HLA-A, -B, -C, -DRB1 allele and haplotype frequencies of the Korean population and performance characteristics of HLA typing by next-generation sequencing.

INTRODUCTION: Human leukocyte antigen (HLA) identification at the allelic level is important for haematopoietic stem cell transplantation (HSCT). Next-generation sequencing (NGS) resolves ambiguous alleles by determining the phase of the polymorphisms. The aim of this study was to validate the software for HLA-SBT (sequence-based typing), assess Korean allele frequency, and characterise the performance of NGS-HLA typing. METHODS: From the 2009 to 2016 registry, 1293 unrelated healthy donors with a complete dataset of previously characterised HLA-A, -B, -C, and -DRB1 loci were selected and assessed for frequency, haplotype inference, and relative linkage disequilibrium. For performance characteristics of NGS-HLA, alleles included in 1293 cases and ambiguous or alleles assigned as new by SBT-HLA software, or unassigned alleles were included. A total of 91 and 41 quality control samples resulted in 1056 alleles (132 samples × 4 loci × 2 diploid) for analysis. The GenDx NGSgo kit was used for NGS-HLA typing using the Illumina MiSeq platform. RESULTS: A panel of 132 samples covered 231 alleles, including 53 HLA-A, 80 HLA-B, 43 HLA-C, and 55 HLA-DRB1 by HLA-SBT typing. Comparison of SBT-HLA and NGS-HLA typing showed 99.7% (1053/1056) concordance and discrepant cases were resolved by manual evaluation. Typing by NGS resulted in 67 HLA-A, 112 HLA-B, 71 HLA-C, and 72 HLA-DRB1 alleles. A total of 132 ambiguous, 4 new, and 1 unassigned alleles by HLA-SBT were resolved by NGS-HLA typing. CONCLUSIONS: NGS-HLA typing provided robust and conclusive results without ambiguities, and its implementation could support HSCT in clinical settings.

Ubiquitin C decrement plays a pivotal role in replicative senescence of bone marrow mesenchymal stromal cells.

Human bone marrow-mesenchymal stromal cells (hBM-MSCs) undergo cellular senescence during in vitro culture. In this study, we defined this replicative senescence as impaired proliferation, deterioration in representative cell characteristics, accumulated DNA damage, and decreased telomere length and telomerase activity with or without genomic abnormalities. The UBC gene expression gradually decreased during passaging along with the reduction in series of molecules including hub genes; CDK1, CCNA2, MCM10, E2F1, BRCA1, HIST1H1A and HIST1H3B. UBC knockdown in hBM-MSCs induced impaired proliferation in dose-dependent manner and showed replicative senescence-like phenomenon. Gene expression changes after UBC knockdown were similar to late passage hBM-MSCs. Additionally, UBC overexpession improved the proliferation activity of hBM-MSCs accompanied by increased expression of the hub genes. Consequently, UBC worked in higher-order through regulation of the hub genes controlling cell cycle and proliferation. These results indicate that the decrement of UBC expression plays a pivotal role in replicative senescence of hBM-MSCs.

Practical informativeness of short tandem repeat loci for chimerism analysis in hematopoietic stem cell transplantation.

OBJECTIVE: Short tandem repeat (STR) loci are most frequently used for chimerism analysis after hematopoietic stem cell transplantation (HSCT). The aim of this study was to evaluate the practical informativeness of STR chimerism by integrating theoretical and analytical points. METHODS: Theoretical and practical informativess of 16 STR loci were evaluated from 1249 pairs of recipients and donors who were prepared for HSCT. RESULTS: Theoretical informativeness was influenced by genetic diversity including allele frequency and heterozygosity, and was higher in the unrelated HSCT group (90.5±5.3%) compared to the related HSCT group (66.2±4.4%). Practical informativeness was lower than theoretical (6.1±1.7%) because several STR loci were excluded due to stutter peaks and less reliable results, especially in type II-2 donor-recipient match pattern with no recipient-specific allele. We simulated an efficient STR combination for reliable chimerism analysis. Eight informative STR loci were required to analyze chimerism with at least one practically informative locus in the related HSCT group (D18S51, FGA, D2S1338, D13S317, D8S1179, D21S11, D16S539 and D7S820) while only three loci were needed in the unrelated group (D2S1338, FGA and D18S51). A minimum set of 2, 4 or 7 STR loci were required to provide at least 1, 3 or 5 practically informative loci in 95% of the unrelated HSCT group while 3, 8 or 12 loci were required in the related HSCT group. CONCLUSION: We deducted the practical informativeness of STR chimerism, identified the major influencing factors on the practical informativeness of each STR locus, and successfully simulated the efficient STR combination for reliable chimerism analysis.

FLT3 expression and IL10 promoter polymorphism in acute myeloid leukemia with RUNX1-RUNX1T1.

We investigated the correlation between FLT3 expression and IL10 gene promoter polymorphism in acute myeloid leukemia with RUNX1-RUNX1T1 and their clinical significance. FLT3 mRNA expression was measured by real-time quantitative PCR (qPCR) and immunohistochemical staining (IHC) on bone marrow (BM) leukemic cells. IL10 gene promoter polymorphisms including rs1800896 (G-1082A), rs1800871 (C-819T), and rs1800872 (C-592T) were genotyped by direct sequencing. Among 45 enrolled patients, 32 (71.1 %) exhibited FLT3 overexpression, whose FLT3 mRNA level was higher than normal cut-off value (0.02). The IHC results also consisted with FLT3 mRNA expression data achieved by qPCR. The FLT3 mRNA level was significantly different among 3 IHC staining groups (P < 0.0001); 0.031 ± 0.041, 0.106 ± 0.097 and 0.588 ± 0.573 in IHC negative, intermediate and positive group, respectively. Interestingly, the FLT3 expression level was correlated with the percentage of BM CD34 positive cells (R = 0.360, P = 0.016). The elevated FLT3 expression at initial BM were decreased after remission and maintained lower than the cut-off level. FLT3 expression was not dependent on IL10 gene promoter polymorphisms. FLT3 overexpression itself did not demonstrate significant effects on overall survival (OS). However, it is notable that IL10 rs1800896 GA genotype tended to have a lower estimated mean OS (20.1 months) compared to GG genotype (54.6 months), but the statistical significance was not derived because of limited number of patients in this study (P = 0.072). Further studies including more type of leukemia and patients may be helpful to understand the relations between cytokine genotype and FLT3 expression and their prognostic impact.

Polymorphisms of the heart-type fatty acid-binding protein as a prognostic factor in patients with atherosclerosis.

The amount of heart-type fatty acid-binding protein (FABP3) in cardiomyocytes may affect the prognosis of patients with coronary atherosclerosis. In this study, we examined whether single nucleotide polymorphisms (SNPs) of the FABP3 gene were prognostic factors in patients with coronary atherosclerosis. We enrolled 100 patients with myocardial infarction and coronary atherosclerosis and 100 healthy individuals to assess the genotypes of four SNPs of the FABP3 gene (RS2271072, RS16834408, RS2279885, and RS10914367). The MI patients with coronary atherosclerosis exhibited a higher frequency (16%) of the C/G-G/G haplotype of RS2271072 and RS10914367 than healthy individuals (7%). Koreans with the C/G-G/G haplotype for these SNPs had a higher risk of MI than did Koreans with the G/G-A/A haplotype. We calculated an odds ratio of 2.83 (95% confidence interval: 1.01-7.96). In conclusion, the C/G-G/G haplotype at RS2271072 and RS10914367 SNPs of FABP3 might be a prognostic factor in patients with coronary atherosclerosis.

Ribosomal protein mutations in Korean patients with Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by hypoproliferative anemia, associated physical malformations and a predisposition to cancer. DBA has been associated with mutations and deletions in the large and small ribosomal protein genes, and genetic aberrations have been detected in ∼50-60% of patients. In this study, nine Korean DBA patients were screened for mutations in eight known DBA genes (RPS19, RPS24, RPS17, RPS10, RPS26, RPL35A, RPL5 and RPL11) using the direct sequencing method. Mutations in RPS19, RPS26 and RPS17 were detected in four, two and one patient, respectively. Among the mutations detected in RPS19, two mutations were novel (c.26T>A, c.357-2A>G). For the mutation-negative cases, array-CGH analysis was performed to identify copy-number variations, and no deletions involving the known DBA gene regions were identified. The relative mRNA expression of RPS19 estimated using real-time quantitative PCR analysis revealed two- to fourfold reductions in RPS19 mRNA expression in three patients with RPS19 mutations, and p53 protein expression analysis by immunohistochemistry showed variable but significant nuclear staining in the DBA patients. In conclusion, heterozygous mutations in the known DBA genes RPS19, RPS26 and RPS17 were detected in seven out of nine Korean DBA patients. Among these patients, RPS19 was the most frequently mutated gene. In addition, decreased RPS19 mRNA expression and p53 overexpression were observed in the Korean DBA patients, which supports the hypothesis that haploinsufficiency and p53 hyperactivation represent a central pathway underlying the pathogenesis of DBA.

Differences in hepatic gene expression as a major distinguishing factor between Korean native pig and Yorkshire.

There are phenotypic differences between Korean native pig (KNP) and Yorkshire (YS) breeds due to different interests in selection. YS has been selected for industrial interests such as a growth rate and lean meat production, while KNP has been maintained as a regional breed with local interests such as disease resistance and fat content in and between muscle. A comparison of gene expression profiles from liver tissue reflected overall long-term effects of artificial selection for these two pig breeds. Based on minimum positive false discovery rate (less than 10%) and fold change (|FC|>1.5), 73 differentially expressed genes (DEGs) were identified. Functional analysis of these DEGs indicated clear distinctions in signaling capacity related to epidermal growth factor (EGF), extracellular structure, protein metabolism, and detoxification. Hepatic DEGs demonstrated the importance of hormonal and metabolic capabilities to differences between these two pig breeds.

Transcriptional alteration of p53 related processes as a key factor for skeletal muscle characteristics in Sus scrofa.

The pig could be a useful model to characterize molecular aspects determining several delicate phenotypes because they have been bred for those characteristics. The Korean native pig (KNP) is a regional breed in Korea that was characterized by relatively high intramuscular fat content and reddish meat color compared to other western breeds such as Yorkshire (YS). YS grew faster and contained more lean muscle than KNP. We compared the KNP to Yorksire to find molecular clues determining muscle characteristics. The comparison of skeletal gene expression profiles between these two breeds showed molecular differences in muscle. We found 82 differentially expressed genes (DEGs) defined by fold change (more than 1.5 fold difference) and statistical significance (within 5% of false discovery rate). Functional analyses of these DEGs indicated up-regulation of most genes involved in cell cycle arrest, down-regulation of most genes involved in cellular differentiation and its inhibition, down-regulation of most genes encoding component of muscular-structural system, and up-regulation of most genes involved in diverse metabolism in KNP. Especially, DEGs in above-mentioned categories included a large number of genes encoding proteins directly or indirectly involved in p53 pathway. Our results indicated a possible role of p53 to determine muscle characteristics between these two breeds.