ΜΕΛΕΤΗ Α΄ Στόχος: Να διερευνηθεί αν ο κινητικός υπότυπος της αστάθειας κορμού και δυσχέρειας της βάδισης (ΑΚΔΒ) σχετίζεται με τη γνωστική δυσλειτουργία που εμφανίζουν οι ασθενείς με νόσο του Parkinson (NP) χωρίς άνοια. Μέθοδοι: Χορηγήσαμε μια συστοιχία επιλεγμένων νευροψυχολογικών δοκιμασιών σε δύο ομάδες μη ανοϊκών ασθενών με ήπια έως μέτριας βαρύτητας νόσο κατηγοριοποιημένους είτε στον υπότυπο της ΑΚΔΒ είτε σε υπότυπο μη ΑΚΔΒ, καθώς και σε μια ομάδα υγιών μαρτύρων. Οι ομάδες εξισώθηκαν κατά το δυνατόν όσον αφορά δυνητικούς συγχυτικούς παράγοντες που επηρεάζουν τις νευροψυχολογικές επιδόσεις. Αποτελέσματα: Δε διαπιστώθηκαν σημαντικές διαφορές μεταξύ των δύο ομάδων ασθενών στην επίδοση σε οποιαδήποτε από τις χορηγηθείσες νευροψυχολογικές δοκιμασίες. Παρόλα αυτά, σε σχέση με τους μάρτυρες υπήρξε μια τάση διαφοροποίησης ως προς το κυρίαρχο πρότυπο της γνωστικής δυσλειτουργίας. Η ομάδα με τον υπότυπο της ΑΚΔΒ είχε βραδύτερες επιδόσεις σε μια δοκιμασία ψυχοκινητικής ταχύτητας και γνωστικής ευελιξίας, ενώ η ομάδα με υπότυπο της νόσου μη ΑΚΔΒ είχε χειρότερες επιδόσεις στις μετρήσεις της λεκτικής μάθησης και της οπτικοχωρικής αντίληψης. Συμπεράσματα: Ο υπότυπος της ΑΚΔΒ δε συσχετίσθηκε με σοβαρότερα γνωστικά ελλείμματα και έτσι είναι πιθανό οι μηχανισμοί της γνωστικής δυσλειτουργίας να είναι, έως ένα ορισμένο βαθμό, κοινοί ανεξάρτητα από τον κινητικό υπότυπο της νόσου. ΜΕΛΕΤΗ Β Στόχος: Να διερευνηθεί αν υπάρχει συσχέτιση μεταξύ της ΝΡ και του γονιδίου CHRNA4, το οποίο κωδικοποιεί την α4 υπομονάδα του α4β2 νικοτινικού υποδοχέα της ακετυλοχολίνης (nAChR). Mέθοδοι: Στη μελέτη συμμετείχαν 100 ασθενείς με ΝΡ και 105 μάρτυρες, εξισωμένοι ως προς την ηλικία και το φύλο και ανήκοντες στην ίδια πληθυσμιακή ομάδα με τους ασθενείς. Ο γενετικός δείκτης που εξετάσθηκε είναι ένας μονονουκλεοτιδικός πολυμορφισμός στο 5ο εξόνιο του γονιδίου CHRNA4 (dbSNP rs1044396). Έγινε απομόνωση DNA γονιδιώματος από περιφερικό αίμα και ακολούθησε ανάλυση μεγέθους περιοριστικών τμημάτων μετά από αλυσωτή αντίδραση πολυμεράσης και κατάτμηση των προϊόντων αυτής με το ένζυμο Hha I. Μια υποομάδα 42 ασθενών υποβλήθηκαν επίσης σε λεπτομερή κλινική και νευροψυχολογική εκτίμηση. Η στατιστική ανάλυση για τη σύγκριση της συχνότητας των αλληλομόρφων και των γονοτύπων μεταξύ των ομάδων έγινε με τη δοκιμασία χ2, και τον ακριβή έλεγχο Fisher εάν έστω ένα κελί είχε n<5. Υπολογίστηκαν οι σχετικοί κίνδυνοι και τα κατά 95% διαστήματα αξιοπιστίας τους που αντιστοιχούσαν στα αλληλόμορφα και τους γονότυπους. Χρησιμοποιήθηκε η λογιστική ανάλυση παλινδρόμησης εάν ήταν απαραίτητη η προσαρμογή για την ηλικία ή το φύλο. Αποτελέσματα: Οι συχνότητες των γονοτύπων στην ομάδα των ασθενών (TT 34%; CT 58%; CC 8%) σε σύγκριση με τις συχνότητες των γονοτύπων στην ομάδα των μαρτύρων (TT 28.6 %; CT 47.6%; CC 23.8 %) παρουσίασαν στατιστικά σημαντική διαφορά (χ2 = 9.48, df = 2, p = 0.009). Η ομοζυγωτία CC συσχετίσθηκε με χαμηλότερο κίνδυνο παρουσίας της ΝΡ (CC vs φορείς T: OR = 0.28; 95% CI = 0.12–0.65; p = 0.002; στατιστική ισχύς 93.1%). Παρατηρήθηκε επίσης απόκλιση στην κατανομή των αλληλομόρφων μεταξύ των ασθενών και των μαρτύρων. Υπήρχε σημαντικά χαμηλότερη συχνότητα του αλληλόμορφου C μεταξύ των ασθενών (37%) σε σχέση με τους μάρτυρες (47.6%) (χ2 = 4.73; df = 1; OR=0.65; 95% CI = 0.44–0.96; p = 0.03). Η ανάλυση διαστρωμάτωσης έδειξε ότι η διαφορά στην κατανομή των γονοτύπων μεταξύ ασθενών και μαρτύρων ήταν στατιστικά σημαντική και συγκριτικά μεγαλύτερη στο θήλυ φύλο σε σχέση με το άρρεν φύλο και στους ασθενείς με εκδήλωση ΝΡ σε όψιμη ηλικία ( > 50 ετών) σε σχέση με αυτούς που εμφάνισαν πρώιμης έναρξης νόσο (< 50 ετών). Οι ασθενείς με ΝP που ανιχνεύθηκαν να φέρουν το γονότυπο CC και υποβλήθηκαν σε νευροψυχολογική αξιολόγηση έτειναν να έχουν καλύτερα διατηρημένες τις γνωστικές λειτουργίες που σχετίζονται με την προσοχή και την ταχύτητα επεξεργασίας των πληροφοριών. Συμπεράσματα: Η παρουσία του αλληλομόρφου C (dbSNP rs1044396) του γονιδίου CHRNA4 συνδέεται με μειωμένο κίνδυνο ΝΡ κατά 35%. Επίσης, τα άτομα με το γονότυπο CC εμφανίζουν σχεδόν τρισήμισυ (3,5) φορές χαμηλότερο κίνδυνο νόσου του Parkinson. Η ποικιλομορφία του γονιδίου CHRNA4 φαίνεται ότι σχετίζεται ιδιαίτερα με την επιρρέπεια εκδήλωσης της ΝΡ με ηλικιακά όψιμη έναρξη της νόσου, και επίσης με τις γνωστικές λειτουργίες των ασθενών χωρίς άνοια, ειδικά αυτές που εξαρτώνται από την προσοχή και την οπτικοκινητική αντίληψη. / Study A Aim: To investigate whether there is an association of the postural instability and gait difficulty (PIGD) motor subtype with cognitive dysfunction in non-demented Parkinson’s disease (PD) patients. Methods: We administered a battery of selected neuropsychological tests to assess attention, psychomotor speed, executive functions (set shifting ability and inhibitory control), visuospatial perception and visual constructive ability to two groups of non-demented patients with mild to moderate disease classified either as PIGD or as non-PIGD subtype and to a group of healthy controls. Groups were matched on potential confounders of neuropsychological performance. Results: No significant differences were revealed between the two groups of patients in the performance of any of the administered neuropsychological tests. However, relative to controls there was a tendency towards a differential pattern of cognitive dysfunction. The PIGD group had slower performance in a test of psychomotor speed and cognitive flexibility, whilst the non-PIGD group performed worse in measures of verbal learning and visuo-spatial perception. Conclusions: The PIGD subtype was not associated with more severe cognitive deficits and may to a certain extent share common mechanisms of cognitive dysfunction with non-PIGD subtypes. Study B Aim: to investigate whether there is an association between PD and a variation in the CHRNA4 gene coding for the α4 subunit, the primary subunit of the α4β2 brain nicotinic acetylcholine receptors. Methods: Patients (N=100) and controls (N=105), matched on the basis of sex, age and ethnicity, were genotyped for a single nucleotide polymorphism at cDNA position 1860 lying within the 5th exon of the CHRNA4 gene. DNA was extracted from peripheral blood samples and genotyping was done by PCR-based restriction fragment length polymorphism analysis. A subset of 42 patients also received detailed clinical and cognitive assessments. Comparisons of allele and genotype frequencies between groups were performed using the χ2 test, and the Fisher exact test if one cell had n<5. The relative risk for genotypes and alleles was estimated through calculation of odds ratios (ORs) with 95% confidence intervals (CIs). Logistic regression analysis was used if adjustment for age or sex was necessary. Results: The genotype frequencies in the patients group (TT 34%; CT 58%; CC 8%) vs. the genotype frequencies in the control group (TT 28.6 %; CT 47.6%; CC 23.8 %) demonstrated a statistically significant difference (χ2 = 9.48, df = 2, p = 0.009). CC homozygosity was associated with a lower risk of PD (CC vs T carriers: OR = 0.28; 95% CI = 0.12–0.65; p = 0.002). Also, the allelic distribution was significantly different between patients and controls. There was a significantly lower frequency of the C allele among the patients with PD (37%) as compared with the controls (47.6%) (χ2 = 4.73; df = 1; OR=0.65; 95% CI = 0.44–0.96; p = 0.03). Stratified analysis showed that the difference in the genotypic distribution between cases and controls was significant among females but did not reach significance among males. The frequency of CC homozygotes was also significantly lower in the group of patients with late onset PD than in the controls, but it was not significantly different between the early onset group of patients and the controls. CC homozygotes also tended to have better performance than T carriers on measures of attention and psychomotor speed (Trail Making Test part A and Symbol Digit Modalities Test). Conclusions: The presence of the C allele at SNP rs1044396 of the CHRNA4 gene is associated with a decreased risk for PD by 35%. Moreover, the CC genotype lowers the risk for PD by ~ 3.5 fold. Variation in the CHRNA4 gene may particularly influence susceptibility for late onset PD and further be associated with measurable effects on overt cognitive performance of yet not-demented PD patients, specifically the part loading on attentional capacities.

Introdução: A grande variabilidade individual em resposta a fármacos antitabagismo sugere que tratamentos específicos podem ser mais efetivos em determinados subgrupos de fumantes. No contexto de medicina personalizada, o principal objetivo do presente estudo foi avaliar se polimorfismos nos genes CHRNA4, CHRNB2, CYP2B6 e ANKK1 estão associados com a resposta às terapias de cessação tabágica em pacientes provenientes de um programa de assistência ao fumante. Métodos: Estudo de coorte com 483 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona em monoterapia ou coadministrada com terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. Os polimorfismos CHRNA4 (rs1044396 e rs2236196), CHRNB2 (rs2072660 e rs2072661) e ANKK1 (rs1800497) foram genotipados pela análise da curva de melting e os polimorfismos CYP2B6 *9 (rs3745274), *4 (rs2279343), *5 (rs3211371) foram genotipados por restrição enzimática. Resultados: Os pacientes com o genótipo CC para o polimorfismo CHRNA4 (rs10443196) obtiveram menor taxa de sucesso no tratamento com vareniclina (29,5%) em comparação com os portadores dos genótipos CT ou TT (50,9%) (P=0,007; n=167). Os genótipos CT ou TT foram associados com maior odds ratio para o sucesso (OR=1,67; IC 95%=1,10-2,53; P=0,02), em um modelo multivariado. Os pacientes com o genótipo AA para o polimorfismo CYP2B6 (rs2279343) obtiveram maior taxa de sucesso no tratamento com bupropiona (48,0%) em comparação com portadores dos genótipos AG ou GG (35,5%) (P=0,05; n=237). O genótipo AA foi associado com maior odds ratio para o sucesso no tratamento (OR=1,92; IC 95%=1,08-3,42; P=0,03), em um modelo multivariado. Não foram observadas diferenças significativas nos escores FTND e Issa com relação aos polimorfismos estudados. Conclusão: Os polimorfismos CHRNA4 (rs1044396) e CYP2B6 (rs2279343) estão associados com a cessação tabágica em indivíduos tratados com vareniclina e bupropiona, respectivamente. Sugere-se que estes polimorfismos influenciam a resposta farmacológica e podem ser importantes para o desenho de uma farmacoterapia individualizada / Background: The large individual variability in response to drugs for smoking cessation suggests that specific treatments can be more effective in particular subgroups of smokers. In the context of personalized medicine, the main aim of the present study was to evaluate whether the CHRNA4, CHRNB2, CYP2B6 and ANKK1 polymorphisms are associated with response to smoking cessation therapies in patients from a smoker assistance program. Methods: This cohort study enrolled 483 smoking patients patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion in monoterapy or plus nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. The CHRNA4 (rs1044396 and rs2236196), CHRNB2 (rs2072660 and rs2072661) and ANKK1 rs1800497 polymorphisms were genotyped by high resolution melting analysis and the CYP2B6 *9 (rs3745274), *4 (rs2279343) and *5 (rs3211371) were genotyped by restriction fragment lenght polymorphisms. Results: Patients with CHRNA4 rs1044396 CC genotype had lower success rate in treatment with varenicline (29.5%) compared with carriers of CT or TT genotypes (50.9%) (P=0.007, n=167). The CT or TT genotypes were associated with higher odds ratio for success (OR=1.67, 95%CI=1.10-2.53, P=0.02), in a multivariate model. Patients with CYP2B6 rs2279343 AA genotype had higher success rate in treatment with bupropion (48.0%) compared with carriers of AG or GG genotypes (35.5%) (P=0.05, n=237). The AA genotype was associated with higher odds ratio for success (OR=1.92, 95%CI=1.08-3.42, P=0.03), in a multivariate model. We did not observe significant differences in the FTND and Issa scores according to the studied polymorphisms. Conclusion: The CHRNA4 rs1044396 and CYP2B6 rs2279343 are associated with smoking cessation in individuals on varenicline and bupropion terapies, respectively. We suggest that these polymorphisms influence the pharmacological response of these drugs and it might be important in the design of individualized pharmacotherapy

Introdução: A grande variabilidade individual em resposta a fármacos antitabagismo sugere que tratamentos específicos podem ser mais efetivos em determinados subgrupos de fumantes. No contexto de medicina personalizada, o principal objetivo do presente estudo foi avaliar se polimorfismos nos genes CHRNA4, CHRNB2, CYP2B6 e ANKK1 estão associados com a resposta às terapias de cessação tabágica em pacientes provenientes de um programa de assistência ao fumante. Métodos: Estudo de coorte com 483 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona em monoterapia ou coadministrada com terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. Os polimorfismos CHRNA4 (rs1044396 e rs2236196), CHRNB2 (rs2072660 e rs2072661) e ANKK1 (rs1800497) foram genotipados pela análise da curva de melting e os polimorfismos CYP2B6 *9 (rs3745274), *4 (rs2279343), *5 (rs3211371) foram genotipados por restrição enzimática. Resultados: Os pacientes com o genótipo CC para o polimorfismo CHRNA4 (rs10443196) obtiveram menor taxa de sucesso no tratamento com vareniclina (29,5%) em comparação com os portadores dos genótipos CT ou TT (50,9%) (P=0,007; n=167). Os genótipos CT ou TT foram associados com maior odds ratio para o sucesso (OR=1,67; IC 95%=1,10-2,53; P=0,02), em um modelo multivariado. Os pacientes com o genótipo AA para o polimorfismo CYP2B6 (rs2279343) obtiveram maior taxa de sucesso no tratamento com bupropiona (48,0%) em comparação com portadores dos genótipos AG ou GG (35,5%) (P=0,05; n=237). O genótipo AA foi associado com maior odds ratio para o sucesso no tratamento (OR=1,92; IC 95%=1,08-3,42; P=0,03), em um modelo multivariado. Não foram observadas diferenças significativas nos escores FTND e Issa com relação aos polimorfismos estudados. Conclusão: Os polimorfismos CHRNA4 (rs1044396) e CYP2B6 (rs2279343) estão associados com a cessação tabágica em indivíduos tratados com vareniclina e bupropiona, respectivamente. Sugere-se que estes polimorfismos influenciam a resposta farmacológica e podem ser importantes para o desenho de uma farmacoterapia individualizada / Background: The large individual variability in response to drugs for smoking cessation suggests that specific treatments can be more effective in particular subgroups of smokers. In the context of personalized medicine, the main aim of the present study was to evaluate whether the CHRNA4, CHRNB2, CYP2B6 and ANKK1 polymorphisms are associated with response to smoking cessation therapies in patients from a smoker assistance program. Methods: This cohort study enrolled 483 smoking patients patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion in monoterapy or plus nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. The CHRNA4 (rs1044396 and rs2236196), CHRNB2 (rs2072660 and rs2072661) and ANKK1 rs1800497 polymorphisms were genotyped by high resolution melting analysis and the CYP2B6 *9 (rs3745274), *4 (rs2279343) and *5 (rs3211371) were genotyped by restriction fragment lenght polymorphisms. Results: Patients with CHRNA4 rs1044396 CC genotype had lower success rate in treatment with varenicline (29.5%) compared with carriers of CT or TT genotypes (50.9%) (P=0.007, n=167). The CT or TT genotypes were associated with higher odds ratio for success (OR=1.67, 95%CI=1.10-2.53, P=0.02), in a multivariate model. Patients with CYP2B6 rs2279343 AA genotype had higher success rate in treatment with bupropion (48.0%) compared with carriers of AG or GG genotypes (35.5%) (P=0.05, n=237). The AA genotype was associated with higher odds ratio for success (OR=1.92, 95%CI=1.08-3.42, P=0.03), in a multivariate model. We did not observe significant differences in the FTND and Issa scores according to the studied polymorphisms. Conclusion: The CHRNA4 rs1044396 and CYP2B6 rs2279343 are associated with smoking cessation in individuals on varenicline and bupropion terapies, respectively. We suggest that these polymorphisms influence the pharmacological response of these drugs and it might be important in the design of individualized pharmacotherapy