Preproposal About BRCA1&2 Germline Mutations In Qatar

Brca1 And Brca2 Germline Muations Screening In Qatari Ovarian Cancer

Discuss about the Prepoposal about Brca1&2 Germline Mutations In Qatar.

Incidence rate of ovarian cancer in the Arab Countries  

Our assumption is that Qatari women diagnosed ovarian cancer have at least one BRAC1 or BRAC2 mutation.

Ovarian cancer has been a global challenge and the illness has cost the lives of many women worldwide and this has attracted the attention of the researchers to research about the BRCA1&2 Germline mutations screening in Qatari ovarian cancer families

The research will examine the total number of cases of ovarian cancer among Qatari women and find ways to mitigate the spread of the disease.

• To look into the extent of BRCA1 and BRCA2 germline mutations in Qatari women with familial ovarian cancer
• To increase the awareness of the Ovarian cancer amongst the people living in Qatar
• To give the foundation for the diagnosing the disease early, treating it soon, and preventing the disease maybe by providing vaccine or advising the Qatar women to keep off some practices which would enhance the spread of the disease. 

We will carry out an experiment to test all the women diagnosed with ovarian cancer with BRCA ½ testing, and we will carry out the process in Hamad Corporation in the Molecular genetic laboratory. We will transfer all the specimens to the laboratory, and we shall ensure that the lab will be compatible with the local transferring guidelines. We will also provide that the lab will be in a position to refrigerate and monitor the specimen.

The current research was about the Qatari women who have been diagnosed with the disease and the onset of the illness in familial cases which were chosen from victims in National Centre for Cancer Care and Research and advised to go for counselling in Hamad Medical Corporation concerning the genetic history of the disease (Denic & Agarwal 2018).  

Before doing the genetic testing of the disease, we will obtain approval from the patients and the Ethical Committee.

We will use the criteria below in selecting the patients who will be used for the study: 

1. Women who have a genetic history of the disease (Bujassourn Bugrein & Al-Sulaiman 2017).
2. The number of cases of the disease in first degree relatives, i.e., if the circumstances are above two (Cini et al. 2016).
3. The number of cases of bilateral breast cancer
4. The people infected with the disease before they attained 40 years. 

The researchers used the criterion of testing the genetic history of the patients who had BRCA1 and BRCA2. The researchers tested them at any age since the age was not a major factor here (Tung et al. 2018). 

We will use limited sampling to come up with the sample groups from the whole community of the patients who have ovarian cancer in Qatar. We will study the sample group keenly and examine them for any cases of the ovarian cancer mutations which are not present in the whole population. We will use the probability methods to select the sample groups which will make it easy to generalize the data based on the statistical inferences results.

What will be performed is as the following:

• Firstly, the population of interest will be taken into account for the research, and in our case, the community is the Ovarian cancer patients from Qatar
• We will use the medical files to retrieve information about all the ovarian cancer patients. This will assist in the coming up with the sample groups, and from the data collected from NCCCR, we realize that the approximate number of patients with the ovarian cancer is 1000 of them.
• The systematic random sampling will be used to determine the patients who will be used for the research study. Before sampling the units, the researchers must first determine the sample size. To ease the determination of the sample size, the researchers must set the confidence level of the sample frame as 95% and know the standard deviation of the sample. Using their experience and the information about the standard deviation and the confidence level the best sample size would be 278 patients. The researchers will use the sample frame and withdraw blood from the selected patients and use the molecular diagnosis technique to analyze the mutations of the disease amongst the people of Qatar.   

Research Question/Hypothesis

Measures are the sources of the actual data for the research. The following causes of data will be very crucial will be very critical for this study:

1. The DNA of each patient that will be used to conduct the research.
2. The researchers will need to use some tools that will be very important in bringing out the genetic history of the disease among the patients. Some of these tools will be the DNA sequencing machine also called the illumine NGS sequencing machine and many others (Corso et al. 2018).

These measures and materials will be very crucial in facilitating the research.

The measures will also contain the elements below:

• The mutation of BRCA1&2 genes which are the main variables that the researchers will take into account
• The measures rely on the mutations of the BRCA1&2 genes.
• The rate of the occurrence of the ovarian cancer amongst relatives will be considered as the independent variable.
• Age could be the only confusing factor and the researchers must look at the best suiatable place to include it. 

Validity and Reliability assessment of measures:

The current research institute for this project is the NCCCR in collaboration with the HMC. The NCCCR has proved to discriminate familial cases of the disease from the no-history in the family. The NCCCR also correlates with other measures.  

Maxwell® 17 blood DNA is a ready-made purification kit that purifies the DNA.  In this case, the catalog number that was selected was AS1010 and the scientists gave the use manual in the link below to assist the researchers in isolating the DNAs (https://worldwide.promega.com/products/dna-purification-quantitation/genomic-dna-purification/maxwell-16-blood-dna-purification-kit/?catNum=AS1010)

The purity of the DNA was evaluated with NanoDrop 1000 Spectrophotometer V3.8. The ratio of sample absorbance at 260 and 280 nm. That ratio of the absorbance between 260 and 280 nm access and determine how pure the DNA sample was. A purity of between 1.8 and 2.0 nm is used. The virtue of something is what is generally accepted for further training.  NanoDrop 1000 Spectrophotometer V3.8User’s Manual for Thermo scientific company is followed for testing (Zhang et al. 2014).

(https://tools.thermofisher.com/content/sfs/manuals/nd-1000-v3.8-users-manual-8%205×11.pdf)

The electronic Oncomine™ BRCA Research Assay kit is compromised two pools, pool1 and pool2, AmpliSeq™ oligonucleotide primers (Christie et al. 2017). The research kit makes libraries, and the two repositories have the sole purpose of generating the amplicon libraries for the NGS (Next-Generation Sequencing) on Ion S5™ platforms (catalog number: A32841). This trial is exact and exhaustive for amplifying the sample of all the exotic parts of the human BRCA1 and BRCA2 genes (Pennington et al. 2014). This form of the attempt/assay has the purpose of preparing an automatic library using the Ion Chef™ System (catalog number: 4484177). The template preparation adjusts the amplification of the library. The process generates a clonal ion sphere particle recovery and also enriches it (Couch et al. 015). A chip loads in the ion chef machine and the loading process prepares the chip for the next step of sequencing (Li et al. 2015). (For choosing the correct disk to go to 510™& Ion 520™& Ion 530™Kit – Chef USER GUIDE) 

The ion Genestudio S5 is one of the Next-Generation Sequencing Systems which is implemented for the targeted sequencing. The ion gene S5 NGS has applied in sequencing all enriched libraries (Castéra et al. 2014). The sequencing is consummated by the replacement of all the consumables, initialization of the instrument, and lastly, before you start sequencing, load the chip that contains the samples of the patients. After the initialization of the sequencing process, two sequences run per initialization. (Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef). Catalogue number:A34461.

Background information

The five samples that were used produced specific variants in BRCA1 and BRCA2 when they were used in Sanger sequencing. The five examples used the big dye terminator kit (BigDye™ Terminator v3.1 Cycle Sequencing Kit, catalogue number: 4337455) to test the NGS technology and the outcome was the same.  The Oncomine™ BRCA was then valid for the sequencing of the different variants in the patients’ tests, and it was clear that the machine would produce the correct results (Kuehn et al. 2014).   

The Hamad Medical Corporation’s medical records department keep the medical records of every patient who seek consultation and medication from them (Bu et al. 2016). The information of all patients is secluded from being viewed by the third party or any other unauthorized people. All the staff working at the Hamad Medical Corporation have a duty of keeping all the personal information of all the patients confidential, and they learn in this in the course of their study. The medical reports of all the patients are also protected from disclosure to the third parties and exposure to unauthorized people. The commitment is part of their ethical and professional code of conduct. This code of conduct requires that the patients and their families have a right to their information being private and confidential. The ethics also demand that the staff protect this patients’ information and ensure that it is readily available when they need it.

Some software that were available online were used to predict the pathogenicity of the missence variants and their consequences were also studied (Dudley et al. 2015). The three software are:

• PolyPhen-2
• (Polymorphism Phenotyping v2). (https://genetics.bwh.harvard.edu/pph2/)
• Mutation taster(https://www.mutationtaster.org/)
• SIFT (https://sift.jcvi.org/ )

Recently, the studies concerning the study and research of molecular genetics of BRCA1 and BRCA2 germline mutations have been few, and the cases that have been reported concerning it are few (Rahman & Zayed 2018). The knowledge of the spread and the extent of BRCA1 and BRCA2 mutations is crucial for the population. The researchers will investigate about 1000 families with the genetic history of ovarian cancer for the germline mutations in BRCA1 and BRCA2 genes associated with the hereditary ovarian cancer (Song et al. 2015). Every patient with his/her relatives will be required to fill and sign consent forms and ethical approvals. The patients and their families will obtain the consent forms and ethical approvals from the Institutional Review Board at the HMC.

In order to carry out the research effectively, the researchers need experienced and specialized doctors who deal with cancer therapy. The doctors need a clean and a well-equipped laboratory

Overall Objective

The researchers will use the applied research method because the method will help us come up with the final remedy of how to mitigate the spread of ovarian cancer.

• It analyses and solves social and real life problems
• It investigates the scientific knowledge of the situation and will eventually help to heal the ovarian cancer condition.

• The method is expensive because it is done on large scale 

Corso, G., Feroce, I., Intra, M., Toesca, A., Magnoni, F., Sargenti, M., … & Veronesi, P. (2018). BRCA1/2 germline missense mutations: a systematic review. European Journal of Cancer Prevention, 27(3), 279-286.

Zhang, J., Walsh, M. F., Wu, G., Edmonson, M. N., Gruber, T. A., Easton, J., … & Wilkinson, M. R. (2015). Germline mutations in predisposition genes in pediatric cancer. New England Journal of Medicine, 373(24), 2336-2346.

Pennington, K. P., Walsh, T., Harrell, M. I., Lee, M. K., Pennil, C. C., Rendi, M. H., … & Agnew, K. J. (2014). Germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian, fallopian tube, and peritoneal carcinomas. Clinical Cancer Research, 20(3), 764-775.

Kuehn, H. S., Ouyang, W., Lo, B., Deenick, E. K., Niemela, J. E., Avery, D. T., … & Frucht, D. M. (2014). Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4. Science, 1255904.

Dudley, B., Brand, R. E., Thull, D., Bahary, N., Nikiforova, M. N., & Pai, R. K. (2015). Germline MLH1 mutations are frequently identified in Lynch syndrome patients with colorectal and endometrial carcinoma demonstrating isolated loss of PMS2 immunohistochemical expression. The American journal of surgical pathology, 39(8), 1114-1120.

Song, H., Dicks, S. J. R., Tyrer, J. P., Intermaggio, M. P., Hayward, J., Edlund, C. K., … & Anderson, C. (2015). Contribution of germline mutations in the RAD51B, RAD51C, and RAD51D genes to ovarian cancer in the population. Journal of Clinical Oncology, 33(26), 2901.

Ramus, S. J., Song, H., Dicks, E., Tyrer, J. P., Rosenthal, A. N., Intermaggio, M. P., … & Anderson, C. (2015). Germline mutations in the BRIP1, BARD1, PALB2, and NBN genes in women with ovarian cancer. JNCI: Journal of the National Cancer Institute, 107(11).

Christie, E. L., Fereday, S., Doig, K., Pattnaik, S., Dawson, S. J., & Bowtell, D. D. (2017). Reversion of BRCA1/2 germline mutations detected in circulating tumor DNA from patients with high-grade serous ovarian cancer. Journal of Clinical Oncology, 35(12), 1274-1280.

Tung, N., Battelli, C., Allen, B., Kaldate, R., Bhatnagar, S., Bowles, K., … & Krejdovsky, J. (2015). Frequency of mutations in individuals with breast cancer referred for BRCA1 and BRCA2 testing using next?generation sequencing with a 25?gene panel. Cancer, 121(1), 25-33.

Bu, R., Siraj, A. K., Al?Obaisi, K. A., Beg, S., Al Hazmi, M., Ajarim, D., … & Al?Kuraya, K. S. (2016). Identification of novel BRCA founder mutations in Middle Eastern breast cancer patients using capture and Sanger sequencing analysis. International journal of cancer, 139(5), 1091-1097.

Rahman, S., & Zayed, H. (2018). Breast cancer in the GCC countries: A focus on BRCA1/2 and non-BRCA1/2 genes. Gene.

Cini, G., Mezzavilla, M., Della Puppa, L., Cupelli, E., Fornasin, A., D’Elia, A. V., … & Maestro, R. (2016). Tracking of the origin of recurrent mutations of the BRCA1 and BRCA2 genes in the North-East of Italy and improved mutation analysis strategy. BMC medical genetics, 17(1), 11.

Denic, S., & Agarwal, M. M. (2017). Breast cancer protection by genomic imprinting in close kin families. BMC medical genetics, 18(1), 136.

Bujassoum, S. M., Bugrein, H. A., & Al-Sulaiman, R. (2017). Genotype and Phenotype Correlation of Breast Cancer in BRCA Mutation Carriers and Non-carriers. J Cancer Sci Ther, 9, 358-362.

Couch, F. J., Hart, S. N., Sharma, P., Toland, A. E., Wang, X., Miron, P., … & Slettedahl, S. (2015). Inherited mutations in 17 breast cancer susceptibility genes among a large triple-negative breast cancer cohort unselected for family history of breast cancer. Journal of clinical oncology, 33(4), 304.

Castéra, L., Krieger, S., Rousselin, A., Legros, A., Baumann, J. J., Bruet, O., … & Baert-Desurmont, S. (2014). Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes. European Journal of Human Genetics, 22(11), 1305.

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