alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

Wenning, M.R.S.C.; Kimura, E.M.; Costa, F.F.; Saad, S.T.O.; Gervásio, S.; de Jorge, S.B.; Borges, E.; Silva, N.M.; Sonati, M.F.

doi:10.1590/S0100-879X2000000900008

Abstract

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha)20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, <FONT FACE="Symbol">aa</FONT>NcoI, alphaIcalpha and alphaTSaudialpha) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha)20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(<FONT FACE="Symbol">aa</FONT>)T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (alpha47Asp->His) associated with the -alpha3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (alpha53Ala->Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys) associated with the alpha+-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (alpha74Asp->Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (alpha7Lys->Glu), 1 (Caucasian) was heterozygous for Hb Westmead (alpha122His->Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

alpha-globin genes; alpha-globin structural variants; alpha-thalassemia; hemoglobin H; Hb H disease; hemoglobin variants; hemoglobinopathies

Braz J Med Biol Res, September 2000, Volume 33(9) 1041-1045

a-Globin genes: thalassemic and structural alterations in a Brazilian population

M.R.S.C. Wenning¹, E.M. Kimura¹, F.F. Costa², S.T.O. Saad², S. Gervásio¹, S.B. de Jorge¹, E. Borges¹, N.M. Silva¹ and M.F. Sonati¹

Departamentos de ¹Patologia 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

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with a-chain structural alterations were studied to identify the a-globin gene mutations present in the population of Southeast Brazil. The -a^3.7, --^MED and -(a)^20.5 deletions were investigated by PCR, whereas non-deletional a-thalassemia (a^Hpha, a^NcoIa, aa^NcoI, a^Ica and a^TSaudia) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(a)^20.5/-a^3.7genotype, one had the --^MED/-a^3.7 genotype, one had the --^MED/a^Hpha genotype and three showed interaction of the -a^3.7 deletion with an unusual, unidentified form of non-deletional a-thalassemia [-a^3.7/(aa)^T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (a47Asp®His) associated with the -a^3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (a53Ala®Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (a78Asn®Lys) associated with the a⁺-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (a74Asp®Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (a7Lys®Glu), 1 (Caucasian) was heterozygous for Hb Westmead (a122His®Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, a26Ala®Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

Key words: a-globin genes, a-globin structural variants, a-thalassemia, hemoglobin H, Hb H disease, hemoglobin variants, hemoglobinopathies

Introduction

The hemoglobinopathies are a heterogeneous group of genetic disorders caused by mutations affecting the globin-chain genes. Generically, these mutations can be classified as structural alterations which result in the production of abnormal proteins, as alterations in synthesis which modify the normal a/ß globin chain ratio (thalassemias), or as persistent production of fetal hemoglobin (Hb) during adult life (hereditary persistence of fetal Hb) (1,2). The hemoglobinopathies represent a public health problem, particularly in the Mediterranean area, in the Middle East and in parts of India, Africa and Southeast Asia (3,4).

The high degree of racial admixture among native Indians and African and European descendants in the Brazilian population has produced elevated frequencies of Hb alterations, which reflect the diversity of racial origins in each region of the country (5). Clinically, Hb S, Hb C and ß-thalassemia are the most important (6), although a⁺-thalassemia (-a^3.7 deletion) is the most frequent alteration, occurring in 20-25% of the Black population (7). Although sporadic cases of Hb H disease and a-chain structural variants have been found (6,8), the a-globin genes have not been systematically investigated. In this study, seven unrelated subjects with Hb H disease and 27 individuals with structural a-globin alterations were investigated in order to identify the mutations present in the population of southeastern Brazil. The Hb H disease patients and the carriers of abnormal Hbs who had hematological alterations were initially screened at the outpatient clinics of the UNICAMP University Hospital and then referred to the Clinical Pathology Laboratory for investigation and diagnosis. Non-symptomatic carriers were detected in a screening program carried out in the same laboratory.

Material and Methods

Peripheral blood samples were collected into Vacutainers (Becton-Dickinson, Corkeysville, MD, USA) with EDTA as anticoagulant and hematological data were obtained with an automated cell counter (Cell Dyn 3500, Abbott Laboratories, Chicago, IL, USA).

Hb analyses were carried out by electrophoresis on cellulose acetate strips at pH 8.9, in agar gels at pH 6.0 (1), and by globin chain electrophoresis on acrylamide gels at acid pH (9). Hb A₂ was measured spectrophotometrically after elution from cellulose acetate strips (1) and Hb F was determined by alkali denaturation (10). The stability of each variant was checked by the n-butanol, isopropanol and heat tests (11). Heinz bodies were investigated by incubation with methyl violet and Hb H was demonstrated by incubation with brilliant cresyl blue (11).

DNA was isolated from peripheral blood leukocytes by organic extraction. Direct sequencing was performed with the Sequenase kit version 2.0 (United States Biochemical Corporation, Cleveland, OH, USA), after selective amplification of the a-globin genes by the polymerase chain reaction (PCR) (12) and single strand separation with magnetic beads (Dynal Inc., Oslo, Norway). Whenever possible, the mutations were confirmed by sequencing the opposite strand and by familial studies and restriction enzyme analyses.

The most common deletions causing a-thalassemia (-a^3.7, -a^4.2, --^MED, -(a)^20.5, --^SEA) were screened by PCR (13-15). The five most frequent non-deletional mutations causing a-thalassemia (a^Hpha, a^NcoIa, aa^NcoI, a^Ica and a^TSaudia) were investigated with the restriction enzymes HphI, NcoI and MseI, respectively (3,16,17) and by specific nested PCR (a^TSaudi) (18).

Among the seven Hb H disease patients, all of Italian descent, four had the following genotypes: -(a)^20.5/-a^3.7 (two cases), --^MED/ -a^3.7 and --^MED/a^Hpha. The other three had an unusual unidentified form of a-thalassemia which seemed to be non-deletional [-a^3.7/(aa)^T] since both genes were present. No mutation was detected following sequencing from the promoter region to the poly A signal. These results are shown in Table 1.

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Among the 27 individuals with structural alterations, 15 (Caucasians of Italian descent) had Hb Hasharon (a47Asp®His), associated with the -a^3.7 deletion (-a^Hasharon): one was homozygous (-a^Hasharon/-a^Hasharon) and 14 were heterozygous (-a^Hasharon/aa). One of these 14 individuals had a concomitant new ß-globin variant, Hb Rio Claro (ß34Val® Met) (19). Four adults (Caucasians of Italian descent) were heterozygous for Hb J-Rovigo (a53Ala®Asp) and one of them was also a ß-thalassemia carrier. Hb Stanleyville-II (a78 Asn®Lys) was found in four individuals (3 Blacks and 1 Caucasian, all heterozygous), always in association with the -a^3.7deletion (-a^Stanleyville). One Black infant from the north of Brazil had Hb G-Pest (a74Asp®Asn), a very rare Hb described initially in a Hungarian family (20). One boy of Portuguese descent had Hb Kurosaki (a7Lys®Glu), another very rare Hb described only once in a Japanese woman (21). Hb Westmead (a122 His®Gln), a relatively common silent variant in southern China (22,23), was found in a Caucasian adult of Italian descent. Hb Campinas (a26Ala® Val) was identified in a Caucasian boy whose ethnic origin was unknown. This electrophoretically silent variant, not described previously, results from a base substitution at the 26th codon of the a₂ gene (GCG®GTG) (24). These structural alterations are summarized in Table 2.

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Results

The thalassemic mutations found in four of the seven Hb H disease patients (-a^3.7, --^MED, -(a)^20.5 and a^Hpha) are the most frequent in Mediterranean populations: the first three are common deletions which remove 3.7-, 18- and 20.5-kb fragments of DNA from the a-globin gene cluster, respectively, and the latter is a common non-deletional form which removes five nucleotides from the consensus sequence of the splicing donor site of IVS-I (3). All of the Hb H disease patients were of Italian descent. In contrast, the remaining three patients showed the association of the -a^3.7 deletion with an unusual form of a-thalassemia which leaves both a-genes intact, but without expression. This situation may result from an alteration in the a-major regulatory element (a-MRE or HS-40), a major positive regulatory region located 40 kb upstream of the x₂-globin gene cap site (25). A few large deletions are known to remove this locus control region and thereby silence the a-globin genes, which remain structurally intact (3).

Among the structural alterations, Hb Hasharon, the most frequent change, and Hb J-Rovigo, are of Italian origin, whereas Hb Stanleyville-II is of African origin and Hb G-Pest was first described in a Hungarian family in 1972 (20). The last one was found here for the first time in the Brazilian population. Hb Kurosaki was initially described in a 70-year-old Japanese woman (21), being the case described here the second reported in the world and the first one in Brazil. Hb Westmead was discovered in a Chinese female in 1980 (23) and is common in Guangxi, a province in southern China (22). In the case described here, the first in Brazil, the carrier was of Italian descent. Hbs G-Pest, Kurosaki and Westmead were detected in a screening program, but in carriers with ethnic origins different from those of the original descriptions, suggesting de novo mutations. Hb Campinas was a novel silent variant encountered in a Caucasian boy of unknown descent (24).

Clinically and hematologically, Hbs J-Rovigo, G-Pest, Kurosaki, Westmead and Campinas caused no abnormalities, since the carriers were asymptomatic. Hb Hasharon and Hb Stanleyville-II carriers had mild microcytic and hypochromic red blood cells, probably because of the association with a-thalassemia.

The present study on 34 persons represents the most extensive analysis to-date of individuals with a-globin gene mutations in Brazil. The results confirmed the strong Italian and African influences in the region of Brazil examined, and reflected the intense immigration that took place in the last century and at the beginning of the twentieth century.

Discussion

Acknowledgments

We thank Dr. Stephen Hyslop, Department of Pharmacology, School of Medical Sciences, UNICAMP, for reviewing the language of the manuscript.

Address for correspondence: M.F. Sonati, Departamento de Patologia Clínica, FCM, UNICAMP, Caixa Postal 6111, 13083-970 Campinas, SP, Brasil. Fax: +55-19-289-3273. E-mail: sonati@fcm.unicamp.br

Research supported by FAPESP (No. 96/1118-8 and No. 97/11725-1) and CNPq (No. 520059/95-6). Received December 17, 1999. Accepted July 6, 2000.

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. 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.
4. Working Party of the General Haematology Task Force of the British Committee for Standards in Haematology (1998). Guideline. The laboratory diagnosis of haemoglobinopathies. British Journal of Haematology, 101: 783-792.
5. Zago MA & Costa FF (1985). Hereditary haemoglobin disorders in Brazil. Transaction of the Royal Society of Tropical Medicine and Hygiene, 79: 385-388.
6. Sonati MF, Kimura EM, Grotto HZW, Gervásio SA & Costa FF (1996). Hereditary hemoglobinopathies in a population from southeast Brazil. Hemoglobin, 20: 175-179.
7. Sonati MF, Farah SB, Ramalho AS & Costa FF (1991). High prevalence of a-thalassemia in a Black population of Brazil. Hemoglobin, 15: 309-311.
8. Zago MA, Costa FF & Bottura C (1984). Hemoglobin H disease in three Brazilian families. Revista Brasileira de Genética, VII: 137-147.
9. Alter BP, Goff SC, Efremov GD, Gravely ME & Huisman THJ (1980). Globin chain electrophoresis: a new approach to the determination of the g^Gg^A ratio of globin synthesis. British Journal of Haematology, 44: 527-534.
10. Pembrey ME, MacWade P & Weatherall DJ (1972). Reliable routine estimation of small amounts of foetal haemoglobin by alkali denaturation. Journal of Clinical Pathology, 25: 738-740.
11. Dacie JV & Lewis SM (1995). Practical Haematology 8th edn. Churchill Livingstone, Edinburgh.
12. Dodé C, Rochette J & Krishnamoorthy R (1990). Locus assignment of human a-mutation by selective amplification and direct sequencing. British Journal of Haematology, 76: 275-281.
13. Baysal E & Huisman THJ (1994). Detection of common deletional a-thalassemia-2 determinants by PCR. American Journal of Hematology, 46: 208-213.
14. 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.
15. 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.
16. Traeger-Synodinos J, Kanavakis E, Tzetis M, Kattamis A & Kattamis C (1993). Characterization of nondeletional a thalassemia mutations in the Greek population. American Journal of Hematology, 44: 162-167.
17. Makonkawkeyoon L, Sanguansermsri T, Asato T, Nakashima Y & Takei H (1993). Rapid detection of chain termination mutation in the a₂ globin gene. Blood, 82: 3503-3504.
18. Hall GW, Thein SL, Newland CA, Chisholm JTS, Kanavakis E, Kattamis C & Higgs DR (1983). A base substitution (TŽC) in codon 29 of the a₂-globin gene causes a thalassemia. British Journal of Haematology, 85: 546-552.
19. Grignoli CRE, Wenning MRSC, Sonati MF, Kimura EM, Arruda VR, Saad STO & Costa FF (1999). Hb Rio Claro ß34 (B16) ValŽMet: a novel electrophoretically silent variant found in association with Hb Hasharon a47 (CE5) AspŽHis and a thalassemia 2 (-a^3.7). Hemoglobin, 23: 177-182.
20. Brimhall B, Durest M, Hollan SR, Stenzel P, Szelčnyl J & Jones RT (1974). Structural characterization of hemoglobin J-Buda [a61(E10) LysŽAsn] and G-Pest [a74(EF3) AspŽAsn]. Biochimica et Biophysica Acta, 336: 344-360.
21. Harano T, Harano K, Imai K, Murakami T & Matsubara H (1995). Hb Kurosaki [a7(A5) LysŽGlu]: a new a chain variant found in a Japanese woman. Hemoglobin, 19: 197-201.
22. Jiang NH, Liang S, Wen XJ, Liang R, Su C & Tang Z (1991). Hb Westmead: an a2-globin gene mutation detected by polymerase chain reaction and StuI cleavage. Hemoglobin, 15: 291-295.
23. Gu Y-C, Gu L-H, Wilson JB, Cepreganova B, Ramachandran M, Walker ELD & Huisman THJ (1991). Hb Westmead [a122(H5) HisŽGln], Hb E [ß26(B8) GluŽLys], and a-thalassemia-2 (3.7 kb deletion) in a Laotian family. Hemoglobin, 15: 297-302.
24. Wenning MRSC, Silva NM, Jorge SB, Kimura EM, Costa FF, Torsoni MA, Ogo SH & Sonati MF (2000). Hb Campinas [a26(B7)AlaŽVal]: a novel, electrophoretically silent, variant. Hemoglobin, 24: 143-148.
25. Higgs DR, Wood WG, Jarman AP, Sharpe J, Pretorius IM & Ayyub H (1990). A major positive regulatory region located far upstream of the human a-globin gene locus. Genes and Development, 4: 1588-1601.

Correspondence and Footnotes

Publication Dates

Publication in this collection
01 Sept 2000
Date of issue
Sept 2000

History

Received
17 Dec 1999
Accepted
06 July 2000

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. 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.

[4] 4. Working Party of the General Haematology Task Force of the British Committee for Standards in Haematology (1998). Guideline. The laboratory diagnosis of haemoglobinopathies. British Journal of Haematology, 101: 783-792.

[5] 5. Zago MA & Costa FF (1985). Hereditary haemoglobin disorders in Brazil. Transaction of the Royal Society of Tropical Medicine and Hygiene, 79: 385-388.

[6] 6. Sonati MF, Kimura EM, Grotto HZW, Gervásio SA & Costa FF (1996). Hereditary hemoglobinopathies in a population from southeast Brazil. Hemoglobin, 20: 175-179.

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

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

[9] 9. Alter BP, Goff SC, Efremov GD, Gravely ME & Huisman THJ (1980). Globin chain electrophoresis: a new approach to the determination of the g^Gg^A ratio of globin synthesis. British Journal of Haematology, 44: 527-534.

[10] 10. Pembrey ME, MacWade P & Weatherall DJ (1972). Reliable routine estimation of small amounts of foetal haemoglobin by alkali denaturation. Journal of Clinical Pathology, 25: 738-740.

[11] 11. Dacie JV & Lewis SM (1995). Practical Haematology 8th edn. Churchill Livingstone, Edinburgh.

[12] 12. Dodé C, Rochette J & Krishnamoorthy R (1990). Locus assignment of human a-mutation by selective amplification and direct sequencing. British Journal of Haematology, 76: 275-281.

[13] 13. Baysal E & Huisman THJ (1994). Detection of common deletional a-thalassemia-2 determinants by PCR. American Journal of Hematology, 46: 208-213.

[14] 14. 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.

[15] 15. 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.

[16] 16. Traeger-Synodinos J, Kanavakis E, Tzetis M, Kattamis A & Kattamis C (1993). Characterization of nondeletional a thalassemia mutations in the Greek population. American Journal of Hematology, 44: 162-167.

[17] 17. Makonkawkeyoon L, Sanguansermsri T, Asato T, Nakashima Y & Takei H (1993). Rapid detection of chain termination mutation in the a₂ globin gene. Blood, 82: 3503-3504.

[18] 18. Hall GW, Thein SL, Newland CA, Chisholm JTS, Kanavakis E, Kattamis C & Higgs DR (1983). A base substitution (TŽC) in codon 29 of the a₂-globin gene causes a thalassemia. British Journal of Haematology, 85: 546-552.

[19] 19. Grignoli CRE, Wenning MRSC, Sonati MF, Kimura EM, Arruda VR, Saad STO & Costa FF (1999). Hb Rio Claro ß34 (B16) ValŽMet: a novel electrophoretically silent variant found in association with Hb Hasharon a47 (CE5) AspŽHis and a thalassemia 2 (-a^3.7). Hemoglobin, 23: 177-182.

[20] 20. Brimhall B, Durest M, Hollan SR, Stenzel P, Szelčnyl J & Jones RT (1974). Structural characterization of hemoglobin J-Buda [a61(E10) LysŽAsn] and G-Pest [a74(EF3) AspŽAsn]. Biochimica et Biophysica Acta, 336: 344-360.

[21] 21. Harano T, Harano K, Imai K, Murakami T & Matsubara H (1995). Hb Kurosaki [a7(A5) LysŽGlu]: a new a chain variant found in a Japanese woman. Hemoglobin, 19: 197-201.

[22] 22. Jiang NH, Liang S, Wen XJ, Liang R, Su C & Tang Z (1991). Hb Westmead: an a2-globin gene mutation detected by polymerase chain reaction and StuI cleavage. Hemoglobin, 15: 291-295.

[23] 23. Gu Y-C, Gu L-H, Wilson JB, Cepreganova B, Ramachandran M, Walker ELD & Huisman THJ (1991). Hb Westmead [a122(H5) HisŽGln], Hb E [ß26(B8) GluŽLys], and a-thalassemia-2 (3.7 kb deletion) in a Laotian family. Hemoglobin, 15: 297-302.

[24] 24. Wenning MRSC, Silva NM, Jorge SB, Kimura EM, Costa FF, Torsoni MA, Ogo SH & Sonati MF (2000). Hb Campinas [a26(B7)AlaŽVal]: a novel, electrophoretically silent, variant. Hemoglobin, 24: 143-148.

[25] 25. Higgs DR, Wood WG, Jarman AP, Sharpe J, Pretorius IM & Ayyub H (1990). A major positive regulatory region located far upstream of the human a-globin gene locus. Genes and Development, 4: 1588-1601.