|Year : 2019 | Volume
| Issue : 4 | Page : 204-207
Association of the cyclin D1 G870A polymorphism with risk of multiple myeloma in a group of Egyptian patients
Dina El Dahshan1, Raafat AbdelFattah2
1 Clinical Pathology Department, Faculty of Medicine, Beni-Suef University, Egypt
2 Medical Oncology Department, National Cancer Institute, Cairo University, Cairo, Egypt
|Date of Submission||18-May-2019|
|Date of Acceptance||20-Jun-2019|
|Date of Web Publication||20-Jul-2020|
Dina El Dahshan
Associate Professor of Clinical Pathology, Clinical Pathology Department, Faculty of Medicine, Beni-Suef University, Compound Zayed Dunes building 89, entrance 2 El Sheikh Zaye, 6 October City, Beni-suef,
Source of Support: None, Conflict of Interest: None
Background Multiple myeloma (MM) is a clonal bone marrow disease characterized by the neoplastic transformation of mature B cells. Single-nucleotide polymorphisms in the Cyclin d1 CCND1 gene were reported with an increased susceptibility to various human cancers such as breast cancer, bladder cancer, colorectal cancer, and lung cancer.
Aim The aim of this study was to detect the distribution of CCND1 G870A genotypes in a group of MM patients and in controls in an attempt to reveal the possible association between the presence of the polymorphism (G870A) and the risk of MM in an Egyptian population.
Patients and methods The PCR-restriction fragment length polymorphism technique was used for the detection of CCND1 G870A polymorphism in 56 MM patients and 70 healthy individuals as a control group.
Results The homozygous GG genotype was present in 10.7% of patients, heterozygous AG in 48.2%, and homozygous AA in 41.1%. Carriers of CCND1 G870A polymorphism did not show increased risk to MM [AG genotype odds ratio (OR)=0.928, P=0.899 and GG genotype OR=1.295, P=0.499]. Patients aged at least 60 years had a higher frequency of mutant CCND1 than controls (88.2 and 64.3%), but this difference did not reach statistical significance (OR=4.167, P=0.128).
Conclusion Carriers of the pleomorphic genotype of CCND1 G870A did not show increased susceptibility to MM. Larger population-based studies are needed to validate the association of CCND1 G870A polymorphism with MM.
Keywords: cyclin D1, gene polymorphism, multiple myeloma, risk
|How to cite this article:|
El Dahshan D, AbdelFattah R. Association of the cyclin D1 G870A polymorphism with risk of multiple myeloma in a group of Egyptian patients. Egypt J Haematol 2019;44:204-7
|How to cite this URL:|
El Dahshan D, AbdelFattah R. Association of the cyclin D1 G870A polymorphism with risk of multiple myeloma in a group of Egyptian patients. Egypt J Haematol [serial online] 2019 [cited 2021 Sep 21];44:204-7. Available from: http://www.ehj.eg.net/text.asp?2019/44/4/204/290227
| Introduction|| |
Multiple myeloma (MM) is a clonal bone marrow disease characterized by the neoplastic transformation of mature B cells. It is a heterogeneous disease both at the clinical and genetic level. Recent studies in molecular cytogenetic and gene profiling have revealed part of the pathogenesis of MM . Despite the advances in MM treatment, the prognosis is poor, and the genetic and molecular mechanisms underlying MM development are still not clear.
Deregulation of the cell cycle plays a pivotal role in oncogenesis. During mitosis, cyclin-dependent kinases (CDKs), CDK4 and CDK6, in protein complexes with cyclin D1 (CCND1), control the cell transformation from the G1 to S phase . A series of transcriptions of downstream genes are activated by tumor suppressor protein retinoblastoma phosphorylation, which is catalyzed by cyclin D1. The G>A polymorphism (G870A) of the CCND1 gene leads to the formation of an alternative splice site and alteration of the protein structure of the carboxy terminal domain, resulting in cell cycle dysregulation at checkpoint (G1/S) and impairing the DNA repair capacity ,. This explains that inhibition of cyclin D1 causes cell cycle arrest, while its overexpression accelerates the G1 phase transition of the cell cycle, leading to increased cell proliferation, which might eventually lead to cancer occurrence ,,.
Previous studies reported that abnormal expression of cyclin D1 was related to different human malignancies, that is, whether it was due to its amplification, translocation, or inversion ,,. Moreover, different single-nucleotide polymorphisms (SNPs) in the CCND1 gene were reported with various human cancers. The SNP G870A at the exon 4 splice site was associated with an increased susceptibility to breast cancer , bladder cancer , colorectal cancer  and lung cancer .
However, it is still unclear whether the susceptibility to MM is linked to the CCND1 SNPs. In this case–control study, we analyzed the CCND1 G870A polymorphism frequencies in patients with MM and in controls in an attempt to find a possible relation between the presence of the polymorphism (G870A) and the occurrence of MM in a group of Egyptian patients.
| Patients and methods|| |
Patients and controls
This study was conducted on 56 (37 male individuals and 19 female individuals, aged 23–68 years, with a mean age of 51.80±10.64 years) MM patients. Patients were recruited from the Department of Oncology, Beni-Suef University Hospital and Sheikh Zayed Specialized Hospital. Seventy age-matched and sex-matched healthy individuals of both sexes served as controls. Informed consents were obtained from patients and controls in advance. The study was approved by the Research Ethical Committee of Faculty of Medicine, Beni-Suef University and was conducted in accordance with the Helsinki Declaration of Human Rights. The patients’ characteristics and clinical data are shown in [Table 1].
From each patient and control, 3–5 ml of venous blood was collected on EDTA and stored at −20°C until DNA extraction was performed using QIAamp DNA Blood Mini kit (Hilden, Germany) following the manual’s instructions. DNA quality and quantity were estimated using NanoDrop 2000 c (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The extracted DNA was then stored at −80°C until the time of use.
Cyclin D1 genotyping
The CCND1 G870A polymorphism was detected using PCR-restriction fragment length polymorphism. Primers for amplification of CCND1 were as follows: 5′-GCA GTG CAA GGC CTG AAC CT-3′ (sense) and 5′-GGG ACA TCA CCC TCA CTT AC-3′ (antisense). The products were then analyzed by electrophoresis on 1.5% agarose gel. The restriction enzyme ScrFI at 37°C (New England Biolabs Inc., Beverly, Massachusetts, USA) was used, and patients’ genotype was detected according to their pattern of electrophoresis. Three electrophoretic patterns were encountered as one of the following: wild-type AA product at 167 bp, heterozygous AG at 167, 146 and 22 and homozygous mutant GG at 146 and 22 only .
Data were coded and entered using the statistical package SPSS version 24. Data were summarized using mean and SD for quantitative variables and frequencies (number of cases) and relative frequencies (percentages) for categorical variables. Comparisons between quantitative variables were carried out using unpaired t-test . For comparing categorical data, the χ2 test was performed. Exact test was used instead when the expected frequency was less than 5 . Genotype and allele frequencies were calculated in the disease and the control groups. Odds ratio (OR) with 95% confidence intervals (CIs) was calculated using logistic regression . P values less than 0.05 were considered as statistically significant.
| Results|| |
The mean age in our cases was 51.80±10.64 years, while the control group had a mean age of 49.13±10.68 years. Patients’ and controls’ age and sex are listed in [Table 1].
The CCND1 G870A wild type (AA), heterozygous (AG) and homozygous (GG) were detected in 41.1, 48.2 and 10.7% of MM patients, respectively. In the control group, the genotype distribution was 45.7% (AA), 41.4% (AG) and 12.9% (GG). Comparing the distribution of different genotypes between MM patients and the control group did not show statistical significance. The frequency of genotypes in both groups is shown in [Table 2].
|Table 2 Cyclin D1 genotype and allele frequency in multiple myeloma patients and controls|
Click here to view
The presence of the CCND1 G870A polymorphism did not show association with MM susceptibility. Heterozygous genotype AG was (OR=0.928, P=0.899) and the homozygous genotype GG was (OR=1.295, AQ8 P=0.499).
Analysis of allele frequency and its distribution showed that the mutant G allele had a higher frequency in patients (34.8%) than in controls (33.6%), but it was not statistically significant (P=0.835; [Table 2]).
Moreover, it is worth noting that we studied the susceptibility to MM among patients carrying the CCND1 mutant genotypes as two groups. One group was aged at least 60 years, and the second group’s age was less than 60 years. Patients aged at least 60 years had a higher frequency of mutant CCND1 than controls (88.2 and 64.3%), but this difference did not reach statistical significance (OR=4.167, P=0.128). Thus, neither of the two groups revealed an increased risk of MM ([Table 2]).
| Discussion|| |
Regulation of the cell cycle has a critical role in the evolution of cancer by the controlling of cell proliferation, differentiation, and apoptosis ,. A series of activation of CDK complexes are responsible for the cell cycle transition from the G1 phase to the S phase. Cyclin D1, which is located on 11q13, plays a pivotal role in this transition through the G1 restriction point . The identified polymorphisms in the CCND1 exceeds a couple of hundred SNPs, and the most studied is the A/G (adenine to guanine) substitution at nucleotide 870 .
Over the past decade, the CCND1 870G>A polymorphism has been widely reported as a risk factor for different cancers, with most of the studies linking the 870A to cancer risk . For example, many studies have reported the association of the CCND1 G870A genetic polymorphism with increased susceptibility to colorectal, lung, breast, and bladder cancers ,,,. Most of the previous studies reported the role of the AA genotype in various cancers’ risk, unlike in MM, wherein the GG genotype contributed to the risk of the disease .
In this study, we investigated cyclin D1 CCND1 G870A polymorphism in 56 patients of primary MM and analyzed a potential association, if any, with the risk of the disease. In addition, we investigated the susceptibility to the disease in two age groups of MM patients and whether it was related to the presence of the polymorphism.
The observed frequencies of genotypes resulting from mutation at CCND1 G870 were as follows: 41.1% AA, 48.2% AG and 10.7% GG. These results were not in complete agreement with the work of Wang et al. , who studied the same polymorphism in 67 MM patients. They reported a different distribution of the genotypes, wherein the AA genotype was in 30% of patients, GA in 28% and GG in 42%. This study reported higher prevalence of the homozygous polymorphic genotype GG than that found in our patients (42 and 10.7%). However, it is worth noting that this difference was present in controls too; 26% of Wang et al.  controls harbored the GG genotype versus only 12.9% in our controls.
Our results showed that patients carrying the CCND1 G870A polymorphism whether heterozygous or homozygous, did not show increased susceptibility to MM (OR=0.928, P=0.899 and OR=1.295, P=0.499) respectively. This lack of association findings is conflicting with results reported by Weinhold et al. , who proposed in their report that CCND1 G870A polymorphism is a risk factor for MM in two groups of European patients. Moreover, this association was reported by Wang et al.  in Chinese patients with MM. The different ethnic and genetic background of our patients and European and Chinese patients might explain these diverse results.
Under normal conditions, the plasma cells are arrested in the cell cycle at the G1 phase. On the contrary, the MM cells have proliferation and self-renewal capabilities; this implies that their physiological cell cycle control has changed. Cyclin D1 is a key role regulator of the G1/S transition, and its overexpression was found in MM. Moreover, it is involved in the MM t(11;14) in which the Ig heavy chain fuses with cyclin D1 locus ,. This key role of cyclin D1 in MM was not demonstrated in our group of patients, unlike the studies by Wang et al.  and Weinhold et al. , whose results strongly supported the theory.
To our knowledge, Wang et al.  were the only ones investigating the effect of age on the association of CCND1 polymorphism and MM susceptibility. They concluded that increased risk to the disease in the GG genotype was more apparent in patients older than 60 years (OR=3.297; 95% CI=1.058–10.267, P=0.036). However, in the current study, we could not conclude such a verbal conclusion, even after trying to stratify the number of patients older than 60 years carrying the GG genotype (OR=4.167; 95% CI=0.664–26.138, P=0.128). This group unlike that of the Wang et al.  report did not show stronger susceptibility to the disease in our work. However, it is necessary to clarify that the distribution of CCND1 G870A genotypes among our population appears different from the Chinese population included in the Wang et al. work . This difference in distribution was also evident in control group, which might partially explain that different ethnic groups exhibit variable genotyping distribution. Yet, more work in large, population-based studies and genome wide studies is needed to validate the association of CCND1 G870A polymorphism with MM and other hematological diseases.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Sawyer JR. The prognostic significance of cytogenetics and molecular profiling in multiple myeloma. Cancer Genet
Cai F, Chen P, Chen L, Biskup E, Liu Y, Chao P et al.
Human RAD6 promotes G1-S transition and cell proliferation through upregulation of cyclin D1 expression. PLoS One
Shih LC, Tsai CW, Tsai MH, Tsou YA, Chang WS, Li FJ et al.
Association of cyclin D1 genotypes with nasopharyngeal carcinoma risk. Anticancer Res
Tsai MH, Tsai CW, Tsou YA, Hua CH. Significant association of cyclin D1 single nucleotide polymorphisms with oral cancer in taiwan. Anticancer Res
Nagasawa S, Onda M, Sasajima K, Makino H, Yamashita K, Takubo K, Miyashita M. Cyclin D1 overexpression as a prognostic factor in patients with esophageal carcinoma. J Surg Oncol.
Chen X, Zhao T, Li L, Xu C, Zhang X, Tse V et al.
CCND1 G870A polymorphism with altered cyclin D1 transcripts expression is associated with the risk of glioma in a chinese population. DNA Cell Biol
Cui J, Shen L, Wang Y. Specific CCND1 G870A alleles associated with breast cancer susceptibility: a meta-analysis of 5528 cases and 5353 controls. Asian Pac J Cancer Prev
Wang Y, Li Z, Liu N, Zhang G. Association between CCND1 and XPC polymorphisms and bladder cancer risk: a meta-analysis based on 15 case-control studies. Tumour Biol
Yang Y, Wang F, Shi C, Zou Y, Qin H, Ma Y. Cyclin D1 G870A polymorphism contributes to colorectal cancer susceptibility: evidence from a systematic review of 22 case-control studies. PLoS One
Zhou C, An H, Hu M, Liu Q, Geng P, Xu J, Sun B, Liu C. The cyclin D1 (CCND1) G870A polymorphism and lung cancer susceptibility: a meta-analysis. Tumour Biol
Grossman AB, Simpson DJ, Fryer AA, Wass JA, Pfeifer M, Kros JM, Clayton RN, Farrell WE. Cyclin D1 (CCND1) genotype is associated with tumour grade in sporadic pituitary adenomas. Carcinogenesis
Chan YH. Biostatistics 102: quantitative data − parametric & non-parametric tests. Singapore Med J
Chan YH. Biostatistics 103: qualitative data − tests of independence. Singapore Med J
Chan YH. Biostatistics 202: logistic regression analysis. Singapore Med J
Zhan F, Sawyer J, Tricot G. The role of cytogenetics in myeloma. Leukemia
Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature
Ekholm SV, Reed SI. Regulation of G(1) cyclin-dependent kinases in the mammalian cell cycle. Curr Opin Cell Biol
Betticher DC, Thatcher N, Altermatt HJ, Hoban P, Ryder WD, Heighway J. Alternate splicing produces a novel cyclin D1 transcript. Oncogene
Knudsen KE, Diehl JA, Haiman CA, Knudsen ES. Cyclin D1: polymorphism, aberrant splicing and cancer risk. Oncogene
Weinhold N, Johnson DC, Chubb D, Chen B, Försti A, Hosking FJ et al.
The CCND1 c.870G>A polymorphism is a risk factor for t(11;14)(q13;q32) multiple myeloma. Nat Genet
Wang S, Huang Y, Su R, Fang Z, Han M. Cyclin D1 G870A polymorphism is associated with an increased risk of multiple myeloma. Genet Mol Res
Specht K, Haralambieva E, Bink K, Kremer M, Mandl-Weber S, Koch I et al.
Different mechanisms of cyclin D1 overexpression in multiple myeloma revealed by fluorescence in situ
hybridization and quantitative analysis of mRNA levels. Blood
[Table 1], [Table 2]