Detection of a Hb A2‐Melbourne (HBD: c.130G>A) combined with β‐thalassemia in a Chinese individual

Abstract Background Thalassemia is common in Southeast Asian countries, including China. Hb A2‐Melbourne is a rare hemoglobin variant and has never been reported in China. Here, we report a Hb A2‐Melbourne combined with β‐thalassemia in Chinese individuals which is the second case described in the published reports. Methods Complete blood counts (CBC) of a 28‐year‐old female showed signs of thalassemia during a routine screening. Hemoglobin analysis was subsequently performed using capillary electrophoresis (CE) and high‐performance liquid chromatography (HPLC). Four common deletional α‐thalassemia detection was carried out using a gap‐polymerase chain reaction (PCR). PCR and reverse dot‐blot were used to detect three non‐deletional α‐thalassemia and 17 types of point mutations in β‐thalassemia. Finally, it was identified by Sanger sequencing. Her husband also had CBC, hemoglobin analysis, and genetic diagnosis. Results CBC of the couple showed Hb 103 and 139 g/L, mean corpuscular volume 58 and 63.1 fL, mean corpuscular hemoglobin 19.7 and 20.4 pg, respectively. Hemoglobin analysis revealed Hb X 2.4%, Hb A2 2.8% by CE and Hb X 2.9%, Hb A2 2.4% by HPLC in the female. The results of her husband were Hb A93.5%, Hb A2 5.7%, Hb F 0.8% by CE. Genetic analysis of both spouses detected the same CD 41/42 mutations in β‐globin gene. Sanger sequencing of female identified a mutation of the δ‐globin gene (HBD:c.130G>A), corresponding to Hb A2‐Melbourne. Conclusion Hb A2‐Melbourne can lead to misdiagnosis of β‐thalassemia. δ‐globin gene mutation must be carefully examined in routine thalassemia screening.


Methods:
Complete blood counts (CBC) of a 28-year-old female showed signs of thalassemia during a routine screening. Hemoglobin analysis was subsequently performed using capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Four common deletional α-thalassemia detection was carried out using a gap-polymerase chain reaction (PCR). PCR and reverse dot-blot were used to detect three non-deletional α-thalassemia and 17 types of point mutations in β-thalassemia.
Finally, it was identified by Sanger sequencing. Her husband also had CBC, hemoglobin analysis, and genetic diagnosis. Hemoglobin analysis revealed Hb X 2.4%, Hb A 2 2.8% by CE and Hb X 2.9%, Hb A 2 2.4% by HPLC in the female. The results of her husband were Hb A93.5%, Hb A2 5.7%, Hb F 0.8% by CE. Genetic analysis of both spouses detected the same CD 41/42 mutations in β-globin gene. Sanger sequencing of female identified a mutation of the δ-globin gene (HBD:c.130G>A), corresponding to Hb A 2 -Melbourne.
Conclusion: Hb A 2 -Melbourne can lead to misdiagnosis of β-thalassemia. δ-globin gene mutation must be carefully examined in routine thalassemia screening.

K E Y W O R D S
capillary electrophoresis (CE), Hb A 2 -Melbourne, high-performance liquid chromatography (HPLC), thalassemia, δ-globin gene mutation

| INTRODUC TI ON
β-thalassemia is one of the most common hereditary disorders in the world. 1 In southern China, the carrier rate of β-thalassemia is high with a prevalence of 6.43% in Guangxi and 2.54% in Guangdong. 2,3 Hb A 2 , composed of two α chains and two δ chains, is a minor component of the hemoglobin present in normal adult red blood cells, accounting for 2.5%-3.5% of the total hemoglobin in healthy individuals. 4 Increased Hb A 2 is the most significant characteristic in β-thalassemia carriers. 4 Therefore, Hb A 2 determination plays a key role in screening for β-thalassemia. However, some individuals with silent β-globin gene mutations or with triplicate α-globin genotypes (ααα/αα) may be misdiagnosed during screening programs which show normal or reduced Hb A 2 . 5 Besides, δ-globin gene mutation may influence the Hb A 2 levels in the blood. Individuals with compound heterozygotes β-thalassemia with a δ-globin gene mutation may have normal Hb A 2 levels and therefore be overlooked as β-thalassemia heterozygotes. Twenty-one δ-globin gene mutations were detected in the Chinese population. However, Hb A 2 -Melbourne has not yet been described. 6 When variants of the δ-globin gene are not accurately identified and not counted into the total Hb A 2 , then β-thalassemia also may be misdiagnosed. Herein, this study is the first time that Hb A 2 -Melbourne combined with β-thalassemia has been reported in Chinese population.

| Complete blood counts and hemoglobin analysis
This study was approved by the Ethics Committee of Guangxi Zhuang Autonomous Region People's Hospital. Signed informed consent was obtained from patients.

| Sanger DNA sequencing
As previously described by Dongzhi Li et al, two pairs of primers were designed to sequence the δ-globin gene. 6 (Table 1).

| D ISCUSS I ON
Mutations that occur on the δ-globin gene can affect the structure or the expression of the δ-globin chain. It will produce a second and usually visible Hb A 2 variant with stable structural defects (as Hb A 2 -Melbourne in this study). If the structure of the variant is unstable, the mutation will behave as a thalassemic defect (δ-thalassemia) and be undetectable using CE or HPLC. 7 Although most of the δ-globin variants are pathologically innocuous, coinheritance with β-thalassemia or α-thalassemia could lead to misdiagnosis. 8,9 The coexistence of a δ-globin gene defect will decrease the Hb A 2 level of the β-thalassemia or α-thalassemia carrier to a normal range. 10 Therefore, it is essential to be aware of the existence of δ-globin gene defect in routine thalassemia screening.
As shown in the Figures 1 And 2, in addition to Hb A 2 , a small peak of an abnormal Hb variant was observed on both CE and HPLC.
Based on its value and location, we speculated that it is an Hb A 2 variant. So, the value of Hb A 2 variant should be added to the total Hb A 2 value (Hb A 2 of CE: 2.8% + 2.4% = 5.2%, Hb A 2 of HPLC: 2.9% + 2.4% = 5.3%) which was an increased Hb A 2 level. With her red cell indices, it suggested that female was a suspected Hb

ACK N OWLED G M ENTS
The

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.