# Pathogenic Analysis of Two SLC22A5 Variants That Alter RNA Splicing in Patients with Primary Carnitine Deficiency

## TL;DR

This study identifies and validates two SLC22A5 gene variants that disrupt RNA splicing, improving diagnosis of primary carnitine deficiency.

## Contribution

The first functionally validated synonymous variant in SLC22A5 is reported, expanding the known pathogenic variant spectrum.

## Key findings

- The c.450C>T variant causes partial exon 2 skipping in RNA splicing.
- The c.394-1G>A variant leads to intron 1 retention and exon 2 skipping.
- Both variants were reclassified as likely pathogenic and pathogenic based on functional evidence.

## Abstract

Functional analysis of SLC22A5 variants 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 synonymous SLC22A5 variant that has not been previously reported, and the other patient harbored a classical splice site variant. The splicing patterns of the two SLC22A5 variants 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 both SLC22A5 variants 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 the SLC22A5 gene that has been functionally validated to affect RNA splicing, thus enriching the variant spectrum of SLC22A5 and aiding accurate PCD diagnosis.