AIP Conference Proceedings 2092, 040022 (2019); https://doi.org/10.1063/1.5096755 2092, 040022
© 2019 Author(s).
Polymorphism detection of gene
CDH1(-160C>A) in head and neck cancer patient and Indonesian healthy individual population
Cite as: AIP Conference Proceedings 2092, 040022 (2019); https://doi.org/10.1063/1.5096755 Published Online: 09 April 2019
Nisa Ajeng Puspitasari, Yurnadi Hanafi Midoen, Nurtami Soedarsono, Niniarty Djamal, Triana Marchelina, and Elza Ibrahim Auerkari
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Polymorphism Detection of Gene CDH1(-160C>A) in Head and Neck Cancer Patient and Indonesian Healthy
Individual Population
Nisa Ajeng Puspitasari
1, Yurnadi Hanafi Midoen
2, Nurtami Soedarsono
1, Niniarty Djamal
1, Triana Marchelina
1, Elza Ibrahim Auerkari
1,a)1Department of Oral Biology, Faculty of Dentistry, Universitas Indonesia, Jalan Salemba Raya No. 4, Jakarta Pusat, 10430, Indonesia
2Department of Medical Biology, Faculty of Medicine, Universitas Indonesia, Jalan Salemba Raya No. 4, Jakarta Pusat, 10430, Indonesia
Corresponding author: a)[email protected]
Abstract. E-cadherin (CDH1) gene plays a role in cellular interaction to maintain connection between cells. Loss of CDH1 gene function can affect the development of cancer. Genetic polymorphism CDH1 (-160C> A) is associated with head and neck cancer (HNC). The primary goal of this study is to detect genetic polymorphisms of CDH1 (-160C> A) in HNC patients and Indonesian healthy individual population. The number of samples used is 100 samples consisting of stored DNA samples of 50 healthy individuals and 50 HNC patients. CDHI1 polymorphism was analyzed by the PCR- RFLP method using the HincII restriction enzyme and visualized with electrophoresis. As the result, genetic polymorphisms of CDH1 (-160C> A) were detected in HNC patients by 78% and in healthy individuals by 68%. Neck and head cancer samples has a CC (non-polymorphic) percentage of genotype of 22%, CA ((heterozygous) genotype of 52%, and AA ((homozygous) genotype of 26%.
Healthy individual samples has a CC percentage of genotype of 32%, CA genotype of 56%, and AA genotype of 12%. This study shows that genetic polymorphism of CDH1 (-160C> A) increases the risk of HNCs in the Indonesian population.
INTRODUCTION
Cancer is one of the major diseases that cause death in the world. Based on GLOBOCAN data, the International Agency for Research on Cancer (IARC) in 2012, about 8.2 million people had died from cancer. In addition, based on the Riset Kesehatan Dasar (Riskesdas) in 2013, the prevalence of cancer in Indonesia had reached 1.4% in all age groups. In Indonesia, cancer is the number seven causes of death with a 5.7% percentage of all causes of death.
Cancer of the head and neck ranks sixth from a variety of cancers in the world with a prevalence of more than 550,000 new cases and has a death rate of 300,000 every year. In Indonesia, head and neck cancer has a high rate with a prevalence of 4.7 per 100,000 population with the most frequent incidence of squamous cell carcinoma of the head and neck region with a percentage of 90%. This occurs in the epithelial lining of the oral cavity, oropharyngeal, larynx, and hypopharynx.
Etiology of head and neck cancer involves several risk factors that are external and internal factors.
External factors include tobacco and alcohol consumption. Individuals who consume alcohol and tobacco plus a bad lifestyle have a higher risk of getting cancer than individuals with a healthy lifestyle. In addition, recent research suggests that head and neck cancer is associated with infection of human papilloma virus (HPV) 16 and 18.
Internal factors of risk of head and neck cancer are genetic factors. Several studies have published that there is a relationship between genetic polymorphism and carcinogenic metabolism. This genetic
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polymorphism may affect the susceptibility of individuals to carcinogenic substances, so that individuals are more susceptible to disease. Research by Bondy et al. showed that families have an important role in the risk of developing cancer. In addition, genetic changes involving tumor suppressor gene p16 and p53 are in great demand by researchers in conducting research on head and neck cancer.
In addition to the p16 and p53 genes, there is another gene that play a role in the disease of head and neck cancer which is E-cadherin (CDH1). The CDH1 gene has a role in regulation and cell communication to maintain intercellular relationship. Loss of function from CDH1 may have an effect on the development of cancer and may affect individual susceptibility to head cancer of the neck.
Various studies on CDH1 genetic polymorphisms associated with risk of head and neck cancer in the esophageal area (Akbas et al and Zhang et al.) and the oral cavity area (Chien et al.) have been performed with different results. However, there is no data on the genetic polymorphism in the population of head cancer patients in Indonesia. Therefore, this study was conducted to obtain preliminary data on whether there is a genetic polymorphism of CDH1 (-160C> A) with risk of head cancer of the neck in the population in Indonesia, so that genetic polymorphism of CDH1 (-160C> A) can be used as one of biomarkers to establish whether a person is at risk for head and neck cancer.
METHODS
This research is a laboratory research using descriptive analysis. The research sample used was biological material stored in the form of DNA extraction from blood of 50 head and neck cancer patients and 50 Indonesian healthy population, which was stored in the laboratory of Oral Biology Faculty of Dentistry, University of Indonesia. The research steps undertaken to detect the genetic polymorphism features of CDH1 (-160C> A) in patients with head and neck cancer and also in healthy individuals were as follows:
1. Amplification of DNA by PCR Method
The first step was dissolving primers with TE buffer. Forward primer was dissolved with 236 μL TE buffer while reverse primer was dissolved with 272 μL TE buffer. The specific primer used was forward: 5'- GCCCCGACTTGTCTCTCTAC-3' and reverse: 5'-GGCCACAGCCAATCAGCA-3'. The next step was to make PCR mix in one PCR tube containing 25 μL reactant consisting of 0.5 μL (1ng / μl) DNA, 12.5 μL PCR mastermix, 0.75 μL (10μmol) primary forward, 0.75 μL (10μmol) reverse primer, and 10.5 μL ddH2O.
Then, PCR mix was mixed by pipetting and spinning down to be homogeneous. After that, PCR mix was amplified using a PCR machine with an initial denaturation condition of 94°C for 3 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 61°C for 30 seconds, and extension at 72°C for 30 seconds.
All steps were done at 30 cycles. After the extension, the final extension was 72°C for 5 minutes.
2. Electrophoresis and Visualization of PCR Products
The first step in conducting electrophoresis was making 1.5% agarose gel in 60 ml total volume of 60 ml of TAE 1x solution in Erlenmeyer tube. This mixture was introduced into the microwave with high temperature for 5 minutes. After that, the Erlenmeyer tube was lifted and left to no heat by placing it on a shaker orbital at 90 rpm. When stirring, 1 μL GelRedTM Nucleid Acid Gel Stain was added to the agarose mixture until evenly distributed. After that, the agarose mixture was poured into a gel agarose mold and let hardened for 10-15 minutes. After hardening, the gel comb was lifted so as to form a well mold of 15 or 20.
Then the hardened gel was transferred into an electrophoresis tank filled with a 1x TAE buffer solution until completely submerged.
3. Digestion with Restriction Enzyme
After obtaining the appropriate PCR product visualization results (447 bp), restriction enzymes were incorporated into PCR products. Restriction enzyme used in CDH1 gene were HincII. The composition of the RFLP mix consisted of 10 μL PCR products, 0.1 μL HincII enzyme, 2 μL buffer, and 5.9 μL ddH2O.
Then, RFLP mix was mixed by pipetting and spinned down until become homogeneous. After mixing evenly, tubes containing the RFLP mix were inserted into the incubator at 37°C for 16 hours. After 16 hours, the RFLP product was removed from the incubator and inactivated with thermoblock at 65°C for 20 minutes.
4. RFLP Product Electrophoresis and Visualization
The RFLP results were electrophoresed using 2% agarose gel. The gel is prepared by mixing 1.2 grams of agarose powder with 60 mL TAE 1x in Erlenmeyer tube. Then, the agar mixture was heated using a microwave for 5 minutes and placed on top of the orbital shaker (90 rpm) and added by 1 μL GelRedTM Nucleid Acid Gel Stain after the temperature decreased. After that, the agarose mixture was poured into agarose mold and let hardened for 10-15 minutes. The gel mold was then transferred into the electrophoresis tank. After that, the RFLP product was placed at each well and one well was used to be filled with ladder
DNA. Electrophoresis was run with a voltage of 60 V and 400 mA for 40 minutes. The electrophoresis results were visualized using Gel Doc. The RFLP product visualization would showed three different results, namely three pieces of ribbon (447 bp, 367 bp, and 80 bp), two band pieces (367 bp and 80 bp), and one band (447 bp). In the first result there was an RFLP product with some DNA cut off by a restriction enzyme and partly not cut off or part of the DNA had a variation of the base order so that it was seen as three pieces of ribbon. In the second result there was an RFLP product with all DNA cut off by a restriction enzyme so that it was seen as two band pieces. In the third result there was an RFLP product with all DNA not cut off by restriction enzymes so that only one piece of ribbon was seen.
5. Data Analysis
The data that has been obtained is then analyzed by descriptive process. The results are displayed in the form of narration and tables.
RESULTS
In the PCR product visualization results can be seen one band of 447 bp as follows.
FIGURE 1. Product Visualization Results of PCR Gen CDH1 (-160C> A)
Figure 1 shows the visualization results of electrophoresed PCR products. In the first column there is a ladder DNA of 50 bp. In the next columns are samples of numbers 1, 2, and 3 showing PCR results at 447 bp. The last column is a negative control.
After that, the PCR product that has shown 447 bp is then done by RFLP and following results are obtained.
FIGURE 2. Product Visualization Results RFLP Gen CDH1 (-160C> A)
Figure 2 shows the visualization results of the RFLP products that have been electrophoresed. In the first column there is a ladder DNA of 50 bp. In the second column, one band shows at 447 bp indicating CC (homozygous) genotype. In the third column, three bands of 447 bp, 367 bp, and 80 bp show the CA genotype (heterozygote). In the fourth column, two bands of 367 bp and 80 bp show the genotype AA (homozygous). In the fifth column is a negative control.
The results of the distribution of genetic polymorphisms of CDH1 (-160C> A) in patients with head cancer of the neck and healthy individuals of Indonesian population can be seen in the following table.
447 bp
1 2 3 Kontrol(-)
447 bp 367 bp 80 bp 1 2 3 Kontrol
(-)
CC CA AA
TABLE 1. Distribution of Genetic Polymorphisms CDH1 (-160C> A)
Polymorphism Head and Neck Cancer Healthy Individual Genotype Polymorphic
Non-polymorphic 78%
22% 70%
30%
Allele Polymorphic Non-polymorphic
52%
48%
40%
60%
Table 1 shows the percentage of polymorphic genotypes in cancer patients of head and neck (78%) and polymorphic genotypes in healthy individuals (70%). The polymorphism distribution seen from polymorphic alleles in patients with head and neck cancer was 52% and polymorphic alleles in healthy individuals was 40%.
In the genetic polymorphism of CDH1 (-160C> A) there are three genotypes: CC (homozygous) genotype, CA (heterozygous) genotype, and AA (homozygous) genotype. Below is the genotype frequency distribution table and allele of genetic polymorphism CDH1 (-160C> A).
TABLE 2. Genotype Distribution and Allele Genetic Polymorphism of CDH1 (-160C> A) Head and Neck
Cancer Healthy
Individual Genotype
CC CA AA
11 (22%) 26 (52%) 13 (26%)
16 (32%) 28 (56%) 6 (12%)
Allele C
A 48 (48%)
52 (52%) 60 (60%)
40 (40%)
Table 2 shows the genotype and allele frequencies in both groups. Genetic polymorphism CDH1 (-160C>
A) changes from C allele to allele A. C allele is a wild type allele (non-polymorphic) and allele A is a mutant allele (polymorphic). Therefore, the genotype CA and AA are polymorphic genotypes whereas the CC genotype is a non-polymorphic genotype. The most frequent genotype frequency was CA genotype with 52% percentage in head and neck cancer patients and 56% in healthy individuals. The most frequent allele frequency in head and neck cancer patients is the A allele with a percentage of 52%. In addition, the most frequent allele frequencies in healthy individuals are C alleles with a percentage of 60%.
DISCUSSION
Research on genetic polymorphism of CDH1 (-160C> A) was performed on head and neck cancer patients and also in healthy individuals of Indonesia population. Neck and head cancer samples had a CC percentage of genotype of 22%, CA genotype of 52%, and AA genotype of 26%. Healthy individual samples had a CC percentage of genotype of 32%, CA genotype of 56%, and AA genotype of 12%. This study showed polymorphism results in neck head cancer samples (78%) were higher than healthy individual samples (68%).
Several previous studies on genetic polymorphisms of CDH1 (-160C> A) associated with risk of neck and head cancer showed different results. Research by Zhang et al. (2008) on the genetic polymorphism of CDH1 (-160C> A) in the Chinese population consisted of 333 samples of head and neck cancer and 343 healthy individual samples. Cervical head cancer samples had CC percentage of genotype 63.1% and genotype CA + genotype AA of 36.9%. Healthy individual samples had a CC genotype percentage of 66.5%
and genotype CA + genotype AA of 33.5%. This study showed the percentage of polymorphism in head and neck cancer sample (36.9%) higher than the sample of healthy individuals (33.5%).
Another study conducted by Akbas et al. (2013) on the genetic polymorphism of CDH1 (-160C> A) in the Turkish population consisted of 98 samples of head and neck cancer and 105 healthy individual samples.
Head and neck cancer samples had CC percentage of genotype of 32%, CA genotypes of 57%, and AA genotype of 11%. Healthy individual samples had a CC percentage of CC genotype of 42%, CA genotype of 51%, and AA genotype of 7%. This study showed the percentage of polymorphism in head and neck cancer samples (68%) higher than the sample of healthy individuals (58%).
In addition, research on genetic polymorphism of CDH1 (-160C> A) performed by Chien et al. (2012) in the Taiwan population consisted of 347 samples of head cancer of the neck and 251 healthy individual samples. Head and neck cancer samples had CC genotype percentage of 35.16%, CA genotype of 47.84%, and AA genotype of 17%. Healthy individual samples had CC genotype percentage of 48.61%, CA genotype of 43.28%, and AA genotype of 7.57%. This study showed the percentage of polymorphism in head and neck cancer samples (64.84%) higher than the sample of healthy individuals (50.85%).
Research by Zhang et al. (2008), Chien et al. (2012), and Akbas et al. (2013) showed that the percentage of genetic polymorphisms in head and neck cancer patients were higher than healthy individuals. This is in accordance with the results of research on genetic polymorphism CDH1 (-160C> A) in the Indonesian population. In addition, the most dominant genotype frequency in both groups in the Indonesian population was the CA genotype. This is in accordance with research conducted by Akbas et al. (2013) in the Turkish population. However, the study by Chien et al. (2012) in the population of Taiwan showed different results, the dominant genotype in head and neck cancer patients was CA genotype, whereas the dominant genotype in healthy individuals was the CC genotype. In the study of Zhang et al. (2008) in the Chinese population, the dominant genotype in both groups was the CC genotype. Based on the comparison of research results on genetic polymorphisms of CDH1 (-160C> A) in some populations, the distribution pattern of genetic polymorphisms CDH1 (-160C> A) is not affected by race because both in Mongoloid races (Indonesia, China and Taiwan) as well as in Caucasian race (Turkey) showed the same result. Variations from the results of the study may be influenced by some risk factors such as tobacco use, alcohol consumption, or other risk factors.
This study is a preliminary study that aims to look at the feature of genetic polymorphism CDH1 (-160C>
A) in patients with head and neck cancer and also in healthy individuals of the Indonesian population.
However, this study has limited sample size and lack of demographic data, so further research on the relevance of CDH1 genetic polymorphism (-160C> A) to head and neck cancer patients with demographic variables and other cancer risk factors is necessary.
CONCLUSION
Based on this research, it can be concluded that there is a genetic polymorphism of CDH1 (-160C> A) in patients with head and neck cancer and in healthy individuals of Indonesian population. There are three genotypes of genetic polymorphisms CDH1 (-160C> A) which are wild type homozygotes (CC), mutant homozygous (AA), and mutant heterozygotes (CA), in head and neck cancer patients and healthy individuals of the Indonesian population. Two alleles of genetic polymorphism CDH1 (-160C> A) are detected that are wild type alleles (C) and mutant alleles (A) in head and neck cancer patients and healthy individuals of Indonesian population
.
SUGGESTION
Further study about relevance of CDH1 genetic polymorphism (-160C> A) to the risk of head and neck cancer which accompanied by demographic data is needed to be done.
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