Exome sequencing identifies a REEP1 mutation involved in distal hereditary motor neuropathy type V

Abstract

The distal hereditary motor neuropathies (dHMNs) are a heterogeneous group of neurodegenerative disorders affecting the lower motoneuron. In a family with both autosomal-dominant dHMN and dHMN type V (dHMN/dHMN-V) present in three generations, we excluded mutations in all genes known to be associated with a dHMN phenotype through Sanger sequencing and defined three potential loci through linkage analysis. Whole-exome sequencing of two affected individuals revealed a single candidate variant within the linking regions, i.e., a splice-site alteration in REEP1 (c.304-2A>G). A minigene assay confirmed complete loss of splice-acceptor functionality and skipping of the in-frame exon 5. The resulting mRNA is predicted to be expressed at normal levels and to encode an internally shortened protein (p.102_139del). Loss-of-function REEP1 mutations have previously been identified in dominant hereditary spastic paraplegia (HSP), a disease associated with upper-motoneuron pathology. Consistent with our clinical-genetic data, we show that REEP1 is strongly expressed in the lower motoneurons as well. Upon exogeneous overexpression in cell lines we observe a subcellular localization defect for p.102_139del that differs from that observed for the known HSP-associated missense mutation c.59C>A (p.Ala20Glu). Moreover, we show that p.102_139del, but not p.Ala20Glu, recruits atlastin-1, i.e., one of the REEP1 binding partners, to the altered sites of localization. These data corroborate the loss-of-function nature of REEP1 mutations in HSP and suggest that a different mechanism applies in REEP1-associated dHMN.

Figure 1

Pedigree Structure, Clinical Images, REEP1 Sanger Sequencing, and Minigene Assay (A) Partial pedigree. Gender information was removed to preserve anonymity. White and black symbols indicate unaffected and affected individuals, respectively. Asterisks mark family members whose DNA was available and was applied in linkage analysis. Indicated below the identifiers are the results of REEP1 Sanger sequencing (WT: wild-type allele, mut: mutant allele). The arrow denotes the index case. (B) Wasting of the thenar (B1) and interosseus dorsalis I (B2) muscles in individual III-3. (C) Distal-lower-limb atrophy in individual IV-5. (D) Pes cavus foot deformity in individual III-5. (E) Exemplary results of Sanger sequencing of REEP1 exon 5. The stippled line indicates the intron/exon boundary. The arrow marks nucleotide c.304-2 (NM_022912). (F) Minigene assay. RT-PCR on HeLa cells transfected with the indicated constructs shows that mutation c.304-2A>G results in skipping of REEP1 exon 5.

Figure 2

Expression of Reep1 in the Spinal Cord at Different Rostrocaudal Levels Alternating spinal cord sections (10 μm) from 4-month-old adult mice were stained with thionine or hybridized with a Reep1-specific RNA probe. Thionine labels all neuronal cell bodies, thereby defining the gray matter, whereas the signal from Reep1 in situ hybridization is restricted to large somata in the ventral horns.

Figure 3

Subcellular Distribution of Overexpressed REEP1 and Atlastin-1 HeLa cells were transfected with the indicated constructs. Untagged REEP1 was detected by immunofluorescence, atlastin-1 by fluorescence of the DsRed tag. Nuclei were stained with DAPI. The scale bar represents 20 μm. (A) Subcellular targeting of wild-type REEP1 is differentially altered by the indicated pathogenic mutations. Wild-type REEP1 localizes to a cytoplasmic network representing ER (upper panel, see also Figure S3A). REEP1 p.Ala20Glu is exclusively found in numerous small, sometimes hollow structures (middle panel). REEP1 p.102_139del, in addition to being found in the ER network (see also Figure S3B), accumulates in 5–20 compact structures, the largest of which occupy a perinuclear position (lower panel). (B) Atlastin-1, in single transfections, is targeted to a highly branched ER network.²⁷ (C) Cotransfections of wild-type REEP1 and atlastin-1 result in a spectrum of distribution patterns. Shown are two extremes, i.e., a cell in which REEP1 distribution appears unaltered whereas some atlastin-1 is redistributed to small peripheral spots (upper panel) and a cell in which atlastin-1 and much of REEP1 coaccumulate in cytoplasmic punctate structures, which are evenly distributed throughout the cytoplasm (lower panel). (D) Mutation p.102_139del, but not p.Ala20Glu, impacts on the subcellular distribution of atlastin-1. Whereas cotransfection of REEP1 p.Ala20Glu leaves the atlastin-1 pattern unaltered (upper panel), REEP1 p.102_139del recruits atlastin-1 to the large perinuclear structures also observed in single transfections of this mutant protein (lower panel, compare lower panel in A).