Assessing the clinical utility of protein structural analysis in genomic variant classification: experiences from a diagnostic laboratory

Abstract

Background The widespread clinical application of genome-wide sequencing has resulted in many new diagnoses for rare genetic conditions, but testing regularly identifies variants of uncertain significance (VUS). The remarkable rise in the amount of genomic data has been paralleled by a rise in the number of protein structures that are now publicly available, which may have clinical utility for the interpretation of missense and in-frame insertions or deletions. Methods Within a UK National Health Service genomic medicine diagnostic laboratory, we investigated the number of VUS over a 5-year period that were evaluated using protein structural analysis and how often this analysis aided variant classification. Results We found 99 novel missense and in-frame variants across 67 genes that were initially classified as VUS by our diagnostic laboratory using standard variant classification guidelines and for which further analysis of protein structure was requested. Evidence from protein structural analysis was used in the re-assessment of 64 variants, of which 47 were subsequently reclassified as pathogenic or likely pathogenic and 17 remained as VUS. We identified several case studies where protein structural analysis aided variant interpretation by predicting disease mechanisms that were consistent with the observed phenotypes, including loss-of-function through thermodynamic destabilisation or disruption of ligand binding, and gain-of-function through de-repression or escape from proteasomal degradation. Conclusions We have shown that using in silico protein structural analysis can aid classification of VUS and give insights into the mechanisms of pathogenicity. Based on our experience, we propose a generic evidence-based workflow for incorporating protein structural information into diagnostic practice to facilitate variant classification.

Candidate variants in DNA replication and repair genes in early-onset renal cell carcinoma patients referred for germline testing

“…The I759 residue makes twice the number of hydrophobic contacts as the wildtype V759 with the long helix below. The variant could lock this strand in an overly rigid position that compromises DNA polymerization steps that involve flexibility [ 35 , 36 ], consistent with the biochemical results observed in Fig. 3 A.…”

“…3 B (left gel), and Supplementary Table 4 ). Thus, a significant destabilization in a relatively rigid region may impact enzyme function, as supported by [ 35 , 36 ]. Interestingly, the activity of the G209V compares well with wt for a normal primer template and might even be more processive (Fig.…”

PhenoScore: AI-based phenomics to quantify rare disease and genetic variation

“…For neurodevelopmental disorders, these assays are scarce since they need to be developed on a gene-per-gene basis, and for these rare disorders, this is usually not cost-effective and solely done for research purposes. Other methods to assess genetic variants include protein structural analysis [61], which however still relies on the availability of relevant protein structures. Our approach theoretically works for any (genetic) condition with a recognizable phenotype, provided there are sufficient individuals for training the algorithm, and that HPO data and 2D-facial photos are available.…”

Molecular, Biochemical, and Clinical Characterization of Thirteen Patients with Glycogen Storage Disease 1a in Malaysia

“…e PDB file was first repaired using the FoldX RepairPDB command, and the repaired PDB was subjected to mutagenesis using the BuildModel command. We used the same criteria described by Caswell et al [17] to interpret the change in free energy of the mutated structure compared to the wild-type structure. Both protein structures (containing native or mutated residues) were visually inspected using PyMOL.…”

“…e Cys109 residue is located in an extended, unstructured loop region of G6Pase which has a high predicted Local Distance Difference Test (pLDDT) score of 92.99 (Figure 3). is score exceeds the recommended threshold of 70 for the AF-2 model, therefore structural analysis performed using this model is likely to generate reliable predictions [17]. Missense3D predicted that replacement of Cys with Arg would abolish the disulphide bond formed between Cys109 and residue Cys254.…”