Nghiên cứu tần suất, đặc điểm lâm sàng và đột biến gen bệnh thalassemia ở trẻ dân tộc tày và dao tỉnh tuyên quang TT TIẾNG ANH

THAI NGUYEN UNIVERSITY THAI NGUYEN UNIVERSITY OF MEDICINE AND PHARMACY  DO THI THU GIANG RESEARCH ABOUT FREQUENCY, CLINICAL FEATURES AND GENETIC MUTATION OF THALASSEMIA AMONG CHILDREN WHO ARE TAY AND DAO ETHNIC AT TUYEN QUANG PROVINCE Speciality: Pediatrics Code number: 9720106 ABSTRACT OF A DISSERTATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY THAI NGUYEN, 2021 INTRODUCTION Thalassemia is name of a disease which is an inherrited hemolytic anemia and an inherrited hemoglobinopathy caused by a decrease or complete losing of the synthesis of a type globin chain in the hemoglobin molecule that affect the growth of red blood cells and cause chronic anemia since infancy Thalassemia distributed throughout the world, as well as in Asia According to the estimation of World Health Oganization (2008), annually, there are approxiamate 300.000 infants with severe thalassemia In Vietnam, according to study of Nguyen Anh Tri, thalassemia is a leading cause of hemolytic anemia in children There is a difference in the proportion of people with thalassemia gene between regions and between ethnic groups Severe disease has severe anemia, megalosplenia, reduced ability to study and work, and reduced life expectancy Patients are a burden to the family and society The current treatment of severe thalassemia is still very difficult and expensive Most children is not received adequate treatment, most children die within the first 10 years of life In Tuyen Quang, there are 23 ethnic groups living together, in which the Tay ethnic group is 185464 people, accounting for 25.6%, followed by Kinh ethnic and Dao ethnic group with 100,000 people, accounting for 13.8% which is the third amount This is a mountainous province with many ethnic minority, low education, population mobility, especially in secluded districts, and limited economic conditions It is a favorable condition for the prevalence and widespread of hereditary hemoglobinopathy Thalassemia In recent years, at Tuyen Quang Provincal General Hospital the proportion of children who is diagnosed and periodic treated thalassemia increase gradually, most of them is in the district The districts which involve Tay and Dao Ethnic people is Ham Yen, Chiem Hoa, Na Hang, etc To determinied incidence and clinical featutes as well as point out genetic mutation of Thalassemia in community of ethnic minorities in general and Tay, Dao ethnic children in particular at Tuyen Quang, we conduct this study: “Research about frequency, clinical features and genetic mutation of thalassemia among children who are Tay and Dao ethnic at Tuyen Quang province”, with following objectives: To determine the incidence of Thalassemia in children of Tay and Dao ethnic groups at Tuyen Quang province Describe clincal, subclinical features and genetic mutation of Thalassemua among children of Tay and Dao ethnic groups at Tuyen Quang province THE NECESSARY OF DISSERTATION In the world, there are more than 200 countries and territories which are affected by this disease In Vietnam, there are about 5-10 million people having the disease gene Nowadays, common treatment methods are red blood cell transfusion and iron chelation If Thalassemia patients is diagnosed early, well treatment and caring, they will be less complication and prolong life However, the best prevention is a limitation to birth thalassemia children Around the world and in Vietnam, there were a number of studies about anemia and thalassemia among children Tuyen Quang province involve 23 ethnic groups with different customs However, it has been no study to determine incidence of Thalassemia in community, clinical symptom, and genetic mutation features among Thalassemia patients who are children of Tay and Dao ethnic at Tuyen Quang province The study of dissertation is necessary, current, scientific meaning which is not only for Thalassemia and Pediatric but also the disseration with interdisciplinary coordination to solve genetic problems of Thalassemia and quality of population THE NEW CONTRIBUTIONS OF DISSERTATION This study has determined the incidence of Thalassemia among children of Tay and Dao ethnic group at Tuyen Quang The study has initally diagnostic clasified β Thalassemia type by analysis and hemoglobin electrophoresis, and screening HbE by DCIP test This study also pointed out α gene mutation which is common in community This study has described clinical features, genetic mutation features of Thalassemia among children of Tay and Dao ethnic groups at Tuyen Quang province THE STRUCTURE OF DISSERTATION The dissertation was presented in 136 pages (not involved reference and appendixes, include: introduction (03 pages), literature review (37 pages), subject and research methods (23 pages), research results (32 pages), discussion (38 pages), conclusion (2 pages), recommendation (1 page) The dissertation has 36 tables, graphes and figures There are 138 reference documents with 55 Vietnamese documents and 83 English document Chapter LITERATURE REVIEW 1.1 Thalassemia concept Thalassemia (Also known as “Marine Anemia” or “Cooley Anemia”) was discovered by Thomas B Cooley in 1925 Thalassemia is a disease which is an inherrited hemolytic anemia caused by a decrease or complete losing of the synthesis of a type globin chain, and depend on deficiency of synthesis chain alpha (α) globin or beta (β) globin, is called alpha thalassemia or beta thalassemia 1.2 Thalassemia and diseases of abnormal hemoglobin Thalassemia is not a single disease but a group of disorders, each resulting from a genetic abnormality of globin production Conditions that form part of a spectrum of diseases are known as hemoglobinopathies 1.3 Thalassemia disease Thalassemia involve genetic deficiencies in the sinthesis of one and more globin chain The result is imbalance of globin chain production, inefficient erythropoiesis, hemolysis, and varying severity of anemia, since adults have almost of hemoglobin A (HbA1), so the significant globin is alpha and beta chains Normally, the body synthesizes α and β chains equally, the ratio of α/β is 1/1 The disease caused by a decrease or failure to produce the α chain is α Thalassemia, the disease caused by a decrease or failure to produce the β chain is β Thalassemia.These are the two main and common diseases of Thalassemia 1.4 Pathogenesis of thalassemia 1.4.1 Pathogenesis Thalassemia: The maincomponent of Hb in a normal people is HbA1 (95-96%), the main task of Hb is transporting oxygen to tissues The HbA1 connection between the α and β chains base on electrostatic force The α chain carries a positive electrical charge, the β chain carries a negative electrical charge The negative electrical charge of the β chain is stronger than that of the globin δ chain and the γ chain, so the normal person has an α-β connection that is more advantageous than the α to δ and γ connection A common phenomenon of Thalassemia syndrome is a deficiency of one polypeptide chain of the globin which causes a relative excess of the other The deficiency that occurs in the β chain is called βthalassemia disease, when the β chain decreases, the α chain connected with β decreases, the residual α chain increases connection with the δ chain and γ chain In contrast, deficiency that occurs in the α chain is called α-thalassemia, when the α globin chain decreases, the β globin increases connection with the remaining globin, and the connection of α chain to δ and γ is reduced 1.4.2 Consequence of thalassemia Chronic anemia: Hypochromic anemia, red blood cells with changes in shape, color, quality Hyperplasia of bone marrow: the typical face of thalassemia Iron overload: Complications of iron overload diversed manifest such as: in liver and heart, endocrine complications and in many other organs Some manifestations of organ damage caused by iron overload: - Heart: Ventricular dilatation, myocardial hypertrophy, heart failure - Liver: Increase fibrosis and cirrhosis - Endocrine: slow growth, delayed puberty, diabetes mellitus - Hyperpigmentation 1.5 Mechanism of inheritance of Thalassemia disease Mechanism of inheritance: Thalassemia is an autosomal recessive inherited disorder The disease only manifest phenotype in progeny if it is in recesssive homozygous state In heterozygous state (people carried the diseased gene), thalassemia patient doesn’t have symptoms The gene synthesized the globin chain is located on both chromosomes, chromosome 11 and chromosome 16, and the inheritance of the disease gene on two chromosomes is independent It led to variety of clinical type of thalassemia, including α Thalassemia, β Thalassemia and alpha - beta Thalassemia combination The disease may be caused by disease genes passed from parents to children or may be caused by new mutations Depending on the degree of genetic mutation, there are different forms of the disease Chapter SUBJECTS AND RESEARCH METHODOLOGY 2.1 Research population * Eligible criteria Objective 1: - Age rank in 2-6 years old; - Children is purebred Tay or Dao ethnic groups; - Have residence registration at the Commune People's Committee according to the State's regulations for at least generation in the research place Ojective 2: - Patients is purebred Tay or Dao ethnic groups who was examined and treated at Pediatric Department, Tuyen Quang Provincal General Hospital; -Patients were tested gene at National Institute of Hematology - Blood Transfusion or Vietnam National Children’s Hospital to study about genetic mutation * Exclude criteria Objective 1:Patient’s parents don’t allow to participate in research Objective 2: Patients receive blood transfusion for less than month, patients with hemolytic anemia due to other causes or have other acute diseases The patient did not consent to participate in the study 2.2 Place and time of research * Research place - Objective 1: district (Yen Son, Son Duong, Chiem Hoa, Ham Yen, Na Hang, Lam Binh) and Tuyen Quang City, Tuyen Quang Province - Objective 2: Pediatric Department, Tuyen Quang Provincal General Hospital *Research time: From January 2017 to December 2017 2.3 Research methodology 2.3.1 Research design Objective 1: Cross-sectional descriptive study Objective 2: Cross-sectional descriptive case series study 2.3.2 Sample size Objective 1: Calculate the descriptive sample size using the sample size formula for a ratio p (1-p) n = Z2(1-α/2) (εp)2 (n: sample size, p: proportion of ethnic minority people carried diseased gene according to research of Duong Ba Truc is 22.6% p=0,226; ε = 0,17 (relative accurary); α = 0.05, Z1-α/2 = 1,96; -> n=455 Added more 10% to prevent loss sample objectively (add 46 children) So, total sample size was 501 children In this study, there was 505 children who were allowed to participate to the research Objective 2: Sample size was chosen conveniently from population of Thalassemia patients at Pediatric Department, Tuyen Quang Provincal General Hospital, and chose 151 Thalassemia patients of Tay or Dao Ethnic groups from January 2017 to December 2017 who met eligible criteria Using sample size formula to calculate number of patients carried mutated gene: (N: total of patients 151; n: Minimum sample size; 1,96: Value from standart distribution table with significant value α = 0.05; p: the proportion of people carried disease gene According to Tran Tuan Anh, percentage of people carried mutated gene CD41/42 is 27,2%; p=0,272; d: error 0.13; -> n = 35 Based on this formula, chose 35 patients to test and determine mutated gene 2.3.3 Sampling technique Objective 1: Chose districts and a city From each district, commues were chosen ramdomly In each commune, researcher chose villages which have the highest proportion of purebred Tay or Dao ethnic groups Writing list of all children who are from to 16 years old at these communes Using computer software to choose 505 children corresponding with ratio of each ethnic Eligible criteria, purebred children (both parents are Tay or Dao ethnic), the child is not blood-relationship, does not have acute or chronic diseases, did not give/receive blood transfusion in the months prior to the time of the survey and agreed to participate research Objective 2: Using clinical sampling technique - Chose 151 thalassemia children of Tay or Dao ethnic who admited to diagnose and treat congenital hemolysis during the study period from 1/2017 -12/2017 - Chose conveniently 35 patients to determine mutated gene caused Thalassemia 2.4 Research varriable 2.4.1 Dermographic features - Age, Tay or Dao ethnic - Education of father or mother is the highest education in national education system, parent’s jobs, married status, relation in generation - Family income: According to national standart of poors in the period 2016-2020 2.4.2 Diagnostic criteria - Clinical: Have symptoms of chronic hemolytic anemia (Anemia, hepatomegaly, splenomegaly, bone deformities, mental and physical retardation, endocrine disorders, iron overload symptom) - Subclinical: Hematology test - Red blood cell forms was described by normal microscopy - Red blood cell index such as hemoglobin, hematocrit, red blood cell amount, Mean Cell Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Red cell Distribution Width (RDW), performed by Beckman Counter LH786 Hemoglobin electrophoresis - Hemoglobin components such as HbA1, HbA2, HbF, HbE were performed by high pressure liquid chromatography (HPLC), by Variant machine Gene sequencing technique, identifying β-globin mutated gene: - This technique was conducted with samples which can not detected mutated gene by Multiplex - PCR and ARMS - PCR techniques - GAP PCR technique: Detecting deletion mutations of the entire βglobin gene Biochemical test Iron biochemical tests such as Ferritin, serum iron, ALT, AST, Billirubin at Department of Biochemistry, Tuyen Quang Provincal General Hospital Abnormal hemoglobin composition on electrophoresis - Abnormal hemoglobin occur: HbS, HbE, HbH, Hb Bart's HbCS, Beta thalassemia heterozygous: HbA2 > 3.5% and/or HbF > 1%; HbA decrease - Beta thalassemia/HbE: HbA2 > 3,5% HbE > 5% - Beta thalassemia homozygous: HbA2> 3,5%; HbF increase; HbA very low or absent - δβ Thalassemia with HbA2 < 3,5%, HbF increase to – 15% Iron storage is normal or increased - Increase: Ferritin > 400 ng/ml, Normal: Ferritin from 13 – 400 ng/ml 2.4.3 Specimen collection technique Objective 1: 3ml whole blood is anticoagulated with EDTA and transported to the laboratory within hours after collection for testing Diagnosing alpha thalassemia gene based on GAP PCR test Objective 2: 3ml of whole blood anticoagulated with EDTA from 151 Thalassemia children for total red blood cell analysis and hemoglobin electrophoresis 35 in 151 blood samples were refrigerated at 40C and transported to the National Institute of Hematology and Blood Transfusion (within hours) to perform a diagnostic test for thalassemia based on PCR test for common mutations of Vietnamese people 2.4.4 Laboratory technique MCV index was obtained from the red blood cells total analysis performed by an automatic analyzer system and hemoglobin composition analysis was performed at Tuyen Quang General Hospital Multiplex GAP PCR technique was used to determine deletion mutation of alpha globin gene, include α0 (type SEA, THAI, -FILL) or α+ (type -α3.7 -α4.2) The above techniques were performed at the Department of Immunology - Genetics, Thai Nguyen National Hospital Multiplex ARMS-PCR technique is used to identify common mutations causing beta thalassemia This test was performed at the National Institute of Hematology and Blood Transfusion Chapter RESEARCH RESULTS 3.1 Determination of Thalassemia gene carrier frequency in children of Tay and Dao ethnic groups in Tuyen Quang province Table 3.1 General information of subjects Ethnic Features Age Gender Total Tay Dao Total n % n % n % 1-6 40 7,9 20 4,0 60 11,9 -11 23 4,6 21 4,2 44 8,7 11-15 287 56,8 114 22,6 402 79,4 Mean ± SD: 11,7± 3,67, (Min: years old, Max 15 years old) Male 144 28,5 60 11,9 204 40,4 Female 206 40,8 95 18,8 301 59,6 350 69,3 155 30,7 505 100 Comment: The average age of community children was 11.7±3.67, children in 11-15 years old group accounts for 79.6% Male children accounted for 40.4%, female children were 59.6% Table 3.2 Thalassemia screening test results in 505 subjects Thalassemia MCV3,5% Comment: Mild anemia manifested in phenotypes such as heterozygous α0 thal, heterozygous β thal and heterozygous Heterozygous β/α combination All phenotypes showed hypochromic microcytosis, mean MCV, MCH were less than cut-off point Table 3.6 Distribution of type of single Alen mutation in children carring α-Thalassemia gene Type of alen mutation α-thalassemia SEA -α3.7 -αCS -α4.2 Total Type Number (n) Percent (%) α0 α+ α+ α+ 62 19 86 72,09 22,1 2,31 3,5 Comment: SEA type mutation accounted for 72,09% Type of alen mutation of -α3.7 was 20,9% Moreover, in community, there was small deletion mutation -α4.2 with 4,7%, type mutation of -αCS accounted for 2,3% 3.2 Clinical features, subclinical features amd genetic mutation features of Thalassemia among children of Tay and Dao ethnic groups at Department of Pediatric, Tuyen Quang General Hospital 12 Table 3.7 Age and gender with ethnic of Thalassemia children group treated at hospital Ethnic Gender* Age ** Male Female Total 1-6 6-11 ≥11 Total Tay n 55 39 94 40 34 20 94 Dao % 36,4 25,8 62,3 26,5 22,5 13,3 62,3 n 33 24 57 34 14 57 % 21,9 15,9 37,7 22,5 5,9 9,3 37,7 Total n % 88 58,3 63 41,7 151 100 74 49,0 43 28,5 34 22,5 151 100 * Comparing in gender between Tay and Dao ethnic, Chi square = 0,006, p = 0,941 Comparing in age groups between Tay and Dao ethnic, Chi square = 7,462, p >0,05 ** Comments: Tay ethnic group accounted for 62.3%, Dao ethnic group accounted for 37.7%, male accounted for 58.3%, female accounted for 41.7% The lowest group was over 11 years old with 22.5% Table 3.8 Physical development features Varriable Tay n Dao % Weight* Normal 59 62,8 Reducing SD 28 29,8 Reducing SD 7,4 Height** Normal 73 77,7 Reducing SD 19 20,2 Reducing SD 2,1 Height by age/Weight by age (Welcome) 0,5 ** Comparing in height between Tay and Dao ethnic children, Chi square = 0,746,, p 0,05 ** Comparing in cardiovascular between Tay and Dao ethnic children, Chi square = 2,531, p > 0,05 Comment: Hematomegaly accounted for 37,1%, Megalosplenia accounted for 48,4%, most of level and 3, there were 13,2% Splenectomy There was 60,9% patient with abnormal cardiovascular, tachycardia was 24,5% Table 3.11 Characteristics of hematological indicators of Thalassemia children of Tay and Dao ethnic groups Ethnic RBC test RBC (T/l) Hemoglobin (g/l) Hematocrit (%) MCV(fl) MCH(pg/l) MCHC(g/l) RDW Tay Mean±SD 3,09±1,36 65,9±21,4 0,206±0,066 69,42±9,98 22,57±3,93 321,84±22,96 52,0±37,73 Dao Mean±SD 2,62±1,09 60,5±17,75 0,181±0,052 73,16±5,04 24,24±3,83 327,32±18,91 63,18±10,4 Total Mean±SD 2,91±1,29 63,86±20,22 0,196±0,062 70,83±6,12 23,20±3,97 323,91±21,62 56,37±27,34 Comment: The number of red blood cells, hemoglobin, hematocrit, MCV, MCH decreased, was signs severe anemia Table 3.12 Blood biochemical features in the group of 151 patients Ethnic Tay Biochemical index X±SD Glucose (mmol/l) ALT(U/L) AST(U/L) Conjugated Billirubin (μmol/l) Indirect Bilirubin (μmol/l) Iron (μmo/L) 5,3 ±1,2 62,76 ±92,51 71,89 ±70,74 84,21 ±81,86 14,77 ±13,3 23,52 ±12,27 Dao Total n 72 22 16 78 45 49 16 % 47,6 14,5 10,6 51,7 29,8 32,5 10,6 n 48 19 38 36 21 % 31,7 5,9 12,6 25,2 23,8 13,9 5,3 n 120 31 35 116 81 70 24 % 79,4 20,5 23,2 76,8 53,6 46,4 15,9 78 51,7 49 32,5 127 84,1 Normal Increase 58 38,5 37 23,8 95 62,3 37 24,4 20 13,3 56 37,7 Normal Increase 56 38 37,1 25,2 30 27 19,9 17,9 86 65 57,0 43,0 Normal Increase Normal Increase Normal Increase Normal Increase 15 Ferritin (ng/dl) 2867,2 ±2706,5 Normal Increase 16 78 10,6 51,7 49 5,3 32,5 24 127 15,9 84,1 Comment: 20,5% patient with hyperglycemia, 76,8% increased ALT and there was a difference between Tay and Dao ethnic (p < 0,05) Average serum ferritin was 2867,2 ng/dl, 84,1% patient increased Bilirubin, with mean value was 84,21±81,86 3.2.2 Analysis of genetic mutations on 35 Thalassemia children Table 3.13 Classification of Thalassemia Ethnic Type* α Thal β Thal βThal/HbE, α-β Thal combination Total Tay Dao Total n 11 % 11,4 31,4 n 11 % 2,9 31,4 n 22 % 14,3 62,8 17,1 5,7 22,9 21 60 14 40 35 100 * Chi square = 2,500, p < 0,5 Comment: The proportion of β-Thalassaemia was (62.8%), Tay and Dao ethnic groups are similar (31.4%) The precentage of β-Thalassaemia/HbF, α-β Thal combination was 22.9% Bảng 3.14 Frequency of alleles single mutation Type thal α-Thalassemia β-Thalassemia No detection Total Type allele SEA deletion 3.7 deletion (HbCS) 4.2 deletion CD95 (A+) CD 41/42(-TCTT) CD17 CD71/72(A+) IVSI-I CD26 - Phenotype α α+ α+ α+ β+ β0 β0 β0 β0 β+ - n(%) Percent(%) 4(36,4) 3(27,2) 2(18,2) 2(18,2) 1(2,2) 18(40,1) 13(28,9) 6(13,3) 1(2,2) 6(13,3) 14 5,7 4,3 2,9 2,9 1,4 25,7 18,5 8,6 1,4 8,6 20,0 100 Comment: - There were 10 types of alen single mutation which was detected in 70 surveyed alen There were 10 alen which hadn’t detected, accounted for 20% 16 Table 3.15 Distribution of phenotypes and combinations of mutant genes Phenotypes and Type of thal n % combinations of genes αThalassemia βThalassemia α-βthal combination Total Heterozygous a+ thal -α3.7/αα Heterozygous α0/a+ thal SEA/-α 3.7 SEA/αCSα Heterozygous β+thal β/βCD95 Homozygous β0/β0 βCD17/βCD17 Heterozygous combinated β0/β0 βCD41/42/βCD17 βCD41/42βCD71/72 βCD17/βCD71/72 βCD41/42/βIVSI-I DHT β/β0 thal β/βCD 41/42 β/βCD17 Heterozygous combinated β0/βE βCD71/72/βCD26 βCD17/βCD26 βCD41/41/βCD26 Heterozygous α thal combinated β thal βCD41/42/β; αα/-α4.2 2,9 5,7 5,7 2,9 2,9 3 8,6 8,6 5,7 2,9 20,0 11,4 2 5,7 5,7 5,7 35 5,7 100 Comment: There were types of Thalassemia phenotypes and 16 common genetic mutations detected by the Panel test - Genetic mutation α-thalassemia included: (Genotype α3.7/αα was patient, accounted for 2,9%, Genotype–SEA/αα and Genotype -SEA CS /α α were 5,7% In group of patients with β-Thalassemia: (Homozygous a combination βCD17//βCD17 accounted for 2,9%, Genotype βCD41/42/βIVSI-I accounted for 2,9%) 17 - Heterozygous combinated β0/β0 had four combinations Both combination βCD41/42/βCD17 and combination βCD41/42βCD71/72 accounted for 8,6% Combination βCD17/βCD71/72 had patients with 5,7%, Heterozygous combinated HbE β0/βE had patients with combinations βCD71/72/βCD26,, βCD17/βCD26 βCD41/41/βCD26 accounted for 7,1% in total mutation β-globin - Heterozygous α-Thalassemia combinated with Heterozygous βThalassemia had patients with combination of gene was βCD41/42/β; αα/-α4.2 with same percentage 5,7% Chapter DISCUSSION 4.1 Epidemiological, hematological and genetic characteristics of 505 ethnic minority children Through the survey of 505 ethnic minority children in Tuyen Quang province, the research results (Table 1) showed that the average age of the children group was 11.7±3.67 years old Female children accounted for 59.6% The Tay ethnic was the majority (69.3%) followed by the Dao (30.7%) In this study, we used index of MCV < 80fl combinated with DCIP test to select Thalassemia and HbE simutaneously, according to recommendation of the Ministry of Health The percentage of children who had positive result for screening tests was 35.2% (Table 2) To screen continuously by hemoglobin electrophoresis and PCR with positive cases in screening tests, the research team discovered that the common gene carrier proportion among ethnic minority children in Tuyen Quang was 28.1% In which, 52.1% of cases carried alpha-thalassemia single gene, 41.6% in beta Thalassemia and remained 6.3% with alpha-beta Thalassemia combination (Table 3) Thus, with the application of PCR technique to screen all cases of small red blood cells, this study discovered more than ½ of the cases of people carrying the alpha Thalassemia gene, including the combination Our study has limited this drawback, 74 cases carrying the α-Thalassaemia gene were identified in the total of 142 cases carrying thalassemia gene Survey of mutant genotype based on hemoglobin electrophoresis and PCR test detected 11 types of mutant gene combinations which formed phenotypes carrying Thalassemia gene Hematological index in the group of children carrying disease gene showed mild to moderate anemia and gradually increased from 18 heterozygous α0, β0 Thalassemia groups to double heterozygous groups such as HbH or β-thalassemia/HbE Heterozygous groups α+, β+ thalassemia not show anemia symptoms This is consistent with the clinical features and genetic mechanism of Thalassemia There were 52 cases of heterozygous β Thalassemia (with an increase in HbA2 rate over 3.5%) However, the community research had not been able to confirm whether the genotype is β0 or β+ thalassemia Examine type of single Alen mutation in children carring αThalassemia gene (table 6), the results showed mutation type of -SEA accounted the highest percentage in all type of mutation in đứng globin alpha gene(72,09%), followed by mutation of -α3.7 (22,1%), αCSα (2,31%), -α4.2(3,5%) The mutation causing HbE status on the globin beta gene was 6.7% Đột biến gây tình trạng HbE gen globin beta 6,7% The study results were similar to studies in the Tay ethnic group, in the Northeast region with the mutation of SEA was 47,3%, -α3.7 (9,9%) Other specific mutations in the beta globin gene were not performed in this study The results of this study showed that the percentage of patients carrying Thalassemia gene in children of Tay and Dao ethnic groups in Tuyen Quang was high, and in α Thalassemia group was more than β Thalassemia and HbE It is necessary to conduct more research on the cost-effectiveness of rapid HbE screening tests (such as KKU-DCIP) to give clear recommendations for specific regions and ethnic groups With genetic testing, we suggest that it is necessary to continue to screen for mutations in the globin beta gene to have better data of frequency distribution of single allele, to help estimate the incidence and set up a mutation screening panel which is suitable to the Northeast region 4.2 Clinical, subclinical features amd genetic mutation features 4.2.1 Clinical and subclinical features 4.2.1.1 Clinical features For the total study group, the incidence of male patients was higher than that of female patients The age group from 1-5 years old was the highest proportion in all age groups admitting for treatment, It is due to patients are detected late after years of age and patients with early detection are often severe 19 Patient with weight reducing 2SD was 29,8% Reducing 3SD was 10,6% and patients of Tay ethnic was 7,4% and Dao ethnic was 15,8% There was no difference between Tay and Dao ethnic with p >0,05 Heigth reducing SD was 20,6%; reducing 3SD was 2,6% Due to the patient's hemolysis led to anemia and iron overload, iron contamination in the organs and endocrine glands The rate of children with dark skin accounted for 67.9%, compared to the study of Nguyen Cong Khanh 58.24% Patients with slow physical growth with insufficient height and weight, not only affect their appearance but also affect their quality of life in the long time The skull deformity of the forehead tumor was 15.9%, Low nasal bridge was the majority with 63.6%, the exposed upper teeth was 22.5%, creating the image of "Thalassaemia face" in clinical The phenomenon of increased bone marrow activity, including flat bones that have stopped working after birth In addition, due to the disorder of bone metabolism, fracture was 7.9% This study in pediatric patients with skeletal deformities is unknown This is an important symptom of iron infection Extramedullary hematopoiesis is also a cause of hepatomegaly Hepatomegaly had high precentage (37,1%) Patients with hepatomegaly are often accompanied by a megalosplenia Patients presenting with early disease have early detection of HbH, βThalassaemia and βthalassemia/HbE had severe symptoms, over time the degree of hepatomegaly was greater and more complications Splenomegaly is one of the significant clinical symptoms of chronic hemolytic anemia It is caused by rupture of red blood cells in the spleen Splenomegaly accompanied by hypersplenism, increased red blood cell rupture, aggravated anemia In this study, the proportion of patients with splenomegaly accounted for 48.4% Patients did not have splenomegaly (38.4%) The number of patients with splenectomy was only 13.2% Patients with no cardiovascular symptoms were 39.1% Tachycardia (40.2%); Heart failure (29.3%) due to severe iron overload The number of patients with tachycardia was 52.3% which is the first manifestation of anemia response 4.2.1.2 Subclinical characteristics 20 Red blood cell index: The number of red blood cells decreased, the average number of red blood cells was 2.91±1.29 Small red blood cells, MCV 70.83±6.12 fl MCH decreased by 23.20±3.9 pg/l MCHC was 323.91±21.62 RDW increased by 25.61±3.98 g/l Inefficient increased erythropoiesis also contributes to explain the erythrocyte deformity The increased erythropoiesis is not effective, so it cannot compensate for the hemolysis, causing clinical anemia In hemolytic anemia, the bone marrow responded by increased erythropoiesis, giving reticulocytes to peripheral blood earlier, reticulocytes exist in the peripheral blood longer The results of some biochemical tests (Table 12) showed that glycemia was increased and there was a large range, the lowest was 2.2 mmol/L and the highest was 10.0 mmol/L For liver function tests, ALT and AST are enzymes which is origin from cells So if cells are damaged ALT, AST will increase ALT is a lot in the liver; The liver is damaged due to many reasons, the most common cause being iron overload in the liver and accompanying infections In our study, ALT increased to 62.76±42.51 U/L At the same time, AST has a value of 71.89±60.74 U/L Bilirubin test showed that there was an increase in conjugated and indirect bilirubin in the pediatric patient The average bilirubin was 84.21±81.86 µmol/l; The average indirect bilirubin was 14.77±13.3 According to the results of our study, the serum iron was 23.52±12.27 Serum ferritin is a easy test and more likely prognostic According to the results of our study, serum ferritin increased, with average value of 3867.2 ± 2760.5ng/ml, in which the highest ferritin increase was 15345ng/ml which can cause toxicity due to iron overload Many complications occurred in this patient population There are some necessary solutions to prolong the patient's life and make the life of Thalassemia patients similarly to normal people 4.2.2 Genetic mutation features In 35 patient tesing thalassemia gene, there were 10 types of alen single mutation which was detected in 70 surveyed alen In which the allele type α-Thalassaemia with α0 were mutations in the deletion group, mutations that delete α gobin genes on the same chromosome 16 also was known as Hemoglobin H disease, α0 thalassemia in this study are mutations SEA accounting for alleles α-thalassemia mutant gene (5.7%) Three patients had phenotype α+ 21 thalassemia; α3.7 accounted for 4.3% Similarly, the percentage of α4.2 deletion mutation gene was 2.9% The study found that the heterozygous α3.7 gene was a healthy person has a disease gene, a mutation with the α3.7/αα combination that loses only one gene is hidden form or person with the αThalassaemia gene accounted for 2.9%, but if patients required blood transfusion, they may have another hemoglobin disease The mutation on the HbA2 gene (c.427 T>C) make a mistake in last gene, so the synthesized α globin chain was extended by 31 amino acids This structure is unstable and most of the formed HbCS was degraded, the rate in peripheral blood was