Liddle Syndrome Caused by Loss-of-Function Mutations in the Ubiquitin Ligase NEDD4L

Background: Liddle Syndrome (Pseudohyperaldosteronism) is an autosomal dominant form of hereditary hypertension characterized by early-onset hypertension, hypokalemia, low blood aldosterone and renin levels, and metabolic alkalosis. In most patients it is caused by a pathogenic variant in the PY motifs (PPxY) of the b (SCNN1B) or g (SCNN1G) subunits of the amiloride-sensitive Epithelial Na+ Channel, ENaC. Mutant ENaC exhibits increased retention and function at the plasma membrane in the distal nephron, increased reabsorption of luminal Na+, increased circulatory blood volume and hypertension. These ENaC variants impair its binding to NEDD4L, an E3 ubiquitin ligase comprised of C2-WW(4)-HECT domain architecture. Impaired NEDD4L binding to ENaC leads to reduced cell surface ENaC ubiquitination, impaired channel endocytosis and degradation, thus explaining the increased retention of ENaC at the plasma membrane. Methods: Targeted exome sequencing was used, which included SCNN1A, SCNN1B, SCNN1G and NEDD4L. The patient's variants on either allele of NEDD4L were generated by site-directed mutagenesis and tested for self-ubiquitination in vitro, substrate ubiquitination against a model substrate and against cell-surface αβγENaC in kidney Hek293T cells. Cell surface ENaC stability was analyzed in parallel. Results: Unlike most Liddle syndrome patients with pathogenic ENaC variants, a subset of patients have normal ENaC. Here we describe the discovery of novel compound heterozygous pathogenic variants in the NEDD4L gene in a patient with Liddle syndrome and normal ENaC genes. Both parents are unaffected carriers. The maternal allele is a frameshift variant that yields a truncated NEDD4L protein devoid of the entire catalytic HECT domain. The paternal allele is a missense variant in the HECT domain that exhibits a severe loss of its enzymatic activity. This results in a dramatic reduction in ENaC ubiquitination, thereby increasing the stability of this channel at the plasma membrane. Conclusions: This is the first demonstration of a novel recessive form of Liddle syndrome caused by loss of function of the ENaC suppressor NEDD4L. On this basis, it will be important to include NEDD4L in commercial hypertension gene panels to facilitate the diagnosis of other Liddle syndrome patients with normal SCNN1 (ENaC) genes. Funding: Government Support - Non-U.S.

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