Recently, the Undiagnosed Disease Network identified a possibly pathogenic allele, UBTF p.E210K (Glu210Lys) that is heterozygous, in a subject that exhibited dystonia, stuttering, progression to mutism, neu- rodevelopmental delay and a high-pitched voice. There is a fundamental gap in understanding the genetic, cellular and biochemical relationship between this allele and these pathogenic phenotypes. It is an important problem to explicitly identify (validate) the pathogenic allele and understand the mechanism that leads from the allele to the disease because that information justifies and shapes the direction of future studies such as development of therapeutic molecules. Upstream Binding Transcription Factor (UBTF; also called UBF) is a transcription factor that exists as two isoforms, UBTF1 and UBTF2. UBTF binds DNA using its HMGB boxes and Glu210 follows a conserved hydrophobic core of HMGB box 2 in both isoforms. While UBTF1 regulates rRNA transcription by RNA pol I, UBTF2 regulates mRNA transcription by RNA pol II. The observed pathologic phenotypes overlap with those reported for isolated and secondary dystonias, and associated neurodevelopmental disorders. When examined, there is evidence that UBTF binds near the promoter of genes associated with dystonia, cell cycle progression and response to DNA damage. Based on this information, there is a critical need to: 1) develop an animal model with the variant E210K UBTF allele and 2) characterize alterations in UBTF activity due to the E210K mutation. The objective of this application is to generate UBTF mouse models with etiologic and face validity and determine how the E210K mutation alters UBTF's functions. Our central hypothesis is that the UBTF E210K allele is a gain-of-function mutation. Based on this framework of data related to UBTF1/2, the following specific aims will be pursued: a) Characterize phenotypes associated with mouse models harboring variant UBTF alleles; and b) Determine the molecular effects of the E210K mutation of previously defined roles of UBTF1 in the nucleolus and UBTF2 on expression of dystonia- associated and cell-cycle genes. In the first aim, conditional UBTF-knockout and UBTF E210K mice will be developed. Without a mammalian model system, the genetic relationship between UBTF E210K and the undiagnosed disease cannot be validated. In the second aim, the effect of the E210K mutation on DNA binding, transcriptional activation of putative target promoters and nucleolar morphology will be examined. Insights into the functional consequence of the E210K mutation may facilitate a deeper understanding of UBTF and cellular processes such as rRNA transcription, cell-cycle control and neurodevelopmental processes. The approach is innovative because it represents a departure from the status quo and shifts focus to UBTF's ability to regulate transcription by RNA pol II and identifies a previously unknown role for UBTF in neurological disease. These contributions will be significant because 1) they would constitute a successful application of personalized medicine to diagnose disease mechanisms and 2) validate the prediction that UBTF E210K is a pathogenic allele. 1
The proposed research is relevant to the public health because it addresses the genetic and functional link be- tween the phenotypes of a subject in the Undiagnosed Disease Program and the variant allele predicted to pathogenic. Thus, the proposed research is relevant to the part of NIH's mission that pertains to the mechanistic understanding of whether and how particular genes and their specific variants are involved in disease pathogenesis using a model mammalian organism, and whether and how genetic variation affects gene expression and function particularly as it relates to the disease phenotype. The impact of validating UBTF E210K as a pathogenic allele is that it will influence the direction of future genetic and translational studies related to UBTF, provide evidence that UBTF variants are causally-associated with neurological disorders and identifies UBTF and/or UBTF as potential targets for intervention. 1
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