Complex Interaction of Hb Q-Thailand with α and β Thalassemia in a Hakka Family CURRENT STATUS:

Background HbQ-Thailand is an α-globin chain variant that results from a point mutation at codon 74 of the α1-globin gene on chromosome 16p. It commonly appears with a leftward single α-globin gene deletion (-α 4.2 ). There have been few reports regarding the interaction between HbQ-Thailand and other globin gene disorders. Here we found and diagnosed it in the Hakka population of the Fujian Province, China. The study provides an important reference for the clinic diagnose and genetic counseling of thalassemia and hemoglobin diseases. Methods Fresh peripheral blood samples were collected from the proband and her family members testing hematological parameters, hemoglobin components, thalassemia gene, and hemoglobin variants. Results The proband (II1) and her sister (II5) manifested in the obvious microcytic hypochromic anaemia. The CE electropherogram of II1 showed an abnormal band in the migration time at 185 s, which was confirmed as HbQ-Thailand. Another exception band appeared at 250 s of migration time and was proved to be HbE by sequence analysis method. The CE electropherogram of I1 and II3 showed an anomalous band HbE. The mother of the proband (I2) and the III4 and III5 of the family members showed a HbQ-Thailand. The gene results showed that the father (I1) also carried α- and β-thalassemia genes. His genotype was -- SEA and β codons26 ; -- SEA was inherited to II1, II 3, II5, III 1, and III2, and β codons26 was inherited to II1 and II3. The mother (I2) carries the -α 4.2 gene, which was inherited to II1, II5, III4, and III5. Conclusion It was complex to diagnose when the thalassemia combined with several abnormal haemoglobin disorders, and we may use various methods to mutual confirmation. Here we found and diagnosed a rare hemoglobin disease in the Hakka population of the Fujian Province. The study provides an important reference for the clinic diagnose and genetic counseling. were identified by sequence analysis method.


Introduction
Hemoglobinopathies are a group of inherited autosomal recessive disorders, including the alteration of hemoglobin quantity (thalassemias) and quality (Hb variants) [1]. They are widely distributed in Mediterranean countries, the Middle East, Africa, and Southeast Asia, including southern China [2][3][4].
HbQ-Thailand [α74(EF3)Asp→His(α1),GAC CAC, HBA1:C.223G C], also known as Hb G-Taichung, Mahidol, Kurashiki-I, and Asabara [5], is an α-globin chain variant that results from a point mutation at codon 74 of the α1-globin gene on chromosome 16p. It commonly appears with a leftward single αglobin gene deletion (-α 4.2 ). The heterozygotes of HbQ-Thailand may show minor clinical symptoms or may be asymptomatic, whereas the double heterozygosity of it and thalassemia may lead to several clinical diseases [6,7]. However, there have been few reports regarding the interaction between HbQ-Thailand and other globin gene disorders. Furthermore, the association among these disorders has important implications in clinical manifestation, laboratory diagnosis, and genetic counseling.
The Hakka people are intriguing Han Chinese populations that primarily reside in southern China, including Guangdong, Fujian, Jiangxi, and Taiwan. However, their cultural background, lifestyle, and customs are different from those of the southern Hans, and they have unique localisms [8]. Here we found a rare family of Hakka people who carry the genes of thalassemia/HbE (HBB:c.79G A) and HbQ-Thailand, which is an under described condition at present.

Results
The proband, a 35-year-old Chinese female from Longyan City of Fujian Province, displayed anemia with the following hematological parameters: RBC 6.27 × 10 12 /L, Hb 93 g/L, MCV 45.9 fl, MCH 148 pg, and MCHC 323 g/L. The automated capillary electrophoresis (CE) (capillarys 2; Sebia, France) showed three dominant peaks, which revealed Hb variants and thalassemia. Most notable observation was that CE revealed a lack of adult hemoglobin, which is the main component in normal adult hemoglobin. Therefore, the proband and her twelve family members were referred to our laboratory for further investigation after obtaining informed consent. They are all Hakkas from Longyan City of Fujian Province. The pedigree of this family is shown in Fig. 1.
The 13 family members had no history of blood transfusion. Fresh peripheral blood samples were collected from all family members using a automated blood cell analyzer for hematological parameters analysis (Bc-5390; Mindray, China), and the results are shown in Table 1. The proband (Ⅱ1) and her sister (Ⅱ5) manifested in the obvious microcytic hypochromic anaemia. Analysis of hemoglobin components and variants was performed using a CE hemoglobin analyzer (capillary2™; Sebia, France). The results are shown in Table 1, and the electropherogram is shown in Fig. 2. The CE electropherogram of Ⅱ1 [ Fig. 2 (a)] did not show a normal HbA band, however, there was an abnormal band in the migration time at 185 s, which was confirmed as HbQ-Thailand [9]; the corresponding HbQA 2 appeared in the migration time of 260 s. Another exception band appeared at 250 s of migration time and was proved to be HbE [10] inherited from his father. The CE electropherogram of Ⅰ1  Table 1 The hematological and molecular data of the family members under study. The α-thalassemia deletions (--SEA /, -α 4.2 /, and -α 3.7 /) commonly found in Chinese populations were typed by gap-PCR. The three non deletional mutations(α cs α/,α Qs α/, and αα ws /) and 17 species of βmutant genes were performed by RDB assay in this subject.The results showed that the father (Ⅰ1) also carried α-and β-thalassemia genes. His genotype was --SEA and β codons26 ; --SEA was inherited to Ⅱ1, Ⅱ 3, Ⅱ5, Ⅲ 1, and Ⅲ2, and β codons26 was inherited to Ⅱ1 and Ⅱ3. The mother (Ⅰ2) carries the -α 4.2 gene, which was inherited to Ⅱ1, Ⅱ5, Ⅲ4, and Ⅲ5. The hemoglobin variant was identified by sequence analysis method. Ⅱ1 and Ⅱ5 were HbQ-Thailand homozygotes; Ⅰ2, Ⅲ4 and Ⅲ5 were HbQ-Thailand heterozygotes, and the sequencing results are shown in Fig. 3.  Table 1.

Discussion
HbQ-Thailand is a hemoglobin variant caused by a point mutation in the α-chain of hemoglobin, commonly associated with the left side deletion of the -α 4.2 [9]. This is verified by the presence of the HbQ-Thailand in the 5 family members carrying the deletion of -α 4.2 . HbQ-Thailand is generally asymptomatic, or the presence of the deletion of -α 4.2 may cause microcytic hypochromic anaemia [11]. Family members Ⅰ2, Ⅲ4, and Ⅲ5 carry HbQ-Thailand variant, accompanied with the deletion of - forming HbH (β4) and the HB-Bart's (γ4); thus, the CE electropherogram shows two fast electrophoresis bands. In contrast, the proband (Ⅱ1) is also carrying the β codons26 point mutation. In this individual, the protein α-chain synthesis decreased and simultaneously, the β-chain synthesis decreased; hence, her anemia severity was less than that of the younger sister (Ⅱ5). Thus, the combination of α-and β-thalassemia, leads to more mildly anemia than a α-thalassemia.
In general, HbQ-Thailand (α Q 2 β 2 ) is shown as a special peak presented at the Z7 region in the CE electropherogram, while HbQA 2 (α Q 2 δ 2 ) is shown as a special peak presented at the zone1 region It is noteworthy that when the alpha chain is mutated, the mutated alpha chain binds to the δ chain, forming a HbQA 2 (α Q 2 δ 2 ), which may cause false reduction of HbA2 (α 2 δ 2 ), resulting in an αthalassemia misdiagnosis or β-thalassemia missed diagnosis. Therefore, in the CE electropherogram, HbA 2 and HbQA 2 should be superimposed for the evaluation of the thalassemia. In general, in αthalassemia patients with decreased α-chain synthesis, hemoglobin analysis showed a decline in HbA 2 (α 2 δ 2 ). β-thalassemia may lead to a increasing in the level of Hb A 2 (α 2 δ 2 ), because of the reduction of β-chain and the increasing of δ-chain and γ-chain accordingly. However, when β-thalassemia was combined with α-thalassemia and/or α -chain variant, the compound of α-chain and β-chain was reduced concurrently, which could lead to a normal HbA 2 ratio. It may cause potential pitfalls for thalassemia. So, when screening of the thalassemia by hemoglobin analysis, we must pay attention,

Conclusion
It was complex to diagnose when the thalassemia combined with several abnormal haemoglobin disorders, and we may use various methods to mutual confirmation. Here we found and diagnosed a rare hemoglobin disease in the Hakka population of the Fujian Province. The study provides an important reference for the clinic diagnose and genetic counseling.

Materials And Methods
Fresh peripheral blood samples were collected from the proband and her family members using a automated blood cell analyzer for hematological parameters analysis (Bc-5390; Mindray, China).