| Abstract|| |
Background: Alpha globin chain variants are clinically significant since they directly influence the structure and function of the hemoglobin (Hb) molecules they constitute, either in combination with normal beta globin chains or with variant beta chains, thereby altering the morbidity and mortality associated with the resultant hemoglobinopathies. We describe here two unrelated families from Madhya Pradesh who had a nondeletional alpha-chain variant, HbO Indonesia (CD116 G → A). Members of one of the two families also had coinheritance of sickle hemoglobin (HbS). Aims: The aim was to study the phenotype of HbO Indonesia and its interaction with HbS. Materials and Methods: Hb electrophoresis, high-performance liquid chromatography (HPLC), covalent reverse dot blot hybridization, amplification refractory mutation system, multiplex polymerase chain reaction, and direct gene sequencing were used to identify and characterize the variant Hbs. Results: The abnormal Hb moved in HbS region in Hb electrophoresis at alkaline pH but gave an abnormal peak in HPLC with a retention time (RT) of 4.86–4.89 min. In two members of the family with coinheritance of HbS, it produced small additional abnormal Hb peaks (4.6% in heterozygous and 11.9% in homozygous member) in HPLC with a longer RT (5.15–5.17 min) possibly resulting from a combination of HbO Indonesia alpha chain with HbS beta chain. Conclusions: It appears that depending on the zygosity of HbS, HbO Indonesia would subtract a variable amount of HbS beta chain from the total pool, thereby potentially reducing the clinical severity of HbS disease. HbO Indonesia per se does not cause anemia or alter the red cell indices.
Keywords: HbO Indonesia, interaction between HbO Indonesia and sickle hemoglobin, sickle hemoglobin
|How to cite this article:|
Gupta AD, Nadkarni A, Mehta P, Goriwale M, Ramani M, Chaudhary P, Mehrotra V, Colah R. Phenotypic expression of HbO Indonesia in two Indian families and its interaction with sickle hemoglobin. Indian J Pathol Microbiol 2017;60:79-83
|How to cite this URL:|
Gupta AD, Nadkarni A, Mehta P, Goriwale M, Ramani M, Chaudhary P, Mehrotra V, Colah R. Phenotypic expression of HbO Indonesia in two Indian families and its interaction with sickle hemoglobin. Indian J Pathol Microbiol [serial online] 2017 [cited 2017 Jul 23];60:79-83. Available from: http://www.ijpmonline.org/text.asp?2017/60/1/79/200030
| Introduction|| |
The inherited hemoglobin (Hb) disorders are the most common single-gene disorders in India. The globin chain variants are grouped into various categories, depending on the type of the mutation present. The majority of the Hb variants result from a single-base substitution in the globin genes. A few alpha chain variants cause changes in the three-dimensional structure of the Hb complex that impair the formation of the Hb dimer and/or tetramer leading to the formation of highly unstable free alpha Hb species.
A number of Hb-alpha chain variants were first reported from India. HbJ Meerut and HbQ India are examples of such Hb variants. Alpha chain variants that were reported from other countries have also been encountered, albeit rarely, in Indians. HbO Indonesia is one such variant. This variant was first reported in Bugis population in Indonesia. Individuals with HbO Indonesia have normal Hb level and red cell indices, and in high-performance liquid chromatography (HPLC), this Hb elutes at 4.9 min. Information on the interaction of this Hb variant with beta chain variants is meager. Here, we describe two unrelated families with HbO Indonesia, an alpha chain variant rarely encountered in India. In one of the two families, the variant Hb was found to be associated with both homozygous and heterozygous states of sickle hemoglobin (HbS) in two members, thereby allowing us to study the spectrum of phenotypic interaction between the two abnormal Hbs.
| Materials and Methods|| |
Blood samples of the two patients (AY and PDA) belonging to two different families were first received in our laboratory at different time points for Hb analysis. Only some of the family members of both the families could be subsequently investigated after both AY and PDA were diagnosed to have abnormal Hbs on examination [Table 1]. Both families are from the Central Indian state of Madhya Pradesh but belong to different castes.
|Table 1: Findings of hematological tests, hemoglobin analyses, and molecular assays in the members of the two families|
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Five milliliters of blood was collected from the patient in evacuated tubes with dipotassium ethylenediaminetetraacetic acid as an anticoagulant. The complete blood count (CBC) was performed on an automated Hematology Analyzer (LH750, Beckman Coulter, FL, USA). The percentage of HbA, HbA2, and HbF was measured by HPLC on the Variant II Hb Testing System (Bio-Rad Laboratories, Hercules, CA, USA). The Hb variants too were detected and quantified by HPLC. Hb electrophoresis was carried out on cellulose acetate at alkaline pH (8.9).
DNA extraction was carried out using a standard method. Beta globin gene mutations were characterized using covalent reverse dot blot hybridization  and by amplification refractory mutation system. Screening for deletional alpha thalassemia was done by multiplex polymerase chain reaction  and nondeletional alpha thalassemia by direct DNA sequencing (Applied Biosystems, Foster City, USA).
| Results|| |
The hematological and molecular findings in five members from the two families who were investigated by us are summarized in [Table 1].
Complete blood count
CBC was available in both the members of family 1 and in two of the three members in family 2. The red cell parameters were highly variable in the members of the two families. CL (24 years old, female) in family 1 had a normal CBC in spite of having HbS trait and an additional abnormal Hb produced by the variant alpha chain under discussion, while her child (AY, 2 years old, male) was anemic and his red cell parameters were hypochromic microcytic. SA (53 years, male) of family 2 had microcytic red cell indices while his sister SJ (56 years old) had macrocytic red cell indices. The latter seems to be related to red cell aging since in her case, the test was performed 5 days after collection of the blood sample. CBC data of their father (PDA, 86 years, male) were not available.
High-performance liquid chromatography
The HPL chromatogram in family 1 showed the presence of HbS in both the mother (CL) and the child (AY) [Figure 1]. CL was heterozygous for HbS (HbA [76.1%] and moderately high HbS [33.1%]) and had two small abnormal Hb peaks - at RT 4.86 min (6.6%) and 5.15 min (4.6%). Her child (AY) was homozygous for HbS (high HbS [64.5%], high HbF [19.3%], and absence of HbA) and had an abnormal Hb peak (11.9%) with a retention time (RT) of 5.17 min in addition to HbS. AY did not have any peak with RT 4.86–4.89 min that was seen in her mother and the other members of family 2 [Figure 1]. In the second family, all the three members showed the presence of only one abnormal Hb peak with RT of 4.86–4.89 min in addition to HbA. The percentage of this abnormal Hb was low in all cases (10.1%, 12.2%, and 16.9%) and was in the range observed with known alpha globin chain variants such as HbO Indonesia. There was no HbS in this family. HbA2 levels were low in all cases in family 2 while it was normal in members of family 1. The contour of HbA2 peak in both the members of family 1 suggested contribution by glycated HbS to the higher level of the HbA2 encountered in this family compared to that in members of family 2.
|Figure 1: High-performance liquid chromatogram of members of the two families showing the various abnormal hemoglobin peaks with their retention times. High-performance liquid chromatography in CL shows two abnormal hemoglobin peaks at 4.86 min (marked by an arrow) and 5.15 min in addition to sickle hemoglobin S while in her son (AY), there is only one abnormal hemoglobin peak at 5.17 min. The latter peak seems to have resulted from a combination between the variant alpha chain and the sickle beta chain. All the three members of family 2 show the same abnormal hemoglobin peak (marked by the arrow) ranging from 10% to 17% with a retention time of 4.86–4.89 min|
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Cellulose acetate electrophoresis in alkaline pH (8.9) showed that the alpha-chain variant is a slow-moving Hb that moves with HbS since only one slow moving (abnormal) Hb band was seen in HbS region in electrophoresis in patients with double heterozygosity for this variant and HbS.
Genotyping of alpha and beta globin genes was performed in CL, AY (family 1), and SA (family 2). CL was a heterozygote for HbS gene while AY was homozygous HbS. SA's beta globin gene was normal. The alpha globin gene sequencing of all the three showed heterozygosity for G → A substitution at codon 116 (glutamic acid to lysine) giving rise to an α-chain variant HbO Indonesia [Figure 2].
|Figure 2: DNA sequencing showing the presence of HbO Indonesia CD116 [GAG → AAG, Glutamic Acid → Lysine] in CL of family 1|
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| Discussion|| |
Structural Hb variants mainly result from a point mutation in a globin gene that produces a single amino acid substitution in the globin chain. Approximately 1200 human Hb variants have been described. The mutation could result in either no physiological abnormality or clinical problem or can lead to instability of the Hb molecule resulting in hemolytic anemia. The study on structural variants has provided important insights into globin gene transcriptional regulation and translation. The structural Hb variants identified in the Indian population, both the rare and the more common ones, included both alpha chain and beta chain variants. From a biological and clinical point of view, hemoglobinopathies resulting from the combination of variant alpha chains with normal or abnormal beta chains constitute a very heterogeneous group. Many variants which are without any clinical effect are accidentally detected during screening programs. The only reason that leads to characterization of these mutations is to eliminate problems resulting from a possible interaction with HbS or with a thalassemic syndrome. In an Indian study, Nair et al. reported five alpha-chain variants (Hb Jackson, Hb J Paris-I, Hb J Meerut, Hb Sun Prairie, HbO Indonesia). An important finding in their study was that most of the patients had co-inherited a beta thalassemia mutation or a beta globin variant along with an alpha chain variant, a situation similar to that encountered in family 1 in our study. The authors concluded that one needs to undertake systematic investigations to avoid missing any of the rare alpha globin chain variants while screening for the beta globin gene disorders. HbO Indonesia was also identified during a newborn screening program in India. Schneider et al. reported an alpha-chain variant, Hb Oleander, in a black family having mutation at the same codon as observed in our cases, i.e., (CD116) HBA2:c. 349 GAG>CAG (or HBA1) replacing glutamic acid by glutamine. Like HbO Indonesia, Hb Oleander moves in the HbS region at alkaline pH. Another alpha-chain variant, Hb Ube-4, HBA2:c. 350 GAG>GCG (or HBA1) at codon 116 was reported in a Japanese and a Korean family., This variant moves slightly faster than HbS at alkaline pH.
HbO Indonesia is also known as Buginese-X or Hb Oliviere. It has been reported earlier in the Buginese from Sulawesi, Makassar, Iranian, South African, Italian, and Chinese populations., So far, only two cases have been reported from India., These cases did not have any clinical symptom, but electrophoresis showed a slow-moving band, and an abnormal Hb peak was seen on HPLC in the HbC window. In contrast, members of family 1 in our report demonstrate the interaction of HbO Indonesia with HbS, not reported earlier in Indian publications referred to above.
In the present study, CL of family 1 had two abnormal Hb peaks on HPLC – 6.6% at RT 4.86 min and 4.6% at RT 5.15 min in addition to HbS [Figure 1]. The Hb peak at RT 5.15–5.17 min (HbC window) in CL and her son (AY) seems to have been produced by a combination of the alpha chain of HbO Indonesia with the beta chain of HbS. On the other hand, the abnormal Hb peak observed in the mother, CL (family 1), at RT 4.86 min, and in all the three members of the second family seems to represent an abnormal Hb resulting from the combination of the variant/defective alpha chain with normal beta chain. The levels of HbO Indonesia in members of family 2 (that did not have HbS) were in the range reported earlier. These individuals also had a very small peak next to the HbA2 peak. The former peak is supposed to represent a variant HbA2 resulting from the combination of alpha chain of HbO Indonesia with normal delta chain as observed in earlier reports.
The fact that CL, who also had HbS (heterozygous) along with the alpha chain variant, had a normal CBC confirms the earlier observation that HbO Indonesia does not seem to alter red cell number and indices and/or cause anemia. Therefore, the microcytic anemia observed in AY in family 1 could have resulted from homozygous sickle cell anemia and that in SA in family 2 could be due to other etiologies such as iron deficiency. In contrast, the red cell indices in patient SJ of family 2 indicated macrocytosis. This could have resulted from aging of the blood sample since the same was analyzed 5 days after its collection and is unlikely due to the alpha-chain variant in question. It is well documented that aging of blood in transit results in an increase in red cell volume and related indices, for example, mean corpuscular volume and mean corpuscular hemoglobin concentration, as observed in this case.
| Conclusion|| |
Thus, our study highlights the contribution of HPLC as a primary diagnostic tool and emphasizes the role of careful analysis and interpretation of HPLC chromatograms in conjunction with genetic analysis of the putative variant globin gene before making a diagnostic conclusion. It also underlines the importance of adopting a planned strategy for complete and extensive investigation of suspected cases of alpha and beta globin gene defects.
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Conflicts of interest
There are no conflicts of interest.
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Amar Das Gupta
SRL Limited, Prime Square Building, Plot No. 1, Gaiwadi Industrial Estate, S. V. Road, Goregaon (West), Mumbai - 400 062, Maharashtra
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]