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To be classified as (likely) pathogenic, a variant would usually have a strong molecular effect (i.e.,
nonsense, frameshift, affect splicing and/or missense with a known molecular phenotype), display very
low population frequency, verified to be de novo, display a known matching phenotype and have been
previously reported in ID or NDD case. However, since most ID variants are de novo, they are also novel,
and thus unlikely to have been reported and/or studied at the molecular level, particularly if they are
missense SNVs. For example, one of the most commonly mutated genes in patients with ID, ARID1B,
comprises only 1% of all ID cases. Moreover, since there are many associated genes and the phenotype
in ID patients is highly variable and overlapping, it is extremely difficult to decide between variants with
similar evidence criteria but located in different genes. For CNV, where genomic intervals deviate from the
normal diploid state, the molecular effect is easier to gauge since the whole gene (or a significant part of it)
is usually deleted or duplicated, conferring a gene dosage effect. CNVs are also more likely to be unique for
the patient but there are some hot spot CNVs, mainly the ones related to syndromic ID, such as the 7q11.23
deletion that is associated with Williams Beuren syndrome, the 17p11.2 deletion associated with Smith-
Magenis Syndrome, and the reciprocal duplication, associated with Potocki-Lupski Syndrome, among
[25]
others .

In this context, it is very important that the whole family (or at least the mother/father/patient trio) is
analyzed, therefore providing direct evidence of family segregation and a straightforward filtering of the
variants observed in the proband, in order to yield a proper molecular diagnosis and make interpretation
of variants easier.

FUNCTIONAL UNDERSTANDING OF ID-RELATED GENETIC VARIANTS
Once a new genetic variant is identified, understanding its relationship with the biological molecular
mechanism is the next important step. Concomitant with the explosion of genomic information came
a revolution of tools that enabled the genetic modification of genomes. CRISPR/Cas and its associated
technologies are versatiles and make gene and genomic editing much easier than before. Model organisms
have been very helpful for studying the effect of a single genetic modification at the level of the organism.
Despite the tremendous complexity of ID in humans, it is possible to look for conservation and relevant
phenotypes to comprehend the ID-related pathophysiology in model organisms. Hence, now more than
ever, model organism studies have become instrumental for understanding the molecular mechanisms
[26]
underlying ID . This includes mice, which have historically been used to learn about disease biology and
to find potential therapeutic strategies, and fruit flies and zebrafish, which have been introduced as disease
models for ID as well.

Several extremely useful tools already exist to assess basic processes that inform on gene function, associate
a particular locus with ID, and enable dissection of both functional variant types and combinations of
variants (biallelic or multilocus) with ID. When a novel genetic variant is identified in a patient, it is very
important to define whether the variant is within a known coding region or elsewhere in the genome for
this is fundamental to determining future steps [Figure 1]. If the variant is located in a coding region,
the next big question is whether it is located within a gene already related to ID If it is a new candidate
gene, many different types of evidence can be used to identify functionally associated ID genes. This “guilt
by association” concept predicts that if two gene products work in the same pathway or process, then
[27]
mutations in these genes probably have overlapping phenotypic consequences . For example, genes that
encode physically interacting proteins, which are co-regulated or co-evolving, are more likely to work
in a common process. In addition, studies on single gene mouse models of ID reveal that the effects of
these mutations converge onto similar or related etiological pathways, highlighting common pathological
nodes that can help in the understanding of new ID related genes . The huge collection of mutant model
[28]
organisms and the literature can be reviewed to study ID related phenotypes, keeping in mind the mode
of inheritance demonstrated in humans when choosing the model to study. Towards this end, existing