Fig 1.
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.
Fig 2.
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.
Table 1.
Pathogenicity assessment of CERT1 variants identified in the present and previous studies.
Fig 3.
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 λPPase 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.
Fig 4.
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 μm and 1 μm (inset).
Fig 5.
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 μm and 1 μm (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).