Purpose: During a project on the molecular epidemiology of beta- and alpha-thalassemia in Southern Italy we identify two new thalassemic splicing mutants. The first was a beta-globin gene mutant characterized by duplication of tetranucleotide "AG/CT" at the junction IVS-II/exon 3, generating a pre-mRNA containing two 3' splice sites in the intron 2. The second was a silent mutation GGC>GGT (Gly>Gly) at codon 22 of the alpha-1 globin gene. It generates a splice donor site sequence (CTG/GTGAGT) at codons 22/23 with a score-splicing consensus (0.99) higher than the normal site (0.89). We would define the effect of the mutations on the splicing. Experimental description: To assess the presence of the variant beta- or alpha-globin chains we performed Hb analysis by HPLC and globin chain in vitro biosynthesis from reticulocytes. To evaluate the presence of normal and anomalous mRNA produced from the mutant alleles we analyzed globin mRNA from reticulocytes and from Peripheral Blood Stem Cell (PBSC) differentiated in vitro performing the following studies: a) separation on acrylamide gel of cDNA amplicomers containing P32 a-dCTP; b) sequencing analysis of amplicomer eluted by the acrylamide gel; c) measurement ratio of mRNA synthesized by the two beta-globin alleles taking advantage from a polymorphism at codon 2 C>T associated, with the presence (betaC)/absence (betaT) of the Alw44I restriction site. The new beta-thalassemic allele was associated with the codon 2 betaC. Results: Duplication of tetranucleotide "AG/CT": The beta-globin cDNA analysis did not reveal any trace of the mutant allele, but only cDNA from the normal allele indicating that our mutant did not contribute to the synthesis of (normal or mutant) beta-globin mRNA. Experimental analysis to prove the presence of anomalous mRNA highlighted two faint heteroduplex bands, absent in the normal control. Their sequencing showed to be aberrant cDNA with the "AGCT" insertion at the cod 105 of the beta-globin cDNA: moreover the semi quantitative analyses indicated to be about 1.5% of the normal one. In the anomalous mRNA the insertion causes a frameshift and synthesis of an abnormal truncated beta-chain (139 residues). Variant chains were not detected by HPLC in fresh blood or in the precipitate from test for unstable Hbs, most probably unable to form Hb variant because the severe conformational changes. The alpha/beta ratio was of beta-thalassemic type. Alpha-1 globin gene codon 22 GGC>GGT: Two cDNA of length shorter than normal were detected from PBSC and from reticulocytes of the carriers. The first one was mRNA abnormally spliced at codon 15; the other was 28 nucleotides shorter than normal because of an abnormal splicing at codon 22, leading to a frameshift and to the formation of the stop codon TGA at codon 39 (the same generated by abnormal splicing at codon 15). The anomalous/normal cDNA ratio in erythroid cells and reticulocytes was 0.08 and 0.02, respectively, for the first cDNA; 0.76 and 0.08, respectively, for the other. In normal subjects limited amounts of the cDNA splicing at codon 15 were detected. Conclusions: Duplication of tetranucleotide "AG/CT": Data reported herein supports the hypothesis that the cod 105 +AGCT is a novel beta-globin gene mutation that impairs the production of normal mRNA, but causes synthesis of abnormally spliced mRNA, which is present only in trace amounts (1.5%) and cannot be translated into a functional ?-chain. Alpha-1 globin gene codon 22 GGC>GGT generates a new 5' splicing site in the alpha1 exon1 that completely substitutes the normal one. The mRNA abnormally spliced at cod 22 accumulates in the nucleus but it is rare in the cytoplasm of cells in culture and reticulocytes, most likely due to the non-sense mediated decay (NMD). No role could be assigned to the nonsense-associated alternative splicing (NAS) because no activation of cryptic splice site was detected. In conclusion these data confirm that mRNA analysis from reticulocytes and PBSC differentiated in vitro is an excellent method for the molecular characterization of new thalassemic mutants especially for splicing mutants. Moreover the studies on these new mutants revealed a very important aspect: in some case the level of anomalous spliced mRNA could be very low, the detection of which requiring more sophisticated approach. Reference: Musollino et al, Ann Hematol. 2012;91(11):1695-701.
Towards a complete definition of the thalassemia defect: molecular characterization of new splicing mutants
Romeo Prezioso;Giuseppina Lacerra
2016
Abstract
Purpose: During a project on the molecular epidemiology of beta- and alpha-thalassemia in Southern Italy we identify two new thalassemic splicing mutants. The first was a beta-globin gene mutant characterized by duplication of tetranucleotide "AG/CT" at the junction IVS-II/exon 3, generating a pre-mRNA containing two 3' splice sites in the intron 2. The second was a silent mutation GGC>GGT (Gly>Gly) at codon 22 of the alpha-1 globin gene. It generates a splice donor site sequence (CTG/GTGAGT) at codons 22/23 with a score-splicing consensus (0.99) higher than the normal site (0.89). We would define the effect of the mutations on the splicing. Experimental description: To assess the presence of the variant beta- or alpha-globin chains we performed Hb analysis by HPLC and globin chain in vitro biosynthesis from reticulocytes. To evaluate the presence of normal and anomalous mRNA produced from the mutant alleles we analyzed globin mRNA from reticulocytes and from Peripheral Blood Stem Cell (PBSC) differentiated in vitro performing the following studies: a) separation on acrylamide gel of cDNA amplicomers containing P32 a-dCTP; b) sequencing analysis of amplicomer eluted by the acrylamide gel; c) measurement ratio of mRNA synthesized by the two beta-globin alleles taking advantage from a polymorphism at codon 2 C>T associated, with the presence (betaC)/absence (betaT) of the Alw44I restriction site. The new beta-thalassemic allele was associated with the codon 2 betaC. Results: Duplication of tetranucleotide "AG/CT": The beta-globin cDNA analysis did not reveal any trace of the mutant allele, but only cDNA from the normal allele indicating that our mutant did not contribute to the synthesis of (normal or mutant) beta-globin mRNA. Experimental analysis to prove the presence of anomalous mRNA highlighted two faint heteroduplex bands, absent in the normal control. Their sequencing showed to be aberrant cDNA with the "AGCT" insertion at the cod 105 of the beta-globin cDNA: moreover the semi quantitative analyses indicated to be about 1.5% of the normal one. In the anomalous mRNA the insertion causes a frameshift and synthesis of an abnormal truncated beta-chain (139 residues). Variant chains were not detected by HPLC in fresh blood or in the precipitate from test for unstable Hbs, most probably unable to form Hb variant because the severe conformational changes. The alpha/beta ratio was of beta-thalassemic type. Alpha-1 globin gene codon 22 GGC>GGT: Two cDNA of length shorter than normal were detected from PBSC and from reticulocytes of the carriers. The first one was mRNA abnormally spliced at codon 15; the other was 28 nucleotides shorter than normal because of an abnormal splicing at codon 22, leading to a frameshift and to the formation of the stop codon TGA at codon 39 (the same generated by abnormal splicing at codon 15). The anomalous/normal cDNA ratio in erythroid cells and reticulocytes was 0.08 and 0.02, respectively, for the first cDNA; 0.76 and 0.08, respectively, for the other. In normal subjects limited amounts of the cDNA splicing at codon 15 were detected. Conclusions: Duplication of tetranucleotide "AG/CT": Data reported herein supports the hypothesis that the cod 105 +AGCT is a novel beta-globin gene mutation that impairs the production of normal mRNA, but causes synthesis of abnormally spliced mRNA, which is present only in trace amounts (1.5%) and cannot be translated into a functional ?-chain. Alpha-1 globin gene codon 22 GGC>GGT generates a new 5' splicing site in the alpha1 exon1 that completely substitutes the normal one. The mRNA abnormally spliced at cod 22 accumulates in the nucleus but it is rare in the cytoplasm of cells in culture and reticulocytes, most likely due to the non-sense mediated decay (NMD). No role could be assigned to the nonsense-associated alternative splicing (NAS) because no activation of cryptic splice site was detected. In conclusion these data confirm that mRNA analysis from reticulocytes and PBSC differentiated in vitro is an excellent method for the molecular characterization of new thalassemic mutants especially for splicing mutants. Moreover the studies on these new mutants revealed a very important aspect: in some case the level of anomalous spliced mRNA could be very low, the detection of which requiring more sophisticated approach. Reference: Musollino et al, Ann Hematol. 2012;91(11):1695-701.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
