The Hellenic type of nondeletional hereditary persistence of fetal hemoglobin results from a novel mutation (g.-109G>T) in the HBG2 gene promoter

Nondeletional hereditary persistence of fetal hemoglobin (nd-HPFH), a rare hereditary condition resulting in elevated levels of fetal hemoglobin (Hb F) in adults, is associated with promoter mutations in the human fetal globin (HBG1 and HBG2) genes. In this paper, we report a novel type of nd-HPFH due to a HBG2 gene promoter mutation (HBG2:g.-109G>T). This mutation, located at the 3′ end of the HBG2 distal CCAAT box, was initially identified in an adult female subject of Central Greek origin and results in elevated Hb F levels (4.1%) and significantly increased Gγ-globin chain production (79.2%). Family studies and DNA analysis revealed that the HBG2:g.-109G>T mutation is also found in the family members in compound heterozygosity with the HBG2:g.-158C>T single nucleotide polymorphism or the silent HBB:g.-101C>T β-thalassemia mutation, resulting in the latter case in significantly elevated Hb F levels (14.3%). Electrophoretic mobility shift analysis revealed that the HBG2:g.-109G>T mutation abolishes a transcription factor binding site, consistent with previous observations using DNA footprinting analysis, suggesting that guanine at position HBG2/1:g.-109 is critical for NF-E3 binding. These data suggest that the HBG2:g-109G>T mutation has a functional role in increasing HBG2 transcription and is responsible for the HPFH phenotype observed in our index cases

Molecular and protein study of hereditary persistence of fetal hemoglobin in adults

Ιn the present study the cis-sequences of the γ-globin genes were studied for the presence of elevated fetal hemoglobin in patients with various thalassemias [β-, δβ- thalassemia and hereditary persistence of fetal hemoglobin (HPFH)], so that the interaction mechanisms in the expression of the globin genes could be understood. 160 patients with β-thalassemia were studied, including patients with β-thalassemia and elevated HbF (52 subjects), δβ- thalassemia (63 subjects) and HPFH (30 subjects). The results can be seen below:a) the distribution of thalassemic mutations in Central Greece are similar to the ones observed in the rest of the country. The –MED deletion of the α-globin gene and deletion/inversion of the Turkish type are the most frequent genetic redistribution. The -101 (CT) mutation of the β-globin gene promoter is present in significantly high frequency in Central Greece.b) Here, we provide evidence for three new gene conversion events in the 5’ regulatory region of the human fetal globin genes. The sequence variations are transcriptionally silent polymorphisms that do not contribute to increased fetal hemoglobin levels, suggesting that the 5’ regulatory region of the human fetal globin genes is a gene conversion hot spot that prevent globin gene promoter sequence diversification, further underlining the need for two functional fetal globin genes in fetal erythropoiesis.c) a novel nd-HPFH mutation in the HBG2 gene promoter (HBG2:g.-109G>T). This mutation results in elevated HbF levels (4.1, 14.3%), significantly increased Gγ-globin chain production (79.2%) and markedly improved hematological indices. HBG2:g.-109G>T mutation abolishes a transcription factor binding site suggesting that the HBG2:g-109G>T mutation has a functional role in increasing HBG2 transcription and is responsible for the HPFH phenotype observed in this case. d) One case of an adult compound heterozygote for nd-HPFH Cretan type in combination with β0-thalassemia (IVSI-1 GA) characterized by reduced HbA2 levels and elevated HbF levels than the ones observed in the presence of the Αγ -158 C>T mutation in heterozygosity with the majority of β-thalassemia mutations suggesting that the Αγ -158 C-->T Αγ mutation has a significant role in Aγ-globin gene transcription. A possible explanation for this difference might be the closer physical proximity of the Αγ-globin gene to the adult globin genes, compared to the Gγ-globin gene.e) it is the first published record of a compound heterozygote cases of HPFH/δβ-thalassemia. Genotypic analysis showed compound heterozygotes for Αγ-117GA mutation (nd-HPFH), and the Turkish type δβ-thalassemia, which is quite frequent in area of central Greece. HbF levels were considerably higher compared to typical Greek nd-HPFH and Turkish db-thalassemia heterozygotes and HbA2 levels were markedly reduced. Our data suggest that the interaction of nd-HPFH with db-thalassemia results in a clinical picture similar to db-thalassemia carriers, despite the markedly increased HbF levels.Μελετήθηκαν οι cis-αλληλουχίες του γ-γονιδίου σφαιρίνης για την ταυτοποίηση μεταλλαγών υπεύθυνων για την παρουσία αυξημένης εμβρυϊκής αιμοσφαιρίνης σε ασθενείς με θαλασσαιμικά σύνδρομα [β-θαλασσαιμία, δβ-θαλασσαιμία και κληρονομική παραμονή εμβρυϊκής αιμοσφαιρίνης (HPFH)] με σκοπό την κατανόηση των μηχανισμών αλληλεπίδρασης έκφρασης των γονιδίων σφαιρίνης. Μελετήθηκαν 160 ασθενείς με β-θαλασσαιμία και αυξημένη HbF (52 άτομα), δβ-θαλασσαιμία (63άτομα), HPFH (30 άτομα). Τα αποτελέσματα είναι τα εξής:α) η κατανομή των θαλασσαιμικών μεταλλαγών στην περιοχή της Κεντρικής Ελλάδος είναι παρόμοια με της υπόλοιπης Ελλάδος. Η έλλειψη –MED του α γονιδίου σφαιρίνης και η έλλειψη/αναστροφή Τουρκικού τύπου αποτελούν τις πιο συχνές γενετικές αναδιατάξεις, ενώ η μετάλλαξη -101 (CT) του υποκινητή του β γονιδίου σφαιρίνης παρουσιάζει υψηλή συχνότητα στην κεντρική Ελλάδα.β) Περιγράφηκαν τρεις νέες περιπτώσεις γονιδιακής αναστροφής στην 5’ ρυθμιστική περιοχή των εμβρυϊκών γονιδίων σφαιρίνης. Οι αλλαγές αυτές αποτελούν μεταγραφικά σιωπηλούς πολυμορφισμούς που δε συμβάλλουν στην αύξηση των επιπέδων της εμβρυϊκής αιμοσφαιρίνης υποδεικνύοντας ότι η 5' ρυθμιστική περιοχή των ανθρώπινων εμβρυϊκών γονιδίων σφαιρίνης είναι ένα ‘’hotspot’’ γονιδιακής αναστροφής που εμποδίζει τη μεγάλη ποικιλότητα της αλληλουχίας του υποκινητή των γονιδίων σφαιρίνης, υπογραμμίζοντας περαιτέρω την ανάγκη για δύο λειτουργικά εμβρυϊκά γονίδια σφαιρίνης στο στάδιο της εμβρυϊκής ερυθροποίησης.γ) περιγράφηκε ένας νέος τύπος nd-HPFH που οφείλεται σε μετάλλαξη στην περιοχή του υποκινητή του HBG2 γονιδίου (HBG2: g.-109G>T). Η μετάλλαξη αυτή, που βρίσκεται στο 3’ άκρο του δεύτερου CCAAT κουτί του γονιδίου HBG2, έχει ως αποτέλεσμα αυξημένα επίπεδα HbF και Gγ-σφαιρινικής αλυσίδας. Ηλεκτροφορητική ανάλυση μετατόπισης κινητικότητας έδειξε ότι η μετάλλαξη αυτή καταργεί μια θέση πρόσδεσης του NF-E3 μεταγραφικού παράγοντα. Τα δεδομένα αυτά υποδεικνύουν ότι η HBG2: g-109G>T μετάλλαξη έχει λειτουργικό ρόλο ενισχύοντας τη μεταγραφή του HBG2 και είναι υπεύθυνη για το φαινότυπο HPFH.δ) μία περίπτωση διπλού ετεροζυγώτη για την nd-HPFH Κρητικού τύπου σε συνδυασμό με β0-θαλασσαιμία (IVSI-1 GA) με ελάχιστα επηρεασμένα αιματολογικούς δείκτες, συγκρινόμενα με αυτά του τυπικού ετεροζυγώτη για τη μεταλλαγή IVSI-1 και αυξημένα επίπεδα HbF. Η αύξηση της HbF είναι μεγαλύτερη από αυτή που παρατηρείται παρουσία της Αγ -158 C>T μεταλλαγής σε ετεροζυγωτία με την πλειονότητα των β-θαλασσαιμικών μεταλλαγών καταδεικνύοντας το λειτουργικό ρόλο της Αγ-158C>T μεταλλαγής στη μεταγραφή του Αγ-γονιδίου. ε) αναφέρεται για πρώτη φορά στη διεθνή βιβλιογραφία παράδειγμα διπλής ετεροζυγωτίας HPFH/δβ-θαλασσαιμίας. Πρόκειται για διπλούς ετεροζυγώτες για τη μεταλλαγή -117GA του Αγ-γονιδίου, υπεύθυνη για nd-HPFH, και τη δβ-θαλασσαιμία Τουρκικού τύπου που απαντάται συχνά στην Κεντρική Ελλάδα. Τα επίπεδα HbF ήταν σημαντικά υψηλότερα από τα αντίστοιχα των τυπικών ετεροζυγωτών για nd-HPFH και δβ-θαλασσαιμία, ενώ τα επίπεδα HbA2 ήταν σημαντικά μειωμένα. Βέβαια, τα αιματολογικά στοιχεία ήταν αντίστοιχα ενός τυπικού ετεροζυγώτη για δβ-θαλασσαιμία. Τα υψηλά επίπεδα της HbF δεν κατάφεραν να βελτιώσουν την αιματολογική εικόνα των διπλών ετεροζυγωτών nd-HPFH/δβ, συνεπώς, η συνύπαρξη nd-HPFH και δβ-θαλασσαιμίας οδηγεί σε κλινική εικόνα όμοια με αυτή των δβ-ετεροζυγωτών, παρά τα υψηλά επίπεδα της HbF

Mechanistic aspects of the water-gas shift reaction on alumina-supported noble metal catalysts: In situ DRIFTS and SSITKA-mass spectrometry studies

Steady-state isotopic transient kinetic analysis (SSITKA) experiments coupled with mass spectrometry were performed for the first time to study essential mechanistic aspects of the water–gas shift (WGS) reaction over alumina-supported Pt, Pd, and Rh catalysts. In particular, the concentrations (μmol g−1) of active intermediate species found in the carbon-path from CO to the CO2 product gas (use of 13CO), and in the hydrogen-path from H2O to the H2 product gas (use of D2O) of the reaction mechanism were determined. It was found that by increasing the reaction temperature from 350 to 500 °C the concentration of active species in both the carbon-path and hydrogen-path increased significantly. Based on the large concentration of active species present in the hydrogen-path (OH/H located on the alumina support), the latter being larger than six equivalent monolayers based on the exposed noble metal surface area (θ > 6.0), the small concentration of OH groups along the periphery of metal-support interface, and the significantly smaller concentration (μmol g−1) of active species present in the carbon-path (adsorbed CO on the noble metal and COOH species on the alumina support and/or the metal-support interface), it might be suggested that diffusion of OH/H species on the alumina support towards catalytic sites present in the hydrogen-path of reaction mechanism might be considered as a slow reaction step. The formation of labile OH/H species is the result of dissociative chemisorption of water on the alumina support, where the role of noble metal is to activate the CO chemisorption and likely to promote formate decomposition into CO2 and H2 products. It was found that there is a good correlation between the surface concentration and binding energy of CO on the noble metal (Pt, Pd or Rh) with the activity of alumina-supported noble metal towards the WGS reaction

Kinetic and mechanistic studies of the water–gas shift reaction on Pt/TiO2 catalyst

Summarization: A detailed kinetic and mechanistic study of the water–gas shift (WGS) reaction on a 0.5 wt% Pt/TiO2 catalyst has been carried out. The dependence of kinetic reaction rate on the partial pressures of reactants (CO, H2O) and products (H2, CO2), and the concentration and chemical structure of active and inactive reaction intermediates that are found in the “hydrogen-path” and “carbon-path” of the reaction have been investigated in the 200–270 °C range. The most likely mechanistic pathway of the WGS reaction over the present catalytic system is discussed. It has been found that the reaction rate increases with an increase in the concentration of CO or H2O in the feed stream, while it decreases significantly with the addition of H2 in the feed stream. On the contrary, the kinetic reaction rate was found to be practically independent on the concentration of CO2 in the feed stream. The experimental reaction rates that were estimated were fitted to an empirical power-law rate expression from which the kinetic reaction orders with respect to CO, H2O, CO2, and H2 were estimated to be 0.5, 1.0, ∼0.0, and −0.7, respectively. An apparent activation energy of 10.8 kcal/mol was also estimated. The formation of formate and carbonate surface species over the TiO2 support under WGS reaction conditions was proved via SSITKA–DRIFTS experiments. However, these reaction intermediates must be considered as inactive (spectator) species for the steady-state WGS reaction. Additional transient experiments that involved 18O-isotope provided strong support for the red-ox mechanism as the prevailing one on the present Pt/TiO2 catalyst, where labile oxygen and oxygen vacancies of TiO2 near the metal–support interface can participate in the reaction path of the WGS reaction.Presented on: Journal of Catalysi