Insight into the specificity and severity of pathogenic mechanisms associated with missense mutations through experimental and structural perturbation analyses

Abstract

Most pathogenic missense mutations cause specific molecular phenotypes through protein destabilization. However, how protein destabilization is manifested as a given molecular phenotype is not well understood. We develop here a structural and energetic approach to describe mutational effects on specific traits such as function, regulation, stability, subcellular targeting or aggregation propensity. This approach is tested using large-scale experimental and structural perturbation analyses in over thirty mutations in three different proteins (cancer-associated NQO1, TTR related with amyloidosis and AGT linked to primary hyperoxaluria type I) and comprising five very common pathogenic mechanisms (loss-of-function and gain-of-toxic function aggregation, enzyme inactivation, protein mistargeting and accelerated degradation). Our results revealed that the magnitude of destabilizing effects, and particularly, their propagation through the structure to promote disease-associated conformational states largely determine the severity and molecular mechanisms of disease-associated missense mutations. Modulation of the structural perturbation at a mutated site is also shown to cause switches between different molecular phenotypes. When very common disease-associated missense mutations were investigated, we also found that they were not among the most deleterious possible missense mutations at those sites, and required additional contributions from codon bias and effects of CpG sites to explain their high frequency in patients. Our work sheds light on the molecular basis of pathogenic mechanisms and genotype-phenotype relationships, with implications for discriminating between pathogenic and neutral changes within human genome variability from whole genome sequencing studies.

The Impact of Mitochondrial Deficiencies in Neuromuscular Diseases

“…This approach has been developed in SMA and DMD and approved by FDA. In SMA, an ASO that switches the splicing to include an exon in the pre-mRNA of SMN2 gene has become the first treatment for SMA approved by FDA, commercialized by Biogen (MA, USA) under the name Nusinersen [ 67 ].…”

“…Once inside neurons, SMN1 cDNA constitutively expresses, with long-term expression in differentiated cells after a single dose, but not in mitotically active cells. This drug has shown efficacy in infantile SMA patients and is commercialized by Avexis under the name Zolgensma [ 67 , 156 ]. Other gene therapies using CRISPR/Cas-mediated approaches in NMD have shown efficacy in models of DMD, CMD, LGMD, DM1, and FSHD [ 157 ], becoming promising therapeutic approaches for further transference into clinical settings.…”

Naturally-Occurring Rare Mutations Cause Mild to Catastrophic Effects in the Multifunctional and Cancer-Associated NQO1 Protein

“…Our limited capacity for establishing large-scale genotype-phenotype correlations can be explained by different reasons: (i) the relationships between molecular, pathogenic and organismal effects are complex, even for diseases with simple Mendelian inheritance [1,7,8]; (ii) genotype-phenotype correlations are improved when experimental data for molecular effects of mutations are available, but current predictive tools still underperform experimental characterization, particularly for mild to moderate phenotypes [8][9][10][11]; (iii) although many diseases are caused by loss-of-function mutations [12], an operational definition of loss-of-function is difficult because human proteins display many functional and regulatory traits. In this context, genotype-phenotype correlations require an integrated understanding on how a single mutation may affect multiple molecular functions simultaneously and, consequently, how these molecular effects translate into pathogenic and fitness consequences [1,[13][14][15][16][17][18][19][20][21][22][23].…”

“…The molecular mechanisms by which missense mutations cause loss-of-function are many-fold [24] and include accelerated protein degradation [5,9,11,25,26], enhanced protein aggregation [14,15,21], catalytic and regulatory alterations [21,[27][28][29] and altered biomacromolecular interactions [10,30]. Importantly, the common molecular origin for all these coexisting mechanisms appears to be the structural and energetic perturbation caused by missense mutations [5,8,11,19,24,26]. These perturbations may differently contribute to the molecular phenotype associated with a given missense variant [19].…”

AAV-Delivered Tulp1 Supplementation Therapy Targeting Photoreceptors Provides Minimal Benefit in Tulp1−/− Retinas

“…With FDA/EMA approval of Luxturna TM , the first ocular gene therapy in 2017/18, interest in development of gene therapies for IRDs is substantial. In preclinical proof of principle studies, animal models of IRDs ( Ziccardi et al, 2019 ; Collin et al, 2020 ) have been extremely useful and ultimately contributed to paving the way toward clinical trials for many retinal gene therapies ( ClinicalTrials.gov ); reviewed by Dalkara et al (2016) and Trapani and Auricchio (2019) . Many preclinical studies have highlighted the value of adeno associated virus (AAV) as a means of gene delivery and have demonstrated benefit at molecular, histological and functional levels of AAV-delivered gene therapies (among others, Millington-Ward et al, 2011 ; Chadderton et al, 2013 ; Schön et al, 2017 ; Cideciyan et al, 2018 ).…”