Research project: “The epidemiology of Parkinson’s disease in Croatia and the influence of genetic factors and microbiota on the progression and treatment outcomes of the disease”
IP-2019-04
Project is funded by the Croatian Science Foundation
Research centers:
Clinical Hospital Centre Rijeka (Croatia, primary research center), Clinical Hospital Centre Zagreb (Croatia), University Medical Center Ljubljana (Slovenia), Karolinska Institutet (Sweden)
Background of the project
Parkinson’s disease (PD) is the second most common neurodegenerative disease, which affects 2-3% of the general population above 65 years. The disease rarely occurs in people younger than 50 years, with the incidence and prevalence gradually increasing tenfold depending on the age of the person. It is thus thought that, due to the aging of the general population, the prevalence of PD will also increase. Men are twice as likely affected than women. However, there are significant differences depending on the geographical location, race and ethnicity. The real cause behind the disease is still unknown, but the role of genetic and environmental factors has already been established. Certain genetic forms of the disease make up for a small percentage, so it is thought that environmental factors have a more significant impact on the development of the disease. It is known that the incidence of Parkinson’s disease is higher in people exposed to significant quantities of pesticides and traumatic brain injury, while there is a smaller incidence in smokers and people consuming higher quantities of caffeine.
Moreover, the intestines, along with oral microbiota, are more and more brought up as a significant factor in the pathogenesis of the disease, mostly because of the discovered imbalance of the intestinal flora in people suffering from PD. At this point, it is unknown whether the dysbiosis of the intestinal microbiota precedes the disease, or it is the result of the disturbed neuronal communication of the liver-brain axis. A more significant genetic impact of PD is seen in closed populations like the Ashkenazi Jews in Israel, which demonstrate a higher prevalence for mutations of the LRRK2 ( Leucine-rich repeat serine/threonine-protein kinase 2) and GBA (glucocerebrosidase) genes, which have a significant role in the emergence of PD. There has been no studies covering the genetic mutations in PD patients in Croatia so far.
A specific pathophysiological event in Parkinson’s disease is the loss of neurons in the substantia nigra with the accumulation of the intracellular protein α-synuclein in the entire central nervous system as a result. The disease is not accompanied by a general brain tissue atrophy, instead it affects specific regions populated with dopaminergic neurons. The lack of these neurons leads to a general lack of dopamine in the brain, which in turn leads to the classic motoric, but also non-motoric symptoms of the disease. It is essential to notice that PD is asymptomatic for an extended period since the symptoms start manifesting only when there is a loss of more than 70-80% of dopaminergic neurons. The disease is also accompanied by the dysfunction of glial cells, which are cells usually in charge of keeping homeostatic conditions in the central nervous system. This presents with neuroinflammation and lowered function of microglial cells regarding their function in monitoring the microenvironment, which in turn leads to phagocytosis and the destruction of neurons. On the other hand, there is a lack of a nutritive and reparative reaction in astrocytes, glial cells involved in neuroprotection and injury repair in the CNS. nfortunately, there is still no specific biomarker known for PD, however alterations of neurodegenerative, nutritive, and immunological markers in these disorders enable experimental monitoring of disease progression. Recent research showed a link between intestinal microflora nad glial cells in physiological conditions, but also in the pathogenesis of various neurodegenerative ailments. Currently, considering the development of PD, it is is thought that the first changes, especially the accumulation of alfa-synuklein, occur inside the intestinal nervous system, which is in direct contact with the intestinal microflora. It is in this manner that the intestinal microbiota could modulate the pathogenesis of PD and contribute to the interindividual variability of the clinical presentation. There is a lack of studies focused on drug-naive patients, but research has shown changes in the composition and function of microbiota even before treatment. It is because of this that prospective studies in de novo PD patients are needed to shed light on the mean effect of dysbiosis on disease progression.
Planned execution of the project
The planned duration of the research is four years (48 months), in which both of the phases will be conducted simultaneously
- Epidemiological genetic research (first phase)
The planned duration of the first phase of research is 20 months. In the first nine months, throughout Croatia, several participants who are members or work with the Croatian Parkinson’s disease patient’s association will be recruited. At the end of the first year of the research, the participants will have their whole exomes sequenced with the help of next-generation sequencing (NGS), to find genetic variants in Croatian patients. Through the second year of research, result analysis and dissemination is planned, as well as counseling in participants with a confirmed familial variant of the disease. For this purpose, the participants will be asked for blood samples for genetic analysis. The target number of subjects in this phase of the research is 50. The participants will also be asked to fill out several structured study forms, which should provide an estimation of the genetic background of the participant, along with information on their biorhythm, sleep patterns, diet, life goals, and general stress.
- Prospective clinical trial (second phase)
The clinical study will span over four years (48 months). Participant selection will be conducted in the Clinical Hospital Centre Rijeka and Clinical Hospital Centre Zagreb throughout the first year of the clinical study, while in the following two and a half years the state of the PD afflicted patients and the control group will be prospectively monitored in the following time intervals : diagnosis and initial therapy ; 6 months, 12 months, 24 months and 30 months since their initial therapy in PD patients, that is , in case of the healthy control group, since their first visit. The result analysis and dissemination are planned in the last two years of research.
Every participant in the study will have their exome sequenced with NGS, with the focus mostly being on parts that have a known impact on the pathogenesis of PD. Both the patients and healthy control population will also have their microbiota composition determined with NGS, as well as their functional state through the analysis of the short-chain fatty acid composition, known metabolic products of the microbiota. Additionally, inflammatory states, glial function, and disease progression will be monitored through biomarkers in serum and plasma (Table 2), as well as pathological protein conformations in the blood. The disease progression will also be monitored with transcranial B-mod sonography and magnetic brain resonance. It is for these methods that the participants will be asked to give a blood, stool, and saliva sample.
The exome sequencing will be conducted at the beginning (baseline), while the analysis of the composition and function of microbiota, along with biomarker analysis and disease progression through neuro-radiological methods will be conducted at every control examination. Starting with the first control examinations, the participants will be asked to fill out several structured study questionnaires, which should provide an estimation of the genetic background of the participant, along with information on their biorhythm, sleep patterns, diet, life goals, and general stress. Moreover, the cognitive state of the participants will be evaluated through validated tests like the Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA), while the general non-motoric symptoms will be evaluated with the help of the Non-Motor Symptoms Questionnaire (NMSQ) and the Non-Motor Symptoms Scale (NMSS). Patients will also be evaluated with the help of the UPDRS (Unified Parkinson’s disease rating scale), the validated and scale most prominently used in longitudinal PD studies.
Objectives of the project
The proposed project spans two phases conducted simultaneously. The first phase encompasses an epidemiologic study of patients with PD throughout the Republic of Croatia. The goal of the first phase is to determine the representation of familiar and sporadic forms of PD in Croatia and, with the help of next-generation sequencing, to identify the genetic variants in patients with the familiar form of PD. With this research, we expect to get the first genetic-epidemiologic information on Parkinson’s disease in Croatia, as well as identify risky genetic variants which contribute to the expression of the disease in Croatian patients, which would contribute to shedding light on the etiopathogenesis of PD.
Primary goal (O1):
- determining which genetic mutations are the ones most represented in the Croatian population afflicted with the familiar form of PD
The second phase of the study encompasses a planned prospective clinical research which will be conducted at the Clinical Hospital Center Rijeka and Clinical Hospital Center Zagreb and it includes drug-naive patients with Parkinson’s disease, as well as a healthy control group.
Primary goal (O2):
- determining the influence of genetic factors and microbiological factors on the disease’s progression as well as on the treatment outcomes
Specific goals :
- determining how many patients in the general population of PD patients present with a genetic disorder and which genes have a role in that disorder
- determining the composition of intestinal and oral microbiota both in the patient test group and the healthy control group
- evaluating the effects of standard PD treatment on the composition of microbiota, neurodegeneration progression and the activity of neuroinflammation in the CNS
- examining whether there is a link between the physiological and the pathophysiological function of microbiota, using markers of disease progression and glial activity
- analyze potential pathological conformation protein forms that could be used as a biomarker in the early stages of the disease and a biomarker of disease progression
The estimated duration of the project is 4 years.
Expected results and impact of the project
The first phase of the study will provide the first epidemiologic data on the familial form of PD, as well as the mutations most represented in patients with PD in Croatia. Additionally, the prospective clinical study will contribute to enlightening the intertwined effects of genetic and environmental factors in the emergence and progression of the disease, as well as their effect on treatment outcome. Intestinal and oral microbiota composition analysis will determine whether there is a difference between PD and the healthy population while using the short-chain fatty acid profile will determine the metabolic differences between the two groups. Analyzing the markers of CNS homeostasis, inflammation, and neuroglial function will determine the progression of the disease and also correlate them to genetic factors as well as the microbiota function and composition. Analyzing the pathological conformation forms of a-synuclein could lead to the discovery of novel biomarkers in the early stages of the disease, as well as to follow the progression of the disease and determine treatment outcomes.
Completion of the project will improve our understanding of the interconnected effects of genetic and environmental factors in the emergence and progression of PD, as well as the effects on treatment outcomes, which could altogether lead to the development of newer, more specific therapy aimed at genetic and environmental factors. The impact on society stems from the fact that it is the first epidemiologic genetic study of PD in Croatia, which could help in determining the incidence of familial forms of the disease, as well as the most emergent mutations behind them.
We plan on publishing the results of the research in leading journals (Q1) on extrapyramidal diseases. Along with the publication of the results in said journals, we also plan on presenting the results on international conferences, as well as organize an international conference. Additionally, the project has four young researchers working on it, which will grow scientifically through project activities, and two of them will have this research as a topic of their doctorate thesis.
