Role of nonsense-mediated decay and nonsense-associated altered splicing in the mRNA pattern of two new α-thalassemia mutants

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.…”

“…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 ].…”

Hb Aalesund (HBA2: c.400_406delAGCACCG), an Unstable α-Globin Variant Found in a Norwegian Patient Causing Moderate Hemolytic Anemia and Falsely High Hb A1c Using Ion Exchange High Performance Liquid Chromatography

“…Normally, transcripts that contain a PTC undergo nonsense-mediated decay (NMD) and rapidly degrade, thus eliminating abnormal transcripts that could have a dominant negative effect [8,9]. If the PTC is located in the last exon or in the 3' end of the penultimate exon, less than 50-55 base pairs from the final intron, the PTC will escape the surveillance system and a truncated protein will be translated [9].…”