Myanmar Health Sciences Research Journal
Original Articles :
Myanamr Health Research Registration 2020; 32(2): 105-111.
DOI: https://doi.org/10.34299/mhsrj.00989

Molecular Diagnosis of Haemoglobin E Among Pregnant Women Attending 300-Bedded Pyin Oo Lwin General Hospital

Khin Moe Aung, Nandar Ko,Thei Thei Moe Han, Myint Myint Khaing, Htay Htay Kywe, Myitzu Tin Oung, Htwe Htwe, & Khin Saw Aye

Myanmar Health Sciences Research Journal, 2020; 32(2):105-111

ABSTRACT

Haemoglobin E (HbE) is the most common Hb variant found among Southeast Asian populations. Myanmar also has high prevalence of important haemoglobinopathies: HbE - 1 to 28.3% accounting for 1 to 4.9 births per 1000 infants with a major haemoglobinopathy. This study was conducted to determine the prevalence of HbE among pregnant women attending 300-bedded PyinOoLwin General Hospital. This hospital-based cross-sectional descriptive study was done in 2019.
A total400 apparently healthy pregnant women (17 to 44 years) with the mean age 27.51 years (SE - 6.14 years) were involved. Four milliliters of venous blood were collected andhaematological parameters were determined by automated haematology analyzer (Pentra-60) and blood film examinations were done. Then, hypochromic microcytic anaemia were assessed for serum ferritin by fully automated immunology analyzer (Mini Vidas) and molecular diagnosis of HbE was identified by using ARMS-PCR reaction. Data entry and analysis was done by SPSS software 20.0 version. The overall prevalence of anaemia was 39.0% (156/400) in which iron deficiency anaemia (IDA) was occurred in 70 women (17.5%) and the rest (86, 21.5%) were not iron deficient anaemia, respectively. The overall percentage of HbE in this study was found to be 27.6% (43/156) among the HMA pregnant women. The ratio of IDA: non-IDA cases among the pregnant women were 1: 1.2. Therefore, this study highlighted that one of the main causes of anaemia was haemoglobinopathy and remains a common health problem among pregnant women. For prevention of thalassaemia major in maternal child health care units in our country, prenatal screening and family counseling of beta and alpha thalassaemia carriers inpregnant women is highly recommended.


RESULT
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The range of age distribution among participants was from 17 to 44 years and the mean age was 27.51 years (SE -6.14 years). Back- ground subjects’ characteristics revealed that total 400pregnant women were Bamar

Table 1.Prevalence of anaemia among pregnant women(400 women)

Variables

Anaemic women (Hb below 11.0 g/dl-WHO)

Percent

Mean

Std.

deviation

First trimester (n=22)

5.5

11.6

0.9

Second trimester (n=223)

55.8

10.8

1.2

Third trimester (n=155)

38.7

11.2

1.2

(305, 76.3%), Shan (26, 6.5%), Indian (43, 10.8%) and others (26, 6.5%).In this study, 305 samples were Bamar and 95 were other races such as Kachin, Chin and Kayin. Bamar dominated the study populations followed by Indian. The number of pregnant women who attended the antenatal clinic during the first, second and third trimester were 22 cases (5.5%), 223 cases (55.80%) and 155 cases (38.80%), respectively(Table 1).Regarding the gravida, there were 195(48.8%) primigravida women, 196(49.0%) gravida two to four women and 9(2.3%) gravida five and above women, respectively.

Table 2.Comparison of haematological parameters and serum erritin level among pregnant women

Variables

Pregnant women (Mean ± SD)

Iron

deficiency

anaemia

(n= 70)

HbE

(n=86)

Normal(n=244)

Haemoglobin(g/dl)

9.7±1.0

10.1±0.7

11.7±0.6

RBCcount(109/L)

4.37±0.45

4.46±0.49

4.55±1.93

Mean corpuscular
volume (fl)

72 ±8

73±6

82± 6

Meancorpuscular
haemoglobin(pg)

22.2±2.7

22.9±2.3

26.2±2.6

Mean corpuscula
haemoglobin

31±0.9

30.9±1.2

32±1.7

Concentration(%)

8.73±4.13

43.74±42.21

28.95±44.35

Serum ferritinlevel
(ng/ml)

9.7±2.5

9.7±3.1

9.6±2.9

WBC (109/L) plate- let(109/L)

274±73

312±83

249±69

The prevalence of the anaemia among pregnant women in this study were 156 women (39.0%) in which iron deficiency anaemia (IDA) was 70 women (17.5%) and the rest (86 women, 21.5%) were not iron deficient anaemia, respectively. Among the 156 anaemicpregnant women, 11 had normochromic normocytic and 145 had hypochromic microcytic anaemia. Seventy out of 145 hypochromic microcytic anaemic women were iron deficiency anaemia.

After that, these 86 women were determined for heterozygous beta thalassemia including haemoglobin E and found out 43 women (10.8%) with presence of HbE by ARMS-PCR reaction.The mean haemoglobin level and absolute red cell values as well as serum ferritin levels of pregnant women were described in Table 2.

 

12     3      4       5      6      7      8

 

Lane 1

=

100 bp DNA ladder

Lane 2, 3,5,6, 7

=

Positive samples (310bp)

Lane 4

=

negative sample

Lane 8

=

negative control

 

Fig.





1. Molecular identification of haemoglo-bin E by ARMS-PCR using 1 µl DNA and 1.5% agarosegel electrophoresis

The prevalence rate of anaemia among the second trimester group was significantly higher than the other groups (p value <0.05). Regarding the gravida, 58/156 (37.2%) primigravida women had anaemia and for the other group (gravida two and above) 55/156 (35.2%) had anaemia (p value>0.05). Among the 156 cases of hypochromic microcytic anaemic pregnant women, the overall percentage of HbE in this study was found to be 27.6% (43/156) among the HMA pregnant women by ARMS-PCR reaction(Fig.1).

 




INTRODUCTION
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Human beta globin (HBB) gene maps in the short arm of chromosome 11 position 15.5 containing the delta globin gene, the embryonic epsilon gene, the fetal A-gamma and G-gamma genes, and a pseudogene. Mutations in HBB cause genetic disorders such as β-thalassaemia as well as sickle cell disease, haemoglobin C (HbC) and haemoglobin E (HbE).1HbE is the second most prevalent haemoglobinopathyafter HbS showing the highestprevalence in Southeast Asia.2HbE is defined by the heterozygous condition associated with one, normal adult haemoglobin (HbA) β gene and one  variant (HbE) βgene. HbEis common in Southeast Asia. Thailand and Myanmar have an overall prevalence of around 14-15 percent.3 The most common combination of beta-thalassaemia with abnormal Hb or structural Hb variant with thalassemic properties is HbE/b-thalassaemia which is most prevalent in Southeast Asia where the carrier frequency is around 50 percent.4

Haemoglobin E has the clinical phenotype of a mild form of β-thalassaemia, and is most frequent in Southeast Asia, particularly eastern Thailand and Laos. The combination of HbE with β-thalassaemia spans thalassaemiaphenotypes, from a conditionindistinguish able from thalassaemi amajor to a mild form of thalassaemia intermedia5. HbE/β-thalassaemiais the commonest severe form of thalassaemia in Southeast Asia and parts of the Indian subcontinent. HbE is inefficiently synthesized and hence, when it is inherited together with β-thalassaemia, there is a marked deficiency of β-chain production.6

One of the codon-26 mutation in exon 1, the βE allele, leads to deficient production of βE-globin, which contains a Glu®Lys substitution at amino acid 26. Many patients with thalassaemiahaemoglobin E disease seen in Myanmar, unlike thalassaemia major, may survive till adult life and full physical and sexual development is possible in a significant number.7Although not always transfusion dependent, haemoglobin values are in the 4-9g/dL range, with an average of 6-7 g/dL. Although very little is known about the natural history of this disorder, it is clear that in many parts of Southeast Asia and India it causes a very high mortality in early life.6 The prevalence of HbE trait was reported as 1 to 28% among various races of Myanmar.7 Anaemia during pregnancy is a common problem. The main causes of anaemia are thalassaemia carrier and iron deficiency anaemia. The prevalence of anaemia has changed from the past which may be due to  the decline of iron deficiency anemia from adequate dietary intake and nutritional supplements.8

Pregnancy itself is known to be a state of oxidative stress and may technically bring about haemolytic crisis episodes. Compli- cations of pregnancy in HbE disease birth weight baby, preterm deliveries and include intrauterine growth restriction, low increased susceptibility to infections. But, the course in pregnancy in HbErait and HbE disease is relatively benign with no significant adverse effects on either foetal or maternal prognosis.There is an improvement of PCR permitting rapid analysis of any known mutation in genomic DNA, known as amplification refractory mutation system (ARMS). ARMS is a technique allowing the direct analysis of any locus of interest and thus generally applicable to any inherited disease provided sufficient sequence data is available.

An important practical consideration with this approach (as with other PCR-based strategies) is that it is unnecessary to prepare high quality DNA suitable for restriction enzyme.10 ARMS is suitable for its rapid, simple,cost-effective, and safe (non-radioactive method) for detection of most common β-mutation worldwide. The other technique includes reverse dot blot, real-time PCR and oligonucleotide microarray analysis have been described for the rapid analysis of thalassemia and hemoglobinopathies; however, these techniques are more sophisticated and expensive than current screening tests.11Therefore, accurate determination of HbE was important by using effective diagnosis protocol for the HbE carrier screening in Myanmar.

The management of HbE trait and disease includes only folic acid but in HbE/β thalassaemia, periodic blood transfusions may be required to maintain the haemoglobin level.The purpose of this study is to assess distribution of HbE among these populations in which it highlights the increasing prevalence of HbE variants in upper Myanmar. Moreover, this study may be useful for early diagnosis and important to avoid unnecessary prescription of iron salts and then, the results of this study may provide the data for further research.It can reduce the therapeutic problem with ferrous sulphate tablet and syrup for the anaemia patients. This study can detect the prevalence of heterozygous beta thalassaemia in pregnant mothers, and will get update information on the prevalence of heterozygous beta thalassaemia in this community for our country.


SUPPLEMENTARY MATERIAL
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This hospital-based cross-sectional descriptive study was done at Pathology Research Division, Department of Medical Research (PyinOoLwin Branch) and 300-bedded PyinOoLwin General Hospital. A total of 400 pregnant women who were attending at first visit to ante-natal clinics had given the written informed consent were recruited by using systematic sampling procedure from January to December 2019. Then, 2mlof venous blood in EDTA tube and 2 ml of blood in plain tube were collected. Firstly, haematological parameters were determined by automated haematology analyzer(Pentra-60) and blood film examinations were done. Then, hypochromic microcytic anaemia were assessed for serum ferritin by fully automated immunology analyzer (Mini Vidas) and molecular diagnosis of HbE was conducted by using ARMS-PCR reaction.

The CD 26 (G-A) of oligonucleotide specific primer was used. DNA amplification was carried out using dNTPs, 10X buffers (Gold), and enzyme Gold Taq polymerase in a total volume of 20µl. PCR mix 19ul and 1 ug of DNA were used. The PCR consists of 25-35 cycle of; denaturation at 95•C for 1minute, annealing at 60•C for 30 seconds, extension at 72•C for 2minutes and final extension step at 72•C for 10minutes.Data entry and analysis was done by SPSS software 20.0 version.

Ethical consideration

The approval was obtained from the Institutional Research Board of the Department of Medical Research before the study was conducted.


DISCUSSION
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In this study, the ratio of iron deficiency anaemia(IDA) and non-IDA cases among the pregnant women were 1: 1.2. It may be concluded that IDA is still the leading cause of nutritional deficiency anaemia and HbE is one of the main causes of anaemia and remains a common health problem among pregnant women and also haemoglobinopathy remains now a global problem.

Among the 156 cases of hypochromic micro-cyticanaemic pregnant women, 5 different races were identified. From this study, Bamarethinic pregnant women with HMA cases were mostly detected. Among the HMA non-IDA pregnant women (86 cases), there were 80.2% (69/86) of Bamar, 4.6% of Indian, and 7.0% of Shan. So, HMA non- IDA cases were also more commonly detected in Bamar ethnic of this study. Among the 86 non-IDA cases, those with first trimester were 4.6% (4/86), those in second trimester were 66.3% (57/86) and those in third trimester were 29.1% (25/86).
So, most of them took AN care at second trimester of gestation.

In this study, the pregnant women in gravida (G-1) were 50.0% (43/86) and those with gravida2 to 4 were 38 cases (44.2%) and multigravida (G5 and above) was 5 cases (5.8%) among the non-IDA (86) cases. Number of gravidity is also one of the important factors for both pregnant woman and her baby. Gravidity is also important because some have no child due to missed or repeated abortion or still-birth in their past medical history. This might be due to their underlying disease or hemoglobinopathy or thalassaemia. The pregnant women took iron supplementation from antenatal care clinics. Sometimes unnecessary iron medication was taken in unknown causes of hypochromic microcytic anaemia patients as they did not know about their underlying causes of HMA. In those cases, this was very important because of getting iron overload and hemosiderosis if she has normal or increased ferritin levels.

In this study, the degrees of anaemia in non-IDA were assessed as mild, moderate and severe anaemia according to WHO classification (WHO 2001). Mild anaemia was seen in 80 cases (93%) and moderate anaemia 3 cases 3.5% and severe anaemia of 3.5% were detected in non-IDA cases. So, there were not so many severe anaemia cases in non-IDA pregnant women. Hematological parameters of total RBC count, MCV, MCH and MCHC were assessed in non-IDA (86) cases. There were 64.0% of cases with MCV level of 70-80 fl, RBC population of ≥4×106/L in 87.2%, MCH levels <25 pg in 89.5% (77/86), and MCHC levels of 30-33g/dl in 88.4% of non-IDA pregnant women. So, the hematological parameters in non-IDA cases were not markedly reduced except MCV levels.

Among the 43 cases of HbE disorders, mild anaemia 42 cases (97.7%), and moderate anaemia1 case(2.3%) and no severe anaemia were detected. There were 65.1% of cases with MCV level of 70-80 fl, RBC population of ≥4×106/L in 86%, MCH levels <25 pg in 86% (37/43), and MCHC levels of30-33g/dl in 95.3%. So, the hematological parameters were not markedly reduced except MCV levels and total RBC population is normal or high in HbE cases.

The RBC count is also useful as a diagnostic tool because the thalassaemia produces a microcytic anaemia with an associated increase in the RBC population. San-chaisuriyaet al described that the primary screening method for all forms of thalassemia relied on hematologic index cutoffs, which involved an accurate blood count using an electronic cell counter like hematology autoanalyzer. Individuals with mean corpuscular volume (MCV) values less than 80 fL and mean corpuscular hemoglobin (MCH) values less than 27 pg should be examined further to confirm or exclude the diagnosis for α-thalassemia and β-thalassemia.12

The distribution of HbE extends from Eastern India through Southeast Asia and has its highest incidence in Thailand, Laos, and Cambodia. The highest frequency of the HbE allele was reported in eastern Thailand near the Vietnamese border-an area termed the ‘‘Hemoglobin E Triangle’’- where the carrier rate is as high as 25% to 30%. The HbE variant also is seen sporadically in parts of China and the Indonesian islands.14

Haemoglobin E in Myanmar was first reported in 195613and the prevalence of haemoglobin E trait was reported as 1% to 28% among the various races of Myanmar.15,16,17The overall percentage of HbE in this study was found to be 27.6% (43/156) among the HMA pregnant women. In Myanmar 2000, peripheral bloods of 132 children who had been clinically diagnosed as thalassemia major and were taking frequent and regular blood transfusion at the daycare room, Yangon Children’s Hospital, Myanmar. Eighty-four out of 132 samples (63.6%) showed large peak in DEAE-HPLC and thick clear band in IEF representing the presence of HbE.18

A cross-sectional study was conducted on 239 schoolage children from Thanlyin and Insein Townships, Yangon Region, Myanmar in 2017. The prevalence of anemia was 46.4% among them and out of 85 children with hypochromic microcytic anaemia revealed that only 3 children (3.5%) had iron deficiency, HbE trait31(36.5%) and also HbE disease only 3(3.5%) by cellulose acetate hemoglobin (Hb)electrophoresis.19So, there should beawareness on the consequencesof anemia during pregnancy and nutritional counseling on consumption of iron-rich foods and iron/foliate supplementation are highly recommended. For prevention of thalassaemia major in maternal child health care units in our country, prenatal screening and family counseling of beta and alpha thalassaemia carriers in pregnant women is important.

Recommendations

-To get early detection, effective screening and easily available screening tests like blood for complete picture are needed.

-Blood for complete picture should be done

in all pregnant women during taking antenatal care so that HMA cases can be detected at first.

- After giving iron supplementation in those HMA cases, then PBF examination and blood for CP should be rechecked again.
If there is no improved in red cells parameters, thalassaemia is more likely especially in thalassaemia ethnic areas.

- The thalassaemia screening tests should be added as compulsory testing in AN care so that if someone has suspected for thalassaemia, her partner should also be investigated to detect for carrier status. By doing the genetic counseling and testing in couples, the occurance of disease frequencies for either alpha or beta thalassaemia can be well prevented.

- It is especially in young reproductive aged group. So important to get early genetic diagnosis, they will know about theirs carrier status.



ACKNOWLEDGMENT
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The authors would like to thank Medical Superintendent,300-bedded PyinOoLwin General Hospital and all the staff of Pathology Research Division.We also wish to express our heartfelt thanks to all pregnant women,without whom this study would not be accomplished.


CONFLICT OF INTEREST
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The authors declare that they have no competing interests.


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