Copyright © 2018 Authors. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Engineering & Technology, 7 (4.37) (2018) 1-11 International Journal of Engineering & Technology Website: www.sciencepubco.com/index.php/IJET Research paper New Finding in KRAS Mutated Structure Detected in Colorectal Cancer CRC Tumors Causing Uncontrolled Cell Proliferation in Iraqi Patients Rehab Subhi Ramadhan 1*, Rebah Najah Jabbar AL-Gafari 2 1 Department of Medical Biotechnology, College of Biotechnology, Al-Nahrain University, Baghdad, Iraq 2 Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq *Corresponding author E-mail: rebahalgafari@gmail.com Abstract A total 200 tumor samples were collected from patients suffering from colorectal CRC at Al-Amal hospital. Their ages ranged from 40 – 70 years old and distributed according to gender as 130 male and 70 female. We found that CRC was more common in male than female, and more frequent in elderly persons especially in age group of 61 – 70 years old. From 12 specifically designed primers that were able to amplify KRAS gene in those patients’ blood samples, only 2 gave a specific band when tumor sample was used. Molecular analysis of the sequence obtained from those two primers showed the presence of 83 missense mutations in both of them, 20 in the first sequence were associated with pathogenic effect on KRAS gene, 3 frame shift, and 3 insertions, while in the second sequence obtained from pri- mer PK2 4 frame shift mutations, and 4 insertions were identified. The impact finding is that in both sequences, open reading frames (ORFs) were developed that affected cell proliferation dramatically and produced truncated functionless proteins causing KRAS to cease control over cell cycle. Keywords: KRAS; Oncogene; colorectal cancer; pathogenetic mutation. 1. Introduction Colorectal cancer (CRC) is the second leading cause of cancer- related death. The development of CRC is a multistep process characterized by accumulation of genetic alterations that have long been considered to occur in a stepwise process (Russo et al., 2005. Along the progression from normal colonic epithelial cells, small adenoma, advanced adenoma, and finally to carcinoma, the KRAS oncogene mutation has a role in a significant proportion of CRCs Castagnola and Giaretti 2005. KRAS has been reported to be mu- tated in about 30% of colorectal adenomas and 30% to 50% of CRCs. The KRAS gene encodes a 21-kDa small protein that is activated transiently as a response to extracellular stimuli or sig- nals such as growth factors, cytokines, and hormones via cell sur- face receptors. On its activation, the KRAS protein also is capable of turning off the signaling pathway by catalyzing hydrolysis of guanosine triphosphates (GTP) to guanosine diphosphates Down- ward 2003. The most common KRAS mutations in codons 12 and 13 are activation mutations, leading to continuous activation of downstream pathways. The most frequently observed types of mutations in KRAS in all human cancers are G > A transition and G > T transversion Haigis et al., 2008. Although the precise mo- lecular and cellular mechanisms that constitute the oncogenic effects of activating KRAS mutations remain incompletely under- stood, in vitro and animal studies showed that KRAS regulated genes involve cytokine signaling, cell adhesion, cell survival, proliferation, apoptosis, and colon development Malumbres and Barbacid, 2003. The RAS oncogene has a well-established role in cell growth and regulation; and its protein product affects many cellular functions including cell proliferation, apoptosis, migration, fate specification, and differentiation. There are three known hu- man isoforms, NRAS, HRAS, and KRAS. Over 90% of pancreatic adenocarcinomas Almoguera , 1988 , 30%–50% of colorectal cancers De Roock et al, 2008, 55% of thyroid cancers, 35% of lung cancers, and 35% of rhabdomyosarcomas harbor mutated RAS genes Kranenburg 2005. Although HRAS was historically the most studied RAS gene, it is actually the isoform least mutated in human cancers Baines, and Xu, D.; Der 2011. In fact, KRAS mutations comprise 86% of all RAS mutations. Mutations in KRAS occur with the greatest frequency in all human cancers (21.6%), followed by NRAS (8.0%), and HRAS (3.3%). In recent studies, a lethal genetic change was identified at exon 2 in KRAS gene in Iraq. The study showed the accumulation of 22 pathogenic SNPs which eventually led to CRC in these patients (Subhi et al., 2018). 2. Materials and Methods Collection of tissue Samples: Tissue samples were collected at Al-Amal hospital from 200 patients after they diagnosed with colorectal cancer using bio marker (CA19-9) and subjected to tumor excision. Their ages ranged between (40-70) years as the majority of individuals attending the hospital for treatment. DNA extraction from blood: The gSYNC™ DNA Extraction Kit from Geneaid (Taiwan) was used for this purpose as instructed by the manufacturer. The extraction procedure mainly depends upon spin column technique which gave DNA purity of 1.8-2 and con- centration of 80-120 ng/sample. Primers used for DNA amplifica-