A novel splice site variant in DEGS1 leads to aberrant splicing and loss of DEGS1 enzyme activity, a VUS resolved

Purpose: variants have been reported in individuals with autosomal recessive hypomyelinating leukodystrophy 18 (HLD18; MIM# 618404). We sought to resolve a 5' +4/+5 splice site variant of uncertain significance found in three individuals with HLD features. Methods: We used next-generation DNA and transcriptome sequencing, cell-based splicing assays, and tandem mass spectrometry to detect and characterize the splice site variant. We then performed RNA structure probing and conventional antisense oligonucleotide screening to investigate molecular mechanisms for potential therapeutic intervention. Results: 5' splice site variant, c.825+4_825+5delAGinsTT (NM_003676.4) was identified in all three participants. Although the gene has been associated with autosomal recessive hypomyelinating leukodystrophy, the variant has not been previously reported in any available databases or literature. We show that the splice site variant: 1) was sufficient to induce exon two skipping in most detected transcripts; 2) resulted in structural changes to the 5' and 3' splice site regions using RNA structure probing; and 3) corresponds to plasma sphingolipid profiles consistent with loss of sphingolipid delta(4)-desaturase activity. Discussion: variant c.825+4_825+5delAGinsTT is pathogenic and suggested a mechanistic model to explain how exon two skipping is induced.