α-Thalassemia Associated with Hb Instability: A Tale of Two Features. The Case of Hb Rogliano or α1 Cod 108(G15)Thr→Asn and Hb Policoro or α2 Cod 124(H7)Ser→Pro.

Abstract

We identified two new variants in the third exon of the α-globin gene in families from southern Italy: the Hb Rogliano, α1 cod108 ACC>AAC or α1[α108(G15)Thr→Asn] and the Hb Policoro, α2 cod124 TCC>CCC or α2[α124(H7)Ser→Pro]. The carriers showed mild α-thalassemia phenotype and abnormal hemoglobin stability features. These mutations occurred in the G and H helices of the α-globin both involved in the specific recognition of AHSP and β1 chain. Molecular characterization of mRNA, globin chain analyses and molecular modelling studies were carried out to highlight the mechanisms causing the α-thalassemia phenotype. The results demonstrated that the α-thalassemia defect associated with the two Hb variants originated by different defects. Hb Rogliano showed an intrinsic instability of the tetramer due to anomalous intra- and inter-chain interactions suggesting that the variant chain is normally synthesized and complexed with AHSP but rapidly degraded because it is unable to form the α1β1 dimers. On the contrary in the case of Hb Policoro two different molecular mechanisms were shown: the reduction of the variant mRNA level by an unclear mechanism and the protein instability due to impairment of AHSP interaction. These data highlighted that multiple approaches, including mRNA quantification, are needed to properly identify the mechanisms leading to the α-thalassemia defect. Elucidation of the specific mechanism leads to the definition of a given phenotype providing important guidance for the diagnosis of unstable variants.

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

“…The sequencing of the α1-globin gene cDNA from reticulocytes revealed the presence of mutated cDNA ( Figure 1 C). The anomalous peak T was of similar intensity to the normal C. To assess if the mutant mRNA underwent a quality control system triggering a degradation pathway and to evaluate the level of mutant cDNA, we performed the semi-quantitative analysis of the homoduplex and heteroduplex cDNA bands following DG-DGGE separation [ 21 , 22 ]. As shown in Figure 1 E, the four bands had similar intensity.…”

“…As shown in Figure 1 E, the four bands had similar intensity. Analysis of the data by the KodaK Carestream MI software indicated that the amount of Hb Bernalda mRNA in the reticulocytes from peripheral blood was comparable to the normal α2-globin mRNA ( Figure 1 E) [ 21 ].…”

“…Proper amplification-refractory mutation system (ARMS-PCR) assays for the definition of the heterozygous Hb Bernalda, Hb Caserta, and Hb Sun Prairie genotypes were set up [ 13 ]. The analysis of the three SNPs, RsaI 5’ of the α2-globin gene (rs2541669), α2 + 14 (HBA2:c.−24C>G rs772829778), and α2 + 861 at the 3’ UTR of HBA2 (HBA2:c.* 136A>G, rs2685121) were performed as previously reported [ 19 , 20 , 21 , 22 ]. The analysis of the polymorphism in the promoter of the UGT1A1 gene was carried out by sequencing.…”

“…Purification of mRNAs, from reticulocyte-enriched peripheral blood cells and from peripheral blood stem cells (PBSC) were differentiated in vitro, as well as reverse transcriptions were performed according to references [ 21 , 22 ]. The qualitative and semi-quantitative cDNA analyses, specifically sequencing, separation on acrylamide gel, BstNI restriction enzyme digestion, and double-gradient denaturing gradient gel electrophoresis (DG-DGGE) separation, were performed as already reported [ 21 , 22 ].…”

“…The separation and differentiation in vitro of the PBSCs, from one normal subject and one carrier of Hb Southern Italy, and the separation and extraction of the RNA from the nuclei and cytoplasm were performed as published elsewhere [ 21 , 22 ].…”