Đặc điểm dịch tễ học, đột biến gen bệnh thalassemia ở phụ nữ người dân tộc tày và thử nghiệm giải pháp can thiệp tại 6 xã, huyện định hóa, tỉnh thái nguyên tt tiếng anh

With 53ethnic groups with genotypes, the frequency of mutant genes may vary among ethnic groups, thestudy of the frequency of gene transfer in each ethnic group is essential for the deve

NGUYEN KIEU GIANG

EPIDEMIOLOGICAL AND MUTATION GENE OF THALASSEMIA

IN TAY WOMEN AND INTERVENTION SOLUTIONS AT 6

COMMUNITIES, DINH HOA DISTRICT, THAI NGUYEN PROVINCE

Specialization: Social Hygiene and Health Organization

Code: 62 72 01 64

NGUYEN KIEU GIANG

EPIDEMIOLOGICAL AND GENE OF THALASSEMIA IN TAY WOMEN AND INTERVENTION SOLUTIONS AT 6 COMMUNITIES,

DINH HOA DISTRICT, THAI NGUYEN PROVINCE

Specialization: Social Hygiene and Health Organization

Code: 62 72 01 64

Science instructor 2: Professor Hoang Khai Lap

THAI NGUYEN - 2019

INTRODUCTION TO DOCTORAL THESIS

1 Necessity of the thesis

Thalassemia is a haemolytic anemia that is inherited due to abnormalities in the synthesis ofhemoglobin resulting in anemia at different levels According to the World Health Organization(WHO), about 7% of the world's population carries the Thalassemia gene, with between 300,000and 400,000 newborns with severe hemolytic anemia

In Vietnam, according to incomplete statistics, more than 12 million people are carrying thegene for thalassemia common in all provinces nationwide In particular, the proportion of peoplecarrying the thalassemia gene in ethnic minority groups is higher than the Kinh majority With 53ethnic groups with genotypes, the frequency of mutant genes may vary among ethnic groups, thestudy of the frequency of gene transfer in each ethnic group is essential for the development of thethalassemia prophylaxis program in Viet Nam Among ethnic minorities in Vietnam, the Tay ethnicgroup has a population of more than 1.6 million, topping the top 10 ethnic groups in the country.The rate of 13.2% carrying the thalassemia gene in the Tay group migrated to the south wasreported in 2010 However, the frequency of gene transfer, mutant genotype and frequency ofalleles in the East group in East North has not been fully reported

Thai Nguyen is a province in the midland and mountainous area of the North East, inhabited

by a number of ethnic minority groups, of which the highest proportion is the Tay ethnic group.However, there is no consistent data on the incidence of disease genes and mutant genotypes, asthese are very useful information for thalassemia prevention in the region In addition, thedevelopment and testing of a preventive screening program - counseling at the grassroots level israrely studied in Vietnam

2 Objectives

1 Identification of some epidemiological and moleculer epidemiological characteristics of thalassemia in Tay ethnic minority women in the reproductive age in Dinh Hoa district, Thai Nguyen province in 2015.

2 Experiment and evaluate the effectiveness of thalassemia screening, monitoring and counseling in Dinh Hoa district primary health care.

3 Practical signification and new contributions of the thesis

New contributions to science

Application of molecular biology assay to analyze the rate of hemoglobin in determining thefrequency of carrying thalassemia gene among Tay ethnic group in Thai Nguyen area The 27%carrying gene thalassemia has never been reported in previous studies in this population In particular,the frequency of carrying alpha thalassemia gene was reported in the Tay group at the site of study at16.3%

Successfully developed and applied screening, counseling and prenatal diagnosis inthalassemia prophylaxis at communi health station Screening and counseling provided directly bythe health staff of communi The process of transporting and receiving blood samples closely, theimplementation of disease diagnosis, genotypic diagnosis at the provincial level has solved theproblem of access and availability of the health system

Contribute to the reality

The identification of mutant genotypes and the frequency of alleles in the Tay population atthe study sites were significant in the prevention of thalassemia This information will be used todevelop a thalassemia diagnostic panel for the Northeastern region in general, to estimate theincidence of new heavy organisms in the population and to contribute data to the development ofthe genetic map thalassemia in Vietnam

The intervention model for thalassemia is feasible with a contingency cost estimate of ~ 1%for a new case compared to the cost of treating patients with new thalassemia major.

The study has transferred the OF screening technique to Dinh Hoa district health center,transfer Transfer of thalassemia screening and counseling model for Dinh Hoa district Transfer ofHbE screening technique to Thai Nguyen Blood Transfusion Center

4 The structure of the thesis

The thesis is presented in 121 pages, including: back ground (2 pages), overview (40 pages),subjects and research methods (17 pages), research results (23 pages), discussion (35 pages),conclusions (2 pages), recommendations (1 pages)

The thesis consists of 37 table, 22 picture In the references 105, 80 documents in English, 25documents in Vietnamese, 42 documents published in the last 5 years (70documents published inthe last 10 years) The appendix includes the data collection tools, the sample list and theintervention images of the research team

CHAPTER I LITERATURE OVERVIEW 1.1 The concept of thalassemia

The term "thalassemia" or "haemolytic haemorrhage" refers to a group of pathologicalconditions characterized by a reduction in the synthesis of one of the two globin chains (α or β) that) thatconstitutes the normal human hemoglobin molecule (HbA, α2β) that2), resulting in a reduction in thehemoglobin synthesis in the endothelium, which is manifested externally by anemia

1.2 Genetic mechanism of thalassemia

According to the inheritance characteristics of chromosomal recesses and the status ofdisease in the previous life, there are five cases of hereditary diseases to the next

In cases where both parents have homozygous disease, 100% of cases are homozygous Incase of a homozygous, a heterozygote: 50% of children with homozygotes, 50% of children withheterozygote In cases both perents have heterozygous: 50% of heterozygous children, 25% ofnormal children, 25% of homozygouts In case of an ordinary person, a heterozygote: 50% ofchildren with heterozygosity, 50% of normal children In case of an ordinary person, a homosexual:100% of children have heterozygous disease

1.3 Some epidemiological problems of thalassemia

Characteristics of age groups: The different forms of disease manifested and affected in age

groups are different Depending on age and other clinical characteristics, the physician maypreliminarily diagnose the condition so that a proper diagnosis is identified for the diagnosis Age,gender is also one of the criteria to consider when developing a thalassemia screening program

Ethnic characteristics: Many studies in the world claim that ethnic minority groups tend to

carry genes much higher than that of majority ethnic groups This is explained by the epidemiologicaland cultural characteristics of ethnic minority groups Ethnic minorities also tend to communicatewith the same locality and with the language group, which involves geographic proximity This is one

of the factors that increase the rate of thalassemia gene in ethnic minority communities

Gender: Gender is considered in the context of anemia and the complications of anemia

caused by thalassemia Both male and female clinical conditions are affected by anemia and similarcomplications However, due to the specificity of pregnancy only in women, pregnant women withthalassemia have many health risks, risk during birth as well as postpartum complications

Migration: Thalassemia is the first known disease in the Mediterranean, where it is described as

prevalent in epidemic-endemic countries such as North Africa, the Middle East, India, South China andSoutheast Asia, however, today the disease is reported in most countries and regions around the world.Studies indicate that inter-territorial migration is the cause of widespread and diversified genomesworldwide

Inbreeding: Inbreeding is the marriage between people who have blood relations, usually

between cousins or sometimes uncles and nieces Inbreeding leads to inbreeding, which increases thelikelihood of two people carrying the same gene to marry, and increases the likelihood of thalassemiahomozygosity in the offspring

Estimating the incidence of newborn infants in the population: Hardy-Weinberg's law is used

to calculate the number of children born with thalassemia in 1,000 live births The frequency of genotypes,estimated by the Hardy-Weinberg formula, is as follows: The frequency of genes is constant overgenerations and allele frequencies in the latter are always proportional to p2: 2pq: q2 The condition ofthis argument is: (1) no mutation occurs resulting in new allele; (2) all individuals have the sameopportunity in marriage and reproduction; (3) no significant immigration from other fully populatedpopulations

1.4 Studies on the epidemiology and molecularities of thalssemia

1.4.1 In the World

According to the World Health Organization (WHO), about 7% of the world's populationcarries the Thalassemia gene, with between 300,000 and 400,000 newborns with severe hemolyticanemia

Figure 1 1 Map of alpha and beta thalassemia mutations in the world1.4.2 In Viet Nam

Tablet 1 1 The contribution carriers thalassemia gene in Viet Nam

Local -Ethnic Sampl

es

Rat

Kinh (Hue) 1100 5,6 Phan Le Minh Tuan

(2014)

8

Pham Ngoc Dung (2011)

3

Phan Thi Thuy Hoa (2010)

Tay, Dao (Thai Nguyen)

Muong (Hoa Biinh)

E de

2M’Nong

2

1.5 The screening and diagnostic tests of thalassemia

1.5.1 The screening tests of thalassemia

Screening of thalassemia by OF test

Screening of HbE by DCIP

Screening of thalassemia by CBC test – MCV, MCH

1.5.2 The diagnostic tests of thalassemia

Diagnostic the types of thalassemia by analysis the rate of hemoglobin typing

Diagnostic the thalassemia mutation gene by moleculer analysis

1.6 Prevention of thalassemia

Currently, there are three cases of thalassemia that are listed as preventive measures:

• homozygous beta thalassemia, or Cooley disease

• Thalassemia / HbE (beta 0 thalassemia - HbE)

• Homozygous alpha 0 thalassemia, or Bart's Hydrops Fetalis

1.6.1 Approach to develop a thalassemia prophylaxis program

Thalassemia affects three groups of people in the community: common people, people withthalassemia, people with thalassemia Prophylactic treatment with thalassemia should have acomprehensive impact on these groups to prevent the risk in these groups at the time of marriage.After marriage and pregnancy, the diagnosis of fetal disease (CTS, pre-embryo transfer diagnosis )

is the last resort to be prevented

The approach to prevention for each target group is different With the community in mind, when

it is not yet known whether individual carriers carry genes, the first step is to test the gene carryingthe gene There are many screening stages: screening through cord blood storage Film screening atschool age, college students Humanitarian Blood Screening Clinic Periodic health screening Check thesubject of marriage registration Screening pregnant women in the early months

1.6.2 Thalassemia Prevention Programs in the World

Screening program for pregnant women

Pre-Marriage Screening Program

Other thalassemia prophylaxis programs

1.6.3 Research on screening and prenatal screening in Vietnam

Vietnam has no national thalassemia prophylaxis program, thalassemia interventions aremainly focused on screening models, TSH A small number of authors propose interventions based

on CT scans in community hospitals or community-based IEC

(A)Figure 1 2 Thalassemia screening procedures

at An Giang Hospital

(B)Screening, counseling, prenatal diagnosis at

Tu Du Hospital, Ho Chi Minh City

CHAPTER II SUBJECT AND METHODODOLOGY 2.

2.1.3 Location and time of study

2.1.3.1 Study location: Dinh Hoa district, Thai Nguyen province

2.1.3.2 Time of the study: June 2015 to June 2017 and divided into two phases (1) Cross-sectional (2)

Interventions (Figure 2.2)

Figure 2 1 Time of the study2.1.4 Methodology

2.1.4.1 Research design

Design of the cross sectional (Phase I) Design of Interventional studies (Phase 2)

2.1.4.2 Calculation of sample size, sample selection

Use the method of calculating sample size for population and random sampling for sectional studies 300 Tay women were selected for descriptive study With intervention research

cross-on basic health staff, sample size and method of target selecticross-on, ccross-onvenience 12 health workers in

6 communes of Dinh Hoa district were allocated to the intervention group 36 health workers in theremaining 18 communes were allocated to the control group

2.1.4.3 Research indicators

Group of epidemiological indicators: age, sex, occupation, near-geographical marriage, blood marriage, economic

in-Hematological indexes: RBC, Hb, HCT, MCV, MCH,

Group screening tests: OF, DCIP

Group of indicators K.A.P: point and classify knowledge, attitude, practice

Indicators of communication: Number of consultations, number of consultations, number ofcounseling materials distributed, number of couples agreeing with counseling

2.1.4.4 Research content

Descriptive study: To determine the proportion of thalassemia carriers gene, genotype, and

frequency of alleles Tay minority ethnic women aged 15-49 in Dinh Hoa, Thai Nguyen were selectedfor the study Subjects were interviewed using a questionnaire that was available to collect

epidemiological information The intravenous infusion of blood is also collected and sent to ThaiNguyen Hematology and Blood transfution Center for screening, complete blood cell andhemoglobin electrophoresis 0.5 ml of each blood samples was separated into a new tube for DNAanalysis of thalassemia gene mutations.

Intervention study: The intervention process is divided into 3 phases.

Phase 1: pre-intervention investigation; Determining the status of health workers in K.A.P, depth interviews with health leaders to identify the need for disease prevention, possible challengeswhen implementing the intervention and plan to overcome

in-Phase 2: Setting up a Steering Committee for interventions for thalassemia prevention andtraining for commune health staff; Training of commune health workers with the followingcontents:

Add, update knowledge about thalassemia

Definition, classification of thalassemia

Epidemiology of carrier genes in Vietnam

Symptoms, diagnosis, treatment thalassemia

Prevention of thalassemia

Training on testing and screening procedures in Dinh Hoa district

Training on the technique of screening for carriers of thalassemia gene in clinics using OF test

Training on improving genetic counseling skills in congenital haemorrhagic fever:

Laboratory counseling: screening, diagnostic tests; Laboratory tests; Interpretation of testresults; The medical facility may carry out tests; Cost of testing

Genetic counseling with test results at specific stages of screening

Consultation on termination of pregnancy with the following contents: Indications, methods ofimplementation, accidents, legal procedures, medical facilities can be implemented, costs

Phase 3: Thalassemia screening for pregnant women <12 weeks at the clinic and monitoringand counseling for health workers (Figure 2.2)

Figure 2 2 Thalassemia screening and counseling strategiesThalassemia screening for pregnant women by the OF test was performed at communehealth stations in combination with prenatal management program Pregnant women under 12weeks of age who are registered for pregnancy management at the commune health stations will beinformed about thalassemia and will be invited to participate in the screening program Subjects

agreed to screen will be tested at the right at the station and counseling test results immediatelyafter the test results Researchers will review and evaluate the screening process and counseling ofhealth workers, which will provide necessary technical support and support to health workers.

Blood samples of couples at risk after screening will be transferred to Blood TransfusionCenter - Thai Nguyen National Hospital (Province level) to carry out diagnostic tests for geneticstatus of couples and risk identification muscle for fetus Fetuses who are at risk for thalassemiamajor will be advised to make a diagnosis to determine the disease of the fetus

The prenatal diagnosis of fetus (identified as risk) will be performed at the Center for AppliedMedical Research in Medicine and Pharmacy - Hanoi Military Medical Academy (National level).Pregnancy cure is advised for the fetus is diagnosed with severe disease after the results of the prenataldiagnosis of fetus

All results of the tests (after screening) are sent to the commune health staff All counselingactivities were conducted by trained health staff in intervention communes

The consultative process used in stage 3 (Figure 2.2)

2.1.4.5 Diagnostic criteria for thalassemia, index of red blood cells and K.A.P

Diagnostic criteria carrying the thalassemia gene

Beta thalassemia gene: Electrophoresis Hb with HbA2> 3.5% and / or HbF> 2% DNA testsdetect mutations in the beta globin gene

Alpha thalassemia gene: Electrophysiologic Hb findings of abnormal hemoglobin mutations inalpha globin genes: HbCS, HbH, HbPs DNA tests detect mutations in the alpha globin gene

Normal index type Red Blood Cells

There are 11 questions related to the views of commune health workers in the prevention ofthalassemia Each question was assessed on a Likert scale (1 - Strongly agree, 2 - Agreed, 3 - Notclear, 4 - Disagree, 5 - Strongly disagree) Each answer with the correct point is a point, the point isnot true or not clear 0 points The maximum score for the part of the attitude was 11 points and wasassessed at 2 levels: achieves when the knowledge score was ≥50% of the total score (≥5.5 points),Not achieved when the knowledge score was <5.5 points

Evaluate Thalassemia Prevention with practical evaluation questions The maximum point forpractical questions is 27 points and is evaluated at two levels achieves when practicum point ≥ 50% ofmaximum score (≥13.5 points), Not achieved when knowledge score <13.5 points

2.1.5 Data processing methods

- Percentage is used to describe classifiers, hierarchies

- Mean and standard deviations are used to describe continuous variables

- Use statistical test χ2 (When squared) with significance level p <0.05 to:

+ Investigate the difference in the rate of thalassemia gene according to epidemiological indicators.+ Difference in K.A.P criteria between control group and intervention group

- Use the McNemar's statistic test to compare K.A.P changes before and after intervention onthe same group of health workers

CHAPTER III THE RESULTS 3.1 Epidemiological characteristics, characteristics of the thalassemia gene of the study subjects and the value of screening tests in the community

Tablle 3.1 Epidemiological characteristics of study subjects (n = 300)

General information Numbers Rate (%) Age

Immigration status before marriage

Marriages inbreeding

Comment: 27.3% of respondents aged 15 to 24 years old, 60.4% of respondents were in the 25 to 34 age group, 35years and over were 12.3% The majority of mothers with lower secondary education (92.6%), 7.4% have primary orlower education The percentage of mothers with farming occupations is 67.3%, followed by civil servants (24.7%) andother occupations (8%) The rate of poor households is 25.3%, near poor 45.0%, the remaining is non-poor households29.7% The majority of married women in the same district (88%), including 38% in the commune, 50% in othercommunes 12% of married women in other districts There are 9 cases of inbreeding in which 1 case of marriage in 3generations

Table 3 5 Anemia classification of subjects studied

Erythrocyte morphology

Anemia classification

Microcytic and / or hypochromic

2 00 2 0,7

2

65,3

17

34,7

49

16,3

3

15,1

185

84,9

218

72,7

8

3 2,7

2 02

6 7,3

3 00

1 00

Comment: 100% red blood cells of patients with moderate to major anemias are microcytic andhypochromic With minor anemia is 65%

Table 3.6 Frequency of carrying thalassemia gene at the study site

Comment: Frequency of carrying thalassemia gene at the study site is 27%

Table 3.7 Thalassemia gene carrier classification

Classification N

umber

Rate (%)

In 81* person carrier gene

In 300 Tay woman

* The persons carrying thalassemia gene are diagnostic by electrophoresis and PCR test

Comment: In the 300 Tay woman, the rate carrier thalassemia gene is 27%: α thalassemia 16,3%, β) thatthalassemia 7,3 %, HbE & α – β) that thalassemia 1,7% In the numbers carrying thalassemia gene, alphathalassemia is 66,6% (60,4% heterozygote, 6,2% heterozygote combinal α/β) that)

Table 3.8 Distribution of phenotype and genotypes thalassemia

* 11 cases carrier gene unknown genotype mutant in the screening panel.

Comment: The 14 combined genotypes of eight mutant phenotypes were identified in 70 of the identified thalassemia genes

Table 3.9 Distribution gene mutant according hematology index

Table 3.10 Rate and classification of mutant alleles among surveyed alleles

Mutations allele type T

Frequ ency allele

Rate (%) in allele mutations

28 (A-> G), Cd 26 (G-> A) HbE Regarding the frequency of single alleles, type SEA has anoutstanding frequency accounting for 47.3%, followed by other mutations

Table 3.11 Estimate the number of children with new thalassemia major at the study site

Genotype n Frequency

allele

Frequency new thalassemia major Estimated

/1000 babies born lived

Estimated /5000 babies born lived

α0 Thalhomozygous

β) that Thalhomozygous

β) that Thal/ HbE

Table 3.12 Classification carriers of thalassemia gene by marital status

otal

Carriers of thalassemia gene

Table 3.13 Classification carrying thalassemia gene according to anemia