He acknowledged that ascertainment and reporting bias likely play a role because patients without disease manifestation won’t come to attention, thus he suspects this phenomenon is significantly under-identified.
Introducing Monoallelic Expression
Dr. Bogunovic noted several mechanisms by which genes are not expressed from both alleles—genomic imprinting, X-inactivation, transcriptional bursting and the focus of this talk, monoallelic expression.
To explain MAE, he explained that all cells have a paternal and maternal allele, meaning genetically they are heterozygous. However, somewhere during development, the cell commits to propagation of either the maternal or paternal allele. This commitment is stable through mitosis and only expressed in a single cell type, such as T cells. Merging the concepts of MAE and incomplete penetrance, he demonstrated a pedigree whereby a mother, father and son all carried a mutation. The son had fully penetrant disease, the father had partial expressivity and the mother did not express disease. The healthy mother dominantly expressed the healthy allele, and the father skewed toward mutant expression.
Dr. Bogunovic explained that the origin of this concept came from studying a patient from New York with immune dysregulation, found to have a de novo mutation in Janus kinase 1 (JAK1), which is downstream to more than 25 different cytokines. Single-cell RNA sequencing on JAK1 surprisingly found that 50% of cells only expressed the mutant allele and not the wild-type allele. This deviated from the traditional dogma that JAK1 should be fully expressed in a biallelic pattern. These findings led to the hypothesis that monoallelic expression can lead to phenotypic variability in IEIs.
Excited by this possibility, Dr. Bogunovic and his team decided to take peripheral blood mononuclear cells (PBMCs) from 10 healthy donors in New York, sorted the cells into T cells, made large populations of T cell clones and validated a mechanism to determine if allelic bias was somatically acquired and stable. Using this clonal population, the team found evidence of MAE in IEI genes (e.g., JAK1, STAT1, NFKB1) even in healthy individuals. They found that about 5% of all genes and about 4% of IEI genes undergo MAE.
To validate their findings, the team examined JAK1 MAE via digital droplet polymerase chain reaction (PCR) for six different clones, as well as for a heterozygous variant in PLCG2 from one of the ten donors, repeating what they saw in RNA sequencing. Importantly, they found that PLCG2 MAE is stable in clones cultured over weeks.
