Beta-thalassemia: from genotype to phenotype

Beta-thalassemias are heterogeneous autosomal recessive hereditary anemias characterized by reduced or absent β globin chain synthesis. The resulting relative excess of unbound α globin chains precipitate in erythroid precursors in the bone marrow, leading to their premature death and, hence, to ineffective erythropoiesis. β-thalassemia phenotypes are variable, ranging from the severe transfusion dependent thalassemia major to the mild form of thalassemia intermedia. Patients with the major form of the disease have severe anemia, microcytic and hypochromic anemia, hepatosplenomegaly, and usually come to medical attention within the first two years of life. Without treatment, affected children have severely compromised growth and development and shortened life expectancy. Treatment with a regular transfusion program and chelation therapy, aimed at reducing transfusional iron overload, allows for normal growth and development and extends life expectancy into the third to fifth decade.¹ Individuals with thalassemia intermedia present later in life, have milder anemia (that never or only rarely requires transfusion), liver and spleen enlargement, typical bone modifications, and mild to moderate jaundice.² Occasionally patients with thalassemia intermedia are completely asymptomatic until adult life with only mild anemia. The major and intermedia forms of the disease are the two extremes of a wide range of clinical variability. Each group includes a continuous scale of severity, as demonstrated by the variability in age at which thalassemia major patients need transfusion; from months to years of life.

Beta-thalassemias are also very heterogeneous at the molecular level, with more than 200 disease-causing mutations so far identified; a complete updated list is available at the Globin Gene Server Web Site - http://globin.cse.psu.edu/. In most cases, mutations are single nucleotide substitutions, deletions or insertions of single nucleotides or small oligonucleotides leading to frameshift. Their diversity and the consequent variable degree of globin chain imbalance are the main determinants for milder phenotypes, the coinheritance of homozygosity or compound heterozygosity for mild β-thalassemia alleles being responsible for a consistent residual output of β chains from the affected β globin locus. However, much of the phenotypic variability is also explained by other genetic determinants capable of reducing the α/non-α chain imbalance thereby resulting in a lesser degree of α chain precipitation.³

One of the first discovered mechanisms able to reduce this imbalance is the coinheritance with homozygous β-thalassemia of α-thalassemia determinant. In this case, the severity of the clinical phenotype correlates with the α globin chain deficiency and with the improved α/non-α chain imbalance as a consequence of reduced α chain output.⁴

A substantial decrease in α/non-α chain imbalance can also be obtained through the coinheritance of genetic determinants able to sustain a continuous production of gamma chains which, binding the excess α chains, result in a persistent fetal hemoglobin (Hb F) production measurable in adult life. In delta-β0 thalassemia, this ability is due to deletions of variable extent within the β globin cluster,⁵ while in other cases it depends on the co-transmission of point mutations at A-gamma or G-gamma promoters (−196 C→T A-gamma; −158 C→T G-gamma).^6,7 A mild phenotype may also be determined by coinheritance of genetic determinants associated with increased gamma chain production mapping outside the β globin cluster. Different polymorphisms at the BCL11A gene on 2p16.1⁸ and HBS1L-MYB intergenic region on 6q23.3 have been described.^9,10 Several polymorphisms within intron 2 of the BCL11A gene have been strongly associated with Hb F levels: rs766432, rs4671393, rs1427407 and rs11886868, all in high linkage disequilibrium (LD) with each other.^11–15 Other independent signals in the same area were also identified with rs10189857 and rs7599488, in high LD, as well as rs7606173 and rs6706648,¹³ also in high LD with each other.^12,13 In the HBS1L-MYB intergenic region, different SNPs have been described as being associated with Hb F variations in different studies: rs9399137, as well as rs4895441, rs9402686 and rs28384513.^10,12 However, evidence has been reported of other contributing loci that have not been validated in recent genome-wide association studies, such as the 8q26-28 and Xp22.2–22.3 loci.^16,17

Together with these discoveries, the interest for prediction of Hb F levels and β-thalassemia phenotype has naturally grown in recent years, and the three loci previously mentioned have now been reported to be responsible for 20 to 50% of the Hb F trait variance in patients with β-thalassemia or sickle cell disease, and in healthy Europeans.¹⁸ Meanwhile, Galanello et al. reported the impact of variants in the BCL11A and HBS1L-MYB loci together with α gene defects on the clinical severity of β0-39-thalassemia, quantifying their overall contribution to 75% of the variation differences between β0-39-thalassemia major and intermedia phenotypes.¹⁴

The work by Badens et al., presented in this issue of the Journal, extends previous studies by integrating the −158 C→T G-gamma polymorphism and β0/β+ status, in addition to rs11886868 in the BCL11A gene, rs9389268 in the HBS1L-MYB intergenic region and α globin genes defects, to define β-thalassemia severity.¹⁹ Multivariate analysis including these five genetic modifiers was carried out and an accurate prediction has been made regarding major/intermedia status in more than 80% of patients. The heterogeneity of this 106 patient cohort, with thirty different β globin gene mutations, might introduce variability not accountable for in the model, but it certainly provides a large amount of information on the prediction ability of the β0/β+ status. Also, while it is likely that future studies will better define the genetic polymorphisms that modulate the effect of the BCL11A and HBS1L-MYB intergenic region loci and eventually uncover causal variants, results from Badens et al. already provide clinically relevant information to practitioners, clarifying the impact of genetic modifiers on the clinical severity of the disease. Furthermore, future studies will probably expand this predictive ability, including the effect of the different strongest independent predictors known to date for each gene (or even identified causal variants), and will eventually relate genetic modifiers to a more detailed measurement of clinical severity.

Very few other complex disease phenotypes can be explained in such depth, and prediction of patient risk as a function of their personal genetic background already offers support in clinical settings for β-thalassemia as opposed to most complex diseases. Extended molecular diagnosis can be carried out in patients affected by homozygous β-thalassemia to define their genotype for different modifiers and to better understand their phenotypic modulation abilities. Before long, β-thalassemia modifiers should allow us to redefine, on a genetic basis, the phenotypic definition actually in use.

Linkage analysis and genome-wide association studies have greatly contributed to such results, and next generation sequencing might further improve prediction ability and eventually guide the development of new therapies. At present, recent studies on Hb F modifier genes have produced mixed results: while a 3bp deletion associated with Hb F levels has been recently identified in the HBS1L-MYB intergenic region,²⁰ no such results have been obtained with the sequencing of the BCL11A gene. We hope that finally, in addition to providing detailed information to help promote enhanced β-thalassemia pre-natal screening, achieving these objectives will also help to identify the mechanisms responsible for fetal hemoglobin control, since reactivation of fetal hemoglobin can provide major therapeutic benefits to people affected by β-hemoglobinopathies.