Use of first nucleotide change technology to determine the frequency of factor V Leiden in a population of Australian blood donors

Activated protein C resistance (APCR), the most common risk factor for venous thrombosis, is the result of a G to A base substitution at nucleotide 1691 (R506Q) in the factor V gene. Current techniques to detect the factor V Leiden mutation, such as determination of restriction length polymorphisms, do not have the capacity to screen large numbers of samples in a rapid, cost- effective test. The aim of this study was to apply the first nucleotide change (FNC) technology, to the detection of the factor V Leiden mutation. After preliminary amplification of genomic DNA by polymerase chain reaction (PCR), an allele-specific primer was hybridised to the PCR product and extended using fluorescent terminating dideoxynucleotides which were detected by colorimetric assay. Using this ELISA-based assay, the prevalence of the factor V Leiden mutation was determined in an Australian blood donor population (n = 500). A total of 18 heterozygotes were identified (3.6%) and all of these were confirmed with conventional MnlI restriction digest. No homozygotes for the variant allele were detected. We conclude from this study that the frequency of 3.6% is compatible with others published for Caucasian populations. In addition, the FNC technology shows promise as the basis for a rapid, automated DNA based test for factor V Leiden.

Genetic and gene expression analyses of the polycystic ovary syndrome candidate gene fibrillin-3 and other fibrillin family members in human ovaries

Several studies have demonstrated an association between polycystic ovary syndrome (PCOS) and the dinucleotide repeat microsatellite marker D19S884, which is located in intron 55 of the fibrillin-3 (FBN3) gene. Fibrillins, including FBN1 and 2, interact with latent transforming growth factor (TGF)-β-binding proteins (LTBP) and thereby control the bioactivity of TGFβs. TGFβs stimulate fibroblast replication and collagen production. The PCOS ovarian phenotype includes increased stromal collagen and expansion of the ovarian cortex, features feasibly influenced by abnormal fibrillin expression. To examine a possible role of fibrillins in PCOS, particularly FBN3, we undertook tagging and functional single nucleotide polymorphism (SNP) analysis (32 SNPs including 10 that generate non-synonymous amino acid changes) using DNA from 173 PCOS patients and 194 controls. No SNP showed a significant association with PCOS and alleles of most SNPs showed almost identical population frequencies between PCOS and control subjects. No significant differences were observed for microsatellite D19S884. In human PCO stroma/cortex (n = 4) and non-PCO ovarian stroma (n = 9), follicles (n = 3) and corpora lutea (n = 3) and in human ovarian cancer cell lines (KGN, SKOV-3, OVCAR-3, OVCAR-5), FBN1 mRNA levels were approximately 100 times greater than FBN2 and 200–1000-fold greater than FBN3. Expression of LTBP-1 mRNA was 3-fold greater than LTBP-2. We conclude that FBN3 appears to have little involvement in PCOS but cannot rule out that other markers in the region of chromosome 19p13.2 are associated with PCOS or that FBN3 expression occurs in other organs and that this may be influencing the PCOS phenotype.

Human-specific E. coli single nucleotide polymorphism (SNP) genotypes detected in a South-East Queensland waterway, Australia

The World Health Organization recommends that the majority of water monitoring laboratories in the world should test for E. coli daily since thermotolerant coliforms and E. coli are key indicators for risk assessment of recreational waters. Recently, we developed a new SNP method for typing E. coli strains, by which human-specific genotypes were identified. Here, we report the presence of these previously described specific SNP profiles in environmental water, sourced from the Coomera River, located on South East Queensland, Australia, over a period of two years. This study tested for the presence of human-specific E. coli to ascertain whether hydrologic and anthropogenic activity plays a key role in the pollution of the investigated watershed or whether the pollution is from other sources. We found six human-specific SNP profiles and one animal-specific SNP profile consistently across sampling sites and times. We have demonstrated that our SNP genotyping method is able to rapidly identify and characterise human- and animal-specific E. coli isolates in water sources.

Evaluation of novel Streptococcus pyogenes vaccine candidates incorporating multiple conserved sequences from the C-repeat region of the M-protein

A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates.

High-resolution DNA melt curve analysis of the clustered, regularly interspaced short-palindromic-repeat locus of Campylobacter jejuni

A novel method for genotyping the clustered, regularly interspaced short-palindromic-repeat (CRISPR) locus of Campylobacter jejuni is described. Following real-time PCR, CRISPR products were subjected to high-resolution melt (HRM) analysis, a new technology that allows precise melt profile determination of amplicons. This investigation shows that the CRISPR HRM assay provides a powerful addition to existing C. jejuni genotyping methods and emphasizes the potential of HRM for genotyping short sequence repeats in other species

Repeat speeding offenders : time to stop thinking of them as just ‘unlucky normal’ drivers

- Speeding and crash involvement in Australia - Speeding recidivist research in Queensland - Challenges from an Australian perspective - Defining speeding - Community attitudes to speeding - Auditor-General reviews of speed camera programs - Implications for future speed management

Repeat speeding offenders : results from research in Queensland, Australia

- Speeding and crash involvement in Australia - Speeding recidivist research in Queensland - Challenges from an Australian perspective - Auditor-General reviews of speed camera programs - Implications for future speed management

A Kallikrein 15 (KLK15) single nucleotide polymorphism located close to a novel exon shows evidence of association with poor ovarian cancer survival

KLK15 over-expression is reported to be a significant predictor of reduced progression-free survival and overall survival in ovarian cancer. Our aim was to analyse the KLK15 gene for putative functional single nucleotide polymorphisms (SNPs) and assess the association of these and KLK15 HapMap tag SNPs with ovarian cancer survival. Results In silico analysis was performed to identify KLK15 regulatory elements and to classify potentially functional SNPs in these regions. After SNP validation and identification by DNA sequencing of ovarian cancer cell lines and aggressive ovarian cancer patients, 9 SNPs were shortlisted and genotyped using the Sequenom iPLEX Mass Array platform in a cohort of Australian ovarian cancer patients (N = 319). In the Australian dataset we observed significantly worse survival for the KLK15 rs266851 SNP in a dominant model (Hazard Ratio (HR) 1.42, 95% CI 1.02-1.96). This association was observed in the same direction in two independent datasets, with a combined HR for the three studies of 1.16 (1.00-1.34). This SNP lies 15bp downstream of a novel exon and is predicted to be involved in mRNA splicing. The mutant allele is also predicted to abrogate an HSF-2 binding site. Conclusions We provide evidence of association for the SNP rs266851 with ovarian cancer survival. Our results provide the impetus for downstream functional assays and additional independent validation studies to assess the role of KLK15 regulatory SNPs and KLK15 isoforms with alternative intracellular functional roles in ovarian cancer survival.

Development and characterisation of tri- and tetra-nucleotide polymorphic microsatellite markers for skipjack tuna (Katsuwonus pelamis)

Skipjack tuna (katsuwonus pelamis) (SJT) is the largest tuna fishery in all the major oceans around the world, and the largest marine fishery in Sri Lanka. Knowledge of genetic population structure and effective population size of SJT in the Indian Ocean and other major oceans, however, is still lacking for better management practices and conservation strategies. We developed microsatellite genetic markers using SJT around Sri Lanka in the Indian Ocean, and characterise one tri- and seven tetra-nucleotide microsatellite loci isolated from enriched genomic libraries from SJT, to provide tools for addressing both conservation and fisheries management questions. An analysis of these eight microsatellite markers in two populations of SJT from eastern Sri Lanka (n = 44) and the Maldives Islands (n = 53) showed that all eight microsatellites were polymorphic with an average number of alleles per locus of 11.80 (range 5-27). Expected heterozygosities at marker loci ranged from 0.450 to 0.961. These markers are being used currently to characterise population structure and extent of natural gene flow in SJT populations from the eastern and western Indian Ocean. No significant linkage disequilibrium was detected among any loci pairs.