Research in Rare Disease: From Genomics to Proteomics

Rare Disease Research in the Genomic Era

A rare disease is defined as a condition affecting fewer than 1 in 2,000 people in Canada and Europe or fewer than 200,000 people in the United States. Although each individual disorder is rare, collectively they affect a significant portion of the population. Approximately 25 million people in the United States are affected by a rare disorder. ¹ In addition to the physical and emotional hardship on patients and their families, rare genetic diseases have an immense cost for healthcare systems and societies. ^{2 –6} There are over 7,000 known rare diseases and more being discovered each year. ⁷ Since the advent of whole-exome sequencing, the pace of gene variant discovery for rare diseases has profoundly increased. ⁷ Approximately 50% of rare diseases have a firm genetic basis, and many of these have a simple Mendelian pattern of inheritance consistent with a monogenic origin. ⁷ Despite our knowledge of the genetic mutations that cause rare diseases, little is known of the cellular consequences of these genetic mutations. Functional studies on rare disease have fallen behind gene discovery since advances in genome sequencing have far outpaced the throughput in functional follow-up studies. In addition, the pharmaceutical and biotech industries have few incentives to invest in researching and developing drugs for diseases that affect a limited patient population. Thus, the development of effective treatments for rare diseases remains a challenge. To date, only 5% of rare disorders have a Food and Drug Administration (FDA)-approved treatment. ⁸

Drug Repurposing in Rare Disease

The most realistic approach to combat rare disease is to repurpose existing drugs. Drug repurposing cuts down the cost of drug development from an estimated $1.8 billion/FDA approval to an estimated $8.4 million, ^9,10 while also decreasing the time it takes to develop an FDA-approved drug from an average of 10–17 to 3–12 years. ¹¹ There are several rare diseases that have been successfully subject to drug repurposing. ^{12 –14} A recent example of human disease that has benefitted from drug repurposing is the Hutchinson–Gilford progeria syndrome (HGPS), a premature aging syndrome. HGPS is caused by a truncated and mislocalized variant of lamin A, a component of the nuclear lamina. ^15,16 In less than 10 years, the farnesyltransferase inhibitor, lonafarnib, was shown to reverse the abnormal nuclear morphology phenotype of patient-derived cell lines. ¹⁷ Furthermore, the compound was shown to ameliorate symptoms in patients in a clinical trial. ¹² This success in clinical trials for progeria is just one example of the promise of drug repurposing in rare disease. It is likely that many more rare diseases could benefit from drug repurposing, but the challenge is to identify which. Repurposing requires an understanding of the functional consequences of pathological genetic variation and a phenotype that can be used as a readout in drug screens. Unfortunately, such information is not available for the vast majority of rare diseases.

A Proteomic Approach to Rare Disease

Rare disease has typically been studied at the cellular level on a case by case basis. More recently, a study systematically phenotyped a library of 2,890 rare disease-causing variants as well as their respective wild-type alleles. ¹⁸ This human mutation open reading frame (hmORF) collection was used to characterize how missense mutations rewire biomolecular interaction networks. Interestingly, most missense mutations did not result in simple loss-of-function proteins. Rather, a large fraction of mutations caused perturbations in the cellular interactome. This study allowed us to understand how rewired cellular interaction networks critically contribute to human disease.

Although this systematic characterization of the hmORF collection provided us with valuable information on protein/protein interactions and protein stability, there is still much more to learn from this library. What could be causing each protein to lose or gain protein interactions? What other molecular mechanisms could be causing disease for the proteins that retain all of their interactions? How can we translate this information into treatments for people affected by rare disease? To address all of these questions, I aimed to study the protein localization (including protein aggregation) of each variant in the hmORF collection.

The Effect of Protein Localization in Rare Disease

Cellular organelles limit the access of proteins to interacting partners and biomolecules, allowing each compartment of the cell to be associated with a unique range of biochemical processes. Thus, proper protein localization is crucial for protein function and a mutation that causes protein mislocalization can lead to disease by protein inactivation, by a disruption of crucial protein/protein interactions, and/or by toxic gain of function. In addition, protein localization is ideal for such a large-scale phenotyping approach because it is relevant irrespective of the phenotype of the disease. A broad range of human diseases have previously been demonstrated to be caused by protein mislocalization ¹⁹ and protein aggregation. ^14,20 One example of a well understood mutant protein is ΔF508-CFTR, a protein that causes cystic fibrosis (CF). ΔF508-CFTR is the most common protein mutation where folding in the endoplasmic reticulum (ER) is disrupted, resulting in ER retention of the mutant protein followed by degradation by the ER-associated degradation pathway. ²¹ Interestingly, if properly trafficked and stabilized in the plasma membrane, ΔF508-CFTR is able to retain some ability to transport chloride. ²² Thus, protein mislocalization is the direct cause of this form of CF. Furthermore, protein localization is an ideal phenotype to study since localization data can provide a platform for future efforts in drug repurposing/discovery by screening for changes in protein localization of disease-causing mutant proteins compared to their wild-type counterparts.

Toward Understanding Rare Disease

When I joined the laboratory of Dr. Mikko Taipale at the University of Toronto, the aim was to take advantage of the previously mentioned hmORF collection ¹⁸ of 2,890 pathogenic protein variants underlying over 1,100 diverse Human Gene Mutation Database (HGMD) annotated phenotypes ²³ and systematically phenotype them using immunofluorescence and high-content automated confocal microscopy. I am comparing the localization, aggregation, and effects on subcellular morphology of each wild-type protein to its mutant counterpart. The data collected from this study, combined with previously generated data ¹⁸ on protein/protein interactions, protein/DNA interactions, and protein stability, will generate a unique, publicly available database for molecular phenotypes in rare disease. This project will generate unparalleled insights into the molecular mechanisms by which mutations lead to disease and provide the groundwork for future drug discovery efforts in these neglected diseases.