Introduction

Thalassemia is an autosomal recessive genetic disorder characterized by impaired hemoglobin synthesis. It is primarily classified into α-thalassemia and β-thalassemia, with the underlying genetic etiologies typically involving deletions of the α-globin genes located on the short arm of chromosome 16 and point variants in the β-globin gene situated on the short arm of chromosome 11. However, non-deletional variants have been reported in α-thalassemia, and certain deletional variants have been identified in β-thalassemia as well. Thalassemia is a globally prevalent monogenic disorder, with a particularly high incidence in regions bordering the Mediterranean, Southeast Asia, India, and coastal southern China. In southern Chinese provinces such as Guangdong, Guangxi, Fujian, Hainan, and Guizhou, α-thalassemia is predominantly associated with gene deletions, whereas β-thalassemia more commonly results from point variants, reflecting distinct regional variant patterns.1

α-thalassemia is categorized into the silent carrier state, mild type, Hemoglobin H (Hb H) disease, and Hemoglobin Bart’s (Hb Bart’s) hydrops fetalis syndrome, according to the severity of clinical manifestations. In contrast, β-thalassemia is classified into mild, intermediate, and severe forms. The clinical expression of α-thalassemia is primarily determined by variants affecting the four α-globin genes located on chromosome 16, whereas the severity of β-thalassemia is influenced by the phenotypic expression of the β-globin alleles, specifically β0 and β⁺.2,3

Individuals with mild α-thalassemia or β-thalassemia are often asymptomatic, though anemia may be precipitated by factors such as malnutrition or infection.2,4 Hb H disease is typically associated with clinical features including anemia, splenomegaly, jaundice, and growth retardation.2 Hb Bart’s hydrops fetalis syndrome is associated with intrauterine death in affected infants, and despite medical intervention, survival remains extremely rare, with significant maternal health risks.5 Patients with intermediate β-thalassemia may present with anemia, mild skeletal abnormalities, and hepatosplenomegaly.6 In patients with severe β-thalassemia, symptoms typically emerge postnatally and may include anemia, jaundice, splenomegaly, characteristic craniofacial features, and growth retardation, with progressive disease manifestations observed over time.4 Given that thalassemia is inherited in an autosomal monogenic pattern, genotypes correlate with specific clinical classifications: individuals with the silent carrier state or mild α-thalassemia are typically α⁺ heterozygotes; Hb H disease is commonly observed in α0 heterozygotes. Hb Bart’s hydrops fetalis syndrome generally occurs in α0 homozygotes, resulting from inheritance of α0 alleles from both parents.2,7 Individuals with mild β-thalassemia are usually β⁺ heterozygotes; severe β-thalassemia may involve either β⁺ or β0 homozygotes; intermediate β-thalassemia is typically associated with β0 or β⁺ heterozygotes.8 Long-term transfusion therapy in patients with moderate to severe thalassemia may result in iron overload, leading to complications such as hepatic fibrosis, diabetes mellitus, and other endocrine dysfunctions.9 Iron chelation therapy has demonstrated efficacy in preventing iron overload; however, it is not universally accessible in all clinical settings.10

Existing literature indicates that genetic counseling and testing for thalassemia among individuals of reproductive age can be effective in reducing the incidence of births affected by thalassemia, thereby decreasing the associated burden on families and society while contributing to improved population health in the region.11 Complete blood count (CBC) testing is essential for characterizing the hematological phenotype of individuals with Mediterranean anemia.12 Prior studies have demonstrated that children with β-thalassemia in Chongqing City exhibit differences in hematological parameters associated with specific genotypes.13 Chongqing’s thalassemia molecular landscape remains understudied despite its public health burden. Recent research has indicated that hematological indices, including hemoglobin (Hb), mean corpuscular volume, mean corpuscular Hb, mean corpuscular Hb concentration, and HbA2, may vary in regular or irregular patterns according to thalassemia genotype and disease severity.14 Examination of the combined relationship between genotype and CBC parameters may enhance the recognition of severe anemia and high-prevalence thalassemia genotypes, thereby clarifying priorities for thalassemia prevention and clinical management. This study was designed to analyze the molecular epidemiological characteristics of thalassemia-associated genes in the University Town of Chongqing, with the objective of supporting timely identification of affected patients and carriers and providing evidence to guide genetic counseling and reproductive risk assessment in the region.

Materials and Methods Data

Molecular testing for Mediterranean anemia was performed on 1,012 patients at the University City Hospital affiliated with Chongqing Medical University between June 2020 and February 2023. Most cases were referred through prenatal screening, genetic counseling clinics, or due to abnormal findings in blood tests or Hb electrophoresis screening. The cohort included 172 males and 840 females, with ages ranging from 0–80 years. CBC data collected during the same period were used to categorize individuals into two groups: the thalassemia group, consisting of individuals who tested positive for Mediterranean anemia gene variants, and the control group, composed of individuals with negative genetic test results for thalassemia. Pediatric cases were excluded from both groups. The control group consisted of a healthy adult population with no hematological abnormalities on routine CBC testing and negative results for thalassemia-associated gene variants. The red blood cell parameters of 100 healthy people in the same period were compared,the control group included 55 females and 45 males.

Reagents and Instruments

At the Mediterranean Anemia Molecular Diagnostic Center, the Guangzhou Cap Mediterranean Anemia Gene Detection Kit (based on dot blot hybridization) was used for the molecular diagnosis of Mediterranean anemia, in combination with the HB 2012A Medical Nucleic Acid Hybridization Instrument. The Shanghai Hongshi SLAN96P fully automated Medical PCR Analysis System was used for polymerase chain reaction analysis. Routine blood examinations were conducted using the Sysmex XN-2800 Hematology Analyzer (Sysmex Corporation, Japan) and the BC7500 fully automated Hematology Analyzer (Mindray Bio-Medical Electronics Co., Ltd).

Methods

Molecular testing reports from all patients diagnosed with Mediterranean anemia at the institution between June 2020 and February 2023 were collected and analyzed. Biotin-labeled oligonucleotide primers (0.2 μM) were used to simultaneously amplify the deletion and variant regions of the α-globin gene and the variant region of the β-globin gene. The amplified products were then subjected to flow-through hybridization on nylon membranes containing thalassemia-specific probes for various deletions or variants using a flow-through hybridization system. The results were interpreted via chemical color development. Guangzhou Kaipu thalassemia extraction kit and matching gene amplification reagent were used (kit20243401082), According to the kit instructions.

The objective of this analysis was to determine the frequency and distribution of common α-thalassemia and β-thalassemia gene variants. Detection rates of Mediterranean anemia-associated genes were calculated separately for male and female participants. Females were predominantly referred for prenatal screening, whereas males were generally tested only in the presence of clinical suspicion (eg., microcytosis) or family history. Statistical analyses were performed using SPSS version 27.0 software. The chi-squared (χ2) test was used to assess differences in detection rates between sexes, with a significance threshold set at p < 0.05 (power analysis, α=0.05, power=0.8). A non-parametric independent samples test was conducted to compare hematological parameters between the Mediterranean anemia group and a healthy control group to assess the impact of thalassemia gene variants on hematological phenotypes. For continuous variables with a skewed distribution, data were expressed as median values with interquartile ranges [25th percentile, 75th percentile]. The Mann–Whitney U-test was applied to assess differences between groups. A p < 0.05 (power analysis, α=0.05, power=0.8) was considered indicative of statistical significance.

The study protocol was approved by the Ethics Committee of the University Town Hospital of Chongqing Medical University on January 31, 2024 (No. LL-202340). All data were fully anonymized prior to analysis. Informed consent was obtained from all participants and/or their legal guardians. All procedures were conducted in accordance with relevant guidelines and regulations.

Results Types of α-Thalassemia Genes

A total of 123 carriers of α-thalassemia gene variants were identified in the present investigation, including one case of –SEA/–HK α-thalassemia, resulting in a detection rate of 12.1% (123/1012). Five types of α-thalassemia gene abnormalities were detected: SEA deletion (73 cases), 3.7 kb deletion (34 cases), 4.2 kb deletion (9 cases), CS variant (3 cases), and QS variant (4 cases). The SEA and 3.7 kb deletions represented the most frequently observed α-thalassemia variants in this cohort (Table 1).

Table 1 Detected α-Thalassemia Mutations and Their Composition in Mediterranean Anemia

Types of β-Thalassemia Genes

A total of 116 carriers of β-thalassemia gene variants were identified, resulting in a detection rate of 11.4% (116/1012). Eleven distinct β-thalassemia gene variants were detected: CD41–42 (39 cases), CD43 (1 case), IVS-II-654 (32 cases), CD17 (33 cases), –28 (4 cases), β^E (1 case), CD71–72 (2 cases), CD27–28 (1 case), IVS-I-1 (1 case), Int (1 case), and CD31 (1 case). The most frequently observed β-thalassemia variants were CD41–42, CD17, and IVS-II-654 (Table 2).

Table 2 Detected β-Thalassemia Mutations and Their Composition in Mediterranean Anemia

Distribution of Thalassemia Genes in Females and Males

A total of 186 female patients were identified as carriers of Mediterranean anemia-related gene variants, resulting in a detection rate of 22.1% (186/840). The most common α-thalassemia genotypes among females were –SEA deletion and –3.7 kb deletion, followed by –4.2 kb deletion, QS variant, and CS variant. Among male patients, 54 were found to carry Mediterranean anemia-associated genes, yielding a detection rate of 30.2% (54/172). The predominant α-thalassemia genotype detected in males was the –SEA deletion. The proportion of the –SEA deletion genotype was significantly higher in males than in females (Figure 1).

Figure 1 Detected α-thalassemia mutations and their composition in females and males.

Among female carriers of β-thalassemia gene variants, the most frequently observed variants were CD41–42, IVS-II-654, and CD17. In addition, three cases involving the –28 variant were identified, while single cases were recorded for the CD43 variant, βE variant, CD27–28 variant, IVS-I-1 variant, and CD31 variant each classified as less common variants. In male patients, the most frequent β-thalassemia genotypes were CD17, CD41–42, and IVS-II-654, while CD71–72, –28, and Int variants were observed less commonly.

The proportions of CD41–42 and IVS-II-654 variants were significantly higher in females than in males, whereas CD17 and CD71–72 variants were more frequently observed in males (Figure 2).

Figure 2 Detected β-thalassemia mutations and their composition in females and males.

Sex Differences in Mediterranean Anemia Detection Rates

The detection rates of thalassemia among different sex groups are presented in Table 3. A χ2 was conducted to compare detection rates between males and females, yielding a statistically significant result (p < 0.05). The carrier rates were 30.2% in males and 21.7% in females, indicating a significantly higher detection rate of Mediterranean anemia–associated gene variants in males compared to females.

Table 3 Comparison of Thalassemia Detection Rates Between Different Gender Groups

Comparison of Red Blood Cell Parameters in Mediterranean Anemia and Control Groups

The red blood cell parameters of 100 healthy individuals from the same period (control group) were compared with those of the thalassemia group. Owing to skewed distribution, the data are expressed as medians with interquartile ranges [25th percentile, 75th percentile]. Results from the non-parametric test indicated statistically significant differences (p < 0.05) in several hematological parameters between the thalassemia group and the control group. The red blood cell count (RBC) and red cell distribution width (RDW) were significantly higher in the thalassemia group, whereas Hb, hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) values were significantly lower compared to those in the control group (p < 0.05) (Table 4).

Table 4 Comparison of Hematological Parameters Between the Thalassemia Group and Control Group

Discussion

Mediterranean anemia is an Hb synthesis disorder for which gene-based diagnosis is considered the gold standard.12 Commonly used methods for the detection of thalassemia-associated gene variants include real-time quantitative polymerase chain reaction, DNA probe hybridization, reverse dot blot hybridization, and gene sequencing. Among these methods, real-time quantitative PCR and reverse dot blot hybridization are the most widely used and are particularly suitable for identifying α-thalassemia gene deletions and β-thalassemia point variants.13–15

Chongqing, as a central urban area in Western China, experiences substantial population mobility, contributing to a distribution of Mediterranean anemia gene variants that differs from those observed in other southern regions. In the present study, among 1,012 patients who underwent genetic testing for Mediterranean anemia at the study hospital, 123 cases of α-thalassemia gene carriers were identified, corresponding to a detection rate of 12.1%. The frequency of deletion-type α-thalassemia gene variants was considerably higher than that of point variants, with –SEA and –3.7 kb deletions being the most frequently observed. The –SEA deletion was more prevalent, a pattern that differed slightly from the distribution reported in the Guangxi region, but was consistent with findings from Sichuan, Fujian, and Hainan provinces.16–19 In contrast, data from the Guiyang region indicated that the most common α-thalassemia gene was the –3.7 kb deletion, followed by the –SEA and –4.2 kb deletions, differing from the distribution observed in this study.20

In this survey, 116 β-thalassemia gene carriers were identified, corresponding to a detection rate of 11.4%. Among the 11 β-thalassemia gene variants detected, the most prevalent types were CD41–42, CD17, and IVS-II-654, findings that are consistent with the results reported by Wu et al and similar to those observed in the Sichuan region.17,21 However, the distribution of variant-type β-thalassemia genes differed from that reported in Guangxi,Fujian, Hainan and Guizhou provinces.16,18–22

The detection rate of Mediterranean anemia gene variants among male patients in this region was 30.2% (54/172), which was higher than the 22.0% (185/840) observed in the female population. Although this difference was statistically significant (p < 0.05), the higher detection rate in males is likely attributable to referral bias rather than a true biological difference between the sexes. The distribution of Mediterranean anemia gene types was similar across sexes, with the –SEA deletion identified as the most common α-thalassemia variant, and CD41–42, IVS-II-654, and CD17 variants as the most frequently observed β-thalassemia variants. The proportion of the –SEA deletion genotype was significantly higher in males compared to females. In contrast, the proportions of the CD41–42 and IVS-II-654 variants were significantly higher in females, whereas the CD17 and CD71–72 variants were more frequently observed in males.

These findings indicate a potential correlation between genotype distribution and sex. However, a substantial number of individuals may remain undiagnosed due to the limited geographic coverage and sample size of this study. Given that couples heterozygous for thalassemia have a 25% probability of conceiving offspring affected by severe disease, this represents a considerable burden on both families and public health systems. Therefore, broad-based screening for Mediterranean anemia gene carriers is recommended among newborns, school-aged children, and individuals of reproductive age in this region to facilitate early identification of carriers. The implementation of prenatal diagnostic services and genetic counseling is essential for the effective prevention of severe thalassemia births.

The red blood cell parameters of 100 healthy individuals from the same period were used as a control group, albeit with acknowledged limitations. This study demonstrated that RBC count and RDW values were generally elevated in thalassemia patients compared to these healthy controls. This finding may be explained by a compensatory increase in RBC production in response to decreased Hb levels, as well as an increase in abnormally shaped erythrocytes, which contributes to elevated RDW values.23 In contrast, Hb, Hct, MCV, MCH, and MCHC were significantly lower in patients with thalassemia than in the control group. Decreased MCV and MCH are considered hallmark hematological features of the thalassemia phenotype.12

However, similar hematological characteristics, specifically, reduced MCV and MCH, are observed in individuals with iron deficiency anemia (IDA). Therefore, the diagnosis of IDA should be supported by additional laboratory assessments, including serum ferritin, transferrin saturation, and total iron-binding capacity. The absence of serum ferritin data limits differentiation between thalassemia and iron deficiency anemiain the present study. Iron supplementation therapy may be used diagnostically to determine if hematological indices normalize, thereby aiding in the differentiation between thalassemia and IDA.24 Hb serves as a critical indicator in the evaluation of anemia severity. In individuals with thalassemia, Hb levels are typically reduced, indicating varying degrees of anemia ranging from mild to severe.12 Although Hct reflects the proportion of red blood cells within the total blood volume, it is subject to multiple physiological influences and is therefore not considered a reliable marker of the thalassemia phenotype. Additionally, individual RBC and RDW parameters have limited utility as standalone screening markers for thalassemia. In recent years, several members of the study team have developed formulas based on red blood cell indices that have demonstrated good performance in distinguishing between IDA and thalassemia and in supporting thalassemia screening efforts. However, the establishment of specific cut-off values tailored to different populations remains necessary.25 Besides, the Mentzer Index (MCV/RBC ratio) is a cost - free way for clinicians to use the data presented here to spot carriers. We also acknowledge that HPLC or hemoglobin electrophoresis data were not performed in this study, which is a limitation for fully characterizing hemoglobin phenotypes.

Conclusion

In summary, the detection rate and distribution of thalassemia gene variants in the University City area of Chongqing demonstrate distinct regional characteristics. Implementation of screening for thalassemia gene variants among newborns, school-aged children, and individuals of reproductive age in this region may facilitate early identification of carriers and support reproductive health initiatives. Therefore, screening that integrates RBC indices with targeted molecular testing is recommended for high-risk regions, although population-wide school screening requires further validation.

Abbreviations

Hb H, Hemoglobin H;Hb Bart’s, Hemoglobin Bart’s;CBC, Complete blood count;RBC, red blood cell count;RDW, red cell distribution width;Hct, hematocrit;MCV, mean corpuscular volume;MCH, mean corpuscular hemoglobin;MCHC, mean corpuscular hemoglobin concentration;IDA, iron deficiency anemia.

Data Sharing Statement

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Ethics Approval and Consent to Participate

The study was conducted in accordance with the Declaration of Helsinki (as was revised in 2013). The study was approved by Ethics Committee of the University-Town Hospital of Chongqing Medical University (Approval Number: LL-202340). All participants provided written informed consent for their involvement in the study. In cases where participants were under the age of 18, informed consent was additionally obtained from their parents or legal guardians.

Acknowledgments

We are particularly grateful to all the people who have given us help on our article.

Funding

China Postdoctoral Science Foundation (Certificate No. 2023MD734129); Chongqing Young and Middle-aged Medical High-end Talent Project (YXGD202416); Chongqing Science Foundation Project (Certificate No. CSTB2023NSCQ-BHX0004); Science and Technology Research Program of Chongqing Municipal Education Commission (Grant Nos. KJQN202300473 and KJQN202400470).

Disclosure

The authors declare that they have no competing interests.

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