AlteredPLP1splicing causes hypomyelination of early myelinating structures

Abstract

ObjectiveThe objective of this study was to investigate the genetic etiology of the X-linked disorder “Hypomyelination of Early Myelinating Structures” (HEMS).MethodsWe included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients’ fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs.ResultsAll patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio.InterpretationBrain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus–Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing.

Functional Characterization of DNA Methylation in the Oligodendrocyte Lineage

“…Consistent with the detection of normal lineage specification, we did not detect any upregulation of neuronally enriched gene categories. Because OL differentiation is characterized by alternatively spliced events (Kevelam et al, 2015; Nave et al, 1987), we interrogated our RNA-Seq dataset in control and mutant cells (Figure 7A). It has been suggested that DNA methylation in specific genomic regions is critical for exon skipping and intron retention splicing events (Gelfman et al, 2013; Yearim et al, 2015).…”

“…It is likely that aberrant splicing might change protein conformation (Kevelam et al, 2015; Yura et al, 2006), and lead to the potential accumulation of incorrectly folded proteins inducing endoplasmic reticulum (ER) stress. Consistent with this possibility we observed dilated ER in mutant oligodendrocytes, with characteristic and unique electron-dense inclusions (Figure 7F), suggestive of protein accumulations and activation of ER stress response.…”

Leukodystrophies: a proposed classification system based on pathological changes and pathogenetic mechanisms

“…Peripheral neuropathy, central hypomyelination, Waardenburg-Hirschsprung [ 36 ] c. Early-onset neuronal degenerative disorders c. Cx47-related Pelizaeus-Merzbacher-like disease [ 36 ] 1. Gangliosidosis GM1 and GM2 [ 75 , 250 ] d. Hypomyelination of early myelinated structures [ 104 ] 2. Infantile neuronal ceroid lipofuscinosis [ 79 ] Demyelination 3.…”

The role of the clinician in the multi‐omics era: are you ready?

“…The lab improved their methods, went back to all their PLP1 negative patients and found more patients with cryptic alterations in this region. Further, careful review of the neuro-imaging of this patient contributed to the discovery of a novel PLP1 related phenotype termed hypomyelination of early myelinating structures (HEMS) caused by abnormal PLP1/DM20 alternative splicing due to exon 3 and intron 3 mutations (Kevelam et al 2015 ), further clarifying the precise relationship between gene mutation and phenotype and contributing to disease stratification. In vivo technologies such as nuclear magnetic resonance spectroscopy, radiolabeled isotype analysis to measure metabolic flux, and continuous glucose monitoring offer unique opportunities for deep phenotyping.…”