Single nucleotide polymorphism (SNP) - genotyping of Community Acquired Methicillin-Resistant Staphylococcus aureus, including the subtyping of PVL toxin producers using Real-Time PCR

Staphylococcus aureus is a common pathogen that causes a variety of infections including soft tissue infections, impetigo, septicemia toxic shock and scalded skin syndrome. Traditionally, Methicillin-Resistant Staphylococcus aureus (MRSA) was considered a Hospital-Acquired (HA) infection. It is now recognised that the frequency of infections with MRSA is increasing in the community, and that these infections are not originating from hospital environments. A 2007 report by the Centers for Disease Control and Prevention (CDC) stated that Staphylococcus aureus is the most important cause of serious and fatal infections in the USA. Community-Acquired MRSA (CA-MRSA) are genetically diverse and distinct, meaning they are able to be identified and tracked by way of genotyping. Genotyping of MRSA using Single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring MRSA, specifically ST93 (Queensland Clone) dissemination in the community. It has been shown that a large proportion of CA-MRSA infections in Queensland and New South Wales are caused by ST93. The rationale for this project was that SNP analysis of MLST genes is a rapid and cost-effective method for genotyping and monitoring MRSA dissemination in the community. In this study, 16 different sequence types (ST) were identified with 41% of isolates identified as ST93 making it the predominate clone. Males and Females were infected equally with an average patient age of 45yrs. Phenotypically, all of the ST93 had an identical antimicrobial resistance pattern. They were resistant to the β-lactams – Penicillin, Flu(di)cloxacillin and Cephalothin but sensitive to all other antibiotics tested. Virulence factors play an important role in allowing S. aureus to cause disease by way of colonising, replication and damage to the host. One virulence factor of particular interest is the toxin Panton-Valentine leukocidin (PVL), which is composed of two separate proteins encoded by two adjacent genes. PVL positive CA-MRSA are shown to cause recurrent, chronic or severe skin and soft tissue infections. As a result, it is important that PVL positive CA-MRSA is genotyped and tracked. Especially now that CA-MRSA infections are more prevalent than HA-MRSA infections and are now deemed endemic in Australia. 98% of all isolates in this study tested positive for the PVL toxin gene. This study showed that PVL is present in many different community based ST, not just ST93, which were all PVL positive. With this toxin becoming entrenched in CA-MRSA, genotyping would provide more accurate data and a way of tracking the dissemination. PVL gene can be sub-typed using an allele-specific Real-Time PCR (RT-PCR) followed by High resolution meltanalysis. This allows the identification of PVL subtypes within the CA-MRSA population and allow the tracking of these clones in the community.

Cigarette smoking in young adults : the influence of the HTR2A T102C polymorphism and punishment sensitivity

Background: The C allele of a common polymorphism of the serotonin 2A receptor (HTR2A) gene, T102C, results in reduced synthesis of 5-HT2A receptors and has been associated with current smoking status in adults. The -1438A/G polymorphism, located in the regulatory region of this gene, is in linkage disequilibrium with T102C, and the A allele is associated with increased promoter activity and with smoking in adult males. We investigated the contributions of the HTR2A gene, chronic psychological stress, and impulsivity to the prediction of cigarette smoking status and dependence in young adults. Methods: T102C and -1438A/G genotyping was conducted on 132 healthy Caucasian young adults (47 smokers) who completed self-report measures of chronic stress, depressive symptoms, impulsive personality and cigarette use. Results: A logistic regression analysis of current cigarette smoker user status, after adjusting for gender, depressive symptom severity and chronic stress, indicated that the T102C TT genotype relative to the CC genotype (OR = 7.53), and lower punishment sensitivity (OR = 0.91) were each significant predictive risk factors. However, for number of cigarettes smoked, only lower punishment sensitivity was a significant predictor (OR = 0.81). Conclusions: These data indicate the importance of the T102C polymorphism to tobacco use but not number of cigarettes smoked for Caucasian young adults. Future studies should examine whether this is explained by effects of nicotine on the serotonin system. Lower punishment sensitivity increased risk of both smoking and of greater consumption, perhaps via a reduced sensitivity to cigarette health warnings and negative physiological effects.

Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

Background Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia. Conclusion We describe an Australian origin for B. pseudomallei, characterized by a single introduction event into Southeast Asia during a recent glacial period, and variable levels of lateral gene transfer within populations. These patterns provide insights into mechanisms of genetic diversification in B. pseudomallei and its closest relatives, and provide a framework for integrating the traditionally separate fields of population genetics and phylogenetics for other bacterial species with high levels of lateral gene transfer.

Common variation in the fibroblast growth factor receptor 2 gene is not associated with endometriosis risk

BACKGROUND Endometriosis is a polygenic disease with a complex and multifactorial aetiology that affects 8-10% of women of reproductive age. Epidemiological data support a link between endometriosis and cancers of the reproductive tract. Fibroblast growth factor receptor 2 (FGFR2) has recently been implicated in both endometrial and breast cancer. Our previous studies on endometriosis identified significant linkage to a novel susceptibility locus on chromosome 10q26 and the FGFR2 gene maps within this linkage region. We therefore hypothesized that variation in FGFR2 may contribute to the risk of endometriosis. METHODS We genotyped 13 single nucleotide polymorphisms (SNPs) densely covering a 27 kb region within intron 2 of FGFR2 including two SNPs (rs2981582 and rs1219648) significantly associated with breast cancer and a total 40 tagSNPs across 150 kb of the FGFR2 gene. SNPs were genotyped in 958 endometriosis cases and 959 unrelated controls. RESULTS We found no evidence for association between endometriosis and FGFR2 intron 2 SNPs or SNP haplotypes and no evidence for association between endometriosis and variation across the FGFR2 gene. CONCLUSIONS Common variation in the breast-cancer implicated intron 2 and other highly plausible causative candidate regions of FGFR2 do not appear to be a major contributor to endometriosis susceptibility in our large Australian sample.

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.

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.

Mutations in exon 3 of the beta-catenin gene are rare in melanoma cell lines

Mutations in exon 3 of the CTNNB1 gene encoding beta-catenin have been reported in colorectal cancer cell lines and tumours. Although one study reported mutations or deletions affecting beta-catenin in 20% of melanoma cell lines, subsequent reports detected a much lower frequency of aberrations in uncultured melanomas. To determine whether this difference in mutation frequency reflected an in vitro culturing artefact, exon 3 of CTNNB1 was screened in a panel of 62 melanoma cell lines. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect intragenic deletions affecting exon 3. One out of 62 (1.6%) cell lines was found to carry a mutation, indicating that aberration of the Wnt-1/wingless pathway through activation of beta-catenin is a rare event, even in melanoma cell lines.

Pharmacological consequence of the A118G μ opioid receptor polymorphism on morphine-and fentanyl-mediated modulation of Ca2+ channels in humanized mouse sensory neurons

Background: The most common functional single nucleotide polymorphism of the human OPRM1 gene, A118G, has been shown to be associated with interindividual differences in opioid analgesic requirements, particularly with morphine, in patients with acute postoperative pain. The purpose of this study was to examine whether this polymorphism would modulate the morphine and fentanyl pharmacological profile of sensory neurons isolated from a humanized mouse model homozygous for either the 118A or 118G allele. Methods: The coupling of wild-type and mutant μ opioid receptors to voltage-gated Ca channels after exposure to either ligand was examined by employing the whole cell variant of the patch-clamp technique in acutely dissociated trigeminal ganglion neurons. Morphine-mediated antinociception was measured in mice carrying either the 118AA or 118GG allele. RESULTS:: The biophysical parameters (cell size, current density, and peak current amplitude potential) measured from both groups of sensory neurons were not significantly different. In 118GG neurons, morphine was approximately fivefold less potent and 26% less efficacious than that observed in 118AA neurons. On the other hand, the potency and efficacy of fentanyl were similar for both groups of neurons. Morphine-mediated analgesia in 118GG mice was significantly reduced compared with the 118AA mice. Conclusions: This study provides evidence to suggest that the diminished clinical effect observed with morphine in 118G carriers results from an alteration of the receptor's pharmacology in sensory neurons. In addition, the impaired analgesic response with morphine may explain why carriers of this receptor variant have an increased susceptibility to become addicted to opioids. © 2011 the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins. Anesthesiology.

A novel duplication polymorphism in the FANCA promoter and its association with breast and ovarian cancer

The FANCA gene is one of the genes in which mutations lead to Fanconi anaemia, a rare autosomal recessive disorder characterised by congenital abnormalities, bone marrow failure, and predisposition to malignancy. FANCA is also a potential breast and ovarian cancer susceptibility gene. A novel allele was identified which has a tandem duplication of a 13 base pair sequence in the promoter region. Methods: We screened germline DNA from 352 breast cancer patients, 390 ovarian cancer patients and 256 normal controls to determine if the presence of either of these two alleles was associated with an increased risk of breast or ovarian cancer. Results: The duplication allele had a frequency of 0.34 in the normal controls. There was a nonsignificant decrease in the frequency of the duplication allele in breast cancer patients. The frequency of the duplication allele was significantly decreased in ovarian cancer patients. However, when malignant and benign tumours were considered separately, the decrease was only significant in benign tumours. Conclusion: The allele with the tandem duplication does not appear to modify breast cancer risk but may act as a low penetrance protective allele for ovarian cancer.

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.

A novel DRD2 single-nucleotide polymorphism associated with schizophrenia predicts age of onset : HapMap tag-single-nucleotide polymorphism analysis

Background: Dopamine D2 receptor (DRD2) is thought to be critical in regulating the dopaminergic pathway in the brain which is known to be important in the aetiology of schizophrenia. It is therefore not surprising that most antipsychotic medication acts on the Dopamine D2 receptor. DRD2 is widely expressed in brain, levels are reduced in brains of schizophrenia patients and DRD2 polymorphisms have been associated with reduced brain expression. We have previously identified a genetic variant in DRD2, rs6277 to be strongly implicated in schizophrenia susceptibility. Methods: To identity new associations in the DRD2 gene with disease status and clinical severity, we genotyped seven single nucleotide polymorphisms (SNPs) in DRD2 using a multiplex mass spectrometry method. SNPs were chosen using a haplotype block-based gene-tagging approach so the entire DRD2 gene was represented. Results: One polymorphism rs2734839 was found to be significantly associated with schizophrenia as well as late onset age. Individuals carrying the genetic variation were more than twice as likely to have schizophrenia compared to controls. Conclusions: Our results suggest that DRD2 genetic variation is a good indicator for schizophrenia risk and may also be used as a predictor age of onset.

Candidate gene analysis for quantitative traits using the transmission disequilibrium test : the example of the melanocortin 4-receptor in pigs

Population-wide associations between loci due to linkage disequilibrium can be used to map quantitative trait loci (QTL) with high resolution. However, spurious associations between markers and QTL can also arise as a consequence of population stratification. Statistical methods that cannot differentiate between loci associations due to linkage disequilibria from those caused in other ways can render false-positive results. The transmission-disequilibrium test (TDT) is a robust test for detecting QTL. The TDT exploits within-family associations that are not affected by population stratification. However, some TDTs are formulated in a rigid-form, with reduced potential applications. In this study we generalize TDT using mixed linear models to allow greater statistical flexibility. Allelic effects are estimated with two independent parameters: one exploiting the robust within-family information and the other the potentially biased between-family information. A significant difference between these two parameters can be used as evidence for spurious association. This methodology was then used to test the effects of the fourth melanocortin receptor (MC4R) on production traits in the pig. The new analyses supported the previously reported results; i.e., the studied polymorphism is either causal of in very strong linkage disequilibrium with the causal mutation, and provided no evidence for spurious association.

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 novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays

Background Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation. Methods The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing. Results The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS −786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele). Conclusions Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants.