Abstract
Alpha thalassemia (α-thal) is relatively common worldwide. Most carriers are defective in either one or two alpha globin genes out of four functional ones, with deletions being more common than point mutations. The hematologic features are very important for the selection of the appropriate molecular tests while determining the genotype. The aim of this study was to compare hematologic features of patients with various types of α globin mutations. Hematological indices including red blood cells (RBC), hemoglobin concentration (Hb), mean cell volume (MCV), mean cell hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC) and percentage of Hemoglobin (HBA1, HBA2 and HBF) of seven-hundred and twenty two patients presenting ten different α-thal genotypes were considered. All patients showed reduced MCV and/or MCH values.Moreover, MCV and MCH were lower in patients with two functional alpha globin genes in comparison to patients with one mutated alpha globin gene (P value<0.001). In conclusion, MCV and MCH valuescan be helpful for the selection of the appropriate molecular tests to determine the genotype of alphathalassemia carriers.
Key Words: Alpha thalassemia, mean cell volume, mean cell hemoglobin, mutation
Alpha thalassemia (α-thal) is one of the hemoglobinopathy that is characterized by a quantitative reduction of the α globin chains (1-2). α-thal is most common in Southeast Asia but is also prevalent in the Mediterranean, Middle East, India, and sub-Saharan Africa, with carrier frequencies ranging from 15% to 30% (3).
Each person have a pair of α globin genes, α1 and α2, on chromosome 16 (4). There are two main classes of α thalassaemia. α0 thalassaemias in which both α globin genes of a chromosome are deleted and α+ thalassaemias in which only one of the α globin genes is lost or inactive. In this later case α+ thalassaemia is also represented as (αTα/αα) (5). Individuals with only one globin deletion(-α/αα), are silent carriers and asymptomatic. Dysfunction of two α globin genes (-α/-α or --/αα ) produces mild anemia, while deletion/mutation of three α globin genes (-α/--) causes a more severe anemia characterized by production of Hb H, infection/inflammation induced hemolysis and acute or chronic cholecystitis (6-7). Complete absence of α globin genes (--/--) results in Hemoglobin Bart’s hydrops fetalis which is characterized by severe intrauterine anemia resulting in fetal hydrops and, in almost all cases, intrauterine death (8). α-thal trait diagnosis is based on microcytosis (MCV < 80 fL, MCH < 27 pg) and normal hemoglobin HB A2 level (<3.5%) (9).
α-thal most frequently results from deletion of one (-α) or both (--) α genes from the chromosome (10). 3.7 kb deletion (-α3.7) and 4.2 kb deletion (-α4.2) are the most common causes of α+ thalassaemia and Mediterranean deletion (--Med), South East Asia deletion(--SEA) are frequent causes of α0 thalassaemia (11). Also point mutations in critical regions of the alpha globin genes can cause disease, so-called, non-deletional α thalassaemia including: polyadenylation site mutations polyA1(AATAAA > AATAAG), polyA2 (AATAAA > AATGAA) and IVS-I donor site [GAG GTG AGG>GAG G-----](-5 nt) and termination codon mutations, Hb Constant Spring (10, 12).
The accurate characterization of the hematologic features is very important for the selection of the appropriate molecular tests to determine the carrier genotype. The basic hematological tests usually used, include: the measurement of the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) value and the quantity of Hb A2 and Hb F (13). Wide α-thal alleles have been identified inαglobin genes however limited studies were performed for considering their interaction and possible genotype-phenotype correlation. In this study, we compared hematologic features and Hb profiles of Iranian patients with various types of α globin mutations.
Materials and methods
Seven-hundred and twenty two adult α-thal carriers (376 males and 346 females) presenting with mild anemia were included in the study. They originated from northern provinces of Iran. Patients had been screened for α globin mutations using Gap-PCR, Reverse dot blot or restriction enzyme digestion (14,15). Of them, 215 patients had one point mutation (polyA1:14, polyA2:133, -5 nt:48, Hb Constant Spring:32), 446 patients had one deletion mutation ((-α3.7: 367, -α4.7: 54, --Med : 62), 61 patients had two mutations (-α3.7/-α3.7: 50, -α3.7/-α4.2: 6, -α3.7 /α PA2α: 7, -α3.7/ αCSPα: 5).
Patients were analyzed for hematological indices, including red blood cells (RBC), hemoglobin concentration (Hb), mean cell volume (MCV), mean cell hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC) and percentage of HB A1 and HB A2; and HB F (for some cases). The data were analyzed using the SPSS 16 software. Descriptive statistics including mean and standard deviation were used to describe hematologic indices of each thalassemia genotype.
Results
Ten different α-thal genotypes in Seven-hundred and twenty two patients were compared based on hematological indices. Hematologic data of both male and female patients were considered together because there were no significant differences in hematologic indices between the 2 sexes (Tables 1 and 2) with exception of RBC and HB level that were higher in males (Tables 3 and 4).
Table 1.
Variation (mean ± SD) in Hematologic Features of α-thal carriers with one defected alpha globin gene
| MCV (fl) | MCH (pg) | MCHC ( g/dl) | A1(%) | A2 (%) | HBF (%) | |
|---|---|---|---|---|---|---|
| αPA1α /αα | 74.86±4.15 | 23.35±1.10 | 31.34±0.79 | 96.86±0.36 | 2.22±0.66 | 0.67±0.12 |
| αPA2α /αα | 76.81±3.32 | 24.65±1.47 | 31.96±1.38 | 96.87±0.61 | 2.49±0.44 | 0.71±0.27 |
| α5NTα /αα | 76.42±4.60 | 24.14±1.33 | 31.93±1.12 | 96.83±0.69 | 2.54±0.38 | 0.73±0.45 |
| αCSPα /αα | 75.62±4.14 | 24.20±1.67 | 32.47±1.18 | 96.84±0.58 | 2.77±0.45 | 0.63±0.29 |
| -α3.7/ αα | 76.82±5.02 | 24.70±1.60 | 32.11±1.21 | 96.68±2.75 | 2.53±0.40 | 0.70±1.00 |
| -α4.2/ αα | 77.01±3.96 | 24.58±2.16 | 31.86±1.64 | 96.82±0.52 | 2.40±0.35 | 0.85±0.39 |
Table 2.
Variation (mean ± SD) in Hematologic Features of α-thal carriers with two defected alpha globin genes
| MCV(fl) | MCH(pg) | MCHC(g/dl) | A1(%) | A2(%) | HBF(%) | ||
|---|---|---|---|---|---|---|---|
| --MED /αα | 66.48±4.62 | 20.21±1.37 | 30.66±1.52 | 96. 28±0.55 | 2.44±0.46 | 0.74±0.26 | |
| -α3.7/ -α3.7 | 72.76±4.55 | 22.46±1.73 | 31.32±1.35 | 96.82±0.59 | 2.44±0.44 | 0.69±0.25 | |
| -α3.7/ -α4.2 | 70.71±5.17 | 22.43±1.85 | 31.41±1.20 | 97.20±0.70 | 2.25±0.53 | 0.50±0.17 | |
| -α3.7 /α PA2α | 70.33±2.89 | 21.91±0.68 | 30.95±1.13 | 97.16±0.56 | 2.56±0.47 | 0.90±0.20 | |
| -α3.7/ αCSPα | 69.35±3.10 | 22.00±1.54 | 31.56±1.22 | 97.33±1.44 | 1.85±0.97 | 0.50±0.20 | |
Table 3.
Variation (mean ± SD) in red blood cells (RBC) counts and Hemoglobin (Hb) levels of α-thal carriers with one defected alpa globin gene
| n | RBC(×1012/L) | HB(g/dl) | |||
|---|---|---|---|---|---|
| αPA1α /αα | Male Female |
5 8 |
6.25±0.29 5.29±0.40 |
14.60±0.50 12.33±0.82 |
|
| αPA2α /αα | Male Female |
70 55 |
5.77±0.43 5.09±0.42 |
14.30±1.01 12.39±1.14 |
|
| α5NTα /αα | Male Female |
19 24 |
5.86±0.56 5.09±0.54 |
14. 27±1.10 12.30±1.31 |
|
| αCSPα /αα | Male Female |
14 14 |
5.62±0.37 5.03±0.35 |
14.15±0.84 12.93±2.80 |
|
| -α3.7/ αα | Male Female |
179 155 |
5.83±0.47 5.10±0.42 |
14.42±1.19 12.49±1.00 |
|
| -α4.2/ αα | Male Female |
25 26 |
5.82±0.50 5.13±0.32 |
14.08±2.34 12.55±0.84 |
|
Table 4.
Variation (mean ± SD) in red blood cells (RBC) counts and Hemoglobin (Hb) levels of α-thal carriers with two defected alpa globin genes
| n | RBC(×1012/L) | HB(g/dl) | ||
|---|---|---|---|---|
| --MED /αα | Male Female |
24 30 |
6.22±0.83 5.49±0.48 |
12.62±1.81 11.13±.89 |
| -α3.7/ -α3.7 | Male Female |
27 18 |
5.97±0.45 5.37±0.518 |
13.55±1.28 11.98±1.03 |
| -α3.7/ -α4.2 | Male Female |
1 5 |
5.29 5.28±0.47 |
13.40 11.46±0.55 |
| -α3.7 /α PA2α | Male Female |
4 2 |
6.28±0.26 5.62±0.02 |
13.63±0.32 12.05±0.21 |
| -α3.7/ αCSPα | Male Female |
3 1 |
5.75±0.59 5.08 |
12.73±0.63 10.80 |
Patients who had one mutated alpha globin gene, showed reduced MCV and MCH values. Moreover, MCV and MCH were lower in patients with two functional alpha globin genes in comparison to patients with one mutated alpha globin gene (P value<0.001). Figures 1 and 2 show the variations of MCV and MCH values respectively for the studied subjects. Although MCHC values were slightly lower in patients presenting one mutation compared to those having two functional alpha globin genes, but this difference was not significant (Tables 1and 2). Other hematological indices such as HbA2 and HbF levels as well as RBC and total Hblevel in either sexes showed no significant differences between patients presenting one and those presenting two alpha globin gene defects.
Fig 1.
Variations (mean ± SD) of MCV values in α-thal carriers
Fig2.
Variations(mean ± SD) of MCH values in α-thal carriers
Discussion
Alpha Thalassemia is a serious disease for many of Mediterranean, Middle East and Southeast Asian countries (3). It is estimated that in Iran nearly 25% of the annual blood production is used for thalassemic patients. Moreover, progresses in treatment of disease and increase in the life expectancy of thalassemic patients is expected to increase the prevalence of thalassemic population (16). Premarital screening programs for identification of carrier of thalassemia and prenatal diagnosis of disease for couples at risk of having affected child, is an effective way for control of thalassemia (17) that was carried out in some countries like Iran (18-19).
The key to identifying the globin genes mutations in carriers and affected patients is an understanding of the genotype/ phenotype relationships of the various globin gene mutations and the effects of interaction when several mutations are co-inherited.
Consideration of hematological indices of α-thal genotypes indicated that all thalassemia genotypes may not be associated with mild hematological phenotypes (Tables 1 and 2). Although there were statistically significant differences in MCV, and MCH between genotypes, it was difficult to distinguish the different conditions based on hematological data. However comparison of MCV and MCH between patients with two functional alpha globin genes and patients who had one defected alpha globin gene, showed noticeable decreases (P value<0.001). Therefore MCV and MCH can be useful for prediction and distinction of the genotype of α-thal patients. Limited phenotype/genotype studies of α-thal patients showed similar results (20-21).
Although hematologic features of thalassemic newborns did not show similar reduction of MCV and MCH, but there was similar difference in MCV and MCH of subjects with two functional alpha globin genes and patients who had one defected alpha globin gene (22). We found no significant differences between genotypes and other hematological indices such as Hb level, MCHC and RBC counts in this study.
In conclusion, analysis of MCV and MCH can be efficiently helpful for the selection of the appropriate molecular tests to determine the genotype of alpha thalassemia carriers.
Conflict of interest: none declared.
References
- 1.Ribeiro DM, Sonati MF. Regulation of human alpha-globin gene expression and alpha-thalassemia. Genet Mol Res. 2008;7:1045–53. doi: 10.4238/vol7-4gmr472. [DOI] [PubMed] [Google Scholar]
- 2.Bernini LF, Harteveld CL. Alpha-thalassaemia. Baillieres Clin Haematol. 1998;11:53–90. doi: 10.1016/s0950-3536(98)80070-x. [DOI] [PubMed] [Google Scholar]
- 3.Turnpenny T, Sian E, editors. Emery's elements of medical genetics. Philadelphia: Elsevier Inc; 2012. Hemoglobin and the hemoglobinopathies; pp. 160–1. [Google Scholar]
- 4.Higgs DR, Gibbons RJ. The molecular basis of alpha-thalassemia: a model for understanding human molecular genetics. Hematol Oncol Clin North Am. 2010;24:1033–54. doi: 10.1016/j.hoc.2010.08.005. [DOI] [PubMed] [Google Scholar]
- 5.Waye JS, Chui DH. The alpha-globin gene cluster: genetics and disorders. Clin Invest Med. 2001;24:103–9. [PubMed] [Google Scholar]
- 6.Patrinos GP, Antonarakis SE. Human hemoglobin. In: Speicher MR, Motulsky AG, Antonarakis SE, editors. Vogel and motulsky’s human genetics. Berlin: Springer Inc; 2010. pp. 383–4. [Google Scholar]
- 7.Fucharoen S, Viprakasit V. Hb H disease: clinical course and disease modifiers. Hematology Am Soc Hematol Educ Program. 2009:26–34. doi: 10.1182/asheducation-2009.1.26. [DOI] [PubMed] [Google Scholar]
- 8.Langlois S, Ford JC, Chitayat D, et al. Carrier screening for thalassemia and hemoglobinopathies in Canada. J Obstet Gynaecol Can. 2008;30:950–71. doi: 10.1016/S1701-2163(16)32975-9. [DOI] [PubMed] [Google Scholar]
- 9.Singer ST. Variable clinical phenotypes of alpha-thalassemia syndromes. Scientific World Journal. 2009;9:615–25. doi: 10.1100/tsw.2009.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Harteveld CL, Higgs DR. Alpha-thalassaemia. Orphanet J Rare Dis. 2010;5 doi: 10.1186/1750-1172-5-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Higgs DR, Weatherall DJ. The Alpha Thalassaemias. Cell Mol Life Sci. 2009;66:1154–62. doi: 10.1007/s00018-008-8529-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Hadavi V, Taromchi AH, Malekpoui M, et al. Elucidating the spectrum of alpha-thalassemia mutations in Iran. Haematol-Hematol J. 2007;92:992–3. doi: 10.3324/haematol.10658. [DOI] [PubMed] [Google Scholar]
- 13.Old JM. Screening and genetic diagnosis of haemoglobin disorders. Blood Rev. 2003;17:43–53. doi: 10.1016/s0268-960x(02)00061-9. [DOI] [PubMed] [Google Scholar]
- 14.Akhavan-Niaki H, Pourtaghi M, Firouzjahi AR, et al. Prevalence of common point mutations of alpha globin gene in Babol, Iran (2005-09) J Gorgan Uni Med Sci. 2012;14:75–81. [Google Scholar]
- 15.Akhavan-Niaki H, Banihashemi A, Mostafazadeh A, et al. Simultaneous detection of Hb constant spring (alpha142, TAA>CAA, alpha2) and the alpha2 IVS-I donor site (-TGAGG) deletion by a simple polymerase chain reaction-based method in Iran. Hemoglobin. 2012;36:124–30. doi: 10.3109/03630269.2012.657728. [DOI] [PubMed] [Google Scholar]
- 16.Abolghasemi H, Amid A, Zeinali S, et al. Thalassemia in Iran: epidemiology, prevention, and management. J Pediatr Hematol Oncol. 2007;29:233–8. doi: 10.1097/MPH.0b013e3180437e02. [DOI] [PubMed] [Google Scholar]
- 17.Vichinsky E. Complexity of alpha thalassemia: growing health problem with new approaches to screening, diagnosis, and therapy. Ann N Y Acad Sci. 2010;1202:180–7. doi: 10.1111/j.1749-6632.2010.05572.x. [DOI] [PubMed] [Google Scholar]
- 18.Haddow JE. Couple screening to avoid thalassemia: successful in Iran and instructive for us. J Med Screen. 2005;12:55–6. doi: 10.1258/0969141053908375. [DOI] [PubMed] [Google Scholar]
- 19.Samavat A, Modell B. Iranian national thalassaemia screening programme. BMJ. 2004;329:1134–7. doi: 10.1136/bmj.329.7475.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Lin HJ, Shih MC, Peng CT, et al. Hematological features and molecular lesions of hemoglobin gene disorders in Taiwanese patients. Int J Lab Hematol. 2010;32:1–7. doi: 10.1111/j.1751-553X.2008.01095.x. [DOI] [PubMed] [Google Scholar]
- 21.Galanello R, Cao A. Gene test review. Alpha-thalassemia.Genet Med. 2011;13:83–8. doi: 10.1097/GIM.0b013e3181fcb468. [DOI] [PubMed] [Google Scholar]
- 22.Tritipsombut J, Sanchaisuriya K, Fucharoen S, et al. Hemoglobin profiles and hematologic features of thalassemic newborns: application to screening of alpha-thalassemia 1 and hemoglobin E. Arch Pathol Lab Med. 2008;132:1739–45. doi: 10.5858/132.11.1739. [DOI] [PubMed] [Google Scholar]