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Pintos-Morell et al. Rare Dis Orphan Drugs J 2024;3:12 https://dx.doi.org/10.20517/rdodj.2023.52 Page 7 of 15

In general, the reference NBS laboratory carries out the first-tier test mainly using the MS/MS method.
Minimizing the number of “false positives”, i.e., those subjects who are shown positive for the screening test
but who are not ill, is relevant in terms of containing healthcare costs and reducing the social impact of a
positive test that can have emotional implications for parents. For this purpose, second-tier tests have been
developed, always carried out on the same DBS and with a higher specificity, for an initial evaluation of the
first screening tests, either positive or negative. Usually, when a result is obtained, a re-test is performed
with the same sample to establish its reference range, and if there is divergence, the same test is repeated
using another DBS of the same screening sample. The selected second tier test may be performed within the
rest of DBS from the original screening test (i.e., amino acids, succinylacetone, total homocysteine, genomic
sequencing), or it may be necessary to collect urine (i.e., organic acids, orotic acid, acylglycines) sometimes
with a confirmatory second screening sample. Most of the time, the referral is organized after the re-test or
the triplicate sample without waiting for the result of the second tier. However, in some cases of vitamin B12
deficiency, the increased level of C3 may not be sufficiently discriminative and it might be wise to wait for
the second tier for MMA/PA before referral takes place. In some countries, genetic diagnostic confirmation
tests are performed after the second tier or as an alternative to these for disorders such as fatty acid beta-
oxidation defects and some organic acidurias. The blood spot is taken from all live births, including live
births with subsequent death between 48 and 72 h of life, for which the sampling is carried out peri-mortem,
communicating this circumstance to the regional NBS laboratory.

As already described in the introduction, a wide range of IMDs cannot or cannot easily be identified due to
a lack of clear biochemical markers in the DBS. Table 2 presents examples of treatable IMDs without a
sufficiently robust biochemical marker where NGS is used as a confirmatory test or where NGS as first-tier
could be the best option for the screening. The number of diseases in this category heavily depends on
several factors, and the complete list is over the scope of this report. Indeed, the list of IMDs beyond the
Wilson & Jungner criteria (1968) is influenced by the discussion on treatability versus actionability of the
disease, knowledge on the natural course of the disease, ease to differentiate patients presenting early from
individuals that may have a later presentation, in which sometimes the definition of “later presentation” is
unclear (e.g., presentation after one year, ten years, or as late as adulthood), and also by several other
factors.

Present situation with NGS as a first-tier test in NBS
A wide range of pilot studies based on NGS are ongoing or planned around the world to demonstrate the
technical feasibility of NGS as a first-tier test for NBS [27,32] . The majority of these studies addresses only a
single or a few aspects related to the use of genomic methodologies in NBS, such as technical, interpretative,
social, ethical, and economic challenges, to name a few [38,39] .

If NGS will be used as the first tier, there is a need to build a database with the NGS data and their
biomarkers -if performed - and the conclusion of whether it is a 4-5 genetic variant, or still a VUS or (likely)
benign. This will require solutions for long-term storage of data. At present, biomarker-based NBS (Bio-
NBS) samples are stored for a minimum of 5 years (following established European ISO regulations). They
can be revised in various situations, e.g., when looking for causes of an unexpected death in childhood. In
this case, parents or the individuals themselves (if the related data or collected samples are stored for
patients older than 16 years of age) should give their consent for data sharing and storage. This process
would encourage the construction of large data libraries with NGS information but should be limited to the
genetic diseases agreed on before, and it must comply with the bio-bank storage regulations and informed
consent specifications for genomic samples.