Analysis of Genetic Variation in Circadian Rhythm Genes and Risk of Ovarian Cancer.

Background Disruption in biological clock due to genetic variations is associated with increased occurrence of cancers such as breast, ovary, prostate, gastrointestinal and hematological malignancies. Circadian rhythm genes regulate the process of ovulation in the ovaries and are highly expressed in ovarian tumors; whereas disturbance in the circadian rhythm pathway is signicantly associated with causative risk factors (i.e. endometriosis, PCOS, etc.) of ovarian cancer. Nevertheless, very few studies have been conducted till date where candidate SNPs of circadian rhythm genes proved as the main prognosticators of ovarian cancer risk and intrusiveness. The main purpose of this study was to investigate some common single nucleotide polymorphisms (SNPs) in circadian rhythm genes (rs475715 of BMAL1/ARNTL, rs1026071, and rs228644 of PER3, rs3792152 of REV1, and rs7302060 of TIMELESS) as causative markers of ovarian cancer risk of in the population of Jammu and Kashmir in India. Our study included a total of 600 samples (200 cases and 400 age and sex-matched controls). Analysis of the genotype data from the selected SNPs indicated most signicant association of rs3792152 of REV1 (OR=1.6, with 95% CI=0.12-1.2, p=0.0003) and rs4757151 of BMAL1/ARNTL (OR=1.847, with 95% CI=1.406-2.426, p=9.15E-06) with the ovarian cancer. The functional putative analysis revealed a signicant regulatory effect of both these variants on other genes. These results suggest that some SNPs in circadian rhythm genes, particularly BMAL1/ARNTL and REV1, might be associated with the risk of ovarian cancer in the J&K population of North India.


Abstract
Background Disruption in biological clock due to genetic variations is associated with increased occurrence of cancers such as breast, ovary, prostate, gastrointestinal and hematological malignancies. Circadian rhythm genes regulate the process of ovulation in the ovaries and are highly expressed in ovarian tumors; whereas disturbance in the circadian rhythm pathway is signi cantly associated with causative risk factors (i.e. endometriosis, PCOS, etc.) of ovarian cancer. Nevertheless, very few studies have been conducted till date where candidate SNPs of circadian rhythm genes proved as the main prognosticators of ovarian cancer risk and intrusiveness. The main purpose of this study was to investigate some common single nucleotide polymorphisms (SNPs) in circadian rhythm genes (rs475715 of BMAL1/ARNTL, rs1026071, and rs228644 of PER3, rs3792152 of REV1, and rs7302060 of TIMELESS) as causative markers of ovarian cancer risk of in the population of Jammu and Kashmir in India.

Results
Our study included a total of 600 samples (200 cases and 400 age and sex-matched controls). Analysis of the genotype data from the selected SNPs indicated most signi cant association of rs3792152 of REV1 (OR=1.6, with 95% CI=0.12-1.2, p=0.0003) and rs4757151 of BMAL1/ARNTL (OR=1.847, with 95% CI=1.406-2.426, p=9.15E-06) with the ovarian cancer. The functional putative analysis revealed a signi cant regulatory effect of both these variants on other genes.

Conclusion
These results suggest that some SNPs in circadian rhythm genes, particularly BMAL1/ARNTL and REV1, might be associated with the risk of ovarian cancer in the J&K population of North India.

Background
Ovarian cancer (OC) is one of the most common cancer globally, with more than 239,000 newly diagnosed cases and 152,000 deaths each year [1]. OC ranks 3rd in gynecological malignancies after cervix and uterine cancer [2]. OC has the worst prognosis and the peak death rate. Even though ovarian cancer has a lesser pervasiveness in assessment with breast cancer, it is three times more fatal, and it is foretold that, by the year 2040 [3], the death rate of OC will increase signi cantly.
The high death rate in OC patients is mainly due to, asymptomatic and undisclosed progression of the ovarian tumor, deferred beginning of signs and symptoms, and the absence of suitable screening that affects diagnosis of the progressive stages [3]. Despite countless developments in early diagnosis and treatment, OC survival has shown only borderline increase due to the intricacy and heterogeneity of molecular pathways involved, speci cally in invasion, relapse of ovaries, and metastasis. Hence, it is crucial to investigate biomarkers to clarify the molecular processes for enlightening the diagnosis of OC. In women, the clock genes under the in uence of hormones regulate the ovulation [4]. It was reported that estradiol hormone in ovary is under the in uence of gonadotropins which may regulate the expression of clock genes associated with ovarian cancer [5]. From the previous ndings, the accruing indication has recommended that circadian clock disturbance is an in uential aspect of tumor instigation and proliferation. Epidemiological studies have proved that night workers have raised risk of various cancers (ovary, breast, prostate, and rectal cancer) [5][6][7][8] signifying a probable functional association between the biological clock and cancer formation.
It has been proved that abnormal expression of circadian rhythm gene is strongly associated with various types of cancers [9]. Various Preceding reports have also con rmed that the anomalous expression of biological clock genes is strongly correlated with the prognosis of cancer patients [10,11]. It has been reported that circadian genes play an active role in tumor formation and cancer cell proliferation [10].
Single-nucleotide polymorphism (SNP) is considered as an important genetic biomarker for the early prediction of risk, their response to treatment, and the proliferation of cancer cells [12]. Reported studies have proved that there were several SNPs of circadian rhythm genes (BMAL1, PER3, PER2, CRY, TIMELESS, REVI) which are signi cantly associated with the development and progression of various types of cancers (breast ovary, prostate, etc.) [13][14][15][16][17]. Moreover, developing evidence has revealed that SNPs of circadian genes are signi cantly involved in cancer predisposition [18,19]. In the present study, we replicated and assessed the effects of candidate variants of circadian rhythm genes (BMAL1 rs475715, PER3 rs228644, REV1 rs379215,2 and TIMELESS rs7302060) as a case-control study from J&K region of India, which was previously found to be associated with ovarian cancer in North American population by Jim et al [20] Methods

Sample collection
This case's control study from the J&K population was conducted in the school of Biotechnology, SMVDU, Katra. The cases included females with a histologically con rmed ovarian cancer. The cases have been obtained from various hospitals and clinics of J&K. Controls were age-matched to the cases. The cases with no familial history of cancer were included in the study. All subjects included in this study were unrelated women of J&K whereas their descendants have lived in the J&K for at least 5 generations. Overall, 600 samples including 200 incident cases of ovarian cancer and 400 population-based controls were enrolled in the study. Written informed consent was obtained from all subjects recruited in this study. During sample collection, pre-designed questionnaire was used to get the information which included age, BMI, hormonal status, age at menarche, menopausal status, histology of tumors, oral contraceptive use and breast nodules (Table 1) from both cases and controls. Patients who undertook radio/chemotherapy were excluded from the study.  Table S3.
From the 600 women (200 cases and 400 controls) who provided blood, su cient DNA was extracted by using manufacturer protocol (Qiagen DNA isolation Kit cat no. 51206). All these subjects were genotyped by the (Sequenom Mass ARRAY platform) using the 384 well chip according to the standardized protocol which were replicated from the study [21].
Software Sequenom Typer was used for the analysis and management of data.

Statistical analysis
The odds ratios (OR) with their 95% con dence intervals (CI) as well as hardy Weinberg equilibrium were estimated by Plink v1.07. Clinical characteristics of cases and controls were compared using the chi-square t-test for variables (Table 1). For the results, SNPs that were not following hardy Weinberg equilibrium (H.W.E) were not included in further analysis.
Logistic regression & strati cation analysis was used to estimate the risk or to estimate the association with Odds Ratio at 95% con dence interval and respective level of signi cance as p-value by using SPSS software. The power of the study was calculated statistically by PS software version 3.1.2 [22].
Putative visualization of variants in the human genome was also done by freely available Insilco tools (SNIPA and Haploreg) [23] [24]. Both tools were used to found the functional annotations i.e., to predict the expression quantitative trait locus (eQTL) of risk associated variants and to nd the high linkage disequilibrium SNPs (r2 > 0.8) with selected candidate variants.

Results
Four histopathological types of cases were analyzed. The sample characterized are discussed in Table 1 Figure S4).

Discussion
In this case-control study, we reported the signi cant association of ovarian cancer with the variants of circadian rhythm gene pathway, predominantly with already reported ovarian cancer risk associated circadian gene variants (BMAL1 / ARNTL rs475715 and rs1026071, PER3 rs228644, REV1 rs3792152, and TIMELESS rs7302060) [20]. We examined variation in the four most common genes of the circadian pathway (REV1, ARNTL/BMAL1, TIMELESS, and PER3) as prognosticators of ovarian cancer risk and invasiveness. We found that two out of ve variants were associated with the risk of ovarian cancer. Speci cally, the risk of ovarian cancer was associated with variant rs475715 of BMAL1/ARNTL & rs3792152 of REV1, whereas other variant rs228644 of PER3, rs7302060 of TIMELESS, and rs1026071 of ARNTL were found to be not associated with ovarian cancer in our studied region.
Biological clock in humans called circadian clock / circadian rhythm, autonomously oscillate with a period near 24 hours. The mechanism of the circadian clock is based on the positive/negative response circlets which are produced by core circadian clock genes. The monitoring feedback loop of circadian rhythm consists of PER, CRY, CLOCK, and BMAL1 proteins having a function of regulations in the transcription/translation process. The heterodimer (BMAL1/CLOCK) complex inhibiting or repressing the PER/CRY genes activity in the nucleus region where the monitoring feedback loop formation is completed after the complex (BMAL1/CLOCK) formation which regulates the transcription of Rev-erbα and Rora (nuclear receptors) [25].
Findings regarding the circadian rhythm seem to be initiated by both transcriptional and post-transcriptional mechanisms which induce gene expression [26,27]. The transcription of PER and CRY genes is initiated by two transcription factors CLOCK and BMAL1 / ARNTL. After reaching the grave concentration the PER / CRY reduces the effect of CLOCK/ BMAL1 facilitated initiation of their particular genes in a negative feedback loop. It was found that both complex PER / CRY and CLOCK/ BMAL1 intricate with each other and bound to chromatin. The regular daily oscillations in clock gene is contributed by protein degradation, phosphorylation, and nuclear entry [28]. The regulation of CRY and PER gene expression generates genetic and biochemical evidence [29] but the regulation of the CLOCK/ BMAL1 gene is very much less known. Various studies reported that PER and CRY gene establishes a positive feedback loop in the process of BMAL1 transcription [28][29][30] REV1 (REV1-DNA directed polymerase) is a nuclear receptor that acts as a transcriptional repressor in the circadian pathway, where activates and inhibits the transcription of the BMAL1 gene [31]. The BMAL1 transcription is regulated by REV-ERB alpha, thus it acts as a connector link through which components of negative and positive limb constitute to form a molecular link. It determines the length of the period and phase-shifting properties of the biological clock [30]. It was proved that BMAL1 de cient cells due to DNA damage lead to arrest in cell cycle and reveal a possible modulatory effect on tumor suppressor genes i.e. P53. It has been reported that the knockdown of the BMAL1 gene induces cell growth, reduced programmed cell death which appears to play a role in carcinogenesis [32].
Our study is the rst replicative case-control association study of clock genes. Jin et al [20] reported that the gene expression of the BMAL1 gene has been controlled by cMYC where the overexpression of cMYC leads to the downregulation of BMAL1. So, it has been suggested that BMAL1 gene variants were signi cantly associated with the risk of ovarian cancer. The circadian gene variants are associated with prostate cancer which was reported in the GWAS study [33]. In some populations of the world, it has been reported that night workers have disturbed biological clocks where the ndings of various studies proved the night working women's and men's are more prone to cancers (Breast, ovary, and prostate cancer) [19,[33][34][35]. Our results indicated that BMAL1 and REV1: rs475715 & rs3792152 both intronic variants, were associated with ovarian cancer risk. Functional prediction implicated that rs47515 has direct eQTL effect and regulates the expression of C2orf15, LIPT1, LYG1, MITD1, REV1, TSGA10, and TXNDC9 genes, whereas rs3792152 regulates the RN7SKP151 & BTBD10 with BMAL1 gene. These ndings suggest that it is located within a region that directly affects expression potentially through the modulation of the histone markers in the enhancer region. Yeh et al, 2014 [36] proved that in the ovarian cancer cell (in CP70 and MCP2) the H3K27 (histone mark) is supplemented in the promoter region of ARNTL / BMAL1 gene, Whereas the presence of inhibitor (GSK126) of EZH2 region reestablished the expression of ARNTL/ BMAL1 gene in ovarian cancer cells (in CP70 and MCP2). They also con rmed that there is a sensitivity of chemotherapy drug (cisplatin) in ovarian cancer cells after increasing the expression of the ARNTL / BMAL1 gene. With these ndings, it was con rmed that BMAL1 /ARNTL may act as a tumor suppressor by regulating the p53 tumor suppressor pathway in ovarian cancer [20,36]. So, our results climax the implication of circadian rhythm gene variation in ovarian cancer susceptibility and suggest an early role for the BMAL1 and REV1 gene in ovarian cancer pathogenesis.
Our results also suggest that the circadian gene variant may play a signi cant role in the etiology of ovarian cancer. From the literature survey, the identi cation of a signi cant association between circadian genes and ovarian cancer is still unpredictable. To the best of our knowledge, the case-control association studies between circadian gene variants and ovarian cancer were investigated in very few studies (20,(25)(26)(27)(28)(29). Few of them have found an association with ovarian cancer and other cancers at the variant level [20,[37][38][39]. Even though various previous research studies have implicated circadian genes in the progression of cancers in women. Nevertheless, our study possibly requires more variants of the circadian rhythm pathway to highlight signi cant associations between certain circadian genes and the risk of aggressive ovarian cancer.

Conclusion
This study forti es the current indication supporting the premise of a link between circadian rhythm genes and ovarian cancer risk. Additional studies with larger sample size as well as functional validation of risk associated gene variants is warranted to con rm those ndings. Besides these it has been reported that women working during nightshifts are associated with several cancer but its probable role in nding the association between circadian genes and ovarian cancer risk should not be justi ed till date so this study proposes the need for further studies to investigate the association of carcinogenic effects of circadian disruption in relation with environmental factors, such as night-workers, regular exposure to stressed conditions, irregular diet patterns, and electromagnetic (EM) waves, which disturb circadian rhythm or biological clock by uctuating the melatonin levels.

Ethical Approval
This study was approved by the Institutional Review Board committee of SMVDU with wide reference no. SMVDU/IERB/14/28. Experimental protocols conducted in this study strictly followed the guidelines set by the Institutional Ethical Review Board (IERB) SMVDU.