Novel large deletions in the human alpha-globin gene cluster: Clarifying the HS-40 long-range regulatory role in the native chromosome environment

Globin genes, which encode the protein subunits of hemoglobin (Hb), are organized in two different gene clusters and present a coordinated and differential pattern of expression during development. Concerning the human alpha-globin gene cluster (located at chromosome region 16p13.3), four upstream highly conserved elements known as multispecies conserved sequences (MCS-R1-4) or DNase I hypersensitive sites (HSs) are implicated in the long-range regulation of downstream gene expression. However, only the absence of the MCS-R2 site (HS-40) has proven to drastically downregulate the expression of those genes, and consequently, it has been regarded as the major and crucial distal regulatory element. In this study, Multiplex Ligation-dependent Probe Amplification was used to screen for deletions in the telomeric region of the short arm of chromosome 16, in an attempt to explain the alpha-thalassemia or the HbH disease present in a group of Portuguese patients. We report four novel and five uncommon deletions that remove the alpha-globin distal regulatory elements and/or the complete alpha-globin gene cluster. Interestingly, one of them occurred de novo and removes all HSs except HS-10, while other eliminates only the HS-40 site, the latter being replaced by the insertion of a 39 nucleotide orphan sequence. Our results demonstrate that HS-10 alone does not significantly enhance the alpha-globin gene expression. The absence of HS-40 in homozygosity, found in a patient with Hb H disease, strongly downregulates the expression of alpha-globin genes but it is not associated with a complete absence of alpha-globin chain production. The study of naturally occurring deletions in this region is of great interest to understand the role of each upstream regulatory element in the native human erythroid environment.