Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes

The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson’s Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59–119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.

Molecular characterisation of environmental enterococci derived from water samples and assessment of associated public health hazards

Enterococci are versatile Gram-positive bacteria that can survive under extreme conditions. Most enterococci are non-virulent and found in the gastrointestinal tract of humans and animals. Other strains are opportunistic pathogens that contribute to a large number of nosocomial infections globally. Epidemiological studies demonstrated a direct relationship between the density of enterococci in surface waters and the risk of swimmer-associated gastroenteritis. The distribution of infectious enterococcal strains from the hospital environment or other sources to environmental water bodies through sewage discharge or other means, could increase the prevalence of these strains in the human population. Environmental water quality studies may benefit from focusing on a subset of Enterococcus spp. that are consistently associated with sources of faecal pollution such as domestic sewage, rather than testing for the entire genus. E. faecalis and E. faecium are potentially good focal species for such studies, as they have been consistently identified as the dominant Enterococcus spp. in human faeces and sewage. On the other hand enterococcal infections are predominantly caused by E. faecalis and E. faecium. The characterisation of E. faecalis and E. faecium is important in studying their population structures, particularly in environmental samples. In developing and implementing rapid, robust molecular genotyping techniques, it is possible to more accurately establish the relationship between human and environmental enterococci. Of particular importance, is to determine the distribution of high risk enterococcal clonal complexes, such as E. faecium clonal complex 17 and E. faecalis clonal complexes 2 and 9 in recreational waters. These clonal complexes are recognized as particularly pathogenic enterococcal genotypes that cause severe disease in humans globally. The Pimpama-Coomera watershed is located in South East Queensland, Australia and was investigated in this study mainly because it is used intensively for agriculture and recreational purposes and has a strong anthropogenic impact. The primary aim of this study was to develop novel, universally applicable, robust, rapid and cost effective genotyping methods which are likely to yield more definitive results for the routine monitoring of E. faecalis and E. faecium, particularly in environmental water sources. To fullfill this aim, new genotyping methods were developed based on the interrogation of highly informative single nucleotide polymorphisms (SNPs) located in housekeeping genes of both E. faecalis and E. faecium. SNP genotyping was successfully applied in field investigations of the Coomera watershed, South-East Queensland, Australia. E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles respectively. This study showed the high longitudinal diversity of E. faecalis and E. faecium over a period of two years, and both human-related and human-specific SNP profiles were identified. Furthermore, 4.25% of E. faecium strains isolated from water was found to correspond to the important clonal complex-17 (CC17). Strains that belong to CC17 cause the majority of hospital outbreaks and clinical infections globally. Of the six sampling sites of the Coomera River, Paradise Point had the highest number of human-related and human-specific E. faecalis and E. faecium SNP profiles. The secondary aim of this study was to determine the antibiotic-resistance profiles and virulence traits associated with environmental E. faecalis and E. faecium isolates compared to human pathogenic E. faecalis and E. faecium isolates. This was performed to predict the potential health risks associated with coming into contact with these strains in the Coomera watershed. In general, clinical isolates were found to be more resistant to all the antibiotics tested compared to water isolates and they harbored more virulence traits. Multi-drug resistance was more prevalent in clinical isolates (71.18% of E. faecalis and 70.3 % of E. faecium) compared to water isolates (only 5.66 % E. faecium). However, tetracycline, gentamicin, ciprofloxacin and ampicillin resistance was observed in water isolates. The virulence gene esp was the most prevalent virulence determinant observed in clinical isolates (67.79% of E. faecalis and 70.37 % of E. faecium), and this gene has been described as a human-specific marker used for microbial source tracking (MST). The presence of esp in water isolates (16.36% of E. faecalis and 19.14% of E. faecium) could be indicative of human faecal contamination in these waterways. Finally, in order to compare overall gene expression between environmental and clinical strains of E. faecalis, a comparative gene hybridization study was performed. The results of this investigation clearly demonstrated the up-regulation of genes associated with pathogenicity in E. faecalis isolated from water. The expression study was performed at physiological temperatures relative to ambient temperatures. The up-regulation of virulence genes demonstrates that environmental strains of E. faecalis can pose an increased health risk which can lead to serious disease, particularly if these strains belong to the virulent CC17 group. The genotyping techniques developed in this study not only provide a rapid, robust and highly discriminatory tool to characterize E. faecalis and E. faecium, but also enables the efficient identification of virulent enterococci that are distributed in environmental water sources.

A DRD2 and ANKK1 haplotype is associated with nicotine dependence

To test the importance of the dopamine D2 receptor (DRD2) region in nicotine dependence, 150 smokers and 228 controls were genotyped for the DRD2 C957T, -141delC and ANKK1 TaqIA polymorphisms (rs6277, rs1799732 and rs1800497, respectively). The -141delC SNP did not show any association but both the C957T and TaqIA SNPs showed association at the allele, genotype, haplotype and combined genotype levels. The 957C/TaqI A1 haplotype was more than 3.5 times as likely to be associated with nicotine dependence compared with the 957T/TaqI A1 haplotype (P = 0.003). Analysis of the combined genotypes of both SNPs revealed that individuals who were homozygous for the 957C-allele (CC) and had either one or two copies of the TaqI A1-allele were 3.3 times as likely to have nicotine dependence compared to all other genotype combinations (P = 0.0003) and that these genotypes accounted for approximately 13% of the susceptibility to nicotine addiction in our population. Our findings suggest that the DRD2 C957T polymorphism and the ANKK1 TaqIA polymorphism are key contributors to the genetic susceptibility to nicotine dependence.

The identification of therapeutic targets in metastatic melanoma

Metastatic melanoma, a cancer historically refractory to chemotherapeutic strategies, has a poor prognosis and accounts for the majority of skin cancer related mortality. Although the recent approval of two new drugs combating this disease, Ipilimumab and Vemurafenib (PLX4032), has demonstrated for the first time in decades an improvement in overall survival; the clinical efficacy of these drugs has been marred by severe adverse immune reactions and acquired drug resistance in patients, respectively. Thus, understanding the etiology of metastatic melanoma will contribute to the improvement of current therapeutic strategies while leading to the development of novel drug approaches. In order to identify recurrently mutated genes of therapeutic relevance in metastatic melanoma, a panel of stage III local lymph node melanomas were extensively characterised using high-throughput genomic technologies. This led to the identification of mutations in TFG in 5% of melanomas from a candidate gene sequencing approach using SNP array analysis, 24% of melanomas with mutations in MAP3K5 or MAP3K9 though unbiased whole-exome sequencing strategies, and inactivating mutations in NF1 in BRAF/NRAS wild type tumours though pathway analysis. Lastly, this thesis describes the development of a melanoma specific mutation panel that can rapidly identify clinically relevant mutation profiles that could guide effective treatment strategies through a personalised therapeutic approach. These findings are discussed in respect to a number of important issues raised by this study including the current limitation of next-generation sequencing technology, the difficulty in identifying ‘driver’ mutations critical to the development of melanoma due to high carcinogenic exposure by UV radiation, and the ultimate application of mutation screening in a personalised therapeutic setting. In summary, a number novel genes involved in metastatic melanoma have been identified that may have relevance for current therapeutic strategies in treating this disease.

NOS1AP is associated with increased severity of PTSD and depression in untreated combat veterans

Background Post traumatic stress disorder (PTSD) and depressive disorder are over represented in combat veterans. Veterans with both disorders have an increased risk of suicide. The nitric oxide synthase 1 adaptor protein (NOS1AP) gene, which modulates stress-evoked N-methyl-D-aspartate (NMDA) activity, was investigated in combat veterans. Methods A comprehensive genetic analysis of NOS1AP and its association with PTSD was investigated in Vietnam combat veterans with PTSD (n=121) and a group of healthy control individuals (n=237). PTSD patients were assessed for symptom severity and level of depression using the Mississippi Scale for Combat-Related PTSD and the Beck Depression Inventory-II (BDI). Results The G allele of NOS1AP SNP rs386231 was significantly associated with PTSD (p = 0.002). Analysis of variance revealed significant differences in BDI-II and Mississippi scores between genotypes for rs386231 with the GG genotype associated with increased severity of depression (p = 0.002 F = 6.839) and higher Mississippi Scale for Combat-Related PTSD scores (p = 0.033). Haplotype analysis revealed that the C/G haplotype (rs451275/rs386231) was significantly associated with PTSD (p = 0.001). Limitations The sample sizes in our study were not sufficient to detect SNP associations with very small effects. In addition the study was limited by its cross sectional design. Conclusions This is the first study reporting that a variant of the NOS1AP gene is associated with PTSD. Our data also suggest that a genetic variant in NOS1AP may increase the susceptibility to severe depression in patients with PTSD and increased risk for suicide.

KPNA3 variation is associated with schizophrenia, major depression, opiate dependence and alcohol dependence

KPNA3 is a gene that has been linked to schizophrenia susceptibility. In this study we investigated the possible association between KPNA3 variation and schizophrenia. To investigate a wider role of KPNA3 across psychiatric disorders we also analysed major depression, PTSD, nicotine dependent, alcohol dependent and opiate dependent cohorts. Using a haplotype block-based gene-tagging approach we genotyped six KPNA3 single nucleotide polymorphisms (SNPs) in 157 schizophrenia patients, 121 post-traumatic stress disorder patients, 120 opiate dependent patients, 231 alcohol dependent patients, 147 nicotine dependent patients and 266 major depression patients. One SNP rs2273816 was found to be significantly associated with schizophrenia, opiate dependence and alcohol dependence at the genotype and allele level. Major depression was also associated with rs2273816 but only at the allele level. Our study suggests that KPNA3 may contribute to the genetic susceptibility to schizophrenia as well as other psychiatric disorders.

Genetic polymorphisms in the human tissue kallikrein (KLK) locus and their implication in various malignant and non-malignant diseases

The Kallikrein (KLK) gene locus encodes a family of serine proteases and is the largest contiguous cluster of protease-encoding genes attributed an evolutionary age of 330 million years. The KLK locus has been implicated as a high susceptibility risk loci in numerous cancer studies through the last decade. The KLK3 gene already has established clinical relevance as a biomarker in prostate cancer prognosis through its encoded protein, prostate-specific antigen. Data mined through genome-wide association studies (GWAS) and next-generation sequencing point to many important candidate single nucleotide polymorphisms (SNPs) in KLK3 and other KLK genes. SNPs in the KLK locus have been found to be associated with several diseases including cancer, hypertension, cardiovascular disease and atopic dermatitis. Moreover, introducing a model incorporating SNPs to improve the efficiency of prostate-specific antigen in detecting malignant states of prostate cancer has been recently suggested. Establishing the functional relevance of these newly-discovered SNPs, and their interactions with each other, through in silico investigations followed by experimental validation, can accelerate the discovery of diagnostic and prognostic biomarkers. In this review, we discuss the various genetic association studies on the KLK loci identified either through candidate gene association studies or at the GWAS and post-GWAS front to aid researchers in streamlining their search for the most significant, relevant and therapeutically promising candidate KLK gene and/or SNP for future investigations.

Optimally increasing secure connectivity in multihop wireless ad hoc networks

We consider the problem of how to maximize secure connectivity of multi-hop wireless ad hoc networks after deployment. Two approaches, based on graph augmentation problems with nonlinear edge costs, are formulated. The first one is based on establishing a secret key using only the links that are already secured by secret keys. This problem is in NP-hard and does not accept polynomial time approximation scheme PTAS since minimum cutsets to be augmented do not admit constant costs. The second one is based of increasing the power level between a pair of nodes that has a secret key to enable them physically connect. This problem can be formulated as the optimal key establishment problem with interference constraints with bi-objectives: (i) maximizing the concurrent key establishment flow, (ii) minimizing the cost. We show that both problems are NP-hard and MAX-SNP (i.e., it is NP-hard to approximate them within a factor of 1 + e for e > 0 ) with a reduction to MAX3SAT problem. Thus, we design and implement a fully distributed algorithm for authenticated key establishment in wireless sensor networks where each sensor knows only its one- hop neighborhood. Our witness based approaches find witnesses in multi-hop neighborhood to authenticate the key establishment between two sensor nodes which do not share a key and which are not connected through a secure path.

Design and analysis of key management schemes for distributed wireless sensor networks

Secure communications in distributed Wireless Sensor Networks (WSN) operating under adversarial conditions necessitate efficient key management schemes. In the absence of a priori knowledge of post-deployment network configuration and due to limited resources at sensor nodes, key management schemes cannot be based on post-deployment computations. Instead, a list of keys, called a key-chain, is distributed to each sensor node before the deployment. For secure communication, either two nodes should have a key in common in their key-chains, or they should establish a key through a secure-path on which every link is secured with a key. We first provide a comparative survey of well known key management solutions for WSN. Probabilistic, deterministic and hybrid key management solutions are presented, and they are compared based on their security properties and re-source usage. We provide a taxonomy of solutions, and identify trade-offs in them to conclude that there is no one size-fits-all solution. Second, we design and analyze deterministic and hybrid techniques to distribute pair-wise keys to sensor nodes before the deployment. We present novel deterministic and hybrid approaches based on combinatorial design theory and graph theory for deciding how many and which keys to assign to each key-chain before the sensor network deployment. Performance and security of the proposed schemes are studied both analytically and computationally. Third, we address the key establishment problem in WSN which requires key agreement algorithms without authentication are executed over a secure-path. The length of the secure-path impacts the power consumption and the initialization delay for a WSN before it becomes operational. We formulate the key establishment problem as a constrained bi-objective optimization problem, break it into two sub-problems, and show that they are both NP-Hard and MAX-SNP-Hard. Having established inapproximability results, we focus on addressing the authentication problem that prevents key agreement algorithms to be used directly over a wireless link. We present a fully distributed algorithm where each pair of nodes can establish a key with authentication by using their neighbors as the witnesses.

Complexity of increasing the secure connectivity in wireless ad hoc networks

We consider the problem of maximizing the secure connectivity in wireless ad hoc networks, and analyze complexity of the post-deployment key establishment process constrained by physical layer properties such as connectivity, energy consumption and interference. Two approaches, based on graph augmentation problems with nonlinear edge costs, are formulated. The first one is based on establishing a secret key using only the links that are already secured by shared keys. This problem is in NP-hard and does not accept polynomial time approximation scheme PTAS since minimum cutsets to be augmented do not admit constant costs. The second one extends the first problem by increasing the power level between a pair of nodes that has a secret key to enable them physically connect. This problem can be formulated as the optimal key establishment problem with interference constraints with bi-objectives: (i) maximizing the concurrent key establishment flow, (ii) minimizing the cost. We prove that both problems are NP-hard and MAX-SNP with a reduction to MAX3SAT problem.

Transcriptomics approaches to identify genes and markers for production traits in giant freshwater prawn Macrobrachium rosenbergii

This study used next generation sequencing technologies to investigate growth in a cultured crustacean. The objective was to identify and characterise specific gene loci that contribute important phenotypic variation to growth as well as to develop a large set of SNP markers in candidate genes for assessing correlations between specific mutations and individual growth performance. The genomic dataset generated provides a fundamental resource for application in future crustacean stock improvement programs. Ultimately, the data can be applied to development of culture lines with improved growth performance.

Association of a GRIA3 gene polymorphism with migraine in an Australian case-control cohort

BACKGROUND: The excitatory neurotransmitter glutamate has been implicated in both the hyperexcitability required for cortical spreading depression as well as activation of the trigeminovascular system required for the allodynia associated with migraine. Polymorphisms in the glutamate receptor ionotropic amino-3-hydroxy-5-methyl-4-isoxazole-propionin acid 1 (GRIA1) and GRIA3 genes that code for 2 of 4 subunits of the glutamate receptor have been previously associated with migraine in an Italian population. In addition, the GRIA3 gene is coded within a previously identified migraine susceptibility locus at Xq24. This study investigated the previously associated polymorphisms in both genes in an Australian case-control population. METHODS: Variants in GRIA1 and GRIA3 were genotyped in 472 unrelated migraine cases and matched controls, and data were analyzed for association. RESULTS: Analysis showed no association between migraine and the GRIA1 gene. However, association was observed with the GRIA3 single nucleotide polymorphism (SNP) rs3761555 (P = .008). CONCLUSION: The results of this study confirmed the previous report of association at the rs3761555 SNP within the migraine with aura subgroup of migraineurs. However, the study identified association with the inverse allele suggesting that rs3761555 may not be the causative SNP but is more likely in linkage disequilibrium with another causal variant in both populations. This study supports the plethora of evidence suggesting that glutamate dysfunction may contribute to migraine susceptibility, warranting further investigation of the glutamatergic system and particularly of the GRIA3 gene.

High-resolution loss of heterozygosity screening implicates PTPRJ as a potential tumor suppressor gene that affects susceptibility to non-Hodgkin's Lymphoma

We employed a Hidden-Markov-Model (HMM) algorithm in loss of heterozygosity (LOH) analysis of high-density single nucleotide polymorphism (SNP) array data from Non-Hodgkin’s lymphoma (NHL) entities, follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). This revealed a high frequency of LOH over the chromosomal region 11p11.2, containing the gene encoding the protein tyrosine phosphatase receptor type J (PTPRJ). Although PTPRJ regulates components of key survival pathways in B-cells (i.e., BCR, MAPK, and PI3K signaling), its role in B-cell development is poorly understood. LOH of PTPRJ has been described in several types of cancer but not in any hematological malignancy. Interestingly, FL cases with LOH exhibited down-regulation of PTPRJ, in contrast no significant variation of expression was shown in DLBCLs. In addition, sequence screening in Exons 5 and 13 of PTPRJ identified the G973A (rs2270993), T1054C (rs2270992), A1182C (rs1566734), and G2971C (rs4752904) coding SNPs (cSNPs). The A1182 allele was significantly more frequent in FLs and in NHLs with LOH. Significant over-representation of the C1054 (rs2270992) and the C2971 (rs4752904) alleles were also observed in LOH cases. A haplotype analysis also revealed a significant lower frequency of haplotype GTCG in NHL cases, but it was only detected in cases with retention. Conversely, haplotype GCAC was over-representated in cases with LOH. Altogether, these results indicate that the inactivation of PTPRJ may be a common lymphomagenic mechanism in these NHL subtypes and that haplotypes in PTPRJ gene may play a role in susceptibility to NHL, by affecting activation of PTPRJ in these B-cell lymphomas.

A genome-wide analysis of 'Bounty' descendants implicates several novel variants in migraine susceptibility

Migraine is a common neurological disease with a complex genetic aetiology. The disease affects ~12% of the Caucasian population and females are three times more likely than males to be diagnosed. In an effort to identify loci involved in migraine susceptibility, we performed a pedigree-based genome-wide association study of the isolated population of Norfolk Island, which has a high prevalence of migraine. This unique population originates from a small number of British and Polynesian founders who are descendents of the Bounty mutiny and forms a very large multigenerational pedigree (Bellis et al.; Human Genetics, 124(5):543-5542, 2008). These population genetic features may facilitate disease gene mapping strategies (Peltonen et al.; Nat Rev Genet, 1(3):182-90, 2000. In this study, we identified a high heritability of migraine in the Norfolk Island population (h (2) = 0.53, P = 0.016). We performed a pedigree-based GWAS and utilised a statistical and pathological prioritisation approach to implicate a number of variants in migraine. An SNP located in the zinc finger protein 555 (ZNF555) gene (rs4807347) showed evidence of statistical association in our Norfolk Island pedigree (P = 9.6 × 10(-6)) as well as replication in a large independent and unrelated cohort with >500 migraineurs. In addition, we utilised a biological prioritisation to implicate four SNPs, in within the ADARB2 gene, two SNPs within the GRM7 gene and a single SNP in close proximity to a HTR7 gene. Association of SNPs within these neurotransmitter-related genes suggests a disrupted serotoninergic system that is perhaps specific to the Norfolk Island pedigree, but that might provide clues to understanding migraine more generally.

An X chromosome association scan of the Norfolk Island genetic isolate provides evidence for a novel migraine susceptibility locus at Xq12

Migraine is a common and debilitating neurovascular disorder with a complex envirogenomic aetiology. Numerous studies have demonstrated a preponderance of women affected with migraine and previous pedigree linkage studies in our laboratory have identified susceptibility loci on chromosome Xq24-Xq28. In this study we have used the genetic isolate of Norfolk Island to further analyse the X chromosome for migraine susceptibility loci. An association approach was employed to analyse 14,124 SNPs spanning the entire X chromosome. Genotype data from 288 individuals comprising a large core-pedigree, of which 76 were affected with migraine, were analysed. Although no SNP reached chromosome-wide significance (empirical α = 1×10−5) ranking by P-value revealed two primary clusters of SNPs in the top 25. A 10 SNP cluster represents a novel migraine susceptibility locus at Xq12 whilst a 11 SNP cluster represents a previously identified migraine susceptibility locus at Xq27. The strongest association at Xq12 was seen for rs599958 (OR = 1.75, P = 8.92×10−4), whilst at Xq27 the strongest association was for rs6525667 (OR = 1.53, P = 1.65×10−4). Further analysis of SNPs at these loci was performed in 5,122 migraineurs from the Women’s Genome Health Study and provided additional evidence for association at the novel Xq12 locus (P<0.05). Overall, this study provides evidence for a novel migraine susceptibility locus on Xq12. The strongest effect SNP (rs102834, joint P = 1.63×10−5) is located within the 5′UTR of the HEPH gene, which is involved in iron homeostasis in the brain and may represent a novel pathway for involvement in migraine pathogenesis.