Chronic hepatitis B is a serious infectious liver disease that often progresses to liver cirrhosis and hepatocellular carcinoma; however, clinical outcomes after viral exposure vary enormously among individuals. Through a two-stage genome-wide association study using 786 Japanese chronic hepatitis B cases and 2,201 controls, we identified a significant association of chronic hepatitis B with 11 SNPs in a region including HLA-DPA1 and HLA-DPB1. We validated these associations by genotyping two SNPs from the region in three additional Japanese and Thai cohorts consisting of 1,300 cases and 2,100 controls (combined P = 6.34 x 10(-39) and 2.31 x 10(-38), OR = 0.57 and 0.56, respectively). Subsequent analyses revealed risk haplotypes (HLA-DPA1(*)0202-DPB1(*)0501 and HLA-DPA1(*)0202-DPB1(*)0301, OR = 1.45 and 2.31, respectively) and protective haplotypes (HLA-DPA1(*)0103-DPB1(*)0402 and HLA-DPA1(*)0103-DPB1(*)0401, OR = 0.52 and 0.57, respectively). Our findings show that genetic variants in the HLA-DP locus are strongly associated with risk of persistent infection with hepatitis B virus.

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Ogataea thermomethanolica TBRC656 is a thermotolerant methylotrophic yeast suitable for heterologous protein expression at various temperatures. However, the lack of efficient methods for targeted gene mutagenesis limits strain engineering in this yeast. In this study, we applied a CRISPR-Cas9-based tool for targeted gene mutagenesis in O. thermomethanolica. The putative unfolded protein response regulator OtHAC1, and the OtMAL1 (maltase) and OtMAL2 (maltose permease) genes involved with sucrose and maltose utilization were targeted for CRISPR-Cas9 mutagenesis. Plasmids were constructed for integrative and episomal expression of CRISPR-Cas9 elements in O. thermomethanolica in which Cas9 and gRNA are transcribed from the alcohol oxidase (AOX) promoter. The expression of these genome-editing elements is controlled by derepression with glycerol and gRNA are flanked by self-cleaving ribozymes. For integrative system, OtHAC1, OtMAL1 and OtMAL2 were disrupted at 63%, 97% and 93%, respectively. In addition, OtMAL1 was also disrupted with episomal system at 92%. These findings indicate that the CRISPR-Cas9 system described herein is thus applicable for studying gene function and strain engineering in yeast O. thermomethanolica.