Breast and Ovarian Cancer Risk due to Prevalence of BRCA1 and BRCA2 Variants in Pakistani Population: A Pakistani Database Report

Introduction. Pakistani population has a very rich anthrogeneological background with waves of migration from neighboring regions. Incidence rates of breast and ovarian cancer in Pakistan are on such a rapid rise that it is necessary to check the contributory factors, genetic and nongenetic. An insight into the prevalence data emphasizes the formulation of a BRCA1 and BRCA2 database for the Pakistani population. Method. In this study conducted by authors, data from diagnosed cases of both sporadic and inherited female breast and ovarian cancer cases was gathered after performing molecular genetic analysis by screening for alterations in the coding sequence of the BRCA gene. The region of interest was analyzed by the aid of various molecular biology tools such as automated DNA sequencer. Bioinformatics software was used to interpret the results, and database was prepared. Results. Mutational screening of the exons in all the samples of our study group did not reveal any pathogenic mutation. These results along with the results of the previous Pakistani studies for both BRCA1 and BRCA2 genes were summed up to prepare a Pakistani database. Percentage involvement of these genes was estimated. Nine percent of these cancers show alterations in BRCA1 gene while 3 percent have shown BRCA2 variants. The remaining 88 percent of breast and ovarian cancers can be attributed to the involvement of other genes.


Introduction
Breast cancer is the most prevalent cancer in the world, and ovarian cancer is the sixth most common cancer in the world [1]. Inherited mutations in the breast cancer susceptibility gene 1 (BRCA1) [MIM 113705] and breast cancer susceptibility gene 2 (BRCA2) [MIM 600185] are associated with a high risk of developing breast and ovarian cancers in females of different age and ethnic groups. These well-defined high-penetrance genes show loss-of-full function germ line mutations in hereditary cases and decreased expression in sporadic tumors [2][3][4]. Approximately, 5 to 10 percent of breast cancer [5] and at least 10 percent of ovarian cancers [6] are hereditary. Sequence variation in the BRCA1 gene accounts for 45 percent of inherited breast cancer and more than 90 percent of inherited breast and ovarian cancer [3], and both genes combined account for only 25 percent of familial risk [7].
The spectrum of mutations in these genes varies between populations, with some showing a high frequency of unique mutations [8]. Many such alterations may be recurrent, often being identified in isolated populations as the results of a founder effect [5] and may be the basis of differences in cancer risk between populations [9]. Ashkenazi Jewish, Norwegian, Dutch, and Icelandic people have a higher rate of certain genetic alterations in BRCA1 [10].

BRCA1 and BRCA2 Screening in Asia. Most of BRCA1
and BRCA2 research has been focused on the Caucasian populations; however, the allelic frequency of higher penetrance genes in the Asian population may be higher than that in Caucasian population [11]. Immigrant Asian women especially from South Asia who are settled in the West show high rates of these cancers. Risch et al. [12] reported greater frequencies of mutations among cases of ovarian cancer that were of Indo-Pakistani descent than among British or mixed northern-or western-European ethnicity in Ontario, Canada.
Although 60 percent of the world's population resides in the Asian continent, and the fact that the Chinese, Malays, and Indians are three major Asian haplogroups, genetic predisposition to hereditary diseases and the applicability of genetic testing in these diverse ethnic groups is still unclear [13]. These regions have high rates of both these cancers especially the developing South Asian countries.

BRCA1 and BRCA2 Screening in Pakistani Population.
Amongst the Asian countries, Pakistan has one of the highest rates of breast and ovarian cancer [14] with breast cancer being the most common and ovarian the third most common malignancy amongst Pakistani women [15] and the most common cancer of gynecological origin in Pakistan [16]. A nine-year study period at a tertiary care cancer institution in Karachi, Pakistan showed that breast cancer was the most common cancer in females accounting for 38.2 percent of total cancer cases, at rates almost highest in Asia and ovarian cancer accounting for 4.9 percent [17]. Incidence rates in this region are on such a rapid rise that it is necessary to check the contributory factors, genetic and nongenetic. An insight into the above data emphasizes the formulation of a BRCA1 and BRCA2 database for the Pakistani population.

Methodology
Study conducted by authors was carried out at the Centre for Research in Applied and Experimental Medicine, National University of Science and Technology, Rawalpindi. This study focused on mutational screening of BRCA1 gene in diagnosed cases of both hereditary and sporadic breast and ovarian cancer. In course of BRCA1 gene screening, all exonintron boundaries were sequenced.
These patients were interviewed after reviewing the history of their disease. Questionnaire was filled out containing information of their personal, family, and disease history. Ethnicity of each candidate was noted with the aim of finding founder effect mutations clustered to a specific ethnic group.
Organic phenol-chloroform extraction method [18] was used to extract the genome. Amplification of all the exonintron boundaries of BRCA1 gene by polymerase chain reaction (PCR) was performed using the oligonucleotide primers designed from the intronic sequences of the gene. Purified products were subjected to DNA sequencing by Automated Genetic Analyzer, Beckman Coulter CEQ8000, Genetic Analysis System. Alignment and mutational analysis was done using Bioedit software [19]. The sequencing chromatograms of the affected individuals were compared with the corresponding control full length gene sequences from UCSC Genome Browser [20] database to identify the aberrant nucleotide base pair change. Ensembl [21] database was used to recheck the gene sequence for cDNA and mRNA information.
Previously, four Pakistani studies depicted the mutational spectrum of these two genes. Two major pioneering investigations were accomplished by Liede et al. [22] at the National Cancer Institute, Karachi and Jinnah Hospital, Lahore and by Rashid et al. [14] at the Shaukat Khanum Memorial Hospital, Lahore. A comparatively small study focusing on mutational analysis of BRCA1 gene was performed by Malik et al. [23] at the COMSATS Institute of Information Technology, Islamabad.
Liede et al. [22] conducted a case-control study of 341 case subjects with breast cancer, 120 case subjects with ovarian cancer, and 200 female control subjects. Human genomic DNA was isolated from peripheral blood. Exonintron boundaries were screened by protein-truncation testing (PTT). Direct sequencing was used for confirmation of all mutant bands detected by PTT.
Rashid et al. [14] selected patients from 176 breast and/or ovarian cancer families who were diagnosed with invasive breast or epithelial ovarian cancer. Genomic DNA was extracted from blood samples. The entire coding regions of the BRCA1 and BRCA2 genes were screened using single strand conformational polymorphism (SSCP) analysis, denaturing high pressure liquid chromatography (DHPLC) analysis, and the protein truncation (PTT) assay [14]. Automated DNA Sequencing was performed for each sample revealing variants by SSCP, DHPLC, or PTT.
150 cases of unilateral breast cancer patients were selected by Malik et al. [23]. After DNA extraction, Single strand conformational polymorphism (SSCP) was done for exons of BRCA1, and sequence analysis was performed for putative sequence variant.

Results
The results of samples recorded on BIORAD gel documentation system showed good yield and amplification of the DNA with the primers. The chromatograms of BRCA1 gene from affected patients ( Figure 1) after mutational screening of the exons in all the samples of our study group revealed no pathogenic sequence variant correlating with breast or ovarian cancer pathogenesis. This is suggestive of the need to focus on the role of other high-or low-penetrance genes in breast and ovarian cancer predisposition in Pakistani population.
By adding present and previous results regarding both BRCA1 and BRCA2 genes, a database with new percentage prevalence was formed. The role of BRCA1 and BRCA2 in the pathogenesis of breast and ovarian cancer in Pakistan was noted, and the most prevalent Pakistani mutations were highlighted.

Percentage of BRCA1 BRCA2 Mutations in Breast and
Ovarian Cancer Patients in the Pakistani Population. Liede et al. [22] identified 42 sequence variants harboring 31 BRCA1 mutations and 11 BRCA2 variants in their study group of 341 women with breast cancer and 120 women with ovarian cancer. Rashid et al. [14] studied 176 Pakistani breast and ovarian cancer patients selected on family history and on age of diagnosis and identified a total of 30 sequence variants and among them 23 deleterious mutations in BRCA1 and 7 mutations BRCA2 gene. Malik et al. [23] detected a total of 6 variants in their samples (Tables 1 and 2). Taking all studies together, a total of 643 probands have been ascertained .  T  T  TT  T  T  T  T  T  T   T   T  T  T  T  T  T   T T T  T  T  T T T  T  T  T  T  T T  T   T  T  T  T   T  T  T  T  T  T  T  T  T  T  T  T  T  T  T  T Figure 1: A representative chromatogram generated by automated genetic analyzer, Beckman Coulter CEQ TM, 8000 sequencing of Ex 13 of BRCA1 gene from an affected patient. Region between the two arrows indicates the exonic sequence. Flanking region is intronic sequence.
Mutations in BRCA1 and BRCA2 were found, respectively, in nine and three percent of breast and ovarian cancer patients ( Figure 2). These results are authenticated by Risch et al. [12] who reported a high frequency of BRCA1 mutations among cases of ovarian cancer that were of Indo-Pakistani descent (14%) along with Jewish (21%) and Italian (17%) ancestry in a population-based study of 649 cases of ovarian cancer in Ontario, Canada.

Most Prevalent BRCA1 and BRCA2 Mutations in the
Pakistani Population. Twenty one distinct BRCA1 mutations were observed by Liede et al. [22] which are mostly insertions, deletions, or point mutations and exon 11, the largest exon of the gene, was mainly found to be the disease causing region. One intronic variant in exon 14 (IVS14-1G → A) was also noted ( Table 1). Ten distinct types of BRCA2 variants were detected by these researchers, 9 in exon 11, and only one in exon 22, the 9140delA mutation in a Muhajir breast cancer patient ( Table 2). The BRCA2 3337C → T mutation was found in two patients. Five BRCA1 mutations (2080insA, 3889delAG, 4184del4, 4284delGA, and IVS14-1ArG) and one BRCA2 mutation (3337C → T) were identified in multiple unrelated case subjects and represented candidate founder mutations. Rashid et al. [14] reported a total of twenty-two distinct mutations, 15 distinct variants in BRCA1, and 7 distinct variants in BRCA2. Among these were 12 frame shift mutations, 8 nonsense mutations, and 2 splice-site mutations (Tables 1 and 2). The most commonly observed BRCA1 mutation was the 4627C → A, nonsense mutation identified in 5 families. Four mutations, 185delAG, 185insA, 4627C → A, and 5622 C → T, were recurrent; these    accounted for 52 percent of all identified BRCA1 mutations. Malik et al. [23] observed two types of mutations in BRCA1, one insertion and one nonsense variant (Tables 1 and  2). Mutational analysis of all these studies emphasizes the justification of genetic testing for predisposing BRCA1 germ line mutations for any Pakistani family with multiple female breast and/or ovarian cancer cases [14]. In total, BRCA1 variant 4627C → A was observed in 22 percent of the probands showing recurrent mutations and features as the most prevalent BRCA1 mutation in Pakistani population (Figure 3), while 15 percent of these patients showed the 4184del4 BRCA1 variant which appears to be the second most prevalent mutation in Pakistani population ( Figure 4). BRCA2 mutational analysis revealed 3337C → T and 5057delTG to be equally prevalent (50 percent each) in the study participants showing recurrent BRCA2 variants.   The two major studies of Liede et al. [22] and Rashid et al. [14] included ethnicity as a vital determinant of mutational screening. In the 54 BRCA1 mutations of both of these studies, 31 (57%) were found in the Punjabi ethnic group. Amongst the 18 BRCA2 variants of both these studies, 6 (33%) were of Punjabi origin. Hence, the Punjabi ethnic group showed maximum sequence variants in the BRCA gene variants (Table 3).

Candidate Founder Mutations of BRCA1 and BRCA2 for the Pakistani Population.
Twelve of the 21 BRCA1 mutations (57 percent) and 8 of the 10 BRCA2 mutations (80 percent) detected by Liede et al. [22] were unique to the Pakistani population [22], while Rashid et al. [14] detected ten mutations (33 percent) which were unique to the Pakistani population, which is comprised of 5 BRCA1 mutations (33 percent) and 5 BRCA2 mutations (71 percent). The finding of thirteen recurrent BRCA1 and two recurrent BRCA2 mutations in some members of the Pakistani population could allow very economical screening for such mutations in specific ethnic groups in the country which could be of great benefit to public health measures. The most prevalent BRCA1 mutation of both studies combined, 4627C → A, was found in six Punjabi patients increasing the likelihood of finding this variant in Punjabi ethnicity. Four probands showed the 4184del4 BRCA1 variant out of which three were Punjabi and one Sindhi. All the four carriers of the BRCA1 185insA and the two carriers of the BRCA1 5622 C → T were also of Punjabi ethnicity. This predisposition of the Punjabis to maximum BRCA variants can lead to focusing on the specific sequence variants of this ethnic group. All the carriers of the BRCA1 2080insA mutation were of Pathan ethnicity. Rashid et al. [14] also found 185delAG in two non-Jewish unrelated Pakistani carriers of Pathan ethnicity, whose ancestors originated from the same geographic region in the North-West Frontier Province of Pakistan. These two mutations can be the focus of candidate founder mutations in this ethnic group. The two cases with the BRCA1 4284delAG mutation belonged to the Muhajir ethnic group, again signifying the importance of focusing on this mutation in this particular ethnic group.
In BRCA2 screening, Liede et al. [22] found the recurrent 3337C → T variant in two Memon breast cancer patients pointing towards the significance of finding this mutation in other Memon patients as well. 5057delTG BRCA2 mutation was a finding of both studies; Rashid et al. [14] detected this mutation in a Punjabi patient, while Liede et al. [22] detected it in a minority ethnic group, a Parsi ovarian cancer patient.

Possible Management Strategies.
Ever since the discovery of breast and ovarian cancer genes, many advances in clinical research have been made which provide the rationale for moving genetic testing of these genes into clinical practice [25]. BRCA1 and BRCA2, like other genes, have not only served as molecular markers for hereditary breast cancer risk screening but also become important indicators for breast cancer prevention, treatment, and prognosis [26]. Genetic testing is gaining acceptance worldwide and has been established throughout North America and much of Europe. Genetic counseling, especially in country like Pakistan having one of the highest rates of consanguinity [27,28], is practiced by many health professionals; however, its expansion into the health policy and training regimes of health care providers is needed. The awareness, however, of genetic testing as a tool for preventive and treatment-oriented management of these tumors is limited. Formulation of BRCA mutational database can serve as a cost-effective tool to identify individuals at high risk such as unaffected carrier relatives who have a defective allele that can be transferred to offspring. Such tests are strategized on the basis of most prevalent mutations in a particular population/ethnic group that can save both time and money. Till then the most effective options for breast cancer risk reduction include prophylactic bilateral mastectomy [29] prophylactic oophorectomy, and oral administration of Tamoxifen [26] in some countries. If genetic testing for these genes becomes streamlined, these prophylactic procedures and other preventive strategies will be warranted to become routine for mutation carriers along with vigilant medical checkups, especially in an economically challenged country like Pakistan.

Conclusions
The need for larger collaborative studies between medical professionals and molecular biologists cannot be emphasized enough. This is necessary for gaining further insight into mutational spectra and ethnic distribution of different types of mutations, in a search for founder effect mutations, which can become a part of cancer screening policies in Pakistan. The high prevalence of these cancers and the presence of recurrent mutations of these genes in the Pakistani population, especially the observation of a high percentage of BRCA1 variants in ovarian cancer cases, emphasize the need for improving genetic counseling strategies and make genetic testing a part of screening policies. The more work done on the genomics of this disease with relevance to the Pakistani population, the closer a genetic cure targeted for this specific population can be found.