Characterization of a Newly Discovered Human Alpha-Globin Gene.
Authors: Goh Sung-Ho , Lee Y. Terry , Bhanu Natarajan , Gherman Robert , Cam Margaret C. , Miller Jeffery L. 
Published: April 2020

It is well known that the globin genes are organized within the human genome as two gene clusters on chromosomes 16 and 11 (α cluster: ζ2-ζ1-α2-α1-𝛉 and β cluster: ε-Gγ-Aγ-δ-β). Switching in the expression patterns of those genes during human ontogeny generally follows a pattern determined by their genomic arrangement, but the genetic mechanism is not defined. In an effort to better understand the γ-to-β globin switching phenomenon, gene expression patterns from reticulocyte RNA were studied. cDNA were generated from the reticulocytes of 28 separate donors (14 cord blood; 14 adult blood) and analyzed using Affymetrix HG-U133 arrays. Quantitative PCR was used to confirm the array expression patterns. Among those cDNA identified as having expression patterns that were relatively decreased among adult reticulocytes, we were surprised by expression associated with a cluster of expressed sequence tags (EST) located within the α-globin locus in the region between the ζ1 and α2 genes. In the context of the 44,229 arrayed probes, high-level expression from that EST cluster was detected (ranked &lt; 25th). Informatic analysis revealed alignment with the second and third exons previously described for the Ψα2 globin gene, but no significant similarity with the first exon of Ψα2. Based upon these comparisons, we used adult human reticulocyte RNA to amplify a cDNA clone for further study (GenBank: AY698022; named μ-globin). Sequencing of that clone revealed an insert that encodes a predicted 423 nt. open reading frame that is one amino acid residue shorter than the α2-globin gene. While the mature μ-globin mRNA results from splicing of two introns, it lacks a canonical Kozak sequence. BLASTP and CLUSTALW analyses revealed the highest level of homology with the avian αD-globin protein with a sequence identity of 55% (78/141 amino acids). In addition, the predicted heme- and globin-binding amino acids of μ-globin and avian αD-globin are largely conserved. Experimentally, we analyzed the expression level of the μ-globin transcripts using quantitative real-time PCR. Non-erythroid tissues including whole brain, white blood cells, and the Jurkat cell line demonstrated only background levels. The expression level of μ-globin (copies per ng cDNA) was 3.04x10(4) ± 1.68x10(3) in fetal liver, 1.71x10(5) ± 9.51x10(4) in cord reticulocytes, 1.15x10(4) ± 1.19x10(3) in bone marrow, and 2.17x10(4) ± 6.84x10(3) in adult reticulocytes. Based upon the consistent decrease in μ-globin expression between fetal and adult erythroid tissues, we measured the expression of γ-globin in those tissues for comparison. The expression level of γ-globin (copies per ng cDNA) was 2.49x10(6) ± 1.48x10(5) in fetal liver, 1.49x10(8) ± 7.88x10(7) in cord reticulocytes, 3.84x10(3) ± 3.83x10(2) in bone marrow, and 5.70x10(5) ± 5.80x10(5) in adult reticulocytes. To determine if μ-globin expression is regulated during erythropoiesis, we assayed primary human erythroid progenitor cells cultured in erythropoietin. The expression pattern of μ-globin was similar to the other globin genes increasing from background levels on day 0 to maximum levels in committed erythroblasts (3.56x10(5) ± 1.99x10(4) copies per ng cDNA). These results suggest that the human genome encodes a previously unrecognized α-globin that most closely resembles avian αD-globin. Expression of that gene is decreased during the fetal-to-adult transition of human ontogeny and regulated during erythropoiesis.