A Brugada syndrome proband with compound heterozygote SCN5A mutations identified from a Chinese family in Singapore.

AIMS: Brugada syndrome (BrS) is a rare heritable ventricular arrhythmia. Genetic defects in SCN5A, a gene that encodes the α-subunit of the sodium ion channel Nav1.5, are present in 15-30% of BrS cases. SCN5A remains by far, the highest yielding gene for BrS. We studied a young male who presented with syncope at age 11. This proband was screened for possible disease causing SCN5A mutations. The inheritance pattern was also examined amongst his first-degree family members. METHODS AND RESULTS: The proband had a baseline electrocardiogram that showed Type 2 BrS changes, which escalated to a characteristic Type I BrS pattern during a treadmill test before polymorphic ventricular tachycardia onset at a cycle length of 250 ms. Mutational analysis across all 29 exons in SCN5A of the proband and first-degree relatives of the family revealed that the proband inherited a compound heterozygote mutation in SCN5A, specifically p.A226V and p.R1629X from each parent. To further elucidate the functional changes arising through these mutations, patch-clamp electrophysiology was performed in TSA201 cells expressing the mutated SCN5A channels. The p.A226V mutation significantly reduced peak sodium current (INa) to 24% of wild type (WT) whereas the p.R1629X mutation abolished the current. To mimic the functional state in our proband, functional expression of the compound variants A226V + R1629X resulted in overall peak INa of only 13% of WT (P < 0.01). CONCLUSION: Our study is the first to report a SCN5A compound heterozygote in a Singaporean Chinese family. Only the proband carrying both mutations displayed the BrS phenotype, thus providing insights into the expression and penetrance of BrS in an Asian setting.

Visual DNA detection and SNP genotyping using asymmetric PCR and split DNA enzymes.

We describe a method to detect DNA sequences visually through a color change reaction using DNAzymes. We successfully applied the assay for the detection of Salmonella and Mycobacterium DNA, as well as for genotyping single base differences from within human genomic DNA samples. Our approach adopts a split probe targeting system, designed with G-rich sequences, which reassembles in the presence of target DNA, producing G-quadruplexes with catalytic activity. Asymmetric PCR is first performed to amplify the target region into single-stranded copies, with primer ratios tailored for optimum amplification. This is followed by direct addition of the visual probes, substrates, and reagents to produce a color change within 15 min should the desired target sequences be present. This approach hence offers a rapid readout, ease-of-use, and handling convenience, especially at the point-of-care.

Direct visual detection of Salmonella genomic DNA using gold nanoparticles.

Gold nanoparticles (AuNPs) are well recognized tools for visual DNA detection. Most reports on their use have however been restricted to synthetic or PCR amplified DNA sequences. Herein, we describe a visual DNA detection method that can detect unamplified genomic DNA sequences specifically, using simple reagents and AuNPs, without the need for PCR. This strategy applies thiolated probes and AuNPs to detect genomic DNA. The thiolated probes, in the presence of target Salmonella genomic sequences, cause the AuNPs to aggregate irreversibly, producing a red to purple colorimetric change. As little as 608 000 copies (at 37 fM) of the Salmonella genome were thus detected visually, by eye, without the need for a power source or sophisticated instrumentation. This method thus opens in-roads to direct visual detection, bringing sophisticated DNA analysis capabilities to the point of need.

Visual SNP genotyping using asymmetric PCR and split DNA enzymes.

Harnessing and applying genomic technologies in resource limited environments demand a next generation of platforms, which are convenient, quick and easy to apply. We describe here a visual colour change assay that can be applied to SNP genotyping, which unlike traditional methods, does not adopt complicated procedures or expensive instrumentation, desirable features in bringing genetic capabilities outside the laboratory. Our strategy involved a two-step method that first enriched target genomic regions using asymmetric PCR, followed by direct in situ application of split DNA probes. In the presence of target sequences that perfectly matched the complementary probes, the split probes reassembled active DNAzymes, which catalysed a colour change reaction. A single-base mismatch (indicative of a polymorphism) prevented this reassembly and colour change, providing the means for accurate SNP calling. This is the first report, to our knowledge, that demonstrates successful visual SNP genotyping of actual human DNA samples using DNAzymes.

Applications of microarrays in pathogen detection and biodefence.

The microarray is a platform with wide-ranging potential in biodefence. Owing to the high level of throughput attainable through miniaturization, microarrays have accelerated the ability to respond in an epidemic or crisis. Extending beyond diagnostics, recent studies have applied microarrays as a research tool towards understanding the etiology and pathogenicity of dangerous pathogens, as well as in vaccine development. The original emphasis was on DNA microarrays, but the range now includes protein, antibody and carbohydrate microarrays, and research groups have exploited this diversity to further extend microarray applications in the area of biodefence. Here, we discuss the impact and contributions of the growing range of microarrays and emphasize the concepts that might shape the future of biodefence research.