PU.1, encoded by the gene SPI1, is a pioneer transcription factor that determines hematopoietic cell fate. Although PU.1 deficiency is embryonically lethal in mice and SPI1 mutations are common in human myeloid leukemias, a primary PU.1-mediated disease has not been previously described. We have identified four unrelated agammaglobulinemic subjects each harboring different, novel, non-synonymous heterozygous SPI1mutations. We sought to determine if and how these mutations caused disease.
PU.MA subjects and their family members were identfied and genotyped via whole exome sequencing. Peripheral blood and bone marrow samples were analyzed by flow cytometry, RNA-sequencing and immunohistochemical stains. Mutant PU.1 proteins were identified with immunoblots and interactions with other transcriptional partners explored with co-immunoprecipitation. Nuclear translocation of mutant PU.1 into HEK293 nuclei was measured via con-focal microscopy. Mutant PU.1 binding to PU.1-box nucleotide sequences was assessed with electrophoretic mobility shift assays. Custom PU.1-reporter lines measured the transcriptional effects of PU.1. mutants.
PU.MA subjects lacked blood IgM-expressing B cells. Bone marrow analyses demonstrated pro-B cell arrest. Two mutant SPI1variants encoded proteins with single amino acid substitutions in the ETS DNA-binding domain. A frame-shift variant produced a truncated PU.1 protein and a stop-gain variant failed to be expressed. Expressed mutant proteins homodimerized and IRF4 heterodimerized normally, entered the nucleus efficiently but did not bind the PU.1 consensus nucleotide sequence. Mutant PU.1 failed to drive transcription in reporter lines but did not interfere with wild type PU.1’s ability to do so.
We identified and molecularly describe a new autosomal dominant form of agammaglobulinemia caused by developmental arrest of PU.1 mutated pro-B cells.