EUROMEDITERRANEAN BIOMEDICAL JOURNAL

2020,15 (09) 40–46

(FORMERLY: CAPSULA EBURNEA)

Original article

ASSOCIATION OF RS2069502 CYCLIN-DEPENDENT KINASE 4 GENE WITH BREAST

CANCER

Dalya Shakir Al-owaidi, Moaed E. Algazally, Alaa Sadeq Alawaad

Department of Biochemistry - College of Medicine, Babylon University, Hillah, Iraq

ARTICLE INFO ABSTRACT

Article history: The goal of this project was to evaluate the association of candidate genetic variant rs2069502 of
Received 28 January 2020 the CDK4 gene with breast cancer and the effect of variant genotypes on the serum concentration of
Revised 22 March 2020 CDK4 enzyme and consequently, on the occurrence of breast cancer and breast cancer subtypes in
Accepted 06 April 2020 the studied population. A total of 80 breast cancer patients were divided into 4 subtypes, Luminal
A, Luminal B, Her2/neu enriched and TPN, while 80 healthy individuals were enrolled as controls.
Keywords: Our results revealed that there were no significant differences between breast cancer patients and
Breast Cancer, Molecular control groups and among breast cancer subtypes in the serum concentration of CDK4 (p-value
Classification, Cyclin-Dependent >0.05). An allelic and genotypic association of rs2069502 with breast cancer showed that there
Kinase 4, Retinoblastoma protein, were no significant allele frequency differences, in both alleles A and G, between breast cancer
Elongation Transcription Factor 2, patients and the control group(p-value > 0.05). Association of rs2069502 genotypes with breast
cyclin-dependent kinase inhibitor 2a. cancer and under different inheritance models revealed that there was no significant association
between rs2069502 genotypes and breast cancer (p-value> 0.05). The association of rs2069502
genotypes with CDK4 serum concentration showed the presence of a significant difference between
A/A and A/G, A/A and G/G in the CDK4 level (p-value < 0.05). But A/G and G/G did not show
any significant difference in the CDK4 level. The rs2069502 CDK4 gene did not show any
significant association with breast cancer except, in the genotyping frequency among breast cancer
subtypes.

© EuroMediterranean Biomedical Journal 2020

1. Introduction cyclin proteins, together they form a complex functioning as a kinase

checkpoint that regulates the progression of the cell during its cycle [5].
Breast cancer is the most common disease in women that lead to mortality The cyclin-CDK complexes regulate the progression of the cell cycle that
worldwide[1]. Breast cancer begins in the lobules or in the milk ducts [2]. leads to (G0) the resting state, (G1) growth phase, (S) phase of DNA
Breast cancer is the first most common cause of death among women in replication, and finally to the (M)phase when cell division occurs. Cell
the Babylon province of Iraq. The number of breast cancer patients cycle will be interrupted when abnormalities in one of the phases occur,
admitted to the Babylon Oncology center between 2017 and 2018 was 242 leading to initiate a signal until the problem is solved [6]. In mammalian
and 187 respectively[3].Breast Cancer is considered the major cause of cells, there are at least 7 CDKs [7]. During G1, an important target of the
death among women between the ages of 40 and 59[4]. CDKs is the retinoblastoma protein (RB), and the G1 cyclin-CDK
The cell cycle is a complex process that includes cell growth and complexes phosphorylate RB on multiple residues[8].
proliferation, as well as the regulation of DNA repair. During the cell Hypophosphorylated RB binds the E2F transcription factor, making it
cycle there are several regulatory proteins involved in the G1, S, G2 and unavailable for transcription. Once the cyclin-CDKs phosphorylate RB,
M phases of mitosis, leading to the synthesis of 2 daughter cells. which releases E2F for the transcription of proteins necessary for the cell
Cell cycle phases have central proteins that include cyclin-dependent cycle progression [9].
kinases CDKs, part of the family of serine/threonine kinases, and the

* Corresponding author: Dalya Shakir, [email protected]

DOI: 10.3269/1970-5492.2020.15.9
All rights reserved. ISSN: 2279-7165 - Available on-line at www.embj.org
 EUROMEDITERRANEAN BIOMEDICAL JOURNAL 2020, 15 (09) 40-46 41

The action of growth factors on cells during G0 and G1. During the G1 2. Material and Methods
phase of the cell cycle, the restriction point occurs when the cells no
longer respond to dragging growth factors [10]. Growth factors stimulate The subjects who participated in this study were 80 breast cancer patients
the cells to enter into the cell cycle from G0. The removal of growth between the ages of 30-80. The subjects were divided into 4 groups
factors at the early stages of G1 will result in cells returning to G0. according to the molecular classification of breast cancer, using an
However, in the later stages of G1 when the cells have passed through the immunohistochemistry technique for the expression of estrogen receptor
restriction point, despite the removal of the growth factor, the cells will (ER), progesterone receptor (PR), and Her2-enriched proteins:
continue to progress to the S phase. Therefore, the restriction point can be -Luminal A: 37 female patients with breast cancer
defined as the time after which the cell is committed to entering into the -Luminal B: 19 female patients with breast cancer
cell cycle. The restriction point is regulated largely by RB [11]. -Her2\neu+ enriched: 15 female patients with breast cancer
Cell division protein kinase 4, or human cyclin-dependent kinase 4, is -Triple-negative: 9 female patients with breast cancer
an enzyme encoded by the CDK4 gene. CDK4 is a catalytic subunit of the All patients of these groups did not receive any type of treatment
protein kinase complex (CDK4-Cyclin D complex) that is important (hormonal or chemotherapy), which means the collection of blood
for G1 progression through the cell cycle, therefore the activity of CDK4 samples was done pre-dose and without using any medication to exclude
is limited to the G1-S phase.CDK4 activity is controlled by D-type cyclins the effect of these drugs on the biochemical and genetic results.
as regulatory subunits, and p16INK4a as a CDK inhibitor. CDK4 with its The control group enrolled in this study included 80 healthy females
partner CDK6 are responsible for the phosphorylation of the examined at the consulting clinic of breast cancer early detection in the
retinoblastoma protein RB. The cyclin D-CDK4 complex, especially the Hilla teaching Hospital.
ser/thr-kinase component of that complex, inhibits RB1 retinoblastoma as The control group matched the breast cancer patients’ group in age. The
a member of the RB protein family by phosphorylation, and regulates the mean value and SD of age for breast cancer patients and controls were
transition of G1/S during the cell cycle. Once RB1is phosphorylated, the (51.89±12.18), (49.2±11.49) respectively. No significant differences in
dissociation of the transcription factor E2F from the RB1/E2F complexes BMI were found among patient subtypes (p-value > 0.05) and the mean
occurs ,targeting the subsequent gene transcription. These genes are values; standard deviations were Luminal A (31.33±4.03), Luminal B
responsible for the progression through the G1 phase, figure (1) explains (29.84±5.05), Her2-enriched (28.31±6.68) and TPN (28.91±5.51).
the roles of CDKs in the cell cycle (Figure 1).
Ethical approval
The project proposal and sampling method were approved by the
Research Ethics Committee of the college of medicine at the Babylon
University. Also, the project received the permission for ethical research
in the Babylon Center of Oncology/Marjan medical city on 15/4/2018.

Sampling
Venous blood samples were collected from control and breast cancer
patients;8 ml of blood was drawn from each subject by vein puncture, 3ml
was put into EDTA purple tube and stored in deep freeze (-20˚C), to be
used later for the genetic testing. The remaining 5 ml of blood were
dispensed in separating gel tubes, stored at room temperature for 30 min
allowing the blood to clot, and then centrifuged at 2000g for
approximately 15 min. The serum was placed into a small Eppendorf tube
and stored in deep freeze (-20°C), which was later used for biochemical
testing of CDK4 enzyme levels using the ELISA technique.

Determination of Cyclin-Dependent Kinase CDK4 Concentration

Sandwich ELISA kits by Sunlong (China) were used in this study. Known
concentrations of Human CDK-4 Standard and its corresponding reading
OD were plotted on the log scale (x-axis) and the log scale (y-axis)
respectively (Figure 2). The concentration of Human CDK-4 in the
sample was determined by plotting the sample’s OD on the Y-axis. The
Figure 1. Role of CDK4 and CDK6 in Cell Cycle.
actual concentration was calculated by multiplying for the dilution factor.

Mutations in the CDK4 gene, D-type cyclins, p16 INK4a and RB are
Genetic marker Selection
associated with numerous types of cancer, including breast cancer[12].
The SNP: rs2069502 genetic marker was selected based on previous
Our aim was to establish the correlation between rs2069502 CDK4
studies[13][14][15] and by using several databases and software such as
genotypes and breast cancer in Iraqi women.
(NCBI, ensemble, HUGE navigator, GWASdb).
 EUROMEDITERRANEAN BIOMEDICAL JOURNAL 2020, 15 (09) 40-46 42

PCR Primer Design HRMA Analysis

For the targeted genomic region we used the protocol described by[16], One μl of 50XEva green stain was added to real-time products and then
summarized as a flow chart (Table1). The product length was set to 100- subjected to high resolution melting analysis (HRMA), the HRMA profile
200 bp, which was suitable for HRM analysis. The specificity of each is listed in Table 2. The HRMA data were analyzed using Novallele
primer pair was tested by blasting the primers against the human genome (Canon Biomedical USA). The HRMA analysis revealed that our samples
(RefSeq reprehensive genome). Furthermore, each primer was tested for segregated into 3 well-defined clusters.
its ability to form a secondary structure and dimer using the Oligcalc
online software. The amplicon sequences of each primer pair were Genotyping of Genetic Marker rs206950
retrieved from the NCBI sequence viewer (V 3.30.0) as a FAST format, Two random samples from each cluster produced by HRMA were
the amplicon melting curve was simulated using the Umelt online analyzed using conventional PCR and then sequenced according to the
software. company manual (Macrogen, South Korea). After sequencing, the HRMA
analysis of rs2069502 CDK4 genotypes were explained in figure 3. A
chromatogram confirmed the 3 different HRMA patterns, showing an
obvious form of zygosity status in the 3 groups of investigated samples.
The observed variant exhibited three different distributions in the
analyzed samples, normal homozygous status (AA),mutant heterozygous
status (AG) and homozygous status (GG), as seen in figure4.

Statistical Analysis
The general statistical parameters such as mean, standard deviation,
percentage, and descriptive plots were carried out using Microsoft® Excel
Table 1. Accepted primers sets for the selected markers rs2069502
2010 software. The phenotypic Odds ratio was calculated, according to
Altman,[20]using medcalc.net software. While phenotypic means and
standard deviation were compared by student t-test using the
IPM®SPSS® software. The genetic association parameters were
calculated using SNPStats® online software, while genotypes association
was calculated using Fisher’s exact test with the Quickcalcs software from
GraphPad ®.

Table 2. High-Resolution Melting Analysis HRMA Profile

Figure 2. Human CDK-4 Standard Curve

DNA Extraction, spectrophotometry, and Electrophoresis

We followed the standard protocol for DNA isolation[17]and
implemented a modification of the original procedure described by Gross-
Bellard et al [18]. DNA was extracted. DNA quantity and quality were
measured by nano-drop, using the scanning ability of a diode array from
200 to 320 NM wavelength and by calculating the 260/280 and 260/230
ratios. If the 260/280 ratio of the sample was less than 1.8 and /or 260/230
ratio less than 2, re-extraction was performed on the sample. The integrity
and molecular weight of extracted DNA were determined using agarose
gel electrophoresis[19].

Real-time PCR
The reaction mixture and amplification profile were optimized by
Figure 3. Normalized Curve of HRMA analysis of rs2069502 CDK4
applying different primer concentrations and different annealing
temperatures, producing the most efficient and specific amplicon that genotypes
yielded a uniform and clear melting curve.
 EUROMEDITERRANEAN BIOMEDICAL JOURNAL 2020, 15 (09) 40-46 43

3. Results

Our results revealed that there were no significant differences between

breast cancer patients and the control groups as far as CDK4
concentrations, expressed as pg/ml [(3956.7±5494.08),
(3133.14±1031.56) mean±SD respectively, p-value > 0.05]. Also, there
were no significant difference in CDK4 concentration between breast
cancer patients subtypes(p-value > 0.05),the means and standard deviation
of CDK4 (pg/ml) for breast cancer patients subtypes were Luminal A
(2993.91±1273.46), Luminal B (3285.15±1238.0), Her2-enriched
(3154.90±986.10) and TPN (2938.55±1554.0).
Our result revealed that there was no significant difference in both alleles
A and allele G frequencies between breast cancer patients and control
group (p-value >0.05). Allele frequency for patients and control are listed
in table 3.The result from Hardy-Weinberg equilibrium model revealed
that the genotype frequency in both control and breast cancer patient
groups were (p-value =0.640) and (p-value =0.17) respectively,
confirming the principle. Table 4 lists the genotype frequency of the 2
Figure 4. The Pattern of The Observed Substitution Mutation Within
groups and the test result for Hardy-Weinberg equilibrium model.
the DNA Chromatogram of The Targeted 138 bp Amplicons within
An association of rs2069502 with breast cancer and under different
the CDK4 gene. The observed substitution mutation is highlighted
models of inheritance was further tested, revealing that there was no according to its position in the PCR products.
significant association between rs2069502 genotypes and breast cancer,
and the results of the genotypic association are listed in the table 5. The
results in table 6 show a significant difference in the levels CDK4
between A/A and A/G genotypes, and significant difference in the CDK4
level between A/A and G/G genotypes (p-value < 0.05). These results
show that homozygote A/A was associated with a higher serum
concentration of CDK4. While A/G and G/G did not show any association
with serum level of CDK4 (p-value >0.05). Figure 5 illustrates the
comparison between rs2069502 genotypes in serum CDK4 concentration.

Figure 5. Comparison Between rs2069502 Genotypes in Serum CD4

Concentration.

Table 3. Alleles frequency and alleles association of rs2069502

Table 4. rs2069502 genotypes frequency and the P-values of exact test Table 5. Association of rs2069502 genotypes with breast cancer under
for the deviation from Hardy-Weinberg (HW) equilibrium different models of inheritance
 EUROMEDITERRANEAN BIOMEDICAL JOURNAL 2020, 15 (09) 40-46 44

We measured the actual concentration of CDK4,and the results revealed

that there was no significant differences in CDK4 concentration between
breast cancer patients and control groups, and among breast cancer patient
subtypes.
Some studies reported that the enzymatic activity of CDK4 may be
elevated in the breast cancer patients, leading to the continuous cell
proliferation of breast tissue and promoting the occurrence of breast
cancer phenotype.
Therefore, the enzymatic activity and enzyme kinetics should be
considered in future studies of breast cancer in Iraq. In addition, the
measurement of enzymes using another technique, such as gene
expression profiling, should be used instead of measuring concentration
Table 6. Association of rs2069502 Genotypes and CDK4 serum level. using the Elisa technique.
We genotyped a highly polymorphic SNP rs2069502, and after a
mutational analysis of selected polymorphism, the results showed that no
4. Discussion and Conclusions significant allele frequency difference was found in both alleles A and G
of rs2069502 between breast cancer patients and the control group. Kristy
In this study the average age of breast cancer patients was 51.89 ±12.18. ED and et al. were reported in their British population-based study that
Women that reach menopause at the age of 55 have two times the risk for there was no evidence of association for rs2069502 of the CDK4 gene
developing breast cancer compared with woman that reach menopause at with breast cancer [28]. An association of rs2069502 genotypes with
the age of 45 [21]. breast cancer and under different models of inheritance, revealed that
An increased threat of developing breast cancer has been regularly there was no significant association between rs2069502 genotypes and
connected with early age at menarche and late age at menopause when the breast cancer, but A/A genotype showed slight distribution differences
period of exposure to high concentrations of endogenous estrogens was between breast cancer patients and the control group, where the
increased. It is estimated that premenopausal and postmenopausal breast genotyping frequency was 18 and 10 respectively. Also, p-value = 0.094
cancer risk will be reduced by 7% and 3% respectively for every year of is close enough to the level of significance of 0.05, which perhaps could
menstrual cycle delay, especially after the age of 12 [22]. be proven by using a cohort of more than 80 patients, meaning that a
Estrogen production in postmenopausal women is constantly compared to large-scale study should be done to confirm our results.
cyclic premenopausal women. Genetic alterations and mutations in more than half of human tumors
Consequently, the exposure of breast tissue (epithelium and stroma) to occur in the components of the RB/CDK4/cyclin D/p16INK4a pathway [29].
estrogen is continuous in postmenopausal women, leading to an increase CDK4 mutation leads to loss of the binding ability of CDK4 to p16INK4a,
in the hazards of mutations in rapidly proliferating breast tissues[23][24]. inducing the selectivity of tumor growth and increase the activation of
Also, in this study no major changes in age were shown among breast CDK4 in cancer [30]. The CDK4 gene is amplified and over expressed in
cancer patients, classified according to IHC. The IHC in the molecular breast carcinoma [31]. Furthermore, in advanced tumors inducing
classification assessment is extremely useful and should be requested as a senescence to prevent tumor progression by targeting CDK4 alleles was
part of routine diagnosis and for treating patients with breast carcinoma. very important, therapeutic strategies of the pharmacological importance
Luminal A patients often have low-grade tumors and a good prognosis. of CDK4 will be highlighted [31]. As presented in this study, the CDK4
Luminal B, HER2, and TPN types are widely recognized to have poorer gene had a substantial role in cellular proliferation and may have an
survival rates and tumors with higher grades. At diagnosis, patient age important role in many types of cancer. Furthermore, many discrepancies
was an important prognostic factor, young age at diagnosis was correlated were found in studies about the pathological role of the CDK4 gene and
with a worse prognosis. may be attributed to the ethnic and environmental factors.
Breast cancer in younger women had a reduced mRNA expression of ER Our results revealed the presence of a significant difference in the serum
and PR, and an increased expression of HER2. CDK4 level between A/A and G/G, A/A and A/G genotypes. Meaning
WHO proposed the classification of BMI as [25]: that homozygotes A/A was associated with a higher serum level of
1. Overweight grade1 25.0–29.9 kg/m2 CDK4.In some studies, an association between gene expression of the
2. Overweight grade 2 30.0–39.9 kg/m2 CDK4 gene and high CDK4 protein levels was reported in glioblastoma
3. Overweight grade 3 40.0 kg/m2 multiforme and breast carcinoma [32]. Han-X. A. and et.al reported that
Our results show that the means of BMI in breast cancer patient subtypes the immunohistochemical analysis of the tumor tissue specimens of 15
occur between grade 1 and 2 overweight. Obesity is the most significant types of breast cancer with CDK4 gene amplification, and their results
factor for developing breast cancer in postmenopausal women[26]. showed of over expression of the gene and increased levels of the gene
Advanced age leads to the increased synthesis of estrone from product [33].
androstenedione, and would be associated with an increase in adipose
tissue activity [27].
 EUROMEDITERRANEAN BIOMEDICAL JOURNAL 2020, 15 (09) 40-46 45

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genotypes on the functional level of the CDK4 protein. This information Uromodulin rs13333226 and Angiotensinogen rs699 genes variants
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weight DNA from mammalian cells. Eur J Biochem. 1973;36(1):32–
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38.
19. Sambrook J, Russell DW. Molecular cloning: A laboratory manual,
the third edition. Cold spring harbor laboratory press, cold spring
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