β-thalassaemia is primarily caused by point mutations and, less commonly, by deletions or insertions in the β-globin gene locus.¹ This gene is highly heterogeneous, with approximately 954 documented variants, over 300 of which are associated with β-thalassaemia.² β-thalassaemia major arises from the complete absence of β-globin chain production, while β-thalassaemia trait (minor) and β-thalassaemia intermedia involve a reduction in β-globin synthesis. All three forms result in decreased production of adult haemoglobin (HbA),^3,4,5 leading to anaemia of varying severity, depending on the mutation type and zygosity.⁴

While β-thalassaemia is globally distributed, its prevalence varies. Historically, it has been endemic in the Mediterranean, the Middle East, and Southeast Asia, with the highest carrier frequencies reported in regions such as Cyprus, Sardinia, and parts of Southeast Asia.^6,7 The global carrier rate is estimated at approximately 1.5%.⁵ However, patterns are shifting as a result of increased migration and interethnic marriages, leading to cases in previously low-prevalence areas.^5,7

In West Africa, particularly Nigeria, β-thalassaemia was once considered rare. However, recent studies suggest a rising prevalence. In Nigeria, population-based screening studies have estimated a β-thalassaemia trait carrier rate of up to 25%,^8,9,10 while neighbouring countries such as Liberia and others in sub-Saharan Africa have reported estimation rates ranging from 9% to 20%.^11,12 These estimates are commonly based on elevated HbA₂ (> 3.5%) and HbF (> 1%) levels determined by high-performance liquid chromatography (HPLC). In contrast, earlier methods such as starch-block electrophoresis, used by Esan, reported a prevalence of < 1%, using a diagnostic cutoff of HbA₂ > 3.8%.¹³

Sickle cell disease (SCD), which also results from a point mutation in the β-globin gene, may co-exist with β-thalassaemia. Among Nigerian patients with SCD, HPLC-based screening suggest a prevalence of co-inherited β-thalassaemia trait between 6% and 10%.^14,15 However, a molecular-based study involving children under five in Northern Nigeria reported a much lower prevalence of 0.4% among children with SCD, and none among those without.¹⁶ The β-thalassaemia trait (β⁺-thal), when co-inherited with SCD, tends to ameliorate the clinical severity of SCD. Individuals with this compound heterozygous genotype (HbSβ⁺-thal) typically experience a milder disease course and fewer vaso-occlusive crises, similar to the HbSC genotype.¹⁷ Hence, it is expected to be at a higher prevalence in individuals with SCD compared to the general population, contrary to recent reports.

Diagnosis of β-thalassaemia trait in Nigeria commonly relies on HbA₂ and HbF thresholds as the first initial screening process, but several confounding factors can elevate HbA₂ levels, including α-thalassaemia,^16,18 vitamin B12 or folate deficiency, antiretroviral therapy, and hyperthyroidism.¹⁹ Notably, α-thalassaemia itself is prevalent in Nigeria, with rates exceeding 35%.^18,20 Nigeria bears the highest burden of SCD globally, yet the true prevalence and genetic characteristics of β-thalassaemia trait in this population remain uncertain. This study was therefore conducted to investigate the β-globin gene architecture in individuals with and without SCD in Nigeria by integrating molecular sequencing, variant annotation, and haplotype analysis to determine the prevalence and genetic spectrum of β-thalassaemia trait and to assess whether this differs significantly between the two groups. This study is among the first to use molecular genotyping to re-evaluate the prevalence of β-thalassaemia trait in Nigerians.

The β-globin gene is highly polymorphic, harbouring numerous mutations that give rise to various haemoglobinopathies. These disorders are often population-specific, with unique combinations of mutations occurring in different regions of the world.^7,26 β-thalassaemia, for instance, is caused by over 300 known mutations in the β-globin gene.^2,27 In our genotyping study of 123 SCD patients in the main cohort, no known β-thalassaemia mutations were detected. Only one individual, an SCD patient in the separate sub-cohort, was found to carry a pathogenic β⁺ thalassaemia mutation (rs33915217C > A), which is typically associated with β-thalassaemia intermedia in the homozygous or compound heterozygous state. This sub-cohort was analysed because of elevated HbA₂ and/or HbF levels, which in clinical practice often lead to presumptive diagnosis of β-thalassaemia trait based on HPLC alone.

The rs33915217 SNP has three rare alleles: C > A, C > G, and C > T². All three disrupt normal splicing at the 5′ splice site, impairing mRNA processing and resulting in reduced or absent β-globin production.^2,28 The A allele, found in our study, is particularly rare, and has previously been reported only in two individuals from Europe—one heterozygous and one homozygous.²⁸ To our knowledge, this is the first report of this allele in both an African individual and an SCD patient. It was absent from the 1000 Genomes Phase 3 data set but is recorded at extremely low frequency (0.000009)²⁴ in the gnomAD exome (non-Finnish European) data set.

The rs111851677 variant (IVS-I-108), found in three individuals without SCD in the heterozygous state, has been previously reported in β-thalassaemia carriers from Cuba,²⁹ Iran,³⁰ and Greece.³¹ Though its clinical impact is debated, it may impair RNA splicing because of its location near the 3′ splice site.^29,32 In our study, however, individuals carrying this variant showed normal haematological profiles, suggesting a benign role.

Our findings support earlier studies from Nigeria^13,16 that reported β-thalassaemia mutations to be rare (< 1%) in the population. This contrasts with recent reports suggesting a prevalence of 6% to 10% in SCD patients and 20% to 25% in the general Nigerian population.^9,10,14,15 The discrepancy likely stems from methodological differences, as these studies relied solely on haematologic indices and HPLC thresholds (HbA₂ > 3.0% – 3.5%, HbF > 1%) to infer β-thalassaemia trait without molecular confirmation. Such screening-based approaches are prone to overestimation because of conditions that elevate HbA₂, including α-thalassaemia,^16,18 vitamin B₁₂ or folate deficiency, antiretroviral therapy, and thyroid disorders.¹⁹ In our study, all the individuals (except one) in both the main and separate cohorts with elevated HbA₂ levels had no detectable β-thalassaemia mutations on genetic testing. Similar findings were reported by Inusa et al.,¹⁶ who found no underlying β-thalassaemia mutations despite elevated HbA₂ levels (up to 7.4%). This underscores the need for standardised diagnostic methods integrating molecular genotyping.

Given the high prevalence of α thalassaemia in Nigeria (estimated at 35% – 45%),^18,20 this could contribute significantly to spurious elevations in HbA₂ and possible misdiagnosis of β-thalassaemia trait. Interestingly, since β-thalassaemia trait can ameliorate SCD symptoms, its frequency would be expected to be higher in SCD patients than in the general population. However, recent HPLC-based studies report the reverse,^9,10 further emphasising the limitations of relying solely on haematologic indices for diagnosis.

Apart from rs33915217 (β-thalassaemia), rs334 (HbS), and rs33930165 (HbC), all other variants identified in our study were non-pathogenic intronic SNPs. Notably, rs713040G—which has a higher global frequency than its reference allele—was found in 100% of SCD patients, compared to ~80% in non-SCD, African, and global populations. Similarly, rs33930165 (HbC mutation) was observed in 1.8% of the non-SCD cohort, aligning with its expected prevalence in African populations (0.013–0.028).²⁴ The rs334 (HbS mutation) had a frequency of 0.085 in the non-SCD group, which is consistent with African averages and lower than in the Yoruba in Ibadan reference population.

Other intronic variants found in this study (rs10768683, rs7480526, rs7946748, rs113152027, rs1609812) displayed population frequencies consistent with African ancestry. One rare 3′ untranslated region variant, rs537944366, was detected in only one individual. This variant, absent in the Yoruba in Ibadan and only recorded in Mende in Sierra Leone and gnomAD non-Finnish Europeans, is being reported for the first time in a Nigerian (Yoruba) population.

Our haplotype analysis revealed two ancestral haplotypes: a major haplotype (frequency: 0.850), typical of African populations, and a minor haplotype (frequency: 0.116), atypical for Africans and more common in South and East Asian populations. These haplotypes are differentiated by four SNPs in linkage disequilibrium (rs713040, rs10768683, rs7946748, rs1609812). The minor ancestral haplotype and its variants were found in 27 (24%) non-SCD participants in the heterozygous state; no participant had the homozygous state. Hardy-Weinberg expectations suggest a frequency too low to detect given the current sample size.

Only the major haplotype (TAGGAGAACG) was associated with HbS and HbC mutations. Interestingly, the SCD patient with the β-thalassaemia intermedia mutation was heterozygous for both haplotypes, linking the pathogenic variant to the minor ancestral haplotype. The rs33915217 β-thalassaemia mutation was found on the minor haplotype, supporting the idea of genetic admixture or historical gene flow into this population. This pattern suggests that HbS and HbC mutations in this population have likely evolved on a common African haplotypic background and have undergone minimal recombination.

Our findings challenge the assumption of a high prevalence of β-thalassaemia trait in Nigeria. The findings also validate the robustness of the sequencing and analysis pipeline. Seven SNPs from our data showed the same linkage disequilibrium pattern in Yoruba in Ibadan individuals from the 1000 Genomes Project,³³ reinforcing the accuracy of our haplotype deductions.