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Alpha chain hemoglobins with electrophoretic mobility similar to that of hemoglobin S in a newborn screening program

Abstract

OBJECTIVE: To characterize alpha-chain variant hemoglobins with electric mobility similar to that of hemoglobin S in a newborn screening program. METHODS: βS allele and alpha-thalassemia deletions were investigated in 14 children who had undefined hemoglobin at birth and an electrophoretic profile similar to that of hemoglobin S when they were six months old. Gene sequencing and restriction enzymes (DdeI, BsaJI, NlaIV, Bsu36I and TaqI) were used to identify hemoglobins. Clinical and hematological data were obtained from children who attended scheduled medical visits. RESULTS: The following alpha chain variants were found: seven children with hemoglobin Hasharon [alpha2 47(CE5) Asp>His, HbA2:c.142G>C], all associated with alpha-thalassemia, five with hemoglobin Ottawa [alpha1 15(A13) Gly>Arg, HBA1:c.46G>C], one with hemoglobin St Luke's [alpha1 95(G2) Pro>Arg, HBA1:c.287C>G] and another one with hemoglobin Etobicoke [alpha212 84(F5) Ser>Arg, HBA212:c.255C>G]. Two associations with hemoglobin S were found: one with hemoglobin Ottawa and one with hemoglobin St Luke's. The mutation underlying hemoglobin Etobicoke was located in a hybrid α212 allele in one child. There was no evidence of clinically relevant hemoglobins detected in this study. CONCLUSION: Apparently these are the first cases of hemoglobin Ottawa, St Luke's, Etobicoke and the α212 gene described in Brazil. The hemoglobins detected in this study may lead to false diagnosis of sickle cell trait or sickle cell disease when only isoelectric focusing is used in neonatal screening. Additional tests are necessary for the correct identification of hemoglobin variants.

Hemoglobins, abnormal; Anemia, Sickle Cell; Neonatal screening; alpha-thalassemia; Polymerase chain reaction


ORIGINAL ARTICLE

Alpha chain hemoglobins with electrophoretic mobility similar to that of hemoglobin S in a newborn screening program

Marcilene Rezende Silva; Shimene Mascarenhas Sendin; Isabela Couto de Oliveira Araujo; Fernanda Silva Pimentel; Marcos Borato Viana

Universidade Federal de Minas Gerais - UFMG, Belo Horizonte, MG, Brazil

Corresponding author Corresponding author: Marcos Borato Viana Departamento de Pediatria da UFMG Av. Alfredo Balena, 190 - Sala 267 30130-100 Belo Horizonte, Brazil Fax: 55-31-34099770 vianamb@gmail.com

ABSTRACT

OBJECTIVE: To characterize alpha-chain variant hemoglobins with electric mobility similar to that of hemoglobin S in a newborn screening program.

METHODS: βS allele and alpha-thalassemia deletions were investigated in 14 children who had undefined hemoglobin at birth and an electrophoretic profile similar to that of hemoglobin S when they were six months old. Gene sequencing and restriction enzymes (DdeI, BsaJI, NlaIV, Bsu36I and TaqI) were used to identify hemoglobins. Clinical and hematological data were obtained from children who attended scheduled medical visits.

RESULTS: The following alpha chain variants were found: seven children with hemoglobin Hasharon [alpha2 47(CE5) Asp>His, HbA2:c.142G>C], all associated with alpha-thalassemia, five with hemoglobin Ottawa [alpha1 15(A13) Gly>Arg, HBA1:c.46G>C], one with hemoglobin St Luke's [alpha1 95(G2) Pro>Arg, HBA1:c.287C>G] and another one with hemoglobin Etobicoke [alpha212 84(F5) Ser>Arg, HBA212:c.255C>G]. Two associations with hemoglobin S were found: one with hemoglobin Ottawa and one with hemoglobin St Luke's. The mutation underlying hemoglobin Etobicoke was located in a hybrid α212 allele in one child. There was no evidence of clinically relevant hemoglobins detected in this study.

CONCLUSION: Apparently these are the first cases of hemoglobin Ottawa, St Luke's, Etobicoke and the α212 gene described in Brazil. The hemoglobins detected in this study may lead to false diagnosis of sickle cell trait or sickle cell disease when only isoelectric focusing is used in neonatal screening. Additional tests are necessary for the correct identification of hemoglobin variants.

Keywords: Hemoglobins, abnormal; Anemia, Sickle Cell; Neonatal screening; alpha-thalassemia; Polymerase chain reaction

Introduction

Hemoglobinopathies are a heterogeneous group of diseases caused by a disruption in the normal pattern of expression of genes encoding the globin chains. They are classified fundamentally into two groups: a) structural variants, in which one or more amino acids are replaced in one of the polypeptide chains and b) thalassemias, in which an imbalance occurs in the production of one or more globin chains(1).

The hemoglobin variants result from substitutions of amino acids in the α, β, γ or δ chain tetramers of hemoglobins (Hbs) A, F and A2. The variants are caused by changes of DNA nucleotides, such as deletions, insertions or point mutations in one of the globin genes(2).

Hb S is the most frequent variant in humans. A point mutation in the b globin gene leads to the exchange of a single amino acid in the sixth position of the polypeptide chain (b6;glutamic acid → valine). Cells with Hb S undergo the sickling phenomenon, caused by low oxygen tension, acidosis and dehydration(3).

In the Globin Gene Server (http://globin.bx.psu.edu/cgi-bin/hbvar/counter) there were 1153 Hb variants registered as of 20 October 2012. The consequences of the structural changes upon the physicochemical properties of the molecule are dependent on the nature of the mutation and the place where it occurs. The consequences can be hemolytic anemia, when the change determines instability of the hemoglobin tetramer, altered oxygen transport if there is an increase or decrease in the affinity of Hb for oxygen or reduced synthesis of a globin chain, resulting in a form of thalassemia(4).

Carriers of "rare" Hb variants are most often asymptomatic. Associated with other hemoglobinopathies and thalassemias, these Hbs may result in a serious illness(5-7). Furthermore, there are dozens of Hb variants that can be mistaken for Hb S because they have similar isoelectric points, leading to a false diagnosis of sickle cell disease or trait if not properly confirmed(2, 6-10).

In this study some alpha chain Hb variants detected in a neonatal screening program over ten years, whose electrophoretic profile was similar to that of Hb S, were characterized. Mutations were identified by molecular methods and clinical implications are described.

Methods

The original sample from which the present study is derived consisted of 118 children who had indeterminate Hb results at birth and who, at six months of life, had an Hb profile identical or similar to that of Hb S. They were identified by the Neonatal Screening Program in Minas Gerais, Brazil from June 1998 to June 2008. Hb Stanleyville-II, in 96 children, has already been thoroughly described(11). The present report comprises 14 patients, including one with Hb Etobicoke for which further information is provided, in addition to what was previously reported(12). Beta chain variants are not part of the current study.

All families signed written consent forms when they returned for this cross-sectional study. The study was approved by the Research Ethics Committees of the institutions involved and conducted according to the Declaration of Helsinki (revision 2008).

Isoelectric focusing (IEF) and high performance liquid chromatography (HPLC) were performed in neonatal screening. At six months of life, only IEF was carried out as the confirmatory method.

Genomic DNA was used to detect the βS allele through allele-specific polymerase chain reaction (PCR)(13) or through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with the restriction endonuclease DdeI. Deletion al alpha thalassemia types 3.7and 4.2 were detected using multiplex gap-PCR(14).

The Hb Hasharon mutation was detected using PCR-RFLP with the restriction enzyme TaqI. The product of the multiplex gap-PCR, which had detected the presence of alpha-thalassemia, was used in the restriction enzyme reaction to identify the Hb Hasharon mutation. The primers of the multiplex gap-PCR were used in two different reactions, one that amplified LIS1 and HBA2 genes and another that amplified the hybrid gene -α3.7 and LIS1 gene (Table 1).

Gene sequencing was necessary to identify the Hb variants in the other children. HBA1 and HbA2 genes were amplified with primers as described previously(15). Exon 1 and part of exon 2 of the alpha genes were sequenced by nested PCR with the S1 and S18 primers(16). The rest of exon 2 and exon 3 were sequenced with the forward primer S3 (for α1 and α2)(16) and reverse primers 3.7R1 for α2 and 3.7R2 for α1 genes(15) (Table 1). Sequencing was performed on the ABI Prism 3130 (Applied Biosystems).

After sequencing, specific PCR-RFLP tests were designed for the easy diagnosis of the respective mutations. Methodological details on the diagnosis of Hb Stanleyville-II have been described previously(17).

To assess the clinical relevance of the detected Hbs and to provide information for the families about the results of the study, medical consultations were scheduled for the 14 children in this study; only nine attended. Blood counts for the child and family members were processed using Coulter T-890 automated blood analyzer. Hb electrophoresis in agarose gel was carried out in acid and alkaline media (SPIFE kits, Helena Laboratories, Beaumont, TX). The relative concentration of Hb variants was estimated by scanning the gels from electrophoresis in the alkaline medium.

Results

Of the 14 children in this study, seven carried Hb Hasharon [alpha2 47 (CE5) Asp> His, HbA2: c.142G> C] and had co-inherited alpha-thalassemia genes (six with genotypeαα/-a3.7;Hasharon and one with -a3.7/-a3.7;Hasharon). In the PCR-RFLP test, LIS1 fragment (2350 base pairs - bp) is cleaved into two fragments (1307 bp and 1043 bp) by the TaqI enzyme. Among the fragments generated by the restriction enzyme, one had 894bp; this was present only in those patients who had the mutation encoding Hb Hasharon (Figure 1A-B).



Five children had Hb Ottawa [alpha1 15 (A13) Gly> Arg, HBA1: c.46G> C], one Hb St. Luke's [alpha1 95 (G2) Pro> Arg, HBA1: c.287C> G] and another Hb Etobicoke [alpha212 84 (F5) Ser> Arg, HBA212: c.255C> G] as shown in the sequencings (Figure 2A-C). Figures 1C, 3A and 3B-C illustrate, respectively, the restriction reactions for Hb Etobicoke (endonucleaseBsu36I), Hb St. Luke's (NlaIV endonuclease) and Hb Ottawa(endonuclease BsaJI).


Of the Hbs identified in this study, two were associated with Hb S: Hb Ottawa and Hb St. Luke's (one case each). Hb Etobicoke was found in a child who had a hybrid α212 allele. Table 2 summarizes the alpha chain variants described in this study.

All these Hbs have isoelectric points similar to that of Hb S. In IEF at birth, all these children had an Hb fraction in the position of Hb S and another fraction between Hb S and Hb C, hence their initial classification was of indefinite hemoglobin profiles. The IEF was repeated at six months of life. The results of four children with Hb Hasharon and two with Hb Ottawa were communicated by the screening program as sickle cell trait (false positive Hb AS). In the other children, the results were communicated as heterozygous "rare" Hbs in the position of Hb S. On retesting for the present study, children with Hb Hasharon were interpreted as having an Hb fraction slightly slower (cathodically) than Hb S, less than 0.5 mm from the control position. For children with Hb Ottawa and Hb St Luke's, the Hb fraction moved slightly faster(anodically) than Hb S, also less than 0.5 mm from the control position. In the case of Hb Etobicoke, the position was exactly the same as Hb S.

In Hb electrophoresis on agarose gels (Figure 4), made as part of the medical consultation of these children, Hb Etobicoke and Hb Ottawa had Hb AS profiles in alkaline medium and Hb AA in acid pH. The child with Hb St. Luke'sco-inherited with Hb S had Hb AS profile in both pHs, because in alkaline pH Hb St. Luke's overlaps with Hb S and in acid medium with Hb A. Hb Hasharon migrates like Hb S both in alkaline and acid media.


All the seven children of the present study with Hb Hasharon had alpha thalassemia associated. The relative proportions of Hb Hasharon (Table 3), estimated in gel electrophoresis in alkaline medium was, as expected, a third for cases with heterozygous alpha thalassemia (αα/-α3.7;Hasharon) and almost 50% for the single homozygous child (-α3.7/-α3.7;Hasharon). No association with alpha-thalassemia was found in the children with Hb Etobicoke, Hb Ottawa and Hb St Luke's. The relative proportion of the Hb variant stood at around 25%. The values of mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) were generally lower than normal when there was co-inheritance of alpha thalassemia. They were normal for the child's age in the other cases. Total Hb concentrations were all above the minimum reference value for the children's ages.

African ancestry was recognized by all families of the present study. There were no reported clinical symptoms attributable to the presence of these Hb variants in any child.

Discussion

Among the children studied, six had been considered by the newborn screening program as having undetermined Hb at birth and being heterozygous for Hb S (sickle cell trait) at sixth months of life, both results using IEF. All were born in the first six years of the program. Resulting from acquired experience, children were considered in the subsequent years as carriers of "rare" Hbs to be further investigated, which materialized in the present study and in others(11, 12, 17). The comparison of the IEF gels at birth and at six months of life helps to correct the interpretation of data. The observation of an Hb fraction between the positions of Hb S and Hb C in the neonatal IEF, Variant corresponding to the dimer ag, draws attention to the possibility of alpha chain variants with electrophoretic mobility similar to that of Hb S. As the production of g chains decreases, children appear to be heterozygous for Hb S (aªbS) when they are six months old, when in fact, they have an alpha chain variant Hb (αVariantβª). Sickling and solubility tests are clearly negative in these cases, but require whole blood for testing. As this study demonstrates, the unequivocal identification of the Hb variant requires molecular methods.

There was no evidence of clinical relevance of the Hbs detected in this study, even when there was an association of Hb St. Luke's with Hb S in one child and of Hb Etobicoke with the α212 hybrid gene in another. According to the literature, Hb Hasharon would not cause clinical or hematological abnormalities unless co-inherited with alpha thalassemia(19, 20) as reported by other Brazilian studies(18, 19, 21, 22). Patients with Hb Ottawa and Hb St. Luke's were considered normal upon clinical and hematological evaluation(8, 23, 24). The mutation for Hb Etobicoke occurs at codon 84 (F5) of the HBA gene (Ser>Arg). Although the nature and molecular location of the amino acid substitution would indicate the possibility of abnormal properties of the Hb, no hematologic or clinical changes have been observed(25).

Due to the small number of cases of each Hb variant, the ethnic origin of these variants could not be determined in this study, in contrast to Hb Stanleyville-II, which is clearly linked to African ancestry(11). Although there were reports of African ancestors in all 14 families in this study, we cannot overlook other ethnic origins as Brazil is markedly genetically heterogeneous, resulting from the admixture of Amerindian, European and African populations(26). As examples, according to the Globin Gene Server(27), Hb Hasharon has been described in people with Italian and Jewish ascent, Hb Ottawa (or Siam)in residents of Canada, Thailand and China, Hb Etobicoke in descendants of the Irish and Japanese(28) and Hb St Luke's in the Maltese population.

PCR-RFLP tests were useful in the diagnosis or confirmation of the alpha chain variants found in the present study. Directly using the amplicons of gap-PCR for alpha thalassemia in a specific RFLP test for Hb Hasharon facilitates diagnosis, making it simple, fast and economical, without the need for sequencing the HBA genes. In this method, however, it was essential to first identify patients with Hb Ottawa, Hb Etobicoke and Hb St Luke's. With standardization, it is simple to use restriction reactions carried out in this study when alpha chain variants with electrophoretic mobility similar to that of Hb S are suspected in screening tests.

Conclusion

This study describes the first cases of Hb St. Luke's, Hb Ottawa and Hb Etobicoke, and the α212 hybrid gene in Brazil. These Hb variants can lead to false diagnosis of sickle cell trait or sickle cell disease if only isoelectric focusing is used in neonatal screening programs. Additional methods are needed for the correct differential diagnosis and molecular analysis's essential for the identification of the Hb variant. There was no evidence of clinical relevance for the Hbs detected in the present study, even in a child with an association of Hb St. Luke's and Hb S. Standardized PCR-RFLP tests for the diagnosis of Hb Ottawa, Hb St Luke's and Hb Etobicoke were devised which are simple, fast and economical making gene sequencing unnecessary.

Acknowledgments

The authors would like to thank all family members for agreeing to participate in the study and Cibele Velloso-Rodrigues, Beatriz Carvalho, and Terezinha D'Avila, of Fundação Hemominas, for their technical assistance. This work was supported by FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil) and Nupad (Núcleo de Ações e Pesquisa em Apoio Diagnóstico, UFMG). Marcos Borato Viana has a research grant from CNPq, Brazil.

Submitted: 9/17/2012

Accepted: 11/9/2012

Conflict-of-interest disclosure: The authors declare no competing financial interest

Erratum

In the article by Silva MR et al. entitled “Alpha chain hemoglobins with electrophoretic mobility similar to that of hemoglobin S in a newborn screening program” which appeared in the volume 35 number 2 issue of the Revista Brasileira de Hematologia e Hemoterapia, a correction should be noted. The legend of Figure 3 part A. should read:

Figure 3 - Products of PCR and PCR-RFLP for the detection of Hb St Luke’s (A) and Hb Ottawa (B and C) A) Lanes 1 to 4 show amplicons derived from the exon 2 of the HBA1 gene (651 bp). Lanes 5 to 8 show the restriction products from samples 1 to 4 with the endonuclease NlaIV. Lanes 6 and 8 illustrate two samples with the mutation that encodes Hb St Luke’s. The fragment of 171 bp is not present in the wild gene

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  • Corresponding author:

    Marcos Borato Viana
    Departamento de Pediatria da UFMG
    Av. Alfredo Balena, 190 - Sala 267
    30130-100 Belo Horizonte, Brazil
    Fax: 55-31-34099770
  • Publication Dates

    • Publication in this collection
      05 June 2013
    • Date of issue
      2013

    History

    • Received
      17 Sept 2012
    • Accepted
      11 Sept 2012
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