Electrophoresis features and genotypes of Hb Bart’s hydrops fetalis Đặc điểm điện di huyết sắc tố và kiểu gene hội chứng thai tích dịch do Hb Bart's bsvotason bs võ tá sơn bác sĩ võ tá sơn

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Electrophoresis features and genotypes of Hb
bart’s hydrops fetalis
Youqiong Li, Liang Liang, Mao Tian, Ting Qing & Xin Wu
To cite this article: Youqiong Li, Liang Liang, Mao Tian, Ting Qing & Xin Wu (2019):
Electrophoresis features and genotypes of Hb bart’s hydrops fetalis, Scandinavian Journal of
Clinical and Laboratory Investigation, DOI: 10.1080/00365513.2019.1703211
To link to this article: https://doi.org/10.1080/00365513.2019.1703211
Published online: 14 Dec 2019.
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2. ORIGINAL ARTICLE
Electrophoresis features and genotypes of Hb bart’s hydrops fetalis
Youqiong Lia,b
, Liang Liangb
, Mao Tianb
, Ting Qingb
and Xin Wub
a
Center of Reproductive and Genetic, People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China; b
Center of Prenatal
Diagnostic, People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China
ABSTRACT
Hydrops fetalis syndrome (Hb Bart’s disease) is one of the common fetal development abnormalities
and the most severe form of a-thalassemia. It causes fetal death during the third trimester or shortly
after birth. The aim of this study is to investigate the feature of Hb Bart’s disease by Capillary electro-
phoresis (CE) and its genotypes. A total of 38 cases of suspected fetal Hb Bart’s disease were analyzed
by B-ultrasound and CE, including 17 with 24–28 weeks of pregnancy, 9 with 29–32 weeks of preg-
nancy, and 12 with 33–38 weeks of pregnancy. All cases were then identified by DNA analysis. The
minimum concentration of Hb Bart’s was 72.2% and the highest 90.9%. Hb Bart’s fraction increased
while that of Hb Portland decreased with the duration of pregnancy. In order to avoid serious mater-
nal morbidity, it is necessary to diagnose Hb Bart’s disease as soon as possible and CE can be used as
a method of rapid diagnosis.
ARTICLE HISTORY
Received 15 April 2019
Revised 3 December 2019
Accepted 8 December 2019
KEYWORDS
Hydrops fetalis syndrome;
Hb Bart’s disease; capillary
electrophoresis (CE);
thalassemia
Introduction
a-Thalassemia is the most common inherited disorder of
hemoglobin synthesis in southern China, characterized by
reduction or absence of the a-globin chains due to deletion
or mutation of a-globin genes [1]. The most severe form of
a-thalassemia is Hb Bart’s disease (Hb Bart’s hydrops fetalis)
because all four a-globin gene loci (–/–) are lost. Hydrops
fetalis syndrome is one of the common fetal development
abnormalities. The fetus shows systemic skin edema, lymph-
atic cysts, enlarged heart and chest, placental thickening,
and other structural abnormalities revealed by B-ultrasound
[2]. The Hb Bart’s hydrops fetalis belongs to non-immune
edema and is the most common type in Guangxi Zhuang
Autonomous Region in the south of China [3]. Pregnancies
with Hb Bart’s hydrops fetalis cause fetal death during the
third trimester or shortly after birth, and serious maternal
morbidity frequently occurs [2]. Therefore, it is very import-
ant to detect the Hb Bart’s hydrops fetalis as early
as possible.
Capillary electrophoresis (CE) is a powerful technique for
screening hemoglobinopathies [4,5] as well as for hemoglo-
bin A1c quantification (HbA1c) [6–8]. It is also used for Hb
Bart quantification in cord blood for screening a-thalas-
semia [9,10]. There is no published study on the electro-
phoregram features correlated to the genotypes of Hb Bart’s
hydrops fetalis in China. In this study, we describe the diag-
nosis Hb Bart’s hydrops fetalis using CE and genotype
investigation of in Guangxi, which is the area of China
where thalassemia is most common [11].
Materials and methods
Samples collection
A total of 38 suspected cases of Hb Bart’s hydrops fetalis
revealed by B-ultrasound were selected from the routine
prenatal examination. After obtaining informed consent
from the pregnant women, 2 mL of cord blood sample were
collected in EDTA tubes from the detached umbilical vein
for hemoglobin and genetic analysis. Other extracted data
include age, gestational week at the moment of fetal diagno-
sis and the thalassemic status of the couples. Ethics approval
of the study protocol was obtained from the Ethics
Committee of Guangxi Zhuang Autonomous
Region hospital.
Hemoglobin analysis
Hemoglobin separation and quantification were performed
on an automated capillary electrophoresis system (Capillarys
2; Sebia, Paris, France). The principle of the system is that
charged molecules are separated in a specific alkaline buffer
by their electrophoresis mobility. The results were displayed
using the Phoresis program with software version 6.2, which
separates the electrophoregram into 15 zones. Each peak of
hemoglobin appears in a specific zone, such as the Hb
Bart’s positioned in zone 12. In absence of Hb A and/or Hb
A2, the zones are not displayed, and fractions are not iden-
tified since peak identification is based on Hb A and A2
positions. In order to obtain the identification of the peaks,
the blood sample must be diluted volume to volume with
CONTACT Youqiong Li liyouqiong327@163.com Center for reproductive and genetic, People’s Hospital of Guangxi Zhuang Autonomous Region, 6
Taoyuan Road, Nanning, Guangxi 530021, China.
ß 2019 Medisinsk Fysiologisk Forenings Forlag (MFFF)
SCANDINAVIAN JOURNAL OF CLINICAL AND LABORATORY INVESTIGATION
https://doi.org/10.1080/00365513.2019.1703211

3. the normal Hb A2 control. Under this condition, the pat-
tern is centered, and peaks are identified.
Genetic analysis
Once the hemoglobin analysis was completed, Genomic
DNA was extracted from leukocytes by using thalassemia
PCR kit (Shenzhen Ying Sheng-Tang Bio-tech Ltd.,
Shenzhen, China) according to manufacturer’s instructions.
The gap polymerase chain reaction (Gap-PCR) was used to
identify the four common Chinese deletion (NG_
000006.1:g.26264_45564del19301, named–SEA; NG_000007.
3:g.64384_76993del12610, named–THAI; NG_000006.1:g.
34164_37967del3804,named –a3.7; –a4.2). b-thalassemia
mutation common in Chinese people (including HBB:c.–
78A > G, named –28 M; HBB:c.–79A > G, named –29 M;
HBB:c.–80T > C, named –30 M; HBB:c.–82C > A, named
–32 M; HBB:c.45_46insG, named CDs 14/15 M;HBB:
c.52A > T, named CD17M; HBB:c.79G > A, named CD26M;
HBB:c.84_85insC, named CDs 27/28 M; HBB:c.94delC,
named CD31M; HBB:c.126_129delCTTT, named CDs
41/42 M; HBB:c.216_217insA, named CDs 71/72 M; HBB:
c.92 þ 1G > T, named IVS-I-1M; HBB:c.92 þ 5G > C, named
IVS-I-5M; HBB:c.316-197C > T, named IVS-II-654M;
HBB:c.–50A > C, named CAP þ 1M; HBB:c.2T > G, named
Int M.) were detected by PCR and reverse dot-blot assay
(PCR-RDB).
Results
Genotype investigation
A total of 38 cases were diagnosed with Hb Bart’s hydrops
fetalis by B-ultrasound in this study. Among these cases, 17
(44.7%) were in 24–28 weeks of pregnancy, 9 (23.7%) were
in 29–32 weeks of pregnancy, 12(31.6%) were in
33–38 weeks of pregnancy. The mean maternal age was
26.9 ± 3.1 years and the mean gestational age was
29.8 ± 3.8 weeks. All pregnant women were recommended
for invasive testing: cord bloods were obtained by puncture
for DNA analysis.
It was found that 37 (97.4%) cases were
homozygous–SEA deletion, genotype named–SEA/–SEA.
Only one case (2.6%) was found by association of the –SEA
deletion and the –Thai deletion.
Hemoglobin patterns
In normal newborns, two peaks of Hb A and Hb F are
observed in Z9 and Z7 zones respectively (Figure 1 left). In
heterozygous a-thalassemia, an additional peak of Hb Bart
is present in Z12 zone (Figure 1 right). In Hb H disease, the
fractions present are: Hb A, F, Hb Bart’s (at higher level
than in heterozygous a-thalassemia) and Hb H in Z15 zone
(Figure 2). In Hb Bart’s disease, neither Hb A nor Hb F is
present but other peaks are observed: Hb H (Z15 zone), Hb
Bart’s (Z12 zone), Epsilon 4 (Z11 zone), Hb Gower1 (Z9
zone), Hb Portland (Z7 zone) (Figure 3) [12]. Epsilon, Hb
Gower1 and Hb Portland are embryonic hemoglobins which
are only detected in embryonic period and in Hb
Bart’s disease.
In our study, the lowest value of Hb Bart’s was 72.2%.
The average value of Hb Bart’s was 78.2 ± 2.5% in second
trimester which was higher than that observed in Hb H dis-
ease (32.1 ± 3.1%) [9]. Surprisingly, the highest value of Hb
Figure 1. Hb analysis of normal (left) and a-thalassemia fetus (right,-a3.7 heterozygote) by CE in cord blood.
Figure 2. Hb analysis of Hb H disease (–SEA/-a3.7) by CE in cord blood.
2 Y. LI ET AL.

4. Bart’s (only the 26th gestational week) was 90.9% and this
was also the only case whose genotype corresponds
to –SEA/–Thai (the genotype of the other cases were
all –SEA/–SEA).
Discussion
Hb Bart’s (c4) is a non-functional hemoglobin for oxygen
transfer and Barts hydrops fetalis almost always results in
death in utero or in the early neonatal period [13].
Moreover, Hb Bart’s disease is frequently associated with
serious maternal morbidity and even mortality [3]. This was
documented in one of our patients, a pregnant woman who
was admitted to the Intensive Care Unit because of a critical
condition during her third trimester. Fortunately, she was
saved after termination of her pregnancy.
Early diagnosis can prevent these complications. Even
though prevention is propagated and provided by hospitals
and governments, some people still do not conduct screen-
ing and prenatal diagnosis of thalassemia in Guangxi
Zhuang Autonomous Region, especially those who live in
rural areas. When they come to hospital for prenatal exam-
ination, it is already in the second or third trimester. At this
point, the sooner the diagnosis is carried out, the better is
the prognosis for pregnancies. It is therefore important to
use a method which is rapid and accurate.
Usually, molecular diagnosis is used as confirmatory
laboratory test, but it often requires at least a week (this is
the time required for testing process in Chinese hospital
clinical laboratory). For affected pregnancies, the result can
not be obtained early enough to prevent maternal
morbidity. Since 2010, CE is used to make rapid diagnosis
of Hb Bart’s disease in our hospital. As shown in Figure 3,
Hb Bart’s disease displays a special pattern of embryonic
hemoglobin fractions which is different from Hb H disease
and heterozygote a-thalassemia (Figures 1 and 2). CE, which
separates Hb fractions and allows their quantification, can
measure Hb H, Hb Bart’s, Epsilon 4, Hb Gower1 and Hb
Portland directly from the electrophoregrams. Embryonic
hemoglobin fractions (Epsilon4, Hb Gower1 and Hb
Portland) can only be detected in the second or third tri-
mester of pregnancy in the case of Hb Bart’s disease.
Therefore, it can be diagnosed by CE quicker than by gen-
etic analysis. By high performance liquid chromatography
(HPLC), Hb Bart’s and H are eluted in the void volume and
therefore can only be distinguished when their concentra-
tions are very high (>5%). Even when they are visualized,
the quantification is not possible [14].
Table 1 shows the mean values for the five peaks in this
study. As gestational weeks increased, Hb Bart’s value
increased and that of Hb Portland decreased. The higher
Hb Bart’s value, the more severe the fetal hypoxia and the
easier the fetus will die early. Hb Portland is a functional
Hb and able to transfer small amount of oxygen. The higher
Hb Portland value, the longer the fetal survival will be [15].
Four cases of Hb Bart’s disease were described by
Srivorakun et al. [16]. However, our data are more detailed
and include 38 cases along with their correspond-
ing genotypes.
In summary, our results have shown that the Epsilon4,
Hb Gower1 and Hb Portland were also specific Hb fractions
for Hb Bart’s disease, in addition to the Hb Bart’s which is
significantly elevated (at least at 72.2% at 30 gestational
weeks). It was difficult to obtain a cutoff value due to sam-
ple size limitation. Diagnosis of Hb Bart’s disease using the
CE system can be a simple and rapid alternative approach
of testing cord blood. Most of the genotypes were mainly
–SEA/–SEA, and it was consistent with SEA deletion which
was the common mutation in our region.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Health Department Research Fund of
Guangxi [Z2011449] and Key Research and Development Program of
Guangxi [AB17292089].
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