Identification of missense mutations in genes related to cancer pathways in glioma

Glioma is the most common primary brain tumour of the central nervous system. Many genetic alterations and mutations have been identified in glioma using various approaches. We performed DNA sequencing on the tumours of 16 patients with Grade I, II, III and IV glioma. The AmpliSeq Cancer Primers Pool was used to generate the amplicons. The targeted-ion sphere particles were prepared using the Ion One Touch and Ion Enrichment systems. DNA sequencing was performed on the Ion Torrent Personal Genome Machine (PGM) and the data were analysed using the Torrent Suite Software. In total, 14 mutations were identified in the following genes: KDR (Q472H), MLH1 (V384D), MET (N375S), PTPN11 (E69K), BRAF (V600E), TP53 (D149E, E154K, V157F), IDH1 (R132H), PIK3CA (H1047R), CSF1R (c1061_1061 ins A), KIT (M541L), PTEN (c1373_1373 del A) and PDGFRA (E556V). In addition, there were four novel mutations identified; TP53 (E154K, and D149E), CSF1R (c1061_1061 ins A) and PDGFRA (E556V). The pathogenicity prediction showed that only three mutations were pathogenic: PTPN11 (E69K), BRAF (V600E) and Tp53 (E154K). These mutations result in changes of the proteins’ structure and could affect their functions. Pathway analyses suggested that these genes are closely related to the pathogenesis of GBM through several pathways such as proliferation and invasion, metabolism and angiogenesis. In conclusion, PGM in combination with the AmpliSeq Cancer Panel could be utilised as a potential molecular diagnostic tool not only for glioma but also for other cancers

Positive TPMT genotype-phenotype correlation underscores importance of TPMT genotyping for personalized thiopurine dosing

This study explored TPMT genotype-phenotype correlation in a group of acute lymphoblastic leukemia (ALL) patients to investigate the potential of TPMT genotyping for personalized thiopurine dosing. Genotyping for G238C (TPMT*2), G460A (TPMT*3B) and A719G (TPMT*3C) loci was determined in 89 subjects via PCR, while TPMT activity was measured using HPLC. TPMT*3C was the only mutant allele detected in 4 heterozygous carriers. These patients had significantly lower (23.0 nmol/g Hb/h) TPMT activity compared to wildtype patients (51.0 nmol/g Hb/h) (p = 0.003). Positive correlation between TPMT genotype and phenotype projects the possibility of using TPMT genotyping as a guide prior thiopurine drug administration.

Epigenome-wide DNA methylation profiling in colorectal cancer and normal adjacent colon using Infinium Human Methylation 450K

The aims were to profile the DNA methylation in colorectal cancer (CRC) and to explore cancer-specific methylation biomarkers. Fifty-four pairs of CRCs and the adjacent normal tissues were subjected to Infinium Human Methylation 450K assay and analysed using ChAMP R package. A total of 26,093 differentially methylated probes were identified, which represent 6156 genes; 650 probes were hypermethylated, and 25,443 were hypomethylated. Hypermethylated sites were common in CpG islands, while hypomethylated sites were in open sea. Most of the hypermethylated genes were associated with pathways in cancer, while the hypomethylated genes were involved in the PI3K-AKT signalling pathway. Among the identified differentially methylated probes, we found evidence of four potential probes in CRCs versus adjacent normal; HOXA2 cg06786372, OPLAH cg17301223, cg15638338, and TRIM31 cg02583465 that could serve as a new biomarker in CRC since these probes were aberrantly methylated in CRC as well as involved in the progression of CRC. Furthermore, we revealed the potential of promoter methylation ADHFE1 cg18065361 in differentiating the CRC from normal colonic tissue from the integrated analysis. In conclusion, aberrant DNA methylation is significantly involved in CRC pathogenesis and is associated with gene silencing. This study reports several potential important methylated genes in CRC and, therefore, merit further validation as novel candidate biomarker genes in CRC

Genome-wide open chromatin methylome profiles in colorectal cancer

The methylome of open chromatins was investigated in colorectal cancer (CRC) to explore cancer-specific methylation and potential biomarkers. Epigenome-wide methylome of open chromatins was studied in colorectal cancer tissues using the Infinium DNA MethylationEPIC assay. Differentially methylated regions were identified using the ChAMP Bioconductor. Our stringent analysis led to the discovery of 2187 significant differentially methylated open chromatins in CRCs. More hypomethylated probes were observed and the trend was similar across all chromosomes. The majority of hyper- and hypomethylated probes in open chromatin were in chromosome 1. Our unsupervised hierarchical clustering analysis showed that 40 significant differentially methylated open chromatins were able to segregate CRC from normal colonic tissues. Receiver operating characteristic analyses from the top 40 probes revealed several significant, highly discriminative, specific and sensitive probes such as OPLAH cg26256223, EYA4 cg01328892, and CCNA1 cg11513637, among others. OPLAH cg26256223 hypermethylation is associated with reduced gene expression in the CRC. This study reports many open chromatin loci with novel differential methylation statuses, some of which with the potential as candidate markers for diagnostic purposes

Molecular Characterization of Somatic Alterations in Dukes’ B and C Colorectal Cancers by Targeted Sequencing

Despite global progress in research, improved screening and refined treatment strategies, colorectal cancer (CRC) remains as the third most common malignancy. As each type of cancer is different and exhibits unique alteration patterns, identifying and characterizing gene alterations in CRC that may serve as biomarkers might help to improve diagnosis, prognosis and predict potential response to therapy. With the emergence of next generation sequencing technologies (NGS), it is now possible to extensively and rapidly identify the gene profile of individual tumors. In this study, we aimed to identify actionable somatic alterations in Dukes’ B and C in CRC via NGS. Targeted sequencing of 409 cancer-related genes using the Ion AmpliseqTM Comprehensive Cancer Panel was performed on genomic DNA obtained from paired fresh frozen tissues, cancer and normal, of Dukes’ B (n = 10) and Dukes’ C (n = 9) CRC. The sequencing results were analyzed using Torrent Suite, annotated using ANNOVAR and validated using Sanger sequencing. A total of 141 somatic non-synonymous sequence variations were identified in 86 genes. Among these, 64 variants (45%) were predicted to be deleterious, 38 variants (27%) possibly deleterious while the other 39 variants (28%) have low or neutral protein impact. Seventeen genes have alterations with frequencies of ≥10% in the patient cohort and with 14 overlapped genes in both Dukes’ B and C. The adenomatous polyposis coli gene (APC) was the most frequently altered gene in both groups (n = 6 in Dukes’ B and C). In addition, TP53 was more frequently altered in Dukes’ C (n = 7) compared to Dukes’ B (n = 4). Ten variants in APC, namely p.R283∗, p.N778fs, p.R805∗, p.Y935fs, p.E941fs, p.E1057∗, p.I1401fs, p.Q1378∗, p.E1379∗, and p.A1485fs were predicted to be driver variants. APC remains as the most frequently altered gene in the intermediate stages of CRC. Wnt signaling pathway is the major affected pathway followed by P53, RAS, TGF-β, and PI3K signaling. We reported the alteration profiles in each of the patient which has the potential to affect the clinical decision. We believe that this study will add further to the understanding of CRC molecular landscape

TCGA-My: A Systematic Repository for Systems Biology of Malaysian Colorectal Cancer

Colorectal cancer (CRC) ranks second among the most commonly occurring cancers in Malaysia, and unfortunately, its pathobiology remains unknown. CRC pathobiology can be understood in detail with the implementation of omics technology that is able to generate vast amounts of molecular data. The generation of omics data has introduced a new challenge for data organization. Therefore, a knowledge-based repository, namely TCGA-My, was developed to systematically store and organize CRC omics data for Malaysian patients. TCGA-My stores the genome and metabolome of Malaysian CRC patients. The genome and metabolome datasets were organized using a Python module, pandas. The variants and metabolites were first annotated with their biological information using gene ontologies (GOs) vocabulary. The TCGA-My relational database was then built using HeidiSQL PorTable 9.4.0.512, and Laravel was used to design the web interface. Currently, TCGA-My stores 1,517,841 variants, 23,695 genes, and 167,451 metabolites from the samples of 50 CRC patients. Data entries can be accessed via search and browse menus. TCGA-My aims to offer effective and systematic omics data management, allowing it to become the main resource for Malaysian CRC research, particularly in the context of biomarker identification for precision medicine

SARS-CoV-2 genomic surveillance in Malaysia: displacement of B.1.617.2 with AY lineages as the dominant Delta variants and the introduction of Omicron during the fourth epidemic wave

Objectives: This study reported SARS-CoV-2 whole genome sequencing results from June 2021 to January 2022 from seven genome sequencing centers in Malaysia as part of the national surveillance program. Methods: COVID-19 samples that tested positive by reverse transcription polymerase chain reaction and with cycle threshold values <30 were obtained throughout Malaysia. Sequencing of SARS-CoV-2 complete genomes was performed using Illumina, Oxford Nanopore, or Ion Torrent platforms. A total of 6163 SARS-CoV-2 complete genome sequences were generated over the surveillance period. All sequences were submitted to the Global Initiative on Sharing All Influenza Data database. Results: From June 2021 to January 2022, Malaysia experienced the fourth wave of COVID-19 dominated by the Delta variant of concern, including the original B.1.617.2 lineage and descendant AY lineages. The B.1.617.2 lineage was identified as the early dominant circulating strain throughout the country but over time, was displaced by AY.59 and AY.79 lineages in Peninsular (west) Malaysia, and the AY.23 lineage in east Malaysia. In December 2021, pilgrims returning from Saudi Arabia facilitated the introduction and spread of the BA.1 lineage (Omicron variant of concern) in the country. Conclusion: The changing trends of circulating SARS-CoV-2 lineages were identified, with differences observed between west and east Malaysia. This initiative highlighted the importance of leveraging research expertise in the country to facilitate pandemic response and preparedness

SARS-CoV-2 genomic surveillance in Malaysia: displacement of B.1.617.2 with AY lineages as the dominant Delta variants and the introduction of Omicron during the fourth epidemic wave

Objectives This study reported SARS-CoV-2 whole genome sequencing results from June 2021 to January 2022 from seven genome sequencing centers in Malaysia as part of the national surveillance program. Methods COVID-19 samples that tested positive by reverse transcription polymerase chain reaction and with cycle threshold values <30 were obtained throughout Malaysia. Sequencing of SARS-CoV-2 complete genomes was performed using Illumina, Oxford Nanopore, or Ion Torrent platforms. A total of 6163 SARS-CoV-2 complete genome sequences were generated over the surveillance period. All sequences were submitted to the Global Initiative on Sharing All Influenza Data database. Results From June 2021 to January 2022, Malaysia experienced the fourth wave of COVID-19 dominated by the Delta variant of concern, including the original B.1.617.2 lineage and descendant AY lineages. The B.1.617.2 lineage was identified as the early dominant circulating strain throughout the country but over time, was displaced by AY.59 and AY.79 lineages in Peninsular (west) Malaysia, and the AY.23 lineage in east Malaysia. In December 2021, pilgrims returning from Saudi Arabia facilitated the introduction and spread of the BA.1 lineage (Omicron variant of concern) in the country. Conclusion The changing trends of circulating SARS-CoV-2 lineages were identified, with differences observed between west and east Malaysia. This initiative highlighted the importance of leveraging research expertise in the country to facilitate pandemic response and preparedness