Genetic Influences on Cognition in Idiopathic Parkinson's Disease

Cognitive impairment in Parkinson's disease was clearly reported in the medical literature [1, 2]. This impairment is common to some degree even in nondemented Parkinson’s disease patients (PD-ND) and eventually progresses to dementia in 24 to 31% of patients [3]. The cognitive changes in PD are characterized by a frontalsubcortical impairment with decreased attention and executive function leading to progressive impairment in prefrontal tasks, visuospatial skills, and memory. Still, 20 to 25% of PDND patients may exhibit a pattern of cortical impairment with memory tasks and confrontation naming defects, and cognitive findings associated with cortical pathology, such as language errors, develop in many patients with PD with dementia (PDD) [4]. The identification of the biomarkers for cognitive impairment in patients with PD will allow better assessment of the patients' prognosis. Some genes, such as apolipoprotein E (ApoE) and microtubule-associated protein tau (MAPT), are of particular interest because of their known association with dementia in other neurodegenerative diseases, such as Alzheimer’s disease (AD) and atypical parkinsonian syndromes, including progressive supranuclear palsy and corticobasal degeneration (Morley et al., 2012) [5]. The aim of this study is to determine the role of genetic factors associated with cognitive decline in Parkinson’s disease (PD). We examined whether variations in apolipoprotein E (ApoE) and microtubule-associated protein tau (MAPT) genotypes are associated with cognitive decline in PD.


Introduction
Cognitive impairment in Parkinson's disease was clearly reported in the medical literature [ , ].
This impairment is common to some degree even in nondemented Parkinson's disease patients (PD-ND) and eventually progresses to dementia in 24 to 31% of patients [ ].
The cognitive changes in PD are characterized by a frontalsubcortical impairment with decreased attention and executive function leading to progressive impairment in prefrontal tasks, visuospatial skills, and memory. Still, 20 to 25% of PD-ND patients may exhibit a pattern of cortical impairment with memory tasks and confrontation naming defects, and cognitive findings associated with cortical pathology, such as language errors, develop in many patients with PD with dementia (PDD) [ ].
The identification of the biomarkers for cognitive impairment in patients with PD will allow better assessment of the patients' prognosis. Some genes, such as apolipoprotein E (ApoE) and microtubule-associated protein tau (MAPT), are of particular interest because of their known association with dementia in other neurodegenerative diseases, such as Alzheimer's disease (AD) and atypical parkinsonian syndromes, including progressive supranuclear palsy and corticobasal degeneration (Morley et al., ) [ ].
The aim of this study is to determine the role of genetic factors associated with cognitive decline in Parkinson's disease (PD). We examined whether variations in apolipoprotein E (ApoE) and microtubule-associated protein tau (MAPT) genotypes are associated with cognitive decline in PD.

Subjects and Methods
The current study is a pilot study. In this pilot study, we applied a prospective design, to evaluate cognitive changes between groups defined by genotype differences.

Subjects.
This study included 50 patients recruited from neurology outpatient clinic and Neurology Department in Kasr AL-Ainy Hospital, Cairo, Egypt, who fulfilled the criteria for diagnosis of idiopathic Parkinson's disease based on British Brain Bank criteria [ ].
Exclusion criteria: patients with secondary parkinsonism (drug-induced, posttraumatic, or postinfectious) or atypical parkinsonism; patients with severe dementia MMSE <11 or with major language disturbance and severe physical, auditory, or visual impairment affecting their ability to complete testing [ ]; patients with Geriatric Depression Scale (GDS) ≥ 10; patients with evidence from the history, 2 Neurology Research International physical examination, or investigations for any concomitant medical or metabolic illness known to affect cognition, e.g., thyroid, parathyroid, hepatic, and renal disease; patients with marked tremor, which interferes with the imaging session and produces movement artifacts, were excluded.
After assessment of cognitive functions by Mini-Mental State Examination and Parkinson's Disease-Cognitive Rating Scale (PD-CRS), the patients were divided into 2 groups; Group I included 14 patients with IPD and no cognitive impairment; Group II included 36 patients with IPD and cognitive impairment. Cognitive impairment was considered if there was an abnormality in more than one cognitive domain, representing a decline from premorbid level.
The study was approved by Neurology Department Board in Cairo University and follows the principles outlined in the Declaration of Helsinki. Verbal informed consent was obtained from all patients prior to the commencement of the study after a structured interview clarifying the aim and steps of the study.

Methods.
All patients in this study were submitted to the following: thorough history taking and neurological examination according to the standardized sheet of Neurology Department, Kasr Al-Ainy Hospital.
All patients were evaluated for Parkinson's disease severity while on their best state using Unified Parkinson's Disease Rating Scale (UPDRS) [8].
Genotyping: all the patients were genotyped for the Apo E and MAPT polymorphism.
Steps of gene polymorphism detection by real-time PCR: (A) DNA extraction and (B) genotyping.
Genotyping was performed using real-time polymerase chain reaction with TaqMan allelic discrimination assay (Applied Biosystems, USA). Genotyping for (rs1052553, C 7563736) MAPT allelic variant and ApoE alleles (rs7412, C 904973 10 and rs429358, C 3084793 20) single nucleotide polymorphisms was performed using real-time polymerase chain reaction.
PCR amplification protocol: a predesigned primer/probe set for the genotypes was used (Applied Biosystems, USA). Probes were synthesized with reporter dye FAM or VIC covalently linked at the 5 and a quencher dye MGB linked to the 3 end of the probe (Applied Biosystems, USA).
PCR mix: DNA amplification was carried out in a 25 l total volume containing 12.5 l Taqman master mix, 1.25 primer/probe, 1 l DNA, and 10.25 H2O.
Cycling conditions: real-time PCR was performed using Applied BioSystem step one plus Real-Time PCR System (Applied BioSystem, CA, USA) with the following conditions: after a denaturation time of 10 min at 95 ∘ C, 45 cycles at 92 ∘ C for 15s, and then 60 ∘ C for 90s for annealing and extension were carried out and fluorescence was measured at the end of every cycle and at the endpoint.  summarized using mean and standard deviation for quantitative variables and percent for qualitative variables. Categorical variables were compared with chi square test, and continuous variables were compared with t-test (normal distribution) or Mann-Whitney tests (nonnormal distribution). A value < 0.05 was set as significant and <00.1 as very significant.

Results
The current study included 50 patients divided into 2 groups with their demographic and baseline characteristics described in Table 1.
The results showed the presence of significant differences concerning the age, scores of UPDRS, MMSE, and PDCRS between the 2 groups, representing that the group with cognitive changes included older patients with higher UPDRS score.

PDCRS and Genotypes in Groups I and II Patients at 0 and 3 Months.
Group I: in ApoE 4 carrier mean PDCRS total score was 78.85 at 0 months, which is slightly better than in 4 noncarrier 77.57 (p value =0.89). MAPT H1/H1 haplotype    Figure 6. A high statistical significance was found between changes in PDCRS frontosubcortical, PDCRS posterior-cortical, PDCRS total, and ApoE 4 carrier/noncarrier (p-value < 0.001) with PDCRS score worse with ApoE 4 carriers.
The mean in PDCRS frontosubcortical scores changed by 1.80 points in MAPT H1/H1 haplotype and by 1.58 points other haplotypes. The mean PDCRS posterior-cortical scores changed by 1 point in MAPT H1/H1 haplotype and by 1.06 points in other haplotypes. The mean PDCRS total changed by 2.80 points in MAPT H1/H1 haplotype and by 2.65 points in other haplotypes.

Comparison of Apolipoprotein 4 Carrier between Groups I and II Patients in Different Rating
Scales. The cognitive performance was better in ApoE 4 carriers in Group I than in Group II as evident by the mean MMSE score and mean PDCRS (total) score at 0 months, as illustrated in Figure 7. A statistical significance was detected between both groups (p-value=<0.001 and 0.017, respectively). The motor functions were worse in ApoE 4 carriers in Group II than in Group I as evident by the mean UPDRS score; a statistical significance was detected between both groups (p-value=0.041).   performance was better in MAPT H1/H1 haplotype in Group I than in Group II as evident by the mean MMSE score ( Figure 8) and mean UPDRS (total) score (at 0 months); a statistical significance was detected between both groups regarding MMSE (p-value=0.053) but not with PDCRS. The motor functions were worse in MAPT H1/H1 haplotype in Group II than in Group I as evident by the mean UPDRS score, but no statistical significance was detected between both groups. The change in mean UPDRS I and mean UPDRS total changes in "ApoE 4 carrier or MAPT H1/H1" and in "ApoE 4 carrier and MAPT H1/H1" were not statistically significant as shown in Table 4. The mean change in PDCRS frontosubcotical in "ApoE 4 carrier/or MAPT H1/H1" group was 2.45 ± 3.41 points, while in the "ApoE 4 carrier and MAPT H1/H1" it was 6.14 ± 3.13 points (p=0.019).
The change in PCDRS posterocortical was not statistically significant as illustrated in Table 4. There was no statistical significant correlation between duration of illness and both UPDRS and PDCRS, as illustrated in Table 5.

Discussion
Cognitive impairment is a common and functionally significant problem in PD, with a cumulative prevalence of dementia as high as 75%-90% [10].
The MMSE score in our study patients ranged from 12 to 30. In group I, the mean MMSE decreased by 0.93 points in 3 months, while in Group II the mean MMSE decreased 6 Neurology Research International  The UPDRS (total) score in patients of this study ranged from 10 to 72. In Group I, the mean UPDRS increased by 5.14 points after 3 months of follow-up. In Group II, the mean UPDRS increased by 3.98 points after 3 months of followup. A statistically significant difference was observed between Groups I and II as regards the UPDRS at baseline assessment and after 3 months of follow-up (p-value; 0.018 and 0.025, respectively).
In a study performed by Campos et al., for factors predicting dementia, they found that motor performance as assessed by the (UPDRS-Part III) was a strong predictor, whereas, neither age nor Hoehn and Yahr scale (H&Y) scoring was predictors [ ]. On the contrary, Stavitsky et al. ( ) found that UPDRS (total) and sleep did not predict a future memory decline [16].
In this study, the decline in cognition after 3 months in Group I patients was 4.14 points (in PDCRS) in relation to worsening of motor symptoms by 1.85 points (in UPDRS III) and in Group II patients where decline in cognition was 2.67 points (in PDCRS) in relation to worsening of motor symptoms by 0.75 points (in UPDRS III); an inverse relationship is observed between UPDRS III (motor) and PDCRS (total), as the UPDRS III score increases (motor symptoms worsen) the PDCRS score decreases (cognitive symptoms worsen). A statistical significant correlation between UPDRS III and PDCRS (total) (p-value=0.003) in all PD patients was  [18] who reported a significant association between the severity of motor symptoms in patients with PD and the development of cognitive dysfunction. Such association was explained by the presence of frontostriatal circuits dysfunction in patients with PD which in turn is responsible for producing both the motor symptoms and cognitive impairment [19]. On the other hand, Cooper et al.
[ ]and Abo-El-Naga [ ] failed to establish a relationship between motor function and cognitive impairment in patients with PD. The investigators defended their findings by concluding that cognitive dysfunction in early PD patients reflected neuropathological changes that are distinct from those responsible for the motor disorder.
There is a continuous search for biological markers of cognitive dysfunction in Parkinson's disease, which encouraged the investigation of the genetic effect; some of the studied genes were previously reported to be associated with cognitive impairment in PD, through affecting the dopaminergic systems. Other genes, ApoE and MAPT, are of particular interest because of their known association with dementia in other neurodegenerative diseases, such as Alzheimer's disease (AD) and atypical parkinsonian syndromes, including progressive supranuclear palsy and corticobasal degeneration [ ].
Relatively few studies examined the MAPT H1 haplotype as a risk factor for cognitive impairment in PD. All PD patients in our study were genotyped for MAPT polymorphism. In Group I, the H1/H1 haplotype (n=7) represents 50%, while in Group II the H1/H1 haplotype (n=5) represents a much smaller proportion 13.9% to other haplotypes (n=31) which represent 86.1%. A statistical significance was found between different haplotypes (P-value=0.020) among Groups I and II patients. Contrary to our findings, Ezquerra et al. [ ] found no difference in H1 frequency between PD patients with and without dementia. The results of the current study showed a faster rate of decline in MMSE scores in individuals with the MAPT H1 variant. Although no statistical significance was found between change in MMSE and MAPT haplotypes, yet, the mean MMSE change was higher in patients with MAPT H1/H1 haplotype compared to those with other haplotypes, being 1.14 (group I) and 1.40 (Group II) points in the former and 0.75 (group I) and 0.61 (Group II) points in the later. A longer follow-up with such decline rates might show a significant decline in MMSE score in MAPT H1/H1.
We also concluded that the changes in PDCRS frontosubcortical, PDCRS posterior-cortical, PDCRS total were worse in the MAPT H1/H1 haplotype compared to other haplotypes as measured by PDCRS without statistical significance. These Neurology Research International 7 results are similar to some extent to a study conducted by Williams-Gray et al., [ ], which included a cohort of 122 PD patients who were followed up for 5 years; they found that MAPT-H1 haplotype was associated with a more rapid decline in Mini-Mental State Examination score and was a significant risk factor for conversion to dementia. On the contrary, Morley et al. ( ) [ ] did not find a significant association between MAPT haplotype and rates of cognitive decline.
We found that worsening of cognition was more with ApoE 4 carrier than with 4 noncarrier where a statistical significance was found between change in PDCRS and ApoE 4 carrier/noncarrier as the mean PDCRS total change by 7.43 (in Group I) and 6 (in Group II) points The observed association between ApoE and cognitive decline may not be specific to PD and it could be observed in otherwise healthy older individuals. This was previously discussed by Morley et al., [5] who could not establish a confirmed association.
In conclusion, patients in Group I with no cognitive dysfunction showed statistically significant worsening in PDCRS frontosubcortical but not in PDCRS posterior-cortical, while patients in Group II with cognitive dysfunction showed significant worsening in PDCRS frontosubcortical and PDCRS posterior-cortical, and this result of early involvement of the frontosubcortical tests rather than posterior-cortical which is affected later in the disease with development of dementia. In this study, 27 PD patients have ApoE 4 carrier and/or MAPT H1/H1. PD patients with ApoE 4 carrier or MAPT H1/H1 represent larger proportion (75%) compared to PD patients with both ApoE 4 carrier and MAPT H1/H1 (n=7) represents 25%. A statistical significance was found between change in MMSE (p-value < 0.048) and ApoE 4 carrier and MAPT H1/H1. A statistical significance was found between change in PDCRS frontosubcortical (p-value=< 0.019), PDCRS total (p-value=< 0.031), and carriers of both ApoE 4 and MAPT H1/H1 with scores of cognitive functions being worse with carriers of both ApoE 4 and MAPT H1/H1 more than carriers of either ApoE 4 or MAPT H1/H1.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.