Fig 1: Functional characterization of kidney stone–associated DGKδ variants.(A) CaSR-mediated SRE and (B) NFAT-RE responses to changes in extracellular calcium concentration [Ca2+]e in HEK-CaSR-DGK cells stably transfected with WT or the kidney stone–associated variants I91V, H190Q, I221N, T319A, V464I, R900H, or R1181W. Transfection with kidney stone–associated DGKD variants led to a reduction in SRE and NFAT-RE responses compared with cells transfected with WT DGKD. (C) Effect of 100 nM cinacalcet (cin) treatment on SRE responses at 3.5 mM [Ca2+]e and (D) NFAT-RE responses at 10 mM [Ca2+]e in HEK-CaSR-DGK cells transfected with the kidney stone–associated variants. Treatment with cinacalcet increased SRE-mediated responses of all variants but had no effect on NFAT-RE responses except for cells transfected with the R900H variant. (E) CaSR-mediated SRE and (F) NFAT-RE responses to changes in [Ca2+]e in HEK-CaSR cells following DGKδ KD (red), which led to a reduction in SRE responses without a change in NFAT-RE responses, compared with WT (black). (G) Effect of 5 nM cinacalcet treatment on SRE responses at 3.5 mM [Ca2+]e in HEK-CaSR cells following DGKδ KD. Treatment with cinacalcet rectified impaired SRE-mediated responses. Mean fold change responses ± SEM are shown for 4 biologically independent experiments. A 2-way ANOVA with Dunnett’s correction for multiple comparisons was used to compare points on the dose response curve with reference to WT. These data provide evidence that KSD is associated with impaired CaSR signal transduction, which can be ameliorated with cinacalcet. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus WT.
Fig 2: Genetic associations of KSD and serum calcium and phosphate concentrations.(A) Meta-analysis of GWASs of data from the UK Biobank and FinnGen including data on 24,167 kidney stone cases and 876,673 controls. Manhattan plot shows genome-wide P values (–log10) plotted against the chromosomal position. Horizontal red line indicates the genome-wide significance threshold (5.0 × 10−8). Loci are labeled with the following primary candidate genes: CASZ1, ALPL, CLDN19, HORMAD1, PTGS2, SLC41A1, SLC30A10, GCKR, RBKS, CYP1B1, THADA, DGKD, COL7A1, WNT5A, HEG1, CASR, ADRAC2, ABCG2, UGT8, ISL1, PDE4D, TMEM171, SLC34A1, FLOT1, HLA-DQA, KCNK5, VEGFA, TFAP2B, PKHD1, RRAGD, ASCC3, L3MBTL3, TCF21, SLC22A2, HIBADH, AQP1, TRPV5, PRKAG2, TMEM252, TRPM6, AOPEP, PARD3, AMPD3, SIK, PRICKLE1, DGKH, CLDN10, PRKD1, AP4E1, PDE8A, UMOD, FTO, ZFPM1, MAP2K4, CDK12, ARHGAP27, ARL17B, SOX9, BCAS3, PTGER1, STAP2, GIPR, ZNF28, CYP24A1, NRIP1, CLDN14, GNAZ, H1-0, and CHADL. Thirty-three of the loci (underlined) have not previously been associated with KSD. (B) Locus zooms from GWASs of KSD and albumin-adjusted serum calcium, serum phosphate, and PTH concentrations at loci, with evidence from regional MR that the risk of KSD is increased via serum calcium and phosphate concentrations and where genetic associations of KSD and serum calcium, phosphate, and PTH concentrations colocalize. (C–E) Associations of genotype with KSD (C), serum calcium concentration (D), and serum phosphate concentration (E) in the DiscovEHR cohort (n = 11,451 kidney stone cases and 86,294 controls). Mean serum calcium (D) and phosphate (E) measurements ± SEM adjusted for KSD case status. Note, in some cases, the SEM is small and obscured by the graphical icon. Associations of combinations of DGKD-, CYP24A1-, and SLC34A1- risk alleles were not assessed for serum phosphate due to a lack of directional concordance. These findings provide evidence that the variants rs838717, rs10051765, and rs6127099 are causal risk factors for KSD acting via reduced CaSR signal transduction, increased urinary phosphate excretion, and impaired vitamin D inactivation, respectively. Het, heterozygous; Hom, homozygous.
Fig 3: Drug target MR.Forest plot of the predicted effects of modulating albumin-adjusted serum calcium concentrations via DGKD, CASR, or CYP24A1 or serum phosphate concentrations via SLC34A1. Gene positions are defined via Ensembl ± 300 kbp. There were insufficient genetic instruments to undertake analyses of modulating serum calcium or phosphate concentrations via DGKD or SLC34A1 using a threshold for genetic independence (r2) of 0.01. These data indicate that reducing serum calcium via DGKD, CASR, or CYP24A1, or increasing serum phosphate via SLC34A1 would decrease the risk of KSD.
Fig 4: Family trees of DiscovEHR kindreds (A–F) were identified as harboring DGKD variants.Squares represent male family members, circles female family members, and ? indicates missing data. Individuals’ ages (years) are shown below the symbols, and the age of the individual at the first record of a kidney stone episode is shown in parentheses.
Supplier Page from OriGene Technologies for DGKD Human shRNA Plasmid Kit (Locus ID 8527)