Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency

Abstract

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1Δ yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. HM and WES reveal SGPL1 mutations as causing SRNS with ichthyosis and facultative adrenal insufficiency or neurologic defects (NPHS type 14).

(A) Nonparametric lod scores across the human genome in 3 siblings of consanguineous family A280 with SRNS, acanthosis, and ichtyosis with facultative adrenal insufficiency. The x axis shows single-nucleotide polymorphism positions on human chromosomes concatenated from p-ter (left) to q-ter (right). Genetic distance is given in cM. The SGPL1 locus (arrowhead) is positioned within the maximum nonparametric lod peak on chromosome 10. (B) Exon structure of human SGPL1 cDNA. SGPL1 contains 15 exons. Positions of start codon (ATG) and of stop codon (TGA) are indicated. (C) Domain structure of SGPL1. The extent of the PLP-dependent transferase domain is shown. (D) Five homozygous (HOM) and 4 compound-heterozygous SGPL1 mutations (het) detected in 7 families with NPHS type 14. Family numbers (underlined), mutations, and predicted translational changes are indicated (see also Tables 1 and 2). (E) Evolutionary conservation of altered amino acid residues of SGPL1. Note that c.395A>G also resulted in p.Ile88Thrfs*25 through exon 5 skipping. (F) Ptosis in individual A280-22. (G) Skin image from individual A280-22 showing brownish black desquamation on sebostatic skin with multiple radial papules with a blueish/black erythema and central calcinosis. (H and I) Median (H) and ulnar nerve (I) paralysis in individual A280-22. (J) H&E-stained epidermal section from individual A280-22 showing acanthosis/orthokeratotic hyperkeratosis (black arrowhead) and calcinosis (white arrowhead). (K) Renal histology (silver staining) of individual A280-22, showing FSGS. Scale bars: 100 μm.

Figure 2. Biological and biochemical consequences of recessive SGPL1 mutations.

(A–F) Kidney sections of Sgpl1–/– mice (A) and Sgpl1+/+ mice (B) were stained with anti-SGPL1 (red) and WT1 antibodies (green). (B–F) Coimmunofluorescence of SGPL1 with marker proteins (green) in Sgpl1+/+ kidney: podocyte (B, WT1), ER (C, BiP), podocyte foot processes (D, synaptopodin), mesangial cells (E, α–smooth muscle actin), and endothelial cells (F, CD31). Scale bars: 25 μm. (G and H) Structural modeling of SGPL1 mutations. The 2 monomers of the SGPL1 homodimer are shown in the drawing in blue and orange, respectively. (G) p.Arg222Gln; (H) p.Ser346Ile. (I) Coimmunoprecipitation to assess dimerization of WT vs. mutant SGPL1 proteins (see also Supplemental Figure 5). Coimmunoprecipitation is representative of 3 experiments. (J) Mislocalization of variant SGPL1 proteins upon overexpression in HEK293T cells. BiP (red), or Golgi marker GOLGB1 (red), and anti-Myc antibody (green). Scale bars: 10 μm. (K) SGPL1 enzyme activity levels in transformed HEK293T cells. HEK293T cells expressing a GFP indicate endogenous SGPL1 activity levels (a vs. b and a vs. d, P < 0.0025; c vs. d, P = 0.013; a vs. c, no significant difference). (L) SGPL1 protein expression and enzyme activity levels in fibroblasts from 2 control individuals (Ctrl 1 and 2), normal human foreskin fibroblasts (Fk), and 4 individuals with SGPL1 mutations. (a vs. b, P < 0.0001.) Results are from the averages of triplicates in K and L. (M) Immunofluorescence of SGPL1 in fibroblasts. BiP (green), Golgi marker GM130 (blue), and anti-SGPL1 antibody (red). Scale bars: 25 μm. (N) PHS toxicity test. Ability to complement dpl1Δ deletion on medium containing PHS was tested for human SGPL1 WT and mutants. (O) Synthetic lethality test. Human WT and p.Glu132Gly SGPL1 expressing RH4863 survived on 5-FOA plates. However, p.Arg222Gln and p.Ser346Ile mutants did not allow for survival of DPL1 (SGPL1) deficient strains.

Figure 3. Effect of Sgpl1 knockdown on RHO GTPase activity in RMCs and patient fibroblasts.

(A) Effect of SGPL1 knockdown on rat RMCs using the xCELLigence system. RMC transfected with 2 different SGPL1 siRNAs exhibited decreased serum-induced migration rate (blue and red lines) compared with scrambled siRNA control (black line). (B) Diminished cell migration rate in patients with SGPL1 mutations. Using the xCELLigence system, fibroblasts from individuals with SGPL1 mutations (blue, red, and orange lines) showed decreased migration rate compared with control (black solid line). (C) Active GTP-bound RAC1 and CDC42 precipitated from RMCs transfected with scrambled (Scr) or SGPL1 siRNA using a GST-PAK1 (CRIB) pulldown assay. Compared with control cells, RMCs transfected with SGPL1 siRNA exhibited a significant decrease in relative CDC42 and RAC1 activity. The efficiency of knockdown by siRNA was confirmed by immunoblotting with an anti-SGPL1 antibody (second to lowest panel). (D) Active GTP-bound RHOA precipitated from RMCs using a GST-rhotekin (RBD) pulldown assay. RMCs transfected with scrambled control siRNA versus SGPL1 siRNA exhibited no significant differences in relative RHOA activity. C and D represent 3 experiments each. (E) Effect of S1PR antagonists on RMC migration rate. SGPL1 knockdown caused decreased migration rate (red line) (Supplemental Figure 3B), which was partially rescued by VPC23109 (green line), but not by JTE013 (orange line). VPC23109 is an antagonist that selectively inhibits S1PR1 and S1PR3, whereas JTE013 is an antagonist for S1PR2. Each cell index value corresponds to the average of more than triplicates and SD is in only 1 direction for clarity in A, B, and E.

Figure 4. SGPL1 missense mutations fail to rescue the phenotype of the Drosophila SGPL1 ortholog (Sply) KO.

Human SGPL1 mutations p.Glu132Gly, p.Arg222Gln, and p.Ser346Ile are equivalent to DrosophilaSply mutations p.Glu119Gly, p.Arg210Gln, and p.Ser335Ile. (A) Viability defects of Sply null hemizygous and Sply mutant flies. Viability was calculated as the percentage of Sply null hemizygous offspring of heterozygous parents. Values are normalized to the viable control Df(2R)BSC433/Df(2R)247. More than 650 flies per genotype; 6 independent experiments. (B) Western blot of HA-tagged Sply in third instar larvae (top panel) and immunofluorescence of third instar garland nephrocytes stained for HA (purple) (bottom panel). Membrane and nuclei were labeled with HRP (green) and Hoechst (blue), respectively. Five or more larvae/genotype; 3 independent experiments. Scale bar: 10 μm. (C) Foot process density in Sply null hemizygous and Sply mutant third instar garland nephrocytes. TEM images and quantification. Six or more nephrocytes/genotype; 2 independent experiments. Scale bars: 200 nm. Statistical analysis performed by Bonferroni’s test following ANOVA. ***P < 0.0005; *P < 0.05. All graphs show mean ± SEM. ns, not significant.

Figure 5. Defects of vesicular transport and sphingosine metabolism in the SGPL1 drosophila ortholog (Sply) KO.

(A) Albumin uptake in Sply null hemizygous and Sply mutant third instar garland nephrocytes. Nephrocytes were incubated for 2.5 minutes with albumin-FITC (green), fixed and stained for the membrane marker HRP (red). Ten or more larvae/genotype; 3 independent experiments. Scale bar: 10 μm. (B) Lipid droplets in Sply null hemizygous and Sply mutant third instar garland nephrocytes, assessed by Bodipy staining. Six or more larvae/genotype; 2 independent experiments. Scale bar: 10 μm. (C) Sphingoid bases accumulation in Sply null hemizygous and Sply mutant third instar larvae assessed by LC/MS. Note that p.Arg210Gln mutant rescues with less efficiency than Sply WT (p.Arg210Gln vs. Sply WT, P = 0.009 for sphingosines and P = 0.02 for ceramides, t test). n = 6 independent experiments. For Sply null, Sply WT, and p.E119G, 1 analysis was removed due to poor quality chromatography. Control corresponds to WT larvae. Statistical analysis performed by Dunnet’s (C) post-hoc tests following ANOVA or Dunn’s post-hoc test following Kruskal-Wallis (A). ***P < 0.0005; **P < 0.005; *P < 0.05. All graphs show mean ± SEM.