OP49 – 2560: PLP1 mutations affecting PLP1/DM20 alternative splicing causes hypomyelination of early myelinating structures

Objectives Inherited leukodystrophies represent a diagnostic challenge, as many patients remain without definitive diagnosis. In this study we investigated the genetic etiology of the recently described X-linked childhood disorder “Hypomyelination of Early Myelinating Structures” (HEMS). In HEMS, brain structures normally myelinating early, are hypomyelinated, which is in contrast to known hypomyelination disorders, like Pelizaeus-Merzbacher disease (PMD). Methods We included patients diagnosed with HEMS by brain MRI criteria and affected siblings. Exome sequencing was used to search for causal mutations in 16 patients of 10 families. In silico analysis of effects of the mutations on splicing and secondary RNA folding was performed. Also, gene splicing was examined in RNA prepared from patients' fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. Results All patients had unusual hemizygous mutations in a specific region of the PLP1 gene. These mutations were located in exon 3B (2 silent, 1 missense and 1 deletion), which is splice out in isoform DM20 or in intron 3 outside the splice donor sequence (5 mutations). Four mutations are located deep in intron 3; a region that is not sequenced with Sanger sequencing. These mutations effect the secondary PLP1 RNA structure and decreased the PLP1/DM20 ratio. The other mutations, except for the deletion that truncated PLP1 but not DM20, were also predicted to alter PLP1/DM20 alternative splicing. Splicing studies in fibroblasts' and transfected cells confirmed a decreased PLP1/DM20 ratio. Conclusions We show that specific mutations in the PLP1 gene cause a hypomyelination pattern which contrasts the pattern seen in PMD, also caused by PLP1 mutations. This indicates that the PLP1/DM20 ratio influences early myelination. The mutations present in HEMS patients provide a challenge for PLP1 diagnostic sequencing and support the need to include intron 3.