Patients Homozygous For Codon 8 (–AA) Frame-Shift β⁰-Thalassemia Mutation With Markedly Increased HbF

Patients with β-thalassemia major caused by homozygosity or compound heterozygosity for β⁰-thalassemia mutations are severely anemic and require chronic RBC transfusions. We presented last year in these meetings an Iraqi-American family, in whom 21-year old non-identical twin brothers were found to be homozygous for codon 8 (–AA) β⁰-thalassemia mutation, and heterozygous for α2 IVSI donor splice site 5 bp deletion, the α^Hph thalassemia mutation. Both were clinically well except for splenomegaly, and were never transfused. Their hemoglobin concentrations were 12-13g/dL, MCV 72-73fL, reticulocyte counts 1.5-1.9%, and HbF (α₂γ₂) 97-98%. Extensive genotyping was done in an attempt to decipher the genetic variants responsible for the marked impairment of γ-globin gene silencing in the twins. Both were found to harbor 5 of the possible 6 minor alleles among the 3 known major HbF quantitative trait loci (QTL), rs7482144 (Xmn I polymorphism) 5’ to the ^Gγ-globin gene, rs9399137 in the HBSIL-MYB intergenic polymorphism, and rs766432 in the 2^nd intron of BCL11A. Interestingly, their mother and sister were both heterozygous for codon 8 (–AA) β⁰-thalassemia mutation, heterozygous for α^Hph thalassemia mutation, and possessed 5 of the possible 6 minor alleles among the 3 major HbF QTL. Their hemoglobin concentrations were 12-13g/dL, and MCV 74-76fL, yet their HbF was merely 4.3-5.4%. These findings raise the possibility that robust γ-globin mRNA accumulation and HbF synthesis occur only when β-globin mRNA is markedly decreased as in homozygosity for codon 8 (–AA) β⁰-thalassemia mutation. Since then, we have studied additional patients homozygous for codon 8 (–AA) mutation with different ethnic backgrounds: 6 from northern Iraq, 6 from Turkey, and 2 from Morocco. These patients were severely anemic, and most were regularly transfused. All but one were homozygous for the SNP rs7482144 minor allele (Xmn I polymorphism). For the other 2 HbF QTL represented by rs9399137 and rs766432, these patients had an average of 1 to 2 copies of the minor alleles, fewer than those present in the twin brothers. Of the 5 patients tested thus far, they did not have α-thalassemia. These observations support the notion that genetic propensity for increased HbF expression and/or concomitant inheritance of α-thalassemia mutation can moderate the disease severity of β-thalassemia major. But these do not fully account for the mild anemia and markedly increased HbF found in the twin brothers. We have now completed genome-wide exome sequencing on one of the twins. No mutation was found in many transcription factors known to be associated with erythroid cell differentiation or globin gene expression including GATA1, FOG1, GATA2, BCL11A, SOX6, KLF1, MYB, Mi2β. Mutations were found and confirmed by Sanger sequencing in several candidate genes of potential interest in erythropoiesis such as SP1, NR2C1, NOTCH2, LFNG, BRIP1, KAT6A, SMARCA2, ILF3. These exonic mutations, all non-synonymous single nucleotide variants except for one frameshift insertion mutation are either novel or have very low allele frequency as reported in the SNP database. Work is in progress to ascertain possible dysfunction of these encoded proteins, and their roles in erythropoiesis and globin gene expression.