Abstract
Background/Aim: This study investigated whether genetic variations in cyclin D1 (CCND1) are associated with susceptibility to childhood acute lymphoblastic leukemia (ALL). Materials and Methods: A total of 266 childhood ALL cases and 266 healthy controls were genotyped for CCND1 rs9344 and rs678653. Results: There was a significant difference in the genotypic distribution of rs9344 between childhood ALL patients and healthy controls (p=0.0077). Compared to the AA genotype, AG and GG genotypes were associated with significantly decreased risks of childhood ALL with odds ratio (OR) of 0.65 [95% confidence interval (CI)=0.44-0.94, p=0.0234] and 0.45 (95%CI=0.26-0.78, p=0.0040), respectively. Supporting this, allelic frequency distributions between childhood ALL patients and controls was significantly different (OR=0.68, 95%CI=0.53-0.88, p=0.0025). There was no significant difference in the genotypic and allelic distributions of rs678653 between cases and controls. Conclusion: CCND1 rs9344, but not rs678653, may serve as a predictive marker of susceptibility for childhood ALL.
Acute lymphoblastic leukemia (ALL) is a kind of cancer of blood cells characterized by the development of abnormally huge numbers of immature lymphocytes. It is the most common type of malignancy among children worldwide (1). Despite the fact that in most cases the cause is unknown, the associations of genetic contributors to the risk of childhood ALL have been investigated, however, they remain largely unknown (2-6). With the development of early detection, diagnosis, therapy and caring systems, the survival rate for children has significantly increased from under 10% in the 1960’s to 90% in 2015 (1). However, it is necessary to reveal the underlying etiology for precision medicine.
Cyclin D1 protein is encoded by the CCND1 gene, which locates on the long arm of chromosome 11 (band 11q13) (7). It is expressed in all adult human cells with the exception of bone marrow stem cells (7, 8). Normally, cyclin D1 teams up with cyclin dependent kinase 4 (CDK4) to inhibit retinoblastoma (Rb) phosphorylation, allowing E2F to transcribe genes required for entry into the S phase of the cell cycle (9). Inactive pRb allows cell cycle progression through the G1/S transition and DNA synthesis (9). Cyclin D1-CDK4 also enables the activation of cyclin E-CDK2 complex by sequestering p21 and p27, allowing cells to enter the S phase of the cell cycle (9). According to literature, cyclin D1 levels are elevated by various growth factors and hormones (10-12), and degraded by the proteasome (13).
In other types of cancer, such as non-small cell lung cancer (14, 15), head and neck carcinoma (16-18), pancreatic cancer (19), and breast cancer (20-22), cyclin D1 over-expression has been reported to correlate with early cancer onset and tumor progression (9). At the same time, dysregulation of cyclin D1 expression has been found to be caused by various mechanisms including mutations (13), over-expression of oncogenic factors (23-26), disruption of nuclear exportation (27) and proteolysis (28). However, no conclusive finding has been obtained in childhood ALL. We hypothesized that CCND1 rs9344 and rs678653 polymorphisms may determine personal susceptibility to childhood ALL and thus, aimed at examining the association of these two polymorphic sites with childhood ALL risk.
Materials and Methods
Childhood ALL and control participants. Briefly, childhood ALL cases were ascertained by expert pediatric clinicians in addition to pathologic confirmation. All of the subjects completed a questionnaire with the help of their parents or guardians, and provided about 5 ml blood samples for genotyping. Control group members were selected in accordance with the methodology established by the Health Examination Cohort matching well for age and gender over the similar period of 2005 to 2010 as we have previously published (5, 29, 30). All the participants were Taiwanese.
DNA extraction and genotyping exam. Genomic DNA from the blood samples was extracted (Qiagen, Valencia, CA, USA) within 24 h after the sampling. The genotypes of CCND1 rs9344 and rs678653 were determined via the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) methodology as we have previously published (31, 32).
Statistical analysis. Student’s t-test was used to examine the continuous age index between the case and control groups. Pearson’s χ-square test without Yates’ correction was used to assess the distribution of CCND1 rs9344 and rs678653 genotypes. At the same time, Hardy-Weinberg equilibrium (HWE) was assessed among the controls. Crude odds ratios (OR) and 95% confidence intervals (CI) were used for estimating significance. p-Values less than 0.05 were considered statistically significant.
Results
Basic characteristics of the case and control groups. Overall, the Taiwanese ALL population collected in this study consisted of 266 Taiwan childhood ALL cases and the same number of age- and gender-matched healthy subjects. As shown in Table I, age and gender were well matched. The average age was 7.0±4.4 years for the ALL cases and 8.3±4.8 for the controls (p=0.6483). The numbers of boys and girls were 148 and 118, respectively, in both the case and control groups (Table I).
The association between CCND1 rs9344 genotypes and childhood ALL risk. The genotypic frequencies of the CCND1 rs9344 polymorphic site among childhood ALL cases and age- and gender-matched controls are shown in Table II. The distributions of CCND1 rs9344 genotypes among the control group were in accordance with HWE, and with a p-value of 0.1986. There was a significant difference in the CCND1 rs9344 genotypic distribution between cases and controls (p for trend=0.0077). In detail, compared to the AA genotype, the AG and GG genotypes were associated with significantly decreased risks of childhood ALL with an adjusted OR of 0.65 (95%CI=0.44-0.94, p=0.0234) and 0.45 (95%CI=0.26-0.78, p=0.0040), respectively. From the results of the dominant model analysis, we can observe that individuals carrying the AG+GG genotypes had a decreased risk of childhood ALL (adjusted OR=0.60, 95%CI=0.42-0.86, p=0.0051, Table II) compared to the AA genotype (Table II). Another piece of evidence derives from the analysis of allelic frequency distribution; the “G” allele seemed to associate with a significantly decreased risk of childhood ALL compared to the “A” allele (OR=0.68, 95%CI=0.53-0.88, p=0.0025, Table III).
The association between CCND1 rs678653 genotypes and childhood ALL risk. The genotypic frequencies of the CCND1 rs678653 polymorphic site among childhood ALL cases and age- and gender-matched controls are shown in Table IV. The distributions of CCND1 rs678653 genotypes among the control group were also in accordance with HWE (p-value=0.5258). In contrast to CCND1 rs678653, there was no significant difference in the genotypic distribution of CCND1 rs678653 polymorphism between childhood ALL cases and controls (p for trend=0.8912). There was no association of childhood ALL risk with CCND1 rs678653 polymorphism in genotypic (Table IV) or allelic analysis (Table V).
Discussion
Dysregulation of the cell cycle in various human cancers has been frequently observed; cyclins and CDK functions are primarily affected, leading to uncontrolled proliferation, genomic instability, and chromosomal instability (33-35). The A allele of CCND1 rs9344 (G870A) was hypothesized by Knudsen to favor production of an alternate transcript encoding CCND1b, which lacks the PEST motif that is critical for the degradation of cyclin D1 (36). In consequence, transcript b, produced by those carrying the A allele, will have a longer life span than the transcript produced by those carrying the G allele (36). However, there is no solid evidence yet in leukemia, not in childhood leukemia, in support of this hypothesis.
In the current study, we found that the A allele of CCND1 rs9344 is more prevalent than the G allele among Taiwanese, and it seems to associate with an elevated risk of childhood ALL (Table III). The AG and GG genotypes of CCND1 rs9344 are good predictive markers for a decreased risk of childhood ALL (Table II). On the contrary, the genotypes of CCND1 rs678653 seem to contribute little to childhood ALL risk prediction (Table IV and Table V). This agrees with the hypothesis of Knudsen, however, the detail mechanisms require further investigation.
We summarized the literature regarding ALL risk and CCND1 rs9344 genotypes in Table VI as for the ethics, investigated population size, and the highlights of their findings. In 2001, Howe and Lynas first investigated the association between CCND1 rs9344 and ALL in a British population (37). However, the sample size of their pilot study was too small (19 cases and 13 controls) to derive any solid conclusion. In 2005, Hou et al. examined the contribution of CCND1 rs9344 to childhood ALL in a Chinese population of 183 children with ALL and 190 healthy controls. This was the largest population investigated at the time and AA genotype of CCND1 rs9344 was found to have a tendency of increasing 3.29-fold the risk for ALL compared with the AG+GG genotypes (38). In 2013, Bedewy et al. surveyed only 25 ALL cases and 15 controls of an African population and found no association between CCND1 rs9344 genotype and adult ALL risk (39). In 2015, Xue et al. hypothesized that CCND1 rs9344 genotype may contribute to the elimination rate and hepatotoxicity of methotrexate in children (40). They found a significant association between the AA genotype of CCND1 rs9344 and an increased risk of methotrexate hepatotoxicity, but no association with the risk of delayed methotrexate elimination (40). Since they did not examine any control subjects, they did not discuss whether CCND1 rs9344 can serve as a good predictor for childhood ALL risk. In 2020, El Menshawy et al. collected 100 individuals from Egypt (50 ALL cases and 50 controls) and investigated the contribution of CCND1 rs9344 genotype to methotrexate cytotoxicity, and survival rates. They found that the AA genotype was associated with a higher risk of developing ALL compared to the control group; no notable association was found between CCND1 genotypes, methotrexate hepatotoxicity and ALL survival rates (41). The meta-analysis of the publications of Howe, Hou, and Bedewy, found a significant association between the CCND1 rs9344 genotype and leukemia (42). Generally, although we collected a relatively large sample of childhood ALL and suggested a potent predictive marker, these findings should be validated in more and larger populations. It is worth noticing that CCND1 rs9344 A variant carriers have been reported to have a lower survival rate compared to carriers of the G variant (43). CCND1 rs9344 may not only serve as a predictor of childhood ALL susceptibility, but also of a poorer outcome, such as lower survival rate. At the same time, we have to distinguish adult and childhood ALL (41).
In conclusion, our study, based on a relatively large and representative population, provided evidence that the AG and GG genotypes of CCND1 rs9344 were significantly associated with decreased risk of childhood ALL in Taiwan. Our study adds another piece of evidence indicating that the CCND1 may play a critical role in cell cycle regulation and determine susceptibility to human cancers. We also provided an updated summary of the role of CCND1 rs9344 in cancer risk prediction. More studies in adult and childhood leukemia across different and larger populations are warranted to reveal the role of CCND1 rs9344 in cancer prediction and precision medicine.
Acknowledgements
The Authors are grateful to Yu-Chen Hsiau for her excellent technical assistance. The study was supported mainly by a grant from Taoyuan General Hospital (grant number: PTH109011) to Dr. Hsu. The funders had no role in study design, data collection, statistical analysis, preparation of the manuscript, or decision to publish the manuscript.
Footnotes
↵* These Authors contributed equally to this study.
Authors’ Contributions
Conceptualization, P.C.H., J.S.P., C.C.C.; Data curation, W.S.C., Y.T.C, T.L.H.; Methodology, J.S.Y., Y.C.W.; Statistics, J.C.C, Y.N.H., C.W.T.; Project administration, J.S.P., D.T.B.; Supervision, D.T.B., C.W.T.; Validation, C.C.C.; Writing—original draft, P.C.H., J.S.P.; Writing—review and editing, D.T.B. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received July 30, 2021.
- Revision received August 9, 2021.
- Accepted August 11, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.