Rina Okada

Comparison of Hepatocellular Carcinoma miRNA Expression Profiling as Evaluated by Next Generation Sequencing and Microarray

MicroRNA (miRNA) expression profiling has proven useful in diagnosing and understanding the development and progression of several diseases. Microarray is the standard method for analyzing miRNA expression profiles; however, it has several disadvantages, including its limited detection of miRNAs. In recent years, advances in genome sequencing have led to the development of next-generation sequencing (NGS) technologies, which significantly advance genome sequencing speed and discovery. In this study, we compared the expression profiles obtained by next generation sequencing (NGS) with the profiles created using microarray to assess if NGS could produce a more accurate and complete miRNA profile. Total RNA from 14 hepatocellular carcinoma tumors (HCC) and 6 matched non-tumor control tissues were sequenced with Illumina MiSeq 50-bp single-end reads. Micro RNA expression profiles were estimated using miRDeep2 software. As a comparison, miRNA expression profiles for 11 out of 14 HCCs were also established by microarray (Agilent human microRNA microarray). The average total sequencing exceeded 2.2 million reads per sample and of those reads, approximately 57% mapped to the human genome. The average correlation for miRNA expression between microarray and NGS and subtraction were 0.613 and 0.587, respectively, while miRNA expression between technical replicates was 0.976. The diagnostic accuracy of HCC, p-value, and AUC were 90.0%, 7.22×10−4, and 0.92, respectively. In summary, NGS created an miRNA expression profile that was reproducible and comparable to that produced by microarray. Moreover, NGS discovered novel miRNAs that were otherwise undetectable by microarray. We believe that miRNA expression profiling by NGS can be a useful diagnostic tool applicable to multiple fields of medicine.

Ultradeep Sequencing for Detection of Quasispecies Variants in the Major Hydrophilic Region of Hepatitis B Virus in Indonesian Patients

ABSTRACT Quasispecies of hepatitis B virus (HBV) with variations in the major hydrophilic region (MHR) of the HBV surface antigen (HBsAg) can evolve during infection, allowing HBV to evade neutralizing antibodies. These escape variants may contribute to chronic infections. In this study, we looked for MHR variants in HBV quasispecies using ultradeep sequencing and evaluated the relationship between these variants and clinical manifestations in infected patients. We enrolled 30 Indonesian patients with hepatitis B infection (11 with chronic hepatitis and 19 with advanced liver disease). The most common subgenotype/subtype of HBV was B3/adw (97%). The HBsAg titer was lower in patients with advanced liver disease than that in patients with chronic hepatitis. The MHR variants were grouped based on the percentage of the viral population affected: major, ≥20% of the total population; intermediate, 5% to <20%; and minor, 1% to <5%. The rates of MHR variation that were present in the major and intermediate viral population were significantly greater in patients with advanced liver disease than those in chronic patients. The most frequent MHR variants related to immune evasion in the major and intermediate populations were P120Q/T, T123A, P127T, Q129H/R, M133L/T, and G145R. The major population of MHR variants causing impaired of HBsAg secretion (e.g., G119R, Q129R, T140I, and G145R) was detected only in advanced liver disease patients. This is the first study to use ultradeep sequencing for the detection of MHR variants of HBV quasispecies in Indonesian patients. We found that a greater number of MHR variations was related to disease severity and reduced likelihood of HBsAg titer.

Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes

BackgroundClarithromycin (CLR) is the key drug in eradication therapy of Helicobacter pylori (H. pylori) infection, and widespread use of CLR has led to an increase in primary CLR-resistant H. pylori. The known mechanism of CLR resistance has been established in A2146G and A2147G mutations in the 23S rRNA gene, but evidence of the involvement of other genetic mechanisms is lacking. Using the MiSeq platform, whole-genome sequencing of the 19 clinical strains and the reference strain ATCC26695 was performed to identify single nucleotide variants (SNVs) of multi-drug resistant efflux pump genes in the CLR-resistant phenotype.ResultsBased on sequencing data of ATCC26695, over one million sequencing reads with over 50-fold coverage were sufficient to detect SNVs, but not indels in the bacterial genome. Sequencing reads of the clinical isolates ranged from 1.82 to 10.8 million, and average coverage ranged from 90.9- to 686.3-fold, which were acceptable criteria for detecting SNVs. Utilizing the conventional approach of allele-specific PCR, point mutations in the 23S rRNA gene were detected in 12 clinical resistant isolates, but not in 7 clinical susceptible isolates. All sequencing reads of CLR-resistant strains had a G mutation in an identical position of the 23S rRNA gene. In addition, genetic variants of four gene clusters (hp0605-hp0607, hp0971-hp0969, hp1327-hp1329, and hp1489-hp1487) of TolC homologues, which have been implicated in multi-drug resistance, were examined. Specific SNVs were dominantly found in resistant strains.ConclusionsGene clusters of TolC homologues are involved in CLR susceptibility profiles in individual H. pylori strains. Whole-genome sequencing has yielded novel understanding of genotype-phenotype relationships.

A deep-sequencing method detects drug-resistant mutations in the hepatitis B virus in Indonesians.

Objective: The long-term administration of a nucleos(t)ide analogue (NA) for the treatment of chronic hepatitis B may encourage the emergence of viral mutations associated with drug resistance. Minor populations of viruses may exist before treatment, but are difficult to detect because of technological limitations. Identifying minor viral quasispecies should be useful in the clinical management of hepatitis B virus (HBV) infection. Methods: Six treatment-naïve Indonesian patients with chronic HBV infection participated in this study. The polymerase region of the HBV genome, including regions with known drug-resistant mutations, was subjected to capillary sequencing and MiSeq sequencing (Illumina). Mutations were analyzed with Genomics Workbench software version 6.0.1 (CLC bio). Results: The mean mapping reads for the six samples was 745,654, and the mean number of amplified fragments ranged from 17,926 to 25,336 DNA reads. Several known drug-resistant mutations in the reverse transcriptase region were identified in all patients, although the frequencies were low (0.12-1.06%). The proportions of the total number of reads containing mutations I169L/M, S202R, M204I/L or N236S were >1.0%. Conclusion: Several known NA-resistant mutations were detected in treatment-naïve patients in Indonesia using deep sequencing. Careful management of such patients is essential to prevent drug-resistant mutations from spreading to other patients.

696 Whole-Genome Sequencing Detects Novel Virulence Variants of Helicobacter pylori cagA Gene and Its Relationship With Oncogenic cagA

The H. pylori cag type 4 secretion system (T4SS) translocates CagA into host cells which can lead to NF-κβ activation. The T4SS component CagY is essential for CagA translocation. cagY harbors two repeat motifs which engender considerable genetic diversity to this locus. We used Mongolian gerbils to select for Hp derivative strains that differed in intensity of host interactions to define the biology of long-term colonization and genetic alteration of virulence factors within the gastric niche. Gerbils were initially challenged with the carcinogenic Hp strain 7.13 for 12 weeks; two derivative isolates, 7.13-1 and 7.13-2, were then obtained from 2 independent gerbils. In vitro, strain 7.13-1 induced significantly higher levels of NF-κβ activation and CagA translocation in AGS gastric epithelial cells compared to strain 7.13-2 (fold/uninfected: 100±7 vs. 26±7, respectively; p<0.001) .We next infected new populations of gerbils with parental 7.13 and the two polar 7.13 Hp derivatives for up to 16 weeks. There were no differences in colonization among the groups, although all animals that developed adenocarcinoma harbored decreased bacterial burdens. Strain 7.131 induced premalignant lesions in 25%, 50%, 62.5%, and 75% at 4, 8, 12, and 16 weeks respectively. In contrast, the incidence of dysplasia and adenocarcinoma was only 25% and 12.5% with the 7.13 parental strain or the 7.13-2 derivative 16 weeks post-infection. In vivo-readapted strains from animals infected with parental 7.13 and 7.13-1 maintained their ability to induce high levels of NFκβ activation similar to the input strains, except for strains isolated from animals that developed cancer, which were attenuated in the ability to activate NF-κβ and translocate CagA. In contrast, readapted isolates from animals infected with strain 7.13-2 induced increased levels of NFκβ activation and CagA translocation in vitro, which was concordant with increased levels of inflammation in the corresponding gerbils. To define mechanisms that mediate these phenotypes, we examined cagY rearrangements by RFLP. We identified two distinct patterns, one specific for parental 7.13 and 7.131 and the other for strain 7.13-2. Readaptation revealed that a proportion of parental 7.13 (25%) and 7.13-1 (37.5%) isolate derivatives altered their cagY pattern, developing a 7.132-type pattern in the presence of cancer which was accompanied by decreases in NFκβ activation and CagA translocation. In contrast, 62.5% of Hp readapted isolates from animals infected with strain 7.13-2 changed their cagY pattern to one that mirrored the original 7.13 parental strain and those changes were linked to increases in inflammation, NFκβactivation and CagA translocation. Collectively, these results suggest Hp can modify function of the T4SS by alternating cagY genotype in response to the neoplastic landscape within gastric mucosa.

Quasispecies variant of pre-S/S gene in HBV-related hepatocellular carcinoma with HBs antigen positive and occult infection

BackgroundHepatocellular carcinoma (HCC) can develop in patients who are negative for the hepatitis B surface antigen (HBsAg) in serum but positive for hepatitis B virus (HBV) DNA in the liver, referred to as occult HBV infection (OBI). Previous reports showed that HBV variants in OBI-related HCC are different from those in HBsAg-positive HCC. In the present study, HBV quasispecies based on the pre-S/S gene in OBI-related HCC patients were examined by high throughput sequencing and compared with those in HBsAg-positive HCC.MethodsNineteen tissue samples (9 OBI-related and 10 HBsAg-positive non-cancerous tissues) were collected at the time of surgery at Kobe University Hospital. The quasispecies with more than 1% variation in the pre-S/S region were isolated and analysed by ultra-deep sequencing.ResultsThere were no significant differences in the major HBV populations, which exhibit more than 20% variation within the entire pre-S/S region, between OBI-related HCC and HBsAg-positive HCC. However, the prevalences of major populations with pre-S2 region mutations and of minor populations with polymerized human serum albumin-binding domain mutations were significantly higher in OBI-related HCC than in HBsAg-positive HCC. Moreover, the major variant populations associated with the B-cell epitope, located within the pre-S1 region, and the a determinant domain, located in the S region, were detected frequently in HBsAg-positive HCC. The minor populations of variants harbouring the W4R, L30S, Q118R/Stop, N123D and S124F/P mutations in the pre-S region and the L21F/S and L42F/S mutations in the S region were detected more frequently in OBI-related HCC than in HBsAg-positive HCC.ConclusionsUltra-deep sequencing revealed that the B-cell epitope domain in the pre-S1 region and alpha determinant domain in the S region were variable in HBsAg-positive HCC, although the quasispecies associated with the pre-S2 region were highly prevalent in OBI-related HCC.Trial registrationRef: R000034382/UMIN000030113; Retrospectively registered 25 November 2017.