Analysis of HapMap tag-SNPs in dysbindin (DTNBP1) reveals evidence of consistent association with schizophrenia

Dystrobrevin binding protein 1 (DTNBP1), or dysbindin, is thought to be critical in regulating the glutamatergic system. While the dopamine pathway is known to be important in the aetiology of schizophrenia, it seems likely that glutamatergic dysfunction can lead to the development of schizophrenia. DTNBP1 is widely expressed in brain, levels are reduced in brains of schizophrenia patients and a DTNBP1 polymorphism has been associated with reduced brain expression. Despite numerous genetic studies no DTNBP1 polymorphism has been strongly implicated in schizophrenia aetiology. Using a haplotype block-based gene-tagging approach we genotyped 13 SNPs in DTNBP1 to investigate possible associations with DTNBP1 and schizophrenia. Four polymorphisms were found to be significantly associated with schizophrenia. The strongest association was found with an A/C SNP in intron 7 (rs9370822). Homozygotes for the C allele of rs9370822 were more than two and a half times as likely to have schizophrenia compared to controls. The other polymorphisms showed much weaker association and are less likely to be biologically significant. These results suggest that DTNBP1 is a good candidate for schizophrenia risk and rs9370822 is either functionally important or in disequilibrium with a functional SNP, although our observations should be viewed with caution until they are independently replicated.

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.

Association of inducible nitric oxide synthase with asthma severity, total serum immunoglobulin E and blood eosinophil levels

Background Nitric oxide is released by immune, epithelial and endothelial cells, and plays an important part in the pathophysiology of asthma. Objective To investigate the association of inducible nitric oxide synthases (iNOS) gene repeat polymorphisms with asthma. Methods 230 families with asthma (842 individuals) were recruited to identify and establish the genetic association of iNOS repeats with asthma and associated phenotypes. Serum nitric oxide levels in selected individuals were measured and correlated with specific genotypes. Multiple logistic regression analysis was performed to determine the effect of age and sex. Results A total of four repeats—a (CCTTT)n promoter repeat, a novel intron 2 (GT)n repeat (BV680047), an intron 4 (GT)n repeat (AFM311ZB1) and an intron 5 (CA)n repeat (D17S1878)—were identified and genotyped. A significant transmission distortion to the probands with asthma was seen for allele 3 of the AFM311ZB1 gene (p = 0.006). This allele was also found to be significantly associated with percentage blood eosinophils (p<0.001) and asthma severity (p = 0.04). Moreover, it was functionally correlated with high serum nitric oxide levels (p = 0.006). Similarly, the promoter repeat was found to be associated with serum total immunoglobulin (Ig)E (p = 0.028). Individuals carrying allele 4 of this repeat have high serum IgE (p<0.001) and nitric oxide levels (p = 0.03). Conclusion This is the first study to identify the repeat polymorphisms in the iNOS gene that are associated with severity of asthma and eosinophils. The functional relevance of the associated alleles with serum nitric oxide levels was also shown. Therefore, these results could be valuable in elucidating the role of nitric oxide in asthma pathogenesis.

Association between polymorphisms in the progesterone receptor gene and endometriosis

The progesterone receptor (PR) is a candidate gene for the development of endometriosis, a complex disease with strong hormonal features, common in women of reproductive age. We typed the 306 base pair Alu insertion (AluIns) polymorphism in intron G of PR in 101 individuals, estimated linkage disequilibrium (LD) between five single-nucleotide polymorphisms (SNPs) across the PR locus in 980 Australian triads (endometriosis case and two parents) and used transmission disequilibrium testing (TDT) for association with endometriosis. The five SNPs showed strong pairwise LD, and the AluIns was highly correlated with proximal SNPs rs1042839 (Δ2 = 0.877, D9 = 1.00, P < 0.0001) and rs500760 (Δ2 = 0.438, D9 = 0.942, P < 0.0001). TDT showed weak evidence of allelic association between endometriosis and rs500760 (P = 0.027) but not in the expected direction. We identified a common susceptibility haplotype GGGCA across the five SNPs (P = 0.0167) in the whole sample, but likelihood ratio testing of haplotype transmission and non-transmission of the AluIns and flanking SNPs showed no significant pattern. Further, analysis of our results pooled with those from two previous studies suggested that neither the T2 allele of the AluIns nor the T1/T2 genotype was associated with endometriosis.

Mutation analysis of the CDKN2A promoter in Australian melanoma families

Approximately 50% of all melanoma families worldwide show linkage to 9p21-22, but only about half of these have been shown to contain germ line CDKN2A mutations. It has been hypothesized that a proportion of these families carry mutations in the noncoding regions of CDKN2A. Several Canadian families have been reported to carry a mutation in the 5' UTR, at position -34 relative to the start site, which gives rise to a novel AUG translation initiation codon that markedly decreases translation from the wild-type AUG (Liu et al., 1999). Haplotype sharing in these Canadian families suggested that this mutation is of British origin. We sequenced 1,327 base pairs (bp) of CDKN2A, making up 1,116 bp of the 5' UTR and promoter, all of exon 1, and 61 bp of intron 1, in at least one melanoma case from 110 Australian families with three or more affected members known not to carry mutations within the p16 coding region. In addition, 431 bp upstream of the start codon was sequenced in an additional 253 affected probands from two-case melanoma families for which the CDKN2A mutation status was unknown. Several known polymorphisms at positions -33, -191, -493, and -735 were detected, in addition to four novel variants at positions 120, -252, -347, and -981 relative to the start codon. One of the probands from a two-case family was found to have the previously reported Q50R mutation. No family member was found to carry the mutation at position -34 or any other disease-associated mutation. For further investigation of noncoding CDKN2A mutations that may affect transcription, allele-specific expression analysis was carried out in 31 of the families with at least three affected members who showed either complete or "indeterminate" 9p haplotype sharing without CDKN2A exonic mutations. Reverse transcription polymerase chain reaction and automated sequencing showed expression of both CDKN2A alleles in all family members tested. The lack of CDKN2A promoter mutations and the absence of transcriptional silencing in the germ line of this cohort of families suggest that mutations in the promoter and 5' UTR play a very limited role in melanoma predisposition.

Quality of life and genetics in men with prostate cancer

As family history has been established as a risk factor for prostate cancer, attempts have been made to isolate predisposing genetic variants that are related to hereditary prostate cancer. With many genetic variants still to be identified and investigated, it is not yet possible to fully understand the impact of genetic variants on prostate cancer development. The high survival rates among men with prostate cancer have meant that other issues, such as quality of life (QoL), have also become important. Through their effect on a person’s health, a range of inherited genetic variants may potentially influence QoL in men with prostate cancer, even prior to treatment. Until now, limited research has been conducted on the relationship between genetics and QoL. Thus, this study contributes to an emerging field by aiming to identify certain genetic variants related to the QoL found in men with prostate cancer. It is hoped that this study may lead to future research that will identify men who have an increased risk of a poor QoL following prostate cancer treatment, which will aid in developing treatments that are individually tailored to support them. Previous studies have established that genetic variants of Vascular Endothelial Growth Factor (VEGF) and Insulin-like Growth Factor 1 (IGF-1) may play a role in prostate cancer development. VEGF and IGF-1 have also been reported to be associated with QoL in people with ovarian cancer and colorectal cancer, respectively. This study completed a series of secondary analyses using two major data-sets (from 850 men newly diagnosed with prostate cancer, and approximately 550 men from the general Queensland population), in which genetic variants of VEGF and IGF-1 were investigated for associations with prostate cancer susceptibility and QoL. The first aim of this research was to investigate genetic variants in the VEGF and IGF-I gene for an association with the risk of prostate cancer. It was found that one IGF-1 genetic variant (rs35765) had a statistically significant association with prostate cancer (p = 0.04), and one VEGF genetic variant (rs2146323) had a statistically significant association with advanced prostate cancer (p = 0.02). The estimates suggest that carriers of the CA and AA genotype for rs35765 may have a reduced risk of developing prostate cancer (Odds Ratio (OR) = 0.72, 95% Confidence Interval (CI) = 0.55, 0.95, OR = 0.60, 95% CI = 0.26, 1.39, respectively). Meanwhile, carriers of the CA and AA genotype for rs2146323 may be at increased risk of advanced prostate cancer, which was determined by a Gleason score of above 7 (OR = 1.72, 95% CI = 1.12, 2.63, OR = 1.90, 95% CI = 1.08, 3.34, respectively). Utilising the widely used short-form health survey, the SF-36v2, the second aim of this study was to investigate the relationship between prostate cancer and QoL prior to treatment. Assessing QoL at this time-point was important as little research has been conducted to evaluate if prostate cancer affects QoL regardless of treatment. The analyses found that mean SF-36v2 scale scores related to physical health were higher by at least 0.3 Standard Deviations (SD) among men with prostate cancer than the general population comparison group. This difference was considered clinically significant (defined by group differences in mean SF-36v2 scores by at least 0.3 SD). These differences were also statistically significant (p<0.05). Mean QoL scale scores related to mental health were similar between men with prostate cancer and those from the general population comparison group. The third aim of this study was to investigate genetic variants in the VEGF and IGF-1 gene for an association with QoL in prostate cancer patients prior to their treatment. It was essential to evaluate these relationships prior to treatment, before the involvement of these genes was potentially interrupted by treatment. The analyses found that some genetic variants had a small clinically significant association (0.3 SD) to some QoL domains experienced by these men. However, most relationships were not statistically significant (p>0.05). Most of the associations found identified that a small sub-group of men with prostate cancer (approximately 2%) reported, on average, a slightly better QoL than the majority of the prostate cancer patients. The fourth aim of this research was to investigate whether associations between genetic variants in VEGF and IGF-1 and QoL were specific to men with prostate cancer, or were also applicable to the general male population. It was found that twenty out of one-hundred relationships between the genetic variants of VEGF and IGF-1 and QoL health-measures and scales examined differed between these groups. In the majority of the relationships involving VEGF SNPs that differed, a clinically significant difference (0.3 or more SD) between mean scores among the genotype groups in prostate cancer patients was found, while mean scores among men from the general-population comparison group were similar. For example, prostate cancer participants who carried at least one T allele (CT or TT genotype) for rs3024994 had a clinically significant higher (0.3 SD) mean QoL score in terms of the role-physical scale, than participants who carried the CC genotype. This was not seen among men from the general population sample, as the mean score was similar between genotype groups. The opposite was seen in regards to the IGF-1 SNPs examined. Overall, these relationships were not considered to directly impact on the clinical options for men with prostate cancer. As this study utilised secondary data from two separate studies, there are a number of important limitations that should be acknowledged including issues of multiple comparisons, power, and missing or unavailable data. It is recommended that this study be replicated as a better-designed study that takes greater consideration of the many factors involved in prostate cancer and QoL. Investigation into other genetic variants of VEGF or IGF-1 is also warranted, as is consideration of other genes and their relationship with QoL. Through identifying certain genetic variants that have a modest association to prostate cancer, this project adds to the knowledge surrounding VEGF and IGF-1 and their role in prostate cancer susceptibility. Importantly, this project has also introduced the potential role genetics plays in QoL, through investigating the relationships between genetic variants of VEGF and IGF-1 and QoL.

Development and validation of novel methods for microbial source tracking based on Escherichia coli as an indicator of water quality

Microbial pollution in water periodically affects human health in Australia, particularly in times of drought and flood. There is an increasing need for the control of waterborn microbial pathogens. Methods, allowing the determination of the origin of faecal contamination in water, are generally referred to as Microbial Source Tracking (MST). Various approaches have been evaluated as indicatorsof microbial pathogens in water samples, including detection of different microorganisms and various host-specific markers. However, until today there have been no universal MST methods that could reliably determine the source (human or animal) of faecal contamination. Therefore, the use of multiple approaches is frequently advised. MST is currently recognised as a research tool, rather than something to be included in routine practices. The main focus of this research was to develop novel and universally applicable methods to meet the demands for MST methods in routine testing of water samples. Escherichia coli was chosen initially as the object organism for our studies as, historically and globally, it is the standard indicator of microbial contamination in water. In this thesis, three approaches are described: single nucleotide polymorphism (SNP) genotyping, clustered regularly interspaced short palindromic repeats (CRISPR) screening using high resolution melt analysis (HRMA) methods and phage detection development based on CRISPR types. The advantage of the combination SNP genotyping and CRISPR genes has been discussed in this study. For the first time, a highly discriminatory single nucleotide polymorphism interrogation of E. coli population was applied to identify the host-specific cluster. Six human and one animal-specific SNP profile were revealed. SNP genotyping was successfully applied in the field investigations of the Coomera watershed, South-East Queensland, Australia. Four human profiles [11], [29], [32] and [45] and animal specific SNP profile [7] were detected in water. Two human-specific profiles [29] and [11] were found to be prevalent in the samples over a time period of years. The rainfall (24 and 72 hours), tide height and time, general land use (rural, suburban), seasons, distance from the river mouth and salinity show a lack of relashionship with the diversity of SNP profiles present in the Coomera watershed (p values > 0.05). Nevertheless, SNP genotyping method is able to identify and distinquish between human- and non-human specific E. coli isolates in water sources within one day. In some samples, only mixed profiles were detected. To further investigate host-specificity in these mixed profiles CRISPR screening protocol was developed, to be used on the set of E. coli, previously analysed for SNP profiles. CRISPR loci, which are the pattern of previous DNA coliphages attacks, were considered to be a promising tool for detecting host-specific markers in E. coli. Spacers in CRISPR loci could also reveal the dynamics of virulence in E. coli as well in other pathogens in water. Despite the fact that host-specificity was not observed in the set of E. coli analysed, CRISPR alleles were shown to be useful in detection of the geographical site of sources. HRMA allows determination of ‘different’ and ‘same’ CRISPR alleles and can be introduced in water monitoring as a cost-effective and rapid method. Overall, we show that the identified human specific SNP profiles [11], [29], [32] and [45] can be useful as marker genotypes globally for identification of human faecal contamination in water. Developed in the current study, the SNP typing approach can be used in water monitoring laboratories as an inexpensive, high-throughput and easy adapted protocol. The unique approach based on E. coli spacers for the search for unknown phage was developed to examine the host-specifity in phage sequences. Preliminary experiments on the recombinant plasmids showed the possibility of using this method for recovering phage sequences. Future studies will determine the host-specificity of DNA phage genotyping as soon as first reliable sequences can be acquired. No doubt, only implication of multiple approaches in MST will allow identification of the character of microbial contamination with higher confidence and readability.

Comparative study of coagulase negative Staphylococci (CoNS) from clinical isolates, skin and nasal sources

Staphylococci are important pathogenic bacteria responsible for a range of diseases in humans. The most frequently isolated microorganisms in a hospital microbiology laboratory are staphylococci. The general classification of staphylococci divides them into two major groups; Coagulase-positive staphylococci (e.g. Staphylococcus aureus) and Coagulase-negative staphylococci (e.g. Staphylococcus epidermidis). Coagulase-negative staphylococcal (CoNS) isolates include a variety of species and many different strains but are often dominated by the most important organism of this group, S. epidermidis. Currently, these organisms are regarded as important pathogenic organisms causing infections related to prosthetic materials and surgical wounds. A significant number of S. epidermidis isolates are also resistant to different antimicrobial agents. Virulence factors in CoNS are not very clearly established and not well documented. S. epidermidis is evolving as a resistant and powerful microbe related to nosocomial infections because it has different properties which independently, and in combination, make it a successful infectious agent, especially in the hospital environment. Such characteristics include biofilm formation, drug resistance and the evolution of genetic variables. The purpose of this project was to develop a novel SNP genotyping method to genotype S. epidermidis strains originating from hospital patients and healthy individuals. High-Resolution Melt Analysis was used to assign binary typing profiles to both clinical and commensal strains using a new bioinformatics approach. The presence of antibiotic resistance genes and biofilm coding genes were also interrogated in these isolates.

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