First study to describe a novel HbA2: c.400A > C mutation and Hb Dongguan heterozygote in two unrelated Chinese families

ABSTRACT Objectives Here we report two rare α-globin chain variants in two unrelated families: Hb Val de Marne [α133(H16) Ser > Arg (AGC > CGC); HBA2: c.400A > C] and Hb Dongguan [α52(E6) Ser > Cys (TCT > TGT); HBA1: c.158C > G]. Notably, HBA2: c.400A > C is an unreported new variant in the third exon of the α2 gene, and simple heterozygous unstable Hb Dongguan haematological characteristics are proposed for the first time. Methods Hb analysis was performed by using capillary electrophoresis (CE). Twenty-three common mutations were detected using a suspension array system. Mutations were identified by DNA sequencing. Results The CE results showed an abnormal peak with incomplete separation from Hb A at zone 8 in two members of Family 1. DNA sequencing confirmed the presence of Hb Val de Marne [α133(H16) Ser > Arg (AGC > CGC); HBA2: c.400A > C]. Five members of Family 2 exhibited an abnormal peak at zone 11, and DNA sequencing confirmed the presence of Hb Dongguan [α52(E6) Ser > Cys (TCT > TGT); HBA1: c.158C > G]. Conclusions The discovery of HBA2: C.400A > C expands the existing spectrum of α-globin variants. The carriers of simple heterozygous Hb Dongguan generally do not have obvious clinical symptoms. The information in this study will help clinicians understand the screening, molecular diagnosis and clinical significance of Hb variants.


Introduction
Haemoglobinopathy is a common single-gene disease. Haemoglobin (Hb) variants are mainly composed of missense mutations (single amino acid substitutions) in the globin chain, resulting in abnormal or 'varied' Hb tetramers, affecting the molecular conformation and function of Hb, such as destabilizing Hb and altering Hb-O2 affinity. Nevertheless, most variants are stable and clinically silent [1,2]. To date, 1414 naturally occurring human Hb variants have been recorded in the HbVar database (http://globin.bx.psu.edu/hbvar); among them, 103 kinds of Hb with high oxygen affinity and 155 unstable Hbs. Guangdong Province, located in southern China, have been identified with a high incidence of thalassemia. The identification and diagnosis of new or unstable mutations are of great significance for laboratory research and clinical genetic counselling. In recent years, an increasing number of unstable variants and variants with the same protein change but different α1or α2-globin gene mutations have been reported [3][4][5]. Here, we present two rare α-globin chain variants. The

Materials and methods
Family 1 and Family 2 were from Guangzhou, Guangdong Province. The proband in Family 1 ( Figure 1, Family 1; II-1) was a 35-year-old female who came to our hospital for genetic counselling due to fertility problems and then underwent thalassemia screening. The proband in Family 2 ( Figure 1, Family 2; II-2) was a 28year-old woman who came to our hospital for routine thalassemia screening in the maternity insurance program. These two families were unrelated. All studies were approved by the Ethics Committee of Guangdong Women and Children Hospital.
Peripheral blood samples were collected using EDTA as an anticoagulant. Red blood cell indices were analysed using a Sysmex XN5000 automated haematology analyser (Sysmex Corporation, Kobe, Japan). Hb analysis (separation and quantitation of Hb subtypes) was performed using an automated capillary 2 electrophoresis system (Sebia, Lisses, France). All human materials were collected after informed consent was obtained from both families.
Genomic DNA was isolated from peripheral blood leukocytes using the Lab-Aid 820 automation system (Xiamen Zeesan Biotech Company, Fujian, China). To exclude the presence of twenty-three common mutations in individuals from southern China, including 3 deletions and 3 non-deletion mutations associated with α-thalassemia and 17 point mutations associated with β-thalassemia, molecular analysis was carried out in our laboratory using a suspension array system as previously described [6].

Results
Routine haematology studies were carried out for all patients in the study, and the results are shown in   Hb analysis of the proband showed an obvious anomalous peak with incomplete separation from the Hb A peak, which together accounted for 96.5% of the total; the Hb A 2 peak accounted for 2.8%, and the Hb A 2 -variant peak accounted for 0.7% ( Figure  2a). Her father was also a carrier with 96.9% Hb A + Hb variant, 2.5% Hb A 2 , and 0.6% Hb A 2 -variant.
Initial molecular screening of the proband and her parents determined that they were negative for α-thalassemia and β-thalassemia by the suspension array system. DNA sequencing of the α-globin genes revealed that both I1 and II1 were heterozygous for one previously undescribed single nucleotide substitution in which adenine (A) had mutated to cytosine As shown in Figure 2b, Hb analysis of the proband using a CE system identified, in addition to Hb A, Hb F and HbA 2 + Hb A 2 -variant, an anomalous peak at zone 11. The quantitative Hb assessment of her parents and affected siblings is summarized in Table  2. Her father and her siblings were all carriers of Hb Dongguan with a normal red blood cell index, except for her sister (II1), who had typical microcytic hypochromic anaemia and a high red blood cell distribution width (RDW) of 19.6%. It is suspected that this individual had iron deficiency anaemia. This finding indicates that the heterozygotes for Hb Dongguan were generally asymptomatic, but the carriers had abnormal peaks ranging from 12% to 16% in zone 11 using CE. We used the suspension array system to screen for routine thalassemia with negative results.
DNA sequencing of the α-globin genes revealed that all members except the mother had a substitution at codon 52 of the α1-globin gene (TCT > TGT), resulting in replacement of serine with cysteine, and this mutation had been previously reported as the unstable Hb Dongguan variant (Figure 3b).

Discussion
Many Hb variants caused by missense mutations confer no physical abnormalities or clinical problems, making these variants easy to ignore. However, as laboratory personnel or clinicians, we must gain insight into how these substitutions interact with other positions on the gene and whether these mutations lead to alterations in oxygen affinity, changes in binding stability between globin chains, structural defects with thalassemia effects, or changes in physical behaviour. This acknowledgement has led an increasing number of laboratories to focus on identifying mutations and determining their clinical significance.
The mutation causing amino acid changes at codon 133 [α133(H16) Ser > Arg] corresponds to that found in Hb Val de Marne [4,[8][9][10][11], which was reported to present normal haematologic parameters, with 16% abnormal Hb [8]. Additionally, the Hb stability of Hb Val de Marne was normal [8,9]. However, the oxygen affinity was increased, while the haem-haem interaction was slightly reduced and the chloride ion binding and Bohr effect remained normal. The reason may be that serine (a133(H16)) is the residue at the C-terminus of the α-globin chain, which is in contact with the haem pocket (Phe a98(C5)) and other internal residues (Lys a99(G6), Ser a102(G9) and Ser a138 (H21)), and this replacement of Ser by Arg may help oxygen or water interact with the haem iron. Regarding the molecular characteristics of Hb Val de Marne, Edmond Shiu-Kwan Ma et al. [4] first revealed an AGC > AGA mutation at codon 133 of the α2 gene in a Chinese family. Based on amino acid analysis, replacing serine at 133 of the codon of the α2 gene with arginine requires only a single mutation in the codon, which can be A→C (our report), C→G, or C→A (2004).
In our study, the proband of Family 1 is the first with a base A→C change at codon 133 of the α2 gene, and HBA2: c.400A > C is an unreported new single nucleotide substitution in the third exon of the α2 gene. The haematologic parameters of the carriers are consistent with those described in the literature. These normal parameters can easily lead to underestimation of this mutation in patients. We identified an obvious abnormal peak by CE, which was not completely separated from the Hb A peak. This property has never been described. CE appears to have a good ability to discriminate this variant in laboratory analysis.
Regarding the second variant in Family 2 that we reported, Hb Dongguan was first described in a 67year-old Chinese male, who was compound heterozygous for Hb Dongguan and --SEA α-thal deletion with an anomalous peak accounting for 37.8% of the total Hb and presented typical microcytic hypochromic anaemia [12]. However, the previous study did not provide a haematological phenotype of the simple    that the isopropanol test is strongly positive [12], indicating that Hb Dongguan is unstable in nature.
Although the heterozygous state of the two variants in this study did not lead to obvious symptoms of anaemia, these variants did cause some functional changes, such as affinity changes and the generation of unstable Hb according to the literature [12] and protein function prediction.

Conclusions
In this study, we characterized a novel α-globin gene mutation Hb Val de Marne [α133(H16) Ser > Arg (AGC > CGC); HBA2: c.400A > C] and performed haematological and molecular characterization. This variant was detected for the first time was localized by using CE. In addition, this study presents the haematological and molecular characterization of individuals carrying only the unstable variant Hb Dongguan. Although the two Hb variants in our study are unlikely to produce significant haemolytic processes or anaemia in the simple heterozygous state, understanding the haematologic characteristics and Hb composition of these variants and obtaining a clear diagnosis of the type of variant are necessary for laboratory research and clinical genetic counselling. Moreover, it cannot be ignored that these asymptomatic variants may produce more relevant haematologic phenotypes if they are associated with α 0 -thal defects, the clinical significance of which remains to be discovered by studying more cases in the future.