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Full Molecular Screening of Colon Cancer in Patients Inherited Familial/Highly Germline BRCA1 and BRCA2 Mutations


 Background: A significant body of research has presented evidence regarding an increasing number of cases of early colon cancer among young people. The present study aimed to assess the inherited germinal mutations in breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) coding areas in four consanguineous cases in generations of a single Saudi family. Methods: The sample consisted of a single Saudi family of four generations in Riyadh, Saudi Arabia. The family has been diagnosed with colon cancer and inherited germline mutations in BRCA1 and BRCA2. DNA sequencing was used to examine the entire coding BRCA1 and BRCA2 sections. The colon samples were immunohistochemically and histologically analysed using BRCA1 and BRCA2 antibodies and H&E Staining Kit (Hematoxylin and Eosin), respectively. Results: A total of 21 scenarios at-risk consanguineous cases were mutations carriers of the BRCA genes (2 BRCA2 and 4 BRCA1), comprised of 3 affected consanguineous cases with malignant colon cancer. The BRCA nucleotide sequencing revealed substantial mutation’s cases in the BRCA1 genes (frameshift mutations situated within exon 11 and exon 2, where no such mutations were present in the BRCA2 genes). A novel replacement mutation within BRCA2 (exon 18) of 139 C > A was obtained with (45.83%) in females and (31.26%) in males. Immunohistochemical staining showed positive staining for BRCA1 and BRCA2. Conclusions: The major factor responsible for colon cancer incidences among the family is associated with the inherited mutations of the germline in BRCA2 and BRCA1 through repeated endogamy of consanguineous cases of the carriers of the mutation.Trial registrationRetrospectively registered.


Background
Colon cancer is a heterogeneous disease with at least three distinct subtypes: colitis-associated, hereditary and sporadic colon cancer. Hereditary colon cancer accounts for roughly 10-15% of all colon cancers [1].
Breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) genes act as caretaker genes that maintain genome integrity and tumour suppressor's genes [3,5]. The rate of BRCA2 and BRCA1 gene mutations vary in different regions and countries [2,4], and they account for 5-10% of colon and breast cancer [3]. The BRCA1 gene is found on chromosome 17, encompassing 24 exons encoding the protein of 1,863 various amino acids.
However, the BRCA2's gene is found on chromosome 13 encompassing 27 exons encoding the protein of 3,418 amino acids [6]. More than 700 BRCA1's mutations and more than 600 BRCA2's mutations are reported in the coding regions [7]. Estimations indicated that several patients with BRCA2 and BRCA1 genes inherited mutations susceptible to 3-fold increased risks of developing colon, breast, and pancreatic cancer [8,15]. In addition, conventional histopathology, molecular analysis and immunohistochemistry provide enormous information regarding types of cancer [9][10][11][12]. The highly favourable methods for identifying sequences are heteroduplex analysis and single-strand conformation polymorphism analysis as they are sensitive and simple in detecting germinal mutations [12].
Patients with hereditary familial colon cancer have germline BRCA2 and BRCA1 mutations, in the form of adenomas colonic mucosa. During the process, patients obtain germline mutations within the BRCA2 and BRCA1 genes [7,8]. Consequently, the cellular mechanism associated with these inherited genes' changes the tissues of both breast and colon tumorigenesis [4,14,15].
Although BRCA1/2 have been studied extensively, Saudi data is still insu cient. This study aims to report the speci c germinal mutations of BRCA2 and BRCA1, within the entire coding regions for highly/familial inherited mutations, to investigate the incidence of colon cancer in consanguineous cases of four generations in a Saudi Arabian family. The study aims to assess other related cofactors that increase colon cancer and other types of cancer among the consanguineous cases within the family.

Methods
Ethics statement and human participants

Case selection and samples collection
The study collected data of 24 consanguineous cases of affected carriers and patients from a single Saudi family (of four generations) in Riyadh, Saudi Arabia. Three biopsy samples were obtained from 24 relatives affected by malignant colon cancer in August 2020 at the King Fahad Medical City in Riyadh, Saudi Arabia. Table 1 summarised the benchmarks of demographic pro les of all the consanguineous cases. Table 2 shows the tumour, node, metastasis (TNM) classi cations of malignant colon cancer of the participating consanguineous cases of the affected patients.   [17].

PCR and Sanger sequencing for BRCA2/ BRAC1 mutations detection
The conventional Sanger sequencing technique was used for the entire BRCA2 and BRCA1 coding sections. The primer three programs were used to design the primers of all the coding exons of BRCA2 and BRCA1 genes. Table 3a shows the primers' sequences for BRCA1. Table 3b shows the primers' sequences for BRCA2. The NucleoFast ® 96 PCR Clean-up Kit puri ed PCR products, according to the manufacturer's instructions.
The sequencing reactions were conducted using the BigDye ® Terminator v1.1 Cycle Sequencing Kit for every PCR product (puri ed). The sequencing reaction products were puri ed using the MontageTM SEQ96 Sequencing Reaction Kit, followed by electrophoresis using the Applied Biosystems 3130 Genetic Analyzer, similar to the previous study [18].  Quantitative genome analysis Sequence data was con rmed visually using the snap gene viewer. BRCA2 (accession number: NP_000050.2) and GenBank BRCA1 human sequences (accession number: NP_009225.1) were referenced to the NCBI Reference Sequences Database.

Pedigree analysis
Factors contributing to colon cancer are carriers of BRCA2 and BRCA1 gene mutations, age-of-onset, tumours type, endogamy and other histories of cancer. Cause and date of death determined the presence of colon cancer for all the descendants of traceable ancestors of the patients. Cancer incidence and pedigree data were registered on a computerised database. The traceable consanguineous cases were patients alive between 2019 and 2020.

Tissue and histological preparation
Tissue specimens consisted of small biopsies i.e. 7-10 mm in diameter, obtained from consanguineous cases of the affected patients with colon cancer. Afterwards, tissue specimens underwent transfer to xation of 10% buffered formalin. After processing tissue in an automated processor of a 24-hour schedule, blocks of para n were sectioned and attained sections of 3-5 µm using conventional histopathology. Moreover, the conventional histopathology applied the Hematoxylin and Eosin (H&E) technique, as described by [19].
Immunohistochemical stating for BRCA1&2 Following the Immunohistochemical expression of BRCA1 and BRCA2 for screening and con rming inherited germline's mutations, BRCA1 & BRCA2 de ciency is linked to biological mechanisms. Immunohistochemical research was conducted in samples of all the consanguineous cases subjects with para n-embedded colon cancer. Sections 5 µm tick were obtained from para n-embedded wax blocks were put in slides (saline-glass) and later air-dried a night at 37°C, depara nised in xylene, followed by rehydration in some graded alcohol

Results
Deleterious BRCA2 and BRCA1 gene mutations of the participating consanguineous cases In addition, BRCA gene mutations were analysed ( Table 4). The BRCA1 gene had 4 mutations in the Kit exon 11 and 2. However, in the BRCA2 gene, two mutations are in the Kit exon 18. Moreover, a substantial amount of BRCA1 gene mutations were the frameshift mutations situated within exon 11 and exon 2.
Overall, the BRCA1 mutations in females and males were 11.06% and 19.8%, respectively. Nevertheless, the BRCA2 mutation's frequency among females and males was 31.8% and 48.62%, respectively.
On contrary, the sequencing of nucleotides for BRCA2 and BRCA1 showed the absence of frameshift mutations within the BRCA2 gene. However, two substituting mutations in exon 18 indicated novel mutations 139 C > A (31.26%) in males and (45.83%) in females. Analysed in this study is the substituting mutation for 8415 G >T (32.29%) in males and (51.42%) in females, which is the common germinal mutation (Figure 1 and Table 4). From the 24 consanguineous people, 21 consanguineous cases are at the risks of BRCA genes mutations when dealing with extended pedigrees. Blood samples were collected from members of the family to identify the colon cancer incidences and their prior history of breast cancer. Their BRCA2 and BRCA1 were screened with nucleic acids to identify mutations within the proband. For this study, the ratio of relatives was 9 males and 15 females for BRCA2 and BRCA1 genes' mutations carriers (21 out of 24, 87.5%) and the germline major genetic disorder with (P = 0.00). Figure 2 shows the summary of the cancer incidence within the family members. Based on the pedigree analysis, the metachronous tumours comprised of three colon cancers, in which the fourth, third and rst generations had a family history of breast and pancreatic cancers. In addition, two cases of breast cancers were recorded. A deceased female was diagnosed with breast cancer. The second case is the breast cancer in the second generation of ve affected consanguineous cases with colon and breast cancers in a family. Figure 2 shows the female patients affected with breast cancer, diagnosed at the age of 53 years based on case #6 during the second generation. The proband revealed the inherited case of cancer from the father, (in which the colon cancer was diagnosed at the age of 79 years, in the rst generation). In contrast, a female cousin (had colon cancer diagnosed at the age of 17 years, case #1 in the fourth generation), having a paternal aunt (who was diagnosed with colon cancer at the age of 34 years, case #3 in the third generation).

Histopathology
Histopathological specimens were obtained from colon cancer patients who carried BRCA2 and BRCA1 germline mutations, substantial mitotic numbers and pleomorphism with lesser gland formation instances. In addition, irregular histological formations were noted with high-level tumours grades. A level of proliferation disorganisations was present (Figure 3, b), as compared to the normal histological section of the colon (Figure 3, a).
Immunohistochemical stating for BRCA2 and BRCA1 BRCA2 and BRCA1 expression was examined using immunohistochemistry (Figure 4), revealing strong intensities of staining by BRCA1 and moderate staining by BRCA2 antibodies with tumour tissues of patients with colon cancer. Overexpression of BRCA1 was markedly noted in all the three colons tumours' samples. Nuclear staining, showing over 20% reactions of positivity with the antibodies, is classi ed as positive reactions ( Figure 4). In addition, the expression of BRCA2 was recorded in all the colon tumours' samples. Moreover, the procedure of antigen retrieval was calculated following the lastly recorded numerical H-scores for every case and obtained by multiplying the scores in respect of the classi cation as follows: over 50% (Strong), 20-40% (moderate), 1-20% (weak) and 0% (none).

Discussion
The history of colorectal cancer is associated with factors that include age-of-onset, BRCA2 and BRCA1 gene mutations, other cancers' history, endogamy, tumours type, cause and date of death. Other associated cofactors are gender, obesity (BMI ≥ 25 kg), history of chronic colitis and history of smoking, revealed in all the descendants of the earliest traceable ancestors for patients having colorectal cancer. The main causes and co-factors received consideration in this research in examining the most impactful factors in the inheritance of germline mutations within BRCA2 and BRCA1 genes and the colon cancer incidence in four single family's generations.
The BRCA2 and BRCA1 mutations are regarded as the key factor that increases the colon cancer distribution [20,21] and tumorigenesis of the breast [15,22,23, 26] at times of increased cells' division mitotic numbers, a reversal for the distribution of the proliferating cell [24,25].
All the participating family members i.e., consanguineous cases of affected patients and carriers in this research, are inherited of a single novel mutation within the BRCA2 gene (139 C > A) of exon 18, which is the highly common germinal mutation analysed and reported in the prior research [15]. From the ndings of both genomes ( Figure 1, Table 4) and pedigree (Figure 2), all consanguineous cases showed germline mutations BRCA2 and BRCA1, passed by repeated endogamy of a family with a history of breast and pancreatic cancers, thereby leading to an increase in the risk of colon cancer [15,18,20], pancreatic [20,21] and breast cancer [15,22,23]. However, one female family member showed germline mutations within BRCA2 and BRCA1 and married an unrelative male with no such mutations. They have twin daughters with no germline mutations of BRCA2 and BRCA1.
In addition, colon tissues' immunohistochemical data indicated overexpression of BRCA1 while it positively stained the BRCA2. These ndings were consistent with previous studies [27,28], showing a substantial relationship between expressions of BRCA1/2 and the advanced stage of the tumour as indicated in (Figure 3, b and a).
In this current research, variables that increased colon cancer's incidence rates are age, gender, history of chronic colitis, obesity (BMI ≥ 25 kg) and smoking history.
For gender, Wang et al., (2020) showed that it was imperative to incorporate gender as a major factor when diagnosing and treating colon cancer. Sex-speci c tools of colon cancer's diagnoses in women help in developing ways of increasing the chemotherapy rate in women [26,29].
In contrast, the age factor plays a crucial role in colon cancer incidences. Several studies have shown the importance of healthy lifestyles in reducing colon cancer for the teenage group [26]. This present study revealed a similar impact of healthy lifestyles on two associated factors such as obesity 8/24 (33.33%) and history of chronic colitis 7/24 (29.16%) [30]. Many studies con rmed that unhealthy foods with hot spices, fast foods with unhealthy nutritional values and meals without fresh fruits and vegetables could increase the incidences of obesity and chronic colitis, particularly when there was a family cancer history [31 -33]. These issues could increase breast and colon cancers incidences at a younger age [26].
As shown in Table 4, the smoking history was recorded as 4/24(16.7%). However, none of the smokers had colon cancer. Nonetheless, smoking history was used in this study in assessing all the potential cofactors that could increase breast and colon cancer incidence within a single-family as previously reported [31,33].

Conclusion
This study proposes that highly germline mutations through inheritance of BRCA2 and BRCA1 and increase of breast and colon cancer over the four generations were associated with the repeated endogamy between consanguineous cases who carried germinal mutations of BRCA2 and BRCA1. Nevertheless, other factors such as a history of chronic colitis and obesity could result in early-onset of colon cancer for young people as indicated in the consanguineous cases in the family's fourth generation. Availability of data and material All relevant data are presented within the paper.
Competing interests Figure 1 Inherited mutations in BRCA2 and BRCA1 genes' frequencies for consanguineous cases in four generations of a Saudi family Pedigree indicating inherited characteristics of a family having deleterious BRCA2 and BRCA1 mutations through four generations, entailing transmission of breast and colon cancers due to paternal and maternal lineages. Photomicrographs for H&E section (stained) with the colon cancer of the relative patients using scale bar (100 μm) with section stained from a patient with colon cancer of BRCA2's mutations within exon 18 and BRCA1's mutations and within exon 2 (a) exon 11. However, BRCA2 mutations are in exon 18 (b). In addition, views of sections of colon cancer are noted with ampli ed cell activity (mitotic), variable pleomorphism grades, proliferation dysregulation, no formation of the gland, necrosis and cell pleomorphism (nuclear) with patterns of asymmetry for colon cells, as well as production of mucin and full of red blood cells.

Figure 4
Immunostained sections' photomicrographs from patients with colon cancer, with BRCA2 and BRCA1 antibodies, were obtained by a light microscope using a scale bar: 100 μm. Immunohistochemical slide for staining with BRCA1's mutants showed BRCA1 protein's overexpression in tissue from the patient of colon cancer diagnosed with the T2 -Tumour that affected the muscularis propria (a). The slide stained using BRCA2 antibody revealed positive staining within BRCA2 mutants' patient of colon cancer tissue and diagnosed as having T1 -Tumor that affected the submucosa (b).