Hb Koln [β98(FG5) [GTG → ATG, Val → Met]: The first report from India

Abstract

Background

The group of unstable hemoglobins are associated with congenital non-spherocytic hemolytic anemia due to instability of the hemoglobin molecule. They often lead to formation of the characteristic inclusion bodies or Heinz bodies.

Aim

To identity the cause of mild anemia, reticulocytosis, and hepatosplenomegly in a case of non-spherocytic hemolytic anemia.

Materials and methods

A 34-year-old female patient originating from Maharashtra, western India presented with mild anemia and jaundice which had persisted since childhood. Investigations included a complete blood count, screening for red cell membrane protein defects, Hb analysis by high-performance liquid chromatography (HPLC) and cellulose acetate electrophoresis (pH 8.9), heat instability test and DNA sequencing.

Results

Hemoglobin analysis by HPLC showed an abnormal peak in the Hb C window (9.8%) with a retention time of 4.90 minutes. Cellulose acetate electrophoresis (pH 8.9) showed a slow moving band (6.15%) between Hb A₂ and Hb S. The heat instability test was positive. DNA analysis of α globin genes showed absence of both deletional and non- deletional α thalassemia. DNA sequencing of the β globin gene revealed heterozygosity for a mutation at codon 98 [GTG → ATG, Val → Met], which gives rise to Hb-Koln.

Conclusion

Hb Koln is the commonest unstable Hb variant reported from many populations in the world. However, this is the first report of this unstable Hb variant from India.

Keywords:

• Hb Koln
• Unstable Hb variant
• Non-spherocytic hemolytic anemia
• India

Introduction

The group of unstable hemoglobins are associated with congenital non-spherocytic hemolytic anemia due to instability of the hemoglobin molecule. They often lead to formation of the characteristic inclusion bodies or Heinz bodies. The clinical presentation of patients varies widely from an asymptomatic or mildly anemic condition to a severe disorder. The severity can be largely attributed to the underlying molecular defect. More than 100 different unstable hemoglobins leading to hemolytic anemia have been described so far. Hemoglobin Koln (Hb Koln) is the most common unstable hemoglobin variant with a dominant inheritance pattern. It results from a replacement of valine by methionine at β98 (FG5) causing increased oxygen affinity and instability possibly due to heme depletion.^1,2 Here we report the first case of Hb Koln in an Indian patient referred for diagnosis of non-spherocytic hemolytic anemia.

Materials and methods

The proband was a 34-year-old female born of a non-consanguineous marriage originating from Sholapur district in the state of Maharashtra in western India. She had a history of mild anemia and jaundice since childhood but had no history of receiving any blood transfusions. There was no family history of chronic anemia or jaundice. She presented with mild anemia, reticulocytosis, and mild hepatosplenomegaly.

Red cell indices were measured on an automated cell counter (Sysmex K-1000). Peripheral smear examination and reticulocyte count were done by standard methods. Eosin-5-maleimide dye test using flow cytometry was used to screen for red cell membrane protein defects.³ Hemoglobin analysis was done using cation exchange high-performance liquid chromatography (HPLC) on the Variant Hemoglobin Testing System (BioRad Laboratories, Hercules, CA, USA) and by cellulose acetate electrophoresis (pH 8.9). Heat and isopropanol instability tests were undertaken to screen for unstable hemoglobins. Screening for 8 common α thalassemia deletions (−α^3.7, −α^4.2, –^SEA,–^SA,–^FIL,–^MED,–^THAI,–^20.5) was done using multiplex PCR.⁴ DNA Sequencing of the α and β globin genes was done using the ABI Prism 310 Sequencer (Applied Biosystems, Foster City, CA, USA) to characterize the hemoglobin variant.

Results

The peripheral smear showed hypochromia, target cells and a reduction in the number of platelets. The hematological and molecular findings in the patient are summarized in Table 1. The proband had a hemoglobin level of 11.9 g/dl with a reticulocyte count of 9.0% and showed the absence of Heinz bodies. She had reduced platelet counts (119 × 10³/μl) with a normal prothrobin time. Flow cytometric analysis of red cell membrane proteins by using eosin-5-maleimide dye showed the absence of red cell membrane protein defects. Hemoglobin analysis by HPLC showed an abnormal peak in the Hb C window – 9.8% with a retention time of 4.90 minutes (Fig. 1). Cellulose acetate electrophoresis (pH 8.9) showed a slow moving band (6.15%) between Hb A₂ and Hb S.

Figure 1. Cation-exchange HPLC analysis on the Variant Hb Testing System; HPLC analysis showed an abnormal peak in the C window – 9.8% with a retention time of 4.90 minutes.

Table 1. Hematological and molecular data of the proband

DNA analysis of the α globin genes showed absence of both deletional and non-deletional α thalassemia. DNA sequencing of the β globin gene revealed heterozygosity for a mutation at codon 98 [GTG → ATG, Val → Met], which gives rise to Hb-Koln (Fig. 2).

Figure 2. DNA sequencing electropherogram of the proband showing the Hb Koln mutation [β98 (FG5) [GTG → ATG, Val → Met]. The arrow indicates the position of the nucleotide substitution GTG (valine) > ATG (methionine).

Discussion

One hundred and forty-five unstable hemoglobins have been identified so far (http://globin.bx.psu.edu/cgi-bin/hbvar/counter). About 100 of these variants lead to hemolysis or abnormal oxygen affinity while the other variants have no hematological abnormalities but show instability in the in vitro test. The unstable hemoglobin variants have a variable phenotype depending on the position of the mutation and whether they disturb the structure of the globin chain or hemoglobin molecule.⁵ Hb Koln is the most common of the unstable hemoglobin variants and it was first described by Pribilla in 1962.⁶ The structural abnormality of the variant was later demonstrated by Carrell et al. in 1966.⁷ One of the main characteristics of unstable hemoglobins is the presence of Heinz bodies. The severity of the anemia is related to the degree of the resulting hemoglobin instability. Numerous Heinz bodies usually appear in the peripheral blood after splenectomy. In the present case, we did not find any Heinz bodies in the red cells possibly due to the active function of the patient's spleen. There are few Hb variants like Hb O Arab, Hb Constant Spring, Hb O Indonesia, Hb Agenogi, and Hb Siriraj which elute in the Hb C window.^8,9 The α chain variants usually have a lower percentage of the abnormal hemoglobin compared to the β chain variants. In view of the low percentage of this variant (9.8%), we first sequenced the α globin gene which was normal. Sequencing of the β globin gene thereafter, led to the identification of Hb Koln. Our patient showed mild thrombocytopenia. A similar finding has been observed in other patients of Hb Koln earlier.^10,11 The decrease in platelet count in our patient was most likely due to splenic sequestration of platelets. This is supported by the observation of Pedersen et al., in their hypersplenic patient where thrombocytopenia was corrected after splenectomy.¹⁰

Hemoglobin Koln has been previously reported in persons of Northern European ancestry^6,7,11 and in Malay,¹² Jewish,¹³ American Blacks,¹⁰ Japanese,¹⁴ Czechoslovakian,¹⁵ and Korean patients.¹⁶ This hemoglobinopathy has a widespread distribution although it is being reported from India for the first time.

Conclusion

Hb Koln is the commonest unstable Hb variant reported from many populations in the world. However, this is the first report of this unstable Hb variant from India.

Acknowledgment

This work was supported by the Indian Council of Medical Research.