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Aguiar. Rare Dis Orphan Drugs J 2024;3:13 https://dx.doi.org/10.20517/rdodj.2023.56 Page 19 of 29

there are several challenges and limitations that need to be overcome to keep pace with advancements and
ultimately realize clinical applicability. These hurdles include the complexity of proteome (a large number
of structural and biochemical differences of proteins), a very wide range of protein concentrations, complex
sample preparation and data analysis (most reported biomarkers remain unidentified), and limited
sensitivity. Additionally, the capacity for quantitative measurements is not yet at the level required for
routine diagnostics in a clinical setting. Furthermore, it remains unclear how clinicians will use the sensitive
data since even small changes in physiology, such as food ingestion or going up a flight of stairs, can have a
[220]
significant impact on the metabolome .
Despite these limitations, plasma and urinary metabolomics showed that Gb3, lyso-Gb3, and
galabiosylceramide isoforms/analogs were elevated in FD patients [47,221-225] . However, these candidate
biomarkers still requires validation that they overcome the lyso-Gb3 limitations. This validation includes
the evaluation of late-onset females and patients with variants of unknown significance and correlation with
clinical manifestations and treatment responses. Thus, properly designed longitudinal studies are warranted.

The proteomic approach has also been applied in the search for biomarkers of FD in plasma, peripheral
blood mononuclear cells, and urine. Urinary proteome is the most extensively studied in FD, with a few
studies with up to 66 patients reporting several proteins with altered expression [226-231] . A panel of biomarkers
including 40 proteins were able to show proteins that are related to early/preclinical phase of FD, to
monitoring kidney injury, and to heart involvement; however, the actual prognostic significance of these
[231]
panels was not clearly depicted and should be evaluated further .

Designing studies to identify new biomarkers is also challenging and the design should be tailored to the
specific objective of the study/biomarker: diagnostic purposes, identify preclinical involvement, clarify the
prognosis, or assess the response to the treatment. Per example, to assess the prognosis and the response to
DST, a complex design is warranted [Figure 3]. To identify prognostic biomarkers, several premises should
be taken into account in the study design, including longitudinal and prospective design, follow-up
duration, and a clear definition of adverse events and disease progression. In studies assessing the response
of a specific biomarker to DST, beyond the described aspect, it is paramount to clarify the definitions of
treatment response and failure. Defining the duration of a biomarkers study is difficult, because FD is a
slowly progressive disorder with a wide spectrum of severity/phenotypes and a low event rate.

Several protein and lipid experimental biomarkers are under investigation in Fabry disease [Table 5], mainly
related to inflammation, endothelial dysfunction, cardiac fibrosis, glomerulosclerosis, and tubulointerstitial
fibrosis. Furthermore, there are also few reports on proteomic and metabolomics analysis.

CONCLUSION
The identification of biomarkers for identification of preclinical involvement, prognostic evaluation, and
response to treatment is an urgent need in FD. The available biomarkers of total disease burden, such as
plasma lyso-Gb3, have several limitations in prognostic evaluation and monitoring treatment response.

Thus, accurate longitudinal studies are needed to identify new biomarkers and their prognostic value.
Furthermore, FD has a very complex physiopathology, and certainly, no single biomarker is able to
characterize all the pathways involved, so composite scores of clinical and laboratory variables should be the
only method to assess each patient’s prognosis and response to treatment.