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Background Community-acquired pneumonia (CAP) is one of the most common infectious diseases and is a significant cause of mortality and morbidity globally. A microbial cause was not determined in a sizable percentage of patients with CAP; there are increasing data to suggest regional differences in bacterial aetiology. We devised a multiplex real-time PCR assay for detecting four microorganisms (Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae and Burkholderia pseudomallei) of relevance to CAP infections in Asia. Methods Analytical validation was accomplished using bacterial isolates (n=10–33 of each target organism for analytical sensitivity and n=117 for analytical sensitivity) and clinical validation using 58 culture-positive respiratory tract specimens. Results The qPCR assay exhibited 100% analytical sensitivity and analytical specificity, and 100% clinical sensitivity and 94–100% clinical specificity. The limit of detection and efficiency for the multiplex PCR assay were 3–33 CFU/mL and 93–110%, respectively. The results showed that the PCR-based method had higher sensitivity than traditional culture-based methods. The assay also demonstrated an ability to semiquantify bacterial loads. Conclusion We have devised a reliable laboratory-developed multiplex qPCR assay, with a turnaround time of within one working day, for detection of four clinically important CAP-associated microorganisms in Asia. The availability of a test with improved diagnostic capabilities potentially leads to an informed choice of antibiotic usage and appropriate management of the patient to achieve a better treatment outcome and financial savings.

Purification of bacteria from human blood samples is essential for rapid identification of pathogens by molecular methods, enabling faster and more accurate diagnosis of bloodstream infection than conventional gold standard blood culture methods. The inertial microfluidic method has been broadly studied to isolate biological cells of interest in various biomedical applications due to its label-free and high-throughput advantages. However, because of the bacteria's tininess, which ranges from 0.5 μm to 3 μm, they are challenging to be effectively focused and sorted out in existing inertial microfluidic devices that work well with biological cells larger than 10 μm. Efforts have been made to sort bacterial cells by utilizing extremely small channel dimensions or employing a sheath flow, which thus results in limitations on the throughput and ease of operation. To overcome this challenge, we develop a method that integrates a non-Newtonian fluid with a novel channel design to allow bacteria to be successfully sorted from larger blood cells in a channel dimension of 120 μm × 20 μm without the use of sheath flows. The throughput of this device with four parallel channels is above 400 μL per minute. The real-time polymerase chain reaction (qPCR) analysis indicates that our inertial sorting approach has a nearly 3-fold improvement in pathogen recovery compared with the commonly used lysis-centrifugation method at pathogen abundances as low as 102 cfu mL-1. With the rapid and simple purification and enrichment of bacterial pathogens, the present inertial sorting method exhibits an ability to enhance the fast and accurate molecular diagnosis of bloodstream bacterial infection.

Introduction: Onychomycosis is a debilitating, difficult-to-treat nail fungal infection with increasing prevalence worldwide. The main etiological agents are dermatophytes, which are common causative pathogens in superficial fungal mycoses. Conventional detection methods such as fungal culture have low sensitivity and specificity and are time-consuming.Aim: The main objective of this study was to design, develop and validate a real-time probe-based multiplex qPCR assay for the detection of dermatophytes and Fusarium species.Methodology: The performance characteristics of the qPCR assays were evaluated. The multiplex qPCR assays targeted four genes (assay 1: pan-dermatophytes/Fusarium spp.; assay 2: Trichophyton rubrum/Microsporum spp.). Analytical validation was accomplished using 150 fungal isolates and clinical validation was done on 204 nail specimens. The performance parameters were compared against the gold standard (fungal culture) and expanded gold standard (culture in conjunction with sequencing).Results: Both the single-plex and multiplex qPCR assays performed well especially when compared against the expanded gold standard. Among the 204 tested nail specimens, the culture method showed that 125 (61.3 %) were infected with at least one organism, of which 40 yielded positive results for dermatophytes and Fusarium spp. These target organisms detected include 20 dermatophytes and 22 Fusarium spp. The developed qPCR assays demonstrated excellent limit of detection, efficiency, coefficient of determination, analytical and clinical sensitivity and specificity.Conclusion: The multiplex qPCR assays were reliable for the diagnosis of onychomycosis, with shorter turn-around time as compared to culture method. This aids in the planning of treatment strategies to achieve optimal therapeutic outcome.

Background and Aim Perturbance in the composition of human gut microbiota has been associated with metabolic disorders such as obesity, diabetes mellitus, and insulin resistance. The objectives of this study are to examine the effects of ethnicity, central obesity, and recorded dietary components on potentially influencing the human gut microbiome. We hypothesize that these factors have an influence on the composition of the gut microbiome. Methods Subjects of Chinese (n = 14), Malay (n = 10), and Indian (n = 11) ancestry, with a median age of 39 years (range: 22–70 years old), provided stool samples for gut microbiome profiling using 16S rRNA sequencing and completed a dietary questionnaire. The serum samples were assayed for a panel of biomarkers (interleukin‐6, tumor necrosis factor alpha, adiponectin, cleaved cytokeratin 18, lipopolysaccharide‐binding protein, and limulus amebocyte lysate). Central obesity was defined by waist circumference cut‐off values for Asians. Results There were no significant differences in Shannon alpha diversity for ethnicity and central obesity and no associations between levels of inflammatory cytokines and obesity. The relative abundances of Anaerofilum (P = 0.02), Gemellaceae (P = 0.02), Streptococcaceae (P = 0.03), and Rikenellaceae (P = 0.04) were significantly lower in the obese group. From principle coordinate analysis, the effects of the intake of fiber and fat/saturated fat were in contrast with each other, with clustering of obese individuals leaning toward fiber. Conclusion The study demonstrated that there were differences in the gut microbiome in obese individuals. Certain bacterial taxa were present in lower abundance in the group with central obesity. Fiber and fat/saturated fat diets were not the key determinants of central obesity., The objectives of this study are to examine the effects of ethnicity, central obesity, and dietary components on the human gut microbiome. Asian subjects (n = 35) provided stool samples for gut microbiome profiling using 16S rRNA sequencing and completed a dietary questionnaire. The relative abundances of Anaerofilum (P = 0.02), Gemellaceae (P = 0.02), Streptococcaceae (P = 0.03), and Rikenellaceae (P = 0.04) were significantly lower in the obese group.

Purpose The emergence of antiseptic resistance and/or antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) may result in failure of decolonization treatments. Plasmid-encoded efflux pump genes qacA/B and qacC (smr) confer tolerance to chlorhexidine and quaternary ammonium compounds. The objective of this study was to develop and validate a multiplex real time-PCR assay for detection of antiseptic-resistance genes, apply the assay on 200 MRSA isolates and explore if carriage of these genes was associated with resistance to topical antibiotics. Methodology A SYBR-Green based multiplex real time-PCR assay was developed to detect qacA/B, qacC, and mecA (internal control) simultaneously. The multiplex assay was compared against conventional single-plex PCR followed by agarose gel electrophoresis , using DNA from the first 73 MRSA isolates, followed by multiplex testing of the remaining 127 MRSA isolates. All 200 MRSA isolates were tested for susceptibility to mupirocin , retapamulin , neomycin , bacitracin and octenidine. The genetic diversity of the isolates was investigated by s pa-typing. Results The concordance between multiplex and conventional PCR, in assignments of qacA/B and qacC status were 99%(72/73) and 100%(73/73) respectively. Among 200 MRSA isolates, 48(24%) and 44(23%) were found to harbour qacA/B and qacC genes, respectively. These isolates remained susceptible to many common decolonization agents, except mupirocin. The predominant spa-types were t020 and t1081 (41 and 32 isolates respectively). Conclusion The real-time assay performed acceptably for the detection of qac genes. A high prevalence of antiseptic-resistance genes were detected in the MRSA isolates in our population and appeared to be associated with spa-type t1081.

Introduction The objectives of this study were to examine the effects of ethnicity, gender and a proton pump inhibitor (PPI), omeprazole, on the human gut microbiome. PPIs are commonly used for the treatment of acid-related disorders. We hypothesised that PPI therapy might perturb microbial communities and alter the gut microbiome. Methods Healthy subjects of Chinese (n = 12), Malay (n = 12) and Indian (n = 10) ancestry, aged 21-37 years, were enrolled. They provided a baseline stool sample (Day 1) and were then given a course of omeprazole at therapeutic dose (20 mg daily) for seven days. Stool samples were collected again on Day 7 and 14 (one week after stopping omeprazole). Microbial DNA was extracted from the stool samples, followed by polymerase chain reaction, library construction, 16S rRNA sequencing using Illumina MiSeq, and statistical and bioinformatics analyses. Results The findings showed an increase in species richness (p = 0.018) after omeprazole consumption on Day 7, which reverted to baseline on Day 14. There were significant increases in the relative abundance of Streptococcus vestibularis (p = 0.0001) and Veillonella dispar (p = 0.0001) on Day 7, which diminished on Day 14. Faecalibacterium prausnitzii, Sutterella stercoricanis and Bacteroides denticanum were characteristic of Chinese, Malays and Indians, respectively. Lactobacillaceae and Bacteroides xylanisolvens were the signature taxa of male and female subjects, respectively. Conclusion The study demonstrated alterations in the gut microbiome following omeprazole treatment. This may explain the underlying pathology of increased risk of Clostridium difficile infections associated with omeprazole therapy.

Drug transporters are differentially expressed in many polarized tissues. The varied distribution and expression of transporters determines the net transcellular transport and influences the disposition of many clinically used drugs. ATP-binding cassette (ABC) and Solute Carrier (SLC) transporters interact dynamically to mediate the passage of drugs across cells. The variable expression of drug transporters could be attributed to genetic and non-genetic factors, which accounts for the differences in drug response among individuals, in terms of both efficacy and adverse effects.The authors provide the background of ABC and SLC transporters, and highlight the fact that their expression is cell-specific and the study of a transporter in isolation is not an adequate measure of its function. The technologies and approaches to characterize the function of transporters, as well as the genetic and non-genetic factors underlying their variable expression in specific cells and among individuals were reviewed.Many studies have utilized tissue homogenization techniques to isolate mRNA for quantifying transporter expression levels. We highlight that transporter expression is cell-specific and mRNA expression does not always reflect its total functionality. In addition, transporter expression in immortalized cell lines may not mirror its expression in the target tissue site.

The major histocompatibility complex (MHC) containing the classical human leukocyte antigen (HLA) Class I and Class II genes is among the most polymorphic and diverse regions in the human genome. Despite the clinical importance of identifying the HLA types, very few databases jointly characterize densely genotyped single nucleotide polymorphisms (SNPs) and HLA alleles in the same samples. To date, the HapMap presents the only public resource that provides a SNP reference panel for predicting HLA alleles, constructed with four collections of individuals of north-western European, northern Han Chinese, cosmopolitan Japanese and Yoruba Nigerian ancestry. Owing to complex patterns of linkage disequilibrium in this region, it is unclear whether the HapMap reference panels can be appropriately utilized for other populations. Here, we describe a public resource for the Singapore Genome Variation Project with: (i) dense genotyping across ∼ 9000 SNPs in the MHC; (ii) four-digit HLA typing for eight Class I and Class II loci, in 96 southern Han Chinese, 89 Southeast Asian Malays and 83 Tamil Indians. This resource provides population estimates of the frequencies of HLA alleles at these eight loci in the three population groups, particularly for HLA-DPA1 and HLA-DPB1 that were not assayed in HapMap. Comparing between population-specific reference panels and a cosmopolitan panel created from all four HapMap populations, we demonstrate that more accurate imputation is obtained with population-specific panels than with the cosmopolitan panel, especially for the Malays and Indians but even when imputing between northern and southern Han Chinese. As with SNP imputation, common HLA alleles were imputed with greater accuracy than low-frequency variants.

Novel organic cation transporter 2 (OCTN2) is a multispecific, bidirectional, pH-dependent organic cation transporter. It can function as a carnitine co-transporter with higher affinity for carnitine than OCTN1 but also functions as a uniporter for other cations. Drugs such as verapamil, pyrilamine and beta-lactam antibiotics have been characterized as substrates of OCTN2 and/or inhibitors of carnitine transport. This study identified variants of the SLC22A5 gene in two distinct ethnic groups of the Singaporean population (n=192) by DNA sequencing. Twenty-eight genetic variants of SLC22A5, including 13 that were novel, were found: 14 were located in the coding exons, 10 in the introns, 1 in the promoter region, 2 in the 5'-untranslated region and 1 in the 3'-untranslated region. Among the novel nonsynonymous variants, Asp122Tyr was predicted to be functionally significant. Functional nonsynonymous variants detected include Ser467Cys and Arg254X; the latter resulted in a premature stop codon and is predicted to result in a truncated protein that is less than half the molecular mass of wild-type OCTN2. These data constitute fundamental information of value for future pharmacogenetic studies in Asian populations on drugs that are substrates of OCTN2.

Ribavirin is found to be absorbed in the intestine through the human concentrative nucleoside transporter 2 (hCNT2). Cellular uptake of ribavirin was strongly inhibited by purine nucleoside in an in vitro study. This study aims to examine the effects of dietary purine on the pharmacokinetics of orally administered ribavirin in vivo. Twenty healthy participants were enrolled in a randomized, 2-period crossover study. Participants were administered a single 600-mg oral dose of ribavirin after either a high-purine meal or a low-purine meal. Serial blood samples were collected predose and over 144 hours after dosing. Ribavirin concentrations were measured by liquid chromatography/tandem mass spectrometry. In comparison with corresponding plasma values of ribavirin following a high-purine meal, C(max), AUC(0-144) and AUC(0-infinity) of ribavirin following a low-purine meal were 136% (90% confidence internal [CI]: 120%-155%), 134% (90% CI: 118%-153%), and 139% (90% CI: 120%-159%), respectively. This study indicates that dietary purines have an effect on ribavirin absorption. Dosage regimens of ribavirin might need to be adjusted according to the purine content of the meal.

The novel organic cation transporter 1 (OCTN1) is a multispecific, bidirectional and pH-dependent organic cation transporter with low carnitine transport activity. It is a transporter of the physiological substance ergothioneine and mediates the transport of a variety of organic cations such as tetraethylammonium, pyrilamine and quinidine. This study identifies genetic variations of the SLC22A4 gene in two distinct ethnic groups of the Singaporean population (n=192) by DNA sequencing. Twenty four genetic variants of SLC22A4, including 14 found to be novel. 16 in the coding exons (10 nonsynonymous and 6 synonymous variations) and 8 in the introns. Among the novel nonsynonymous variations, Arg63His, Arg83Pro, Met344Lys and Ile500Asn were predicted to be functionally significant. These data should provide fundamental and useful information for pharmacogenetic studies on drugs that are substrates of OCTN1 in Asians.

MRP2 is a drug transporter that is responsible for the gastrointestinal absorption and biliary excretion of a wide variety of endogenous and xenobiotic compounds, including many clinically used drugs. This study aims to identify genetic variations of ABCC2 gene in three distinct ethnic groups of the Singaporean population (n = 288). The coding region of the gene encoding the transporter protein was screened for genetic variations in the study population by denaturing high-performance liquid chromatography and DNA sequencing. Twenty-two genetic variations of ABCC2, including 8 novel ones, were found: 1 in the 5' untranslated region, 10 in the coding exons (8 nonsynonymous and 2 synonymous variations), and 11 in the introns. Three novel nonsynonymous variations: 2686G > A (Glu896Lys), 4240C > T (His1414Tyr) and 4568A > C (Gln1523Pro) were detected in single heterozygous Malay, Chinese, and Indian subjects, respectively. Among the novel nonsynonymous variations, 4240C > T and 4568A > C were predicted to be functionally significant. These data would provide fundamental and useful information for pharmacogenetic studies on drugs that are substrates of MRP2 in Asians.

OATP1B1 is a liver-specific transporter that mediates the uptake of various endogenous and exogenous compounds including many clinically used drugs from blood into hepatocytes. This study aims to identify genetic variations of SLCO1B1 gene in three distinct ethnic groups of the Singaporean population (n=288). The coding region of the gene encoding the transporter protein was screened for genetic variations in the study population by denaturing high-performance liquid chromatography and DNA sequencing. Twenty-five genetic variations of SLCO1B1, including 10 novel ones, were found: 13 in the coding exons (9 nonsynonymous and 4 synonymous variations), 6 in the introns, and 6 in the 3' untranslated region. Four novel nonsynonymous variations: 633AG (Ile211Met), 875CT (Ala292Val), 1837TC (Cys613Arg), and 1877TA (Leu626Stop) were detected as heterozygotes. Among the novel nonsynonymous variations, 633AG, 1837TC, and 1877TA were predicted to be functionally significant. These data would provide fundamental and useful information for pharmacogenetic studies on drugs that are substrates of OATP1B1 in Asians.

Contemporary chemotherapeutic treatments incorporate the use of several agents in combination. However, selecting the most appropriate drugs for such therapy is not necessarily an easy or straightforward task. Here, we describe a targeted approach that can facilitate the reliable selection of chemotherapeutic drug combinations through the interrogation of drug-resistance gene networks. Our method employed single-cell eukaryote fission yeast (Schizosaccharomyces pombe) as a model of proliferating cells to delineate a drug resistance gene network using a synthetic lethality workflow. Using the results of a previous unbiased screen, we assessed the genetic overlap of doxorubicin with six other drugs harboring varied mechanisms of action. Using this fission yeast model, drug-specific ontological sub-classifications were identified through the computation of relative hypersensitivities. We found that human gastric adenocarcinoma cells can be sensitized to doxorubicin by concomitant treatment with cisplatin, an intra-DNA strand crosslinking agent and suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. Our findings point to the utility of fission yeast as a model and the differential targeting of a conserved gene interaction network when screening for successful chemotherapeutic drug combinations for human cells.

What is already known about this subject • Genetic variability of the FcγRIIIa 176 F/V polymorphism has been widely studied in patients with systemic lupus erythomatosus and rheumatoid arthritis, as well as the general population of various White groups. • Its implications in disease pathogenesis and response to therapeutics have been well documented. • This study aimed to profile its polymorphism pattern in the Asian population, thus it serves as useful reference controls in disease association studies and tailoring therapy to ethnic-specific populations. What this study adds • In this study, we have established the genetic variability profile of the FcγRIIIa 176 F/V polymorphism in three distinct Asian groups (Chinese, Malays and Indians) and also supplemented existing data based on ethnic-specific healthy controls. • The polymorphism pattern of Malays was found to differ significantly from Chinese and Indians, which was also extended to almost all other healthy controls of various ethnic groups published elsewhere. • Such differences could have implications in disease susceptibility and pathogenesis, as well as response to drug therapeutics. Aims To determine and compare the distribution of the FcγRIIIa 176 F/V polymorphism across three ethnically distinct populations (Chinese, Asian Indians and Malays) in Singapore. Methods The FcγRIIIa 176 F/V polymorphism was genotyped by direct sequencing from genomic DNA samples obtained from normal healthy Chinese, Asian Indians and Malays (n = 192 from each population). Results The allelic frequencies of the high binding affinity FcγRIIIa 176 V allele for Chinese, Asian Indians and Malays were 35%, 33% and 46%, respectively (F allele frequencies were 65%, 67% and 54%, respectively). Genotype distributions were found to conform to the Hardy–Weinberg law (P > 0.05) in each group. χ2 comparisons revealed significant differences in the genotype distributions of the FcγRIIIa 176 V/F polymorphism of Malays from the other two populations (Chinese and Asian Indians). However, no significant difference in the genotype distributions of the FcγRIIIa 176 V/F polymorphism was observed between Chinese and Asian Indian populations. Conclusions The genotype distributions of the FcγRIIIa 176 V/F polymorphism in healthy Malays are significantly different from both Chinese and Indians. These observations provide the fundamentals on which future disease associations may be built and also present important implications for the design of therapeutic regimens amongst various ethnic groups.

South Asia possesses a significant amount of genetic diversity due to considerable intergroup differences in culture and language. There have been numerous reports on the genetic structure of Asian Indians, although these have mostly relied on genotyping microarrays or targeted sequencing of the mitochondria and Y chromosomes. Asian Indians in Singapore are primarily descendants of immigrants from Dravidian-language–speaking states in south India, and 38 individuals from the general population underwent deep whole-genome sequencing with a target coverage of 30X as part of the Singapore Sequencing Indian Project (SSIP). The genetic structure and diversity of these samples were compared against samples from the Singapore Sequencing Malay Project and populations in Phase 1 of the 1,000 Genomes Project (1 KGP). SSIP samples exhibited greater intra-population genetic diversity and possessed higher heterozygous-to-homozygous genotype ratio than other Asian populations. When compared against a panel of well-defined Asian Indians, the genetic makeup of the SSIP samples was closely related to South Indians. However, even though the SSIP samples clustered distinctly from the Europeans in the global population structure analysis with autosomal SNPs, eight samples were assigned to mitochondrial haplogroups that were predominantly present in Europeans and possessed higher European admixture than the remaining samples. An analysis of the relative relatedness between SSIP with two archaic hominins (Denisovan, Neanderthal) identified higher ancient admixture in East Asian populations than in SSIP. The data resource for these samples is publicly available and is expected to serve as a valuable complement to the South Asian samples in Phase 3 of 1 KGP., Author Summary Indians of South Asia has long been a population of interest to a wide audience, due to its unique diversity. We have deep-sequenced 38 individuals of Indian descent residing in Singapore (SSIP) in an effort to illustrate their diversity from a whole-genome standpoint. Indeed, among Asians in our population panel, SSIP was most diverse, followed by the Malays in Singapore (SSMP). Their diversity is further observed in the population's chromosome Y haplogroup and mitochondria haplogroup profiles; individuals with European-dominant haplogroups had greater proportion of European admixture. Among variants (single nucleotide polymorphism and small insertions/deletions) discovered in SSIP, 21.69% were novel with respect to previous sequencing projects. In addition, some 14 loss-of-function variants (LOFs) were associated to cancer, Type II diabetes, and cholesterol levels. Finally, D statistic test with ancient hominids concurred that there was gene flow to East Asians compared to South Asians.

We analyzed the coding regions of the cardiac calcium-handling genes, ryanodine receptor 2 (RyR2) and calsequestrin 2 (CASQ2) for genetic variants in a healthy Chinese population (n = 95) and in a cohort of 28 sudden unexplained death victims. Mutations in RyR2 and CASQ2 have been shown to alter calcium homeostasis during excitation–contraction coupling and predispose individuals to fatal cardiac arrhythmias. The genetic screening was accomplished by denaturing high-performance liquid chromatography and DNA sequencing methods. Genetic analysis revealed the following nonsynonymous genetic variations: two reported RyR2 polymorphisms; 5654G>A (G1885E) and 5656G>A (G1886S), two reported CASQ2 polymorphisms; 196A>G (T66A) and 226G>A (V76M) and one novel CASQ2 mutation; 529G>C (E177Q). The functional significance of the novel CASQ2 mutation has not been evaluated and characterized. This study shows that multiple genetic variations of the RyR2 and CASQ2 genes exist in the two study populations. The inter-individual genetic variability may underlie the different susceptibility of individuals to developing ventricular tachycardia. The research results will be valuable for which future work involving clinical and forensic samples can be based upon to distinguish potential disease-associated mutations from common polymorphisms.

Drug resistance is a major hurdle to the success of chemotherapy. The permeability glycoprotein (P-gp) is an important factor dictating drug access to the cells, as it controls the efflux of chemotherapeutic agents against the concentration gradient. Pmd1, a P-gp-like protein, was recently isolated as a doxorubicin resistance gene in fission yeast. Although the null mutant of pmd1 (Δ pmd1) exhibited sensitivity to doxorubicin, it showed an unexpectedly high resistance to the drug at relatively high concentrations. The data presented here suggest that this is due to the presence of cooperative processes that can complement and counteract drug cytotoxicity in the absence of Pmd1. One such factor, Rav1, is an essential factor in controlling the assembly of the pH-regulating transporter vacuolar-ATPase (V-ATPase) in fission yeast. The simultaneous disruption of Pmd1 and Rav1 resulted in a prominent accumulation of doxorubicin in the cytoplasm of cells, accompanied by a decline in cell viability. With concurrent treatment of pharmacological inhibitors in human cervical cancer cells, P-gp and V-ATPase were further shown to act synergistically to sensitize cells to doxorubicin also in the human cells. Furthermore, a novel Cornichon-like protein SPAC2C4.05 (herein named as Cor1) was demonstrated for the first time to be involved in the interaction with P-gp and V-ATPase to counteract doxorubicin-dependent cytotoxicity. Therefore this study identified a molecular cooperation between multiple membrane transporter proteins that confers chemoresistance to cells against the chemical insult of doxorubicin. Interestingly, this network exhibited differential effects to doxorubicin as compared with its close epimeric analog epirubicin, suggestive of the intricacy of the drug response regulated by this synergistic interaction. A model is discussed on how the versatility of this network can differentiate closely related chemical drug structures yet allow for the robustness to counteract a vast range of drugs.

Atrial fibrillation (AF) is the most common sustained arrhythmia with poor prognosis, reaching epidemic proportions in the ageing population. The condition affects more than five million people worldwide and is a significant clinical problem. Of particular interest is a subset of younger patients (

The sudden unexplained death syndrome is a tragic and distressing event, which may arise due to an underlying fatal cardiac arrhythmia, which may be associated with a defective ion channel. These electrical abnormalities of the heart do not present with gross structural changes, rendering it a challenging task to the forensic pathologist in ascertaining the cause of death during autopsies. The attempts to identify genetic variations in the key ion channels known to cause arrhythmias have only been able to reveal the presence of putative mutations in a small percentage of the cases. It is thus imperative to look beyond the discovery of single-nucleotide polymorphisms to other forms of genetic variations (such as copy number variations and structural rearrangements), as well as alternative mechanisms that may underlie the molecular pathogenesis of arrhythmias and electrocardiographic abnormalities causing sudden death. Additionally, the various technological platforms for genetic analysis are also described in this article. Key Concepts: Sudden unexplained death syndrome is an important clinical problem. Sudden death may be the result of a fatal cardiac arrhythmia. Arrhythmias are associated with genetic mutations in ion channel genes. Genetic mutations often exhibit incomplete penetrance. Genetic mutations may have functional impact on the ion channel. Single-nucleotide polymorphisms only account for a fraction of the cases. Copy number variations and chromosomal aberrations may play a role in sudden death pathology. The role of alternative mechanisms such as epigenetics in arrhythmias/sudden death remains to be ascertained. The technology for identifying genetic variations is advancing rapidly. Genome-wide sequencing is expected to become increasingly feasible. Keywords: sudden unexplained death syndrome; arrhythmias; genetic variants; ion channels; genome-wide association studies; candidate gene sequencing; next-generation sequencing

The human concentrative nucleoside transporter 2 (hCNT2) plays a major role in the intestinal absorption of naturally occurring nucleosides as well as some nucleoside analog drugs. To determine if single nucleotide polymorphisms (SNPs) in the promoter region of hCNT2 affect gene expression, we examined approximately 1 kb upstream the hCNT2 transcription start site. Ninety Chinese samples were screened and seven SNPs were identified: -115T>G, -146T>A, -264A>G, -564G>A, -861A>C, -880T>C and -906C>T. Based on these seven variants and their relative positions, eight haplotypes were identified using PHASE v2.1.1. Three naturally occurring haplotypes were cloned into the pGL3-Basic vector and transfected into HEK293 cells. Dual luciferase assay revealed that haplotype 4 (GTAGACC) and 7 (GAGAACT) exhibited significantly lower expression levels compared to the published haplotype 1 (TTAGATC). Results from our in-vitro study showed that the hCNT2 promoter region haplotype may modulate gene expression and cause different drug responses.

Sudden cardiac death (SCD) can be caused by a number of reasons. Previous works have identified the genetic causes, such as alterations in the DNA sequence, for many of these diseases. We hypothesize that some patients may show genomic imbalances and changes in the gene copy number leading to genetic instability. To clarify this, we analysed DNA samples from SCD victims using comparative genomic hybridization (CGH), a molecular cytogenetic technique that permits the genome-wide screening of chromosomal imbalances, and telomere length measurement. DNA derived from peripheral blood and heart tissue of 14 SCD cases and six apparently healthy control individuals were subjected to CGH analysis. Telomere length measurements were done by the Southern blotting method. Eight out of 14 SCD cases exhibited changes in DNA/gene copy number. CGH analysis showed variation in the gene copy number of some of the genes associated with potassium (KCNAB1, KCNH2, and KCNA4) and calcium (RyR2, ATP2A2) ions which are involved in maintaining the ionic balance of the heart. Alterations in TERC and TERT genes were also detected in SCD victims. In nine SCD victims shorter telomeres were detected. This might have resulted from excessive cellular proliferation and/or oxidative stress in these individuals. Copy number changes observed and telomere shortening detected in SCD cases would possibly explain at least some of the causes of SCD at early ages in humans. Identification of biomarkers of SCD is of great importance and thus the present study will facilitate the identification of some of the biomarkers.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • An increasing number of drugs on the market or under development have been identified as substrates of the ATP-binding cassette drug efflux transporter breast cancer resistance protein (BCRP; ABCG2), which can affect the pharmacokinetics of drugs by reducing absorption and/or increasing biliary elimination. • ABCG2 C421A, a single nucleotide polymorphism associated with decreased protein expression/transport activity in vitro and higher anti-cancer drug concentrations in carriers of the C421A polymorphism, may contribute to the intersubject pharmacokinetic variability of BCRP substrates. • Predicting the potential influence of BCRP on drug disposition or drug interactions is challenging because of the lack of a well-characterized, BCRP-selective clinical probe substrate. • Nitrofurantoin is potentially a suitable clinical BCRP probe substrate based on preclinical and clinical information available (e.g. in vitro transport studies, Bcrp knockout mouse studies, inhibition studies in rats, and milk secretion studies in rats and humans). WHAT THIS STUDY ADDS • The ABCG2 C421A SNP had no effect on oral nitrofurantoin plasma and urine pharmacokinetic parameters in healthy male Chinese subjects. • Nitrofurantoin does not appear to be a useful clinical BCRP probe. AIMS A number of drugs are substrates or inhibitors of the efflux transporter breast cancer resistance protein (BCRP; ABCG2), which can limit systemic exposure by reducing absorption and/or increasing biliary elimination. The identification of a BCRP-selective clinical probe drug would provide a useful tool to understand the effect of genetic polymorphisms and transporter-based drug interactions on drug pharmacokinetics. The aim of this study was to assess the utility of nitrofurantoin as a clinical probe substrate for BCRP activity by evaluating the impact of genetic variation on nitrofurantoin pharmacokinetics. METHODS Nitrofurantoin pharmacokinetics were studied in an open-label, single-oral dose (100 mg) study in 36 male Chinese subjects who were pre-screened for ABCG2 421 CC, CA and AA genotypes (n = 12 each). Plasma and urine concentrations of nitrofurantoin were determined by LC/MS/MS and LC/UV respectively. anova was used to compare pharmacokinetic parameters among genotypes. RESULTS There were no significant differences in nitrofurantoin pharmacokinetics among the genotypic cohorts. The geometric mean nitrofurantoin plasma AUC(0-∞) (95% confidence interval) values were 2.21 (2.00, 2.45), 2.42 (2.11, 2.78) and 2.32 (1.99, 2.70) µg h ml−1 and half-life values were 0.79 (0.59, 1.0), 0.76 (0.64, 0.89) and 0.72 (0.62, 0.84) h for ABCG2 421 genotypes CC, CA and AA, respectively. The percentage of dose excreted unchanged in the urine was 43, 44 and 39%, respectively. CONCLUSIONS The ABCG2 C421A polymorphism had no effect on nitrofurantoin plasma and urine pharmacokinetic parameters in healthy Chinese subjects. These results indicate that nitrofurantoin is not a suitable clinical probe substrate for assessing BCRP activity.

The human concentrative nucleoside transporter (hCNT2), also known as SLC28A2, plays an important role in the cellular uptake across intestinal membrane of some naturally occurring nucleosides and nucleoside analogs. This study aims to determine the genetic variability of hCNT2 (SLC28A2) in three major Asian ethnic groups residing in Singapore: Chinese, Malay and Indian, and functionally characterize the variants of hCNT2. Healthy participants (n=96) from each group were screened for genetic variations in the exons of hCNT2 (SLC28A2) using denaturing high performance liquid chromatography and sequencing analyses. A total of 23 polymorphisms were identified in the exonic and flanking intronic regions, and ethnic differences in single nucleotide polymorphism frequencies were evident. Five novel nonsynonymous variants (L12R, R142H, E172D, E385K, M612T) were constructed by mutagenesis and functionally characterized in U-251 cells. Expression of these variants in U-251 cells revealed that all except E385K can uptake various substrates of hCNT2: inosine, ribavirin and uridine.

Aims To determine the genetic variability of long QT syndrome (LQTS)-associated genes (KCNQ1, HERG, KCNE1 and KCNE2) among three distinct ethnic groups in the Singapore population. Methods Genomic DNA samples from up to 265 normal healthy Chinese, 118 Malay and 139 Indian volunteer subjects were screened for genetic variations in the coding region of the LQTS-associated genes using denaturing high-performance liquid chromatography and sequencing analyses. Results In total, 37 single nucleotide polymorphisms (SNPs) were identified in the coding exons of the LQTS-associated potassium ion channel genes, seven of which were novel nonsynonymous polymorphisms. SNPs 356GA (exon 1 of KCNQ1), 2624CT and 2893GA (exon 11 of HERG), 3164GA, 3322CG and 3460GA (exon 14 of HERG), and 79CT (exon 3 of KCNE2) resulted in Gly119Asp, Thr875Met, Gly965Arg, Arg1055Gln, Leu1108Val, Gly1154Ser and Arg27Cys amino acid substitutions, respectively. In addition, 16 intronic variants were detected. The functional consequence of these variants has not been studied and their association with risk of LQTS is unclear. Conclusions There exist multiple genetic polymorphisms of the LQTS-associated genes in the three distinct Asian populations. Though the functional significance of many of these SNPs is unknown, this interindividual and interethnic genetic variability may underlie the different susceptibilities of individuals to developing LQTS.