PB2402 BETA‐GLOBIN GENE CLUSTER HAPLOTYPES ASSOCIATED WITH HBF VARIATION IN β‐THALASSEMIA CARRIERS OF PORTUGUESE ORIGIN
Authors: L. Manco, C. Bento, L. Relvas, T. Maia, M. L. Ribeiro
Journal: HemaSphere (2019)

Background: Genetic association studies showed that HbF levels variation in haemoglobinopathies, such as sickle cell disease and β‐thalassemia (β‐thal), are under the influence of single nucleotide polymorphisms (SNPs) in several genomic loci. The first described polymorphism influencing gamma globin gene expression was XmnI in HBG2 promoter −158C &gt; T (rs7482144). Several reports showed that β‐thal carriers with HbF raised levels have a significantly higher rs7482144 minor allele frequency than patients with low HbF levels. The functional significance of HBG2 ‐XmnI mutation has never been clearly defined and it is possible that it reflects a marker in linkage disequilibrium (LD) with other functional polymorphism elsewhere in the β‐globin gene cluster. It has been suggested that the role assigned to XmnI could be played by the rs16912979 A &gt; G polymorphism in the palindromic region of 5′HS4 β‐globin locus control region (Neishabury et al., Blood Cells Mol Dis. 2012;48:1‐5). Aims: The main goal of this work was to define haplotype backgrounds in β‐globin gene cluster that could explain HbF variation in a population sample of β‐thal carriers. Methods: We studied 79 Portuguese β‐thal carriers (39 males, 40 females), aged between 2 to 70 years old, with HbF levels ranging from 0.2% to 5.1%. HbA2 levels were 3.4 to 5.8% (mean 4.71). Six different HBB mutations were identified at heterozygous state, the two most commons were c.92+6T &gt; C [IVSI‐6 (T &gt; C)] (n = 30) and c.118C &gt; T [CD39 CAG &gt; TAG)] (n = 23). The remaining mutations were c.48G &gt; A [CD15 TGG &gt; TGA] (n = 11), c.92 + 1G &gt; A [IVS‐I‐1 (G &gt; A)] (n = 10), c.93‐21G &gt; A [IVSI‐110 G &gt; A] (n = 3) and c.20delA [CD 6 ‐A] (n = 2). HbA2 and HbF levels were determined by HPLC. The two SNPs rs16912979 and rs7482144 were genotyped by PCR‐RFLP and the HBB gene mutations were identified by Sanger sequencing. Associations of SNPs with HbF levels were assessed by linear regression models using PLINK. Informed consent was provided by all the participants. Results: The two SNPs HBG2 ‐XmnI rs7482144 and 5′HS4 rs16912979 were in moderate LD (r 2 = 0.65): from a total of 41 rs16912979 G alleles, 73.2% were linked to an rs7482144 T‐allele. Linear regression, in the additive model, showed significant association between the XmnI rs7482144 minor T‐allele and increased levels of HbF (β = 0.648; P = 0.0004). A marginal association was found between the 5’HS4 rs16912979 minor G‐allele and HbF (β = 0.648; P = 0.039), however, no significant association was found after conditioning on XmnI rs7482144 (P = 0.192). The common mutation HBB : c.118C &gt; T [CD39] was found strongly associated with increased levels of HbF (β = 0.948; P = 6.4e‐5), remaining significant after conditional analysis on XmnI rs7482144 (P = 0.0002). Haplotypes combining the HbF‐associated alleles on SNPs rs16912979 and rs7482144 (haplotype GT) showed a significant association with raised levels of HbF (P = 0.0008), explaining 13.7% of HbF levels variation. Combining rs16912979, rs7482144 and mutation HBB : c.118T, three haplotypes were found significantly associated with HbF: GTT (β = 1.346; P = 0.00089), ACT (β = 0.784; P = 0.017) and GTC (β = 0.492; P = 0.032), explaining 13.4%, 7.2% and 5.9% of HbF levels variance, respectively. Summary/Conclusion: Our data suggest that in Portuguese β‐thal carriers the HBG2 ‐XmnI polymorphism and HBB : c.118C &gt; T [CD39] mutation are associated with HbF levels, showing independent effects. The marginally significant effect on HbF levels variation of SNP 5’HS4 rs16912979 appear to be mediated by the effect of XmnI polymorphism.