Identification and molecular characterization of a novel 163 kb deletion: The Italian (ϵγδβ)⁰-thalassemia

<p><b>Objective and importance</b>: To verify the presence of β-thalassemia in subjects showing hematologic phenotype of α-thalassemia, conduct normal molecular sequence analysis of the α-globin genes, and detect the absence of the most frequent α-thalassemia deletions.</p> <p><b>Clinical presentation:</b> A patient from Apulia (Southern Italy) was referred to our institution for the occasional founding of hypochromic polyglobulia and microcytic red blood cells associated with normal levels of Hb A2 and Hb F and normal iron parameters.</p> <p><b>Intervention and technique</b>: The patient has been investigated using Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), quantitative real-time PCR, restriction analysis, and gap-PCR. A novel deletion, the Italian (ϵγδβ)⁰-thalassemia, has been identified. The 5′ breakpoint was within a LINE element of 80 kb 3′ of the ε-globin gene, and the 3′ breakpoint was within a 160-bp palindrome of about 30 kb 5′ of the β-globin gene. The breakpoint region was characterized by the presence of a microhomology (5′-TCT-3′) and of an insertion of 43 bp owing to the duplication of the 160-bp palindrome. Comparison of the Hb and Hb A2 values of (ϵγδβ)⁰-thalassemia from the literature with those of (molecularly known) thalassemia carriers indicated a higher level of Hb A2 with respect to α-thalassemia and a lower level of Hb with respect to β⁰-thalassemia carriers.</p> <p><b>Conclusion</b>: In this study, we report the first (ϵγδβ)⁰-thalassemia case identified in Italy. To avoid misdiagnosis of β-thalassemia, we suggest verifying the presence of large deletions of the β-globin gene cluster in subjects showing a higher border line level of Hb A2 and a lower level of Hb.</p

α-Thalassemia Associated with Hb Instability: A Tale of Two Features. The Case of Hb Rogliano or α1 Cod 108(G15)Thr→Asn and Hb Policoro or α2 Cod 124(H7)Ser→Pro.

<div><p>We identified two new variants in the third exon of the α-globin gene in families from southern Italy: the Hb Rogliano, α1 cod108 ACC>AAC or α1[α108(G15)Thr→Asn] and the Hb Policoro, α2 cod124 TCC>CCC or α2[α124(H7)Ser→Pro]. The carriers showed mild α-thalassemia phenotype and abnormal hemoglobin stability features. These mutations occurred in the G and H helices of the α-globin both involved in the specific recognition of AHSP and β1 chain. Molecular characterization of mRNA, globin chain analyses and molecular modelling studies were carried out to highlight the mechanisms causing the α-thalassemia phenotype. The results demonstrated that the α-thalassemia defect associated with the two Hb variants originated by different defects. Hb Rogliano showed an intrinsic instability of the tetramer due to anomalous intra- and inter-chain interactions suggesting that the variant chain is normally synthesized and complexed with AHSP but rapidly degraded because it is unable to form the α1β1 dimers. On the contrary in the case of Hb Policoro two different molecular mechanisms were shown: the reduction of the variant mRNA level by an unclear mechanism and the protein instability due to impairment of AHSP interaction. These data highlighted that multiple approaches, including mRNA quantification, are needed to properly identify the mechanisms leading to the α-thalassemia defect. Elucidation of the specific mechanism leads to the definition of a given phenotype providing important guidance for the diagnosis of unstable variants.</p></div

Total ion current (TIC) of the LC-MS analysis and electrospray mass spectra of variant and normal α-chains.

<p>Liquid chromatography-mass spectrometry analysis (LC/MS) of globin chains precipitated from the hemolysate of a Hb Policoro carrier after a 20 minutes of 17% isopropanol incubation. The anomalous globin chain eluting before the normal β-globin is marked as α^V.</p

Environment of Thr/Asn108α and Ser/Pro124α in the structure of the human hemoglobin tetramer and of AHSP-alpha globin chain complex structure.

<p><b>A:</b> Structural representation of the environment of Thr108α in the structure of the human hemoglobin tetramer. <b>B:</b> Structural representation of the environment of Asn108α in the plausible structure of human hemoglobin T108N mutant. In panels A and B, the α- and β-chains are coloured in cyan and yellow, respectively. <b>C:</b> Structural representation of the environment of Thr108α in the structure of the complex between AHSP and the α-chain of human hemoglobin. <b>D:</b> Structural representation of the environment of Asn108α in the structure of the plausible hypothetical complex between AHSP and the α-chain variant of human hemoglobin. There are no major differences in side chain orientation between <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115738#pone.0115738.g004" target="_blank">Fig. 4C and 4D</a> if one exclude the flipping of the His112α imidazole ring. <b>E:</b> Structural representation of the environment of Ser124α in the structure of the complex between AHSP and the α-chain of human hemoglobin. <b>F:</b> Structural representation of the environment of Pro124α in the structure of the plausible hypothetical complex between AHSP and the α-chain variant of human hemoglobin. In panels C-E, the α-chain of HbA is coloured in cyan, whereas AHSP is in green. In all the panels, the nitrogens are coloured in blue, oxygens in red and hydrogen bonds are indicated as dashed lines.</p

Hematologic, biochemical data and α-genotype of the families with the new Hb Policoro or α2 cod 124 TCC>CCC.

<p><i>Ret</i>: <i>Reticolocytes; I</i>.<i>B</i>.: <i>Inclusion Body; N</i>: <i>normal; Hb Pol</i>: <i>Hb Policoro; Pos</i>: <i>Positive; Abs</i>: <i>Absent; Neg</i>: <i>Negative; nt</i>: <i>not tested</i>.</p><p>Hematologic, biochemical data and α-genotype of the families with the new Hb Policoro or α2 cod 124 TCC>CCC.</p

Location of residues discussed in the manuscript on the overall structure of the complex between the α-chain and AHSP and on the structure of the αβ dimer.

<p><b>A:</b> Cartoon representation of the three-dimensional structure of the complex between the α-chain and AHSP (PDB code 1Y01) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115738#pone.0115738.ref003" target="_blank">3</a>]. The α-chain of HbA is highlighted in cyan, whereas the AHSP molecule is in green. In this structure the position of residues 1, 74, 81–91 and 140–142 in the α-globin chain of HbA has not been determined. For this reason the structural representation lacks these residues. <b>B:</b> Cartoon representation of the three-dimensional structure of the αβ dimer from the structure of the tetrameric human deoxy hemoglobin (PDB code 2HHB) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115738#pone.0115738.ref023" target="_blank">23</a>]. α- and β-chains are highlighted in cyan and yellow, respectively.</p

Liquid chromatography-mass spectrometry analysis (LC/MS) of the hemolysate from a Hb Rogliano carrier.

<p><b>A</b>: Total ion current (TIC) of the LC-MS analysis of the globin chains. The anomalous globin chain eluting before the normal α-globin is marked as α^V. The arrows indicated the electrospray mass spectra of the variant and the normal α-globin chains. <b>B</b> and <b>C:</b> Reverse-phase HPLC separation of globin chains. <b>B</b>: normal control; <b>C</b>: Hb Rogliano carrier. The variant α-globin chain (α^V) is indicated by an arrow.</p

Molecular characterization of the Hb Policoro.

<p><b>A:</b> DGGE of the fragment III of the α-globin genes containing the codon 124. Lanes 1 and 2: normal subjects, Lane 3: Hb Policoro heterozygote. <b>B:</b> DNA sequence of the α2-globin gene of the proband from codon 122 to codon 126; the arrow indicates the mutation. <b>C:</b> ARMS for the screening of carriers for Hb Policoro: the control amplicon was of 714 bp, the amplicon specific for the mutation was 255 bp long. Lanes 1, 2, 4: Hb Policoro heterozygotes; Lane 3: normal subject; lane 5: negative control. <b>D:</b> ARMS with the normal primer at codon 124 for the genotyping of the Hb Policoro carriers: the control amplicon was of 714 bp, the amplicon specific for the cod 124 normal allele was 139 bp long. Lanes 1, 2: Hb Policoro heterozygotes; Lane 3: compound heterozygote for the Hb Policoro and the -α^3.7 deletion; Lane 4:-α^3.7 deletion heterozygote; Lane 5: normal subject; Lane 6: negative control. The ARMS-PCR conditions were: hot start 95° for 10'; PCR: 94° for 45'', 63° for 45'', 72° for 45'', for 30 cycles.</p