واملستخدم (Hb A٢) الدم خضاب مستوى Hb A٢ خضاب الدم مستوى كان ملعرفة فيما إذا هذه الدراسة أجريت DNA). ألهدف: ) (دي ان أي) ال في طفرة حالة في thalassemia ) وكذلك ل (HPLC) الفعالية عالي خاص حتليل جهاز Hb A٢ بواسطة قياس الطريقة: مت عمان. في ثاالسيميا البيتا حاملي على للتعرف مناسبة وسيلة كان واثنني فقط ، ٣٫٥ من مستوى Hb A٢ أعلى لديهم كان ١٥٨ شخصا في صفات البيتا ثاالسيميا مت كشف ثاالسيميا. للبيتا مؤكد ١٦٠ حامل يكون عوز احلديد غياب ٣٫٥. اخلالصة: في Hb A٢ الى أعلى من مستوى ارتفع العالج، بعد بعوز احلديد. انهما مصابني ذلك واكتشف أقل من لديهم عمان. سلطنة في ثاالسيميا البيتا صفة Hb A٢ مؤشرا دقيقا لوجود S H A H I N A D A A R , D AV I D G R AV E L L 28 complement of α chains, most β-thalassaemia heter- ozygotes can be diagnosed by an increase in Hb A2. However, the presence of coexistent α-thalassaemia could theoretically lower the Hb A2 level. The Om- ani population is known to have a high prevalence of α-thalassaemia and a substantial number of the population are carriers of either Haemoglobin S or β- thalassaemia.4,5 Pre-marital screening and counselling are now encouraged. All blood samples at the Sultan Qaboos University Hospital (SQUH) that have a low MCV and MCH are routinely analysed by high per- formance liquid chromatography (HPLC). For these reasons, it is important to know the range of Hb A2 among β-thalassaemia carriers in this population and, in particular, to observe if the presence of α-thalassae- mia has lowered the levels Hb A2 to overlap with the normal range. This study also compared Hb A2 levels in β0 and β+ heterozygotes. M E T H O D DNA analysis (for beta globin gene mutations and α- thalassaemia) had previously been performed on 117 unrelated Omani patients with homozygous β tha- lassaemia being treated at SQUH in the Sultanate of Oman.6 For the present study, blood samples from 160 Omani obligate carriers (parents of known β-tha- lassaemia homozygotes) were collected into K EDTA and analysed by HPLC Biorad variant, β-thalassaemia ‘short’ programme. The samples included 28 parents heterozygous for Hb Dhofar (β58 C-G, β29 C-T) a known β-thalassaemia mutation, 86 from parents of patients homozygous for either β0 or β+ mutations and 46 par- ents of patients who were compound heterozygotes for β0/ β+. 40 of these samples had been previously analysed for the presence of α thalassaemia. Ten (25%) had no α-thalassaemia, 14 (35%) had a single gene de- letion and 16 (40%) had two alpha genes deleted.7 R E S U L T S 158 of 160 samples from obligate β-thalassaemia car- riers, including Hb Dhofar, had Hb A2 levels above 3.5% (mean 4.8%, range 3.6%-6.7%) (Figure 1). The two exceptions had Hb A2 levels of 3.4%; they were both found to be severely iron deficient and after six months of treatment with haematinics, the Hb A2 lev- els had risen to 3.6% and 3.5% respectively. Hb Dhofar elutes in the ‘D’ window, very close to Hb A2 and raises the baseline of Hb A2 resulting in a falsely low reading (Figure 2). In these cases an ac- curate value for Hb A2 was obtained by elution after electrophoresis on cellulose acetate. The range of Hb A2 in Hb Dhofar simple heterozygotes was 3.6%-5.5%, (mean 4.5%± 0.47%). Of the 160 samples, 69 were from known β+ heterozy- gotes and 26 from known β0 heterozygotes. Of the sixty- nine β+ samples, all but two had an Hb A2 level above 3.5% (range 3.6%-6.5%); the two exceptions were found to have iron deficiency. The mean level in this group was 4.9% ± 0.65%. The range of Hb A2 in the twenty-two β 0 samples was 3.7% - 7.1% (mean 4.9% ± 0.62%). Mutations No. Alleles % Total Type IVS 1-5 (G-C) 146 62.4 β+ Codon 44 –C 26 11.1 β0 Hb Dhofar (β29 β58) 17 7.3 β+ IVS 1-3 –25bp 14 6.0 β0 619 del 8 3.4 β0 IVS II –1 (G-A) 7 3.1 β0 218 93.3 β0 The remaining 16 alleles were spread between 10 mutations found in very low frequency, namely, IVS 1-1( G-A) (βo), codon 39 (C-T) (βo), codon 5 –CT (βo), HbE (β+), IVS1-110( G-A) (βo), codon 15 ( G-A) (βo), codon 30 ( G-C) (βo), codon 36/37 -T(βo), Codon 37( G-A) (βo), IVS 1-128 ( G-A) (β+) Table 1. Analysis of β-thalassaemia mutations in Omanis (234 alleles from 117 unrelated homozygous beta thalassaemia individuals) D I A G N O S I S O F B E TA -TH A L A S S A E M I A C A R R I E R S I N T H E S U LTA N AT E O F O M A N 29 D I S C U S S I O N A large number of β-thalassaemia mutations have been identified worldwide; some of these result in a total absence of beta globin chain production (β0 mu- tations) while others cause a variable decrease in beta globin chain production (β+ mutations). In the present study, there was no significant difference in Hb A2 lev- els between the carriers of β+, β0 or Hb Dhofar. This concurs with the separate findings of investigators who have found that despite the immense heterogeneity of β-thalassaemia mutations, the level of Hb A2 in het- erozygotes is remarkably similar. Huisman analysed data from six hundred β-thalassaemia heterozygotes and found Hb A2 levels to be between 4.5% and 5.5% in persons with β0 or severe β+ thalassaemia and be- tween 3.6% and 4.2% in those with milder mutations.2 Altay and Gurgey obtained similar data from Turkish individuals.8 It is only when very mild β+ alleles are present that the Hb A2 level may drop. Other than the single allele of Hb E, (which poses no diagnostic difficulty), none of the mutations thus far identified in the Sultanate can be classified as mild Table 1.6 How- ever, it should be noted that our studies have all been hospital based; there may be a number of milder beta thalassaemia mutations in the general population that have yet to be identified. A complicating issue in the accurate identification of β-thalassaemia trait is that the coexistence of iron deficiency may suppress the level of Hb A2 leading to misdiagnosis. The association of iron deficiency with lowered Hb A2 is not a constant finding. Wasi et al 9 describe normal levels of Hb A2 in β-thalassaemia trait individuals who were also iron deficient but Alperin et al10 found that the more severe the anaemia the lower the Hb A2 level. In contrast, Madan found the mean Hb A2 to be significantly higher in those patients with iron deficiency anaemia than in those without in a group of Indian β-thalassaemia heterozygotes.11 Our results suggest that iron deficiency lowers the Hb A2 level in some individuals. The effect of concurrent α-thalassaemia on Hb A2 is also a potential pitfall in the diagnosis of the β-thalas- saemia carrier state, as in parts of the world where β- thalassaemia is found, α-thalassaemia is often present in high prevalence.12-14 Martinez and Menendez have shown that lower amounts of Hb A2 may be formed when the supply of α chains is limited15 but a recent study in Singapore has shown that routine haemato- logical tests can confirm β-thalassaemia carrier status in the simultaneous presence of α and β-thalassaemia.16 In addition, the results of a study in Sardinian children conducted by Galanello et al showed that when α- thalassaemia coexists with β-thalassaemia, although the mean cellular volume (MCV) and mean cellular haemoglobin (MCH) in heterozygote β-thalassaemics may fall within the normal range, the levels of Hb A2 are always increased compared to the normal.17 Thus, in regions where both α and β-thalassaemia are preva- lent the diagnosis of heterozygous β-thalassaemia should not be excluded on the basis of normal indices Figure 1. Hb A2 in oblgate β-thalassaemia carriers S H A H I N A D A A R , D AV I D G R AV E L L 30 and Hb A2 must always be evaluated. Statistically, at least 75% of Omanis are expected to have either single gene or two-gene deletional α- thalassaemia.4 The same ratio was found in a study group of Omani thalassaemia major patients, with 36% having no α-thalassaemia, 32% having a single alpha gene deletion and 32% having two alpha genes deleted.7 A smaller study of 40 of the obligate carriers showed 10 (25%) to have no α-thalassaemia, 14 (35%) with a single gene deletion and 16 (40%) with two al- pha gene deletion. The results of the present study show that, other than the two carriers with iron deficiency, all the ob- ligate carriers had Hb A2 levels over the diagnostic range for β-thalassaemia trait. C O N C L U S I O N For the purposes of genetic screening and premarital counselling it is reassuring to find that, despite the high prevalence of α-thalassaemia in the Omani popula- tion, the levels of Hb A2 in β-thalassaemia carriers are above the normal range and that β-thalassaemia trait in Omanis can be diagnosed by the presence of Hb A2 equal to or above 3.5% as long as there is no concomi- tant iron deficiency. In order to minimise false nega- tives, all individuals with Hb A2 of 3.3%-3.4%, or who have anemia, should be screened for iron deficiency; if this is present, the Hb A2 level should be repeated after adequate treatment with hematinics. R E F E R E N C E S 1. Kunkel HG, Ceppellini R, Muller-Eberhard U, et al. Ob- servations on the Minor Basic Hemoglobin Component in the Blood of Normal Individuals and Patients with Thalassemia. J Clin Invest 1957; 36: 1615-1625. 2. Huisman T. Levels of HbA2 in Heterozygotes and Ho- mozygotes for Beta-Thalassemia mutations: Influence of Mutations in the CACCC and ATAAA Motifs of the Beta-Globin Gene Promoter. Acta Haematol 1997; 98: 187-194. 3. Steinberg MH, Adams JG III. Hemoglobin A2: Origin, Evolution, and Aftermath. Blood 1991; 78: 2165-2177. 4. White JM, Christie BS, Nam D, Daar S, Higgs DR. Fre- quency and Clinical Significance of Erythrocyte Genetic Abnormalities in Omanis. J Med Genet 1993; 30: 396- 400. 5. Al-Riyami AA, Suleiman AJ, Afifi M, Al-Lamki ZM and Daar S. A Community –Based Study of Common Figure 2. HPLC of Hb Dhofar trait Analyte ID % Time A0 76.9 2.40 A2 1.7 3.65 D-Window 11.3 4.04 D I A G N O S I S O F B E TA -TH A L A S S A E M I A C A R R I E R S I N T H E S U LTA N AT E O F O M A N 31 Hereditary Blood Disorders in Oman. East Mediterr Health J 2001; 7(6): 1004-1011. 6. Daar S, Hussein HM, Merghoub T and Krishnamoor- thy R. Spectrum of ‚ β-thalassemia Mutations in Oman. Ann N Y Acad Sci 1998; 850: 404-406. 7. Daar S. MD thesis Haemoglobinopathies in the Sultan- ate of Oman. In: University of Bristol UK, 2000. 8. Altay C, Gurgey A. β-thalassemia Intermedia in Tur- key. Ann N Y Acad Sci 1990; 612: 81-89. 9. Wasi P, Disthasongchan P, Na-Nakorn S. The Effect of Iron Deficiency on the Levels of Hemoglobins A2 and E J. Lab Clin Med 1968; 71: 85-91. 10. Alperin JB, Dow PA, Petteway MB. Hemoglobin A2 Levels in Health and Various Hematologic Disorders. Am J Clin Pathol 1977; 67: 219-226. 11. Madan N, Sikka M, Sharma S, Rusia U. Phenotypic Ex- pression of Hemoglobin A2 in β-thalassemia Trait with Iron Deficiency. Ann Hematol 1998; 77: 93-96. 12. Ganeshaguru K, Acquaye JK, Samuel AP, et al. Preva- lence of Thalassaemias in Ethnic Saudi Arabians. Trop Geogr Med 1987; 39: 238-243. 13. El-Kalla S, Mathews AR. A Significant‚ β-thalassemia Heterogeneity in the United Arab Emirates. Hemo- globin 1997; 21:237-247. 14. El-Kalla S, Baysal E. α-thalassemia in the United Arab Emirates. Acta Haematol 1998; 100: 49-53. 15. Martinez G, Menendez R. Differences in Affinity of β and δ hemoglobin Chains for α Chains. Biochim Bio- phys Acta 1983; 743: 256-259. 16. Law HY, Chee MKL, Tan GP, Ng ISL. The Simultane- ous Presence of α and β thalassaemia Alleles: A Pitfall in thalassaemia Screening. Community Genet 2003; 6: 14-21. 17. Galanello R, Lilliu F, Bertolino F, et al. Percentile Curves for Red Cell Indices of β0-thalassaemia Heterozygotes in Infancy and Childhood (1991). Eur J Paediatr; 150: 413-415.