Genotype Distribution and Clinical Characteristics of Thalassemia Patients Needing Transfusion in Yangjiang, Western Guangdong

Thalassemia, the most prevalent inherited disease in the world, particularly affects regions such as the Mediterranean, Middle East, Southeast and South Asia, and southern China [1‒3]. Thalassemia arises from a diverse group of genetic abnormalities linked to reduced hemoglobin (Hb) chain synthesis [4]. This imbalance leads to ineffective erythropoiesis and chronic hemolysis. α-thalassemia results from a deficiency of the α-chain of Hb, while reduced synthesis of the Hb β-chain leads to β-thalassemia. The condition typically manifests in early childhood and persists throughout the patient’s lifetime [5]. Thalassemia is categorized into three main types based on clinical symptoms and laboratory test results: minor thalassemia, intermediate thalassemia and major thalassemia. Intermediate and major thalassemia are further classified as transfusion-dependent thalassemia (TDT) and non-transfusion-dependent thalassemia (NTDT), depending on severity. The severity ranges from mild anemia to moderate and severe anemia. Patients with minor thalassemia are usually asymptomatic or experience mild anemia. The symptoms of intermediate thalassemia can vary and may include paleness, growth retardation, lethargy and fatigue but do not depend on blood transfusion. Major thalassemia symptoms typically appear after 6 months of age, due to the transition from HbF to HbA. Unlike minor thalassemia, major thalassemia patients may present clinical features such as paleness caused by extramedullary hematopoiesis, shortness of breath, irritability, failure to thrive, severe hemolytic anemia, skeletal abnormalities, hepatomegaly, and splenomegaly [1, 2].

A previous meta-analysis found higher prevalence of α-thalassemia (7.88%) in southern China, with the most common α-globin gene mutation being --^SEA, while the prevalence of β-thalassemia was 2.21%, with the most common β-globin gene mutation being HBB:c.126—129delCTTT(CD41/42). The prevalence of α + β-thalassemia was 0.48% [6]. TDT patients require lifelong blood transfusions from an early age, while NTDT patients only require occasional transfusions, such as during pregnancy or surgery [7, 8]. Individuals with thalassemia major have severe anemia and hepatosplenomegaly and typically require medical attention within the first 2 years of life [2]. Advancements in treatment have extended the life expectancy of thalassemia patients to over 50 years with regular blood transfusions and iron chelation medication [9]. However, total body iron overload due to transfusions is a potential burden, and monitoring serum ferritin (SF) levels is important for assessing iron overload [10]. Adherence to iron chelation medication is crucial for managing iron overload in thalassemia patients [11]. This study aims to analyze the genotypes of TDT patients and evaluate the prevalence of iron overload in this population.

Between January 2019 and February 2024, a retrospective analysis was conducted on 84 patients at Yangjiang People’s Hospital in Guangdong, China. Data on patient characteristics, medical history, symptoms, laboratory test results at admission, and final clinical outcomes were collected from the hospital system for analysis.

Inclusion criteria: The study focused on patients who tested positive for thalassemia genes and had a history of blood transfusions. Specifically, those with complete data spanning 3 years for investigating biochemical markers were included.

Transfusion criteria: Transfusion was deemed necessary for internal medicine patients with Hb levels below 60 g/L, surgical patients with Hb levels below 70 g/L, and patients experiencing hypoxic symptoms with Hb levels below 100 g/L. Adolescents with thalassemia should have a minimum Hb level of 90 g/L.

Research design: A retrospective analysis of hospitalized patients diagnosed with thalassemia and requiring blood transfusions at People’s Hospital of Yangjiang was conducted, with statistical analysis performed on the mutation gene classification and genotype of these thalassemia patients. → Statistical analysis was performed on the relationship between the genotype of thalassemia patients requiring blood transfusions and the frequency of transfusions, as well as the Hb concentration levels before transfusion. → The distribution of genotypes in patients with TDT was analyzed. → The clinical symptoms of patients with TDT were analyzed, and the genotypes of those who have been under complete observation for 3 years were further analyzed. → The iron overload status in patients with TDT who have been under continuous observation for 3 years was analyzed. → The relationship between SF levels and liver function in patients with TDT was explored. The liver function biochemical indicators were discussed in two parts: 1. The changes in the liver function indicators at the highest and lowest SF concentrations was analyzed. 2. The changes in the liver function indicators at the beginning and end of a 3-year observation period for patients with TDT was analyzed. → Conclusions was drawn based on the above research analysis.

Ferritin levels were measured using the chemiluminescence method on the i2000SR device from Abbott (Pennington, NJ, USA). Hb, mean corpuscular volume (MCV), and mean corpuscular Hb (MCH) were analyzed on the Sysmex XN-9000 Hematology Analyzer (Japan). Genetic testing was conducted with kits from Hybribio Co., LTD (China). All tests were performed with the manufacturer’s reagents and in accordance with the provided instructions. This study was approved by the Ethics Committee of Yangjiang People’s Hospital (2023003).

Genomic DNA was extracted from peripheral blood leukocytes of the study participants using the DNA Prep Kit manufactured by Guangdong Hybribio Limited Corporation in Chaozhou, Guangdong Province, China. The concentration of the extracted DNA was measured using the NanoDrop™ One/One C Microvolume UV-Vis Spectrophotometer from Thermo Fisher Scientific in Rockford, IL, USA, at a wavelength of 260 nm. The purity of the DNA was assessed by examining the ratio of absorbance at 260 nm–280 nm. These DNA samples were used for the subsequent PCR analysis. The genotypes of all samples were characterized using M-PCR/RDB II [12].

All data were analyzed using the SPSS version 27.0 statistical package. The normality of distribution of the parameters was assessed using the Kolmogorov-Smirnov test, and the homogeneity of variances was verified by Levene’s test. For non-normally distributed values for two groups, comparisons were made using Wilcoxon signed-rank test, and the correlation between SF and alanine aminotransferase (ALT)/aspartate aminotransferase (AST) was assessed using Spearman correlation analysis. The data were presented as median ± interquartile ranges (M[P25-P75]), and p < 0.05 was considered statistically significant.

A retrospective analysis was conducted on 84 thalassemia cases requiring transfusions at People’s Hospital of Yangjiang from January 2019 to February 2024. The cases included 38 males and 46 females ranging in age from 1 year and 3 months to 72 years. In our study, we identified six types of α-gene deletions, such as --^SEA, -α^3.7, -α^4.2, HBA2: c.427T>C (α^CS), HBA2: c.377T>C (α^QS), and HBA2: c.369C>G (α^WS), and eight types of β-gene mutations, namely HBB: c. 126_129delCTTT (CD41-42), HBB: c.316–197C>T (IVS-II-654), HBB: c.‒78A>G (β^-28), CD17 (HBB: c.52A>T), HBB: c.216_217insA (CD71-72), HBB: c.92+1G>A, c.92+1G>T (IVS-I-1), HBB: c. 84_85insC (CD27-28), and HBB: c.79G>A (β^E).

Out of the 84 transfusion cases, 22 (26.2%) had α-thalassemia, with the most common gene mutation being --^SEA (11.3%). The most frequent genotype observed was --^SEA/α^CSα (10.5%). Additionally, 50 cases (59.5%) were diagnosed with β-thalassemia, with CD41-42 as the most prevalent gene mutation (28.3%), followed by IVS-II-654 (23.0%). The predominant genotype was CD41-42/-28 (12.6%), followed by CD41-42/N (11.6%). Furthermore, there were 12 cases (14.3%) with both α-globin and β-globin genotypes, where the most common combination was -α^3.7/αα, CD41-42/-28 (3.6%) (Table 1).

In this study involving 84 cases of blood transfusion, it was observed that 56 patients required more than 6 transfusions annually and were classified as having TDT. These patients, with ages ranging from 1 to 42 years and an average age of 12, comprised 45 children and 11 adults.

Among the 56 TDT patients, there were 31 compound heterozygotes of β-thalassemia, 4 β-thalassemia homozygotes, 4 β-thalassemia heterozygotes, 11 with combined α-thalassemia and β-thalassemia, and 6 compound heterozygotes of α-thalassemia. The remaining 28 patients received occasional transfusions due to illness, with ages ranging from 1 to 72 years and an average age of 29. This group included 13 compound heterozygotes of α-thalassemia, 3 α-thalassemia heterozygotes, 3 compound heterozygotes of β-thalassemia, 8 β-thalassemia heterozygotes, and 1 patient with α-thalassemia combined with β-thalassemia (Table 2).

Prior to transfusion, Hb levels indicated that 18 patients had ≤60 g/L (21.4%, 18/84), 44 had levels between 60 and 90 g/L (52.4%, 44/84), and 22 had ≥90 g/L (26.2%, 22/84). Regarding the genotype distribution among the TDT patients, out of the 56 individuals, 6 had α-thalassemia (10.71%, 6/56), 39 had β-thalassemia (69.64%, 39/56), and 11 had α compound β-thalassemia (19.64%, 11/56). The predominant α-thalassemia genotype was found to be --^SEA/α^CSα (4 cases), while the most common β-thalassemia genotype was CD41-42/-28 (8 cases) (Table 3).

Among the 84 thalassemia patients receiving blood transfusions, 56 have TDT, requiring over 6 transfusions annually. Within this group, clinical symptoms and genotype distributions were analyzed.

Hepatomegaly was present in 24 patients (42.9%, 24/56), splenomegaly in 28 patients (50.0%, 28/56), and both conditions in 17 patients (30.4%, 17/56). Three patients (5.4%, 3/56) underwent splenectomy.

Among the 56 TDT patients, 48 were tracked over a 3-year period. Of these, 6 were α-thalassemia heterozygotes, with the prevalent genotype being --^SEA/α^CSα, β/β (6.3%). 34 patients had β-thalassemia, with genotypes including CD41-42/-28 (7 cases) and CD41-42/IVS-II-654 (6 cases). Additionally, 8 patients exhibited combined α and β thalassemia.

A family within the cohort consisted of 3 children requiring transfusions. One daughter and one son had the genotype -α^3.7/αα, CD41-42/-28, while the other son had αα/αα, CD41-42/-28. The father had αα/αα, CD41-42/β genotype, and the mother had α^3.7/αα, −28/β genotype (Table 1).

Among the cohort of 48 patients tracked over a 3-year period, consisting of 21 males and 27 females aged between 4 and 42 years (with an average age of 12.0 years), iron overload was evaluated based on SF levels: 8 patients (16.7%, 8/48) had ferritin levels below 1,000 ng/mL, 13 (27.1%, 13/48) between 1,000 and 2,500 ng/mL, and 27 (56.3%, 27/48) above 2,500 ng/mL. In total, 40 patients (83.3%, 40/48) in this group displayed iron overload (SF >1,000 ng/mL).

A retrospective analysis was conducted to explore the correlation between SF levels and liver function in thalassemia patients undergoing blood transfusion therapy. Patients were divided into two groups based on their SF levels: the highest SF level group and the lowest SF level group. Changes in liver function markers, including alanine aminotransferase (ALT), AST, and total bilirubin (TBil), were assessed in both groups. Significant differences in ALT, AST, and TBil levels were observed between the two groups, as determined by the Wilcoxon test (Table 4). It was revealed that 36 patients (75.0%, 36/48) had a history of abnormal liver function (ALT ≥80). In addition, a retrospective analysis was conducted on 48 TDT patients who had been receiving blood transfusions and iron chelation therapy for over 3 years. We reviewed the levels of four biochemical markers (SF, ALT, AST, and TBil) over the past 3 years. The results showed that, compared to 3 years ago, there were no significant changes in the levels of ALT and AST in the most recent evaluation (p > 0.05). Nonetheless, notable variances were observed in SF and TBil levels, with TBil exhibiting an escalation and SF experiencing a significant decline (p < 0.05) over the same period (Table 5). Regarding the correlation between SF levels and liver function markers, when SF was at its lowest, no significant correlation was found with ALT, AST, and TBil (p > 0.05). Conversely, when SF was at its highest, a significant correlation was identified with ALT and AST (p < 0.05), with correlation coefficients of 0.387 and 0.328, respectively (Table 6).

The study concluded that iron chelation therapy in TDT patients did not yield satisfactory results over the past 3 years. Although ferritin levels showed a slight decrease, SF levels remained elevated in both groups.

The study analyzed the distribution of thalassemia genes in 84 patients needing transfusion to gain insights into the genetic profile of this hemolytic anemia and its clinical manifestations. Specifically, our research delved into the genotypes of thalassemia patients requiring blood transfusions. Among the participants, the predominant genotype observed was --^SEA, with -α^3.7 following closely behind, consistent with commonly documented α-thalassemia genotypes in mainland China [6, 13, 14] and Taiwan [15]. In addition, our prior investigation has shown that --^SEA/αα is the predominant genotype of α-thalassemia, in line with findings from another study [13].

Furthermore, eight types of β-gene mutations were identified, with CD41-42 and IVS-II-654 being the most common genotypes observed, particularly in the northern region of Guangdong Province [12, 14]. Notably, our previous research in Yangjiang City highlighted CD41-42, IVS-II-654, and -28 as the predominant β-thalassemia genotypes in the region [13], representing a substantial proportion of cases requiring transfusions. The incidence of β-thalassemia was relatively high among the patients in this study, with 12 patients being diagnosed with α-thalassemia combined with β-thalassemia, most of whom were dependent on transfusions for management.

A separate study from Taiwan analyzed the distribution of α-globin genotype in patients with Hb H disease, identifying --^SEA/-α^3.7 and --^SEA/-α^4.2 as the most common genotypes [15]. Similarly, a meta-analysis of epidemiological studies indicated that --^SEA was the most prevalent α-thalassemia genotype in mainland China [6]. The geographic distribution of thalassemia indicated a higher prevalence in the south of China, with --^SEA, -α^3.7, and -α^4.2 being the main genotypes of α-thalassemia, and IVS-II-654 (C>T), CD41-42 (-TCTT), −28 (A>G), and CD17 (A>T) being the primary mutations of β-thalassemia in Meizhou [14]. This consistency in thalassemia genotypes in the southern coast of China reinforces previously reported findings.

Thalassemia is classified into NTDT and TDT based on disease severity and the necessity of regular blood transfusions. NTDT patients do not require frequent blood transfusions, only requiring intermittent transfusions during specific circumstances such as infection, pregnancy, or surgery causing a decrease in Hb levels. In contrast, TDT patients lack adequate Hb production to sustain life without regular transfusions. The predominant genotypes in TDT patients are homozygous or compound heterozygous, leading to severe anemia necessitating regular transfusions for daily activities and growth [16].

Thalassemia intermediate presents with milder anemia, allowing individuals to survive without transfusions or with infrequent transfusions. However, patients with homozygous (β⁺/β⁰, β⁰/β⁰) or compound heterozygous (β⁺/β⁺) for β-thalassemia mutations experience more severe syndrome, requiring more frequent transfusions compared to patients with β⁰β and β⁺β genotypes [17].

The pre-transfusion Hb level varies based on transfusion frequency. Adult patients typically have lower pre-transfusion Hb due to long-term tolerance to low levels, while children require a minimum Hb level of 90 g/L for growth maintenance. Our study revealed that 21.4% of patients had Hb levels ≤60 g/L, and 26.2% had levels ≥90 g/L before transfusion.

Additionally, thalassemia syndrome classification is dependent on clinical severity and transfusion requirements, rather than genotype. Genotype variations influence transfusion frequency, with milder genotypes initiating transfusions later in life and requiring less blood [18]. Different α-thalassemia genotypes impact MCV, MCH, and HbA₂ levels [18].

Our study identified that the most prevalent genotype among TDT patients was compound heterozygote for β-thalassemia, diagnosed predominantly before age 1 year and initiating transfusions early. Non-TDT patients mostly exhibited a compound heterozygote genotype for α-thalassemia, with diagnoses occurring later in life.

The majority of thalassemia patients present symptoms within the first few months of life. Our study revealed that 67.8%(38/56) of patients received a thalassemia diagnosis before the age of 1 year, and the age at first blood transfusion varied from 5 months to 4 years, among these patients, 24 cases showed hepatomegaly (42.9%, 24/56), and 28 cases showed splenomegaly (50%, 28/56), 17 cases experienced both hepatomegaly and splenomegaly, consistent with previous findings [17]. Individuals with thalassemia major often experience severe anemia and hepatosplenomegaly, typically necessitating transfusions within the first 2 years of life [19].

According to the findings of this study, the proportion of α-thalassemia among TDT patients was 10.71% (6/56). In a study investigating molecular characteristics of thalassemia in children aged 1–10 years, α-thalassemia incidence was reported as 10.66%, while β-thalassemia incidence was 4.90% [20]. Among thalassemia cases, 0.98% presented with both α- and β-thalassemia. The most prevalent mutation and genotype were ^--SEA and ^--SEA/αα, respectively. The predominant β-mutation was CD41-42 (-CTTT), accounting for 46.05% of all β-mutations [21].

Iron overload in TDT patients is characterized by a SF level above 1,000 ng/mL [22]. Chelation therapy is recommended to commence after the completion of 10–20 blood transfusions or when the SF level surpasses 1,000 ng/mL [3]. Our study revealed suboptimal utilization of iron chelation therapy, with 83.3% (40/48) of patients exhibiting ferritin levels exceeding 1,000 ng/mL and 56.3% (27/48) surpassing 2,500 ng/mL. Liver iron concentration, assessable through MRI, serves as the gold standard for estimating total body iron burden due to its accuracy and non-invasive nature. However, the costliness of MRI imaging poses a barrier for many thalassemia children from low-income backgrounds. Consequently, SF level evaluation remains the predominant and cost-effective method for evaluating transfusion-induced iron overload.

Thalassemia patients with SF levels exceeding 2,500 ng/mL face a mortality risk of over 80% linked to cardiac complications [23]. A positive correlation was observed between the SF level and the extent of hepatic iron overload in children who underwent blood transfusions [24].

A population-based cohort study unveiled the early detection of liver cirrhosis in specific thalassemia patients, frequently diagnosed during early childhood. It was noted that 75.0% of the patients exhibited a history of liver function abnormalities [22]. Previous investigations have demonstrated a concurrent presence of splenomegaly in patients with hepatomegaly or jaundice [25].

In TDT patients, 75.0% (36/48) have a record of abnormal liver function, and the development of liver disease with fibrosis may progress to liver cirrhosis and hepatocellular carcinoma, especially in the presence of concomitant chronic hepatitis, posing severe complications [8]. In this retrospective study, SF levels during previous blood transfusion therapy and liver function indicators were examined in TDT patients at their peak. The analysis revealed no significant relationship between the lowest SF concentration and ALT or AST levels. However, at peak SF levels, a positive correlation with ALT and AST was observed, with correlation coefficients of 0.387 and 0.328, respectively, indicating a low level of correlation.

Additional studies suggest a positive correlation between SF and iron levels with ALT and AST [26]. Furthermore, patients with severe iron overload displayed significantly elevated ALT levels compared to those with normal cardiac iron levels, while AST levels in the normal iron load group were notably lower than those in the mild, moderate, and severe iron load groups within the liver [27]. No correlation was found between TBil levels and SF [26]. Studies also indicate a positive relationship between SF and ALT/AST, yet a weak correlation exists between SF and TBil levels [28].

Our investigation illustrated no significant difference in ALT and AST levels between the most recent measurements and those recorded 3 years prior. However, SF levels from 3 years ago were higher than those from the latest measurements. It is worth noting that, based on the levels of SF concentration, we grouped the subjects and found that the group with higher SF levels had relatively lower TBil concentrations, while the group with lower SF levels had relatively higher TBil concentrations. In addition, the recent TBil concentration is higher than 3 years ago, and it shows an opposite trend to that of SF, ALT, and AST. Therefore, a larger sample size multicenter study is needed to further investigate the variation of TBil.

One study indicated that elevated SF levels strongly indicate the severity of liver and cardiac iron load, with no association between high-serum ALT or AST levels and liver iron load [29]. Nevertheless, this study presents several limitations, including its retrospective nature, single-center analysis, small sample size, and occasional absence of standard iron chelation. Moreover, discrepancies between patient phenotypes and genotypes suggest the necessity for additional molecular investigations. In conclusion, TDT is a prevalent condition requiring regular blood transfusions to support growth and development. Our study emphasizes iron overload as a common complication in these patients, stressing the crucial role of standardized iron chelation for their long-term survival.

We extend our gratitude to the individuals who contributed to this study, namely the research assistants and blood donors.

The research conducted adhered to the principles outlined for human studies and demonstrated ethical practices in line with the World Medical Association Declaration of Helsinki. Approval for the study protocol was granted by the Ethics Committee of People’s Hospital of Yangjiang under reference number 2023003. Informed consent was obtained from participants, their legal guardian, or next of kin.

The authors affirm no conflicts of interest.

This research received financial support from the High Level Development Plan of People’s Hospital of Yangjiang under Grant No. G2020007 and the High Level and Key Health Research Project of Yangjiang (No. 2023001). The funding entities were not involved in data preparation or manuscript drafting.

Li-Ye Yang conceptualized and designed the study, as well as oversaw manuscript revisions. Zhi-Xiao Chen conducted data analysis and manuscript composition. Rong-Huo Liu, Jian-Cheng Huang, Jia-Min Mo, Yan-Qing Zeng, and Yu-Chan Huang executed the experiments. All authors reviewed and approved the final manuscript.

All data relevant to this study are provided in this article. Further inquiries can be directed to the corresponding author.