Rinaldi Carlo, Schmidt Thomas, Situ Alan J, Johnson Janel O, Lee Philip R, Chen Ke-Lian, Bott Laura C, Fadó Rut, Harmison George H, Parodi Sara, Grunseich Christopher, Renvoisé Benoît, Biesecker Leslie G, De Michele Giuseppe, Santorelli Filippo M, Filla Alessandro, Stevanin Giovanni, Dürr Alexandra, Brice Alexis, Casals Núria, Traynor Bryan J, Blackstone Craig, Ulmer Tobias S, Fischbeck Kenneth H
Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland.
Department of Biochemistry and Molecular Biology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles.
JAMA Neurol. 2015 May;72(5):561-70. doi: 10.1001/jamaneurol.2014.4769.
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.
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.
Mutation in the neuronal isoform of carnitine palmitoyl-transferase (CPT1C) gene.
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.
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.
与遗传性痉挛性截瘫（HSPs）相关的基因家族正在迅速扩大，这主要归功于下一代DNA测序方法的广泛应用。然而，许多患者仍无法获得基因诊断。
确定一种新型纯合常染色体显性HSP的遗传原因。
设计、地点和参与者：我们对一家前往三级转诊中心评估HSP的家庭进行了检查和随访，持续了十年，直至2014年8月。对来自同一家庭的4名患者进行了全外显子组测序，并与连锁分析相结合。采用桑格测序法确认候选变异在该家庭其余受影响和未受影响成员中的存在，并对其他HSP患者进行筛查。纳入了一个三代家庭中的5名受影响者和6名未受影响者，该家庭患有遗传起源不明的纯成人发病常染色体显性HSP。此外，还筛查了163名遗传原因不明的纯HSP无关参与者。
肉碱棕榈酰转移酶（CPT1C）基因神经元亚型的突变。
我们在CPT1C的外显子3中鉴定出核苷酸替换c.109C>T，该替换决定了基因产物中一个进化保守的半胱氨酸残基被精氨酸替换的碱基替换。该变异与疾病表型严格共分离，在在线单核苷酸多态性数据库和另外712名对照参与者的外显子组中均未出现。我们发现，定位于内质网的CPT1C在运动神经元中表达，并与atlastin-1相互作用，atlastin-1是一种由ATL1基因编码的内质网蛋白，已知在纯HSP中发生突变。核磁共振波谱研究表明，该突变改变了蛋白质构象，并在细胞中过表达时降低了脂滴的平均数量（标准差）（213.0[46.99]对81.9[14.2]；P<0.01）和大小（0.29[0.01]对0.26[0.01]；P<0.05）。我们还观察到，与野生型小鼠相比，从Cpt1c-/-小鼠分离的原代皮质神经元中的平均（标准差）脂滴减少（1.0[0.12]对0.44[0.05]；P<0.001），这表明该突变存在显性负效应机制。
本研究扩展了常染色体显性HSP的遗传学，据我们所知，这是首次将CPT1C突变与人类疾病联系起来。CPT1C突变与脂滴生物合成变化的关联支持了脂质介导的信号转导改变在HSP发病机制中的作用。
