# Phenotypic Definition and Genotype-Phenotype Correlates in PMPCA-Related Disease
## 3 Results
### In Silico Molecular Modeling and Simulation  
All _PMPCA_ mutations reported to date are missense variants, variably distributed throughout the protein, except for one frameshift variant very recently reported (Figure 3). To attempt performing genotype-phenotype correlates, we performed in silico modeling of a few missense variants detected in patients with severe, intermediate, and mild phenotypes (Figure 4). We compared the behavior of wild type (wt) versus mutant proteins to measure the impact on the protein structure and stability induced by each variant.  
Figure 3: Established peptidase mitochondrial processing alpha (PMPCA) domain structure and location of missense mutations previously reported. Mutations identified in homozygous state are indicated with H, while heterozygous mutations identified together in a compound heterozygous state are equally marked (*, #, $ or *). For each mutation, severity of related phenotype is indicated with a color code.  
Figure 2: Protein levels of alpha subunit of mitochondrial processing peptidase (\(\alpha\)-MPP) in the two Italian patients with intermediate phenotypes versus controls (average of three healthy subjects). (**A**) Representative Western blot of \(\alpha\)-MPP and \(\beta\)-actin (as normalizer). (**B**) Densiometric analysis from three independent experiments. Protein levels in patients are represented as percentage of control (average of three healthy controls) set at 100. Error bars indicate standard error of the mean (SEM). * \(p<0.05\); ** \(p<0.01\).  
The wt heterodimer exhibited increased values of RMSD for the first 50 ns and then reached a plateau, fluctuating in a range of 0.18-0.24 nm, compared with the reference starting structure. All mutants exhibited less stable and higher RMSD values than wt but p.Val256 Met, which overlapped the wt profile (Figure 4A). Focusing on the glycine-rich loop, all mutants showed more stable RMSD values than wt (Figure 4B), with p.Arg185 Trp, p.Arg362 Gly, and p.Ala377 Thr being the most dissimilar with \(\sim\)0.1 nm lower RMSD values. Regarding the DCCM profiles, variants p.Val256 Met, p.Trp278 Leu, p.Gly356 Ser, and p.Arg362 Gly showed a dramatic increase in all atomic motions (Figure 4C), reflecting an evident gain in the overall mobility compared to the wt system, while p.Arg185 Trp and p.Ala377 Thr were similar to wt. The regions spanning residues 300-400 of PMPCB and 260-460 of PMPCA (the latter containing the GRL) exhibited anticorrelated movements, which were lost in mutants p.Arg185 Trp, p.Val256 Met, p.Gly356 Ser, and p.Ala377 Thr, finally suggesting a decreased flexibility of the glycine-rich region in these mutants.  
Figure 4: Root-mean-square deviation (RMSD) profiles of the C\({}_{\alpha}\) atoms of the wild type (wt) (black) and all mutant systems for the whole heterodimer (**A**) and the glycine-rich loop (**B**). **(C)** Dynamic cross-correlation maps (DCCMs) of wt (lower triangular of each matrix) and all PMPCA mutants (upper triangular). Perfect correlations are highlighted in red (direct) or violet (inverse). Green box highlights the wt anticorrelated movements between residues 300 and 400.