Liver-Humanized NSG-PiZ Mice Support the Study of Chronic Hepatitis B Virus Infection and Antiviral Therapies.

Hepatitis B virus (HBV) is a pathogen of major public health importance that is largely incurable once a chronic infection is established. Only humans and great apes are fully permissive to HBV infection, and this species restriction has impacted HBV research by limiting the utility of small animal models. To combat HBV species restrictions and enable more in vivo studies, liver-humanized mouse models have been developed that are permissive to HBV infection and replication. Unfortunately, these models can be difficult to establish and are expensive commercially, which has limited their academic use. As an alternative mouse model to study HBV, we evaluated liver-humanized NSG-PiZ mice and showed that they are fully permissive to HBV. HBV selectively replicates in human hepatocytes within chimeric livers, and HBV-positive (HBV+) mice secrete infectious virions and hepatitis B surface antigen (HBsAg) into blood while also harboring covalently closed circular DNA (cccDNA). HBV+ mice develop chronic infections lasting at least 169 days, which should enable the study of new curative therapies targeting chronic HBV, and respond to entecavir therapy. Furthermore, HBV+ human hepatocytes in NSG-PiZ mice can be transduced by AAV3b and AAV.LK03 vectors, which should enable the study of gene therapies that target HBV. In summary, our data demonstrate that liver-humanized NSG-PiZ mice can be used as a robust and cost-effective alternative to existing chronic hepatitis B (CHB) models and may enable more academic research labs to study HBV disease pathogenesis and antiviral therapy. IMPORTANCE Liver-humanized mouse models have become the gold standard for the in vivo study of hepatitis B virus (HBV), yet their complexity and cost have prohibited widespread use of existing models in research. Here, we show that the NSG-PiZ liver-humanized mouse model, which is relatively inexpensive and simple to establish, can support chronic HBV infection. Infected mice are fully permissive to hepatitis B, supporting both active replication and spread, and can be used to study novel antiviral therapies. This model is a viable and cost-effective alternative to other liver-humanized mouse models that are used to study HBV.

Using Somatic Mutations from Tumors to Classify Variants in Mismatch Repair Genes.

Present guidelines for classification of constitutional variants do not incorporate inferences from mutations seen in tumors, even when these are associated with a specific molecular phenotype. When somatic mutations and constitutional mutations lead to the same molecular phenotype, as for the mismatch repair genes, information from somatic mutations may enable interpretation of previously unclassified variants. To test this idea, we first estimated likelihoods that somatic variants in MLH1, MSH2, MSH6, and PMS2 drive microsatellite instability and characteristic IHC staining patterns by calculating likelihoods of high versus low normalized variant read fractions of 153 mutations known to be pathogenic versus those of 760 intronic passenger mutations from 174 paired tumor-normal samples. Mutations that explained the tumor mismatch repair phenotype had likelihood ratio for high variant read fraction of 1.56 (95% CI 1.42-1.71) at sites with no loss of heterozygosity and of 26.5 (95% CI 13.2-53.0) at sites with loss of heterozygosity. Next, we applied these ratios to 165 missense, synonymous, and splice variants observed in tumors, combining in a Bayesian analysis the likelihood ratio corresponding with the adjusted variant read fraction with pretest probabilities derived from published analyses and public databases. We suggest classifications for 86 of 165 variants: 7 benign, 31 likely benign, 22 likely pathogenic, and 26 pathogenic. These results illustrate that for mismatch repair genes, characterization of tumor mutations permits tumor mutation data to inform constitutional variant classification. We suggest modifications to incorporate molecular phenotype in future variant classification guidelines.