Allelic Bias Contributes to Incomplete Penetrance of NK Cell Deficiency

Natural killer cell deficiencies (NKDs) can be caused by partial loss-of-function variants in the CDC45-MCM-GINS (CMG) DNA replicative helicase complex. While these variants have been identified and attributed to be the monogenic causes of inborn errors of immunity (IEI), family members with the same variants often exhibit incomplete penetrance with NK cells detectable in the peripheral blood to varying degrees. Here, we demonstrate the effects of NKD-causing compound heterozygous variants in GINS4, a critical member of the GINS tetramer that forms the CMG complex and is required for DNA replication and cell cycle progression. Two siblings with the same inherited biallelic GINS4 variants but differing severities of clinical disease were previously identified and characterized. To better understand the effect of these variants on NK cell maturation, induced pluripotent stem cell (iPSC) lines were generated from the siblings, their biological parents, and an unrelated healthy control. We profiled efficiency in NK cell differentiation, proliferative dynamics, and sensitivity to replication stress. Immunophenotyping and RNA-Seq were used to track the emergence of NK and non-NK cell populations over time from iPSCs. The efficiency of NK cell differentiation correlated with clinical severity of NKD. iPSCs from the individual with more severe NKD failed to generate NK cells, and we identified impairment in cell proliferation after NK cell lineage specification as the cause of this failure. These phenotypes could be rescued by CRISPR correction of both variants, demonstrating the monogenic nature of this deficiency. In contrast, iPSCs from the less severely affected NKD individual were able to generate NK cells more efficiently, despite having the same GINS4 variants. Further analysis of RNA species from iPSC-derived and primary NK cells revealed distinct allelic bias in GINS4 in the NK cells of the two individuals. NK cells from the more severely affected individual expressed more of the GINS4 allele shown to have greater destabilizing effect on protein. Conversely, the less severely affected individual’s NK cells were biased toward a missense allele with less damaging effects. Therefore, this study identifies allelic bias as a nongenetic factor that contributes to the phenotypic variations of monogenic diseases.