• Users Online: 545
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 2  |  Page : 79-82

Report of three Bengali cases with Hemoglobin E variant in Najran


1 Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt
2 Department of Family and Community Medicine, College of Medicine, Najran University, Najran, Saudi Arabia
3 Department of Pediatrics, College of Medicine, Najran University, Najran, Saudi Arabia
4 Department of Cl. Hematopathology, College of Medicine, Najran University, Najran, Saudi Arabia

Date of Submission07-Aug-2020
Date of Decision11-Sep-2020
Date of Acceptance18-Sep-2020
Date of Web Publication06-Aug-2021

Correspondence Address:
Dr. Mohamed Samir M. Khalil
Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/joah.joah_132_20

Rights and Permissions
  Abstract 


INTRODUCTION: Differential diagnosis of hemoglobin (Hb) variants eluting in the A2 window on high-performance liquid chromatography (HPLC) is of particular importance. Of particular importance is Hb E, which is the most common and the most significant variant. The aim of this study was to study a rare variant infrequently seen in our countries during the routine work.
METHODS: Sixteen Bengali workers at Najran University Hospital, Saudi Arabia, came to the laboratory for routine investigation. CBC, routine blood chemistry, and Hb separation by HPLC were performed.
RESULTS: Three cases out of 16 showed an abnormal Hb peaked on the A2 window on HPLC consistent with the diagnosis of Hb E. Two of them had Hb E of 29.1% of each. The third case had Hb E around 86%. In addition, one case was consistent with β thalassemia trait had increased Hb A2 of 5.7%. Five cases were borderline.
CONCLUSION: HbE could be adequately differentiated from other Hb variants eluting in the A2 window on HPLC by the percentage of the variant, its retention time, the mild clinical presentation, and the ethnic origin of the patient.

Keywords: Hemoglobin A2, hemoglobin E, high-performance liquid chromatography, Kolkata, variant


How to cite this article:
M. Khalil MS, Al-Qahtani AM, AlQahtani JM, El Khawanky MM. Report of three Bengali cases with Hemoglobin E variant in Najran. J Appl Hematol 2021;12:79-82

How to cite this URL:
M. Khalil MS, Al-Qahtani AM, AlQahtani JM, El Khawanky MM. Report of three Bengali cases with Hemoglobin E variant in Najran. J Appl Hematol [serial online] 2021 [cited 2021 Dec 2];12:79-82. Available from: https://www.jahjournal.org/text.asp?2021/12/2/79/323321




  Introduction Top


The δ globin gene is expressed at low levels, approximately 2.0%–3.0% of the total hemoglobin (Hb) in normal individuals. Over 1352 human Hb variants are known up-to-date.[1] The diagnosis of variants elutes in the A2 window on high-performance liquid chromatography (HPLC) is of particular importance because increased Hb A2 (around 5%–6%) in the presence of microcytosis and hypochromia of red blood cells (RBCs) is the main characteristic parameter in diagnosing most β-thalassemia carriers.

The increase in Hb A2 may refer to other structural variants that also migrate in the Hb A2 window on HPLC,[2] and DNA-based diagnosis is necessary before genetic counseling is undertaken. Of particular importance is Hb E which is the most common and the most significant abnormal Hb eluting in the A2 window on HPLC. HbE arises from a point mutation in codon 26 in which the normal GAG codon is mutated to an AAG, resulting in a change from glutamic acid to lysine. The βE mutation activates this cryptic splice site so that it begins to function in a rather efficient way. The result is that about 40% of the splices result in termination at an out-of-frame stop codon. The normally spliced RNA contains the βE mutation and produces only about 60% of the normal amount of β-globin.[3]

HbE is a mild allele, almost with nearly normal clinical presentation in heterozygote and mild hemolytic anemia in homozygote state.[4] HbE is the most prevalent variant in Southeast Asia (Thailand, Myanmar, Cambodia, Laos, and Vietnam). In India, it is clustering in Kolkata and Assam where the carrier frequency is 2%–5% and 6%–51%, respectively.[5],[6]

In this study, we have faced with three cases consistent with the diagnosis of HbE trait and HbE disease discovered during a routine investigation of Bengali workers at the Najran University Hospital, KSA. They had abnormal Hb peaks masking Hb A2 on HPLC. An adequate presumptive diagnosis was done based on the RT, clinical picture, and ethnic origin of the patients. The Hb A2 expression and the diagnosis of β thalassemia carriers in these cases and in the other cases were also discussed.


  Materials and Methods Top


Sixteen male Bengali workers at Najran University Hospital, Saudi Arabia, came to the laboratory for routine investigations. Complete peripheral blood pictures (CBCs) were carried out using Sysmex XS 500i (Sysmex, https://www.sysmex.com/). Serum biochemical analysis was carried out using COBAS C311 (Roche, https://www.roche.com/). Hb separation was carried out by HPLC using the D-10 instrument (Bio-Rad Laboratories Hercules, California, USA). One case, showed a marked increase in expression of abnormal Hb on the A2 window, was repeated on the Variant II HPLC system ((Bio-Rad) with the use of the Variant II Thalassemia Short Program (Bio-Rad Laboratories Hercules, California, USA).


  Results Top


In this study, 16 Bengali male workers from Kolkata (also written Colcutta) (the capital of the Indian state of West Bengal) were investigated. One case was consistent with β thalassemia carrier. It showed increased Hb A2 of 5.7% with microcytic hypochromic RBCs and increased RBC distribution width (RDW) [Case 1, [Table 1] and [Figure 1]a. Five cases were borderline. Although they had Hb A2 of 4% or slightly more, they had normal total Hb, nearly normal RBC indices, and normal RDW [Cases 2–6, [Table 1] and [Figure 1]b]. Family study, further investigations, and DNA analysis were required to reach to a definite diagnosis. This agrees with Rosnah et al.[7] who found that normal HbA2 level may reach 4%.
Table 1: Descriptive data of 6 cases with increased/ borderline hemoglobin A2

Click here to view
Figure 1: (a) A case consistent with β thalassemia carrier. (b) Borderline case

Click here to view


HbE is the most common variant eluting in the A2 window on HPLC. It is a mild allele, almost with nearly normal clinical presentation in heterozygote and mild hemolytic anemia in homozygote state.[4] HbE is the most prevalent variant in Southeast Asia (Thailand, Myanmar, Cambodia, Laos, and Vietnam). In India, it is clustering in Kolkata and Assam where the carrier frequency is 2%–5% and 6%–51%, respectively.[5],[6] In this study, three cases with abnormal peak in the A2 window were consistent with the diagnosis of Hb E. Two cases (Cases 7 and 8) [Table 2] showed an abnormal peak of 29.1% for each. They gave nearly the same chromatograph of Hb separation on HPLC [Figure 2]a. They had no complaint and with clinically stable picture, only microcytic hypochromic RBCs.
Table 2: Descriptive data of the three cases with abnormal peaks in the A2 window on high-performance liquid chromatography

Click here to view
Figure 2: High-performance liquid chromatography of the three cases with abnormal peaks on the A2 window. (a) Case 7 or 8 (b and c) case 9

Click here to view


The third case (case 9) [Table 2], [Figure 2]b and [Figure 2]c and [Figure 3], showed abnormal Hb at 86% expression on HPLC, was separated by two instruments (D-10 instrument and Variant II) for confirmation. It showed a mild clinical presentation, a mild microcytic hypochromic anemia, an increase in RDW, a mild hemolysis (mild increase in unconjugated bilirubin), and a mild increase in Hb F (2.7%). Other small Hb peaks were attributed to posttranslational modification of Hb E. This picture was consistent with HbE disease.[8] This patient gave a history of a hemolytic attack once during a high fever period. This was consistent with the reported instability of Hb E in oxidative stress and high fever.[9]
Figure 3: Peripheral blood smear of case 9

Click here to view


In Eβ0 thalassemia, Hb E is usually around 50%, yet it can be differentiated with ease by its severe clinical presentation and the marked increase in Hb F.[9] Eβ+ thalassemia is milder and characterized by the presence of Hb A.[10] Retention time of the presumed HbE (3.44 and 3.51 min) [Figure 2] was clearly different from that of Hb A2 (3.24 and 3.25 min) [Figure 1] on D-10 HPLC. It could be specific for HbE and can be used in differentiation, although it needs more evaluation.


  Discussion Top


The majority of hemoglobinopathy present in the western and eastern provinces of Saudi Arabia, particularly in the southwestern province. Abuzenadah et al.[11] reported a great heterogeneity at the molecular level in the western province and attributed this to the large population of immigrants there. Hb E was one of the seven common β-thalassemia alleles reported. The prevalence of Hb E trait was the least frequent (0.85%) compared to the other investigated Hb disorders in Jedda (57 Hb E carrier from 6750 cases studded).[12] Only three cases (2.4% of cases studied) at Taif city were reported by Dahlawi et al.[13] Najran city, located in the south of KSA, had the least prevalence of β Thalassemia trait (2.4%) and zero β thalassemia disease in a study done by Alsaeed ES et al.[14] 2011–2015. As far as we know, there is no report about Hb E in Najran.

Although HbE is not prevalent in KSA, physician awareness by diagnosis and the clinical course is important, especially because of the pilgrimage and pilgrims, and the presence of sustainable employment, especially from East Asian countries. Better understanding of Hb phenotypes is crucial in the diagnosis and would help in simple and cheap DNA diagnosis by ARMS PCR or restriction digest rather than the expensive and laborious sequencing.

In this study, we focused on the phenotype of three cases consistent with the diagnosis of HbE and rare variants, two heterozygotes, and one homozygote. The abnormal Hb was masking the Hb A2 on HPLC. The ethnic origin of the patients was crucial in confirming the diagnosis of Hb E. It was impossible to determine the Hb A2 expression on HPLC in these cases. The diagnosis of a mild β thalassemia carrier is compromised and only can be excluded by DNA diagnosis in such cases. The awareness of such dilemma is important, especially as regards the premarriage programs in KSA and the Middle East countries where Hb E is not prevalent. Furthermore, this study highlighted the importance of the clinical picture, ethnic origin, family history, and the DNA techniques in the diagnosis of hemoglobinopathies.


  Conclusion Top


HbE could be adequately differentiated from other Hb variants eluting in the A2 window on HPLC by the percentage of the variant, its retention time, the mild clinical presentation, and the ethnic origin of the patient. Adequate presumptive diagnosis in hemoglobinopathy is a preliminary step to a definite diagnosis by DNA analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Giardine B, Borg J, Viennas E, Pavlidis C, Moradkhani K, Joly P, et al. Updates of the HbVar database of human hemoglobin variants and thalassemia mutations. Nucleic Acids Res 2014;42:D1063-9.  Back to cited text no. 1
    
2.
Bain BJ. Other significant haemoglobinopathies. In: Haemoglobinopathy Diagnosis. 2nd ed. Malden, MA: Blackwell Publishing; 2006. p. 201-11.  Back to cited text no. 2
    
3.
Gelehrter TD, Collins FS, Ginsburg D. Molecular genetics of human diseases: Hemoglobinopathies. In: Principles of Medical Genetics. 2nd ed. Baltimore: Williams and Wilkins; 1998.  Back to cited text no. 3
    
4.
Weatherall DJ. The inherited diseases of hemoglobin are an emerging global health burden. Blood 2010;115:4331-6.  Back to cited text no. 4
    
5.
Weatherall DJ, Clegg JB. Inherited haemoglobin disorders: An increasing global health problem. Bull World Health Organ 2001;79:704-12.  Back to cited text no. 5
    
6.
Mukhopadhyay D, Saha K, Sengupta M, Mitra S, Datta C, Mitra PK. Spectrum of hemoglobinopathies in West Bengal, India: A CE-HPLC study on 10407 subjects. Indian J Hematol Blood Transfus 2015;31:98-103.  Back to cited text no. 6
    
7.
Rosnah B, Shahida NS, Nazri MH, Marini R, Noor Haslina MN, Shafini MY, et al. The diagnosis of beta thalassemia with borderline HbA2 level among kelantan population. J Blood Disord Transfus 2017;8:396 9.  Back to cited text no. 7
    
8.
Maheshwari U, Samant P, Agarwal A, Kadam S, Dhar R. A case report On HbE homozygous haemoglobinopathy. Int J Recent Sci Res 2015;6:5840-2.  Back to cited text no. 8
    
9.
Strader MB, Kassa T, Meng F, Wood FB, Hirsch RE, Friedman JM, et al. Oxidative instability of hemoglobin E (β26 Glu→Lys) is increased in the presence of free α subunits and reversed by α-hemoglobin stabilizing protein (AHSP): Relevance to HbE/β-thalassemia. Redox Biol 2016;8:363-74.  Back to cited text no. 9
    
10.
Vichinsky E. Hemoglobin E syndromes. Hematol Am Soc Hematol Educ Program 2007;1:79-83.  Back to cited text no. 10
    
11.
Abuzenadah AM, Hussein IM, Damanhouri GA, A-Sayes FM, Gari MA, et al. Molecular basis of β-thalassemia in the western province of Saudi Arabia: Identification of rare β-thalassemia mutations. Hemoglobin 2011;35:346-57.  Back to cited text no. 11
    
12.
Al-Jaouni SK. Prevalence of thalassemia disorders and hemoglobinopathies in Jeddah, Western Saudi Arabia. J Appl Hematol 2010;1:43-6.  Back to cited text no. 12
    
13.
Dahlawi HA, Zaini RG, Almehmadi M, Zamzami O. Hemoglobinopathies among Saudi adults at Taif city, Saudi Arabia. Biosci Res 2019;16:1391-4.  Back to cited text no. 13
    
14.
Alsaeed ES, Farhat GN, Assiri AM, Memish Z, Ahmed EM, Saeedi MY, et al. Distribution of hemoglobinopathy disorders in Saudi Arabia based on data from the premarital screening and genetic counseling program, 2011-2015. J Epidemiol Glob Health 2018;7:S41-7.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed700    
    Printed12    
    Emailed0    
    PDF Downloaded69    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]