Severe Impairment of γ-Globin Gene Silencing in an Asymptomatic Adult Patient Homozygous for the Codon 8 (–AA) Frame-Shift β⁰-Thalassemia Mutation

Abstract 1022

Beta-thalassemia is caused by β-globin gene mutations that result in either markedly decreased (β⁺) or absent (β⁰) β-globin chain production. Patients who are either homozygous or compound heterozygous for β-thalassemia mutations are severely anemic and require chronic RBC transfusions. Concomitant inheritance of an α -thalassemia mutation or increased HbF (α ₂γ₂) expression can ameliorate the disease severity. Surprisingly, many patients homozygous for the codon 8 (–AA) β⁰-thalassemia mutation are mildly anemic with over 95% HbF [Altay & Gürgey, Ann NY Acad Sci 612:81, 1990]. We now report one such patient, a 20-year old man of Iraqi ancestry who was found to have splenomegaly but was otherwise well. His hemoglobin was 12.4 g/dL, MCV 77 fL, reticulocyte count 3.2%. Hemoglobin analysis by HPLC revealed HbF 98%, HbA₂ 2%. Multiplex ligation-dependent probe amplification (MLPA) of his β-globin gene cluster confirmed that he did not have a large deletion as found in some patients with hereditary persistence of fetal hemoglobin (HPFH), nor γ-globin gene quadruplication. In addition, he was heterozygous for the α 2 IVSI donor splice site 5 bp deletion (–GTGAG), the α ^Hph thalassemia mutation. To determine the genetic basis for his persistently high HbF expression, his γ-globin gene promoters were sequenced and no HPFH point mutation was found. We next assessed the status of his 3 major HbF quantitative trait loci (QTL). He was homozygous for SNP rs7482144 C>T minor allele (Xmn I polymorphism) at 158 bp 5' to the ^Gγ-globin gene on chr 11p15; homozygous for rs9399137 minor allele in the HBSIL-MYB intergenic polymorphism (HMIP) on chr 6q23, homozygous for the 3-bp deletion which is in linkage disequilibrium with rs9399137 minor allele [Farrell et al, Blood 117:4935, 2011]; and heterozygous for rs766432 minor allele in the 2^nd intron of BCL11A on chr 2p16. Thus he had alleles associated with elevated HbF in all 3 QTL. No mutation was found in his KLF1 genes. Within HS2 in his β LCR is the motif (TA)₉ (CA)₂ (TA)₂ CG (TA)₁₀ which is found in Senegal β-globin haplotype 3 [Öner et al, Blood 79:813, 1992]. The Corfu deletion removes part of the δ-globin gene and ∼6 kb upstream flanking sequence, encompassing the 2-kb region reported to be necessary for γ-globin gene silencing [Sankaran et al, NEJM 365:807, 2011], and is often in LD with the IVSI-5 G>A severe β⁺-thalassemia mutation. Corfu heterozygotes have slightly increased HbF, but Corfu homozygotes have HbF ∼95%. We sequenced 6.5 kb upstream of δ-globin gene in our patient, and found homozygosity throughout and only 2 nucleotide differences from the GRCh37/hg19 assembly sequence: C>T (rs3759074) at 2,065 bp, and T>G (rs7948416) at 718 bp upstream of δ globin gene transcription start site. No deletion was found. At the repressor protein BP1 binding site 530 bp upstream of the β-globin gene, our patient had the common reference sequence, (AC)₂ (AT)₇ T₇. Among 13 subjects heterozygous for codon 8 (–AA) β⁰-thalassemia mutation, their Hb was 11.6 ± 1.8 g/dL, HbF 2.8 ± 2.6% [Öner et al, Hemoglobin 14:1, 1990]. We studied two unrelated codon 8 (–AA) heterozygotes. One was a 37-year old woman with Hb 9.8, HbA 88%, HbF 5.7%. She was homozygous for the Xmn I polymorphism, and heterozygous for the HbF QTL on chr 6q23 and 2p16. The other was a 24-year old woman with Hb 11.2, HbA 90%, HbF 7.5%. She was homozygous for the Xmn I polymorphism, and heterozygous for the HbF QTL on chr 2p16. These results support the hypothesis that determinant(s) in cis to the β-globin gene cluster, including the Xmn I QTL and related functional motif(s), in concert with HMIP and BCL11A QTL can sustain high-level γ-globin gene transcription in adults. Robust γ-mRNA accumulation and HbF expression occur only when β-mRNA is markedly decreased due to nonsense mediated decay in the codon 8 (–AA) homozygote, as has been shown in patients homozygous for the Corfu deletion [Chakalova et al, Blood 105:2154, 2005] and in experimental model system [Russell, Eur J Haematol 79:516, 2007]. Our findings could have implications for the therapeutic design to induce HbF expression in β-hemoglobinopathies.