Jiwon Hong

TLR2, TLR4 and TLR9 polymorphisms and Crohn's disease in a New Zealand Caucasian cohort.

Background and Aim:  Toll‐like receptors (TLRs) have been identified as susceptibility genes for Crohns disease (CD) in some, but not all, studies. Here we examined the association between candidate disease‐susceptibility polymorphisms in the TLR2, TLR4 and TLR9 genes and CD in a New Zealand Caucasian population.

Polymorphisms in the organic cation transporter genes SLC22A4 and SLC22A5 and Crohn's disease in a New Zealand Caucasian cohort.

Polymorphisms in the organic cation transporter (OCTN) genes SLC22A4 (OCTN1; polymorphism 1672C/T) and SLC22A5 (OCTN2; polymorphism −207G/C) at the inflammatory bowel disease (IBD) 5 locus comprise a two‐allele haplotype (SLC22A‐TC) associated with increased risk for Crohns disease (CD). In this study, we examined the contribution of the disease susceptibility haplotype SLC22A‐TC to CD in a New Zealand Caucasian population. The frequencies of the gene polymorphisms 1672C/T and −207G/C were examined in 182 patients with CD and 188 ethnically matched controls by PCR‐RFLP analysis. There was a significant difference in the allele frequency (0.444 vs 0.519; P = 0.041) of the 1672T polymorphism in the SLC22A4 gene between controls and patients with CD. In contrast, there was no significant difference (0.497 vs 0.552; P = 0.135) for the −207C polymorphism in the SLC22A5 gene. The homozygote SLC22A‐TC diplotype was significantly associated with an increased risk for CD (odds ratio 2.19), and the SLC22A‐TC haplotype was associated with increased risk (P = 0.0007) of ileocolonic involvement. The population‐attributable risk for the SLC22A‐TC haplotype is 15.1%. Thus, SLC22A‐TC is associated with an increased risk of CD and disease phenotype in our New Zealand CD cohort.

Polymorphisms of CARD15/NOD2 and CD14 genes in New Zealand Crohn's disease patients.

Polymorphisms in the CARD15/NOD2 gene, which encodes a cytosolic protein involved in bacterial recognition, are associated with development of Crohns disease (CD). Other potential susceptibility genes such as CD14 may compound the risk of developing CD. We examined the frequency of the three major CARD15 risk alleles (3020insC/L1007fsinsC, G908R and R702W), and a functional polymorphism (–159C/T) in the promoter of the CD14 gene in 185 CD patients in New Zealand and 187 ethnically matched controls. The frequencies of the 3020insC (8.1 vs 0.8%, P < 0.0001), G908R (3.5 vs 2.4%, P = 0.37) and R702W (7.3 vs 5.1%, P = 0.21) alleles in CD patients and controls, respectively, were similar to those described in Australia, and the ancestral countries of Scotland, Ireland and the UK. Only the 3020insC polymorphism was found to be a significant risk factor for CD in our New Zealand cohort (odds ratio = 10.91 [95% confidence intervals 3.30–36.08]; P < 0.0001 for heterozygotes), but not a single patient was homozygous for the 3020insC polymorphism. The T allele (51 vs 50%, P = 0.77) and TT genotype (26 vs 24%, P = 0.84) frequencies of the –159C/T CD14 gene promoter polymorphism did not significantly differ between CD patients and controls. In summary, our findings provide evidence that the CARD15 3020insC risk allele influences disease susceptibility in a small proportion (<17%) of New Zealand CD patients, whereas there was no evidence that the CD14 –159C/T polymorphism is associated with CD.

Uropathogenic Escherichia coli Releases Extracellular Vesicles That Are Associated with RNA

Background Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection. Methods and Results Using the uropathogenic Escherichia coli (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different ‘size’ RNA populations (<50nt, 50-200nt and 200nt+) isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of csrC. It was estimated that 1% of MV RNA cargo is delivered into cultured cells. Conclusions These data add to the growing evidence of pathogenic bacterial MV being associated a wide range of RNAs. It further raises the plausibility for MV-RNA-mediated cross-kingdom communication whereby they influence host cell function during the infection process.

IL4 , IL10 , IL16 , and TNF polymorphisms in New Zealand Caucasian Crohn’s disease patients

Dear Editor: Crohn’s disease (CD; OMIM 266600) is a multi-factorial, polygenic disease characterized by differential production of a number of cytokines that potentially contribute to the inflammatory response within the gut. Gene transcription of tumor necrosis factor (TNF; previously known as TNF-α; OMIM 191160; IBD3 locus) is increased in the intestinal mucosa of CD patients even in remission, and high levels of TNF are present in the serum, intestinal mucosa, and stool samples of CD patients. The −308G/A polymorphism (also known as TNF2) in the promoter region of the TNF gene was associated with increased levels of TNF in the plasma of cancer patients [Warzocha et al. (1998) Genetic polymorphisms in the TNF locus influence non-Hodgkin’s lymphoma outcome. Blood 91:3574–3581]. Peripheral blood mononuclear cells from CD patients homozygous for the AA alleles secrete more TNF than those of GG homozygotes, upon stimulation with T cell activators. The A allele is more frequent in subgroups of CD patients with colonic, fistulizing, or steroid-dependent disease. A functional TNF gene polymorphism −863C/A that reduced circulating levels of TNF was positively associated with CD and disease location in Japan [Negoro et al. (1999) CD is associated with novel polymorphisms in the 5′-flanking region of the TNF gene. Gastroenterology 117:1062–1068]. Expression of the pro-inflammatory cytokine interleukin-16 (IL-16; OMIM 603035) is increased in a number of chronic inflammatory diseases including CD. A −295T/C polymorphism in its gene promoter was found to be associated with CD in Germany [Glas et al. (2003) The -295T-to-C promoter polymorphism of the IL-16 gene is associated with CD. Clin Immunol 106:197–200]. Conversely, reduction in expression of the anti-inflammatory cytokine IL-10 (OMIM 124092) is implicated in CD. Administration of recombinant human IL-10 has a clinical benefit to CD patients. Among at least 23 polymorphisms reported in the promoter region of the IL10 gene to date, −1082G/A, −819C/T, and −592C/A, which give rise to three wellconserved haplotypes (GCC, ACC, and ATA) are the most investigated [Turner et al. (1997) An investigation of polymorphism in the interleukin-10 gene promoter. Eur J Immunogenet 24:1–8]. The -1082A allele was significantly associated with decreased IL-10 production in CD patients and controls. The Gly15Arg mutation in the leader sequence of the IL10 gene, which reduces secretion of IL10 by 50%, was present in a family with multiple individuals affected with CD, but not in healthy controls [van der Linde et al. (2003) A Gly15Arg mutation in the interleukin-10 gene reduces secretion of interleukin-10 in Crohn disease. Scand J Gastroenterol 38:611–617]. IL-4 Int J Colorectal Dis (2008) 23:335–337 DOI 10.1007/s00384-007-0338-3

Isolation of membrane vesicles from prokaryotes: a technical and biological comparison reveals heterogeneity

ABSTRACT Prokaryotes release membrane vesicles (MVs) with direct roles in disease pathogenesis. MVs are heterogeneous when isolated from bacterial cultures so Density Gradient Centrifugation (DGC) is valuable for separation of MV subgroups from contaminating material. Here we report the technical variability and natural biological heterogeneity seen between DGC preparations of MVs for Mycobacterium smegmatis and Escherichia coli and compare these DGC data with size exclusion chromatography (SEC) columns. Crude preparations of MVs, isolated from cultures by ultrafiltration and ultracentrifugation were separated by DGC with fractions manually collected as guided by visible bands. Yields of protein, RNA and endotoxin, protein banding and particle counts were analysed in these. DGC and SEC methods enabled separation of molecularly distinct MV populations from crude MVs. DGC banding profiles were unique for each of the two species of bacteria tested and further altered by changing culture conditions, for example with iron supplementation. SEC is time efficient, reproducible and cost effective method that may also allow partial LPS removal from Gram-negative bacterial MVs. In summary, both DGC and SEC are suitable for the separation of mixed populations of MVs and we advise trials of both, coupled with complete molecular and single vesicle characterisation prior to downstream experimentation.

Polymorphisms in NFKBIA and ICAM-1 genes in New Zealand Caucasian Crohn's disease patients.

Background and Aim:  Crohns disease (CD) is a complex polygenetic inflammatory disease of the bowel. NFKBIA (IκBα) is a downstream regulator of NF‐κB, which is an effector of the major Crohns susceptibility gene CARD15/NOD2. ICAM‐1 is an integrin ligand and vascular addressin, which contributes to the trafficking of pathogenic leukocytes into the inflamed bowel. Here we examined whether the NFKBIA 2758G/A and ICAM‐1 K469E variants, recently shown to be CD susceptibility genes in Caucasian populations in Germany and the United Kingdom, are associated with CD in Caucasian patients in New Zealand.

Nucleic Acid from Saliva and Salivary Cells for Noninvasive Genotyping of Crohn's Disease Patients

CARD15 genes carrying the 3020insC frameshift polymorphism encode a truncated CARD15 protein that is unresponsive to bacterial muramyl dipeptide, and are strongly associated with increased susceptibility to Crohns disease (CD). In this study we established that CARD15 gene sequences encompassing the major 3020insC polymorphism could be readily amplified from the DNA found in saliva. In addition, CARD15 RNA sequences can be readily derived from the cellular component of saliva, which is primarily comprised of buccal epithelial cells. Our results demonstrate that saliva is a readily accessible source of DNA and RNA for genotyping CD patients for variants of the CARD15 gene, representing an alternative source of nucleic acid to that obtained from venous blood.

Peroxisome proliferator-activated receptor-γ gene polymorphisms and Crohn’s disease

Dear Editor: Recently, Sugawara et al. identified peroxisome proliferator-activated receptor-γ (PPARG) as a susceptibility gene for Crohn’s disease (CD) [Sugawara et al. (2005) Linkage to peroxisome proliferator-activated receptor-gamma in SAMP1/YitFc mice and in human Crohn’s disease. Gastroenterology 128:351–60]. PPAR-γ is a transcription factor belonging to the nuclear receptor superfamily. It initiates transcription of genes involved in diverse biological processes and inhibits the nuclear factor kappa B (NF-κB) signalling pathway. PPARG contains three promoters that yield the four mRNA isoforms PPARγ1, PPARγ2, PPARγ3, and PPARγ4 encoding two proteins. Sugawara et al. reported that two minor alleles of PPARG, namely, SNP1 [G12350898A (rs2067819)] and SNP2 [G12359887A (rs3892175)], were more common in control subjects than in CD patients. We examined the association between PPARG and CD in New Zealand by genotyping G12350898A (rs2067819), the polymorphism Pro12Ala (C34G) (rs1805192) in PPARγ2 and the PPARγ3 promoter polymorphism C681G (position −595 from ATG in exon B). A total of 182 Caucasian CD patients and 188 ethnicallymatched control subjects were genotyped by PCR-RFLP analysis using NlaIII for C681G, BsaAI for G12350898A, and BstUI for Pro12Ala. Statistical analysis was performed using the Chi-square or Fisher’s exact test. Haplotype frequencies were estimated using SHEsis (http://202.120.7.14/analysis/my Analysis.php). Linkage disequilibrium was estimated using Arlequin Ver. 2.000 (http://anthro.unige.ch/ arlequin). Genotype frequencies were analysed by the Hardy–Weinberg equilibrium (HWE) program (Institute of Human Genetics, Technical University Munich, Munich, Germany; URL: (http://ihg.gsf.de/ cgi-bin//hw/hwa1.pl)), using the Fisher’s exact test to estimate P-values. There was no significant difference in the genotype frequencies of the CD patients vs control subjects for PPARγ C681G [GG 0.632 (n=115) vs 0.590 (n=111), GA 0.308 (n=56) vs 0.346 (n=65), AA 0.060 (n=11) vs 0.064 (n=12)], PPARγ G12350898A [CC 0.681 (n=124) vs 0.649 (n=122), CG 0.264 (n=48) vs 0.293 (n=55), GG 0.055 (n=10) vs 0.058 (n=11)], and PPARγ Pro12Ala [CC 0.775 (n=141) vs 0.777 (n=146), CG 0.209 (n=38) vs 0.213 (n=40), GG 0.017 (n=3) vs 0.011 (n=2)], respectively. There was no evidence that any of the genotype distributions deviate from the Hardy– Weinberg equilibrium. There was no significant difference in the allele E. Leung . J. Hong . P. Vishnu . G. W. Krissansen (*) Department of Molecular Medicine & Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand e-mail: gw.krissansen@auckland.ac.nz Tel.: +64-9-3737599 Fax: +64-9-3737674

Colony-stimulating factor-1 receptor gene polymorphisms and Crohn’s disease

Dear Editor: Zapata-Velandia et al. identified a single nucleotide polymorphism (A2033T) in an intron in the gene (CSF1R) encoding colony-stimulating factor-1 receptor (CSF1R), and revealed that the latter polymorphism was more common in American Crohn’s disease (CD) patients than in healthy controls, suggesting that CSF1R is a susceptibility gene for CD [Zapata-Velandia et al. (2004) Association of the T allele of an intronic single nucleotide polymorphism in the colony-stimulating factor 1 receptor with Crohn’s disease: a case-control study. J Immune Based Ther Vaccines 2:6]. CSF1R (OMIM 164770) is a receptor tyrosine kinase expressed in the superficial epithelium of the ileum and colon, which stimulates the proliferation, differentiation, and survival of monocytes, and macrophages. We examined the association between CSF1R and CD in New Zealand by genotyping the A2033T polymorphism in the eleventh intron of CSF1R. A total of 182 Caucasian CD patients and 188 ethnicallymatched control subjects were genotyped by PCR-RFLP analysis using the primer set described by ZapataVelandia et al. Statistical analysis was performed using the Chi-square or Fisher’s exact test. There was no significant difference in the genotype, allele, and carriage frequencies of the CSF1R 2033T polymorphism in controls vs CD patients [genotype AA 0.734 (n=138) vs 0.764 (n=139), AT 0.255 (n=48) vs 0.225 (n=41), TT=0.011 (n=2) vs 0.011 (n=2), P=0.80; allele T 0.138 vs 0.124, P=0.55; carriage of T allele 0.266 vs 0.236, P=0.51), respectively]. There was no significant difference (P=0.35 and 0.72, respectively) in the CSF1R 2033T allele frequencies of patients who did (0.093) or did not (0.129) carry the 3020insC polymorphism in the CD susceptibility gene CARD15 in comparison to control subjects (0.138) [for genotyping of the CARD15 3020insC allele refer to Leung et al. (2005) Polymorphisms of CARD15/ NOD2 and CD14 genes in New Zealand Crohn’s disease patients. Immunol Cell Biol 83:498–503]. There was no evidence that any of the genotype distributions deviate from Hardy–Weinberg equilibrium. There was no significant difference in the allelic distribution of the 2033T polymorphism with respect to disease location, behavior, or need of surgery in 177 CD patients for whom clinical data was obtained. In summary, there was no evidence that the 2033T variant is a major risk factor for CD in New Zealand. Post priori power calculations in which E. Leung . J. Hong . P. Vishnu . G. W. Krissansen (*) Department of Molecular Medicine & Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand e-mail: gw.krissansen@auckland.ac.nz Tel.: +64-9-3737599 Fax: +64-9-3737674