Association between NMD3 and symptoms of Parkinson’s disease in Chinese patients

Background Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder that is characterized by motor symptoms such as tremor, rigidity, slowness of movement and problems with gait. Large-scale meta-analyses of genome-wide association studies (GWAS) have identified few susceptibility loci in patients with sporadic PD. The aim of this study was to investigate the association between NMD3 single nucleotide polymorphism (SNP) and symptoms in PD patients in South China. Methods A total of 217 PD patients were recruited in this study and genotyped by using the SNaPshot technique and the polymerase chain reaction. All subjects were evaluated by the Mini-Mental State Examination (MMSE), Beijing version Montreal Cognitive Assessment (MoCA), Sniffin’ Sticks 16 (SS-16), Hamilton Anxiety Rating Scale, Hamilton Depression Rating Scale, 39-item Parkinson’s Disease Questionnaire (PDQ-39) and MDS Unified PD Rating Scale (MDS-UPDRS). Results NMD3 rs34016896 (C > T) carriers have worse cognitive function than wild types (MMSE: p = 0.042, NMD3 wild type: 27.44 ± 2.89, NMD3 carriers: 26.31 ± 3.79; MoCA: p = 0.005, NMD3 wild type: 23.15 ± 4.20, NMD3 carriers: 20.75 ± 6.68). Conclusions The recessive and overdominant model of NMD3 rs34016896 was associated with cognitive impairment in PD patients.


Background
Parkinson's disease (PD) is one of the most common neurodegenerative diseases, affecting approximately 1.7% of people over the age of 65, and the annual incidence ranges from 1.5 to 8.7/100,000 in the People's Republic of China [1]. The pathological features of PD are the abnormal aggregation of α-synuclein and the loss of dopaminergic neurons in the substantia nigra [2]. Both acquired and inherited risk factors have been implicated in the death of dopaminergic neurons [3]. Genetic factors play a crucial role in the pathogenesis of sporadic PD. Genome-wide association studies (GWAS) have identified several susceptibility loci for PD [4][5][6]. Genes such as LRRK2, SNC A, etc. have been associated with the pathogenesis of PD [7,8]. Marie Y. Davis reported that GBA variants predicted a more rapid progression of cognitive dysfunction and motor symptoms in patients with PD [9].
Recently, variants at NMD3 were found to be related to substantia nigra neuronal loss and PD susceptibility [10,11]. The minor allele frequency of NMD3 rs34016896 was 0.41 in the Chinese PD population and 0.45 in the Chinese healthy population [11]. NMD3 encodes a ribosomebinding protein. Nmd3 is a structural mimic of eIF5A and activates the cpGTPase Lsg1 during 60S ribosome biogenesis [12]. Associations of NMD3 rs34016896 with clinical pathological phenotypes have been discovered. JM Shulman and colleagues found that NMD3 rs34016896 was related to the severity of nigral neuronal loss and not to Lewy bodies [13]. However, the function of NMD3 in PD is unknown. In addition, it is necessary to investigate the association between NMD3 and clinical symptoms of PD, which could indicate the pathogenesis of PD.
In this study, we attempted to discover the clinical hallmarks of NMD3 rs34016896 (C > T) in southern Chinese PD patients.

Study population
PD patients were recruited from the outpatients clinic of Ren Ji Hospital (South Campus) and diagnosed by movement disorder specialists based on diagnostic criteria outlined by the Movement Disorders Society (MDS) [14]. For the PD patients, Hoehn-Yahr staging and their disease duration were recorded. A family history of PD was also recorded. Parkinsonism patients with secondary causes, such as inflammatory, drug-induced, vascular and toxin-induced parkinsonism, were excluded. Patients with parkinsonism with other neurodegenerative diseases, such as Wilson's disease, progressive supranuclear palsy, cerebral-basal degeneration and multiple system atrophy, were also excluded. This study was approved by the ethics committee of Ren Ji Hospital.

Evaluation
Each PD patient included in this study received an evaluation including the following rating scales: Unified PD Rating Scale provided by the Movement Disorders Society (MDS-UPDRS) was used to assess the status of PD [15]. The Mini-Mental State Examination (MMSE) and Beijing version of the Montreal Cognitive Assessment (MoCA) were adopted to assess cognitive function. The Sniffin' Sticks 16 (SS-16) was used to assess olfactory function [16]. The Hamilton Anxiety Rating Scale and Hamilton Depression Rating Scale were used to assess anxiety and depression, respectively. The non-motor symptoms scale (NMSS) was used to assess non-motor symptoms. The Scales for Outcomes in Parkinson's disease-Autonomic questionnaire (SCOPA-AUT) was used to assess autonomic symptoms. The 39-item Parkinson's Disease Questionnaire (PDQ-39) was used to assess the quality of life of the PD patients. The researchers received strict training regarding the use of these scales before assessing the PD patients. We also documented the presence (yes/no) of the following symptoms that were assessed by two individual neurologists: hallucination, apathy, excessive daytime sleepiness, pain, frequent urination, constipation, postural hypotension, sialorrhea, restless legs syndrome (RLS), delusion, double vision, decreased attention, decreased recent memory, nycturia, sexual dysfunction, hypogeusia, change in weight, daytime sweatiness, nocturnal sweatiness, urgent urination or urinary incontinence, sensitivity to light, sensitivity to cold, sensitivity to hot, anxiety, depression, and probable rapid eye movement sleep behaviour disorder (RBD). Probable RBD was diagnosed via the RBD screening questionnaire [17]. We followed the method for detecting NMD3 rs34016896 described by Li and colleagues [18].

Statistics
The R stats (version 3.5.1) and CATT (version 2.0) packages were used to perform the statistical analyses. Student's t tests were performed to evaluate the differences in numeric variables between NMD3 carriers and wild types. For comparing categorical variables, chi-square tests were performed. The Cochran-Armitage test was used to assess ordinal variables between NMD3 carriers and wild types and an additive model of NMD3. Logistic regression was used to assess the association between the additive model/dominant model/recessive model/ overdominant model of NMD3 and clinical phenotypes. The odds ratio (OR) and its 95% confidence intervals (CI) were also used. We also adjusted for age, sex and Hoehn-Yahr staging results.

Results
There were 217 PD patients included in this study. SNPs of two PD patients failed to be detected. In all, there were 39 NMD3 wild types and 178 NMD3 carriers in our study. The minor allele frequency in our group was 0.41, which is similar to a previous genetic association study in southeastern China [11]. There was no difference in age, sex, disease duration, family history, and Hoehn-Yahr staging between the two groups. In the NMD3 wild-type group, the age was 55.69 ± 10.24 years (mean ± SD), and 17 (43.59%) females were included in this group. In the NMD3 carrier group, the average age was 57.03 ± 10.16 years (mean ± SD), and 73 (41.01%) females were included in this group. There were no   (Table 3). Under the overdominant model, an association with apathy and delusion were found (apathy: p = 0.011, OR: 2.05, CI: 1.19-3.57, before adjustment; p = 0.012, OR: 2.06, CI: 1.18-3.64, after adjustment; delusion: p = 0.048, OR: 3.66, CI: 1.14-16.31, before adjustment; p = 0.035, OR: 4.35, CI: 1.25-20.99, after adjustment.). A trend for an association between hallucination and the overdominant model was found (p = 0.071, OR: 2.32, CI: 0.97-6.17, before adjustment; p = 0.086, OR: 2.26, CI: 0.93-6.13, after adjustment) ( Table 4).
We performed the Bonferroni correction following multiple comparisons to adjust p values. After the correction, there were no remaining statistically significant results.

Discussion
Our study found that NMD3 carriers had worse cognitive function. The additive model of NMD3 was associated with hallucination, postural hypotension and delusion. The dominant model of NMD3 was associated with postural hypotension. The recessive model of NMD3 was associated with hallucination, apathy, postural hypotension and delusion. The overdominant model of NMD3 was associated with apathy and delusion. Trends for associations between the additive model of NMD3 and apathy, decreased recent memory and change in weight were found. Trends for associations between the recessive model of NMD3 and decreased attention, decreased recent memory, hypogeusia and change in weight were found. Trends for associations between the overdominant model of NMD3 and hallucination were found. To our knowledge, this is the first study to investigate the association between NMD3 and its clinical symptoms in Chinese PD patients. NMD3 encodes a cytoplasmic protein for stable 60S ribosomal subunits [19,20]. The function of ribosomes in the pathogenesis of PD remains unknown. A study revealed that parkin-PARIS (parkin-interacting substrate) played a deleterious role in rRNA transcription in PD patients, which indicated that ribosomes might be involved in the pathogenesis of PD [21]. A possible hypothesis of the function of NMD3 is that the dysfunction or dysregulation of ribosomes produces proteins relevant to PD. More relationships between the eukaryotic ribosome and PD should be discovered. JM Shulman and colleagues found that NMD3 rs34016896 was associated with nigral neuronal loss and not with Lewy bodies [13]. This research indicated that NMD3 rs34016896 was associated with nigral neurodegeneration rather than the formation of Lewy bodies. Neuronal loss, especially dopaminergic neuronal loss, is associated with the pathogenesis of PD. However, we have little information regarding the details of the type of neurons for neuronal loss. To date, there have been no studies on the clinical phenotype based on the detailed pathological phenotypes. It is difficult to elucidate the impact of neuronal loss on clinical phenotypes. Further research on NMD3-related neuronal loss could uncover the presence of those relevant symptoms.
The strengths of our study are that PD were assessed by a structured scale, which is widely accepted. The diagnosis was based on MDS criteria. We also covered a wide-range assessment examining motor function, nonmotor symptoms and quality of life in PD patients.
This study has some weaknesses and limitations. First, we did not perform objective clinical methods, such as electrophysiology, to assess symptoms. Second, we did not perform stratifications due to the small sample size. Third, the sample of our study was small, and our study was a single-centre study. More multicentre and larger studies are warranted.

Conclusions
In conclusion, NMD3 carriers had worse cognitive function. The additive model of NMD3 was associated with hallucination, postural hypotension and delusion. The dominant model of NMD3 was associated with postural hypotension. The recessive model of NMD3 was associated with hallucination, apathy, postural hypotension and delusion. The overdominant model of NMD3 was associated with apathy and delusion. More larger and multicentre studies are warranted. The recessive and overdominant model of NMD3 rs34016896 was associated with cognitive impairment in PD patients.