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mediate severity, three of the four a-globin genes are Sciences, version 15.0 (SPSS Inc., Chicago, IL, USA).
affected. Although the clinical severity of HbH disease The data are expressed as median values with ranges
can be quite variable, genotypes involving point for continuous variables and numbers with percent-
mutations of the a2-globin gene (aTa/–) result in a ages for categorical variables. Nonparametric tests
more severe phenotype as compared with genotypes were used to compare continuous variables (the
involving gene deletions (-a/–) [1]. Mann–Whitney test or the Kruskal–Wallis test) or cat-
As a result of the highly deficient globin chain egorical variables (Pearson’s chi-squared test) across
synthesis in HbH disease, there are insufficient a globin subgroups of patients. These tests were followed by a
chains available for binding to b-globin chains, leading post hoc Dunn test. P values are two-sided and con-
to the formation of unstable homotetramers (HbH = b4) sidered significant when <0.05.
, which are the most clear diagnostic parameter and the Medical data were collected from each patient.
cause of pathogenesis. Theoretically, b-thalassemia trait Informed consent was obtained from all patients or
is considered an ameliorating factor of HbH disease and parents of pediatric participants.
several isolated cases have been reported. In these cases,
the disease was masked by the reduction or absence of
RESULTS
HbH [2]. In a previous study, we also found that
co-inherited b-thalassemia heterozygosity slightly, but
The cohort
not significantly, alleviated anemia in patients with
nondeletional HbH disease [3]. Here, we further sum- Of the 314 patients with HbH disease, 141 were male
marize the clinical characteristics of patients with HbH and 173 were female. The average age was 17.0 years,
disease in the presence of b-thalassemia heterozygosity and 148 cases (47.1%) were younger than 14 years of
in a larger cohort of patients. age.
From October 2009 to March 2011, we performed The 20 kb deletion that is typical for a-thalassemia
hemoglobinopathy analysis and full genotyping on patients in Southeast Asia (–SEA) was the only a defect
3256 patients from the province of Guangxi, Southern found in our cohort. Fifteen patients were diagnosed as
China. HbH-WS (–SEA/aWSa). One hundred and sixty-six
Hemoglobin levels and red blood cell indices were patients were diagnosed with HbH-CS (–SEA/aCSa), and
determined using an auto-hematology analyzer (Cell eight patients were diagnosed with HbH-QS (–SEA/aQSa),
Dyn 1700, Abbott Park, IL, USA). HbA, HbA2, and both of which are classified as nondeletional HbH dis-
HbF levels were determined using a Bio-Rad Variant ease. One hundred and twenty-five patients were diag-
II high-performance liquid chromatography (HPLC) nosed with deletional HbH disease, including 82 cases of
system (Variant; Bio-Rad, Hercules, CA, USA). The –SEA/-3.7a and 43 cases of –SEA/-4.2a. Anemia in 15
three most common deletional a-thalassemia genes patients with HbH-WS was mild [3], and therefore these
(including –SEA, -3.7a, and -4.2a) were detected by patients were analyzed separately.
multiplex gap-polymerase chain reaction. The three
common nondeletional a-thalassemia genes [Constant
The b-thal defects
Spring variant (aCS), Quong Sze variant (aQS), and
Westmead variant (aWS)] and the 17 most common Twenty-seven of the 314 HbH cases (8.6%) were het-
b-thalassemia genes were detected by reverse dot- erozygous for b-thalassemia. Among these patients, 17
blotting with a genetic diagnostic kit (Yi Sheng Tang were diagnosed with a b0 mutation (eight cases with
Biological Products, Shenzhen, China) [3]. A group of CD41-42(-TCTT), eight cases with CD17(AfiT), and
314 patients, with three of the four a-globin genes one case with CD14-15(+G)). The remaining 10
affected and diagnosed with HbH disease, were exam- patients carried the b+ mutation [six cases with IVS-2-
ined for b-globin gene defects. Statistical analyses 654(CfiT), two cases with )28(AfiG), and two cases
were performed using the Statistical Package for Social bE(CD 26 GfiA)].
0.778
0.095
0.694
0.482
0.772
0.597
M, male; F, female; Hb, Hemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin
1.00
heterozygosity
P
b-thalassemia heterozygosity significantly increased
b-thalassemia
the hemoglobin level and delayed the age of diagnosis
26.8 ± 8.5
7.6
2.6
3.7
17
22
Without
in patients with nondeletional HbH disease (Table 1).
(n = 12)
±
±
±
±
±
Conversely, neither the hemoglobin level nor the age
66.5
22.3
23.1
117
289
5/7
of diagnosis differed significantly in patients with dele-
tional HbH disease. For patients with HbH-WS, b-thal-
HbH-Westmead
b-thalassemia
assemia slightly decreased the Hb level, but this was
25.3 ± 4.9
2.5
1.9
1.6
11
26
not significant.
(n = 3)
±
±
±
±
±
With
294
98
68.3
21.1
21.9
1/2
Table 1. Clinical characteristics of patients with different types of HbH disease with or without b-thalassemia trait
Effects of coexisting b-thalassemia on HPLC analysis
0.217
0.725
0.452
0.004
0.105
0.010
0.131
Owing to practical reasons, only 218 patients of 314 were
analyzed on HPLC. All samples were tested within 68 h
P
of being taken. In the three patients with HbH-WS, no
b-thalassemia
HbH fraction was visible either in presence or absence of
19.7 ± 13.8
7.1
3.8
6.3
12
20
(n = 117)
b-thalassemia. Fourteen of the remaining 24 cases of
Without
89 ±
58.9 ±
18.8 ±
281 ±
20.9 ±
HbH disease combined with b-thalassemia were analyzed
50/67
Deletional HbH disease
on HPLC; the HbH fraction was visible in only six cases
(42.9%). This percentage is significantly lower than the
proportion observed among HbH patients without b-thal- b-thalassemia
13.5 ± 11.9
assemia (180/192, 93.8%) (v2 = 38.6, P < 0.001). When
4.1
1.2
1.7
09
15
regrouped according to deletional or nondeletional HbH
(n = 8)
±
±
±
±
±
With
49.4
16.2
17.5
300
92
disease, b-thalassemia still significantly decreased the
amount of free b and the presence of the fraction on 4/4
HPLC (Table 2). HbA2 levels were as low as 3.6% while
0.078
0.798
0.043
0.000
0.021
0.000
0.376
HbE was reduced to 16.6%. In 10 cases with HbH-CS,
P
13.7 ± 11.2
2.1
17
17
(n = 158)
Without
77 ±
70.5 ±
20.0 ±
198 ±
19.4 ±
73/85
DISCUSSION
In our cohort, 8.6% of the patients with HbH disease
With b-thalassemia
10.4
1.9
16
17
(n = 16)
18.9
8/8
MCHC (g/l)
RDW (%)
MCV (ll)
Hb (g/l)
Table 2. Results of high-performance liquid chromatography analysis in patients with different types of HbH disease
with or without ß-thalassemia trait
tions were confined to patients with the more severe [8]. The present study showed that the HbH fraction
nondeletional HbH disease. As expected, reduction of tends to disappear in the presence of b-thalassemia trait
the unstable b4 tetramer alone is not enough to allevi- and that HbA2, still elevated in combinations of b-thal
ate hemolysis in these cases. The improved Hb levels in with (–/aa) or (-a/-a), tends to reach almost normal lev-
patients with nondeletional HbH diseases can also be els in the presence of HbH disease (–/-a). Moreover,
ascribed to a lower formation of HbCS tetramers. HbH- abnormal fractions, such as HbE, HbS, or HbC, tend to
CS was responsible for the overwhelming majority of be much depleted or disappear for preferential binding
nondeletional HbH disease (95.7%). HbCS is caused by of the reduced alpha chains with the normal b, rather
a mutation in the stop codon of the a2-globin gene, than with the abnormal one. All these have obvious
which results in 31 additional amino acids. Oxidized consequences for the diagnosis when only HPLC is used.
HbCS is deposited on the membranes of red blood cells The HbA2 level was only 3.6% in three patients, a
and accelerates the damage to these cells [5, 6]. level which is in the ‘gray zone’ and insufficient to
In HbH-WS, however, concomitant b-heterozygos- diagnose the b-thalassemia trait in most laboratories.
ity tends to aggravate rather than alleviate anemia. The cutoff value of HbA2 for the determination of b-
HbH-WS is prevalent in the Guangxi Province. Unlike thalassemia without considering the total picture and
other types of nondeletional HbH disease, anemia in the molecular analysis remains controversial [9].
HbH-WS is only mild or even absent [3]. In these Although 3.5% is the generally accepted cutoff point
cases, the b-globin defect adds to the severity of the in many studies, some authors have used an HbA2 of
a-globin gene defects when the HbH-WS genotype more than 4.0% to diagnose b-thalassemia trait with
combines with a b-thalassemia trait. certainty [10]. Therefore, in areas with a high preva-
Additionally, we noticed a significant decrease in lence of both a- and b-thalassemia, it may be more
the mean corpuscular volume (MCV) levels in cases reasonable to consider a cutoff value of 3.5% when
of HbH disease in the presence of b-thalassemia trait, anemia and low MCV persist in spite of normal iron
both in deletional and nondeletional HbH disease and in the absence of alpha thalassemia defects.
patients, in spite of the decreased imbalance of the b/
a ratio and probably due to the lower Hb content
ACKNOWLEDGEMENTS
(mean corpuscular hemoglobin) in the red cell.
High-performance liquid chromatography is an This research was supported by the National Basic
important technology in thalassemia screening. How- Research Program of China (973 Program, No.
ever, HPLC diagnostics become complex when HbH dis- 2010CB530406) and the Natural Science Foundation
ease is combined with b-heterozygosity [7], HbS, or HbC of Guangxi (No. 2011GXNSFA018196).