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Year : 2020  |  Volume : 63  |  Issue : 4  |  Page : 663-665
Hb Yaizu: A rare beta-globin chain variant posing diagnostic dilemma in high-performance liquid chromatography


1 Sections of Hematology, Coagulation and Flow Cytometry, SRL Limited, Mumbai, Maharashtra, India
2 Department of Hematogenetics, National Institute of Immunohematology, Mumbai, Maharashtra, India

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Date of Submission31-Oct-2019
Date of Decision18-Feb-2020
Date of Acceptance23-Feb-2020
Date of Web Publication28-Oct-2020
 

How to cite this article:
Gupta AD, Daruwalla MR, Pawar R, Sidhwa K, Hariharan P, Nadkarni A. Hb Yaizu: A rare beta-globin chain variant posing diagnostic dilemma in high-performance liquid chromatography. Indian J Pathol Microbiol 2020;63:663-5

How to cite this URL:
Gupta AD, Daruwalla MR, Pawar R, Sidhwa K, Hariharan P, Nadkarni A. Hb Yaizu: A rare beta-globin chain variant posing diagnostic dilemma in high-performance liquid chromatography. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Nov 25];63:663-5. Available from: https://www.ijpmonline.org/text.asp?2020/63/4/663/299332




Morbidity associated with genetic abnormalities of hemoglobin (Hb) can be widely variable. An accurate diagnosis of Hb variants is necessary for distinguishing cases requiring medical intervention and preventive measures, including genetic counseling, from those that are clinically silent.[1],[2] HbS is a commonly encountered pathogenic hemoglobinopathy in India. With the increasing use of high-performance liquid chromatography (HPLC) as the primary mode of screening for hemoglobinopathies, non-HbS Hb variants eluting in the HbS window pose diagnostic challenges. Hb Yaizu (Codon 79 GAC > AAC; Beta 79 [EF3] Asp > Asn) is one such hemoglobin.[3],[4] Very few cases of this rare beta-globin chain variant have been reported in the literature since the first case published from Japan in 1995.[3] Persons with this mutation are asymptomatic and have normal hematological parameters.[3],[4] Herein, we describe a family with this defect that highlights the importance of a systematic approach to the diagnosis of hemoglobinopathies that mimic HbS in HPLC. This possibly is the first case report of Hb Yaizu from India.

The propositus (55-year-old), his wife (30-year-old), and son (6-month-old) hail from Vishakhapatnam, Andhra Pradesh.

Complete blood count (CBC) was carried out on an automated analyzer, LH780 (Beckman Coulter, Miami, FL, USA). The Hb abnormality was first identified by HPLC (Variant II, Bio-Rad, Hercules, CA, USA). Sickling (using 1% sodium metabisulfite solution), solubility, and heat stability tests were performed using standard methods. Further analysis of the abnormal Hb was carried out by cellulose acetate electrophoresis at an alkaline pH (8.9). The molecular defect was characterized by DNA sequencing. The genomic DNA was extracted by the conventional phenol-chloroform technique and the beta-globin gene variation was detected by automated DNA sequencing (3130 XL genetic analyzer, Applied Biosystems, Foster City, USA).

Blood counts were normal in the propositus and his wife [Table 1]. The son had microcytic hypochromic anemia with the red cell parameters suggesting iron deficiency. The abnormal Hb eluted as sharp peaks (39.4% and 34.2%) in the HbS window with retention times (RT) of 4.60 and 4.57 min in the index case and his son, respectively [Figure 1]. HPLC did not show any abnormal Hb in the wife. The HbA2 was elevated (4.7%) in the propositus and his son (4.8%). Interestingly, the HbA2 peaks in both showed a hump in its descending slope similar to that seen in cases of HbS. HbA2 was normal (2.9%) in the wife. HbF was mildly elevated (8%) in the son which was commensurate with his age (6 months). The sickling test was negative in the father and the son. In Hb electrophoresis done at alkaline pH, the abnormal Hb (Hb X) moved slightly faster than Hb S [Figure 2] and [Table 1] but slower than Hb S at acidic pH. DNA sequencing confirmed the abnormal Hb in the index case and his son as Hb Yaizu (Codon 79 GAC > AAC; Beta 79 [EF3] Asp > Asn) [Figure 3]. None of the family members had thalassemic defects on gene sequencing.
Table 1: Red cell parameters, HPLC findings, and gene sequencing data in the patient and his family members

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Figure 1: HPLC histogram in the child (upper panel) and the propositus showing Hb Yaizu at a retention time of 4.57 and 4.60 min, respectively. Also, note the hump in the descending limb of the HbA2 peak in both. The child had mildly elevated HbF, consistent with his age

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Figure 2: Cellulose acetate electrophoresis of Hb Yaizu in alkaline pH (Lane 1) showing a slow-moving Hb band (horizontal thick arrow) that is faster than HbS (Lane 5). Lanes 2, 3, and 4 show normal Hb patterns. The predominant band in all lanes is HbA. The direction of movement of Hb bands is shown by the vertical arrow

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Figure 3: Hb Yiazu - beta-globin gene mutation in the propositus: Codon 79 G > A, heterozygous

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As reported earlier,[5],[6] several non-HbS hemoglobins, both alpha and beta-globin chain variants, elute out in the narrow HbS window (RT 4.3–4.70 min) in HPLC. With HPLC increasingly becoming the primary screening modality for hemoglobinopathies, this fact poses a major challenge in distinguishing the non-HbS variants, especially the clinically silent ones, from HbS. The high prevalence of sickling disorders in many parts of India further compounds the problem and can lead to misdiagnosis of such non-HbS variants as HbS. This fact is amply demonstrated by us in this family with Hb Yaizu (Codon 79 GAC > AAC; Beta 79 [EF3] Asp > Asn) - a beta-globin chain variant that elutes out in the HbS window in HPLC. The presence of a hump on the descending limb of an elevated HbA2 peak in both our cases affected by this hemoglobinopathy further pointed to the possibility of HbS on initial screening of the HPLC chromatogram. Therefore, as shown by us, elution of an abnormal Hb peak in the HbS window calls for a step-wise diagnostic approach. Most of the suspected cases of HbS get confirmed by simple laboratory tests such as sickling and solubility tests and do not require molecular assays. In non-HbS variants, however, these tests are not helpful and DNA sequencing becomes mandatory for diagnosis. The slight difference in the RT of Hb Yaizu in the father and the child (4.6 vs. 4.57 min) could be due to the inter-assay variation of the HPLC system.

The reasons for the elevated level of HbA2 and the presence of a hump in the descending arm of the HbA2 peak in the propositus and his son could be similar to that observed in HPLC of HbS cases, i.e., formation of adduct Hb (glycated Hb Yaizu in this case) and elution of the glycated abnormal Hb along with HbA2 in HPLC.[7],[8] Furthermore, the increased HbA2 in these cases may be also a reflection of preferential binding of normal delta globin chains with the normal alpha-globin chain as proposed to explain the elevated HbA2 in patients with HbS.[7] Mildly elevated HbF in the son of the index case is consistent with his age.

Although Hb Yaizu per se is clinically silent, it would be interesting to observe the outcome of its interaction with the other (pathogenic) hemoglobinopathies; both beta and alpha chain variants. A case in point is the increased severity of the disease in double heterozygotes for HbS and HbD Punjab compared to HbS trait patients. Independently, HbD Punjab heterozygotes are asymptomatic and have a normal hematological profile while HbS trait patients have mild disease.[1]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Wajcman H, Moradkhani K. Abnormal haemoglobins: Detection and characterization. Indian J Med Res 2011;134:538-46.  Back to cited text no. 1
[PUBMED]  [Full text]  
2.
Colah RB, Surve R, Sawant P, D'Souza E, Italia K, Phanasgaonkar S, et al. HPLC studies in hemoglobinopathies. Indian J Pediatr 2007;74:657-62.  Back to cited text no. 2
    
3.
Harano T, Harano K, Katsuki T. Hb Yaizu [beta 79(EF3) Asp 1 Asn]: A new beta chain variant found in a Japanese female. Hemoglobin 1995;19:21-5.  Back to cited text no. 3
    
4.
Atalay EO, Atalay A, Koyuncu H, Öztürk O, Köseler A, Özkan A, et al. Rare hemoglobin variant Hb Yaizu observed in Turkey. Med Princ Pract 2008;17:321-4.  Back to cited text no. 4
    
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Das Gupta A, Hariharan P, Daruwalla M, Sidhwa K, Pawar R, Nadkarni A. Hemoglobin titusville [×2 codon 94 G>A] – A rare alpha globin chain variant causing low oxygen saturation. Indian J Hematol Blood Trans 2019;35:593-5.  Back to cited text no. 5
    
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Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: A study of 60,000 samples in a clinical diagnostic laboratory. Clinical Chemistry 2004;50:1736-47.  Back to cited text no. 6
    
7.
Griffin PJ, Sebastiani P, Edward H, Baldwin CT, Gladwin M, Gordeuk V, et al. The genetics of hemoglobin A2 regulation in sickle cell anemia. Am J Hematol 2014;89:1019-23.  Back to cited text no. 7
    
8.
Fonsecaa SF, Amorimb T, Purificac A, Goncalvesc M, Boa-Sorteb N. Hemoglobin A2 values in sickle cell disease patients quantified by high performance liquid chromatography and the influence of alpha thalassemia. Brazilian J Hematol Hemotherapy 2015;37:296-301.  Back to cited text no. 8
    

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Correspondence Address:
Amar Das Gupta
603 Janimal Tower, Sector 17, Plot 42, Vashi, Navi Mumbai - 400 703, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_851_19

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