1 THESIS INTRODUCTION Essentials of the topic Thalassemia (thal) and hemoglobinopathies (Hb variant) are the most common inherited monogenic disorders in the world, causing anemia due to congenital hemolysis Southeast Asia has four common types: α-thalassemia, β-thalassemia, hemoglobin E (Hb E) and Hb Constant Spring (HbCS) with a prevalence vary between 4.5 to 40%; to 9%; to 8%; Hb E can be up to 50-60% depending on region The clinical manifestations of thalassemia syndrome vary from asymptomatic to transfusion dependent, even death in the fetus The Vietnamese-Khmer is one of the largest ethnic minority in the country with about 1.3 million people living in the Mekong Delta The rate of Hb E in the Khmer is from 20 to 30%; that of β-thal is about 1.7%, causing high risk of severe genetic combinations Preventing the severe forms, we need information on the frequency of gene, distribution of gene mutations, clinical and hematological characteristics of the disease in the community Therefore, we conducted this dissertation with the following objectives: Determine the prevalence of thalassemia and hemoglobinopathies, the rate of globin gene mutations in the Khmer ethnic group in the Mekong Delta Describe some clinical and hematological characteristics of thalassemia and hemoglobinopathies in the Khmer ethnic group in the Mekong Delta Meaning and urgency of the topic Thalassemia and hemoglobinopathy are groups of the most common causes of congenital hemolysis in the world and Southeast Asia, with marked geographical and racial features Vietnam has a high prevalence of thalassemia and Hb E, especially Hb E The Khmer minority is the most populous ethnic groups in the Mekong Delta, living mainly in rural areas with a high risk of developing severe disease according to literature The study of the prevalence and characteristics of thalassemia and hemoglobinopathy in the Khmer community contributes to make a sketch of thalassemia and hemoglobinopathies maps in our country, thus providing data for screening, genetic counseling and prenatal diagnosis It aims to reduce the incidence of severe disease and carriers in the community Therefore, the topic is urgent New contributions The dissertation provides information on thalassemia and hemoglobinopathies in the Khmer ethnic group in the Mekong Delta as follows: - Rate of thalassemia and hemoglobinopathies carriers, and rate of globin gene mutation types in the community - Identification of Hb Tak variant for the first time, especially in the Khmer group in Vietnam and its some hematological characteristics - Describe clinical and hematological characteristics of αthalassemia combined Hb E and Hb E - Define the role of red blood cells indexes, OF test and DCIP test in the screening of thalassemia and hemoglobinopathíe in the Khmer ethnic group in the Mekong Delta Structure of the thesis: The thesis consists of 133 pages, introduction pages, overview 32 pages, subjects and research method 26 pages, results 28 pages, discussion 42 pages, conclusion page, recommendation page There are 37 tables, 10 charts, 16 figures, flowcharts and 158 references (36 in Vietnamese and 122 in English) Chapter OVERVIEW Hemoglobin (Hb) is a tetramer of four polypeptide chains (also called globin chains) attached to four heme bases that help the red blood cells to carry oxygen The globin chain in hemoglobin is of the same pair In postpartum period, the main Hb type is HbA (α2β2) accounting for 97-98%; HbA2 (α2δ2) 2-3%, the main Hb during pregnancy is HbF (α2γ2), but in adults there’s only trace 1.1 Classification of thalassemia and hemoglobinopathies Hemoglobinopathy consists of two major groups: thalassemia syndrome: a group of diseases due to reduce of globin chains, called α-, β-, γ-, δ-, δβ-, or εγδβ-thal depending on sort of defective globin chains; hemoglobin variant (hemoglobinopathy): due to structural defects of the globin chain, one or two amino acids in the chain are replaced by other amino acids Types of thalassemia syndrome can be combined together or combined with hemoglobin variants to create diverse clinical phenotypes 1.2 Characteristics of thalassemia and hemoglobin E 1.2.1 β -thalassemia β-thalassemia minor (heterozygous β-thalassemia): no symptoms or mild anemia Peripheral total blood count (TBC) shows increased red cell count (RBC), low MCV and MCH, normal RDW, hypochromic microcytosis, target cells, basophilic stippling or contracted cells… could be present Hemoglobin analysis shows increased HbA2 by 3.5%, depending on gene mutation types, 30-50% of cases combines with increased HbF from 2% to 7% HbF increase may be due to hereditary persistence of fetal hemoglobin (HPFH), δβ-thalassemia β-thalassemia major: blood transfusion dependance, clinical presentation occurs in about 6-24 months with severe anemia, jaundice, hepatosplenomegaly Bone marrow hypertrophy results in typical skull and face deformity Patients are regularly treated with blood transfusions and iron chelation with many serious blood transfusion associated complications In blood tests, low Hb level from to g/dL, markly low MCV, MCH, high RDW; hypochromic microcytosis, irregularly shaped, anisocytosis, fragments, and nucleated red cells The Hb component changes according to β thalassemia genotype β0/β0 without HbA, 92-95% of HbF; β0/β+ or β+/β+ HbA from 10 to 30%, sometimes up to 35%; in β thalassemia major, HbA2 may be normal, increased or even decreased β-thalassemia intermedia: clinical manifestations may range from asymptomatic as β-thalassemia minor to transfusion dependent as β-thalassemia major Age of onset is one of the most useful indicators of homozygous or compound heterozygous βthalassemia 1.2.2 α -thalassemia α-thal minor/trait: caused by a deletion of one or two α globin gene, or by non-deletion mutations (-α/αα, /αα, -α/-α or αTα/αα, ααT/αα), without clinical manifestations and slight changes in red blood cell indexes, may not be detected or diagnosed only during routine or prenatal screening Loss of two α globin genes, low MCV and MCH, increased RBC Analysis of umbilical cord, Hb analysis can determine 1-10% of Hb Bart's HbH disease: consists of a single functional α globin gene ( /-α, or /α Tα.) HbH disease due to deletion has a fairly similar phenotype, whereas HbH disease due to non-deletion mutation varies significantly In general, it is difficult to predict Hb H phenotype if basing only on genotype especially in cases of nondeletion mutation In TBC, HbH patients usually have low Hb, MCV and MCH, but high RBC and high reticulocyte Peripheral blood smear, shows hypochromic microcytosis, irregularly shaped, anisocytosis, fragments, and nucleated red cells Brilliant Cresyl Blue coloration shows HbH inclusion in most red cells Hb Bart's level at birth is about 19-27%, and gradually decreases and will be replaced with HbH, but almost at lower level Hb Bart's syndrome: genotype is ( / ), which is the most severe α thalassemia type Fetus is almost death at about 23-38 th week because of severe complications Hb analysis shows 100% of Hb Bart's 1.2.3 Hb E disease Hb E is the most common Hb variant in Southeast Asia HbE can interact with α thalassemia, β thalassemia, or other Hb variants resulting in multiple thalassemic syndromes with complex clinical and hematological phenotypes Heterozygous Hb E alone or coupling with α +-, α0thalassemia usually does not present clinical manifestation, RBC indexes and morphology slightly change EABart's disease manifests as thalassemia intermedia Homozygous Hb E presents mild anemia, hematologic changes as in β thalassemia, such as hypochromic microcytosis, anisocytosis, presence of target cells Hb E rate in heterozygosity is about 25-30%, and about 85-99% in homozygote Hb E disease that combines with α-thalassemia reducing Hb E synthesis Diagnosis of Hb E combined with α thalassemia requires DNA analysis Compound heterozygous Hb E/β-thal presents from mild to severe clinical manifestations due to effects of many factors 1.3 Research of thalassemia and hemoglobinopathy in Viet Nam and in the world 1.3.1 Frequency of gene carriers In the world, α thalassemia, β thalassemia, Hb E and Hb CS are common groups, Hb E can be considered as a hallmark of Southeast Asia with frequency in some regions up to 50 to 60% Hb E in combination with thalassemia as well as with other abnormal Hb leads to extremely diverse compound heterozygosities Hb diseases studies in Viet Nam reported types of Hb diseases including α thalassemia, β thalassemia, Hb E and combinations The rate of β thalassemia and Hb E in the ethnic groups in Viet Nam in studies conducted in the North: Tay from 4.1 to 11.0% and 1.0 to 1.4; Dao 4,6 to 9,8%; Muong 20.6% and 12.3%; Thai 11.4% and 20.03%, in the Central: Van Kieu 2.56% and 23.0%; E de 0.3-1.0% and 27.741%; M'Nong 0.2-26.4% and in the South: Kinh 1.7% and 0.7-8.9%, HbE rate in the Cham ethnic group 29%, S'Tieng 35.6-55,9% α thalassemia was rarely studied 1.3.2 Clinical features and hematological changes Generally, studies on clinical features and hematologic changes in Vietnam have focused only on hospitalized severe thalassemia patients, very few studies on thal and Hb E carriers In 1999, Bui Van Vien noted the clinical characteristics of HbE diseases widely varied depending on thal types: heterozygous and homozygous HbE showed no clinical manifestation, only 20.4-52.3% of heterozygote has mild anemia In β thalassemia carriers, Hoang Van Ngoc noted that only 13.9% of skin pallor, 2.3% of mucosa pallor in Tay and Dao children in Thai Nguyen While Vu Thi Bich Van recorded a 69.7% skin pallor In these subjects, RBC was significantly higher than noncarriers, with MCV and MCH significantly low, decreased HbA levels 1.3.3 Type of mutation In Viet Nam, there are common mutations that cause 9598.2% of cases of β-thal include Cd17 (A> T), Cd41/42 (-TCTT), -28 (A> G), Cd71 IVS1-1 (G> T), IVS1-5 (G> C), IVSII-654 (C> T) and Cd26 (G> A) causing Hb E disease In addition, Cd95 (+ A) is a mutation quite characteristic for the Vietnamese were also recorded In particular, the mutations Cd26 (G> A), Cd17 (A> T), and Cd41/42 (-TCTT) are the most common The most common α thalassemia mutations in Viet Nam are: SEA, -α3.7, αCSα (Hb Constant Spring), -α4.2, less frequent are deletion THAI, DUTCH 2, ATRA 16, and αQSα The highest proportion of Hb Constant Spring gene carriers recorded in women of Co Tu ethnic group (20.5%) There are no studies in Viet Nam investigating the type of globin gene mutation in Khmer people Thalassemia minor and Hb E are not significantly symptomatic, with only slightly microcytosis and without or very mild anemia Severe forms including Hb Bart's hydrop fetalis, β thalassemia major and HbE/β thalassemia present marked hematologic and biochemical changes on blood tests The mutation types of β globin and α globin genes are extremely diverse and vary from region to region However, studies have documented that each nation has only a few common types of gene mutations that make it possible to select effective screening techniques Chapter SUBJECTS AND METHODS 2.1 Research subjects Research subjects were Khmer people living in Mekong Delta provinces from August 2011 to December 2014 2.1.1 Selection criteria The subjects were members in Khmer family including of at least generations: grandparents, parents, and themselves living in the Mekong Delta provinces for more than year, irrespective of age, gender or blood-relation, who agreed to participate in the study 2.1.2 Exclusion criteria - Not included subjects who did not agree to participate, or did not consent to their children involved into the study - Not included subjects with acute hepatitis, cirrhosis, other diagnosed hematological diseases, too elderly subjects who were forgetful - Subjects with poor quality blood samples to conduct the study investigations at all stages 2.2 Research methods 2.2.1 Study design: cross sectional description study 2.2.2 Sample size: Sample size: 1087 In reality: 1273 subjects 2.2.3 Sampling: multiple-stage sampling 2.2.3.1 Study populations selection - Based on the distribution characteristics of Khmer people in the area, four selected provinces were Soc Trang, Tra Vinh, Bac Lieu and Hau Giang - Based on the rate of Khmer population in the provinces, the following study clusters were selected: Soc Trang, Tra Vinh clusters / provinces; Bac Lieu and Hau Giang clusters / provinces In total: 14 clusters 2.2.3.2 Subjects selection Based on the Khmer list of residents in the clusters provided by the local authorities, used R software to select random numbers, plus 10% loss, determined blood relations and sent a letter of invitation to participate to study 2.2.4 Research contents 2.2.4.1 Clinical variables: Clinical examination to identify pallor skin, pallor mucosa, jaundice, icteric eyes, facial deformities 2.2.4.2 Paraclinical variables: Osmotic fragility test (OF test) with self-modulating NaCl solution 0.35%, Dichlorophenol indophenol (DCIP) test purchased from Thailand RBC indexes on peripheral blood smears were performed at Can Tho University Hospital Hemoglobin electrophoresis with Sebia's capillary electrophoresis at Medic Hoa Hao - Ho Chi Minh City, determined the proportion of HbA, HbA2, HbF, HbH, Hb Bart's, HbE and other abnormal Hb Molecular diagnostic biology techniques on the globin gene were performed at laboratory of the Thalassemia Research Center, Mahidol University and Khon Khaen University, to identify deletions of α globin genes: -α3.7, -α4.2, SEA, and THAI; Hb CS; mutations on β globin gene: 28, Cd 17, Cd 19, Cd 26 HbE, Cd 35, IVS-I-5, Cd 41/42, Cd 71/72, IVS-II-654; three variants of the β globin gene: Hb S, Hb Tak and Hb D-Punjab; 05 deletions causing increased HbF, including: GγAγ(δβ)0-thal, HPFH-6, Indian Gγ(Aγδβ)0-thal, Chinese G A γ( γδβ)0-thal and SEA HPFH 2.2.5 Data processing methods Data were processed by computer using SPSS statistical software X 22.0 version to calculate statistical values such as: mean ( ), standard deviation (SD), minimum value - maximum value, percentage Compare two means with t-test Compare two rates with χ2 test Statistical representation with a 95% confidence interval, p 5% cases of HbTak, Hb variant appeared in the HbF region at a level of more than> 30% α-thal associated HbE: α-thal combined with βE/β βE/β reduced the level of HbE over βE/β βE / β alone, at variable levels depending on the lost number of globin- α gene, the difference was statistically significant with p = 0.000 βE/βE βE / βE combined with α-thal did not significantly alter HbE levels compared with βE/βE βE / βE alone 3.3.2.4 Characteristics of Hb Tak cases The study identified cases of HbF increasedabove > 30% We conducted a survey of some family members of two subjects to find out the characteristics of this variant In Tthe first family, there was surveyed members surveyed, the second family was surveyedand members in the second family (including the subjects) - There were carriers of Hb Tak gene carriers in the first family and carriers Hb Tak gene carriers in the second family Among them, one member of the first family was detected compound heterozygous Hb Tak / Hb E in combination with α3.7α3.7 / αα - Subjects bearingAffected Hb Tak subjectsgene had normal or high RBC, generally Hb level was generally normal or high Hb level, red blood cell indexes were within normal range - On the electrophoresis, Hb Tak gene carriers cases showed an HbF increase of above> 30%, more increasinghigh level when combined with HbE, HbA2 within the normal range or slightly increased 15 Table 3.19 Red blood cell indexes and Hb content of the subjects in the two Hb Tak genealogiesfamily pedigrees RBC indexes Subje Gend Ag ct er e RBC (x1012/ L) Hb content (%) HG RD HC MC MC MCH B WT V H C (g/d CV (%) (fL) (pg) (g/dL) L) (%) 2.2 M 44 4.,85 15,7 44,9 92,6 32,4 35,0 11,4 1.2 F 84 3,48 10,7 32,8 94,3 30,7 32,6 13,0 2.3 M 52 4,60 14,5 42,6 92,6 31,5 34,0 11,9 2.4 M 40 5,69 18,0 49,8 87,5 31,6 36,1 12,2 3.2 M 5,89 14,7 44,1 74,9 25,0 33,3 10,8 3.3 M 11 4,78 12,8 39,8 83,2 26,7 32,1 10,2 3.4 F 14 5,53 14,5 44,5 80,3 26,2 32,6 10,4 2’.2 F 61 5,70 17,5 50,3 88,2 30,7 34,8 12,1 2’.3 F 63 5,07 15,2 45,8 90,3 30,0 33,2 11,6 3’.2 F 33 5,55 12,9 41,2 74,2 23,2 31,3 12,1 4’.1 F 5,14 10,8 36,2 70,4 21,0 29,8 12,2 4’.2 F 13 4,98 14,2 41,9 84,2 28,5 33,8 11,2 A A F 62, 97, 97, 61, 34, 3,1 Genoty pe E 0,0 2,2 0,0 2,3 35, 3,0 57, 5, 37, 0,0 97, 0,0 2,6 63, 32, 4,0 64, 32, 3,2 64, 32, 3,2 4 67, 29, 3,4 97, 0,0 2,6 62, 34, 3,3 βTak/β β/β β/β βTak/β βTak/βE β/β βTak/β βTak/β βTak/β βTak/β βAβ βTak/β 3.3.2.5 Red blood cell indexes and their roles in thalthalassemia screening and hemoglobinopathies screening Hb diseases We studied the role of RBC indexes and some tests in thalassemia and hemoglobinopathiesthal screening and Hb diseases screening as follows: - MCV and MCH were the two most valuable indexes in thalthalassemia screening and HbE MCV value below A) causing HbE disease, accounted for 29.0%, codon 17 (A> T) 0,7%, codon 41/42 (-TCTCT) 0.3%, and IVSII-654 accounted for 0.1% Our results are similar tocomparable with study other results on the β globin-β gene mutation types in ethnic minorities in Viet Nam Phan Le MinhTuan Phan Le Minh and Nguyen Khac Han Hoan Nguyen Khac Han all identified (δβ) thal / βA, two cases with HbF level of 7.7% and 15.7%, in which DNA analysis was done for screening common mutations causing increased HbF level cases, including GγAγ ((δβ)0 0-thal, HPFH-6, Indian Gγ (Aγδβ)0 0-thal, Chinese Gγ (Aγδβ)0 0-thal and SEA HPFH, but the results were all negative, therefore (δβ)thal/βA could be due to a rare mutation We specifically detectednoted a β globin -β variant in the Khmer population of Mekong Delta, accounted for 0.2% - the HbTak mutation HbTak is a variant of the β globin - β chain that has high affinity for oxygen There are no studies that have documented this status of mutationgene carrier status in Vietnam Ten types of combined HbE / α-thal mutations have been identified In which significantly are the genotypes of -α3.7 / αα, αCSα / αα, -α3.7 / -α3.7, and SEA / αα combining with HbE The authors noted similarity in Cambodian subjects, with a greater diversity of gene combination Sanguansemsri T also found that 0.8% of cases of 3-α- 22 genes carrier (αααanti3.7 and αααanti4.2) Tran Thi Thuy Minh Tran Thi Thuy noted that 5.5% of the Ede and M'Nong communities had a less diversity combination of HbE and α-thal mutations than the Khmer in Mekong Delta 4.3 Clinical and hematological characteristics of thalthalassemia genotypes and hemoglobinothiesHb diseases 4.3.1 Clinical characteristics of thalassemia and hemoglobinopathiesHb diseases The main characteristics recorded was pallore skin accounting for 32.2%, pale mucosa accounting for 44.7% Our results showed that the proportion of pallore skin and pale mucosa was higher than those of the Tay and Dao ethnic groups (13.9% pallorblue and 2.32% pale mucosaskin) in study of Hoang Van Ngoc ’Hoang Vans study in Thai Nguyen province , much less than Vu Thi Bich Van Vu Thi with 69.7% pallore skin 4.3.2 Hematological characteristics of thal and hemoglobinopathiesHb diseases We named all genotypes -α3.7 / αα; -α4.2 / αα; and αCSα / αα which are one1 defective α -globin -α gene defect (or α+ + -thal) group Group includesing genotypes SEA / αα; -α3.7 / -α3.7, -α3.7 / αCSα which were two defective α2- globin-α gene defect (or α00-thal) Total peripheral blood cell count We studied the indexes RBC, HGB, MCV, MCH and RDW indexes in two groups of children and adults In general, the results were similar to the literature Isolated α-thal, β-thal and HbE disease in both adults and children all have had average RBC within the upper normal range, the maximum was high, indicating increased RBC production for compensation Anemia was mild or not present MCV, MCH were low, especially low in α0-thal, β-thal, βE/β that combined with SEA deletion mutation; EABart's disease and βE E/ βEE (p