Comparison of HPLC results of patients with hemoglobin D with DNA sequence analysis: Detection of compound heterozygosity HbD/β-Thalassemia traits

Ayse Aktas; Latife Yilmazsamli; Belkiz Ongen Ipek; Eren Vurgun; Mustafa Sahin; Okan Dikker; Huseyin Dag

doi:10.5281/zenodo.14183773

Authors

Ayse Aktas Department of Medical Biochemistry, Istanbul Prof. Dr. Cemil Taşcıoğlu City Hospital, University of Health Sciences, Istanbul 34384, Turkey https://orcid.org/0009-0008-1301-562X
Latife Yilmazsamli Department of Medical Biochemistry, Istanbul Prof. Dr. Cemil Taşcıoğlu City Hospital, University of Health Sciences, Istanbul 34384, Turkey https://orcid.org/0009-0002-4240-7215
Belkiz Ongen Ipek Department of Medical Biochemistry, Istanbul Prof. Dr. Cemil Taşcıoğlu City Hospital, University of Health Sciences, Istanbul 34384, Turkey https://orcid.org/0000-0002-2998-263X
Eren Vurgun Department of Medical Biochemistry, Istanbul Prof. Dr. Cemil Taşcıoğlu City Hospital, University of Health Sciences, Istanbul 34384, Turkey https://orcid.org/0000-0002-2288-1123
Mustafa Sahin Department of Medical Biochemistry, Hitit University Faculty of Medicine, Çorum 19030, Turkey https://orcid.org/0000-0001-6073-563X
Okan Dikker Department of Medical Biochemistry, Istanbul Prof. Dr. Cemil Taşcıoğlu City Hospital, University of Health Sciences, Istanbul 34384, Turkey https://orcid.org/0000-0002-9153-6139
Huseyin Dag Department of Pediatrics, Istanbul Prof. Dr. Cemil Taşcıoğlu City Hospital, University of Health Sciences, Istanbul 34384, Turkey https://orcid.org/0000-0001-7596-7687

DOI:

https://doi.org/10.5281/zenodo.14183773

Keywords:

DNA analysis, Hemoglobin D, Hemoglobin variant analysis, HPLC, β-Talassemia

Abstract

Objective: In our study, we aimed to compare the results of patients with near-total hemoglobin D variants detected by the HPLC method with DNA sequence analysis.

Materials and methods: In our laboratory, hemoglobin variant analysis was performed with an HPLC analyzer (Adams A1C HA8180T, Arkray, Inc., Kyoto, Japan) from EDTA-tubed whole blood samples of three male patients. PCR-DNA Sanger sequence analysis was then performed on these samples for a definitive diagnosis.

Results: We found that three male patients had hemoglobin D variants close to the total by HPLC method (HbD levels are 97.92%, 93.2%, and 97.6%, and HbA levels are 0% for all patients, respectively). In two patients, we did not detect hemoglobin A2 levels by HPLC, while in one patient, we detected <3.5% HbA2. According to the results of PCR-DNA sequence analysis, we found that all three patients had heterozygous Hemoglobin D, characterized by the p.Glu121Gln (c.364G>C) mutation. In addition, the patients had c.92 +5G>C, IVS2-1G>A (c.315+1G>A), and p.Lys9Valfs*14(c.25_26del) mutations, which were pathologically identified and consistent with heterozygous β-thalassemia carriage. Thus, we found that three patients had compound heterozygous HbD/β-thalassemia.

Conclusion: The presence of hemoglobin D is close to total hemoglobin D in percentage, according to the HPLC result, but does not always indicate homozygous hemoglobin DD. In addition, β-thalassemia carriage may be missed from the laboratory results of those who are carriers of the hemoglobin D variant and have nonincreasing hemoglobin A2 levels. In these patients, it should be clarified whether they are β-thalassemia carriers by genetic methods. In this way, the birth of babies with β-thalassemia major can be prevented.

References

Harteveld CL, Achour A, Arkesteijn SJG, Ter Huurne J, Verschuren M, Bhagwandien-Bisoen S, et al. The hemoglobinopathies, molecular disease mechanisms and diagnostics. Int J Lab Hematol. 2022;44(1):28-36.

Dikker O, Vardar M, Sandıkçı R, Basatb, Sucu V, Vurgun E, et al. Abnormal hemoglobin variants detected by HPLC method in Okmeydanı training and research hospital medical biochemistry laboratory. Okmeydani Med J. 2016;32(4):185-9.

Pandey S, Mishra RM, Pandey S, Saxena R. Molecular characterization of hemoglobin D Punjab traits and clinical-hematological profile of the patients. Sao Paulo Med J. 2012;130(4):248-51.

Origa R. β-Thalassemia. Genet Med. 2017;19(6):609-19.

Panyasai S, Sakkhachornphop S, Pornprasert S. Diagnosis of compound ceterozygous Hb Tak/β-thalassemia and HbD-Punjab/β-thalassemia by HbA2 levels on capillary electrophoresis. Indian J Hematol Blood Transfus. 2018;34(1):110-4.

Taghavi Basmanj M, Karimipoor M, Amirian A, Jafarinejad M, Katouzian L, Valaei A, et al. Co-inheritance of hemoglobin D and β-thalassemia traits in three Iranian families: clinical relevance. Arch Iran Med. 2011;14(1):61-3.

Denic S, Souid A-K. Hemoglobin D-Punjab homozygotes and double heterozygotes in premarital screening: Case presentations and mini review. Eur J Med Health Sci. 2021;3(1):90-4.

Theodoridou S, Alemayechou M, Perperidou P, Sinopoulou C, Karafoulidou T, Kiriakopoulou G. Compound heterozygosity for Hb D-Punjab / β-thalassemia and blood donation: case report. Turk J Haematol. 2009;26(2):100-1.

Dikker, O, Vardar M, Usta M, Dağ H. Hemoglobin Variant Analysis Methods. Okmeydani Med J. 2016;32(3):161-6.

Crossley BM, Bai J, Glaser A, Maes R, Porter E, Killian ML, et al. Guidelines for Sanger sequencing and molecular assay monitoring. J Vet Diagn Invest. 2020;32(6):767-75.

Zakerinia M, Ayatollahi M, Metal R. Hemoglobin D(HbD Punjab/ Los Angeles and HbD Iran) and co-inheritance with a- and beta-thalassemia in southern Iran. Iran Red Crescent Med J. 2011;22(7):493–8.

Adekile AD, Kazanetz EG, Leonova JY, Marouf R, Khmis A, Huisman TH, et al. Co-inheritance of Hb D-Punjab (codon 121; GAA-->CAA) and beta (0)-thalassemia (IVS-II-1;G-->A). J Pediatr Hematol Oncol. 1996;18(2):151-3.

Van Delft P, Lenters E, Bakker-Verweij M, de Korte M, Baylan U, Harteveld CL, et al. Evaluatingfive dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnic populations. Int J Lab Hematol. 2009;31(5):484–95.

Keren DF, Hedstrom D, Gulbranson R, Ou CN, Bak R. Comparison of Sebia Capillarys capillary electrophoresis with the Primus high-pressure liquid chromatography in the evaluation of hemoglobinopathies. Am J Clin Pathol. 2008;130(5):824-31.

Owaidah TM, Al-Saleh MM, Al-Hellani AM. Hemoglobin D/beta-thalassemia and beta-thalassemia major in a Saudi family. Saudi Med J. 2005;26(4):674-7.

Belhoul KM, Bakir ML, Abdulrahman M. Misdiagnosis of Hb D-Punjab/β-thalassemia is a potential pitfall in hemoglobinopathy screening programs: a case report. Hemoglobin. 2013;37(2):119-23.

Ozdemır S, Oruc MA, Yazıcıoglu B, Turkan S. Premarital hemoglobinopathy screening program results of a province in the Black Sea region of Turkey: three years' experience. Postgrad Med. 2023;135(8):818-23.