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.

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.

Hepatitis B virus (HBV) is a pathogen of major public health importance that is largely incurable once a chronic hepatitis B (CHB) infection is established. Only humans and great apes are fully permissive to HBV replication, and this species restriction has impacted HBV research by limiting the utility of small animal models of HBV. To combat the species restriction of HBV and enable more HBV studies in vivo, liver-humanized mouse models have been developed that harbor primary human hepatocytes (PHH) and are fully 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 and can develop CHB. Mice were infected with a precore mutant clinical isolate that has now been serially passaged through 3 generations of mice without loss of fitness. HBV selectively replicates in hCK18+ human hepatocytes within chimeric livers, and HBV+ mice secrete infectious virions and HBsAg into blood, while also harboring covalently closed circular DNA (cccDNA). HBV+ mice remain viremic for at least 169 days, which should enable the study of new curative therapies targeting CHB and respond to antiviral entecavir therapy. The extended duration of viremia is sufficient to enable the study of established and new therapeutic approaches targeting CHB. Furthermore, HBV+ PHH in NSG-PiZ mice can be transduced by the hepatotropic AAV3b and AAV.LK03 vector capsids, which should enable the study of curative gene therapies that target CHB. In summary, our data demonstrates that liver humanized NSG-PiZ mice can be used as a robust and cost-effective alternative to existing CHB models and may enable more academic research labs to study HBV disease pathogenesis and antiviral therapy in a setting that is fully permissive to ongoing replication.