PMC7751862_01
Clinical features of a patient with severe ID.. (A) Perinatal information and systemic symptoms of the patient. SD, standard deviation. (B) Brain MRI (T2-weighted imaging) of the patient at 23 years of age (axial imaging). The MRI revealed general cerebral atrophy, especially in the frontal lobe, and hypoplasia of the corpus callosum. (C) Coronal MRI imaging. (D) Patient photograph showing the coarse face, thick eyebrows, hypotelorism, long palpebral fissures, midface hypoplasia, bulbous nose, wide ala nasi, short philtrum, thin upper lip, and hypertrichosis.
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She was born after a full-term delivery with normal birth parameters after an uneventful pregnancy (Fig 1A). The progression of her leukodystrophy, especially in the frontal lobe, was confirmed by MRI at 23 years of age (Fig 1B and 1C). On physical examination, she exhibited several systemic complications and distinctive facial features (Fig 1A and 1D) (for additional clinical information, see S1 Table). This is consistent with the fact that all the previously reported CERT1 variants that cause ID or autism spectrum disorder (ASD) were missense variants, and not truncation variants (Fig 2D and Table 1).
An ID-associated de novo CERT1 mutation.. (A) WES sequencing quality. (B) Filtering pipeline of variants identified by trio WES. A total of 1864 variants with a minor allele frequency (MAF) of less than 0.01 were found in the patient. After excluding low-quality variants and variants found in the control healthy population, 16, 5, and 21 variants were identified as de novo heterozygous, homozygous, and compound heterozygous, respectively. (C) Trio Sanger sequence of the CERT1 variant (NM_0031361.3:c.403T>C:p.[Ser135Pro], Chr5[GRCh37]:g.74722249A>G) identified in our patient. (D) Variant distribution of the CERT protein structure (NP_112729.1), which includes the PH domain, SRM, FFAT motif, and steroidogenic acute regulatory protein-related lipid transfer (START) domain.
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At 20 years of age, we conducted trio WES analysis of the patient and her parents and identified a novel heterozygous missense variant in CERT1 (NM_001130105.1:c.787T>C:p.[Ser263Pro]) (Fig 2A and 2B). Trio Sanger sequencing confirmed that this was a de novo variant (Fig 2C) that was absent in the control healthy population found in the genome aggregation database (gnomAD; https://gnomad.broadinstitute.org/) and the Japanese multi omics reference panel (jMorp; https://jmorp.megabank.tohoku.ac.jp/202001/variants). This is consistent with the fact that all the previously reported CERT1 variants that cause ID or autism spectrum disorder (ASD) were missense variants, and not truncation variants (Fig 2D and Table 1).
The ID-associated de novo mutation in CERT1 affects the phosphorylation status of CERT in LCLs.. (A) The trio-derived LCLs were analyzed by Western blotting with the indicated primary antibodies. Hyperphosphorylated (hyper-p-) and de/hypophosphorylated (d/hypo-) CERT are shown (left). The de/hypophosphorylated levels of CERT in the trio-derived LCLs were quantified by densitometric scanning of the band intensities (right). The data comprise the mean +- SEM; n = 3 (**, p < 0.01; n.s., not significant). (B) Trio LCL lysates were incubated with or without lambdaPPase and analyzed by Western blotting with the indicated primary antibodies. The white and black arrowheads represent completely de/hypophosphorylated CERT and CERT/L, respectively. (C) Trio LCLs were cultured with L-[U-14C]serine for 16 hr. Metabolically labelled lipids separated on a TLC plate were visualized (representative image, left) and labelled SM was quantified (right). PE, phosphatidylethanolamine; PS, phosphatidylserine. The data comprise the mean +- SEM; n = 4.
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Moreover, Western blotting of lymphoblastoid cell (LCL) extracts derived from our ID patient and her parents showed that the hyperphosphorylated form of CERT was more abundant than the de/hypophosphorylated forms in both the mother and father, while the de/hypophosphorylated forms were more abundant than the hyperphosphorylated form in the patient (Fig 3A). A faint band comprising the hyperphosphorylated form of CERT/L was also observed in all trio LCLs (Fig 3A). The de/hypophosphorylated forms of CERT/L were not clearly observed, presumably due to their size overlap with the hyperphosphorylated form of CERT (Fig 3A). The upper and lower bands correspond to the de-phosphorylated forms of CERT/L and CERT, respectively (Fig 3B). It should also be noted that no protein derived from the putative CERT1 isoform 3 transcript was detected in LCLs (Fig 3A and 3B) and HCT116 cells (S3B Fig). Under our experimental conditions, no significant difference was observed in the de novo SM synthesis among the trio LCLs, although the synthesis of SM in patient-derived LCLs was slightly higher than that in the parents (Fig 3C). Like the endogenous CERT in HCT116 cells, the ectopically expressed HA- or mVenus-tagged WT CERT displayed a major hyperphosphorylated form (Fig 4A and S3C Fig). Metabolic labelling of SM, but not other sphingolipids (i.e., ceramide and glucosylceramide) nor glycerophospholipids (i.e., phosphatidylserine and phosphatidylethanolamine), was markedly reduced in CERT1 KO cells, compared with parental HCT116 cells (Fig 4B and S3D Fig).
The CERT S135P mutant enhances de novo SM synthesis in HCT116 cells.. (A) WT (HCT116 WT) and CERT1 KO HCT116 cells stably expressing various HA-CERT constructs were analyzed by Western blotting. (B) WT and CERT1 KO HCT116 cells stably expressing various mVenus-CERT constructs were cultured with L-[U-14C]serine for 16 hr. Metabolically labeled lipids were analyzed by TLC. The data comprise the mean +- SEM; n = 4 (*, p < 0.05). (C) WT and CERT1 KO HCT116 cells stably expressing the indicated mVenus-CERT constructs were cultured with 250 ng/ml of lysenin for 1 hr. The cell viability was measured with a lactate dehydrogenase (LDH) cytotoxicity assay. The data comprise the mean +- SEM; n = 4 (*, p < 0.05; **, p < 0.01). (D) CERT1 KO HCT116 cells stably expressing the indicated mVenus-CERT constructs were analyzed by immunofluorescence microscopy. The data are representative of two independent experiments. Areas enclosed by rectangles are enlarged (insets). Scale bars, 10 mum and 1 mum (inset).
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Like the endogenous CERT in HCT116 cells, the ectopically expressed HA- or mVenus-tagged WT CERT displayed a major hyperphosphorylated form (Fig 4A and S3C Fig). Metabolic labelling of SM, but not other sphingolipids (i.e., ceramide and glucosylceramide) nor glycerophospholipids (i.e., phosphatidylserine and phosphatidylethanolamine), was markedly reduced in CERT1 KO cells, compared with parental HCT116 cells (Fig 4B and S3D Fig). Cells expressing the CERT mutants exhibited higher lysenin sensitivity (Fig 4C), which indicated that the intracellular CERT S135A, S135C, and S135P mutants exhibited higher activity than the WT CERT. When mVenus-tagged CERT variants were expressed in HCT116 CERT1 KO cells, the WT CERT was diffused throughout the cytoplasm with a partial enrichment in the peri-nuclear regions, which are adjacent to a 130 kDa cis-Golgi matrix protein (GM130) (Fig 4D). Interestingly, all the CERT S135 variants exhibited a punctate distribution pattern, and part of the puncta co-stained with the vesicle-associated membrane protein-associated protein (VAP)-A (Fig 4D).
The S135 mutations in CERT enhance its de novo SM synthesis activity.. (A) WT and CERT1 KO HCT116 cells stably expressing various mVenus-CERT constructs were analyzed by Western blotting with the indicated primary antibodies. (B) CERT1 KO HCT116 cells stably expressing various mVenus-CERT constructs were fixed and analyzed by immunofluorescence microscopy. GM130 and VAP-A were immuno-stained with specific antibodies. The data are representative of two independent experiments. The areas enclosed by rectangles are enlarged (insets). Scale bars, 10 mum and 1 mum (inset). (C) WT and CERT1 KO HCT116 cells stably expressing various mVenus-CERT constructs were cultured with 250 ng/ml of lysenin for 1 hr. Next, cell viability was measured with an LDH cytotoxicity assay. The data comprise the mean +- SEM; n = 3 (***, p < 0.001). (D) The lipids in WT and CERT1 KO HCT116 cells stably expressing various mVenus-CERT constructs were metabolically labelled with L-[U-14C]serine. The labeled lipids were analyzed by TLC and visualized using an image analyzer. The data comprise the mean +- SEM; n = 3 (*, p < 0.05; ***, p < 0.001).
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The punctate distribution of the CERT S135P mutant was abrogated by an additional mutation of either G67E (i.e., G67E/S135P) or deletion of the FFAT-motif (i.e., S135P/DeltaFFAT) (Fig 5A and 5B). When the intracellular activity of CERT was assessed by both the lysenin sensitivity assay and de novo SM synthesis, the enhanced activity of CERT conferred by the S135P mutation was partially blocked by the additional mutation of G67E or DeltaFFAT (Fig 5C and 5D).