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mutations in patients with other brain malformations, such as double-cortex syndrome or
polymicrogyria [32,33] .

Classical karyotype studies
Despite new technological advances, it should be noted that chromosome analysis still has a role in the
study of patients with URDs. Chromosome analysis remains particularly valuable in the detection of mosaic
abnormalities; the clarification of unbalanced translocations, rings, and complex rearrangements; and the
detection of balanced rearrangements . Patients with de novo apparently balanced rearrangements might
[34]
[35]
harbor cryptic deletions or potential gene disruptions, which would require further testing .
MULTI-OMICS APPROACHES
Massively parallel technologies appear as powerful approaches to achieve diagnosis integrated with both
DNA sequencing and phenotypic data. Different biological molecules can be approached with omics
techniques beyond DNA, such as RNA, metabolites, and proteins or epigenetic modifications. The analysis
of these molecules offers complementary data that could help to understand diseases mechanism and
provide new diagnostic tools. A general drawback of multi-omics approaches is the difficulty in obtaining
the most informative tissue for the analysis when it is not blood or skin fibroblasts.

Epigenomics
Epigenomics is the analysis of the entire set of epigenetic modifications on the genetic material of a cell,
generally intending to identify alterations in DNA methylation or histone modification in diagnosing
specific disorders. DNA methylation is one of the most commonly studied epigenetic modifications, and
aberrant DNA methylation has long been associated with several RDs, such as Angelman and fragile X
syndromes. Several methods have been developed to assess genome-wide DNA methylation in peripheral
blood [36-38] . These methods have proven particularly useful to diagnose patients for whom there is a clinical
suspicion of an imprinting disorder or Mendelian disorders caused by mutations in genes that code for
proteins involved in epigenetics (e.g., Coffin-Siris, Kabuki, and Sotos syndromes) [36,37] . The potential utility
[38]
of diagnostic clinical genomic DNA methylation testing in patients with RDs has been recently reported .
Using a clinical genome-wide methylation test for patients with RDs, 35% of patients in a targeted cohort
were positive for a diagnostic episignature versus 11% of patients in a screening cohort, proving that this
approach can be applied for the untargeted study of patients with URDs .
[38]
Transcriptomics
The utility of analyzing the patient’s transcriptome for RDs has already been proven. Single-gene RNA
sequencing has been carried out to elucidate the role of candidate genetic variants of uncertain significance
[39]
(VUS) by combining reverse transcriptase to create cDNA and RT-PCR to measure RNA . Performing
RNA sequencing (RNA-seq) of the entire transcriptome in a single run can facilitate the detection of
[40]
aberrant expression, aberrant splicing, and mono-allelic expression . RNA-seq has proven particularly
useful in specific clinical settings, such as the analysis of muscle biopsies from patients with rare muscle
disorders and cultured fibroblasts from patients with mitochondrial disorders, or recessive conditions for
which only one mutation has been identified [41-43] . Some studies have assessed the utility of RNA-seq, in
combination with ES/GS, as a diagnostic tool for URDs of diverse disease categories, with an additional
yield of 17%-18% [44,45] . The main challenges of RNA-seq are the accessibility of appropriate tissues for
mRNA extraction, the large amount of sequencing required to detect changes, and the necessity of large
[46]
control cohorts .