PMP22 exon 4 deletion causes ER retention of PMP22 and a gain‐of‐function allele in CMT1E

Abstract

ObjectiveTo determine whether predicted fork stalling and template switching (FoSTeS) during mitosis deletes exon 4 in peripheral myelin protein 22 KD (PMP22) and causes gain‐of‐function mutation associated with peripheral neuropathy in a family with Charcot–Marie–Tooth disease type 1E.MethodsTwo siblings previously reported to have genomic rearrangements predicted to involve exon 4 of PMP22 were evaluated clinically and by electrophysiology. Skin biopsies from the proband were studied by RT‐PCR to determine the effects of the exon 4 rearrangements on exon 4 mRNA expression in myelinating Schwann cells. Transient transfection studies with wild‐type and mutant PMP22 were performed in Cos7 and RT4 cells to determine the fate of the resultant mutant protein.ResultsBoth affected siblings had a sensorimotor dysmyelinating neuropathy with severely slow nerve conduction velocities (<10 m/sec). RT‐PCR studies of Schwann cell RNA from one of the siblings demonstrated a complete in‐frame deletion of PMP22 exon 4 (PMP22Δ4). Transfection studies demonstrated that PMP22Δ4 protein is retained within the endoplasmic reticulum and not transported to the plasma membrane.ConclusionsOur results confirm that that FoSTeS‐mediated genomic rearrangement produced a deletion of exon 4 of PMP22, resulting in expression of both PMP22 mRNA and protein lacking this sequence. In addition, we provide experimental evidence for endoplasmic reticulum retention of the mutant protein suggesting a gain‐of‐function mutational mechanism consistent with the observed CMT1E in this family. PMP22Δ4 is another example of a mutated myelin protein that is misfolded and contributes to the pathogenesis of the neuropathy.

Myelin protein zero mutations and the unfolded protein response in Charcot Marie Tooth disease type 1B

“…In order to obtain wild‐type human MPZ we performed 2 mm punch skin biopsies from a normal control, without CMT, using methods previously described11 and the biopsy was immediately placed into RNALater (Cat. #:1017980, QIAGEN GmbH, Hilden, Germany).…”

Rare PMP22 variants in mild to severe neuropathy uncorrelated to plasma GDF15 or neurofilament light

“…Rare PMP22 variants have been previously associated with a range of phenotypes, including CMT-type neuropathy denoted as CMT1E, HNPP, and severe congenital neuropathy known as DSS [ 37 ]. Exon 4 deletion has been reported previously in one sibling pair, then as part of a larger 17 kb deletion [ 24 , 36 ]. Our results expand the spectrum of rare PMP22 mutations and exemplify their genotype–phenotype correlations and their effects on plasma biomarkers.…”

“…Nerve CSA was larger than in axonal CMT2K or even in CMT1A, where nerve CSA is known to be enlarged [ 14 ]. The previously reported pair of sisters with a 17 kb deletion of PMP22 , which also led to the exclusion of exon 4, had similar severe early-onset disease, although the older sister appeared somewhat less severely affected than individual B as she was able to walk with orthoses at age 15 [ 24 ]. The PMP22 exon 4 deletion differs from most other known indel or splice variants because it produces an in-frame change and therefore does not lead to mRNA instability.…”

“…The PMP22 exon 4 deletion differs from most other known indel or splice variants because it produces an in-frame change and therefore does not lead to mRNA instability. Interestingly, loss of exon 4 leads to an aberrant protein, which is trapped in the ER [ 24 ]. p.His12 in turn is positioned in the transmembrane domain 1 (TM1).…”

“…Expression of GDF15 is responsive to integrated stress response [ 18 ], while NFL is a structural component of neurons that is released upon axon degeneration [ 23 ]. Rare variants in PMP22 that cause severe CMT1E or DSS, such as exon 4 deletion [ 24 ] and point mutations [ 25 , 26 ], induce protein misfolding and ostensibly a strong stress response with commensurate nerve enlargement [ 24 ]. However, whether the severe forms of disease correlate with a further elevation of these blood biomarkers is not known.…”