Calcium-sensing receptor signaling is amplified in patients with a FAM111A gene mutation
Introduction: A nine-year old female presented to the Stollery Emergency Department with seizures and was found to have low blood calcium (Ca2+) levels (0.72 mM ionized), low parathyroid hormone (PTH) and increased urinary Ca2+ excretion. She was diagnosed with Autosomal Dominant Hypocalcemia (ADH), a childhood disorder characterized by low blood Ca2+ and inappropriately low PTH. She had no mutations in the genes known to cause ADH (Ca2+-sensing receptor (CASR) and G-protein subunit alpha 11 (GNA11)). Consequently, we performed whole exome sequencing and a trio analysis that identified a novel FAM111A gene mutation (c.1454G>A, p.C485Y). FAM111A is a putative serine protease with functions largely unknown. However, FAM111A gene mutations are known to cause Kenny Caffey syndrome (KCS) and Osteocraniostenosis (OCS), conditions characterized by low blood Ca2+, low PTH, short stature and bony abnormalities. The molecular mechanism mediating these phenotypes are unknown. However, we hypothesize that mutations in FAM111A cause increased CASR signaling, resulting in low blood Ca2+ and high urine Ca2+ levels. The CASR is a G-protein coupled receptor, which upon activation by extracellular Ca2+, initiates signaling cascades that increase intracellular Ca2+ levels. CASR activation also increases the expression of the tight-junction protein claudin-14 (CLDN14). In the kidney, CLDN14 blocks Ca2+ reabsorption leading to increased urine Ca2+ excretion and lower blood Ca2+. Our objective was to determine if FAM111A wild-type (WT) and mutants affect CASR signaling. Methods: HEK293 cells were transfected with empty vector (EV) as a control, CASR and EV, FAM111A WT with EV or CASR and FAM111A WT or mutants (C485Y, ADH; Y511H, KCS; R569H, KCS; T338A, OCS; P527R, OCS; D528G, OCS; S541A, inactivated protease). We performed Fura2 AM imaging of intracellular Ca2+ in transfected cells in the presence of increasing extracellular Ca2+ (0.5-11.3 mM) levels. Also, as an indication of CASR activity, we measured CLDN14 expression in transfected cells incubated in 0.5 mM or 5 mM extracellular Ca2+, via a dual luciferase assay. Results: The change in the peak intracellular Ca2+ concentration was approximately two times higher (p<0.05) in cells with CASR plus EV or FAM111A mutants, compared to EV alone or FAM111A WT plus CASR. Similarly, luciferase activity as an indication of CLDN14 levels, was significantly higher (p<0.05) in CASR plus EV compared to EV and FAM111A WT plus CASR. Some mutants (C485Y, T338A, P527T, D528G, S542A) had similar CLDN14 levels to CASR plus EV, while others (Y511H, R569H) showed similar levels to FAM111A plus CASR. Conclusions: FAM111A WT attenuates CASR activity. All FAM111A mutations assessed, enhanced CASR activity when measured by an increase in intracellular Ca2+. Some mutants increase CLDN14 expression, while others may affect a different CASR signaling pathway. Elucidating the mechanism of how FAM111A affects CASR activity and Ca2+ homeostasis will provide a better understanding of our patient's condition as well as those with KCS or OCS.