Case Report: Neonatal lethality caused by novel compound heterozygous missense variants of SLC6A9 gene

The homozygous or compound heterozygous missense variants in the SLC6A9 gene (OMIM*601019) are associated with autosomal recessive glycine encephalopathy with normal serum glycine (OMIM#617301). Fewer than ten SLC6A9 variants have been reported to date. Neonates harboring pathogenic variants in the SLC6A9 gene exhibited a mortality rate of up to 75% during the first 7 months of life. We reported the first documented case of neonate death in China attributable to compound heterozygous missense SLC6A9 variants.

Trio-based whole-exome sequencing (trio-WES) was performed on genomic DNA (gDNA) of amniotic fluid cells and parental peripheral blood samples. Two mutant SLC6A9 expression vectors, namely pCMV-Flag-Neo(EGFP)-SLC6A9-m1 and pCMV-Flag-Neo(EGFP)-SLC6A9-m2 (SLC6A9-m1/m2), and wild-type pCMV-Flag-Neo(EGFP)-SLC6A9-wt (SLC6A9-wt) mammalian expression vectors were constructed. 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.

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.

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.