Nghiên cứu tần suất, đặc điểm thalassemia và các bệnh hemoglobin trong cộng đồng dân tộc khmer ở đồng bằng sông cửu long tóm tắt LUẬN án TIẾNG ANH

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THESIS INTRODUCTION
1. 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%; 1
to 9%; 1 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:
1. Determine the prevalence of thalassemia and hemoglobinopathies,
the rate of globin gene mutations in the Khmer ethnic group in the
Mekong Delta.
2. Describe some clinical and hematological characteristics of
thalassemia and hemoglobinopathies in the Khmer ethnic group in
the Mekong Delta.
2. Meaning and urgency of the topic
Thalassemia and hemoglobinopathy are groups of the most
common causes of congenital hemolysis in the world and Southeast


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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.
3. 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.
4. Structure of the thesis: The thesis consists of 133 pages, introduction
2 pages, overview 32 pages, subjects and research method 26 pages,
results 28 pages, discussion 42 pages, conclusion 1 page,
recommendation 1 page. There are 37 tables, 10 charts, 16 figures, 3
flowcharts and 158 references (36 in Vietnamese and 122 in English).


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Chapter 1
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,

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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 3 to 7 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,


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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


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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 8 common mutations that cause 9598.2% of cases of β-thal include Cd17 (A> T), Cd41/42 (-TCTT), -28


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(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 2
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 3 generations: grandparents, parents, and themselves living in
the Mekong Delta provinces for more than 1 year, irrespective of age,
gender or blood-relation, who agreed to participate in the study.


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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 5 clusters
/ provinces; Bac Lieu and Hau Giang 2 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.


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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 <0.05
was considered statistically significant.
2.3. Ethical issues
Research was done only with consent of the subjects, and of
the parents if the subject were children. The subject private
information was kept completely confidential. Research steps did not
affect to the health of the subjects.


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Chapter 3
RESEARCH RESULTS
From August 2011 to December 2016, we collected
information and blood samples of 1273 subjects. The results are as
follows:
3.1. General characteristics of subjects
Of the 1273 subjects, 199 were children, accounting for 15.6%;
1074 were adults, accounting for 84.4%.
Median age of carriers group was 41 years old (2 to 89), this of
group without mutation was 42 years old (1 to 92). The proportion of
men and women in the group with and without mutation was
45.7%/54.3% and 46.5%/53.5%, respectively. The majority of them
were farmers and hirelinglaborers, accounting for over 60.0% of
sample. The main religion of the Khmers was Buddhism, accounting
for more than 43.6%. Most of them were from rural areas, accounting

for 91,4% of samples.
3.2. Prevalence of thalassemia and hemoglobinopathies, rate of
globin gene mutations in the Khmer ethnic group in the Mekong
Delta
3.2.1. Prevalence of thalassemia and hemoglobinopathies
22,4% HbE

60,6%
without mutation

39,4%
with
mutation

9,1% α-thal
6,5% α-thal/HbE
1,3% β-thal
0,1% β-thal/α-thal

Figure 3.1. Frequency of thalassemia and hemoglobinopathies in
the population
Comment: 39.4% of the Khmer ethnic group carry thalassemia
and hemoglobinopathies. There are 05 types compose of HbE


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(22.4%), α thalassemia (9.1%), α thalassemia/HbE (6.5%), β
thalassemia (1.3%) and β-/α-thalassemia (0.1%).
Figure 3.2. Rate of thalassemia and hemoglobinopathies carrier in
the population

Comments: prevalence of HbE is 28,9%, α thalassemia
15,7% and β thalassemia 1,4%
3.2.2. Rate of globin gene mutation types
3.2.2.1. α – thalassemia
Table 3.1. The rate of genotypes and mutation types of globin gene
in the carrier group and in the population
Geno
type

Rate in
mutant
group
(%)

Rate in
the
populat
ion (%)

α-thalassemia
-α/-α
78
--/αα
7,0
-α/-α
10,5
--/αTα
2,5
αTα/ αTα
0,5

--/-α
1,5
β-thalassemia

12,3
1,0
1,6
0,4
0,1
0,2

β0/β

0,9

70,6

β+/β

0,1

5,9

δβ-thal

0,2

11,8

Hb Tak

0,2

11,8

Hemoglobin E
βE/β
26,5
βE/βE
2,4

91,8
8,2

Gene
mutation
type

-α3.7
-α4.2
--SEA
αCSα

No.
of
allele

Rate in
mutant
group

(%)

Rate in
the
populat
ion (%)

157
6
17
28

75,5
2,9
8,2
13,5

12,2
0,5
1,2
2,2

70,6

0,9

Total
Cd 17 (A>T)
Cd 41/42
(-TTCT)

IVSII-654
(C>T)
Undetermined

Hb Tak
[β147(+AC)
]

9
3

0,1

1

5,9

2

11,8

0,2

2

11,8

0,2

Comments: There are 4 mutation types of α globin gene and 6 of

β globin gene. The most common mutation type of α +-thalassemia
was -α3.7 account for 12.2%. In β globin gene, the most prevalent
mutation was HbE with 28.9%, Hb Tak mutation account for 0.2%,


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was detected in Vietnam for the first time.
HbE combined with 4 type of mutation on α globin gene
produced 10 types of different double heterozygotes. The most
popular combined type was βE/β;-α3.7/αα; the next was βE/β;αCSα/αα
and βE/β;--SEA/αα.
3.3. Clinical and hematological characteristics of thalassemia and
hemoglobinopathies in the Khmer ethnic group in the Mekong
Delta
3.3.1. Clinical features of thalassemia and hemoglobinopathies
Pallor accounted for 32.2% and pale mucosa accounted for
44.7% in the genetic carrier group higher than non-mutant group with
p <0.005. Jaundice, conjuntival icterus and facial deformities have
not been reported.
3.3.2. Hematological characteristics of thalassemia and Hb disease
3.3.2.1. RBC indexes of thlassemia and Hb disease
In isolated α-thal: mean value of red blood cells in children and
adults was within normal range or slightly increased, mild anemia or
not, MCV, MCH markedly decreased with p<0.001, especially in
those with two defective α globin gene. RDW was within the normal
range p> 0.05.
In isolated β-thal: mean value of red blood cells was higher when
compared to non-mutant patients with p<0.005, mild anemia or not,
MCV, MCH significantly decreased (p <0.005), RDW was within the
normal range but higher than the non-mutant group (p <0.005). In

two suspected cases of δβthal/βA, red blood cell indexes were within
the normal range, only in children cases MCV and MCH were
decreased. In two cases of Hb Tak, the indexes were within the
normal range except for Hb level, which was slightly increased.
In HbE disease: isolated forms of βE/β or β globin mutation and
βE/βE combination had a higher mean RBC than non-mutant group
with p <0.005; βE/βE combined with an α globin deletion did not alter
RBC compared with solitary βE/βE (p> 0.05). HbE alone or combined


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could have moderate or no anemia. MCV and MCH were generally
low, there were cases which were in the normal range, while β E/β
combined with deletion --SEA; in EABart's disease and βE/βE diseases
MCV, MCH were always low (p <0.001).
3.3.2.2. Red blood cell characteristics on peripheral blood smear
α-thal: the α0-thal carrier group showed the majority of microcytic,
hypochromic erythrocytes and target cells. About 50% of the α +thalassemia carrrier group showed microcytic, hypochromic
erythrocytes.
β-thal: variable signs of microcytic, hypochromic erythrocytes. βthal/β
and βE/βE all had microcytic, hypochromic erythrocytes, especially
91.3% of βE/βE 91.3% cof cases had target cells. βEE / β had 60-80% of
microcytic, hypochromic erythrocytes and few target cells. In two cases
of Hb Tak mutation only revealed only a small number of target cells.
α-thal associated HbE: βEE/β combined with α+-thal mutation, the
rate of erythrocyte abnormalities was lower than that of β EE / β alone.
βEE / β combined with α00-thal, most cases did not show microcytic,
hypochromic and 100% of cases showed target cells. The EABart's
disease form was characterized by markedly erythrocyte
abnormalities on peripheral blood smears with 100% of anisocytosis,

microcytic, hypochromic erythrocytes and target cells.
3.3.2.3. Characteristics of Hb content on electrophoresis
α-thal: the HbA content on the electrophoresis of α-globin-α gene
carriers was generally within the normal range. HbA levels in α-thal
gene carriers did not differ significantly from the non-mutant group
with p = 0.485 and 0.496. Proportion of HbA2: slight decrease
between the mutant and non-mutant group, in which the α 00-thal
group had a lower proportion of HbA2 than the α+ + -thal group, the
difference was statistically significant (p = 0.000).
β-thal: βthalthal / β had HbA2 level between 4.1-7.0%, with either
increased HbF or not. In βEE / β , HbE level ranging from 15.4 to
27.8%. βEE / βE βE had no HbA, the HbE level was 81.4-95.5%.
βthalthal / β combination with HbCS non-deletion mutation HbCS


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showed the same results as in βthal/ββthal / β, no hemoglobinHb
variant revealed on the electrophoresis. Two suspected cases of δβthal showed Hb F increased above > 5%. 2 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 2 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 7 members surveyed, the second family was surveyedand 5
members in the second family (including the subjects).

- There were 4 carriers of Hb Tak gene carriers in the first family and
4 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.


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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

3

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

3

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
2

A

F

62,
4
97,
8
97,
7
61,
9

34,
3,1
5

Genoty
pe

E

0,0 2,2
0,0 2,3

35,
3,0
1
57, 5, 37,
0,0
1
0
9
97,
0,0 2,6
4
63, 32,
4,0
9
1
64, 32,
3,2
6
2
64, 32,
3,2

4
4
67, 29,
3,4
6
0
97,
0,0 2,6
4
62, 34,
3,3
4
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 <85fL had a
sensitivity of 93.0%, specificity of 73.4%. MCH value below <27 pg
had a sensitivity of 86.8% and a specificity of 77.3%. When
combined MCV <85fL andwith / or MCH <27pg, screening accuracy
would be high with a sensitivity of 96.2% and a negative predictive
value of 96.5%.
- DCIP (dichlorophenol indophenol) used alone had a
sensitivity of 94.6%, a specificity of 97.3%; high positive and


16
negative predictive values (94.1 and 97.8%) in screening for HbE
diseases.
- In thalassemia and hemoglobinopathies screeningthal
screening and Hb diseases, test (OF test) had was of low sensitivity
with (55.5%), DCIP test had was at medium sensitivity with (70.9%).
When combined OF test and / or DCIP test, the sensitivity increased
significantly (83.6%).
- Combined MCV <85 fL and/or MCH <27 pg and/or positive
DCIP test(+), the sensitivity and negative predictive value reached up
99.2%.


17
Chapter 4
DISCUSSION
The information and blood samples collected from 1273 Khmer

subjects residing in Soc Trang, Tra Vinh, Bac Lieu and Hau Giang
provinces were analyzed. We have some discussion as follows:
4.1. General characteristics of the studied subjects
In 1273 subjects, 199 were children accounting for 15.6% and
1074 were adults, accounting for 84.4%. The median age of subjects
was similar between the two groups with and without mutation, and
varied from children to up to> 80 years of age. GenderSex
distribution among groups with and without mutation showed a
slightly higher proportion of females than males, t. However, the
difference was not significantnegligible., and Moreover, thalassemia
and hemoglobinopathiesthalassemia and Hb diseases are autosomal
recessive genetic diseases caused by the alleles on the normal
chromosomes, so that the gene carrierying status is not related to
gendersex. The main occupations of ths studied subjects were the
farmers who made up 51.9% and hirelingsemployers 10.6%, only a
small proportion of them were vendtraders (3.9%). 33.6% of the
samples were non-income subjects such as the elderly, students and
housewives. Over 90% of the population lived in rural areas and less
than only <10% lived in urban areas, most of the Khmer were
Buddhist, therefore our sample was representative of the
community, and also showed thal and HbE gene carrier status
has not affected the longevity of the carrier.
4.2. Prevalence of thalassemia and hemoglobinopathiesthalas and Hb
disease, rate of globin gene mutation in Khmer ethnic group in the
Mekong Delta
4.2.1. Rate of thalassemia and hemoglobinopathiesthal gene and
Hb disease carrier
The study identified 39.4% of the Khmer population in the
Mekong Delta carrying thalassemia and hemoglobinopathiesHb

18
disease mutations. The following mutations were reported: α-thal
(9,1%), β-thal (1,3%), HbE (22,4%), Hb E / α-thal (6.5%) and α- thal
/ β-thal (0.1%). PrevalenceThe common rate of thalassemia and
hemoglobinopathies thal and Hb disease gene carrier in the
community were : HbE was 28.9% of HbE, 15,7% of αthalthalassemia was 15.7% and 1.4% of β-thalassemiathal.
Table 4.1.: Rate of thalassemia and hemoglobinopathiesthal and
HbE disease gene carrier in Vietnamese and Cambodian people
Race

Author- year

Tayày

HV. Ngọc, 2007
O’Riordan S., 2010
BV. Viên, 1999
ĐTM. Cầm, 2000

242
580
266
236

αthal
-

Pako
Van Kieuân

Kiều
E ĐeÊ đê

BQ. Tuyên, 1985
NĐ. Lai, 1985

228
78

-

O’Riordan S., 2010
TTT. Minh, 2015

31,6

S’Tieêng
Chaăm
Khmer in the
Central of
VN
Khmer
Cambodia
Cambodia

O’Riordan S., 2010
Bowman JE, 1971
Bowman JE, 1971

266

195
588
3030
55
220
1273
290

Muongường
Thaiái

LTH Myỹ, 2018
Carnley PB., 2006
MunKongdee T.,
2016

number

Rate (%)
HbE
βthal
9,66
4,1
1,4
20,6
12,3
11,4
20,0
3
8,33

6,14
2,56
23,0
0,8

31,4

-

0,3
-

27,7
35,6
29,1
36,8

15,7

1,4

28,9
28,8
13,933,1

PrevalenceThe rate of HbE in the Khmer of Mekong Delta is
higher than that of the Tay, Muong and Thai ethnic groups in the
North; higher than the Pako and Van Kieu ethnic groups in the
Central, comparable to the E De and lower than the S'Tieng ethnic
group, which was the highest prevalence among of Vietnamese ethnic

populations with the highest rate of genes according to the Bowman
JE study. ((55.9%) and O'Riordan S. (35.6%). In Southeast Asia,


19
prevalencethe rate of HbE gene carrier varies widely by regions and
countries,y: Indonesia 1.0-25% in Indonesia, 3.0-10.0% in Malaysia
3.0-10.0%, 6.0-48.0% in Myanmar 6.0-48.0% , and Thailand 10-53%
in Thailand.
PrevalenceThe rate of β-thalthalassemia gene carrier in the
Khmer of Mekong Delta is 1.4%, higher than that in Cambodia
(Munk Kongdee (0.2%)), more similar to the one from Carnley BP.
(0.8%) and Sanguansermsri T. (1.7%) study. In Vietnam, the status of
β-thalthalassemia gene carrier status on ethnic minorities decreases
gradually from the north to the south.
Prevalence The rate of the α-thalthalassemia gene carrier in the
Khmer ethnic group in our study was 15.7%. This wasis lower than
that of MunKongdee T. (2016) study in Cambodia with 22.6% and
Sanguansermsri T. (2006) with 35.4% of Cambodian subjects in the
study carried the α-thal gene mutations. There may be an impact of
unspecified environmental factors causing this difference, such as
malaria,… The rate of the α-thathalassemia l gene carrier in Vietnam
has not reported sufficiently due to the need for molecular
biotechnology. We only recorded the rates of α-thalthalassemia gene
carrier on some ethnic groups in studies conducted in recent years
such as: Ede 31.6%, M'Nong 17.5%; in particular, 24.0% of Hb
Constant SpringCS is the rate of Hb Constant Spring gene carrier rate
in the community of 3 Ta Oi - Van Kieu and Co Tu ethnic groups in
Thua Thien Hue province. Thus, the rate of α-thalthalassemia gene
carrier of the Khmer ethnic group in the South Vietnam is lower than

that of the above ethnic groups.
4.2.2. The rate of globin genotypes of thalassemia mutation
4.2.2.1. α-thalassemiaa
Study revealed four types of types of α-thal mutation in the
Khmer ethnic group including three deletions - α3.7, -α4.2, --SEA, and 1
non-deletion mutation of αCSα (HbCS) . These types of mutation
might be present in heterozygosity or in combination with together,
or be inherited with the β globin -β gene mutations, in particular with
HbE, which generated diverse genotypes in the community. Nguyen
Khac Han Hoan also noted that thesre are four types of mutation that
cause 98.7% of the α-thalthalassemia disease in pregnant women and
the husbands coming for prenatal screening at Tu Du Hospital.


20
Table 4.2.: The rate of α globin-α mutation types
in some ethnic groups

Most combination of mutant genotypes are recorded in the
Khmer of Mekong Delta. The α + + -thal mutations are predominant
with 12.3% in the community and accounts for 78.0% in the globin-α
mutation subgroup. The highest mutation causing α + + - thal is -α3.7
up to 12.2% in the community and accounts for more than 75.0% of
the α globin - α mutation type. Other ethnic minorities in Viet Nam
also have a relatively high rate of - α.3.7: Ede 18.4%, M'Nong 10.3%.
The -α4.2 mutation rate is only 0.5% and αCSα is 2.2%. Study of
Munkongdee T. on Cambodian community had similar results to
ours,: the most common mutation was -α 3.7, the rate of mutation types
was quite similar with 0.6% -α 4.2, 3 , 8% αCSα and 15.8-16.7% -αa3.7.
However, Munkongdee detectnoted the presence of non-deletion

mutant αPakséα at a rate of 0.4-1.4%. This is one of the limitations of
the thesis, we did not study of Hb Paksé. We only identified one
deletionmutation on the α globin-α gene causing α00-thal which was
a deletion mutation --SEA deletion accounting for 1.2% in the
community. This rate is similar to that of Munkongdee T. (1.0%). -SEA
deletion mutation causes the loss of two α-globin -α geness on the
same chromosome, homozygous (--SEA / --SEA produces) Hb Bart's,
unable to release oxygen to tissues, leading to death in the fetus or
short time after birth. Therefore, the identification of this mutation is
very important in prenatal genetic counseling and prenatal screening.
In addition, non deletion mutation HbCS when combined with -- SEA
deletion mutation can also cause Hb H hydrop fetalis pattern.
However, the rate of -- SEA and HbCS mutation in the Khmer
community was not high, suggesting that the risk of severe illness
could not be high. Compared to the Ede and M'Nong ethnic groups,
the risk is higher due to the HbCS mutation rate of 16.9% and the rate
of mutation --–SEA deletion is 2.1%.


21
4.3.2. β-thalassemia and HbE
Table 4.2. Types of β globin-β gene mutations in studies
Types of mutation
Hoan
Tuấn
Hà
My
NKH. PLM.
LTT.
LTH.Our

Hoan
Tuấn
Hà
s
Codon 26
+
+
+
+
Codon 17
+
+
+
+
Codon 41/42
+
+
+
+
IVS-1-1
+
+
+
IVS-1-5
+
+
IVS-II-654
+
+
+

Codon 95
+
+
Codon 71/72
+
+
-28
+
+
(δβ)thal
+
+
+
Five types of mutation have been noted such as codon 26 (G> 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 5 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 1.
Group 2 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 <0.001). RDW was within normal limits, except for AEBart's
disease.


23

R
B

Figure 4.1. Histogram:RBC valueriation of isolated HbE and combined βthalassemia HbE/β-thalassemia


24
HbE is a variant of the β globin-β, which is classified as β-thal
because it also reduces β chain production, α-thal mutation combined
HbE will reduce the imbalance between the two α globin-α and -β
globin, but when the α globin-α is more affected then the imbalance
becomes uncompensated. Therefore, the average RBC in βE E/ β
forms is higher than in the non-mutant group, however when HbE
combining with one1 damaged α globin-α gene, RBC decreases,
whereas HbE combining with both 2 damaged α globin-α gene,
RBC is higher and highest in EABart's disease. β E/E / βEE also has

high RBC but unmarkedsignificant when combining with α-thal. The
variation of the MCV and MCH indexes follows the same pattern,
but in a downward direction.
Erythocyte morphological characteristics on peripheral blood
smear
On peripheral blood smear, red blood cell morphorology also
exhibited similarity as described in the literature with morphologic
changes in ascending order in thal gene carrier and
hemoglobinopathiesHb disease carrier as follows: HbTak variant,
(δβ)thal/β thal, one defective α globin -α gene defect < two defective α
globin gene globin-α gene defect and βEE / β < β- -thal <βEE / βEE
hypochromic microcytosis, poikilocytosis, target cells, basophilic
stippling cells. Especially βEE / βE E showed markedly high rate of
target cells on the smear and EABart's disease had significant red cell
morphological changes.
HHemoglobinb content on electrophoresis
The Hb content on electrophoresis of thalassemia and
hemoglobinopathiesHb disease was similar as described in the
literature with Hb content was almost unchanged in 1 and 2 damaged
α globin-α gene, HbA2 increased more thanby> 4% and not exceed
10% in β-thal. The HbE level in βE E/ β was about 25-30%, and more
than > 80% in βEE / βEE. HbE combined with α-thal decreased or not
the level of HbE, and the level of HbE decreased directly
proportional to the number of defected α globin genes. In the Khmer
community, determining the concomitant inherited α globin and HbE
mutations need to use DNA analysis.


25

Characteristics of Hb Tak gene carriers
We documented 2 cases with an elevated Hb F over 30%, that
were determined as Hb Tak mutation,
a β globin variant.
Heterozygous Hb Tak carriers had red blood cell indexes within the
normal range, with the exception of normal and high Hb level. This is
a variant Hb that increases the affinity of red blood cells with oxygen
hence heterozygous Hb Tak carriers might have slight erythocytosis
which have been described in the literature. Hb Tak in combination
with HbE, Hb analysis were similar to HbE/β thalassemia. Therefore,
in the Khmer ethnic group, when the hemoglobin electrophoresis was
similar to HbE/β-thal and RBC indexes are normal, HbTak/HbE
could be suspected. Patients with Hb F level over 30% should be
analyzed to identify the mutation.
RBC indexes, some other tests and their roles in thalassemia
and Hb E disease screening
- OF test is considered as one of valuable tests of thalassemia
and HbE screening in the world if the solution is carefully prepared
and standardized.
- When the reaction conditions are secured, the DCIP test has a very
high sensitivity and specificity for HbE screening and is an optimal
selection for screening in communities with high HbE carrier rate.
- Combining MCV <85 fL and/or MCH <27pg and/or DCIP
positive test for thalassemia and hemoglobinopathies screening, the
sensitivity and positive predictive value are highest with 99.2%;
specificity of 66.7%.
CONCLUSIONS
Based on the above results and discussions, we would like to
make the following conclusions:
1. Prevalence of thalassemia and hemoglobinopathies, the rate of

globin gene mutations in the Khmer ethnic group in the Mekong
Delta
1.1. Prevalence of thalassemia and hemoglobinopathies
- Prevalence of thalassemia and hemoglobinopathies in the
Khmer population of Mekong Delta is 39.4% with 05 types: HbE
accounts for 22.4%; α-thal 9.1%; β-thal 1,3%; HbE/α-thal 6.5%; and
β-thal/α-thal 0.1%.