Screening and computational analysis of colorectal associated non-synonymous polymorphism in CTNNB1 gene in Pakistani population

Suhail Razak,Sarwat Jahan,Nousheen Bibi,Tayyaba Afsar,Zahida Parveen,Ali Almajwal,Javid Ahmad Dar

doi:10.1186/s12881-019-0911-y

Abstract

BackgroundColorectal cancer (CRC) is categorized by alteration of vital pathways such as β-catenin (CTNNB1) mutations, WNT signaling activation, tumor protein 53 (TP53) inactivation, BRAF, Adenomatous polyposis coli (APC) inactivation, KRAS, dysregulation of epithelial to mesenchymal transition (EMT) genes, MYC amplification, etc. In the present study an attempt was made to screen CTNNB1 gene in colorectal cancer samples from Pakistani population and investigated the association of CTNNB1 gene mutations in the development of colorectal cancer.Methods200 colorectal tumors approximately of male and female patients with sporadic or familial colorectal tumors and normal tissues were included. DNA was extracted and amplified through polymerase chain reaction (PCR) and subjected to exome sequence analysis. Immunohistochemistry was done to study protein expression. Molecular dynamic (MD) simulations of CTNNB1WT and mutant S33F and T41A were performed to evaluate the stability, folding, conformational changes and dynamic behaviors of CTNNB1 protein.ResultsSequence analysis revealed two activating mutations (S33F and T41A) in exon 3 of CTNNB1 gene involving the transition of C.T and A.G at amino acid position 33 and 41 respectively (p.C33T and p.A41G). Immuno-histochemical staining showed the accumulation of β-catenin protein both in cytoplasm as well as in the nuclei of cancer cells when compared with normal tissue. Further molecular modeling, docking and simulation approaches revealed significant conformational changes in the N-terminus region of normal to mutant CTNNB1 gene critical for binding with Glycogen synthase kinase 3-B (GSK3) and transducin containing protein1 (TrCp1).ConclusionPresent study on Pakistani population revealed an association of two non-synonymous polymorphisms in the CTNNB1 gene with colorectal cancer. These genetic variants led to the accumulation of the CTNNB1, a hallmark of tumor development. Also, analysis of structure to function alterations in CTNNB1 gene is crucial in understanding downstream biological events.

Highlights

Colorectal cancer (CRC) is categorized by alteration of vital pathways such as β-catenin (CTNNB1) mutations, WNT signaling activation, tumor protein 53 (TP53) inactivation, BRAF, Adenomatous polyposis coli (APC) inactivation, KRAS, dysregulation of epithelial to mesenchymal transition (EMT) genes, MYC amplification, etc
This substitution resulted in replacing a hydrophilic neutral serine to a hydrophobic phenylalanine at amino acid position 33 [TCT (Ser) → TTT (Phe)] (Fig. 1) and a polar threonine was converted to non-polar alanine at amino acid position 41 [ACC (Thr) → GCC (Ala)] (Fig. 2), of exon 3 of Beta catenin (CTNNB1) gene
Our observation revealed that β-catenin may have a vital part in the progression of colorectal carcinoma and that activating mutation of the β-catenin gene may substitute bi-allelic APC inactivation in this tumor type in Pakistani Population

Summary

Introduction

Colorectal cancer (CRC) is categorized by alteration of vital pathways such as β-catenin (CTNNB1) mutations, WNT signaling activation, tumor protein 53 (TP53) inactivation, BRAF, Adenomatous polyposis coli (APC) inactivation, KRAS, dysregulation of epithelial to mesenchymal transition (EMT) genes, MYC amplification, etc. The worldwide prevalence of colorectal cancer (CRC) is third among cancer incidences in males and fourth among females [1, 2]. The frequency of CRC is increasing among the native population of Pakistan as observed by a three-fold growth in frequency in males from 2.3 to 6.8% within around 4 years and analogous tendency in females was seen with an increase from 2.5 to 6.7% for the same period [6, 7]. The rate of occurrence of CRC in Pakistan showed an increase due to the lack of surveillance programs and insufficient molecular investigations, suggests a theoretically alarming situation developing in the coming decades [10]

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