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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 449

[50]
patients with epilepsy remains limited, especially among adults with DEEs .

Challenges in the analysis and interpretation of variants of uncertain significance
Even after our rapid increment in knowledge about the diverse genetic mechanisms, we do not understand
yet most of the genomic variations. The American College of Medical Genetics and Genomics (ACMG)
defines a variant of uncertain significance (VUS) as a genetic variant with a probability of pathogenicity
[51]
between 10% and 90% . The proportion of VUS recognized in every report is variable, as are some factors,
such as the indication, the size of the genetic region, and the current knowledge of a function in a particular
[52]
protein . This increment in VUS produces difficulties in patient outcomes and is a major challenge in the
correct diagnosis of DEEs.

The clinical validation of genetic testing outcomes is complex to establish. In several examples, there are
phenotypic expansions of already known disease-gene correlations. When numbers analyzed increase, the
detection of atypical phenotypic presentations of DEE grow, expanding the phenotype and the clinical
variability associated with that specific gene. Usually, the severe forms of the disease are better established,
while milder forms will remain hidden until genetic studies begin to be requested in a larger sample of
[53]
patients with less severe epilepsy forms .

Our knowledge about the gene variant-disease relationship is continuously growing; approximately 300 new
identified associations or new genes are reported each year . These newly described associations may result
[54]
from genes that had never been related to a genetic condition (new disease gene identification) or that were
previously related to a different disease (new phenotypic association). Some laboratories report variants in
genes of uncertain significance (GUS), requiring the search for other individuals with variants in the that
GUS and similar or overlapping clinical features to aid this candidate gene as a disease-causing gene and
ultimately a diagnosis . Some studies elucidate that around 6000-13,000 Mendelian diseases remain to be
[55]
defined . Although this suggests a huge potential for the identification of new disease-gene associations,
[55]
those sometimes cannot be discover using the current diagnostic strategy, thus requiring in some cases the
implementation of functional studies to elucidate the gene variant-disease association . In light of these
[56]
findings, the evaluation and interpretation of studies in DEE patients requires continuous reevaluation and
analysis. These are outlined schematically in Figure 2. Additionally, recent disease-focused specifications for
ACMG guidelines might be helpful in the interpretation of some clinical scenarios, regarding variants
[57]
(especially VUS) with specific genes/disorders .

Suitability and sufficiency of previous genetic testing
“Negative genetic testing” in patients with DEE is often the result of scarce workup. Thus, a large
proportion of negative genetic outcomes in this population results from insufficient screening or obsolete
[58]
evaluation requiring an update . There are three types of genetic studies that are currently considered
insufficient in all types of DEEs: karyotyping, single gene sequencing, and array-CGH. Karyotyping is rarely
indicated in epilepsy (only in cases of suspected ring chromosomes, such as 20 or 14) and should not be
considered as the unique study to be performed in any case . Regarding single- or two-gene sequencing,
[59]
there are also few clinical scenarios where it can be efficient. The most characteristic are Dravet syndrome
or tuberous sclerosis, entities where the predictive capacity for a specific genetic diagnosis (SCN1A or
[60]
TSC1/2) is high . However, in most cases, if this first approach is negative, large panels or exome
sequencing will be appropriate. Array-CGH is a test that has been widely used in DEE patients, but its
diagnostic yield is of only about 10% . Therefore, its negative study should never be considered as
[61]
sufficient in these patients. Whole exome sequencing (WES) is currently considered the most appropriate
technique for genetic diagnosis in DEE, with a diagnostic yield of up to 50% , which improves if trio
[62]
strategies are used (WES also performed in the parents to be able to segregate identified variants of interest),