Mutations of PARK Genes and Alpha-Synuclein and Parkin Concentrations in Parkinson’s Disease

Parkinson’s disease (PD) is a chronic and progressive neurological disorder characterized by resting tremor, rigidity, and bradykinesia, affecting at least 2% of individuals above the age of 65 years. Parkinson’s disease is a result of degeneration of the dopamine-producing neurons of the substantia nigra. Available therapies in PD will only improve the symptoms but not halt progression of disease. The most effective treatment for PD patients is thera‐ py with L-3,4-dihydroxy-phenylalanine (L-dopa) [Olanow, 2008].


Introduction
Parkinson's disease (PD) is a chronic and progressive neurological disorder characterized by resting tremor, rigidity, and bradykinesia, affecting at least 2% of individuals above the age of 65 years. Parkinson's disease is a result of degeneration of the dopamine-producing neurons of the substantia nigra. Available therapies in PD will only improve the symptoms but not halt progression of disease. The most effective treatment for PD patients is therapy with L-3,4-dihydroxy-phenylalanine (L-dopa) [Olanow, 2008].
It is now believed that the cause of PD, are both environmental and genetic factors. During the last two decades, there has been breakthrough progress in genetics of PD. It is known that genetic background of PD is in mutations a number of pathogenic genes PARK, e.g. SNCA, PRKN, UCHL1, DJ-1, PINK1, ATP13A2, and LRRK2 (Polrolniczak et a., 2011,2012). In 2001, Shimura et al. first described the presence in the human brain complex containing Parkin with the glycosylated form of the alpha-synuclein (ASN, alpha-SP22). Moreover, the study by Dorszewska et al. (2012) has been shown, that in the PD patients increased plasma level of ASN was associated by the decreased of Parkin plasma level. It has also shown that configuration: increased plasma level of ASN and decreased of Parkin concentration was associated with earlier onset of PD. It seems that in PD genotypic testing of PARK mutations and analysis of their phenotypes (e.g. ASN, Parkin) may be diagnostic agents for these patients.

Mutations in PRKN, SPR and HTRA2 genes and polymorphism of NACP-Rep1 region of SNCA promoter in the patients with Parkinson's disease
During the last two decades, there has been breakthrough progress in genetics of PD. Currently it is known that genetic background of PD is heterogeneous and mutations in a number of pathogenic genes (e.g. SNCA, PRKN, UCHL1, DJ-1, PINK1, ATP13A2, and LRRK2) have been described as associated with familial (FPD) or as genetic risk factors increasing the risk to develop of sporadic PD (SPD). Some of these genes (like SNCA and PRKN) are fairly well understood while the others (like SPR and HTRA2) are still little known (Corti et al., 2011).
Monogenic forms, caused by a single mutation in a dominantly or recessively inherited gene, are well-established. Nevertheless, they are relatively rare types of PD and account for about 30% of the FPD and 3-5% of the SPD cases. Although 18 specific chromosomal locus (called PARK and numbered in chronological order of their identification) have been reported as more or less convincingly related to FPD (Klein & Westenberger, 2012), the majority of PD cases are SPD (only about 10% of patients report a positive family history) [Thomas & Beal, 2007] (Chung et al., 2011;Spadafora et al., 2003). However, genome-wide association studies have provided convincing evidence that polymorphic variants in some genes contribute to higher risk of SPD (Gao et al., 2009). Moreover, it is suggested that the etiology of PD is multifactorial, which probably results from coocurence of genetic and environmental factors (Klein & Westenberger, 2012).
Summarizing, from the existing studies reported, it is not yet clear how common mutations in few genes, including: PRKN, HTRA2, SPR and SNCA genes contribute to idiopathic PD (Nuytemans et al., 2010). Finally despite previous reports, significance of these genes mutation and polymorphism in pathogenesis of PD (especially SPD) is not clear and is still debated, mainly because of discrepancy of studies results and variance between different ethnic populations. To clarify these issues, more data of genetic analysis are needed while there were only a few reports of genetic studies of PD in Polish populations (Bialecka et al., 2005;Koziorowski et al., 2010). Moreover, little is understood about putative director functional interactions between the genes that cause PD, and a single pathway unifying these factors has not been confirmed (Bras et   The study by Chiba-Falek et al. (2006) has shown that the region NACP-Rep1 of SNCA gene promoter, there is the polymorphic region differenting in dinucleotide repeats count and affecting the level of ASN expression. Moreover, it has been shown that polymorphism of NACP-Rep1 region in promoter of SNCA, are associated with an increased risk of SPD in some population like: German, Australian, American and Polish, but the other multi-population studies have observed no association or reported an inverse association between the risk allele and PD Kruger et al., 1999;Maraganore et al., 2006;Polrolniczak et al., 2012;Tan et al., 2003).
Region NACP-Rep1 contains dinucleotide repeats (TC)x(T)2(TC)y(TA)2(CA)z, which may vary both the number of repeats, and include substitutions of nucleotides. However, it has been proven, that a change in the length of the NACP-Rep1 region more than substitutions, affects the regulation of the expression of ASN (Fuchs et al., 2008;Mellick et al., 2005;Tan et al., 2003). As the most common in humans it has been described five alleles of NACP-Rep1 of the SNCA gene promoter: -1, 0, +1, +2, +3. Generally in the European population the most frequently was allele +1 of NACP-Rep1. It has been also shown, that the allele 0 of NACP-Rep1 region in SNCA promoter is two pairs shorter than allele +1, allele -1 respectively, shorter by 4 bp however alleles 2 and 3 are longer by 2 and 4 bp.
Functional analysis on the two most common NACP-Rep1 alleles +1 and +2 suggested that the +2 allele is associated with an up-regulation of SNCA expression, whereas the +1 variant shows Nerveless, although protective effect of allele +1 rather not currently subject to discussion, but for alleles 0, +2 and +3 it has been suggested both no impact, as well as increasing the risk of PD, and even sometimes the protective action (Maraganore et al., 2006;Spadafora et al., 2003;Tan et al., 2000;Trotta et al., 2012). The following studies by Tan et al. (2000) and Myhre et al. (2008) observed a higher frequency of the +3 allele in PD cases compared with healthy controls while in the study by both Tan et al. (2003) and Spadafora et al. (2003) no significant differences of the various genotypes between PD and controls were found in population of Singapore and Italy. However, the study in Italy population, have also shown evidence of association for allele +2 on NACP-Rep1 (Trotta et al., 2012). In 2006, a meta-analysis of 11 study populations provided strong evidence that the 263bp allele was more frequent in PD cases increasing risk of this disease while the 261bp allele did not differ between PD cases and unaffected controls but the authors suggested, that the lack of association of the +2 allele in the meta-analysis could be due to the large fluctuation in its frequencies observed in the analyzed populations (Maraganore et al., 2006). Therefore the aim of the study was analysis of NACP-Rep1 region in PD patients and in controls in Polish population.

Patients
The studies were conducted on 90 patients with PD [SPD patients, 10 with early onset of PD, EOPD, and 80 with late onset of PD, LOPD patients), including 42 women and 47 men aging 34-82 years. Control group included 113 individuals, 79 women and 34 men, 39-83 years of age. Demographic data of all groups summarized in Table 1.
Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr (Hoehn & Yahr, 1967 [Tan et al., 2003]. One of the primers was labeled with fluorescent marker -FAM. Sizing of the PCR products was performed by capillary electrophoresis on the 3130xl Genetic Analyzer (Applied Biosystems HITACHI, USA) using GeneScan Size Standard 600LIZ (Applied Biosystems, USA) and controls. The results of electrophoresis were analyzed using Peak Scanner Software v.1.0 (Applied Biosystems, USA). Genotypes were differentiated according to the length of the PCR product. Designations of alleles was followed those previously described Xia et al., 2001).
Moreover, random duplicate samples (10%) were genotyped for all assays for quality control with 100% reproducibility.
Statistical analysis. Statistical analysis was performed using Statistica for Windows Software. The level of significance was set at 5%. Chi-square test and Fisher's exact probability test, test for the two components of the structure, univariate odds ratio (ORs) and logistic regression analysis were used to compare the categorical variables and distribution of alleles and genotypes. The allele frequencies of PD patients and controls were evaluated with regards to Hardy-Weinburg equilibrium using standardized formula.

Results
Screening for mutation c.88 G>C of SNCA gene in patients with PD and neurologically healthy controls detected no mutations in both group allow the exclusion of FPD determined by this mutation.
Using PCR amplification and capillary electrophoresis five previously described polymorphic alleles of NACP-Rep1 region in SNCA promoter were identified (designated -1, 0, +1, +2, +3) Xia et al., 2001]. Alleles and genotypes frequencies were in Hardy-Weinburg equilibrium in both groups: PD and controls with the exception of alleles +1 and +2, which frequencies in PD patients differed significantly from the expected frequencies calculated from Hardy-Weinburg equilibrium (exact test; p=0.032 and p=0.006 respectively). The frequency of allele +1 (Table 2) was significantly higher in healthy controls as compared to PD patients (p<0.001). In contrast to the allele +1, the frequency of alleles +2 and +3 were significantly higher in PD patients as compared to controls (p<0.01; p<0.05 respectively). However, the frequency of allele 0 was similar between PD and controls. Moreover, presence of allele -1 was detected only in control subjects (Polrolniczak et al., 2012).
The frequency of +1/+1 genotype was almost fourfold higher in control group than in PD patients (p<0.001) whereas the frequency of the genotype +1/+2 was similar in both groups ( Table 3). Comparisons of +2/+2 genotype frequencies between PD patients and control group revealed no significant differences but the frequency of this genotype was almost twofold higher in PD patients as compared to controls (p=0.056). It has been also detected, that the frequency of +2/+3 was significantly higher in PD patients compared to controls and was almost threefold higher in PD patients (p<0.05). Moreover, genotype +1/+3 has been detected only in one PD patient while genotype -1/+1 occurred only in controls (Table 3).  Although the study in Singapore and Italian populations shown no association for alleles +2 and +3 with PD our results confirming the study in populations: German, Italian, Japanese, and multipopulation research detected higher frequency of those alleles in PD patients compared with controls and indicated association of genotypes +2/+2 and +2/+3 with increased risk of PD in Polish population ( It seems that examination of genotypes of region NACP-Rep1 of SNCA promoter may help to explain the pathogenesis of PD, as well as facilitate early diagnosis and determine the degree of risk for this neurodegenerative disease.  The observation, that mutations in the PRKN gene are common in juvenile-(JPD) and EOPD and increasing evidence supporting a direct role for Parkin in LOPD make this gene a particularly compelling candidate for intensified investigation. However, despite previous reports, significance of PRKN mutation and polymorphism in pathogenesis of PD is still not clear.

Mutations in
The aim of the study was to estimate the frequency of PRKN mutation in Polish PD patients and controls.

Patients
According to the inclusion and exclusion criteria a total of 199 subjects were included in this study: 87 SPD patients (10 EOPD -TTTCCCAAATATTGCTCTA-3';  5'-GCAGTGTGGAGTAAAGTTCAAGG-3'  for  exon  2  and  5 Moreover, random duplicate samples (10%) were genotyped for all assays for quality control with 100% reproducibility.
Statistical analysis. Statistical analyses were performed using Statistica for Windows Software. The level of significance was set at 5%. Chi-square test and Fisher's exact probability test, univariate odds ratio (ORs) and logistic regression analysis were used to compare the categorical variables and distribution of alleles. The allele frequencies of PD patients and controls were evaluated with regards to Hardy-Weinburg equilibrium using standardized formula.

Results
Analysis of deletions of exons 2 and 4 PRKN has detected no genetic changes both in PD patients and control group. However, point mutation screening in patients with PD and healthy controls identified 5 missence substitutions which were almost fourfold more frequent in PD patients as compared with controls (p<0.001) [ Table 6]. We also showed, that the presence of PRKN substitution increased risk of PD over six-fold (p<0.001; OR=6.059). All substitutions were non-synonymous and were in heterozygous state. In exon 4 of PRKN two mutations were detected: c.500 G>A transition leads to S167A substitution (with frequency sevenfold higher in PD than in control group; p<0.05) and a novel heterozygous mutation c.520 C>T resulting L174F substitution and occurring only in PD patients. Furthermore, first time in Polish population we detected c.823 C>T (exon 7, R275T; only in PD) and c.930 G>C (exon 8, E310D) substitutions (over threefold more frequently in PD than in controls; p<0.01). Moreover, we detected also a transition c.1180 G>A in exon 11 of PRKN. It has been also shown, that c.500 G>A, c.930 G>C and c.1180 G>A substitutions significantly increased PD risk (  Additionally in 5% PD patients it has been detected more than one mutation in PRKN gene while all control subjects who had substitution in PRKN, had only one mutation ( Table 8). Moreover, we showed, that in the Polish population the most frequently were polymorphisms c.500 G>A, c.1180 G>A and c.930 G>C of PRKN. Simultaneously, it appears that these polymorphisms may have incomplete penetration or lead to preclinical changes in the CNS and increased risk LOPD probably in combination with other genetic or environmental factors, as evidenced by Bardien et al. reports (2009). The other two identified PRKN mutations (c.823 C> T, c.520 C> T) were detected only in PD patients, what may indicate a high penetration of these substitutions (Sinha et al., 2005), while novel mutation c.520 C>T was identified in two patients and led to a relatively early onset of disease before age 40.

Controls
It is suggested, that haploinsufficiency may be considered as a reduction of normal gene expression accompanied by a loss of normal protein activity. Moreover, a lot of reports indicate to the existence of a second, undetected mutation in these patients, perhaps in the promoter or intronic regions (Giasson & Lee, 2001).
Our results, also suggests that the presence more than one heterozygous mutation in the PRKN gene may be necessary to PD manifestation. This hypothesis was first proposed by Abbas et al. (1999) moreover, later reviews generally assume the existence of a second, undetected mutation (Giasson & Lee, 2001). In our study also it is probably that patient who had one mutation in PRKN may have more genetic changes in not tested region of the gene so extension the studies of the other region of PRKN gene is necessary to clarify this issue. On the other hand it can not be ruled that one heterozygous mutation in PRKN may be sufficient to increase risk of PD and induce preclinical changes in substantia nigra (Khan et al., 2005).
Finally, it seems that clinically, PD patients with PRKN substitution generally are characterized by slower progression of the disease compared with PD patients without mutation. Moreover, it has been also observed, that in PD patients with PRKN mutations response to L-dopa therapy has been better than in PD patients without It seems, that point mutation in PRKN gene may be involved in the pathogenesis of LOPD and modulate clinical futures in this disease. It is also probably, that analysis of mutations in PRKN gene may be useful for diagnostic and prognostic process in PD.

Mutations in HTRA2 and SPR in the patients with Parkinson's disease
It seems that presence of mutation in the other genes involved in the pathogenesis of PD like SPR (involved in dopamine biosynthesis) and HTRA2 (involved with mitochondrial pathway of PD) probably may additionally affect the levels of ASN and Parkin through interaction with these proteins ( . It is known, that SPR is involved in dopamine synthesis and likewise ASN probably may be responsible for disturbances in methabolisme of dopamine. The study of Tobin et al. (2007) has shown that expression of SPR was significantly increased in PD patient compared with controls. However mutations in SPR in PD have not been analyzed so far. Moreover, it is known that phosphorylation of SPR increase sensitivity for protease activity and that in human SPR protein phosphorylated is only Ser213 (Fujimoto et al., 2002). Therefore, we decided to search for mutation in codon 213 of SPR in PD cases.

Patients
The studies were conducted on 89 patients with PD (10 EOPD patients, and 79 sporadic LOPD patients), including 41 women and 47 men aging 34-82 years. Control group included 113 individuals, 79 women and 34 men, 39-83 years of age. Demographic data of all groups summarized in Table 9.
Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr (Hoehn & Yahr, 1967 Table 9. Demographic data of patients with PD and control subjects analyzed for HTRA2 and SPR mutations. SDstandard deviation, F -female, M -male.

Results
In Polish population the presence of HTRA2 point mutation was detected in 3% of PD patients (in 2% -c.1195 G>A resulting A141S substitution and in 1% c.421 G>T leads to G399S substitution) and none of controls (Table 10). However, c.1210 C>T mutation of HTRA2 has not occurred both in PD patients and controls.  In 213 codon of SPR gene novel mutation c.637 T>A was identified in 4% patients with PD and 2% controls (Table 11). This substitution is non-synonymous and leads to S213T changes in amino acid chain. However, we did not detected the second analyzed substitution c.637 C>G SPR in any of the subjects.  In PD patients with substitutions in HTRA2 gene it have been observed slower progression of the disease wherein there was statistically significant association (Spearman correlation test) transition c.1195 G>A with decrease stage of disease (p=0.029; r=-0.237) but association of c.421 G>T substitution have not been significant and have remained at the level of trend. Furthermore it have been shown, that using in PD patients with HTRA2 mutations doses of L-dopa were lower than in patients without mutations and the response to therapy was better in presence of substitution. Finally, it seems that identified HTRA2 mutations may be one of PD risk factor, especially since Strauss et al. (2005) demonstrated the presence of olfactory dysfunction in asymptomatic HTRA2 mutation carrier.
Moreover, it seems that mutation c.637 T>A, because of localization, probably may affect phosphorylation of SR and thereby its activity and finally regulate biosynthesis of DA and serotonin (5-HT). However, analysis of expression and functional testing are necessary to explain importance and role of this mutation. Nevertheless, what is important, in our study c. 637 T>A SPR mutation has been significantly associated in Spearman correlation test, with the presence of depressive symptoms in PD patients (p<0.0001; r=0.371) probably by regulating the level of 5-HT (McHugh et al., 2009). Simultaneously, the presence of c.637 T>A of SPR mutation in PD patients have not been associated with differences in progression of the disease, response to L-dopa therapy, amount using L-dopa dose or presence of dementia compared to PD patients without SPR mutation.

Coexistence of mutations in more than one gene (SNCA, PRKN, HTRA2 and SPR) in the patients with Parkinson's disease
Our study indicated, that in PD patients as well as in controls in the Polish population, PRKN mutations most frequently accompanied by the presence of genotype +1/+2. Interesting the coexistence of mutations PRKN with genotypes +2/+2 and +2/+3 have been demonstrated only in patients with PD (Fig. 1). It seems that co-occurrence of point mutation of PRKN and polymorphism of SNCA promoter region may in additive manner increase risk of PD manifestation. Furthermore, in the patients with PD we demonstrated coexistence of point mutations in PRKN and SPR or PRKN and HTRA2 genes (Table 12). However, in controls, coexistence of mutations PRKN and SPR have been observed also in one person. Therefore it seems that in patients with mutation of PRKN and HTRA2 genes, simultaneous incorrect function of two proteins involved in the mitochondria proper functioning (HTRA2 and Parkin) may additionally increase risk of PD manifestation.

Role of alpha-synuclein in pathogenesis of Parkinson's disease
Alpha-synuclein is a protein composed of 140 amino acids and is a part of family of proteins with the β-and γ-synuclein (Clayton & George, 1998). For many years, the structure of ASN was determined as the,,not-folded" chain of amino acids, taking the helical form only in conjunction with the lipids of cell membranes. It was thought that the ASN is a monomer form but the recent studies have shown that under physiological conditions ASN largely takes the form of tetramers, and may take the helical form without connection to the lipid membrane (Bartels et al., 2011).
Immunohistochemical studies have shown that in the cells, there is essentially ASN bonded to both the nuclear membrane, and in the synaptic vesicles (Totterdel & Meredith, 2005). To a lesser extent, ASN occurs in the free form in the cytoplasm.
Functions of ASN are not fully understood, however, due to cellular location of this protein it is suggested, that function of ASN may be related with the synaptic transport (Alim et al., 2002). There are also reports indicating that ASN participate in the process of differentiation and survival of the dopaminergic neuron progenitor cells of the mouse and human (Michell et al., 2007;Schneider et al., 2007). It is obvious that the process of aggregation of the ASN is a negative phenomenon for neural cells not only because of the high toxicity of the resulting aggregates, but also because of the ASN physiological function disorders caused by the reduction of bioavailability of this protein (Conway et al., 2000). It has been shown, that in PD, the process of ASN aggregation may be modulated by a number factors (Fig. 2)

Alpha-synuclein concentration in Parkinson's disease
It has been shown that aggregation of the ASN may be caused among others by multiplication of SNCA gene. Furthermore, it has been shown that triplication of SNCA gene leads to twofold increase of ASN level, while duplication of SNCA gene increases the level of this protein one and a half-fold Singleton et al., 2003). Therefore it is believed that increased level of ASN may be related with PD manifestation Mata et al., 2004). It is also known that over-expression of ASN in neuron facilitates aggregation of this protein even in the presence of the correct structure of ASN. Moreover, elevated expression of SNCA-mRNA levels have been found in the affected regions of PD brain (Chiba-Falek et al., 2006). Increased of ASN level has been also associated with progress and worsening of the disease symptoms (Singleton et al., 2003). However, there are only few reports investigating the level of ASN in the blood of PD patients (Bialek et al., 2011;Fuchs et al., 2008;Lee et al., 2006).
The aim of the study was to estimate the concentration of ASN in plasma of patients with PD and in control group.

Patients
The studies were conducted on 32 patients with PD, including 18 women and 14 men aging 35-82 years. Control group included 24 individuals, 20 women and 4 men, 40-69 years of age. Demographic data of all groups summarized in Table 13.
Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr (Hoehn & Yahr, 1967

Analysis of ASN concentrations
Preparation of samples. Blood samples from these subjects were drawn using EDTA as an anticoagulant in the morning after an overnight fast and the samples were centrifuge for 15 min at 1000xg at 4° C within 30 min and plasma was frozen at −80° C for later use.
Determination of ASN concentration. ASN ELISA was performed using the Enzyme-linked Immunosorbent Assay Kit for Human Synuclein Alpha (Uscn Life Science Inc., China) according to the manufacturer's protocol. The minimal detection limits in this assay is typically less than 4.8 pg/ml. The standard curve concentrations used were 1000; 5000; 250; 125; 62.5; 31.2 and 15.6 pg/ml. The intra-and interassay precision of coefficiences of variation were <10% and <12% respectively. After completion of each assay the plate(s) were read at 450 nm on an EPOCH Multi-Volume Spectrophotometer (BioTek, USA) and the results were analyzed using Gen5 2.1 Software (BioTek, USA).

Results
Detectable concentrations of ASN have been detected in higher percentage of controls than in PD patients. However, in patients with PD has been shown higher concentration of ASN (Table  14). In PD patients, the highest concentrations of ASN were present in two first stages of disease progress (Hoehn and Yahr scale) [ Table 15] and in the first ten years of the disease ( In this study and Bialek et al. (2011) have been shown higher concentration of plasma ASN level in PD patients as compared to controls. However, it seems that aggregation of the ASN in the nerve cells may reduce ASN ability to pass through the blood-brain barrier, which in turn may result in significantly reduced levels of this protein in the peripheral blood. Moreover, a high concentration of ASN has been detected only in the initial period of PD (in two first stages of PD progress in Hoehn and Yahr scale, and in the first ten years of the disease), probably even before the accumulation of deposits in the form of LB in the brain of PD patients. However, in the study by Pchelina et al. (2011) the level of ASN was significantly lower in patients with LRRK2-associated PD compared with SPD and controls what may be caused also by severed ASN aggregation in this group.

Role of Parkin in pathogenesis of Parkinson's disease
Parkin is a cytoplasmic protein which plays a vital role in the proper functioning of the mitochondria and functions as an E3 ligase ubiquitin stimulating protein binding (directed to degradation in the proteasome) with ubiquitin, consequently preventing the cell apoptosis (Zhang et al., 2000). Ubiquitination is a vital cellular quality control mechanism that prevents accumulation of misfolded and damaged proteins in the cell. It is thought that substrates of Parkin include among others synphilin-1, ASN, CDC-rel1, cyclin E, p38 tRNA synthase, Pael-R and synaptotagmin XI. It has been shown in the study by Zhang et al, [2000] Parkin is also responsible for their own ubiquitination and degradation in the proteasome.
Recent studies have shown that Parkin may play a role in decision-making, choosing between two systems of degradation: the proteasome activity (through its ability to promote ubiquitination K48 associated with the proteasome) and macroautophagy (through K63 ubiquitination related to cell signaling and the formation of LB) [Henn et al., 2007;Lim et al., 2006].

Parkin concentration in Parkinson's disease
The aim of the study was to estimate the concentration of Parkin in plasma of patients with PD and in control group.

Analysis of Parkin concentrations
Preparation of samples. Blood samples from these subjects were drawn using EDTA as an anticoagulant in the morning after an overnight fast and the samples were centrifuge for 20 min at 1000xg at 4° C within 30 min and plasma was frozen at −80° C for later use.
Determination of Parkin concentration. Parkin ELISA was performed using the Enzyme-linked Immunosorbent Assay Kit for Human Parkinson Disease Protein 2 (Uscn Life Science Inc., China) according to the manufacturer's protocol. The minimal detection limits in this assay is typically less than 0.058 ng/ml. The standard curve concentrations used were 10; 5; 2.5; 1.25; 0.625; 0.312; and 0.156 ng/ml. The intra-and interassay precision of coefficiences of variation were <10% and <12% respectively. After completion of each assay the plate(s) were read at 450 nm on an EPOCH Multi-Volume Spectrophotometer (BioTek, USA) and the results were analyzed using Gen5 2.1 Software (BioTek, USA).

Results
Detectable concentrations of Parkin have been detected in similar percentage of controls and PD patients. However, in patients with PD has been shown lower concentration of Parkin (Table 17). In PD patients, the highest concentration of Parkin occurred in 2 stage of disease progress with tendency to reduce the concentration in the 3 stage of the disease (Hoehn and Yahr scale) [ Table  18] and in the first ten years of the disease (Table 19). It is known that dysfunction of Parkin may lead to manifestation of PD in several mechanism including mitochondrial and ubiquitination disturbances. It also seems that expression and cellular level of Parkin may be essential factor for proper function of this protein. decreasing with the progress and duration of this disease. It seems that in the early stages of PD development may occur to increase of Parkin expression through the ongoing degenerative process and to the accumulation of pathological proteins-Parkin substrates. However, as the disease progresses, probably, resources of the Parkin running out and occurs weaken its neuroprotective function.

Relationship between alpha-synuclein and Parkin levels in Parkinson's disease
In 2001, Shimura et al. first described the presence in the human brain complex containing Parkin with the glycosylated form of the ASN (alpha-SP22), thus indicating the involvement of Parkin in ASN degradation in ubiquitin-proteasome system [Shimura et al., 2001;Chung et al., 2004]. It has been also shown that dysfunction of the Parkin can lead to ineffective elimination of ASN and the aggregation of this protein [Haass & Kahle, 2001]. In addition, according to the reports, the Parkin may also interact with the dopamine and indirectly influence the aggregation of the ASN in the nerve cell (Oyama et al., 2010). Therefore, it seems that the levels of these two proteins may be related and dependent on each other.

Results
In patients with PD detectable levels of Parkin occurred in a nearly two-fold higher incidence than the ASN (Tables 14, 17).  In this study and studies by Bialek et al. (2011) and Dorszewska et al. (2012) have been shown, that in PD patients increased level of ASN was associated with the decreased level of Parkin in contrast to control group (Tables 20-22). Independently for the analyzed group, the highest levels of ASN have been observed in the subjects who had very low Parkin levels. It suggested that low concentration of Parkin may contribute to increased ASN level in the nerve cells and combined with over-expression of ASN intensify or accelerate neurodegenerative process. Moreover, it has been also shown that configuration: increased plasma level of ASN and decreased of Parkin was associated with earlier onset of this disease.

Mutations in PARK (PRKN, SPR, HTRA2, SNCA) genes and ASN and Parkin concentrations in Parkinson's disease
Our study on Polish population shown that in PD patients PRKN (exons 4, 8, 11) mutations were more than four times frequency as compared to controls. Moreover, in PD patients more frequently occurred genotypes +2/+2 and +2/+3 of the promoter region SNCA gene than in controls. In patients with PD shown higher concentration of ASN while higher Parkin level in controls. In PD patients without mutations in PARK, highest concentration of ASN and Parkin was present in two first stages of disease progress (Hoehn and Yahr scale) and in the first ten years of the disease. However, only in PD patient with mutation in 11 exon of PRKN gene shown presence of Parkin without ASN after ten years of disease duration (Table 19, 22).
It seems that analysis of these pathological proteins with PARK gene mutations may be useful in the diagnostic and monitoring of the PD progress in the future.

Conclusion
In Polish population, in PRKN gene point mutations occur few times more often in PD patients than controls. Control subjects tend to show higher level of plasma Parkin whereas patients suffering from PD tend to generate higher level of plasma ASN. In PD patients without point mutations in PRKN gene Parkin and ASN plasma levels increase until 2 nd stage of disease in Hoehn and Yahr scale and during first 10 years of disease.
Analysis of the variations of PARK gene as well as plasma levels of ASN and Parkin may consist an additional diagnostic factor for PD.