Talal Qadah

In vitro characterization of the α-thalassemia point mutation HBA2:c.95+1G>A [IVS-I-1(G>A)(α2)]

The α-thalassemias are a group of disorders occurring as a result of decreased synthesis of α-globin chains, most commonly due to deletions of α-globin genes. Detection of α-thalassemia (α-thal) caused by point mutations has increased during the past few years and more than 70 different point mutations have been reported for the α1- and α2-globin genes. The mutation at the splice donor site of the first intervening sequence [IVS-I-1 (G>A)] of the α2-globin gene, HBA2:c.95+1G>A, is thought to cause a thalassemic phenotype by interfering with and preventing the normal splicing of pre-mRNA. We developed an in vitro expression system to study α-globin gene point mutations at the molecular and cellular levels. The expression vector carrying the HBA2:c.95+1G>A mutation (α2GIVS-I-1G>A) was created using site-directed mutagenesis of a wild type (WT) construct of the α2-globin gene (α2G2034WT). Gene expression experiments in human bladder carcinoma 5637 cells were carried out using sequence verified WT and mutated clones. Complementary DNA synthesis and polymerase chain reaction (PCR) analysis showed normal α2-globin transcripts from cells transfected with the WT vector, but aberrant transcripts from cells transfected with the mutated vector carrying the splice donor site mutation. In the presence of the G>A mutation, normal splicing does not occur, and a cryptic splice site 49 bp upstream of the normal site is used. The translation of this product produces a premature termination codon, thus resulting in a 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 &agr;-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 &agr;-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 &agr;-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 &agr;-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.

The Effect of Nonsense Mediated Decay on Transcriptional Activity Within the Novel β-Thalassemia Mutation HBB: c.129delT.

Abstract Premature termination codons (PTCs) are caused by mutations in the coding sequences of functional genes resulting in an incorrect assignment of a stop codon. Abnormal and truncated proteins are prevented from being translated due to the rapid degradation of mRNA carrying these mutations by an RNA surveillance mechanism referred to as nonsense mediated decay (NMD). Recently, a novel mutation in a patient from Thailand with the clinical diagnosis of Hb E (HBB: c.79G > A)/β0-thalassemia (Hb E/β0-thal) and whose molecular analysis demonstrated a novel mutation in the β-globin gene, HBB: c.129delT, was reported. The result of this deletion is a frameshift (FSC) resulting in a PTC at codon 60. We have analyzed the impact of this mutation on transcription and translation of the affected β-globin gene using an in vitro model. The quantitative real-time polymerase chain reaction (qReTi-PCR) analysis revealed that this nucleotide mutation resulted in marked mRNA degradation, which we attributed to the NMD mechanism and as such, the expected deleterious truncated HBB was not generated. This result highlights a valuable application of our in vitro gene expression model that can be used to predict possible molecular pathology for any given nucleotide mutations.

Experimental Characterization of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) and Hb Boghé (HBA2: c.177 C > A, p.His > Gln) Reveals Contradictory HBA2 Expression and Translation Patterns Despite Identical Amino Acid Substitutions

Abstract In this study, we describe the clinical features and provide experimental analyses of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) that contrasted with Hb Boghé (HBA2: c.177 C > A, p.His > Gln). Despite the identical amino acid substitution in both variants, Hb Flurlingen shows the phenotype of α-thalassemia (α-thal), whereas Hb Boghé has no impact on α2-globin (HBA2) production. For in vitro transcription analysis, HBA2 expression constructs carrying the HBA2-WT (wild type), Hb Flurlingen and Hb Boghé sequences were generated and expressed in human bladder carcinoma 5637 cells for downstream analyses by quantitative real time-polymerase chain reaction (qReTi-PCR) and immunofluorochemistry (IFC). In silico analysis of secondary folding structures of the HBA2-WT, Hb Flurlingen and Hb Boghé mRNA sequences was performed using Mfold software. The gene transcription and translation analyses revealed that cells transfected with the Hb Flurlingen construct had significantly lower HBA2 transcription (−55.4%, p ≤ 0.01) and reduced protein synthesis when compared to the wild type group. In contrast, cells transfected with the Hb Boghé construct showed no significant changes in HBA2 transcription or translation activities when compared to the wild type group. The in silico prediction of possible effects of these mutations on the folding structures of the HBA2 transcripts showed a change of secondary folding pattern in the Hb Flurlingen transcript when compared to those of HBA2-WT and Hb Boghé. Our experimental findings support the clinical presentation of an α-thalassemic phenotype for Hb Flurlingen in contrast with Hb Boghé, despite identical amino acid substitutions. The results confirm the importance of experimental analysis in establishing the impact of novel base substitutions.

Molecular and Cellular Analysis of a Novel HBA2 Mutation (HBA2: c.94A>G) Shows Activation of a Cryptic Splice Site and Generation of a Premature Termination Codon

Abstract In this study, we describe the clinical features and provide experimental analyses of a novel point mutation affecting the penultimate nucleotide of the first exon of the HBA2 (HBA2: c.94A>G) gene identified in a 26-year-old female who also carries a heterozygous Hb E (HBB: c.79G>A) variant. The aim of the study was to investigate the impact of this point mutation on the transcriptional activity of the HBA2 gene using a combination of an initial in silico prediction followed by in vitro mutagenesis and transcriptional activity assessment. The analyses revealed that the HBA2: c.94A>G point mutation causes the activation of a cryptic splice site located 49 bp upstream of the exon1-intron1 boundary in both HBA2 long and short isoforms, thus generating a frameshift and a premature termination codon between codons 48 and 49 in the second exon. A rapid degradation of the aberrantly spliced transcripts by the nonsense mediated decay (NMD) surveillance system is highly indicative of an α-thalassemia (α-thal) phenotype. However, the abnormal mRNA may not be entirely degraded since the proband presents a slight splenomegaly that could be the sign of extra vascular hemolysis.

Identification and Characterization of Two Novel and Differentially Expressed Isoforms of Human α2- and α1-Globin Genes

In most references, the transcription initiation site for the α2- and α1-globin genes has been described to lie 37 bp upstream of the translation initiation codon, however, a review of data repositories such as GenBank and Ensembl showed a report of the α2-globin transcription initiation site occurring at position –66 relative to the initiation codon. To confirm the occurrence of these isoforms for both the α2- and α1-globin genes and to document their expression levels, we initiated our current investigation. Total RNA from the peripheral blood of 15 healthy volunteers was analyzed using both semi-quantitative-polymerase chain reaction (PCR) and real-time (ReTi-PCR) protocols developed in our laboratory, with primers designed to enable distinction between the α2- and α1-globin transcripts.We observed two distinct PCR products for each of the globin genes. Subsequent DNA sequencing of 11 individual PCR products revealed that the α2- and α1-globin transcripts are present in both a long and a short isoform, initiating at positions –66 and –37, respectively. The shorter (–37) isoform is expressed approximately 10,000–100,000 times more strongly than the longer isoform, demonstrating differential expression within the healthy population. This study, for the first time, confirms the presence of two isoforms for both the α2- and α1-globin genes with varying transcription levels in healthy individuals. The short isoform is expressed at significantly higher levels than the longer isoform for both α2 and α1 genes. Therefore, based on our observations, we propose that despite the contribution of the long isoforms to the total α-globin RNA pool, the short isoforms are the main physiological transcripts.

Molecular characterization of Hb hamilton hill (HBA2: c.388delC), a novel HBA2 variant generating a premature termination codon and truncated HBA2 chain

Abstract In recent years, the identification of α-thalassemias caused by nondeletional mutations has increased significantly due to the advancement of sensitive molecular genetics tools. We report clinical and experimental data for a novel frameshift mutation caused by a single base deletion at position 388 in exon 3 of the α2-globin gene (HBA2: c.388delC; Hb Hamilton Hill), resulting in the phenotype of α-thalassemia (α-thal). Hb Hamilton Hill was identified in an adult female of unknown ethnicity investigated for unexplained microcytosis. Direct DNA sequencing of the HBA2 gene revealed a heterozygous mutation, HBA2: c.388delC, and the molecular effect of this mutation was assessed experimentally using our previously described in vitro model. The experimental analysis involved transfection of a human bladder carcinoma (5637) cell line with expression vectors carrying either HBA2-wild type (HBA2-WT) or HBA2: c.388delC followed by total RNA purification and cDNA synthesis. Both wild type and mutant gene expression was studied and compared at the transcriptional and translational levels using quantitative real time polymerase chain reaction (qReTi-PCR) and immunofluorochemistry (IFC), respectively. Our experimental data showed a significant reduction by 25.0% (p = 0.04) in the transcriptional activity generated from HBA2: c.388delC compared to HBA2-WT. As a result of this base deletion, a frameshift in the open reading frame generates a premature termination codon (PTC) at codon 132 of exon 3 resulting in the formation of a truncated α-globin chain. The truncated α-globin chain, observed by the IFC technique, is most likely unstable and undergoes a rapid turnover resulting in the thalassemic phenotype.

Molecular and cellular analysis of three novel alpha2-globin gene promoter mutations [HBA2:c.-59C>T], [HBA2:c.-81C>A] and [HBA2:c.-91G>A] reveal varying patterns of transcriptional and translational activities

Summary While point mutations affecting the promoter region of &bgr;-globin gene are widely described, there are no well characterised reports of any point mutations currently found in the promoter of the &agr;2-globin (HBA2) gene. We present clinical and experimental data for three novel HBA2 gene core and proximal promoter mutations. Using an in vitro system designed to assess the impact of point mutations, the three novel [HBA2:c.-59C>T], [HBA2:c.-81C>A] and [HBA2:c.-91G>A] promoter mutations identified in three unrelated patients were analysed for HBA2 gene transcriptional and translational activities. Following the generation and transfection of expression vectors carrying each mutation, the HBA2 transcription activity of the promoters from each mutant was analysed with quantitative real time-PCR (qReTi-PCR) technique. Immunofluorochemistry (IFC) was used to analyse HBA2 protein synthesis. The analyses showed that [HBA2:c.-59C>T] and [HBA2:c.-91G>A] mutant constructs caused significant reduction in the HBA2 transcription levels by 53.7% (p = 0.0008) and 36.2% (p = 0.004), respectively, resulting in markedly lower HBA2 protein labelling when compared to the wild type as shown with subsequent IFC analysis. Conversely, the [HBA2:c.-81C>A] construct showed no significant changes in either transcription (p = 0.089) or in protein labelling when compared to the wild type. The equal pAmp transcription levels found in each group confirmed that the observed labelling differences were not due to varying transfection efficiencies. This study emphasises the importance of in vitro studies to establish the impact of base substitutions on the level of gene expression, and the value of these studies in clinicopathological correlation so that appropriate advice can be given in genetic counselling.