Sphingosine-1-phosphate lyase mutations cause primary adrenal insufficiency and steroid-resistant nephrotic syndrome

Abstract

Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G>A (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G>A (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism. Sgpl1-/- mice recapitulated the main characteristics of the human disease with abnormal adrenal and renal morphology. Sgpl1-/- mice displayed disrupted adrenocortical zonation and defective expression of steroidogenic enzymes as well as renal histology in keeping with a glomerular phenotype. In summary, we have identified SGPL1 mutations in humans that perhaps represent a distinct multisystemic disorder of sphingolipid metabolism.

Figure 1. Pedigrees of kindreds 1 to 5 where all affected individuals manifested PAI, with or without SRNS, and were positive for mutations in SGPL1.

Black symbols indicate individuals with PAI alone, half-filled in green indicate those who additionally had steroid-resistant nephrotic syndrome, and green indicate those with SRNS alone. All affected individuals were homozygous for the indicated mutations (patients sequenced have been numbered from 1 to 8), and parents were heterozygous (those sequenced denoted by gray symbols). Mutations for patients 2, 3, and 5 were identified by WES and the remainder by Sanger sequencing of SGPL1.

Figure 2. p.R222Q and p.F545del mutations affect highly conserved areas in SGPL1 and are loss of function, resulting in proteins with reduced lyase activity.

(A) SGPL1 regulates flow of the sphingolipid biochemical intermediates (in green) and carries out the final degradation step in the pathway. (B) Partial alignment of SGPL1 protein sequences, generated by Clustal Omega (48), showing conservation of arginine (R) at position 222 and phenylalanine (F) at position 545, highlighted in yellow, with numbering relative to human sequence. For all but the most distant organisms, these amino acids are conserved. Alignment source accession numbers from ENSEMBL are as follows: Homo sapiens, human, ENSP00000362298; Mus musculus, mouse, ENSMUSP00000112975; Rattus norvegicus, rat, ENSRNOP00000070983; Tetraodon nigroviridis, pufferfish, ENSTNIP00000016065; Xenopus laevis, clawed frog, ENSXETP00000017960; Ciona intestinalis, sea squirt, ENSCINP00000002369; Drosophila melanogaster, fruit fly, FBpp0086158; Caenorhabditis elegans, nematode, B0222.4; and Saccharomyces cerevisiae, yeast, YDR294C. Sequence conservation is beneath the alignment. Asterisks indicate total conservation; colons indicate partial conservation. (C) SGPL1 activities were measured in lysates of Sgpl1^–/– mouse fibroblasts. **P < 0.01, 2-tailed Student’s t test (n = 3). (D) Lysates of Sgpl1^–/– mouse fibroblasts expressing WT or the mutant SGPL1 (25 μg protein/lane) were analyzed by immunoblotting for the presence of the FLAG-tagged protein (representative image, n = 3). (E) SGPL1 mRNA expression in a human tissue panel, analysis using the 2^–ΔΔCT algorithm (n = 3). S intestine, small intestine.

Figure 3. Adrenals from Sgpl1^–/– mice show histological abnormalities, and SGPL1 is expressed in human adrenals.

(A and B) Adrenals from Sgpl1^–/– mice show histological abnormalities. (A) H&E staining of Sgpl1^+/+ and Sgpl1^–/– adrenals. Note the less defined morphological zonation in the Sgpl1^–/– adrenals compared with that from Sgpl1^+/+ mice. Moreover, the characteristic lipid droplets found in the ZF (arrowheads in top-right panel) and visible as large areas in the cytoplasm devoid of eosin staining (as lipids are extracted during the paraffin-embedding procedure) are strongly reduced in Sgpl1^–/– adrenals (n = 3). Cap, capsule. Scale bars: 100 μm (left); 25 μm (middle); 5 μm (right). (B) CYP11A1 and CYP11B2 expression in Sgpl1^+/+ and Sgpl1^–/– adrenals. CYP11A1 staining in Sgpl1^–/– adrenals is less prominent compared with Sgpl1^+/+, while the characteristic patchy expression of aldosterone synthase (CYP11B2) is lost in Sgpl1^–/– adrenals (n = 3). Scale bars: 100 μm (top); 25 μm (bottom). (C–E) Expression of SGPL1 in human adrenals. (C) Western blotting of lysates from human adrenal, HEK293, cells and HEK293 cells overexpressing SGPL1 probed with anti-SGPL1 antibody (representative image of n = 3). (D) SGPL1 expression in the HFA at 19 and 22 Carnegie (Carn) stage as well as at 18 weeks showing widespread expression (n = 1 each). FZ, fetal zone; DZ, definitive zone. Scale bars: 100 μm. (E) SGPL1 expression in the human adult adrenal (n = 3). Note the stronger expression of SGPL1 in the ZR compared with ZG and ZF, while the capsule and medulla (M) are negative. Scale bars: 100 μm (left panel); 25 μm (right 3 panels).

Figure 4. Histological features of the glomeruli.

(A–C) H&E staining of Sgpl1^+/+ kidney showing normal cortical histology (A) and glomeruli with open capillary loops and normal cellularity (B and C, yellow arrowhead). The kidneys from Sgpl1^–/– mice (E–G) have mild mesangial hypercellularity with glomerular hypertrophy (F and G, yellow arrowhead) and large protein casts in the tubules (white arrows). (D and H) Masson’s trichrome stain. Kidneys from Sgpl1^–/– mice (H) show increased glomerular fibrosis (red stain) compared with Sgpl1^+/+ (D). n = 3 in all cases. Scale bars: 100 μm (A and E); 25 μm (B–D and F–H).