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Next-generation DNA sequencing (NGS) in short-read mode has been recently used for genetic testing in various clinical settings. NGS data accuracy is crucial in clinical settings, and several reports regarding quality control of NGS data, focusing mostly on establishing NGS sequence read accuracy, have been published thus far. Variant calling is another critical source of NGS errors that remains mostly unexplored despite its established significance. In this study, we used a machine-learning-based method to establish an exome-wide benchmark of difficult-to-sequence regions using 10 genome sequence features on the basis of real-world NGS data accumulated in The Genome Aggregation Database (gnomAD) of the human reference genome sequence (GRCh38/hg38). We used the obtained metrics, designated “UNMET score,” along with other lines of structural information of the human genome to identify difficult-to-sequence genomic regions using conventional NGS. Thus, the UNMET score could provide appropriate caveats to address potential sequential errors in protein-coding exons of the human reference genome sequence GRCh38/hg38 in clinical sequencing.

Theileria orientalis (T. orientalis) is a bovine protozoal disease similar to malaria in humans. Although the common outcome of malaria in humans and T. orientalis infection in cattle is hepatic disorder, the mechanisms of its development remain unknown. In this study, we investigated hepatocyte injury characterized by accumulation of macrophages with ingested erythrocytes in sinusoid and extramedullary hematopoiesis in cattle and mice experimentally infected with T. orientalis (T. orientalis-infected cattle and T. orientalis-infected mice). Vacuolization of hepatic cells was frequently observed in the vicinity of the aggregated macrophages in the liver sinusoids of T. orientalis-infected mice. A significant percentage of the macrophages accumulated in the liver sinusoids of the severely infected cattle and mice (14.6% and 24.2 to 53.2%, respectively) reacted positively with interleukin-1, interleukin-6 and TNF-α antibodies. Increase in the production of these cytokines was confirmed in T. orientalis-infected cattle and mice by real-time RT-PCR. These findings strongly suggest that increased cytokine production by the macrophages that have phagocytosed T. orientalis-infected erythrocytes causes hepatic disorder in T. orientalis-infected animals.

Next-generation sequencing (NGS) is a revolutionary sequencing technology for analyzing genomes. However, preprocessing methods for mitochondrial DNA (mtDNA) sequencing remain complex, and it is required to develop an authenticated preprocessing method. Here, we developed a simple and easy preprocessing method based on isothermal rolling circle mtDNA amplification using commercially available reagents. Isothermal amplification of mtDNA was successfully performed using both nanoliter quantities of plasma directly and 25 ng of total DNA extracted from blood or tissue samples. Prior to mtDNA amplification, it was necessary to treat the extracted total DNA with Exonuclease V, but it was not required to treat plasma. The NGS libraries generated from the amplified mtDNA provided sequencing coverage of the entire human mitochondrial genome. Furthermore, the sequencing results successfully detected heteroplasmy in patient samples, with called mutations and variants matching those from previous, independent, Sanger sequencing analysis. Additionally, a novel single nucleotide variant was detected in a healthy volunteer. The successful analysis of mtDNA using very small samples from patients is likely to be valuable in clinical medicine, as it could reduce patient discomfort by reducing sampling-associated damage to tissues. Overall, the simple and convenient preprocessing method described herein may facilitate the future development of NGS-based clinical and forensic mtDNA tests.

Background Long-range PCR (LR-PCR) is used to enrich the target regions of the genome. This study aimed to establish the pipeline of targeted gene sequencing using LR-PCR and massively parallel sequencing (MPS). Methods The 14-kb-long MEFV gene, including the entire coding exons, was selected as a target gene and amplified using LR-PCR. The evaluated analytical factors were as follows: LR-PCR conditions, three types of post-PCR cleanup methods, and two types of MPS library preparation methods. Results With regard to LR-PCR conditions, Tks Gflex DNA polymerase at 7-min (30-s/kb) annealing/extension with 100-ng genomic DNA input had the highest yield. Regarding post-PCR purification methods, the magnetic beads-based method had high recovery and purity. In the MPS library preparation methods, the ligation-based method had a higher base coverage in the target (94.58%), uniformity of base coverage (99.95%), and target bases with no strand bias (97.40%). The exonic variants determined by Sanger sequencing were detected by both ligation- and transposon-based methods. Conclusions Various analytical factors were evaluated, and the pipeline of targeted gene sequencing using LR-PCR and MPS was established. These data can enable the optimization of targeted gene sequencing using LR-PCR and MPS in the clinical laboratory.

Overexpression of alternative splicing of far upstream element binding protein 1 (FUBP1) interacting repressor (FIR; poly(U) binding splicing factor 60 [PUF60]) and cyclin E were detected in esophageal squamous cell carcinomas (ESCC). Accordingly, the expression of FBW7 was examined by which cyclin E is degraded as a substrate via the proteasome system. Expectedly, FBW7 expression was decreased significantly in ESCC. Conversely, c-myc gene transcriptional repressor FIR (alias PUF60; U2AF-related protein) and its alternative splicing variant form (FIRΔexon2) were overexpressed in ESCC. Further, anticancer drugs (cis-diaminedichloroplatinum/cisplatin [CDDP] or 5-fluorouracil [5-FU]) and knockdown of FIR by small interfering RNA (siRNA) increased cyclin E while knockdown of FIRΔexon2 by siRNA decreased cyclin E expression in ESCC cell lines (TE1, TE2, and T.Tn) or cervical SCC cells (HeLa cells). Especially, knockdown of SAP155 (SF3b1), a splicing factor required for proper alternative splicing of FIR pre-mRNA, decreased cyclin E. Therefore, disturbed alternative splicing of FIR generated FIR/FIRΔexon2 with cyclin E overexpression in esophageal cancers, indicating that SAP155 siRNA potentially rescued FBW7 function by reducing expression of FIR and/or FIRΔexon2. Remarkably, Three-dimensional structure analysis revealed the hypothetical inhibitory mechanism of FBW7 function by FIR/FIRΔexon2, a novel mechanism of cyclin E overexpression by FIR/FIRΔexon2-FBW7 interaction was discussed. Clinically, elevated FIR expression potentially is an indicator of the number of lymph metastases and anti-FIR/FIRΔexon2 antibodies in sera as cancer diagnosis, indicating chemical inhibitors of FIR/FIRΔexon2-FBW7 interaction could be potential candidate drugs for cancer therapy. In conclusion, elevated cyclin E expression was, in part, induced owing to potential FIR/FIRΔexon2-FBW7 interaction in ESCC.

Background Sanger sequencing is the gold standard for mutational analysis and widely used after high resolution melting (HRM) screening. However, the sensitivity of this method may be insufficient for identifying low frequency mutations. Therefore, for accurate diagnosis, enhanced sensitivity is warranted. Methods We designed a wild-type blocking cycle sequencing method using locked nucleic acid (LNA) probe (LNA-Sanger sequencing) for codons 12 and 13 of KRAS exon 2. We analyzed the sensitivities of HRM, conventional Sanger sequencing, and LNA-Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) reference standard samples with low frequency (5%) mutations in codons 12 and 13. Results Use of LNA probe significantly improved the sensitivity of Sanger sequencing (p = 0.0003). Sensitivities of KRAS mutation tests were as follows: HRM, 5%; conventional Sanger sequencing, 10%; and LNA-Sanger sequencing, 5%. FFPE samples with 5% mutation were accurately diagnosed by LNA-Sanger sequencing, whereas it was difficult to identify the mutations by conventional Sanger sequencing. Conclusions LNA-Sanger sequencing is a facile technique for the enrichment of mutant alleles and useful for the accurate diagnosis of HRM-positive cases with low frequency mutations.

Background Obliterative bronchiolitis (OB) is a known issue during minor histocompatibility antigen (mHA) disparity during lung transplantation. This study evaluated gene expression in a murine orthotropic lung transplantation model using microarray analysis. Methods Left lungs from C57BL/10(H-2b) donor mice were transplanted into mHA-mismatched C57BL/6(H-2b) recipient mice. Three groups (OB, non-OB, and sham controls) were confirmed pathologically and analyzed. Gene expression changes in the lung grafts were determined by microarray and immunohistochemical staining, and genes were verified by quantitative PCR in the lungs and mediastinal lymph nodes (LNs). Results A total of 1343 genes were upregulated in the OB lungs compared to the sham group. Significant upregulation was observed for genes related to innate, e.g. Tlr2 and CCL3 and adaptive immunity, e.g. H2-ab1 and Il-21. Positive labeling for MHC class II antigen was observed in the bronchial epithelium of OB accompanied with B cells. We found increased Tlr2, Ccl3, H2-ab1, Il-21, Ighg3, Ifng, and Pdcd1 mRNA expression in the OB lung, and increased Il-21, Ighg3, and Pdcd1 expression in the OB LNs. Conclusions Adaptive and innate immune reactions were involved in OB after lung transplantation, and genetic examination of related genes could be used for detection of OB.

The multiplex PCR melting analysis method was developed for detecting the five UGT1A1 variants. Multiplexing was achieved using color probes and Tm. The probes for *28/*6, *27, *29, and *7 were discriminated by colors. Although the probes for *28 and *6 had the same colors, their variants were clearly discriminated by probe Tm. The allelic frequencies of each genotype were 0.12 for *28, 0.19 for *6, 0.02 for *27, 0.0 for *29, and 0.005 for *7. We developed a multiplex PCR melting analysis method, which will be useful in molecular diagnostics and pharmacogenetic analyses in clinical laboratories.

The molecular diagnosis of the cancer mutational status is essential for modern clinical laboratory medicine. Mutations in EGFR, KRAS, BRAF, and PIK3CA genes are widely analyzed in solid tumors such as lung cancer, colorectal cancer, breast cancer, and melanoma. The allele-specific polymerase chain reaction, high-resolution melting, and Sanger sequencing are used for detecting and identifying gene mutations in many clinical laboratories. The locked nucleic acid (LNA) is a class of nucleic acid analogs that contain a methylene bridge connecting the 2' oxygen and 4' carbon in the ribose moiety. This methylene bridge locks the ribose group into a C3'-endo conformation. LNA, including an oligonucleotide, increases the thermal stability of hybrid strands. The use of LNA technology in molecular diagnostic methods improves the specificity and sensitivity of assays. This review describes routinely analyzed mutations and molecular diagnostic methods used in the clinical laboratory along with the performance improvement of mutational analysis with LNA.

The molecular diagnosis of the cancer mutational status is essential for modern clinical laboratory medicine. Mutations in EGFR, KRAS, BRAF, and PIK3CA genes are widely analyzed in solid tumors such as lung cancer, colorectal cancer, breast cancer, and melanoma. The allele-specific polymerase chain reaction, high-resolution melting, and Sanger sequencing are used for detecting and identifying gene mutations in many clinical laboratories. The locked nucleic acid (LNA) is a class of nucleic acid analogs that contain a methylene bridge connecting the 2' oxygen and 4' carbon in the ribose moiety. This methylene bridge locks the ribose group into a C3'-endo conformation. LNA, including an oligonucleotide, increases the thermal stability of hybrid strands. The use of LNA technology in molecular diagnostic methods improves the specificity and sensitivity of assays. This review describes routinely analyzed mutations and molecular diagnostic methods used in the clinical laboratory along with the performance improvement of mutational analysis with LNA.

Background: The limited negative predictive value of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has often been discussed. Objective: The aim of this study was to identify a highly sensitive molecular biomarker for lymph node staging by EBUS-TBNA. Methods: Five microRNAs (miRNAs) (miR-200a, miR-200b, miR-200c, miR-141, and let-7e) were selected as biomarker candidates for the detection of nodal metastasis in a miRNA expression analysis. After having established a cutoff level of expression for each marker to differentiate malignant from benign lymph nodes among surgically dissected lymph nodes, the cutoff level was applied to snap-frozen EBUS-TBNA samples. Archived formalin-fixed paraffin- embedded (FFPE) samples rebiopsied by EBUS-TBNA after induction chemoradiotherapy were also analyzed. Results: The expression of all candidate miRNAs was significantly higher in metastatic lymph nodes than in benign ones (p < 0.05) among the surgical samples. miR-200c showed the highest diagnostic yield, with a sensitivity of 95.4% and a specificity of 100%. When the cutoff value for miR-200c was applied to the snap-frozen EBUS-TBNA samples, the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were 97.4, 81.8, 95.0, 90.0, and 94.0%, respectively. For restaging FFPE EBUS- TBNA samples, the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were 100, 60.0, 80.0, 100, and 84.6%, respectively. Among the restaged samples, 4 malignant lymph nodes were false negative by EBUS-TBNA, but they were accurately identified by miR-200c. Conclusions: miR-200c can be used as a highly sensitive molecular staging biomarker that will enhance nodal staging of lung cancer.

BackgroundA genetic diagnosis has been rarely performed in benign familial hyperphosphatasaemia, and molecular mechanism largely remains unclear.ObjectivesWe encountered a case with benign familial hyperphosphatasaemia of intestinal alkaline phosphatase (IAP). To elucidate the molecular mechanism, we performedALPIgene sequencing and in vitro protein expression analysis.MethodsALPIgene was sequenced by long-range PCR and massively parallel sequencing. The soluble and membrane-bound ALP activities of the cultured cell line, transfected with the wild-type or variant-typeALPIgene were analysed by a glycosylphosphatidylinositol (GPI)-cleaving assay.ResultsWe identified a deletion–insertion variant in the C-terminal end of theALPIgene. This variant causes the attenuation of the hydrophobicity in GPI-anchor signal of IAP. An in vitro GPI-cleaving assay demonstrated that the membrane-bound IAP was greatly decreased, whereas the soluble IAP was increased, in the variant IAP.ConclusionsThe C-terminal variant inALPIcauses the benign familial hyperphosphatasaemia of IAP by the attenuation of the membrane-binding capability.

The c‐myc transcriptional suppressor, far‐upstream element (FUSE)‐binding protein (FBP)‐interacting repressor (FIR), is alternatively spliced in colorectal cancer tissue (Matsushita et al., Cancer Res 2006). Recently, the knockdown of SAP155 pre‐mRNA‐splicing factor, a subunit of SF3b, was reported to disturb FIR pre‐mRNA splicing and yield FIRΔexon2, an exon 2‐spliced variant of FIR, which lacks c‐myc repression activity. In the present study, novel splicing variants of FIR, Δ3 and Δ4, were also generated by SAP155 siRNA, and these variants were found to be activated in human colorectal cancer tissue. Furthermore, the expression levels of FIR variant mRNA were examined in the peripheral blood of colorectal cancer patients and healthy volunteers to assess its potency for tumor detection. As expected, circulating FIR variant mRNA in the peripheral blood of cancer patients were significantly overexpressed compared to that in healthy volunteers. In particular, the area under the receiving operating characteristic curve of FIR, FIRΔexon2 or FIRΔexon2/FIR, was greater than those of conventional carcinoembryonic antigen or carbohydrate antigen 19‐9. In addition, FIRΔexon2 or FIR mRNA expression in the peripheral blood was significantly reduced after operative removal of colorectal tumors. Thus, circulating FIR and FIRΔexon2 mRNA are potential novel screening markers for colorectal cancer testing with conventional carcinoembryonic antigen and or carbohydrate antigen 19‐9. Taken together, our results indicate that overexpression of FIR and its splicing variants in colorectal cancer directs feed‐forward or addicted circuit c‐myc transcriptional activation. Clinical implications for colorectal cancers of novel FIR splicing variants are also discussed in the present paper.

Alternative splicing is an important mechanism that links to transcription and contributes to protein diversity. Disturbed alternative splicing is frequently observed in cancers, but its precise mechanism remains largely unknown. FUSE-binding protein (FBP) -interacting repressor (FIR) is a transcriptional repressor of the c-myc gene. Previous studies indicated that a splice variant of FIR, FIRΔexon2, that lacks exon2 in the transcriptional repressor domain, was increased in colorectal cancers, hepatocellular carcinomas, and leukemia cells. Furthermore, FIRΔexon2 activated c-myc transcription by disabling wild-type FIR as a dominant-negative form of FIR. Recently, somatic mutations of the SF3B1 (SAP155) gene, a subunit of the SF3B spliceosome complex, were found in myelodysplastic leukemia. In this study, FIR heterozygous knockout (FIR(+/-)) was established as a dominant-negative model of FIR in the C57BL/6 mouse. FIR(+/-) mice showed an increased c-myc mRNA expression level, particularly in peripheral blood, although FIR(+/-) mice had no apparent pathogenic phenotype. Therefore, an increased c-myc mRNA expression level alone is not enough for leukemogenesis. Nevertheless, FIR(+/-)TP53(-/-) mice generated acute T-cell lymphoblastic leukemia (T-ALL) with increased organ and/or bone marrow invasion. In conclusion, alternative splicing of FIR, generating FIRΔexon2, contributes to not only colorectal carcinogenesis but also leukemogenesis independent of the c-Myc activation pathway. Finally, we will discuss our hypothesis that FIRΔexon2 interferes with FBW7, that FIRΔexon2 inhibits PP1 in the EGFR pathway, and that FIR haploinsufficiency is potentially associated with protein expression through transcriptional and post-transcriptional mechanisms.

Background Proteomic approaches may provide new insights into pathological conditions associated with alcoholism. The aim of this study was to conduct a proteomic analysis of liver tissue and serum in chronically alcohol-fed rats using agarose 2-dimensional gel electrophoresis (2-DE) and 3-step serum proteome analysis. Methods A total of 12 rats were pair-fed nutritionally adequate liquid diet containing ethanol as 36% of the total energy or an isocaloric control diet for 2 months. Rat liver homogenates and cytosol fractions were subjected to agarose 2-DE. Serum samples were subjected to 3-step serum proteome analysis involving immunodepletion of abundant proteins followed by fractionation using reverse-phase high-performance liquid chromatography and 1-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Candidate proteins were digested with trypsin and identified using mass spectrometry. Observed differences in protein expression levels were confirmed using Western blotting. Results A total of 46 protein spots were found to be differentially expressed in the liver homogenates and cytosol fractions of alcohol-fed rats relative to pair-fed controls. The most notable change was down-regulation of a 29-kDa protein, which was subsequently identified as carbonic anhydrase III (CA III). Down-regulation of this protein in alcohol-fed rats was confirmed by Western blotting. The messenger RNA level of CA III was decreased as well. In rat serum, a total of 41 proteins were differentially expressed. Of these proteins, only betaine–homocysteine methyltransferase (BHMT) was also found to be differentially expressed in the liver. Conclusions A combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats revealed that the expression of CA III is significantly down-regulated in the liver of alcohol-fed rats. Our results also showed that BHMT expression is up-regulated in both the liver and serum of alcohol-fed rats.

Hereditary colorectal cancer accounts for approximately 4-5% of all colorectal cancers. The causative genes for familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer are large, making comprehensive analyses difficult. Therefore, high-throughput and practical methods are required to make an early diagnosis of hereditary colorectal cancers and identify high-risk individuals. For this purpose, we developed a novel gene scanning method by high-resolution melting (HRM) analysis.High-resolution melting (HRM) analysis is a promising prescreening method for nucleic acid sequence variants because of its high sensitivity and high-throughput capability. We evaluated HRM for screening APC, MLH1, MSH2, and MSH6 genes for point mutations, small deletions, and insertions. Simultaneously, we evaluated quantitative polymerase chain reaction-HRM (qPCR-HRM) for screening the MSH2 gene for large rearrangements.All 28 point mutations and 1 large rearrangement were successfully detected by qPCR-HRM analysis.A fast and reliable mutation detection strategy with HRM and qPCR-HRM was used to diagnose hereditary colorectal cancers. Because this method is simple and economical, it may be useful in diagnostic laboratories.

Background: The currently available biological markers are limited in sensitivity and specificity for screening of excessive alcohol drinkers. We recently purified and identified 3 potential markers for alcoholism (5.9 and 7.8 kDa peptides and 28 kDa protein) by using surface-enhanced laser desorption/ionization (SELDI-TOF MS) technology. In the present study, we determined the diagnostic values of these novel markers in screening habitual drinkers with moderate alcohol consumption (hereafter habitual drinkers) in the general population as compared with 2 conventional markers: γ-glutamyltransferase (GGT) and carbohydrate-deficient transferrin (CDT). Methods: Serum samples obtained from a total of 128 Japanese who sought regular medical checkup were analyzed by SELDI-TOF MS. Results: The relative intensities of the 5.9 and the 28 kDa peak of habitual drinkers were significantly different from those in nondrinkers even in GGT nonresponders. The sensitivity and specificity were the highest in 5.9 kDa. When 5.9, 28 kDa, and GGT were combined, 96.8% of the habitual drinkers were successfully screened with a specificity of 60.9%. Conclusion: The results of this study clearly indicate that the 5.9 kDa peak is a promising novel biological marker for moderate alcohol consumption.

Alternative splicing is a fundamental process of gene regulation that contributes to protein diversity, a common phenomenon in the mammalian genome. Alternative splicing events not only happen in the normal gene regulation process, but are also closely related to certain diseases, including cancer. In this review, we briefly demonstrate the proof of concept (POC) of the relationship between alternative splicing and DNA damage, and describe the associations among alternative splicing and cancer pathogenesis, DNA damage, and gastrointestinal cancers. We discuss whether alternative splicing leads to genetic instability, which is considered to be a driving force for tumorigenesis. FUSE-binding protein (FBP) -interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2), upregulates c-myc transcription by inactivating wild-type FIR. FIR+/- mice exhibited marked c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Because the single knockout of TP53 generates thymic lymphoma, FIR+/-TP53-/- mice developed T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion and showed a poor prognosis. After describing the POC of alternative splicing of FIR in DNA damage and carcinogenesis, clinical application for cancer diagnosis and treatment by FIR/FIRΔexon2 was briefly summarized. Chiba University has prepared a biobank to support studies to develop biomarker detection, molecular diagnosis, and "Omics" research. In conclusion, alternative splicing of FIR, generating FIRΔexon2, potentially contributes to not only colorectal carcinogenesis, but also leukemogenesis, and a better understanding of the role and mechanism of alternative splicing in tumorigenesis may reveal new directions for cancer biomarker detection.

A number of methods of genotyping single nucleotide polymorphisms (SNPs) are currently available, ranging from the traditional restriction fragment length polymorphism (RFLP) and single-stranded conformational polymorphism (SSCP) to more sophisticated technologies. We determined the utility of three novel methods by genotyping aldehyde dehydrogenase-2 (ALDH2).DNA was isolated from blood samples of 241 control subjects and 74 patients with esophageal cancer. The utility of three novel genotyping methods-melting curve analysis using a LightCycler, SNaPshot analysis using an ABI PRISM 310 genetic analyzer, and denaturing high-performance liquid chromatography using a WAVE DNA fragment analysis system-were compared with that of conventional fluorescent-based polymerase chain reaction (PCR)-SSCP using an ALF express DNA sequencer.The frequency of the mutant ALDH2*2 allele was significantly higher in patients with esophageal cancer (27.7%) than in control subjects (16.2%; p0.01; habitual alcohol drinkers). The melting curve analysis was accurate, more rapid, and easier to use than the SNaPshot analysis or denaturing high-performance liquid chromatography analysis. Fluorescent-based PCR-SSCP proved useful for analyzing a large number of samples.Melting curve analysis using the LightCycler is suitable for the genotyping of small numbers of samples in a routine clinical setting; fluorescent-based PCR-SSCP analysis using the ALF express DNA sequencer can be used for large-scale genotyping in epidemiologic studies.

Background: There are remarkable interindividual differences in the expression of cytochrome P-4502E1(CYP2E1), which in turn may alter susceptibility to alcohol-related diseases and various cancers. We recently characterized the tandem repeat polymorphism in the 5'-flanking region of the human CYP2EI gene and found that subjects with the homozygous mutant-type (A4/A4) may be at higher risk of developing esophageal cancer. In this study, we determined how this tandem repeat polymorphism alters transcriptional activities of the human CYP2EI gene by transfection studies. Methods: The -5'-flanking region (-2562 base pair to +9 base pair) of the CYP2EI gene from three individuals of different genotypes (A2/A2, A2/A4, A4/A4) was amplified by polymerase chain reaction. The polymerase chain reaction products placed in front of a luciferase reporter gene were transfected into human hepatoblastoma cells, human esophageal cancer cells, and human uterus cancer cells. Transcriptional activities were determined by the dual-luciferase assay. When indicated, ethanol (50 mM) was included in the culture medium. CYP2EI messenger RNA levels in peripheral lymphocytes were measured by the real-time reverse transcription-polymerase chain reaction using the LightCycler system. Results: The construct including the tandem repeat region exhibited luciferase activities in both A2 and A4 type. It was of note that the activity produced by the A4 allele was significantly greater than that by A2 allele in HeLa cells (p < 0.001). CYP2EI messenger RNA levels in peripheral blood lymphocytes were comparable between the two genotypes. Conclusion: Transcriptional activity of the mutant allele of the tandem repeat polymorphism in the 5'-flanking region of the CYP2E1 gene is greater than that of the wild type.

Cytochrome P-450 (CYP) enzymes have been shown to play important roles in chemical carcinogenesis. Reports have indicated that polymorphism in CYP1A1, CYP1A2, CYP2A6, CYP2E1, and CYP2D6 are associated with an altered risk of developing cancers of the lung, bladder, liver, and colon. CYP2E1 plays an important role in alcohol metabolism and participates in the metabolic activation of various carcinogens, such as N-nitrosodimethylamine. Recently, we found repeated sequences that consist of four alleles (A1, A2, A3, and A4) and six genotypes (A1/A2, A2/A2, A2/A3, A2/A4, A3/A4 and A4/A4) of the CYP2E1 gene. In the present study, we investigated whether these polymorphisms are associated with lung cancer and esophageal cancer.DNA was isolated from blood samples of 192 healthy control subjects, 85 patients with lung cancer, and 82 patients with esophageal cancer in Japanese males. DNA samples were amplified by polymerase chain reaction in the 5'-flanking region of the CYP2E1 gene and in exon 12 in the ALDH2 gene. The tandem repeat polymorphisms were examined using DNA fragment analysis. Missense mutations in exon 12 of the ALDH2 gene were examined using fluorescence-based single strand conformational change polymorphism analysis. Written informed consent was obtained from all the subjects.In the CYP2E1 gene 5'-flanking region polymorphism, patients with esophageal cancer showed significantly higher frequency of the A4/A4 genotype compared with the control subjects (p = 0.02), but no difference was found in patients with lung cancer. The frequencies of the mutant ALDH2*2 allele were significantly higher in patients with esophageal cancer (27.7%) than in healthy control subjects (7.3%; p0.0001; habitual alcohol drinkers).The distribution of genotypes and allele frequencies of the tandem repeats of the 5'-flanking region of the CYP2E1 gene was significantly different in patients with esophageal cancer.

Objectives Recent studies have demonstrated that, in advanced colorectal carcinoma (CRC) patients, extended RAS (in KRAS exons 2–4 and NRAS exons 2–4) and BRAF mutations are negative predictors for anti-EGFR treatment efficacy and negative prognostic factor, respectively. Thus, high-throughput and cost-effective methods for identification of the mutation status are required. Design and methods We developed a PCR-high-resolution melting (HRM)-based method for screening extended RAS and BRAF mutations, and relative frequency of mutations in formalin-fixed paraffin-embedded samples of CRC was analyzed. Results Among 93 CRC samples, 29 harbored mutations in KRAS exon 2, and 9 harbored mutations in BRAF exon 15. Analysis of 55 KRAS exon 2 and BRAF exon 15 wild-type CRC samples identified the following mutations: 1/55 in exon 3 and 2/55 in exon 4 of KRAS; 1/55 in exon 2, 3/55 in exon 3, and 0/55 in exon 4 of NRAS. Conclusions Our PCR-HRM method will enable rapid determination of the extended RAS and BRAF mutation status prior to anti-EGFR treatment in the clinical setting.

Proton pump inhibitors (PPIs) are commonly used in ICUs. Here, we report a severe case of rhabdomyolysis associated with omeprazole. A 20-year-old man, who previously had been healthy, visited a hospital with epigastric pain. An upper gastrointestinal endoscopy revealed a duodenal ulcer in an active stage. He was admitted to the hospital and received intravenous omeprazole (20 mg) twice a day. On day 14 of admission, he developed muscular pain, predominantly in the lower extremities, and had elevated serum creatinine phosphokinase (CPK) (28,314 IU/L; normal is less than 25 IU/L) (Figure 1). The patient was transferred to the hospital’s ICU on day 16, since the serum CPK (112,240 IU/L) and myoglobin (25,082 ng/mL; normal is less than 154 ng/mL) levels were extremely high. After potential causes of elevated CPK were considered, omeprazole-associated rhabdomyolysis seemed the most probable diagnosis. We discontinued intravenous omeprazole administration and started aggressive fluid repletion, continuous renal replacement therapy, and urine alkalization. The CPK and myoglobin levels successively decreased and reached within the normal range on day 31. The patient recovered completely and was discharged on day 38.

Background: Poly-adenosine diphosphate (ADP)-ribosylation is involved in a variety of biological processes, which include DNA repair, malignant transformation, and apoptosis. It is of interest how this reaction is altered after long-term alcohol intake. Therefore, we determined long-term alcohol effects on hepatic poly-ADP-ribosylation in the rat. Methods: Male Sprague Dawley® rats (four pairs) were pair-fed a nutritionally adequate liquid diet that contained ethanol as 36% of total energy and an isocaloric control diets for 4 weeks. Liver tissue homogenates and nuclear fractions were subjected to ADP-ribosylation with [ 32 P]nicotinamide adenine dinucleotide. The ADP-ribosylated proteins were separated by SDS-PAGE, followed by autoradiography. Expression of poly-ADP-ribose polymerase (PARP) also was evaluated by Western blotting. Results: Incubation of rat liver homogenates in ADP-ribosylation reaction mixture resulted in a radiolabeling of a 116 kDa protein, most likely auto-ribosylation of PARP. This poly-ADP-ribosylation was increased significantly (p < 0.025) after long-term alcohol intake. This alcohol effect was reproducible in nuclear fractions as well. Expression levels of PARP, however, were comparable between alcohol-fed rats and their pair-fed controls. Conclusion: Poly-ADP-ribosylation, an important posttranslational modification of nuclear proteins, was increased significantly after chronic alcohol consumption in the rat.

Background: Recently, a restriction fragment length polymorphism in the regulatory region of the CYP2EI gene was identified. It has been suggested that the polymorphism is associated with the elevated activity of the cytochrome P-450 2E1 (CYP2E1) enzyme in obese or alcoholic subjects. However, significance of the polymorphism in connection with alcoholism has not been studied. In the present study, we have characterized these repeated sequences in the 5'-untranslated region of the CYP2E1 gene in Japanese subjects and North American white subjects and investigated whether these polymorphisms are associated with drinking habits and alcoholism. Methods: DNAs were isolated from blood samples of 192 Japanese nonalcoholics and 202 alcoholics as well as 125 North American white nonalcoholics. DNA samples were amplified by polymerase chain reaction and subjected to a fluorescent-based single-strand conformational change polymorphism analysis, DNA fragment analysis, and polymerase chain reaction-direct sequencing. Results: Four alleles (A1-A4) were found, which were differentiated by the six subunits (L1, L2, L3, L4, S1, S2) based on the size difference and nucleotide replacement. A2 and A4 alleles were observed in the two ethnic groups (A2: Japanese subjects, 0.752; white subjects, 0.976; A4: Japanese subjects, 0.227; white subjects, 0.016). However, A1 allele was found only in North American white subjects (0.008), and A3 allele was detected only in Japanese subjects (0.021). Allele frequencies were significantly different between the two ethnic groups (p < 0.0001). Distribution of genotypes between Japanese nonalcoholics and alcoholics were not significantly different. Also, no significant difference for the allele frequencies was observed between Japanese moderate drinkers and heavy drinkers. Conclusions: Our data suggested no association among the polymorphic repeats, drinking behavior, and alcoholism. Allele frequencies were significantly different between Japanese subjects and North American white subjects.

Background: Prevalence of transfusion-transmitted virus (TTV) infection among blood donors and in patients with liver diseases in China was studied. Methods: DNA was extracted from serum and amplified by seminested polymerase chain reaction with reported primer sets from a conserved region of the TTV genome. Results: TT Virus DNA was detected in 55 of 196 blood donors (28%); 31% (40 of 127) in the north and 22% (15 of 69) in the south. TT Virus DNA was also detected in 14 of 31 patients (45%) with non-A–non-G fulminant hepatitis and in eight of 25 patients (32%) with non-A–non-G chronic hepatitis. The rate of TTV viraemia in these patients with liver disease was comparable to that in blood donors. TT Virus DNA sequencing of 12 isolates showed that the prevalence of genotype 2 was significantly higher than that reported in Japan (66.7 vs 2.6%, P < 0.001). Furthermore, genotyping assays based on restriction fragment length polymorphism were carried out on all 88 TTV DNA-positive samples. It was found that 42 isolates (47.7%) belonged to genotype 1 and 40 (45.5%) to genotype 2. It was of particular interest that the prevalence of genotype 1 in patients with non-A–non-G fulminant hepatitis was significantly higher than that in blood donors (10/14 vs 22/55, P < 0.05). Conclusions: The data indicate that TTV infection is common in China and that the pathogenic potential of TTV toward the liver (if any) may differ between genotypes.

ADP-ribosylation is a posttranslational protein modification catalyzed by two classes of enzymes: mono-ADP-ribosyltransferase and poly-ADP-ribose polymerases. We previously demonstrated that long-term alcohol intake remarkably enhanced an endogenous ADP-ribosylation of a 58 kDa protein in rat liver and also identified the 58 kDa protein as phosphoglucomutase (PGM). To assess biological significance of this phenomenon, we tested the effects of long-term alcohol intake on PGM activities in connection with posttranslational modification of the protein. ADP-ribosylation of PGM was mono- rather than poly-ADP-ribosylation. Also, nonenzymatic binding of ADP-ribose was excluded. It was of note that ADP-ribosylation of exogenous PGM was remarkably increased by adding rat liver plasma membranes, and that the extent of the increase was greater in alcohol-fed rats than in pair-fed controls. Furthermore, PGM activities were significantly increased after long-term alcohol intake concomitant with increased ADP-ribosyltransferase activities toward PGM. In view of the variety of roles of PGM in the liver, such as carbohydrate metabolism and Ca2+ homeostasis, it is tempting to speculate that increased ADP-ribosylation of PGM may play a role in long-term alcohol effects on hepatocytes.

Background: Excessive alcohol consumption is a health risk that can lead to a variety of medical and social problems. Carbohydrate Deficient Transferrin (CDT) is now widely used for detection of chronic alcohol abuse and monitoring sobriety in alcoholics. We assessed the diagnostic performance of the direct immunoassay for %CDT (N Latex CDT) in Japanese habitual drinkers at their annual medical check-up. We also tested whether this direct assay is influenced by the presence of non-alcoholic liver disease including non-alcoholic steatohepatitis and HCVrelated hepatocellular carcinoma. Methods: We performed the N Latex CDT assay using a Siemens BN-II nephelometric analyzer to measure CDT concentration. The reference intervals of %CDT and gamma glutamyl transferase (GGT) activities were obtained based on their measurements in 160 apparently healthy nondrinkers. Habitual drinkers were divided into light drinkers (less than 40g ethanol/day) and heavy drinkers (more than 60g ethanol/day) Furthermore, %CDT levels in a total of 184 patients with non-alcoholic chronic liver diseases including Hepatitis C Virus (HCV) related liver cirrhosis, Non-Alcoholic Steatohepatitis (NASH) were determined. Results: In the heavy drinkers, the positive rates of %CDT and GGT were 58.3% and 41.7%, respectively. The combination assay of %CDT and GGT resulted in 75% sensitivity and 95% specificity. It was notable that 57% of GGT non-responders were detected by CDT measurements. The mean serum level of %CDT was not influenced by the presence of NASH, but was increased in patients with moderate-severe HCV-related liver cirrhosis. The %CDT levels were also high in patients with hepatocellular carcinoma with liver cirrhosis, but were not related to their tumor stages. Conclusions: The %CDT determined by automated N Latex CDT may be useful biomarker complementary to GGT to detect excessive habitual drinkers in Japan. However, caution should be taken to interpret the results in advanced non-alcoholic liver cirrhosis.

Spinocerebellar ataxia type 31 (SCA31) is defined by the presence of an insertion mutation containing a TGGAA repeat within the intron of the brain-expressed, associated with NEDD4 (BEAN) gene. Detecting this mutation is conventionally done by southern blotting or DNA sequencing, but these methods are technically demanding and not easily implemented in clinical diagnosis. Here, we adapted repeat-primed PCR (RP-PCR) to develop a clinical genetic test for SCA31 using only the PCR process to detect the TGGAA repeat within the insertion mutation. Pentanucleotide RP-PCR and subsequent DNA fragment analysis demonstrated characteristic ladder peaks with a 5-bp periodicity, originating from the TGGAA repeat, in 100% of samples (n=14) from SCA31 patients in whom the presence of the TGGAA repeat had been verified by DNA sequencing. No peaks were observed in a normal control and two non-SCA31 patients, in whom the TGGAA repeat was absent. This method is valuable for genetic diagnosis of SCA31 in clinical practice.

After hepatitis B virus (HBV) e antigen (HBeAg) seroconversion, HBV-DNA continues to replicate, and HBeAg-negative patients still face the risk of liver disease progression. We investigated the predictive factors for alanine aminotransferase (ALT) elevation, antiviral drug use, and hepatocellular carcinoma (HCC) occurrence in HBeAg-negative patients.Age, sex, ALT, platelet counts, HBV-DNA levels, genotype, antidiabetic drug use, body mass index, smoking, and alcohol consumption were analyzed for a total of 244 HBV carriers who were HBeAg-negative.Of 244 HBeAg-negative patients, 158 (64.8%) showed normal ALT levels at baseline. Multivariate Cox hazard regression analysis identified high HBV-DNA levels and high ALT at baseline as independent risk factors for ALT elevation in the patients with normal ALT at baseline. The threshold ALT and HBV-DNA levels were determined to be 31 IU/L and 5.3 log copies/mL, respectively. Seventeen (7.0%) patients used antiviral drugs. Multivariate Cox hazard regression analysis identified high HBV-DNA levels (threshold, 5.7 log copies/mL), the use of antidiabetic drugs, and daily alcohol consumption at baseline as an independent risk factor for the use of antiviral drugs in HBeAg-negative patients. In 10 patients (4.1%), HCC was detected, and a low platelet count (threshold, 10.0×10(4)/mm(3)) was associated with the occurrence of HCC.This study identified predictors of future active liver disease in HBeAg-negative patients, i.e. ALT elevation, unavoidable use of antiviral drugs, and occurrence of HCC.

To determine the risk factors for the occurrence of hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV) infection.A total of 620 patients who tested positive for hepatitis B surface antigen and were referred to Chiba University Hospital between February 1985 and March 2008 were included in the study and the following characteristics were analyzed: age, gender, status of hepatitis B e antigen, alanine aminotransferase level, HBV DNA level, and number of platelets (PLTs).HCC was detected in 30 cases during the follow-up period (5.4 +/- 5.1 years). Multivariate analysis revealed that age40 years [compared with patients aged40 years; odds ratio (OR) = 4.28; 95% confidence interval (CI) = 1.68-10.9] and PLT level206,000/microl (compared with patients with a higher PLT level; OR = 8.50; 95% CI = 1.98-36.2) were predictive factors for HCC occurrence. In patients aged40 years, the HBV DNA level (compared with5.0 log copies/ml; OR = 4.22, 95% CI = 1.13-15.8) and PLT level (compared with patients with196,000/microl PLTs; OR = 15.6, 95% CI = 2.06-118.3) were predictive factors for HCC occurrence.Advanced age and low PLT level were risk factors for HCC occurrence in patients with HBV infection. In patients aged40 years, viral load was also a risk factor for HCC.

Development of useful biomarkers is pivotal for prediction of micro-metastasis, recurrence probability and/or prognosis of the patients. Recent studies have revealed that cancer-specific alternative splicing can be valuable for cancer cell detection. Among them, FUSE-binding protein-interacting repressor, FIR, has been reported to repress c-myc transcription and its exon2-spliced variant, FIRDelta(exon)2, is unable to repress c-myc by competing with authentic FIR in vivo and in vitro. Moreover FIRDelta(exon)2 was frequently discovered in human primary colorectal cancers, but not in the adjacent normal tissues, indicating its cancer-related expression. Thus, the expression level of FIRDelta(exon)2 mRNA in the colorectal cancer tissues as tumor marker candidates is examined. Further, to determine the interacting proteins, FIR-flag or FIRDelta(exon)2-flag stably expressing HeLa cells have been established by G418 selection and nuclear proteins were co immunoprecipitated with flag-conjugated magnetic beads. Those co-immunoprecipitated proteins with FIR or FIRDelta(exon)2 are candidates of tumor makers. In addition, substances that interfers FIR mRNA splicing should be anti-cancer drugs. Together, FIR splicing variant, FIRDelta(exon)2 mRNA or proteins and its interacting proteins are applicable for novel screening tumor markers in colorectal cancer detection.

Background: The human cytochrome P-4502E1 (CYP2E1) has been known to be that are associated with alcohol-related diseases and various cancers. Difference in susceptibility in enzyme induction of the CYP2E1 by environmental factors such as ethanol may be associated with the risk of these diseases. In the 5′-flanking region of the human CYP2E1 gene, there were tandem repeat polymorphism (CYP2E1*1C*,1D) and RsaI polymorphism (CYP2E1*5A*,5B), which have been reported to be related to these diseases. Thus, we focused relationship between these polymorphisms and transcriptional regulation. The aim of this study was to determine the role of these polymorphisms for the transcriptional regulation of the human CYP2E1 gene in general transcription and during enzyme induction. Methods: To determine the role of these polymorphisms, various constructs of the 5′-flanking region of the human CYP2E1 gene were prepared and transfected into HeLa cells and HepG2 cells treated or not with pyrazine, a well-known CYP2E1 inducer. Results: The transcriptional activity of CYP2E1*1D was higher than that of CYP2E1*1C in HeLa cells. The −1,306 base pairs (bp) construct (−1,306 to +9) increased transcriptional activity compared with the 2.5 kb full-length construct. The suppressive effect of transcriptional activity by deletion of the region including CYP2E1*1C was greater than that by CYP2E1*1D in HeLa cells. The −580 bp construct (−580 to +9) decreased transcriptional activity compared with the −1,306 bp construct. The −1,306 bp construct also showed a similar tendency during pyrazine treatment. Conclusions: The region including the tandem repeat polymorphism was suggested to regulate transcription suppressively. Besides, it was speculated that the transcriptional activity of CYP2E1*1D was higher than that of CYP2E1*1C because transcriptional suppression of CYP2E1*1D was weaker than that of CYP2E1*1C. Also, it was confirmed that the region including RsaI polymorphism was associated with the promotion of transcription.

OBJECTIVE Familial Mediterranean fever (FMF) had been considered a rare disease in Japan, but since the identification of the causative gene (MEFV) for pyrin/marenostrin in 1997, the occurrence of FMF has been successively reported. We reviewed the clinical features of 7 patients diagnosed with FMF by gene analysis. METHODS During April 2003 and March 2005, we investigated the clinical symptoms, treatment and MEFV types of 7 FMF patients who consulted the General Outpatient Clinic of Chiba University Hospital. RESULTS Six patients were in their 20-30s, and one was 54 years of age. There were 4 males and 3 females including a mother and child, and an adult male and his female cousin. Three were solitary incidences. In addition to intermittent fever, 4 patients had chest pain, 1 had abdominal pain, and 1 had chest or abdominal pain. The frequency of attacks was once per 3 months to 1 year in the early stage of the disease, but it slowly increased with disease progression. Leukocytosis and C-reactive protein (CRP) elevation were noted during attacks in all patients. On investigation of MEFV, heterozygosity for the compound pyrin L110P-E148Q/M694I, E148Q/M694I, L110P/E148Q and heterozygosity for pyrin variant M694I alone were detected. Daily administration of colchicine at 0.5 mg prevented attacks in 6 patients, however 1 patient required 1.0 mg for adequate prevention. CONCLUSIONS Although the incidence is rare, internists should be aware of the characteristic symptoms of FMF: periodic fever and serositis symptoms, and its presence in Japan despite the disease name.

Cytochrome P450 2E1 (CYP2E1) catalyzes the metabolic activation of the procarcinogen, N-nitrosodimethylamine, and cytotoxic carbon tetrachloride compounds. A tandem repeat polymorphism in the 5'-flanking region of the CYP2E1 gene was investigated in non-small cell lung carcinoma (NSCLC) patients to clarify the relationship between CYP2E1 gene polymorphism and lung cancer susceptibility. Blood samples were taken from 236 healthy control subjects (192 males and 44 females) and 111 patients (78 males and 33 females) who underwent surgery for NSCLC in Japan. DNA was isolated from these samples and the 5'-flanking region of the CYP2E1 gene was amplified by polymerase chain reaction and examined for tandem repeat polymorphisms using DNA fragment analysis. Sequence analysis confirmed the presence of three alleles, A2, A3, and A4 (361, 367, and 457 bp, respectively), with four genotypes observed in the lung cancer group and five genotypes in the control group. There was a statistically significant difference in genotype distribution between the lung adenocarcinoma and control group (P=0.0088, A4/A4 vs. non-A4/A4). In the lung adenocarcinoma group, the univariate risk estimates for the A4/A4 subgroup compared to the most common subgroup (A2/A2) was 4.300 (95% confidence interval = 1.358-13.618, P=0.0131). We conclude that the A4/A4 genotype of the 5'-flanking region of CYP2E1 was significantly more frequent in lung adenocarcinoma cases than in healthy controls and, therefore, may be involved in the development of lung adenocarcinoma.

The fibroblast growth factors (FGFs) are involved in hematopoiesis and tumorigenesis. However, little is known about the contribution of the FGFs identified within the past 10 years to leukemogenesis. To elucidate whether these FGFs (FGF-8, -9, -10, -11, -12, -13, -14, -16, -17, -18, -19, -20, and -21) are expressed in leukemic cells, we performed RT-PCR analyses using 28 cell lines. The members of a fetal-oncogenic subfamily, FGF-8/-17/-18, were often expressed (53.5%, 25.0%, and 32.1%) with the co-expression of their receptors. Realtime quantitative-PCR analysis showed that FGF-8/-17 were aberrantly expressed in patients with acute leukemia. Moreover, cell proliferation assays revealed the proliferation activity of FGF-17 on leukemic cells expressing its receptors. These results demonstrated that certain recently identified FGFs play an important role in the growth of leukemic cells, possibly with an autocrine mode of action, and that these FGFs will become novel biomarkers for hematopoietic tumors.

Detection of virus genomes and their mutants by molecular technology is essential in clinical practice for patients with hepatitis B virus(HBV) and hepatitis C virus(HCV) related liver diseases. We here briefly summarize genome structures of HBV-DNA and HCV-RNA. Also, we describe representative methods to detect and quantify HBV-DNA and HCV-RNA.

Background and Aim: Hepatitis B virus (HBV) variants with mutations in the S gene would pose a substantial risk to the community as current HBV vaccines are not effective in preventing infection with them. The majority of such vaccine escape mutants so far reported have been found while studying vertical transmission of HBV; the vaccine failure rate in connection with vaccine escape mutants in adults is not clear at the moment. The purpose of this study was to evaluate the efficacy of immunization against HBV in the adult population by analysis using polymerase chain reaction (PCR) to detect HBV-DNA, and also to elucidate the type of mutation encountered in vaccine failure cases. Method: A total of 176 adult restaurant employees in China, who had been vaccinated according to the food epidemic law, were enrolled in a standard vaccination program. Their serum HBV-DNA was determined before and 1 year after the completion of the vaccination program. In those infected with HBV, despite having received the HBV vaccine, direct sequencing within the S gene of the amplified samples was conducted. Results: Although only two cases were found to be hepatitis B surface antigen (HBsAg) positive 1 year after the completion of the vaccination program, six subjects (3.4%) were found to be HBV-DNA positive assessed by a nested PCR. Four out of these six cases had a point mutation within the ‘a’ determinant; they were Gly-145-Ala, and Ile/Thr-126-Asn/Ser. Conclusion: The HBV vaccine failure rate assessed by using PCR analysis was 3.4% (six of 176) in the Chinese adult population undergoing the HBV vaccination program. Hepatitis B virus variants with missense mutation within the ‘a’ determinant were responsible in most cases.

Cytochrome P-4502E1 (CYP2E1) is a major component of the microsomal ethanol-oxidizing system (MEOS) and is also involved in the metabolism of a variety of foreign compounds, including carcinogens. It has been shown that there are interindividual variations in the expression of human CYP2E1. Gene-environmental interactions have been suggested to account for the difference. In this study, we screened nine exons and exon-intron junctions of the human CYP2E1 gene for detecting allelic variants in genomic DNA samples obtained from 115 Japanese controls, 96 alcoholics, and 44 patients with alcoholic liver diseases. A novel missense mutation in exon 2 (V72L) was found in Japanese controls, but the frequency was low (2.6%). In addition, two novel silent mutations (T303T and F420F), together with one mutation in intron 2, were found. However, no association of these mutations with alcoholism and alcoholic liver diseases was found. Our data indicate that nucleotide replacement in the open reading frame of CYP2E1 gene is not a major factor for interindividual differences in expression of CYP2E1 and susceptibility to alcohol-related disorders.

7046 Background: Genetic information is essential for molecular targeted therapy for personalized medicine, although tissue sampling for genetic analysis remains challenging. In this study, we investigated the utility of bronchoscopic ultra-micro samples compared with conventional histological materials for multiple gene analyses for NSCLC. Methods: Patients with NSCLC proven by on-site cytological examinations during bronchoscopic survey were eligible. Following conventional needle aspiration biopsy by flexible bronchofiberscope (from primary lesion) or convex probe endobronchial ultrasound (from lymph nodes), the used needle was rinsed with 20 ml of saline and this ultra-micro sample (MS) was used for both cytological diagnosis and genetic analysis. DNA and RNA were extracted from each sample. Gene mutations [EGFR (epidermal growth factor receptor), K-ras, BRAF] and anaplastic lymphoma kinase (ALK) fusion genes were examined by high resolution melting analysis and direct sequencing. The results of cytological diagnoses and gene profiles using MS were compared to conventional histological diagnoses. Results: A total of 79 lesions (30 primary and 49 metastatic nodes) from 76 patients were eligible. Adenocarcinoma (N = 46), squamous cell carcinoma (N = 25), and NSCLC (N = 8) were pathologically confirmed in histological lesion cores; however, only 29 malignancies (37%) were detected with MS. DNA and RNA could be extracted from all histological samples and MS. In 35 cases, genetic disorders were identified, including EGFR mutations (N = 12), K-ras mutations (N = 5), BRAF mutation (N = 1), ALK fusion genes (N = 3), and silent mutations (N = 13); these results were identical for both histological samples and MS. Conclusions: Bronchoscopic ultra-micro samples (biopsy needle rinse fluids) are useful for multiple genetic examinations.

Introduction: Although genetic information is essential for molecular targeted therapy for personalized medicine, tissue sampling for genetic analysis remains challenging. We investigated the utility of bronchoscopic sampling in non–small-cell lung cancer (NSCLC) patients compared with conventional histological materials for multiple genetic analyses. Methods: Patients with NSCLC proven by onsite cytological evaluation during bronchoscopic survey were eligible for this study. After conventional needle aspiration biopsy by flexible bronchofiberscopy of primary lesions or convex-probe endobronchial ultrasound of lymph nodes, the used needle was rinsed with saline, and the ultra-microsample (uMS) was used for cytological diagnosis and genetic analysis. Gene mutations and fusion genes were examined by high-resolution melting analysis and direct sequencing. The results from the uMS and those from conventional histological samples were compared. Results: A total of 134 lesions (48 primary and 86 metastatic) were analyzed. Adenocarcinoma ( n = 80), squamous-cell carcinoma ( n = 43), and NSCLC ( n = 11) samples were pathologically confirmed in histological cores; however, malignancies were detected in only 45 (34%) of the corresponding uMS. In 62 samples, genetic disorders, including epidermal growth factor receptor ( n = 21), K-ras ( n = 11), and BRAF mutations ( n = 1); anaplastic lymphoma kinase ( n = 5), receptor tyrosine kinase ( n = 1), and RET fusion genes ( n = 1); and silent mutations ( n = 22), were identified. In total, 1474 molecular tests were performed, and 1464 tests (99.3%) were identical for both histological samples and uMS. Conclusion: Bronchoscopic uMS (biopsy needle rinsed fluids) are useful for multiple genetic examinations in NSCLC.

// Kazuyuki Matsushita 1,2,* , Kouichi Kitamura 1,2,* , Bahityar Rahmutulla 1 , Nobuko Tanaka 1 , Takayuki Ishige 1,2 , Mamoru Satoh 1 , Tyuji Hoshino 3 , Satoru Miyagi 4 , Takeshi Mori 5 , Sakae Itoga 2 , Hideaki Shimada 6 , Takeshi Tomonaga 7 , Minoru Kito 8 , Yaeko Nakajima-Takagi 4 , Shuji Kubo 9 , Chiaki Nakaseko 10 , Masahiko Hatano 11 , Takashi Miki 12 , Masafumi Matsuo 5,13 , Masaki Fukuyo 14 , Atsushi Kaneda 14 , Atsushi Iwama 4 and Fumio Nomura 1,2 1 Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Inohana, Chiba, Japan 2 Division of Laboratory Medicine, Chiba University Hospital, Inohana, Chiba, Japan 3 Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana, Chiba, Japan 4 Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Inohana, Chuo-ku, Chiba, Japan 5 Department of Pediatrics, Graduate School of Medicine, Kobe University, Kusunoki-cho, Kobe, Japan 6 Department of Surgery, School of Medicine, Toho University, Omori-nishi, Ota-ku, Tokyo, Japan 7 Laboratory of Proteome Research, National Institute of Biomedical Innovation, Saito-Asagi, Ibaraki, Osaka, Japan 8 Oriental Yeast Co., Ltd. Azusawa, Itabashi-ku, Tokyo, Japan 9 Department of Genetics, Hyogo College of Medicine, Mukogawa-cho, Nishinomiya, Hyogo Prefecture, Japan 10 Department of Haematology, Chiba University Hospital, Inohana, Chiba, Japan 11 Department of Biomedical Science, Graduate School of Medicine, Chiba University, Inohana, Chiba, Japan 12 Department of Medical Physiology, Graduate School of Medicine, Chiba University, Inohana, Chiba, Japan 13 Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, Arise, Ikawadani, Nishi, Kobe, Japan 14 Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Inohana, Chiba, Japan * These authors contributed equally to this work Correspondence: Kazuyuki Matsushita, email: // Keywords : FBP interacting repressor (FIR), splicing variant, haplo-insufficiency, leukemia, P53, T-ALL Received : November 27, 2014 Accepted : December 27, 2014 Published : December 31, 2014 Abstract FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon 2/ FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout ( FIR +/− ) C57BL6 mice were generated. FIR complete knockout ( FIR −/− ) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR +/− mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2 / FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR +/- TP53 -/- generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR +/− TP53 −/− compared with that in FIR +/+ TP53 −/− mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion, the alternative splicing of FIR, which generates FIRΔexon2, may contribute to both colorectal carcinogenesis and leukemogenesis.