Loss of LGR4/GPR48 causes severe neonatal salt wasting due to disrupted WNT signaling altering adrenal zonation
Cécily Lucas, Kay-Sara Sauter, Michael Steigert, Delphine Mallet, James Wilmouth, Julie Olabe, Ingrid Plotton, Yves Morel, Daniel Aeberli, Franca Wagner, Hans Clevers, Amit V. Pandey, Pierre Val, Florence Roucher-Boulez, Christa E. Flück
Cécily Lucas, Kay-Sara Sauter, Michael Steigert, Delphine Mallet, James Wilmouth, Julie Olabe, Ingrid Plotton, Yves Morel, Daniel Aeberli, Franca Wagner, Hans Clevers, Amit V. Pandey, Pierre Val, Florence Roucher-Boulez, Christa E. Flück
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Research Article Endocrinology

Loss of LGR4/GPR48 causes severe neonatal salt wasting due to disrupted WNT signaling altering adrenal zonation

Abstract

Disorders of isolated mineralocorticoid deficiency, which cause potentially life-threatening salt-wasting crisis early in life, have been associated with gene variants of aldosterone biosynthesis or resistance; however, in some patients no such variants are found. WNT/β-catenin signaling is crucial for differentiation and maintenance of the aldosterone-producing adrenal zona glomerulosa (zG). Herein, we describe a highly consanguineous family with multiple perinatal deaths and infants presenting at birth with failure to thrive, severe salt-wasting crises associated with isolated hypoaldosteronism, nail anomalies, short stature, and deafness. Whole exome sequencing revealed a homozygous splice variant in the R-SPONDIN receptor LGR4 gene (c.618-1G>C) regulating WNT signaling. The resulting transcripts affected protein function and stability and resulted in loss of Wnt/β-catenin signaling in vitro. The impact of LGR4 inactivation was analyzed by adrenal cortex–specific ablation of Lgr4, using Lgr4fl/fl mice mated with Sf1:Cre mice. Inactivation of Lgr4 within the adrenal cortex in the mouse model caused decreased WNT signaling, aberrant zonation with deficient zG, and reduced aldosterone production. Thus, human LGR4 mutations establish a direct link between LGR4 inactivation and decreased canonical WNT signaling, which results in abnormal zG differentiation and endocrine function. Therefore, variants in WNT signaling and its regulators should systematically be considered in familial hyperreninemic hypoaldosteronism.

Authors

Cécily Lucas, Kay-Sara Sauter, Michael Steigert, Delphine Mallet, James Wilmouth, Julie Olabe, Ingrid Plotton, Yves Morel, Daniel Aeberli, Franca Wagner, Hans Clevers, Amit V. Pandey, Pierre Val, Florence Roucher-Boulez, Christa E. Flück

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Figure 2

Protein expression and functional testing of Wnt/β-catenin signaling of the 2 LGR4 variants.

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Protein expression and functional testing of Wnt/β-catenin signaling of ...
Human fibroblasts and HEK cells were used. (A) Western blot analysis for LGR4 protein expression in patient and control fibroblasts. A representative blot of 3 independent experiments is shown. β-Actin was used as a loading control. The molecular weight (kDa) of a protein standard is given. M, marker; Co, control fibroblasts; Pat, patient fibroblasts. (B) RSPO1-activated, LGR4-mediated Wnt signaling in HEK293 cells. Cells were transfected with WT and mutant LGR4 nt-24 and nt-72 plasmids (including a mock control) and reporter vectors TOP-Flash and Renilla. Signaling was stimulated by RSPO1 and assessed by the Dual-Luciferase assay (Promega). Results are expressed as relative LUC activities (RLU). Mean ± SD of 3 independent experiments is shown. *P < 0.01, Student’s t test. (C and D) Interaction of RSPO1 with membrane-localized WT and variant LGR4. HEK293 cells were transfected with HA-tagged LGR4 plasmids (pcDNA3 LGR4wt, nt-24 bp, nt-72 bp) and incubated with conditioned RSPO1-GFP SN medium (previously produced in HEK cells transfected with pSpark- RSPO1-GFP). Cells were fixed with Carnoy’s solution. Staining was performed with antibody anti–HA-Tag (green) first and antibody anti-mouse Alexa Fluor 594 (red) second. Immunofluorescent microscopy was used to detect the cellular distribution of the tagged proteins as well as their colocalization (Zeiss LSM 710). Three independent experiments were analyzed. Representative images of confocal analysis at original magnification, ×40 are shown for WT and variants of LGR4. Scale bars: 5 μm (top 2 images in middle column and right); 10 μm (left and bottom image in the middle column). Quantification of colocalized LGR4 and RSPO1 was performed by Imaris (Bitplane AG).