α-thalassemia trait caused by frameshift mutations in exon 2 of the α2-globin gene: HBA2:c.131delT and HBA2:c.143delA.

We describe two frameshift mutations associated with an α-thalassemia (α-thal) phenotype, identified in three unrelated individuals investigated for persistent microcytosis. The first mutation, HBA2:c.131delT, is located in codon 43, and the second, HBA2:c.143delA, is located in codon 47. Both are due to single base pair deletions that cause a frameshift and a premature termination codon (PTC) at positions 48/49. The presence of a PTC at this position has been documented to result in nonsense mediated mRNA decay that would account for the thalassemic phenotype.

A molecular tool to assess the pathological relevance of alpha-globin DNA variants.

AIM: While the phenotype for heterozygous beta-thalassaemia is straightforward, it is more difficult to confirm a causative relationship for mutations in the alpha-globin genes. The aim of this study was to generate an in vitro system to evaluate the pathological relevance of α-globin mutations. METHODS: The novel variant HBA1:c.301-3C>G was used as a model. In silico analysis predicted an aberrant acceptor splice site in the mutant sequence. Subsequent in vitro studies included generation of and transfection of an expression vector carrying the HBA1:c.301-3C>G mutation, RNA purification, reverse-transcription polymerase chain reaction (RT-PCR) and cDNA sequencing. Immunofluorochemistry (IFC) with antibodies specific to the N- and or C- terminal of the α-globin protein was used in protein detection. RESULTS: In vitro molecular characterisation of this point mutation confirmed the preferential utilisation of a cryptic splice site at intron 2 of the pre-mRNA, resulting in a shift in the reading frame causing a premature termination codon (PTC) at codons 101/102 and generation of a truncated protein. CONCLUSION: We have described here a molecular tool to study mutations that affect α-globin pre-mRNA splicing and translation. We confirm in silico predictions of the consequences of the HBA1:c.301-3C>G mutation, proving aberrant RNA splicing and the production of a truncated α-globin protein.

Molecular and cellular characterization of a new α-thalassemia mutation (HBA2:c.94A>C) generating an alternative splice site and a premature stop codon.

The identification of α-thalassemia (α-thal) due to point mutations has been increasing significantly with the advancement of molecular diagnostic tools. We describe here the molecular and cellular characteristics of the thalassemia mutation HBA2:c.94A>C, a novel point mutation affecting the α2-globin gene, causing a mild α-thal phenotype in a male patient of undisclosed ethnicity, investigated for unexplained microcytosis. The detected mutation is located at the penultimate nucleotide (nt) of the first exon which we postulated might affect pre mRNA splicing. While an in silico analysis did not predict any aberrant splice variants, experimental analysis using our in vitro model for gene expression studies showed utilization of a cryptic splice site at codon 15 that resulted in an aberrant splice variant. As a result, a frameshift in the reading frame of the mature mRNA was produced, leading to the formation of a premature termination codon (PTC) between codons 48 and 49 in exon 2. This in turn leads to nonsense mediated mRNA decay (NMD) and the phenotype of α-thal.

Hb Lynwood [α107(G14) (-T) (α2) HBA2:c.323delT)] in conjunction with the α(3.7) deletion produces a moderately severe α-thalassemia phenotype.

We describe a novel frameshift mutation associated with an α-thalassemia (α-thal) phenotype in a patient of Sudanese origin investigated for persistent microcytosis. In addition to the α(3.7) deletion, a novel mutation on the α2 gene was detected: HBA2:c.323delT. This mutation causes a frameshift at codon 107 of the α2 gene. The result is a disturbed amino acid sequence for the following 24 amino acids, and a premature termination codon at position 132.

Hb East Timor [ß80(EF4)Asn→His, AAC>CAC (HBB c.241A>C)], a variant hemoglobin associated with normal hematology.

Routine hemoglobin (Hb) analyses identified a new ß-globin variant in a family from East Timor. The red cell indices were within normal limits for all affected family members. The variant is due to a missense mutation at amino acid codon 80 (AAC>CAC) which results in the substitution of histidine for asparagine.