Analysis of the 5' ends of SLC4A11 transcripts in the corneal endothelium

Fuchs corneal dystrophy (FECD) and congenital hereditary endothelial dystrophy (CHED) are corneal endothelial pathologies associated with SLC4A11 gene variants. To date, the principal findings on the expression and function of SLC4A11 in the corneal endothelium have been based on studies of three major transcripts (variants 1-3), which differ in their 5 ends and have different N-terminal fragments of encoding proteins. We aimed to address gaps and resolve contradictions regarding the expression of SLC4A11 protein-coding transcripts and the location of major transcription initiation regions in the corneal endothelium. To identify the most represented 5 ends of transcripts and the primary transcription start points for the SLC4A11 gene, rapid analysis of cDNA 5 ends (5 RACE) was conducted on RNA samples from five healthy donors and five patients with FECD. Two-step PCR with nested primers was used for specific amplification of 5 transcript regions. Instead of a gene-specific primer, we used a poly-T primer in the reverse transcription step. Two sets of gene-specific primers (GSP) were utilized: GSP1 (located in exon 2 of transcripts 3 and 6) and GSP2 (located in exon 3 of transcript 3 and 6).