Mapping eQTLs in the Norfolk Island genetic isolate identifies candidate genes for CVD risk traits

Cardiovascular disease (CVD) affects millions of people worldwide and is influenced by numerous factors, including lifestyle and genetics. Expression quantitative trait loci (eQTLs) influence gene expression and are good candidates for CVD risk. Founder-effect pedigrees can provide additional power to map genes associated with disease risk. Therefore, we identified eQTLs in the genetic isolate of Norfolk Island (NI) and tested for associations between these and CVD risk factors. We measured genome-wide transcript levels of blood lymphocytes in 330 individuals and used pedigree-based heritability analysis to identify heritable transcripts. eQTLs were identified by genome-wide association testing of these transcripts. Testing for association between CVD risk factors (i.e., blood lipids, blood pressure, and body fat indices) and eQTLs revealed 1,712 heritable transcripts (p < 0.05) with heritability values ranging from 0.18 to 0.84. From these, we identified 200 cis-acting and 70 trans-acting eQTLs (p < 1.84 × 10(-7)) An eQTL-centric analysis of CVD risk traits revealed multiple associations, including 12 previously associated with CVD-related traits. Trait versus eQTL regression modeling identified four CVD risk candidates (NAAA, PAPSS1, NME1, and PRDX1), all of which have known biological roles in disease. In addition, we implicated several genes previously associated with CVD risk traits, including MTHFR and FN3KRP. We have successfully identified a panel of eQTLs in the NI pedigree and used this to implicate several genes in CVD risk. Future studies are required for further assessing the functional importance of these eQTLs and whether the findings here also relate to outbred populations.

Genome-wide analysis in a murine Dnmt1 knockdown model identifies epigenetically silenced genes in primary human pituitary tumors

DNA methylation at promoter CpG islands (CGI) is an epigenetic modification associated with inappropriate gene silencing in multiple tumor types. In the absence of a human pituitary tumor cell line, small interfering RNA-mediated knockdown of the maintenance methyltransferase DNA methyltransferase (cytosine 5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20. Sustained knockdown induced reexpression of the fully methylated and normally imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite restriction analysis (COBRA) revealed that reexpression of Nnat was associated with partial CGI demethylation, which was also observed at the H19 differentially methylated region. Subsequent genome-wide microarray analysis identified 91 genes that were significantly differentially expressed in Dnmt1 knockdown cells (10% false discovery rate). The analysis showed that genes associated with the induction of apoptosis, signal transduction, and developmental processes were significantly overrepresented in this list (P < 0.05). Following validation by reverse transcription-PCR and detection of inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and S100A10) were analyzed in primary human pituitary tumors, each displaying significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For two of these genes, NNAT and S100A10, decreased expression was associated with increased promoter CGI methylation. Induced expression of Nnat in stable transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is the first report of array-based "epigenetic unmasking" in combination with Dnmt1 knockdown and reveals the potential of this strategy toward identifying genes silenced by epigenetic mechanisms across species boundaries.

An investigation of functional diversity of endogenous cellulase and expression of other digestive enzyme genes in freshwater crayfish (Cherax) species

The project evaluated potential of soluble cellulose as a cheap feed ingredient for major farmed Australian freshwater crayfish species testing their growth performance, digestive enzyme activity and digestive enzyme gene expression patterns. Test animals showed an innate capacity to utilise a range of carbohydrate sources including complex structural polysaccharides. Results suggest that more plant-derived ingredient can be incorporated in formulated low-cost feeds for the culture industry.

Managing identity transitions during radical change in nuclear engineering organizations

This study reports the construction and reconstruction of identities of new and existing employees during a significant transition phase of a nuclear engineering organization. We followed a group of new and existing employees over the period of three years, during which the organization constructed a greenfield nuclear facility with new generational technologies whilst in parallel, decommissioned the older reactor. This change led to the transfer and integration of existing trade-based employees with the newly recruited, primarily university educated graduates in the new site. Three waves of interview data were collected, in conjunction with the cognitive mapping of social grouping and photo elicitation portrayed the stories of different group of employees who either succeeded or failed at embracing their new professional identity. In contrast with the new recruits who constructed new identities as they join this organization, we identify and report on the number of enabling and disabling factors that influence the process of professional identity construction and reconstruction during gamma change.

Transcription factories : gene expression in unions?

Transcription is a fundamental step in gene expression, yet it remains poorly understood at a cellular level. Visualization of transcription sites and active genes has led to the suggestion that transcription occurs at discrete sites in the nucleus, termed transcription factories, where multiple active RNA polymerases are concentrated and anchored to a nuclear substructure. However, this concept is not universally accepted. This Review discusses the experimental evidence in support of the transcription factory model and the evidence that argues against such a spatially structured view of transcription. The transcription factory model has implications for the regulation of transcription initiation and elongation, for the organization of genes in the genome, for the co-regulation of genes and for genome instability.

Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing

Increasing evidence suggests that chromatin modifications have important roles in modulating constitutive or alternative splicing. Here we demonstrate that the PWWP domain of the chromatin-associated protein Psip1/Ledgf can specifically recognize tri-methylated H3K36 and that, like this histone modification, the Psip1 short (p52) isoform is enriched at active genes. We show that the p52, but not the long (p75), isoform of Psip1 co-localizes and interacts with Srsf1 and other proteins involved in mRNA processing. The level of H3K36me3 associated Srsf1 is reduced in Psip1 mutant cells and alternative splicing of specific genes is affected. Moreover, we show altered Srsf1 distribution around the alternatively spliced exons of these genes in Psip1 null cells. We propose that Psip1/p52, through its binding to both chromatin and splicing factors, might act to modulate splicing.

Identification of genes important for growth of asymptomatic bacteriuria Escherichia coli in urine

Escherichia coli is the most important etiological agent of urinary tract infections (UTIs). Unlike uropathogenic E. coli, which causes symptomatic infections, asymptomatic bacteriuria (ABU) E. coli strains typically lack essential virulence factors and colonize the bladder in the absence of symptoms. While ABU E. coli can persist in the bladder for long periods of time, little is known about the genetic determinants required for its growth and fitness in urine. To identify such genes, we have employed a transposon mutagenesis approach using the prototypic ABU E. coli strain 83972 and the clinical ABU E. coli strain VR89. Six genes involved in the biosynthesis of various amino acids and nucleobases were identified (carB, argE, argC, purA, metE, and ilvC), and site-specific mutants were subsequently constructed in E. coli 83972 and E. coli VR89 for each of these genes. In all cases, these mutants exhibited reduced growth rates and final cell densities in human urine. The growth defects could be complemented in trans as well as by supplementation with the appropriate amino acid or nucleobase. When assessed in vivo in a mouse model, E. coli 83972carAB and 83972argC showed a significantly reduced competitive advantage in the bladder and/or kidney during coinoculation experiments with the parent strain, whereas 83972metE and 83972ilvC did not. Taken together, our data have identified several biosynthesis pathways as new important fitness factors associated with the growth of ABU E. coli in human urine.

Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species

Background Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. Results Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. Conclusions The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.

Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

Markers of genetic susceptibility in human environmental hygiene and toxicology : the role of selected CYP, NAT and GST genes

Inherited genetic traits co-determine the susceptibility of an individual to a toxic chemical. Special emphasis has been put on individual responses to environmental and industrial carcinogens, but other chronic diseases are of increasing interest. Polymorphisms of relevant xenobiotic metabolising enzymes may be used as toxicological susceptibility markers. A growing number of genes encoding enzymes involved in biotransformation of toxicants and in cellular defence against toxicant-induced damage to the cells has been identified and cloned, leading to increased knowledge of allelic variants of genes and genetic defects that may result in a differential susceptibility toward environmental toxicants. "Low penetrating" polymorphisms in metabolism genes tend to be much more common in the population than allelic variants of "high penetrating" cancer genes, and are therefore of considerable importance from a public health point of view. Positive associations between cancer and CYP1A1 alleles, in particular the *2C I462V allele, were found for tissues following the aerodigestive tract. Again, in most cases, the effect of the variant CYP1A1 allele becomes apparent or clearer in connection with the GSTM1 null allele. The CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squameous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has also pointed to interactive effects. Of particular interest for the industrial and environmental field is the isozyme CYP2E1. Several genotypes of this isozyme have been characterised which seem to be associated with different levels of expression of enzyme activity. The acetylator status for NAT2 can be determined by genotyping or by phenotyping. In the pathogenesis of human bladder cancer due to occupational exposure to "classical" aromatic amines (benzidine, 4-aminodiphenyl, 1-naphthylamine) acetylation by NAT2 is regarded as a detoxication step. Interestingly, the underlying European findings of a higher susceptibility of slow acetylators towards aromatic amines are in contrast to findings in Chinese workers occupationally exposed to aromatic amines which points to different mechanisms of susceptibility between European and Chinese populations. Regarding human bladder cancer, the hypothesis has been put forward that genetic polymorphism of GSTM1 might be linked with the occurrence of this tumour type. This supports the hypothesis that exposure to PAH might causally be involved in urothelial cancers. The human polymorphic GST catalysing conjugation of halomethanes, dihalomethanes, ethylene oxide and a number of other industrial compounds could be characterised as a class theta enzyme (GSTT1) by means of molecular biology. "Conjugator" and "non-conjugator" phenotypes are coincident with the presence and absence of the GSTT1 gene. There are wide variations in the frequencies of GSTT1 deletion (GSTT1 *0/0) among different ethnicities. Human phenotyping is facilitated by the GST activity towards methyl bromide or ethylene oxide in erythrocytes which is representative of the metabolic GSTT1 competence of the entire organism. Inter-individual variations in xenobiotic metabolism capacities may be due to polymorphisms of the genes coding for the enzymes themselves or of the genes coding for the receptors or transcription factors which regulate the expression of the enzymes. Also, polymorphisms in several regions of genes may cause altered ligand affinity, transactivation activity or expression levels of the receptor subsequently influencing the expression of the downstream target genes. Studies of individual susceptibility to toxicants and gene-environment interaction are now emerging as an important component of molecular epidemiology.

Evidence of associations between cytokine gene polymorphisms and quality of life in patients with cancer and their family caregivers

PURPOSE/OBJECTIVES: To identify latent classes of individuals with distinct quality-of-life (QOL) trajectories, to evaluate for differences in demographic characteristics between the latent classes, and to evaluate for variations in pro- and anti-inflammatory cytokine genes between the latent classes. DESIGN: Descriptive, longitudinal study. SETTING: Two radiation therapy departments located in a comprehensive cancer center and a community-based oncology program in northern California. SAMPLE: 168 outpatients with prostate, breast, brain, or lung cancer and 85 of their family caregivers (FCs). METHODS: Growth mixture modeling (GMM) was employed to identify latent classes of individuals based on QOL scores measured prior to, during, and for four months following completion of radiation therapy. Single nucleotide polymorphisms (SNPs) and haplotypes in 16 candidate cytokine genes were tested between the latent classes. Logistic regression was used to evaluate the relationships among genotypic and phenotypic characteristics and QOL GMM group membership. MAIN RESEARCH VARIABLES: QOL latent class membership and variations in cytokine genes. FINDINGS: Two latent QOL classes were found: higher and lower. Patients and FCs who were younger, identified with an ethnic minority group, had poorer functional status, or had children living at home were more likely to belong to the lower QOL class. After controlling for significant covariates, between-group differences were found in SNPs in interleukin 1 receptor 2 (IL1R2) and nuclear factor kappa beta 2 (NFKB2). For IL1R2, carrying one or two doses of the rare C allele was associated with decreased odds of belonging to the lower QOL class. For NFKB2, carriers with two doses of the rare G allele were more likely to belong to the lower QOL class. CONCLUSIONS: Unique genetic markers in cytokine genes may partially explain interindividual variability in QOL. IMPLICATIONS FOR NURSING: Determination of high-risk characteristics and unique genetic markers would allow for earlier identification of patients with cancer and FCs at higher risk for poorer QOL. Knowledge of these risk factors could assist in the development of more targeted clinical or supportive care interventions for those identified.

Progesterone regulation of implantation-related genes: new insights into the role of oestrogen

Genomic profiling was performed on explants of late proliferative phase human endometrium after 24-h treatment with progesterone (P) or oestradiol and progesterone (17 beta-E-2+P) and on explants of menstrual phase endometrium treated with 17 beta-E-2+P. Gene expression was validated with real-time PCR in the samples used for the arrays, in endometrium collected from early and mid-secretory phase endometrium, and in additional experiments performed on new samples collected in the menstrual and late proliferative phase. The results show that late proliferative phase human endometrium is more responsive to progestins than menstrual phase endometrium, that the expression of several genes associated with embryo implantation (i.e. thrombomodulin, monoamine oxidase A, SPARC-like 1) can be induced by P in vitro, and that genes that are fully dependent on the continuous presence of 17 beta-E-2 during P exposure can be distinguished from those that are P-dependent to a lesser extent. Therefore, 17 beta-E-2 selectively primes implantation-related genes for the effects of P.