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.

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.

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.

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.

A First Clinical and Molecular Study of Rare IVS-II-806 (G > C) (HBB:c.316-45G > C) Variant in the ß-globin Gene: A Possibly Benign Variant.

Introduction: ß-thalassemia is a common genetic disease affecting a single gene, disease with a high incidence in South China. We hereby, aim to provide the clinical and hematological features of a rare ß-globin gene variant in the Chinese population. Methods: Ten subjects from three unrelated Chinese families were enrolled in this study. Hematological analysis and thalassemia gene testing were preformed to screen for common α and ß-thalassemia variants. Gap-polymerase chain reaction (Gap-PCR) and DNA sequencing were utilized to examine the rare or novel thalassemia variants. Results: Six cases were identified carrying the rare IVS-II-806 (G > C) (HBB:c.316-45G > C) variant in the ß-globin gene. The proband in family 1 carry three rare ß-globin gene mutations including CD39 (C > T), IVS-II-81 (C > T) and IVS-II-806 (G > C) combined with a --SEA/αα deletion, exhibiting the ß-thalassemia trait. Further pedigree investigation indicated that the genotype of the proband in family 1 was --SEA/αα, ßCD39 (C>T), IVS-II-81(C>T)/ßIVS-II-806(G>C). Meanwhile, the twin girls in family 1 carrying the IVS-II-806 (G > C) mutation demonstrated a normal hematological phenotype. In family 2, the proband and his sister carry the IVS-II-806 (G > C) mutation, eliciting high levels of Hb A2 and slightly low levels of MCV and MCH. Moreover, the proband in family 3 carrying the same mutation exhibited a slightly low MCV level as well. Conclusions: In this study, clinical and hematological analysis of the IVS-II-806 (G > C) mutation was first conducted within the Chinese population, with results indicating that it may be a benign variant.

Development of field-applicable endogenous internally controlled recombinase-aided amplification (EIC-RAA) assays for the detection of human papillomavirus genotypes 6 and 11 using sample releasing agent.

Objective: Human papillomavirus (HPV) 6 and 11 are the two most common low-risk HPV subtypes, accounting for more than 90% of condyloma acuminatum. A simple, accurate and rapid screening method to be applied in community-level hospitals is in high demand. Methods: Endogenous internally controlled recombinase-assisted amplification (EIC-RAA) assays for HPV6 and 11 were performed in a single closed-tube at 39 °C within 30 min. The sensitivity and specificity of EIC-RAA were examined using recombinant plasmids and pre-tested HPV DNA. A total of 233 clinical samples were collected, and the DNA was extracted by traditional multi-step extraction, or sample releasing agent, before analysis by EIC-RAA. For comparison, HPV detection via Quantitative real-time PCR (qPCR) was also performed. Results: The sensitivity of EIC-RAA analysis was 10 copies/reaction for HPV6, 100 copies/reaction for HPV11, and 100 copies/reaction for the human ß-globin gene. No cross-reaction was observed with other HPV subtypes. Clinical performance of the EIC-RAA assay achieved a 100% of concordance rate with the commercial HPV qPCR kit. Further, the EIC-RAA assay achieved a 100% of concordance rate when using multi-step extracted DNA and sample releasing agent-processed DNA. Summary: The EIC-RAA assay for HPV6 and 11 detection possesses the advantages of accuracy, simplicity and rapidity, and demonstrates great potential to be used in community-level hospitals for field investigation.

A Novel 5 kb Deletion in the ß-Globin Gene Cluster Identified in a Chinese Patient.

ß-Thalassemia (ß-thal), a highly prevalent disease in tropical and subtropical regions of Southern China, is caused mainly by point mutations in the ß-globin gene cluster. However, large deletions have also been found to contribute to some types of ß-thal. We identified a novel 5 kb deletion in the ß-globin cluster in a Chinese patient using multiplex ligation-dependent probe amplification (MLPA), and characterized it with single molecule real-time (SMRT) sequencing, gap-polymerase chain reaction (gap-PCR) and Sanger sequencing. The deletion was located between positions 5226189 and 5231091 on chromosome 11 (GRCh38), extending from 4 kb upstream of the 5' untranslated region (5'UTR) to the second intron of the ß-globin gene. The patient with this deletion presented with microcytosis and hypochromic red cells, as well as relatively high Hb F and Hb A2 levels. Our research indicated that SMRT sequencing is a useful tool for accurate detection of large deletions. Our study broadens the spectrum of deletional ß-thalassemias and provides a perspective for further study of the function of the ß-globin cluster.

A homotetrameric hemoglobin expressed in alveolar epithelial cells increases blood oxygenation in high-altitude plateau pika (Ochotona curzoniae).

The plateau pika (Ochotona curzoniae) is native to the Qinghai-Tibet Plateau. In this study, the gene that encodes a heme-binding protein in the pulmonary surfactant (PS) of the pika is identified. The protein is a homotetrameric hemoglobin (δ4) encoded by HBD (δ). HBD is expressed in alveolar epithelial type II (ATII) and type I (ATI) cells, upregulated by hypoxia. δ4 is secreted into alveolar cavities through osmiophilic multilamellar bodies. HBD expression is downregulated by RNAi, which significantly increases hypoxia-inducible factor 1α expression in lung tissue and red blood cells and hemoglobin and blood lactate concentrations but significantly decreases arterial partial pressure of oxygen (PaO2). Our results indicate that plateau pikas physiologically show hypoxemia when HBD expression is downregulated. Therefore, specific HBD expression in the lungs helps plateau pikas to obtain oxygen under hypoxia by maintaining higher PaO2. These findings provide insights into the adaptive mechanisms of plateau pikas to withstand high-altitude environments.

Case Report: The third-generation sequencing confirmed a novel 7.2 Kb deletion at ß-globin gene in a patient with rare ß-thalassemia.

Background: Thalassemia was the most common monogenic diseases worldwide, which was caused by mutations, deletions or duplications in human globin genes which disturbed the synthesis balance between α- and ß-globin chains of hemoglobin. There were many classics methods to diagnose thalassemia, but all of them had limitations. Although variations in the human ß-globin gene cluster were mainly point mutations, novel large deletions had been described in recent years along with the development of DNA sequencing technology. Case report: We present a case of 32-year-old male with abnormal hematological results. However, 23 genotypes of the most common thalassemia were not detected by two independent conventional platforms. Finally, using multiplex ligation-dependent probe amplification (MLPA), third-generation sequencing (TGS) and Gap PCR detection methods, we first confirmed the case with a novel 7.2 Kb deletion (Chr11:5222800-5230034, hg38) located at HBB gene. Conclusion: Our results showed that TGS technology was a powerful tool for thalassemia breakpoint detection, had promising potentiality in genetic screening of novel thalassemia, especially for the novel deletions in globin genes.

Combined Gap-Polymerase Chain Reaction and Targeted Next-Generation Sequencing Improve α- and ß-Thalassemia Carrier Screening in Pregnant Women in Vietnam.

Vietnam has a high thalassemia burden. We collected blood samples from 5880 pregnant Vietnamese women during prenatal health checks to assess thalassemia carrier frequency using combined gap-polymerase chain reaction (gap-PCR) and targeted next-generation sequencing (NGS). Thalassemia carriers were identified with prevalence of 13.13% (772), including 7.82% (460) carriers of α-thalassemia (α-thal), 5.31% (312) carriers of ß-thalassemia (ß-thal), and 0.63% (37) concurrent α-/ß-thal carriers. Deletional mutations (368) accounted for 80.0% of α-thal carriers, of which, --SEA (Southeast Asian) (n = 254; 55.0%) was most prevalent, followed by the -α3.7 (rightward) (n = 66; 14.0%) and -α4.2 (leftward) (n = 45; 9.8%) deletions. Hb Westmead (HBA2: c.369C>G) (n = 53) and Hb Constant Spring (Hb CS or HBA2: c.427T>C) (in 28) are the two most common nondeletional α-globin variants, accounting for 11.5 and 6.0% of α-thal carriers. We detected 11 different ß-thal genotypes. Hb E (HBB: c.79G>A) (in 211) accounted for 67.6% of ß-thal carriers. The most common ß-thal genotypes were associated with mutations at codon 17 (A>T) (HBB: c.52A>T), codons 41/42 (-TTCT) (HBB: c.126_129delCTTT), and codon 71/72 (+A) (HBB: c.217_218insA) (prevalence 0.70%, 0.68%, and 0.2%, respectively). Based on mutation frequencies calculated in this study, estimates of 5021 babies in Vietnam are affected with clinically severe thalassemia annually. Our data suggest a higher thalassemia carrier frequency in Vietnam than previously reported. We established that combining NGS with gap-PCR creates an effective large-scale thalassemia screening method that can detect a broad range of mutations.

Hb Kalundborg [ß79(EF3)Asp→Glu; HBB: c.240C>a], a Possible Low-affinity Hemoglobin Variant Detected during Hb A1c Measurement.

A previously unknown hemoglobin (Hb) variant was detected during measurement of glycosylated Hb (Hb A1c) after the introduction of a new high performance liquid chromatography (HPLC) apparatus. Subsequent DNA sequencing revealed a heterozygous single nucleotide substitution at codon 79 (C>A) on the ß-globin gene changing an amino acid [ß79(EF3)Asp→Glu; HBB: c.240C>A]. The new Hb variant was named Hb Kalundborg after the place of origin of the proband. Heterozygosity for this mutation appears to have no clinical significance in itself except for a possibly slightly lower oxygen affinity. However, it interferes with Hb A1c measurement by HPLC, causing a falsely high Hb A1c concentration when using the G11 apparatus with clinical implications possibly to follow.

Hb F Levels in ß-Thalassemia Carriers and Normal Individuals: Known and Unknown Quantitative Trait Loci in the ß-Globin Gene Cluster.

In the already identified quantitative trait loci (QTL), modulating Hb F levels are cis-acting haplotypes of the ß-globin gene cluster itself, although the single nucleotide polymorphisms (SNPs) accounting more for the association, remain uncertain. In this study, the role in Hb F production of previously reported candidate SNPs within the ß-globin gene cluster was reexamined, along with a yet poorly studied variation in the BGLT3 gene. In a sample of ß-thalassemia (ß-thal) carriers, we succeeded in replicating the significant association between increased Hb F levels and rs7482144 (C>T) (HBG2 XmnI), which is the most well-established variation in the cluster influencing the trait. This SNP was found to be in strong linkage disequilibrium (LD) with a variation in the HBBP1 gene [rs10128556 (G>A)], which consistently revealed a similar association signal. Remarkably, much stronger than the latter associations were those involving both rs968857 (T allele) (3' HBBP1) and rs7924684 (G allele) (BGLT3), two SNPs that were also in strong LD. As the pattern of LD detected in the ß-globin gene cluster does not correlate with a tight linkage between markers, complex interactions between SNPs at the cluster seem to modulate Hb F. Seeing that no such associations were detected in normal subjects, the question can be raised on whether, under erythropoiesis stress, epigenetic mechanisms contribute to change the regulation of the entire ß-globin gene cluster. In conclusion, we provide statistical evidence for a new player within the ß-globin gene cluster, BGLT3, that in cooperation with other regions influences Hb F levels in ß-thal carriers.

Diagnostic Dilemma of Hemoglobinopathies Using High Performance Liquid Chromatography Alone: A Case Report from a Resource-Constrained Setting.

Hemoglobinopathies are quite common in India, and multiple awareness and screening initiatives exist for detection of thalassemia in the population. One of the most common and successfully used method for thalassemia screening is the high performance liquid chromatography (HPLC) test. However, in spite of its excellent usefulness as a screening tool, there are situations where HPLC alone may not be able to make an accurate diagnosis. Here we highlight a fairly common situation where HPLC alone failed to confirm the diagnosis. A detailed family and transfusion history along with clinical examination and investigations, such as a complete hemogram, HPLC, along with molecular studies would have aided in diagnosis. Another cause of concern raised by this case is that the most common mutation in our population, such as IVS-I-5 (G>C), HBB: c.92+5G>C, was not represented in the HPLC, and thus, was missed during the preconception screening process, leading to a chain of events.

Structural switching/polymorphism by sequential base substitution at quasi-palindromic SNP site (G → A) in LCR of human ß-globin gene cluster.

The human ß-globin gene Locus Control Region (LCR), a dominant regulator of globin gene expression contains five tissue-specific DNase I-hypersensitive sites (HSs). A single nucleotide polymorphism (SNP) (A â†’ G) present in HS4 region of locus control region (LCR), have shown a notable association between the G allele and the occurrence of ß-thalassemia. This SNP site exhibiting a hairpin - duplex equilibrium manifested in A â†’ B like DNA transition has previously been reported from this laboratory. Since, DNA is a dynamic and adaptable molecule, so any change of a single base within a primary DNA sequence can produce major biological consequences commonly manifested in genetic disorders such as sickle cell anemia and ß-thalassemia. Herein, the differential behavior of sequential single base substitutions G â†’ A on the quasi-palindromic sequence (d-TGGGGGCCCCA; HPG11) has been explored. A combination of native gel electrophoresis, circular dichroism (CD), and UV-thermal denaturation (Tm) techniques have been used to investigate the structural polymorphism associated with various variants of HPG11 i.e. HPG11A2 to HPG11A5. The CD spectra confirmed that all the HPG11 variants exhibit a hairpin - duplex equilibrium. Oligomer concentration dependence on CD spectra has been correlated with A â†’ B DNA conformational transition. However, as revealed in gel electrophoresis, HPG11A2 â†’ A5 exhibit the formation of a tetramolecular structure (four-way junction) at higher oligomer concentration. UV-melting studies also supported the melting of hairpin, duplex and four-way junction structure. This polymorphism pattern may possibly be significant for DNA-protein recognition, in the process of regulation of LCR in the ß-globin gene.

Long noncoding RNAs transcribed downstream of the human ß-globin locus regulate ß-globin gene expression.

The five ß-like globin genes (ε, Gγ, Aγ, δ and ß) at the human ß-globin gene locus are known to be expressed at specific developmental stages, although details of the underlying mechanism remain to be uncovered. Here we used an in vitro transcription assay to clarify the mechanisms that control this gene expression. We first tested nuclear RNA from HeLa cells using RT-qPCR and discovered a long noncoding RNAs (lncRNAs) within a 5.2-kb region beginning 4.4 kb downstream of the ß-globin gene coding region. We investigated nuclear RNA from K562 cells using a primer-extension assay and determined the transcription start sites (TSSs) of these lncRNAs. To clarify their functional role, we performed knockdown (KD) of these lncRNAs in K562 cells. Hydroxyurea (HU), which induces differentiation of K562 cells, increased haemoglobin peptide production, and the effect was enhanced by KD of these lncRNAs, which also enhanced upregulation of the γ-globin expression induced by HU. To confirm these results, we performed an in vitro transcription assay. Noncoding single-stranded RNAs inhibited ß-globin expression, which was upregulated by GATA1. Furthermore, lncRNAs interacted with GATA1 without sequence specificity and inhibited its binding to its target DNA response element in vitro. Our results suggest that lncRNAs downstream of the ß-globin gene locus are key factors regulating globin gene expression.

Genetic research and clinical analysis of ß-globin gene cluster deletions in the Chinese population of Fujian province: A 14-year single-center experience.

BACKGROUND: Heterozygotes of HPFH and 뫧 thalassemia are clinically asymptomatic or have mild hemoglobin (Hb) values. However, when both HPFH and δß-thalassemia are coinherited with heterozygous ß-thalassemia, patients may progress to a clinical phenotype of thalassemia intermedia or thalassemia major. The purpose of this study was to characterize the genotypes and analyze the phenotypes of these disorders in Fujian Province, to offer advice for genetic counseling and accurate prenatal diagnosis in this region. A total of 55 001 subjects were participated in thalassemia screening. 142 subjects with HbF levels ≥10%, before the blood transfusion, were selected for further investigation. METHODS: Multiplex ligation-dependent probe amplification (MLPA) and Gap-PCR were used to screen for three ß-globin gene cluster deletions: Chinese G γ(A γδß)0 thalassemia and Southeast Asia HPFH (SEA-HPFH) deletion and 1357 bp deletion (NG-000007.3:g.69997-71353 del 1357). RESULTS: A total of 142 patients with HbF (≥10%) were enrolled to characterize the molecular basis of ß-globin gene cluster deletions in our study; 22 cases 0.04% (22/55 001) were definitively diagnosed with ß-globin gene cluster deletions. Ten cases were heterozygous for the Chinese G γ(A γδß)0 -thal mutations, 10 cases were heterozygous for SEA-HPFH, and one case was compound heterozygous for SEA-HPFH and the α-thal mutation. The 1357 bp deletion (NG-000007.3:g.69997-71353 del 1357) was detected in one case. Moreover, the hemoglobin A2  levels in patients who were heterozygous for Chinese G γ(A γδß)0 -thal were statistically lower than in cases with SEA-HPFH deletion(p < 0.05). CONCLUSION: In Fujian Province, the prevalence of common ß-globin gene cluster deletions was 0.04%. What's more, the most common ß-globin cluster deletions are the Chinese G γ(A γδß)0 and SEA-HPFH.

Genotype-Phenotype Study of ß-Thalassemia Patients in Sabah.

ß-thalassemia is a serious public health problem in Sabah due to its high prevalence. This study aimed to investigate the effects of different types of ß-globin gene mutations, coinheritance with α-globin gene mutations, XmnI-Gγ, and rs368698783 polymorphisms on the ß-thalassemia phenotypes in Sabahan patients. A total of 111 patients were included in this study. The sociodemographic profile of the patients was collected using a semi-structured questionnaire, while clinical data were obtained from their medical records. Gap-PCR, ARMS-PCR, RFLP-PCR, and multiplex PCR were performed to detect ß- and α-globin gene mutations, as well as XmnI-Gγ and rs368698783 polymorphisms. Our data show that the high prevalence of ß-thalassemia in Sabah is not due to consanguineous marriages (5.4%). A total of six different ß-globin gene mutations were detected, with Filipino ß°-deletion being the most dominant (87.4%). There were 77.5% homozygous ß-thalassemia patients, 16.2% compound heterozygous ß-thalassemia patients, and 6.3% ß-thalassemia/Hb E patients. Further evaluation on compound heterozygous ß-thalassemia and ß-thalassemia/Hb E patients found no concomitant α-globin gene mutations and the rs368698783 polymorphism. Furthermore, the XmnI-Gγ (-/+) genotype did not demonstrate a strong impact on the disease phenotype, as only two of five patients in the compound heterozygous ß-thalassemia group and two of three patients in the ß-thalassemia/Hb E group had a moderate phenotype. Our findings indicate that the severity of the ß-thalassemia phenotypes is closely related to the type of ß-globin gene mutations but not to the XmnI-Gγ and rs368698783 polymorphisms.