A Mutation in a GATA-1 Binding Site 5′ to the Gγ-Globin Gene (nt -567, T&gt;G) May Be Associated with Increased Levels of Fetal Hemoglobin.
Authors: Luo H.Y. , Mang D. , Patrinos G.P. , Wu C.J.Y. , Eung S.H. , Pourfarzad F. , Whitacre J. , Rosenfield C.G. , Daoust P.R. , Braun A. , Grosveld F.G. , Steinberg M.H. , Chui D.H.K. 
Published: April 2020

Abstract
The identification of single nucleotide polymorphisms (SNPs) associated with increased fetal hemoglobin (Hb F) levels in adults provide important insights to the regulation of γ-globin gene expression, and the modulation of Hb F production in severe β hemoglobinopathies. Fourteen known point mutations located between nucleotide (nt) −110 to −205 5′ to the Gγ- and Aγ-globin genes are associated with hereditary persistence of fetal hemoglobin (HPFH). These likely affect the interactions between transcription factors and proximal promoter elements. We investigated both parents and 2 sons of an Iranian-American family, in whom the father and his younger son had elevated Hb F levels (See table). The mother and her older son were heterozygous for the (−α3.7) single α-globin gene deletion, likely accounting for their borderline microcytosis. Extensive nt sequencing of the β-globin gene and promoters of Gγ- and Aγ-globin genes was carried out. No known β-thalassemia mutation was detected in any of the 4 family members. None of the known HPFH point mutations was present. The C&gt;T SNP (Xmn I) at nt −158 5′ to the Gγ-globin gene that has been associated with increased Gγ-globin gene expression was also not found. However, a novel T&gt;G substitution was detected at nt −567 5′ to the Gγ-globin gene in the father and his younger son, but not in the mother and her older son. This SNP alters a putative GATA-1 binding sequence, AGATAA to AGAGAA. Haplotyping of the Gγ Aγβ region in the family showed that the T&gt;G SNP in the father and his younger son resides on the same GγAγβ haplotype. This SNP was not present in 15 individuals of diverse racial and ethnic origins, in 186 Thai individuals, and in 133 of 134 Iranians living in Tehran. To our knowledge, this SNP has not been previously reported in the literature. To begin to study the functional significance of this SNP, gel mobility shift analysis was done with two 40 nt long oligomers, one with the wild type GATA sequence and the other with the mutated GAGA sequence, using uninduced mouse erythroleukemia (MEL) cell nuclear extracts. The mutant GAGA sequence results in a complete loss of GATA-1 binding. The region from nt −382 to −730 5′ to the Aγ-globin gene was reported to be related to Aγ-globin gene silencing (Stamatoyannopoulos et al, Mol Cell Biol 13:7636, 1993). The nt −567 T&gt;G SNP is located within the comparable region of the Gγ-globin gene, which is highly homologous to the Aγ-globin gene. Among the 4 family members under study, no other SNPs are found in the same region. Taken together, these observations raise the possibility that the T&gt;G SNP at nt −567 5′ to the Gγ-globin gene is associated with elevated Hb F, that might be caused by a novel mechanism, i.e., incomplete silencing of the Gγ-globin gene, resulting from the abolished GATA-1 binding. Additional clinical and functional studies will be needed to further document the effect of this SNP upon Gγ-globin gene expression and to ascertain that this SNP represents a HPFH mutation.
Hematological Data of Family
Father Mother Son Son Age 52 44 13 9 Hb 15.7 14.1 13.2 13.8 MCV 82 77 75 75 Hb A2 2.5 % 3.0 % 3.4 % 3.3 % Hb F 10.2 % 0.7 % 0.7 % 5.9 %