METHODS: Potential MAR/SAR sites were predicted in the AF9 gene by using MAR/SAR prediction tools. Normal nasopharyngeal epithelial cells (NP69) and NPC cells (TWO4) were treated with BA at neutral and acidic pH. Inverse-PCR (IPCR) was used to identify chromosome breaks in SAR region (contains MAR/SAR) and non-SAR region (does not contain MAR/SAR). To map the chromosomal breakpoints within the AF9 SAR and non-SAR regions, DNA sequencing was performed.
RESULTS: In the AF9 SAR region, the gene cleavage frequencies of BA-treated NP69 and TWO4 cells were significantly higher than those of untreated control. As for the AF9 non-SAR region, no significant difference in cleavage frequency was detected between untreated and BA-treated cells. A few breakpoints detected in the SAR region were mapped within the AF9 region that was previously reported to translocate with the mixed lineage leukaemia (MLL) gene in an acute lymphoblastic leukaemia (ALL) patient.
CONCLUSIONS: Our findings suggest that MAR/SAR may be involved in defining the positions of chromosomal breakages induced by BA. Our report here, for the first time, unravelled the relation of these BA-induced chromosomal breakages to the AF9 chromatin structure.
METHODS: The performance of a custom, in-house designed 22-gene NGS panel was technically validated using reference standards across two independent replicate runs. The panel was subsequently used to screen a total of 10 clinical MPN samples (ET n = 3, PV n = 3, PMF n = 4). The resulting NGS data was then analysed via a bioinformatics pipeline.
RESULTS: The custom NGS panel had a detection limit of 1% variant allele frequency (VAF). A total of 20 unique variants with VAFs above 5% (4 of which were putatively novel variants with potential biological significance) and one pathogenic variant with a VAF of between 1 and 5% were identified across all of the clinical MPN samples. All single nucleotide variants with VAFs ≥ 15% were confirmed via Sanger sequencing.
CONCLUSIONS: The high fidelity of the NGS analysis and the identification of known and novel variants in this study cohort support its potential clinical utility in the management of MPNs. However, further optimisation is needed to avoid false negatives in regions with low sequencing coverage, especially for the detection of driver mutations in MPL.
RESULTS: This work describes a computational methodology to achieve this analysis, with data of dengue, West Nile, hepatitis A, HIV-1, and influenza A viruses as examples. Our methodology has been implemented as an analytical pipeline that brings significant advancement to the field of reverse vaccinology, enabling systematic screening of known sequence data in nature for identification of vaccine targets. This includes key steps (i) comprehensive and extensive collection of sequence data of viral proteomes (the virome), (ii) data cleaning, (iii) large-scale sequence alignments, (iv) peptide entropy analysis, (v) intra- and inter-species variation analysis of conserved sequences, including human homology analysis, and (vi) functional and immunological relevance analysis.
CONCLUSION: These steps are combined into the pipeline ensuring that a more refined process, as compared to a simple evolutionary conservation analysis, will facilitate a better selection of vaccine targets and their prioritization for subsequent experimental validation.
METHODS: The study included scoping review and key stakeholder interviews in four focus countries - Indonesia, Malaysia, Singapore, and Thailand. The current landscape of PM adoption was evaluated based on an assessment framework of six key themes - healthcare system, governance, access, awareness, implementation, and data. Six PM programs were evaluated for their financing and implementation mechanisms.
RESULTS: The findings revealed SEA has progressed in adopting PM especially Singapore and Thailand. A regional pharmacogenomics research network has been established. However, PM policies and programs vary significantly. As most PM programs are champion-driven and the available funding is limited, the current PM distribution has the potential to widen existing health disparities. Low PM awareness in the society and the absence of political support with financial investment are fundamental barriers. There is a clear need to broaden opportunities for critical discourse about PM especially for policymakers. Multi-stakeholder, multi-country strategies need to be prioritized in order to leverage resources and expertise.
CONCLUSIONS: Adopting PM remains in its infancy in SEA. To achieve an effective PM adoption, it is imperative to balance equity issues across diverse populations while improving efficiency in healthcare.