Abstract

Functional analysis ofSLC22A5variants can improve diagnostic accuracy in patients with primary carnitine deficiency (PCD). Herein, we performed a genetic analysis of three neonates with PCD. Two of the patients harbored a novel synonymousSLC22A5variant that has not been previously reported, and the other patient harbored a classical splice site variant. The splicing patterns of the twoSLC22A5variants were evaluated using three in silico tools, and in vitro minigene analysis was performed to verify the impact of variants on RNA splicing mechanisms. All three in silico tools predicted that bothSLC22A5variants could alter normal RNA splicing. Functional studies using minigene assays demonstrated that the c.450C>T (p.F150=) leads to partial exon 2 skipping, and c.394-1G>A leads to intron 1 retention and exon 2 skipping. Intron 1 retention of 65 nucleotides and exon 2 skipping were confirmed by sequencing cDNA amplification products. These results, along with functional evidence, led to reclassification of c.450C>T (p.F150=) and c.394-1G>A as likely pathogenic and pathogenic, respectively. This is the first reported synonymous variant in theSLC22A5gene that has been functionally validated to affect RNA splicing, thus enriching the variant spectrum ofSLC22A5and aiding accurate PCD diagnosis.