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Year : 2014  |  Volume : 57  |  Issue : 4  |  Page : 626-628
Diagnosis of a rare double heterozygous Hb D Punjab/Hb Q India hemoglobinopathy using Sebia capillary zone electrophoresis

Department of Electrophoresis, Thyrocare Technologies Limited, Turbhe, Navi Mumbai, Maharashtra, India

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Date of Web Publication11-Oct-2014


In India, hemoglobinopathies constitute a major genetic disorder and hemoglobin variants such as Hb S, Hb D Punjab, and Hb E are the most common ones. Other variants include Hb Q India, Hb Lepore, Hb J Meerut, Hb D Iran, etc. These variants show heterozygous state along with beta thalassemia. However, compound heterozygosities among these variants are very rare. Ethylenediaminetetraacetic acid whole blood sample received for routine thalassemia screening was subjected to alkaline electrophoresis using automated capillary zone electrophoresis. Suspecting the presence of rare variants, further analysis was carried out using Bio-Rad D10 and Tosoh G8 high-performance liquid chromatography (HPLC) systems. Capillary zone electrophoretograms showed the presence of peaks in zone Hb A, Hb D, a fused peak in Hb A2, and a small peak in Z1 zone. Bio-Rad and Tosoh chromatograms also indicated the presence of four peaks which are identified as Hb A, Hb D Punjab, Hb Q India, and hybrid of Hb D Punjab/Hb Q India. A peak in Hb D zone of capillary was due to co-migration of Hb D Punjab and Hb Q India variants. Small peak in Z1 zone indicated the presence of alpha chain variant Hb Q India. The findings were further confirmed by HPLC results and molecular genetic studies. The present study reports for the 1 st time a rare hemoglobinopathy of double heterozygosity for Hb D Punjab, Hb Q India on Capillarys 2 Flex Piercing analyzer and is forth reported case for this rare hemoglobinopathy.

Keywords: Capillary electrophoresis, Hb D Punjab, Hb Q India, hemoglobinopathies, high-performance liquid chromatography

How to cite this article:
Parab S, Sakhare S, Sengupta C, Velumani A. Diagnosis of a rare double heterozygous Hb D Punjab/Hb Q India hemoglobinopathy using Sebia capillary zone electrophoresis . Indian J Pathol Microbiol 2014;57:626-8

How to cite this URL:
Parab S, Sakhare S, Sengupta C, Velumani A. Diagnosis of a rare double heterozygous Hb D Punjab/Hb Q India hemoglobinopathy using Sebia capillary zone electrophoresis . Indian J Pathol Microbiol [serial online] 2014 [cited 2023 Nov 30];57:626-8. Available from:

   Introduction Top

Hemoglobinopathies are the genetic disorders leading to the formation of abnormal hemoglobin variants. Most of them result due to mutations in the alpha or beta globin chains which alter the structure of hemoglobin molecules. Most of the hemoglobin variants does not show any clinical symptoms in heterozygous condition but can lead to severe anemia in homozygous states or combination with thalassemias.

Hb Q India is caused by mutation GAC->CAC in codon 64 of alpha-1 globin gene and is genetically silent. This variant normally occurs in heterozygous form with the occurrence rate of 0.4% in Indians. [1] Hb Q variant does not show any clinical symptoms as the change in the amino acid from Asp to His only leads to change in the charge and does not affect the structural properties of the hemoglobin molecule. [2]

Hb D Punjab or Hb D Los Angeles is a type of beta globin gene mutation at 121 codon resulting in replacement of amino acid glutamic acid to glutamine (Glu->Gln). Hb D Punjab in the homozygous state presents with mild hemolytic anemia and mild to moderate splenomegaly. [3] Hb D Punjab trait is a harmless condition showing normal hemoglobin and red blood cell indices. Heterozygosities for Hb D Punjab have been reported to show the prevalence of 1.1% in North Indian population. [1]

Double heterozygous condition for Hb D Punjab and Hb Q India is very rare. We are presenting here the forth case of rare compound heterozygosity for Hb D Punjab and Hb Q India.

   Case report Top

The whole blood ethylenediaminetetraacetic acid sample was received in our laboratory for detection of hemoglobin variants by electrophoresis. Capillary zone electrophoresis for this sample was carried out using automated Capillarys 2 Flex Piercing analyzer, Sebia, France following the manufacturer's instructions. Electrophoresis was done using alkaline buffer pH 9.4, which was provided by the manufacturer. Hemoglobins were detected at 415 nm and electrophoretograms were recorded providing identification of hemoglobins in specific zones.

Due to the presence of unknown peaks sample was analyzed further on two different high-performance liquid chromatography (HPLC) systems (Tosoh automated glycohemoglobin Analyzer G8, Tosoh, Japan using β-thalassemia analysis mode and Bio-Rad D10 analyzer, Bio-Rad Laboratories, California, USA).

Genomic DNA analysis was performed commercially at Department of Genetics, Sir Ganga Ram Hospital, New Delhi to confirm the presence of Hb D Punjab and Hb Q India. Hb D Punjab was analyzed by polymerase chain reaction (PCR) and restriction enzyme digest, while confirmation of Hb Q India was performed by PCR-based amplification refractory mutation system (ARMS) technique.

Routine hematological examinations were carried out using automated cell counter, Sysmex XN-1000 (Kobe, Japan).

Capillary zone electrophoresis indicated the presence of four peaks as follows: Hb A (47.5%), Hb D (42.3%), Hb A2 + unknown peak in Hb C zone and Z1 peak [Figure 1]. Identification and confirmation of variants were done in conjunction with HPLC results.
Figure 1: Capillary zone electrophoresis pattern indicates peaks in Hb D, Hb A2/Hb C zone, and additional small peak in Z1 zone indicating alpha chain variant

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High-performance liquid chromatography chromatogram on Tosoh analyzer showed following four major peaks, Hb A (39.2%), Hb D Punjab (30.7%) with retention time 3.98 in Hb D window, peak for Hb Q India (11.2%) with retention time 4.59 in Hb S window, and combined peak for hybrid of Hb D Punjab/Hb Q India (8.2%) at retention time of 4.88 in Hb C window [Figure 2]a.
Figure 2

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Bio-Rad HPLC results also show similar findings as Tosoh analyzer [Figure 2]b. It also showed four major peaks at retention times (minutes) of 1.74, 3.93, 4.48, and 4.72 indicating the presence of Hb A, Hb D Punjab, Hb Q India and hybrid of Hb D Punjab/Hb Q India, respectively.

This patient showed normal hematological parameters such as hemoglobin (12.2 g/dL), mean corpuscular volume (MCV - 92.3 FL), mean corpuscular hemoglobin concentration (MCHC - 33.0 g/dL), and red cell count (4.01 × 10 6 /μL).

Polymerase chain reaction analysis confirmed the replacement of base G to C at 121 position on beta globin gene resulting in heterozygosity for Hb D Punjab. Also, replacement of Asp to His at position no. 64 on alpha chain confirmed the presence of Hb Q India.

   Discussion Top

Detection of unknown variants of hemoglobin using alkaline electrophoresis or HPLC alone is a difficult task for a reference laboratory. Confirmation of variants can only be possible with the findings of both the techniques along with blood cell counts and clinical history.

Both previously published reports for double heterozygosity of Hb D Punjab/Hb Q India were detected on Bio-Rad HPLC analyzer. [4],[5] We are 1 st time reporting it on capillary zone electrophoresis. Electrophoretogram on Capillarys 2 showed a peak of 42.4% in Hb D zone. This is a combined peak for Hb D Punjab and Hb Q India as both these variants for Hb D Punjab and Hb Q India co-elute in Hb D zone. Furthermore, a small peak of 0.5% in Z1 zone confirms the presence of alpha chain variant of Hb Q India (ααHbQ Indiadd). [6]

Third peak (9.6%) in Hb A2/Hb C zone was of Hb A2 merging with an unknown variant. This unknown variant probably can be the hybrid molecule of Hb D Punjab/Hb Q India. Alkaline electrophoresis pattern reported by Higgins et al. showed that this hybrid Hb D Punjab/Hb Q India migrated in Hb C zone. [4]

High-performance liquid chromatography findings are consistent with previously reported cases of double-heterozygosity for Hb D Punjab/Hb Q India. Both HPLC systems showed Hb D Punjab variant of approximately 30% suggesting heterozygosity for Hb D Punjab (ααββHbD Punjab ). Furthermore, a sharp peak in S window indicates the presence of Hb Q India (ααHbQ Indiaββ). As per previous reports, a peak in C window is a hybrid variant of Hb Q India/Hb D Punjab (ααHbQ IndiaββHbD Punjab ).

DNA sequencing of alpha and beta globin genes is a definitive method for confirmation of Hb variants and especially point mutations. PCR studies using ARMS technique confirmed the presence of double heterozygosity for Hb Q India/Hb D Punjab.

   References Top

Rao S, Kar R, Gupta SK, Chopra A, Saxena R. Spectrum of haemoglobinopathies diagnosed by cation exchange-HPLC & modulating effects of nutritional deficiency anaemias from North India. Indian J Med Res 2010;132:513-9.  Back to cited text no. 1
[PUBMED]  Medknow Journal  
Abraham R, Thomas M, Britt R, Fisher C, Old J. Hb Q-India: an uncommon variant diagnosed in three Punjabi patients with diabetes is identified by a novel DNA analysis test. J Clin Pathol 2003;56:296-9.  Back to cited text no. 2
Pandey S, Mishra RM, Pandey S, Shah V, Saxena R. Molecular characterization of hemoglobin D Punjab traits and clinical-hematological profile of the patients. Sao Paulo Med J 2012;130:248-51.  Back to cited text no. 3
Higgins T, Schnabl K, Savoy M, Rowe P, Flamini M, Bananda S. A novel double heterozygous, HbD Punjab/HbQ India, hemoglobinopathy. Clin Biochem 2012;45:264-6.  Back to cited text no. 4
Mutreja D, Tyagi S, Tejwani N, Dass J. Double heterozygous hemoglobin Q India/hemoglobin D Punjab hemoglobinopathy: Report of two rare cases. Indian J Hum Genet 2013;19:479-82.  Back to cited text no. 5
[PUBMED]  Medknow Journal  
Greene DN, Pyle AL, Chang JS, Hoke C, Lorey T. Comparison of Sebia Capillarys Flex capillary electrophoresis with the BioRad Variant II high pressure liquid chromatography in the evaluation of hemoglobinopathies. Clin Chim Acta 2012;413:1232-8.  Back to cited text no. 6

Correspondence Address:
Suhas Sakhare
Thyrocare Technologies Limited, D-37/1, TTC MIDC, Turbhe, Navi Mumbai - 400 703, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.142709

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