Huizing et al. Page 4 acid metabolism occurs in different cellular compartments and is divided into three A uthor Man processes, i.e., biosynthesis, salvage and degradation (Fig 2). De novo enzymatic sialic acid biosynthesis occurs mainly in the cytosol but also includes a nuclear step and a negative feedback-inhibition mechanism [31–34]. Free sialic acid salvage from degradation of recycled glycans occurs in lysosomes and free sialic acid exits lysosomes into the cytosol uscr through the SLC17A5 membrane transporter [1, 35, 36]. Catabolic degradation of sialic acid into N-acetylmannosamine (ManNAc) and pyruvate by N-acetyl-neuraminate pyruvate lyase ipt (NPL), also known as sialic acid aldolase, occurs in the cytosol [37, 38]. It remains unclear how free sialic acid biosynthesis, salvage and degradation pathways are regulated and contribute to steady state free sialic acid levels. Studies of inborn errors in free sialic acid metabolism have clarified some aspects (Fig 2) [38–40]. Apart from FSASD, there are two other sialic acid metabolism disorders, sialuria and NPL deficiency, associated A with significantly increased urinary free sialic acid (Table 1). The dominant disorder (French uthor Man type) sialuria (MIM 269921) is due to a monoallelic mutation in the allosteric site of UDP- GlcNAc 2-epimerase/ManNAc kinase (GNE), the initial and rate-limiting enzyme in sialic acid synthesis. The mutation prevents feedback inhibition of GNE by CMP-sialic acid, leading to constitutive production of cytoplasmic free sialic acid and resulting in excessive uscr urinary free sialic acid excretion (100–1000x normal) and increased cytoplasmic free sialic ipt acid in fibroblasts and lymphoblasts (Fig 2, Table 1) [27, 33, 34, 41]. Sialuria has been described in only 11 cases worldwide and presents with relatively mild organomegaly, coarse facial features and varying degrees of developmental delay [33, 41, 42]. NPL deficiency (MIM 611412) is due to biallelic mutations in the NPL gene, leading to decreased cytoplasmic free sialic acid degradation and increased urinary (~ 10x normal) and red blood cell (50–100x normal) free sialic acid levels, but no detectable free sialic acid accumulation A in fibroblasts [38]. NPL deficiency, so far described in only 2 siblings, presents with a uthor Man progressive cardiac myopathy and mild skeletal myopathy. These findings are likely not due to cytosolic accumulation of sialic acid, since they are absent from sialuria subjects with much greater elevations in cytoplasmic free sialic acid compared with NPL deficiency [38]. uscr The apparent rarity of these 3 inborn errors of sialic acid metabolism, all characterized by ipt elevated urinary free sialic acid, can be due to failure to diagnose these diseases because of unfamiliarity with these disorders, the nonspecific nature of the clinical features and, importantly, absence of routine testing for urinary sialic acid. Once increased free sialic acid is detected, these conditions can be easily distinguished by molecular genetic testing of SLC17A5 (for FSASD), GNE (for sialuria) or NPL (for NPL deficiency) and/or determining the cellular localization (cytoplasmic versus lysosomal) of free sialic acid (Table 2). A predominantly lysosomal localization indicates a FSASD; cytoplasmic localization indicates A uthor Man sialuria or NPL deficiency. Of note, other causes of mild elevation in urinary free sialic acid may exist. 3. FSASD Diagnosis uscr FSASD should be considered in probands with a clinical presentation of global ipt developmental delay or cognitive impairment, particularly affecting speech development, and regression combined with coarse facies, failure to thrive, organomegaly, truncal Neurosci Lett. Author manuscript; available in PMC 2021 June 11.