Effect of Mutations on mRNA and Globin Stability: The Cases of Hb Bernalda/Groene Hart and Hb Southern Italy

Abstract

We identified two unstable variants in the third exon of α-globin genes: Hb Bernalda/Groene Hart (HBA1:c.358C>T), and Hb Caserta (HBA2:c.79G>A) in cis to Hb Sun Prairie (HBA2:c.391G>C), also named Hb Southern Italy. These mutations occurred in the H helix of the α-globin that is involved in heme contacting, specific recognition of α-hemoglobin-stabilizing protein (AHSP), and α1β1 interactions. The carriers showed α-thalassemia phenotype, but one also jaundice and cholelithiasis. Molecular identification of clusters of families in Southern Italy encouraged molecular characterization of mRNA, globin chain analyses, molecular modeling studies, and comparison with globin variants to understand the mechanisms causing the α-thalassemia phenotype. A normal amount of Hb Bernalda/Groene Hart mRNA were found, and molecular modeling highlighted additional H bonds with AHSP. For Hb Southern Italy, showing an unexpected α/β biosynthetic ratio typical of the β-thalassemia type, two different molecular mechanisms were shown: Reduction of the variant mRNA, likely due to the No-Go Decay for the presence of unused triplet ACG at cod 26, and protein instability due to the impairment of AHSP interaction. The UDP glucuronosyltransferase 1A (UGT1A1) genotyping was conclusive in the case of jaundice and cholelithiasis. Multiple approaches are needed to properly identify the mechanisms leading to unstable variants and the effect of a mutation.

Impact of α-Globin Gene Expression and α-Globin Modifiers on the Phenotype of β-Thalassemia and Other Hemoglobinopathies: Implications for Patient Management

“…Finally, it is of interest to note that several α-globin chain variants have been described, which are prone to structural instability associated with structural alterations and impaired interaction with AHSP [ 69 , 70 , 71 ]. This is the case of the recently described α-globin frameshift mutants with stop codon in the last exon at codon 102 (Hb Campania) or at codon 133 (Hb Sciacca) [ 71 ].…”

Functional analysis of three new alpha-thalassemia deletions involving MCS-R2 reveals the presence of an additional enhancer element in the 5’ boundary region

“…The effects of the MCS-R2 deletions on the α-globin genes transcription were investigated by measuring the ratio of the mRNA synthesized by the two α2-globin genes through expression analysis by the restriction enzyme (RE) NlaIV recognizing the SNP +14C>G on the α2 globin cDNA [26][27][28][29].…”

“…α2-globin alleles in heterozygous subjects. The digested fragments were separated on a NuSieve 3:1 agarose 3.5% gel (FMC, Rockland, Maine, USA), and a semi-quantitative analysis of the bands was performed with the Kodak software Carestream MI, as already reported [26][27][28][29]. A corrective factor was used to normalize for the loss of a shorter band of 11 bp and the efficiency of digestion of the NlaIV was performed (S1 Fig) . The data on cDNAs samples were confirmed two times on different mRNA extractions.…”

mRNA Analysis of Frameshift Mutations with Stop Codon in the Last Exon: The Case of Hemoglobins Campania [α1 cod95 (−C)] and Sciacca [α1 cod109 (−C)]

“…The amplicomers were digested, according to the manufacturer's recommendations, with 10 U of RE, and the restricted bands were size-fractionated in a 3.5% NuSieve 3:1 agarose gel (FMC, Rockland, Maine, U.S.A.) at 70 V for 2-3 h. After separation on the NuSieve gel, a semiquantitative analysis of the bands was performed with the Kodak software Carestream MI (Carestream Health, Inc, Rochester, NY, U.S.A.). The ratio of undigested/digested bands (that is, mutated/wild type (WT) DNA and cDNA) was obtained as previously reported [13][14][15][16].…”

“…In particular, the inability of the Hb Campania α-chain to interact with the AHSP chaperone causes strong instability (Figure 2, Figure S1A-C and S1D-F). The mutant α-chains impairing the interactions with AHSP are usually characterized by a rapid degradation and for this reason often are not identified [15,16]. The Hb Campania has not been identified in the peripheral blood of patients, and this confirms that the variant chain Hb Campania is unstable and could be rapidly degraded and not involved in the formation of the hemoglobin tetramer.…”