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Proteomics and metabolomics
The analysis of proteins and the proteome can provide information about the activity, interaction, location,
and composition of protein complexes of clinical interest. On the other hand, metabolomic profiles have
been used as biomarkers for disease progression and response to treatment, and they are now applied in
diagnostics to determine the functional consequences of a given VUS. Mass spectrometry is the most widely
used platform in proteomics and metabolomics. Proteomics is the quantitative and qualitative analysis of
the entire set of proteins in a given specimen, generally to identify proteins that are consistently modified or
[47]
present at abnormal concentrations in specific disorders . Proteomic strategies have been applied to
investigate the pathophysiology of metabolic disorders, such as methylmalonic acidemia [48,49] . Metabolomics
is the quantitative and qualitative analysis of all metabolites derived from sugars, lipids, proteins, and
nucleic acids in a given specimen . The most successful application of targeted metabolomics analysis is
[50]
[51]
the newborn screening of inborn errors of metabolism . Overall, significant work needs to be done to
implement proteomics or metabolomics in the untargeted study of patients with URDs in routine clinical
practice.

IN SILICO BIOLOGY AND EXPERIMENTAL FUNCTIONAL STUDIES
One of the main challenges for genetic diagnosis using NGS is the interpretation of the pathogenicity of
variants, particularly when the phenotype is a source of uncertainty (e.g., reduced penetrance and variable
expressivity) or when a variant is classified as VUS or localizes in a yet-to-be-discovered disease gene.
Indeed, during the analysis and interpretation of NGS data in a particular patient, the probability of
detecting a VUS is higher than the probability of detecting a pathogenic variant . The ACMG has
[52]
elaborated standards and guidelines to classify genetic variants into five criteria-based categories using
different types of variant evidence (e.g., population data, computational data, functional data, and
[53]
segregation data) . In addition, certain bioinformatic tools facilitate the classification process (e.g.,
A v a i l a b l e f r o m : V a r s o m e , h t t p s : / / v a r s o m e . c o m / ; F r a n k l i n , A v a i l a b l e f r o m :
https://franklin.genoox.com/clinical-db; CADD, Available from: https://cadd.gs.washington.edu/), but each
geneticist must provide specific knowledge to be able to classify a certain variant . Sometimes, segregation
[13]
studies can clear up doubts, but those variants that are not reclassified remain as VUS until further studies
allow their pathogenicity to be determined.

In silico prediction of the pathogenicity of a VUS can be improved with several tools such as literature
[13]
review and data mining, pathogenicity predictors, and 3D protein modeling . After that, functional
genomics can be used for the validation of the genetic variant through molecular and cellular experiments
(e.g., subcellular localization studies, expression levels, and specific studies related to protein function) [13,54] .
At Sant Joan de Déu (SJD) Hospital and Research Institute, we developed the in-house Translational
Diagnostics Program (TDP) to functionally validate both candidate VUS and variants found in patients
with phenotype-genotype incongruity [13,55] . The TDP uses different tools of experimental and computational
biology to analyze VUS and determine the function and possible pathophysiological alteration of the
encoded protein. The objective is to delineate the impact of VUS by combining four stages including the in-
depth and precision phenotyping with functional genomics, for the validation of the genetic variant through
molecular and cellular experiments. Using this pipeline in RDs patients, we can assist the process of
reclassifying variants concerning a patient’s phenotype and improve the diagnostic deficit.

OTHER STRATEGIES FOR DIAGNOSIS AND DISEASE-RELATED GENE DISCOVERY
In recent years, the greater integration of genomic science and medicine has made it possible to explore
other strategies to achieve the diagnosis of patients or to define new genes as responsible for the disease. The
director’s board of the ACMG released a position statement on how responsible sharing of genomic variant