Mutational Analysis of EZH2 Gene in Patients with Colorectal Adenoma Reveals a Genetic Variant Associated with Risk of Malignant Transformation

Abstract

Background: Several studies have revealed that chromatin modifications lead to activation or repression of multiple genes including oncogenes and tumor suppressor genes. Inactivation mutation in EZH2 gene would result in activation of oncogenes. The aim of this case-control study was to identify mutations in the EZH2 gene, to study their prevalence among Jordanian patients with colorectal adenoma and to determine how these mutations could be related to colorectal cancer (CRC) progression.

Methods: EZH2 gene sequencing was done by Sanger method for 100 DNA samples, extracted from blood of 50 patients, and 50 controls. Sequencing results were analyzed by Chromaspro and mutational effects were predicted by Mutation Taster bioinformatics tool.

Results: Four variants were identified in Jordanian patients with adenoma; Two novel variantsc.1941T>A and c.2201G>C and two reported variants, g.73038C>T and g.75508A>C. g.73038C>T is the most common germline variant identified in this study. A significant association between the presence of c.2201G>C mutation and colorectal adenoma was found (p value < 0.05).

Conclusion: The present study identified several variants in EZH2 gene among Jordanians with colorectal adenoma.

Keywords: Colorectal polyps; DNA Methylation; EZH2; Mutation.

Figure 1

Schematic Representation of DesignAed EZH2 Primers. EZH2 5' untranslated region, 6 exons and exons-intron boundaries were amplified using 6 primer sets

Figure 2

The Association between g.73038C>T and Colorectal Adenoma Malignancies. Strong association between g.73038C>T and Colorectal Adenoma (p-value=0.00002).

Figure 3

The Mutational Analysis of Exon 16 of EZH2 Gene.(a) Schematic representation of EZH2 showing exon 16. (b) The reference sequence of exon 16 obtained from Gene Bank (NG_032043) and the highlighted sequence is for exon 16. The PCR was run using the forward (F) and reverse (R) primers (sequence above and below the block arrows). The expected PCR products are 350bp. The red arrow shows the position of g.73038c>t variation and the green arrow shows the position of c.1941T>A variation. (c) 2% gel electrophoresis for exon 16. The exon was successfully amplified and 350bp bands were visualized by 2% agarose gel. The size was compared to 100 ladder (the first lane) and negative control (lane1) was used. Lanes from 2-6 are representative patients samples and lanes from 7-18 are representative samples from controls. (d) Representative partial chromatogram for exon16 normal control showing wild type sequence (wt/wt), (e) heterozygous g.73038C>T (wt/vt), and (f) homozygous (vt/vt) of g.73038C>t, respectively. (g) Representative partial chromatogram for exon16 from patients illustrating wild type sequence (wt/wt), and (h) heterozygous c.1941T>A (wt/vt)

Figure 4

The Mutational Analysis of Exon 18 of EZH2 Gene. (a) Schematic representation of EZH2 showing exon 18. (b) The reference sequence of exon 18 obtained from Gene Bank (NG_032043) and the highlighted sequence is for exon 18. The PCR was run using the forward (F) and reverse (R) primers (sequence above and below the block arrows). The expected PCR products are 250bp. The red arrow shows the position of g.75508A>C variation. (c) 2% gel electrophoresis for exon 18. The exon was successfully amplified and 250bp bands were visualized by 2% agarose gel. The size was compared to 100 ladders (the first lane) and negative control (lane1) was used. Lanes from 2-6 are representative patients samples and lanes from 7-19 are representative samples from controls. (d) Representative partial chromatogram for exon18 normal control showing wild type sequence (wt/wt). (e) Representative partial chromatogram for exon18 from patients illustrating heterozygous, g.75508A>C, variant (wt/vt). (f) Representative partial chromatogram for exon18 patient showing variant sequence (vt/vt)

Figure 5

The Mutational Analysis of Exon 20 of EZH2 Gene.(a) Schematic representation of EZH2 showing exon 20. (b) The reference sequence of exon 20 obtained from Gene Bank (NG_032043) and the highlighted sequence is for exon 20. The PCR was run using the forward (F) and reverse (R) primers (sequence above and below the block arrows). The expected PCR products are 747bp. The red arrow shows the position of c.2201GG>C mutation. (c) 2% gel electrophoresis for exon 20. The exon was successfully amplified and 747 bp bands were visualized by 2% agarose gel. The size was compared to 100 ladders (the first lane) and negative control (lane1) was used. Lanes from 2-6 are representative patients samples and lanes from 7-19 are representative samples from controls. (d) Representative partial chromatogram for exon20 normal control showing wild type sequence (wt/wt). (e) Representative partial chromatogram for exon20 from patients illustrating heterozygous, c.2201G>C, variant (wt/vt). C.2201G>C is noval mutation found in Jordanian patients with colorectal adenoma