KSEF RDE: Long QT Gene to Function Screens that Clarify Variants of Uncertain Significance

Grants and Contracts Details


Genome-wide studies have identified large numbers of sequence variants linked to a wide range of diseases. A current challenge is to identify which of the many variants have direct causal links to disease physiology. Inevitably gene-to-function screens will play critical roles in making these connections. Once the identities of genes underlying specific diseases are discovered, this in turn provides new insights into pathophysiological processes, identifies new targets for therapeutics, and allows the development of suitable genetic tests. Where efficient gene-to-function screens will really come into their own, however, is in the characterization of the many private mutations that will be discovered when patients submit to these tests. Genes associated with many diseases tend to exhibit extremely high allelic diversity so that sequence based genetic testing very often identifies large numbers of previously undiscovered mutations whose roles in disease causation are unclear. Despite this uncertainty, patients with private mutations often receive a positive diagnosis and a possible risk of being mis- or over-treated. This risk can be easily avoided if assays are performed to test the physiological functions of a variant before a management decision is made. Furthermore, work in the Delisle lab on Congenital Long QT syndrome (LQTS) has shown that functional testing of private mutations can help identify promising therapeutics, explain complex disease presentations, and provide fundamental insights into protein structure-function. Genetic testing for LQTS based on the disease linked KCNQ1 and KCNH2 K+ ion channel genes has identified at least 60 private mutations whose impacts on disease physiology are yet to be established. To facilitate characterization of the large numbers of private mutations that present in sequence based genetic tests, our goal is to develop an efficient gene-to-function assay system that allows parallel analysis of large numbers of variant alleles. A major challenge lies in the ability to synthesize and express numerous variants for functional testing. We have devised an elegant solution to this problem. Briefly, we will utilize mutagenic PCR and next generation sequencing to generate saturated, sequence-indexed clone libraries comprising thousands of gene variants. In parallel we will optimize existing biochemical and electrophysiological assays to facilitate high throughput screening of the libraries. As a proof of principle, we will use the system for functional screening of private mutations associated with LQTS. It should be noted, however, that the methods for library production and the assay systems will be broadly applicable to other diseases. Our innovative approach will allow functional assays to be performed not only on existing variants but also on novel mutants that may present in future genetic tests. This “anticipatory” screening approach has the potential to transform sequence based genetic tests into true diagnostic assays.
Effective start/end date7/1/1412/31/16


  • KY Science and Technology Co Inc: $30,000.00


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