Abnormal Splicing of GALC Transcripts Underlies Unusual Cases of Krabbe Disease

<b>Background/Objectives</b>: Krabbe disease (KD) is a hereditary lysosomal disorder whose hallmark is progressive demyelination, with variable involvement of the central nervous system. It is caused by pathogenic variants in the <i>GALC</i> gene that disrupt the function of its gene product, the lysosomal enzyme galactosylceramidase. We analyzed two unrelated cases (one early infantile and one adult) with a clinical suspicion of KD. <b>Methods</b>: We used a combination of biochemical techniques (high-performance liquid chromatography-tandem mass spectrometry), NGS (resequencing gene panels), splicing assays, and molecular modeling to identify and analyze the pathogenicity of the variants underlying the disorder. <b>Results</b>: The two probands were compound heterozygotes for disease-causing variants in the <i>GALC</i> gene, encoding the lysosomal hydrolase galactosylceramidase. Three of the variants were novel and caused aberrant splicing, either by exon skipping (c.908+5G>A and c.1034-1G>C) or by inclusion of a cryptic, deep intronic pseudoexon (c.621+772G>C). The fourth variant was a known missense change (c.956A>G, p.(Tyr319Cys)) with conflicting interpretations of pathogenicity in the databases. <b>Conclusions</b>: We demonstrated the pathogenicity of the three novel splicing variants, all with strong impact on galactosylceramidase function. We also concluded that the c.956A>G missense variant is a hypomorph usually underlying the later-onset, milder phenotypes of KD. Our results stress the importance of integrated approaches combining clinical, biochemical, and genetic testing to obtain a definitive diagnosis of lysosomal diseases.