Filomena Seuanes

Novel large deletions in the human α-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.

Mutational spectrum of delta‐globin gene in the Portuguese population

The phenotype of increased Hb A2 typical of β‐thalassaemia (β‐thal) carriers can be reduced to normal or borderline values because of the co‐inheritance of a δ‐globin gene (HBD, MIM #142000) mutation, which may lead to misinterpretation of diagnostic results. To know the spectrum of δ‐globin mutations in the Portuguese population we performed a mutational analysis of the δ‐globin gene in a group of 51 Portuguese β‐thal carriers presenting microcytosis, hypochromia and a normal/borderline Hb A2 level and in another group of 15 individuals suspected to have δ‐globin structural abnormalities. The heterozygosity for the β+IVS‐I‐6T→C (HBB:c. 92+6T>C) mutation was the main cause for the mentioned atypical β‐thal carrier phenotype. Furthermore, eight individuals were double heterozygous for one common β‐thal mutation and the δ+Cd27G→T mutation (Hb A2–Yialousa; HBD:c.82G>T). One of them also presented a novel δ‐globin gene promoter mutation,−80G→A (HBD:c.−130G>A), responsible for about 25% decrease of the promoter activity in transient expression assays. One the other hand, in the other group of 15 individuals suspected to have δ‐globin structural abnormalities observed by biochemical methods, some known Hb A2 variants were identified – Hb A2′ (HBD:c.49G>C), Hb A2‐Babinga (HBD:c.410G>A), and Hb A2‐Wrens (HBD:c.295G>A), and the novel Hb A2‐Fogo [δ64(E8)(Gly→Ser); (HBD:c.193G>A)]. This novel Hb A2 variant was observed segregating in linkage with Hb E (HBB:c.79G>A) in a three generation family. In conclusion, six different δ‐globin mutations were found, being two of them new molecular defects. All δ‐alleles identified were found linked to the expected β‐globin cluster haplotype. All mutations caused a low Hb A2 level and through this could lead to misdiagnosis when inherited together with a β‐thal allele.