Genotype Spectrum and Hematological Features of β–Thalassemia with or without Different Forms of α-Thalassemia in Shenzhen


 Background: The effects of different forms of α–thalassemia on β-thalassemia phenotype has not been clearly described thus far. Methods: Genotype spectrum and hematological features of 873 female diagnosed as β-thalassemia carriers with or without different forms of α-thalassemia was retrospectively analyzed. Results: Thirteen kinds of genotypes were found in the 755 β-thalassemia carriers, including four kinds of β+-thalassemia, eight kinds of β0 -thalassemia and one kind of βE-thalassemia. The values of hemoglobin (Hb), mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) decreased and red blood cell (RBC) increased in the order of βE, β+- and β0 group (p＜0.05). Nine genotypes were determined from the 43 carriers with concurrent α- and β+-thalassemia and seventeen genotypes were determined from the 75 carriers with concurrent α- and β0 -thalassemia. Significant higher Hb, MCV and MCH values were noted in β+–thalassemia or β0–thalassemia co-inherited with α+-thalassemia or α0-thalassemia as compared to the only β+–thalassemia or β0–thalassemia heterozygosity with normal alpha globin gene (p＜0.05). Moreover, the values of Hb, MCV and MCH increased much more when β0–thalassemia co-inheritance of α0-thalassemia than that of α+-thalassemia (p＜0.05). Conclusion: The β-thalassemia presented diverse molecular heterogeneity and hypochromic microcytosis at various degrees. Co-inherited with α–thalassemia could alleviate phenotype of anemic in β+- or β0-thalassemia, and β0-thalassemia demonstrated milder phenotype with two deletion or mutation in α-globin gene than one.


Background
Thalassemia is an autosomal recessive hereditary diseases, it is widely distributed in the tropical and subtropical areas including Mediterranean region, Middle East, Indian subcontinent, east and southeast Asia [1]. It is characterized by decreased or absent of α or β-globin chain synthesis that resulting in ineffective erythropoiesis and excessive peripheral hemolysis. There are two main types of thalassemia, α-thalassemia and β-thalassemia. The clinical manifestations range widely from mild hypochromic anemia to lifelong transfusion-dependent anemia [2,3]. Most of fetuses with hemoglobin Bart's Hydrops fetalis died in the middle or late period of pregnancy or just after birth. β-thalassemia major presented with severe anemia from infancy and it is usually treated by regular blood transfusion, iron therapy and hematopoietic stem cell transplantation [4]. It is an effective way to avoid the birth of children with severe thalassemia by prenatal diagnosis of fetal gene, early detection and termination of pregnancy.
According to slight reduction or complete absence of β-globin chain, β-thalassemia is divided into two types, β +and β 0 -thalassemia [5]. Because of the complex interaction of several genetic modifiers and environment, different clinical manifestations were presented even for the same genotype [6]. Several modifiers (including the severity of β-thalassaemia mutation, concomitant α-thalassemia and HbF levels) have strong impacts on ameliorating the clinical severity of β-thalassemia [7]. Carries co-inheritance of different types of α-thalassaemia could ameliorate the phenotype of β-thalassemia at various degrees, which could result in β-thalassemia ignored and is a huge challenge for the screening program. It would be important to detect the genotype and phenotype characterizations of β-thalassemia in high prevalence regions. Previous studies in China were mainly focus on the molecular epidemiological investigation of β-thalassemia carriers, but limited data were available about the effects of different forms of α-thalassemia on β-thalassemia phenotype in a cohort of carriers [8][9][10][11]. In this study, we prospectively evaluated a cohort of 873 β-thalassemia carriers in order to elucidate the molecular heterogeneity and interaction of α-thalassemia on clinical severity of β-thalassemia.

Study Population
A total of 898 females who were diagnosed as β-thalassemia carriers by DNA analysis due to abnormal hematological parameters or hemoglobin A 2 (HbA 2 ) when attending at antenatal or premarital outpatient were included in Shenzhen Baoan Women's and Children's Hospital in Guangdong Province, China. The age of βthalassemia carriers ranged from 18 to 45 years. The genotype and hematological parameter of all the subjects were analyzed retrospectively. Informed consent was signed by each participant. This study was approved by the Ethics Committee of Shenzhen Baoan Women's and Children's Hospital, Jinan University.

Hematological Measurements
For each participant, 2 ml of peripheral blood sample was collected into EDTA-K 2 anti-coagulated tube and sent for hematological analyses within 3 hours. By using automatic LH750 blood cell analyzer (Beckman Coulter, USA), hematological parameters including red blood cell (RBC), hemoglobin (Hb), mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were measured according to the manufacturer's procedures.

Dna Analysis
2 ml of peripheral blood samples were collected into sodium citrate anti-coagulated tubes for gene detection. Genomic DNA was extracted from peripheral blood with commercially available kit (Yaneng Biotech, Shenzhen, China) according to the manufacturer's procedures. The DNA concentration was determined by NanoDrop spectrophotometry (Thermo Scientific, USA). Three nondeletional mutations of α-thalassemia and seventeen common nondeletional mutations of β-thalassemia were detected by reverse dot-blot hybridization after being amplified by the polymerase chain reaction (PCR),and three deletional mutations were carried out by gap PCR (Yaneng Biotech, Shenzhen, China).

Statistical analysis
Statistical analyses were conducted with SSPS 12.0 software. Mean and standard deviation were used to describe the distribution of hematological parameters of the subjects. One-Way analysis of variance (ANOVA) was used to test the difference of RBC, Hb, MCV and MCH among groups with different genotypes. P-value 0.05 was considered as statistically significant.
The values of Hb, MCV and MCH decreased and RBC increased in the order of β E , β + -and β 0 -group (P < 0.05, Table 1). The hematological parameters of the different genotypes of β + -and β 0 -thalassemia carriers were summarized in Table 2. In most of cases, β + -and β 0 -thalassemia manifested typical hypochromic microcytosis with low Hb, MCV and MHC values. However, we didn't do statistical analysis for each kind of mutations in β + -or β 0 -thalassemia group because of insufficient samples number in some genotypes.
Significant higher Hb, MCV and MCH values were noted in β + -thalassemia co-inherited with α + -thalassemia or α 0 -thalassemia as compared to the only β + -thalassemia heterozygosity with normal alpha globin gene (control) (p 0.05). There were no significant differences observed of Hb, MCV and MCH values between β + -thalassemia co-inherited with α + -thalassemia and α 0 -thalassemia (p 0.05). Moreover, there were no significant differences of RBC value among the three groups (p 0.05). Table 3 The genotype spectrum of β + -thalassemia combined with α-thalassemia  Genotypes and hematological parameters of β 0 -thalassemia combined with α-thalassemia Seventeen genotypes were observed from the 75 carriers with concurrent α-and β 0 -thalassemia in Table 5. The most common combined genotype was β CD41-42 /β N simultaneously with --SEA /αα that accounted for 30.7% (23/75). The hematological parameters for β 0 -thalassemia combined groups were summarized in Table 6. The average values of Hb, MCV and MCH decreased in the order of β 0 -thalassemia co-inheritance of α 0 -thalassemia group, α + -thalassemia group and control group (p 0.05). However, there were no significant differences of RBC value among the three groups (p 0.05). Table 5 The genotype spectrum of β 0 -thalassemia combined with α-thalassemia  a P , b P and c P meaned significant differences respectively from α 0 -thalassemia group, α + -thalassemia group and control group.
In this study, the values of Hb, MCV and MHC decreased and RBC increased in the order of β E , β + -and β 0thalassemia group. A previously study in Thai people also reported that β + -thalassemia was associated with relatively milder phenotype than β 0 -thalassemia according to hematological characteristics [16], which was consistent with our results. It's worth noting that β E -thalassemia manifested borderline RBC, Hb, MCV and MCH values which were nearly close to normal values. However, the clinical manifestations varied widely from mild form of thalassemia intermedia to severe thalassemia major when β E -thalassemia co-inherited with other type of β-thalassemia [17]. Some severe patients would present with hepatosplenomegaly, severe anemia, skeletal disease and need receive intermittent or regular blood transfusions [18]. For this reason, HbA 2 parameter should be considered in case of β E /β N being ignored in the β-thalassemia screening in high risk couples. In most of cases, β + -and β 0 -thalassemia manifested typical anemia characteristic. However, some kind of mutations such as β −28 /β N , β −29 /β N and β CAP /β N manifested mild phenotypes could also compromise carrier screening. It is well known that more than 200 kinds of β-thalassemia mutations have been recognized and the predominant modifier of thalassemia severity is depending on the severity of β-thalassaemia mutation [5]. But we didn't do statistical analysis for each kind of mutations because of limited numbers of certain genotypes. We will collected more samples in each type in order to obtain comprehensive data in future studies.
Among the 873 β-thalassemia carriers, 118 β-thalassemia carriers were co-inheritance of α-thalassemia, indicating the high frequency in Shenzhen. The overall prevalence of β-thalassemia co-inherited with αthalassemia in mainland China is about 0.48% [19], but the general prevalence in Guangdong Province is 8.1% [9]. A total of 26 different genotypes were detected, and the most common two genotypes were β IVS−Ⅱ−654 /β N with --SEA /αα and β CD41-42 /β N with --SEA /αα. This pattern is in accordance with the finding that --SEA /αα was the most common genotype of α-thalassemia in Guangdong Province [10].
The major form of hemoglobin tetramer in normal red blood cells in adult is HbA, which consists of two α and β chains, mutation or defect in the α-or β-globin chain results in imbalanced globin chain synthesis and ineffective erythropoiesis. Absolute excess production of α-chain could increase the imbalance state and exacerbate the clinical course of β-thalassaemia [20,21]. Conversely, decrease of α chain or increase of other substitution of βchain such as γ-chain could reduce chain imbalance of hemoglobin in β-thalassemia [22]. This could be supported by our results that significant higher Hb, MCV and MCH values in β + -thalassemia or β 0 -thalassemia co-inherited with α-thalassemia were detected compared to the only β + -thalassemia or β 0 -thalassemia heterozygosity with normal alpha globin gene. It meaned obviously that β-thalassemia co-inherited of αthalassemia demonstrated slighter phenotype as compared to β-thalassemia heterozygote could be misdiagnosed in clinical screening. The phenotype severity of β-thalassaemia depending on the variable extent of α or β-globin chain imbalance. It is known that α + -thalassemia is characterized with one a-globin gene deleted or reduced and α 0 -thalassemia with two, which could have different influences on clinical characteristics of βthalassaemia. In this study, we observed that β 0 -thalassemia demonstrated milder phenotype with α 0thalassemia compared with α + -thalassemia, but was not detected in β + -thalassemia group. A Study in Thailand reported that α-thalassemia ameliorated the clinical severity of β-thalassemia, but β + -thalassemia coexistent of α 0 -thalassemia showed no significant improvement of MCV and MCH campared with pure β-thalassemia heterozygote [23], which was different from our results. This could be explained by the fact that only the β −28 /β N genotype were contained in β + -thalassemia group and merely five samples were concluded in α 0 -thalassemia group, and the epidemic genotypes in Thailand are also different from China.

Conclusions
In summary, most genotypes of β-thalassemia present typical hypochromic microcytosis and co-inheritance of α-thalassemia could alleviate phenotype of anemic in β + -or β 0 -thalassemia at various degrees. Although βthalassemia carries co-inherited with α-thalassemia ameliorated clinical severity, they are still at risk of having babies with severe thalassemia such as Hemoglobin-H disease, hemoglobin Bart's Hydrops fetalis or βthalassemia major when married with thalassemia heterozygote. Accurate diagnosis is essential to prevent βthalassaemia carriers with mild phenotype or borderline hematological parameters be ignored. Therefore, our results are meaningful for carrier screening, genetic counseling and clinical outcome predicting.