Mutation screening of the BRCA1 gene in sporadic breast cancer in the Central of Iran

Accepted Manuscript Mutation screening of the BRCA1 gene in sporadic breast cancer in the Central of Iran

Mandana Behbahani, Mokhtar Nosrati, Hassan Mohabatkar PII: DOI: Reference:

S2214-5400(18)30045-8 doi:10.1016/j.mgene.2018.04.005 MGENE 430

To appear in:

Meta Gene

Received date: Revised date: Accepted date:

7 February 2018 5 April 2018 5 April 2018

Please cite this article as: Mandana Behbahani, Mokhtar Nosrati, Hassan Mohabatkar , Mutation screening of the BRCA1 gene in sporadic breast cancer in the Central of Iran. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Mgene(2017), doi:10.1016/j.mgene.2018.04.005

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ACCEPTED MANUSCRIPT Mutation screening of the BRCA1 gene in sporadic breast cancer in the Central of Iran

Mandana Behbahani1 *, Mokhtar Nosrati1, Hassan Mohabatkar1

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*Corresponding author Tel.: +98 311 7934327; Fax: +98 311 7932342.

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Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran

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E-mail addresses: [email protected]

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Abstract The purpose of the work was to study mutation screening of BRCA1 in Iranian patients with sporadic breast cancer. We carried out a mutational analysis of BRCA1 gene in 101 breast cancer patients from a population in Central of Iran. The comparison of DNA of paraffin-embedded breast cancer tissue from patients was studied, and breast tissue from 30 unrelated normal

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women without cancer was selected as controls. The entire BRCA1 coding sequence was

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amplified by PCR with primers especially designed for comprehensive mutation screening by

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single-strand conformation polymorphism (SSCP) analysis. The PCR products revealing abnormal SSCP migration pattern were sequenced. Then, in silico investigation was performed

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to identify the effects of new mutations on stability and function of BRCA 1.A total of ten nucleotide alterations were observed in the breast cancer tissue DNA. Six cases of single

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nucleotide changes in BRCA1 were detected in the study without records in the BIC database consisted four polymorphism in exon 11 (1543 Del G, 1597 C>T, 2246 Del T, 2612 C>G), Del

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one missense mutation in exon 7 (490 C>T), and one deletion mutation in exon 10 (743

C). No nucleotide alterations were detected in the controls. In addition, the results of in silico

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analysis indicated that three mutations including 2612C>G, 1543 Del G and 743 Del C were not recorded in the especial database. Furthermore, the results of molecular docking studies

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confirmed that 2612C>G could change physicochemical properties of BRCA1.The findings of the present study suggest that screened mutations may have an important role on the incidence of

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breast cancer in women.

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Keywords: Breast cancer; SSCP; Mutation.

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1. Introduction

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Breast cancer is one of the most common malignancies affecting women, with a lifetime risk of 10% and remains the leading form of cancer in women in Iran (Radmard 2010). The cancer is one of the most frequent malignancies among Iranian women with prevalence of 120/100000 (Tazhibi, Dehkordi et al. 2014). Based on the statistics of 2005-2006 about 10% of all breast cancer cases in Iran are reported in Isfahan (Otaghvar, Hosseini et al. 2015). Approximately 5– 10% of breast cancers are of hereditary origin, and to date two major breast cancer susceptibility genes, BRCA1 and BRCA2 are identified. It was initially suggested that these two genes would be responsible for most cases of inherited breast cancer (Ramus and Gayther 2009). Since the first cloning and sequencing of the BRCA1 gene (Miki, Swensen et al. 1994), the spectrum of disease associated mutations in various populations has been investigated in detail. The BRCA1 gene encodes a 1863-amino acid protein with a single large region, exon 11, encoding some 60%. The gene is highly polymorphic, with many common single-base exon changes. The mutations of BRCA1 are distributed widely over the entire gene; thus, it is necessary to screen the entire gene in order to detect mutations of BRCA1. Most mutations in the BRCA1 gene that are identified to date are point mutations or small insertions and deletions scattered throughout coding sequences (Brown and Solomon 1994). In addition, a significant correlation between the location of the mutation and the ratio of breast to ovarian cancer the incidence is reported (Gayther, Warren et al. 1995). Various methods based on gel electrophoresis are traditionally used in mutation detection to

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separate the wild-type sequence from the mutant ones. The methods based on gel separation are simple to run and do not require large investments in equipment. Single strand conformation

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polymorphism (SSCP) is a commonly used method for mutation screening. It enables rather quick and reliable detection of variations in DNA sequence, but does not provide information

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about the type or exact location of the variation. Generally, for identification of the alterations detected in screening, the DNA fragments must be sequenced (Holmila and HusgafvelPursiainen 2006). Experimental determination of gene alternations is tedious and costly. Currently, bioinformatics methods are highly regarded to determine new mutations and to predict their effects on physicochemical property of proteins (Mooney, Krishnan et al. 2010, Hassan, Omer et al. 2016, Quan, Lv et al. 2016). Some bioinformatics methods such as molecular docking, molecular dynamics and homology modeling could predict the mutation type, as well as the effect of mutations on function, stability and the structure of proteins with high accuracy (Doss, George et al. 2014, Nagasundaram, Zhu et al. 2015). The purpose of the work was to 3

ACCEPTED MANUSCRIPT study the mutation screening of BRCA1 in the Iranian patients with sporadic breast cancer using the PCR–SSCP sequencing method. In this study, we analyzed mutation screening of BRCA1 in Iranian patients with sporadic breast cancer using the PCR–SSCP sequencing method. Furthermore, the screened mutations in BRCA 1 are compared with recorded data in special database and the effects of the mutations on the stability and function of protein is studied using

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some special server as well as molecular docking method.

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2. Material and methods

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2.1. Patient samples and DNA extraction

Patient samples were drawn from four medical centers in Isfahan, Iran. We retrieved 101

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formalin-fixed, paraffin-embedded tissue blocks from women with breast cancer diagnosed, the age of 25−57 years for the years 2006 and 2007. The sample size was determined based on

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power desired 85%, significance level desired 5% and margin of error 5% using online sample size calculator at http://www.raosoft.com/samplesize.html.The samples were analyzed for

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BRCA1 mutations. Sections (8 μm) of each paraffin-embedded tissue were cut using a standard microtome with a fresh disposable blade, and each section was placed in a microfuge tube. Each

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section was deparaffinized by extraction with 500 μL of xylene for 30 minutes at room temperature. Samples were suspended in 300 μL of 500 mM Tris-HCl, pH 9.0, containing 20

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mM EDTA, 10 mM NaCl, 1% (w/v) sodium dodecyl sulfate, and 150 μg of proteinase K. Samples were incubated at 56°C overnight, 100°C for 10 minutes, and cooled to room

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temperature. Samples were extracted twice with 300 μL of phenol, pH 8.0 chloroform (1:1, v/v) by centrifugation at 13,000g for 2 minutes to separate the lower phenol/chloroform phase from

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the upper aqueous phase containing DNA. The upper phase was transferred to a new tube and incubated with 100 μL of 10 M ammonium acetate, 3 μL of glycogen (1 mg/mL), and 900 μL of ethanol at -80°C for 30 minutes. Samples were centrifuged at 13,000g for 15 minutes at 4°C, and recovered pellets were washed with 500 μL of 70% ethanol at 4°C. The pellets were air dried and solubilized with 50 μL of 10 mM Tris-HCl, pH 7.5, containing 1 mM EDTA. DNA was also isolated from a healthy human placental tissue (DiCioccio and Siniscalco 1975) to serve as a normal control for mutation analysis of BRCA1. The preparation of tissue sections, isolation of DNA, and subsequent amplification of DNA by the polymerase chain reaction were conducted in separate rooms. 4

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2.2. Polymerase chain reaction The sequences of twenty-one primer pairs used to amplify exons, were obtained from the Human Genome Database (GDB) (http://www.gdb.org/), and primers were purchased from Invitrogen Germany. Amplification of DNA fragments was performed in a Corbett thermocycler in 10 ml of solution containing 50 mM KCl, 10 mM Tris–HCl (pH 9.0 at 25°C), 0.1% Triton X-100, 1.5

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mM MgCl2, 125 mM each dNTP, 10 pmol of each primer, 0.4 units of Tag DNA polymerase

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(roche), and 100 ng of genomic DNA. The DNA solution was pre-heated to 94°C for 5 min and

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then subjected to 35 cycles of varying temperatures and times: each cycle consisted of 30 s at 94 °C, 30 s at 48–62 °C, and 1 min at 72 °C, with a final extension of 5 min at 72 °C. The quality

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of amplification was determined by separation of the PCR products on a 2% agarose gel (in

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1×TAE) at 120 V for 40–50 min.

2.3. Single-strand conformation polymorphism analysis (SSCP) PCR products were diluted two-fold with a formamide denaturing buffer (95% formamide,

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10mM NaOH, 0.05% bromophenol blue, 0.05% xylene cyanol), heated at 95 ◦C for 5 min,

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chilled on ice for at least 5 min and 7µl were loaded onto a non-denaturing 10% polyacrylamide gel (49:1 acrylamide: bisacrylamide). Electrophoresis was run at 70 V, in 0.5X Tris-borate-

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EDTA buffer for 15 h, at approximately 24 ◦C, and silver stained as described elsewhere (Bassam, Caetano-Anollés et al. 1991). Patterns of single-stranded DNA were checked for

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abnormal mobility. Abnormal DNA fragments detected in SSCP analysis were eluted from the dried gels and amplified by PCR for 30 cycles with sequence primers and were subjected to

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direct sequencing.

2.4. DNA sequencing

All fragments showing an abnormal migration on SSCP were sequenced on both strands. The sequencing reactions were performed with a dye-labeled terminators kit using the cycle sequencing method. Both separations of DNA fragments and sequence analysis were performed in an ABI PRISM automated sequencer (Model 310 version 3.0, ABI-CE1 version 3.0).

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2.5. In silico analysis 2.5.1. Sequence analysis of mutations

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To determine the new mutations of BRCA1, the screened mutations were compared with mutations documented in BRCA1 mutations databases available at http://arup.utah.edu/database/BRCA/.

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2.5.2. Mutation effect on protein stability

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The effect of the mutations on total protein stability was evaluated using MUpro server at http://mupro.proteomics.ics.uci.edu. This server is a set of machine learning programs to predict

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how a single-site amino acid mutation affects protein stability. The outputs of the server are two types of A scores based on two different algorithms consisting Support Vector Machine and

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Neural Network. A score less than 0 means the mutation decreases the protein stability. 2.5.3. Mutation effect on functional domain(s)

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To determine the effect of the SNP on functional domain of BRCA1, the protein was scanned to define the important domains using PinSnps server at http://fraternalilab.kcl.ac.uk/PinSnps/faces/index.xhtml;jsessionid=41C253486D12E08E20BEB1 E6473B5754 . At first, amino acid sequence and three-dimension structure of the domain was retrieved from NCBI (https://www.ncbi.nlm.nih.gov/) and protein data bank (https://www.rcsb.org) respectively. Then, the domain, which was affected by the mutation, was selected for further analysis. The effect of determined SNP(s) on the domain stability was studied using instability index in Protparam server at http://web.expasy.org/protparam/, I-Mutant server at http://folding.biofold.org/i-mutant/i-mutant2.0.html. and MUpro server.

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2.5.4. Molecular docking

Due to the important role of BRCA1 in the repair of DNA damages, the effect of screened SNP(s) on the binding affinity of the functional domain of BRCA1 with DNA was investigated using hex 8.0.0 docking software. At first, the coding DNA sequence (CDS) regions of the most important genes in breast cancer consist of P53, Bcl2 and Caspase 9 were stimulated using Avogadro 1.2 software. Secondly, molecular docking was carried out between mentioned DNAs and mutated structure of BRCA1 DNA domain with the following parameters: FFT Mode – 3D fast life, Distance Range – 40, Twist range – 360, Correlation type – Shape only, Grid Dimension – 0.6, Receptor range – 180 and Ligand Range – 180. 6

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3. Results In the present analysis, mutation screening was performed using SSCP for exons 2-12 of the BRCA1 gene. A total of 16 samples with aberrant bands or different bands were observed in

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exon 5, exon 7, exon 10 and exon 11 in the 101 breast cancer patients. Aberrant bands or

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different bands were not observed in the controls. Samples were showing a variant band after

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SSCP analysis were sequenced using an ABI3100 DNA sequencer according to the manufacturer’s instructions. Sequencing was performed on both DNA strands. Any mutation detected was confirmed by repeating both PCR analysis and sequencing. A summary of the

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genetic analyses of the BRCA1 cancers is shown in Table 1.

Six mutation carriers were diagnosed in exon 11 including four missense mutations (3113A>G,

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2612C>T, 1597 C>T, 2612 C>G) and two deletions (1543 Del G and 2246 Del T). The mutations in exon 11 were found in ten patients older than 40 years. Mutation 490 C>T was

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localized in BRCA1 exon 7 and it was present in two patients younger than 35 years. Two missense mutations in exon 5: 287 T >A and 273 G> C, was found in two patients older than 40

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years. One BRCA1 mutation in exon 10: 743 Del C, was found in only in one 63-year-old female.

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Six cases of single nucleotide changes in BRCA1 gene detected in the study without records in the BIC database consisted of four polymorphism in exon 11 (1543 Del G, 1597 C>T, 2246

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Del T, 2612 C>G), one missense mutations in exon 7 (490 C>T), one deletion mutation in exon 10 (743 Del C). In our series BRCA1

patients older

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than 40 years old.

mutations occurred more frequently in

3.1. Sequence analysis of mutations The results showed that three mutations consisting one missense mutation in nucleotide 2612 (exon NO. 11) which changed proline to arginine as well as two deleterious mutations in nucleotides 1543 (exon 11) and 743 (exon 10) were not already reported in the database. Due to the formation of defective protein, which was caused by deleterious mutations, and the lack of the reliable tool to check deleterious mutation, the screened missense mutation was selected for further analysis. 7

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3.2. Mutation effect on protein stability The results from two different algorithms including support vector machine and Neural Network confirmed that protein stability was decreased in the mutations with a confidence score of -0.53 and -0.72 respectively.

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3.3. Mutation effect on functional domain(s)

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The protein scanning results demonstrated the missense mutation in nucleotide 2612 was located in EIN3 domain and could affect the binding of BRCA1 to the DNA. The mutant and wild-type domains are depicted in Figure 1. The effect of the mutation on EIN3 domain stability is presented in Table 1. The results demonstrated that EIN3 stability was decreased after occurring the mutation. 3.4. Molecular docking

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The results of molecular docking study between mutant and wild type domains of EIN3 with coding regions of some important genes in cancer genes are shown in Table 2. The results showed that total interaction energy was decreased in all studied complexes, which demonstrate tangible drop of binding affinity of the domain to DNA after the mutation occurs.

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4. Discussion

Breast cancer (BC) is the most commonly diagnosed cancer in Iranian women, and is the leading

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cancer cause of death in this population. Both environmental factors and genetics have an impact on the risk of Breast cancer. Approximately 90% of breast cancers are sporadic while the

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remaining 10% are inheritable (Miki, Swensen et al. 1994). The percentage of total mutation rate in BRCA1 gene in our series was 15.8 % and the missense

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mutation rate was 6.9 %. Four cases of single nucleotide changes in BRCA1 gene detected in the study, already reported in southern Chinese (Haitian, Yunfei et al. 2008) and Iranian populations (Pietschmann, Mehdipour et al. 2005) consisted two missense mutations in exon 11 (3113A>G, 2612C>T) and two missense mutations in exon 5(287 T >A and 273 G> C). Additional sequence variants detected in this study included four polymorphisms in exon 11 (1543 Del G, 1597 C>T, 2246

Del T, 2612 C>G), one missense mutation in exon 7 (490 C>T), and one

deletion mutation in exon 10 (743 Del C). To our knowledge, this report is the first to include information on polymorphisms in BRCA1 in the central population of Iran. Moreover, these Iranian population-based polymorphisms could be used as potential markers. 8

ACCEPTED MANUSCRIPT Li et al. (1999) screened for BRCA1 and BRCA2 mutations in 18 families with multiple cases of breast cancer from Southern Taiwan using SSCP, and found two BRCA1 mutations in three of the families (16%) (Li, Tseng et al. 1999). Zhang (2008) analyzed BRCA1 genes in 144 probands from sporadic breast cancer patients in southern Chinese populations and he detected 16 cases of single nucleotide changes in BRCA1 gene (Haitian, Yunfei et al. 2008). Gorovenko et al. (2007) consider incidence of the familial breast cancer (FBC) and BRCA mutation in Iranian and

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Ukrainian BC patients and he detected 5382 insC mutation in BRCA1 gene in 9% cases in

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Iranian breast cancer patients and 9% in Ukrainian breast cancer patients (Gorovenko,

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Podolskaya et al. 2007). Ghaderia et al. (2001) reported a novel point mutation in exon 16 which was observed among the Iranian breast cancer patients and control subjects. This A/G mutation

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caused the substitution of Glutamine 1612 with Glycine, with an allele frequency of 38.6 and 52.8% in patients and controls, respectively (Ghaderi, Talei et al. 2001).

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More recent studies have attempted to test apart from the genetic and environmental variables by

factor (McGuire, Felberg et al. 2004).

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focusing on women with BRCA mutations, with increased parity as the most consistent protective

Molecular analysis of BRCA1 in different populations has demonstrated a very large mutational

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spectrum and variable mutation prevalence related to the different techniques employed, to the selection criteria, and to the ethnic origin of the patients. While most of the studies on BRCA

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gene mutations has focused on the western population and patients with a family history of breast cancer, 29 only a relatively small number of investigations on the role of the BRCA genes

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are undertaken in Asian sporadic breast cancer populations (De Leon Matsuda, Liede et al. 2002, Whittemore, Balise et al. 2004, Musolino, Bella et al. 2007).

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Due to high accuracy, cost and time effectiveness of bioinformatics methods, recently there is lots of interest for in silico evaluation of gene features, mutations and structural and functional properties of mutant proteins. In this regard in the recent years, many in silico studies are performed to analyze the mutant proteins and gene polymorphism. Selaha et al showed that a missense mutation of PCDH15 is related to Usher syndrome in a consanguineous Pakistani family(Saleha, Ajmal et al. 2016). Naveed and colleague reported that occurring 42 missense SNPs in STEAP2 could play key roles in the up-regulation of STEAP2 and progression of prostate cancer (Naveed, Tehreem et al. 2016). Joshi et al indicated that two functional SNPs in human TRPC6 gene are associated with steroid resistant nephrotic syndrome (Joshi, Koringa et 9

ACCEPTED MANUSCRIPT al. 2015). In another study, it was found that occurring some mutations in TNF-α gene could destabilize the molecular interactions of TNF-α with other molecules in immune system(Dabhi and Mistry 2014). Singh

et al predicted the effects of non-synonymous single nucleotide

polymorphisms in the human Quinone Oxidoreductase 1 (NQO1) and indicated that the SNPs could destabilize the amino acid interactions and hydrogen bond networks of NQO1(Singh, Deka et al. 2017).The results of our study indicated that three mutations especially 2612 C>G

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can reduce stability and function of BRCA1 which may lead to disability of DNA repair and

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progression of breast cancer.

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In conclusion, our study showed that SSCP analysis could be an ideal platform for identifying both somatic and germline genetic variants that lead to cancer. They will provide a basis for

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DNA-based cancer classification and help to define the genes being modulated, improving understanding of cancer genesis and potential therapeutic targets based on biochemistry

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functions of allelic variants that could oppose cancer progress. We think that the genetic screening of patients with breast cancer could be conducted not only for mutational analysis but

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also for polymorphisms analysis because of the need to understand their possible role. Furthermore, based on the results from the in silico analysis, it was found that the screened

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mutation could affect the stability and function of BRCA1. Therefore, the screened mutation could be good a candidate for more evaluation about its role in breast cancer progression as well

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as the cancer diagnosis in primary stage.

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5. Acknowledgements

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The authors would like to acknowledge the University of Isfahan for the financial support of this study.

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Fig 1. The mutant (a) and wild type (b) of BRCA1 DNA binding domain (EIN3)

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Table 1. The results of prediction of mutation effect on EIN3 domain stability. Protparam

I-Mutant

Instability index

DDG

Mutant-EIN3

52.9

-0.23

Wild-EIN3

52.2

+0.45

Server

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C.S.SVM

C.S.NT

-0.53

-0.72

+0.34

+0.46

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Domain

MUpro

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Table 2. Results of molecular docking studied between wild and mutant EIN3 domain with important gene in breast cancer. The mutation was decreasing binding affinity of the domain to studied genes.

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Gene

P53

Bcl2

Caspase 9

-443.25

-594.76

-612.76

-441.12

-593.27

-610.33

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Wild-EIN3

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Mutant-EIN3

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Domain

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ACCEPTED MANUSCRIPT Highlights:

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Mutation screening of BRCA1 in Iranian patients was performed. The entire BRCA1 coding sequence was amplified by PCR. Mutated BRCA1 was studied using in silico approaches. Six cases of single nucleotide changes in BRCA1 were detected. In silico analysis confirmed that 2612C>G could affect BRCA1.

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