Individualized therapy of patients suffering from β-hemoglobinopathies treated with hydroxyurea: molecular analysis and evaluation of pharmacogenomic markers

Patients with β-hemoglobinopathies exhibit a variety of phenotypes, due to mutations located in the β-gene encoding the β-globin chain of HbA and, due to genetic variations located in modifying genes encoding important transcription factors, that act in trans to the human β-globin cluster and regulate its expression. Several studies have shown that elevated levels of fetal hemoglobin improve the clinical symptoms in adult patients with β-hemoglobinopathies.In the present study, mutations in the β-globin gene as well as genetic variants located in FLT1, ARG2, NOS2A, KLF10, MAP3K5, PDE7B, ASS1, NOS1 and TOX genes were determined. These modifying genes act in trans and seem to be involved in regulating the expression of γ-globin genes in adult patients with β-hemoglobinopathies. We recruited and genotyped 38 transfusion-dependent β-thalassemia patients (TDT), 7 non-transfusion-dependent β-thalassemia patients (NTDT), 53 healthy individuals as well as 42 compound heterozygotes with sickle cell disease/β-thalassemia of Hellenic origin. The latter group of patients was treated with hydroxyurea in order to increase their HbF levels and, as a result, improve their clinical phenotype. However, not all patients respond to this medication. The aim of the present study was to highlight genetic variants in the above-mentioned modifying genes that could be used as potential (pharmaco)genomic biomarkers. These biomarkers are associated with β-thalassemia patients’ ability to produce high or low HbF levels, thus, improving or not their clinical phenotype, as well as the patients’ response to HU treatment. Furthermore, we aimed to validate previous studies conducted in our laboratory (LPIT), as far as the above-mentioned biomarkers are concerned, including their data to ours, thus increasing our statistical sample and the validity of our results.β-globin gene mutations were determined by PCR analysis and Sanger sequencing. In order to determine the genetic variants in the above-mentioned modifying genes, PCR analysis was carried out followed either by Sanger sequencing or by RFLP or Heteroduplex Analysis. Furthermore, some cases were analyzed by allele-specific PCR.Our study revealed a statistically significant correlation between STR-5’-GCGCG-3’ of MAP3K5 gene, rs944725 of NOS2A gene and rs10483801 of ARG2 gene with hydroxyurea response in β-thalassemia/sickle cell disease compound heterozygotes. Furthermore, our study revealed a statistically significant correlation between rs2182008 of FLT1 gene, rs3191333 of KLF10 gene and rs10483801 of ARG2 gene with β-thalassemia intermedia phenotype-NTDT patients, who can produce elevated HbF levels without carrying HPFH syndromes, thus improving their clinical phenotype.Ο κλινικός φαινότυπος των ασθενών με β-αιμοσφαιρινοπάθειες ποικίλλει, γεγονός που οφείλεται αφενός στις μεταλλάξεις που εντοπίζονται στο γονίδιο που κωδικοποιεί τη β-αλυσίδα της αιμοσφαιρίνης HbΑ και αφετέρου σε τροποποιητικά γονίδια που κωδικοποιούν σημαντικούς μεταγραφικούς παράγοντες που δρούν in trans ως προς το σύμπλεγμα των γονιδίων της β-σφαιρίνης, ρυθμίζοντας την έκφραση των γονιδίων του. Μελέτες έχουν δείξει ότι η αυξημένη παραγωγή της εμβρυϊκής αιμοσφαιρίνης μπορεί να βελτιώσει την κλινική εικόνα των ενήλικων ασθενών με β-αιμοσφαιρινοπάθειες. Στην παρούσα διδακτορική διατριβή προσδιορίστηκαν οι μεταλλάξεις και οι γενετικές παραλλαγές, αφ’ενός στο γονίδιο της β-σφαιρίνης και αφ’ετέρου στα τροποποιητικά γονίδια FLT1, ARG2, NOS2A, KLF10, MAP3K5, PDE7B, ASS1, NOS1 και TOX που δρουν in trans και εμπλέκονται και στη ρύθμιση της έκφρασης των γονιδίων της γ-σφαιρίνης σε ενήλικες ασθενείς με β-αιμοσφαιρινοπάθειες. Μελετήθηκαν ενήλικες ασθενείς ελληνικής καταγωγής και συγκεκριμένα, 38 ασθενείς με εξαρτώμενη μεταγγίσεων β-θαλασσαιμία, 7 ασθενείς με μη εξαρτώμενη μεταγγίσεων β-θαλασσαιμία, 42 ασθενείς με μικροδρεπανοκυτταρική αναιμία και 53 υγιή άτομα, ως μάρτυρες. Στους μικροδρεπανοκυτταρικούς ασθενείς χορηγείται υδροξυουρία προκειμένου να αυξήσουν τα επίπεδα της εμβρυϊκής τους αιμοσφαιρίνης και κατά συνέπεια να βελτιώσουν τον κλινικό τους φαινότυπο. Παρ’ όλα αυτά δεν ανταποκρίνονται όλοι οι ασθενείς σε αυτή τη φαρμακευτική αγωγή. Ο σκοπός της παρούσας διδακτορικής διατριβής ήταν η μελέτη (φαρμακο)γονιδιωματικών δεικτών. Η παρουσία αυτών των δεικτών σχετίζεται με τη δυνατότητα α) των ασθενών με β-θαλασσαιμία να παράγουν υψηλά επίπεδα HbF, απουσία του HPFH συνδρόμου, εμφανίζοντας φαινότυπο μη εξαρτώμενης μεταγγίσεων β-θαλασσαιμίας–ενδιάμεση β-θαλασσαιμία ή ασθενών που δεν έχουν αυτή τη δυνατότητα εμφανίζοντας κλινικό φαινότυπο βαριάς β-θαλασσαιμίας εξαρτώμενη από μεταγγίσεις και β) των μικροδρεπανοκυτταρικών ασθενών να ανταποκρίνονται στη θεραπευτική αγωγή με υδροξυουρία. Απώτερος στόχος της παρούσας μελέτης ήταν και η επιβεβαίωση των συγκεκριμένων γενετικών παραλλαγών στα προαναφερθέντα τροποποιητικά γονίδια ως φαρμακογονιδιωματικών δεικτών, συμπεριλαμβάνοντας και αποτελέσματα προηγούμενων μελετών του εργαστηρίου Ε.Φ.Ε.Θ, προκειμένου να αυξηθεί το στατιστικό δείγμα και η εγκυρότητα των αποτελεσμάτων. Ο προσδιορισμός των μεταλλάξεων του γονιδίου της β-σφαιρίνης έγινε με ενίσχυση του γονιδίου με αλυσιδωτή αντίδραση της πολυμεράσης (PCR) και ακολούθως αλληλούχηση κατά Sanger. Οι γενετικές παραλλαγές προσδιορίστηκαν είτε με αλληλούχηση κατά Sanger, είτε με κατάτμηση με συγκεκριμένα ένζυμα περιορισμού ή με εφαρμογή της μεθόδου ανάλυσης των ετεροδιμερών (Heteroduplex Analysis, HDA), αφού είχε προηγηθεί ενίσχυση με PCR των συγκεκριμένων τμημάτων των τροποποιητικών γονιδίων που φέρουν τις συγκεκριμένες γενετικές παραλλαγές, είτε με την εφαρμογή της αλυσιδωτής αντίδρασης της πολυμεράσης ειδικής για το αλληλόμορφο (Allele Specific PCR).Τα αποτελέσματα της παρούσας μελέτης ανέδειξαν σημαντική συσχέτιση μεταξύ της μικρής επαναλαμβανόμενης εν σειρά αλληλουχίας - STR-5’-GCGCG-3’ στον υποκινητή του MAP3K5 γονιδίου με την ανταπόκριση στη θεραπεία με υδροξυουρία των ασθενών με μικροδρεπανοκυτταρική αναιμία. Επίσης, σημαντική συσχέτιση βρέθηκε μεταξύ των μονονουκλεοτιδικών πολυμορφισμών rs944725 στο NOS2A γονίδιο και rs10483801 στο ARG2 γονίδιο, με την ανταπόκριση στη θεραπεία με υδροξυουρία των ασθενών με μικροδρεπανοκυτταρική αναιμία. Επιπλέον, βρέθηκε ισχυρή συσχέτιση των μονονουκλεοτιδικών πολυμορφισμών rs2182008 στο FLT1 γονίδιο, του rs3191333 στο KLF10 γονίδιο και του rs10483801 στο ARG2 γονίδιο με το φαινότυπο ενδιάμεσης β-θαλασσαιμίας που εμφανίζουν οι ασθενείς με μη εξαρτώμενη μεταγγίσεων β-θαλασσαιμία, οι οποίοι έχουν την ικανότητα να παράγουν αυξημένα επίπεδα εμβρυϊκής αιμοσφαιρίνης, απουσία του HPFH συνδρόμου, βελτιώνοντας τον κλινικό τους φαινότυπο

Clinical implementation of preemptive pharmacogenomics in psychiatryResearch in context

Summary: Background: Pharmacogenomics (PGx) holds promise to revolutionize modern healthcare. Although there are several prospective clinical studies in oncology and cardiology, demonstrating a beneficial effect of PGx-guided treatment in reducing adverse drug reactions, there are very few such studies in psychiatry, none of which spans across all main psychiatric indications, namely schizophrenia, major depressive disorder and bipolar disorder. In this study we aim to investigate the clinical effectiveness of PGx-guided treatment (occurrence of adverse drug reactions, hospitalisations and re-admissions, polypharmacy) and perform a cost analysis of the intervention. Methods: We report our findings from a multicenter, large-scale, prospective study of pre-emptive genome-guided treatment named as PREemptive Pharmacogenomic testing for preventing Adverse drug REactions (PREPARE) in a large cohort of psychiatric patients (n = 1076) suffering from schizophrenia, major depressive disorder and bipolar disorder. Findings: We show that patients with an actionable phenotype belonging to the PGx-guided arm (n = 25) present with 34.1% less adverse drug reactions compared to patients belonging to the control arm (n = 36), 41.2% less hospitalisations (n = 110 in the PGx-guided arm versus n = 187 in the control arm) and 40.5% less re-admissions (n = 19 in the PGx-guided arm versus n = 32 in the control arm), less duration of initial hospitalisations (n = 3305 total days of hospitalisation in the PGx-guided arm from 110 patients, versus n = 6517 in the control arm from 187 patients) and duration of hospitalisation upon readmission (n = 579 total days of hospitalisation upon readmission in the PGx-guided arm, derived from 19 patients, versus n = 928 in the control arm, from 32 patients respectively). It was also shown that in the vast majority of the cases, there was less drug dose administrated per drug in the PGx-guided arm compared to the control arm and less polypharmacy (n = 124 patients prescribed with at least 4 psychiatric drugs in the PGx-guided arm versus n = 143 in the control arm) and smaller average number of co-administered psychiatric drugs (2.19 in the PGx-guided arm versus 2.48 in the control arm. Furthermore, less deaths were reported in the PGx-guided arm (n = 1) compared with the control arm (n = 9). Most importantly, we observed a 48.5% reduction of treatment costs in the PGx-guided arm with a reciprocal slight increase of the quality of life of patients suffering from major depressive disorder (0.935 versus 0.925 QALYs in the PGx-guided and control arm, respectively). Interpretation: While only a small proportion (∼25%) of the entire study sample had an actionable genotype, PGx-guided treatment can have a beneficial effect in psychiatric patients with a reciprocal reduction of treatment costs. Although some of these findings did not remain significant when all patients were considered, our data indicate that genome-guided psychiatric treatment may be successfully integrated in mainstream healthcare. Funding: European Union Horizon 2020

A 12-gene pharmacogenetic panel to prevent adverse drug reactions: an open-label, multicentre, controlled, cluster-randomised crossover implementation study

© 2023Background: The benefit of pharmacogenetic testing before starting drug therapy has been well documented for several single gene–drug combinations. However, the clinical utility of a pre-emptive genotyping strategy using a pharmacogenetic panel has not been rigorously assessed. Methods: We conducted an open-label, multicentre, controlled, cluster-randomised, crossover implementation study of a 12-gene pharmacogenetic panel in 18 hospitals, nine community health centres, and 28 community pharmacies in seven European countries (Austria, Greece, Italy, the Netherlands, Slovenia, Spain, and the UK). Patients aged 18 years or older receiving a first prescription for a drug clinically recommended in the guidelines of the Dutch Pharmacogenetics Working Group (ie, the index drug) as part of routine care were eligible for inclusion. Exclusion criteria included previous genetic testing for a gene relevant to the index drug, a planned duration of treatment of less than 7 consecutive days, and severe renal or liver insufficiency. All patients gave written informed consent before taking part in the study. Participants were genotyped for 50 germline variants in 12 genes, and those with an actionable variant (ie, a drug–gene interaction test result for which the Dutch Pharmacogenetics Working Group [DPWG] recommended a change to standard-of-care drug treatment) were treated according to DPWG recommendations. Patients in the control group received standard treatment. To prepare clinicians for pre-emptive pharmacogenetic testing, local teams were educated during a site-initiation visit and online educational material was made available. The primary outcome was the occurrence of clinically relevant adverse drug reactions within the 12-week follow-up period. Analyses were irrespective of patient adherence to the DPWG guidelines. The primary analysis was done using a gatekeeping analysis, in which outcomes in people with an actionable drug–gene interaction in the study group versus the control group were compared, and only if the difference was statistically significant was an analysis done that included all of the patients in the study. Outcomes were compared between the study and control groups, both for patients with an actionable drug–gene interaction test result (ie, a result for which the DPWG recommended a change to standard-of-care drug treatment) and for all patients who received at least one dose of index drug. The safety analysis included all participants who received at least one dose of a study drug. This study is registered with ClinicalTrials.gov, NCT03093818 and is closed to new participants. Findings: Between March 7, 2017, and June 30, 2020, 41 696 patients were assessed for eligibility and 6944 (51·4 % female, 48·6% male; 97·7% self-reported European, Mediterranean, or Middle Eastern ethnicity) were enrolled and assigned to receive genotype-guided drug treatment (n=3342) or standard care (n=3602). 99 patients (52 [1·6%] of the study group and 47 [1·3%] of the control group) withdrew consent after group assignment. 652 participants (367 [11·0%] in the study group and 285 [7·9%] in the control group) were lost to follow-up. In patients with an actionable test result for the index drug (n=1558), a clinically relevant adverse drug reaction occurred in 152 (21·0%) of 725 patients in the study group and 231 (27·7%) of 833 patients in the control group (odds ratio [OR] 0·70 [95% CI 0·54–0·91]; p=0·0075), whereas for all patients, the incidence was 628 (21·5%) of 2923 patients in the study group and 934 (28·6%) of 3270 patients in the control group (OR 0·70 [95% CI 0·61–0·79]; p <0·0001). Interpretation: Genotype-guided treatment using a 12-gene pharmacogenetic panel significantly reduced the incidence of clinically relevant adverse drug reactions and was feasible across diverse European health-care system organisations and settings. Large-scale implementation could help to make drug therapy increasingly safe. Funding: European Union Horizon 2020