Proteotyping Gene Dosage Effects in Genetic Diseases

and Objectives Gene dosage imbalance is a general working hypothesis for studying the genetic diseases, such as human syndromes, because the DNA copy number variations (CNVs) are causal for many genetic diseases. However, recent important studies have showed that many CNVs resulting in mRNA changes only weakly influence corresponding protein abundances. Herein we show by proteome‐wide turnover analysis that CNV's substantially remodel the proteome. In the presentation we will discuss specific instances of CNV's varying in the extent and magnitude of affected loci, and disease phenotypes. These examples include trisomy 21 (T21) in human Down Syndrome, and Williams Syndrome (WBS) and 7dup‐ASD syndrome caused by 7q11.23 hemideletion or hemiduplication. Methods We utilized SWATH mass spectrometry (SWATH‐MS) to accurately quantify protein degradation through a pulse SILAC experiment (pSILAC) up to 24 hours. For T21, we used fetal skin fibroblasts derived from a pair of monozygotic twins discordant for T21. To validate the results, we also analyzed fibroblasts from 11 unrelated T21 individuals bearing Down Syndrome and 11 controls. For WBS, patient‐derived induced pluripotent stem cells were analyzed. Data were processed by OpenSWATH workflow. The transcript profiling was measured by RNA‐Seq. Results and Discussion In the case of Down Syndrome, we investigated the effect of T21 at the levels of transcript quantity, proteome quantity and protein turnover rate. We quantified 4056 unique proteins for expression and ~2200 proteins by pSILAC experiment for turnover analysis in both normal and T21 twins. The T21/normal fold‐change correlation between transcript and protein levels was extremely low, indicating substantial post‐transcriptional regulation and buffering effects in T21. Overall, the protein degradation was faster in trisomy cells than the controls. Remarkably, those Chr21 encoded proteins that are members of heteromeric protein complexes were largely exempt from responding to CNV alterations, primarily through accelerated protein degradation. Moreover, we found that both mitochondrial and cytosolic ribosomal proteomes were degraded heavily in T21, but different degree of translational regulation shaped their final, divergent expression levels. In the case of WBS, we found that proteins with significant differences in turnover at the pluripotent state are components of pathways involved in translation, ubiquitination, splicing and basal metabolism. Conclusions Our data suggests that protein‐specific turnover presents a primary mechanism of proteome remodeling in response to DNA dosage alterations and thus highlighting a proteomic understanding of “gene dosage imbalance” in genetic disorders. Support or Funding Information Y.L. was supported by the European Research Council (ERC, consolidator grant number 616441‐DISEASEAVATARS). The group of RA was supported by was supported by ERC grants Proteomics v3.0 (AdG‐233226 Proteomics v.3.0) and AdG‐670821 Proteomics 4D), and the Swiss National Science Foundation (SNSF) grant number: 31003A_166435.