Possible Association of PER2/PER3 Variable Number Tandem Repeat Polymorphism Variants with Susceptibility and Clinical Characteristics in Pancreatic Cancer

Abstract

Objective:

Pancreatic cancer (PC) is a serious disease with poor outcomes, and its prevalence has been increasing steadily. The circadian rhythm (CR) is involved in multiple physiological events and maintains homeostasis. Alterations in the CR elevate the risk of developing cancer. The present case-control research was carried out to estimate the possible association between PERIOD2/PERIOD3 (PER2/PER3) gene variable number tandem repeat polymorphism (VNTR) variants and PC in the Turkish population.

Materials and Methods:

A total of 198 subjects (78 patients with PC and 120 healthy controls) were enrolled in this work. Genomic DNA was collected from peripheral blood mononuclear cells, and genotypic analyses was performed using a polymerase chain reaction (PCR) method. Odds ratio (OR) with a 95% confidence interval (95% CI) was calculated using the χ² test.

Results:

The frequency of the 4R (4 repeats)/3R (3 repeats), 3R/3R genotypes, and 3R allele of PER2 VNTR in patients with PC was significantly higher than in the control group (p = 0003, p = 0.00004, respectively). PER2 VNTR 4/5 genotype was related to perineural invasion (p = 0.040). The genotype and allele distribution of PER3 VNTR variant did not show any statistical difference between the two groups (p > 0.05). The PER2/PER3 VNTR 4/5-4R/3R combined genotype was increased in the patient group (p = 0.013), while 4/5-4R/4R combined genotype was increased in the control group (p = 0.0001).

Conclusions:

Our work has indicated that PER2 VNTR 3R allele may play a crucial role in the pathogenesis of PC in Turkish patients, which may become a useful marker for predicting the development of PC. Furthermore, the PER2 VNTR genotype seems to be related to perineural invasion in PC.

Introduction

Pancreatic cancer (PC) is a serious disease with poor outcomes, and its prevalence has been increasing steadily. PC is among the major causes of cancer-related mortality, ranking fourth in the United States and fifth in Europe (Sonoyama et al., 2011). Pancreatic adenocarcinoma and its related types account for 90% of all PCs (Feldmann et al., 2007). Although there have been marked advances in this field, PC remains to be one of the most aggressive and therapy-resistant types of malignancy, with the lowest 5-year survival rate of all types of malignancies (Jemal et al., 2009).

The poor outcome is mainly due to the delayed recognition of the disease along with the high invasiveness and drug resistance of the tumor. The current advancements in the investigation of the molecular and genetic changes in PC have not yet been reflected into significant improvement in patient survival or reduced mortality. There is a crucial need for new treatment targets and molecular markers in PC management to attain higher treatment efficiency. The cause of sporadic PC remains unclear. However, growing evidence reveals that pancreatic carcinogenesis involves a complex interaction between genetic mutations, epigenetic changes, and environmental risk factors (Bardeesy and DePinho, 2002).

Some biochemical pathways can result in carcinogenesis. Among these circadian rhythms (CRs) in physiological and behavioral processes take place. Numerous works have proved that circadian clock gene deregulation plays a task in the occurrence of cancer and other diseases. Impairment in CRs enhances tumor progression, and restoring its restoration is expected to improve prognosis.

The CR organizers aim clock-controlled genes to produce physiological clock at all essential cellular metabolism levels in both suprachiasmatic nucleus (SCN) neurons and peripheral organs, leading to a molecular rhythmicity that expresses 3-10% of all mRNAs in tissues, that is due to time-dependent communication between the circadian modulators with certain gene promoter sequences, transcription factors, or transcriptional initiation, elongation, and termination complexes, as well as the main factors regulating chromatin remodeling (Duong et al., 2011; Padmanabhan et al., 2012). Clock-controlled genes that were investigated include the essential regulators of cell proliferation, metabolism, senescence, and DNA damage response (Fu and Kettner, 2013).

Both lower expression and polymorphism of the core circadian genes PER1, PER2, and PER3 are often encountered in the human mammillary gland, uterus, prostate, pancreatic, colorectal, and NSCLC, as well as hepatocellular carcinoma, squamous cell carcinoma of the head and neck, glioma, acute myeloid lymphoma, and chronic myeloid lymphoma compared to normal host tissues (Fu and Kettner, 2013).

The circadian system regulates various biological functions during 24 h, including sleep/activity, body temperature, heart rate, blood glucose level, cortisol synthesis, and oxidative stress (Valenzuela et al., 2016). In mammalian animals, this system involves a central clock found in the SCN of the hypothalamus and in a series of peripheral oscillators like the liver, lungs, adrenal glands, fibroblast cells, and other tissues (Valenzuela et al., 2016). Some clock genes, such as PER1, PER2, PER3, CRY1, CRY2, BMAL1, CLOCK, and casein kinase 1 delta/epsilon (CK1D and CK1E), interact in translational-transcriptional autoregulatory feedback loops to produce CRs (Hirayama and Sassone-Corsi, 2005).

Any alteration in the Clock gene regulation can affect any part of the organism, including the cardiovascular, gastrointestinal, endocrine, central nervous systems (CNSs), sleep-wake cycles, and cellular processes that are very important in carcinogenesis (e.g., cell proliferation, DNA damage response, and apoptosis) (Gery and Koeffler, 2010). The PER (period circadian clock) gene family is deemed to be the core group member of the circadian clock genes family, which possesses the PER1/2/3 genes as subgroups. Changes in PER gene manifestation have been implied in the genesis and prognosis of cancer (Zhao et al., 2016).

PER1/2 expression levels have been stated to be considerably suppressed in PC (Relles et al., 2013), gastric (Zhao et al., 2014), colorectal (Karantanos et al., 2013; Momma et al., 2017), prostate (Chu et al., 2008; Cao et al., 2009), breast cancer (Winter et al., 2007; Dai et al., 2011), head and neck squamous cell carcinoma (HNSCC) (Rahman et al., 2019), nonsmall cell lung cancer (NSCLC) (Liu et al., 2014), chronic lymphocytic leukemia (Rana et al., 2014), and melanoma (De Assis et al., 2018). It was reported that PER3 expression is considerably suppressed in PC, colorectal cancer, HNSCC, NSCLC, and hepatocellular carcinoma (Deng and Yang, 2019).

Because CR alteration is a known risk factor for several cancers, we aimed to determine the relationship of PER2/PER3 gene variable number tandem repeat polymorphism (VNTR) variants with development, clinical, and pathological characteristics of PC in a Turkish population using polymerase chain reaction (PCR) method for PER2/PER3 VNTR variant genotyping (Shumay et al., 2012).

Materials and Methods

Study population

The patient group was composed of 78 unrelated patients (49 males and 29 females; mean age (±SD) = 66.8 ± 9.9 years) with a clinical diagnosis of PC recruited from those who were treated and followed up in the Department of Medical Oncology, Education and Research Hospital, Gaziosmanpasa University, Tokat, Turkey. The diagnosis of PC was confirmed by immuno-histo-pathological examination of biopsy or resected tissue specimens as reported by the World Health Organization classification. A total of 120 unrelated healthy subjects (71 male and 49 female; mean age (±SD) = 65.4 ± 8.4 years) were enlisted as control group. Controls were matched with patients by sex and age. All controls were confirmed to be free of any malignancy. All cases and controls were Turkish.

The demographical and clinical characteristics (age, gender, smoking, age at diagnosis, tumor diameter, tumor localization, tumor subtypes, tumor differentiation, number of metastatic lymph nodes, and perineural invasion) of the patients were obtained using questionnaires and medical records. Written informed consent was obtained from all patients and healthy controls. The study protocols were performed in accordance with the principles of the Declaration of Helsinki. The experimental study protocol and process were assessed and approved by the Local Research Ethics Committee of Tokat Gaziosmanpaşa University, registered under the number 18-KAEK-262.

Genotyping

The genomic DNA was isolated from peripheral lymphocytes using DNA Isolation Kit (PureLink Genomic DNA Mini Kit; Invitrogen). The isolated DNA was used for description of the following polymorphic PER2/PER3 genes. PCR method was used for PER2/PER3 VNTR variant genotyping (Shumay et al., 2012). The studied VNTR region chr2: 239 184 578-239 185 009 has been defined by Shumay et al. (2012) and that rs10462023, rs13391269, and rs2304673 single nucleotide polymorphisms (SNPs) have been defined in regions close to this VNTR.

Statistical analysis

Data were analyzed using the Statistical Package for Social Sciences (SPSS) software version 20.0 for Windows (SPSS, Inc., Chicago, IL). Mean and standard deviation were used for the presentation of continuous quantitative variables. Frequencies and percentages were used for categorical data. The PER2/PER3 VNTR overall genotype distribution was compared by chi-square (χ²) test, and the specific genotype and allele distributions were compared using Fisher's exact test. The odds ratios (ORs) and 95% confidence intervals (CIs) were used to determine the relationships between the PER2/PER3 VNTR allelic and genotypic variants and their occurrence in the patients. A p-value smaller than 0.05 was considered statistically significant.

Sample size estimations (Priori) and power analysis (Post hoc)

The sample size was calculated for Chi-square test, which was used to test the main hypothesis of our study. As a result of the sample size analysis conducted using previous study knowledge (Relles et al., 2013), it was found that minimum 150 individuals needed to be involved in the study in order to reveal the significant differences in the groups using 80% power (1-β = 0.80) and α = 0.05 error (95% confidence interval). Post hoc power analysis was performed to determine the power of the study with a type 1 error value of 0.05 for the basic hypotheses that were found to be statistically significant. The G*power (version 3.1.9.7) package was used for sample size estimation and post hoc power analysis.

Results

A total of 78 PC patients and 120 age- and gender-matched healthy controls were genotyped for PER2/PER3 gene variants. Demographic and clinical characteristics of the study participants are shown in Table 1.

Table 1.

Baseline Characteristics of Pancreatic Cancer Patients and Controls

Characteristics	PC patients n = 78	Controls n = 120	P
Gender, male/female, n (%)	49/29 (62.8/37.2)	71/49 (59.2/40.8)	0.657
Age, years	66.85 ± 9.988	65.47 ± 8.419	0.300
Age of diagnosis, years	65.32 ± 10.410
Tumor diameter, mm	40.69 ± 19.122
Tumor localizations, n (%)
Head	58/78 (74.4)
Body	14/78 (17.9)
Tail	6/78 (7.7)
Tumor subtype, n (%)
Ductal Adenocarcinoma	67/78 (85.9)
Mucinous Adenocarcinoma	6/78 (7.7)
IPMN	3/78 (3.8)
Neuroendocrine	1/78 (1.3)
Other	1/78 (1.3)
Tumor differentiation, n (%)
Good differentiation	7/29 (24.1)
Medium differentiation	17/29 (58.6)
Low differentiation	5/29 (17.2)
Lymph node metastasis, Yes/No, n (%)	51/12 (81.0/19.0)
Perineural invasion, Yes/No, n (%)	24/18 (57.1/42.9)
Smoking, Yes/No, n (%)	20/58 (25.6/74.4)

Data were analyzed by analysis of variance and χ² test. Mean plus standard deviation values are presented for age, age of diagnosis, and tumor diameter.

PC, pancreatic cancer; IPMN, intraductal papillary mucinous neoplasm.

The distribution of genotypes and allele frequencies of PER2/PER3 VNTR variants in the patient and control groups are shown in Table 2 and Table 3. There was a significant difference in genotype and allele frequencies of PER2 VNTR variant between patients and control subjects. The power for genotypes was 96.8%, and power for alleles was 98.7%. PER2 VNTR 4R (4 repeats)/3R (3 repeats) and 3R/3R genotype were more common in patient group (p = 0003). In addition, 3R allele increased in patient group (p = 0.00004). The genotype and allele distribution of PER3 VNTR variant did not show any statistical difference between patients and controls (p > 0.05).

Table 2.

Genotype and Allele Frequencies of PER2 Variable Number Tandem Repeat Polymorphism Variant in Pancreatic Cancer Patients and Controls

PER2 VNTR	PC patients n = 78 (%)	Controls n = 120 (%)	p	Power
Genotypes
4R/4R	49 (62.8)	102 (85.0)	0.0003	96.8%
4R/3R	21 (26.9)	15 (12.5)
3R/3R	8 (10.3)	1 (0.8)
3R/2R	—	2 (1.7)
Alleles
4R	119 (76.3)	219 (91.3)	0.00004	98.7%
3R	37 (23.7)	19 (7.9)
2R	—	2 (0.8)

Data were analyzed by χ². The results that are statistically significant are typed in bold.

PER2, period circadian clock 2; VNTR, variable number tandem repeat polymorphism.

Table 3.

Genotype and Allele Frequencies of PER3 Variable Number Tandem Repeat Polymorphism Variant in Pancreatic Cancer Patients and Controls

PER3 VNTR	PC patients n = 78 (%)	Controls n = 120 (%)	p	OR (CI 95%)
Genotypes
4/4	32 (41.0)	36 (30.0)	0.100
4/5	34 (43.6)	71 (59.2)
5/5	12 (15.4)	13 (10.8)
4/4: 4/5 + 5/5	32 (41.0): 46 (59.0)	36 (30.0): 84 (70.0)	0.110	0.62 (0.34-1.12)
4/4 + 4/5: 5/5	66 (84.6): 12 (15.4)	107 (89.2): 13 (10.8)	0.346	1.50 (0.64-3.47)
Alleles
4	98 (62.8)	143 (59.6)	0.519	0.87 (0.58-1.32)
5	58 (37.2)	97 (40.4)	0.519	0.87 (0.58-1.32)

Data were analyzed by χ².

PER3, period circadian clock 3.

Furthermore, we also analyzed whether any differences existed in the clinical and pathological features of patients based on the genotype distributions of PER2/PER3 VNTR variants (Table 4). There was a significant difference between the PER2 variant and a clinical feature. PER2 VNTR 4/5 genotype was related with perineural invasion (p = 0.040).

Table 4.

Clinical and Pathological Characteristics of Pancreatic Cancer Patients Stratified According to PER3 and PER2 Variable Number Tandem Repeat Polymorphism Variants

	PER3 VNTR				PER2 VNTR
Characteristics	4/4 (n = 32)	4/5 (n = 34)	5/5 (n = 12)	p	4R/4R (n = 49)	4R/3R (n = 21)	3R/3R (n = 8)	p
Gender, n (%)
Male	21 (65.6)	20 (58.8)	8 (66.7)	0.812	32 (65.3)	11 (52.4)	6 (75.0)	0.445
Female	11 (34.4)	14 (41.2)	4 (33.3)		17 (34.7)	10 (47.6)	2 (25.0)
Age, years	66.19 ± 8.551	65.47 ± 9.765	72.50 ± 12.817	0.098	66.71 ± 10.243	68.43 ± 8.675	63.50 ± 11.940	0.494
Age of diagnosis, years	65.12 ± 8.135	63.41 ± 10.540	71.25 ± 13.752	0.079	65.08 ± 11.020	67.00 ± 8.462	62.37 ± 11.661	0.551
Tumor diameter, mm	40.62 ± 20.275	40.03 ± 17.736	42.75 ± 21.244	0.916	42.04 ± 20.545	36.48 ± 16.324	43.50 ± 17.071	0.493
Tumor localization, n (%)				0.179				0.639
Head	21 (65.6)	29 (85.3)	8 (66.7)		34 (69.4)	18 (85.7)	6 (75.0)
Body	7 (21.9)	5 (14.7)	2 (16.7)		11 (22.4)	2 (9.5)	1 (12.5)
Tail	4 (12.5)	—	2 (16.7)		4 (8.2)	1 (4.8)	1 (12.5)
Tumor subtype, n (%)
Ductal adenocarcinoma	30 (93.8)	26 (76.5)	11 (91.7)	0.157	40 (81.6)	20 (95.2)	7 (87.5)	0.877
Mucinous adenocarcinoma	—	6 (17.6)	—		4 (8.2)	1 (4.8)	1 (12.5)
IPMN	1 (3.1)	1 (2.9)	1 (8.3)		3 (6.1)	—	—
Neuroendocrine	—	1 (2.9)	—		1 (2.0)	—	—
Other	1 (3.1)	—	—		1 (2.0)	—	—
Tumor differentiation, n (%)
Good differentiation	3 (37.5)	2 (12.5)	2 (40.0)	0.385	5 (23.8)	2 (28.6)	—	0.586
Medium differentiation	3 (37.5)	12 (75.0)	2 (40.0)		11 (52.4)	5 (71.4)	1 (100.0)
Low differentiation	2 (25.0)	2 (12.5)	1 (20.0)		5 (23.8)	—	—
Lymph node metastasis, n (%)
Yes	18 (85.7)	25 (83.3)	8 (66.7)	0.367	36 (81.8)	13 (76.5)	2 (100.0)	0.700
No	3 (14.3)	5 (16.7)	4 (33.3)		8 (18.2)	4 (23.5)	—
Perineural invasion, n (%)
Yes	6 (35.3)	14 (77.8)	4 (57.1)	0.040	16 (64.0)	7 (63.6)	1 (16.7)	0.096
No	11 (64.7)	4 (22.2)	3 (42.9)	(Power 61.7%)	9 (36.0)	4 (36.4)	5 (83.3)
Smoking, n (%)
Yes	10 (31.2)	8 (23.5)	2 (16.7)	0.573	14 (28.6)	4 (19.0)	2 (25.0)	0.704
No	22 (68.8)	26 (36.5)	10 (83.3)		35 (71.4)	17 (81.0)	6 (75.0)

Data were analyzed by Anova or χ² tests.

The results that are statistically significant are shown in boldface.

PC, pancreatic cancer; PER2, period circadian clock 2; PER3, period circadian clock 3.

In addition, we studied the risk related to the combined genotypes for the PER2 and PER3 VNTR variants (Table 5). Our results also revealed a significant difference between the combined genotypes in the patient and control groups. PER3/PER2 VNTR 4/5-4R/4R combined genotype was higher in the control group (p = 0.0001). 4/5-4R/3R combined genotype increased in the patient group (p = 0.013).

Table 5.

Comparative Analysis of Combined Genotypes of Patients and Controls

	Patients (n = 78)		Controls (n = 120)		p	Power
Genotypes	n	%	N	%	p	Power
PER3-PER2 VNTR
4/4-4R/4R		28.2	29	24.2	0.525
4/4-4R/3R	5	6.4	6	5.0	0.900
4/4-3R/3R	5	6.4	1	0.8	0.071
4/5-4R/4R	19	24.4	62	51.7	0.0001	96.8%
4/5-4R/3R	12	15.4	6	5.0	0.013	70.0%
4/5-3R/3R	3	3.8	—	—	—
4/5-3R/2R	—	—	3	2.5	—
5/5-4R/4R	8	10.3	10	8.3	0.646
5/5-4R/3R	4	5.1	3	2.5	0.550

Data were analyzed by χ² or fisher's exact tests. The results that are statistically significant are typed in bold.

Discussion

In this article, we investigated whether the PER2 VNTR (in intron 3) and PER3 VNTR variants are related to the risk of PC and evaluated its relationship with clinical and pathological findings. In most trials studying the task of the molecular clock in human cancers, disturbance or variations of several or all whole circadian genes are encountered. The PER2 gene belongs to the period family type of genes containing PER1, PER2, and PER3 and is mostly found in the CNS, including the SCN and the peripheral nervous systems. The PER2 is localized on chromosome 2 at 2q37.3 (Toh et al., 2001). The PER2 gene is vital in controlling the CR and plays a crucial function in tumor suppression (Oda et al., 2009).

PER2 mutant mice exhibited a shorter circadian period compared to wild type (WT) mice and diminished PER1 expression in the SCN, implying that PER2 controls PER1 (Kim et al., 2018). Downregulation of PER2 enhances β-catenin protein levels and its target cyclin D, resulting in cell proliferation in colon cancer cell lines and intestinal and colonic polyp development. This indicates that the PER2 gene product suppresses tumorigenesis in the small intestine and colon by downregulation of β-catenin and β-catenin target gene signaling pathways (Wood et al., 2009). Other article stated that overexpression of mPER2 in mouse tumor cells caused apoptotic cell death and the suppression of tumor growth (Hua et al., 2006). Relles et al. (2013) found that expression levels of PER1, PER2, PER3, Cry1, Cry2, Tipin, Tim, CK1e, Bmal-ARNTL, and Clock genes were reduced in the pancreatic ductal adenocarcinoma tissue in comparison with their matched nearby tissue.

The PER2 gene can turn on the c-Myc signaling pathways producing a genomic imbalance and cell growth. PER2 malfunction can also disrupt p53-mediated apoptosis and cause genomic instability and the accumulation of deficient cells (Benna et al., 2018). The PER2 locus has elevated levels of interpopulation genetic differentiation relative to other loci, indicating a task for geographically limited positive selection (Cruciani et al., 2008).

The SNPs in the PER2 gene have been linked with aberrant circadian parameters, chronotypes, depression, and also with increased alcohol intake; however, no correlation with cocaine dependence was encountered in a study that investigated some PER2 SNPs (Shumay et al., 2012). It was declared that PER2 rs934945 variant T allele was associated with reduced predisposition to soft tissue sarcoma (Benna et al., 2018). Although these VNTRs are often classified and neglected as “junk” DNA, the number of repetitions of the sequence can differ within and between individuals, thus making these VNTRs a polymorphic entity.

The PER3 VNTR (rs57875989) consists of four or five copies of a 54-bp sequence encoding 18 amino acids (Nadkarni et al., 2005). The 5-repeat variant supplements some potential phosphorylation motifs to the gene, and PER3's that interoperate with circadian activity could be increased in those people (Wirth et al., 2013). The 5-repeat PER3 allele is linked with a rather penetrant phenotype that has morning circadian preference, overt cognitive decline as a reaction to sleep deficiency, discrepancies in rate or schedule of melatonin or cortisol secretion, and a propensity for depressive manifestations or an earlier commencement of bipolar disorder (ALExANDER et al., 2015).

In a study, it was found that the PER3 5/5 variant carriers were related to increased amount of interleukin-6, B-type natriuretic peptide, and lower vitamin A levels (Lipkova et al., 2014b). Golalipour et al. (2017) reported that PER3 4/4 genotype and 4-repeat allele were connected with multiple sclerosis. No significant difference was observed in allele periodicities of PER3 VNTR between the obese and the nonobese individuals (Bienertova-Vasku et al., 2014). In addition, there was no significant difference between chronic heart failure patients and controls regarding PER3 VNTR allele and genotype frequencies (Lipkova et al., 2014a).

Geng et al. (2015) found a moderate increase in risk of cancer among individuals with the PER3 VNTR 5 allele compared to individuals with the 4 allele in a meta-analysis. Alexander et al. (2015) indicated that the individuals carrying the 5-repeat PER3 VNTR variant may be more susceptible to colorectal adenoma formation. Yeğin et al. (2020) reported that PER3 VNTR variant was not associated with bladder carcinoma. But they found that the survival times of patients decreased in the patient group (progression and cystectomy positive) for PER3 4/4 genotype and (recurrence, progression, and cystectomy positive) for PER3 4/5 genotype.

In this study, the statistical analyses showed that genotype and allele distribution of PER2 were statistically significantly different between the control and patient groups. PER2 VNTR 4R/3R, 3R/3R genotype and 3R allele increased in patient group (p < 0.05) (Table 2). So PER2 VNTR 3R allele seems to be a risk factor in PC formation. Our study was unable to show any significant association of the PER3 VNTR genotype and allele distribution with PC (p > 0.05) (Table 3). Then, we also analyzed if any differences existed in the demographical and clinical characteristics of patients according to allele frequencies. We found that PER2 VNTR 4/5 genotype increased in patients with perineural invasion (p < 0.05) (Table 4). In PER3/PER2 combined analysis, 4/5-4R/3R combined genotype was more prevalent in patient group (p < 0.05) (Table 5).

There are some limitations of this study. First, we investigated only two variants involved in the pathway of PER2/PER3; other regulatory genes in the clock gene family signaling pathway may also play a role in the genesis of PC. Second, even though the PER3 VNTR variant that we examined was not found to be associated with PC, other variants of PER2/PER3 gene have not been examined yet. Third, owing to the rather small sample size, the frequencies of some homozygous variants were low in groups and consequently diminished the statistical power. Finally, absence of assessment of expression levels of PER2/PER3 is also a limitation of this study.

Conclusion

In conclusion, our work has indicated that PER2 VNTR 3R allele may play a significant task in the development of PC in Turkish patients, which may become a useful marker for predicting the development of PC. Furthermore, the PER2 VNTR genotype seems to be associated with perineural invasion in PC. These findings imply that continued research into clock gene variants will be an important source of information in understanding the pathogenesis of PC. Further multicenter, well-designed studies with larger populations that evaluate gene-environment interactions are necessary to endorse our results.

Data Availability Statement

The data sets generated during the current study are available from the corresponding author upon request.

Statement of Licensing Committee and Institution

Ethics committee approval for this study was obtained from the Local Research Ethics Committee registered under the number 18-KAEK-262. All methods were performed in accordance with the relevant guidelines and regulations of the institution. Written informed consent was obtained from subjects and patients who participated in this study.

Footnotes

Authors' Contribution

H.D.: Conceptualization, Software, Validation, Formal analysis, Data Curation, Writing—Review & Editing, Funding acquisition.

S.Y.: Methodology, Software, Validation, Formal analysis, Writing—Review & Editing, Visualization, Supervision.

A.F.N.: Methodology, Software, Validation, Resources, Writing—Original Draft, Supervision, Project administration.

E.D.: Validation, Formal analysis, Data Curation, Writing—Review & Editing, Supervision.

O.G.: Conceptualization, Validation, Formal analysis, Resources, Data Curation, Writing—Review & Editing, Project administration.

Author Disclosure Statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article. The author confirms on behalf of the other authors that there is no conflict of interest to declare.

Funding Information

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References

Alexander

, Burch

, Steck

, et al. (2015) Case-control study of the PERIOD3 clock gene length polymorphism and colorectal adenoma formation. Oncol Rep, 33:935-941.

Bardeesy

, DePinho

(2002) Pancreatic cancer biology and genetics. Nat Rev Cancer, 2:897-909.

Benna

, Rajendran

, Spiro

, et al. (2018) Associations of clock genes polymorphisms with soft tissue sarcoma susceptibility and prognosis. J Transl Med, 16:338.

Bienertova-Vasku

, Novak

, Zlámal

, et al. (2014) The PER3 VNTR polymorphism is a predictor of dietary composition in the Central European population. Biol Rhythm Res, 45:747-757.

Cao

, Gery

, Dashti

, et al. (2009) A role for the clock gene per1 in prostate cancer. Cancer Res, 69:7619-7625.

Chu

, Zhu

, Yu

, et al. (2008) Variants in circadian genes and prostate cancer risk: a population-based study in China. Prostate Cancer Prostatic Dis, 11:342-348.

Cruciani

, Trombetta

, Labuda

, et al. (2008) Genetic diversity patterns at the human clock gene period 2 are suggestive of population-specific positive selection. Eur J Hum Genet, 16:1526-1534.

Dai

, Zhang

, Cao

, et al. (2011) The role of polymorphisms in circadian pathway genes in breast tumorigenesis. Breast Cancer Res Treat, 127:531-540.

De Assis

LVM

, Moraes

, Magalhães-Marques

, et al. (2018) Non-metastatic cutaneous melanoma induces chronodisruption in central and peripheral circadian clocks. Int J Mol Sci, 19:1065.

10.

Deng

, Yang

(2019) Current status of research on the period family of clock genes in the occurrence and development of cancer. J Cancer, 10:1117.

11.

Duong

, Robles

, Knutti

, Weitz

(2011) A molecular mechanism for circadian clock negative feedback. Science, 332:1436-1439.

12.

Feldmann

, Beaty

, Hruban

, Maitra

(2007) Molecular genetics of pancreatic intraepithelial neoplasia. J Hepatobiliary Pancreat Sug, 14:224-232.

13.

, Kettner

(2013) The circadian clock in cancer development and therapy. Prog Mol Biol Transl Sci, 119:221-282.

14.

Geng

, Ou

, Li

, et al. (2015) Genetic association between PER3 genetic polymorphisms and cancer susceptibility: a meta-analysis. Medicine (Baltimore), 94:e568.

15.

Gery

, Koeffler

(2010) Circadian rhythms and cancer. Cell Cycle, 9:097-1103.

16.

Golalipour

, Maleki

, Farazmandfar

, Shahbazi

(2017) PER3 VNTR polymorphism in Multiple Sclerosis: a new insight to impact of sleep disturbances in MS. Mult Scler Relat Disorders, 17:84-86.

17.

Hirayama

, Sassone-Corsi

(2005) Structural and functional features of transcription factors controlling the circadian clock. Curr Opin Genet Dev, 15:548-556.

18.

Hua

, Wang

, Wan

, et al. (2006) Circadian gene mPer2 overexpression induces cancer cell apoptosis. Cancer Sci, 97:589-596.

19.

Jemal

, Siegel

, Ward

, et al. (2009) Cancer statistics, 2009. CA Cancer J Clin, 59:225-249.

20.

Karantanos

, Theodoropoulos

, Gazouli

, et al. (2013) Association of the clock genes polymorphisms with colorectal cancer susceptibility. J Surg Oncol, 108:563-567.

21.

Kim

, De La Pena

, Cheong

, Kim

(2018) Neurobiological functions of the period circadian clock 2 gene, Per2. Biomol Ther, 26:358.

22.

Lipkova

, Bienertova-Vasku

, Spinarova L

(2014a) Per3 VNTR polymorphism and chronic heart failure. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 158:080-083.

23.

Lipkova

, Splichal

, Bienertova-Vasku

, et al. (2014b) Period3 VNTR polymorphism influences the time-of-day pain onset of acute myocardial infarction with ST elevation. Chronobiol Int, 31:878-890.

24.

Liu

, Xu

, Jiang

, Li

(2014) Aberrant expression of Per1, Per2 and Per3 and their prognostic relevance in non-small cell lung cancer. Int J Clin Exp Pathol, 7:7863.

25.

Momma

, Okayama

, Saitou

, et al. (2017) Expression of circadian clock genes in human colorectal adenoma and carcinoma. Oncol Lett, 14:5319-5325.

26.

Nadkarni

, Weale

, Von Schantz

, Thomas

(2005) Evolution of a length polymorphism in the human PER3 gene, a component of the circadian system. J Biol Rhythms, 20:490-499.

27.

Oda

, Katayose

, Yabuuchi

, et al. (2009) Clock gene mouse period2 overexpression inhibits growth of human pancreatic cancer cells and has synergistic effect with cisplatin. Anticancer Res, 29:1201-1209.

28.

Padmanabhan

, Robles

, Westerling

, Weitz

(2012) Feedback regulation of transcriptional termination by the mammalian circadian clock PERIOD complex. Science, 337:599-602.

29.

Rahman

, Kraljević Pavelić

, Markova-Car

(2019) Circadian (de) regulation in head and neck squamous cell carcinoma. Int J Mol Sci, 20:2662.

30.

Rana

, Munawar

, Shahid

, et al. (2014) Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia. Mol Biol Rep, 41:95-103.

31.

Relles

, Sendecki

, Chipitsyna

, et al. (2013) Circadian gene expression and clinicopathologic correlates in pancreatic cancer. J Gastrointest Surg, 17:443-450.

32.

Shumay

, Fowler

, Wang

, et al. (2012) Repeat variation in the human PER2 gene as a new genetic marker associated with cocaine addiction and brain dopamine D2 receptor availability. Transl Psychiatry, 2:e86.

33.

Sonoyama

, Sakai

, Mita

, et al. (2011) TP53 codon 72 polymorphism is associated with pancreatic cancer risk in males, smokers and drinkers. Mol Med Rep, 4:489-495.

34.

Toh

, Jones

, He

, et al. (2001) An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome. Science, 291:1040-1043.

35.

Valenzuela

, Vera

, Venegas

, et al. (2016) Evidences of polymorphism associated with circadian system and risk of pathologies: a review of the literature. Int J Endocrinol, 2016:2746909.

36.

Winter

, Bosnoyan-Collins

, Pinnaduwage

, Andrulis

(2007) Expression of the circadian clock genes pert, per2 in sporadic, familial breast tumors. Neoplasia, 9:797-800.

37.

Wirth

, Burch

, Violanti

, et al. (2013) Association of the Period3 clock gene length polymorphism with salivary cortisol secretion among police officers. Neuro Endocrinol Lett, 34:27-37.

38.

Wood

, Yang

, Hrushesky

(2009) Clock genes and cancer. Integr Cancer Ther, 8:303-308.

39.

Yeğin

, Özen

, Altinisik

, et al. (2020) PER3 VNTR genotypes may predict overall survival in bladder cancer patients in the Turkish population. Mugla J Sci Technol, 6:120-135.

40.

Zhao

, Zeng

, Yang

, et al. (2014) Prognostic relevance of Period1 (Per1) and Period2 (Per2) expression in human gastric cancer. Int J Clin Exp Pathol, 7:619-630.

41.

Zhao

, Zheng

, Yang

, et al. (2016) The clock gene PER1 plays an important role in regulating the clock gene network in human oral squamous cell carcinoma cells. Oncotarget, 7:70290-70302.