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Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a neurodegenerative disorder caused by the ATXN3 CAG repeat expansion. Preimplantation genetic testing for monogenic disorders (PGT-M) of SCA3/MJD should include reliable repeat expansion detection coupled with high-risk allele determination using informative linked markers. One couple underwent SCA3/MJD PGT-M combining ATXN3 (CAG) n triplet-primed PCR (TP-PCR) with customized linkage-based risk allele genotyping on whole-genome-amplified trophectoderm cells. Microsatellites closely linked to ATXN3 were identified and 16 markers were genotyped on 187 anonymous DNAs to verify their polymorphic information content. In the SCA3/MJD PGT-M case, the ATXN3 (CAG) n TP-PCR and linked marker analysis results concurred completely. Among the three unaffected embryos, a single embryo was transferred and successfully resulted in an unaffected live birth. A total of 139 microsatellites within 1 Mb upstream and downstream of the ATXN3 CAG repeat were identified and 8 polymorphic markers from each side were successfully co-amplified in a single-tube reaction. A PGT-M assay involving ATXN3 (CAG) n TP-PCR and linkage-based risk allele identification has been developed for SCA3/MJD. A hexadecaplex panel of highly polymorphic microsatellites tightly linked to ATXN3 has been developed for the rapid identification of informative markers in at-risk couples for use in the PGT-M of SCA3/MJD.

Background: Current strategies for preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) rely mainly on next-generation sequencing (NGS) and microarray platforms, which are robust but require expensive instrumentation. We explored the suitability of third-generation single-molecule sequencing as a PGT-A/SR screening platform for both aneuploidy and segmental imbalance., Methods: Single-cell and multicell replicates from aneuploid or segmentally unbalanced cell lines (n = 208) were SurePlex-amplified, randomized, and subjected to (a) Nanopore-based single-molecule sequencing (Oxford Nanopore Technologies) and (b) NGS using a leading commercial PGT-A solution (Illumina VeriSeq PGS). Archival SurePlex-amplified trophectoderm biopsy samples (n = 96) previously analyzed using the commercial kit were blinded and reanalyzed using Nanopore., Results: Nanopore-based PGT-A identified the specific aberration in 95.45% (84/88) and 97.78% (88/90) of single-/multicells with an aneuploidy or segmental imbalance (10-30.5 Mb), respectively. Comparison against the commercial kit's results revealed concordances of 98.86% (87/88) and 98.89% (89/90) for the aneuploid and segmentally unbalanced (10-30.5 Mb aberration) samples, respectively. Detection sensitivity for smaller segmental imbalances (5-5.8 Mb aberration, n = 30) decreased markedly on both platforms. Nanopore-based PGT-A reanalysis of trophectoderm biopsy samples was 97.92% (94/96) concordant with the commercial kit results., Conclusion: Up to 24 SurePlex-amplified single-cell, multicell, or trophectoderm samples could be sequenced in a single MinION flow-cell for subsequent preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) analysis, with results obtainable in ≤3 days and at per-sample costs that are competitive with commercial offerings. Nanopore's third-generation single-molecule sequencing represents a viable alternative to current commercial NGS-based PGT-A solutions for aneuploidy and segmental imbalance (≥10 Mb) screening of single-/multicell or trophectoderm biopsy samples., (American Association for Clinical Chemistry 2023.)

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Although alpha fetoprotein (AFP) remains a commonly used serological marker of HCC, the sensitivity and specificity of AFP in detecting HCC is often limited. Exosomal RNA has emerged as a promising diagnostic tool for various cancers, but its use in HCC detection has yet to be fully explored. Here, we employed Machine Learning on 114,602 exosomal RNAs to identify a signature that can predict HCC. The exosomal expression data of 118 HCC patients and 112 healthy individuals were stratified split into Training, Validation and Unseen Test datasets. Feature selection was then performed on the initial training dataset using permutation importance, and the predictive performance of the selected features were tested on the validation dataset using Support Vector Machine (SVM) Classifier. A minimum of nine features were identified to be predictive of HCC and these nine features were then evaluated across six different models in an unseen test set. These features, mainly in the immune, platelet/neutrophil and cytoskeletal pathways, exhibited good predictive performance with ROC-AUC from 0.79-0.88 in the unseen test set. Hence, these nine exosomal RNAs have potential to be clinically useful minimally invasive biomarkers for HCC.

Background: The popular statistics-based Genome-wide association studies (GWAS) have provided deep insights into the field of complex disorder genetics. However, its clinical applicability to predict disease/trait outcomes remains unclear as statistical models are not designed to make predictions. This study employs statistics-free machine-learning (ML)-optimized polygenic risk score (PRS) to complement existing GWAS and bring the prediction of disease/trait outcomes closer to clinical application. Rheumatoid Arthritis (RA) was selected as a model disease to demonstrate the robustness of ML in disease prediction as RA is a prevalent chronic inflammatory joint disease with high mortality rates, affecting adults at the economic prime. Early identification of at-risk individuals may facilitate measures to mitigate the effects of the disease., Methods: This study employs a robust ML feature selection algorithm to identify single nucleotide polymorphisms (SNPs) that can predict RA from a set of training data comprising RA patients and population control samples. Thereafter, selected SNPs were evaluated for their predictive performances across 3 independent, unseen test datasets. The selected SNPs were subsequently used to generate PRS which was also evaluated for its predictive capacity as a sole feature., Results: Through robust ML feature selection, 9 SNPs were found to be the minimum number of features for excellent predictive performance (AUC > 0.9) in 3 independent, unseen test datasets. PRS based on these 9 SNPs was significantly associated with (P < 1 × 10 -16 ) and predictive (AUC > 0.9) of RA in the 3 unseen datasets. A RA ML-PRS calculator of these 9 SNPs was developed ( https://xistance.shinyapps.io/prs-ra/ ) to facilitate individualized clinical applicability. The majority of the predictive SNPs are protective, reside in non-coding regions, and are either predicted to be potentially functional SNPs (pfSNPs) or in high linkage disequilibrium (r2 > 0.8) with un-interrogated pfSNPs., Conclusions: These findings highlight the promise of this ML strategy to identify useful genetic features that can robustly predict disease and amenable to translation for clinical application., (© 2023. The Author(s).)

Objective: To develop a hypothesis-free model that best predicts response to MTX drug in RA patients utilizing biologically meaningful genetic feature selection of potentially functional single nucleotide polymorphisms (pfSNPs) through robust machine learning (ML) feature selection methods., Methods: MTX-treated RA patients with known response were divided in a 4:1 ratio into training and test sets. From the patients' exomes, potential features for classifier prediction were identified from pfSNPs and non-genetic factors through ML using recursive feature elimination with cross-validation incorporating the random forest classifier. Feature selection was repeated on random subsets of the training cohort, and consensus features were assembled into the final feature set. This feature set was evaluated for predictive potential using six ML classifiers, first by cross-validation within the training set, and finally by analysing its performance with the unseen test set., Results: The final feature set contains 56 pfSNPs and five non-genetic factors. The majority of these pfSNPs are located in pathways related to RA pathogenesis or MTX action and are predicted to modulate gene expression. When used for training in six ML classifiers, performance was good in both the training set (area under the curve: 0.855-0.916; sensitivity: 0.715-0.892; and specificity: 0.733-0.862) and the unseen test set (area under the curve: 0.751-0.826; sensitivity: 0.581-0.839; and specificity: 0.641-0.923)., Conclusion: Sensitive and specific predictors of MTX response in RA patients were identified in this study through a novel strategy combining biologically meaningful and machine learning feature selection and training. These predictors may facilitate better treatment decision-making in RA management., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The autosomal dominantly inherited and genetically heterogeneous spinocerebellar ataxias (SCAs) exhibit highly similar clinical presentations. Many are caused by repeat expansions, of which at least 8 involve CAG repeats. Repeat expansion detection is the only method to confirm disease status in symptomatic individuals. We present a novel strategy to simultaneously screen for the presence of CAG repeat expansion in the genes responsible for SCAs 1, 2, 3, 6, 7, 12, and dentatorubral-pallidoluysian atrophy using a simplified single-tube assay., Methods: The method employs differentially labeled locus-specific primers and a common triplet-primed primer. Amplified products from each locus are distinguished by a combination of the product size and the fluorophore tag. The upper size limit of the normal allele range was used as the cutoff for distinguishing normal from potentially affected samples, with repeat expansion detected by presence of electrophoretic peaks extending beyond the cutoff., Results: Blinded evaluation of the assay on 60 genotype-known DNA samples correctly detected repeat expansion in the expected SCA repeat locus for all 31 DNA samples., Conclusions: In principle, this strategy can be applied to the simultaneous screening of any group of disease genes sharing the same repetitive units and/or their reverse complement., (© American Association for Clinical Chemistry 2022.)

Methylated FMR1 full-mutation expansions cause fragile X syndrome. FMR1 premutation carriers are susceptible to other late-onset conditions, and women with premutation are at risk of transmitting a fully expanded FMR1 allele to offspring. Identification of individuals with actionable FMR1 genotypes (full-mutation males and females, and premutation females at risk for primary ovarian insufficiency and/or having fragile X-affected offspring) can enable timely access to intervention services and genetic counseling. This study presents a rapid, first-tier test based on melting curve analysis of methylation-specific triplet-primed PCR amplicons (msTP-PCR MCA) for concurrent detection of FMR1 CGG-repeat expansions and their methylation status. The msTP-PCR MCA assay was optimized on 20 fragile X reference samples, and its performance was evaluated on 111 peripheral blood-derived DNA samples from patients who have undergone prior molecular testing with PCR and/or Southern blot analysis. The msTP-PCR MCA assay detected all samples with a methylated FMR1 CGG-repeat expansion, and had sensitivity, specificity, positive predictive value, and negative predictive values of 100%, 92.06%, 91.1%, and 100%, respectively. The msTP-PCR MCA assay identified premutation/full-mutation mosaicism down to 1%, detected skewed inactivation in females with FMR1 expansions, and enabled selective identification of all individuals with an actionable FMR1 genotype. The msTP-PCR MCA assay may aid in fragile X screening of at-risk populations and newborns and voluntary carrier screening of women of reproductive age., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Major challenges in large scale genetic association studies include not only the identification of causative single nucleotide polymorphisms (SNPs), but also accounting for SNP-SNP interactions. This study thus proposes a novel feature engineering approach integrating potentially functional coding haplotypes (pfcHap) with machine-learning (ML) feature selection to identify biologically meaningful, possibly causative genetic factors, that take into consideration potential SNP-SNP interactions within the pfcHap, to best predict for methotrexate (MTX) response in rheumatoid arthritis (RA) patients., Methods: Exome sequencing from 349 RA patients were analysed, of which they were split into training and unseen test set. Inferred pfcHaps were combined with 30 non-genetic features to undergo ML recursive feature elimination with cross-validation using the training set. Predictive capacity and robustness of the selected features were assessed using six popular machine learning models through a train set cross-validation and evaluated in an unseen test set., Findings: Significantly, 100 features (95 pfcHaps, 5 non-genetic factors) were identified to have good predictive performance (AUC: 0.776-0.828; Sensitivity: 0.656-0.813; Specificity: 0.684-0.868) across all six ML models in an unseen test dataset for the prediction of MTX response in RA patients., Interpretation: Majority of the predictive pfcHap SNPs were predicted to be potentially functional and some of the genes in which the pfcHap resides in were identified to be associated with previously reported MTX/RA pathways., Funding: Singapore Ministry of Health's National Medical Research Council (NMRC) [NMRC/CBRG/0095/2015; CG12Aug17; CGAug16M012; NMRC/CG/017/2013]; National Cancer Center Research Fund and block funding Duke-NUS Medical School.; Singapore Ministry of Education Academic Research Fund Tier 2 grant MOE2019-T2-1-138., Competing Interests: Declaration of Competing Interest CGL, KPL, CCK, SSC, AJWL, and LJL declare that they have a pending Coversheet IP application., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

As the COVID-19 pandemic rages unabated, and with more infectious variants, vaccination may offer a way to transit out of strict restrictions on physical human interactions to curb the virus spread and prevent overwhelming the healthcare system. However, vaccine hesitancy threatens to significantly impact our progress towards achieving this. It is thus important to understand the sentiments regarding vaccination for different segments of the population to facilitate the development of effective strategies to persuade these groups. Here, we surveyed the COVID-19 vaccination sentiments among a highly educated group of graduate students from the National University of Singapore (NUS). Graduate students who are citizens of 54 different countries, mainly from Asia, pursue studies in diverse fields, with 32% expressing vaccine hesitancy. Citizenship, religion, country of undergraduate/postgraduate studies, exposure risk and field of study are significantly associated with vaccine sentiments. Students who are Chinese citizens or studied in Chinese Universities prior to joining NUS are more hesitant, while students of Indian descent or studied in India are less hesitant about vaccination. Side effects, safety issues and vaccine choice are the major concerns of the hesitant group. Hence, this study would facilitate the development of strategies that focus on these determinants to enhance vaccine acceptance.

Moderate to hyper-expansion of trinucleotide repeats at the FRAXA and FRAXE fragile sites, with or without concurrent hypermethylation, has been associated with intellectual disability and other conditions. Unlike molecular diagnosis of FMR1 CGG repeat expansions in FRAXA, current detection of AFF2 CCG repeat expansions in FRAXE relies on low-throughput and otherwise inefficient techniques combining Southern blot analysis and PCR. A novel triplet-primed PCR assay was developed for simultaneous screening for trinucleotide repeat expansions at the FRAXA and FRAXE fragile sites, and was validated using archived clinical samples of known FMR1 and AFF2 genotypes. Population samples and FRAXE-affected samples were sequenced for the evaluation of variations in the AFF2 CCG repeat structure. The duplex assay accurately identified expansions at the FMR1 and AFF2 trinucleotide repeat loci. On Sanger sequencing of the AFF2 CCG repeat, the single-nucleotide polymorphism variant rs868914124(C) that effectively adds two CCG repeats at the 5'-end, was enriched in the Malay population and with short repeats (<11 CCGs), and was present in all six expanded AFF2 alleles of this study. All expanded AFF2 alleles contained multiple non-CCG interruptions toward the 5'-end of the repeat. A sensitive, robust, and rapid assay has been developed for the simultaneous detection of expansion mutations at the FMR1 and AFF2 trinucleotide repeat loci, simplifying screening for FRAXA- and FRAXE-associated disorders., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. Here, we present a novel strategy to identify key circRNA signatures of clinically relevant co-expressed circRNA-mRNA networks in pertinent cancer-pathways that modulate prognosis of HCC patients, by integrating clinic-pathological features, circRNA and mRNA expression profiles. Through further integration with miRNA expression profiles, clinically relevant competing-endogenous-RNA (ceRNA) networks of circRNA-miRNA-mRNAs were constructed. At least five clinically relevant nodal-circRNAs, co-expressed with numerous genes, were identified from the circRNA-mRNA networks. These nodal circRNAs upregulated proliferation (except circRaly) and transformation in cells. The most upregulated nodal-circRNA, circGPC3, associated with higher-grade tumors and co-expressed with 33 genes, competes with 11 mRNAs for two shared miRNAs. circGPC3 was experimentally demonstrated to upregulate cell-cycle and migration/invasion in both transformed and non-transformed liver cell-lines. circGPC3 was further shown to act as a sponge of miR-378a-3p to regulate APSM (Abnormal spindle-like microcephaly associated) expression and modulate cell transformation. This study identifies 5 key nodal master circRNAs in a clinically relevant circRNA-centric network that are significantly associated with poorer prognosis of HCC patients and promotes tumorigenesis in cell-lines. The identification and characterization of these key circRNAs in clinically relevant circRNA-mRNA and ceRNA networks may facilitate the design of novel strategies targeting these important regulators for better HCC prognosis.

The autosomal dominantly inherited spinocerebellar ataxias (SCAs) can be caused by dynamic mutations of short tandem repeats within various genes. Because of the significant clinical overlap among the various SCA types, molecular screening of multiple genetic loci by fluorescent PCR and capillary electrophoresis is necessary to identify the causative repeat expansion. We describe a simple, rapid, and inexpensive strategy to screen for CAG repeat expansion mutations at the ATXN1, ATXN2, and ATXN3 loci using melting curve analysis of triplet-primed PCR products. Plasmid DNAs of known repeat sizes were used to generate threshold melt peak temperatures, which rapidly and effectively distinguish samples carrying an expanded allele from those carrying nonexpanded alleles. Melting curve analysis-positive samples were confirmed by capillary electrophoresis sizing of the triplet-primed PCR products. All three assays achieved 100% sensitivity, with 95% CIs of 67.86% to 100% (SCA1), 74.65% to 100% (SCA2), and 91.58% to 100% (SCA3). The SCA1 assay also achieved 100% specificity (95% CI, 97.52%-100%), whereas the SCA2 and SCA3 assays achieved specificity of 99.46% (95% CI, 96.56%-99.97%) and 99.32% (95% CI, 95.70%-99.96%), respectively. These screening assays provide robust and highly accurate detection of expanded alleles and are amenable to large-scale screening while minimizing the need for capillary electrophoresis sizing for every sample., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Statins can cause muscle symptoms resulting in poor adherence to therapy and increased cardiovascular risk. We hypothesize that combinations of potentially functional SNPs (pfSNPs), rather than individual SNPs, better predict myalgia in patients on atorvastatin. This study assesses the value of potentially functional single nucleotide polymorphisms (pfSNPs) and employs six machine learning algorithms to identify the combination of SNPs that best predict myalgia. Methods: Whole genome sequencing of 183 Chinese, Malay and Indian patients from Singapore was conducted to identify genetic variants associated with atorvastatin induced myalgia. To adjust for confounding factors, demographic and clinical characteristics were also examined for their association with myalgia. The top factor, sex, was then used as a covariate in the whole genome association analyses. Variants that were highly associated with myalgia from this and previous studies were extracted, assessed for potential functionality (pfSNPs) and incorporated into six machine learning models. Predictive performance of a combination of different models and inputs were compared using the average cross validation area under ROC curve (AUC). The minimum combination of SNPs to achieve maximum sensitivity and specificity as determined by AUC, that predict atorvastatin-induced myalgia in most, if not all the six machine learning models was determined. Results: Through whole genome association analyses using sex as a covariate, a larger proportion of pfSNPs compared to non-pf SNPs were found to be highly associated with myalgia. Although none of the individual SNPs achieved genome wide significance in univariate analyses, machine learning models identified a combination of 15 SNPs that predict myalgia with good predictive performance (AUC >0.9). SNPs within genes identified in this study significantly outperformed SNPs within genes previously reported to be associated with myalgia. pfSNPs were found to be more robust in predicting myalgia, outperforming non-pf SNPs in the majority of machine learning models tested. Conclusion: Combinations of pfSNPs that were consistently identified by different machine learning models to have high predictive performance have good potential to be clinically useful for predicting atorvastatin-induced myalgia once validated against an independent cohort of patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ooi, Raechell, Ying, Koh, Jin, Yee, Lee, Chong, Tan, Liu, Lee and Drum.)

Although numerous long non-coding RNAs (lncRNAs) were reported to be deregulated in Hepatocellular Carcinoma (HCC), experimentally characterized, and/or associated with patient's clinical characteristics, there is, thus far, minimal concerted research strategy to identify deregulated lncRNAs that modulate prognosis of HCC patients. Here, we present a novel strategy where we identify lncRNAs, which are not only de-regulated in HCC patients, but are also associated with pertinent clinical characteristics, potentially contributing to the prognosis of HCC patients. LOC101926913 (LOC) was further characterized because it is the most highly differentially expressed amongst those that are associated with the most number of clinical features (tumor-stage, vascular and tumor invasion and poorer overall survival). Experimental gain- and loss-of-function manipulation of LOC in liver cell-lines highlight LOC as a potential onco-lncRNA promoting cell proliferation, anchorage independent growth and invasion. LOC expression in cells up-regulated genes involved in GTPase-activities and downregulated genes associated with cellular detoxification, oxygen- and drug-transport. Hence, LOC may represent a novel therapeutic target, modulating prognosis of HCC patients through up-regulating GTPase-activities and down-regulating detoxification, oxygen- and drug-transport. This strategy may thus be useful for the identification of clinically relevant lncRNAs as potential biomarkers/targets that modulate prognosis in other cancers as well., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Long non-coding RNAs (lncRNAs) are often aberrantly expressed in Hepatocellular Carcinoma (HCC). We hypothesize that lncRNAs modulate HCC prognoses through differential deregulation of key lncRNAs affecting important gene network in key cancer pathways associated with pertinent clinical phenotype. Here, we present a novel approach integrating lncRNA-mRNA expression profiles with clinical characteristics to identify lncRNA signatures in clinically-relevant co-expression lncRNA-mRNA networks residing in pertinent cancer pathways. Notably one network, associated with poorer prognosis, comprises five up-regulated lncRNAs significantly correlated (|Pearson Correlation Coefficient|≥ 0.9) with 91 up-regulated genes in the cell-cycle and Rho-GTPase pathways. All 5 lncRNAs and 85/91 (93.4%) of the correlated genes were significantly associated with higher tumor-grade while 3/5 lncRNAs were also associated with no tumor capsule. Interestingly, 2/5 lncRNAs that are correlated with numerous genes in this oncogenic network were experimentally shown to up-regulate genes involved in cell-cycle and transcriptional regulation. Another network comprising 4 down-regulated lncRNAs and 8 down-regulated metallothionein-family genes are significantly associated with tumor invasion. The identification of these key lncRNAs signatures that deregulate important network of genes in key cancer pathways associated with pertinent clinical phenotype may facilitate the design of novel therapeutic strategies targeting these 'master' regulators for better patient outcome.

Circulating factors have been implicated in the pathogenesis of minimal change disease (MCD), and may have direct effects on cholesterol metabolism. This study investigated the pathogenesis of hypercholesterolemia in an IL-13 overexpression rat model of MCD prior to the onset of proteinuria, so as to establish the direct contribution of IL-13, especially with regard to hepatic cholesterol handling. In this model of MCD, the temporal relationship between hypercholesterolemia and proteinuria was first identified. Plasma proprotein convertase subtilisin/kexin type 9 (Pcsk9) and liver ATP-binding cassette sub-family G member 5 (Abcg5) were measured using ELISA. Liver Ldlr and liver X receptor alpha (Lxra) were quantified with Western blot. Abcg5-mediated cholesterol efflux in IL-13-stimulated rat primary hepatocytes was measured using taurocholate as cholesterol acceptor. The role of Lxra was validated using a luciferase assay in Lxre-luciferase-transfected IL-13-stimulated hepatocytes. IL-13-transfected rats developed hypercholesterolemia prior to proteinuria, with 35% of rats hypercholesterolemic but only 11% proteinuric by Day 20 (P = 0.04). These pre-proteinuric hypercholesterolemic rats showed elevations in total and LDL-cholesterol, but not hypertriglyceridemia or hepatic steatosis. The hypercholesterolemia was associated with increased hepatic Pcsk9 synthesis and enhanced circulating Pcsk9 levels, which correlated strongly with plasma total cholesterol (r = 0.73, P<0.001). The hypercholesterolemia was also contributed by decreased Abcg5 expression and activity, due to reduced Lxra expression. Lxra expression correlated with plasma total cholesterol levels (r = -0.52, P = 0.01), and overexpression of pLxra in rat hepatocytes abrogated the IL-13-mediated down-regulation of Lxre-driven gene expression. In conclusion, we have shown that IL-13 induced changes in hepatic cholesterol handling in a cytokine-induced rat model of MCD, resulting in hypercholesterolemia which can precede the onset of proteinuria., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Drug response variations amongst different individuals/populations are influenced by several factors including allele frequency differences of single nucleotide polymorphisms (SNPs) that functionally affect drug-response genes. Here, we aim to identify drugs that potentially exhibit population differences in response using SNP data mining and analytics. Ninety-one pairwise-comparisons of >22,000,000 SNPs from the 1000 Genomes Project, across 14 different populations, were performed to identify 'population-differentiated' SNPs (pdSNPs). Potentially-functional pdSNPs (pf-pdSNPs) were then selected, mapped into genes, and integrated with drug-gene databases to identify 'population-differentiated' drugs enriched with genes carrying pf-pdSNPs. 1191 clinically-approved drugs were found to be significantly enriched (Z > 2.58) with genes carrying SNPs that were differentiated in one or more population-pair comparisons. Thirteen drugs were found to be enriched with such differentiated genes across all 91 population-pairs. Notably, 82% of drugs, which were previously reported in the literature to exhibit population differences in response were also found by this method to contain a significant enrichment of population specific differentiated SNPs. Furthermore, drugs with genetic testing labels, or those suspected to cause adverse reactions, contained a significantly larger number (P < 0.01) of population-pairs with enriched pf-pdSNPs compared with those without these labels. This pioneering effort at harnessing big-data pharmacogenomics to identify 'population differentiated' drugs could help to facilitate data-driven decision-making for a more personalized medicine.

Population variation in disease and other phenotype are partly attributed to single nucleotide polymorphisms (SNPs) in the human genome. Due to selection pressure, two individuals from the same ancestral population have more genetic similarity compared to individuals from further geographic regions. Here, we elucidated the genomic population differentiation pattern, by interrogating >22,000,000 SNPs. Majority of population-differentiated (pd) SNPs (~95%), including the potentially functional (pf) (~84%) subset reside in non-genic regions, compared to the proportion of all SNPs (58%) found in non-genic regions. This suggests that differences between populations are more likely due to differences in gene regulation rather than protein function. Actin Cytoskeleton, Axonal Guidance and Protein Kinase A signaling pathways are enriched with genes carrying at least three pdSNPs (enriched pdGenes), while Antigen Presentation, Hepatic Fibrosis and Huntington Disease Signalling pathways are over-represented by enriched pf-pdGenes. An inverse correlation between chromosome size and the proportion of pd-/pf-pdSNPs was observed. Smaller chromosomes have relatively more of such SNPs including genes carrying these SNPs. Genes associated with common diseases and enriched with these pd-/pfpdSNPs are localized to 11 different chromosomes, with immune-related disease pd/pf-pdGenes mainly residing in chromosome 6 while neurological disease pd/pf-pdGenes residing in smaller chromosomes including chromosome 21/22. The associated diseases were reported to show population differences in incidence, severity and/or etiology. In summary, this study highlights the non-sporadic nature of population differentiation footprint in the human genome, which can potentially lead to the identification of genomic regions that play roles in the manifestation of phenotypic differences, including in disease predisposition and drug response., Competing Interests: The authors have declared that no competing interests exist.

Next-generation sequencing has uncovered thousands of long noncoding RNAs (lncRNA). Many are reported to be aberrantly expressed in various cancers, including hepatocellular carcinoma (HCC), and play key roles in tumorigenesis. This review provides an in-depth discussion of the oncogenic mechanisms reported to be associated with deregulated HCC-associated lncRNAs. Transcriptional expression of lncRNAs in HCC is modulated through transcription factors, or epigenetically by aberrant histone acetylation or DNA methylation, and posttranscriptionally by lncRNA transcript stability modulated by miRNAs and RNA-binding proteins. Seventy-four deregulated lncRNAs have been identified in HCC, of which, 52 are upregulated. This review maps the oncogenic roles of these deregulated lncRNAs by integrating diverse datasets including clinicopathologic features, affected cancer phenotypes, associated miRNA and/or protein-interacting partners as well as modulated gene/protein expression. Notably, 63 deregulated lncRNAs are significantly associated with clinicopathologic features of HCC. Twenty-three deregulated lncRNAs associated with both tumor and metastatic clinical features were also tumorigenic and prometastatic in experimental models of HCC, and eight of these mapped to known cancer pathways. Fifty-two upregulated lncRNAs exhibit oncogenic properties and are associated with prominent hallmarks of cancer, whereas 22 downregulated lncRNAs have tumor-suppressive properties. Aberrantly expressed lncRNAs in HCC exert pleiotropic effects on miRNAs, mRNAs, and proteins. They affect multiple cancer phenotypes by altering miRNA and mRNA expression and stability, as well as through effects on protein expression, degradation, structure, or interactions with transcriptional regulators. Hence, these insights reveal novel lncRNAs as potential biomarkers and may enable the design of precision therapy for HCC., (©2019 American Association for Cancer Research.)

Background: Hepatocellular carcinoma is the second most deadly cancer with late presentation and limited treatment options, highlighting an urgent need to better understand HCC to facilitate the identification of early-stage biomarkers and uncover therapeutic targets for the development of novel therapies for HCC., Methods: Deep transcriptome sequencing of tumor and paired non-tumor liver tissues was performed to comprehensively evaluate the profiles of both the host and HBV transcripts in HCC patients. Differential gene expression patterns and the dys-regulated genes associated with clinical outcomes were analyzed. Somatic mutations were identified from the sequencing data and the deleterious mutations were predicted. Lastly, human-HBV chimeric transcripts were identified, and their distribution, potential function and expression association were analyzed., Results: Expression profiling identified the significantly upregulated TP73 as a nodal molecule modulating expression of apoptotic genes. Approximately 2.5% of dysregulated genes significantly correlated with HCC clinical characteristics. Of the 110 identified genes, those involved in post-translational modification, cell division and/or transcriptional regulation were upregulated, while those involved in redox reactions were downregulated in tumors of patients with poor prognosis. Mutation signature analysis identified that somatic mutations in HCC tumors were mainly non-synonymous, frequently affecting genes in the micro-environment and cancer pathways. Recurrent mutations occur mainly in ribosomal genes. The most frequently mutated genes were generally associated with a poorer clinical prognosis. Lastly, transcriptome sequencing suggest that HBV replication in the tumors of HCC patients is rare. HBV-human fusion transcripts are a common observation, with favored HBV and host insertion sites being the HBx C-terminus and gene introns (in tumors) and introns/intergenic-regions (in non-tumors), respectively. HBV-fused genes in tumors were mainly involved in RNA binding while those in non-tumors tissues varied widely. These observations suggest that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts may occur during tumorigenesis., Conclusions: Transcriptome sequencing of HCC patients reveals key cancer molecules and clinically relevant pathways deregulated/mutated in HCC patients and suggests that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts likely occur during the process of tumorigenesis.

Hepatocellular carcinoma (HCC) is the fifth most common cancer with high mortality, due to late diagnosis and limited treatment options. Blood miRNAs, which circulate in a highly stable, cell-free form, show promise as novel potential biomarkers for early detection of HCC. Whole miRNome profiling was performed to identify deregulated miRNAs between HCC and normal healthy (NH) volunteers. These deregulated miRNAs were validated in an independent cohort of HCC, NH and chronic Hepatitis B (CHB) volunteers and finally in a 3 rd cohort comprising NH, CHB, cirrhotic and HCC volunteers to evaluate miRNA changes during disease progression. The associations between circulating miRNAs and liver-damage markers, clinicopathological characteristics and survival outcomes were analysed to identify prognostic markers. Twelve miRNAs are differentially expressed between HCC and NH individuals in all three cohorts. Five upregulated miRNAs (miR-122-5p, miR-125b-5p, miR-885-5p, miR-100-5p and miR-148a-3p) in CHB, cirrhosis and HCC patients are potential biomarkers for CHB infection, while miR-34a-5p can be a biomarker for cirrhosis. Notably, four miRNAs (miR-1972, miR-193a-5p, miR-214-3p and miR-365a-3p) can distinguish HCC from other non-HCC individuals. Six miRNAs are potential prognostic markers for overall survival.

Myotonic dystrophy type 1 (DM1) is caused by expansion of the DMPK CTG trinucleotide repeat. Disease transmission to offspring can be avoided through prenatal diagnosis or preimplantation genetic testing for monogenic disorders (PGT-M). We describe a robust strategy for DM1 PGT-M that can be applied to virtually any at-risk couple. This strategy utilizes whole-genome amplification, followed by triplet-primed PCR (TP-PCR) detection of expanded DMPK alleles, in parallel with single-tube haplotype analysis of 12 closely linked and highly polymorphic microsatellite markers. Bidirectional TP-PCR and dodecaplex marker PCR assays were optimized and validated on whole-genome amplified single lymphoblasts isolated from DM1 reference cell lines, and tested on a simulated PGT-M case comprising a parent-offspring trio and three simulated embryos. Bidirectional DMPK TP-PCR reliably detects repeat expansions even in the presence of non-CTG interruptions at either end of the expanded allele. Misdiagnoses, diagnostic ambiguity, and couple-specific assay customization are further minimized by the use of multi-marker haplotyping, preventing the loss of potentially unaffected embryos for transfer.

PubMed was text mined to glean insights into the role of Hepatitis B virus (HBV) in hepatocellular carcinoma (HCC) from the massive number of publications (9249) available to date. Reports from ∼70 countries identified >1300 human genes associated with either the Core, Surface or X gene in HBV-associated HCC. One hundred and forty-three of these host genes, which can potentially yield 1180 biomolecular interactions, each were reported in at least three different publications to be associated with the same HBV. These 143 genes function in 137 pathways, involved mainly in the cell cycle, apoptosis, inflammation and signalling. Fourteen of these molecules, primarily transcriptional regulators or kinases, play roles in several pathways pertinent to the hallmarks of cancers. 'Chronic' was the most frequent word used across the 9249 abstracts. A key event in chronic HBV infection is the integration of HBV into the host genome. The advent of cost-effective, next-generation sequencing technology facilitated the employment of big-data analytics comprehensively to characterize HBV-host integration within HCC patients. A total of 5331 integration events were reported across seven publications, with most of these integrations observed between the Core/X gene and the introns of genes. Nearly one-quarter of the intergenic integrations are within repeats, especially long interspersed nuclear elements (LINE) repeats. Integrations within 13 genes were each reported by at least three different studies. The human gene with the most HBV integrations observed is the TERT gene where a total of 224 integrations, primarily at its promoter and within the tumour tissue, were reported by six of seven publications. This unique review, which employs state-of-the-art text-mining and data-analytics tools, represents the most complete, systematic and comprehensive review of nearly all the publications associated with HBV-associated HCC research. It provides important resources to either focus future research or develop therapeutic strategies to target key molecules reported to play important roles in key pathways of HCC, through the systematic analyses of the commonly reported molecules associated with the various HBV genes in HCC, including information about the interactions amongst these commonly reported molecules, the pathways in which they reside as well as detailed information regarding the viral and host genes associated with HBV integration in HCC patients. Hence this review, which highlights pathways and key human genes associated with HBV in HCC, may facilitate the deeper elucidation of the role of HBV in hepato-carcinogenesis, potentially leading to timely intervention against this deadly disease., (© 2018 Cambridge Philosophical Society.)

Background: Hepatocellular carcinoma (HCC) is the cancer with the second highest mortality in the world due to its late presentation and limited treatment options. As such, there is an urgent need to identify novel biomarkers for early diagnosis and to develop novel therapies. The availability of next-generation sequencing (NGS) data from tumors of liver cancer patients has provided us with invaluable resources to better understand HCC through the integration of data from different sources to facilitate the identification of promising biomarkers or therapeutic targets., Findings: Here, we review key insights gleaned from more than 20 NGS studies of HCC tumor samples, comprising approximately 582 whole genomes and 1,211 whole exomes mainly from the East Asian population. Through consolidation of reported somatic mutations from multiple studies, we identified genes with different types of somatic mutations, including single nucleotide variations, insertion/deletions, structural variations, and copy number alterations as well as genes with multiple frequent viral integration. Pathway analysis showed that this curated list of somatic mutations is critically involved in cancer-related pathways, viral carcinogenesis, and signaling pathways. Lastly, we addressed the future directions of HCC research as more NGS datasets become available., Conclusions: Our review is a comprehensive resource for the current NGS research in HCC, consolidating published articles, potential gene candidates, and their related biological pathways.

Background: Genetic polymorphisms can contribute to phenotypic differences amongst individuals, including disease risk and drug response. Characterization of genetic polymorphisms that modulate gene expression and/or protein function may facilitate the identification of the causal variants. Here, we present the architecture of genetic polymorphisms in the human genome focusing on those predicted to be potentially functional/under natural selection and the pathways that they reside., Results: In the human genome, polymorphisms that directly affect protein sequences and potentially affect function are the most constrained variants with the lowest single-nucleotide variant (SNV) density, least population differentiation and most significant enrichment of rare alleles. SNVs which potentially alter various regulatory sites, e.g. splicing regulatory elements, are also generally under negative selection. Interestingly, genes that regulate the expression of transcription/splicing factors and histones are conserved as a higher proportion of these genes is non-polymorphic, contain ultra-conserved elements (UCEs) and/or has no non-synonymous SNVs (nsSNVs)/coding INDELs. On the other hand, major histocompatibility complex (MHC) genes are the most polymorphic with SNVs potentially affecting the binding of transcription/splicing factors and microRNAs (miRNA) exhibiting recent positive selection (RPS). The drug transporter genes carry the most number of potentially deleterious nsSNVs and exhibit signatures of RPS and/or population differentiation. These observations suggest that genes that interact with the environment are highly polymorphic and targeted by RPS., Conclusions: In conclusion, selective constraints are observed in coding regions, master regulator genes, and potentially functional SNVs. In contrast, genes that modulate response to the environment are highly polymorphic and under positive selection.

FAT10, which is also known as diubiquitin, has been implicated to play important roles in immune regulation and tumorigenesis. Its expression is up-regulated in the tumors of Hepatocellular Carcinoma (HCC) and other cancer patients. High levels of FAT10 in cells have been shown to result in increased mitotic non-disjunction and chromosome instability, leading to tumorigenesis. To evaluate whether the aberrant up-regulation of the FAT10 gene in the tumors of HCC patients is due to mutations or the aberrant methylation of CG dinucleotides at the FAT10 promoter, sequencing and methylation-specific sequencing of the promoter of FAT10 was performed. No mutations were found that could explain the differential expression of FAT10 between the tumor and non-tumorous tissues of HCC patients. However, six single nucleotide polymorphisms (SNPs), including one that has not been previously reported, were identified at the promoter of the FAT10 gene. Different haplotypes of these SNPs were found to significantly mediate different FAT10 promoter activities. Consistent with the experimental observation, differential FAT10 expression in the tumors of HCC patients carrying haplotype 1 was generally higher than those carrying haplotype II. Notably, the methylation status of this promoter was found to correlate with FAT10 expression levels. Hence, the aberrant overexpression of the FAT10 gene in the tumors of HCC patients is likely due to aberrant methylation, rather than mutations at the FAT10 promoter., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Molecular diagnosis of Huntington disease (HD) is currently performed by fluorescent repeat-flanking or triplet-primed PCR (TP-PCR) with capillary electrophoresis (CE). However, CE requires multiple post-PCR steps and may result in high cost in high-throughput settings. We previously described a cost-effective single-step molecular screening strategy employing the use of melting curve analysis (MCA). However, because it relies on repeat-flanking PCR, its efficiency in detecting expansion mutations decreases with increasing size of the repeat, which could lead to false-negative results. To address this pitfall, we have developed an improved screening assay coupling TP-PCR, which has been shown in CE-based assays to detect all expanded alleles regardless of size, with MCA in a rapid one-step assay. A companion protocol for rapid size confirmation of expansion-positive samples is also described. The assay was optimized on 30 genotype-known DNAs, and two plasmids pHTT(CAG)26 and pHTT(CAG)33 were used to establish the threshold temperatures (TTs) distinguishing normal from expansion-positive samples. In contrast to repeat-flanking PCR MCA, TP-PCR MCA displayed much higher sensitivity for detecting large expansions. All 30 DNAs generated distinct melt peak Tms which correlated well with each sample's larger allele. Normal samples were clearly distinguished from affected samples. The companion sizing protocol accurately sized even the largest expanded allele of ~180 CAGs. Blinded analysis of 69 clinical samples enriched for HD demonstrated 100% assay sensitivity and specificity in sample segregation. The assay targets the HTT CAG repeat specifically, tolerates a wide range of input DNA, and works well using DNA from saliva and buccal swab in addition to blood. Therefore, rapid, accurate, reliable, and high-throughput detection/exclusion of HD can be achieved using this one-step screening assay, at less than half the cost of fluorescent PCR with CE.

Background: Preimplantation genetic diagnosis (PGD) of myotonic dystrophy type 1 (DM1) currently uses conventional PCR to detect nonexpanded dystrophia myotonica protein kinase ( DMPK ) alleles or triplet-primed PCR to detect the CTG-expanded alleles, coupled with analysis of linked microsatellite markers to increase diagnostic accuracy. We aimed to simplify the process of identification and selection of informative linked markers for application to DM1 PGD., Methods: An in silico search was performed to identify all markers within 1-1.5 Mb flanking the DMPK gene. Five previously known (D19S559, APOC2, D19S543, D19S112, and BV209569) and 7 novel (DM45050, DM45178, DM45209, DM45958, DM46513, DM46892, and DM47004.1) markers with potentially high heterozygosity values and polymorphism information content were selected and optimized in a single-tube multiplex PCR panel., Results: Analysis of 184 DNA samples of Chinese and Caucasian individuals (91 from unrelated, anonymized cord blood of Chinese babies born at the National University Hospital, Singapore, and 93 Caucasian DNA samples from the Human Variation Panel HD100CAU) confirmed the high polymorphism indices of all markers (polymorphism information content >0.5), with observed heterozygosity values ranging from 0.62-0.93. All individuals were heterozygous for at least 6 markers, with 99.5% of individuals heterozygous for at least 2 markers on either side of the DMPK CTG repeat. The dodecaplex marker assay was successfully validated on 42 single cells and 12 whole genome amplified single cells., Conclusions: The DM1 multiplex PCR panel is suitable for use in DM1 PGD either as a standalone linkage-based assay or as a complement to DMPK CTG repeat expansion-mutation detection., (© 2017 American Association for Clinical Chemistry.)

Background: DMPK CTG-repeat expansions that cause myotonic dystrophy type 1 (DM1) can be detected more rapidly, cost-effectively, and simply by combining triplet-primed PCR (TP-PCR) with melting curve analysis (MCA). We undertook a detailed technical validation study to define the optimal operational parameters for performing bidirectional TP-PCR MCA assays., Methods: We determined the assays' analytic specificity and sensitivity, assessed the effect of reaction volumes, DNA diluents, and common contaminants on melt peak temperature, determined the assays' sensitivity in detecting low-level mosaicism for repeat expansion, and evaluated their performance on two real-time PCR platforms., Results: Both assays were highly specific and sensitive, and performed optimally under a broad range of parameters. Bidirectional TP-PCR MCA analysis also reduces the risk of generating false-negative results associated with the rare CCG-interruptions that may be present at either end of expanded alleles., Conclusion: The DMPK TP-PCR MCA is a highly specific, sensitive, and significantly cost-saving screening tool for DM1.

Background: The tumour suppressor p53 is a central player in transcription regulation and cell fate determination. By interacting with p53 and altering its sequence-specific binding to the response elements, the hepatitis B virus X protein (HBx) was reported to re-direct p53 regulation of some genes., Results: Coupling massively parallel deep sequencing with p53 chromatin immunoprecipitation, we demonstrate that HBx modulates global p53 site selection and that this was strongly influenced by altered interaction with transcription co-factors/co-regulators as well as post-translational modifications. Specifically, HBx attenuated p53-TBP-RB1 transcription complex recruitment and interaction and this was associated with hyper-phosphorylation of p53 at serine 315 by HBx. Concurrently, HBx enhanced p53 DNA occupancy to other response elements either alone by displacing specific transcription factors such as CEBPB and NFkB1, or in complex with distinct interacting co-factors Sp1, JUN and E2F1. Importantly, re-wiring of p53 transcription regulation by HBx was linked to the deregulation of genes involved in cell proliferation and death, suggesting a role of HBx in errant cell fate determination mediated by altered p53 site selection of target genes., Conclusions: Our study thus presents first evidence of global modes of p53 transcription alteration by HBx and provides new insights to understand and potentially curtail the viral oncoprotein., (Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Purpose: To develop a single-tube polymerase chain reaction (PCR) panel of highly polymorphic markers for preimplantation genetic diagnosis (PGD) of fragile X syndrome (FXS)., Methods: An in silico search was performed to identify all markers within 1 Mb flanking the FMR1 gene. Selected markers were optimized into a single-tube PCR panel and their polymorphism indices were determined from 272 female samples from three populations. The single-tube assay was also validated on 30 single cells to evaluate its applicability to FXS PGD., Results: Thirteen markers with potentially high polymorphism information content (PIC) and heterozygosity values were selected and optimized into a single-tube PCR panel together with AMELX/Y for gender determination. Analysis of 272 female samples confirmed the high polymorphism (PIC > 0.5) of most markers, with expected and observed heterozygosities ranging from 0.31 to 0.87. More than 99% of individuals were heterozygous for at least three markers, with 95.8% of individuals heterozygous for at least two markers on either side of the FMR1 CGG repeat., Conclusion: The tetradecaplex marker assay can be performed directly on single cells or after whole-genome amplification, thus supporting its use in FXS PGD either as a standalone linkage-based assay or as a complement to FMR1 mutation detection.Genet Med 18 9, 869-875.

Population-based screening for CGG-repeat expansions in the fragile X mental retardation 1 (FMR1) gene that cause fragile X syndrome can now be performed more cost-effectively and simply by combining direct triplet-primed PCR (dTP-PCR) with melting curve analysis (MCA). We have now performed a detailed technical validation to define the operational parameters for achieving robust and reliable performance of the FMR1 dTP-PCR MCA assay. We compared the assay's performance on 2 real-time PCR platforms and determined its analytic sensitivity and specificity. We also assessed the assay's performance on DNA isolated from different sources, the effect of differences in CGG-repeat length and AGG-interruption pattern on melt peak temperature (Tm), and the effect of common substances found in DNA solutions on Tms. The assay performed well in distinguishing normal from expansion-carrying samples. The assay had detection sensitivity down to 1 ng and an analytical specificity beyond 150 ng. In addition to peripheral blood DNA, analysis could also be performed on DNA from saliva, buccal swabs, and dried blood spots. Salt increased Tms, glycogen contamination had minimal effect, whereas AGG interruptions lowered Tms. The FMR1 dTP-PCR MCA screening assay is highly sensitive and specific, performs well using DNA from different sources, and is robust and reproducible when reagent concentrations are maintained across all tested samples., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Preimplantation genetic diagnosis (PGD) of Huntington disease (HD) generally employs linkage analysis of flanking microsatellite markers to complement direct mutation testing, as well as for exclusion testing. Thus far, only 10 linked markers have been developed for use in HD PGD, with a maximum of 3 markers coamplified successfully. We aimed to develop a single-tube multiplex PCR panel of highly polymorphic markers to simplify HD PGD., Methods: An in silico search was performed to identify all markers within 1 Mb flanking the huntingtin (HTT) gene. Selected markers were optimized in a single-tube PCR panel, and their polymorphism indices were determined in 2 populations. The panel was tested on 63 single cells to validate its utility in PGD., Results: We identified 102 markers in silico, of which 56 satisfied the selection criteria. After initial testing, 12 markers with potentially high heterozygosity were optimized into a single-tube PCR panel together with a 13th more distally located marker. Analysis of DNA from 183 Chinese and Caucasian individuals revealed high polymorphism indices for all markers (polymorphism information content >0.5), with observed heterozygosities ranging from 0.5-0.92. All individuals were heterozygous for at least 5 markers, with 99.5% of individuals heterozygous for at least 2 markers upstream and downstream of the HTT CAG repeat., Conclusions: The tridecaplex marker assay amplified reliably from single cells either directly or after whole genome amplification, thus validating its standalone use in HD exclusion PGD or as a complement to HTT CAG repeat expansion-mutation detection., (© 2016 American Association for Clinical Chemistry.)

Chronic hepatitis B virus (HBV) infection is one of the most associated factors in hepatocarcinogenesis. HBV is able to integrate into the host genome and encode the multi-functional hepatitis B virus x protein (HBx). Although the mechanism between HBx and carcinogenesis is still elusive, recent studies have shown that HBx was able to influence various signaling pathways, as well as epigenetic and genetic processes. This review will examine and summarize recent literature about HBx's role in these various processes.

To date, studies of the roles of microRNAs (miRNAs) in hepatocellular carcinoma (HCC) have either focused on specific individual miRNAs and a small number of suspected targets or simply reported a list of differentially expressed miRNAs based on expression profiling. Here, we seek a more in-depth understanding of the roles of miRNAs and their targets in HCC by integrating the miRNA and messenger RNA (mRNA) expression profiles of tumorous and adjacent non-tumorous liver tissues of 100 HCC patients. We assessed the levels of 829 mature miRNAs, of which 32 were significantly differentially expressed. Statistical analysis indicates that six of these miRNAs regulate a significant proportion of their in silico predicted target mRNAs. Three of these miRNAs (miR-26a, miR-122, and miR-130a) were down-regulated in HCC, and their up-regulated gene targets are primarily associated with aberrant cell proliferation that involves DNA replication, transcription and nucleotide metabolism. The other three miRNAs (miR-21, miR-93, and miR-221) were up-regulated in HCC, and their down-regulated gene targets are primarily involved in metabolism and immune system processes. We further found evidence for a coordinated miRNA-induced regulation of important cellular processes, a finding to be considered when designing therapeutic applications based on miRNAs.

Background: Accurate determination of the CAG repeat number is crucial for diagnostic and predictive testing for Huntington disease (HD). Current PCR-based assays can accurately size up to ∼110 HTT CAG repeats., Objective: To develop an improved assay capable of detecting larger CAG repeat expansions., Methods: A triplet-primed PCR (TP-PCR) assay was optimized and validated on 14 HD reference DNAs, including a sample carrying a large expansion of ∼180 CAG repeats., Results: All 14 HD reference samples showed 100% concordance with the previously verified allele sizes. For alleles under 45 CAGs, identical repeat sizes were obtained, while alleles larger than 46 CAGs were sized to within ±1 CAG. The improved TP-PCR assay successfully detected the ∼180 CAG repeat allele in an affected sample., Conclusion: This method extends the detection limit of large expanded alleles to at least ∼175-180 CAG repeats, thus reducing the likelihood of requiring Southern blot analysis for any HD-affected sample., (© 2016 S. Karger AG, Basel.)

Pleiotropic pro-inflammatory cytokines, TNF-α and IFN-γ (TI), play important yet diverse roles in cell survival, proliferation, and death. Recent evidence highlights FAT10 as a downstream molecule in the pathway of inflammation-induced tumorigenesis through mediating the effect of cytokines in causing numerical CIN and protecting cells from cytokines-induced cell death. cDNA microarray analysis of cells treated with TI revealed 493 deregulated genes with FAT10 being the most up-regulated (85.7-fold) gene and NF-κB being the key nodal hub of TI-response genes. Silibinin is reported to be a powerful antioxidant and has anti-C effects against various carcinomas by affecting various signaling molecules/pathways including MAPK, NF-κB and STATs. As NF-κB signaling pathway is a major mediator of the tumor-promoting activities of TI, we thus examine the effects of silibinin on TI-induced FAT10 expression and CIN. Our data showed that silibinin inhibited expression of FAT10, TI-induced chromosome instability (CIN) as well as sensitizes cells to TI-induced apoptosis. Significantly, silibinin suppressed intra-tumorally injected TNF-α-induced tumor growth. This represents the first report associating silibinin with FAT10 and demonstrating that silibinin can modulate TI-induced CIN, apoptosis sensitivity and suppressing TNF-α-induced tumor growth., (© 2015. Published by The Company of Biologists Ltd.)

Glutaminase (GLS), which converts glutamine to glutamate, plays a key role in cancer cell metabolism, growth, and proliferation. GLS is being explored as a cancer therapeutic target, but whether GLS inhibitors affect cancer cell-autonomous growth or the host microenvironment or have off-target effects is unknown. Here, we report that loss of one copy of Gls blunted tumor progression in an immune-competent MYC-mediated mouse model of hepatocellular carcinoma. Compared with results in untreated animals with MYC-induced hepatocellular carcinoma, administration of the GLS-specific inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) prolonged survival without any apparent toxicities. BPTES also inhibited growth of a MYC-dependent human B cell lymphoma cell line (P493) by blocking DNA replication, leading to cell death and fragmentation. In mice harboring P493 tumor xenografts, BPTES treatment inhibited tumor cell growth; however, P493 xenografts expressing a BPTES-resistant GLS mutant (GLS-K325A) or overexpressing GLS were not affected by BPTES treatment. Moreover, a customized Vivo-Morpholino that targets human GLS mRNA markedly inhibited P493 xenograft growth without affecting mouse Gls expression. Conversely, a Vivo-Morpholino directed at mouse Gls had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cell-autonomous dependence on GLS for cancer therapy.

Premutation and full-mutation hyperexpansion of CGG-triplets in the X-linked Fragile X Mental Retardation 1 (FMR1) gene have been implicated in fragile X-associated tremor/ataxia syndrome, fragile X-associated primary ovarian insufficiency, and fragile X syndrome (FXS), respectively. The currently available molecular diagnostic tests are either costly or labour-intensive, which prohibits their application as a first-line FMR1 test in large-scale population-based screening programs. In this study, we demonstrate the utility of a simplified closed-tube strategy for rapid first-line screening of FXS based on melt peak temperature (Tm) analysis of direct triplet-primed polymerase chain reaction amplicons (dTP-PCR MCA). In addition, we also evaluated the correlation between Tm and CGG-repeat size based on capillary electrophoresis (CE) of dTP-PCR amplicons. The assays were initially tested on 29 FMR1 reference DNA samples, followed by a blinded validation on 107 previously characterised patient DNA samples. The dTP-PCR MCA produced distinct melt profiles of higher Tm for samples carrying an expanded allele. Among the samples tested, we also observed a good correlation between Tm and CGG-repeat size. In the blinded validation study, dTP-PCR MCA accurately classified all normal and expansion carriers, and the FMR1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dTP-PCR CE results. This simple and cost-effective MCA-based assay may be useful as a first-line FXS screening tool that could rapidly screen out the large majority of unaffected individuals, thus minimising the number of samples that need to be analysed by Southern blot analysis.

Chronic infection with hepatitis B virus (HBV) is a risk factor for developing hepatocellular carcinoma (HCC). The life cycle of HBV is complex and has been difficult to study because HBV does not infect cultured cells. The HBV regulatory X protein (HBx) controls the level of HBV replication and possesses an HCC cofactor role. Attempts to understand the mechanism(s) that underlie HBx effects on HBV replication and HBV-associated carcinogenesis have led to many reported HBx activities that are likely influenced by the assays used. This review summarizes experimental systems commonly used to study HBx functions, describes limitations of these experimental systems that should be considered, and suggests approaches for ensuring the biological relevance of HBx studies., (© 2014 by the American Association for the Study of Liver Diseases.)

Aim: Molecular diagnosis of fragile X syndrome demands assessment of fragile X mental retardation 1 (FMR1) CGG repeat size and methylation status, while predicting disease transmission risk requires determination of AGG interruption pattern. There is currently no single assay that provides all three categories of information. We describe a single-tube methylation-specific triplet-primed PCR assay for concurrently assessing methylation state, repeat size and structure of CGG repeat(s)., Methods: Differentially labeled primers specific for methylated and unmethylated FMR1 alleles were used to amplify bisulfite-modified DNA, followed by capillary electrophoresis. Twenty-four reference DNAs and 107 patient samples were analyzed to evaluate assay performance., Results: Repeat size, AGG interruption pattern and methylation state were correctly identified in all tested samples. The assay also detected skewed X-inactivation when present in females, and somatic mosaicism in fragile X males., Conclusion: When used in a molecular diagnostic setting, this novel assay could significantly minimize the need to reflex patient samples for Southern analysis.

Instability and expansion of the DMPK CTG repeat cause myotonic dystrophy type 1 (DM1), the most common adult-onset neuromuscular disorder. Overlapping clinical features between DM1 and other myotonic disorders necessitate molecular confirmation for definitive diagnosis. Preconception screening could improve reproductive planning especially in DM1-affected women, who show diminished ovarian reserve and unfavorable in vitro fertilization-preimplantation genetic diagnosis outcome. We optimized triplet-primed PCR and melting curve analysis on 17 DNAs from DM1-affected/unaffected cell lines. A blinded test was performed on 60 genotype-known clinical samples. Plasmid constructs pDMPK(CTG)35 and pDMPK(CTG)48 were used to establish threshold temperatures separating DM1-affected from unaffected samples. Postscreen triplet-primed PCR amplicon sizing was achieved by short-cycle labeled-primer extension followed by capillary electrophoresis. Triplet-primed PCR melting curve analysis melt peak temperatures of unaffected and DM1-affected samples were lower and higher than the control plasmids' melt peak temperatures, respectively. Capillary electrophoresis of post-melting curve analysis amplicons was completely concordant with the screening results. Triplet-primed PCR melting curve analysis is a simple and cost-effective screening tool for rapid identification of DM1. The companion confirmation protocol allows quick determination of CTG repeat size when required. This strategy avoids the need to perform capillary electrophoresis sizing on all test samples, limiting capillary electrophoresis analysis to only a subset of cases that are screen-positive., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

FAT10 (HLA-F-adjacent transcript 10) is a ubiquitin-like modifier that is commonly overexpressed in various tumors. It was found to play a role in mitotic regulation through its interaction with mitotic arrest-deficient 2 (MAD2). Overexpression of FAT10 promotes tumor growth and malignancy. Here, we identified the MAD2-binding interface of FAT10 to be located on its first ubiquitin-like domain whose NMR structure thus was determined. We further proceeded to demonstrate that disruption of the FAT10-MAD2 interaction through mutation of specific MAD2-binding residues did not interfere with the interaction of FAT10 with its other known interacting partners. Significantly, ablation of the FAT10-MAD2 interaction dramatically limited the promalignant capacity of FAT10, including promoting tumor growth in vivo and inducing aneuploidy, proliferation, migration, invasion, and resistance to apoptosis in vitro. Our results strongly suggest that the interaction of FAT10 with MAD2 is a key mechanism underlying the promalignant property of FAT10 and offer prospects for the development of anticancer strategies.

5-Fluorouracil (5-FU) and its pro-drug Capecitabine have been widely used in treating colorectal cancer. However, not all patients will respond to the drug, hence there is a need to develop reliable early predictive biomarkers for 5-FU response. Here, we report a novel potentially functional Single Nucleotide Polymorphism (pfSNP) approach to identify SNPs that may serve as predictive biomarkers of response to 5-FU in Chinese metastatic colorectal cancer (CRC) patients. 1547 pfSNPs and one variable number tandem repeat (VNTR) in 139 genes in 5-FU drug (both PK and PD pathway) and colorectal cancer disease pathways were examined in 2 groups of CRC patients. Shrinkage of liver metastasis measured by RECIST criteria was used as the clinical end point. Four non-responder-specific pfSNPs were found to account for 37.5% of all non-responders (P<0.0003). Five additional pfSNPs were identified from a multivariate model (AUC under ROC = 0.875) that was applied for all other pfSNPs, excluding the non-responder-specific pfSNPs. These pfSNPs, which can differentiate the other non-responders from responders, mainly reside in tumor suppressor genes or genes implicated in colorectal cancer risk. Hence, a total of 9 novel SNPs with potential functional significance may be able to distinguish non-responders from responders to 5-FU. These pfSNPs may be useful biomarkers for predicting response to 5-FU.

Hepatocellular Carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. However, the role of epigenetic changes such as aberrant DNA methylation in hepatocarcinogenesis remains largely unclear. In this study, we examined the methylation profiles of 59 HCC patients. Using consensus hierarchical clustering with feature selection, we identified three tumor subgroups based on their methylation profiles and correlated these subgroups with clinicopathological parameters. Interestingly, one tumor subgroup is different from the other 2 subgroups and the methylation profile of this subgroup is the most distinctly different from the non-tumorous liver tissues. Significantly, this subgroup of patients was found to be associated with poor overall as well as disease-free survival. To further understand the pathways modulated by the deregulation of methylation in HCC patients, we integrated data from both the methylation as well as the gene expression profiles of these 59 HCC patients. In these patients, while 4416 CpG sites were differentially methylated between the tumors compared to the adjacent non-tumorous tissues, only 536 of these CpG sites were associated with differences in the expression of their associated genes. Pathway analysis revealed that forty-four percent of the most significant upstream regulators of these 536 genes were involved in inflammation-related NFκB pathway. These data suggest that inflammation via the NFκB pathway play an important role in modulating gene expression of HCC patients through methylation. Overall, our analysis provides an understanding on aberrant methylation profile in HCC patients.

FAT10 (HLA-F-adjacent transcript 10) is an ubiquitin-like modifier, which has been implicated in immune response and cancer development. In particular, the hypothesis of FAT10 as a mediator of tumorigenesis stems from its ability to associate with a spindle checkpoint protein Mad2 during mitosis and cause aneuploidy, a hallmark of cancer cells. Furthermore, FAT10 is overexpressed in several carcinomas types, including that of liver and colon. Nevertheless, direct evidence linking FAT10 to cell malignant transformation and progression is lacking. Here, we demonstrate that high FAT10 expression enhanced the proliferative, invasive, migratory and adhesive functions of the transformed cell line, HCT116. These observations were consistently demonstrated in an immortalized, non-tumorigenic liver cell line NeHepLxHT. Importantly, FAT10 can induce malignant transformation as evidenced from the anchorage-independent growth as well as in vivo tumor-forming abilities of FAT10-overexpressing NeHepLxHT cells, whereas in rapidly proliferating HCT116, increased FAT10 further augmented tumor growth. FAT10 was found to activate nuclear factor-κB (NFκB), which in turn upregulated the chemokine receptors CXCR4 and CXCR7. Importantly, small interfering RNA depletion of CXCR7 and CXCR4 attenuated cell invasion of FAT10-overexpressing cells, indicating that the CXCR4/7 is crucial for the FAT10-dependent malignant phenotypes. Taken together, our data reveal novel functions of FAT10 in malignant transformation and progression, via the NFκB-CXCR4/7 pathway.

ATP-binding cassette (ABC) proteins in the placenta regulate fetal exposure to xenobiotics. We hypothesized that functional polymorphisms in ABC genes influence risk for non-syndromic oral clefts (NSOC). Both family-based and case-control studies were undertaken to evaluate the association of nine potentially functional single-nucleotide polymorphisms within four ABC genes with risk of NSOC. Peripheral blood DNA from a total of 150 NSOC case-parent trios from Singapore and Taiwan were genotyped, as was cord blood DNA from 189 normal Chinese neonates used as controls. In trios, significant association was observed between the ABCB1 single-nucleotide polymorphisms and NSOC (P<0.05). Only ABCB1 rs1128503 retained significant association after Bonferroni correction (odds ratio (OR)=2.04; 95% confidence interval (CI)=1.42-2.98), while rs2032582 and rs1045642 showed nominal significance. Association with rs1128503 was replicated in a case-control analysis comparing NSOC probands with controls (OR=1.58; 95% CI=1.12-2.23). A comparison between the mothers of probands and controls showed no evidence of association, suggesting NSOC risk is determined by fetal and not maternal ABCB1 genotype. The two studies produced a combined OR of 1.79 (95% CI=1.38-2.30). The T-allele at rs1128503 was associated with higher risk. This study thus provides evidence that potentially functional polymorphisms in fetal ABCB1 modulate risk for NSOC, presumably through suboptimal exclusion of xenobiotics at the fetal-maternal interface.

MicroRNA-224 (miR-224) is frequently over-expressed in liver and colorectal cancers. We and others have previously described the role of miR-224 over-expression in cell proliferation in vitro but we have yet to identify the relevant miR-224 direct target. In this study, we further demonstrated that miR-224 up-regulation promotes cell proliferation using both in vitro assays and in vivo tumor growth models. We systematically screened for high confidence miR-224 targets by overlapping in silico predicted targets from multiple algorithms and significantly down-regulated genes in miR-224-expressing cells from whole genome expression microarrays. A total of 72 high confidence miR-224 targets were identified and found to be enriched in various cancer-related processes. SMAD family member 4 (SMAD4) is experimentally validated as the direct cellular target through which miR-224 promotes cell proliferation. The clinical relevance of our experimental observations was supported by a statistically significant inverse correlation between miR-224 and SMAD4 transcript expression in tumor versus paired adjacent non-tumorous tissues from HCC patients (p<0.001, r= -0.45, R(2) =0.122). Furthermore, miR-224 up-regulation and SMAD4 down-regulation is significantly associated with poorer patient survival (p<0.05). In summary, miR-224/SMAD4 pathway is a clinically relevant pathway to provide new insights in understanding HCC. (191 words).