Fig 1.
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.
A: Cartoon representation of the three-dimensional structure of the complex between the α-chain and AHSP (PDB code 1Y01) [3]. 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: Cartoon representation of the three-dimensional structure of the αβ dimer from the structure of the tetrameric human deoxy hemoglobin (PDB code 2HHB) [23]. α- and β-chains are highlighted in cyan and yellow, respectively.
Fig 2.
Molecular characterization and cDNA analysis of the Hb Rogliano.
A: Pedigree of the family. The arrow indicates the proband. B: DGGE of the fragment III of the α-globin genes containing the codon 108. Lanes 1 and 2: Hb Rogliano heterozygotes, Lane 3: normal subject. C and D: DNA and cDNA sequence of the α1-globin gene of the proband from codon 106 to codon 110; the arrow indicates the mutation. E and F: The fragment of 190 bp (DNA) and 230 bp (cDNA), digested with the restriction enzyme BstEII, and separated on a Nu Sieve 3:1 agarose 4% gel. E: Lane 1: 50 bp ladder; Lane 2: DNA of the Hb Rogliano carrier; Lane 3: DNA of the control subject; Lane 4: negative control; Lane 5: undigested sample. F: Lane 1: 50 bp ladder; Lane 2: cDNA of the Hb Rogliano carrier; Lane 3: cDNA of the control subject; Lane 4 PCR RT- on the Hb Rogliano carrier; Lane 5: undigested sample. The fragments’ lengths are reported on the left and right.
Table 1.
Hematologic, biochemical data, α-genotype and SNP analysis of the members of the family with the new Hb Rogliano or α1 cod 108 ACC>AAC.
Fig 3.
Liquid chromatography-mass spectrometry analysis (LC/MS) of the hemolysate from a Hb Rogliano carrier.
A: 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 αV. The arrows indicated the electrospray mass spectra of the variant and the normal α-globin chains. B and C: Reverse-phase HPLC separation of globin chains. B: normal control; C: Hb Rogliano carrier. The variant α-globin chain (αV) is indicated by an arrow.
Fig 4.
Environment of Thr/Asn108α and Ser/Pro124α in the structure of the human hemoglobin tetramer and of AHSP-alpha globin chain complex structure.
A: Structural representation of the environment of Thr108α in the structure of the human hemoglobin tetramer. 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. C: Structural representation of the environment of Thr108α in the structure of the complex between AHSP and the α-chain of human hemoglobin. D: 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 Fig. 4C and 4D if one exclude the flipping of the His112α imidazole ring. E: Structural representation of the environment of Ser124α in the structure of the complex between AHSP and the α-chain of human hemoglobin. F: 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.
Table 2.
Hematologic, biochemical data and α-genotype of the families with the new Hb Policoro or α2 cod 124 TCC>CCC.
Fig 5.
Molecular characterization of the Hb Policoro.
A: 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: DNA sequence of the α2-globin gene of the proband from codon 122 to codon 126; the arrow indicates the mutation. C: 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. D: 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 -α3.7 deletion; Lane 4:-α3.7 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.
Fig 6.
Total ion current (TIC) of the LC-MS analysis and electrospray mass spectra of variant and normal α-chains.
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 αV.
Fig 7.
cDNA analysis of the Hb Policoro.
A: cDNA sequence of the α2-globin gene of the proband from codon 122 to codon 126; the arrow indicates the mutation. B: DGGE of the cDNA fragment containing the exon three of the α2-globin gene. Lanes 1: normal subject, Lane 2: Hb Policoro heterozygote. C: DGGE of the cDNA fragment containing the exon three of the α2-globin gene. Lanes 1, 2, 3: total (1), nuclear (2) and cytoplasmic (3) cDNA from PBSC at 11 day of culture of a Hb Policoro heterozygote. Lanes 4: normal subject, Lane 5, 6: cDNA from reticolocytes of Hb Policoro heterozygote.