A Novel Frameshift Mutation of HBB Causing Dominant ß-Thalassemia in a Chinese Individual.

We reported a rare ß-thalassemia patient, a 41-year-old Chinese male with small cell hypopigmentation anemia, jaundice and splenomegaly as the main clinical symptoms. By using Next-Generation Sequencing (NGS), we identified a novel de novo HBB mutation(c.358_365dup, p.Phe123Alafs*39) which resulted in an abnormally prolonged ß-globin chain comprising 159 amino acid residues. The secondary and three-dimensional structures of the ß-globin predicted that the novel prolonged ß-globin chain has a considerable risk of instability in the hemoglobin, and leads to clinical phenotype. This study contributes to the enrichment of the genetic pathogenic mutation database for thalassemia and underscores the significance of NGS in the screening of mutations for thalassemia families.

Divergent humoral responses between males and females against 24 kDa excretory-secretory protein of Haemonchus contortus and influence of ovine ß-globin polymorphism.

Lambs harboring the Hb-AA ß-globin haplotype present improved cell-mediated responses and increased resistance against Haemonchus contortus infection. The aim of the present study was to compare the effect of sex and ß-globin haplotypes on specific humoral responses and phenotypes of resistance during H. contortus infection in Morada Nova sheep. As expected, females displayed stronger resistance during the first and second experimental challenges. Differential systemic humoral immune responses were observed comparing sex groups, in which higher levels of specific antibodies targeting 24 kDa excretory-secretory (ES24) protein of H. contortus of IgG and IgM antibodies were respectively observed as predominant isotypes in males and females. The IgM levels were significantly correlated with phenotypes of resistance, evaluated by packed cell volume and fecal egg counts. To our knowledge this is the first study reporting divergent humoral responses profiles to H. contortus infection between male and female sheep. The impact of ß-globin haplotypes was less pronounced in females compared to males. Notably, only males showed significant weight differences across haplotypes, with Hb-AA lambs being the heaviest. Additionally, Hb-AA males had significantly higher PCV (indicating better red blood cell health) and lower FEC (indicating lower parasite burden). These findings suggest a more pronounced effect of ß-globin polymorphisms on H. contortus infection in males, potentially due to their generally weaker resistance compared to females. This study highlights the importance of sex and ß-globin haplotypes in shaping immune responses to H. contortus infection. Specifically, IgM antibodies targeting the ES24 protein appear to play a crucial role in host-parasite interactions and may hold promise for therapeutic development.

Newborn Screening for ß-Thalassemia Identifies a Complex Genotype Involving a Novel ß-Globin Gene Mutation (HBB:c.336dup).

Newborn screening identified a Chinese-Canadian infant who was positive for possible ß-thalassemia (ß-thal). Detailed family studies demonstrated that the proband was a compound heterozygote for the Chinese Gγ(Aγδß)0-thal deletion and a novel frameshift mutation within exon 3 (HBB:c.336dup), and heterozygous for the Southeast Asian α-thal deletion (--SEA/αα). This case illustrates the importance of follow-up molecular testing of positive newborn screening results to confirm the diagnosis and define risks for future pregnancies.

ß0-Thalassemia Caused by a Novel Nonsense Mutation [HBB:c.199A > T].

We report two hemoglobinopathy cases involving a novel ß-thalassemia (ß-thal) nonsense mutation, HBB:c.199A > T. One patient had Hb S/ß-thal, and a second unrelated patient had Hb D-Punjab/ß-thal. The HBB:c.199A > T mutation introduces a premature termination codon at amino acid codon 66 (AAA→TAA) in exon 2, resulting in typical high Hb A2 ß0-thal.

Splice Acceptor Mutation [HBB:c.93-2A > T] in a Patient with Hb S/ß0-Thalassemia.

We report a case of Hb S/ß0-thalassemia (Hb S/ß0-thal) in a patient who is a compound heterozygote for the Hb Sickle mutation (HBB:c.20A > T) and a mutation of the canonical splice acceptor sequence of IVS1 (AG > TG, HBB:c.93-2A > T). This is the fifth mutation involving the AG splice acceptor site of IVS1, all of which prevent normal splicing and cause ß0-thal.

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.

A mass spectrometry assay for detection of endogenous and lentiviral engineered hemoglobin in cultured cells and sickle cell disease mice.

Sickle cell disease (SCD) results from a sequence defect in the ß-globin chain of adult hemoglobin (HbA) leading to expression of sickle hemoglobin (HbS). It is traditionally diagnosed by cellulose-acetate hemoglobin electrophoresis or high-performance liquid chromatography. While clinically useful, these methods have both sensitivity and specificity limitations. We developed a novel mass spectrometry (MS) method for the rapid, sensitive and highly quantitative detection of endogenous human ß-globin and sickle hß-globin, as well as lentiviral-encoded therapeutic hßAS3-globin in cultured cells and small quantities of mouse peripheral blood. The MS methods were used to phenotype homozygous HbA (AA), heterozygous HbA-HbS (AS) and homozygous HbS (SS) Townes SCD mice and detect lentiviral vector-encoded hßAS3-globin in transduced mouse erythroid cell cultures and transduced human CD34+ cells after erythroid differentiation. hßAS3-globin was also detected in peripheral blood 6 weeks post-transplant of transduced Townes SS bone marrow cells into syngeneic Townes SS mice and persisted for over 20 weeks post-transplant. As several genome-editing and gene therapy approaches for severe hemoglobin disorders are currently in clinical trials, this MS method will be useful for patient assessment before treatment and during follow-up.

C2H2 Zinc Finger Transcription Factors Associated with Hemoglobinopathies.

In humans, specific aberrations in ß-globin results in sickle cell disease and ß-thalassemia, symptoms of which can be ameliorated by increased expression of fetal globin (HbF). Two recent CRISPR-Cas9 screens, centered on ∼1500 annotated sequence-specific DNA binding proteins and performed in a human erythroid cell line that expresses adult hemoglobin, uncovered four groups of candidate regulators of HbF gene expression. They are (1) members of the nucleosome remodeling and deacetylase (NuRD) complex proteins that are already known for HbF control; (2) seven C2H2 zinc finger (ZF) proteins, including some (ZBTB7A and BCL11A) already known for directly silencing the fetal γ-globin genes in adult human erythroid cells; (3) a few other transcription factors of different structural classes that might indirectly influence HbF gene expression; and (4) DNA methyltransferase 1 (DNMT1) that maintains the DNA methylation marks that attract the MBD2-associated NuRD complex to DNA as well as associated histone H3 lysine 9 methylation. Here we briefly discuss the effects of these regulators, particularly C2H2 ZFs, in inducing HbF expression for treating ß-hemoglobin disorders, together with recent advances in developing safe and effective small-molecule therapeutics for the regulation of this well-conserved hemoglobin switch.

Hemoglobin ß-Globin Variants In Hispanic Patients: An Institutional Experience From Dallas, Texas.

Hemoglobinopathies are the most common single-gene disorders in humans. There are 1,424 variants of human hemoglobin described with 951 involving the ß-globin gene. Ancestry and geography play a significant role in the incidence and nature of hemoglobinopathies, with African, Asian, and Mediterranean populations and their descendants being amongst the most affected. Investigation of variants in individuals of Hispanic descent is needed to reflect the changing demographics of the United States. Hemoglobin ß-globin evaluation through gel electrophoresis, high-performance liquid chromatography, and HBB gene sequencing was performed on patients from Texas hospitals between 2010 and 2015 and demographic parameters (age, sex, ethnicity) was subsequently analyzed. A total of 846 patients underwent hemoglobinopathy evaluation. A ß chain variant was detected in 628 of the 846 total patients. Hispanic patients represented 37% (314/846 patients), which were equally distributed between females (50%; 156/314) and males (50%; 156/314). A ß-globin chain variant was found in 67% of Hispanic patients with a distribution across 10 variants seen in greater than 1% of patients. For hemoglobin variants, an understanding of the regional and ethnic prevalence will improve patient care through more effective screening and identification of the variant, early diagnosis, and appropriate treatment if necessary, and better genetic counseling.

Non-Viral Episomal Vector Mediates Efficient Gene Transfer of the ß-Globin Gene into K562 and Human Haematopoietic Progenitor Cells.

ß-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPß-globin for the physiological ß-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the ß-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the ß-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce ß-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPß-globin provides the basis for the development of therapeutic EV for gene therapy of ß-thalassemias.

Beta Globin Gene Cluster Haplotypes in Beta Thalassemia in the Kurdistan Region of Iraq.

ß-thalassemia is a prevalent inherited red cell disorder in the Kurdistan region of Iraq. To determine the chromosomal background of the frequent ß-thalassemia mutations in the latter region, we investigated the ß-globin gene cluster haplotypes in 202 ß-thalassemia chromosomes. Haplotypes analysis utilized restriction fragment length polymorphism-PCR of seven restriction sites through the ß-globin gene cluster. It was observed that IVS-II-1 (G > A) was mainly associated with haplotype III (68.8%), IVS-1-110 (G > A), codon 8/9 (+G) and codon 44 (-C) with haplotype I (in 90.0%, 100%, and 62.5% respectively), IVS-1-6 (T > C) with haplotype VI (97.4%), codon 8 (-AA) with haplotype IV (75%), codon 5(-CT) and IVS1.1 (G > A) with haplotype V (55.6% and 58.3% respectively), while codon 39 (C > T) and IVS1.5 (G > C) were mainly associated with haplotype VII (85.7% and 75% respectively). These observations support the notion that while some mutations may have originated in the Kurdistan region, others were more likely brought in by gene flow from neighboring countries or the Indian subcontinent. The association of some ß-thalassemia defects with more than one haplotype may be due to mutations or recombination events.

A novel gain-of-function PIP4K2A mutation elevates the expression of ß-globin and aggravates the severity of α-thalassemia.

Haemoglobin H (Hb H) disease (intermediate status of α-thalassemia) shows marked phenotypic variability from asymptomatic to severe anaemia. Apart from the combined ß-thalassemia allele ameliorating clinical severity, reports of genetic modifier genes affecting the phenotype of Hb H disease are scarce which bring inconvenience to precise diagnosis and genetic counselling of the patients. Here, we present a novel mutation (c.948C>A, p.S316R) in the PIP4K2A gene in a female Hb H disease patient who displayed moderate anaemia and a relatively high Hb H level. Haematological analysis in her family members revealed that individuals carrying this mutation have upregulated ß-globin expression, leading to a more imbalanced ß/α-globin ratio and more Hb H inclusion bodies in peripheral red blood cells. According to functional experiments, the mutant PIP4K2A protein exhibits enhanced protein stability, increased kinase activity and a stronger regulatory effect on downstream proteins, suggesting a gain-of-function mutation. Moreover, introduction of the S316R mutation into HUDEP-2 cells increased expression of ß-globin, further inhibiting erythroid differentiation and terminal enucleation. Thus, the S316R mutation is a novel genetic factor associated with ß-globin expression, and the PIP4K2A gene is a new potential modifier gene affecting the α-thalassemia phenotype.

Case report: A novel 10.8-kb deletion identified in the ß-globin gene through the long-read sequencing technology in a Chinese family with abnormal hemoglobin testing results.

Background: Thalassemia is a common inherited hemoglobin disorder caused by a deficiency of one or more globin subunits. Substitution variants and deletions in the HBB gene are the major causes of ß-thalassemia, of which large fragment deletions are rare and difficult to be detected by conventional polymerase chain reaction (PCR)-based methods. Case report: In this study, we reported a 26-year-old Han Chinese man, whose routine blood parameters were found to be abnormal. Hemoglobin testing was performed on the proband and his family members, of whom only the proband's mother had normal parameters. The comprehensive analysis of thalassemia alleles (CATSA, a long-read sequencing-based approach) was performed to identify the causative variants. We finally found a novel 10.8-kb deletion including the ß-globin (HBB) gene (Chr11:5216601-5227407, GRch38/hg38) of the proband and his father and brother, which were consistent with their hemoglobin testing results. The copy number and exact breakpoints of the deletion were confirmed by multiplex ligation-dependent probe amplification (MLPA) and gap-polymerase chain reaction (Gap-PCR) as well as Sanger sequencing, respectively. Conclusion: With this novel large deletion found in the HBB gene in China, we expand the genotype spectrum of ß-thalassemia and show the advantages of long-read sequencing (LRS) for comprehensive and precise detection of thalassemia variants.

Molecular Basis and Hematologic Phenotype of Hemoglobin H Disease Combined with Two Rare ß-Globin Mutations.

In area where α-thalassemia and ß-thalassemia are prevalent, the coinheritance of hemoglobin H disease (Hb H disease) and ß-thalassemia are not uncommon and could result in complex thalassemia intermedia syndromes. In this study, we investigate the hematological and molecular characteristics of two previously undescribed cases that co-inherited Hb H disease and rare ß-globin gene (HBB) mutations found in Chinese populations. Proband I was a boy with Hb H disease in association with IVS-II-5(G > C) (HBB:c0.315 + 5G > C) mutation. Proband II was a boy with a combination of Hb H and Hb Zengcheng [ß114(G16) Leu > Met; HBB:c.343C > A]. Both of them had mild hypochromic microcytic anemia, and neither had ever received a blood transfusion. In both cases, the level of Hb A2 was within normal range, and no Hb H was detected, but a small amount of Hb Bart's was observed in proband I. Routine DNA analysis detected the deletional Hb H disease in both cases. IVS-II-5(G > C) (HBB:c0.315 + 5G > C) and Hb Zengcheng (HBB:c.343C > A) mutations were found by DNA sequencing of ß-globin gene. The co-inheritance of Hb H disease with rare ß-thalassemia may result in an atypical pattern of Hb H disease, and further investigation of rare genotypes should be conducted to avoid missed diagnosis.

Rare and New Mutations of B-Globin in Azari Population of Iran, a Considerable Diversity.

Background: Thalassemia, as the most common single-gene genetic disorder, is related to a defect in the synthesis of one or more hemoglobin chains. More than 200 mutations have been identified in the ß-globin gene. Globally, every susceptible racial group has its own specific spectrum of the common mutations that are well-known to a particular geographic region. On the other hand, varying numbers of diverse rare mutations may occur. Materials and Methods: The subjects of the study included 2113 heterozygote or homozygote ß-thalassemia cases selected among couples who participated in the Iranian national thalassemia screening program from January 2011 to November 2019. Molecular characterization of the ß-thalassemia mutation was initially carried out by the amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) technique for common mutations, followed by sequencing, Gap PCR, and Multiple ligation-dependent probe amplification (MLPA) methods - in cases not detected by the ARMS-PCR. Results: The existence of 39 rare and new point mutations and 4 large deletions were described in our cohort. Sicilian (-13,337bp) deletion, CD36/37 (-T), and CD15 TGG>TGA were encountered more often than the others in a decreasing order, in terms of frequency. The least frequent mutations/deletions were deletion from HBD exon 1 to HBB promoter, 619 bp deletion, Deletion from up HBBP1-Exon3 HBBP1 and up HBB-0.5Kb down HBB, CAP+8 C>A, CD37 (G>A), CD6 (-A), IVSI-2 (T>C), IVSII-705 T>G, and IVSII-772 (G>A). Each occurred once. Five mutations/variants were also determined which have not been reported previously in Iran. Conclusion: According to the findings of the study, the Northwestern Iranian population displayed a wide variety of thalassemia allelic distributions. Identification of rare and new mutations in the ß-thalassemia in the national population is beneficial for screening programs, genetic counseling, and prenatal diagnosis.

Hb Ryazan: An Elongated C-Terminal ß-Chain Due to a New Frameshift Mutation, HBB: c.396delG p.Val133Trpfs*25.

We identified a novel abnormal hemoglobin variant caused by a frameshift mutation at nucleotide position 396 in exon 3 of the ß-globin gene (HBB): NM_000518:c.396delG. This variant causes an emergence of alternative amino acid sequence starting at codon 133 and a new stop codon formed in the 3' untranslated region (3'UTR) of the HBB gene at amino acid position 158. This ß-globin gene variant was identified in a woman with a long history of hemolytic anemia. We named this variant Hb Ryazan after the proband's city of origin.

HBB Gene Mutations and Their Pathological Impacts on HbE/ß-Thalassaemia in Kuala Terengganu, Malaysia.

BACKGROUND: ß-thalassaemia is a disorder caused by mutations in the ß-globin gene, leading to defective production of haemoglobins (Hb) and red blood cells (RBCs). It is characterised by anaemia, ineffective erythropoiesis, and iron overload. Patients with severe ß-thalassaemia require lifelong blood transfusions. Haemoglobin E beta-thalassaemia (HbE/ß-thalassaemia) is a severe form of ß-thalassaemia in Asian countries. More than 200 alleles have been recognised in the ß-globin region. Different geographical regions show different frequencies of allelic characteristics. In this study, the spectrum of ß-thalassaemia (ß-thal) alleles and their correlation with iron overload, in HbE/ß-thalassaemia patients, ß-thalassaemia trait, and HbE trait were studied. METHODS: Blood samples (n = 260) were collected from 65 ß-thalassaemia patients, 65 parents (fathers and/or mothers) and 130 healthy control individuals. Haematological analyses, iron profiles, and serum hepcidin levels were examined for all participants. DNA was extracted from patients' and their parents' blood samples, then subjected to PCR amplification. Multiplex amplification refractory mutation system PCR (MARMS-PCR) was conducted for eighteen primers to detect the mutations. RESULTS: There was severe anaemia present in HbE/ß-thalassaemia patients compared to their parents and healthy controls. The ferritin and iron levels were significantly increased in patients compared to their parents and healthy controls (p = 0.001). Two common mutations were detected among the patient group and three mutations were detected among their parents, in addition to seven novel mutations in HbE/ß-thalassaemia patients (explained in results). CONCLUSION: Some mutations were associated with severe anaemia in ß-thalassaemia patients. The detection of mutations is a prognostic marker, and could enhance the appropriate management protocols and improve the haematological and biochemical statuses of ß-thalassaemia patients.

To Study the ß-Globin Haplotype Pattern of Descent of a Set of Linked Alleles Occurring on the Same Chromosome in the Northern Province of India.

OBJECTIVE: To study the five mutations commonly prevalent in North India, i.e., IVS-I-5 (G→C), 619 bp deletion, IVS-I-1 (G→T), codon 41/42 (-TTCT), and codon 8/9 (+G), in the beta thalassemia (ß-thalassemia) major children. The specific ß-thalassemia mutations of different haplotype patterns of the ß-globin gene cluster will also be determined. METHODS: A total of 125 children diagnosed with ß-thalassemia major visiting the Department of Pediatrics of King George's Medical University were involved in the study. As per the QIAamp (Qiagen, Hilden, Germany) manufacturer guidelines, genomic DNA was isolated from whole blood. To identify the haplotype pattern within the ß-globin gene cluster, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used. The respective restriction endonucleases used were Hind III/GƔ, Hinc II/Ψß, Hinf I/ß, Ava II/ß, and BamHI for the haplotype analysis in the ß-globin pattern of descent of a set of linked alleles occurring on the same chromosome. RESULTS: Among the five common mutations, 73 patients had IVS-I-5 (G→C), 28 patients had 619 bp deletion, 17 patients had IVS-I-1 (G→T), five patients had Cd 41/42 (-TTCT), and two patients had Cd 8/9 (+G) mutations. Fifteen haplotypes (haplotypes 1-15) were identified in 125 ß-thalassemia major children. Among the five haplotypes observed in the IVS-I-5 (G→C) mutation, the H1 haplotype was most predominant with a frequency of 27.2%, followed by the H2, H4, H3, and H10 haplotypes in the given population. In 619 bp deletion, IVS-I-1 (G→T), codon 41/42, and codon 8/9, haplotype H9, H12, H11, and H5 were seen, respectively. CONCLUSION: ß-thalassemia was found to be the most common in the northern province of Uttar Pradesh. The linkage of ß-globin gene haplotypes with ß-thalassemia mutations was explored in the northern province of Uttar Pradesh. The population of different natives is being mixed up due to migration and industrialization. These were some reasons for the occurrence of haplotypic heterogeneity. This haplotype heterogeneity was correlated with the origin of these mutations found to be unlike the origin of common ones from different provinces.

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.

Novel Promoter Mutation (HBB:C.-139_-138del) Associated with ß-Thalassemia Trait Detected by Next-Generation Sequencing in Southern China.

Here we report a novel ß-globin gene mutation in the promoter (HBB:c.-139_-138delAC) detected by next-generation sequencing (NGS). The proband was a 28-year-old Chinese male, living in Shenzhen City, Guangdong Province, who originates from Hunan Province. The red cell indices were almost normal, with a slightly decreased Red Cell volume Distribution Width(RDW). Capillary electrophoresis (CE) showed the Hb A (93.1%) value was below normal, while the Hb A2 (4.2%) and Hb F (2.7%) values were both beyond normal. A set of genetic tests of the α and ß-globin genes were then performed to determine whether the subject carried any causative mutations. The results of NGS revealed a two-base pair deletion at position -89 to -88(HBB:c.-139_-138delAC）in the heterozygous state, which was subsequently confirmed by Sanger sequencing.