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Page 106 Marti et al. J Transl Genet Genom 2020;4:104-13 I http://dx.doi.org/10.20517/jtgg.2020.10

as 20 kb long, including heterozygous deletions and duplications. However, a significant number of patients
remained undiagnosed and consequently, physicians moved on to target sequencing of disease associated
genes, or more recently WES. Target sequencing and WES allow identification of SNVs as well as small
[18]
indels (2-20 bp) providing a diagnostic yield of 25% for children with ID . The main difference between
[19]
target and WES is the per sample cost, which tends to be lower for the target approach . However, due to
the aforementioned high phenotypic variability, sequencing only a limited number of genes can reduce the
overall diagnostic yield.

The reduction in sequencing costs in the last decades has enabled WGS to be added to the diagnostic
armamentarium. WGS has the potential to identify all forms of genetic variants, SNVs, indels, as well as
CNVs. Recent studies demonstrated the advantages of WGS over both CGH and WES for the identification
of novel mutations, with an overall diagnostic yield between 40%-60% for children with ID. The genetic
heterogeneity of ID [17,20] makes WGS possibly the most cost-effective approach in terms of diagnostic
yield and sequencing costs. However, it is important to note that WGS has larger costs related to data
processing and storage, as well as analysis - which is much more challenging - compared to CGH or WES.
As an example, while WES provides about 100,000 SNVs, WGS yields over 3 million variants per sample,
of which only one (or a few) are likely to be relevant to the case. Moreover, WGS will require appropriate
counseling, including appropriate management of any incidental finding.

INTERPRETATION OF GENETIC VARIANTS IN ID PATIENTS
One of the main challenges in the molecular diagnosis of ID concerns the identification and, most
importantly, assignment of any found variant as responsible for the observed phenotype. This task, which
requires the annotation, interpretation and selection of variants for each case, is usually performed in
a multidisciplinary context, with the involvement of bioinformaticians, molecular geneticists and the
responsible physician, and is referred to as Clinical Genomics Interpretation. The complexity of the task is
related to the chosen technique. While sequencing of gene panels - already focused on ID associated genes
- delivers a few hundred variants, CGH results in thousands of CNVs, WES yields up to 100,000 variants,
and finally for WGS, over a million.

The first step of variant filtering (i.e., reduction of the number of potential candidates) involves focusing
on ID related genes. OMIM, an Online Catalog of Human Genes and Genetic Disorders, lists 1330
independent genes associated with the words “intellectual disability” - double the number of ID-related
[21]
genes listed in 2015 - with a variety of functions and modes of inheritance . The latest update on 4
[22]
December 2019 of the SysID-database (https://sysid.cmbi.umcn.nl/) currently contains 1291 primary
ID genes, and 1140 candidate ID genes. This huge number and functional diversity of ID-related genes
contributes to the challenge of identifying new genes or genetic variants related to ID unequivocally.

Another important filtering criterion is related to genetic variation properties. Current state of the art
[23]
techniques classify variants according to the American College of Medical Genetics criteria , which
involves determination of several evidence criteria (or level), which then add up to a final score that
determines whether the variant is (likely) benign, (likely) pathogenic or of uncertain significance (i.e., a
variant of unknown significance or VoUS). Although there are more than 25 different criteria, they can
arguably be grouped into those related to: (1) predicted molecular effect; (2) observed frequency in healthy
individuals; (3) familial segregation; (4) genotype to phenotype relationships; and (5) previous reports.
Ideally, the combination of genomic techniques and use of appropriate filtering criteria should result in the
identification and report of a (likely) pathogenic variant. Yet, as will be explained below, this is particularly
difficult when dealing with ID related variants. Databases such as IDGenetics, (http://www.ccgenomics.cn/
[24]
IDGenetics/) , a genetic database for ID that provides integrated genetic, genomic and biological data,
can facilitate the interpretation of ID related genetic variants.