A Proteomic Variant Approach (ProVarA) for Personalized Medicine of Inherited and Somatic Disease

Abstract

The advent of precision medicine for genetic diseases has been hampered by the large number of variants that cause familial and somatic disease, a complexity that is further confounded by the impact of genetic modifiers. To begin to understand differences in onset, progression and therapeutic response that exist among disease-causing variants, we present the proteomic variant approach (ProVarA), a proteomic method that integrates mass spectrometry with genomic tools to dissect the etiology of disease. To illustrate its value, we examined the impact of variation in cystic fibrosis (CF), where 2025 disease-associated mutations in the CF transmembrane conductance regulator (CFTR) gene have been annotated and where individual genotypes exhibit phenotypic heterogeneity and response to therapeutic intervention. A comparative analysis of variant-specific proteomics allows us to identify a number of protein interactions contributing to the basic defects associated with F508del- and G551D-CFTR, two of the most common disease-associated variants in the patient population. We demonstrate that a number of these causal interactions are significantly altered in response to treatment with Vx809 and Vx770, small-molecule therapeutics that respectively target the F508del and G551D variants. ProVarA represents the first comparative proteomic analysis among multiple disease-causing mutations, thereby providing a methodological approach that provides a significant advancement to existing proteomic efforts in understanding the impact of variation in CF disease. We posit that the implementation of ProVarA for any familial or somatic mutation will provide a substantial increase in the knowledge base needed to implement a precision medicine-based approach for clinical management of disease.

CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine

“…Studies using genome-and proteome-wide association analyses have identified proteostasis components that could be targeted to rescue CFTR in F508del-expressing cells (Wang et al, 2006;Simpson et al, 2012;Tomati et al, 2018a). Furthermore, the recent identification of differences in WT-and F508del-CFTR interactomes unveiled several targets that could be exploited to rescue F508del (Pankow et al, 2015) and potentially other misfolding CFTR mutants (Hutt et al, 2018).…”

HDAC inhibitors rescue multiple disease-causing CFTR variants

“…To provide insight into the mechanistic impact of HDACi on proteostatic environment of hBE cells, we performed a western blot analysis to monitor any changes in expression or activation of markers of a number of known CF-linked cellular pathways. These included the expression level of HDAC7, previously shown to be affected by HDACi treatment (74,99) and the eukaryotic translation Initiation Factor 3a (eIF3a), whose silencing has been shown to mediate significant correction of F508del-CFTR based on our recent proteomic and molecular analysis (100,101). Additionally, we monitored the expression and phosphorylation status of eIF2α and HSF1, markers of the UPR and HSR pathways, respectively, which have been shown to be chronically activated in F508del-expressing cells and impact the severity of the disease state (42).…”

HDAC Inhibitors Rescue Multiple Disease-Causing CFTR Variants

“…CFBE41o-, F508del CFBE41o-and WT-HBE41o-were stably transfected with pENTR1A-CFTR plasmids were sequenced and recombined into pAD-CMV-V5-DEST using LR-clonase II (Invitrogen). pAD-CMV-CFTR plasmid were then sent to ViroQuest for adenovirus production [42,113]. 80% confluent CFBE41o-were transduced for 5 h in OPTI-MEM in the presence of 10 µg/ml of polybrene (EMD Milipore Corp) with adenovirus carrying CFTR at a multiplicity of infection of 200.…”

“…To perform a side-by-side comparison of the different CF variants, we utilized parental CFBE41o-cells, which are genotypically F508del/F508del, and do not express detectable CFTR mRNA or protein. The choice of these CFBE41o-null cell lines provide a cellular environment optimized for the expression, folding, trafficking and functional regulation of CFTR [104]. To characterize the 10 variants described above in response to HDACi in the presence or absence of Lumacaftor, we transduced the CFBE41o-null cells with adenoviral particles carrying the different CFTR variant cDNAs.…”