Tumor Suppressor Gene Mutations in Iraqi Women with Breast Cancer and Their Relatives

 

Ahmed J, Mohammed

Department of Clinical Laboratory Science /Faculty of Pharmacy/ University of Kufa /Najaf Governorate, Iraq

 

ABSTRACT:

Background: Breast cancer is characterized by various malignant tumors arise from breast tissues,age, genetic factors,diet and environmental factors,reproductive and hormonal factors, some types of benign breast disease andsocio-economic factors are factors correlated to an enlarged risk of breast cancer.Tumor suppressor genes are genes whose loss of roleresults in the elevation of malignancy. Aim of the Study: The present study was conducted to verify the tumor suppressor gene mutations in breast cancer women and their relatives. Material and Methods: Three groups of samples were included. The first consisted of 100 blocks of formalinfixed, paraffinembedded(FFPE) breast cancer tissuesof women(group 1). The second contained blood samples of 46 breast cancerwomen(group 2). The third comprised blood samples of46 apparently healthy women who were relatives to the breast cancer patients of group two(group 3). The ages of patients of samples tissue were 46.78±11.5 years, while for samples of blood patients were 47.34±11.18 years. The ages of the healthy relatives were 40.79±9.84 years. Five tumor suppressor gene mutations were examined.BRCA1 185delAG, BRCA1 5382insC and BRCA2 6174delT mutations were evaluated by mutagenically separated polymerase chain reaction, while   CHEK2 1100delC and Tp53 exon 7 mutations were analyzed by RFLP. Results: The amplification of BRCA1gene for185 delAG mutation revealed amplicons of  335 bp for the wild type and 354 bp for the mutant gene, whereas for 5382 insC mutation exhibited 2 amplicons  of  sizes 271 bp for the wild type and 295 bp for the mutant gene. The analysis of BRCA26174 delT mutation indicated 2 amplicons of 151 bp for the wild type and 171 bp for the mutant gene. Tp53 exon 7 mutation analysis highlighted an amplicon of 125 bp size. Digestion of thisamplicon with the Hae III enzyme resulted in 2 fragments with sizes of 83 bp and 42 bp for the wild type. CHEK2 1100 delC mutation evaluation showed an amplicon of 116 bp size. Digestion of this amplicon with Sca I enzyme exhibited 2 fragments with sizes of 92 bp and 24 bpfor the wild type. Data analysis demonstrated that 27 (27%) out of the 100 enrolled breast cancer patients of tissue samples have mutations in tumor suppressor genes, 11 (23.9%) out of 46enrolled breast cancer patients of blood samples have such mutations, while 8 (17.4%) out of 46 relatives have these mutations.  Nine (9%) patients of group1 were indicated to have BRCA1 185delAGmutation, while 4 (8.7%) and 3 (6.5%)women from groups 2 and3 exhibited the same mutationrespectively.  BRCA15382insC mutation was identified in 7 (7%), 3 (6.5%) and 1 (2.2%) women of groups 1, 2 and 3 respectively. The BRCA2 6174delT mutation was observed in 5 (6%), 2 (4.3%) and 2 (4.3%)women of groups 1,2 and 3 respectively.  Three (3%) patients of group1, 1 (2.2%) and 1 (2.2%) women from groups 2 and 3 were found to have CHEK2 1100delC respectively.   Tp53exon 7 mutation was evident in 2 (2%), 1 (2.2%) and 1 (2.2%) women of groups 1, 2 and 3 respectively. No patient had more than one mutation. Conclusions: About one quarter of the investigated breast cancer patients have mutations in their tumor suppressor genes and 17% of their relatives have such mutations. Most of breast cancer patients enrolled in this study are sporadic not familial breast cancer.

 

 

 

INTRODUCTION:

It is commonly acknowledged that breast cancer is a heterogeneous disease with amolecular features, wide spectrum of clinicaland pathologic [1–3].

Breast cancer is the most common type of cancer in Iraq [4]. There were 3540 cases of breast cancer in both genders accounting for 19.15% of all newly diagnosed cancer cases, 3464 cases of them were among females and76 cases among males.It is the most common cancer in Iraq, it ranks the first in all the years from 1986-2010. It is also the most common cancer among females with incidence rate about 21.75 per 100,000 female population in 2010, compared to 16.65per 100,000 female population in 2008 [5].

 

Age specific incidence rate of breast cancer about 114.79/100000 female population. Were in the age group (50-54) years, followed 108.42/100000 female population were in the age group between (65-69) years, while the incidence rate 99.23/ 100,000 female population. Was found in the age group (45-49) years, followed by 92.26/100,000 female population. In the age group (60-64) years in 2009 [5].

 

The factors related to an increased risk of breast cancer are age, genetic factors, diet and environmental factors,  reproductive and hormonal factors, some types of benign breast disease and socio-economic factors [6].

 

A probable genetic participation to breast cancer risk is shown via the enlarged rate of these carcinomas among females of a family history, through the examination of some families in which numerous family members are affected with breast carcinoma, in a design corresponding with an inheritance of autosomal dominant carcinoma liability. Established researches of families (linkage analysis) have then identified the occurrence of autosomal dominant tendencies to breasts carcinoma and have given to the identification of several greatly penetrant gene history as the reason of inherited carcinoma hazard in numerous families [7].

 

Breast cancer is a component of some autosomal dominant carcinoma syndromes. The syndromes highest strongly related with both cancers are the BRCA1 or BRCA2 mutation syndromes. Breast cancer is also an usual aspect of Li-Fraumeni syndrome scheduled to Tp53 transmutations and of Cowden syndrome credited to PTEN transmutations [7].

 

 

Additional syndromes of genetic that may contain breasts cancer as a related aspect comprise heterozygous like a variant of hazardous CHEK2 alleles produce an extensive involvement to breast cancer liability and their identification might assistance in the medical managing of patients carrying a CHEK2 mutation [8].

 

 

 

Patients and Relative Groups:

Samples of research were obtained from AL-Sader Medical City in AL-Najaf city from Sept 2015 to December 2016. Three groups of samples were included. The first consisted of 100 formalin-fixed, paraffin-embedded (FFPE) blocks tissues of breast cancer women (group 1). The second contained blood samples obtained from 46women who were diagnosed as cases of confirmedbreast cancer (group 2). The third comprised blood samples obtained from 46 apparently healthy women who were relatives to the breast cancer patients of group two (group 3).

 

The tissue samples patients ageswere 46.77±11.4 years with a 21–71 years range while that forpatientsof blood samples were 47.35±11.17with a 25-66 yearsrange. The relatives group ages were 40.78±9.85 with a 29–55 years range.

 

A feedback form was considered to get the datafrom breast cancer patients and their relatives. The name, sex, age,marital state, address, breast fed, number of children, and laterality of disease were included. Thepractical research part was achieved at the Department of Clinical Laboratory Sciences/ Faculty of Pharmacy/ University of Kufa.

 

Collection of Samples:

Slightslices (up to 25 mg) of paraffin-embedded tissueblocks were carved and transported to a 1.5 ml micro centrifuge tube.Two tubesaliquots were preparedas a minimum and kept at refrigerator while waiting forseparation of DNA.

 

samples of blood were took from patients of breast cancer and their relativesvia vein puncture. The volume of blood werefour milliliters collected in 2 aliquot EDTA tube and kept at (–20 C) tillseparation of DNA.

 

Methods:

The DNA extracted from FFPE by ReliaPrep^™ FFPE gDNA Miniprep System while from frozen blood the DNA extracted by ReliaPrep™ Blood gDNAMiniprep System both kits from Promega^TM.

 

The programmable thermal cycler gradient PCR systemwas used for amplification. Chan et al, 1999 designated a PCR procedure named  mutagenically separated PCR (MS-PCR) polymerase chain reaction (PCR), was done by allele-specific oligonucleotide primers used For recognition of 185del AG and 5382insC in BRCA1and 6174delT in BRCA2 [9, 10]. In this technique, 3 primers (one specific for the wild-type allele, one common, andone specific for the mutant) were considered for a mutation. While for Tp53 a Single strand conformation polymorphism (SSCP) polymorphism was usedthe GT trans version at 3^rdl ocation of codon 249 (AGG to AGT) in exon 7 of the Tp53 gene was distinguished by PCR-RFLP.

 

The programmable thermal cycler gradient PCR systemwas used foramplification. Naina and Alpana, 2009were designatedthe primers [11- 13]. Two primers (forward and reverse) were intended,in this technique, thePCR products were digested with FastDigest HaeIII (BsuRI) (Thermo Scientific #FD0154),theTp53 exon sevenamplicon125 bpdemonstration2separate bands, 42 bp and 83 bp, showinglack of a 249 codon mutation.

 

But theamplification ofCHEK2gene was achievedat a programmable thermal cycler gradient PCRtechnique. To display the CHEK2 1100delC mutation 2groups of PCR primers were used(Table 1). To cancel all other homologous sequences in the genome, precise primers for the CHEK2 exon 10 on chromosome 22 were intended, P12 and P13(Table 1). The PCRamplicons were before re-amplified with nested primers, P14 and P15(Table 1), which were intended to amplify the sectionincluding the spot of CHEK2 1100delC. The forward primer involved1 base replacement (a T to G replacement at site 1097) to create a limitationposition for restriction enzyme ScaI inside the wild-type allele after PCRmagnification. Ifthe 1100delC mutation wasabsence, the productfrom the second amplification was 116 bp cut by ScaI for fragments of 24 and 92 bp, while the mutant producestayed uncut [14, 15]. The PCR products were digested with Thermo Scientific FastDigest ScaI(Thermo Scientific #FD0434).The 116 bp produceof wild-type allele was cut by ScaI to fragments of 24 and 92 bp, (the 24-bp band usually goes out of the gel). While the mutant producestayedcomplete.All the primers are in Table 1.

 

Table 1: The primers of tumor suppressor genes

Gene	Primer	Sequence	Length	Melting Temperature	Amplified fragmentsize
BRCA1 185delAGmutation	Common forward (P1)	5'-GGTTGGCAGCAATATGTGAA-3'	20 mer	58.0ᵒC
	Wild-type reverse (P2)	5'-GCTGACTTACCAGATGGGACTCTC-3'	24 mer	74.0ᵒC	335 bp
	Mutant reverse (P3)	5'-CCCAAATTAATACACTCTTGTCGTGACTTACCAGATGGGACAGTA -3'	45 mer		354 bp
BRCA1 5382insC mutation	Common reverse (P4)	5'-GACGGGAATCCAAATTACACAG-3'	22 mer	64.0ᵒC
	Wild-type forward (P5)	5'-AAAGCGAGCAAGAGAATCGCA-3'	21 mer	62.0ᵒC	271 bp
	Mutant forward (P6)	5'-AATCGAAGAAACCACCAAAGTCCTTAGCGAGCAAGAGAATCACC -3'	44 mer		295 bp
BRCA2 6174delT mutation	Common reverse (P7)	5'-AGCTGGTCTGAATGTTCGTTACT-3'	23 mer	66.0ᵒC
	Wild-type forward (P8)	5'-GTGGGATTTTTAGCACAGCTAGT-3'	23 mer	66.0ᵒC	151 bp
	Mutant forward (P9)	5'-CAGTCTCATCTGCAAATACTTCAGGGATTTTTAGCACAGCATGG-3'	44 mer		171 bp
Tp53gene exon 7	Forward (P10)	5'-TCTCCTAGGTTGGCTCTGACT -3'	21 mer	64.0ᵒC	125 bp
	Revers  (P11)	5'-TCCTGACCTGGAGTCTTCCAG-3'	21 mer	66.0ᵒC
CHEK2 1100delC mutation	forward (P12)	5’-TTAATTTAAGCAAAATTAAATGTC-3’	24 mer	46.0ᵒC
	reverse (P13)	5’-GGCATGGTGGTGTGCATC-3’	18 mer	54.0ᵒC
	forward (P14)	5’-CCCTTTTGTACTGAATTTTAGAGTA-3’	25 mer	51.0ᵒC	116pb
	reverse (P15)	5’-ACAAGAACTTCAGGCGCCAAGTAG-3’	24 mer	59.0ᵒC

 

Statistical Analysis:

Statistical analyses were donebySPSS v. 20.0 software (SPSS Inc., Chicago, IL). Spreading of genotypes was matched by chi-square test. The odds ratios (ORs) and 95% confidence intervals (CIs) were considered. Statistical significance was defined as P< 0.05.

 

RESULTS AND DISCUSSION:

Data analysis demonstrated that 27 (27%) from the hundredregistered breast cancer patients of tissue samples have mutations in tumor suppressor genes, 11 (23.9%) out of 46 enrolled breast cancer patients of blood samples have such mutations, while 8 (17.4%) out of 46 relatives have these mutations.  Nine (9%) patients of group1 were indicated to have BRCA1 185delAG mutation, while 4 (8.7%) and 3 (6.5%) women from groups 2 and 3 exhibited the same mutation respectively (Figure 1).  BRCA15382insC mutation was identified in 7 (7%), 3 (6.5%) and 1 (2.2%) women of groups 1, 2 and 3 respectively(Figure 2). The BRCA2 6174delT mutation was observed in 5 (6%), 2 (4.3%) and 2 (4.3%) women of groups 1,2 and 3 respectively(Figure 3).  Three (3%) patients of group1 and 1 (2.2%) and 1 (2.2%) women from groups 2 and 3 were found to have CHEK2 1100delC respectively(Figure 4).   Tp53 exon 7 mutation was evident in 2 (2%), 1 (2.2%) and 1 (2.2%) women of groups 1, 2 and 3 respectively(Figure 5). No patient had more than one mutation.

 

To understand the impact of mutations on patients with breast cancer and their relatives, the data of women with confirmed mutations were analysed by Chi-square test. A none significant difference of tumor suppressor gene mutations was obtained in patients with breast cancer when compared with those of relatives (Table 2) (Figure 6).

 

Table 2: Distribution of mutations gene in patientsof breast cancer andtheir relatives.

Mutations gene	Patients				Relatives
	Tissue sample (100)	%	Blood sample (46)	%	Blood sample (46)	%
BRCA1 185delAG	9	9%	4	8.7%	3	6.5%
BRCA15382insC	7	7%	3	6.5%	1	2.2%
BRCA2 6174delT	6	6%	2	4.3%	2	4.3%
CHEK2 1100delC	3	3%	1	2.2%	1	2.2%
Tp53 exon 7	2	2%	1	2.2%	1	2.2%
Total	27	27%	11	23.9%	8	17.4%

 

It is clear from the current study that about one quarter of the recruited breast cancer patients had mutations in their tumor suppressor genes. Moreover about 17.4% of relatives of breastcancer patients with blood samples had mutations in theirtumor suppressor genes, they are prone for development of breast cancer. Thus it is very reasonable to advice such women to consult specialist physicians and subjected to repeated examination for early detection breast cancer.

 

It is believed that the incidence of BRCA gene mutationinfluences by ethnicityamong people. Osborne (2004)revealed that certain mutations in genes relatedto breast cancer are more public among certain ethnic or geographic groups. The genetic alterationsarise more frequently in these groups because they have a shared ancestry aboveseveral generations. Therefore different mutations may occur in different ethnic groups [16].

 

The comparison of the results stated formerly with current results demonstrated some controversies.Parkin et al., (2005) who showed47.3% from allpatients ofbreast cancer have mutations in BRCA1and/or BRCA2 genes representing those patients to have hereditary breast cancer [17].Vaidyanathanet al. (2009) have identified  61 young patients from India withmutations, genetic breast cancer in 17 patients (28%) were establish,whileBRCA1 mutations in 15 (24.6%) and BRCA2mutations in 2 (3.28%). Whereas no definiteconnotation between cancer type andBRCA1 or BRCA2 mutations was seen, the occurrence of BRCA1 and BRCA2 mutations among women of India of familial ovarian and breast cancers is establish to be 24.6% and 3.28% respectively [18]. Steffensenet al. (2010) recognized a Danish ovarian and breast cancer family with germ-line mutations in the BRCA1 and BRCA2genes. Remarkably, the father of both  priband, the probands, and the probands 2 children have hereditary both mutations [19]. In research on womenof Israel, it has been declared that breast cancer definite rates of death are analogous between carriers of a BRCA1/2 founder mutation and non-carriers [20].

 

Most of breast cancer patients enrolled in this study are sporadic not familial breast cancer and that is compatible with Sodha, et.al. (2000)  and Allinen, et al. (2001) who concluded that later it became clear СНЕК2 mutations are also present in patients with familial and sporadic breast cancer[21, 22].

 

CONCLUSIONS:

About one quarter of the investigated breast cancer patients have mutations in their tumor suppressor genes and 17% of their relatives have such mutations. Most of breast cancer patients enrolled in this study are sporadic not familial breast cancer.

 

Figure1:PCR product in gel electrophoresis of BRCA1 185delAG exposure on 3%agarose gel via10X TAE buffer for 35 minutes, at 140 V, with 100bp DNA ladder and ethidium bromide stained. Lane 1-3 show genotype mutations. Lane 9 negative control.

 

 

Figure 2: Gel electrophoresis of PCR product for BRCA1 5382insC mutation detection on 3%agarose gel by10X TAE buffer for 35 minutes, at 140 V, with 100bp DNA ladder and ethidium bromide stained. Lane 1-3 show genotype mutations, lane 9 negative control.

 

 

Figure 3: Gel electrophoresis of PCR product for BRCA2 6174delT mutation detection on 3%agarose gel by10X TAE buffer for 35 minutes, at 140 V, with 100bp DNA ladder and ethidium bromide stained. Lane 1-3 shows genotype mutations, lane 9 negative control.

 

Figure 4: Restriction enzyme (Hae III) digestions of Tp53 polymerase chain reaction products of  Tp53 exon 7 amplimer (125 bp) show two distinct bands, 83 bp and 42 bp, indicating absence of a 249 codon mutation (lane 5-8).

Lane (1-4) shows a 249 codon mutation of Tp53 exon 7.

Lane 9 negative control.

Electrophoresis on 1.5% agarose gel, for 30 minutes, at 120 volts used 10X TAE buffer with 100bp DNA Ladder and stained with ethidium bromide.

 

 

Figure 5: Restriction enzyme (Sca I) digestions of CHEK2 PCR products were separated by electrophoresis on 3% agarose gel, for 35 minutes, at 140 volts used 10X TAE buffer with the 100bp DNA Ladder and stained with ethidium bromide. The wild-type allele (lane 1-3) of 116 bp product was cleaved by ScaI to fragments of 92 and 24 bp, (the 24-bp band usually runs out off the gel). Whereas the mutant product remained uncut (lane 4-8).Lane 9 negative control.

 

 

Table 2: Distribution ofgene mutations in patients and their relatives forall groups.

 

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Accepted on 04.05.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(10): 4467-4472.