The calcium-sensing receptor (CaSR) is a G-protein coupled receptor that predominantly signals via Gαq/11-mediated pathways to regulate extracellular calcium (Ca2+e) homeostasis. Germline Gα11 inactivating and activating mutations cause familial hypocalciuric hypercalcaemia type-2 (FHH2) and autosomal dominant hypocalcaemia type-2 (ADH2), respectively. To date, four FHH2 and six ADH2 mutations have been reported. To identify novel variants, we investigated large-scale sequencing databases (ExAc, dbSNP), comprising 60,706 exomes from unrelated individuals and the DiscovEHR cohort, comprising exomes from 51 289 patients with matched phenotyping data. We identified 91 missense variants and selected 14 (n=9 ExAc/dbSNP; n=5 DiscovEHR) variants predicted to be pathogenic for functional analysis. Wild-type (WT) or variant GNA11 expression constructs were transiently expressed in CaSR-expressing HEK293A Gαq/11 knockout cells, with Gα11 protein expression confirmed by Western blots. Functional effects on CaSR-mediated intracellular calcium (Ca2+i) release and MAPK activity were assessed using nuclear factor of activated T-cells response element (NFAT-RE) and serum response element (SRE) luciferase reporter constructs, respectively. Two ExAc/dbSNP variants (Gly51Arg and Arg213Trp) significantly reduced NFAT-RE and SRE activity in response to 8 different increasing concentrations of Ca2+e (up to 4.2-fold; P<0.001), whereas a Gln152His variant significantly increased NFAT-RE and SRE activity (up to 1.6-fold; P<0.01), when compared to WT, consistent with these being loss- and gain-of-function variants, respectively. In addition, three variants (Gly66Asp, Arg147Cys and Ala231Thr) significantly increased NFAT-RE reporter activity (up to 2.4-fold; P<0.001) but had no significant effect on SRE response. Two (Arg37Leu and Arg210Trp) of the five DiscovEHR cohort variants identified in patients with mild hypercalcemia (mean plasma calcium >10mg/dL) significantly decreased NFAT-RE and SRE activity (up to 6.5-fold; P<0.001), thereby indicating they likely represent novel FHH2-causing mutations. Thus, our study, which reveals eight novel, rare Gα11 variants affecting two different components of CaSR-mediated signalling, indicates that the prevalence of FHH2 is ˜4 per 100 000 individuals.