Spectrum of hemoglobinopathies in a tertiary care centre

HbD/HbE hemoglobinopathies; Hemoglobin electrophoresis; Hemoglobinopathies; Sickle cell disorders; Thalassemia; Background: Thalassemia and other structural hemoglobinopathies are the most common single gene disorders throughout the world with the highest frequency in the tropics, subtropics, Mediterranean basin and Southeast Asia. This study aims to provide a better assessment on the spectrum of hemoglobinopathies in our context.


INTRODUCTION
Hemoglobinopathies are characterized by the presence of structurally defective haemoglobin (Hb) due to abnormalities in the formation of globin moiety of the molecule. 1 Hemoglobinopathies fall into two main groups: thalassemia syndromes and structural hemoglobin variants (abnormal hemoglobin). 2 D (Hb D) disease is derived from a point mutation in the β-globin gene with the substitution of glutamine for glutamic acid at 121 st position. 4 Hemoglobin E (Hb E) is produced due to the substitution of lysine for glutamic acid at 26 th position. 5 This study was designed to provide a better assessment on the spectrum of hemoglobinopathies in Nepal. The frequencies of various hemoglobinopathies, age and sex distribution, geographical distribution, ethnic communities commonly affected were analysed and compared between different disorders.

MATERIALS AND METHODS
This descriptive study is a hospital based cross-sectional study carried out in the Department of Pathology, TUTH. The study duration was of one year (November 1, 2014 to October 31, 2015). Ethical approval was obtained from Institutional Review Board of the institute and consent was taken while filling the proforma. 350 patients were initially registered for the work up of hemoglobinopathies. Out of 350 cases, 97 were diagnosed to have hemoglobinopathies. The study included 97 samples for further analysis. Patients with inconclusive/normal Hb electrophoresis results, the repetition of the same case, those who received blood transfusion three months back were excluded from the study. EDTA anti-coagulated blood was used. Hematological indices were derived from Sysmex 500i 5-part coulter counter. Morphological alterations in red cells were studied. Hemoglobin electrophoresis was carried out by cellulose acetate medium at alkaline PH. Other tests as sickling test using sodium metabisulfite, HbH inclusions (Golf balls) demonstration using brilliant cresyl blue stain, and Hb F estimation by Singer's method were performed. Detailed history (like age, sex, caste, ethnicity, and place of origin) was recorded. Parental screening was done to confirm the diagnosis wherever feasible. The data collection was done in pre-designed proforma and data entry was done in SPSS and results were computed using Statistical Package for the Social Sciences (SPSS) version 20.

RESULTS
A total number of 350 blood samples were received in the Department of Pathology for hemoglobin electrophoresis during the study period of one year, out of which 97 cases (27.71%) had hemoglobinopathies.

DISCUSSION
Hemoglobinopathies affect people worldwide including Nepal. However, there is a limitation of scientific study which demonstrates the actual burden of the same. This study made evident that hemoglobinopathies were seen widespread in Nepal. Thalassemia were the most common constituting 57.73% of the total cases, the findings being broadly consistent with the studies done by Mondal   had hemoglobinopathies out of 62 patients revealed higher frequencies of hemoglobinopathies in males. 10 In our context, 49.48% of the hemoglobinopathies were seen in the age group 21-30 years, followed by 23.71% under 10 years of age. Whereas, Tiwari in her study in Nepal had drawn the conclusion of maximum cases in under-10 age group. 11 This could be because in our context parental screening was done for almost all cases of homozygous hemoglobinopathies, and majority of the parents fell into that age range.
Sharma N et al.

Figure 1: Map of Nepal showing various districts affected by hemoglobinopathies. Dang district, in Terai terrain accounted for the majority (26 cases)
DOI : 10.3126/jpn.v10i1.27369   13,14 In addition, the practice of consanguineous marriages also contributes to higher rate of hemoglobinopathies in the Terai region.
The most predominant hemoglobinopathy was β-thalassemia trait (36 cases, 37.11%), the observations being congruent with the studies in Eastern India and in Pakistan. 6,10 Maximum cases were the parents of children with homozygous diseases such as β-thalassemia major, Sickle β-thalassemia and E β-thalassemia. The HbS β-thalassemia patients had a more severe disease with lower Hb level, MCV (8.57 g/dl, 64.17fl) as compared to HbSS (8.86 g/dl, 67.08 fl) which was comparable to the study done in Central India. 15 Our research also touched on a few rarer hemoglobinopathies as hemoglobin D hemoglobinopathies which are seen sporadically in Blacks and Europeans. Fast moving Hb H bands were seen in 5 cases (5.15%) of all the hemoglobinopathies embracing three children and two adults. Furthermore, numerous HbH inclusions were demonstrated with brilliant cresyl blue stain. Nadkarni et al in his paper "Molecular diversity of Hemoglobin H Disease in India" has unequivocally elucidated that Hb H disease is the most severe form of α-thalassemia compatible with life. 16 This statement was consonant with our results as age at diagnosis varied from 11 months to 32 years. E-thalassemia trait and Hb-E disease in our context was 6.19% and 1.03% which was almost similar to the values found in Thailand where the prevalence was 5.2% and 0.9 % respectively. 17 These findings are also akin to other studies that show hemoglobin E hemoglobinopathies to be common among Southeast Asians. 18 Hemoglobinopathies pose considerable economic and psychosocial burden on the affected individuals, families, society and the country, at large. This study through its findings aims to come up with conclusive results that will help to induce changes and amendments in the health sector for instance, screening of the population, identification of carriers and adoption of appropriate measures for prevention of hemoglobinopathies.

LIMITATION
The study being an institutional study, samples in our research may not be representative of the whole nation. Some of the disorders may require HPLC and capillary hemoglobin electrophoresis along with DNA testing for confirmation not available in our institute.

CONCLUSIONS
This study made evident the widespread prevalence of hemoglobinopathies in Nepal. The population of Nepal, essentially those from Terai territory were seen the most significant group affected by hemoglobinopathies. Thalassemia was seen to be the most prevailing hemoglobinopathies followed by sickle cell disorders and HbD/HbE hemoglobinopathies. Tharu/Chaudhary population was seen to be the predominant ethnic tribes affected by the same with many of the cases seen clustered in the families. Hemoglobinopathies pose considerable economic and psychosocial burden on the affected individuals, families, society and the country, at large.  It is very important that the population be screened so that carriers could be detected and informed about the various complications and reproductive risks. In addition, preventive measures could also be adopted in the form of genetic counseling, prenatal diagnosis and termination of the affected babies.