Differential quantification of CYP2D6 gene copy number by four different quantitative real-time PCR assays

Copy number variations (CNVs) in the CYP2D6 gene contribute to interindividual variation in drug metabolism. As the most common duplicated allele in Asian populations is the nonfunctional CYP2D6*36 allele, the goal of this study was to identify CNV assays that can differentiate between multiple copies of the CYP2D6*36 allele and multiple copies of other CYP2D6 alleles. We determined CYP2D6 gene copy numbers in 32 individuals with known CYP2D6 CNVs from the Coriell Japanese-Chinese panel using four quantitative real-time PCR assays. These assays target different regions of the CYP2D6 gene: 5'-flanking region, intron 2, intron 6, and exon 9 (Ex9). The specific target site of the Ex9 assay was verified by sequencing the PCR amplicon. Three of the CYP2D6 CNV assays (5'-flanking region, intron 2, and intron 6) estimated CYP2D6 copy numbers that were concordant for all 32 individuals. However, the Ex9 assay was concordant in only 10 of 32 samples. The 10 concordant samples did not contain any CYP2D6*36 alleles and the 22 discordant samples contained at least one CYP2D6*36 allele. In addition, the Ex9 assay accurately quantified all of the non-CYP2D6*36 alleles in all samples. Ex9 amplicon sequencing indicated that it targets a region of CYP2D6 exon 9 that undergoes partial gene-conversion in the CYP2D6*36 allele. In conclusion, CYP2D6 Ex9 CNV assay can be used to determine the copy number of non-CYP2D6*36 alleles. Selective amplification of non-CYP2D6*36 sequence by the Ex9 assay should be useful in determining the number of functional copies of CYP2D6 in Asian populations

Inferring Haplotypes of Copy Number Variations From High-Throughput Data With Uncertainty

Accurate information on haplotypes and diplotypes (haplotype pairs) is required for population-genetic analyses; however, microarrays do not provide data on a haplotype or diplotype at a copy number variation (CNV) locus; they only provide data on the total number of copies over a diplotype or an unphased sequence genotype (e.g., AAB, unlike AB of single nucleotide polymorphism). Moreover, such copy numbers or genotypes are often incorrectly determined when microarray signal intensities derived from different copy numbers or genotypes are not clearly separated due to noise. Here we report an algorithm to infer CNV haplotypes and individuals’ diplotypes at multiple loci from noisy microarray data, utilizing the probability that a signal intensity may be derived from different underlying copy numbers or genotypes. Performing simulation studies based on known diplotypes and an error model obtained from real microarray data, we demonstrate that this probabilistic approach succeeds in accurate inference (error rate: 1–2%) from noisy data, whereas previous deterministic approaches failed (error rate: 12–18%). Applying this algorithm to real microarray data, we estimated haplotype frequencies and diplotypes in 1486 CNV regions for 100 individuals. Our algorithm will facilitate accurate population-genetic analyses and powerful disease association studies of CNVs

Significant effect of polymorphisms in CYP2D6 and ABCC2 on clinical outcomes of adjuvant tamoxifen therapy for breast cancer patients

Purpose The clinical efficacy of tamoxifen is suspected to be influenced by the activity of drug-metabolizing enzymes and transporters involved in the formation, metabolism, and elimination of its active forms. We investigated relationships of polymorphisms in transporter genes and CYP2D6 to clinical outcome of patients receiving tamoxifen. Patients and Methods We studied 282 patients with hormone receptor–positive, invasive breast cancer receiving tamoxifen monotherapy, including 67 patients who have been previously reported. We investigated the effects of allelic variants of CYP2D6 and haplotype-tagging single nucleotide polymorphisms (tag-SNPs) of ABCB1, ABCC2, and ABCG2 on recurrence-free survival using the Kaplan-Meier method and Cox regression analysis. Plasma concentrations of tamoxifen metabolites were measured in 98 patients receiving tamoxifen 20 mg/d. Results CYP2D6 variants were significantly associated with shorter recurrence-free survival (P = .000036; hazard ratio [HR] = 9.52; 95% CI, 2.79 to 32.45 in patients with two variant alleles v patients without variant alleles). Among 51 tag-SNPs in transporter genes, a significant association was found at rs3740065 in ABCC2 (P = .00017; HR = 10.64; 95% CI, 1.44 to 78.88 in patients with AA v GG genotypes). The number of risk alleles of CYP2D6 and ABCC2 showed cumulative effects on recurrence-free survival (P = .000000055). Patients carrying four risk alleles had 45.25-fold higher risk compared with patients with ≤ one risk allele. CYP2D6 variants were associated with lower plasma levels of endoxifen and 4-hydroxytamoxifen (P = .0000043 and .00052), whereas no significant difference was found among ABCC2 genotype groups. Conclusion Our results suggest that polymorphisms in CYP2D6 and ABCC2 are important predictors for the prognosis of patients with breast cancer treated with tamoxifen

Independent and population-specific association of risk variants at the IRGM locus with Crohn's disease

DNA polymorphisms in a region on chromosome 5q33.1 which contains two genes, immunity related GTPase related family, M (IRGM) and zinc finger protein 300 (ZNF300), are associated with Crohn's disease (CD). The deleted allele of a 20 kb copy number variation (CNV) upstream of IRGM was recently shown to be in strong linkage disequilibrium (LD) with the CD-associated single nucleotide polymorphisms and is itself associated with CD (P < 0.01). The deletion was correlated with increased or reduced expression of IRGM in transformed cells in a cell line-dependent manner, and has been proposed as a likely causal variant. We report here that small insertion/deletion polymorphisms in the promoter and 5′ untranslated region of IRGM are, together with the CNV, strongly associated with CD (P = 1.37 × 10−5 to 1.40 × 10−9), and that the CNV and the 5′-untranslated region variant −308(GTTT)5 contribute independently to CD susceptibility (P = 2.6 × 10−7 and P = 2 × 10−5, respectively). We also show that the CD risk haplotype is associated with a significant decrease in IRGM expression (P < 10−12) in untransformed lymphocytes from CD patients. Further analysis of these variants in a Japanese CD case-control sample and of IRGM expression in HapMap populations revealed that neither the IRGM insertion/deletion polymorphisms nor the CNV was associated with CD or with altered IRGM expression in the Asian population. This suggests that the involvement of the IRGM risk haplotype in the pathogenesis of CD requires gene-gene or gene-environment interactions which are absent in Asian populations, or that none of the variants analysed are causal, and that the true causal variants arose after the European-Asian spli

Identification of Nine Novel Loci Associated with White Blood Cell Subtypes in a Japanese Population

White blood cells (WBCs) mediate immune systems and consist of various subtypes with distinct roles. Elucidation of the mechanism that regulates the counts of the WBC subtypes would provide useful insights into both the etiology of the immune system and disease pathogenesis. In this study, we report results of genome-wide association studies (GWAS) and a replication study for the counts of the 5 main WBC subtypes (neutrophils, lymphocytes, monocytes, basophils, and eosinophils) using 14,792 Japanese subjects enrolled in the BioBank Japan Project. We identified 12 significantly associated loci that satisfied the genome-wide significance threshold of P<5.0×10−8, of which 9 loci were novel (the CDK6 locus for the neutrophil count; the ITGA4, MLZE, STXBP6 loci, and the MHC region for the monocyte count; the SLC45A3-NUCKS1, GATA2, NAALAD2, ERG loci for the basophil count). We further evaluated associations in the identified loci using 15,600 subjects from Caucasian populations. These WBC subtype-related loci demonstrated a variety of patterns of pleiotropic associations within the WBC subtypes, or with total WBC count, platelet count, or red blood cell-related traits (n = 30,454), which suggests unique and common functional roles of these loci in the processes of hematopoiesis. This study should contribute to the understanding of the genetic backgrounds of the WBC subtypes and hematological traits

A Genome-Wide Association Study Identified AFF1 as a Susceptibility Locus for Systemic Lupus Eyrthematosus in Japanese

Systemic lupus erythematosus (SLE) is an autoimmune disease that causes multiple organ damage. Although recent genome-wide association studies (GWAS) have contributed to discovery of SLE susceptibility genes, few studies has been performed in Asian populations. Here, we report a GWAS for SLE examining 891 SLE cases and 3,384 controls and multi-stage replication studies examining 1,387 SLE cases and 28,564 controls in Japanese subjects. Considering that expression quantitative trait loci (eQTLs) have been implicated in genetic risks for autoimmune diseases, we integrated an eQTL study into the results of the GWAS. We observed enrichments of cis-eQTL positive loci among the known SLE susceptibility loci (30.8%) compared to the genome-wide SNPs (6.9%). In addition, we identified a novel association of a variant in the AF4/FMR2 family, member 1 (AFF1) gene at 4q21 with SLE susceptibility (rs340630; P = 8.3×10−9, odds ratio = 1.21). The risk A allele of rs340630 demonstrated a cis-eQTL effect on the AFF1 transcript with enhanced expression levels (P<0.05). As AFF1 transcripts were prominently expressed in CD4+ and CD19+ peripheral blood lymphocytes, up-regulation of AFF1 may cause the abnormality in these lymphocytes, leading to disease onset

Palmitoylethanolamide Ameliorates Carbon Tetrachloride-Induced Liver Fibrosis in Rats

Background: Liver fibrosis is a complex inflammatory and fibrogenic process, and the progression of fibrosis leads to cirrhosis. The only therapeutic approaches are the removal of injurious stimuli and liver transplantation. Therefore, the development of anti-fibrotic therapies is desired. Palmitoylethanolamide (PEA) is an endogenous fatty acid amide belonging to the N-acylethanolamines family and contained in foods such as egg yolks and peanuts. PEA has therapeutic anti-inflammatory, analgesic, and neuroprotective effects. However, the effects and roles of PEA in liver fibrosis remain unknown. Here we investigated the therapeutic effects of PEA in rats with liver fibrosis. Methods: We conducted in vitro experiments to investigate the effects of PEA on the activation of hepatic stellate cells (HSCs, LX-2). Liver fibrosis was induced by an intraperitoneal injection of 1.5 mL/kg of 50% carbon tetrachloride twice a week for 4 weeks. Beginning at 3 weeks, PEA (20 mg/kg) was intraperitoneally injected thrice a week for 2 weeks. Then rats were sacrificed and we performed histological and quantitative reverse-transcription polymerase chain reaction analyses. Results: The expression of α-smooth muscle actin (SMA) induced by transforming growth factor (TGF)-β1 in HSCs was significantly downregulated by PEA. PEA treatment inhibited the TGF-β1-induced phosphorylation of SMAD2 in a dose-dependent manner, and upregulated the expression of SMAD7. The reporter gene assay demonstrated that PEA downregulated the transcriptional activity of the SMAD complex upregulated by TGF-β1. Administration of PEA significantly reduced the fibrotic area, deposition of type I collagen, and activation of HSCs and Kupffer cells in rats with liver fibrosis. Conclusion: Activation of HSCs was significantly decreased by PEA through suppression of the TGF-β1/SMAD signaling pathway. Administration of PEA produced significant improvement in a rat model of liver fibrosis, possibly by inhibiting the activation of HSCs and Kupffer cells. PEA may be a potential new treatment for liver fibrosis

Effects of human amnion-derived mesenchymal stromal cell transplantation in rats with radiation proctitis

Background aims. Mesenchymal stromal cells (MSCs) have been reported to be a promising cell source in cell therapy, and large amounts of MSCs can easily be isolated from human amnion. Therapeutic irradiation for intra-pelvic cancer often causes radiation proctitis; however, there is currently no effective treatment. We therefore investigated the effect of transplantation of human amnion derived MSCs (AMSCs) in rats with radiation proctitis. Methods. Amnion was obtained at cesarean delivery, and AMSCs were isolated and expanded. Sprague-Dawley rats were gamma-irradiated (5 Gy/d) at the rectum for 5 days. On day 5, AMSCs (1 x 10(6) cells) were intravenously transplanted. Rats were killed on day 8. Histological analyses were performed, and messenger RNA expression of inflammatory mediators was measured. In vitro, after gamma-irradiation of rat intestinal epithelial cells (IEC-6), the cells were cultured with AMSC-conditioned medium (CM). The effect of AMSC-CM was evaluated by measurement of caspase-3/7 activity, p53 transcription activity and quantitative reverse-transcription polymerase chain reaction for p53-target genes. Results. Histological examination demonstrated that epithelial injury and infiltration of inflammatory cells in the rectum were significantly suppressed by transplantation of AMSCs. In vitro, the cell injury in IEC-6 cells induced by gamma-irradiation was inhibited by AMSC-CM, which also inhibited the upregulation of p53 transcription activity, caspase-3/7 activity and p21 expression. Conclusions. Transplantation of AMSCs improved radiation proctitis, possibly through inhibition of cell injury and inflammatory reactions. AMSC transplantation should be considered as a new treatment for radiation proctitis

Therapeutic effects of human amnion-derived mesenchymal stem cell transplantation and conditioned medium enema in rats with trinitrobenzene sulfonic acid-induced colitis

Cell therapy with mesenchymal stem cells (MSCs) is expected to provide a new strategy for the treatment of inflammatory bowel disease (IBD). Large amounts of MSCs can be obtained from human amnion. Therefore, we investigated the effect of transplantation of human amnion-derived MSCs (hAMSCs) or enema of conditioned medium (CM) from hAMSCs into rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. In the first experiment, 10-week-old male Sprague-Dawley rats were intravenously injected with hAMSCs (1 × 10^6 cells) 3 h after rectal administration of TNBS (45 mg/kg). In the second experiment, rats with TNBS-induced colitis received CM by enema into the colon for 3 days. Colitis was investigated by endoscopy, histology, immunohistochemistry, and by measuring mRNA expression of inflammatory mediators. Administration of hAMSCs or CM enema significantly improved the endoscopic score. In addition, these two interventions resulted in significantly decreased infiltration of neutrophils and monocytes/macrophages and decreased expression levels of TNF-α, CXCL1, and CCL2. In conclusion, transplantation of hAMSCs and CM enema provided significant improvement in rats with TNBS-induced colitis. CM from hAMSCs and hAMSCs may be new strategies for the treatment of IBD

Oral administration of conditioned medium obtained from mesenchymal stem cell culture prevents subsequent stricture formation after esophageal submucosal dissection in pigs

Background and Aims: Endoscopic submucosal dissection (ESD) for esophageal cancer often causes postoperative stricture when more than three fourths of the circumference of the esophagus is dissected. Mesenchymal stem cells are a valuable cell source in regenerative medicine, and conditioned medium (CM) obtained from mesenchymal stem cells reportedly inhibits inflammation. In this study we evaluated whether CM could prevent esophageal stricture after ESD. Methods: We resected a semi-circumference of pig esophagus by ESD. We prepared CM gel by mixing with 5% carboxymethyl cellulose and endoscopically applied it onto the wound bed immediately after ESD and on days 8 and 15 (weekly CM group) or administered it orally from days 1 to 4 (daily CM group). We also injected triamcinolone acetonide into the remaining submucosa immediately after ESD (steroid group). We killed the pigs on day 8 or day 22 to measure the stricture rate and to perform histologic analysis. Results: Stricture rate in weekly and daily CM groups and steroid groups were significantly lower than in the control group on day 22. Moreover, CM significantly attenuated the number of activated myofibroblasts and fiber thickness on day 22. CM also significantly decreased the infiltration of neutrophils and macrophages compared with the control group on day 8. Conclusions: CM gel prevents esophageal stricture formation by suppressing myofibroblast activation and fibrosis after the infiltration of neutrophils and macrophages. Oral administration of CM gel is a promising treatment for the prevention of post-ESD stricture