Mutation and expression analysis of BRCA2

gene in East Java breast cancer cases

Cite as: AIP Conference Proceedings 2260, 040020 (2020); https://doi.org/10.1063/5.0015899
Published Online: 16 September 2020

I. Kade Karisma Gita Ardana, Zefry Okta Wardana, Vina Rizkiana, et al.

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AIP Conference Proceedings 2260, 040020 (2020); https://doi.org/10.1063/5.0015899 2260, 040020

© 2020 Author(s).
 Mutation and Expression Analysis of BRCA2 Gene in East
Java Breast Cancer Cases
I Kade Karisma Gita Ardana1, a), Zefry Okta Wardana1, 3, 4, Vina Rizkiana1, 3,
Nursasi Handayani1, Hendra Susanto1, Justinus Dwi Pratjojo Wisnubroto2,
Alfiyannul Akhsan2, Vidi Vianney Chrisana Magrit Tanggo2 and Dwi Listyorini1, 3
1
Department of Biology, Faculty of Mathematic and Natural Sciences, Universitas Negeri Malang
Jl. Semarang No. 5, Malang 65145, Indonesia.
2
Department of Oncology, Saiful Anwar Regional Public Hospital
Jl. Jaksa Agung Suprapto No. 2, Malang 65112, Indonesia.
3
Biotechnology Division, Central Laboratory of Mineral and Advanced Material,
Faculty of Mathematics and Natural Sciences, Universitas Negeri Malang
Jl. Semarang No. 5, Malang 65145, Indonesia.
4
PT. GeneCraft Labs Jakarta, Kebon Jeruk Business Park F2-9,
Jl. Meruya Ilir No. 88, Meruya Utara, Jakarta 11620, Indonesia.
a)
Corresponding author: gitaardana1998@gmail.com

Abstract. East, Central, and West Java Provinces have the largest estimated number of people with breast cancer in
Indonesia. Breast cancer can be caused by mutations in the RAD51 domain of BRCA2 genes. The purpose of this study
was to determine the mutations and binding affinity of RAD51 domains of the BRCA2 proteins. This research also
pursued to measure the expression of BRCA2 genes based on TNM stages of breast cancer. Total RNA was isolated from
22 samples provided by Saiful Anwar Regional Public Hospital, East Java, Indonesia. Mutation analysis conducted using
Sanger sequencing. Gene expression analyzed using relative q-PCR with standardized OneWay ANOVA. The results of
this study indicate that the expression of the BRCA2 gene did not increase significantly. Mutation analysis showed that
there was a mutation in the RAD51 domain of the BRCA2 gene in the samples 17 and 19. Mutations that occur did not
affect the binding domain structure area which in turn did not affect the binding affinity between the BRCA2 and RAD51
proteins. Moreover, there were no significant changes throughout the protein structure which in turn did not affect the
binding structure.

INTRODUCTION

Breast cancer is the cancer with the highest prevalence both in Indonesia and globally [1]. Data compiled by
GLOBOCAN and the International Agency for Research on Cancer (IARC) in 2012, showed that there were
14,067,894 new cases of cancer and 8,201,575 cancer related mortality worldwide. New cases of breast cancer are
estimated at ± 1,050,346 every year. The IARC also assumes that in 2020 there will be 1.15 million new cases of
breast cancer with a predicted percentage of 70% of new cases and 55% of deaths in developing countries [1]
Indonesia is a country with highest number of both breast cancer prevalency and mortality in Southeast Asia.
Indonesia estimated has around 48,998 cases of breast cancer recorded with a mortality rate of 19,750 [1]. West
Java, Central Java and East Java are the 3 provinces with the highest contributors to breast cancer related mortality
in Indonesia. Until 2016, 911 women of East Java was diagnosed with breast cancer (1.03%) [2].
Breast Cancer Susceptibility Protein 2 (BRCA2) is a gene that is often studied in breast cancer. This gene is
expressed as a BRCA2 protein that plays a role in maintaining the genome integrity of mammalian cells [2,3,4].
Mutations that occur in the BRCA2 gene have been reported to cause re-occurence of breast cancer accompanied by

The 6th International Conference on Biological Science ICBS 2019

AIP Conf. Proc. 2260, 040020-1–040020-7; https://doi.org/10.1063/5.0015899
Published by AIP Publishing. 978-0-7354-2020-5/$30.00

040020-1
an increase in the probability of contralateral cancer growth [5,6,7]. Studies conducted by Oliver et al. (2009) [8]
show that changes in amino acids in various BRCA2 protein domains cause loss of protein binding affinity with
various other effector proteins such as PALB2, RAD51, and BRCA1 [9,10,11]. The failure to bind BRCA2 with
those proteins causes disruption of DNA repair due to Double Strand Break (DSB) so that cells may divide
uncontrollably [12,13,14]. In case of repairing DSBs, BRCA2 works by binding to the RAD51 protein to do
homologous recombination. DSB repair initiated by the BRCA2 protein is carried out through the Double Holliday
Junction (DHJ) pathway [15]. Mutations that occur in the RAD51 domain of BRCA2 protein can cause a loss of
binding ability between the two proteins [16]. Point mutations, missense, non-sense, and deletions that occur in this
domain are reported to cause about 30-40% of breast cancer events in the age range of 15-80 years [17].
The explanation above shows that, studies on the expression and mutation of the BRCA2 gene are important in
efforts to determine better genetic prognostic factors for breast cancer in Indonesia, especially in East Java.

MATERIALS AND METHODS

This research was conducted in the Biotechnology Laboratory Division, Central Laboratory of Advanced
Minerals and Materials, Faculty of Mathematics and Natural Science, Universitas Negeri Malang. This research was
performed from August 2018 until January 2019.

RNA Isolation and cDNA Synthesis

The fresh biopsy samples were taken from the Oncology Surgical team Saiful Anwar General Hospital, Malang.
Samples were selected using standard breast cancer diagnostic methods. Samples were stored in 70% alcohol and
distributed in cold conditions to the Biotechnology Laboratory Division, Central Laboratory of Advanced Minerals
and Materials, Faculty of Mathematics and Natural Science, Universitas Negeri Malang. This study has been
declared eligible to conduct, with the ethical number: 400/209 / K.3 / 302/2018. Samples were then disrupted using
TissueLyzer® JICA. RNA total isolation was performed using Qiagen Qiazol® with the protocols that are already
provided. Total RNA quantity and quality was then measured using NanoDrop® Spectrophotometer Thermo. RNA
integrity was measured using agarose gel electrophoresis. RNA is then synthesized into cDNA using a ReverTra
Ace® Toyobo Reverse Transcription KIT. The quantity and quality of the cDNA then measured using NanoDrop®
Spectrophotometer Thermo.

qPCR and Gene Expression Measurement

qPCR was performed using QuantiNova® SYBR Green qPCR KIT Qiagen. The qPCR was performed in
RotorGene Q ® Qiagen. Primers that used in the amplification process consist of: BRCA2 F: 5’ TGT TAA GTG
GAG AAA GGG TTT TGC 3’ and BRCA2 R: 5’ CTG GCG CTT TGA AAC CTT GA 3’. -actin used as a control
housekeeping gene with the primers: -Actin F: 5'-CTT CCT TCC TGG GCA TG-3 ', -Actin R: 5'-GTC TTT GCG
GAT GTC-3'. PCR reactions were performed with 40 cycles using the profile that is consist of denaturation at the
temperature of 95° C for 20 seconds, annealing at 60° C for 10 seconds, and extension at 72 o C for 20 seconds. Ct
value is then calculated using the equation of 2Ct to determine the level of gene expression as described in 18. The
value of the expression levels then analyzed using ANOVA to specify the significance of gene expression between
samples and stadium.

Mutation and Protein Modeling Analysis

The electrophoresis was performed using 1% agarose gel with 1% TBE Buffer in 50 volts for 60 minutes. The
amplicon then sent to Fist-Base Malaysia for sequencing. Sequencing result aligned using control sequence from
NCBI. Sequence than translated into amino acid and uploaded into SwissModel webserver for modelling [19]. The
model from the mutant and normal sample than quantitatively aligned to see the changes within the nucleic acid
alteration. The docking was performed using PatchDock whilst the quality of the docking then measured in
FireDock [21,22].

040020-2
 RESULTS
Of the 22 samples analyzed, mutation occurs only in sample 17 and 19 (Fig. 1). Significant mutations were seen
in sample 17 which is a missense mutation. This mutation occurs in the 2041th amino acids which are in the coil gap
region between the Repeat 7 and Repeat 8 domains of the BRCA2 protein (Fig. 2). This mutation causes the serine
amino acid substitution to become proline. Mutations occurred, do not affect the binding affinity of the BRCA2
protein with RAD51 protein (Table 1). Mutations also do not affect the binding structure topology between the
BRCA2 and RAD51 proteins (Fig. 3).

FIGURE 1. Alignment result for 22 samples analyzed. Alignment result shows that mutations only occur in sample 17 and 19.
Mutation type occured in both samples is substitutive.

FIGURE 2. Amino acid alignment from sample 17 and 19. Only sample 17 showed changes within its amino acid sequence.
Substition occured in amino acid number 2041 from serine into prolin. Sample number 19 did not shows any differences
compared to control.

040020-3
 a b

FIGURE 3. Docking result from sample 17 (a) and sample 19 (b). The mutation that occur on sample 17 does not
affect the structure of BRCA2 protein segment (displayed in rainbow) indeed sample 19, so it did not affect the
binding topology of both RAD51 (displayed in gray) and BRCA2 protein.

TABLE 1. The binding affinity score comparation of BRCA2 and RAD51 protein from sample 17 and control sequence from
UniProt (RSCPDB:1nOw). The result showed indifferences between normal and mutant sequences. The atomic contact energy
between the two models remain the same.
ACE
No Receptor Ligand Transformation
(kJ*Mol-1)
RAD51 ATPase
BRCA2_REPEAT8_NO -1.41; 0.73; -1.41;
1 Domain -556.36
RMAL 27.18; 64.90; 6.42
(RSCPDB:1nOw)
RAD51 ATPase
BRCA2_REPEAT8_MU -1.41; 0.73; -1.41;
2 Domain -556.36
TAN17E 27.18; 64.90; 6.42
(RSCPDB:1nOw)
RAD51 ATPase
REPEAT8_CONTROL -1.41; 0.73; -1.41;
3 Domain -556.36
(RSCPDB:1nOw.B.) 27.18; 64.90; 6.42
(RSCPDB:1nOw)

From the results of the 22 samples analyzed, the expression of the BRCA2 gene was significantly different (P-
Value <0.001). The highest expression was shown by sample 3 (Fig. 4). BRCA2 gene expression did not differ
significantly by stage. Stage IVA shows a slightly higher expression compared to another 2 stages analyzed (Fig. 5).

BRCA2
3
FoldChange

0
10
11
12
13
14
15
16
17
18
19
20
21
22
1
2
3
4
5
6
7
8
9

Sample

FIGURE 4. Relative expression of BRCA2 gene from 22 samples.

040020-4
 BRCA2
1.0

0.8

FoldChange
0.6

0.4

0.2

0.0

IIA

IV
III
Stadium

FIGURE 5. Relative expression of BRCA2 based of the sample stadium.

DISSCUSSION
The results of data analysis showed that the mutations occurred in sample 17 did not cause changes in the
Atomic Contact Energy (ACE) bond between the BRCA2 protein and RAD51. Mutant BRCA2 found in sample 17
still has a full key amino acid to bind to the RAD51 protein [20]. Mutations that occur in sample 17 are in the gap
area between BRC Repeat 8 and Repeat 7 so that it does not affect the bond between the BRCA2 protein and the
RAD51 protein [20,21]. The emergence of cancer in this case is thought to be due to mutations that occur in another
domain that has a significant influence on the appearance of breast cancer. RAD51 binding domain of BRCA2
protein is not the only functional domain associated with breast cancer. BRCA2 protein has many other domains that
have a significant relationship to the emergence of breast cancer. There are 8 BRC Repeat [22,23], POLH binding
site [24], and SEM1 binding site [25] which also plays a major role in breast and ovarian cancer cases. Hormonal
regulation pathways [26], genetic regulation pathways such as Wnt and MAPK regulation pathway [27] are also
reported to play a major role in the emergence of breast cancer, therefore the absence of mutations in the RAD51
domain cannot yet be a diagnosis and prognosis factor for the emergence of breast cancer especially in this case
[17,28,29].
BRCA2 gene expression also did not show any difference based on stage level. Differences in expression are
actually apparent by the sample. Of the 22 samples analyzed, sample 3 showed the highest expression pattern. The
BRCA2 gene works as a tumor suppressor gene so that in the event of a tumor its expression tends to be constant
from stage to stage [30]. BRCA2 gene expression increases when genomic damage occurs, especially as the case of
Double Strand Break (DSB) [6,23]. Expression of the BRCA2 gene is said to be increased due to UV induction, its
performance coincided with the BRCA1 and RAD51 proteins [15]. Wang et al (2018) reported that the BRCA2 gene
expression was not able to be a prognostic factor because the expression of this gene was only correlated based on
tumor size.

CONCLUSION
The BRCA2 gene point mutations in the RAD51 domain occur in 2 samples, 17 and 19. The mutations that
occur in samples 17 and 19 not cause changes in the Repeat 8 domain and not affect the bond between the BRCA2
protein and the RAD51 protein. BRCA2 gene expression differs significantly by sample but not significantly by
stage.

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 ACKNOWLEDGMENTS
We thank to dr. Wisnubroto, dr. Alian Akhsa, dr. Prisca, and all the doctor from Surgical Oncogene
Department Saiful Anwar General Hospital for their cooperation in this research. Generous thank also be devoted to
the Pt. GeneCraft Labs who had been willing to lend tools and their methods during this research process.

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