High prevalence of alpha-thalassemia among individuals with microcytosis and hypochromia without anemia

Borges, E.; Wenning, M.R.S.C.; Kimura, E.M.; Gervásio, S.A.; Costa, F.F.; Sonati, M.F.

doi:10.1590/S0100-879X2001000600009

Abstract

In order to determine the contribution of alpha-thalassemia to microcytosis and hypochromia, 339 adult outpatients seen at Unicamp University Hospital (with the exception of the Clinical Hematology outpatient clinics), who showed normal hemoglobin (Hb) levels and reduced mean corpuscular volume and mean corpuscular hemoglobin, were analyzed. Ninety-eight were Blacks (28.9%) and 241 were Caucasians (71.1%). In all cases, Hb A2 and F levels were either normal or low. The most common deletional and nondeletional forms of alpha-thalassemia [-alpha3.7, -alpha4.2, --MED, -(alpha)20.5, alphaHphIalpha, alphaNcoIalpha, aaNcoI and alphaTSAUDI] were investigated by PCR and restriction enzyme analyses. A total of 169 individuals (49.9%) presented alpha-thalassemia: 145 (42.8%) were heterozygous for the -alpha3.7 deletion (-alpha3.7/aa) and 18 (5.3%) homozygous (-alpha3.7/-alpha3.7), 5 (1.5%) were heterozygous for the nondeletional form alphaHphIalpha (alphaHphIalpha/aa), and 1 (0.3%) was a --MED carrier (--MED/aa). Among the Blacks, 56 (57.1%) showed the -alpha3.7/aa genotype, whereas 12 (12.2%) were -alpha3.7/-alpha3.7 and 1 (1.0%) was an alphaHphIalpha carrier; among the Caucasians, 89 (36.9%) were -alpha3.7/aa, 6 (2.5%) had the -alpha3.7/-alpha3.7 genotype, 4 (1.7%) presented the nondeletional form (alphaHphIalpha/aa), and 1 (0.4%) was a --MED carrier. These results demonstrate that alpha-thalassemia, mainly through the -alpha3.7 deletion, is an important cause of microcytosis and hypochromia in individuals without anemia. These data are of clinical relevance since these hematological alterations are often interpreted as indicators of iron deficiency.

alpha-thalassemia; microcytosis; hypochromia; hemoglobinopathies; brazilian population

Braz J Med Biol Res, June 2001, Volume 34(6) 759-762

High prevalence of a -thalassemia among individuals with microcytosis and hypochromia without anemia

E. Borges¹, M.R.S.C. Wenning¹, E.M. Kimura¹, S.A. Gervásio¹, F.F. Costa² and M.F. Sonati¹

Departamentos de¹Patologia Clínica, and ²Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brasil

References

Correspondence and Footnotes ^{Correspondence and Footnotes} Correspondence and Footnotes

Abstract

In order to determine the contribution of a-thalassemia to microcytosis and hypochromia, 339 adult outpatients seen at Unicamp University Hospital (with the exception of the Clinical Hematology outpatient clinics), who showed normal hemoglobin (Hb) levels and reduced mean corpuscular volume and mean corpuscular hemoglobin, were analyzed. Ninety-eight were Blacks (28.9%) and 241 were Caucasians (71.1%). In all cases, Hb A₂ and F levels were either normal or low. The most common deletional and nondeletional forms of a-thalassemia [-a^3.7, -a^4.2, --^MED, -(a)^20.5, a^HphIa, a^NcoIa, aa^NcoI and a^TSAUDI] were investigated by PCR and restriction enzyme analyses. A total of 169 individuals (49.9%) presented a-thalassemia: 145 (42.8%) were heterozygous for the -a^3.7 deletion (-a^3.7/aa) and 18 (5.3%) homozygous (-a^3.7/-a^3.7), 5 (1.5%) were heterozygous for the nondeletional form a^HphIa (a^HphIa/aa), and 1 (0.3%) was a --^MED carrier (--^MED/aa). Among the Blacks, 56 (57.1%) showed the -a^3.7/aa genotype, whereas 12 (12.2%) were -a^3.7/-a^3.7 and 1 (1.0%) was an a^HphIa carrier; among the Caucasians, 89 (36.9%) were -a^3.7/aa, 6 (2.5%) had the -a^3.7/-a^3.7 genotype, 4 (1.7%) presented the nondeletional form (a^HphIa/aa), and 1 (0.4%) was a --^MED carrier. These results demonstrate that a-thalassemia, mainly through the -a^3.7 deletion, is an important cause of microcytosis and hypochromia in individuals without anemia. These data are of clinical relevance since these hematological alterations are often interpreted as indicators of iron deficiency.

Key words: a-thalassemia, microcytosis, hypochromia, hemoglobinopathies, Brazilian population

Introduction

Microcytosis and hypochromia result from deficient hemoglobin (Hb) synthesis in erythroid cells, causing a reduction in both mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) of red blood cells. Without the increase in Hb A₂ levels, these hematological alterations may be due to a-thalassemia, iron deficiency or, occasionally, chronic disease anemia (1,2).

a-Thalassemia is the most common genetic disorder of Hb synthesis in the world, with gene frequencies varying between 1% and 98% throughout the tropics and subtropics, where Plasmodium falciparum is or has been endemic, or in populations which received people from these areas through immigration (3-5). It results from an imbalance in a-globin chain production, which can be reduced (a⁺-thalassemia) or completely abolished (a⁰-thalassemia). Most commonly a-thalassemia results from deletion of one (-a) or both (--) of the duplicated a genes (aa) on chromosome 16p13.3. Less frequently, it is caused by small deletions or point mutations (so-called nondeletional a-thalassemia) involving the predominantly expressed a₂ gene (a^Ta) or rarely the a₁ gene (aa^T), or yet by deletions outside the a cluster which leave the structural genes intact but without expression (3,6).

The a-thalassemia phenotypes range from mild microcytic hypochromic anemia to a hemolytic anemia of variable severity characterized by the presence of Hb H in the case of loss of three functional a genes, or to the hydrops fetalis syndrome, characterized by severe intrauterine anemia and fetal or perinatal death due to the loss of all four a globin genes (1-3). The most common cause of a-thalassemia is a deletion of 3.7 kb of DNA originated by homologous recombination between misaligned chromosomes, which affects both a genes in cis and results in a unique hybrid gene (a₂a₁) (-a^3.7 deletion). The hematological alterations caused by this deletion, also known as rightward a⁺-thalassemia, can be very mild, if not silent (7-9). It is most prevalent in the African and Mediterranean regions. Other relatively frequent causes of a-thalassemia are the -a^4.2 deletion (leftward a⁺-thalassemia) found in Asian and Mediterranean populations, the --^MED and -(a)^20.5 deletions, common causes of a⁰-thalassemia in the Mediterranean region, and the --^SEA deletion, found with high frequency in Southeast Asia (7). Among the most common nondeletional forms, a^HphIa is a pentanucleotide deletion in the splice donor site of IVS-I which abolishes a HphI site in the a₂ gene; a^NcoIa and the aa^NcoI are caused by base substitutions in the translational initiation codon ATG in the a₂ or in the a₁ gene, respectively, which presumably completely abolish translation and can be recognized by the loss of the NcoI site present in the initiation codon, and a third mutation, a^TSAUDIa, caused by a base substitution in the highly conserved polyadenylation signal sequence AATAAA, which must prevent endonucleolytic cleavage and poly A addition to the 3' end of mRNAs (7). They are all encountered in Mediterranean populations. In Brazil, the -a^3.7 deletion has been frequently found in the Black population (10,11), and the --^MED and -(a)^20.5 deletions have sporadically been described (12,13). One family with the nondeletional form a^HphIa has been recently reported (13).

Individuals with microcytosis and hypochromia, without anemia, have been detected in clinical laboratories. In order to determine the contribution of a-thalassemia to such cases, we analyzed 339 adult individuals, followed as outpatients at Unicamp University Hospital (with the exception of the Clinical Hematology outpatient clinics), who showed normal Hb levels together with reduced MCV and MCH and normal or decreased Hb A₂ (and F).

Material and Methods

Subjects

A total of 339 adult individuals (age >14 years), followed as outpatients at Unicamp University Hospital, Campinas, State of São Paulo, Southeast Brazil, with the exception of the Clinical Hematology outpatient clinics, were analyzed. The subjects presented normal Hb levels (Hb ³14 g/dl for men and ³12 g/dl for women) and reduced MCV (£80 fl) and MCH (£27 pg). Ninety-eight individuals were Blacks (28.9%), including Mulattoes and Negroes, and 241 were Caucasians (71.1%). In all cases, Hb A₂ and F levels were normal or decreased (<3.6 and <2.0%, respectively).

The patients studied here formally consented to be investigated for the presence of a-thalassemia.

Methods

Red blood cell indices were electronically determined (Cell Dyn 3500, Abbott Laboratories, Chicago, IL, USA), and Hb analyses were carried out according to Weatherall and Clegg (1).

DNA samples were obtained from peripheral blood leukocytes by organic extraction. With the exception of the --^SEA deletion, all the other deletional and nondeletional forms of a-thalassemia mentioned above were investigated by PCR-based methods. Rightward deletion (-a^3.7) was detected by the method of Dodé et al. (14); --^MED and -(a)^20.5 were investigated as described by Bowden et al. (15), and the -a^4.2 leftward deletion was screened by the method of Oron-Karni et al. (16). Nondeletional forms were investigated according to Hall et al. (17) using the corresponding restriction enzymes (HphI and NcoI) and a specific nested PCR for a^TSAUDI (15).

Serum ferritin levels were determined by an automated chemoluminescent immunoenzymatic method (Immulite, Diagnostic Products Co., Los Angeles, CA, USA) for all a-thalassemic cases to make sure that microcytosis and hypochromia were not due to concomitant iron deficiency.

Results

The results are summarized in Table 1. Among the 339 individuals studied, 169 (49.9%) presented a-thalassemia: 145 (42.8%) were heterozygous for the -a^3.7 deletion (-a^3.7/aa), 18 (5.3%) were homozygous (-a^3.7/-a^3.7), 5 (1.5%) showed the nondeletional form a^HphIa/aa, and 1 (0.3%) was a --^MED carrier (--^MED/aa). Among the 98 Blacks, 56 (57.1%) showed the -a^3.7/aa genotype, while 12 (12.2%) were -a^3.7/-a^3.7 and 1 (1.0%) was a^HphIa/aa; among the Caucasians, 89 (36.9%) were -a^3.7/aa, 6 (2.5%) had the -a^3.7/-a^3.7genotype and 4 (1.7%) were a^HphIa/aa. The --^MED carrier belonged to this racial group (0.4%).

Thumbnail

The serum ferritin levels determined for the a-thalassemia cases were all above the lower normal limits (9 ng/ml for women and 19 ng/ml for men).

Discussion

The present results demonstrated that a-thalassemia, mainly through the -a^3.7 deletion, is an important cause of microcytosis and hypochromia in individuals without anemia, indicating that non-anemic Brazilian Blacks, with low MCV and MCH, have a 70.4% chance of carrying a-thalassemia, whereas this chance is of 41.5% among Brazilian Caucasians, i.e., still high. The -a^3.7 deletion is known to occur at significant frequencies in Black populations (7). Since in Brazil there is an elevated degree of miscegenation, it seems that even in the non-Black population its prevalence is also high.

The present data are of clinical relevance, since microcytosis and hypochromia are often interpreted as indicators of iron deficiency and patients may be mistreated with oral iron therapy (18). In about 50% of the cases analyzed here, the cause of these hematological alterations was a-thalassemia. It is possible that this proportion is still a little higher, because many silent mutations causing a-thalassemia and other not so common deletions may not have been detected.

Acknowledgments

We thank Dr. Helena Z.W. Grotto and Ms. Carmen A.C. Aguiar for helping us with the iron status determinations. We also thank the Statistics Committee, FCM, UNICAMP, especially Dr. Helymar C. Machado, for the statistical and computational analyses.

Address for correspondence: M.F. Sonati, Departamento de Patologia Clínica, FCM, UNICAMP, Caixa Postal 6111, 13083-970 Campinas, SP, Brasil. Fax: +55-19-3788-9434. E-mail: sonati@fcm.unicamp.br

Research supported by FAPESP (Nos. 97/11725-1 and 98/14532-2) and CNPq (No. 520059/95-6). Received August 8, 2000. Accepted April 10, 2001.

1. Weatherall DJ & Clegg JG (1981). The Thalassaemia Syndromes 3rd edn. Blackwell Scientific Publications, Oxford.
2. Bunn HF & Forget BG (1986). Hemoglobin: Molecular, Genetics and Clinical Aspects W.B. Saunders, Philadelphia.
3. Higgs DR, Vickers MA, Wilkie AOM, Pretorius IM, Jarman AP & Weatherall DJ (1989). A review of the molecular genetics of the human a-globin gene cluster. Blood, 73: 1081-1104.
4. Kazazian Jr H (1990). The thalassemia syndromes: molecular basis and prenatal diagnosis in 1990. Seminars in Hematology, 27: 209-228.
5. Harteveld KL, Losekoot M, Heister AJGAM, van der Wielen M, Giordano PC & Bernini LF (1997). a-Thalassaemia in the Netherlands: a heterogeneous spectrum of both deletions and point mutations. Human Genetics, 100: 465-471.
6. Higgs DR (1993). a-Thalassaemia. Baillieres Clinical Haematology, 6: 117-150.
7. Kattamis AC, Camaschella C, Sivera P, Surrey S & Fortina P (1996). Human a-thalassemia syndromes: detection of molecular defects. American Journal of Hematology, 53: 81-91.
8. Bianco I, Cappabianca MP, Foglietta E, Lerone M, Deidda G, Morlupi L, Grisanti P, Ponzini D, Rinaldi S & Graziani B (1997). Silent thalassemias: genotypes and phenotypes. Haematologica, 82: 269-280.
9. Galanello R, Sollaino C, Paglietti E, Barella S, Perra C, Doneddu I, Pirroni MG, Maccioni L & Cao A (1998). a-Thalassemia carrier identification by DNA analysis in the screening for thalassemia. American Journal of Hematology, 59: 273-278.
10. Sonati MF & Costa FF (1990). Hemoglobin Bart's in a Brazilian black population. Brazilian Journal of Medical and Biological Research, 23: 395-396.
11. Sonati MF, Farah SB, Ramalho AS & Costa FF (1991). High prevalence of a-thalassemia in a Black population of Brazil. Hemoglobin, 15: 309-311.
12. Zago MA, Costa FF & Bottura C (1984). Hemoglobin H disease in three Brazilian families. Revista Brasileira de Genética, VII: 137-147.
13. Wenning MRSC, Kimura EM, Costa FF, Saad STO, Gervásio SA, de Jorge SB, Borges E, Silva NM & Sonati MF (2000). a-Globin genes: thalassemic and structural alterations in a Brazilian population. Brazilian Journal of Medical and Biological Research, 33: 1041-1045.
14. Dodé C, Krishnamoorthy R, Lamb J & Rochette J (1993). Rapid analysis of -a^3.7 thalassaemia and aaa^{anti 3.7} triplication by enzymatic amplification analysis. British Journal of Haematology, 82: 105-111.
15. Bowden DK, Vickers MA & Higgs DR (1992). A PCR-based strategy to detect the common severe determinants of a thalassaemia. British Journal of Haematology, 81: 104-108.
16. Oron-Karni V, Filon D, Oppenheim A & Rund D (1998). Rapid detection of the common Mediterranean a-globin deletions/rearrangements using PCR. American Journal of Hematology, 58: 306-310.
17. Hall GW, Thein SL, Newland CA, Chisholm JTS, Kanavakis E, Kattamis C & Higgs DR (1993). A base substitution (T®C) in codon 29 of the a₂-globin gene causes a thalassemia. British Journal of Haematology, 85: 546-552.
18. Pearson HA, Ehrenkranz RA & Rinder HM (2000). Hemosiderosis in a normal child secondary to oral iron medication. Pediatrics, 105: 429-431.

Correspondence and Footnotes

Publication Dates

Publication in this collection
25 May 2001
Date of issue
June 2001

History

Received
08 Aug 2000
Accepted
10 Apr 2001

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] 1. Weatherall DJ & Clegg JG (1981). The Thalassaemia Syndromes 3rd edn. Blackwell Scientific Publications, Oxford.

[2] 2. Bunn HF & Forget BG (1986). Hemoglobin: Molecular, Genetics and Clinical Aspects W.B. Saunders, Philadelphia.

[3] 3. Higgs DR, Vickers MA, Wilkie AOM, Pretorius IM, Jarman AP & Weatherall DJ (1989). A review of the molecular genetics of the human a-globin gene cluster. Blood, 73: 1081-1104.

[4] 4. Kazazian Jr H (1990). The thalassemia syndromes: molecular basis and prenatal diagnosis in 1990. Seminars in Hematology, 27: 209-228.

[5] 5. Harteveld KL, Losekoot M, Heister AJGAM, van der Wielen M, Giordano PC & Bernini LF (1997). a-Thalassaemia in the Netherlands: a heterogeneous spectrum of both deletions and point mutations. Human Genetics, 100: 465-471.

[6] 6. Higgs DR (1993). a-Thalassaemia. Baillieres Clinical Haematology, 6: 117-150.

[7] 7. Kattamis AC, Camaschella C, Sivera P, Surrey S & Fortina P (1996). Human a-thalassemia syndromes: detection of molecular defects. American Journal of Hematology, 53: 81-91.

[8] 8. Bianco I, Cappabianca MP, Foglietta E, Lerone M, Deidda G, Morlupi L, Grisanti P, Ponzini D, Rinaldi S & Graziani B (1997). Silent thalassemias: genotypes and phenotypes. Haematologica, 82: 269-280.

[9] 9. Galanello R, Sollaino C, Paglietti E, Barella S, Perra C, Doneddu I, Pirroni MG, Maccioni L & Cao A (1998). a-Thalassemia carrier identification by DNA analysis in the screening for thalassemia. American Journal of Hematology, 59: 273-278.

[10] 10. Sonati MF & Costa FF (1990). Hemoglobin Bart's in a Brazilian black population. Brazilian Journal of Medical and Biological Research, 23: 395-396.

[11] 11. Sonati MF, Farah SB, Ramalho AS & Costa FF (1991). High prevalence of a-thalassemia in a Black population of Brazil. Hemoglobin, 15: 309-311.

[12] 12. Zago MA, Costa FF & Bottura C (1984). Hemoglobin H disease in three Brazilian families. Revista Brasileira de Genética, VII: 137-147.

[13] 13. Wenning MRSC, Kimura EM, Costa FF, Saad STO, Gervásio SA, de Jorge SB, Borges E, Silva NM & Sonati MF (2000). a-Globin genes: thalassemic and structural alterations in a Brazilian population. Brazilian Journal of Medical and Biological Research, 33: 1041-1045.

[14] 14. Dodé C, Krishnamoorthy R, Lamb J & Rochette J (1993). Rapid analysis of -a^3.7 thalassaemia and aaa^{anti 3.7} triplication by enzymatic amplification analysis. British Journal of Haematology, 82: 105-111.

[15] 15. Bowden DK, Vickers MA & Higgs DR (1992). A PCR-based strategy to detect the common severe determinants of a thalassaemia. British Journal of Haematology, 81: 104-108.

[16] 16. Oron-Karni V, Filon D, Oppenheim A & Rund D (1998). Rapid detection of the common Mediterranean a-globin deletions/rearrangements using PCR. American Journal of Hematology, 58: 306-310.

[17] 17. Hall GW, Thein SL, Newland CA, Chisholm JTS, Kanavakis E, Kattamis C & Higgs DR (1993). A base substitution (T®C) in codon 29 of the a₂-globin gene causes a thalassemia. British Journal of Haematology, 85: 546-552.

[18] 18. Pearson HA, Ehrenkranz RA & Rinder HM (2000). Hemosiderosis in a normal child secondary to oral iron medication. Pediatrics, 105: 429-431.