Project Summary/ Abstract:
Our limited ability to relate genotype to phenotype is a major obstacle for biomedical research and personalized
medicine. Currently only ~2% of germline missense variants have clinical interpretations, and the remainder,
variants of uncertain significance (VUS), offer no information to inform diagnosis or guide treatment. As the
clinical use of whole exome and genome sequencing increases, the number of VUS will skyrocket. Large-scale
functional assays in model organisms are the only methods for variant interpretation currently poised to match
the pace of variant discovery, and here we propose to extend their use to interpret genetic complexity. Our
approach leverages the advent of low-cost, large-scale gene synthesis and the development of high throughput
in vivo assays of protein function in model organisms, such as yeast. We propose a generalizable approach for
determining the functional consequences of polymorphisms in human disease genes, including individual alleles,
combinations of alleles in the same gene, and combinations of alleles in multiple genes in a pathway, on a
massively parallel scale. The quantitative nature of our assay and the structure of our experimental design will
allow us to compare the impact of allele combinations with their individual effects, and thus detect genetic
epistasis (nonlinear genetic interactions) arising from naturally occurring human genetic variation outside of the
limits of outbred human populations. Through this novel approach, we will not only explore the extent to which
nonlinear interactions between human genes are pervasive or rare, but by placing them in the context of protein
and metabolic pathway structure, we will gain insight into their molecular underpinnings. Our study will also
provide an unprecedented amount of information about the contribution of individual amino acids to the function
of the three disease-relevant enzymes in our study, and we will analyze our results in the context of their
published crystal structures. Finally, we will develop new methods and assays that will expand the throughput,
combinatorics, and number of assays available for functional analysis of human variation.
We will pilot our approach using three human genes (OTC, ASS1, and ASL) associated with a class of metabolic
disorders known as urea cycle disorders (UCD). Neonatal UCD is associated with severe enzyme deficiency.
These infants rapidly develop high levels of ammonia, cerebral edema, and symptoms that can include seizures,
coma, and death. Less severe forms may remain undiagnosed into childhood or adulthood. Late onset UCDs
generally involve an environmental trigger (e.g. surgery, pregnancy, or chemical exposure) in individuals with
reduced enzyme function. Diagnosis of the adult onset form is hampered by the fact that it often presents with
symptoms such as episodic psychosis, bipolar disorder and major depression, and without treatment, prognosis
is poor. Thus, knowledge of the functional implications of genetic variation in these genes has the potential to
reduce the morbidity and mortality associated with delayed treatment or underdiagnosis.
