REEP1 Mutations Research Articles

The membrane curvature-inducing REEP1-4 proteins generate an ER-derived vesicular compartment.

The endoplasmic reticulum (ER) is shaped by abundant membrane curvature-generating proteins that include the REEP family member REEP5. The REEP1 subfamily, consisting of four proteins in mammals (REEP1-4), is less abundant and lack a N-terminal region. Mutations in REEP1 and REEP2 cause Hereditary Spastic Paraplegia, but the function of these four REEP proteins remains enigmatic. Here we show that REEP1-4 reside in a unique vesicular compartment and identify features that determine their localization. Mutations in REEP1-4 that compromise curvature generation, including those causing disease, relocalize the proteins to the bulk ER. These mutants interact with wild-type proteins to retain them in the ER, consistent with their autosomal-dominant disease inheritance. REEP1 vesicles contain the membrane fusogen atlastin-1, but not general ER proteins. We propose that REEP1-4 generate these vesicles themselves by budding from the ER, and that they cycle back to the ER by atlastin-mediated fusion. The vesicles may serve to regulate ER tubule dynamics.

Generation of homozygous and heterozygous REEP1 knockout induced pluripotent stem cell lines by CRISPR/Cas9 gene editing

REEP1 is a transmembrane protein in the endoplasmic reticulum (ER) membrane that is involved in shaping and remodeling of the ER. Mutations in REEP1 cause SPG31, an autosomal dominant form of hereditary spastic paraplegia (HSP). Here we show the generation of a homozygous and a heterozygous REEP1 knockout induced pluripotent stem cell line suitable for in vitro disease modelling using the CRISPR/Cas9 editing system.

Oligomeric scaffolding for curvature generation by ER tubule-forming proteins

The reticulons and receptor expression-enhancing proteins (REEPs) in the endoplasmic reticulum (ER) are necessary and sufficient for generating ER tubules. However, the mechanism of curvature generation remains elusive. Here, we systematically analyze components of the REEP family based on AI-predicted structures. In yeast REEP Yop1p, TM1/2 and TM3/4 form hairpins and TM2-4 exist as a bundle. Site-directed cross-linking reveals that TM2 and TM4 individually mediate homotypic dimerization, allowing further assembly into a curved shape. Truncated Yop1p lacking TM1 (equivalent to REEP1) retains the curvature-generating capability, undermining the role of the intrinsic wedge. Unexpectedly, both REEP1 and REEP5 fail to replace Yop1p in the maintenance of ER morphology, mostly due to a subtle difference in oligomerization tendency, which involves not only the TM domains, but also the TM-connecting cytosolic loop and previously neglected C-terminal helix. Several hereditary spastic paraplegia-causing mutations in REEP1 appear at the oligomeric interfaces identified here, suggesting compromised self-association of REEP as a pathogenic mechanism. These results indicate that membrane curvature stabilization by integral membrane proteins is dominantly achieved by curved, oligomeric scaffolding.

Large single-centre cohort of HSP patients in UK: advantages of deep pheno- typing and longitudinal follow-up

AimTo identify clinical, radiological and instrumented gait correlates predictive of natural history in a cohort of HSP patients.BackgroundHSP is a rare heterogeneous group of neurodegenerative disorders. We present our experi- ence managing a large cohort of HSP patients in the regional centre for HSP. We believe Sheffield Teaching Hospitals to have the largest cohort in the North of England.Methodology and ResultsOur cohort comprises 426 patients, 246 male (57.7%) of which 290 were geneti- cally characterized. The commonest mutation is in SPASTIN (SPG4, 38.3%), whilst REEP1 (SPG31) and SPG11 mutations make up 3.1% and 2.1% respectively. Sheffield also holds the largest cohort of SPG7 patients in the UK (n=54) accounting for 18.6% of genetic HSP. The remainder have mutations in KIF5A, Atlastin, WASHC5, KIAA0196, NIPA1, ARSACS, CYP27A1, CYP7B1, AP4S1, GBA2 and L1CAM. Over 240 patients have had detailed MR imaging which has identified novel features in HSP. Repeated instrumented gait monitoring and cognitive assessments have shown novel clinical markers suggesting disease progression.ConclusionFor rare disorders like HSP, specialised clinics optimise comprehensive longitudinal clinical and radiological data collection for large cohorts. This develops local expertise and helps identify novel radiological and clinically relevant biomarkers, enabling better patient care.klprice1@sheffield.ac.uk

Novel homozygous mutations in Pakistani families with Charcot\u2013Marie\u2013Tooth disease

BackgroundCharcot–Marie–Tooth disease (CMT) is a group of genetically and clinically heterogeneous peripheral nervous system disorders. Few studies have identified genetic causes of CMT in the Pakistani patients.MethodsThis study was performed to identify pathogenic mutations in five consanguineous Pakistani CMT families negative for PMP22 duplication. Genomic screening was performed by application of whole exome sequencing.ResultsWe identified five pathogenic or likely pathogenic homozygous mutations in four genes: c.2599C > T (p.Gln867*) and c.3650G > A (p.Gly1217Asp) in SH3TC2, c.19C > T (p.Arg7*) in HK1, c.247delG (p.Gly83Alafs*44) in REEP1, and c.334G > A (p.Val112Met) in MFN2. These mutations have not been reported in CMT patients. Mutations in SH3TC2, HK1, REEP1, and MFN2 have been reported to be associated with CMT4C, CMT4G, dHMN5B (DSMA5B), and CMT2A, respectively. The genotype–phenotype correlations were confirmed in all the examined families. We also confirmed that both alleles from the homozygous variants originated from a single ancestor using homozygosity mapping.ConclusionsThis study found five novel mutations as the underlying causes of CMT. Pathogenic mutations in SH3TC2, HK1, and REEP1 have been reported rarely in other populations, suggesting ethnic-specific distribution. This study would be useful for the exact molecular diagnosis and treatment of CMT in Pakistani patients.

Screening for REEP1 Mutations in 31 Chinese Hereditary Spastic Paraplegia Families.

Background: REEP1 is a common cause of autosomal dominant hereditary spastic paraplegia (HSP) but is rare in China. The pathological mechanism of REEP1 is not fully understood.Methods: We screened for REEP1 mutations in 31 unrelated probands from Chinese HSP families using next-generation sequencing targeting pathogenic genes for HSP and other related diseases. All variants were validated by Sanger sequencing. The proband family members were also screened for variants for the segregation analysis. All previously reported REEP1 mutations and cases were reviewed to clarify the genetic and clinical features of REEP1-related HSP.Results: A pathogenic mutation, REEP1c. 125G>A (p.Trp42*), was detected in a pure HSP family from North China out of 31 HSP families (1/31). This locus, which is located in the second hydrophobic domain of REEP1, is detected in both Caucasian patients with complicated HSP phenotypes and Chinese pure HSP families.Conclusion: REEP1-related HSP can be found in the Chinese population. The 42nd residue is a novel transethnic mutation hotspot. Mutations in this spot can lead to both complicated and pure form of HSP. Identification of transethnic hotspot will contribute to clarify the underlying pathological mechanisms.

Genotype-phenotype associations in hereditary spastic paraplegia: a systematic review and meta-analysis on 13,570 patients.

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of inherited neurodegenerative disorders. Although, several genotype-phenotype studies have carried outon HSPs, the association between genotypes and clinical phenotypes remain incomplete since most studies are small in size or restricted to a few genes. Accordingly, this study provides the systematic meta-analysis of genotype-phenotype associations in HSP. We retrieved literature on genotype-phenotype associations in patients with HSP and mutated SPAST, REEP1, ATL1, SPG11, SPG15, SPG7, SPG35, SPG54, SPG5. In total, 147 studies with 13,570 HSP patients were included in our meta-analysis. The frequency of mutations in SPAST (25%) was higher than REEP1 (3%), as well as ATL1 (5%) in AD-HSP patients. As for AR-HSP patients, the rates of mutations in SPG11 (18%), SPG15 (7%) and SPG7 (13%) were higher than SPG5 (5%), as well as SPG35 (8%) and SPG54 (7%). The mean age of AD-HSP onset for ATL1 mutation-positive patients was earlier than patients with SPAST, REEP1 mutations. Also, the tendency toward younger age at AR-HSP onset for SPG35 was higher than other mutated genes. It is noteworthy that the mean age at HSP onset ranged from infancy to adulthood. As for the gender distribution, the male proportion in SPG7-HSP (90%) and REEP1-HSP (78%) was markedly high. The frequency of symptoms was varied among patients with different mutated genes. The rates of LL weakness, superficial sensory abnormalities, neuropathy, and deep sensory impairment were noticeably high in REEP1 mutations carriers. Also, in AR-HSP patients with SPG11 mutations, the presentation of symptoms including pes cavus, Neuropathy, and UL spasticity was higher. Our comprehensive genotype-phenotype assessment of available data displays that the mean age at disease onset and particular sub-phenotypes are associated with specific mutated genes which might be beneficial for a diagnostic procedure and differentiation of the specific mutated genes phenotype among diverse forms of HSP.

Spastic paraplegia type 31: A novel REEP1 splice site donor variant and expansion of the phenotype variability

Mutations in REEP1 have been identified in three types of neurological disorders, autosomal dominant form of Hereditary Spastic Paraplegia type 31 (SPG31), autosomal dominant distal hereditary motor neuronopathy type VB (HMN5B), and autosomal recessive form of congenital axonal neuropathy and diaphragmatic palsy. Previous studies demonstrated different molecular pathogenesis in SPG31, including loss-of-function, gain-of-function and haploinsufficiency. A four-generation family from Japan, including 12 members, was investigated clinically and genetically. Seven affected members displayed pure spastic paraplegia. Impression of genetic anticipation was observed in the family, including tendency of earlier age-at-onset and increasing severity in subsequent generations. Genetic analysis revealed a heterozygous intronic variant, c.303+2T > A, in REEP1, which segregated with disease, and was also identified in one unaffected member. The variant causes exon 4 skipping leading to frame shift and a truncated transcript identified by complementary DNA sequencing of reverse transcription polymerase chain reaction products. Measurement of REEP1 transcripts in lymphocytes demonstrated a reduction through nonsense mediated mRNA decay (NMD). Our study demonstrated further evidence of allelic heterogeneity in SPG31, mutant REEP1 mRNA dosage effects through NMD and intra-familial phenotype variability.

Hereditary spastic paraplegia type 31: A novel splice site donor mutation and intra-familial phenotypic variability

Background: Mutations in REEP1 have been identified in two types of neurological disorders, autosomal dominant form of Hereditary Spastic Paraplegia type 31 (SPG31) and autosomal dominant distal hereditary motor neuronopathy type VB (HMN5B). Previous studies demonstrated various molecular pathogenesis in SPG31, including loss-of-function, gain-of-function and haploinsufficiency.

Defects in ER–endosome contacts impact lysosome function in hereditary spastic paraplegia

Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER-endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell-derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER-endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.

Mitochondrial morphology and cellular distribution are altered in SPG31 patients and are linked to DRP1 hyperphosphorylation.

Hereditary spastic paraplegia, SPG31, is a rare neurological disorder caused by mutations in REEP1 gene encoding the microtubule-interacting protein, REEP1. The mechanism by which REEP1-dependent processes are linked with the disease is unclear. REEP1 regulates the morphology and trafficking of various organelles via interaction with the microtubules. In this study, we collected primary fibroblasts from SPG31 patients to investigate their mitochondrial morphology. We observed that the mitochondrial morphology in patient cells was highly tubular compared with control cells. We provide evidence that these morphological alterations are caused by the inhibition of mitochondrial fission protein, DRP1, due to the hyperphosphorylation of its serine 637 residue. This hyperphosphorylation is caused by impaired interactions between REEP1 and mitochondrial phosphatase PGAM5. Genetically or pharmacologically induced decrease of DRP1-S637 phosphorylation restores mitochondrial morphology in patient cells. Furthermore, ectopic expression of REEP1 carrying pathological mutations in primary neuronal culture targets REEP1 to the mitochondria. Mutated REEP1 proteins sequester mitochondria to the perinuclear region of the neurons and therefore, hamper mitochondrial transport along the axon. Considering the established role of mitochondrial distribution and morphology in neuronal health, our results support the involvement of a mitochondrial dysfunction in SPG31 pathology.

Whole-exome sequencing in neurologic practice: Reducing the diagnostic odyssey.

The current issue of Neurology® Genetics emphasizes the unparalleled role of next-generation sequencing (NGS) in defining an expanding spectrum of genetic neurologic disorders. Clinically, NGS encompasses the use of large gene panels, whole-exome sequencing (WES), or whole-genome sequencing (WGS). The impact of NGS technology is twofold. First, researchers have discovered novel genes as the cause of neurologic disorders. This research includes the efforts of Martikainen et al.(1) to define further the phenotype of a previously reported SNCA mutation that is associated with autosomal dominant Parkinson disease. Second and more common is the connection of novel phenotypes with previously described genes. Several articles in the current issue highlight the role of NGS in this effort. For example, Schottman et al.(2) identified REEP1 mutations as the cause of a severe axonal neuropathy with a spinal muscular atrophy with respiratory distress (SMARD) phenotype. This gene was previously associated with a hereditary spastic paraplegia phenotype. Similarly, Shieh et al.(3) expand the phenotype associated with mutations in L1CAM to a neuronal migration phenotype.

Hereditary spastic paraplegia-linked REEP1 modulates endoplasmic reticulum/mitochondria contacts.

Mutations in receptor expression enhancing protein 1 (REEP1) are associated with hereditary spastic paraplegias (HSPs). Although axonal degeneration is thought to be a predominant feature in HSP, the role of REEP1 mutations in degeneration is largely unknown. Previous studies have implicated a role for REEP1 in the endoplasmic reticulum (ER), whereas others localized REEP1 with mitochondria. We sought to resolve the cellular localization of REEP1 and further elucidate the pathobiology underlying REEP1 mutations in patients. A combination of cellular imaging and biochemical approaches was used to refine the cellular localization of REEP1. Next, Reep1 mutations associated with HSP were functionally tested in neuritic growth and degeneration assays using mouse cortical culture. Finally, a novel assay was developed and used with wild-type and mutant Reep1s to measure the interactions between the ER and mitochondria. We found that REEP1 is present at the ER-mitochondria interface, and it contains subdomains for mitochondrial as well as ER localization. Knockdown of Reep1 and expression of pathological Reep1 mutations resulted in neuritic growth defects and degeneration. Finally, using our novel split-RLuc8 assay, we show that REEP1 facilitates ER-mitochondria interactions, a function diminished by disease-associated mutations. Our data potentially reconcile the current conflicting reports regarding REEP1 being either an ER or a mitochondrial protein. Furthermore, our results connect, for the first time, the disrupted ER-mitochondria interactions to a failure in maintaining health of long axons in HSPs. Finally, the split-RLuc8 assay offers a new tool to identify potential drugs for multiple neurodegenerative diseases with ER-mitochondria interaction defects.

Mutation and Clinical Characteristics of Autosomal-Dominant Hereditary Spastic Paraplegias in China

Background: Hereditary spastic paraplegias constitute a heterogeneous group of inherited neurodegenerative disorders. To date, there has been no systematic mutation and clinical analysis for a large group of autosomal-dominant hereditary spastic paraplegias in China. Objective: The purpose of this study was to investigate the mutation frequencies and the clinical phenotypes of Chinese spastic paraplegia patients. Methods: Direct sequencing and a multiplex ligation-dependent probe amplification assay were applied to detect the mutations of SPAST and ATL1 in 54 autosomal-dominant hereditary spastic paraplegia probands and 66 isolated cases. Next, mutations in NIPA1, KIF5A, REEP1 and SLC33A1 were detected in the negative patients. Subsets of spastic paraplegia patients were genotyped for the modifying variants. Further, detailed clinical data regarding the genetically diagnosed families were analysed. Results: Altogether, 27 families were diagnosed as SPG4, 3 as SPG3A and 1 as SPG6. No mutations in KIF5A, REEP1 or SLC33A1 were found; 9 SPAST mutations were novel. There was no p.S44L or p.P45Q variant in SPAST and no p.G563A variant in HSPD1 in either the 120 spastic paraplegia patients or the 500 controls. There was a remarkable clinical difference between the SPG4 and non-SPG4 patients and even between genders among the SPG4 patients. Non-penetrance and remarkable gender difference were observed in some SPG4 and SPG3A families. Conclusions: Our data confirm that hereditary spastic paraplegias in China represent a heterogeneous group of genetic neurodegenerative disorders in autosomal-dominant and apparently sporadic forms. Novel genotype-phenotype correlations were established.

HEREDITARY SPASTIC PARAPLEGIA: PATHOGENESIS AND PATHOPHYSIOLOGY

- Hereditary spastic paraplegias constitute a larger group of disorders than expected. - Autosomal dominant types are mainly composed of SPAST, Atlastin (SPG3A) and REEP1 mutations. Genetic testing is suggested mainly for these genes. - The most common autosomal recessive type is SPG11, hereditary spastic paraplegia with thin corpus callosum, but SPG15 shares the same clinical features with SPG11. Genetic testing should be done for both if thin corpus callosum is present in patients. - How different genes with many different biological functions, including axonal transport, mitochondrial functions, fatty acid and cholesterol pathways and DNA repair defects, cause hereditary spastic paraplegia is still unknown.

Loss of Association of REEP2 with Membranes Leads to Hereditary Spastic Paraplegia

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous neurological conditions. Their main pathogenic mechanisms are thought to involve alterations in endomembrane trafficking, mitochondrial function, and lipid metabolism. With a combination of whole-genome mapping and exome sequencing, we identified three mutations in REEP2 in two families with HSP: a missense variant (c.107T>A [p.Val36Glu]) that segregated in the heterozygous state in a family with autosomal-dominant inheritance and a missense change (c.215T>A [p.Phe72Tyr]) that segregated in trans with a splice site mutation (c.105+3G>T) in a family with autosomal-recessive transmission. REEP2 belongs to a family of proteins that shape the endoplasmic reticulum, an organelle that was altered in fibroblasts from an affected subject. In vitro, the p.Val36Glu variant in the autosomal-dominant family had a dominant-negative effect; it inhibited the normal binding of wild-type REEP2 to membranes. The missense substitution p.Phe72Tyr, in the recessive family, decreased the affinity of the mutant protein for membranes that, together with the splice site mutation, is expected to cause complete loss of REEP2 function. Our findings illustrate how dominant and recessive inheritance can be explained by the effects and nature of mutations in the same gene. They have also important implications for genetic diagnosis and counseling in clinical practice because of the association of various modes of inheritance to this new clinico-genetic entity.

Mutation analysis of SPAST, ATL1, and REEP1 in Korean Patients with Hereditary Spastic Paraplegia.

Background and PurposeHereditary spastic paraplegia (HSP) is a genetically heterogeneous group of neurodegenerative disorders that are characterized by progressive spasticity and weakness of the lower limbs. Mutations in the spastin gene (SPAST) are the most common causes of HSP, accounting for 40-67% of autosomal dominant HSP (AD-HSP) and 12-18% of sporadic cases. Mutations in the atlastin-1 gene (ATL1) and receptor expression-enhancing protein 1 gene (REEP1) are the second and third most common causes of AD-HSP, respectively.MethodsDirect sequence analysis was used to screen mutations in SPAST, ATL1, and REEP1 in 27 unrelated Korean patients with pure and complicated HSP. Multiplex ligation-dependent probe amplification was also performed to detect copy-number variations of the three genes.ResultsTen different SPAST mutations were identified in 11 probands, of which the following 6 were novel: c.760A>T, c.131C>A, c.1351_1353delAGA, c.376_377dupTA, c.1114A>G, and c.1372A>C. Most patients with SPAST mutations had AD-HSP (10/11, 91%), and the frequency of SPAST mutations accounted for 66.7% (10/15) of the AD-HSP patients. No significant correlation was found between the presence of the SPAST mutation and any of the various clinical parameters of pure HSP. No ATL1 and REEP1 mutations were detected.ConclusionsWe conclude that SPAST mutations are responsible for most Korean cases of genetically confirmed AD-HSP. Our observation of the absence of ATL1 and REEP1 mutations needs to be confirmed in larger series.

REEP1 and REEP2 proteins are preferentially expressed in neuronal and neuronal-like exocytotic tissues

The six members of the Receptor Expression Enhancing Protein (REEP) family were originally identified based on their ability to enhance heterologous expression of olfactory receptors and other difficult to express G protein-coupled receptors. Interestingly, REEP1 mutations have been linked to neurodegenerative disorders of upper and lower motor neurons, hereditary spastic paraplegia (HSP) and distal hereditary motor neuropathy type V (dHMN-V). The closely related REEP2 isoform has not demonstrated any such disease linkage. Previous research has suggested that REEP1 mRNA is ubiquitously expressed in brain, muscle, endocrine, and multiple other organs, inconsistent with the neurodegenerative phenotype observed in HSP and dHMN-V. To more fully examine REEP1 expression, we developed and characterized a new REEP1 monoclonal antibody for both immunoblotting and immunofluorescent microscopic analysis. Unlike previous RT-PCR studies, immunoblotting demonstrated that REEP1 protein was not ubiquitous; its expression was restricted to neuronal tissues (brain, spinal cord) and testes. Gene expression microarray analysis demonstrated REEP1 and REEP2 mRNA expression in superior cervical and stellate sympathetic ganglia tissue. Furthermore, expression of endogenous REEP1 was confirmed in cultured murine sympathetic ganglion neurons by RT-PCR and immunofluorescent staining, with expression occurring between Day 4 and Day 8 of culture. Lastly, we demonstrated that REEP2 protein expression was also restricted to neuronal tissues (brain and spinal cord) and tissues that exhibit neuronal-like exocytosis (testes, pituitary, and adrenal gland). In addition to sensory tissues, expression of the REEP1/REEP2 subfamily appears to be restricted to neuronal and neuronal-like exocytotic tissues, consistent with neuronally restricted symptoms of REEP1 genetic disorders.

ATL1 and REEP1 mutations in hereditary and sporadic upper motor neuron syndromes

SPAST mutations are the most common cause of autosomal dominant hereditary spastic paraplegias (AD-HSPs), but many spastic paraplegia patients are found to carry no mutations in this gene. In order to assess the contribution of ATL1 and REEP1 in AD-HSP, we performed mutational analysis in 27 SPAST-negative AD-HSP families. We found three novel ATL1 mutations and one REEP1 mutation in five index-patients. In 110 patients with sporadic adult-onset upper motor neuron syndromes, a novel REEP1 mutation was identified in one patient. Apart from a significantly younger age at onset in ATL1 patients and restless legs in some, the clinical phenotype of ATL1 and REEP1 was similar to other pure AD-HSPs.

Exome Sequencing Identifies a REEP1 Mutation Involved in Distal Hereditary Motor Neuropathy Type V

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.