Supplementary file 1_Case Report: Neonatal lethality caused by novel compound heterozygous missense variants of SLC6A9 gene.docx

All vectors were subsequently transformed into HEK293T cells. Assays measuring the relative expression of SLC6A9 gene mRNA and SLC6A9 protein were performed. Concurrently, we conducted a systematic review of previously reported SLC6A9-related cases with accessible clinical data, encompassing relevant genetic testing findings and prenatal/postnatal clinical manifestations.</p>Results<p>Trio-WES identified novel compound heterozygous missense variants c.859C > T (p.Arg287Trp) and c.1120T > C (p.Cys374Arg) in the SLC6A9 gene (NM_201649.4).

In vitro functional studies demonstrated that neither variant caused a reduction in the expression of mutant mRNA, but both led to a significant decrease in the expression of mutant SLC6A9 protein. After reviewing the current literature, we found that fetuses with SLC6A9 gene variants commonly presented with increased nuchal translucency (NT), polyhydramnios and talipes equinovarus (clubfoot). Pathogenic SLC6A9 variants exhibited complete penetrance and a relatively consistent phenotypic spectrum characterized by developmental delay, facial dysmorphism, clubfoot, and a high neonatal mortality rate.

The characteristic phenotype in affected infants was characterized by elevated cerebrospinal fluid (CSF) glycine concentrations, with or without concurrent elevation in serum glycine levels.</p>Conclusion<p>Preliminary evidence from in vitro functional studies suggested that the pathogenic mechanism of SLC6A9-related glycine encephalopathy may be associated with the loss-of-function (LoF) effect caused by SLC6A9 gene variants.

This study also expanded the mutational spectrum of the SLC6A9 gene.</p>