Analysis of genotype–phenotype correlation in patients with α‐thalassemia from Fujian province, Southeastern China

Abstract Background There is a high carrying rate of α‐thalassemia in Fujian province. However, there are few large‐scale studies on the correlation between genotype and phenotype in Fujian province. The purpose of this study was to analyze the phenotype and genotype in a cohort of 2923 patients with α‐thalassemia in Fujian province, so as to provide reference data for screening and diagnosis of α‐thalassemia in Fujian province. Methods The genotype of α‐thalassemia was detected by PCR reverse dot blot assay, gap‐PCR, single PCR, nested PCR, and sequencing. Clinical and hematological indices of 2923 patients were collected, and the correlation between genotype and phenotype was analyzed. Results Among 10,350 patients, 2923 cases were found with α‐thalassemia, with a detection rate of 28.24%. Among them, ‐‐SEA/αα was the most common genotype, accounting for 64.80%. In addition, rare α‐thalassemia genotypes were detected in Fujian province, including ‐‐THAI/αα (0.41%), HKαα/‐‐SEA (0.03%), and the novel α‐thalassemia gene mutation CD5 (GCC>ACC) (HGVS named HBA1: c.16G>A) (0.03%). Patients with deletional genotypes of α‐thalassemia were found to have higher RBC and lower Hb, MCV, MCH, and HbA2 than patients with non‐deletional genotypes of α‐thalassemia (p < 0.05). Conclusion The clinical phenotype of α‐thalassemia is influenced by molecular mechanisms. HBA1: c.16G>A mutation is a novel mutation that was first reported in Fujian province, which enriches the human hemoglobin mutation spectrum.


| INTRODUC TI ON
α-thalassemia is one of the most common autosomal recessive genetic diseases in the world. Its pathogenesis is due to the defect of α-globin gene; the synthesis of α-globin peptide chain was partially or completely inhibited, resulting in hereditary hemolytic anemia. It is one of the most common monogenic diseases with the highest incidence in the world and has attracted extensive attention at home and abroad because of its fatal and disabling nature, which can lead to birth death or birth defects. 1 Around 80%-90% of α-thalassemia is caused by genomic deletion of the α-globin gene cluster on chromosome 16p13.3. 2 There is increasing evidence that around 7.0% of the world's population are carriers of mutations in the α-globin gene. 3 α-thalassemia has obvious regional or population differences and is mainly found in the Mediterranean, Africa, and Southeast Asia. The incidence of α-thalassemia is high in southern China, especially in Sichuan, Yunnan, Guangxi, Guizhou, Guangdong, and Fujian. 4 Fujian province, located in the southeast coastal area of China, is a province with high incidence of α-thalassemia. 5 At present, there is no other specific treatment for severe α-thalassemia except long-term blood transfusion or hematopoietic stem cell transplantation. Therefore, understanding the prevalence of α-thalassemia in high incidence areas, monitoring the population of childbearing age, preventing the birth of children with severe α-thalassemia, and gradually eliminating the prevalence of α-thalassemia in the population through genetic intervention are the only coping methods.
However, there are few large-scale studies on the correlation between genotype and phenotype in α-thalassemia patients in Chinese population. In this study, 10,350 patients from Fujian province were analyzed for genotype and phenotype. Such a study may provide more data for genetic counseling and clinical diagnosis in this region.

| Rare genotype test
--THAI genotype was tested using gap polymerase chain reaction (gap-PCR), and HKαα genotype was tested by single PCR and nested PCR, as described previously. 7,8 For suspected rare types of α-thalassemia, the full-length α1-globin genes and the full-length α2globin genes were amplified using PCR assay and checked. The purified PCR products were subjected to direct sequencing with an ABI 3100 DNA Sequencer (Applied Biosystems; Foster City, CA, USA) as described. 9 Wallis test, presented as median (95% confidence interval). The results with p < 0.05 were considered statistically significant.

| Hematological indices, the hemoglobin components and levels, and SF of patients with different genotypes of α-thalassemia
Hematological indices, the hemoglobin components and levels, and SF concentration in patients with different α-thalassemia genotypes were analyzed in this study, and we found that the red blood cell count (RBC) in patients with deletional genotypes of α-thalassemia was higher than that in patients with non-deletional genotypes of α-thalassemia (5.3 (5.0, 5.8) × 10 12 / L vs 4.9 (4.5, 5.4) × 10 12 /L) (p < 0.05).Conversely, the levels of Hb, MCV, MCH, and HbA2 in patients with deletional genotypes of α-thalassemia were lower than that in patients with non-deletional genotypes of α-thalassemia  (Table 2). Moreover, some indices in different deletional genotypes of αthalassemia and non-deletional genotypes of α-thalassemia were various. Among the deletional genotypes of α-thalassemia, patients with --SEA /αα were found to have higher RBC, HbF, and SF and lower Hb, MCV, MCH, and HbA2 than patients withα 3.7 /αα (p < 0.05).

| Hematological analysis and molecular diagnosis of the novel α-thalassemia gene mutation in Fujian province
In this study, we found a patient showed moderate microcytic hypochromic anemia with Hb 86 g/L, MCV 66.4 fL, and MCH 19.1 pg and decreased level of HbA 2 (1.9%) that was suspected to be a α-

| DISCUSS ION
α-thalassemia is common genetic diseases in human and is a specific recessive genetic disease caused by the obstruction of α-globin peptide chain synthesis. 10 The correlation between genotype and phenotype in 78 patients with α-thalassemia in Hainan province was reported, 11 and our previous study also reported the distribution of genotype in 314 patients with α-thalassemia in Fujian province. 12 In this study, we further analyzed the correlation between genotype and phenotype in a large sample of 2923 cases of α-thalassemia in Fujian province, filling in the gap in this field.
There are two types of α-thalassemia: deletional genotype and non-deletional genotype. In addition to the most common deletional genotype, rare deletional genotype of α-thalassemia has been found in different populations. 13 Up to now, more than 40 different deletional genotypes of α-thalassemia have been described, of which 10 types have been found in China. 14 There are three kinds of deletional genotype of α-thalassemia in clinic: --SEA /αα,α 3.7 /αα, and α 4.2 /αα. Among 2923 cases with α-thalassemia, there were 1894 cases (64.80%) with--SEA /αα, 554 cases (18.95%) withα 3.7 /αα, and 145 cases (4.96%) withα 4.2 /αα, suggesting that the most common genotype of α -thalassemia in this region was --SEA /αα, which was consistent with the reports from Guangxi, Guangdong, Chongqing, and Hainan. 15 The most common non-deletional genotypes of αthalassemia were α QS α/αα (3.63%), followed by α CS α/αα (1.47%) and α WS α/αα (0.99%), which were the same as those in Chongqing and slightly different from those in Guangxi, Guangdong, and Hainan, [16][17][18][19] and were speculated to be related to regional population and other genetic factors. The spectrum of mutations detected in this study is similar to that observed in adjacent provinces, but there are differences in genotype prevalence in different regions. The incidence of α-thalassemia in adjacent provinces of Fujian, such as Guangdong, Guangxi, and Jiangsu, was respectively 8.53%, 15.33%, and 1.48%, TA B L E 1 Genotyping of α-thalassemia in Fujian province   All hematological indices with more than one records are presented as median (95% confidence interval). Bootstrap method is used in computing 95% confidence intervals. *p < 0.05, Kruskal-Wallis test.
and --SEA /αα was dominant in these provinces, and the frequencies were respectively 57.55%, 52.78%, and 83.87%. 20 This is due to the random distribution of α-thalassemia in the world, different ethnic regions have their own distribution characteristics. It is generally believed that populations with the same genetic background are prone to homogenous mutations.
Up to now, there are few reports about HKαα/αα type thalassemia, which mainly occurs in Guangxi and Guangdong of China. 21,22 HKαα allele is a special structure containingα 3.7 fragment and ααα anti4.2 fragment formed by recombination and unequal transposition of the homologous X segment of the α-globin gene cluster. So far, ααα anti4.2 fragments cannot be detected through the routine thalassemia diagnostic kit. The genotype ofα 3.7 /αα, HKαα/α 3.7 , and HKαα/αα is entirely the result ofα 3.7 /αα by gap-PCR. 26 The clinical symptoms of HKαα/αα were much milder than those ofα 3.7 /αα. Therefore, the symptoms of thalassemia associated with HKαα/αα and --SEA were milder than those of hemoglobin H disease associated withα 3.7 and --SEA , and only mild globin production disorder was observed (usu- and the patient was found to be diagnosed as iron deficiency anemia few days later, when follow-up. In addition, 64 cases (2.19%) with concurrent α-and βthalassemia were detected in 2923 positive samples. Patients with concurrent α-and β-thalassemia have been reported to suffer from mild anemia due to a reduction in α-and β-globin chain synthesis, which alleviates the imbalance caused by reduced globin chain synthesis and thus reduces the severity of anemia. 30 In this study, we detected 2.19% concurrent α-and β-thalassemia, the five most common genotypes including --SEA /αα/β IVS-2-654(C→T) /β N (11 cases), α 3.7 /αα/β IVS-2-654(C→T) /β N (11 cases), --SEA /αα/β CD41-42(-CTTT) /β N (11 cases),α 3.7 /αα/β CD41-42(-CTTT) /β N (4 cases), and --SEA /αα/β −28(A→G) / β N (4 cases). While patients with α-and β-thalassemia have milder symptoms, their offspring are more likely than the general population to develop severe thalassemia, and the long-term damage is much greater. Therefore, the clinical diagnosis of concurrent αand β-thalassemia should not be ignored. According to Xiong Fu et al., 31 concurrent α-and β-thalassemia is generally manifested as the phenotypic characteristics of β-thalassemia in Hb analysis, so αthalassemia is easy to be missed and misdiagnosed in clinical diagnosis, which should be paid attention to.
There is significant variation in clinical severity among patients with α-thalassemia, which is indirectly reflected by the span of age at first diagnosis. Age of --SEA /αα was lower than that ofα 3.7 /αα andα 4.2 /αα (p < 0.05); this may be related to the number of inactivated α-globin genes of --SEA /αα were more thanα 3.7 /αα andα 4.2 / αα. Patients with --SEA /αα were associated with more severe anemia and came to the hospital earlier for diagnosis and treatment. Chen Suqin et al. 32 also demonstrated that factors such as age influenced the genotype and phenotype of α-thalassemia. Liebhaber et al. 33 found that the α1-globin gene on the same chromosome as the Hb CS mutation was only half as expressed as theα 4.2 orα 3.7 variants.
Thus, the absence of α-globin peptide chains or the relative excess of β-globin peptide chains is more severe in non-deletional patients.
The hematologic appearance of patients with non-deletional genotype of α-thalassemia differs from that patients with deletional genotype of α-thalassemia; anemia is more severe in patients with non-deletional genotype of α-thalassemia. 34 In our study, patients with deletional genotypes of α-thalassemia were found to have higher RBC and lower Hb, MCV, MCH, and HbA2 than patients with non-deletional genotypes of α-thalassemia (p < 0.05).It may be related to the damage of erythrocyte membrane caused by excess β-chain oxidation and α QS α or α CS α chain oxidation. This results in decreased EPO production and RBC in non-deletional genotypes of α-thalassemia. 35 α-thalassemia mainly affects the synthesis of Hb, which is the main content of red blood cells. Therefore, Hb, MCV, and MCH will show obvious changes. Interestingly, we found that α QS α/αα's Hb was lower thanα 3.7 /αα andα 4.2 /αα, which was partly supported that anemia in the non-deletional genotype was more severe than in the deletional genotype. Patients with --SEA /αα were found to have higher RBC, HbF, and SF, it may be related to --SEA /αα was associated with more γ-globin mutations, and its RBC synthesis requires more iron, so the RBC, HbF, and SF were higher thanα 3.7 / αα. 36 The Hb, HbA2, MCV, and MCH of --SEA /αα were lower than α 3.7 /αα andα 4.2 /αα in the three common deletional genotypes, suggesting that the severity of anemia depends on the number of inactivated α-globin genes.
In conclusion, the clinical manifestations and hematologic phenotypes of α -thalassemia are related to genotype. The clinical phenotype of α-thalassemia is influenced by molecular mechanisms.
HBA1: c.16G>A is a novel mutation that was first reported in Fujian province. The discovery of this novel mutation in α-globin gene has enriched the database of hemoglobin variants, and the detailed genetic analysis and clinical symptom description of this mutation will contribute to the further study of hemoglobin function in the future.

AUTH O R CO NTR I B UTI O N S
Yali Pan, Meihuan Chen, Na Lin, Liangpu Xu, and Hailong Huang designed and prepared the study. YanHong Zhang, Min Zhang, and Lingji Chen collected the literature and the data and prepared the study. All authors approved the final study.