Mutation in CPT1C Associated With Pure Autosomal Dominant Spastic Paraplegia

Abstract

Importance: The family of genes implicated in hereditary spastic paraplegias (HSPs) is quickly expanding, mostly owing to the widespread availability of next-generation DNA sequencing methods. Nevertheless, a genetic diagnosis remains unavailable for many patients.

Objective: To identify the genetic cause for a novel form of pure autosomal dominant HSP.

Design, setting, and participants: We examined and followed up with a family presenting to a tertiary referral center for evaluation of HSP for a decade until August 2014. Whole-exome sequencing was performed in 4 patients from the same family and was integrated with linkage analysis. Sanger sequencing was used to confirm the presence of the candidate variant in the remaining affected and unaffected members of the family and screen the additional patients with HSP. Five affected and 6 unaffected participants from a 3-generation family with pure adult-onset autosomal dominant HSP of unknown genetic origin were included. Additionally, 163 unrelated participants with pure HSP of unknown genetic cause were screened.

Main outcome and measure: Mutation in the neuronal isoform of carnitine palmitoyl-transferase (CPT1C) gene.

Results: We identified the nucleotide substitution c.109C>T in exon 3 of CPT1C, which determined the base substitution of an evolutionarily conserved Cys residue for an Arg in the gene product. This variant strictly cosegregated with the disease phenotype and was absent in online single-nucleotide polymorphism databases and in 712 additional exomes of control participants. We showed that CPT1C, which localizes to the endoplasmic reticulum, is expressed in motor neurons and interacts with atlastin-1, an endoplasmic reticulum protein encoded by the ATL1 gene known to be mutated in pure HSPs. The mutation, as indicated by nuclear magnetic resonance spectroscopy studies, alters the protein conformation and reduces the mean (SD) number (213.0 [46.99] vs 81.9 [14.2]; P < .01) and size (0.29 [0.01] vs 0.26 [0.01]; P < .05) of lipid droplets on overexpression in cells. We also observed a reduction of mean (SD) lipid droplets in primary cortical neurons isolated from Cpt1c-/- mice as compared with wild-type mice (1.0 [0.12] vs 0.44 [0.05]; P < .001), suggesting a dominant negative mechanism for the mutation.

Conclusions and relevance: This study expands the genetics of autosomal dominant HSP and is the first, to our knowledge, to link mutation in CPT1C with a human disease. The association of the CPT1C mutation with changes in lipid droplet biogenesis supports a role for altered lipid-mediated signal transduction in HSP pathogenesis.

Figure 1. Pedigree and Sequence Analysis of CPT1C

A, Pedigree of the family. White indicates unaffected individuals and black indicates affected individuals. Presence (mutation) or absence (wild type) of the mutation in CPT1C is indicated below each individual. B, Schematic diagram of CPT1C. Exons are represented as solid black boxes, noncoding exons are depicted as white boxes, and introns are depicted as bent connectors. The c.108C>G variant (p.Arg37Cys) is indicated. C, Electropherograms show the c.109C>T (p.Arg37Cys) variant (arrowhead) in CPT1C in an affected (heterozygote) and a nonaffected individual. D, Multiple sequence analysis of CPT1C in various species.

Figure 2. Structural Model and Nuclear Magnetic Resonance (NMR) Spectroscopy of CPT1C

A, Structural model of the inhibitory Nα state of CPT1C. This model is based on the Nα state of CPT1A and looks onto the hydrophilic faces of amphiphilic helices α1 and α2. The model depicts Cys 37, which replaces Arg 37. In CPT1C, Glu26 of CPT1A is instead Pro26. B, Superposition of backbone amide-nitrogen correlation NMR spectra of wild-type Nα and Nα(Arg37Cys) of CPT1C. The spectra were recorded in the presence of tetradecyltrimethylammonium chloride (TDAC) at pH 7.4, 25°C, and a hydrogen 1 (¹H) frequency of 700 MHz. C, Nitrogen 15 (¹⁵N) chemical shift differences, Dd(¹⁵N) between Nα and Nα(Arg37Cys), and Nβ and Nβ(Arg37Cys). The borders of the secondary structure elements of Nα, helices α1, α2, and α2′, are indicated. D, Comparison of Nα and Nα(Arg37Cys) secondary carbon α 13 (¹³C^α) chemical shifts, d(¹³C^α), defined as the difference between observed and tabulated random coil ¹³C^α shifts. Positive and negative shifts indicate helical and extended backbone conformations, respectively. DDAC indicates didecyldimethylammonium chloride.

Figure 3. CPT1C Interaction With Atlastin-1

A, COS7 cells cotransfected with 1.5 μg of avian myelocytomatosis (myc)–tagged atlastin-1 and 1.5 μg of HA-tagged CPT1C. Coimmunoprecipitation using an anti-myc antibody (C3956; Sigma) was performed and interactions were detected by Western blot analysis using an anti-hemagglutinin (HA) antibody (clone 16B12, 1:5000; Covance). B, COS7 cells were cotransfected and signal was detected using anti-HA (clone 16B12, 1:5000; Covance) and anti-myc antibody (C3956, 1:5000; Sigma). The nuclear 4′,6-diamidino-2-phenylinodole stain is blue. Scale bar = 20 μm.

Figure 4. p.Arg37Cys CPT1C Is Associated With Reduced Lipid Droplet (LD) Number in COS7 Cells

A and B, COS7 cells were transfected with wild-type or mutant hemagglutinin (HA)-tagged CPT1C and stained with HA antibody (clone 16B12, 1:5000; Covance) and BODIPY 493/503 for LDs (0.1 μg/mL; Invitrogen). The bottom panel shows an enlarged view of the boxed area. Scale bar = 20 μm. C and D, Numbers and areas of LDs were counted blindly in an automated fashion and results were derived from a total of 2949 LDs. Results are given as mean (SEM). E, Relative distribution of LD areas in wild-type and mutant CPT1C. Counting of LDs was performed in a blinded and automated fashion using the software Volocity 3D Image Analysis Software (PerkinElmer). The data were analyzed using GraphPad Prism software version 6 (http://www.graphpad.com) and P ≤ .05 was considered significant. DAPI indicates 4′,6-diamidino-2-phenylinodole. ^aP < .01. ^bP < .05.

Figure 5. Lipid Droplet Number Is Decreased in Cpt1c^−/− Cortical Neurons

A, Cells were obtained from the cortex of wild-type or Cpt1c^−/− E16 embryos and seeded in 48-well plates during 7 days in vitro. For lipid loading, 300μM oleic acid was added for 8 or 16 hours before fixation. Representative images are shown. Images of neural lipids and the nucleus were acquired on a Nikon Eclipse TE-2000E optic microscope using a plan apochromat objective (BODIPY 493/503 and Hoechst dyes [Life Technologies], respectively; original magnification ×20). For quantification, sets of cells were cultured and stained simultaneously and imaged using identical settings. The region of interest was randomly selected using nucleic staining. B, For quantification, the Fiji image processing package was used to determine the integrated density stain per soma in at least 80 cells per condition. The results are shown as the mean of 2 independent experiments ± SEM. ^aP < .001.