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Aledo-Serrano et al. J Transl Genet Genom 2021;5:443-55 https://dx.doi.org/10.20517/jtgg.2021.40 Page 451

Firstly, the patient phenotype is deeply analyzed. Subsequently, the strategy of previous tests should be
reviewed, in order to look for pitfalls that did not lead to a diagnosis. Previous tests should be reviewed to
reveal whether a broad approach (in genes and conditions) was properly performed. For patients with
convincing VUS, we would start by searching for new publications with information showing pathogenicity
(e.g., in ClinVar or HGMD) or benign variation (e.g., high frequency in gnomAD). Some additional
[67]
information might come from segregation or functional studies .
Nowadays, it is not only important to carry out quality genetic studies, but also to have the possibility to
reanalyze them after a period (maximum every 3-5 years). Exome sequencing that is negative today may be
positive in two years. The clinician should demand access to sequences and regular reanalysis of patient
data. In many clinical fields, WES is available as a funded test for most of the patients with complex
[67]
phenotypes . However, it is only 30%-50% successful . Although it is not established which percentage of
[66]
individuals carry pathogenic variants in the protein-coding part of the genome (exome), it is likely to be
much higher than this diagnostic yield. In fact, a negative exome is considered a point of start for the patient
without a diagnosis, as it has been documented that there is a high probability that reanalysis will identify
[68]
diagnoses both in old and new genes .

Reanalysis of exome data provide additional diagnostics for some reasons [55,61,68,69] :

(1) Immediate reanalysis of exome data with geneticist-clinician collaboration has been reported to reach a
diagnosis in known disease genes in 10% of undiagnosed patients. This gap in genetic diagnostic finds its
origin in a poor clinical interpretation of genetic data when interaction between geneticists and clinical
neurologists is lacking.

(2) In a similar way in which new information on disease-associated variants can assist in the reevaluation
of VUS identified in known genes, the approximately 300 disease-gene associations newly described every
year may identify variants that were not evaluated when the genetic testing was performed.

(3) Clinicians may elucidate these new disease-gene correlations on their own. It has been demonstrated
that discovery of new genes through recurrent analysis of exome data can double the diagnostic yield .
[69]
(4) Reanalysis collaborating with the laboratory using distinct bioinformatics approaches can reach a
diagnosis which was missed using the previous strategies.

(5) Reanalysis allows the clinician to modulate different thresholds (e.g., quality or rarity) and use different
bioinformatic tools. It depends on some clinical scenarios, such as mosaic variants mimicking autosomal
recessive pattern of inheritance.

(6) RNA sequencing methods can play a role in some specific cases, with the identification of pathogenic
variants that remain unnoticed or doubtful after WES .
[70]

LIMITATIONS AND FUTURE PERSPECTIVES
Currently, there is a broad spectrum of genetic diagnostic tools suitable for clinical assessment of DEEs.
However, all have technical limitations. Clinicians must embrace that, even if the best selected genetic test
for their patient had been performed, a genetic alteration cannot be certainly ruled out; they may only
reduce its probability. For instance, although NGS (next-generation sequencing) techniques have improved