X-linked reticulate pigmentary disorder (XLPDR) is a rare condition characterized by recurrent infections and a range of skin manifestations. Utilizing whole genome sequencing, we have identified that the cause of XLPDR is an intronic mutation resulting in missplicing of POLA1, an essential cell replication gene encoding the catalytic subunit of DNA polymerase alpha. Moreover, we have uncovered that XLPDR results in a prominent natural killer cell (NK) defect, consistent with the clinical history of recurrent infections. The cutaneous and immunologic changes seen in XLPDR are reminiscent of mutations of the NF-?B activating gene NEMO, a central regulator of immune function. Indeed, we find that XLPDR-derived fibroblasts display severely impaired NF-?B activation, which is recapitulated by RNAi silencing of POLA1, suggesting an unexpected connection between POLA1 and the NF-?B pathway. However, the mechanism by which a mutation in this ubiquitously expressed gene results in the specific manifestations of XLPDR remains unclear. Thus, the project's overall goal is to uncover the cellular and molecular basis of XLPDR and its associated immunodeficiency. Our hypothesis is that the POLA1 protein functions not only in DNA replication, but also plays a direct role in NF-?B activation, and that XLPDR and its associated immunodeficiency result from reduced POLA1 expression in select cell populations. To address these goals, we propose the following Specific Aims: (1) Determine the genetic mechanism of XLPDR: In order to explain the specific phenotype of XLPDR, we hypothesize that missplicing and reduced POLA1 protein occurs preferentially in select cell lineages. In this aim, we will examine this possibility in leukocyte populations from patients'peripheral blood. Furthermore, using a splicing reporter system we will analyze the effect of the XLPDR mutation on POLA1 splicing in cell lines of multiple tissue origins and define in detail the elements required to recapitulate POLA1 missplicing in the corresponding mouse intron. (2) Define the biochemical basis of defective NF-?B activation in XLPDR: In addition to the defect in NF-?B dependent gene expression in XLPDR, we have found a physical interaction between the POLA1 and NEMO proteins. Interestingly, the nuclear entry of NF-?B complexes is not affected by the XLPDR mutation, suggesting that the defect lies in nuclear events required for NF-?B activation. In this aim we will examine the mechanism of assembly and regulation of the POLA1-NEMO complex, and the participation of POLA1 in nuclear events required for NF-?B dependent gene induction. (3) Understand the NK cell deficiency of XLPDR: In this Aim, we will perform a careful evaluation of NK cell function in patients from different families and examine whether direct silencing of POLA1 in wild-type NK cells is sufficient to recapitulate the XLPDR cellular phenotype and generate a cellular model of XLPDR. Altogether, these studies of a rare and orphan immunologic disorder offer a unique opportunity to obtain remarkable and novel insights into immune regulation and signaling pathways of great significance to a number of disciplines.
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