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Mutation spectrum and erythrocyte indices characterisation of α-thalassaemia and β-thalassaemia in Sichuan women in China: a thalassaemia screening survey of 42 155 women
  1. Bei Li1,
  2. Xiao Han1,
  3. Jian Ma1,
  4. Dan Yang2
  1. 1 Medical Laboratory Department, Sichuan Jinxin Women's and Children's Hospital, Chengdu, China
  2. 2 Medical Laboratory Departement, Sichuan Huaxi Shukang Biological Pharmaceutical Co. Ltd, Chengdu, China
  1. Correspondence to Jian Ma, Sichuan Jinxin Women's and Children's Hospital, Chengdu, China; majian_212{at}163.com

Abstract

Aims The present study aims to investigate the mutation spectrum of thalassaemia, and characterise the erythrocyte indices of thalassaemia in a large cohort in Sichuan, which is a province with a high prevalence of thalassaemia in southern China.

Methods The present study was conducted from July 2017 to July 2019. A total of 42 155 women screened for thalassaemia were enrolled. The thalassaemia carriers were screened by erythrocyte analysis and haemoglobin electrophoresis. Then, the screening test results and genetic results were collected.

Results A total of 1109 individuals had thalassaemia gene defects. Among these individuals, 69.7% were α-thalassaemia (α-thal) and 28.6% were β-thalassaemia (β-thal). For α-thal defects, carriers with --SEA had the lowest mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) values. For β-thal defects, carriers with heterozygous haemoglobin E and −28 had the highest MCV and MCH values. In addition, an MCV cut-off of 80 fl and an MCH cut-off of 27 pg was able to detect 99.1% α0 thalassaemia and 99.7% β0/β+ thalassaemia; however, that criterion could result in a great number of false-negative results in α+ carriers.

Conclusion A criterion of MCV <80 fl and MCH <27 pg is recommended for detecting –SEA carriers and β0/β+ carriers, while higher cut-offs are needed to detect α+ carriers.

  • thalassemia
  • epidemiology
  • hematology

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Introduction

Thalassaemia is one of the most common genetic disorders worldwide. Thalassaemia is characterised by hypochromic microcytic anaemia, and a phenotype that varies from almost asymptomatic to lethal haemolytic anaemia.1 There are mainly two types of thalassaemia: α-thalassaemia (α-thal) and β-thalassaemia (β-thal). The α-thal is commonly caused by deletions, and two subtypes are defined by a single gene deletion (-α/αα, α+) or two gene deletions (--/αα, α0). The β-thal is mainly caused by mutations, and two subtypes are defined by totally absent (β0) or partially reduced (β+) production of normal β chains. The most common combination of β-thal with haemoglobin variant is haemoglobin E (HbE). Heterozygotes of thalassaemia may have mild anaemia, while homozygotes may result in severe anaemia, stillbirth or transfusion dependency.2 Therefore, the ability to identify and characterise thalassaemia carriers can assist in genetic counselling and prenatal diagnoses.

It has been reported that approximately 1% to 5% of the global population are carriers of thalassaemia.3 In China, thalassaemia is very prevalent, and the prevalence of α-thal, β-thal and α+β thalassaemia was 7.88%, 2.21% and 0.48%, respectively.4 The greatest disease burden was found in southern China, particularly in Guangxi,5 Guangdong,6 Yunnan,7 Sichuan8 and Chongqing.9 Populations of different regions have different prevalence and spectrum of thalassaemia, and an understanding of the epidemiological characteristics of the disease provides important information on its prevention. In fact, in order to decrease the birth rate of thalassaemia majors, carrier screening and prenatal diagnosis have been widely used in China. However, the screening strategies of thalassaemia varies in different regions due to the lack of national screening guidelines.

In general, the mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) are the most common indicators for thalassaemia screening. In addition, elevated haemoglobin A2 (HbA2) is also a useful marker for the identification of β-thal. Since the normal reference intervals of these indices can be affected by many factors, such as gender, disease status, race and geographic region, it is necessary to determine the proper cut-off of these indices in different regions and populations.

To date, there are few studies related to the prevalence and mutation spectrum of thalassaemia in a large sample size of Sichuan population. In addition, few studies have determined the appropriate cut-off of erythrocyte indices in the Sichuan population, and in carriers of α-thal. Therefore, the present study aimed to investigate the mutation spectrum of thalassaemia, and characterise the erythrocyte indices of α-thal and β-thal defects in a large cohort in Sichuan, which is a province with a high prevalence of thalassaemia in southern China.

Materials and Methods

Study population

A total of 42 155 female individuals (18 to 60 years old, average age=28±4 years) were enrolled. Female individuals with positive screening results were recommended for further molecular diagnosis. A total of 2430 female individuals had molecular results. The molecular diagnosis, which included thalassaemia mutation detection, was performed using gap-PCR and reverse dot blot (RDB) analysis. Finally, a total of 1109 women (1108 heterozygotes and one homozygous HbE) were identified as α-thal and/or β-thal. The flowchart for the study design is presented in figure 1.

Figure 1

Diagnostic flowchart for the detection of thalassaemia in the present study.

Screening strategy

Blood specimens (3 mL) were collected in tubes anticoagulated with ethylenediaminetetraacetic acid (EDTA-K2), and sent to the laboratory for thalassaemia screening. Screening tests included haematological analysis and Hb analysis. The haematological parameters were measured using an automated blood counter (BC-6900; Mindray Bio-Medical Electronics Co, Shenzhen, China). Hb analysis was performed using a capillary electrophoresis system (Helena, Texas, USA). A woman was eligible and suggested for further DNA test if she had a low MCV (≤85 fl) or low MCH (≤27 pg) or high HbA2 level (>3.5%) or Hb variant. Patients with iron deficiency were excluded.

Molecular analysis

In order to test the mutation or deletion of thalassaemia, we collected 5 mL of venous blood from each patient. Three α-thal deletions were detected by gap-PCR. Furthermore, three α-thal mutations and 17 β-thal mutations were detected by RDB analysis (Yaneng Bioscience Co Ltd, Shenzhen, China). All the deletions or mutations we tested are shown in table 1.

Table 1

The α-thalassaemia and β-thalassaemia gene distribution in the present study

Statistical analysis

The SPSS V.17.0 software (IBM, New York, USA) was used for the statistical analysis. The data were analysed by Mann-Whitney U test. P<0.05 was considered statistically significant.

Results

A total of 42 155 women were screened for thalassaemia in our hospital from July 2017 to July 2019. Among them, 2430 women had further molecular results and 1109 women had thalassaemia gene abnormalities. Of 1109 thalassaemia women identified, 69.7% (773/1109) were α-thal carriers or individuals, 28.6% (317/1109) were β-thal carriers, and 1.71% (19/1109) were both α-thal and β-thal carriers or individuals (table 1).

Table 2 shows the ratio of the thalassaemia gene abnormalities, the most common gene defects of α-thal were –α3.7 (45.3%), --SEA (44.0%) and –α4.2 (7.9%). Seventeen β-thal gene mutations were tested, and 10 gene mutations were identified. Among them, CD17 (30.6%) was the most frequent mutation of β-thal, which was followed by IVS-II-654 (23.3%), CD41/42 (22.7%), −28 (7.9%) and heterozygous HbE (7.5%). Women with these five mutations accounted for more than 90% of the β-thal carriers or individuals in this region (table 1). The different types of combinations of α-thal and β-thal gene defects are presented in table 2.

Table 2

The coexistence of α-thalassaemia and β-thalassaemia

The mean value and spread of distributions of erythrocyte indices (red blood cells (RBC), Hb, MCV, MCH and mean cell haemoglobin concentration (MCHC)) and HbA2 were compared among different α-thal gene defects (online supplementary figure 1S) and among different β-thal gene defects (online supplementary figure 2S). In α-thal groups, the carriers with--SEA had the highest RBC values, but had the lowest Hb, MCV, MCH and MCHC values. In the β-thal mutation groups, carriers with heterozygous HbE and carriers with −28 had highest Hb, MCV, MCH and MCHC values. Carriers with heterozygous HbE had the lowest RBC values.

Supplemental material

Supplemental material

The erythrocyte indices and HbA2 were also compared between the α0 group (--SEA) and α+ group (–α3.7 and –α4.2). Results indicate that α0 carriers have significantly lower Hb, MCV, MCH and MCHC levels, when compared with α+ carriers. However, there was no significant difference in total RBC between the two groups. The mean values±SD for MCV in the α0 group and α+ group were 69.4±3.4 fl and 83.7±3.4 fl, respectively. The mean values±SD for MCH in the α0 group and α+ group were 21.7±1.1 pg and 26.9±1.1 pg, respectively (table 3 and online supplementary figure 3S). Carriers with non-deletional α-thal gene mutations (αT) and carriers with α-thal single gene deletion (α+) had similar values for erythrocyte indices and HbA2. Among carriers with α gene mutations (αCS, αQS and αWS), those with αQS had relatively lower MCV and MCH levels.

Supplemental material

Table 3

Comparison of erythrocyte and electrophoretic indices between the α0 group and α+ group

Among the 340 carriers with α0 (SEA) thalassaemia, three women had MCV values >80 fl and MCH values >27 pg at the same time, while only one individual had an MCV value >85 fl and an MCH value >27 pg. This means that an MCV cut-off of 80 fl and an MCH cut-off of 27 pg were able to detect 99.1% ((340–3)/340) of the heterozygous carriers of α0 carriers, while an MCV cut-off of 85 fl and an MCH cut-off of 27 pg were able to detect 99.7% ((340–1)/340) of the heterozygous carriers of α0 deletion. Among the 411 carriers with α+ deletions, 215 individuals (52.3%) had an MCV >80 fl, while 146 (35.5%) carriers with α+– thalassaemia had an MCV >85 fl. It means that 16.8% ((215–146)/411) of α+– thalassaemia carriers had MCV levels between 80 fl and 85 fl, and these carriers could be misdiagnosed as false negatives in certain situations.

Next, the erythrocyte indices and HbA2 were compared among carriers in the β0, β+ and heterozygous HbE groups (table 4 and online supplementary figure 3S). It was found that carriers in the β0 group had the lowest MCV and MCH levels, while carriers in the heterozygous HbE group had the highest MCV and MCH levels. The mean values±SD of MCV for carriers in the β0, β+ and heterozygous HbE groups was 65.3±3.7 fl, 74.8±3.3 fl and 81.5±3.6 fl, respectively. The mean values±SD of MCH for carriers in the β0, β+ and heterozygous HbE groups were 20.4±1.1 pg, 23.7±1.0 pg and 26.7±1.3 pg, respectively. Hence, β0 carriers had the lowest values, while heterozygous HbE carriers had the highest values. Heterozygous HbE (CD26, G>A) carriers had the highest MCV, MCH and Hb levels among β-thal carriers. Women with the −28 (A>G) mutation (β+) had the highest MCH, MCV, MCHC and Hb values, when compared with other types of β0/β+ carriers. It was also found that the MCV cut-off of 80 fl and the MCH cut-off of 27 pg could detect 99.7% (292/293) of the common β0/β+ alleles.

Table 4

Comparison of erythrocyte and electrophoretic indices among the β0, β+ and βE groups

The mean value and spread of distributions of the erythrocyte indices and HbA2 were compared among the α, β-thal and simple β-thal groups. In the present study, major differences were observed in terms of MCV, MCH, MCHC and Hb. However, there was no significant difference in RBC levels between the two groups. Individuals who co-inherited with α, β-thal had a higher mean MCV, MCH, MCHC and Hb levels, when compared with individuals with simple β-thal (table 5).

Table 5

Comparison of erythrocyte and electrophoretic indices between α, β-thalassaemia and β-thalassaemia

Discussion

It is known that provinces in southern China have a high incidence of thalassaemia, while sporadic studies can be found in Sichuan province. The present study is the first comprehensive study carried out to investigate the spectrum of thalassaemia in Sichuan province, with a largest sample size of 42 155 women. Furthermore, this study is also the first to describe the characteristics of erythrocyte indices in the screening of thalassaemia in Sichuan.

In the screened population, at least 2.6% (1109/42 155) were carriers of thalassaemia, and more α-thal carriers (69.7%) than β-thal carriers (28.6%) in Sichuan were found. This is consistent with previous studies in southern China,5 6 9 10 but differs from the data reported in Yunnan,7 where more β-thal than α-thal were found and multiethnicities may play a key role in the high prevalence of β-thal in Yunnan. In the present study, α-thal accounted for 97.2% of thalassaemia carriers and –α3.7 was the most prevalent while this was inconsistent with the data reported by Xia Yu et al.8 Based on their study, the most prevalent type of α-thal is --SEA, instead of –α3.7. The lower cut-off of MCV (MCV <82 fl) in their study should be blamed. In our study, nearly half (49.9%, 205/411) of α+ thalassaemia carriers had MCV values of >82 fl. Hence, a low cut-off of MCV could lead to the false negative screening of α+ (–α3.7 and –α–4.2) thalassaemia.

The spectrum of β-thal mutations in our study were similar to those previously reported in southern China,11 although the exact ratios differed. For example, HbE is very common in Yunnan, and ranks third in β-thal spectrum.7 HbE is also very common in some other countries in Southeast Asia, such as Thailand.12 13 As we know, the screening for thalassaemia in community-based and antenatal programmes is usually initially based on the cut-off values for either MCV or MCH, while clinical guidelines and practices vary. In some strategies, MCV <80 fl is applied as a criterion for thalassaemia screening. However, a study revealed that there are many carriers of α+– thalassaemia in populations with MCV values between 80 fl to 85 fl.14 In order to minimise false negatives, the criterion for molecular diagnosis was extended from an MCV of 80 fl to an MCV of 85 fl in the present study.

In the present study, we find that α0 carriers have significantly lower values for erythrocyte indices, when compared with α+ carriers Among carriers with three mutations (αCS, αQS an αWS), those with αQS had relatively lower MCV and MCH values.

Therefore, α0 thalassaemia with two α gene deletions is associated with typical thalassaemia erythrocyte changes, while α+– thalassaemia with only one α gene deletion exhibits mild erythrocyte changes. Among the 411 carriers with α+ deletions, 215 individuals (52.3%) had an MCV of >80 fl, while 146 (35.5%) carriers with α+– thalassaemia had an MCV of >85 fl. This means that 16.8% ((215–146)/411) of α+– thalassaemia carriers have MCV levels between 80 fl and 85 fl, and that these carriers could be misdiagnosed as false negatives. This further shows that an MCV cut-off of 80 fl is not suitable for α+– thalassaemia detection.

It should be noted that, as some guidelines use as MCH of 25 pg as the cut-off for α0, it is important to also analyse with this cut-off for the benefit of people outside China. Here, we find that among 340 carriers with α0 (SEA) thalassaemia, five women had MCH values between 25 pg and 28 pg. This means that an MCH cut-off of 25 pg was able to detect 98.5 ((340–5)/340) of the heterozygous carriers of α0 carriers. Therefore, we recommend an MCH of 27 pg instead of 25 pg as the cut-off for α0 screening.

Here, it was revealed that β0 carriers have the lowest MCV and MCH values, and that heterozygous HbE carriers have the highest MCV and MCH values. HbE (CD26, G>A), which is a haemoglobin variant, is clinically asymptomatic, with almost normal erythrocyte indices in heterozygotes. Individuals with the −28 (A>G) mutation (β+) had the highest MCV and MCH levels, when compared with the other types of β0/β+ carriers. Based on these results, an MCV cut-off of 80 fl and an MCH cut-off of 27 pg were able to detect 99.7% of the common β0/β+ alleles. It is noteworthy that individuals with the Cap+1 mutation had higher MCV, MCH and Hb values, when compared with patients with other β-thal mutations.

It was found that compared with β-thal carriers, α, β-thal carriers had higher values of MCV and MCH. These results show the coexistence of α-thal mutations with the β-thal resulted in milder anaemia.15 Therefore, it is necessary to detect the α-thal defect in all cases of β-thal with normal MCV or MCH. Therefore, a criterion of MCV <80 fl is recommended for the detection of --SEA carriers and β0/β+ carriers and the cut-off values for MCV should be increased, while it remains inevitable to miss some thalassaemia carriers. The only fail-safe strategy to detect α+ is DNA analysis, and the most economical way for heterozygous HbE carrier screening is electrophoresis. In conclusion, the present study is the first to describe characteristics of erythrocyte indices for thalassaemia carriers in Sichuan province. These results provide a comprehensive view of the mutation spectrum of thalassaemia in Sichuan, which may contribute to thalassaemia’s control and management, where it is prevalent.

Take home messages

  • Among Sichuan women, 69.7% were α-thalassaemia (α-thal) and 28.6% were β-thalassaemia (β-thal).

  • The most common defects of α-thal were –α3.7, --SEA and –α4.2, while for those of β-thal were CD17, IVS-II-654 and CD41/42.

  • For α-thal defects, carriers with --SEA had the lowest mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) values. For β-thal defects, carriers with heterozygous HbE and −28 had the highest MCV and MCH values.

  • An MCV cut-off of 80 fl and an MCH cut-off of 27 pg was able to detect 99.1% α0 thalassaemia and 99.7% β0/β+ thalassaemia; however, that criterion could result in a great number of false-negative results in α+ carriers.

References

Footnotes

  • Handling editor Mary Frances McMullin.

  • Contributors Research conception and study design: BL and JM. Research and data analysis: BL, JM and XH. Manuscript preparation: BL and XH. Clinical data collection: BL, XH and JM. Research sample preparation: DY. All authors have read the journal’s authorship agreement and agree with the statements. Each author contributed important intellectual content during manuscript drafting or revision, and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Patient consent for publication Obtained.

  • Ethics approval All clinical samples and data were collected for routine patient care. This retrospective study was done in accordance with the ethical standards of the Ethics Committees of Sichuan Jinxin Women and Children’s Hospital.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement All data relevant to the study are included in the article or uploaded as online supplementary information.