Abstract
INTRODUCTION: Autosomal recessive polycystic kidney disease (ARPKD) is caused primarily by pathogenic variants in PKHD1, encoding fibrocystin/polyductin. In Cys1(cpk/cpk) (cpk) mice, the kidney and liver lesions closely phenocopy ARPKD. Cys1 encodes cystin, a myristoylated protein that traffics to the primary cilium and nucleus. We recently reported the first patient with ARPKD due to a homozygous CYS1 splicing variant. METHODS: To study this, we utilized high-resolution ultrasound, array Comparative Genomic Hybridization and exon sequencing to identify an ARPKD-like kidney lesion and CYS1 defects in two families. Bioinformatic analyses compared cystin sequence conservation across mammals. Immunofluorescence, optogenetic studies, and 3D culture comparatively evaluated subcellular localization, ciliary trafficking, and cyst formation under cAMP stimulation of wild-type and mutant cystin. Tandem affinity purification/mass spectrometry identified cystin-binding partners. Serine-17 phosphorylation was evaluated functionally. RESULTS: The Family 1 proband carried a homozygous deletion about 24-kb in size encompassing the CYS1 regulatory region and exon 1. In Family 2, two affected siblings had a homozygous CYS1 c.4G>A (p.Gly2Ser) variant affecting Gly2 within the N-terminal myristoylation motif. This region, together with an adjacent arginine-rich stretch flanked by serines-8 (S8) and -17 (S17) residues, comprises a conserved myristoyl-electrostatic switch that can regulate reversible protein-membrane binding. S17 phosphorylation modulates cystin membrane association and intracellular trafficking. In 3D culture, CRISPR/Cas9-generated cystin-null cells or null cells expressing cystin-1(G2S) showed enhanced cyst formation versus their respective controls. cAMP activation by cilia-specific optogenetic stimulation in cultured cells or pharmacologic treatment of ex vivo embryonic kidneys reduced cystin ciliary localization in a protein kinase A-dependent manner. Mass spectrometry identified the phosphatase PPM1A as a cystin-interacting partner, and pharmacologic inhibition reduced cystin S17 dephosphorylation. CONCLUSIONS: Cystin-1 intracellular trafficking and function are regulated by a myristoyl-electrostatic switch mechanism. Cystic kidneys in three individuals with homozygous CYS1 pathogenic variants provide further support that cystin-1 deficiency causes polycystic kidney disease that phenocopies ARPKD.