Multi-chip Meta-analysis of Parkinson's Disease for Clinical Validation of Small Samples of Key Genes in Disease
Conditions: Parkinson's Disease
Sponsor: Zhujiang Hospital

Multi-chip Meta-analysis of Parkinson's Disease for Clinical
Validation of Small Samples of Key Genes in Disease
Unit in charge of clinical research ：Zhujiang Hospital of Southern Medical
University
principal
investigator：Gao Xiaoya, deputy chief physician
Scheme version: No2
Scheme version date: April 3, 2019
Confidentiality statement
All information contained in this study protocol belongs to the investigator
of the project and is only available for review by the ethics committee and relevant
authorities. it is strictly prohibited to give any information to any third party
unrelated to the study without the written consent of the principal investigator
(PI).

Research plan confirmation signature page
Version No.: 2.0
Version date: April 3, 2019
Title: Multi-chip Meta-analysis of Parkinson's Disease for Clinical Validation
of Small Samples of Key Genes in Disease
Consent from the principal investigator on the protocol:
I have read the program carefully, agree with all the necessary information in
the program to conduct the research, and agree to execute as described in the
program.I understand that the research should not be initiated without the approval
of the ethics committee and should fully comply with the relevant regulations of
the unit.
Informed consent and appropriate documentation are required for all
participants in the study.After obtaining the written informed consent signed by
the subjects, the study will be carried out according to the declaration of Helsinki
and the requirements of laws and regulations related to clinical research.
______________________
_________________
_________________
Name of principal investigator
signature
date

Summary of research programme
Title
Multi-chip
Meta-analysis
of
Parkinson's
Disease
for
Clinical
Validation of Small Samples of Key Genes in Disease
Investigator
Gao Xiaoya, deputy chief physician
Main research
units
Zhujiang Hospital of Southern Medical University
research
center
Department
of
Neurology,
Zhujiang
Hospital,
Southern
Medical
University
research
objective
The research team used meta-analytical statistical methods to
integrate the results of different research groups on Parkinson's
disease, using meta-analysis to find key genes related to the
pathogenesis and development of Parkinson's disease, and to make
small clinical results. The verification of the sample, the internal
mechanism of the pathogenesis of Parkinson's disease and provide
guidance and reference for subsequent experimental research.
research
hypothesis
Synaptic
related
functions
may
be
closely
related
with
the
development of Parkinson's disease(PD). Alpha - synuclein (alpha
alpha-synuclein), PPP3CB (protein phosphorylase 3 catalytic subunit
beta) and other genes may be key genes in the pathogenesis of
Parkinson's disease, and their expression may be closely related to
the occurrence and development of PD.
research
design
Observational case-control clinical study
Subject
population
Inclusion criteria: 1. PD group: Patients with primary PD; 2.
Patients
with
Parkinsonism-Plus
syndrome (PPS):
Patients
with
Progressive
superanuclear
palsy (PSP) and multiple system atrophy
(MSA); 3. Healthy controls (HC);
diagnostic
criteria
2015 International Parkinson's and Movement Disorders Association
(MDS) Parkinson's disease diagnostic criteria.
2017 clinical diagnostic criteria for MDS progressive supra palsy;
Giman standard, the 2008 American board of neurology (AAN) MSA
diagnostic consensus.
sample size
The study was divided into PD group, PPS group and healthy control

group, at least 80 cases, 23 cases and 110 cases, respectively.
Main exposure
factors
1.After admission, all patients and the control group took 2 tube
venous blood samples of 12 ml and saliva samples of 20 ml
respectively and sent them to the laboratory for testing.
2.Unified Parkinson’s Disease Rating Scale（UPDRS）,non motor symptom
rating scale (NMSS), Hamilton Depression Scale(HAMD), Hamilton
Anxiety Scale (HAMA), Montreal Cognitive Assessment(MoCA) and
Pittsburgh sleep quality index
(PSQI) were used to assess the
motor and non-motor symptoms of PD patients；The clinical symptoms
of patients with PSP were evaluated by
Progressive
superanuclear
palsy Rating Scale（PSPRS）and MSA
by Unified multiple system atrophy Rating Scale (UMSARS).
Outcome
variable
1.The primary end point: the expression level of alpha synuclein,
PPP3CB
and
miRNA-ceRNA
(microRNA-endogenous
competitive
RNA)
network and its target protein in the disease group and the control
group;
2.Secondary
end
point:
correlation
between
gene,
transcriptome ,target protein expression and clinical indicators of
patients and analysis of risk factors for Disease.
Data
collection
and
management
After the completion of the experiment, the case observation table
and experimental test data will be reviewed by the researcher and
then recovered, and data processing will be carried out. The data
of the case report form was input in double copies, and the American
clinical trial database
was locked after
the examination and
confirmation.
Statistical
methods
Statistical hypothesis: it is assumed that the expression of alpha
synuclein, PPP3CB and other genes in PD patients is different among
PD, PPS and healthy controls, and has a good correlation with clinical
indicators such as the severity of PD.
Main statistical analysis methods: SPSS 10.0 software was used to
analyze the data.The normal distribution of the measurement data is
represented by mean ± SD, and the comparison among the three groups
is represented by one-way ANOVA; the non-normal distribution data
is represented by median (quartile), and Kruskal Wallis rank test;
the count data is represented by chi-square test; the grade data is

represented by Ridit analysis or chi-square test.The value of P <
0.05 was considered statistically significant.ROC curve is used to
analysis genes
diagnostic value, when AUC > 0.5, the closer AUC is
to 1, the better the diagnosis effect.The accuracy of AUC is lower
when it is 0.5-0.7, medium when it is 0.7-0.9 and higher when it is
above
0.9.When
AUC=0.5,
the
diagnostic
method
is
completely
ineffective and has no diagnostic value.AUC<0.5 is not consistent
with the real situation.The risk factors of disease were analyzed
by logistic regression.

Catalog
1. RESEARCH BACKGROUND AND PURPOSE-----------------------------------------5
1.1 RESEARCH BACKGROUND -----------------------------------------------------------------5
1.2 PURPOSE OF THE STUDY.............................................................................................. 5
1.3 RESEARCH HYPOTHESES.............................................................................................. 5
2. STUDY DESIGN ------------------------------------------------------------------------------5
3. SUBJECTS (COHORT SELECTION)............................................................................... 5
3.1 DIAGNOSTIC CRITERIA..............................................................................................5
3.2 INCLUSION CRITERIA-------------------------------------------------------------------- 5
3.3 EXCLUSION CRITERIA ----------------------------------------------------------------------------5
4. OBSERVATION INDEX (VARIABLE DEFINITION AND OBSERVATION TIME AND
NODE).......................................... 5
4.1 GENERAL CLINICAL CHARACTERISTIC INDEXES (INCLUDING DEMOGRAPHIC
INDEXES).......................................... 5
4.2 MAIN EXPOSURE FACTORS / FACTORS AFFECTING PROGNOSIS (INCLUDING CONTROL
SELECTION)........................................... 5
4.3 END POINT OF STUDY-------------------------------------------------------------------- 5
4.4 POTENTIAL CONFOUNDING VARIABLES -----------------------------------------------------------6
5. SAMPLE SIZE ESTIMATION ----------------------------------------------------------------6
6 DATA COLLECTION AND DATA MANAGEMENT
6.1 DATA SOURCE................................................................................................................. 6
6.2 DATA COLLECTION ------------------------------------------------------------------------------6
6.3 DATABASE ----------------------------------------------------------------6
6.4 DATA MANAGEMENT PLAN AND QUALITY CONTROL------------------------------------------- 6
7 STATISTICAL ANALYSIS------------------------------------------------------------------------- 6
8 ETHICAL CONSIDERATIONS -------------------------------------------------------------------7
9 QUALITY CONTROL AND QUALITY ASSURANCE-------------------------------------- 7
10 ORGANIZATION AND IMPLEMENTATION (TIME
SCHEDULE)..........................................7
11 RESPONSIBILITIES AND SIGNATURES OF ALL PARTIES INVOLVED--------------------7
12 REFERENCES ---------------------------------------------------------7

1
research background and purpose
1.1 research background
Parkinson's disease (PD) is a relatively common degenerative disease of the
central nervous system. As society gradually becomes aging, the number of PD patients
is increasing, but its exact pathogenesis is still not fully understood. May be
related to genetic factors, environmental factors, immunological abnormalities,
mitochondrial dysfunction and oxidative stress, ageing, apoptosis and other factors;
the current genetic diagnosis is in the ascendant, making the understanding of the
etiology and pathogenesis of Parkinson's disease more In-depth, provide more basis
and means for the pathogenesis and development of Parkinson's disease, but due to
the number of individual samples, operational norms and platform differences,
different research groups have great differences in the results of gene chip research
on Parkinson's mechanism, resulting in the reliability is poo; in order to improve
the reliability of Parkinson’s disease onset and development gene diagnosis,We will
carry out the research of integrating the results of different research groups by
meta-analysis to find the key genes of Parkinson's disease, and carry out the
clinical verification.
1.2 purpose of the study
The research group uses the statistical method of meta-analysis to integrate
the research results of different research groups on Parkinson’s disease chip
together, uses meta-analysis to find the key genes related to the pathogenesis and
development of Parkinson’s disease, and carries out small sample verification in
clinical, excavates the internal mechanism of Parkinson’s disease and provides
guidance and reference for the follow-up experimental research.
1.3 research hypothesis
Synaptic function may be closely related to the occurrence and development of
PD. Alpha synuclein, PPP3CB and other genes may be the key genes in the pathogenesis
of PD, and their expression is related to the pathogenesis and development of PD
patients.
2
Study design and procedure
Meta analysis and clinical retrospective case-control design were used in this
study.The research period is about 2 years.

3 subjects (cohort selection)
PD patients, PPS patients in the inpatient department or outpatient department
of Neurology Department of Zhujiang Hospital who meet the requirements and the
healthy control population in the physical examination center of Zhujiang Hospital
from November 2018 .
3.1 diagnostic criteria
PD group: patients with Parkinson’s disease were diagnosed according to 2015
International Association of Parkinson’s and Movement Disorders Association (MDS);
PPS group: patients with Parkinson-plus syndrome were diagnosed according to
2017 MDS progressive supranuclear paralysis and 2017 clinical diagnostic criteria
for MDS progressive supra palsy and Giman standard, the 2008 American board of
neurology (AAN) MSA diagnostic consensus.
3.2 inclusion criteria
Inclusion criteria:PD group: Patients with primary PD according to the 2015
International Association of Parkinson’s and Movement Disorders Association (MDS);
2. Patients with Parkinson-plus syndrome:according to 2017 MDS progressive
supranuclear paralysis and 2017 clinical diagnostic criteria for MDS progressive
supra palsy and Giman standard, the 2008 American board of neurology (AAN) MSA
diagnostic consensus. 3. Healthy controls.
3.3 Exclusion criteria
1.Other causes of Parkinson’s syndrome, including cerebrovascular disease,
encephalopathy, trauma and drugs;
2.people with ther neurodegenerative diseases, such as huntington's disease,
lewy body dementia, alzheimer's disease, etc.
3.The person have other disabilities or diseases, such as aphasia, severe
dementia or consciousness disorders, malignant tumors, liver and kidney dysfunction,
serious heart disease or other acute or chronic diseases or life-threatening
diseases.
4.someone who refuse to participate in the study.
3.4 Patient recruitment
Patients with Parkinson’s disease or Parkinson-plus syndrome who met the
requirements after 2018-11 in the Department of Neurology of Zhujiang Hospital, and
healthy controls in the health examination center of Zhujiang Hospital.

4 observation indicators
General clinical data, CT, MRI and other imaging examinations within one week
after admission, and PD group patients, MDS-UPDRS,NMSS, HAMD, HAMA, Moca, PSQI, H
& Y rating scale and other scales were used to assess the movement and non-movement
symptoms of PD patients, PSPRS was used to assess the clinical symptoms of PSP
patients, UMSARS was used to assess the clinical symptoms of MSA patients.
4.1 General clinical characteristics (including demographic indicators)
Age, sex, admission diagnosis, comorbidity, routine examination, etc.
4.2 main exposure factors/factors affecting prognosis(including control selection)
All the participants took 3 tubes of venous blood for gene detection; this study
did not interfere with the clinical treatment of patients, and the clinical treatment
of patients was carried out according to the guidelines and relevant specifications.
4.3 study end point
The primary end points:The expression levels of alpha-synuclein, PPP3CB and
other genes and proteins in peripheral blood and saliva.
Secondary end point: The correlation between the expression level of alpha
synuclein, PPP3CB in peripheral blood and saliva and the clinical characteristics
of PD and PPS, and the analysis of risk factors of PD and PPS.
4.4 potential confounding variables
Patients may have unknown diseases that affect the expression level of alpha
synuclein, PPP3CB
and other genes and proteins or take related drugs.
5 sample size estimation
The patients were divided into PD group (an least 80 cases), PPS group (at least
23 cases) and control group (at least 110 cases).
6 data collection and data management
6.1 data source
The data came from general population data and clinical data, peripheral blood
and saliva test results, PD and PPS patients’related scale evaluation results.
6.2 data collection
1.Make a case observation table, and record the age, gender, race, admission
diagnosis,
complications,
neurological
physical
examination,
laboratory
examination, craniocerebral imaging and other
general demographic,
clinical
symptoms and signs as well as routine examination and examination data of the
patients.

2. Expression level of alpha-synuclein, PPP3CB and other genes and proteins in
peripheral blood and saliva.
3. In PD group, MDS-UPDRS, NMSS, HAMD, HAMA, MoCA, PSQI, HOEHN&YAHR (H-Y) grading
were used to assess the motor and non motor symptoms and disease severity of PD
patients, PSPRS were used to assess the clinical symptoms of PSP patients, UMSARS
was used to assess the clinical symptoms of MSA patients.
6.3 database
The study data will be updated in real time in the US clinical trials database
(Clinical Trials. Gov).
6.4 data Management plan and quality control
The completed case observation form, gene and protein detection data shall be
reviewed by the supervisor and then recovered, and handed over to Chaohao Guan and
Cailing Feng for data processing. The data in the case report form shall be entered
in double copies (Chaohao Guan and Cailing
Feng). After the inspection and
confirmation, the data shall be locked in the US clinical trial database.
7 statistical analysis
(1) statistical software: use SAS 22.0 statistical software for statistical
analysis.
(2) basic principle: Bilateral test was used for all statistical inferences.
The test level with statistical significance was set at 0.05, and the confidence
interval of parameters was estimated to be 95%.As far as possible, parameter method
can be adopted. When the data does not meet the condition of parameter method, data
transformation method can be adopted to make it meet the condition. If it still does
not meet the condition, non-parameter method can be considered.
(3) minimum, maximum, P25, median and p75 are given when necessary;Matching
measurement data to mean and standard deviation of travel value; the median and
average rank are given by the nonparametric method.The frequency distribution and
the corresponding percentage are given by counting data.The frequency distribution
and the corresponding percentage, as well as the median and average rank are given
in the rank data.Qualitative data showed positive rate, positive number and number
of
cases in the denominator.
(4) analysis of baseline data: description and analysis of baseline data
(including demographic indicators, etc.).
(5) one way ANOVA was used to analyze the differences among groups.

(6) ROC curve was used to analyze the correlation between gene and diagnosis.
when AUC > 0.5, the closer AUC is to 1, the better the diagnosis effect.The accuracy
of AUC is lower when it is 0.5-0.7, medium when it is 0.7-0.9 and higher when it
is above 0.9.When AUC=0.5, the diagnostic method is completely ineffective and has
no diagnostic value.AUC<0.5 is not consistent with the real situation.The risk
factors of disease were analyzed by logistic regression.
(7) Logestic regression analysis is used to analyze the risk factors of PD and
PPS , and the differences among each group.
8 ethical considerations
To ensure that this clinical study complies with the declaration of Helsinki
and relevant Chinese regulations on clinical research.Subjects can be selected for
clinical study only after signing the informed consent.The researchers guaranteed
to maintain the privacy of the subjects.
9 quality control and quality assurance
Xiaoya
Gao, the head of clinical trial, Qing Wang, Chaohao Guan, Xiaobo Wei ,
Cailing Feng and
Xiaomei Liang, the fixed members of the project, carried out the
study strictly
according to the requirements of the clinical trial plan. The
applicant appointed 1-2 supervisors ( Lingling Zhang) to supervise and cooperate
with the work of the experimental unit at any time. The person in charge of the project
checked the case record at any time and solved the possible problems in time.
10 organization and Implementation (time schedule)
Two years of clinical trials, statistical processing of clinical trial data and
writing and publishing papers for about one year.
11 responsibilities of all parties involved
Xiaoya Gao, deputy chief physician: project leader, responsible for the design
and development of clinical trial implementation plan and clinical evaluation.
Qing Wang, Chief Physician: CO researcher, participated in the design of
clinical research program.
Chaohao Guan: Assistant Researcher, responsible for sample collection, data
collection and statistics.
Xiaobo Wei: Assistant Researcher, in charge of sample testing, etc.
Cailing Feng postgraduate: Assistant Researcher, responsible for sample
testing and other work.
Xiaomei Liang, postgraduate: Assistant Researcher, in charge of sample testing
and other work.

12 references
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