Molecular Characterization of α- and ß-Thalassemia Among Children Less Than 18 Years Old in Guizhou, China.

BACKGROUND: Thalassemia is an inherited hemolytic disease, the complications and sequelae of which have posed a huge impact on both patients and society. But limited studies have investigated the molecular characterization of α- and ß-thalassemia in children from Guizhou, China. METHODS: Between January 2019 and December 2022, a total of 3301 children, aged 6 months to 18 years, suspected of having thalassemia underwent molecular analysis. RESULTS: Out of the total sample, 824 (25%) children were found to carry thalassemia mutations. The carrier rates of α-thalassemia, ß-thalassemia, and α + ß-thalassemia were determined as 8.1%, 15.6%, and 1.3%, respectively. Approximately 96.5% of the α-thalassemia gene mutations were --SEA (51%), ααCS (20.9%), -α3.7 (19.6%), and -α4.2 (5.0%). The most prevalent mutations of ß-thalassemia were ßCD17(A>T) (41.5%), ßCD41-42(-TTCT) (37.7%), and ßIVS-II-654(C>T) (11.3%). Additionally, we identified rare cases, including one case with ααHb Nunobiki/αα, two cases with triplicated α-thalassemia (one case with ααα/ααα and ßCD41-42/ßN and the other with ααα-3.7/αα and ßE CD26/ßN), and also one case with α Q-Thailandα/-α4.2 and ßCD41-42/ßN. CONCLUSIONS: Our study findings provide important insights into the heterogeneity of thalassemia carrier rates and molecular profiles among children in the Guizhou region. The findings support the development of prevention strategies to reduce the incidence of severe thalassemia in the future.

Prevalence of thalassemia-carrier couples and fertility risk assessment.

Thalassemia is a highly prevalent hematologic disease in Guizhou, China. This study aimed to determine the epidemiological characteristics of thalassemia in couples at childbearing age and assess the neonatal risk of thalassemia in this subpopulation. A cohort of 4481 couples at childbearing age were recruited for thalassemia carrier screening by both traditional hematological tests and next-generation sequencing. Of them, 1314 (14.66%) thalassemia carriers were identified, including 857 (9.76%) α-thalassemia, 391 (4.36%) ß-thalassemia, and 48 (0.54%) composite α and ß-thalassemia. A total of 12 α-globin gene alterations and 16 ß-globin mutations were detected, including four novel thalassemia mutations. SEA was the most common α-thalassemia genotype (26.86%), CD41-42 the most common ß-thalassemia genotype (36.57%), and αα/- α3.7 + CD41-42 the most common composite α- and ß-thalassemia genotype (18.75%). Ethnically, the Zhuang had the highest rate of thalassemia gene carriers among the ethnic groups. Geographically, Qiannan had the highest rate of thalassemia gene carriers. In addition, 38 of the 48 couples with composite α- and ß-thalassemia were high-risk thalassemia carriers, and 4 carrying the -SEA/αα gene needed fertility guidance.

Genetic epidemiology of thalassemia in couples of childbearing age: over 6 years of a thalassemia intervention project.

BACKGROUND: Shenzhen is one of the most populated metropolises in southern China where thalassemia is highly prevalent. The prevention of thalassemia inheritance is an ambition of child-bearing couples. METHODS AND RESULTS: A total of 22,098 peripheral blood samples were collected from 11,049 potentially at-risk couples of childbearing age from Shenzhen. Thalassemia mutations were determined by PCR-based flow-through hybridization. The results identified 45.02% of the participants (9948 out of 22,098) as harboring globin gene mutations, distributed into 18 α-thalassemia alleles detected in 71.48% (7111 out of 9948) and 15 ß-thalassemia alleles detected in 32.68% (3252 out of 9948) of all mutant individuals, among which 415 individuals carried both α- and ß-thalassemia alleles. The most frequent phenotypes for α-globin variations were --SEA/αα (63.37%), -α3.7/αα (18.66%), and -α4.2/αα (7.31%), and those for ß-globin variations were ß41-42/ßN (34.96%), ß654/ßN (28.11%), and ß17/ßN (13.84%). A total of 970 high-risk couples who could possibly give birth to offspring with thalassemia intermedia or major were identified. In addition, the hematological indices were compared among thalassemia genotypes. Significant differences in MCH, MCV, Hb A, and Hb A2 levels among α-thalassemia minor (α+), trait (α0), and intermediate phenotypes (P < 0.05) and between ßE/ßN and the other ß-thalassemia phenotypes (P < 0.05) were found. Moreover, GAP-PCR and next-generation sequencing further identified 42 rare mutations, 13 of which were first reported in the Chinese population. A novel mutation in the ß-globin gene (HBB: c.246 C > A (rs145669504)) was also discovered. CONCLUSIONS: This study presented a comprehensive analysis of thalassemia variations in a population from Shenzhen and may offer valuable insights for thalassemia control and intervention strategies in this area.

Carrier rate of thalassemia among 25,910 high school students in Shaoguan area, China.

OBJECTIVES: As one of the most common hereditary diseases, thalassemia affects a large number of people in China. The aim of this study was to investigate the feasibility of a method based on next-generation sequencing (NGS) for screening of thalassemia carriers among high school students in the Shaoguan area. MATERIALS AND METHODS: The NGS-based method was performed using 25,910 high school students recruited from 38 schools. The screening yield was systematically analyzed. Before screening, a lecture on how the disease is inherited, the symptoms of thalassemia, and how to prevent it was given to 28,780 students. RESULTS: Implying successful delivery of information on the disease, 90.03% (25,910 of 28,780) of the students agreed to join this program for thalassemia screening. A thalassemia carrier rate of 15.99% (4144 of 25,910) was found. Also, 69 rare genotypes (28 of α-thalassemia and 41 of ß-thalassemia) and 9 novel variants were identified. CONCLUSIONS: This NGS-based method provided a feasible platform for high school population thalassemia screening. Combined with a clinical follow-up strategy, it could help eventually to prevent the births of affected children.

[Gene Mutation Types of Thalassemia in Chongzuo Childbearing-age Population of Guangxi Zhuang Autonomous Region of China].

OBJECTIVE: To investigate the gene mutation and genotype distribution of thalassemia in the population of childbearing age in Chongzuo area of Guangxi. METHODS: Six α-thalassemia and 17 ß-thalassemia gene mutations common in Chinese were detected by gap-polymerase chain reaction (gap-PCR) combined with agarose gel eletrophoresis and reserve dot bolt hybridization in 29 266 cases of child-bearing age suspected of thalassemia. RESULTS: A total of 19 128 (65.36%) cases were identified with thalassemia. The detection rate of α-thalassemia, ß-thalassemia and α-combining ß-thalassemia was 45.25% (13 242/29 266), 15.47% (4 526/29 266) and 4.65% (1 360/29 266), respectively. A total carrying rate of 8 kinds of α-thalassemia gene mutations was 26.74% (15 649/58 532), including 12.51% for --SEA, followed by 5.70% for -α3.7, and 0.24% for --Thai. Among 32 α-thalassemia genotypes, the most common five were --SEA/αα, -α3.7/αα, αCSα/αα, -α4.2/αα and αWSα/αα, accounting for 47.27%, 18.31%, 8.56%, 8.52% and 7.91%, respectively, as well as 0.97% for --Thai/αα. A total carrying rate of 13 kinds of ß-thalassemia gene mutations was 10.07% (5 897/58 532), including 3.63% for CD41-42, followed by 2.55% for CD17, and 0.003% for -50 (G>A). Among 17 ß-thalassemia genotypes, the most common six were CD41-42/N, CD17/N, CD71-72/N, CD26/N, 28/N and IVSI-1/N, accounting for 36.15%, 25.81%, 9.43%, 8.18%, 8.09% and 7.75%. The homozygous genotype CD26/CD26 [hemoglobin (Hb): 121 g/L] and -28/-28 (Hb: 56 g/L) were respectively detected in one case, and double heterozygous genotype were detected in 5 cases, including 3 cases of CD41-42/CD26 (Hb: 41 g/L, 51 g/L, 63 g/L, respectively), 1 case of -28/IVSI-1 (Hb: 53 g/L), and 1 case of CD71-72/CD26 (Hb: 89 g/L), in which patients with moderate or severe anemia had a history of blood transfusion. Among 104 α-combining ß-thalassemia genotypes, the most common were --SEA/αα, -α3.7/αα combining CD41-42/N and --SEA/αα combining CD17/N, accounting for 12.13%, 9.63% and 9.26%, respectively. In addition, 1 case of --SEA/-α3.7 combining -28/IVSI-1 (Hb: 83 g/L) and 1 case of -α3.7/αα combining CD41-42/ CD41-42 (Hb: 110 g/L) were detected without history of blood transfusion, while 1 case of αWSα/αα combining CD41-42/CD17 (Hb: 79 g/L) and 1 case of --SEA/αα combining CD17/-28 (Hb: 46 g/L) were detected with history. CONCLUSIONS: The detection rate of thalassemia genes is high and the mutations are diverse in the population of childbearing age in Chongzuo area of Guangxi. The common deletion genotype is --SEA/αα in α-thalassemia and CD41-42/N in ß-thalassemia, and deletion genotype --Thai is not rare. There is a certain incidence of intermediate and severe ß-thalassemia, and most patients require transfusion therapy. The results are beneficial for genetic consultation and intervention of thalassemia.

Molecular spectrum and prevalence of thalassemia investigated by third-generation sequencing in the Dongguan region of Guangdong Province, Southern China.

BACKGROUND: PCR, Sanger sequencing and NGS are often employed for carrier screening of thalassemia but all of these methods have limitations. In this study, we evaluated a new third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) to explore the prevalence of thalassemia in the Dongguan region of southern China. METHODS: 19,932 subjects were recruited for thalassemia screening and hemoglobin testing was performed for each of them. Routine PCR was performed for all the hemoglobin testing-positive subjects and CATSA was conducted for randomly selected subjects from hemoglobin testing-positive and negative subjects. RESULTS: In the 2716 subjects tested both by PCR and CATSA, 2569 had the same results and 147 had discordant results between the two methods. Sanger sequencing, specially designed PCR and MLPA confirmed the results of CATSA were all correct. In total, CATSA correctly detected 787 subjects with variants while routine PCR correctly detected 640 subjects with variants. CATSA yielded a 5.42% (147 of 2716) increment compared with routine PCR. In the 447 hemoglobin testing-negative subjects, CATSA identified pathogenic variants in 12 subjects. Moreover, CATSA identified a novel deletion (chr16:171262-202032) in the α-globin gene cluster. As a result, the deduced carrier frequency of α-thalassemia,ß-thalassemia and α-/ß-thalassemia was 5.62%, 3.85% and 0.93%, respectively. CONCLUSIONS: Our study demonstrated CATSA was a more comprehensive and precise approach than the routine PCR in a large scale of samples, which is highly beneficial for carrier screening of thalassemia. It provided a broader molecular spectrum of hemoglobinopathies and a better basis for a control program in Dongguan region.

Next-generation sequencing analysis of the molecular spectrum of thalassemia in Southern Jiangxi, China.

BACKGROUND: Thalassemia is an extremely prevalent monogenic inherited blood disorder in southern China. It is important to comprehensively understand the molecular spectrum of thalassemia in an area with such a high prevalence of thalassemia before taking appropriate actions for the prevention and treatment of this disorder. Herein, we explored the clinical feasibility of using next-generation sequencing (NGS) for large-scale population screening to illustrate the prevalence and spectrum of thalassemia in Southern Jiangxi. METHODS: Blood samples collected from 136,312 residents of reproductive age in Southern Jiangxi were characterized for thalassemia by NGS. A retrospective analysis was then conducted on blood samples determined to be positive for thalassemia. RESULTS: In total, 19,827 (14.545%) subjects were diagnosed as thalassemia carriers, and the thalassemia prevalence rate significantly varied by geographical region (p < 0.001). A total of 40 α-thalassemia genotypes including 21 rare genotypes were identified, with -@-SEA/αα being the most prevalent genotype. 42 ß-thalassemia genotypes including 27 rare genotypes were identified, with the most common mutation IVS II-654 C > T accounting for 35.257% of these ß-thalassemia genotypes. Furthermore, 74 genotypes were identified among 608 individuals with combined α- and ß-thalassemia. Notably, most individuals with rare thalassemia mutations had mildly abnormal hematologic parameters including microcytic hypochromia. CONCLUSIONS: Our findings demonstrate the great heterogeneity and diverse spectrum of thalassemia in Southern Jiangxi, emphasizing the importance and necessity of persistent prevention and control of thalassemia in this region. Additionally, our findings further suggest that NGS can effectively identify rare mutations and reduce the misdiagnosis rate of thalassemia.

[Association Analysis between Genotype and Phenotype of α,ß-Thalassaemia Carriers in Huizhou Area of Guangdong Province].

OBJECTIVE: To analyze the prevalence, genotype distribution and hematological characteristics of α,ß-thalassaemia carriers in Huizhou area of Guangdong Province. METHODS: 10 809 carriers of simple ß-thalassaemia and 1 757 carriers of α,ß-thalassaemia were enrolled as our study cohort. The hematological parameters were detected by automated blood cell counters and automatic capillary electrophoresis. Suspension array technology, gap-polymerase chain reaction (gap-PCR) and PCR-reverse dot blot were used for the genotyping of thalassaemia carriers. RESULTS: The prevalence of α,ß-thalassaemia in Huizhou area of Guangdong Province was 1.99%. A total of 62 genotypes were detected, and the most prevalent genotype was --SEA/ αα, ßCD41-42/ ßN (19.29%), the next was --SEA/ αα, ßIVS-II-654/ ßN (16.73%). Significant differences in mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were found between different genotype groups for simple ß-thalassaemia and α,ß-thalassaemia. Violin plots showed that carriers with co-inheritance of ß-thalassaemia and mild α-thalassaemia expressed the lightest anemia, and carriers with co-inheritance of ß-thalassaemia and hemoglobin H (Hb H) disease expressed the most severe anemia. CONCLUSION: There is a high prevalence of α,ß-thalassaemia in Huizhou area of Guangdong Province. Because of the lack of specific hematological makers for diagnosis of α,ß-thalassaemia, it is necessary to distinguish it from simple ß-thalassaemia by genotyping of α- and ß-thalassaemia in order to correctly guide genetic counseling and prenatal disgnosis.

Clinical significance of mutational variants in beta and alpha genes in patients with hemoglobinopathies from two large Greek centers: a complex interplay between genotype and phenotype.

Hemoglobinopathies affect patients in the wider Mediterranean area consisting of 4 distinct subgroups: beta thalassemia major (TM), beta thalassemia intermedia (TI), sickle cell disease (SCD) and hemoglobin H disease (alpha thalassemia). The clinical spectrum varies from mild to severe. Complex interactions between genes and environmental factors form the clinical manifestations. There is an unmet need to clarify these multifactorial mechanisms. This is the first Greek study describing mutational alleles (HBB and HBA1/HBA2 gene variants) in 217 patients with hemoglobinopathies of two large centers in Greece (Larissa and Athens) and associating particular genotypes or gene variants with clinical manifestations (transfusion frequency, complications). Thus, the complex interplay between corresponding genotypes and phenotypes was investigated. Our results are in accordance with previous national studies with limited variations, due to regional prevalence of specific gene variants, as expected. It is also a description of the prevalence of hemoglobinopathies in the Greek population. The type and prevalence of beta and alpha globin gene variants differ significantly among countries. We also confirm the well-known observation of many studies that in our beta thalassemic or SCD patients, co-inheritance of variants in the alpha globin genes, leading to absence or reduction of alpha globin synthesis were associated with milder clinical course, whereas the inheritance of additional alpha genes (triplication) led to a more severe clinical phenotype. In cases in whom the genotype and phenotype did not correlate, factors like the function or modification of possible regulatory genes or additional nutritional-environmental effects should be investigated. KEY MESSAGES: • This is the first Greek study, fully molecularly defining the beta and alpha mutational alleles in 217 patients with hemoglobinopathies of two large centers in Greece and correlating particular genotypes or gene variants with clinical manifestations (transfusion frequency, complications). • In the beta thalassemic or SCD patients of our cohort, co-inheritance of variants in the alpha globin genes, leading to absence or reduction of alpha globin synthesis were associated with milder clinical course (confirmation of a well-known previous observation). • The inheritance of additional alpha genes (triplication) led to a more severe clinical phenotype (confirmation of a well known previous observation). • The function or modification of possible regulatory genes should be investigated in cases in whom the genotype and phenotype did not correlate.

Thalassemia and hemoglobinopathy prevalence in a community-based sample in Sylhet, Bangladesh.

BACKGROUND: Inherited blood disorders affect 7% of the population worldwide, with higher prevalences in countries in the "thalassemia belt," which includes Bangladesh. Clinical management options for severely affected individuals are expensive; thus, targeted government policies are needed to support prevention and treatment programs. In Bangladesh, there is a lack of data, in particular community-based estimates, to determine population prevalence. This study aims to estimate the prevalence of a wide range of hemoglobinopathies and their associations with anemia in a community-based sample of women and young children in rural Sylhet, Bangladesh. METHODS: Capillary blood samples from 900 reproductive-aged women and 395 children (aged 6-37 months) participating in the Food and Agricultural Approaches to Reducing Malnutrition (FAARM) trial in two sub-districts of Habiganj, Sylhet Division, Bangladesh were analyzed for alpha thalassemia, beta thalassemia, and other hemoglobinopathies. We examined the association of each inherited blood disorder with hemoglobin concentration and anemia using linear and logistic regression. RESULTS: We identified at least one inherited blood disorder in 11% of women and 10% of children. Alpha thalassemia was most prevalent, identified in 7% of women and 5% of children, followed by beta thalassemia and hemoglobin E in 2-3%. We also identified cases of hemoglobin S and hemoglobin D in this population. Having any of the identified inherited blood disorders was associated with lower hemoglobin values among non-pregnant women, largely driven by alpha and beta thalassemia. Pregnant women with beta thalassemia were also more likely to have lower hemoglobin concentrations. Among children, we found weak evidence for a relationship between hemoglobinopathy and lower hemoglobin concentrations. CONCLUSIONS: We found a high prevalence of alpha thalassemia among both women and children in rural Sylhet, Bangladesh-higher than all other identified hemoglobinopathies combined. Community-based estimates of alpha thalassemia prevalence in Bangladesh are scarce, yet our findings suggest that alpha thalassemia may comprise the majority of inherited blood disorders in some regions of the country. We recommend that future research on inherited blood disorders in Bangladesh include estimates of alpha thalassemia in their reporting for public health awareness and to facilitate couples  counseling.

Revisiting and updating molecular epidemiology of α-thalassemia mutations in Thailand using MLPA and new multiplex gap-PCR for nine α-thalassemia deletion.

α-thalassemia is an inherited blood disorder that is most frequently found in Southeast Asian populations. In Thailand, molecular characterization can diagnose most patients with α-thalassemia; however, several atypical patients are also observed in routine analyses. Here, we characterized α-thalassemia mutations among 137 Hemoglobin H (Hb H) disease patients and three fetuses of Hb Bart's hydrops, a fatal clinical phenotype of α-thalassemia. Specifically, we performed multiplex ligation-dependent probe amplification (MLPA) followed by direct DNA sequencing. We noticed common genotypes in 129 patients and eight patients had rare Hb H disease caused by compound heterozygous α0-thalassemia (--CR or --SA deletion) with α+-thalassemia (-α3.7/-α4.2/αConstant Springα). Furthermore, two affected fetuses had the --SA/--SEA and one had the --CR/--SEA genotypes. Next, we developed and validated a new multiplex gap-PCR and applied this method to 844 subjects with microcytic red blood cells (RBCs) from various parts of Thailand. The frequency of heterozygous α0-thalassemia was dominated by --SEA 363/844 (43%), followed by --THAI 3/844 (0.4%), --SA 2/844 (0.2%), and --CR 2/844 (0.2%) mutations. These findings suggest that aforementioned four mutations should be routinely applied to increase the effectiveness of diagnosis and genetic counseling in this region.

Epidemiology of clinically significant forms of alpha- and beta-thalassemia: A global map of evidence and gaps.

This systematic literature review assessed the global prevalence and birth prevalence of clinically significant forms of alpha- and beta-thalassemia. Embase, MEDLINE, and the Cochrane Library were searched for observational studies published January 1, 2000, to September 21, 2021. Of 2093 unique records identified, 69 studies reported across 70 publications met eligibility criteria, including 6 records identified from bibliography searches. Thalassemia prevalence estimates varied across countries and even within countries. Across 23 population-based studies reporting clinically significant alpha-thalassemia (e.g., hemoglobin H disease and hemoglobin Bart's hydrops fetalis) and/or beta-thalassemia (beta-thalassemia intermedia, major, and/or hemoglobin E/beta-thalassemia), prevalence estimates per 100 000 people ranged from 0.2 in Spain (over 2014-2017) to 27.2 in Greece (2010-2015) for combined beta- plus alpha-thalassemia; from 0.03 in Spain (2014-2017) to 4.5 in Malaysia (2007-2018) for alpha-thalassemia; and from 0.2 in Spain (2014-2017) to 35.7 to 49.6 in Iraq (2003-2018) for beta-thalassemia. Overall, the estimated prevalence of thalassemia followed the predicted pattern of being higher in the Middle East, Asia, and Mediterranean than in Europe or North America. However, population-based prevalence estimates were not found for many countries, and there was heterogeneity in case definitions, diagnostic methodology, type of thalassemia reported, and details on transfusion requirements. Limited population-based birth prevalence data were found. Twenty-seven studies reported thalassemia prevalence from non-population-based samples. Results from such studies likely do not have countrywide generalizability as they tended to be from highly specific groups. To fully understand the global prevalence of thalassemia, up-to-date, population-based epidemiological data are needed for many countries.

Essential genetic modifiers and their measurable impact in a community-recruited population analysis for non-severe hemoglobin E/ß-thalassemia prenatal genetic counseling.

The study aimed to identify essential phenotype-modulating factors among the pre-existence of several important ones and clarify their measurable impact on the clinical severity of hemoglobin (Hb) E/ß-thalassemia in a community-recruited population analysis. This prospective study was designed to compare modifiers between community- (less or no symptoms) and hospital-recruited individuals with Hb E/ß-thalassemia. The formerly included couples previously assessed for prenatal thalassemia at-risk status at 42 community and 7 referral hospitals in Thailand through on-site investigations between June 2020 and December 2021. The control included Hb E/ß-thalassemia patients undergoing transfusions. The Mahidol score classified disease severity. Beta-globin, α0-thalassemia (-SEA, -THAI), α+-thalassemia (-α3.7, -α4.2), Hb Constant Spring (αCS) alleles, rs766432 in BCL11A, rs9399137 in HBS1L-MYB, and rs7482144-XmnI were evaluated. Modifiers were compared between 102 community- and 104 hospital-recruited cases. Alleles of ß+, -SEA, -α3.7, αCS, and a minor allele of rs9399137 were prevalent in the community and mild severity groups (p < 0.05). Multiple linear regression analysis associated modulating alleles with -4.299 (-SEA), -3.654 (ß+), -3.065 (rs9399137, C/C), -2.888 (αCS), -2.623 (-α3.7), -2.361 (rs7482144, A/A), -1.258 (rs9399137, C/T), and -1.174 (rs7482144, A/G) severity score reductions (p < 0.05). Certain modifiers must be considered in routine prenatal genetic counseling for Hb E/ß-thalassemia.

Effect of α+ Thalassemia on the Severity of Plasmodium falciparum Malaria in Different Sickle Cell Genotypes in Indian Adults: A Hospital-Based Study.

There is a paucity of literature on the association of α+-thalassemia, sickle-cell hemoglobin disorders, and malaria in India. This study aimed to understand the effect of α+-thalassemia on the severity of Plasmodium falciparum malaria in adults with respect to sickle-cell genotypes. The study subjects were categorized into 'severe-malaria' and 'uncomplicated-malaria' and age-gender matched 'control' groups. Sickle-cell and α+-thalassemia were investigated in all the recruited subjects. The effect of α+-thalassemia on the severity of malaria was analyzed in HbAA and sickle-cell genotypes (HbAS and HbSS) separately. The prevalence of α+-thalassemia in various groups ranged from 41.5% to 81.8%. The prevalence of α+-thalassemia was lower (OR = 1.64; p = 0.0013) in severe malaria (41.5%) as compared to healthy controls (53.8%) with HbAA genotype. In contrast, in HbAS genotype, the prevalence of α+-thalassemia was higher (OR = 4.11; p = 0.0002) in severe malaria (81.8%) compared to controls (52.2%). In severe malaria with HbAA genotype, there was a significantly higher hemoglobin level and low MCV and MCH level in patients with α+-thalassemia compared to the normal α-globin genotype. Further, the incidence of cerebral malaria, hepatopathy, and mortality was lower in patients (HbAA) with α+-thalassemia as compared to normal α-globin genotype (HbAA). In severe malaria with either HbAS or HbSS genotype, only a few parameters showed statistical differences with respect to α+-thalassemia. Low prevalence of α+-thalassemia in severe malaria with HbAA genotype compared to healthy controls with HbAA genotype indicates the protective effect of α+-thalassemia against severe malaria. However, the high prevalence of α+-thalassemia in patients with HbAS genotype depicts its interference in the protective effect of sickle-cell against severe malaria.

A novel α Globin Gene Cluster Duplication, αααα380 Heterozygous ß0-Thal Variant, Leading to a Blood Transfusion-Dependent Phenotype.

To assess the effectiveness of three-level prevention and control of thalassemia, we routinely collect samples from transfusion-dependent individuals and perform genetic analysis. Here, we report on a 10-year-old boy requiring blood transfusions with routine thalassemia gene test results of αα/αα, and ßCD41/42/ßN, but he had thalassemia-like changes in his appearance and a high need for frequent blood transfusions, suggesting a case of thalassemia major in childhood. Given these equivocal results, samples from the family members were collected for further analysis. A multiplex ligation-dependent probe amplification assay was used to detect a multicopy number variant of the α globin gene cluster in the proband. The variant was detected as a long fragment repeat of 380 Kb using CNV assay technique, which contains the entire α globin gene cluster, describing it as αααα380/αα. Analysis of family members suggested that both the brother and mother of the proband carried the variant, and both MCV and MCH values were reduced in carriers. Individuals carrying multiple copy number variants of the α globin gene cluster exist in the population. Individuals carrying such variants who are also heterozygous for the ß0 thalassemia variant result in an imbalance in the α/ß chain ratio, potentially leading to the creation of individuals with a severe anemia genotype. Most secondary prevention and control laboratories currently do not include variants with increased α gene copy number in their testing, which is one of the blind spots of prevention and control efforts. In order to provide more accurate genetic counseling to test subjects, especially in regions with high rates of thalassemia carriage, testing laboratories should pay attention to individual genotype-phenotype matches to avoid the under-detection of such variants.

Epidemiological Survey of Hemoglobinopathies Based on Next-Generation Sequencing Platform in Hunan Province, China.

Objective: This study was aimed at investigating the carrier rate of, and molecular variation in, α- and ß-globin gene mutations in Hunan Province. Methods: We recruited 25,946 individuals attending premarital screening from 42 districts and counties in all 14 cities of Hunan Province. Hematological screening was performed, and molecular parameters were assessed. Results: The overall carrier rate of thalassemia was 7.1%, including 4.83% for α-thalassemia, 2.15% for ß-thalassemia, and 0.12% for both α- and ß-thalassemia. The highest carrier rate of thalassemia was in Yongzhou (14.57%). The most abundant genotype of α-thalassemia and ß-thalassemia was -α 3.7/αα (50.23%) and ß IVS-II-654/ß N (28.23%), respectively. Four α-globin mutations [CD108 (ACC>AAC), CAP +29 (G>C), Hb Agrinio and Hb Cervantes] and six ß-globin mutations [CAP +8 (C>T), IVS-II-848 (C>T), -56 (G>C), beta nt-77 (G>C), codon 20/21 (-TGGA) and Hb Knossos] had not previously been identified in China. Furthermore, this study provides the first report of the carrier rates of abnormal hemoglobin variants and α-globin triplication in Hunan Province, which were 0.49% and 1.99%, respectively. Conclusion: Our study demonstrates the high complexity and diversity of thalassemia gene mutations in the Hunan population. The results should facilitate genetic counselling and the prevention of severe thalassemia in this region.

Prevalence Rate of Thalassemia Carriers among Individuals with Microcytosis or Hypochromia in Portugal.

INTRODUCTION: Microcytosis and hypochromia result from deficient hemoglobin synthesis in red blood cells and are easily detected in a complete blood count test. These conditions are mainly due to iron nutritional deficiency, but may also result from some genetic diseases, such as thalassemia. The aim of this study was to determine the contribution of ß- and α-thalassemia to these abnormal hematological phenotypes in a representative sample of adult individuals living in Portugal who participated in the first Portuguese National Health Examination Survey (INSEF). MATERIAL AND METHODS: Among the 4808 INSEF participants, 204 had microcytosis, hypochromia or both. The corresponding 204 DNAs were screened for changes in the ß-globin gene by next-generation sequencing and Sanger sequencing. In addition, α-thalassemia deletions within the α-globin cluster were investigated by Gap-PCR and multiplex ligation-dependent probe amplification. RESULTS: In this selected subgroup of INSEF participants, 54 had α-thalassemia (26%), predominantly caused by the -α3.7kb deletion, and 22 were ß-thalassemia carriers (11%) mainly due to point mutations in the ß-globin gene previously known in Portugal. CONCLUSION: Thalassemia trait is a frequent cause of microcytosis or hypochromia in Portugal since this genetic condition was found in 37% of the investigated cases.

A Particular Focus on the Prevalence of α- and ß-Thalassemia in Western Sicilian Population from Trapani Province in the COVID-19 Era.

Thalassemia is a Mendelian inherited blood disease caused by α- and ß-globin gene mutations, known as one of the major health problems of Mediterranean populations. Here, we examined the distribution of α- and ß-globin gene defects in the Trapani province population. A total of 2,401 individuals from Trapani province were enrolled from January 2007 to December 2021, and routine methodologies were used for detecting the α- and ß-globin genic variants. Appropriate analysis was also performed. Eight mutations in the α globin gene showed the highest frequency in the sample studied; three of these genetic variants represented the 94% of the total α-thalassemia mutations observed, including the -α3.7 deletion (76%), and the tripling of the α gene (12%) and of the α2 point mutation IVS1-5nt (6%). For the ß-globin gene, 12 mutations were detected, six of which constituted 83.4% of the total number of ß-thalassemia defects observed, including codon ß039 (38%), IVS1.6 T > C (15.6%), IVS1.110 G > A (11.8%), IVS1.1 G > A (11%), IVS2.745 C > G (4%), and IVS2.1 G > A (3%). However, the comparison of these frequencies with those detected in the population of other Sicilian provinces did not demonstrate significant differences, but it contrarily revealed a similitude. The data presented in this retrospective study help provide a picture of the prevalence of defects on the α and ß-globin genes in the province of Trapani. The identification of mutations in globin genes in a population is required for carrier screening and for an accurate prenatal diagnosis. It is important and necessary to continue promoting public awareness campaigns and screening programs.

Molecular spectrum of α- and ß-thalassemia among individuals of reproductive age in the Zhuhai region of southern China.

INTRODUCTION: Thalassemia is the most common monogenic disease in South and Southeast Asia. An accurate assessment of the relative frequency and composition of thalassemia mutations is important for the design of appropriate strategies to prevent the disease. In this study, we aimed to decode the molecular characterization of thalassemia mutations in Zhuhai region of southern China. METHODS: A total of 8048 individuals who were potential thalassemia carriers were enrolled. Gap-polymerase chain reaction (Gap-PCR) and reverse dot-blot (RDB) hybridization methods were employed to detect common deletional and non-deletional thalassemia mutations. Multiplex ligation dependent probe amplification (MLPA) and Sanger sequencing were used to analyze and verify rare and complex mutations. RESULTS: We diagnosed 3433 individuals as thalassemia carriers or patients. Of these, 2395 (69.76%) individuals with α-thalassemia harbored 13 α-globin gene mutations. The three most common α-thalassemia mutations were --SEA (60.08%), -α3.7 (20.62%) and -α4.2 (9.25%). We diagnosed 903 (26.30%) individuals with ß-thalassemia and identified 20 ß-globin gene mutations, of which the three most frequent were CD41/42 (-TCTT) (38.10%), IVS-II-654 (C>T) (23.69%) and TATAbox-28 (A>G) (15.18%). In addition, we identified 15 rare thalassemia variants. We also summarized the association between the thalassemia genotype and hematological parameters, which demonstrated the broad phenotypic heterogeneity caused by globin gene mutations. CONCLUSION: This is the first survey of thalassemia molecular epidemiology and hematological phenotype in Zhuhai region. It uncovered a high prevalence and complex molecular spectrum of thalassemia. These findings can be used as a basis for thalassemia diagnosis, counseling and prevention management.

Third-Generation Sequencing as a New Comprehensive Technology for Identifying Rare α- and ß-Globin Gene Variants in Thalassemia Alleles in the Chinese Population.

CONTEXT.­: Identification of rare thalassemia variants requires a combination of multiple diagnostic technologies. OBJECTIVE.­: To investigate a new approach of comprehensive analysis of thalassemia alleles based on third-generation sequencing (TGS) for identification of α- and ß-globin gene variants. DESIGN.­: Enrolled in this study were 70 suspected carriers of rare thalassemia variants. Routine gap-polymerase chain reaction and DNA sequencing were used to detect rare thalassemia variants, and TGS technology was performed to identify α- and ß-globin gene variants. RESULTS.­: Twenty-three cases that carried rare variants in α- and ß-globin genes were identified by the routine detection methods. TGS technology yielded a 7.14% (5 of 70) increment of rare α- and ß-globin gene variants as compared with the routine methods. Among them, the rare deletional genotype of -THAI was the most common variant. In addition, rare variants of CD15 (G>A) (HBA2:c.46G>A), CD117/118(+TCA) (HBA1:c.354_355insTCA), and ß-thalassemia 3.5-kilobase gene deletion were first identified in Fujian Province, China; to the best of our knowledge, this is the second report in the Chinese population. Moreover, HBA1:c.-24C>G, IVS-II-55 (G>T) (HBA1:c.300+55G>T) and hemoglobin (Hb) Maranon (HBA2:c.94A>G) were first identified in the Chinese population. We also identified rare Hb variants of HbC, HbG-Honolulu, Hb Miyashiro, and HbG-Coushatta in this study. CONCLUSIONS.­: TGS technology can effectively and accurately detect deletional and nondeletional thalassemia variants simultaneously in one experiment. Our study also demonstrated the application value of TGS-based comprehensive analysis of thalassemia alleles in the detection of rare thalassemia gene variants.