Spectrum of types of thalassemias and hemoglo-binopathies : study in a tertiary level children hospital in Bangladesh

Thalassemias and hemoglobinopathies are the most common hemolytic congenital disorders in Bangladesh as in many parts of the world. This study was done to find the common types of thalassemias and abnormal hemoglobin variants seen in Bangladeshi populations. A total of 4813 samples were analyzed for hemoglobin disorders out of which 2308 (49.95%) showed abnormalities. The samples were analyzed by Bio Rad D 10 Analyzer in 3914 (81.32%) cases, BIORAD VARIANTβ thalassemia short program using the principle of high performance liquid chromatography in 474 (9.85%) cases and by CAPILLARYS 2 FLEX PIERCING utilizing capillary electrophoresis in 425 (8.83%) cases. The samples were analyzed in the Department of Biochemistry and Molecular Biology of Dhaka Shishu (Children) Hospital, Dhaka, Bangladesh. The common hemoglobin disorders seen were β trait 863 (17.94%), Hb E trait 601 (12.50%), Hb E β thalassemia 524 (10.87%), β thalassemia major 192(4.00 %), Hb E disease 99 (2.05%). Other Hb abnormalities detected were Hb D trait 17 (0.35%), Sickle cell trait 4 (0.08%), hereditary persistence of fetal hemoglobin (HPFH) 2 (0.04%), and Hb Lepore, δ β thalassemia, sickle cell β thalassemia, Sickle cell disease, compound heterozygote for HbE+D and Hb Q band one case each (0.02%).


Introduction
An estimate of World Health Organization shows that 7% of the world population are a carrier for hemoglobin disorders and 300,000 to 500,000 children are born with clinically significant hemoglobin disorders every year and majority of them are born in developing countries. 1Inherited disorders of hemoglobin both structural and synthesis are an important cause of morbidity and mortality.World wide 3.4% of mortality rate in children under 5 years age are due to Hb disorders. 2Birth of a single child with a hemoglobin disorder in a family poses enormous psychological trauma along with financial strain for treatment especially in developing countries like Bangladesh.The aim of this study was to determine the types and frequencies of thalassemias and hemoglobinopathies seen in a tertiary level children hospital (Dhaka Shishu (Children) Hospital) in Bangladesh from referred cases of subjects presenting with anemia, abnormal hemograms and for detection of carriers.It is important to know the common Hb variants for diagnostic and preventive planning and to create awareness of physicians to refer cases with abnormal hemograms to rule out hemoglobin disorders.Most of the samples were from children.

Materials and Methods
The study was conducted in the Department of Biochemistry and Molecular Biology of Dhaka Shishu (Children) Hospital, Dhaka, Bangladesh.
A total of 4813 samples were analyzed for hemoglobin disorders over the period of 4 years from August 2011 to September 2015.Two ml of blood were collected in EDTA, and RBC indices were done by using automated Hematology analyzer SIEMEN ADVIAi and Mytic 22AL.Hb A2,HbF and hemoglobin variants levels were obtained by high pressure liquid chromatography (HPLC) by Bio Rad 10 analyzer, BIORAD VARIANT β thalassemia short program and by CAPILLARYS 2 FLEX PIERCING utilizing capillary electrophoresis and tests done according to procedures provided by manufactures.Samples from subjects below one year were excluded as the presence of Hb F, which is seen below one year, may interfere with interpretation of results.Also subjects with a history of recent blood transfusion were advised to come after four months from the last transfusion.Serum iron or ferritin levels were not done as samples were sent to the laboratory to do electrophoresis only by the referring physicians.
β thalassemia trait was the most common β chain carrier abnormality detected in 863 (17.94%) cases.
Hb E trait was the most common abnormal hemoglobinopathy detected in 601 (12.50%) cases.

Discussion
This study shows the types and frequencies of thalassemias and abnormal hemoglobins seen in subjects referred to a tertiary level children hospital for analysis of Hb disorders presenting with anemia, abnormal hemograms or for detection of carriers.
Hb abnormalities were detected in 2308( 47.95%) cases and thus signifying that disease of Hb disorders is a very significant genetic problem in Bangladesh.
Hb Eβ thalassemia was the most common type of thalassemia seen constituting o n l y 524 (10.87%) of cases.][6] Hemoglobin E β thalassemia shows a varied presentation from a mild type of thalassemia to severe type like beta thalassemia major. 7,8 thalassemia major was the second most common type of thalassemia seen.It was seen in 192 cases (4.00%) which was higher than in comparison to the study by Uddin et   3,5,6 It may be due to the fact that most of our samples were from children and its presentation is earlier than Hb E β thalassemia.Thalassemia major occurs due to mutations of β globin genes resulting in the absence or decreased production of the globin genes.
There was one case (0.02%) each of sickle cell β thalassemia and δβ thalassemia.δβ thalassemia was diagnosed in one subject when HPLC report showed complete absence of Hb A and A2 with 100% Hb F. Hb Analysis of parents revealed increased level of Hb F in both with low MCV and MCH and was diagnosed as δβ thalassemia.It needs to be confirmed by DNA analysis, which we could not do due to lack of facilities in our laboratory.
Sickle cell β thalassemia was seen in only 1 case (0.02%).5][6] HbS gene is mainly seen in Central India like Maharashtra, Madhya Pradesh and Gujarat while it is rare in other states of India. 9Hb S gene is characterized by replacement of glutamine to valine at position 6 of beta globin chain. 10][6] β thalassemia trait was seen in 863 cases (17.94%) and its frequency was rather high as most of the cases referred had low Hb or abnormal RBC indices and specially samples were sent to rule out β thalassemia trait from iron deficiency.Nearly 1.5% of the world's population are carriers of β trait. 11High frequency of β trait was also detected by Jain et al and Chaudhury et al in their study as their samples were also referred cases with abnormal hemogram. 4,6n β trait Hb may be lower than normal with elevation of RBC count and low MCV and MCH and a HbA 2 level typically between 4-6%.The blood film shows microcytosis, anisocytosis, poikilocytosis and hypochromia. 12b E is one the most common and important mutations in the world.It was seen in 601 cases (12.50%) in this study.It is caused by the substitution of lysine for glutamic acid at position 26 of the beta globin chain. 13Hb E traits is mainly seen in the eastern half of Indian sub-continent, Bangladesh, Myanmar and east and south east Asia. 14In Hb E trait carriers the red blood cells are either normal or mildly microcytic and few target cell may be seen.The frequency of β trait and Hb E trait in Bangladeshi population is 4.1% and 6.1%. 12,15b E disease was detected in 99 cases (2.05%)The frequency of Hb E disease detected was rather high in the study by Uddin et al. 9.2%, while other studies from West Bengal by Mondal et al. and Chaudhury et al. found 0.39% and 0.12% respectively. 3,5,6Hb E disease is clinically normal, but the blood film shows hypochromia with variable number of target cells.HPLC shows the major hemoglobin to be Hb E constituting 85% to 99% of the total hemoglobin the rest being hemoglobin F. The differential diagnosis is with Hb E βthalassemia and needs the help of clinical features, family studies and sometimes DNA analysis. 16Most of our cases were confirmed by testing of parent's blood showing Hb E trait in both the parents.
Hb D trait also known as D Punjab was detected in 17 (0.35%).Hb D Punjab is seen in northwest India, Pakistan and Iran. 17It is also a common abnormal Hb variant in the population of Denizli of South Eastern Turkey and also in Xinjiang province of China. 18,19Hb D Punjab differs structurally from normal Hb A at 121 position of beta globin chain where glutamine replaces glutamic acid. 20Heterozygous Hb D Punjab is clinically normal and blood film may show some target cells.However its association with Hb S results in moderate to severe clinical manifestations. 21This mutation may have come from migration of people from western part of India during Muslim rule in Bengal.
Other rare Hb disorders detected in this study were sickle cell trait, sickle cell anemia and Hb Lepore and hereditary persistence of fetal hemoglobin (HPFH).HPFH was reported when HbF level between 15% to 30% with normal hemogram.

Conclusions
This study shows that hemoglobin disorders is a significant genetic problem in this country Measures needs to be taken to prevent the births of thalassemic children with hemoglobin disorders like thalassemia by creating awareness, education of medical doctors, screening of population and prenatal diagnosis.Furthermore physicians should be made aware of the fact that all abnormal hemograms shown in cell counter should be referred to laboratories to detect Hb disorders and not brushed aside as iron

Thalassemia
Reports 2017; volume 7:6354 Correspondence: Waqar A. Khan, Department of Biochemistry and Molecular Biology Dhaka Shishu (Children) Hospital, Bangladesh Institute of Child Health, Sher-e-Bangla Nagar, Dhaka 1207, Bangladesh.i a l u s e