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 Crohn's 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.     

Eight novel CARD15 variants detected by DNA sequence analysis of the CARD15 gene in 111 patients with inflammatory bowel disease

We performed a limited DNA sequence analysis of the CARD15 gene in 89 patients with Crohn’s disease (CD), 19 patients with ulcerative colitis (UC), and three patients with indeterminate colitis (IC), who were heterozygous carriers of one of the common CARD15 mutations [c.2104C>T (p.R702W), c.2722G>C (p.G908R), or c.3019_3020insC (p.Leu1007fsX1008)], the c.2462+10A>C variant, or of a new amino acid substitution in the 3′-end of exon 4. CARD15 exons 4, 5, 6, 8, and 11 were amplified by PCR and completely sequenced, thereby theoretically covering 73.9% of the described CARD15 variants and 96.6% of the mutated alleles. Using this approach, eight novel amino acid substitutions [c.1171C>T (p.R391C), c.1387C>G (p.P463A), c.2138G>A (p.R713H), c.2278C>T (p.R760C), c.2368C>T (p.R790W), c.2371C>T (p.R791W), c.2475C>G (p.N825K), and c.2546C>T (p.A849V)] were detected in six CD and two IC patients, and one UC patient. A severe disease phenotype was observed especially in patients who are compound-heterozygous for a common and a novel CARD15 mutation.Schnitzler and Brand contributed equally     

Frequency of CARD15 polymorphisms in patients with Crohn's disease from Toledo, Spain: genotype-phenotype correlation

Crohn's disease (CD) presents a complex multifactorial etiology with genetic and environmental factors contributing to the disorder. Epidemiological studies have shown that three major CARD15 polymorphisms, R702W, G908R, and 1007fs, are associated with CD. We studied the frequencies of these three polymorphisms in patients from Toledo, Spain, and compared them with the frequencies found in studies of other populations. A total of 183 patients with CD and 172 healthy controls from Toledo, Spain, were included in this study. All of these individuals were genotyped for the three CARD15 polymorphisms R702W, G908R, and 1007fs. Frequencies were analyzed to identify any genotype-phenotype associations. The control population exhibited frequencies of CARD15 polymorphisms similar to the results of previous studies, 3.4%, 1.1%, and 2.0% for the R702W, G908R, and 1007fs polymorphisms, respectively, whereas CD patients had allele frequencies of 7.6%, 3.0%, and 4.6%, respectively. Significant associations were found between the presence of R702W and patients carrying two susceptibility variants with early age of onset and stricturing pattern.     

CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease

CARD15/NOD2 encodes a protein involved in bacterial recognition by monocytes. Mutations in CARD15 have recently been found in patients with Crohn disease (CD), a chronic inflammatory condition of the digestive tract. Here, we report the mutational analyses of CARD15 in 453 patients with CD, including 166 sporadic and 287 familial cases, 159 patients with ulcerative colitis (UC), and 103 healthy control subjects. Of 67 sequence variations identified, 9 had an allele frequency >5% in patients with CD. Six of them were considered to be polymorphisms, and three (R702W, G908R, and 1007fs) were confirmed to be independently associated with susceptibility to CD. Also considered as potential disease-causing mutations (DCMs) were 27 rare additional mutations. The three main variants (R702W, G908R, and 1007fs) represented 32%, 18%, and 31%, respectively, of the total CD mutations, whereas the total of the 27 rare mutations represented 19% of DCMs. Altogether, 93% of the mutations were located in the distal third of the gene. No mutations were found to be associated with UC. In contrast, 50% of patients with CD carried at least one DCM, including 17% who had a double mutation. This observation confirmed the gene-dosage effect in CD. The patients with double-dose mutations were characterized by a younger age at onset (16.9 years vs. 19.8 years; P=.01), a more frequent stricturing phenotype (53% vs. 28%; P=.00003; odds ratio 2.92), and a less frequent colonic involvement (43% vs. 62%; P=.003; odds ratio 0.44) than were seen in those patients who had no mutation. The severity of the disease and extraintestinal manifestations were not different for any of the CARD15 genotypes. The proportion of familial and sporadic cases and the proportion of patients with smoking habits were similar in the groups of patients with CD with or without mutation. These findings provide tools for a DNA-based test of susceptibility and for genetic counseling in inflammatory bowel disease.     

Interleukin-1 receptor cluster: gene organization of IL1R2, IL1R1, IL1RL2 (IL-1Rrp2), IL1RL1 (T1/ST2), and IL18R1 (IL-1Rrp) on human chromosome 2q

The family of interleukin-1 receptor-like genes currently has six known members. We have constructed a contig of 10 overlapping human PAC clones that covers 530 kb and includes five of the six family members. The termini of the contig were mapped to the interval between D2S373 and D2S176 (chromosome 2q12) by radiation hybrid mapping. The contig contains the genes (cen --> tel), in the order given, for the type II interleukin-1 (IL-1) receptor (IL1R2), the type I IL-1 receptor (IL1R1), the IL-1 receptor-related protein 2 (IL1RL2), T1/ST2/fit-1 (IL1RL1), and the IL-1 receptor-related protein 1, which has recently been shown to be a component of the IL-18 receptor (IL18R1). We show that all the genes are transcribed in the same direction, with IL1R2 being transcribed toward the cluster. The only known family member that is absent from the human contig is the IL-1 receptor accessory protein gene (IL1RAP), which maps to 3q28.     

Epitope shared by functional variant of organic cation/carnitine transporter, OCTN1, Campylobacter jejuni and Mycobacterium paratuberculosis may underlie susceptibility to Crohn's disease at 5q31

Campylobacter jejuni and Mycobacterium paratuberculosis have been implicated in the pathogenesis of Crohn's disease. The presence of bacterial metabolites in the colonic lumen causing a specific breakdown of fatty acid oxidation in colonic epithelial cells has been suggested as an initiating event in inflammatory bowel disease (IBD). l-Carnitine is a small highly polar zwitterion that plays an essential role in fatty acid oxidation and ATP generation in intestinal bioenergetic metabolism. The organic cation/carnitine transporters, OCTN1 and OCTN2, function primarily in the transport of l-carnitine and elimination of cationic drugs in the intestine. High-resolution linkage disequilibrium mapping has identified a region of about 250kb in size at 5q31 (IBD5) encompassing the OCTN1 and -2 genes, to confer susceptibility to Crohn's disease. Recently, two variants in the OCTN1 and OCTN2 genes have been shown to form a haplotype which is associated with susceptibility to Crohn's. We show that OCTN1 and OCTN2 are strongly expressed in target areas for IBD such as ileum and colon. Further, we have now identified a nine amino acid epitope shared by this functional variant of OCTN1 (Leu503Phe) (which decreases the efficiency of carnitine transport), and by C. jejuni (9 aa) and M. paratuberculosis (6 aa). The prevalence of this variant of OCTN1 (Phe503:Leu503) is 3-fold lower in unaffected individuals of Jewish origin (1:3.44) compared to unaffected individuals of non-Jewish origin (1:1). We hypothesize that a specific antibody raised to this epitope during C. jejuni or M. paratuberculosis enterocolitis would cross-react with the intestinal epithelial cell functional variant of OCTN1, an already less efficient carnitine transporter, leading to an impairment of mitochondrial beta-oxidation which may then serve as an initiating event in IBD. This impairment of l-carnitine transport by OCTN1 may respond to high-dose l-carnitine therapy.     

Influence of polymorphisms in the NOD1/CARD4 and NOD2/CARD15 genes on the clinical outcome of Helicobacter pylori infection

Host immune response influences the clinical outcome of Helicobacter pylori infection leading to ulcer disease, gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. A genetic risk profile for gastric cancer has been identified, but genetic susceptibility to develop MALT lymphoma is still unclear. We investigated the role of NOD1 and NOD2 as intracellular recognition molecules for pathogen-associated molecules in H. pylori infection in vitro and analysed the influence of single nucleotide polymorphisms on susceptibility to ulcer disease and MALT lymphoma. Expression of NOD1 and NOD2 significantly sensitized HEK293 cells to H. pylori-induced NF-kappaB activation in a cag pathogenicity island (cagPAI)-dependent manner. In cells carrying the Crohn-associated NOD2 variant R702W the NF-kappaB response was significantly diminished. NOD1/NOD2 expression levels were induced in the gastric epithelium in H. pylori-positive patients. No mutations were found to be associated with gastritis or gastric ulcer development. However, the R702W mutation in the NOD2/CARD15 gene was significantly associated with gastric lymphoma. Carrier of the rare allele T had a more than doubled risk to develop lymphoma than controls [odds ratio (OR): 2.4, 95% confidence interval (CI): 1.2-4.6; P < 0.044]. H. pylori-induced upregulation of NOD1 and NOD2 in vivo may play a critical role in the recognition of this common pathogen. A missense mutation in the leucine-rich region of CARD15 is associated with gastric lymphoma.     

Analysis of HLA DR, HLA DQ, C4A, FcgammaRIIa, FcgammaRIIIa, MBL, and IL-1Ra allelic variants in Caucasian systemic lupus erythematosus patients suggests an effect of the combined FcgammaRIIa R/R and IL-1Ra 2/2 genotypes on disease susceptibility

Dysfunction in various parts of immune defence, such as immune response, immune complex clearance, and inflammation, has an impact on pathogenesis in systemic lupus erythematosus (SLE). We hypothesised that combinations of common variants of genes involved in these immune functions are associated with susceptibility to SLE. The following variants were analysed: HLA DR3, HLA DQ2, C4AQ0, Fcγ receptor IIa (FcγRIIa) genotype R/R, Fcγ receptor IIIa (FcRγIIIa) genotype F/F, mannan-binding lectin (MBL) genotype conferring a low serum concentration of MBL (MBL-low), and interleukin-1 receptor antagonist (IL-1Ra) genotype 2/2. Polymorphisms were analysed in 143 Caucasian patients with SLE and 200 healthy controls. HLA DR3 in SLE patients was in 90% part of the haplotype HLA DR3-DQ2-C4AQ0, which was strongly associated with SLE (odds ratio [OR] 2.8, 95% CI 1.7–4.5). Analysis of combinations of gene variants revealed that the strong association with SLE for HLA DR3-DQ2-C4AQ0 remained after combination with FcγRIIa R/R, FcγRIIIa F/F, and MBL-low (OR>2). Furthermore, the combination of the FcγRIIa R/R and IL-1Ra 2/2 genotypes yielded a strong correlation with SLE (OR 11.8, 95% CI 1.5–95.4). This study demonstrates that certain combinations of gene variants may increase susceptibility to SLE, suggesting this approach for future studies. It also confirms earlier findings regarding the HLA DR3-DQ2-C4AQ0 haplotype.     

The human OCTN1 (SLC22A4) reconstituted in liposomes catalyzes acetylcholine transport which is defective in the mutant L503F associated to the Crohn's disease

The organic cation transporter (OCTN1) plays key roles in transport of selected organic cations, but understanding of its biological functions remains limited by restricted knowledge of its substrate targets. Here we show capacity of human OCTN1-reconstituted proteoliposomes to mediate uptake and efflux of [(3)H]acetylcholine, the Km of transport being 1.0mM with V(max) of 160nmol?mg(-1)protein?min(-1). OCTN1-mediated transport of this neurotransmitter was time-dependent and was stimulated by intraliposomal ATP. The transporter operates as uniporter but translocates acetylcholine in both directions. [(3)H]acetylcholine uptake was competitively inhibited by tetraethylammonium, γ-butyrobetaine and acetylcarnitine, and was also inhibited by various polyamines. Decreasing intraliposomal ATP concentrations increased OCTN Km for acetylcholine, but V(max) was unaffected. Evaluation of the acetylcholine transporter properties of a variant form of OCTN1, the Crohn's disease-associated 503F variant, revealed time course, Km and V(max) for acetylcholine uptake to be comparable to that of wild-type OCTN1. Km for acetylcholine efflux was also comparable for both OCTN1 species, but V(max) of OCTN1 503F-mediated acetylcholine efflux (1.9nmol?mg(-1)protein?min(-1)) was significantly lower than that of wild-type OCTN1 (14nmol?mg(-1)protein?min(-1)). These data identify a new transport role for OCTN1 and raise the possibility that its involvement in the non-neuronal acetylcholine system may be relevant to the pathogenesis of Crohn's disease.     

Association study of IL28B: rs12979860 and rs8099917 polymorphisms with SVR in patients infected with chronic HCV genotype 1 to PEG-INF/RBV therapy using systematic meta-analysis

Recently, genome-wide associated studies (GWAS) have identified that host genetics IL28B SNPs rs12979860 and rs8099917 were significantly associated with SVR in patients infected with chronic HCV genotype 1 to PEG-INF/RBV therapy. Results from these studies remain conflicting. We conducted this meta-analysis to estimate the overall association of SVR with rs12979860 and rs8099917. We searched the PubMed, Embase, Scholar Google, ISI Web of Knowledge, and Chinese National Knowledge Infrastructure (CNKI) databases for all articles before July 30, 2012. The odds ratio (OR) corresponding to the 95% confidence interval (CI) was used to assess the association. The statistical heterogeneity among studies was assessed with the I² statistics. Begg's test and Egger's test were performed to evaluate the publication bias. Eventually, twenty studies were selected for the meta-analysis. The IL-28B SNPs rs12979860 genotype CC and rs8099917 genotype TT significantly positive associated with SVR in patients infected chronic HCV genotype 1 to PEG-INF/RBV therapy (OR = 4.473, 95% CI = 3.814–5.246, OR = 5.171, 95% CI = 4.372–6.117 respectively). The results suggested that rs12979860 genotype CC and rs8099917 genotype TT could be used as independent predictors of the HCV-1 infected patients.     

Dopamine release is severely compromised in the R6/2 mouse model of Huntington's disease

Recently, alterations in dopamine signaling have been implicated in Huntington's disease. In this work, dopamine release and uptake was measured in striatal slices from the R6/2 transgenic mouse model of Huntington's disease using fast-scan cyclic voltammetry at carbon-fiber microelectrodes. Dopamine release in brain slices from 6-week-old R6/2 mice is substantially reduced (53% of wild type), while dopamine uptake is unaffected. In agreement with this, R6/2 mice injected with the dopamine uptake inhibitor cocaine exhibited a blunted motor activity response (54% of wild type). At 10 weeks of age, an even more dramatic motor activity decrease in response to cocaine injection (21% of wild type) was observed. Moreover, the pre-drug activity of 10-week-old R6/2 mice was significantly reduced (by 37%) compared with 6-week-old R6/2 mice. Striatal dopamine release decreased with age, indicating that progressive alterations in dopaminergic pathways may affect motor activity. The inhibition constants of cocaine and methamphetamine (METH) determined in brain slices differed little between genotype or age group, suggesting that the decreased responses to cocaine and METH arise from compromised dopamine release rather than differences in uptake or drug action. Collectively, these data demonstrate (i) a reduction in the ability of dopamine terminals to release dopamine and (ii) the importance of this attenuation of release on the motor symptoms of Huntington's disease.     

Mutational analysis of OGG1, MYH, MTH1 in FAP, HNPCC and sporadic colorectal cancer patients: R154H OGG1 polymorphism is associated with sporadic colorectal cancer patients

MYH, OGG1 and MTH1 are members of base excision repair (BER) families, and MYH germline mutations were recently identified in patients with multiple adenomas or familial adenomatous polyposis (FAP). A total of 20 APC-negative Korean FAP patients were analyzed for OGG1, MYH and MTH1 germline mutations. A total of 19 hereditary nonpolyposis colorectal cancer (HNPCC), 86 suspected HNPCC, and 246 sporadic colorectal cancer cases were investigated for OGG1 and MYH mutations. A total of 14 R154H OGG1 polymorphisms were identified in hereditary, sporadic colorectal cancers, and normal controls. For the case-control analysis of OGG1 R154H, a total of 625 hereditary or sporadic colorectal cancer patients and 527 normal controls were screened. R154H was a rare polymorphism associated with sporadic colorectal cancer patents (OR: 3.586, P= 0.053). R154H does not segregate with cancer phenotypes. Upon examining the possibility of recessive inheritance of R154H, we could not identify any complementary mutations in OGG1, MYH or MTH1. Samples with R154H were further screened for mutations of K-ras, -catenin, APC, p53, BRAF and the microsatellite instability (MSI) status. Eight somatic mutations were identified in these genes and G:C to T:A transversion mutations were not dominant in samples harboring R154H. This result raises the possibility that OGG1 R154H may function as a low/moderate-penetrance modifier for colorectal cancer development.I.-J. Kim and J.-L. Ku contributed equally to this work     

A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu’s itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.     

Genetic analysis of IREB2, FAM13A and XRCC5 variants in Chinese Han patients with chronic obstructive pulmonary disease

Recently, variants (rs2568494, rs2869967 and rs3821104) in the IREB2, FAM13A and XRCC5 genes were found to be associated with chronic obstructive pulmonary disease (COPD) in non-Asian populations by genome-wide association study (GWAS) analysis. To evaluate whether variants in these genes are related to COPD in Chinese Han population, we investigated COPD patients of Chinese Han ethnicity from Mainland China. Significant differences in genotypic distributions (χ² = 6.319, p = 0.042 for rs2869967; χ² = 6.062, p = 0.048 for rs3821104) and allele distributions (χ² = 4.014, p = 0.045 for rs2869967; χ² = 5.607, p = 0.018 for rs3821104) were observed between patients and control subjects for variants rs2869967 and rs3821104, whereas no statistically significant associations for genotypic and allelic distribution between IREB2 rs2568494 and COPD phenotype (p > 0.05) were identified. Our results support that FAM13A rs2869967 and XRCC5 rs3821104 are associated with COPD in Chinese Han population.     

Combined analysis of polymorphism variants in hMTH1, hOGG1 and MUTYH genes on the risk of type 2 diabetes in the Chinese population

Reactive oxygen species are considered to play a role in the development of type 2 diabetes mellitus (T2DM) and its complications. 8-Oxoguanine, which is one of the major oxidation base lesions produced by reactive oxygen species, may cause G:C to T:A transversion mutations because it can mispair with adenine. hMTH1 (human mutT homolog 1), hOGG1 (human 8-oxoguanine glycosylase 1) and MUTYH (human mutY homolog) genes constitute the 8-oxoG repair pathway. In this study, we screened for the polymorphism variants Val83Met (c.247G>A, rs4866) in hMTH1; c.-53G>C (rs56387615), c.-23A>G (rs1801129) and c.-18G>T (rs1801126) in the 5′-UTR of hOGG1; and AluYb8 insertion in MUTYH (AluYb8MUTYH, rs10527342) and investigated their synergistic effect on the risk of T2DM in the Chinese population. The genotypes were determined by electrophoresis, a high-resolution melting technique and sequencing of PCR products. Our results showed that the c.247G>A variant in the hMTH1 gene increased the risk of T2DM in > 55 years of age groups (OR = 1.579; 95%CI: 1.029–2.421). The set of c.-53G>C, c.-23A>G and c.-18G>T variants detected in the 5′-UTR of the hOGG1 gene and the AluYb8 insertion in the MUTYH gene were each associated with an increased risk of T2DM (OR = 1.507, 95%CI: 1.122–2.024; OR = 1.229, 95%CI: 1.030–1.466, respectively). Combined analysis of the variations among the three genes suggested that the c.247G>A variant in hMTH1 combined with AluYb8MUTYH variant had a synergistic effect on increasing the risk of T2DM (OR = 1.635; 95%CI: 1.147–2.330). This synergy was also observed between the variants in the 5′-UTR of the hOGG1 and the AluYb8MUTYH variant (OR = 1.804; 95%CI: 1.254–2.595). Our results suggest, for the first time, the combined effects of AluYb8MUTYH with either hMTH1 c.247G>A or variants in the 5′-UTR of the hOGG1 on the risk of T2DM.