The thyrotroph embryonic factor gene is a circadian
clock-controlled gene. The rs738499 polymorphism of this gene has
been suggested to be associated with depression and sleep disturbance in
Parkinson’s disease in previous cross-sectional studies. We aimed to investigate
whether this single nucleotide polymorphism is associated with the progression
rates of various motor and non-motor symptoms in patients with Parkinson’s
disease. We recruited 186 patients with Parkinson’s disease for a longitudinal
study. Motor and non-motor symptoms were assessed at baseline and follow-up, and
170 Parkinson’s disease patients completed the clinical evaluation twice with an
average follow-up period of 3.3
Parkinson’s disease (PD) is characterized by progressive loss of dopaminergic neurons in the substantia nigra and Lewy bodies’ formation in the midbrain. These pathological changes result in motor symptoms. Braak’s staging theory  and the presence of multiple non-motor symptoms reflect the complex and multifaceted nature of PD . Non-motor symptoms are often resistant to L-DOPA treatment and will inevitably become a significant factor affecting patients’ function and quality of life in advanced PD stages. Therefore, further exploration of other pathophysiological mechanisms involved in these symptoms holds promise for other effective therapeutic strategies. During the last decade, increasing attention has been paid to the link between circadian rhythms and PD. It has been recognized that the circadian rhythm is disrupted in patients with PD . First, circadian function biomarkers, such as body temperature, blood pressure, and hormone secretion, are dysregulated in PD [4, 5]. Second, patients in advanced stages lose normal motor activity, and actigraphy studies in patients with PD reported flattened diurnal motor activity patterns . Third, patients receiving repeated L-DOPA administration often report a lower response to L-DOPA in the evening . Fourth, common non-motor symptoms of PD, such as sleep-wake disturbances, depression, and visual symptoms, are modulated by circadian rhythms [8, 9].
A large sample cohort study of community-dwelling older men without PD reported that reduced circadian rhythmicity at baseline is associated with an increased risk of developing PD during an 11-year follow-up, suggesting that circadian disruption rhythms might be prodromal to PD . Evidence indicates that the electrical activity of neurons within the master pacemaker of the circadian system, the suprachiasmatic nuclei (SCN), is disrupted at the onset of motor symptoms . Therefore, circadian dysfunction may be a key cause of non-motor symptoms, especially for pre-motor symptoms. Sleep disturbances are common non-motor symptoms reported by 64% of patients with PD , including insomnia, rapid eye movement sleep behavior disorder, restless legs syndrome, sleep-disordered breathing, and excessive daytime sleepiness. Patients often demonstrate sleep alterations during the prodromal phase of PD . It has been hypothesized that circadian and sleep changes may trigger neurodegeneration in the earliest phases of the disease . Therefore, it is important to understand and improve sleep disturbances and circadian rhythm in patients with PD.
Even though there are reciprocal interactions between the dopaminergic system and circadian rhythms, the underlying mechanisms of circadian dysfunction in PD remain elusive. Circadian rhythms are regulated by circadian gene networks consisting of a series of clock genes (such as CLOCK, BMAL1, BMAL2, PER1, PER2, PER3, CRY1, and CRY2) and clock-controlled genes (such as TEF, DEC1, DEC2, and TIMELESS) . Several studies have explored desynchronization at the molecular level. A study examining the expression profile of several principal circadian genes in peripheral leukocytes in whole blood for a 12-hour nighttime period found that the mRNA expression of BMAL1 and BMAL2 was significantly reduced in patients with PD in the evening. Furthermore, the level of BMAL2 correlated with motor severity and sleep quality in the morning [16, 17]. Cai and his colleagues  discovered that genetic polymorphisms in ARNTL and PER1 were significantly associated with PD risk. These reports suggest that circadian rhythm disruption is a result and a cause of the degeneration process. In this case, it would be expected to occur early in the disease’s course (or even preceding disease onset) and exacerbate disease progression. There has been no longitudinal study focusing on the effect of circadian genes on the progression of PD.
The thyrotroph embryonic factor (TEF) gene is one of the downstream genes in the circadian gene network and expresses a broad range of cells and tissues. It encodes a thyrotroph embryonic factor, together with albumin D box-binding protein (DBP), human hepatic leukemia factor (HLF) are three members of the proline and acidic amino-acid-rich basic leucine zipper (PAR bZip) transcription factor family. Different members of the subfamily can readily form heterodimers and share DNA-binding and transcriptional regulatory properties. The thyrotroph embryonic factor can bind to proline and acidic amino-acid-rich response elements (PARRE) in promoters and elevate the transcription levels of light-induced circadian genes NR1D1, NR1D2, CRY1a, CRY2b, and PER2 . The TEF rs738499 polymorphism has been suggested to be associated with depression and sleep disturbance in PD in previous cross-sectional studies [20, 21]. The present study was conducted longitudinally in a cohort of patients with PD to investigate whether TEF is associated with worsening PD patients’ clinical symptoms with an average follow-up period of 3.3 years.
For this longitudinal study, the patients should satisfy the following criteria:
(1) younger than 80 years, (2) Hoehn-Yahr stage (H-Y stage)
To assess disease progression, all patients underwent clinical evaluation at baseline and during each follow-up to confirm the diagnosis and assess disease progression. Motor disability was evaluated using the Unified Parkinson’s Disease Rating Scale  containing Part-III (UPDRS-III), Part-II - Activities of Daily Living (ADL), and Part-V-H-Y stage. We calculated four composite motor scores from UPDRS-III : tremor (items 20 and 21), rigidity (item 22), akinesia (items 19, 23-26, and 31), and axial symptoms (items 18 and 27-30). The non-motor symptoms were assessed using the Mini-Mental State Examination (MMSE) , 24-item Hamilton Rating Scale for Depression (HAMD) , 14-item Hamilton rating scale for anxiety (HAMA) , Parkinson’s Disease Sleep Scale (PDSS) , and Parkinson’s Disease Non-Motor Symptom Quest (NMSQ) . All these assessments were carried out during an ‘on’ period. Dopaminergic medication and levodopa equivalent daily doses (LEDD)  were recorded at every visit. We successfully re-examined 170 patients during their follow-up visits between June 2012 and October 2014. The remaining 16 patients dropped out before the follow-up (four patients withdrew, six could not be contacted, two were too ill, and four patients were deceased).
DNA samples were extracted from citric acid-anticoagulated peripheral blood
specimens using DNA extraction kits (Tiangen Biotech, Beijing, P. R. China)
according to the manufacturer’s recommendations. DNA samples were randomly
distributed into 96-well plates and sent to CapitalBio Corporation (Beijing,
China) for genotyping. Laboratory personnel was blinded to the subject status and
our study objective. The iPLEX Gold SNP genotyping kit, software, and
experimental equipment used by the laboratory were provided by the MassARRAY
platform (Sequenom, San Diego, CA). In brief, an oligonucleotide primer annealed
upstream to the single nucleotide polymorphism (SNP) site was genotyped to
accomplish a locus-specific primer extension reaction. A 1
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS Version 18.0, IBM). The Hardy-Weinberg equilibrium of SNPs was calculated with the SHEsis program . Baseline information between different genotypes (TT vs. TG + GG) was compared using the Mann-Whitney U test for continuous data and a chi-square test for categorical data. Changes between assessments at baseline and follow-up were compared using the Wilcoxon test. Each PD symptom’s progression was defined by the annual change in the scale score and described by median (min-max).
Meanwhile, the annual progression rates of clinical symptoms were compared
between genotypes using the Mann-Whitney U test. The correlations between
genotypes, the baseline clinical variables, and the annual progress ratio of each
scale were analyzed by Spearman’s rank correlation and variables with P
The distribution of genotypes was fitted with the
Hardy-Weinberg equilibrium in the PD sample. The mean age of patients with PD was
|Characteristic||TT||TG + GG||Overall PD|
|(n = 102)||(n = 68)||P-value||(n = 170)||P-value|
|Gender, % male||59.8||57.4||0.750||58.8||–||–|
|Age at onset, years||59.3
|Age at interview, years||69.2
|Years of follow-up||3.3
|Years of education||10.4
|H-Y stage||2.0 (1.0-4.0)||2.0 (1.0-3.0)||0.144||2.0 (1.0-4.0)||2.5 (1.0-4.0)||0.000|
|Values are expressed as mean |
ADL, Activities of Daily Living; APR, annual progression rate; HAMA, 14-item Hamilton Rating Scale for Anxiety; HAMD, 24-item Hamilton rating scale for depression; H-Y stage, Hoehn-Yahr stage; LEDD, levodopa equivalent daily doses; MMSE, Mini-Mental State Examination; n, number of samples; NMSQ, Parkinson’s Disease Non-Motor Symptom Questionnaire; PD, Parkinson’s disease; PDSS, Parkinson’s Disease Sleep Scale; UPDRS-III, Unified Parkinson’s Disease Rating Scale Part-III.
|Annual Progression Rates||Overall PD||TT||TG + GG||Z||P-value|
|(n = 170)||(n = 102)||(n = 68)|
|LEDD, mg||26.1 (-151.6
|H-Y stage||0.1 (-0.9
|Axial symptoms||0.4 (-2.8
|Values are expressed as the median (min-max).
ADL, Activities of Daily Living; APR, annual progression rate; HAMA, 14-item Hamilton Rating Scale for Anxiety; HAMD, 24-item Hamilton rating scale for depression; H-Y stage, Hoehn-Yahr stage; LEDD, levodopa equivalent daily doses; MMSE, Mini-Mental State Examination; n, number of samples; NMSQ, Parkinson’s Disease Non-Motor Symptom Questionnaire; PD, Parkinson’s disease; PDSS, Parkinson’s Disease Sleep Scale; UPDRS-III Unified, Parkinson’s Disease Rating Scale Part-III; Z, Mann-Whitney U-test statistic.
Spearman’s rank correlation showed that the annual decline rate of the PDSS
score was associated with the PDSS score at baseline, the annual progression
rates of HAMA and NMSQ during follow-up, and the TT genotype of TEF rs738499. The H-Y stage’s progression rate was associated with the H-Y stage,
tremor score, rigidity score at baseline, and the annual progression rates of
HAMD, HAMA, NMSQ, ADL, MMSE, tremor, rigidity, akinesia, axial symptoms, and the
TT genotype of TEF rs738499. Table 3 displays all
related factors with P
|Variables||Annual Decline Rate of PDSS||Annual Progression Rate of H-Y stage|
|Years of follow-up||0.056||0.512||-0.107||0.120*|
|APR of LEDD||-0.045||0.666||0.129||0.195*|
|Baseline H-Y stage||-0.047||0.583||-0.567||0.000*|
|APR of tremor||0.007||0.939||0.184||0.017*|
|APR of baseline rigidity||0.024||0.776||0.258||0.001*|
|APR of baseline akinesia||0.016||0.860||0.248||0.001*|
|Baseline axial symptoms||0.156||0.078*||-0.053||0.511|
|APR of axial symptoms||-0.094||0.298||0.386||0.000*|
|APR of ADL||0.097||0.328||0.264||0.005*|
|APR of MMSE||0.070||0.433||0.275||0.001*|
|APR of HAMD||0.153||0.093*||0.208||0.015*|
|APR of HAMA||0.322||0.000*||0.197||0.029*|
|APR of NMSQ||0.245||0.004*||0.199||0.013*|
|ADL, Activities of Daily Living; APR, annual progression rate; HAMA, 14-item Hamilton Rating Scale
for Anxiety; LEDD, levodopa equivalent daily doses; HAMD, 24-item
Hamilton rating scale for depression; H-Y stage, Hoehn-Yahr
stage; MMSE, Mini-Mental State Examination; NMSQ,
Parkinson’s Disease Non-Motor Symptom Questionnaire; PDSS, Parkinson’s Disease Sleep Scale; r, Spearman rank
correlation coefficient; *P |
Stepwise linear regression showed that the progression rate of anxiety, the initial severity of sleep disturbance and axial symptoms, and the TT genotype of TEF rs738499 were independent risk factors affecting the decline rate of PDSS score. These four predictors together could explain the 40.5% variation in the decline rate of PDSS. TEF rs738499 alone accounted for 3.1% of the variance (P = 0.030) (Table 4). Meanwhile, stepwise linear regression revealed that the progression rate of axial symptoms, the TT genotype of TEF rs738499, and the initial severity of anxiety were independent risk factors affecting the H-Y progression rate stage. These three predictors together could explain the 49.1% variation in the progression rate of the H-Y stage. TEF rs738499 alone accounted for 8.3% variance (P = 0.021) (Table 5).
|Annual progression rate of HAMA||0.445||0.189||0.198||0.361||4.366||0.000||1.069|
|Baseline axial symptoms||0.632||0.380||0.092||0.301||3.762||0.000||1.003|
|Beta standardized regression coefficient stands for each
variable’s contribution to the dependent variable; HAMA, 14-item Hamilton rating
scale for Anxiety; PDSS, Parkinson’s Disease Sleep Scale; r, multiple correlation coefficient; r|
|Annual progression rate of axial symptoms||0.619||0.371||0.383||0.651||6.557||0.000||1.008|
|Beta the standardized regression coefficient stands for each
variable’s contribution to the dependent variable; HAMA, 14-item Hamilton rating scale for Anxiety; H-Y stage, Hoehn-Yahr stage; r, multiple correlation coefficient; r|
The present research indicates that the TT genotype in TEF rs738499 correlated with a faster deterioration rate of sleep quality and H-Y stage of PD patients. Moreover, patients with more severe sleep disturbance and axial symptoms at baseline and faster worsening rate of anxiety during the follow-up period presented a faster deterioration rate of sleep quality, and patients with more severe anxiety at baseline and faster worsening rate of the axial symptoms during the follow-up period experienced a quicker progression rate of the H-Y stage.
The sleep-wake cycle is under the dual control of homeostatic and circadian processes. Homeostatic processes are responsible for the sleep rebound following sleep deprivation, and the circadian processes determine the timing of the sleep period via the circadian clock in the SCN. However, little is known about the circadian system’s molecular mechanism in patients’ sleep problems with PD. Several neurotransmitters with circadian rhythmicity, such as dopamine, melatonin, 5-hydroxytryptamine (5-HT), glutamate, and gamma-aminobutyric acid (GABA), have been reported to be implicated in the regulation of sleep-wake behavior [31, 32, 33]. David et al.  found that reduced circulating melatonin levels were associated with reduced slow-wave sleep and reduced rapid eye movement (REM) sleep.
Furthermore, a lower melatonin onset concentration was associated with increased sleep latency in patients with PD. TEF is involved in maintaining neurotransmitter homeostasis (such as 5-HT and dopamine) . We speculate that the association of TEF with the rapid deterioration of sleep quality and H-Y stage might be related to its above-described function, but further studies are needed to provide further evidence.
Our results show that anxiety and axial symptoms were closely related to worse sleep quality and motor staging. The etiology of sleep disturbances in PD is multifactorial and previous studies have shown that anxiety symptoms are a risk factor for sleep quality and motor complications [36, 37]. Axial nocturnal hypokinesia in PD manifests fewer turning-over episodes, turns with smaller degrees, less velocity, and acceleration. Patients with severe nocturnal hypokinesia usually need to take a higher total and bedtime levodopa equivalent dose, and drinking more water at night could result in frequent nocturia that compels the patients to get out of bed, resulting in lower sleep quality [38, 39]. The onset of falls is a warning symptom of H-Y stage aggravation. Spanish researchers  employed an objective technology for rigidity assessment. They reported that axial rigidity was related to the risk of falls in patients with PD. Axial symptoms contribute to the postural instability and gait difficulty (PIGD) subtype of PD. The PROPARK study  reported that PIGD-dominant patients had more severe motor and non-motor symptoms, a more rapid disease progression, and a poorer prognosis PD compared to the tremor-dominant (TD) patients. Another circadian rhythm-related study  showed that agomelatine, acting as a melatonin receptor (MT1 and MT2) agonist and as a serotonin receptor (5-HT2b and 5-HT2c) antagonist, improves not only depressive symptoms in PD but also improves sleep problems and motor symptoms. This confirmed the intimate internal relations of these three clinical phenotypes. Therefore, much closer relationships between sleep and mood disorders and motor symptoms may have a specific impact on the genotypic association.
The longitudinal follow-up is the main strength of this study, and a comprehensive follow-up evaluation of multiple non-motor symptoms of PD was carried out. There are several limitations to this study. First, the sample size was small, and no control group to perform gene-disease association analysis was included. We genotyped the TEF rs738499 polymorphism in 480 PD patients and 500 controls in a previous study. We found that the T allele and TT genotype were significantly higher in patients with PD, but this result was not shown in that study . Second, the participants were not assessed during the same time of the day or the same season for baseline, and follow-up visits as both factors will influence motor and non-motor scores. Therefore, case-control studies with a larger sample size and more strict control of various factors affecting circadian rhythm are needed to explore the role of circadian rhythm in different clinical stages of PD.
This longitudinal study aimed to determine the association between a circadian gene’s polymorphism and PD symptoms progression. It also revealed that TEF might be considered a predictive marker of the progression of sleep disturbances and motor staging in PD. This advances our understanding of the genetic associations in PD and suggests circadian dysfunction may be involved in Parkinson’s disease’s pathophysiology.
ADL, Activities of Daily Living; APR, annual progression rate; ARNTL, Aryl Hydrocarbon Receptor Nuclear Translocator Like (another name of BMAL1 gene); BMAL, brain and muscle ARNT-like protein; CLOCK, circadian locomotor output cycles kaput; CRY, cryptochrome; DEC, Differentiated embryonic chondrocyte; HAMA, 14-item Hamilton rating scale for Anxiety; HAMD, 24-item Hamilton rating scale for depression; H-Y, stage Hoehn-Yahr stage; LEDD, levodopa equivalent daily doses; MMSE, Mini-Mental State Examination; NMSQ, Parkinson’s Disease Non-Motor Symptom Quest; NPAS2, neuronal PAS domain protein 2; NR1D1, Nuclear Receptor Subfamily 1 Group D Member 1; PD, Parkinson’s disease; PDSS, Parkinson’s Disease Seep Scale; PER, period; TEF, Thyrotroph embryonic factor; TIMELESS, Timeless Circadian Regulator; UPDRS-III, Unified Parkinson’s Disease Rating Scale Part-III.
Study concept and design: Hua and Liu; Acquisition of data: Liu, Xu, Yu, Yao and Cui; Analysis and interpretation of data: Hua and Cui; Drafting of the manuscript: Hua; Critical revision of the manuscript for important intellectual content: Liu; Obtained funding: Liu; Administrative, technical, and material support: Chen.
The study was approved by the Ethics Committee of Nanjing Medical University. Informed consent was obtained from each participant.
The authors thank for Sheng-Han Kuo’s guidance in manuscript writing, and thank all the patients participated in this study.
The study was supported by the National Natural Science (81571348), Jiangsu Natural Science Foundation (BK20151077), National key research and development plan (2016YFC1306600, 2017YFC1310302).
The authors declare no competing interests.