Introduction: Alternative splicing helps transcription process to produce many proteins from single gene and has

important roles on tumorogenesis. The BRCA1 gene has almost 11 alternate spliced variants which have been

mentioned as susceptibility factors for initiation and progression of breast cancer. Materials and Methods: The

expression of BRCA1 was analyzed by qualitative and quantitative RT-PCR assays, among 27 samples from

patients with primary breast cancer. Then the variants Δ2, Δ11b, and Δ14 were detected. Results: The BRCA1 gene

found to be expressed among 85.2% of tumors. The distribution of Δ2, Δ11b, and Δ14 variants were 91.3%, 73.9%,

and 78.3%, respectively. Discussion: The findings about three investigated variants emphasize on importance of

BRCA1 alternative splicing for breast tumorogenesis

اخوان, هما

اصغراوا, طاهیرا

حسینی اصل, سید سعید

فغانی, نفیسه

مهدی پور, پروین

وثوقی, ابراهیم

پیری دوگاهه, هادی

English

ArUMS Digital Repository (سامانه اطلاعات زیست پزشکی و سلامت)

Available online at www.scholarsresearchlibrary.com 
 
 
 
 
 
 
 
Scholars Research Library 
 
Annals of Biological Research, 2013, 4 (4):139-143 
(http://scholarsresearchlibrary.com/archive.html)  
ISSN 0976-1233 
CODEN (USA): ABRNBW 
 
 
139 
Scholars Research Library 
BRCA1 alternative splicing in breast tumorogenesis 
 
Ebrahim Vosoughi1, 2, Nafisseh Faghani2, Parvin Mehdipour3, Homa Akhavan2,  
Tahira Asgarova4, Hadi Piri dogahe2 and S Saied Hosseini-Asl2* 
 
1Azerbaijan National Academy of Scinces,Genetic Resources Institute, Baku, Azerbaijan  
2Medical Genetics lab, Imam Hospital, Ardabil University of Medical Sciences, Ardabil, I. R. Iran 
3Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 
4Department of Medical Genetics and Biochemistry Azerbaijan Medical University,Baku, Azerbaijan 
_____________________________________________________________________________________________ 
 
ABSTRACT 
 
Introduction: Alternative splicing helps transcription process to produce many proteins from single gene and has 
important roles on tumorogenesis. The BRCA1 gene has almost 11 alternate spliced variants which have been 
mentioned as susceptibility factors for initiation and progression of breast cancer. Materials and Methods: The 
expression of BRCA1 was analyzed by qualitative and quantitative RT-PCR assays, among 27 samples from 
patients with primary breast cancer. Then the variants ∆2, ∆11b, and ∆14 were detected. Results: The BRCA1 gene 
found to be expressed among 85.2% of tumors. The distribution of ∆2, ∆11b, and ∆14 variants were 91.3%, 73.9%, 
and 78.3%, respectively. Discussion: The findings about three investigated variants emphasize on importance of 
BRCA1 alternative splicing for breast tumorogenesis. 
 
Key words: BRCA1, Breast cancer, alternative splicing 
_____________________________________________________________________________________________ 
 
INTRODUCTION 
 
Breast cancer is one of the most common diseases affecting women worldwide. Its overall mortality each year is 
519,000 deaths [1].Previous investigation studied on Iranian population showed 17 per 100,000 woman years with 
the peak incidence rate in the age group 45-54 years [2].Iranian patients seems to be relatively about 10 years 
younger than other countries [3], indicating more effective role of genetic than environmental factors and may lead 
to study on germ-line and somatic alterations in cancer-related genes. 
 
In human and other higher eukaryotes, alternative splicing is a process to produce many proteins with different but 
related function from a single gene. 
 
In tumorogenesis processes, the changes occurred in tumor suppressor genes, oncogenes, and genes encoding the 
proteins which their function promote the development of tumors, could resulted in progression of the process. 
 
Therefore, identification of tumor- and tissue- specific spliced forms of mRNAs could confer the diagnostic factors 
which are suitable for prognosis and therapeutic interventions [4]. 
 
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The Breast Cancer 1 (BRCA1) gene was cloned in 1994, by Miki et al [5].Mutation screening in its 22 coding exons 
revealed its accounting for ̰ 45% of hereditary breast cancers and ̰ 80% of families with both breast and ovarian 
cancers [6,7,8].In spite of finding rare mutations in sporadic cases, reduced expression and finding splice variants of 
its mRNA, suggest more important role for this protein in breast and/or ovarian carcinogenesis.  
 
Among the identified BRCA1 spliced isoforms, the BRCA1-∆11b was more attractive on the previous studies. The 
BRCA1-∆11b variant was discovered by 
 
Wilson et. al. [9]They described this variant with missing exon 4 and most of exon 11 (position 908 to 4215). The 
BRCA1-∆11b protein sequence is differed from the BRCA1 GenBank sequence at two positions including a three 
nucleotide deletion resulting in the loss of an alanine at position 1453 and a serine to glycine substitution at amino 
acid position 1612. 
 
In the current investigation, three BRCA1 isoforms containing ∆11b (includes deletion occurred on exon 11), ∆2, 
and ∆14 were studied and their relation with tumorogenesis process and some histopathologic data was analyzed. 
 
MATERIALS AND METHODS 
 
Patients 
Institutional guidelines including ethical approval and informed content were considered. The tumor samples from 
27 patients affected with primary invasive breast cancer have been collected from Day Hospital, Tehran. The 
samples were rapidly preserved in liquid nitrogen immediately after surgical excision and then were stored at -80°C. 
 
Expression analysis 
Total RNA was isolated using Trizol reagent (Invitrogen Co.). The accuracy of RNA isolation procedure was 
confirmed by electrophoresis. Two microgram of total RNA was used to create cDNA, by random hexamer and M-
MuLV reverse transcriptase enzyme (Fermentas Co., Canada). 
 
For validation of cDNA synthesis, amplification of β-actin's cDNA according to the previously described primers 
was used. 
 
Qualitative assay 
In routine RT-PCR, detection of RNAs in samples containing slight amounts of extracted RNA or cells with low 
expression levels could be failed by leading to the false-negative finding. Using nested PCR strategy is one of the 
ways to approach the improved detection. 
 
Designing the primers on the exons, could lead to lose the false-positives results developed by DNA contamination. 
With this strategy, amplified fragments created by DNA samples, have longer lengths rather than corresponded 
cDNAs. The primers for amplification of the noted fragments were designed by the Genefisher 2 online software 
[10], and were listed on table 1. 
 
PCR conditions for amplifying exons 2 and 11b was 3 min at 95°C, followed by 
 
30 cycles containing denaturation by 94°C for 30 sec., annealing by 58°C for 45 
 
sec., and extension by 72°C for 30 seconds and eventually final extension by 3 minutes at 72°C. This condition for 
amplifying exon 14 was the same with one exception in annealing temperature which was 54°C. 
 
Diluted first round amplification products (1/10) were used as template for nested PCR. The amplified fragments 
lengths containing alternative variants were noted on table 2. 
After distinguishing different variants on 1% agarose gel electrophoresis, sequencing of the amplified fragments 
confirmed the accuracy of detection. 
 
Quantitative assay 
For measuring BRCA1 expression level in tumor samples, primers and probe designed by Primer Express software 
version 3.0 (Applied Biosystems) were used on ABI 7500 system. Relative gene expression quantification was 
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calculated according to the comparative Ct method using GAPDH with previously reported conditions [11]as an 
endogenous control and RNaseP (Applied Biosystems) as an RNA control. 
 
RESULTS 
 
All of the 27 patients with primary breast cancer were female with average age of 47.15 Yrs. (31-71; std.=± 11.05) 
with invasive ductal carcinoma type. Tumor grade was 7.7%, 46.2%, and 46.2% for grade I, II, and III, respectively. 
The tumor stage IIA was found to be the most common among them. 
 
Approximately 52% of patients have lymph node involvement. Progesterone and estrogen receptors were positive 
among 24% of patients. 
 
Qualitative and quantitative results revealed BRCA1 expression among 85.2% (23 out of tumors). 
 
Alternative splicing variants distribution among tumors was listed on table 3. 
 
By analyzing the relationship between spliced variants and histopathologic data, significant associations were found 
only for variant E11b by considering the lymph node status and disease stage, (tables 4 and 5). 
 
Table 1 . Primers sequences for amplifying exons 2, 11, and 14 of BRCA1 cDNA and quantitative PCR. 
 
Detected fragments Exon on request Step Sequence Primer location 
1-3 
Exon2 First Forwrad:5'-CTTGGTTTCCGTGGCAA-3' Exon 1 Reverse:5'-CTTCTTCAACGCGAA-3' Exon 3 
 Second Forward: 5'- AAGCGCGGGAATTACAGA-3' Exon 1 
Reverse:5'-TTCAACGCGAAGAGCAGA-3' Exon 3 
10-12 Exon 11 
First Forwrad:5'-CAAATCACCCCTCAAGGA-3' Exon 10 
Reverse:5'-CTGAGAGGATAGCCCTGA-3' Exon 12 
Second Forwrad:5'-TCACCCCTCAAGGAACCA-3' Exon 10 Reverse:5'-AGAGGATAGCCCTGAGCA-3' Exon 12 
12-15 Exon 14 
First Forwrad:5'-AGCATCTGGGTGTGAGA-3' Exon 12 Reverse: 5'-CTATCTGCAGACACCTCA-3' Exon 15 
Second Forwrad:5'-GAAACAAGCGTCTCTGA-3' Exon 12 
Reverse:5'-CCTCAAACTTGTCAGCA-3' Exon 15 
Quantization assay BRCA1 Forward 5'-GTCTCCACAAAGTGTGACCACATAT-3' Exon4 Reverse 5'-AGGCCCTTTCTTCTGGTTGAG-3' Exon5 
  Probe 5'-FAM-CAAATTTTGCATGCTGAAA-TAMRA-3' Exon4 Exon5 
 GAPDH 
Forward 5'-GAAGGTGAAGGTCGGAGT-3'  
Reverse 
Probe 
5'-GAAGATGGTGATGGGATTTC-3'  
5'-FAM-CAAGCTTCCCGTTCCAGCC-TAMRA-3'  
 
Table 2. PCR products lengths among different fragments 
 
Fragments 1st  round PCR 2nd round PCR DNA contamination* 
Exon 2 
 
∆2 217 bp 212 bp 994bp 
+2 595 bp 569 bp 1372bp 
Exon 11 
 
∆11b 240 bp 233 bp ~1kb 
Complete exon 11 ~4kb ~4kb ~5kb 
Exon 14 
 
∆14 345bp 313bp ~14kb 
+14 411 bp 379 bp ~14kb 
*PCR product bands created by amplification of DNA molecules which could not been completely removed during the RNA isolation procedure. 
 
Table 3. Spliced variants on exons 2, 11, and 14 among BRCA1 expressed tumors (85.2% of patients) 
 
Variant ∆2 +exon 2 ∆11b Complete exon 11 ∆14 +exon 14 
Frequency 91.3% 8.7% 73.9% 26.1% 78.3% 21.7% 
 
 
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Table 4. Association between E11b spliced variant and stage of disease. 
 
  Stage of disease 
  I IIA IIB IIIA IIIB 
Exon 11 ∆ 11b 27.8% 27.8% 5% 0 5% 
Complete 0 5% 22.2% 5% 0 
P= 0.03 
 
Table 5. Association between spliced E11b and lymph node involvement. 
 
  Lymph node involvement 
  Negative Positive 
Exon 11 ∆11b 44.5% 29.6% 
Complete exon 3.7% 22.2% 
P= 0.04 
 
DISCUSSION 
 
Among the well-known predisposing markers for breast cancer, germline mutations in BRCA1 gene are valuable. 
After the cloning of the BRCA1 gene and distinguishing its coding sequence, the analyzing its sequential changes 
has been emphasized. So far, more than 1,000 mutations in this gene have been identified. Most of them are 
associated with an increased risk of breast cancer. 
 
The BRCA1 as a tumor suppressor gene is involved in repairing damaged DNA. 
 
In the case of damaged BRCA1, damaged DNA could not be repaired properly and so, cells containing damaged 
DNA cannot be destroyed. Therefore, risks for breast cancer will be increased. 
 
In addition to mutations occurred at coding sequences, alteration on BRCA1 expression status have significant roles 
on accuracy of cellular proliferation. As the noted defects accumulated, cells allowed dividing uncontrollably and 
forming tumors. For highlighting the importance of BRCA1 expression level at tumorogenesis, many investigations 
were carried out [12,13,14].Alternative splicing is an essential mechanism for generating proteins with different 
functions from a single gene. Alternative splicing affects an estimated 
 
70% of human genes [15,16].LKB1, KIT, KLF6, HAS1, and BRCA1 are among the investigated genes suffering 
this mechanism [17]. Lu, et al. were among the first investigators on BRCA1 alternative who described its variants 
[18].In the present study, the variants included transcripts lacking exons 2, 11b, and 14. The findings emphasize on 
the importance of BRCA1 alternative splicing for breast tumorogenesis. Among them, the variant ∆11b seems to 
have more attractive roles. More investigations on this variant might lead to emerge therapeutic and diagnostic 
factors on breast cancer. 
 
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Accessed 30.01.2011,

E.Felip, P.Pedro Mendez,

M.Nouraie, A.Ghorbani, M.Alimohammadian, R.Malekzadeh ,

S.Chao, G.Chaojiang, Z.Congyi,

S.Z.Tabei, N.Azarpira, M.A.Vakili,

BRCA1 alternative splicing in breast tumorogenesis

http://eprints.arums.ac.ir/4191/1/BRCA1%20alternative%20splicing%20in%20breast%20tumorogenesis.pdf

BRCA1 alternative splicing in breast tumorogenesis

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