Evaluation of intervention strategy of thalassemia for couples of childbearing ages in Centre of Southern China

Abstract Background To describe the free intervention strategy of thalassemia for childbearing couples in Guangzhou. Methods Routine hematology examinations were conducted for 137,222 couples. Among them, 37,501 couples who had mean corpuscular volume (MCV) <82 fL or mean corpuscular hemoglobin <27 pg were elected for Hb analysis and the deletions of four common α‐thalassemia mutation. Reverse dot blot for common nondeletional α‐thalassemia and β‐thalassemia was selectively used. Three thousand twenty‐two couples randomly selected were offered all those tests as a control group. Sanger sequencing, multiplex ligation‐dependent probe amplification and next‐generation sequencing were used for rare thalassemia. High‐risk couples were offered prenatal diagnosis at 10–13 weeks’ gestation based on informed consent. Results The carrier rates of α‐, β‐, and αβ‐thalassemia and δβ thalassemia/deletional HPFH were 7.7%, 3.02%, 0.5% and 0.059% respectively. Of them, 1.37% were identified as at‐risk couples and 345 couples terminated the pregnancy. No severe α‐ and β‐thalassemia births were observed. In the control group, two β‐ thalassemia carriers and one case with −α3.7/ααQS were misdiagnosed, but all at‐risk couples were found, and we could save 1,523,774 ¥ using our strategy. The cut‐off points of 73.46 fL and 23.25 pg would be useful to find −α+/αT thalassemia. Conclusion The intervention strategy was cost‐effective and offered reference in population thalassemia screening.


| INTRODUC TI ON
Hemoglobinopathies are the most common Mendelian disorders including abnormal hemoglobin variants and thalassemia in the world. 1 α thalassemia major leads to hydrops fetalis or perinatal death. β thalassemia major and some types of abnormal hemoglobin variants such as hemoglobin S, C compound with β thalassemia minor causes severe anemia. Carrier screening and high-risk couple subjected to prenatal diagnosis are the main content of the thalassemia control plan. 2,3 Population-screening programs for hemoglobinopathies have been performed in many countries in globe. 4 The main impact factors for successful population screening programmes included political support, strong publicity in public, mutation spectrum of thalassemia in the local population, and cost effectiveness. 5 Red blood cell indices including mean corpuscular volume (MCV) or mean corpuscular hemoglobin (MCH) and HbA2 quantification were often utilized to find carriers. 6 Cut-off values of hematologic parameters are used in determining whether molecular methods are needed, 7 including Gap-PCR and RDB for known breaking point or mutations and MLPA and next-generation sequencing for rare genotypes of thalassemia.
In China, many places including Guangdong, Guangxi, Hailan, Fujian, Taiwan and Hong Kong performed the carrier screening for thalassemia. [8][9][10][11][12][13] Except Hongkong and Taiwan, the objectives of carrier screening in other places were focused in the molecular spectrum of thalassaemia. 14 Our center established the hospitalbased program and proved to be highly effective in reducing severe thalassemia in pregnant populations, 15 but only few genotypes were included and the program was not detailed. As the centra of South China, a large number of migrants have settled in recent years and migrations may change the mutation spectrum. Pre Gestational Thalassemia Screening program has been carried out for 4 years. The objective of our study is to evaluate the current population screening strategy based on government support and multiple departments included, which can provide evidence for establishing the guideline of population thalassemia screening.

| Ethical study subjects and prenatal screening strategy
Pre Gestational Thalassemia Screening Program was performed through the three-level network of maternal and child health in Internal quality control and external quality assessment (EQC) were performed to ensure precision and consistency of results.
From the 37,501 couples, 74,102 subjects who had MCV < 82 fL or MCH < 27 pg were transported to our center, which belongs to the third-class maternity children hospital for further investigation. All of them were performed Hb analysis using capillary electrophoresis (CE; Serbia, Paris, France). Four common α-thalassemia deletions were also tested using gap-PCR in those samples, including --SEA ,α 3.7 ,α 4.2 and --Thailand . Molecular analysis for common nonrecreational α-thalassemia was performed according to the following conditions (1) the samples without four common deletional α thalassemia but positive for thalassemia screening (MCV < 82 fL, MCH < 27 pg or Hb A2 < 2.5%); (2) the partner with --SEA /αα, --Thailand /αα or Hb H disease; (3) the cases with CS peak observed on electrophoresis; (4) the samples with Hb H or Hb Bart's but negative for common deletional α thalassemia detection; and (5) β-thalassemia carriers. Samples with HbA2 > 3.5% or HbF > 5% were detected by RDB to eliminate seventeen common mutations in the HBB gene.
For cases with HbF levels ≥5%, we used Gap-PCR to screen three types of δβ-thalassemia/deletional HPFH reported in some studies in China, including Chinese G γ( A γδβ) 0 -thalassemia, SEA-HPFH and Taiwanese deletion. Two thalassemia gene detection kits were used to perform these assays (Shenzhen Yishengtang Biological Products Co., Ltd.). Sanger sequencing was carried out for samples with abnormal variants or negative RDB results for common mutations of β-globin gene. Gap-PCR was used for cases with HbA2 levels ≥3.5% but negative for sanger sequencing or Hb F levels ≥5% to screen three common δβ-thal/deletional HPFH. MLPA was used to investigate whether uncommon delusional α or β thalassemia existed.
Next-generation sequencing was used to find deletional point or the subjects with suspected family history of thalassemias. The strategy was presented in Figure 1.

| Molecular analysis for the control group
About 7044 samples from 3522 couples were randomly chosen as the control group. All samples received Hb analysis and common molecular analysis including four common deletional α-thalassemia, three common nondeletional α-thalassemia and seventeen common mutation β-thl.

| Statistical analysis
Nonparametric Mann-Whitney U test was conducted by analyzing the hematological parameters differences between groups. The difference of carrier rate of hemoglobinopathies in four types of districts was conducted by Pearson Chi-square analysis. A p-value of less than 0.05 was regarded as a significant difference. ROC (the receiver operator characteristic) was performed to determine the cut-off value of Hb E, MCH and MCV. All those data were analyzed using SPSS V.19.0 software.

| Screening status in Guangzhou
The overall carrier rate of thalassemia was 11.34%, including 7.76% for α-thalassemia, 3.02% for β-thalassemia and 0.49% for combined α-/β-thalassemia. The carrier rate of δβ/delusional thalassemia was about 0.059%. 0.69% of couples had high risk of delivering a baby with thalassemia major and received genetic counselling. Three hundred and forty-five couples chose to terminate the pregnancy due to affected fetuses. No infants were born with thalassemia major in this screening program. According to the distance from the city center and economic development level, eleven districts in Guangzhou area can be divided into four types: the outer suburb areas, the inner suburban district, the central city district and the economic development zone. The carrier rates of α-thalassemia, β-thalassemia and combined α-/β-thalassemia were highest in the outer suburb areas while it was lowest in the economic development zone ( Figure 2).

| Gene mutation spectrum and hemorheology characteristics of thalassemia
Molecular spectrum of persons with α, β and αβ thalassemia were shown separately as a top-Fifteen list in Table 1. Hematology parameters of them were shown in Table 2. Thirty-two genotypes of α thalassemia, thirty-five genotypes of β thalassemia and seventy-two genotypes of αβ thalassemia were identified in our study. Specifically, the most frequent mutation of β thalassemia −28(A>G) in Nansha District belonging to the economic development zone which was very distinctive from other districts. Beyond that, Ninety-five carriers with Chinese F I G U R E 1 The intervention strategy of thalassemia for couples of childbearing age in Guangzhou G γ( A γδβ) 0 -thalassemia, 53 cases with SEA-HPFH, seven cases with Taiwanese deletion have been diagnosed by gap-PCR. We also identified two cases of δβ thalassemia. One of them was initially reported by our team before while the δβ thalassemia was never described ( Figure S1). α WS α/αα rather than α QS α/αα coexisted with β CD 41-42(-C T T T ) /β N was the most common genotype in carriers with concurrent nontraditional α-and β-thalassemia.
Considering the correlation between parameters and genotypes, we found one case with rare α thalassemia which was described in Figure S2. Twenty-five different types of hemoglobinopathies were found in our study (Table S1).

| Red blood cell indices and hemoglobin analysis using in this strategy
Among the 32985 samples with MCV values >82 fL and MCH values >27 pg in our study, α + thalassemia carriers were common, includingα 3.7 /αα (2.05%) andα 4.2 /αα carriers (0.66%). 8 Figure S3). Hb E level of Hb E/β + thalassemia carriers was higher than 40% while Hb level was more than 90 g/L.

| The result of molecular analysis of the control group
All those samples were detected common mutations in the HBB gene and common nondeletional/deletional α thalassemia. We compared the results and cost using our screening strategy. Using were not detected because their Hb A2 levels were less than 3.5% and Hb F were less than 5%. One case was identified to have a δ sults showed that no severe α-and β-thalassemia births were observed in participants.
According to references and our economic situation, we chose cut-off of 82 fL for MCV and 27 pg for MCH as the primary screening index to identify couples at risk. 16 Although α + -thalassemia carriers would be misdiagnosed using the cut-off values, the couples at risk of having a baby with deletional HbH could be detected because all of couples one of which has the lower MCV or MCH F I G U R E 3 (a) ROC curve analysis for MCV, MCH and HbA2 in distinguishing α +thalassemia carriers and α0-thalassemia carriers got genetic testing for four common deletional α-thalassemias.

Most of the couples at risk of having a baby with nondeletional
HbH could be identified using our strategy. or β 0 /β CAP40-43 (-A A AC) presented mild anemia and one case with β CAP40-43 (-A A AC) /β N presented normal in our study. We inferred that this mutation may not cause β-thalassemia. It needs us do more research. Our study showed that 2.3% of the high-risk fetus for betathalassemia major also had high risk for hydrops fetalis at the same time. It is very essential to screen α-thalassemia in β-thalassemia carriers when the spouse was α-thalassemia carrier. The Hb E level of less than 19.4% could be as the best cut-off value for discriminating Hb E heterozygotes and Hb E.
Hb F > 5% would be useful for detecting δβ-thalassemia and HPFH, which could also cause beta thalassemia intermedia compounded with β thalassemia. Two carriers with rare β-globin gene cluster deletion were also identified in those people. One person with rare ( A γδβ) 0 thalassemia which was first reported by our laboratory in 2016. δβ thalassemia was common in cases with high Hb F and low HbA2. MLPA was so beneficial for us to identify δβ thalassemia carriers and NGS could provide the correct basis to breakpoint which was helpful for prenatal diagnosis.
Although the carriers with common Hb variants usually have normal MCV and MCH level except for Hb E and Hb Q-Thailand carriers, Hb variants compounded with thalassemia will not cause severe anaemia. In our study, all those cases with Hb variants were detected common genotypes of thalassemia. The Hb A2 level was more than 3.5%, and MCV and MCH levels were lower than cut-off value in persons with Hb variants compounded with β thalassemia.
It also identified that the screening values of MCV and MCH in population screening.
Using the strategy, we also described the carrier rates of αthalassemia, β-thalassemia and combined α-/β-thalassemia in Guanghzou. The carrier rates were all lower than those identified in Guangdong province by Aihua Yin 21  and Aihua Yin. 22 The slight difference between them including the constituent ratio for nondeletional α-thalassemia and β-thalassemia.
The detection rate of α QS α/αα was highest while that of α WS α/αα was highest in Guangdong province. Interestingly, we found that α WS compound with CD41-42(-CTTT) was the most frequent genotype of αβ-thalassemia. All αα QS /αα carriers had MCV < 82 fL and MCH < 27pg while most α WS /αα carriers had normal MCV and MCH which may cause the latter to be undiagnosed. Based on those data, we speculated that α WS α/αα was also the most common nondeletional α-thalassemia. The proportion of migrant population is highest ing. The biggest data in our study could provide laboratory support to genetic counselling and intervention strategy of thalassemia.

ACK N OWLED G M ENTS
The authors would like to acknowledge all patients for their cooperation with our study.

CO N FLI C T O F I NTE R E S T S
The authors in our study do not have any conflict of interest, financial or otherwise.

AUTH O R CO NTR I B UTI O N S
Can Liao and Dongzhi Li contributed the central idea, Fan Jiang analyzed most of the data and wrote the initial draft of the paper, Duolian Zuo and Jian Li were responsible for project management,

Molecular experiments were performed by Xuewei Tang and Guilan
Chen. Jianying Zhou and Yanxia Qu were responsible for CE and data analysis. All authors reviewed the results and revised the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data generated and/or analyzed in this work are available from the corresponding author according to request (canliao7981@21cn. com).