Pharmacogenetic markers of adverse events of dopaminergic treatment in Parkinson's disease

Redenšek, Sara (author), Dolžan, Vita (mentor), Trošt, Maja (comentor)

Introduction Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. The main pathological hallmark is the death of dopaminergic neurons in the nigrostriatal pathway, which mainly affects motor function. Therefore dopamine replacement strategy has a central role in PD management. The most prescribed dopaminergic drugs in PD are levodopa and dopamine agonists. Symptomatic dopaminergic treatment is very effective, but poses risk for occurrence of motor and non-motor adverse events. Motor fluctuations and dyskinesia are the most frequent motor adverse events. Motor fluctuations present as oscillations between good and bad motor symptom control, while dyskinesia usually manifest as involuntary movements. They mostly occur after long-term levodopa administration, however they are sometimes observed shortly after treatment initiation. Non-motor adverse events, such as excessive daytime sleepiness and sleep attacks, visual hallucinations, nausea/vomiting, orthostatic hypotension, peripheral oedema, and impulse control disorders, are associated with both levodopa and dopamine agonists. Although some risk factors for adverse events are known, their occurrence and time to occurrence in individual patients cannot be predicted yet. Genetic variability in the dopaminergic and other pathways affecting the development and progression of PD could serve as foundation for research and identification of pharmacogenetic markers of adverse events. Dopaminergic pathway is the most heavily disrupted neurotransmitter pathway in the pathogenesis of PD. Polymorphic genes encode several enzymes (DDC, COMT, MAOB), transporters (SLC6A3, SLC18A2, SLC7A5, SLC22A1), receptors (DRD1-5) and other molecules (SV2C) leading to inter-individual variability in the capacity of dopamine synthesis, transport, degradation, and signalling. Several pharmacogenetic studies searching for associations between single nucleotide polymorphisms (SNPs) of above mentioned genes and response to dopaminergic treatment have already been performed. Dopaminergic treatment outcome may also be influenced by pathways related to molecular pathogenesis of the disease. Neuroinflammation and oxidative stress related pathways contribute to PD pathogenesis. Several SNPs in the IL1&#946;, TNF&#945;, and IL6 have already been associated with PD susceptibility. Genetic variability and/or activities of the inflammasome NALP3, glutathione peroxidase (GPX1), catalase (CAT), superoxide dismutase (SOD2), and nitric oxide synthase (NOS1) have been associated with PD susceptibility. Additionally, genetic variability of the neurodevelopment and apoptosis pathways have also been associated with PD susceptibility. Genetic variability of dopaminergic pathway has been extensively studied, but only individual genes were analysed. Furthermore, pathways affecting PD development and progression may play an important role in the treatment response, but no data on this has been reported so far. Aim of the study and hypotheses The aetiology of adverse events is not completely understood and their occurrence cannot be clearly predicted. Some clinical and pharmacogenetic factors were already identified as potential markers of the adverse events. However, a comprehensive evaluation of the genetic variability in (i) dopaminergic pathway and pathways of (ii) neuroinflammation, (iii) oxidative stress, (iv) neuron development, proliferation, and differentiation, and (v) apoptosis has not been conducted. The aim of this study was to use pathway-based approach to evaluate SNPs from the above listed pathways as potential pharmacogenetic markers of adverse events of dopaminergic treatment. Furthermore, we wanted to construct clinical and clinical-pharmacogenetic models for prediction of time to occurrence of motor complications after initiation of levodopa treatment. In this study we tested the following hypotheses: • Genetic variability in the pathways of transport, metabolism, and mechanism of action of dopamine, levodopa and other dopaminergic substances influences the occurrence and/or time to occurrence of adverse events of dopaminergic treatment in PD. • Genetic variability in pathways affecting the development and progress of PD has an influence on occurrence and/or time to occurrence of adverse events of dopaminergic treatment in PD. • Clinical-pharmacogenetic models can help us to evaluate the simultaneous influence of certain genetic polymorphisms and clinical factors on the occurrence of adverse events of dopaminergic treatment in PD. Participants and methods A total of 231 unrelated PD patients were enrolled in this retrospective study at the Department of Neurology, University Medical Centre Ljubljana, Slovenia between October 2016 and April 2018. Inclusion criteria were (i) diagnosis of PD according to the UK Parkinson Disease Society Brain Bank criteria by an experienced movement disorders specialist, (ii) available clinical data, (iii) at least three months of an ongoing levodopa and/or dopamine agonists treatment duration. The study protocol was approved by the Slovenian Ethics Committee for Research in Medicine (KME 42/05/16). All of the patients signed the informed consent. Each patient donated a sample of their peripheral blood for DNA isolation within withdrawal for other routine laboratory tests. All patients were genotyped for 37 SNPs in the genes involved in the processes of (i) dopaminergic pathway, (ii) neuroinflammation, (iii) oxidative stress, (iv) neuron development, proliferation, and differentiation, and (v) apoptosis. Median and 25th to 75th percentile range were used to describe central tendency and variability of continuous variables, while frequencies were used to describe the distribution of categorical variables. The agreement of genotype frequencies with Hardy-Weinberg equilibrium was examined by Chi-squared test. Logistic regression was used to calculate odds ratios (ORs), 95% confidence intervals (95%CIs), and p-values to examine the associations of selected SNPs and clinical data with the risk for adverse events. Allele with the highest frequency was used for reference. Cox proportional hazards models were used to estimate the association of SNPs and clinical covariates with the time to occurrence of motor complications after levodopa treatment initiation. Results were reported as hazard ratios (HR), 95%CI, and p-values. Models were built using Cox analysis with LASSO (Least Absolute Shrinkage and Selection Operator) penalization due to a large number of explanatory variables in comparison to the number of events. For both models the time-dependent receiver operating characteristic (ROC) curves were constructed, where sensitivity, specificity, and tAUC were assessed. The predictive scoring system was estimated by selecting the threshold that provided the maximized sum of the cross-validated true positive rate and true negative rate. Cross-validation was applied on all the predictive accuracy estimates to avoid biased and overoptimistic results. Statistical analyses were carried out by IBM SPSS Statistics, version 21.0 (IBM Corporation, Armonk, NY, USA) and the R software. Results Within the first part of the study we studied associations between genetic variability of the dopaminergic pathway and the occurrence of adverse events of dopaminergic treatment. We observed some important associations: visual hallucinations were associated with COMT rs165815 C allele (OR=0.34; 95%CI=0.16-0.72; p=0.004), DRD3 rs6280 C allele (OR=1.88; 95%CI=1.00-3.54; p=0.049), and DRD3 rs6280 CC genotype (OR=3.31; 95%CI=1.37-8.03; p=0.008); orthostatic hypotension was associated with DDC rs921451 C allele (OR=1.86; 95%CI=1.07-3.23; p=0.028), DDC rs921451 CT genotype (OR=2.30; 95%CI=1.26-4.20; p=0.007), DDC rs3837091 AGAG- genotype (OR=1.94; 95%CI=1.07-3.51; p=0.028), and SLC22A1 rs628031 AA genotype (OR=2.57; 95%CI=1.11-5.95; p=0.028); peripheral oedema were associated with SLC22A1 rs628031 AA genotype (OR=4.00; 95%CI=1.62-9.88; p=0.003); impulse control disorders were associated with SLC22A1 rs628031 AA genotype (OR=3.16; 95%CI=1.03-9.72; p=0.045); and dyskinesia were associated with SLC22A1 rs628031 GA genotype (OR=0.48; 95%CI=0.24-0.98; p=0.043), and with SLC22A1 rs628031 A allele (OR=0.48; 95%CI=0.25-0.92; p=0.027). In the analysis of the genetic variability of the neuroinflammation pathway only one association showing a nonsignificant trend was observed. The IL1&#946; rs1143623 C allele was associated with lower odds for developing orthostatic hypotension (OR=0.57; 95%CI=0.32-1.00, p=0.050), while heterozygotes had nominally significant lower odds for developing orthostatic hypotension (OR=0.51; 95%CI=0.28-0.93, p=0.028). Genetic variability of the oxidative stress pathway also showed some important associations with the occurrence of adverse events of dopaminergic treatment: peripheral oedema were associated with CAT rs1001179 A allele (OR=0.32; 95%CI=0.15-0.68, p=0.003) and CAT rs1001179 GA genotype (OR=0.32; 95%CI=0.15-0.69, p=0.004); excessive daytime sleepiness and sleep attacks were associated with NOS1 rs2682826 A allele (OR=1.75; 95%CI=1.00-3.06, p=0.048) and NOS1 rs2682826 AA genotype (OR=3.75; 95%CI=1.23-11.45, p=0.020); nausea/vomiting was associated with SOD2 rs4880 T allele (OR=0.49; 95%CI=0.25-0.94, p=0.031) and SOD2 rs4880 CT genotype (OR=0.48, 95%CI=0.24-0.98, p=0.045); impulse control disorders were associated with NOS1 rs2682826 A allele (OR=2.59; 95%CI=1.09-6.19, p=0.032) and NOS1 rs2682826 GA genotype (OR=2.74, 95%CI=1.13-6.64, p=0.025). Variables associated with time to occurrence of motor fluctuations after initiation of levodopa treatment were: age at diagnosis (HR=0.97; 95%CI=0.96-0.99; p<0.001), time from diagnosis to initiation of levodopa treatment (HR=1.36; 95%CI=1.25-1.49; p<0.001), NOS1 rs2293054 (GG: Ref.; AA: HR=0.36; 95%CI=0.13-1.00; p=0.051), DRD2 rs1799732 (CC: Ref.; --: HR=8.89; 95%CI=1.19-66.18; p=0.033), and DRD3 rs6280 (TT: Ref.; CC: HR=2.04; 95%CI=1.16-3.60; p=0.014). Variables associated with time to occurrence of dyskinesia after initiation of levodopa treatment were: age at diagnosis (HR=0.96; 95%CI=0.95-0.98; p<0.001), beta-blockers (HR=0.60; 95%CI=0.36-1.00; p=0.051), time from diagnosis to initiation of levodopa treatment (HR=1.23; 95%CI=1.11-1.37, p<0.001), CAT rs1001179 (GG: Ref.; AA: HR=2.60; 95%CI=1.17-5.79; p=0.019), SOD2 rs4880 (CC: Ref.; TT: HR=0.54; 95%CI=0.30-0.98; p=0.043), SLC22A1 rs628031 (GG: Ref.; GA: HR=0.63; 95%CI=0.40-1.00; p=0.048; AA: HR=0.53; 95%CI=0.29-0.99; p=0.047), DRD2 rs1799732 (CC: Ref.; --: HR=8.66; 95%CI=1.16-64.86; p=0.036), and NRG1 rs3735781 (AA: Ref.; GA: HR=0.65; 95%CI=0.41-1.99; p=0.051). The clinical-pharmacogenetic model for motor fluctuations included age at diagnosis (HR=0.99), time from diagnosis to initiation of levodopa treatment (HR=1.24), COMT rs165815 (HR=0.90), DRD3 rs6280 (HR=1.03), and BIRC5 rs9904341 (HR=0.95). With the ROC curve after five years of treatment (cross-validated AUC=0.70, sensitivity=52.2%, specificity=82.3%). We constructed the ROC curve for data after five years of treatment for the clinical model for comparison (AUC=0.68; sensitivity=48.4%, specificity=81.9%). The clinical-pharmacogenetic model for dyskinesia included sex (HR=1.07), age at diagnosis (HR=0.97), tremor-predominant PD (HR=0.88), beta-blockers (HR=0.95), alcohol consumption (HR=0.99), time from diagnosis to initiation of levodopa treatment (HR=1.15), CAT rs1001179 (HR=1.27), SOD2 rs4880 (HR=0.95), NOS1 rs2293054 (HR=0.99), COMT rs165815 (HR=0.92), and SLC22A1 rs628031 (HR=0.80). With the ROC curve of five year treatment (cross-validated AUC=0.68, sensitivity=54.1%, specificity=66.1%). We constructed the ROC curve for data after five years of treatment for the clinical model for comparison (AUC=0.71; sensitivity=79.8%, specificity=48.4%). Discussion In this study we evaluated the effect of 34 SNPs from five different biological pathways and several clinical parameters on the occurrence and also time of occurrence of adverse events of dopaminergic treatment in PD patients. We observed a protective effect of the COMT rs165815 C allele against visual hallucinations This SNP has never been studied in association with this phenotype. However, COMT rs4680 already showed association with visual hallucinations. On the other hand, DDC genotypes were nominally significantly associated with the elevated odds for occurrence of orthostatic hypotension. It was suggested that the studied SNPs reduce DDC expression or activity, which could lead to lowered concentrations of noradrenalin and consequential vasodilatation. This mechanism may support the occurrence of orthostatic hypotension due to dopaminergic treatment. Furthermore, the SLC22A1 rs628031 appeared to be a promising genetic marker of several adverse events of dopaminergic treatment. It was associated with higher odds for development of orthostatic hypotension, peripheral oedema, and impulse control disorders. It was also associated with lowered odds for development of dyskinesia. Since this SNP appeared to be important in the occurrence of several adverse events, it may play a role in the overall drug action and may thus modify the overall susceptibility to adverse events. DRD3 rs6280 CC genotype was observed as a risk factor for visual hallucinations. This SNP may lead to modifications in intracellular signalling via higher binding affinity Our study showed one, however nonsignificant, association of the orthostatic hypotension with IL1&#946; rs1143623. Some studies already explored and observed an association between inflammation and orthostatic hypotension, but our study was the first to point out an association with a genetic component. This SNP has already showed an association with PD susceptibility. CAT rs1001179 was significantly associated with the occurrence of peripheral edema. The mechanism of this adverse event is not fully understood. Our results suggest that oxidative pathway might be involved in its occurrence. We observed that the NOS1 rs2682826 is associated with the excessive daytime sleepiness and sleep attacks and impulse control disorders. The NOS1 genotype might affect sleep cycle according to some papers. Additionally, NOS1 rs2682826 has already been associated with several psychiatric diseases, such as depression and anxiety. SOD2 rs4880 was associated with lowered odds for nausea/vomiting occurrence. Some studies have already showed association of oxidative stress in the enteric nervous system with development of gastrointestinal complications. We constructed clinical and clinical-pharmacogenetic models for prediction of time to occurrence of motor complications and compared them. These models could serve for construction of algorithms for identification of patients with elevated risk for earlier occurrence of motor complications. The difference in predictive capacities of the clinical and clinical-pharmacogenetic models was not clinically important. The cross-validated AUCs, specificities, and sensitivities for clinical and clinical-pharmacogenetic models for motor fluctuations did not differ significantly. In the case of dyskinesia the clinical-pharmacogenetic model had higher specificity in comparison to the clinical model. This means that the probability of an actual adverse event in the case of a positive result is higher under the clinical-pharmacogenetic model. All of the models correctly predicted the occurrence of the adverse event in the first five years of levodopa treatment in the majority of cases. Conclusions With this study we have shown that genetic variability influences the occurrence and time to occurrence of adverse events of dopaminergic treatment in PD. We have confirmed or at least partially confirmed the following hypotheses: • SNPs of the dopaminergic pathway influence the occurrence of several adverse events of dopaminergic treatment in PD. • IL1&#946; rs1143623 from the neuroinflammation pathway shows a trend towards an association with the occurrence of orthostatic hypotension as an adverse event of dopaminergic treatment in PD. • SNPs from genes from the oxidative stress pathway influence the occurrence of several non-motor adverse events of dopaminergic treatment in PD. • Clinical-pharmacogenetic models may enable a simultaneous evaluation of the influence of clinical and genetic factors on the time of occurrence of motor complications due to levodopa treatment in PD.

• Language: English
• Keywords: Parkinson's disease, pharmacogenetics, dopaminergic treatment, adverse events, genetic polymorphisms, personalized medicine, clinical-pharmacogenetic models
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2019
• PID: 20.500.12556/RUL-118016
• COBISS.SI-ID: 302939136
• Publication date in RUL: 12.08.2020

Secondary language

• Language: Slovenian
• Title: Farmakogenetski označevalci neželenih učinkov dopaminergičnega zdravljenja pri Parkinsonovi bolezni

Abstract

Uvod Parkinsonova bolezen (PB) je druga najbolj pogosta nevrodegenerativna bolezen možganov. Njena glavna patofiziološka značilnost je propad dopaminergičnih nevronov nigrostriatne poti, ki se kaže z motnjo gibanja. Osnovni princip zdravljenja je nadomeščanje dopamina. Najpogosteje predpisana zdravila so levodopa in agonisti dopamina. Zdravljenje je učinkovito, vendar pogosto povzroča motorične in nemotorične neželene učinke. Najpogosteje se pojavljajo motorična nihanja in diskinezije. Motorična nihanja so izmenjevanje faz dobrega in slabega motoričnega stanja. Diskinezije pa se kažejo kot nehoteni zgibki. Ti neželeni učinki se večinoma pojavljajo po nekaj letih zdravljenja z levodopo, redkeje že po nekaj mesecih. Nemotorični neželeni učinki, kot so prekomerna dnevna zaspanost in napadi spanja, vidne halucinacije, slabost/bruhanje, ortostatska hipotenzija, periferni edemi in motnja kontrole impulzov, pa se lahko pojavijo tako po zdravljenju z levodopo kot tudi po agonistih dopamina. Nekaj dejavnikov tveganja za pojav neželenih učinkov je že znanih. Kljub temu se pojava neželenih učinkov in časa do pojava le-teh ne da napovedati. Genetska variabilnost dopaminergične poti in poti, ki vplivajo na pojav in potek bolezni, bi lahko služila kot temelj za raziskovanje in identifikacijo farmakogenetskih označevalcev teh neželenih učinkov. Dopaminergična pot je najbolj okvarjena pot v patogenezi PB. Zaradi genetskih polimorfizmov v genih za presnovne encime (DDC, COMT, MAOB), prenašalce (SLC6A3, SLC18A2, SLC7A5, SLC22A1), receptorje (DRD1-5) in druge molekule (SV2C) se bolniki razlikujejo glede sposobnosti sinteze dopamina, njegovega prenosa, presnove in signaliziranja. Kar nekaj farmakogenetskih študij je raziskovalo in tudi pokazalo povezave med polimorfizmi posameznih nukleotidov zgoraj omenjenih genov in odgovorom na dopaminergično zdravljenje. Odgovor na dopaminergično zdravljenje bi lahko bil povezan tudi z genetsko variabilnostjo v poteh, ki vplivajo na nastanek in potek bolezni. Vnetje in oksidativni stres sta biološki poti, ki pomembno vplivata na razvoj PB. Posamezni polimorfizmi genov IL1&#946;, TNF in IL6 so že bili povezani s tveganjem za PB. Genetska variabilnost in/ali aktivnost inflamasoma NALP3, glutation peroksidaze 1 (GPX1), katalaze (CAT), superoksid dismutaze 2 (SOD2) in NO sintaze (NOS1) sta že bili povezani s tveganjem za pojav PB. Prav tako sta s tveganjem povezani genetska variabilnost nevrorazvojne poti (BDNF, NOTCH4, NRG1) in poti apoptoze (BIRC5). Genetska variabilnost dopaminergične poti v povezavi z odgovorom na dopaminergično zdravljenje je bila že dobro preučena, vendar so študije običajno preiskovale le posamezne gene. Poti, ki vplivajo na razvoj in potek bolezni, bi prav tako lahko vplivale na odgovor na zdravljenje, vendar takšne študije do sedaj še niso bile objavljene. Namen in hipoteze Etiologija neželenih učinkov dopaminergičnega zdravljenja ni povsem jasna. Nekateri klinični in farmakogenetski dejavniki so že bili identificirani kot potencialni označevalci teh neželenih učinkov. Vendar pa celostna obravnava genetske variabilnosti (i) dopaminergične in (ii) vnetne poti, (iii) poti oksidativnega stresa, (iv) razvoja, proliferacije in diferenciacije nevronov in (v) apoptoze še ni bila opravljena. Namen naše študije je bil s pristopom obravnave celotnih bioloških poti odkriti nove potencialne genetske označevalce neželenih učinkov dopaminergičnega zdravljenja. Želeli smo tudi oblikovati klinične in klinično-farmakogenetske modele za napoved časa do pojava motoričnih komplikacij po začetku zdravljenja z levodopo. V študiji smo preverili naslednje hipoteze: • Genetska variabilnost v poteh transporta, presnove in delovanja dopamina, levodope in drugih dopaminergičnih zdravil vpliva na pojav in/ali čas do pojava neželenih učinkov dopaminergičnega zdravljenja Parkinsonove bolezni. • Genetska variabilnost v različnih molekularnih poteh, ki lahko prispevajo k razvoju in poteku bolezni, lahko vpliva na pojav in/ali čas do pojava neželenih učinkov dopaminergičnega zdravljenja Parkinsonove bolezni. • S klinično farmakogenetskim modelom lahko ovrednotimo sočasen vpliv izbranih genetskih polimorfizmov in kliničnih dejavnikov na pojav neželenih učinkov dopaminergičnega zdravljenja Parkinsonove bolezni. Preiskovanci in metode Izvedli smo retrospektivno študijo, v katero smo vključili 231 bolnikov s PB na dopaminergičnem zdravljenju. Bolnike smo vključevali na Nevrološki kliniki Univerzitetnega kliničnega centra Ljubljana med oktobrom 2016 in aprilom 2018. Vključitveni kriteriji so bili naslednji: (i) diagnoza PB, (ii) dostopni klinični podatki in (iii) vsaj tri mesece trajajoče zdravljenje z levodopo in/ali dopaminskimi agonisti v času vključitve v študijo. Protokol študije je odobrila Komisija za medicinsko etiko Republike Slovenije (KME 42/05/16). V študijo so bili vključeni samo bolniki, ki so soglašali s sodelovanjem v študiji, in so ob vključitvi v študijo podpisali pristanek po predhodni poučitvi. Vsakemu bolniku so ob odvzemu krvi za druge laboratorijske preiskave odvzeli tudi vzorec periferne venske krvi za izolacijo DNA. Genotipizirali smo 37 polimorfizmov posameznih nukleotidov 23 genov iz petih različnih bioloških poti: (i) dopaminergična in (ii) vnetna pot, poti (iii) oksidativnega stresa, (iv) razvoja, proliferacije in diferenciacije nevronov in (v) apoptoze. Številske spremenljivke smo predstavili z mediano in intervalom med 25. in 75. percentilom. Za predstavitev porazdelitve kategoričnih spremenljivk smo uporabili frekvence. S &#967;2 testom smo preverili, če genotipi ustrezajo Hardy-Weinbergovemu ravnovesju. Z logistično regresijo smo preverili povezave med polimorfizmi in neželenimi učinki. Povezave med kliničnimi in genetskimi dejavniki ter preučevanimi spremenljivkami smo opisali z razmerjem obetov, 95 % intervalom zaupanja in p vrednostjo. Kot referenco smo uporabili bolj pogosto zastopan alel. Coxov regresijski model sorazmernih tveganj smo uporabili pri ugotavljanju povezave med polimorfizmi in časom do pojava neželenih učinkov. Povezave med kliničnimi in genetskimi dejavniki ter časom do pojava neželenih učinkov smo opisali z razmerjem tveganj, 95 % intervalom zaupanja in p vrednostjo. Za izdelavo modelov smo uporabili Coxovo regresijo z LASSO (ang. Least Absolute Shrinkage and Selection Operator) penalizacijo. Ocenili smo tudi napovedne vrednosti modelov s pomočjo površine pod krivuljo (AUC) časovno odvisne ROC (lastnost delovanja sprejemnika, ang. receiver operating characteristics) krivulje. Določili smo tudi senzitivnost in specifičnost modelov. V točki, kjer je bila vsota senzitivnosti in specifičnosti najvišja, smo določili pražno vrednost za identifikacijo bolnikov s povišanim tveganjem za zgodnejši pojav neželenega učinka. Statistično analizo smo opravili s programskim paketom IBM SPSS (verzija 21.0) in v programskem okolju R. Rezultati V sklopu prve študije smo preučevali povezave med genetsko variabilnostjo dopaminergične poti in pojavom neželenih učinkov dopaminergičnega zdravljenja. Opazili smo nekaj pomembnih povezav, in sicer: vidne halucinacije so bile povezane z aleli COMT rs165815 C (OR=0,34; 95%CI=0,16-0,72; p=0,004), DRD3 rs6280 C (OR=1,88; 95%CI=1,00-3,54; p=0,049) in z genotipom DRD3 rs6280 CC (OR=3,31; 95%CI=1,37-8,03; p=0,008); ortostatska hipotenzija z alelom DDC rs921451 C (OR=1,86; 95%CI=1,07-3,23; p=0,028) in z genotipi DDC rs921451 CT (OR=2,30; 95%CI=1,26-4,20; p=0,007), DDC rs3837091 AGAG- (OR=1,94; 95%CI=1,07-3,51; p=0,028) in SLC22A1 rs628031 AA (OR=2,57; 95%CI=1,11-5,95; p=0,028); periferni edemi z genotipom SLC22A1 rs628031 AA (OR=4,00; 95%CI=1,62-9.88; p=0,003); motnja kontrole impulzov z genotipom SLC22A1 rs628031 AA (OR=3,16; 95%CI=1,03-9,72; p=0,045); diskinezije z genotipom SLC22A1 rs628031 GA (OR=0,48; 95%CI=0,24-0,98; p=0,043) in z alelom SLC22A1 rs628031 A (OR=0,48; 95%CI=0,25-0,92; p=0,027). Pri analizi genetske variabilnosti vnetne poti smo zaznali le eno povezavo, ki nakazuje statistično neznačilen trend, in sicer je bila ortostatska hipotenzija povezana z alelom IL1&#946; rs1143623 C (OR=0,57; 95%CI=0,32-1,00, p=0,050) in z genotipom IL1&#946; rs1143623 GC (OR=0,51; 95%CI=0,28-0,93, p=0,028). Genetska variabilnost poti oksidativnega stresa je prav tako pokazala nekaj pomembnih povezav s pojavom nekaterih neželenih učinkov dopaminergičnega zdravljenja, in sicer: med perifernimi edemi in alelom CAT rs1001179 A (OR=0,32; 95%CI=0,15-0,68, p=0,003) in genotipom CAT rs1001179 GA (OR=0,32; 95%CI=0,15-0,69, p=0,004); med prekomerno dnevno zaspanostjo in napadi spanja in alelom NOS1 rs2682826 A (OR=1,75; 95%CI=1,00-3,06, p=0,048) in genotipom NOS1 rs2682826 AA (OR=3,75; 95%CI=1,23-11,45, p=0,020); med slabostjo/bruhanjem in alelom SOD2 rs4880 T (OR=0,49; 95%CI=0,25-0,94, p=0,031) in genotipom SOD2 rs4880 CT (OR=0,48, 95%CI=0,24-0,98, p=0,045); med motnjo kontrole impulzov in alelom NOS1 rs2682826 A (OR=2,59; 95%CI=1,09-6,19, p=0,032) in genotipom NOS1 rs2682826 GA (OR=2,74, 95%CI=1,13-6,64, p=0,025). Klinični in genetski dejavniki, ki so bili povezani s časom do pojava motoričnih komplikacij po začetku zdravljenja z levodopo, so bili sledeči: starost ob diagnozi (HR=0,97; 95%CI=0,96-0,99; p<0,001), čas od diagnoze do začetka zdravljenja z levodopo (HR=1,36; 95%CI=1,25-1,49; p<0,001), NOS1 rs2293054 (GG: Ref.; AA: HR=0,36; 95%CI=0,13-1,00; p=0,051), DRD2 rs1799732 (CC: Ref.; --: HR=8,89; 95%CI=1,19-66,18; p=0,033) in DRD3 rs6280 (TT: Ref.; CC: HR=2,04; 95%CI=1,16-3,60; p=0,014). Klinični in genetski dejavniki, ki so bili povezani s časom do pojava diskinezij po začetku zdravljenja z levodopa, so bili sledeči: starost ob diagnozi (HR=0,96; 95%CI=0,95-0,98; p<0,001), zdravljenje z antagonisti adrenergičnih receptorjev beta (HR=0,60; 95%CI=0,36-1,00; p=0,051), čas od diagnoze do začetka zdravljenja z levodopo (HR=1,23; 95%CI=1,11-1,37, p<0,001), CAT rs1001179 (GG: Ref.; AA: HR=2,60; 95%CI=1,17-5,79; p=0,019), SOD2 rs4880 (CC: Ref.; TT: HR=0,54; 95%CI=0,30-0,98; p=0,043), SLC22A1 rs628031 (GG: Ref.; GA: HR=0,63; 95%CI=0,40-1,00; p=0,048; AA: HR=0,53; 95%CI=0,29-0,99; p=0,047), DRD2 rs1799732 (CC: Ref.; --: HR=8,66; 95%CI=1,16-64,86; p=0,036) in NRG1 rs3735781 (AA: Ref.; GA: HR=0,65; 95%CI=0,41-1,99; p=0,051). Klinično-farmakogenetski model za napoved časa do pojava motoričnih nihanj je vseboval naslednje dejavnike: starost ob diagnozi (HR=0,99), čas od diagnoze do začetka zdravljenja z levodopo (HR=1,24), COMT rs165815 (HR=0,90), DRD3 rs6280 (HR=1,03) in BIRC5 rs9904341 (HR=0,95). Oblikovali smo tudi ROC krivuljo za oceno napovedne vrednosti po petih letih zdravljenja (AUC=0,70, senzitivnost=52,2 %, specifičnost=82,3 %). Za primerjavo smo oblikovali tudi ROC krivuljo po petih letih zdravljenja za klinični model (AUC=0,68; senzitivnost=48,4 %, specifičnost=81,9 %). Klinično-farmakogenetski model za napoved časa do pojava diskinezij je vseboval naslednje dejavnike: ženski spol (HR=1,07), starost ob diagnozi (HR=0,97), tremorozna oblika PB (HR=0,88), zdravljenje z antagonisti adrenergičnih receptorjev beta (HR=0,95), uživanje alkohola (HR=0,99), čas od postavitve diagnoze do začetka zdravljenja z levodopo (HR=1,15), CAT rs1001179 (HR=1,27), SOD2 rs4880 (HR=0,95), NOS1 rs2293054 (HR=0,99), COMT rs165815 (HR=0,92) in SLC22A1 rs628031 (HR=0,80). Oblikovali smo tudi ROC krivuljo za oceno napovedne vrednosti po petih letih zdravljenja (AUC=0,68, senzitivnost=54,1 %, specifičnost=66,1 %). Za primerjavo smo oblikovali tudi ROC krivuljo po petih letih zdravljenja za klinični model (AUC=0,71; senzitivnost=79,8 %, specifičnost=48,4 %). Razprava V študiji smo raziskali vpliv 34 polimorfizmov posameznih nukleotidov iz petih različnih bioloških poti in nekaterih kliničnih parametrov na pojav in/ali čas do pojava neželenih učinkov dopaminergičnega zdravljenja PB. Genetska variabilnost gena COMT se je izkazala za povezano z znižanim tveganjem za razvoj vidnih halucinacij. Ta polimorfizem v povezavi s tem fenotipom še ni bil preučevan. Je pa povezava z vidnimi halucinacijami že bila opisana pri polimorfizmu COMT rs4680. Genetska variabilnost gena DDC se je izkazala za povezano s povišanim tveganjem za pojav ortostatske hipotenzije. Oba polimorfizma, ki sta bila preučevana, znižata aktivnost encima, kar lahko privede do znižane koncentracije noradrenalina in posledične vazodilatacije. Ta mehanizem bi lahko prispeval k nastanku ortostatske hipotenzije ob dopaminergičnem zdravljenju. Nadalje se je SLC22A1 rs628031 izkazal za potencialnega genetskega označevalca različnih neželenih učinkov dopaminergičnega zdravljenja. Ugotovili smo, da je ta polimorfizem prenašalca levodope povezan s povišanim tveganjem za pojav ortostatske hipotenzije, perifernih edemov in motnje kontrole impulzov. Ta polimorfizem pa je tudi pomembno zmanjšal tveganje za pojav diskinezij. Glede na te rezultate lahko zaključimo, da ima polimorfizem SLC22A1 rs628031 verjetno precej pomemben vpliv na splošno delovanje dopaminergičnih zdravil. Ugotovili smo tudi, da DRD3 rs6280 poveča tveganje za pojav vidnih halucinacij, kar lahko razložimo s tem, da ta polimorfizem vpliva na znotrajcelično signalizacijo preko povečanja vezavne afinitete receptorja DRD3. Naše preučevanje vnetnih poti je pokazalo le eno, sicer nesignifikantno, a vseeno zanimivo povezavo med IL1&#946; rs1143623 in pojavom ortostatske hipotenzije. Nekaj študij je že preučevalo povezave med vnetjem in ortostatsko hipotenzijo, vendar je naša študija prva, ki je pokazala povezanost z genetsko komponentno. Taisti polimorfizem pa se je tudi izkazal za pomembnega pri vplivu na tveganje za pojav PB. Polimorfizem CAT rs1001179 je bil statistično značilno povezan s pojavom perifernih edemov. Mehanizem nastanka perifernih edemov kot neželenega učinka dopaminergičnega zdravljenja še ni znan, vendar ta rezultat lahko nakazuje povezanost z oksidativnim stresom. Opazili smo tudi, da polimorfizem NOS1 rs2682826 vpliva na povišano tveganje za pojav prekomerne dnevne zaspanosti in napadov spanja in za pojav motnje kontrole impulzov. Genotip NOS1 se je že izkazal za povezanega s spalnim ciklom. Ta polimorfizem pa je tudi že bil povezan s pojavom različnih psihiatričnih motenj, kot sta depresija in anksioznost. SOD2 rs4880 je v naši študiji zmanjšal tveganje za pojav slabosti/bruhanja. Nekatere študije so že pokazale povezanost oksidativnega stresa v enteričnem živčnem sistemu s pojavom gastrointestinalnih komplikacij. Oblikovali smo tudi klinične in klinično-farmakogenetske modele za napoved časa do pojava motoričnih komplikacij po začetku zdravljenja z levodopo. Ti modeli bi lahko služili za oblikovanje algoritmov za identifikacijo bolnikov s povišanim tveganjem za zgodnejši pojav motoričnih komplikacij. Izkazalo se je, da se klinični in klinični-farmakogenetski modeli po napovedni vrednosti med seboj klinično pomembno ne razlikujejo. Pri napovedi časa do pojava motoričnih komplikacij med obema modeloma ni bilo razlik v AUC, specifičnosti in senzitivnosti. Pri napovedi časa do pojava diskinezij pa se je izkazalo, da ima klinično-farmakogenetski model višjo specifičnost v primerjavi s kliničnim modelom. To pomeni, da je verjetnost dejanskega pojava neželenega učinka v primeru pozitivnega rezultata višja pri klinično-farmakogenetskem modelu. V splošnem so modeli v večini primerov pravilno napovedali pojav neželenega učinka v prvih petih letih zdravljenja z levodopo. Zaključki Pokazali smo, da genetska variabilnost vpliva na pojav in čas do pojava neželenih učinkov dopaminergičnega zdravljenja. Potrdili oz. vsaj delno potrdili smo naslednje hipoteze: • Polimorfizmi posameznih nukleotidov v genih dopaminergične poti vplivajo na pojav nekaterih neželenih učinkov dopaminergičnega zdravljenja PB. • Polimorfizem vnetne poti IL1&#946; rs1143623 je povezan s pojavom ortostatske hipotenzije kot neželenega učinka dopaminergične terapije PB, vendar ta povezava ni statistično pomembna. • Polimorfizmi posameznih nukleotidov v genih poti oksidativnega stresa vplivajo na pojav nekaterih nemotoričnih neželenih učinkov dopaminergičnega zdravljenja PB. • Klinično-farmakogenetski modeli omogočajo ovrednotenje sočasnega vpliva izbranih kliničnih in genetskih dejavnikov na čas do pojava motoričnih komplikacij po začetku zdravljenja z levodopo.

• Keywords: Parkinsonova bolezen, farmakogenetika, dopaminergično zdravljenje, neželeni učinki, genetski polimorfizmi, personalizirana medicina, klinično-farmakogenetski modeli