Loss of LGR4/GPR48 causes severe neonatal salt wasting due to disrupted WNT signaling altering adrenal zonation
Cécily Lucas, … , Florence Roucher-Boulez, Christa E. Flück
Cécily Lucas, … , Florence Roucher-Boulez, Christa E. Flück
Published December 20, 2022
Citation Information: J Clin Invest. 2023;133(4):e164915. https://doi.org/10.1172/JCI164915.
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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 1

Genetic and structural characterization of a human LGR4 mutation identified in a highly consanguineous family.

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Genetic and structural characterization of a human LGR4 mutation identif...
(A) Family pedigree showing first-degree consanguinity and multiple affected individuals. Squares: male; circles: female; diamonds: unknown sex; triangle: miscarriage. Black symbols represent affected and white symbols represent unaffected individuals. Numbers in symbols represent multiple individuals. The index patient is indicated by a black arrowhead. Asterisks indicate individuals for whom DNA was sequenced. (B) Partial chromatograms showing the LGR4 mutation NM_018490.5:c.618-1 G>C. Intron 5 (red) and exon 6 (blue) are highlighted. The proband is homozygous, parents and 1 brother are heterozygous, and 1 brother had the WT sequence. (C) LGR4 mRNA analysis of proband fibroblasts. The first track represents patient mRNA after reverse transcription, indicating presence of 2 transcripts. The 2 transcripts were separated by cloning and sequenced (2 middle tracks). The bottom track shows cDNA of control fibroblasts. The scheme above shows normal splicing in dark lines and the effect of the mutation on splicing in blue dotted lines. (D) Amino acid sequence of extracellular domain of human LGR4 that binds to RSPOs. Amino acids found deleted in the patient are located in LRR7 (–8 AA) and LRR7/8 (–24 AA) coded by exon 6 of LGR4. (E) Structure of LGR4 and its interaction with RSPOs. (i) Structure of human LGR4 extracellular domain in complex with part of RSPO1 (PDB 4KT1). Amino acids coded by exon 6 are depicted in red. (ii) Human LGR4-RSPO3 complex. RSPO3 shares high structural similarity with RSPO1 and binds to LRR7 and LRR8 of LGR4. (iii and iv) Patient LGR4, missing 8 or 24 amino acids. Several critical hydrogen-bonding residues in LGR4 are missing due to mutations, causing weaker interaction and binding of RSPO3 to mutant LGR4 proteins. (v) A surface view of the LGR4-RSPO3 complex, showing close interaction points. Exon 6–encoded residues are shown in red.