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Magnifico et al. Rare Dis Orphan Drugs J 2023;2:16 https://dx.doi.org/10.20517/rdodj.2023.17 Page 9 of 15

on the diagnostic list. Negative results indicated no detection of any pathogenic or likely pathogenic variants
or any VUS on the diagnostic gene lists. 15/17 (88,2%) of patients affected by metabolic conditions resulted
as GS-NBS positive. In the hearing loss cohort, “inconclusive” findings, not providing definitive results,
were reported (some participants were heterozygous or homozygous for different VUSs in genes associated
with hearing loss). Two false negative results were detected: one patient had a single heterozygous
pathogenic variant in a gene associated with maple syrup urine disease and a patient with Malonyl-CoA
decarboxylase deficiency had a homozygous missense VUS. However, since the authors did not have
sufficient information to better identify the genetic etiology of the patient’s disease, both were reported as
“inconclusive findings”. One patient was a carrier for another condition. 5/28 (17,9%) patients affected by
hearing loss tested GS-NBS positive and two of them had positive screen results unrelated to their
[27]
condition .
After the conclusion of the NC NEXUS project, it has been stated that using a GS approach could not
widely substitute current screening tests. However, genomic information could be useful to perform a
“secondary” or “indication-based” analysis, improving the sensitivity and specificity of NBS for inborn
[27]
errors of metabolism .

In the Netherlands, the NBS (NGSf4NBS) project is a technical feasibility study also aiming at assessing the
ethical, legal, social, and financial aspects to explore the adoption of NGS approaches as a first-tier method
in NBS . The study will proceed in three steps. In Step 1, inherited metabolic disorders eligible for NGS as
[26]
a first-tier test will be identified based on treatability. In Step 2, the feasibility, limitations, and comparability
of different technical NGS approaches and analysis workflows for NBS will be tested. In Step 3, the results
will be incorporated into the current Dutch NBS program, including guidelines for the referral of a child
after a positive NGS test result .
[26]

Methods to evaluate the criteria for inclusion of genes in GS studies
[23]
NBS through WGS and WES should be based on a clear path of clinical utility and/or actionability . The
magnitude of the genomic information generated, and its management are key challenges of introducing GS
in the clinical setting. Other issues that must be taken into account are the definition of a subset of clinically
actionable findings, the use of standardized protocols, and the introduction of appropriate and shared
informed consensus for the families involved. In 2016, Berg et al. defined a semiquantitative metric for
evaluating clinical actionability by assessing five criteria: the severity and likelihood of manifesting a
particular condition, the efficacy and acceptability of the intervention, and the overall knowledge base of the
gene-disease association . The metric did not take into account the individual’s age and sex, the timing of
[2]
the onset of the disease, and the availability and cost of any preventive strategy.

The North Carolina Newborn Exome Sequencing for Universal Screening (NC NEXUS) project
implemented the semiquantitative metric and assessed an age-based framework for evaluating genome-scale
sequencing results in NBS. The age-based, semiquantitative metric categorized gene-disease pairs into
groups based on age of onset or timing of interventions, improving the past method and facilitating the
definition of inclusion criteria in the GS studies .
[5]

Additionally, a list of genes with putative pediatric relevance based on the framework released by the
Clinical Genome Resource (ClinGen) working group has been assessed to manage the return of results in
the BabySeq project. The generation of the gene-disease pair association was curated for the following
criteria: validity of gene-disease association, age of onset, penetrance, and inheritance pattern. Based on the
selected criteria, three categories of classification of gene-disease pairs were defined: category A: genes