Molecular mechanisms of a novel β-thalassaemia mutation due to the duplication of tetranucleotide ‘AGCT’ at the junction IVS-II/exon 3
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We report a new β-thalassaemia allele detected in a young Italian woman, suffering with mild non-haemolytic anaemia (Hb < 10 g/dL) and not showing Hb variant or Heinz bodies. The allele is characterised by duplication of tetranucleotide ‘AG/CT’ (+1344/+1347) including the invariant dinucleotide ‘AG’ of IVS-II acceptor splicing site and the first two nucleotides of codon 105. β-Globin complementary DNA (cDNA) sequencing did not reveal any mutation and qualitative analysis of the reverse transcription PCR reaction showed that only the proximal 3′ splice site present in the duplicated gene is used giving race to an anomalous messenger RNA (mRNA) present in trace (1.5 %) because, most probably, rapidly degraded. In the anomalous mRNA, the insertion causes a frameshift and synthesis of an abnormal truncated β-chain (139 residues), unable to form Hb variant because of the severe conformational changes. The duplication might have arisen from secondary structures generated by quasi-palindromic sequence 5′-CCCA(C)AG/CT(CC)TGGG-3′. Restriction fragment length polymorphism analysis for the β-globin haplotype and familiar segregation analysis indicated that the mutant β-globin gene was associated with the haplotype V.
Keywordsβ-Thalassaemia Tetranucleotide insertion Quantitation of anomalous mRNA β-Globin gene defects
β-Thalassaemia, one of the most frequent hereditary diseases in the Mediterranean area, shows heterogeneous molecular basis comprising about 280 mutations . Out of them, most are point mutations and rarely are micro-insertion/deletion of more than three bases. In particular, only three mutants due to the micro-insertion of four bases have been described for the β-globin gene, all located in the coding region: the +ATCT at codon 48, +TGAT at codon 116, and +GCCT at codon 130/131 [2, 3, 4]. Although the β-thalassaemias are due to β-globin alleles inherited as recessive, in rare cases, some alleles act as ‘dominant’ and are inherited associated with thalassaemia intermedia phenotypes. This is the case of β-globin alleles causing synthesis of highly unstable β-variants or truncated/elongated β-chains .
Here, we report a new β-thalassaemia allele, characterised by the duplication of the tetra nucleotide AG↓CT (IVS-II-849/exon 3 nt 2 or +1344/+1347) at the 3′ splicing junction IVS-II↓exon 3. Splicing in the canonical site could cause a frameshift, leading to the formation of a stop codon in position 138–139. To date, only another mutant involving the intron/exon junction of the β-globin gene has been described: the duplication of 22 bp in the IVS-I/exon 2 junction leading to a frameshift and a premature chain termination at codon 37 [6,7].
We examined the molecular mechanisms of the +AGCT duplication and the presence of normal and aberrant messenger RNA (mRNA) synthesised from the mutant allele to define the effect of the mutation on the splicing.
Haematological data of the proband and other family members
Hb A2 (%)
Hb F (%)
Serum iron (μg/dL)
Total bilirubin (mg/dL)
Direct bilirubin (mg/dL)
Indirect bilirubin (mg/dL)
non α/α biosynthetic ratio
Haematological data and Hb analyses
The haematological data, ferritin and serum iron values were determined with standard methods in the hospitals. Hb analyses were carried out by cation exchange high performance liquid chromatography (HPLC) (Bio-Rad-Diagnostics Group, Hercules, CA, USA).
Globin chain in vitro biosynthesis
Globin chain in vitro biosynthesis in reticulocytes was carried out in the proband and in a normal control, as already reported. Globin chains were separated by reverse-phase HPLC .
Molecular screening for most common β-thalassaemia mutations was carried out by amplification refractory mutation system (ARMS)-PCR . Identification of new or rare point-form mutant was carried out by denaturing gradient gel electrophoresis (DGGE)  and by sequencing  with a standard automatic sequencer (Perkin Elmer, Foster City, CA, USA, Mod 3100). α-Thalassaemia deletions were analysed by gap-PCR [11,12]; point mutations were analysed with multiplex ARMS or DNA sequencing [13,14].
Reticulocytes from peripheral blood were purified by means of 36 % Ficoll–Paque Plus (Amersham-Pharmacia-Biotech, Freiburg, Germany), 0.25 % NaCl and 9 % v/v Selectographin (Schering, Milan, Italy) . Total cellular mRNA was isolated from reticulocytes with Trizol-LS reagent kit (Life-Technologies, Gaithersburg, MD, USA). RT-PCR and cDNA amplification was carried out by Gene-Amp-RNA-PCR-Kit (Perkin Elmer, Foster City, CA, USA) using the primers and the conditions already reported .
Ratio of mRNA synthesised by β-globin alleles
The ratio of mRNA synthesised by the two β-globin alleles was measured in the proband heterozygous for the polymorphism codon 2 C>T, taking advantage of the fact that the β-codon 2 ‘C’ (βC) and β-codon 2 ‘T’ (βT) alleles are associated respectively, with the presence/absence of the Alw44I restriction site. In brief, β-globin cDNA was amplified using dCTP (α-32P) with the primers forward (5′-GCTTACATTTGCTTCTGACACAACTGTG-3′, position −4/+24) and the primer reverse (5′-GGTCCAAGGGTAGACCACCAG-3′, position +297/+277). To reduce the production of the heterodimers (indigestible with restriction enzymes), we carried out a first PCR round of 20 cycles and a second round of two cycles after increasing five times the volume of reaction. The cDNA samples were digested with Alw44I restriction enzyme and run on a 4 % Nu-Sieve 3:1 agarose gel at 100 V for 1/2 h. The gel was dried, and the intensity of the bands was quantified using a phosphoimager with ImageQuant software for unmarked DNA (Molecular Dynamics, Sunnyvale, CA, USA). The ratio between digested and undigested bands represented the βcod2C/βcod2T ratio .
Detection of anomalous mRNA
The PCR and RT-PCR of the β-globin gene—at low number of cycles (24 cycles for DNA, 22 and 27 cycles for the cDNA) and containing 0.2 μl of P32 α-dCTP—were carried out using the primers forward (5′-TTCTGAGTCCAAGCTAGGCC-3′, position +1284/+1303) and the primer reverse (5′-CTTTGCCAAAGTGATGGGCCAGC-3′, position +1372/+1394) generating a DNA amplicon of 111 bp and the primer forward (5′-CACCTTTGCCACACTGAGTG-3′, position +432/+451) with the reverse (5′-CACTGGTGGGGTGAATTCTT-3′, position +1391/+1411) generating a cDNA amplicon of 130 bp. Heteroduplex molecules were generated at the end of each PCR and RT-PCR session by 60 s at 95 °C, 5 min at 56 °C and 5 min at 4 °C. Ten microlitres of the PCR and RT-PCR was separated on a 6.5 % (37.5:1) Acrylamide gel in TEA 1× buffer run at 300 V for 6 h. The gel was dried on a gel drier, and the intensity of each band was quantified using a phosphoimager with ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA). Heteroduplex bands were electro eluted, amplified by PCR and analysed by sequencing.
β-Globin gene haplotype
The analysis of the restriction fragment length polymorphism (RFLP) (+/−) HindIII/Gγ, HindIII/Aγ, HincII/ψβ, HincII/3' ψβ, AvaII/β and BamHI/3′β was carried out on PCR amplified DNA fragments as already reported . RFLP haplotypes associated with the mutated β-globin allele was assembled with family segregation studies.
The prediction of the mutant β-chain was carried out by means of FGENESH 1.0 Software (http://genomic.sanger.uk); its secondary structure was predicted by means of the SOPMA-Software (www.espasy.ch). The software Splice Site Prediction by Neural Network (http://www.fruitfly.org/seq_tools/splice.html) was used for the prediction of splicing sites.
Haematological data and β-/α-globin biosynthesis of the proband and her family members are summarised in Table 1. The haematological alterations of the proband carried out for the first time at age of 11 years were detected in subsequent observations at the age of 27, 31 and 32 years when serum iron was 106, 62 and 22 μg/dL, and ferritin was 12, 18 and 39 ng/mL, respectively (Table 1). The proband’s mother showed β-thalassaemia phenotype, whereas his father and brother and sister all had normal red-cell parameters and haemoglobin electrophoretic profile. In the proband, heat denaturation and isopropanol tests, for detection of unstable haemoglobins, were negative; Heinz bodies were not detected even microscopic observations were made after 1, 2, 3, 6, 12 and 24 h of incubation.
RFLP analysis for the β-globin haplotype indicated that the father was heterozygotes for the haplotype IV/IX and the proband for the haplotype IV/V. Familiar segregation analysis revealed that the mutant β-globin gene was associated with the haplotype V (Table 1).
Analysis using the software for predictions of splice site find out only one putative acceptor splicing site in the DNA—even using a cut-off score of 0.1—including the ‘AG’ at the sequence +1344/+1347 (proximal site) giving rise to anomalous mRNA. Moreover, β-globin cDNA sequencing did not show any mutation (Fig. 1c) and at codon 2 was present only the base ‘T’, associated with the normal β-globin gene.
The proband and family had been analysed during a screening for β-thalassaemia carriers, carried out in the school about 20 years ago (Fig. 1a). She had the same haematological alterations detected by us associated with serum iron 106 mg/dL (data not showed). Out of the family (parents, two sisters and one brother), only the mother had shown the same haematological phenotype of the daughter associated with normal serum iron (Table 1). Most likely the proband had inherited the mutation from the mother who had shown the same phenotype (Hb < 10 g/dL) at the screening carried out about 20 years ago (Table 1) and no endocrine alterations. Independent confirmation of the genotype–phenotype correlation was not possible because no other carrier was present in the family and the only other carrier, the mother of the proband, had died and she had been an only child.
Molecular mechanisms of the ‘AGCT’ duplication
The tetranucleotide sequence ‘AGCT’, present in normal sequence at position +1344/+1347 (or IVS-II-849/exon 3 nt 2), includes the invariant dinucleotide ‘AG’ of the IVS-II acceptor splicing site and the first two nucleotides of the codon 105 (CTC). We detected two ‘AGCT’ in sequence (Fig. 1b). This duplication might be due to possible secondary structure in the ssDNA generated by quasi-palindromic sequence 5′-CCCA(C)AG/CT(CC)TGGG-3′ from IVS-II-844 to exon 3 nt 8.
The duplication of ‘AG↓CT’ sequence generated a pre-mRNA containing two 3′ splice sites in the intron 2 and consequently the duplication could leads to two alternative splice sites. The splicing at the distal ‘AG’ (+1348/+1349) would eliminate the duplicated tetranucleotide and produce only normal mRNA and no phenotypic abnormalities. On the other end, the splicing at the proximal ‘AG’ (+1344/1345), the only one predicted by FGENESH software with a score of 0.99 (score cutoff 0.1), would lead to the production of abnormal mRNA, showing the insertion of +AGCT in the third exon that cause a frameshift from codon 105 and formation of the stop codon TAA after 35 codons.
The sequencing of the β-globin cDNA showed only normal splicing, but to study the contribution of the β-globin mutant allele, at the production of normal and aberrant mRNA, two strategies were used.
The experiment to measure the level of mRNA synthesised from the mutant and normal allele in the carrier for the tetranucleotide duplication indicated that the contribution of mutant allele to production of β-globin mRNA was null or undetectable (Fig. 1d). Experimental analysis to verify the presence of anomalous mRNA demonstrated the presence of trace of aberrant cDNA with the ‘AGCT’ insertion (Fig. 2b, c).
The presence only of trace of aberrant cDNA product from the allele with the tetranucleotide duplication indicated that the distal ‘AG’, producing normal mRNA, is not selected for the splicing, but only the proximal ‘AG’, giving race to an aberrant mRNA, present in trace because most likely rapidly degraded. This observation is in accordance with the prediction of the FGENESH software and with Luukkonen and Seraphin who, using a collection of 3′ splice site mutants on cellular systems, reported that in case of duplicated 3′ splice sites in direct competition, the site proximal to the branchpoint is preferentially used .
In the anomalous mRNA, present in negligible amount, the insertion causes elongation of the third exon (+AGCT), a frameshift from codon 105, formation of the stop codon TAA after 35 codons and, consequently, synthesis of an abnormal truncated β-chain of 139 residues with substitution of 30/35 residues. These substitutions abolish completely the normal chain secondary structure (SOPMA Software, www.espasy.ch).
The new mutant is the first one causing a duplication of the β-globin IVS-II/exon 3 junction. Only another mutant has been described due to a duplication of 22 bp in the 3′ splice site at IVS-I/exon 2 junction of the β-globin gene. RT-PCR showed that only the proximal 3′ splice site present in the duplicated gene was used, leading to a frameshift and a premature chain termination at codon 37. β-Globin mRNA transcribed from the mutant gene was not detected, suggesting that the process of nonsense-mediated mRNA decay may be triggered by the premature termination codon (PTC) .
In close proximity to the new β-globin mutant has been described another frameshift mutant, the codon 104 (-G) at the exon 2/IVS-II boundary that shows similar characteristic respect to codon 105 +AGCT for the undetectable level of aberrant mRNA. Nevertheless the frameshift cause a modification of the C-terminal sequences extended to 156 amino acids instead of 142 and the carrier for codon 104 (-G) resulted in a dominantly inherited β°-phenotype with thalassemia intermedia .
Only three mutant alleles giving rise to a stop codon in position 138–139 have been described until now; the β-codon 106/107 +G , the β-codon 116 +TGAT  and the β-codon 120/121 +A . The mRNA analysis of the first two had not been carried out, whereas mRNA analysis of the β-codon 120/121 +A  showed a ratio α/β = 5.18 (4.44 in control), indicating that the βthal mRNA was stable and confirming that the non-sense mediated decay is not active for mutants showing PTC in the third exon . This means that in the case of the codon 105 +AGCT the degradation of aberrant mRNA is not due to the PTC, but most likely to the ability of the nuclear or cytoplasmic mRNA quality control mechanisms to recognise and degraded anomalous spliced mRNA, for example by interacting with splicing factors that remain loaded on the transcript due to presence of the IVS-II/exon 3 junction .
The phenotype observed in the new mutant is a picture of β°-thalassaemia with biosynthetic ratio of β-thalassemia type (β/α = 0.43) and with LDH, bilirubin and reticulocytosis in the normal range indicating absence of haemolysis and with no sign of mutant haemoglobin in peripheral blood. This phenotype is comparable to that observed in the case of the other three mutants that can give rise to a shorter β-chain of 139 aa and it is in accordance with Thein indicating that, due to the shorter length of the β-globin chain, the new mutant is expected to be associated with a normal phenotype and not with a dominant thalassaemia phenotype as in the case of the codon 104 (-G) . However, the presence of a persistent sideropenic anaemia in the carrier of the new mutation had contributed to render ambiguous the interpretation of the case.
In conclusion, data reported herein support the hypothesis that the codon 105 +AGCT is a novel β-globin gene mutation that impairs the production of normal mRNA, but causes synthesis of abnormal mRNA, which is present only in trace amounts and cannot be translated into a functional β-chain.
We acknowledge the Patient and the family for the collaboration. This study was supported by Ministero Istruzione, Università e Ricerca (MIUR), Legge 488/92, Cluster C02, Project 2.