The α globin gene cluster, which contains embryonic (ζ) and fetal/adult (α) genes, has been highly conserved throughout evolution.1 Four putative regulatory elements (called MCS-R1 to MCS-R4; Figure 1) corresponding to erythroid-specific DNase1 hypersensitive sites (HSs)2 are found upstream (30-160 kb) of the α genes in most mammalian species, but the relative importance of each of these elements in each species has not been fully elucidated. In humans, characterization of natural deletions,3-5 analysis of interspecific hybrids,3,6 stable transfectants,6 and studies of transgenic mice7 all indicate that the most highly conserved element (MCS-2, which corresponds to HS-40 in humans) is the major regulatory element; on their own, the other elements (MCS-1, -3, and -4) cannot drive substantial levels of α globin expression. Targeted deletion of HS-40 in a hybrid containing human chromosome 16 in a mouse erythroleukemia (MEL) background reduces human α globin expression to less than 5% of normal.6 It was therefore surprising to find that deletion of the highly conserved MCS-R2 element (mHS-26) in mice, using homologous recombination, only reduced α globin expression to approximately 50% of normal,8 suggesting that the 4 elements may play different roles in different species and casting some doubt on the value of the mouse as a faithful model of human α globin gene expression.8 At present, the only way to determine the role of individual regulatory elements in situ, in the normal human erythroid environment, is to identify natural mutants in human populations.
We have previously characterized 9 deletions that remove various combinations of the upstream regulatory elements and cause α-thalassemia.5 In all cases, MCS-R1, -R2, and -R3 are removed and in 4 cases, MCS-4 is also deleted. Here we describe a small, approximately 16-kb interstitial deletion that removes only MCS-R1 (HS-48) and MCS-R2 (HS-40) and that caused α-thalassemia in a Filipino girl. The combination of severely reduced red cell indices (hemoglobin [Hb], 113 g/L [11.3 g/dL]; mean corpuscular volume [MCV], 65 fL; and mean corpuscular hemoglobin [MCH], 19.48 pg), normal Hb analysis (HbF < 1% and HbA2 = 2.2%), demonstrable HbH inclusion bodies, and a significantly reduced α/β globin chain synthesis ratio (0.71) suggested that she had α-thalassemia with inactivation of at least 2 of the normal 4 α globin genes. Analysis of interspecific hybrids confirmed that expression from the affected chromosome is reduced to less than 1% of normal (Figure 1). Although we have confirmed that the α genes are structurally normal (data not shown), analysis of the upstream region using multiplex ligation-dependent probe amplification (MPLA) analysis identified a deletion removing MCS-R2 (HS-40).10 DNA sequence across the breakpoint shows that this deletion (αα)ZW extends for 15 997 bp (coordinates, 90 778-106 773 bp) and that it arose via recombination between 2 Alu repetitive elements (Figure 1), as observed for many other deletions in this region of the genome.
This patient provides the first example of an upstream deletion in which MCS-R3 (HS-33) remains intact (Figure 1A), demonstrating that this element alone or in combination with MCS-R4 has no significant positive effect on α globin expression. To fully demonstrate that, in humans, in contrast to mice, HS-40 is the dominant α globin regulatory element, it now only remains to be seen whether MCS-R1 (HS-48) can act alone or in combination with MCS-R2 (HS-40) in human erythroid cells. Future identification of an upstream deletion, including MCS-R2, in which MCS-R1 remains intact, would therefore complete the search for such deletions andhelp clarify the apparent differences between the regulation of α globin expression in mice and humans.