Heung Bum Oh

HIGH PREVALENCE OF HEPATITIS B AND HEPATITIS C VIRUS INFECTIONS IN KOREAN PATIENTS WITH HEMATOPOIETIC MALIGNANCIES

We performed a large case–control study (3,932 cases, 15,562 controls) to investigate the association of hepatitis B virus (HBV) and hepatitis C virus (HCV) with hematopoietic malignancies in Korea, where HBV is endemic. HBV was present in 636 control patients (4.1%), 333 lymphoma patients (12.4%), and 75 leukemia patients (6.0%). HCV infection was present in 173 control patients (1.1%), 76 lymphoma patients (2.8%), and 18 leukemia patients (1.4%). Co-infection of HBV and HCV was present in one (0.007%) control patient, seven lymphoma patients (0.3%), and one leukemia patient (0.08%). HBV infection was associated with increased risks for most subtypes of B and T/NK-cell lymphomas, Hodgkin’s lymphoma, and acute myeloid leukemia. HCV infection was associated with increased risks for diffuse large B cell lymphoma, extranodal marginal zone B cell lymphoma, peripheral T cell lymphoma, and acute lymphoid leukemia B cell early pre-B type. HBV seems to have a more important role than HCV in the pathogenesis of specific hematologic malignancies in Korea.

Direct detection of lamivudine-resistant hepatitis B virus mutants by a multiplex PCR using dual-priming oligonucleotide primers.

Mutations in the YMDD motif of the hepatitis B virus (HBV) polymerase gene increase lamivudine resistance of HBV, highlighting the clinical importance of accurate and sensitive detection of HBV mutants. Using dual-priming oligonucleotide primer technology, an assay that can detect mutations at codons 180 (L528M) and 204 (YVDD, YIDD, and YSDD) by a single-step multiplex PCR was developed. This Seeplex Lami-DR assay was sufficiently sensitive to detect 10(3)HBV/ml and was able to detect minor mutants comprising as little as 2% of the viral population. Mutants were detected in 57 of 65 serum samples (88%) from patients with chronic hepatitis B who had been treated with lamivudine (median, 32 months; range, 1-83 months). The agreement with direct sequencing was only 38.5% (25/65). Discrepancies between these methods resulted from detection of additional mutants by the Seeplex Lami-DR assay, as confirmed by a novel verification analysis. This assay is not only highly accurate and sensitive, but is also simple and cost-effective, requiring no expensive probes, laborious sequencing procedures, or digestion with restriction enzymes. Accordingly, the Seeplex HBV Lami-DR assay should be considered as a first-line, cost-effective tool for detecting viral mutations in patients with chronic hepatitis B receiving lamivudine therapy.

Performance evaluation of the Abbott RealTime HCV Genotype II for hepatitis C virus genotyping

Abstract Background: The Abbott RealTime hepatitis C virus (HCV) Genotype II (Abbott Molecular Inc.) for HCV genotyping, which uses real-time PCR technology, has recently been developed. Methods: Accuracy and sensitivity of detection were assessed using the HCV RNA PHW202 performance panel (SeraCare Life Sciences). Consistency with restriction fragment mass polymorphism (RFMP) data, cross-reactivity with other viruses, and the ability to detect minor strains in mixtures of genotypes 1 and 2 were evaluated using clinical samples. Results: All performance panel viruses were correctly genotyped at levels of >500 IU/mL. Results were 100% concordant with RFMP genotypic data (66/66). However, 5% (3/66) of the samples examined displayed probable genotypic cross reactivity. No cross reactivity with other viruses was evident. Minor strains in the mixtures were not effectively distinguished, even at quantities higher than the detection limit. Conclusions: The Abbott RealTime HCV Genotype II assay was very accurate and yielded results consistent with RFMP data. Although the assay has the advantages of automation and short turnaround time, we suggest that further improvements are necessary before it is used routinely in clinical practice. Efforts are needed to decrease cross reactivity among genotypes and to improve the ability to detect minor genotypes in mixed infections. Clin Chem Lab Med 2010;48:469–74.

MICB polymorphisms and haplotypes with MICA and HLA alleles in Koreans

Major histocompatibility complex (MHC) class I chain-related gene B (MICB) is located within the human MHC class I region. The location of MICB in the MHC region may imply the presence of linkage disequilibrium with polymorphic MICA and human leukocyte antigen (HLA) loci. MICB is also polymorphic; however, MICB polymorphisms have not been investigated in Koreans. Using sequence-based typing (SBT), we estimated the allelic frequencies of MICB and haplotypes with MICA, HLA-B, and HLA-DRB1 at high resolution in a population of 139 unrelated Korean individuals. Eight MICB alleles were identified. The most frequent allele was MICB*005:02/*010 (57.2%), followed by *002 (11.5%), *004 (8.3%), *005:03 (8.3%), and *008 (6.8%). The most common two-locus haplotypes were MICB*005:02/*010-MICA*010 (19.4%), MICB*005:02/*010-DRB1*15:01 (6.5%), and MICB*005:02/*010-B*15:01 (10.4%); the most common three-locus haplotypes were B*15:01-MICA*010-MICB*005:02/*010 (5.8%) and MICA*010-MICB*005:02/*010-DRB1*04:06 (10.4%); and the most common four-locus haplotype was B*15:01-MICA*010-MICB*005:02/*010-DRB1*04:06 (5.8%). This is the first study to provide information about MICB allele frequencies and haplotypes with HLA in Koreans. These study results should help understand mechanisms of disease association between the MICB locus and neighboring loci in Koreans.

No Association between 5-HTTLPR and Harm Avoidance in Korean College Students

There have been numerous studies on the association between 5-HTTLPR (polymorphisms in the promoter region of the serotonin transporter gene) and anxiety-related personality traits, with conflicting results. In this study, we administered Korean version of the Temperament and Character Inventory (K-TCI) to a sample of 158 Korean college students and genotyped for the 5-HTTLPR in order to compare the TCI dimensional scores including harm avoidance according to the 5-HTTLPR genotype and sex. We could not find the association between 5-HTTLPR and harm avoidance and other TCI measures. Considering known allele frequencies differences of 5-HTTLPR among different ethnic groups, further cross-cultural studies with a larger sample would be needed.

Subgenotype and Serotype Analysis of Hepatitis B virus in Korean Chronic Hepatitis B Patients Under Treatment

BACKGROUND Hepatitis B virus (HBV) detected in Korean patients almost belongs to genotype C, which is subdivided into subgenotype C1 (or Cs) and C2 (or Ce). It was recently reported that the risk of hepatocellular carcinoma is different between subgenotype C1 and C2. Thus, we studied the distribution of subgenotypes of HBV in Korean chronic hepatitis B (CHB) patients. METHODS Specimens of 421 patients, who were diagnosed as CHB and underwent antiviral treatment, were used. After sequence analysis for HBV S gene, subgenotype was identified through phylogenetic analysis. Utilizing the same sequence data, the distribution of serotypes was also investigated. RESULTS Among 421 patient specimens, genotype C was found in 419 (99.5%) and genotype B in 2 (0.5%). Among the genotype C strains, 417 strains were C2 subgenotype and 2 strains were mixed subgenotypes. However, C2 was evidently found even in the mixed sequences. Serotypes of 419 HBV with genotype C were classified as follows: adr, 385 (91.9%), adw, 22 (5.3%), ayr, 2 (0.4%) and mixed serotype, 10 (2.3%). Serotype of both HBV with genotype B was adw. CONCLUSIONS It was found that HBV detected in Korean CHB patients under treatment almost all belong to the C2 (Ce) genotype.

Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein

Human fibroblast growth factor 21 (hFGF21) has been characterized as an important regulator of glucose and lipid metabolism homeostasis. Here, to produce hFGF21 efficiently in Escherichia coli, the expression and solubility of hFGF21 were tested and optimised by fusing the protein with one of eight tags: hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b′a′ domain of PDI (PDIb′a′). Each tag increased solubility of the protein when the expression temperature was 18°C. Unlike many other tags that were tested, MBP significantly enhanced the solubility of the protein also in the culture condition at 37°C. Thus, the MBP-hFGF21 construct was further pursued for optimisation of affinity chromatography purification. After tag removal, 8.1 mg of pure hFGF21 was obtained as a final product from 500 mL of starting culture. The protein was then characterised by mass spectroscopy and an in vitro functional assay using NIH-3T3 cells transfected with a β-klotho reporter gene. These characteristics are similar to those of commercial hFGF21. Thus, the MBP tag is useful for efficient prokaryotic production and purification of bioactive hFGF21.

Comparison of quasispecies diversity of HCV between chronic hepatitis c and hepatocellular carcinoma by Ultradeep pyrosequencing.

Backgrounds. Hepatitis C virus (HCV) exists as population of closely related genetic variants known as quasispecies. HCV quasispecies diversity is strongly influenced by host immune pressure on virus. Quasispecies diversity is expected to decline as host immune response to HCV decreases over natural course of progressing from chronic hepatitis C (CHC) to hepatocellular carcinoma (HCC). Methods. Ultradeep pyrosequencing (UDPS) was used to evaluate degree of quasispecies diversity in 49 patients infected with HCV including 26 with CHC and 23 with HCC. Whole structural protein of HCV genome was subjected to UDPS. Results. Shannons indices for quasispecies diversity in HCV E1 were significantly lower in patients with HCC than in those with CHC. 14 amino acid positions differed significantly between two groups. Area under curve of ROC analysis for differentiating HCC from CHC was >0.8 for all of 14 amino acid positions. Conclusion. HCV quasispecies diversity as indicator of declining host immune functions was easily assessed by UDPS technology. Shannons indices in 14 amino acid positions were found to differentiate between patients with CHC and those with HCC. Our data propose that degree of HCV quasispecies measured by UDPS might be useful to predict progression of HCC in chronic HCV patients.

Loss of Mismatched HLA Detected in the Peripheral Blood of an AML Patient who Relapsed After Haploidentical Hematopoietic Stem Cell Transplantation

Dear Editor Loss of patient-specific HLA was recently identified in AML patients who relapsed after haploidentical hematopoietic stem cell transplantation (haploHSCT) [1]. Several subsequent studies were reported [2,3,4], but they were all performed by using bone marrow (BM) specimens. However, peripheral blood (PB) samples are commonly used for hematopoietic chimerism (HC) analysis by short-tandem repeat (STR) amplification after HSCT. Therefore, the peripheral detection of patient-specific HLA loss along with HC analysis could be clinically useful. We report a case in which the loss of mismatched HLA was detected in the PB of an AML patient who relapsed after haploHSCT. A 51-yr-old man was diagnosed as having refractory anemia with excess blasts that progressed to a normal karyotype AML after six months. The patient received haploHSCT from his first daughter two years after the diagnosis of AML, but the disease relapsed 14 months later, and he therefore received a second haploHSCT from his second daughter. The conditioning regimen consisted of fludarabine, busulfan, and antithymocyte globulin. A total of 7.2×106 cells/kg of CD34+ cells was transplanted, and 4.3×108 cells/kg of donor T lymphocytes was infused. One month after retransplantation, HC analysis performed by STR amplification revealed that all cells were derived from the donor, and BM examination showed normocellular marrow with trilineage regeneration. However, the patient eventually experienced a relapse of the disease four months after transplantation, with 76.4% of blasts in his BM. The immunophenotype and cytogenetic features of the BM blasts were the same as those at the time of the primary diagnosis and first relapse. HC analysis of PB revealed that all cells had changed to the patient type (Table 1). Table 1 Loss of mismatched HLA detected in the peripheral blood at relapse by HLA-DQB1 genotyping At the time of the second relapse, HLA-DQB1 typing analysis performed by sequence-based typing in PB revealed a loss of mismatched HLA-DQB1. The remaining specimen volume was insufficient for all HLA-A, -B, -C, -DR, and -DQ analyses. The patients original HLA-DQ type was DQB1*03:01, *03:03, and the donors type was DQB1*03:01, *02:02; however, only DQB1 *03:01 was detected, not the patient-specific type (Table 1). To verify this result, copy number variation was analyzed with BM specimens at relapse by using the HumanCytoSNP-12 bead chip (Illumina Inc. San Diego, CA, USA) and GenomeStudio software v2010.1/v1.6.3 (Illumina Inc.). B allele frequency (BAF) and Log R ratio (LRR) were used for copy number determination. LRR value of 0 represents two copies of alleles. In BAF plots a value of 0.5 indicates a heterozygous genotype, whereas 0 and 1 indicate homozygous genotypes. The percentage of BAF indicates the percentage of B allele raw copy number among the total allele raw copy number. A copy number-neutral loss of heterozygosity (CN-LOH) of 6p was identified, indicating that the acquired uniparental disomy (UPD) had caused the loss of the patient-specific HLA allele (Fig. 1). Additionally, CN-LOH of both 13q12.11-q34 and 21q21.3-q22.3 in all clones and subclones, respectively, were detected (Fig. 1). The patient was treated with salvage chemotherapy but he died three months later. Fig. 1 Copy number variation analysis of a bone marrow specimen at relapse using the HumanCytoSNP-12 bead chip demonstrates a logR ratio of 0 and a B allele frequencies (BAF) of 85%, indicating copy number-neutral loss of heterozygosity (CN-LOH) of 6p in all ... In the present case, acquired UPD on 6p resulted in the mismatched HLA loss. It was reported that 13q of the FLT3 gene was the most frequent site of UPD in relapsed AML [5], and that CN-LOH of 21q of the RUNX1 gene was responsible for the homozygosity of its mutation in normal karyotype AML [6]. Thus, various types of CN-LOH that were identified in the patients leukemic cells are presumed to have occurred at the time of diagnosis or relapse. The loss of patient-specific HLA could lead to disease relapse by allowing them to escape recognition by donor T cells. The HLA types of the primary and second donors were identical. Therefore, the patients CN-LOH could have been detected at the time of primary relapse, although no data were available to confirm this. HC analysis using the detection of individual specific STR is the most commonly used marker for residual disease after allogeneic HSCT, with a sensitivity of less than 5% [7]. Both BM and PB can be used, but the latter is more commonly used because it is easy to collect and the collection procedure is less invasive. With progressive disease relapse, the mixed cellular population can differ between BM and PB, and remnant donor cell clones can lead to discrepancies in HLA results between these samples (Table 1). The case described above demonstrates that HLA typing using PB might be sufficient to estimate the LOH when excess leukemic blasts are present in PB. In conclusion, the present case suggests that HLA typing performed in PB, along with HC analysis, could be clinically useful for the detection of the loss of patient-specific HLA in PB. The mismatched HLA could lead to disease relapse. Additional studies might be necessary to confirm whether detecting the loss of mismatched HLA before overt disease relapse is possible.

HLA-C∗01 is a risk factor for Crohn's disease

Background:A dysregulated mucosal immune response to the intestinal environment in a genetically susceptible host is hypothesized to be critical to the pathogenesis of Crohns disease (CD). Therefore, we examined CD-susceptibility genes involved in the immune response through a genome-wide association study and consecutive genotyping of human leukocyte antigens (HLAs) and killer cell immunoglobulin-like receptors. Methods:An initial genome-wide association study was performed with 275 CD patients and 2369 controls from a Korean population. To validate the loci identified in the genome-wide association study, replication genotyping was performed in a different cohort of 242 CD patients and 1066 controls. Finally, high-resolution genotyping of HLA and killer cell immunoglobulin-like receptor was performed. Results:Four susceptibility loci, a promoter region in tumor necrosis factor (ligand) superfamily member (TNFSF15) and 3 independent regions in HLAs, showed significant associations with CD. Among them, rs114985235 in the intergenic region between HLA-B and HLA-C showed the strongest association, with an increased risk of CD (P = 8.71 × 10−23; odds ratio, 2.25). HLA typing in this region showed HLA-C*01 to be responsible for the association of CD among 43 HLA-B and HLA-C genotypes identified in the Korean population. However, the interaction of HLA-C with killer cell immunoglobulin-like receptor had little effect on the development of CD. Conclusions:We newly identified HLA-C*01 as a prominent CD-susceptibility HLA allotype in the Korean population. In addition, these results confirm that genetic variations in immune response genes, such as HLAs and TNFSF15, are important host factors for the pathogenesis of CD.