Loss-of-Function Homozygous Variant in LPL Causes Type I Hyperlipoproteinemia and Renal Lipidosis

Figures

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  Figure 1. Serum lipoprotein profile difference between healthy control and patient with homozygous LPL (c.292G > A, p.A98T) variant. The lipoprotein electrophoresis results of the healthy control are shown in Figure 1a and Figure 1c. From top to bottom, the 3 bands of β-lipoprotein, pre-β-lipoprotein, and α-lipoprotein have the percentages 46.7%, 24.9%, and 28.4%, respectively. The lipoprotein electrophoresis result of the patient with homozygous LPL (c.292G > A, p.A98T) variant is shown in Figure 1b and Figure 1d. The chylomicron band is shown at the origin and is labeled with ∗. From top to bottom, the 4 bands of β-lipoprotein, chylomicron, pre-β-lipoprotein, and α-lipoprotein have the percentages 19.9%, 22.8%, 13.3%, and 44%, respectively.

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  Figure 2. Pathological findings of renal biopsy specimen from patient with type I hyperlipoproteinemia. A moderate-to-severe increase in mesangial cell number and matrix, and marked foam cell infiltration in a glomerulus stained with periodic acid–Schiff stain and periodic acid-sliver methenamine stain (×200) are shown in Figure 2a and Figure 2b. A partial region of glomerulus positive for Oil red O staining (×200) is shown in Figure 2c. A CD68-positive foam cell in a glomerulus is shown in Figure 2d. Numerous lipid droplets and cholesterol clefts in the cytoplasm of foam cells (marked by white arrow) are shown in Figure 2e and Figure 2f.

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  Figure 3. Homozygous LPL missense mutation identification, mutation site evolutionary conservation analysis, and pedigree analysis of the patient with type I hyperlipoproteinemia. Partial sequences of LPL exon 3 from the patient and a healthy control, respectively are shown in Figure 3A and Figure 3B. The mutation site is marked by the arrow and presented as LPL (c.292G > A, p.A98T). The wild-type LPL amino acid sequence fragments of various species are shown in Figure 3c. The white-colored box shows that the amino acid p.Ala98 is evolutionarily highly conserved. The family pedigree of the patient with type I hyperlipoproteinemia is shown in Figure 3D. An arrow represents the proband (II-1), a square represents a male individual, a circle represents a female individual, blank symbols represent unaffected family members, and black symbols represent patients homozygous for LPL (c. 292G > A, p.A98T). The blood samples of I-1, I-2, and II-2 are not available.

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  Figure 4. Protein structures of LPL (c.292G > A, p.A98T) and wild-type LPL. The 3-dimensional structure modeling of the mutant human LPL protein was built based on the template of the wild-type human LPL in complex with GPIHBP1 (Protein Data Bank entry 6E7K).The whole LPL protein (PDB ID: 6E7K) and the predicted LPL (c.292G > A, p.A98T) protein structures are shown in panels a, b, and c. Panels d, e, and f show the amplification regions of panels a, b, and c, respectively. The substitution of the amino acid Thr for Ala at site 98 may increase the side chain and H-bond number between LYS94 and THR98 or between LEU95 and THR98, causing a clash (shown by the arrow) and a conformational change.

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  Figure 5. Functional analysis of LPL (c.292G > A, p.A98T) mutation in 293T cells. (a) Western blot analysis of the HA-tagged human LPL protein relative expression in various transfected 293T cells, GAPDH is used as the loading control. (b) qPCR analysis of human LPL relative expression in various transfected 293T cells. (c and d) the LPL activity and mass of the cell medium in 3 transfected 293T cells groups. (e) the LPL activity of the cell lysates in 3 transfected 293T cells groups. The experiments are performed with 3 replicates, data are shown as mean ± SD, and significance tested with 1-way ANOVA for all data and indicated as ∗P < 0.05,∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 to the wild-type LPL group.

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  Figure 6. Analyses of LPL expression and lipid uptake capability of LPL (c.292G > A, p.A98T)-transfected RAW264.7 cells. (Ia) Western blot analysis of the HA-tagged human LPL protein relative expression in various transfected RAW264.7 cells, GAPDH is used as the loading control. (Ib) qPCR analysis of human LPL relative expression in various transfected RAW264.7 cells. (Ic) Dil-labeled mean fluorescence intensity in 3 groups. (IIa, IIb, and IIc) the Dil-labeled oxLDL uptake capability of 3 transfected RAW264.7 cells. (IId, IIe, and IIf) the Oil-red O staining of 3 transfected RAW264.7 cells. The experiments are performed with 3 replicates, data are shown as mean ± SD, and significance tested with 1-way ANOVA for all data and indicated as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 to the wild-type LPL group.