Ethics statement and human participants
The current study obtained the approval of the Deanship of Scientific Research at Princess Norah Bint Abdulrahman University. This study was approved by the National Committee of Bioethics, King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia (Study number H-01-R059, IRB LOG number 20-0287). The written acquainted permissions have been obtained from the participants before obtaining their samples.
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 profiles of all the consanguineous cases. Table 2 shows the tumour, node, metastasis (TNM) classifications of malignant colon cancer of the participating consanguineous cases of the affected patients.
Table 1
Benchmark demographic profiles showing clinical characteristics of the participating consanguineous cases in four generations and consanguineous cases of mutated couples
Cases #
|
Age at diagnosis (years)
|
Gender
|
Family history with other types of cancer
|
Diagnosed with colon cancer
|
BRCA1 & BRCA2 mutations
|
Smoking history
|
Chronic colitis history
|
Obesity
(BMI ≥ 25 kg)
|
1
|
79
|
M
|
unknown
|
YES
|
YES
|
-
|
-
|
-
|
2
|
76
|
F
|
-
|
-
|
YES
|
-
|
-
|
YES
|
3
|
59
|
M
|
YES – breast
|
-
|
YES
|
YES
|
YES
|
-
|
4
|
55
|
F
|
YES – pancreatic & breast
|
-
|
YES
|
-
|
-
|
YES
|
5
|
55
|
M
|
YES – breast
|
-
|
YES
|
-
|
-
|
-
|
6
|
53
|
F
|
YES – breast
|
-
|
YES
|
-
|
YES
|
YES
|
7
|
51
|
M
|
YES – breast
|
-
|
YES
|
YES
|
YES
|
-
|
8
|
48
|
F
|
YES – breast
|
-
|
YES
|
-
|
-
|
YES
|
9
|
46
|
F
|
YES – breast
|
-
|
YES
|
-
|
-
|
-
|
10
|
39
|
M
|
YES – breast
|
-
|
YES
|
-
|
YES
|
-
|
11
|
38
|
F
|
YES – pancreatic & breast
|
-
|
YES
|
-
|
YES
|
-
|
12
|
36
|
F
|
YES – breast
|
-
|
YES
|
YES
|
-
|
YES
|
13
|
34
|
F
|
YES – breast
|
YES
|
YES
|
-
|
YES
|
YES
|
14
|
31
|
M
|
YES – breast
|
-
|
YES
|
-
|
-
|
-
|
15
|
30
|
M
|
YES – breast
|
-
|
YES
|
YES
|
-
|
YES
|
16
|
28
|
F
|
YES – breast
|
-
|
YES
|
-
|
-
|
-
|
17
|
24
|
F
|
YES – breast
|
-
|
YES
|
-
|
-
|
-
|
18
|
17
|
F
|
YES – breast
|
YES
|
YES
|
-
|
YES
|
YES
|
19
|
14
|
M
|
YES – breast
|
-
|
YES
|
-
|
-
|
-
|
20
|
12
|
F
|
YES – breast
|
-
|
-
|
-
|
-
|
-
|
21
|
10
|
F
|
YES – breast
|
-
|
YES
|
-
|
-
|
-
|
22
|
7
|
M
|
YES – breast
|
-
|
YES
|
-
|
-
|
-
|
23
|
6
|
F
|
YES – breast
|
-
|
-
|
-
|
-
|
-
|
24
|
6
|
F
|
YES – breast
|
-
|
-
|
-
|
-
|
-
|
Total
|
22/24
|
3/24
|
21/24
|
4/24
|
7/24
|
8/24
|
Table 2
Pathological characteristics (TNM classifications) for the participating consanguineous cases with malignant colon cancer
Stage (TNM Classification)
|
Number of Patients (%)
|
T1 - Tumour affects the submucosa (1-2 cm)
|
2
|
T2 - Tumour affects the muscularis propria (5 cm)
|
1
|
Total
|
3
|
DNA extraction
Blood samples were obtained from all the consanguineous cases of affected carriers and patients from a single Saudi family (of four generations). Then, DNA was extracted using the Qiagen DNA Isolation Kit (Cat No./ID: 69506, Quigen, Hilden, Germany), following the manufacturer’s instructions. The sample concentration and quality of DNA ratio were assessed using the NanoDrop Spectrophotometer system (Thermo Scientific, USA).
Targeted capture sequencing of BRCA2 /BRCA1 mutations
Exon coding sequences for the BRCA2 BRCA1 genes were conducted using the heteroduplex analysis, according to the description by [16] or the single-stranded conformation analysis, according to the description by [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 purified 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 (purified). The sequencing reaction products were purified using the MontageTM SEQ96 Sequencing Reaction Kit, followed by electrophoresis using the Applied Biosystems 3130 Genetic Analyzer, similar to the previous study [18].
Table 3. BRCA2 and BRCA1 PCR primers
a) BRCA1 primers sequence
Exon 2
|
Forward
|
5'-GAAGTTGTCATTTTATAAACCTTT-3'
|
315
bp
|
Reverse
|
5'-TGTCTTTTCTTCCCTAGTATGT-3'
|
Exon 5
|
Forward
|
5'-GCTCTTCGCGTTGAAGAAGT-3'
|
400
bp
|
Revers e
|
5'-GAAGTCTTTTGGCACGGTTT-3'
|
Exon 8
|
Forward
|
5'-CAGCTTGACACAGGTTTGGA-3'
|
499
bp
|
Reverse
|
5'-TTTCTGGATGCCTCTCAGCT-3'
|
Exon 11
|
Forward
|
5'-GAGGACAAAGCAGCGGATAC-3'
|
359
bp
|
Reverse
|
5'- GCTGTAATGAGCTGGCATGA-3'
|
Exon 18
|
Forward
|
5'- GGGAGAAGCCAGAATTGACA-3'
|
354
bp
|
Reverse
|
5'- CTCGCTTTGGACCTTGGTG -3'
|
Exon 23
|
Forward
|
5'- TTCAGGGGGCTAGAAATCTG-3'
|
289
bp
|
Reverse
|
5'- AAGCTCATTCTTGGGGTCCT -3'
|
Exon 24
|
Forward
|
5'- TTCAGGGGGCTAGAAATCTG-3'
|
289
bp
|
Reverse
|
5'- GGGGTATCAGGTAGGTGTCC-3'
|
b) BRCA2 Primers Sequence
Exon 2
|
Forward
|
5'- AGCGTGAGGGGACAGATTTG-3'
|
519
bp
|
|
Reverse
|
5'-GTGGACAGGAAACATCATCTGC -3'
|
Exon 9
|
Forward
|
5'- AGGAGCTGAGGTGGATCCTG -3'
|
980
bp
|
|
Reverse
|
5'- TCAGAATTGTCCCAAAAGAGCT -3'
|
Exon 9
|
Forward
|
5'- GTTCAGCCCAGTTTGAAGCA -3'
|
980
bp
|
|
Reverse
|
5'- TGACACTTGGGTTGCTTGTT -3'
|
Exon 14
|
Forward
|
5'- TGTCCCGAAAATGAGGAAATGG -3'
|
925
bp
|
|
Reverse
|
5'- TGTGAAACTGAAAAGACTCTGCA -3'
|
Exon 18
|
Forward
|
5'- GGTGGATGGCTCATACCCTC -3'
|
809
bp
|
|
Reverse
|
5'-TTTGCTGCTTCCTTTTCTTCC-3'
|
Exon 23
|
Forward
|
5'- GAAGAATGCAGCAGACCCAG -3'
|
751
bp
|
|
Reverse
|
5'- TGTCTCTTGAAAGTGGCCCT-3'
|
Quantitative genome analysis
Sequence data was confirmed 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 fixation of 10% buffered formalin. After processing tissue in an automated processor of a 24-hour schedule, blocks of paraffin 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 confirming inherited germline’s mutations, BRCA1 & BRCA2 deficiency is linked to biological mechanisms. Immunohistochemical research was conducted in samples of all the consanguineous cases subjects with paraffin-embedded colon cancer. Sections 5 µm tick were obtained from paraffin-embedded wax blocks were put in slides (saline-glass) and later air-dried a night at 37°C, deparaffinised in xylene, followed by rehydration in some graded alcohol (70 and 100%). Then, the slides were incubated in 3% methanol/ H2O2 for 10 minutes and washed with phosphate-buffered saline (PBS). Primary antibodies, anti-BRCA1 (MS110, mouse monoclonal, 1:200, Abcam, Cambridge, UK), anti-BRCA2 (MAB2476, mouse monoclonal, 1:500; R&D Systems, Inc. Minneapolis, MN, USA), were used at a diluted ratio of 1:10. Then, the solution was recorded as negative (absent or greatly reduced) of the brown-like nuclear stain, indicating < 20%. However, nuclear staining of > 20% was regarded as positive (American society of clinical oncology/college of American pathologist clinical practice guideline update, 2013). The sections were subjected to incubation for half a day in the primary antibodies BRCA2 and BRCA1 at 4°C and washed with PBS. Consequently, a light microscope was used to examine the immunostaining sections.
Statistical analysis
The Analytical examinations are performed using the SigmaStat programming adaptation 3.5 (Systat Software, San Jose, CA, USA). Then, the results of the quantitative analysis were expressed in standard deviations and mean. P values < 0.05 were regarded as statistically significant.