Huizing et al. Page 7 Mennonite (homozygous for p.Arg39Cys) [66], Italian, Danish, Spanish, Dominican, A uthor Man Kurdish, and Japanese [11]. For a better understanding of the worldwide FSASD prevalence, we used SLC17A5 gene variants listed in the GnomAD database (https://gnomad.broadinstitute.org/; accessed uscr December 2020). Since FSASD is associated with bi-allelic variants in one autosomal gene locus (SLC17A5), and assuming random mating in an indefinitely large population, we ipt 2 2 applied the Hardy-Weinberg principle of population genetics (p + 2pq + q = 1; Table 3, Supp. Table S1) [67–69] to calculate disease prevalence. We aggregated all pathogenic SLC17A5 variants into a single category to use this simple binomial expression (detailed in Supp Table S1). To avoid over-estimating SLC17A5 variant allele frequencies, we did not include intronic A variants more than 2 nucleotides away from exon boundaries, synonymous variants, or any uthor Man missense variant with a ‘benign’ or ‘likely pathogenic’ pathogenicity score (per Variant Effect Predictor in GnomAD) (Supp. Table S1). We also omitted the number of Finnish alleles with the p.Arg39Cys variant (149 alleles), but we included non-Finnish alleles with this variant (79 alleles). This resulted in a conservative estimate of the prevalence of FSASD uscr to be at least 3 per 1,000,000, with a carrier rate of 1/286 individuals (heterozygotes) (Table ipt 3). Assuming this database represents the worldwide population diversity, these data translate to a prevalence of ~23,000 global FASD cases, with ~13,000 in Asia, ~2,000 in Europe and ~1,700 in North America. However, embryonic lethality of severe cases and childhood death of intermediate severe cases [14, 44, 49] will reduce the number of living FSASD cases significantly. Nevertheless, given that ~200 FSASD cases are reported in the literature, these prevalence values confirm suspicions that many FSASD cases go A undiagnosed. Based on GnomAD data of Finnish alleles, we estimate that FSASD due to the uthor Man homozygous p.Arg39Cys variant has a carrier rate of ~1 per 84 individuals in the Finnish population, translating to a prevalence of ~35 FSASD cases per million (~190 FSASD cases) in Finland (Table 3). uscr 6. FSASD Etiology ipt The exact pathophysiology of FSASD remains unknown. Effects of SLC17A5 mutations on sialic acid transport activity, SLC17A5 intracellular localization, and amount of stored free sialic acid have been directly correlated with disease severity and survival [19,83,85]. Loss of function of SLC17A5 due to FSASD-associated mutations was demonstrated by utilizing the SLC17A5 N-terminal dileucine lysosomal targeting motif, DRTPLL (Fig 3) [4, 70]. Newly synthesized SLC17A5 traffics to the plasma membrane, from where it is rapidly A internalized to the endo-lysosomal system by coat proteins recognizing the dileucine uthor Man targeting motif [4]. Expression of SLC17A5 with an altered targeting motif results in plasma membrane expression, allowing for the use of whole cell uptake assays to measure transport activity and intracellular localization [4, 59, 71, 72]. While missense variants associated with uscr more severe phenotypes had absent sialic acid transport activity, the variants associated with a milder phenotype (p.Arg39Cys, p.Lys136Glu) had residual transport activity [4, 59, 71]. ipt Some variants also showed partial Golgi retention [72, 73] or endoplasmic reticulum (ER) retention [59]. These findings confirmed an SLC17A5 loss of function disease mechanism Neurosci Lett. Author manuscript; available in PMC 2021 June 11.