A mutation of EPT1 (SELENOI) underlies a new disorder of Kennedy pathway phospholipid biosynthesis

Abstract

Mutations in genes involved in lipid metabolism have increasingly been associated with various subtypes of hereditary spastic paraplegia, a highly heterogeneous group of neurodegenerative motor neuron disorders characterized by spastic paraparesis. Here, we report an unusual autosomal recessive neurodegenerative condition, best classified as a complicated form of hereditary spastic paraplegia, associated with mutation in the ethanolaminephosphotransferase 1 (EPT1) gene (now known as SELENOI), responsible for the final step in Kennedy pathway forming phosphatidylethanolamine from CDP-ethanolamine. Phosphatidylethanolamine is a glycerophospholipid that, together with phosphatidylcholine, constitutes more than half of the total phospholipids in eukaryotic cell membranes. We determined that the mutation defined dramatically reduces the enzymatic activity of EPT1, thereby hindering the final step in phosphatidylethanolamine synthesis. Additionally, due to central nervous system inaccessibility we undertook quantification of phosphatidylethanolamine levels and species in patient and control blood samples as an indication of liver phosphatidylethanolamine biosynthesis. Although this revealed alteration to levels of specific phosphatidylethanolamine fatty acyl species in patients, overall phosphatidylethanolamine levels were broadly unaffected indicating that in blood EPT1 inactivity may be compensated for, in part, via alternate biochemical pathways. These studies define the first human disorder arising due to defective CDP-ethanolamine biosynthesis and provide new insight into the role of Kennedy pathway components in human neurological function.

Keywords: EPT1 mutation; Kennedy pathway; hereditary spastic paraplegia; phospholipid biosynthesis; whole exome sequencing.

Figure 1

Genetic and clinical summary of the pedigree investigated. (A) The Omani pedigree with a pictorial representation of the homozygous SNP genotypes across the critical interval (red hashed box) with ‘A’ and ‘B’ genotypes indicated by blue and yellow bars. SELENOI/EPT1 genotypes shown in red (plus sign indicates presence of c.335 G > C alteration, negative sign indicates wild-type). (B) Sequence electropherograms showing the DNA encompassing the SELENOI/EPT1 c.335 G > C alteration. (C) Species alignment of EPT1 amino acids encompassing the altered p.Arg112 residue showing stringent conservation within the CDP catalytic motif. (D–G) Clinical features of affected individuals. (D) Photograph of Patient V:1 showing bifid uvula. (E) Retinal photograph from the right eye of Patient V:5 showing: mild retinal vessel tortuosity, a dull macular reflex and a mild retinal pigment epithelium level pigmentary disturbance most noticeable inferiorly. (F and G) Brain MRI scan of Patient V:6 reveals bilateral symmetrical periventricular hyperintensity in the trigon, frontal subcortical white matter and U fibre. All four children displayed similar MRI findings.

Figure 2

Outcomes of the p.Arg112Pro EPT1 sequence alteration. Yeast cells devoid of endogenous ethanolaminephosphotransferase activity were transformed with plasmids bearing wild-type human EPT1 (hEPT1) or EPT1 containing the p.Arg112Pro mutation (hEPT1*) each tagged with a DDK epitope. (A) Schematic representation of the CDP-ethanolamine branch of Kennedy pathway showing the role of EPT1 in PE formation. CK = choline kinase; CPT = choline phosphotransferase; CT = phosphocholine cytidylyltransferase; EK = ethanolamine kinase; ET = phosphoethanolamine cytidylyltransferase; Etn = ethanolamine; LPEAT = lyso-PE acyltransferase; PSD = phosphatidyl serine decarboxylase; PSS = phophatidyl serine synthase. (B) Western blot versus whole cell extracts (WCE), which were fractionated into soluble (S100) and membrane (P100) fractions, and probed using anti-DDK antibodies. Pgk1 and Dpm1 are soluble and membrane fraction loading and fractionation purification controls, respectively. EV = empty vector control. (C) Mid-log phase cells were radiolabelled with ¹⁴C-ethanolamine for 1 h. As a positive control yeast strain HJ001 (cpt1::LEU2) transformed with an empty vector was also radiolabelled; this strain possess the wild-type genomic allele of yeast EPT1 (yEPT1). Cells were processed for lipid extraction and the radioactivity associated with PE and PC was determined.